Solutions Manual for Biomaterials Science - An Introduction to Materials in Medicine 4th Edition by

Solutions Manual for Biomaterials Science - An Introduction to Materials in Medicine 4th Edition by Wagner, William R.; Zhang, Guigen; Sakiyama-Elbert, Shelly E.; Yaszemski, Michael J.

Solutions Manual for Biomaterials Science - An Introduction to Materials in Medicine 4th Edition by Wagner, William R.; Zhang, Guigen; Sakiyama-Elbert, Shelly E.; Yaszemski, Michael J.

Chapter Questions 1. A stainless-steel rod with a circular cross-section has a length of 100 mm and a diameter of 2.0 mm. It is deformed elastically in compression by a force of 500 N applied parallel to its length. The material has a Young’s modulus of 200 GPa, a shear modulus of 77 GPa, and a yield strength of 0.3 GPa. Calculate (A) the amount by which the rod will decrease in length; and (B) the amount by which the rod will increase in diameter.

Solution: (A) Before we solve the problems, it is helpful to convert all units to the SI units. Doing so, we have the length of the rod L = 0.1 m and diameter d = 0.002 m. By the definition of stress, σ = F/A, we calculate the compressive stress as F − 500 (N) σ= = = − 0.159 (GPa) A π (0.002 m)2 4 Since the induced compressive stress is below the yield strength of 0.3 GPa, the rod is deforming elastically. Moreover, because we are dealing with a rod under uniaxial loading, we can use the simplified Hooke’s Law to capture the linear relationship between stress and strain with: σ = Eε and find the induced strain in the rod σ ε = = – 0.159 (GPa) = − 7.962 × 10 − 4 200 (GPa) E Knowing the strain value, the change in length of the rod can be determined by δ = Lε = 0.1 (m) × ( − 7.962) × 10 − 4 = −7.962 × 10 − 5 (m) = − 7.962 × 10 − 2 (mm) The negative value suggests that the length of the rod is getting shorter due to a compressive force.

(B) To determine the change in diameter, we will need to know the Poisson’s ratio. From the known relationship between Yong’s modulus (E) and shear modulus (G), namely,

G=

E 2 (1 + ν)

we have ν=

E 2G

– 1=

200 (GPa) 2 × 77 (GPa)

– 1= 0.299

From the definition of the Poisson’s ratio ε ν = − transverse εlongitudinal and with the longitudinal strain determined earlier, we calculate εtransverse = − νεlongitudinal = – 0.299 × ( − 7.962) × 10 − 4 = 2.378 × 10 − 4 With this, the change in diameter can be determined as δ = dεtransverse = 0.002 (m) × 2.378 × 10 − 4 = 4.757 × 10 − 7 (m) The positive value suggests that the diameter of the rod is becoming wider due to a compressive force.

2. Use a stress-strain curve to depict the determination of the followings: (A) strain energy, (B) resilience, and (C) toughness.

Solution: Using the stress-strain curve of a typical metallic material, we can sketch the followings.

(A) Strain energy is typically obtained by the area under the stress-strain curve at a given stress which is below the elastic yield point.

(B) Resilience is determined by the area under the stressstrain curve up to the point where stress reaches the elastic yield point.

(C) Toughness is determined by the area under the stressstrain curve up to the point where stress reaches the breaking point.

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Solutions Manual for Biomaterials Science - An Introduction to Materials in Medicine 4th Edition by Wagner, William R.; Zhang, Guigen; Sakiyama-Elbert, Shelly E.; Yaszemski, Michael J.

Questions

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3. Referring to the generalized Hooke’s Law relation and the [c] matric given in Fig. 1.2.3.5A for an isotropic

material, derive the reduced stress-strain relation for a 2D plane-strain and plane-stress situation.

along with

For a plane-stress situation, we first express the above generalized stress-strain relations in strain-stress relations by taking the inverse of the generalized Hooke’s Law matrix equation as

Solution:

With the [c] matrix for an isotropic material given in Fig. 1.2.3.5A, we can write its stress and strain relations according to the generalized Hooke’s Law as,

By substituting the given expressions in this chapter for c11 and c12, we have

For a plane-strain situation, by referring to Fig. 1.2.3.8B ε =γ =γ . the following three strain components are zero: z yz xz Plugging in these zero components into the above matrix equation we have

With simplification, we obtain the following reduced stress-strain relation for a 2D plane-strain situation:

Solutions Manual for Biomaterials Science - An Introduction to Materials in Medicine 4th Edition by Wagner, William R.; Zhang, Guigen; Sakiyama-Elbert, Shelly E.; Yaszemski, Michael J.

Referring to Fig. 1.2.3.8A, we have the three zero stress components: σz = τyz = τxz for a plane-stress situation.

Plug- ging in these zero components into this equation we have With simplification, we obtain the reduced strain-stress relation for a 2D plane-stress situation:

or its inverse stress-strain relation

along with 4. For an isotropic elastic material having Young’s modulus of E =210 GPa and Poisson’s ratio of =0.33, find the [c] matrix for 2D simplified plane-stress and plane-strain situations.

Solution: For a plane-strain situation, by plugging the given values for E and into the reduced stress-strain relations found in Question 3, we have

For a plane-stress situation, by plugging the given values for E and into the reduced stress-strain relations found in Question 3, we get

Solutions Manual for Biomaterials Science - An Introduction to Materials in Medicine 4th Edition by Wagner, William R.; Zhang, Guigen; Sakiyama-Elbert, Shelly E.; Yaszemski, Michael J.

Chapter Questions

6. The image given below shows a helical fracture in a femur bone. Explain your observation.

Apparently, the two [c] matrices are not the same. The [c] matrix of the plane-strain situation is stiffened as compared with that of the plane-stress situation. The two terms in the first row are 3.110×1011 GPa and 1.530×1011 GPa for the plane-strain situation, which are approximately 1.32 and 1.97 times higher than the respective terms in the planestress situation (i.e, 2.360×1011 GPa and 0.778×1011 GPa).

This suggests that treating a plane-stress problem by mistake as a plane-strain problem would lead to stiffening the material significantly. On the contrary, treating a plane-strain problem as a plane-stress one would lead to softening the material.

5. The constitutive relations between strains and stresses for a 2D plane-stress simplified problem can be expressed in a reduced 3×3 matrix equation as follows

Solution: Show that when the situation can be further simplified to a 1D problem, a simple relationship between stress and strain as σx = Eεx Will govern the constitutive relation.

Solution: For a 1D situation, we can further assume σy = τxy = 0. By plugging these two zero terms into the given relations, we have

With simplification, we obtain σx = Eεx along with ε = − νσx and γxy = 0 y

A helical fracture line is surely the result of torsional loading. Referring to the Mohr’s circle and stress elements shown in Fig. 1.2.3.6, it is known that under a torsional load, the femur could break either in a shear failure mode or a tensile failure mode. If in a shear-failure mode, the fracture surface should be in a cross-cutting plane perpendicular to the shaft of the femur because the this plane is where the maximum shear stress lies, and if in tensile-failure mode, the fracture surface will follow a helical line due to the existence of the maximum tensile stress following a helical pattern. From the fracture shown, it is believed that the bone fractured under a torsional load. However, the load was applied very likely at a relatively high speed such that the bone behaves more like a brittle material. In this situation, the tensile strength of the bone becomes the weakest link because it will not be able to resist the highest tensile stress generated in the bone, thereby leading to a tensile-stress induced helical fracture due to the high-speed torsional load. This example highlights the fact that regardless the external loading situations, the actual fracture mode in a material is always dictated by the internal stress state and Poisson’s ratio equation

E The first equation is the 1D Hooke’s Law governed constitutive relation, and the first two equations will lead to the Solutions Manual for Biomaterials Science - An Introduction to Materials in Medicine 4th Edition by Wagner, William R.; Zhang, Guigen; Sakiyama-Elbert, Shelly E.; Yaszemski, Michael J.

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νσx − εy = − − E = ν

εx

σx E

the nature of material, as well as the loading speed in the case of materials of viscoelastic nature.

7. Of the two images given below, one showing the stress trajectories obtained from a finite element analysis of a 2D femoral-head-like structure, and the other showing the X-ray microarchitectures in a femoral head. Explain your observations.

Solutions Manual for Biomaterials Science - An Introduction to Materials in Medicine 4th Edition by Wagner, William R.; Zhang, Guigen; Sakiyama-Elbert, Shelly E.; Yaszemski, Michael J.

Questions

dεd dt

=

σ η

Taking the time derivative of the first two equations, we obtain dε dεs dεd and dεs 1 dσ = = + dt E dt dt dt dt With the three derivative expressions, we can eliminate the εs and εd terms, and arrive at the following governing differential equation in time domain for the viscoelastic material: dε dt

Solution: Inside the thin layer of strong cortical (or compact) bone of a femoral head is coarsely structured trabecular (or cancellous, spongy) bony structure. The microarchitecture of the spongy bony structure is not random, instead it appears to follow the stress trajectories inside the femoral head. According to Wolff’s Law, living bones will remodel in adaptation to the external loads they experience. The microarchitecture of the spongy bone is likely the result of the bone remodeling process in meeting the strengthening needs of human/animal growth and daily activities, guided by the stress trajectories dictated by the internal stress state.

8. A viscoelastic material represented by a spring (with modulus of E) and a dashpot (with viscosity of η) connected in series is illustrated below. The spring is regarded as governed by Hooke’s Law and the dashpot by Newton’s Law. Derive the governing differential equation for the material and find the time-dependent stress function in response to a step function of strain.

Solution: Since the spring and dashpot are connected in series, the stress in the material ( ) will be experienced by the spring (σs = σ) and the dashpot (σd = σ), and the strain in the material will be shared by the spring and dashpot in the following relationship:

=

1 dσ E dt

+

σ η

To find the time-dependent stress function in response to a step function of strain, we first multiply the above differential equation by E and rearrange it to dσ dt

+

E

dε σ=E η dt

Then by taking the Laplace transformation of the above equation, we arrive at the following frequency-domain differential equation ( ) E σ (s) s + = Eε (s) s η in which σ (s) and ε (s) are stress and strain in frequency domain, corresponding to the σ (t) and ε (t) in time domain, respectively, and s is the Laplace variable.

Let a step function of strain take the following form ε0 , t ≥ 0 ε (t) = 0 , t <0 then its Laplace transformed function will be ε0 ε (s) = s Substituting this expression into the frequency-domain differential equation with some rearrangement, we obtain the stress response function in frequency-domain as σ (s) = (

Eε0 ) s+ E η

ε = εs + εd

Solutions Manual for Biomaterials Science - An Introduction to Materials in Medicine 4th Edition by Wagner, William R.; Zhang, Guigen; Sakiyama-Elbert, Shelly E.; Yaszemski, Michael J.

By applying Hooke’s Law to the spring, we have: σ εs = E

and by applying the Newton’s Law to the dashpot, we have:

To find the time-dependent stress function, we will just take the inverse Laplace transformation. Do so leads us to the following stress response function in time domain: ( ) E σ (t) = E ε0exp − t η Clearly, the time-dependent stress function in response to a step function of strain is an exponential delaying function. This behavior is commonly referred to as the stress

Solutions Manual for Biomaterials Science - An Introduction to Materials in Medicine 4th Edition by Wagner, William R.; Zhang, Guigen; Sakiyama-Elbert, Shelly E.; Yaszemski, Michael J.

Chapter Questions

relaxation behavior of viscoelastic materials. Obviously, the stress decays exponentially from Eε0 at t = 0 to zero as t → ∞,

and by the combined spring 2 and dashpot (σd + σ2s = σ), and the strain in the material will be shared by spring 1 and the combo of spring 2 and dashpot in the following relationship: ε = ε1s + ε2 By applying Hooke’s Law to the springs, we have: σ = σ1s = E1ε1s and σ2s = E2ε2s = E2ε2 and by applying the Newton’s Law to the dashpot, we have: dεd dε2 σ =η =η dt

d

as illustrated in the plot given below.

9. For a viscoelastic material represented by two springs and a dashpot as illustrated in the given figure, show its governing differential equation as d dt

+

E1 + E2



 =E

d 1 dt

+

E 1E 2





and its time-dependent stress function in response to a step function of strain as

dt

Substituting the last two expressions into σd + σ2s = σ, we have dε2 σ=η +E ε 2 2 dt With further substitution using ε = ε1s + ε2, σ = E1ε1s dσ dε1s and = E ,we obtain dt

1

dt dε σ = η dt2 + E2ε2 = ηdε −dtdε1s + E2 (ε − ε1s) = ( ) ( ) dσ + E ε − σ η dε − 2 dt E dt E 1

1

After rearranging it, we obtain the following timedomain governing differential equation for the viscoelastic material as ( ) E2 η dσ dε σ 1+ + = E2 ε + η E1 E1 dt dt Or in the following form after some further rearrangement, σ+

η

dσ

E1 + E2 dt

=

E1E2 E1 + E2

ε+

E1η

dε

E1 + E2 dt

Solution:

Solutions Manual for Biomaterials Science - An Introduction to Materials in Medicine 4th Edition by Wagner, William R.; Zhang, Guigen; Sakiyama-Elbert, Shelly E.; Yaszemski, Michael J.

52.e5

This is the governing differential equation for the visco- elastic material represented by the given model.

This equa- tion, with some rearrangement, is exactly the same as that given in the question.

To find the time-dependent stress function in response Since spring 2 (E2) is connected in parallel with the dashpot (η), they will experience the same strain, εd = ε2s = ε2.

Moreover, because spring 1 (E1) is in series with the parallel combo of spring 2 and the dashpot, the stress in the material ( ) will be experienced by spring 1 (σ1s = σ)

to a step function of strain, we take the Laplace transformation of the above equation, and arrive at the following(frequency domain differential equation ) ) ( ηs E1E2 E1ηs σ (s) 1 + = ε (s) + E1 + E2

E1 + E2

E1 + E2

in which σ (s) and ε (s) are stress and strain in the frequency domain, corresponding to the σ (t) and ε (t) in the time domain, respectively, and s is the Laplace variable.

Solutions Manual for Biomaterials Science - An Introduction to Materials in Medicine 4th Edition by Wagner, William R.; Zhang, Guigen; Sakiyama-Elbert, Shelly E.; Yaszemski, Michael J.

Questions

Let a step function of strain take the following form ε , t ≥ 0 ε (t) = 0 0 , t <0 then its Laplace transformed function is ε0 ε (s) = s Substituting this expression into the frequency-domain differential equation along with some rearrangement, we obtain ( ) ( ) ε0 E1E2 + E1ηs ε0 E1E2 + E1s σ ( s) = s E1 + E2 ηsE1 + E2 = ( ηE + E ) 2 1+ s s+ 1 E1 + E2 η This equation can be further expressed by the partialfraction decomposition method as ( ) E1E2 + E s 1 η A B σ (s) = ε0 ( E + E ) = ε0 s + = E 1 2 s s+ η s + 1 +ηE2 2 EE1E +2E E E+1 E ε0

1

s

2 +

1

with A =

E1E2 E1 + E2

E2 and B = E +1 E .

1

2

Taking the inverse Laplace transformation of the above equation, we obtain the following stress response function in time domain: ( ) E1 E2 E12 E1 + E2 t ε0exp – σ (t) = ε0 + E1 + E2 E1 + E2 η Apparently, the time-dependent stress function in response to a step function of strain for this viscoelastic material is still an exponential relaxation function. In this case, the stress decays exponentially from E1ε 0 at t = 0 to E1 E2 , not zero, as t → ∞. ε0 E1 + E2

2

s + E1 + E2 η

Solutions Manual for Biomaterials Science - An Introduction to Materials in Medicine 4th Edition by Wagner, William R.; Zhang, Guigen; Sakiyama-Elbert, Shelly E.; Yaszemski, Michael J.

Chapter Questions 1. Scan the table of contents and abstracts from the last three issues of Journal of Biomedical Materials Research or Biomaterials. List all the surface analysis methods used in the articles therein and briefly describe what was learned by using them.

2. How is critical surface tension related to wettability? For the polymers in Table 1.2.4.2, draw the chemical formulas of the chain repeat units and attempt to relate the structures to the wettability. Where inconsistencies are noted, explain those inconsistencies using Table 1.2.4.3.

3. A titanium dental implant was manufactured by the Biomatter Company for the past 8 years. It performed well clinically. For economic reasons, manufacturing of the titanium device was outsourced to Metalsmed, Inc. Early clinical results on this Metalsmed implant, supposedly identical to the Biomatter implant, suggested increased inflammation. How would you compare the surface chemistry and structure of these two devices to see if a difference that might account for the difference in clinical performance could be identified?

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Solutions Manual for Biomaterials Science - An Introduction to Materials in Medicine 4th Edition by Wagner, William R.; Zhang, Guigen; Sakiyama-Elbert, Shelly E.; Yaszemski, Michael J.

Chapter Exercises Consider having two types of homogeneous surface chemistries: one with hydrophilic hydroxyl (-OH) groups and the other with hydrophobic methyl (-CH3) groups. (a) Which surface would more strongly adsorb a protein in air? (b) Which surface would more strongly adsorb a protein in water?

Answers: (a) In air, the –OH surface would more strongly adsorb the protein due to the fact that it would be able to form the strongest type of van der Waals (vdW) interactions (i.e., hydrogen bonds) with the polar groups of the protein, as well as forming moderately strong dipole-induced dipole vdW interactions with the nonpolar groups of the protein. In comparison, the -CH3 surface would only be able to form the moderate and very weak vdW interactions

with the protein (i.e., induced dipole–dipole and induced dipole–-induced dipole interactions, respectively). (b) In water, the -CH3 surface would more strongly adsorb the protein due to the ability of water molecules to form hydrogen bonds with the –OH groups of the surface, which will directly compete with the protein, thus weakening the –OH surface’s bonding with the protein. In contrast, the -CH3 surface would form very strong hydrophobic interactions with the hydrophobic domains of the protein due to the thermodynamically unfavorable interactions between the -CH3 groups of the surface and the surrounding water.

Suggested External Reading Voet, D., Voet, J.G., Pratt, C.W., 2016. Fundamentals of Biochemistry: Life at the Molecular Level, fifth edition, Chapter 2: Physical and Chemical Properties of Water, John Wiley & Sons, pp. 23–41.

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Chapter Exercises 1. Describe the structure of the polyurethane molecule and how it gives rise to the favorable properties of polyurethanes, such as high strength and toughness.

2. Describe the differences between thermosets and thermoplastic elastomers and give examples of each of these types of materials that are useful for biomedical devices.

3. Draw schematics of monodentate and bidentate hydrogen bonding between urethane and urea groups in the hard segments of polyurethanes. Discuss how hydrogen bonding between adjacent hard segments enhances polyurethane properties.

4. Discuss the targeted design properties for resorbable polyurethanes for tissue repair and nonresorbable

polyurethane permanent implants. How can the properties of the polyurethane be tailored to the application by modifying the structure?

5. Draw the schematic for synthesizing a nonresorbable thermoplastic elastomer from MDI, butanediol, and poly(tetramethylene oxide) (PTMO, 2000 g/mol).

Highlight the hard and soft blocks in the polyurethane molecule.

6. Draw the schematic for synthesizing a resorbable polyurethane thermoset from LDI and a polyester triol (900 g/mol).

7. List the optimal mechanical, rheological, biological, and degradation properties of polyurethane elastomers for 3D printing scaffolds for tissue repair using fused deposition modeling.

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Solutions Manual for Biomaterials Science - An Introduction to Materials in Medicine 4th Edition by Wagner, William R.; Zhang, Guigen; Sakiyama-Elbert, Shelly E.; Yaszemski, Michael J.

Chapter Questions 1. Which material property was the basis for Charles Holter’s selection of silicone rubber for the hydrocephalic shunt valve? a. Biostability b. Hemocompatibility c. Thermal stability d. Viscoelasticity e. All of the above

2. Among the following applications where silicones are still in use today, which was the first reported human implantation of a silicone elastomer medical device? a. Breast augmentation b. Bile duct repair c. Hydrocephalus shunt d. Urethra repair

a. Would a standard manufacturing facility be sufficient for the production of the catheter or would either cGMP (good manufacturing practice) or clean room facilities be required? How would each process affect the bio-burden and subsequent sterilization required? b. What types of sterilization techniques can be used with silicone elastomers? c. What are the considerations for each sterilization technique? d. What are the consequences of selecting an inappropriate sterilization technique? e. How would the biomaterial or device be assessed poststerilization? How do you guarantee sufficient sterilization?

Chapter Answers

3. What functions can fillers serve in silicone elastomers? a. Material extender to reduce bulk cost b. Reinforce the cross-linked polymer matrix c. Provide color pigmentation d. Enhance visibility on X-ray e. All of the above

4. Draw the molecular structure represented by the shorthand notation TM2MH. What is its chemical name?

5. What properties of silicones are responsible for their (a) biodurability and (b) biocompatibility?

6. For the following biomaterials design scenarios, select the preferred material and provide your assumptions and justifications for doing so. a. Your customer is designing a syringe. They want to select a material capable of lubricating the interior surface of the syringe to reduce the force required to depress the plunger. Would you select a silicone fluid or a silicone elastomer? b. You have two material options for designing an indwelling urinary catheter: (1) silicone elastomer or (2) thermoplastic polymer, e.g., polyvinyl chloride (PVC).

Which would you select? c. You are designing a wound dressing. It is intended to be used on geriatric patients who are suffering from chronic wounds. The adhesive must be selected such that it provides secure attachment yet gentle release from the skin.

Would you select an acrylic adhesive or a silicone tacky gel adhesive? d. An artificial tendon is being designed and is intended for implantation. You are primarily concerned about the tensile strength of the material. Would you select a silicone elastomer, a polyurethane elastomer, or PEEK (polyether ether ketone)?

7. Case Study: Sterilization of a silicone elastomer (Foley catheter, neonatal catheter, tubing).

One of the most significant challenges to consider when designing a medical device is whether and how to sterilize it. Sterilization, which can help to ensure the safety of the device for implantation, can also drastically affect material properties and ultimate stability. Discuss each of the following topics as they would affect the sterilization of a silicone elastomer being used as a Foley catheter, neonatal catheter, tubing, etc.

Question 1 The most correct answer is c. While hemocompatibility, biostability, and viscoelastic properties are all related to silicone’s successful application in shunts for the treatment of hydrocephalus, thermal stability was the basis for Holter to select silicone rubber for his valve. Steam sterilization deformed other rubbers he tried.

Question 2 The correct answer is b. While many believe that Holter’s silicone hydrocephalic shunts in 1956 were the earliest use, the first reported human implantation of a silicone elastomer medical device was actually a decade before. In April 1946, Dr. Frank Lahey used silicone elastomer materials from the experimental laboratory of General Electric Company called “bouncing clay” for bile duct repair. Silicone materials continue to be used in each of these applications today. The vast majority of today’s breast implants still have shells made of silicone elastomer. Silicone stents and T-tubes remain in use for biliary repair. Valved hydrocephalus shunts of silicone—including the popular Pudenz-Schulte and Holter types—continue to save lives. Silicone tubes and catheters continue to find wide urologic use, including in the surgical repair of epi- and hypospadias.

Question 3 The correct answer is e, all of the above. Fillers such as fumed amorphous silica can be used to both extend the material and reinforce the cross-linked polymer matrix. Silicone-compatible dyes and pigments can modify the color of the elastomer. And additives such as barium sulfate increase radiopacity if necessary so implants are visible on X-ray.

Question 4 The long-hand name for TM2MH is 3-dimethylsiloxy-heptamethyltrisiloxane. Its structure is below: 124.e1

124.e2

Answers

polydispersity of silicone polymers and over the resulting material properties.

Question 6

Question 5 a. The biodurability of silicone materials is attributed to their tremendous thermal and chemical stability, afforded to them by three specific properties: (1) Silicon, when compared to carbon, has much lower electronegativity (χSi ≈ 1.80, χC ≈ 2.55). In the case of the Si–O bond, this electronegativity difference leads to a more polar bond with a high bond energy, which is highly resistant to homolytic scission. This provides improved stability over organic polymers with primarily C–C bonds.

(2) The side methyl groups on the siloxane polymers shield the polar Si–O backbone from strong intermolecular interactions that could result in degradation.

(3) Additionally, the Si–O bonds have a low barrier to rotation (3.3 kJ/mol around a Me2Si–O bond, compared to 13.8 kJ/mol for an H2C–CH2 bond). This enables silicone polymers to adopt the most energetically favorable configuration. b. The biocompatibility of silicones is also due to a combination of unique properties: (1) Due to its hydrophobic nature, any implanted silicone material undergoes rapid protein adsorption (opsonization) to its surface. This can limit the host response to the foreign material.

(2) The cross-linked and/or long-chain silicone polymers used in medical materials will not leach over time in the relatively aqueous environments found within the body. This is due to their hydrolytic stability, which prevents the degradation of these larger silicone molecules into lower molecular weight silicones that would leach more readily. The one exception is in the acidic environment of the stomach, where the elevated level of hydrochloric acid is capable of attacking PDMS over long residence times.

(3) Silicones have high permeability to oxygen, nitrogen, and water vapor due to their high free volume compared to hydrocarbons.

(4) Silicones are widely considered to be hemocompatible, having very low hemolytic potential.

(5) Finally, silicones are able to be manufactured with high levels of purity, with tight controls over the

a. The correct answer is silicone fluid. Silicone elastomers, even when filled with silica that tends to reduce bulk adhesion, remain very tacky and sticky and would not impart a lubricious surface to the interior of a syringe.

Silicone fluids, on the other hand, are able to readily form a film on the interior surface (e.g., barrel) of a syringe due to their critical surface tension of wetting being lower than their own surface tension. This leaves a lubricating surface on the syringe and, additionally, can help prevent blood coagulation. b. The correct answer is the silicone elastomer. Although widely used in urinary catheters due to their low cost, PVC (and other thermoplastic) catheters are less flexible than their silicone counterparts and the plasticizers required to soften the material can have deleterious effects over long-term use. Silicone elastomer catheters have excellent biocompatibility, but can require lubrication to easily insert the catheter into the urinary tract. c. The correct answer is the silicone tacky gel adhesive. In patients with compromised skin, such as the geriatric population, a silicone soft skin adhesive dressing will provide secure attachment of a wound dressing while maintaining a gentle release profile, which is less likely to further damage the skin. Acrylic adhesives are inexpensive, can be coated at low coat weights, and provide strong attachment to the skin. This strong attachment, coupled with acrylic adhesives potentially sensitizing skin over repeated exposures, makes it a less gentle alternative for wound dressings. d. The correct answer is PEEK. The silicone elastomer, even at comparable durometers, will have significantly lower tensile strength than either polyurethane or PEEK. In an artificial tendon application, the implant must be able to match the tensile strength of a tendon to withstand the mechanical loads required in use without failure.

Question 7 a. The cleaner the manufacturing facility, the cleaner the surface of the material will be prior to sterilization. A cleaner surface will require less exposure to harsh sterilization conditions, which will limit the possibility of the sterilization process negatively impacting the robustness of the material/device. This needs to be balanced with the feasibility of manufacturing in these environments, e.g., cost, access. b. Among the most common methods, ethylene oxide, dry heat, and autoclaving can be used to sterilize devices comprised of silicone elastomers. Depending on the material and other parameters, gamma sterilization and e-beam (also known as beta) sterilization can also be used with silicone materials.

Solutions Manual for Biomaterials Science - An Introduction to Materials in Medicine 4th Edition by Wagner, William R.; Zhang, Guigen; Sakiyama-Elbert, Shelly E.; Yaszemski, Michael J.

124.e3

Chapter Answers

c. What are the considerations for each sterilization technique?

(1) Ethylene oxide. Exposure to ethylene oxide will successfully sterilize silicone elastomers. However, complete outgassing of the ethylene oxide gas must be verified. Any remaining EtO and/or byproducts of its reaction with water, e.g., ethylene glycol, can have significant and deleterious effects upon implantation.

The device packaging, therefore, must be permeable to not only EtO, but those other byproducts.

(2) Autoclave/Dry Heat. Due to the thermal and moisture resistance of silicone elastomers, autoclaving is generally considered a well-tolerated sterilization technique for these materials. Similarly, dry heat sterilization is used regularly with silicone elastomers due to their thermal resistance.

(3) Gamma Sterilization/e-Beam. Although these techniques will sterilize the silicone elastomer, they can potentially further cross-link the material and change its mechanical properties. The dose will directly affect the extent of the change. d. One needs to balance the sterilant dose that a material can withstand with the dose it receives. For polymers, in particular, you need to minimize the amount of radiation a material receives while still ensuring a sufficient sterilization effect. An inappropriate sterilization technique will not allow the user this balance, resulting in either an insufficiently sterilized material or a material whose properties have been negatively impacted and that can no longer perform as needed. e. According to ISO 10993-1, a battery of biological evaluation tests shall be performed either on the final product, or representative samples from the final product, or

materials processed in the same manner as the final product (including sterilization). This is important because the processing, especially sterilization, impacts these materials and their properties. The critical performance parameters should also be reevaluated with the sterilized material to ensure no significant change has occurred.

The challenge is to balance sufficient sterilization with maintaining the material properties of the device. Sufficient sterilization ensures that the dosage exceeds the bioburden with adequate safety margin (overkill). Typically, sterilization assurance is performed by either dosimetric or parametric release or a combination of both.

Dosimetric release is defined as the release of terminally sterilized batches or lots of sterile products based on physical data from the irradiation process instead of direct sterility testing. This includes appropriate process qualification to verify that the irradiation cycle provided the required minimum dose to the product. Parametric release is defined as the release of terminally sterilized batches or lots of sterile products based upon compliance with the defined critical parameters of sterilization without having to perform the requirements under pharmacopeial sterility tests, such as USP 〈71〉. Parametric release is a possibility when the mode of sterilization is very well understood, the physical parameters of processing are well defined, predictable, and measurable, and the lethality of the cycle has been microbiologically validated through the use of appropriate biological indicators or, in the case of ionizing radiation, the appropriate microbiological and dosimetric tests. The use of parametric release for sterilization processes requires prior FDA approval.

Chapter Exercises 1. Fluoropolymers are commonly considered “inert.” Teflon PTFE for example does not dissolve in any known solvents. Are fluoropolymers biologically inert? Explain.

Answer: Fluoropolymers of all types and chemistries are well recognized to interact actively with all biological components: proteins, platelets, bacteria, fungus, algae, cells. Tight protein adsorption to fluoropolymer surfaces from different fluids (e.g., serum, plasma, blood, simple buffered protein solutions) is reported. Blood coagulation and complement activation are well documented.

In fact, some fluorinated implants (e.g., ePTFE vascular grafts) are preclotted in patient fluids (e.g., whole blood) to passivate them prior to implantation. Cells and bacteria also adhere and proliferate under both in vitro and in vivo conditions. Fluoropolymers are not biologically inert. They do, however, appear to prefer to adsorb certain proteins from mixtures that then bias their subsequent interactions with other biological components.

PVDF protein blotting membranes are passivated with various masking proteins (e.g., casein) to prevent adsorption of other proteins during protein blotting and transfers. Additionally, many fluorinated surfaces will adsorb high amounts of serum albumin from blood, plasma fractions, and mammalian sera. This “albumin passivation” renders these surfaces resistant to other protein adsorption, cell and platelet adhesion, and blood coagulation activation.

2. PTFE under mechanical loading suffers from creep: describe this problem and its impact on implant

applications where durable mechanical properties under compression or cyclic loading are essential.

Answer: Creep and cold flow are classic problems for PTFE polymers under continuous loading that result in deformation. PTFE failures in certain biomechanical applications experiencing loading (e.g., temporal mandibular knee hip joint bearing surfaces) are notable examples. Despite low coefficients of friction that make PTFE attractive in loadbearing surfaces, PTFE implant experiences to date in mechanical loading have not been promising (witness the early Charnley hip bearing and TMJ joint failures).

3. RGP contact lens materials based on glassy polymethylmethacrylates have included fluorinated copolymers with either silicone polymers or as perfluorohydrocarbons. What is the functional advantage for including fluorinated materials in these contact lenses?

Answer: Due to their polymer chemistry, RGP lenses do not contain significant amounts of water, affecting their mechanical properties, comfort, and oxygen through-lens transmissibility. RGP lenses rely instead on microscopic pores in the lens to transmit oxygen to the cornea. But RGP lenses exhibit limited oxygen permeability, compromising several contact lens properties.

As highly fluorinated materials have intrinsically high oxygen permeability, fluorinated lenses provide an alternative transport mechanism to improve oxygen permeability through the lens compared to the aqueous phase in conventional soft lens materials. Additionally, fluorine content in an RGP lens influences lens flexibility, lipid/ protein deposition properties, and cost considerations.

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Chapter Exercises 1. Identify the major milestones in the evolution of dental composite restorative materials and predict what future modifications may be forthcoming to produce more ideal formulations.

2. Describe the chemistry involved in the initiation and propagation of the polymerization of dimethacrylate monomers and explain the importance of maximizing the extent of the curing reaction.

3. Describe the origin of polymerization stress in dental composite restorations, and explain what chemical modifications to current formulations have been attempted to mitigate it.

4. Identify the chemical difference between the monomers used in self-adhesive and etch-and-rinse dental adhesive formulations, and explain the mechanism of adhesion of each to dentin and enamel tissues.

5. Explain what clinical benefit may be derived from the incorporation of an antimicrobial monomer in a dental resin, and discuss how such monomers demonstrate their activity.

6. Discuss how the monomers used in dental composite and adhesive formulations can be chemically and physically degraded within the oral cavity, and identify potential changes in formulation that can protect the polymerized resin and make it more degradation resistant.

7. Explain chemical approaches to enhance the toughness of dental resins used for composite restorations, as well as the need for improved toughness.

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Chapter Exercises 1. Define the term erosion.

2. What are the major routes of degradation for an orally administered drug? How could the activity of this drug be extended?

3. What factors should be considered when designing a photodegradable polymeric device?

4. How could a nondegradable polymer be useful in a degradable biomaterial?

5. Proteinase K has been added to a PLA device. How quickly will the device degrade? What are the degradation products?

6. How would the rate of degradation change for polymers made with a macrolactone (C8–C17) over PCL? Why does the degradation rate change?

7. What polymer composition, degradation rate, and delivery route would you use for a dental fixative?

8. What are the important factors to consider when designing biodegradable nanoparticles to deliver therapeutics?

9. Research an FDA-approved commercial PLGA product of your choice that was not covered in this chapter. Identify as much information about the polymer properties as you can (G:L ratio, molecular weight, end group chemistry, processing, etc.) and then use this information to explain why this specific design was well suited for the biomaterial’s application.

10. Biochronomer, a poly(ortho ester)-based technology for controlled release applications, is used in two commercially available therapeutics, CINVANTI and SUSTOL, loaded with aprepitant and granisetron, respectively. Aprepitant has a logP value of 4.5 while granisetron has a logP value of 2.6, where a higher logP corresponds to more lipophilic drugs. Assuming the polymer formulation and drug loading are the same in both therapeutics, which treatment would you expect to have faster drug release? Why?

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Questions 1. For the titanium–TiO2 surface, find the Pilling–Bedworth ratio for this system. Would you expect this alloy– oxide film system to result in a passive behavior based on this result? Why or why not?

2. For a metal–solution interface with properties of the solution resistance of 100 Wcm2, polarization resistance of 106 Ωcm2, and constant phase impedance ZCPE = 1/ ((iω)0.89 10−5), plot the Bode diagrams (for log|Z| and θ. Also, plot the Nyquist diagram and the Z″ vs. log ω, A″ vs. log ω. Finally, find the frequencies (in rad/s) associated with the reciprocal of the time constants, τ1 = (RsQ)1/α, τ2 = (RpQ)1/α, and identify these on the log|Z| versus logω Bode plot. Where do these sit?

3. For an oxide film-covered surface, plot the Mott– Schottky plot for ND = 1022 carriers/cm3 and 1012 carriers/cm3. How does the change in carrier density affect the plot and the ability of the charge to transit the oxide?

4. Explain how shot peening results in the development of compressive residual stresses in the surface of a metal and how these stresses enhance the fatigue crack initiation resistance of the alloy?

5. Using the Butler–Volmer equation (see equation in the section “Electrochemical Currents [Evans Diagrams]”) recreate the plot in Fig. 1.3.3.17 (use Excel or Matlab).

Using this diagram show an additional plot and explain why increased cathodic reaction exchange current density will shift the corrosion current and corrosion potential for the anodic reaction.

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Chapter Exercise 1 1. Explain the term “biocompatibility” in metallic biomaterials.

2. What are the two unique behaviors of NiTi alloys that make them promising for a variety of implant applications?

3. Which of the following alloys contain allergic and carcinogenic elements? (a) Ti-13Nb-13Zr (b) Ti-6Al-7Nb (c) Co–20Cr–15W–10Ni

4. Explain “stress shielding” in metallic hip implants and its potential consequences.

5. Which of the following implant materials can possibly exhibit the stress-shielding effect when implanted as hip prostheses? (a) Ti-15Mo (annealed) (b) Ti-13Nb-13Zr (aged) (c) Co-Cr-Mo (ASTM-F75)

6. What are the advantages of using Ti alloys over other metallic biomaterials for orthopedic implant applications?

7. Sort the elastic modulus of the following biomaterials in a descending order: (1) AZ91D magnesium alloy (AZ91D), (2) CP-Ti, (3) Co-Cr-Mo alloy (ASTM-F75) (Co-Cr-Mo), and (4) stainless steel (316L). (a) AZ91D > CP-Ti > Co-Cr-Mo > 316L (b) CP-Ti > AZ91D > Co-Cr-Mo > 316L (c) 316L > AZ91D > CP-Ti > Co-Cr-Mo (d) Co-Cr-Mo > 316L > CP-Ti > AZ91D

8. What are the advantages of using β-type Ti alloys over α-type Ti alloys for orthopedic applications?

9. Which of the following sets of properties should loadbearing implant materials have? (a) High elastic modulus, high fatigue strength, high tensile strength, high wear resistance, and high corrosion resistance (b) Low elastic modulus, high fatigue strength, high tensile strength, high wear resistance, high corrosion resistance (c) High elastic modulus, high fatigue strength, high tensile strength, low wear resistance, high corrosion resistance

Chapter Exercise 2 1. Explain the term “osseointegration” in metallic biomaterials.

2. Which of the following biomaterials exhibit the lowest wear resistance? (a) CP-Ti (Grade 1) (b) Co–20Cr–15W–10Ni (c) Stainless steel (316L)

3. What is the effect of interstitial oxygen atoms on the mechanical properties of Ti alloys?

4. Which of the following Ti alloys exhibit better coldforming ability?

(a) α-type Ti alloys (b) β-type Ti alloys

5. Why is fatigue strength important for metallic biomaterials?

6. Why do surface properties of Ti alloys differ from their bulk properties?

7. Which of the following contains information on critical anodization parameters? (a) Applied potential, type of electrolyte, time, temperature (b) Laser pulse intensity, laser energy, exposure time

8. What are the most commonly used coloring methods for Ti alloys?

9. What are the key steps involved in the additive manufacturing of Ti structures?

Chapter Exercise 3 1. Which of the following materials exhibit shape memory property? (a) CP-Ti (Grade 1) (b) NiTi alloy (c) Stainless steel (316 SS) (d) Co-Cr-Mo (ASTM-F75)

2. Which of the following techniques can fabricate complex Ti geometrical shapes? (a) Powder metallurgy (b) Additive manufacturing (c) Casting

3. What are the complications of using stainless steels for orthopedic applications?

4. Why cannot α-type Ti alloys be strengthened by heat treatment?

5. Porous Ti alloys with the desired porosity ratio and pore size may be produced in the required geometric shape without the use of any space-holder material via: (a) Casting (b) Selective laser sintering (c) Powder metallurgy

6. Which of the following is an additive manufacturing process? (a) Cold rolling (b) Hot isostatic pressing (c) Electron beam melting

7. Co-based alloys have higher wear resistances than Ti alloys? (a) True (b) False

8. Which of the following methods is commonly used to grow oxide nanotubes on Ti alloys? (a) Forging (b) Anodization (c) Additive manufacturing

9. What are the key strategies to avoid stress shielding in orthopedic surgeries?

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Questions 1. Describe the advantages and disadvantages of CrCr alloys for biomedical applications.

2. What are the typical processing routes for CrCo alloys?

3. Why is additive manufacturing becoming popular for making CoCr alloy-based implants?

4. Describe typical biocorrosion testing methods for CoCr alloys. What parameters can influence biocorrosion of CoCr alloys?

5. What kind of microstructures are preferred for CoCr alloys and why? What is the purpose of adding Mo or W in CoCr alloys? What is the role of Cr in CoCr alloys?

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Chapter Questions (a) Explain why “nearly bionert” bioceramics are not completely bionert.

[3 marks] (b) Define the term “bioactive ceramic”.

[2 marks] (c) Explain how the general immune response to implants leads to the mechanism of bone bonding for a synthetic hydroxyapatite (sHA) bone graft.

[5 marks] (d) Explain how biological apatite and stoichiometric sHA differ.

[3 marks] (e) Discuss how the bioactivity mechanism of Bioglass differs from that of sHA. [4 marks] (f) Describe the synthesis chemistry and processing required to produce an sHA powder.

[4 marks] (g) Describe the design criteria for bioactive ceramic scaffold for bone regeneration.

[6 marks] (h) Explain why Actifuse contains some Si [2 marks] (i) Explain why it is challenging to produce porous 45S5 Bioglass.

[2 marks]

Questions with answers Here are the answers (a) Explain why “nearly bionert” bioceramics are not completely bionert.

[3 marks] Answer: They do illicit a response from the body in that the body recognises them as foreign, triggering an inflammatory response (b) Define the term “bioactive ceramic”.

[2 marks] Answer: Historically a hard crystalline material (ceramic) that can form bond with bone. Now modified to one that stimulates a positive biological response from the body. (c) Explain how the general immune response to implants leads to the mechanism of bone bonding for a synthetic hydroxyapatite (sHA) bone graft.

[5 marks] Answer: macrophages recruited: macrophages become osteoclasts; digestion of HA as it is similar to bone mineral; Ca and phosphate ion release; precipitation of Ca and phosphate as HCA; osteoblasts produce new bone

(d) Explain how biological apatite and stoichiometric sHA differ.

[3 marks] Answer: biological apatite is calcium deficient and carbonate substituted (for phosphate) sHA has Ca/P ratio of 1.67 (e) Discuss how the bioactivity mechanism of Bioglass differs from that of sHA. [4 marks] Answer: Bioglass dissolution is Solution mediated via ion exchange, silica rich layer formation. Ions cause Osteostimulation– ions stimulate osteogenic cells to produce more bone (f) Describe the synthesis chemistry and processing required to produce an sHA powder.

[4 marks] Answer: solution precipitation by the equation 10 Ca(OH)2 + 6H3PO4 → Ca10(PO4)6(OH)2 + 18H2O Once aged, the precipitate is thoroughly washed, filtered and dried. (g) Describe the design criteria for bioactive ceramic scaffold for bone regeneration.

Answers [expected to be able to list at least 6 of ]: • Biocompatible • Inflammation?

• No immuno-rejection • No formation of fibrous capsules; • Be easily and cheaply produced to ISO/FDA/CE standards (must be easily sterilised) • Have mechanical properties similar to the host tissue • Act as template for tissue growth in 3D; • Have an interconnected macroporous network for vascularisation, tissue ingrowth and nutrient delivery; • Bond to the host tissue without the formation of scar tissue; • Resorb at the same rate as the tissue is repaired • Provide signals to the cells [6 marks] (h) Explain why Actifuse contains some Si [2 marks] Answer: to increase dissolution rate by introducing some disorder in the crystal structure (particularly at grain boundaries); soluble silica dissolution (Si release) can stimulate osteogenesis (i) Explain why it is challenging to produce porous 45S5 Bioglass.

[2 marks] Answer: the original Bioglass composition will crystallise on sintering, losing its amorphous structure.

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Questions 1. What are the differences in properties between alumina, zirconia, and ZTA and why are these differences important?

2. Materials are often sensitive to thermal history. Why would a batch furnace and a belt furnace yield different thermal histories?

3. What was the original reason for the addition of chrome to the ZTAs used for hip replacements in the early 2000s?

4. What different toughening mechanisms are available in medical-grade zirconia?

5. What are the different hardening and toughening mechanisms found in the ZTAs used in medical devices?

6. In zirconia, what is the transformation that causes toughness and how does it work?

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Chapter Questions and Answers 1. Classify the following carbon materials regarding their dimension (size/grain size) and carbon hybridization DIMENSION (size/grain size)

0D

1D

2D

HYBRIDIZATION RATIO

3D

Sp2 Sp3

Diamond MCD

>1

Sp2 Sp3

X

X

X

X

NCD

X

X

UNCD

X

X

ND

X

X

DLC

X

PyC

X

X

Graphite

X

X

Fullerene

X

CNT Graphene

X X

CF

X X

GNR CD

X

GQD

X

X

X X

CNF

<1

X

X

X

X

X

2. One of the major constraints associated to the application of carbon materials colloids in biological systems is their aggregation in aqueous media. Describe a currently used strategy to improve it, explaining the changes in interfacial properties.

Answer: Due to the high content of carbon atoms, carbon materials exhibit a high hydrophobicity which compromises their solubility and/or dispersion in aqueous media, such as the biological systems. The most common strategies to prevent aggregation are carbon materials oxidation or their functionalization with hydrophilic molecules such as polyethylene glycol, since these will increase their oxygen containing groups and therefore make them more hydrophilic.

3. Classify the following sentences as true (T) or false (F) 3.1. Discovery of PyC, fullerene and G is associated to attribution of three Nobel prizes. F 3.2. For biomedical applications, carbon materials can be used as colloids, free-standing materials, coatings, or composites. T 3.3. PyC, and MCD, NCD and UNCD are synthetized as a film being frequently used as a surface coating of biomaterials. T 3.4. DLC, PyC and ND are carbon materials already applied in commercially available biomaterials. T 3.5. Despite their small size, fullerenes can’t penetrate the mammalian cell membrane. F 3.6. Drugs can only interact with carbon materials by covalent interaction. F 3.7. Due to their high surface area and aspect ratio, CNT can be loaded with high amounts of molecules (e.g., drugs, antibodies, peptides) and used

X X

to deliver them intracellularly, by permeating cell membranes. T 3.8. The good dispersion in water and biocompatibility of CD and GQD are attributed to their small size (<10 nm) and oxygen content. T 3.9. ND present strong absorption and fluorescence in the UV region, and strong Raman signals. T 3.10. GBM outstanding optical properties perspective their application in bioimaging. F 3.11. CNT and GBM have strong absorption of NIR radiation, enabling their use in photothermal therapy. T 3.12. Most colloidal carbon based materials have antibacterial effect by acting on bacterial membrane. T 4. Select the correct answer: 4.1. Carbon-based coatings: a) have been used for decades to enhance biomaterials performance. b) of DLC are used in commercially available orthopedic implants. c) of DLC are used in commercially available stents and ventricular assisted devices. d) Two of the above. e) All of the above.

4.2. Carbon materials composites: a) of CNT and GBM are used to improve the polymer conductivity. b) of CNT and GBM are used to improve the polymer mechanical properties. c) of ND are commercially available for dental applications.

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d) of CF are commercially available for dental applications. e) all of the above.

4.3. Mechanical heart valves that contain carbon materials: a) can be made of PyC. b) are commercially available. c) have good hemocompatibility and therefore do not require systemic anticoagulation therapy. d) Two of the above. e) All of the above.

4.4. Considering carbon materials application in phototherapy: a) ND with ideal crystalline structure are fluorescent. b) Fullerenes present strong absorbance at wavelengths above UV region. c) Graphene decreases fluorescence quenching of dyes and sensitizers. d) CD have poor photostability. e) None of the above.

4.5. Considering application in biosensors, carbon materials: a) can be used as the receptor that detects the molecule. b) can be used as the signaling transducer which converts the physical/chemical signal. c) can be used as a signal processor. d) Two of the above. e) All of the above.

4.6. Considering carbon materials application in antimicrobial therapy: a) Fullerenes have an inherent antiviral action. b) GBM antimicrobial mechanisms of action are different for GBM colloidal dispersions and GBM containing-surfaces. c) The coating production technique influences the antibacterial outcome of the surface d) Two of the above. e) All of the above.

5. Identify the main applications under study or commercially available for the carbon biomaterials presented in the table below:

Drug delivery Phototherapy Imaging Biosensors Antimicrobial Cardiovascular Orthopaedics Neuronal Dental NCD/UNCD ND

X X

X

X

X

X

DLC

X

PyC

X X

X

X

X

X

Graphite

X

Fullerene

X

X

X

X

X

X

X

X

CNT

X

X

X

X

X

X

X

X

GBM

X

X

X

X

X

X

X

X

X

X

X

CF CD/GQD

X

X X

X

6. Using a carbon nanomaterial of your choice, describe its main properties and give an example of a biomedical application, explaining how/why it can be used.

Answer: This question can have numerous possible answers. However, it should be taken into account that carbon nanomaterials have at least one dimension smaller than 100 nm, so only 0D, 1D or 2D carbon materials should be selected. Students are intended to highlight the main properties of one of these nanomaterials and correlate them with a selected biomedical application, explaining the rational for the design of the medical device.

7. Provide example of one success and one failure on carbon-based materials applications in clinics.

Answer: For more than 50 years, pyrolytic carbon has been used in more than 25 different mechanical heart valves (MHV) designs, and nowadays 95% of all MHV have at

X X

X

least one structural component made of this carbon biomaterial. The balance between blood compatibility, physical

and mechanical properties, and durability made PyC the best material option available in MHV. A 17-year follow-up study in 500 patients implanted with Sorin MHV revealed that only five reoperations were necessary, and that there was a total of 15 valve-related deaths.

Failures were observed in diamond-like carbon (DLC) coated implants for orthopaedics. In 2001, Diamond Rota Gliding (Implant Design, Switzerland), a DLC-coated ultra- high molecular weight polyethylene (UHMWPE) knee joint, was commercialized. However, some of the implants showed increased wear and partial delamination, with the residual coating on the upper side of the implant leading to inadequate bone ingrowth. As such, the use of this implant was forbid- den. In 2003, results were published from an 8-year clinical follow-up of 100 patients implanted with DLC-coated Ada- mante (Biomécanique, France) femoral heads, articulating against polyethylene. After 1.5 years, aseptic loosening was found, with implant revision being needed, and after 8 years almost half of the implants had to be replaced.

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Questions 1. What strategies can be used to control the temporal, spatial, and type of released bioactive molecules from blood derivatives?

2. Which advantages and disadvantages do natural polymers offer?

3. What characteristics should an ideal ECM-mimetic hydrogel have?

4. What characteristics of DNA are critical to enable its use as a biomaterial?

5. Identify two different types of supramolecular cross-linking strategies that can be applied to produce reversible hydrogels using polymeric precursors.

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Questions 1. What are the issues with classical cryopreservation (freezing) on tissue preservation? How are these issues addressed with vitrification? What limitations still exist with tissue preservation despite minimizing structural damage to tissue at low temperatures? a. Classical cryopreservation causes immobilization of solute components by freezing of water and formation of ice crystals causing extracellular tearing, cell membrane lysis, and segregation of solute molecules, leading to a toxically high concentration of solute molecules due to displacement from organized crystal structure. b. Vitrification creates a high-viscosity, glass state due to the relatively high concentration of cryoprotectant and rapid freezing leading to immobilization of solute components faster than ice crystal formation can take place. c. Despite improvements in reaching the “frozen” state, standard methods cause uneven thawing throughout large volumes of tissue, causing similar issues with classical cryopreservation as different regions regain mobility of solute components. The cellular response during these freezing and thawing processes also can cause pathological responses that limit the actual restoration of the tissue, however, this requires further evaluation.

2. What is the purpose of tissue cross-linking and crosslinking for decellularized materials? Why does glutaraldehyde still have issues regarding durability and biocompatibility? Give an example of a chemical crosslinking method together with the mechanism of action. a. Tissue cross-linking decreases the rate of enzymatic digestion by the host cells and delays the hyper-acute and acute immune response by masking antigens. For decellularized ECM, besides modulating the degradation rate, cross-linking also tailors the mechanical properties of the material. b. Incomplete binding of glutaraldehyde to the collagen network leads to the presence of cytotoxic aldehyde groups that might be involved in long-term calcification as observed in bioprosthetic heart valves. c. An example of a chemical cross-linker is carbodiimide coupled with N-hydroxysulfosuccinimide. These agents cross-link nonspecifically all proteins by linking the amine and the carboxyl groups by amide bonds.

3. Why is DNA commonly utilized as a surrogate marker of decellularization quality? What are the limitations of this approach? How do methods of antigen removal address some of these limitations? a. DNA is commonly measured as a remnant of cellular content because it has been linked to the activation of inflammatory mechanisms. b. A limitation of this method is that DNA is not the only source of antigenicity that could elicit an inflammatory response for in vivo applications as other remnant cellular components and especially xenogeneic epitopes can elicit immune rejection responses.

c. Antigen removal focuses specifically on assessing content during the decellularization that interacts with antibodies that could elicit immune responses of engrafted tissue-derived materials, thus being a more direct measure of content that could stimulate an immune rejection response.

4. Give an example of three commercially available ECMbased products. Mention the tissue source, together with the application. a. AlloDerm is an acellular human dermal matrix used for full-thickness skin injury, breast reconstruction, and abdominal hernia repair. Oasis is a porcine small intestinal submucosa matrix applied in chronic leg ulcers, myocardial repair, and arterial and venous grafting. MatriStem is an acellular porcine urinary bladder matrix used for deep burns, and acute diabetic or venous ulcerations.

5. What is an advantage of injectable ECM particulate and ECM hydrogels as therapies? a. Injectable therapies can be delivered through less invasive or minimally invasive methods, which can improve the recovery times and safety of the procedure. For hydrogels, in particular, their shear thinning properties allow them to be delivered by catheter methods, allowing delivery to organs that require highly invasive and risky surgeries such as the heart.

6. What current medical issues do tissue-processing methods seek to address? How do these methods address limitations of the standard practices in the medical field? a. Tissue-processing methods seek to improve the resources and treatments for patients with chronic tissue injuries and diseases, especially ones that can only be addressed by eventual organ transplant. b. Tissue-processing methods address the limitations of current medical technology in a variety of ways. These methods hope to improve the preservation of donated organ tissue, expand the available sources of organs used for transplant with xenogeneic-derived substitutes, and develop alternative treatments that can be utilized to promote repair for damaged and diseased tissue in patients.

Burlingham, W.J., Grailer, A.P., Heisey, D.M., Claas, F.H., Norman, D., Mohanakumar, T., Brennan, D.C., De Fijter, H., Van Gelder, T., Pirsch, J.D., Sollinger, H.W., Bean, M.A., 1998. The effect of tolerance to noninherited maternal HLA antigens on the survival of renal transplants from sibling donors. N. Engl. J. Med. 339, 1657–1664.

Hannet, I., Erkeller-Yuksel, F., Lydyard, P., Deneys, V., Debruyere, M., 1992. Developmental and maturational changes in human blood lymphocyte subpopulations. Immunol. Today 13, 215– 218.

Luk, A., Rao, V., Cusimano, R.J., David, T.E., Butany, J., 2015. CorMatrix extracellular matrix used for valve repair in the adult: is there de novo valvular tissue seen? Ann. Thorac. Surg. 99, 2205–2207.

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Questions

Mattis, V.B., Wakeman, D.R., Tom, C., Dodiya, H.B., Yeung, S.Y., Tran, A.H., Bernau, K., Ornelas, L., Sahabian, A., Reidling, J., Sareen, D., Thompson, L.M., Kordower, J.H., Svendsen, C.N., 2014.

Neonatal immune-tolerance in mice does not prevent xenograft rejection. Exp. Neurol. 254, 90–98.

Mold, J.E., Venkatasubrahmanyam, S., Burt, T.D., Michaelsson, J., Rivera, J.M., Galkina, S.A., Weinberg, K., Stoddart, C.A., Mccune, J.M., 2010.

Fetal and adult hematopoietic stem cells give rise to distinct T cell lineages in humans. Science 330, 1695–1699.

Nelson, J.S., Heider, A., Si, M.S., Ohye, R.G., 2016.

Evaluation of explanted CorMatrix intracardiac patches in children with congenital heart disease.

Ann. Thorac. Surg. 102, 1329–1335.

Padalino, M.A., Quarti, A., Angeli, E., Frigo, A.C., Vida, V.L., Pozzi, M., Gargiulo, G., Stellin, G., 2015. Early and mid-term clinical experience with extracellular matrix scaffold for congenital cardiac and vascular reconstructive surgery: a multicentric Italian study. Interact. Cardiovasc. Thorac. Surg.

21, 40–49; Discussion 49.

Rosario-Quinones, F., Magid, M.S., Yau, J., Pawale, A., Nguyen, K., 2015. Tissue reaction to porcine intestinal submucosa (CorMatrix) implants in pe-

diatric cardiac patients: a single-center experience.

Ann. Thorac. Surg. 99, 1373–1377.

Scholl, F.G., Boucek, M.M., Chan, K.C., ValdesCruz, L., Perryman, R., 2010. Preliminary experience with cardiac reconstruction using decellularized porcine extracellular matrix scaffold: human applications in congenital heart disease. World J. Pediatr. Congenit. Heart Surg. 1, 132–136.

Witt, R.G., Raff, G., Van Gundy, J., Rodgers-Ohlau, M., Si, M.S., 2013. Short-term experience of porcine small intestinal submucosa patches in paediatric cardiovascular surgery. Eur. J. Cardiothorac.

Surg. 44, 72–76.

Woo, J.S., Fishbein, M.C., Reemtsen, B., 2016. Histologic examination of decellularized porcine intestinal submucosa extracellular matrix (CorMatrix) in pediatric congenital heart surgery. Cardiovasc.

Pathol. 25, 12–17.

Zaidi, A.H., Nathan, M., Emani, S., Baird, C., Del Nido, P.J., Gauvreau, K., Harris, M., Sanders, S.P., Padera, R.F., 2014. Preliminary experience with porcine intestinal submucosa (CorMatrix) for valve reconstruction in congenital heart disease: histologic evaluation of explanted valves. J. Thorac.

Cardiovasc. Surg. 148, 2216–4, 2225 e1.

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Chapter Questions

to Equation (1.3.7.8) and Figure 1.3.7.7, show the following relations

1. A unidirectionally reinforced carbon fiber-polyetheretherketone (CF/PEEK) composite is investigated for the stem of hip prostheses. The composite contains 30% by volume of the carbon fiber. Properties of carbon fiber and PEEK are summarized in the table below.

Table 1 Properties of Carbon Fiber and PEEK Property

Constituent of the Composite

Young’s Modulus (GPa)

Tensile Strength (MPa)

Density (g/cm3)

Carbon fiber

360

1800

2.10

PEEK

5.0

100

1.40

(A) Calculate the Young’s modulus and tensile strength of this composite according to the composite theory.

(B) If an elderly man having a body weight of 800N has been implanted with a total hip prosthesis made of this composite and he is walking slowly, what is the maximum load borne by carbon fibers and what is the maximum load applied to the polymer matrix in the stem of the hip prosthesis? (You can assume that the direction of the load applied to the stem is parallel to carbon fibers in the stem.)

Solution: (A) At Vf = 30%, we have Vm = 1 - Vf = 70% Young’s modulus of the composite: Ec = EfVf + EmVm= 360×0.3 + 5.0×0.7 = 111.5 GPa Tensile strength of the composite: σc = σfVf + σmVm = 1800×0.3 + 100×0.7 = 610 MPa (B) Knowing P = A, L = L , = E and = , the σ ε εc εf c f σ loads shared by carbon fibers and PEEK can be found as Pf /Pc = σf Af /σc Ac = σf AfLf /σc AcLc= σfVf /σc = Ef εfVf / Ecεc= EfVf /Ec During walking, the maximum force in the vertical direction of the hip is typically 5 times the body weight. The load borne by carbon fibers is

σ f(Vf → 0 )

=

Ef t2 + 2E (1 − νm ) + 1 + t t m 3 (1 + νm ) (3 − νm)

when Vf → 0, or fi → ∞.

Solution: With Vf → 0 or fi → ∞, by applying the L'Hôpital's Rule with respect to fi to the following expressions along with the relationships between the hyperbolic sine/cosine functions and the exponential function, one establishes the following relations 2βt = lim 2t lim 2t cosh 2 βt sinh 2 βt β →∞ β →∞ 1 = lim 2 = lim =0 2βt + e − 2βt cosh 2 βt e β →∞ β →∞ 2 sinh2 βt lim 2 sinh2 βt = lim β →∞ sinh 2 βt β →∞ 2 sinh βt cosh βt = lim sinh βt = lim eβt − e − βt =1 β →∞cosh βt β →∞ eβt + e − βt 2 2 cosh2 βt lim 2 cosh βt = lim β →∞ sinh 2 βt β →∞ 2 sinh βt cosh βt = lim cosh βt = lim eβt + e − βt =1 β →∞ sinh βt β →∞ eβt − e − βt To take advantage of these relations, it is necessary to rearrange Equation (1.3.7.8) slightly, hence one has 2βt (1 + vm) (1 − vm) sinh 2 βt + sinh 2 βt sinh 2 βt 2 2 sinh βt 2t cosh2 βt + + E t2 t sinh 2 βt sinh 2 βt σf = f + (1 + k) Em 2βt ( 1 + v ) m 3 ( 1 + vm) + (3 − vm) sinh 2 βt 2βt (1 + vm) (1 − vm) sinh 2 βt − sinh 2 βt sinh 2 βt 2 cosh2 βt 2t sinh2 βt – − t sinh 2 βt sinh 2 βt + (1 − k) Em 2βt (1 + vm) ( 1 + vm) − (3 − vm) sinh 2 βt

Pf = PcEfVf /Ec = 5×800×360×0.3/111.5 = 3874 N and the load borne by PEEK is Pm = 5×800 – 3874 = 126 N 2. As discussed in this chapter, in the k-mode microbuckling the compressive strength of a uniaxial fiber embedded in a matrix as predicted by Equation (1.3.7.8) will reduce to that of a single-fiber composite case when the fiber volume fraction (Vf) becomes very small. Referring

Plugging in the above derived relationships, one obtains (1 − vm) + 1 + t Eft2 t + (1 + k) Em σf = (1 + vm) (3 − vm) 3 (1 − vm) + 1 + t t + (1 − k) Em (1 + vm) (3 − vm) Apparently, regardless what the k value is, one has

Solutions Manual for Biomaterials Science - An Introduction to Materials in Medicine 4th Edition by Wagner, William R.; Zhang, Guigen; Sakiyama-Elbert, Shelly E.; Yaszemski, Michael J.

Questions

σf(V → 0) =

Eft2

(1 − νm) + 1 + t t + 2Em

(1 + νm) (3 − νm) By taking the critical value of the above expression with respect to t one arrives at the following expression f

3

cr

cr

σ

f(Vf → 0)

=

Eft2cr

+ 2Em

3

(1 − νm) + 1 + tcr tcr (1 + νm) (3 − νm)

This relation supports the converged result for the k=1 and k=−1 cases of microbuckling when Vf → 0, as shown in Figure 1.3.7.7.

3. Use the values provided in Table 1.3.7.1 for a CF/PEEK composite, determine the critical value for the compressive strength of a fiber undergoing microbuckling as predicted by the following expression (1 − νm) + 1 + tcr E t2 f tcr σcr = cr + 2Em f(Vf → 0) (1 + νm) (3 − νm) 3 Solution: For the given expression, its critical value can be determined by finding its minimum with respect to t. So, by taking the first-order derivative of the above expression and setting it to zero, one can find the tcr value for the given Ef, Em and νm: tcr = 0.243. By plugging this tcr value and the known Ef, Em and νm values into the above expression, one arrives at: σfcr =17.417 GPa 4. As shown in Figure 1.3.7.7, for Vf > 0.13, the compressive strength of a uniaxial fiber reinforced composite predicted by the k=1 case of the k-mode microbuckling will be the same as that predicted by Rosen’s theory. Show that σfcr =

Gm ( ) Vf 1 − Vf

when Vf is sufficiently large (e.g., Vf > 0.13 for CF/PEEK composite).

Solution: When Vf is sufficiently large, fi will be a finite value. In this situation, the critical value of the compressive strength of a uniaxial fiber reinforced composite as predicted by Equation (1.3.7.8) will be reached when t → 0. By applying the L'Hôpital's Rule with respect to t to the following expressions, one establishes the following relations

lim sinh 2 βt = lim 2t cosh 2 βt 2βt 2βt t→0 2βt − 2βt = lim cosh 2 βt = lim e + e =1 2 t→0 t→0 2 sinh2 βt 4 sinh βt cosh βt im 4βt lim 2 2 = tl→ t→ 0 0 βt sinh 2 βt = tlim = β cosh 2 βt = β = t lim → 0 2t →0 2 2 2t cosh βt cosh βt lim = lim 2βt t→0 β t→0 = lim =1=1 β β 2 t→0

t→0

Letting k=1 and rearranging Equation (1.3.7.8), one has sinh 2 βt 2βt 2Em 2 sinh βt + 2t cosh2 βt + 2βt 2βt2 + sinh 2 βt (1 + vm) (1 − vm) + (3 − vm) 2βt

(1 + vm)+(1 − vm)

σcr= lim σf = lim f t→0 t→0

Plugging in the above derived relationships Ealong with 1 m the relationships of β = − 1 and G = , one obtains

σcrf = 2Em

Vf

m

2 (1 + m)

(1 + vm) + (1 − vm) + β + 1β (1 + vm) [(1 + vm) + (3 − vm)] Em Gm = = 2 (1 + vm) Vf 1 − Vf Vf 1 − Vf

5. Referring to Figures 1.3.7.6 and 1.3.7.8, find the expression for f1 when Vf → 0 (or fi → ∞) for an arbitrary k value (−1≤k ≤1).

Solution: From Figure 1.3.7.6 one copies the expression for f1 as follows:

(1 + vm) βt2 + (1 − vm) t sinh βt cosh βt + 2 sinh2 βt f1 = (1 + k) (1 + vm) [2βt (1 + vm) + (3 − vm) sinh 2 βt] (1 + vm) βt2 − (1 − vm) t sinh βt cosh βt − 2 sinh2 βt + (1 − k) (1 + vm) [2βt (1 + vm) − (3 − vm) sinh 2 βt] With some rearrangement one further writes

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Chapter Questions

βt2 (1 + vm ) sinh 2βt + (1 − vm) t sin βt cosh βt + 2 sinh2 βt sinh 2βt sinh 2βt f1 = (1 + k) (1 + vm) (1 + vm) 2βt + (3 − vm) sinh 2βt βt2 (1 + vm) sinh 2βt − (1 − vm)

By substituting the limits relations in Question 2 into the above f2 expression along with some elimination and simplification, one obtains 1 ( 1 − vm) + t lim f2 = (1 + k) 2 + (1 − k) β →∞ (1 + vm) (3 − vm) 1 ( 1 − vm) + t (1 − vm) + 2t 2

(1 + vm) (3 − vm)

2

t sin βt cosh βt 2 sinh βt − sinh 2βt sinh 2βt + (1 − k) 2βt (1 + vm) (1 + vm) sinh 2βt − (3 − vm) By substituting the limits relations obtained in Question 2 into the above f1 expression along with some elimination and simplification, one obtains (1 − vm) t + 1 2 lim f1 =(1 + k) + (1 − k) β →∞ (1 + vm) (3 − vm) (1 − vm) t + 1 (1 − v ) t + 2 m 2 = (1 + vm) (3 − vm) (1 + vm) (3 − vm) Apparently, the above expression is not a function of k, indicating that when Vf → 0 (or fi → ∞), the function of f1 is independent of the k parameter.

6. Referring to Figures 1.3.7.6 and 1.3.7.8, find the expression for f2 when Vf → 0 (or fi → ∞) for an arbitrary k value (−1≤k ≤1).

Solution: From Figure 1.3.7.6 one copies the expression for f2 as follows: f = 2

(1 + k)

+ (1 − k)

(1 + vm) βt + (1 − vm) sinh βt cosh βt + 2t cosh2 βt

=

(1 + vm) (3 − vm)

Apparently, the above expression is not a function of k, indicating that when Vf → 0 (or fi → ∞), the function of f2 is independent of the k parameter.

7. The biphasic calcium phosphate (BCP) bioceramic is actually a composite material. Its composition can be 60% of hydroxyapatite (HA) and 40% of tricalcium phosphate (TCP). It is developed for biodegradable implants for human hard tissue repair.

(A) Non-porous BCP rods are now used in in vitro degradation tests. It is found that the strength of this BCP decreases to 70% of the original strength after 10 weeks’ immersion in phosphate buffered saline solution (PBS).

It further decreases to 50% of the original strength after another 10 weeks’ immersion. How many more weeks will it take for the strength of this BCP to be reduced to 5% of the original strength? You can assume that the strength (σ) of this BCP follows the relationship shown below: σ = σo + C ln t where t is the immersion time (week) in PBS, and σo and C are constants.

(B) The degradation rate of BCP can be controlled by changing proportions of HA and TCP in the BCP. In order to increase the degradation rate of BCP, do you need to increase or decrease the percentage of TCP in BCP? Provide the reason for your decision.

(1 + vm ) [2βt (1 + vm ) + (3 − vm ) sinh 2 βt] (1 + vm) βt − (1 − vm) sinh βt cosh βt −2t cosh2 βt (1 + vm) [2βt (1 + vm) − (3 − vm) sinh 2 βt]

With some rearrangement one writes βt (1 + vm) + (1 − vm) sinh 2βt sinh βt cosh βt + 2t cosh2 βt sinh 2βt sinh 2βt f = (1 + k) βt 2 + (3 − v ) (1 + v ) (1 + v ) m m m sinh 2βt βt − (1 − vm) sinh 2βt sinh βt cosh βt 2t cosh2 βt – sinh 2βt sinh 2βt + (1 − k) βt (1 + vm) (1 + vm) − (3 − vm) sinh 2βt

(1 + vm)

Solution: (A) Knowing σ =σo + C ln t by assuming the original strength of BCP before degradation tests as σi we have 0.70σi = σo + C ln 10 = σo + 2.303C and 0.50σi = σo + C ln 20 = σo + 2.996C By taking the difference of the two above equations, we have 0.20σi = −0.693C This leads to C = −0.289σi and σo = 0.70σi – 2.303C = 1.366σi

Solutions Manual for Biomaterials Science - An Introduction to Materials in Medicine 4th Edition by Wagner, William R.; Zhang, Guigen; Sakiyama-Elbert, Shelly E.; Yaszemski, Michael J.

Questions

We can calculate the time required (t0.05) for the strength of BCP to be reduced to 5% of the original strength by solving the following equation: 0.05σi = 1.366σi – 0.289σi ln t0.05 By elimination, we then have ln t0.05 = 1.316 / 0.289 = 4.554

therefore t0.05 = 95.01 weeks t = 95.01 − 24 = 75.01 weeks that is to say, it would take another 75 weeks for the BCP rods to degrade to 5% of the original strength.

(B) Increase the amount of TCP in BCP will do.

HA is perceived to be stable in the human body environment and TCP is a proven biodegradable bioceramic.

Increasing TCP in BCP enhances biodegradation of BCP.

Chapter Exercises 1. Which of the following cell types is least likely to take up microparticles?

A. Dendritic cells B. Macrophages C. Endothelial cells 2. Which of the following routes of administration is not commonly used for drug-loaded microparticles?

A. Oral delivery B. Intravenous injection C. Pulmonary delivery D. Local injection 3. Which is the principal mechanism underlying drug release from poly(ortho ester) microparticles?

A. Surface erosion B. Bulk erosion C. Diffusion D. Swelling 4. Why do microparticles usually exhibit more prolonged release of drugs compared to nanoparticles?

5. What is burst release? What are the potential consequences of burst release? What parameters of polymers and microparticles may affect burst release?

6. Discuss strategies to improve microencapsulation efficiency of proteins. What factors in the microencapsulation process may lead to the loss of biological activity of proteins?

7. A new hydrophobic drug which absorbs light at 580 nm has been developed. PLGA microspheres are used for the encapsulation of this drug by a conventional o/w emulsion and solvent evaporation method. Twenty mg of drug and 200 mg of PLGA polymer were dissolved in methylene chloride and the final volume was made to 2 mL. The absorption of polymer and drug solution at 580 nm was 0.8. The solution was then emulsified in 10 mL PBS buffer with a surfactant, using a homogenizer. Microspheres underwent solvent evaporation, washing and freeze-drying. To quantify drug encapsulation, 50 mg of the 210 mg of drug-loaded PLGA microspheres obtained were redissolved in methylene chloride and the final volume was made to 5 mL. The absorption of the redissolved solution at 580 nm was 0.065. What were the drug encapsulation and encapsulation efficiency in this example?

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Solutions Manual for Biomaterials Science - An Introduction to Materials in Medicine 4th Edition by Wagner, William R.; Zhang, Guigen; Sakiyama-Elbert, Shelly E.; Yaszemski, Michael J.

Chapter Assessment Questions 1. Define NPs, and mention three major types of NPs. 2. Please list the major advantages and disadvantages of polymeric micelles, polymersomes, liposomes, and nanocapsules.

3. Define the term “critical micellar concentration.” List three strategies to reduce the critical micellar concentration.

4. List at least five examples concerning the correlation between NP properties (size, surface charge, morphology, etc.) and functions.

5. Explain different mechanisms to noncovalently and covalently load drugs into NPs and compare their advantages/disadvantages.

6. Summarize the major systemic barriers to anticancer drug delivery and existing approaches to overcome these barriers.

7. List different endolysosomal escape mechanisms mediated by gene-delivery vectors.

8. List different intracellular gene release mechanisms mediated by polycationic vectors.

9. What are passive targeting and active targeting? List three approaches to realize passive targeting or active targeting, respectively.

10. List five different types of internal or external stimuliresponsive mechanisms in drug delivery.

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Chapter Study Questions 1. What are the general selection criteria for polymeric materials for medical fiber production?

2. Describe the main advantages and disadvantages of synthetic and natural materials as a source for producing medical fibers.

3. Give three examples each of synthetic and natural polymers and list examples of biotextile products generated from each polymer.

4. Name one clinical challenge that biotextiles have provided a solution for, and explain what specific structural features and properties are critical to accomplishing the particular tasks or serving the unique functions.

5. Consider a fiber that is 2 m long and weighs 3 g. a. If the fiber is a monofilament fiber, what is the linear density of the fiber in denier and dtex? b. If the fiber is a multifilament yarn made with 60 filaments, what is the linear density of each fiber in denier and dtex? c. Contrast the advantages and disadvantages of monofilaments and multifilament fibers.

6. Assume that you are given a 260-denier monofilament PET yarn to prepare a vascular graft. a. What is the diameter of the yarn used for this graft? b. What are the advantages and disadvantages for each of the following structures for this vascular graft: woven, knit, and braided textiles? c. Pick one of the three structures and describe how you could improve upon the disadvantages, which you have described, by altering the textile or yarn properties.

7. Pick a natural polymer, for example, from this list: chitosan, collagen, gelatin, alginate, and hyaluronic acid.

Research and briefly describe its endogenous bioactivities and its functions in a current or potential biotextile.

What properties of the biopolymer made it ideal for this application? What are the potential disadvantages of the biopolymer in this application?

8. The concept of combining naturally occurring biologic elements with artificially manufactured biotextiles has been observed with multiple products discussed in this chapter, including hybrid textiles intended for use as small vessel stent grafts and stem cell-coated polyester implants for orthopedic surgery. What are some of the advantages of products that combine textiles with biologic elements? What are some potential shortcomings of a hybrid textile compared with either the textile or biologic element alone?

9. Consider the process of producing electrospun polymer nanofibers. a. Describe the basic principle of electrospinning for preparing polymeric nanofiber mesh; use a sketch or schematic to aid your description. b. Explain how polymer solution properties and electrical potential applied may influence the fiber diameter. c. Discuss approaches to control fiber alignment. Give two examples each for electrospun fiber products with either ordered or random alignment. d. Propose one method each for encapsulating a hydrophobic drug (e.g., vancomycin) and a water-soluble drug (e.g., bone morphogenetic protein-2) into electrospun fibers.

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Solutions Manual for Biomaterials Science - An Introduction to Materials in Medicine 4th Edition by Wagner, William R.; Zhang, Guigen; Sakiyama-Elbert, Shelly E.; Yaszemski, Michael J.

Chapter Exercises 1. What are the major challenges for successful structural and functional integration of biomedical implants with native tissues?

There is a clear need for seamless and functional integration of synthetic implants with host tissue. Properties of biomedical implants must be balanced with their specific application. Important considerations include vascularization and innervation, and minimizing fibrous tissue encapsulation. Architectural and biochemical properties of the cell microenvironment, particularly at the cell–material interface, are critically important and tailorable. For example, nanofibers promote cell adhesion, proliferation, and differentiation on the material surface; porous implants facilitate tissue ingrowth and host integration.

2. Many strategies to engineer a functional tissue equivalent propose the use of an appropriate biomaterial that mimics the native ECM. What are the critical considerations of a biomaterial to be considered appropriate?

Key considerations include recapitulating native mechanical properties, physiological response, degradation rate and degradation products, cell infiltration and integration, cost, ease of manufacturing, and minimizing immune response.

These factors can be modulated by selecting materials with appropriate mechanical properties, chemical composition and degradation rates, surface modification, and choosing appropriate fabrication methods for nano-, micro-, and macrolevel architectures.

3. Collagen, in the native ECM, imparts tensile strength to tissues. What aspects of collagen are structurally favorable?

How can this favorable 3D structure–function relationship be recapitulated by synthetic biomaterials? What are the limitations of naturally isolated collagen as a biomaterial?

Type I collagen, which composes a majority of the native ECM, is a protein with a triple-helical fiber structure. Fiber diameters are roughly 50–500 μm. Therefore synthetic polymers must be fabricated with similar fiber diameters.

This is best accomplished by TIPS. Collagen lacks processing control in regards to mechanical properties, degradation rate, batch-to-batch inconsistencies, and pathogenicity.

4. You have synthesized a new biodegradable polymer for potential use in tissue-engineering scaffold applications.

What design criteria would be implicated in: a. Increasing cell adhesion to the scaffold construct? Nanofibrous surface texture. b. Directing cell alignment in the scaffold construct?

Aligned macropores using a tubular porogen.

c. Promote osteogenic differentiation and calcium secretion? Use the increased surface area of a nanofibrous macroporous scaffold to adhere osteogenic factors such as bone morphogenetic proteins or hydroxyapatite.

5. Angiogenesis is critical to influence tissue neogenesis and integration of synthetic biomaterial constructs. What strategies would you employ to enhance vascularization in a synthetic prosthetic implant? Outline an appropriate experiment, with controls, to test this strategy.

To enhance vascularization, blood vessels must be able to form at the surface of and into the implanted construct.

Therefore a macroporous texture is advantageous to allow cell and blood vessel infiltration. To test this hypothesis, smooth and macroporous implants could be implanted into an animal; after adequate time, histology for blood vessel formation can be quantified.

6. A patient presents with a critical size osteochondral defect (bone and cartilage) in their knee as the result of repeated athletic injury. Describe a method for fabricating a tissue-engineering scaffold that is suitable for regeneration of both tissue types in this defect.

Bone and cartilage must be combined in the same tissue-engineering construct in two distinct regions. Considerations may include pore size, pore interconnectivity, and surface decoration.

7. You are a senior staff scientist at a pharmaceutical company, working in the biomedical devices and implants division. The director of research and development has tasked you with leading a phase 1 project of an implantable biosensor for optical blood pressure sensing. What specific features would you propose to increase biocompatibility of the synthetic construct?

To increase long-term device success, you want it to integrate with the surrounding tissue without being encapsulated by fibrous tissue. Therefore ECM-like nanofibers and/ or a porous surface texture are important to allow tissue ingrowth.

8. Implantation of biomaterials leads to a host of reactions that can be characterized by a series of cellular responses. You are tasked with designing a composite temporal mandibular joint implant. In general, how would you circumvent complications associated with host rejection and expedite wound healing?

ECM-like nanofibers and/or a porous surface texture are important to allow tissue ingrowth. Pores, in particular, are advantageous and must be large enough to facilitate cell and tissue infiltration, but small enough to prevent fibrous tissue formation. Nanofibers are important to disrupt capsule formation by disrupting collagen fiber orientation.

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Chapter Review Questions 1. Why does SLA print objects with a smoother surface finish than FDM does?

Higher resolution is possible due to the laser being smaller than the nozzle diameter, and less force being applied to the object during the printing process.

2. Why is DLP a faster printing process than SLA? DLP prints an entire layer of resin at once using a pixel pattern, while SLA prints along a path with a laser.

3. Why is it beneficial that SLA and DLP do not use support materials?

Supporting structures require postprocessing, which makes the printing process longer and may result in damage or breaking of the surface connected to the support.

4. Why is wax often used as the support structure in MJM printing?

Wax has a lower melting temperature than most of the photopolymers used in MJM printing, so it can be melted away while the cured polymer stays intact.

Melting the wax allows for hands-off postprocessing to minimize damage to the object.

5. What is the benefit of using multiple nozzles?

This allows for simultaneous printing of multiple materials in a single print to impart variable characteristics and properties.

6. Compare and contrast the following techniques: EBM, SLS, and LMD. Similarities: Materials that are used to print these techniques include different types of metals, and all techniques can be used to generate implants.

Differences: EBM prints based on an electron beam melting down powder materials, SLS uses a laser beam to melt down powder materials, and LMD uses a laser to melt a metal surface.

7. What are the benefits of using FDM to fabricate biomaterial constructs compared to SLA? What are the limitations?

FDM is an extrusion-based printing technique that can translate to limited use of material and there is rapid turnaround. Furthermore, it was previously used to print thermoplastics but now can be used to print pastes, gels, and other viscous materials. SLA has higher resolution compared to FDM, which might be better for printing biomaterials such as implants or scaffolds, but often requires the use of large volumes of material.

8. What is the advantage of using LMD for metal implants compared to other metal printing techniques such as EMD or SLS? How does this advantage make it a potentially useful technique for biomaterials?

LDM process parameters allow for printing multiple materials (metals) at a time, as compared to single material printing with EMD or SLS. Previous attempts have demonstrated fabrication of implants with coat-

ings of different types that promote antibacterial resistance and mechanical strength in one single fabrication step.

9. What is bioprinting? Please list four bioprinting methods currently used for printing cell-laden bioinks.

Bioprinting is a 3D printing strategy that involves robotic deposition of cell-laden bioinks into custom shapes for facilitating regeneration of tissue. Four bioprinting methods that are currently being used for printing bioinks include microextrusion, inkjet, stereolithography, and laser-assisted bioprinting.

10. Describe major bioink properties that affect the performance of printed scaffolds.

Specific bioink properties that affect the performance of printed scaffolds include ink rheological properties, cross-linking mechanism, degree of hydration, ink mechanical properties, and biological characteristics.

• Rheological properties: The material for printing needs to be viscoelastic and shear thinning to allow for high print fidelity and structural stability. In addition, the viscosity of the ink must be controlled enough to allow for uniform distribution of cells within the ink.

• Cross-linking mechanism: Enables solidification of the ink upon deposition and can be achieved via light, ionic strength, electrostatic interactions, pH, and temperature. The primary criteria to select a cross-linking method should consider how viability of encapsulated cells is affected.

• Degree of hydration: Amount of water that is present in the ink. This can affect viscosity, print fidelity, and scaffold mechanical properties. In addition, modulation of this property can enable control over porosity, which facilitates transport of oxygen and nutrients through the scaffolds.

• Mechanical properties: These properties are dependent on chemical composition, polymer concentration, and cross-linking density. Optimization of these parameters must be investigated thoroughly because they can significantly affect viability/functionality of encapsulated cells.

• Biological characteristics: Presence of biological components can affect how cells encapsulated inside the bioink behave. Depending on what type of cells are encapsulated and printed, the biological properties of the ink (for example, presence or lack of adhesion sites) should be considered and designed.

11. What is the biofabrication window?

The biofabrication window is an important concept that describes the required compromises between the printability of a bioink formulation and cell viability. This allows for rational design of materials to improve the functionality of the cell-laden printed scaffolds.

12. What are the advantages and opportunities afforded by 3D printing techniques utilized in surgical planning and medical training models?

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Solutions Manual for Biomaterials Science - An Introduction to Materials in Medicine 4th Edition by Wagner, William R.; Zhang, Guigen; Sakiyama-Elbert, Shelly E.; Yaszemski, Michael J.

A 3D printed tissue model has the following advantages: 1. Compared with imaging examination techniques, a 3D printed model demonstrates a more straightforward and intuitive spatial relationship of targeted tissues and organs; 2. High-resolution 3D printing can exhibit more details; 3. A 3D printed model helps to reduce the operation time and to achieve more cost-effective management of medical resources.

4. In medical training, 3D printing provides an alternative option with a lower cost and higher availability.

13. In 3D printing of medical implants, what parameters should be taken into consideration? Support your statement with specific examples.

Based on target tissue, one or multiple materials can be used for the fabrication of implants. The mechanical properties and bioactivity of the material should be in compliance with the application as well as its biostability or biodegradability. For example, alloys can be fabricated into bone grafts due to a similar mechanical property and a polymer with high elasticity and flexibility used to mimic the heart valve tissue.

A proper 3D printing method must be chosen to integrate multiple materials and even incorporate bioprinting with cells. The fabrication resolution requirement also differs with different implant applications. The example of the 3D printed heart valve conduit with encapsulated human cells requires a combination of hydrogel printing and bioprinting.

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Chapter Solutions to Problems 1. What are the three main driving forces that affect the competitive adsorption of proteins to biomaterials from complex mixtures such as blood plasma?

2. Calculate the amount of adsorbed protein in a closepacked monolayer for an average protein of molecular weight 100,000. Assume the protein is spherical and has a partial specific volume of proteins of 0.73 mL/g.

3. When various biomaterials are implanted in the body, the responses often depend on the chemistry of the implanted material. Yet we know that the surface is covered with adsorbed protein and thus the biological response is not due to direct interaction with the surface. How then are the differences in responses explained?

4. What are three methods that show the specific effect of adsorbed proteins on cellular interactions with biomaterials?

5. As illustrated in Fig. 2.1.2.5, polystyrene and other hydrophobic surfaces become more wettable after exposure to protein-containing media. However, for certain materials such as cleaned germanium prisms that initially have low contact angles with water (e.g., 11°) and are thus fairly wettable to begin with, exposure to proteincontaining solutions has been reported to cause the contact angle to increase (to around 60°). How can this be explained?

6. Proteins are tightly bound to many biomaterials and adsorption is considered irreversible in that the protein is not washed off in the buffer used for adsorption, even after long soaking periods. Give a molecular explanation of why proteins are so tightly bound.

7. What are the two primary factors that control the kinetics of initial protein adsorption onto a biomaterial surface?

8. Which type of biomaterial surface tends to induce a greater degree of protein unfolding when the protein adsorbs onto the surface: a biomaterial functionalized with nonpolar hydrophobic surface groups (e.g., –CH3 groups) or a biomaterial functionalized with neutral polar hydrophilic groups (e.g., –OH groups)? Explain.

9. Are Vroman effects primarily driven by kinetic or thermodynamic forces?

Chapter Solutions to Problems 1. The three main factors that are believed to control the outcome of competitive adsorption processes are: (1) the intrinsic affinity of each protein for surfaces, meaning that some proteins are more surface active and more adsorptive than others; (2) the chemical nature of the adsorbing surface, due to the fact that the variations in surface affinity among proteins one sees for a given surface are only fixed for that surface, and will differ if another surface is used; (3) the relative bulk

concentration of each protein, meaning that even a protein with high affinity for a surface will not be present in high amounts on the surface if it is present in low bulk concentration compared to the other proteins since the excess concentration of the competing proteins will overcome the affinity differences.

2. Close packing means that the molecules touch each other at their boundaries, so the problem can be solved by calculating the cross-sectional area of one molecule of the protein and dividing that into square centimeters of area, which then gives the number of molecules in that area. Using the molecular weight of the protein allows conversion of the number of molecules into the mass of molecules, and thus you end up getting mass per unit area of a close-packed monolayer.

Protein Monolayer Calculation The specific volume (volume per gram) of a protein can be used to calculate the volume of an individual protein molecule by using the molecular weight and Avogadro’s number.

Vmolecule = Vspecific * M / NAvogadro ( ) = (0.73 mL / g) * 105 g / mole / 6.02×1023 = 1.22×10 − 19 mL / molecule The volume of an individual protein molecule can be used to calculate the radius of the molecule r, assuming it is spherical. ( )1/ 3 r = 3 Vmolecule 4Π = 3.08×10 − 7 cm (This radius estimate can be compared to dimensions of proteins measured by X-ray crystallography. For proteins in the range of 100,000 molecular weight, average length dimensions are around 70 Å, giving a radius of around 35 Å, fairly close to what we just calculated using the specific volume of proteins.)

The radius of the protein molecule can then be used to calculate the projected areas of an individual protein molecule Amolecule, which is the “footprint” or area occupied on the surface by the protein molecules.

Amolecule = Πr2 = 2.97×10 − 13 cm2 With the footprint area, you then can calculate the number of protein molecules that can be fit into a square centimeter nprotein, assuming close packing of circles representing the footprint area occupied by each spherical protein molecule. (This is a small overestimate of the occupiable area, because the tightest packing of circles on a planar surface leaves about 9% area unoccupied, according to a theory by Gauss.) nprotein =1 cm2 / Amolecule = 3.36×1012 molecules

Solutions Manual for Biomaterials Science - An Introduction to Materials in Medicine 4th Edition by Wagner, William R.; Zhang, Guigen; Sakiyama-Elbert, Shelly E.; Yaszemski, Michael J.

Then convert the number of molecules per unit area nprotein into mass of protein per unit area mprotein using Avoga-

dro’s number and the molecular weight of the protein. mprotein = nprotein * M / NAvo = 5.6×10 − 7 g = 0.56 μg Thus, according to this calculation, there should be around 0.56 μg per square centimeter in a close-packed monolayer of a spherical protein of molecular weight 100,000.

3. Due to differences in relative competitive affinity of proteins for various surfaces, the amount of adsorption of each protein varies with surface chemistry. For example, some surfaces have more adsorbed fibrinogen, and others more adsorbed albumin, and since platelets bind only to fibrinogen, surfaces enriched in fibrinogen are more platelet reactive. Thus, the effect of surface chemistry is to vary the composition of the adsorbed protein and thus this is why the reactivity with cells varies with surface chemistry.

4. Three methods to show the role of adsorbed proteins are as follows. a. If surfaces are preadsorbed with various purified proteins, it is found that most inhibit cell adhesion to the surface but a few such as fibronectin, fibrinogen, and vitronectin cause the cell adhesion to be much higher than for other proteins. Proteins that block adhesion are called passivating or blocking proteins, while those that mediate it are called adhesion proteins. b. Preadsorption with complex protein mixtures such as blood plasma selectively deficient in only one protein is a more physiologically relevant way to show if a protein is contributing an important role in platelet adhesion in the presence of many other potential adhesion proteins in plasma. If cell adhesion is greatly reduced when the surface is adsorbed with the protein mixture that is missing one protein such as fibronectin, it means this protein is playing an important role in that it is not replaced by the action of other proteins still in the mixture and on the surface. c. Addition of antibodies specific to a given adhesion protein or to its receptor to a cell suspension incubating with a biomaterial will result in a decrease in adhesion that is proportional to the concentration of added antibody. Because such antibody-blocking studies can be done with surfaces adsorbed with mixtures of proteins such as plasma, yet they selectively interfere in adhesion mediated by only one adhesion protein, they provide information relevant to the functional role of the adhesion protein under conditions closer to the physiologic situation where biomaterials are exposed to complex mixtures of proteins.

5. An increase in contact angle after exposure to proteincontaining media can be explained by adsorption of protein if the adsorbed protein layer is less wettable than the starting surface. For certain solid materials such as cleaned gallium prisms, the interaction is very strong with water and so the contact angle is low. After protein

adsorption occurs, the higher contact angle must mean that adsorbed proteins interact with water less strongly than the starting germanium surface. Thus, while proteins are much more wettable than polystyrene, they evidently are not as wettable as some solid surfaces.

6. The irreversible, tight binding of proteins to surfaces is due to the large size of protein molecules so that many noncovalent bonds are formed between the surface and each molecule, i.e., “multipoint attachment” occurs.

Although each bond is relatively weak and reversible, the chance that all the bonds would be broken simultaneously is low, so multivalent bonding results in strong bonding.

R. Latour adds another way to look at this issue. He notes that because of the large number of contacts between functional groups of a protein and a surface, even weak interactions will hold the protein. For example, if the interactions with the surface are equal in energy to their interactions with water in the bulk phase, and we assign the probability of a surface functional group being bonded to a protein versus a water molecule to be 0.5, the probability for all of the functional groups of an adsorbed protein to dissociate from the protein at the same time would be P = (0.5)n, with n being the number of functional group contacts between the protein and surface. So even if n is as small as 20, the probability that they would all dissociate at the same time is very small.

Thus, even a hydrophilic surface with OH groups (e.g., OH-SAM) will tend to irreversibly adsorb a large protein, while for hydrophobic surfaces that has favorable thermodynamics of adsorption for a nonpolar amino acid residue versus water the likelihood of desorption is even smaller.

7. Since the rate of adhesion of a protein to a biomaterial surface once it contacts the surface is generally much faster than the rate of diffusion of the protein from solution to the biomaterial surface, the kinetics of the initial process of protein adsorption is typically diffusion limited. The rate of diffusion of a protein in solution is directly proportional to the concentration of the protein in solution and inversely proportional to the square root of the protein’s molecular weight. Thus, the concentration of the protein in solution and the size of the protein (i.e., its molecular weight) are the primary factors that determine the initial rate of adsorption of a given protein on a biomaterial surface.

8. Biomaterials functionalized with nonpolar hydrophobic groups will tend to induce a greater degree of protein unfolding than neutral hydrophilic groups.

This occurs because proteins tend to be structured with their nonpolar hydrophobic amino acids buried within the protein with the outer surface of the protein largely containing hydrophilic groups, which enable the protein to be soluble in physiological solution. When proteins adsorb to hydrophobic surfaces, 660.e1 strong hydrophobic interactions tend to cause the

proteins to unfold with their hydrophobic amino acid

Solutions Manual for Biomaterials Science - An Introduction to Materials in Medicine 4th Edition by Wagner, William R.; Zhang, Guigen; Sakiyama-Elbert, Shelly E.; Yaszemski, Michael J.

Chapter Solutions to Problems

residues tightly adsorbed to the surface. This process is driven by the thermodynamic decrease of system free energy that is caused by reducing the solvent-accessible surface area of the hydrophobic surface. However, when proteins adsorb to a hydrophilic surface, the nonpolar amino acid residues within the protein’s core tend to remain buried within the protein with the

protein’s outer hydrophilic residues forming hydrogen bonds with the surface, thus tending to induce a lower degree of unfolding.

9. Vroman effects represent the displacement of one adsorbed protein by a more strongly adsorbing protein, thus being thermodynamically driven to lower the overall free-energy state of the system.

Chapter Exercises • Describe key functions and properties of the extracellular matrix.

• How do cells interact with their surrounding extracellular matrix?

• What are some of the properties and factors that need to be considered when engineering a cell-instructive biomaterial?

• Describe a strategy to design a biomaterial that exhibits increased sequestration of growth factors, such as FGF2 and PDGF. Justify your proposed strategy.

• What are some of the advantages and disadvantages of using naturally derived matrices for biomedical applications? What are some of the advantages and disadvantages of synthetic matrices?

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Solutions Manual for Biomaterials Science - An Introduction to Materials in Medicine 4th Edition by Wagner, William R.; Zhang, Guigen; Sakiyama-Elbert, Shelly E.; Yaszemski, Michael J.

Chapter Exercises Innate Immunity 1. What is the first defense mechanism and what are its salient features?

Answer: Epithelial surfaces of the body provide the first line of dense against foreign materials. The salient features are (1) microbiota, (2) chemical–pH, enzymes, fatty acids, mucosa, surfactants, and cilia, and (3) mechanical–epithelial tight junctions, mucous flow, flow of air, and fluid.

2. What is the first protein complex in the classical pathway of complement activation?

Answer: C1.

3. Name three main pathogen recognition receptors?

Answer: TLRs, NLRs, and RLRs. 4. How many TLRs have been identified to date in humans and mice?

Answer: Humans TLR1–10 and mice TLR1–13.

5. What type of innate immune cells are activated by stress, microbial molecules, or cytokines and are responsible for chemokine-based attraction of APCs? What are their major classifications?

Answer: ILCs. ILC1—IFNγ producing proinflammatory phenotype; ILC2—IL-4 producing tissue repair phenotype; and ILC3—IL-17, IL-22, and GM-CSF producing chemoattractive cells.

Adaptive Immunity 1. What are the major constituents of the adaptive immune response?

Answer: T lymphocytes, adaptive immune cytokines, B lymphocytes, and antibodies comprise the major components of the adaptive immune response.

2. True or false: Antibodies, in general, have four polypeptide chains of similar size that are connected at the “hinge” by disulfide bonds.

Answer: False; antibodies have two light chains and two heavy chains, which are much larger than the former.

3. All of the following are effector mechanisms of antibodies except: a. Neutralization b. Opsonization c. ADCC d. Agglomeration

Answer: d. Agglomeration.

4. Briefly explain what is meant by the term “anergy.”

Answer: Within the molecular communication bridge between APC and T cell (or immune synapse), other molecules also play a role in enhancing intracellular signaling networks that lead to complete activation. These molecules are called costimulators (e.g., CD28 on T cells binding to CD86 on APCs). In their absence, there is insufficient stimulation of T cells resulting in a state of hyporesponsiveness to the antigen or anergy.

5. Outline five general biomaterial-based strategies employed to harness the function of the immune system.

Answer: Any explanation, including five of the following strategies is an acceptable response: a. Material shape, size, chemistry, and other physicochemical properties impact drainage through lymphatics, interactions with APCs, and the intrinsic immunogenicity of many common polymers. b. Biomaterials can be used to control the combinations and relative concentrations of immune cargos reaching APCs and lymphocytes, or, by designing polymers with a desired degradation rate, the cargo delivery kinetics. c. Scaffolds can be used to control the context or density in which antigens and adjuvants are displayed, and as local environments to recruit APCs or lymphocytes (e.g., by incorporation of chemokines). d. T cells, APCs, and other immune cells can be modified with nanoparticles incorporating immune signals to exert autocrine effects on the modified cells, or to exert paracrine effects on target cells and tissue to which the modified cell will migrate (e.g., a tumor). e. Microneedles coated with or incorporating immune cues increase safety and patient compliance by efficiently targeting skin-resident immune cells without pain, generation of medical sharps, or need for refrigeration. f. Biomaterial carriers can be engineered with specific moieties to control cellular entry and intracellular trafficking for directing spatially restricted immune processes (e.g., TLR signaling and antigen processing). g. Stimuli responsive materials can exploit physiological (e.g., changes in pH or temperature) or external (e.g., ultraviolet light) cues to provide environment-specific control within cells, target tissues, or tumors (e.g., access to neoantigens during nanoparticle-enabled local ablation via photothermal exposure). h. Nanoparticles and microparticles can alter how antigens are processed to modulate responses away from proimmune and toward regulation.

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Chapter Questions 1. Identify the typical activators of the (a) classical pathway, (b) lectin pathway, and (c) alternative pathway.

2. Name three approaches to limit complement activation by biomaterials.

3. ISO guidelines suggests two ways to monitor complement activation on blood-contacting materials, what are they?

4. Hemodialysis on complement-activating membranes produces a significant neutropenia within 15 min of initiating therapy. What complement component is responsible for this adverse event?

5. Explain why C3a and C5a levels are higher in serum compared to anticoagulated plasma.

6. Based on surface properties, rank these biomaterials in order of increasing potential to activate complement: Cuprophan hemodialysis (HD) membrane, cellulose triacetate HD membrane, polypropylene oxygenator membrane, nylon apheresis membrane, sulfonated nanomaterials.

7. Omeros is a biotechnology company developing inhibitors to MASP-2 and MASP-3 for the treatment of various pathologies. How would this help control AP activation?

8. During collection of plasma with an apheresis device containing a nylon membrane, both C3a and C5a levels are observed to increase modestly in both the collected plasma and in the blood returned to the donors. Citrate is the standard anticoagulant used in these procedures.

Predict what would happen if heparin would be substituted for citrate in this clinical setting.

Answers to questions: 1. (a) Immune complexes, CRP; (b) mannose-containing polysaccharides, ficolins; (c) yeast or bacterial cell walls, biomaterials. 2. Limit surface nucleophiles for C3b attachment, remove C5a by filtration or binding to negatively charged groups, facilitate control by factor H (e.g., heparin coating), or pharmacologically. 3. C3a and SC5b-9 EIA methods. 4. C5a. 5. Thrombin produced during coagulation cleaves both C3 and C5 producing the anaphylatoxins. 6. Cuprophan > nylon membrane > cellulose triacetate > polypropylene oxygenator membrane > sulfonated materials. 7. MASP-3 cleaves profactor D to active factor D in blood, providing this important AP component.

MASP-2 indirectly affects AP by activating C1, which forms the CP C3 convertase and produces C3b. C3b can then bind factor B and form the AP C3 convertase in an amplification loop. 8. Much higher levels of C3a and C5a would be produced since citrate partially inhibits complement activation.

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Solutions Manual for Biomaterials Science - An Introduction to Materials in Medicine 4th Edition by Wagner, William R.; Zhang, Guigen; Sakiyama-Elbert, Shelly E.; Yaszemski, Michael J.

Chapter Exercises 1. You are developing magnesium alloy-based orthopedic plates and screws designed to support healing of a bone fracture, which then degrades. This avoids the need for a second surgery to remove the plates and screws. The plates need to retain structural integrity for 6 weeks and then degrade over another 6 weeks to a year. What local and systemic toxicity testing would be required by the regulatory agencies to approve the safety of the magnesium alloy implants? What rationale would allow you to eliminate certain toxicity tests (e.g., carcinogenesis, reproductive toxicity) and provide patient selection recommendations instead?

Answer: Dissolution rate and cytotoxic dose response to local cell types; histological evaluation of local site for osteolysis/osteogenesis and gas pocket formation; blood ion level disturbance; histological evaluation of brain, liver, heart, and kidney tissues; immunological testing if alloy contains an allergenic metal or metal of unknown allergenicity; carcinogenesis if the alloy contains a potentially carcinogenic metal or metal of unknown carcinogenesis; etc.

The alloy would not need to be tested for carcinogenesis if it does not contain any potential carcinogenic metals. However, magnesium can promote tumor growth and a recommendation for concern with implanting the device in a cancer patient might be made. Magnesium is beneficial in pregnancy and development. However, if the alloy contains a metal with known reproductive effects, a recommendation might be made to avoid implantation in pregnant women. This is of course relevant to those who would still be pregnant when the device degrades.

2. You have optimized the design and manufacturing process for a novel trunnion for total hip arthroplasty, and have been highly diligent in testing for potential toxicities. The FDA gives approval for its use and you want to maintain an impeccable safety record to grow sales. What instructions would you provide clinicians to ensure that their selection of patients, placement of the trunnion, and postsurgery monitoring reduce risks of failure and/ or systemic toxicity in the patient? Suggest a postimplantation monitoring program that allows you to provide postmarket safety data to the FDA (e.g., Phase III clinical trial data).

Answer: The surgeons might be advised to use the trunnion only with compatible components (e.g., stems and femoral heads) to avoid grinding and release of cobalt particles. Care should be taken in revision surgery where catastrophic failure of the original implants might leave debris that grind the trunnion. The surgeons also might be advised to use adhesives and cements that are compatible. They may be warned that patients with a history or family history of metal allergies, or susceptibility to potential cobalt toxicities (e.g., rheumatic disease, thyroid conditions, cardiac disease), might be prone to device failure and metal-induced disease. Patch testing

for metal allergies might be useful in identifying patients who should not receive the material or should be monitored for symptoms of metal disease. In addition to device failure, postmarket testing might include monitoring for cobalt levels in the blood, muscle and joint pain, neurological symptoms (ataxia, confusion), cardiac abnormalities, thyroid deficiency, and dermatological rashes.

3. Name four ways that metals like cobalt or nickel can cause systemic toxicity and identify the form of the metal that would likely result in toxicity.

Answer: Trace amounts of cobalt are essential cofactors for vitamin B12. However, above trace levels, cobalt can bind to proteins to generate allergenic haptens, can bind inappropriately to regulatory signaling molecules to cause cardiac tissue damage, and can cause electrical disturbance in the heart and brain by competing with ion uptake essential for normal electrical conduction and transduction. Nickel is a nonessential metal and, in addition to forming allergenic haptens and blocking calcium channels, can displace essential metals from their effector sites.

It is the free concentration of the elemental ionic form of the metal that is important.

4. What is required for an element like nickel to elicit sensitization that would result in an allergy to a nickel-based implant? When would a Nitinol implant elicit an allergic reaction?

Answer: Elemental nickel is a weak but highly prevalent allergen. For nickel allergy to occur, the patient must be genetically susceptible and exposed to elemental nickel ions (Ni2+). The nickel ions must bind to proteins to form hapten complexes that are taken into antigen-processing cells and incorporated into major histocompatibility complexes (MHCs) on the surface of antigen-presenting cells. Reexposure results in T cells interacting with the Ni–hapten in the MHC, which stimulates differentiation into cytotoxic T cells and an allergenic reaction promoting B cells. The Nitinol (nickel–titanium) alloys are not allergenic. However, if the implanted device corrodes or generates wear debris, nickel ions might be released and be available to elicit an allergic reaction in a susceptible individual.

5. Zinc is an essential metal, but can produce local and systemic toxicity when in excess and when depleted. What would be necessary for zinc from an implant to be toxic and what would cause another metal leached from an implant to cause zinc toxicity?

Answer: The levels of essential metals like zinc in the circulation and in cells are tightly controlled. The level of free zinc available for toxicity is very low and toxicity occurs only when the levels of zinc overwhelm the capacity of cellular compartments to maintain homeostatic zinc levels or when zinc levels are depleted. Other metals can promote zinc toxicity by displacing zinc from storage or protein-binding sites resulting in an excess of free zinc concentration. Toxicity can also result from the 800.e1

Exercises

metal preventing zinc access to essential sites of action, which simulates zinc deficiency. Finally, the metal may substitute for zinc in essential structural sites and disrupt protein function by changing the zinc-dependent protein structure.

6. You have designed and perfected a metal/ceramic/plastic composite dental implant and proven that each constituent, as well as the final manufactured implant, is biocompatible and safe. The FDA approves a clinical trial and several patients have severe allergic reactions to the implant. What would be the probable cause for these

allergic reactions when your premarket testing showed the implant was completely nonallergenic?

Answer: The most likely culprit would be that an allergenic adhesive or cement was used for the implantation.

A suggestion might be to market the implant with adhesives and cements that have been tested not to generate an allergic reaction to your implant. A secondary culprit would be that mice are not men and all of the premarket testing in animals failed to reveal a human-specific response.

Solutions Manual for Biomaterials Science - An Introduction to Materials in Medicine 4th Edition by Wagner, William R.; Zhang, Guigen; Sakiyama-Elbert, Shelly E.; Yaszemski, Michael J.

Chapter Exercise Questions Question 1 Cardiovascular stents are positioned at a site of arterial stenosis by inserting a catheter containing an undeployed stent at its tip into an artery in the arm or groin area, with the tip of the catheter then guided to the site of the stenosis for deployment of the stent under real-time imaging. Once the stent is deployed, the catheter is then removed with the stent remaining at the site of stenosis to hold the artery open at that location.

Part A. When a catheter is inserted into the artery, will the blood coagulation cascade primarily be activated via the extrinsic or intrinsic pathway at the site of catheter insertion?

Answer: At the site of insertion, the blood vessel wall will be cut, causing blood to be exposed to tissue external to the vasculature, with the blood then exposed to tissue factor. Exposure of the blood to tissue factor will initiate blood coagulation via the extrinsic pathway.

Part B. As the catheter is slid within the artery away from the insertion site, will the blood coagulation cascade primarily be activated by the catheter via the extrinsic or intrinsic pathway?

Answer: Away from the insertion site, the catheter will be exposed to blood within intact arteries without exposure to tissue outside of the vasculature, thus tending to activate the intrinsic blood coagulation pathway.

Question 2 Which blood protein is polymerized and cross-linked at the end of the common pathway of the blood coagulation cascade to form a stable insoluble blood clot?

Answer: Fibrinogen polymerization and cross-linking is the final phase of the common pathway of the blood coagulation cascade to form a stable insoluble fibrin blood clot.

Question 3 To prevent or at least minimize blood coagulation against medical devices that are used during cardiovascular procedures, patients are given either anticoagulant or antiplatelet drugs, or a combination of both. What are the differences in the physiological activities in these two different types of antithrombotic drugs?

Answer: Anticoagulant drugs inhibit the contact activation of the coagulation factors of the blood clotting cascade while antiplatelet drugs suppress platelet activation.

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Chapter Questions 1. What are the main stages in bacterial biofilm formation and give examples of how three of these stages may require different approaches with respect to biofilm control?

2. What are three features of bacteria when they are growing as biofilms that make them much more difficult to kill than when they are growing rapidly as a planktonic culture?

3. Classify the different strategies of biofilm control on the basis of their effects on bacterial cells.

4. Given the following three types of medical devices (urinary catheters, orthopedic implants, and CVCs), identify and list materials used for fabrication of the device.

5. Explain how material surface texture (roughness, topography) affects bacterial adhesion.

6. List three antibiotics used for medical device coating to prevent device-related infections. What are three main challenges with coating strategies?

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Solutions Manual for Biomaterials Science - An Introduction to Materials in Medicine 4th Edition by Wagner, William R.; Zhang, Guigen; Sakiyama-Elbert, Shelly E.; Yaszemski, Michael J.

Chapter Questions 1. List the five ISO 10993 standards that discuss in vitro tests for cell and tissue compatibility.

2. In Part 3 of the ISO 10993 standards, the three areas of testing describe tests for genotoxicity, carcinogenicity, and reproductive toxicity. Name one type of in vitro test in each category and use one to three sentences to describe the basic scientific premise behind each test.

3. In one to three sentences describe Part 4 of the ISO 10993 standards and list the five categories of the type of testing done under this part of the standard.

4. Define toxicant and xenobiotic. What is the main distinction between these terms?

5. You have been asked to test a certain new medical device material for cytotoxicity, and for thoroughness you have been asked to perform two tests. Name two tests for cytotoxicity that you would apply and describe the scientific basis behind each test.

6. In vitro tests to establish cell and tissue compatibility can be broken down into two general areas of testing: (1) standardized screening tests such as those described

in the ISO 10993 standards, and (2) specialized devicespecific or material-specific tests that are often used to evaluate a special bioactive property of the material. In the latter, name one such bioactive material, the molecular/biochemical nature of the bioactivity, and describe the specific bioactive test performed to ascertain bioactivity.

7. What do the acronyms NOAEL, LOAEL, TI, and MOS stand for? Given that a TI for chemical X is 1.0 mg/kg and the worst-case exposure dose of chemical X is 0.0003 mg/kg, calculate the MOS for chemical X. 8. Draw the basic geometry of a normal chromosome, then draw a chromosome that presents an interstitial deletion. What category of in vitro test does this question describe?

9. Describe the molecular mechanism behind immobilized heparin applied to medical devices.

10. Describe two events that could trigger the need for an in vitro assessment of cell or tissue compatibility and describe two of the immediate next steps in the assessment.

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Chapter Study Questions 1 What characteristic of coronary circulation of the pig heart makes it a better cardiac animal model than dogs and sheep? a Answer: Pigs’ hearts are supplied primarily by the right coronary artery, much like humans. Sheep and dogs are supplied primarily by the left coronary artery.

2 What are the four common bone defect models in animals and which is most commonly performed in rodents? Which is the most common in sheep? a Answer: Calvarial defect (rodent most common), long bone or segmental defect, partial cortical defect, and cancellous bone defect. b Answer: The sheep tibia is the most common segmental long bone defect model.

3 A new graduate student in your lab is responsible for handling rabbits on your studies. The student has experience with mice and was briefly trained on rabbit care by the outgoing graduate student. The facility veterinarian notifies you that one of your rabbits has complete paralysis in the back legs. The student acknowledged the rabbit struggled and kicked during the previous day’s physical examination. What is the likely diagnosis and treatment? What could have prevented this from occurring? a Answer: It is likely that this rabbit experienced lower back fracture during handling by the inexperienced researcher. The seventh lumbar vertebra is considered the most frequent site of fracture. This occurs when the hindquarters of the rabbit are not supported, allowing for the powerful hind limb muscles to damage the relatively light and fragile skeleton. Radiologic examination can confirm the diagnosis; however, these clinical signs are characteristic. The injury is painful and not treatable. The animal requires emergency euthanasia for humane reasons. b Answer: Training and a calm unhurried approach are critical for successful handling of any animal in research. Although the facility veterinary staff typically provides training and technical support by

request, the PI is ultimately responsible to assure all staff have training and experience in research procedures, including handling and restraint.

4 What nerve mediates the oculocardiac reflex, which can cause bradycardia and death during surgical procedures on the eye? What drugs are recommended for prevention and treatment? a Answer: The vagus nerve mediates the oculocardiac reflex. b Answer: Topical proparacaine reduces the incidence and severity of the reflex. Glycopyrrolate is effective for treatment.

5. What injectable anesthetic combination is recommended for short-term surgical procedures in rats?

What drug is recommended to speed recovery by antagonizing one of the drugs in the combination? a. Answer: Ketamine combined with either xylazine or medetomidine provides 15–30 min of surgical anesthesia. b. Answer: Atipamezole reverses xylazine or dexmedetomidine.

6. Which injectable analgesic provides 72 h of postoperative pain control in rats? a. Answer: Sustained-release buprenorphine.

7. Which opioid analgesic provides effective pain control for 48–72 h when applied as a topical patch? a. Answer: Fentanyl.

8. What are the three separate functional components required to perform survival aseptic surgery in dogs, pigs, and other USDA “regulated species”? a. Answer: Animal preparation. b. Answer: Surgeon preparation. c. Answer: Surgery room.

9. Which common research animal resembles the human because it lacks the panniculus carnosus causing cutaneous wounds to heal by reepithelialization rather than by contraction? a. Answer: Swine.

10. What anatomic features complicate endotracheal intubation in the pig? a. Answer: Long soft palate. b. Answer: Laryngeal diverticulum.

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Solutions Manual for Biomaterials Science - An Introduction to Materials in Medicine 4th Edition by Wagner, William R.; Zhang, Guigen; Sakiyama-Elbert, Shelly E.; Yaszemski, Michael J.

Chapter Questions • Hydrophobic polymer biomaterials designed both to degrade and to be stable in vivo may produce sub-micron particles during use. What are issues with the host responses that are common to the degradation of both types of polymers?

• Consider the degradation of materials commonly used in medicine that do not have well-defined breakdown mechanisms. Some examples include poly(ethylene glycol), hydroxyapatite, and some polysaccharides. How does the body deal with these common materials?

• A new class of biomaterials is now under development that degrades on cue. The cue might be thermal, photonic, or enzymatic. Ingenious chemical design principles are being applied to create such materials, but how might the body react to the products generated by a sudden breakdown of the structure?

• Learn about new strategies to stabilize materials against degradation, for example, vitamin E loading of orthopedic polymers, and incorporation of poly-isobutylene segments into elastomers.

• Endovascular stents are among the most widely used of all medical devices (Chapter 2.5.2B). A new generation of biodegradable stents is expected to have a huge impact on cardiovascular therapies. Consider how biodegradable poly(lactic acid) or magnesium or iron will perform in the complex intravascular environment.

• For a medical device intended for years of service, especially a device where failure can lead to death, how can we test and qualify the device for the expected period of service? Are there useful in vitro tests? Are there relevant and justified animal models?

• Henry Petroski and other authors have discussed the important role of failure in advancing engineering design.

Consider medical device failure, past and present, associated with degradation, and how these unintended complications will lead to better medical devices. A few examples include the degradation of polyurethane pacemaker leads, the breakdown of a protective sheath on the tailstring of the Dalkon Shield IUD, and the wear debris associated with the oxidation of ultrahigh-molecularweight polyethylene in hip prostheses.

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Chapter Exercises 1. What are the common disease states that might require the use of an artificial lung? What conditions require the most challenging requirements for respiratory support or blood–biomaterial interactions?

2. What is the dominant cause of coagulation in an ECMO circuit? Why is this the primary cause? What are some other causes?

3. Below is the schematic of the extracorporeal circuit used during cardiopulmonary bypass (CPB) when the heart is stopped to allow for surgical correction of cardiac defects (Fig. 2.5.3.24). This circuit is often described as a “heart– lung machine.” a. What are the similarities and differences between CPB and ECMO surgeries and circuits? b. How will these differences increase or decrease activation of coagulation and inflammation? c. The vast majority of publications test commercial anticoagulant surface coatings during CPB or simulated CPB. Discuss the confounding factors present during CPB and how this might lead to misconceptions about the effectiveness of the coatings during ECMO?

4. In the study by Ladd et al., (2008) they evaluated hydrophilic and zwitterionic coatings on a gold surface using four types of biological media: (1) 10% serum, (2) 100% serum, (3) 10% plasma, and (4) 100% plasma (see Fig. 2.5.3.9).

a. What is the fundamental difference between serum and plasma? How might this cause changes in the results? b. Is 10% plasma sufficient for evaluating these coatings?

How might blood present additional challenges? c. Speculate on what these results say about surface coatings tested to examine the adsorption of single dilute proteins, such as albumin or fibrinogen?

5. What would be the total rate of nitric oxide delivery to the bloodstream in mL/min if it is delivered at a flux rate sufficient to significantly inhibit clot formation through a typical oxygenator?

6. Speculate on biocompatibility differences between peritoneal dialysis and hemodialysis. From your reading, what might be the differences in how these two treatment modes affect activation of coagulation and inflammation?

7. The typical hemodialysis blood flow rates were described as 200–400 mL/min through a dialyzer with a surface area of 0.8–2.5 m2. Discuss how both the process of hemodialysis and the dialyzer design might be adjusted to reduce the duration of dialysis. What are the mitigating, negative consequences of these changes?

8. Discuss the similarities and differences between hemodialysis and ECMO and how these might affect the extent of coagulation and inflammation and their effects on the patients using these support modes.

• Figure 2.5.3.24 Schematic showing cardiopulmonary bypass circuit for Exercise 3. (Campbell Ritchie, 2012).

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Chapter Study Questions (1) What are the general mechanical property qualifications for orthopedic biomaterials and what are the major categories of orthopedic implants?

Answer: Generally orthopedic implants must be capable of high strength, fracture toughness, and most importantly corrosion resistance. The two major kinds of orthopedic implants are bone fixation or total joint arthroplasty (to a less extent new kinds of dynamic stabilization devices with articulating components are slowly making their way into the market.

(2) (A) What is the primary problem associated with the longevity of current total joint replacement implants and why? (B) How could this be solved by improved biomaterials?

Answer: (A) Particle and wear debris generation at the articulating surfaces of total joint replacements causes aseptic loosening of the implants through the induction of inflammation around the implant (macrophage-particle overload) which causes a decreased production of new bone and increased bone resorption. (B) More wear-resistant materials at the articulating surface while maintaining current available levels of other mechanical properties.

(3) What are the primary cytokines associated with particle-induced osteolysis and what is the primary cell type involved in this process.

Answer: The primary cytokines associated with osteolysis are TNF-alpha, IL-6, IL-1beta, prostaglandin E2, etc. produced by macrophage interaction with particulate debris from implants.

(4) Name six of the primary orthopedic implant biomaterials?

Answer: (A) Co–base alloys, Ti–base alloys, stainless steel, PMMA, UHMWPE, and alumina.

(5) Name one new orthopedic biomaterial and why it was introduced (i.e., what specific problem/deficit is it attempting to solve?).

Answer: Zirconium alloy was introduced to decrease the amount of metallic wear debris being generated at the bearing surface and as an alternative metal less likely to induce hypersensitivity reactions because of its corrosion resistance and less common presence environmentally.

(6) What is one way in which current designs of orthopedic implants are creating more challenging conditions for biomaterials to overcome and why?

Answer: Orthopedic implant component modularity (more components that fit together in vivo) is creating more interfacial surfaces from which implant debris can be generated through fretting corrosion (micromotion) that interact both locally and systemically.

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Chapter Exercises 1. What factors dictate the effectiveness of osseointegration? Who/what controls each of these factors?

Surgical technique—clinician.

Patient's medical and dental status—patient's physiology.

Bone quality—patient's physiology, with decisions on whether degree of bone quality in an individual patient is a contraindication made by the clinician.

Extent and kinetics of healing—patient's physiology, biomaterial (manufacturer).

Implant design factors—geometry, length, diameter, macroscopic and microscopic shapes of implant (manufacturer).

Minimization of interfacial motion—a large number of biomaterial factors, including material choice, mechanical properties of implant, surface topology, chemistry (manufacturer + clinician's choice of specific implant).

Biomechanics—loading patterns, bone quality/properties, loading (patient's physiology, implant material, design determined by manufacturer, time at which prosthesis is attached and patient can begin loading is determined by clinician).

2. What is the difference between an osteoconductive biomaterial and an osteoinductive biomaterial, and how can you demonstrate that a biomaterial is osteoinductive?

An osteoconductive biomaterial is a material that acts as a scaffold for new bone formation by providing an appropriate environment for attachment, proliferation and function of osteoblasts or their progenitor cells, leading to the formation of new bone matrix.

An osteoinductive biomaterial is a material (or biologic agent) that causes the conversion of nonlineage-committed cells preferentially to bone progenitor cells.

The key to demonstrating that a biomaterial is osteoinductive is to show that bone will form in a site that normally does not form bone (i.e., it is the material that triggers bone formation, not factors from the environment). Experiments conducted in a bony site cannot effectively demonstrate that a material is osteoinductive.

3. It is qualitatively understood that excessive micromotion (relative motion between an endosseous implant and adjacent bone) compromises osseointegration. However, few studies have quantified the effect of micromotion on osseointegration. Design an experiment to determine if there is a threshold in motion for osseointegration.

Key to experiments is designing a system in which motion is controlled, such as by creating a standard sized defect in an animal model and inserting different sized implants, and measuring the relative motion when subjected to a controlled load. Relative motion can then be related to histological findings or contact area to create a calibration curve between displacement and % bone contact or thickness of fibrous tissue as metrics for osseointegration.

4. You are starting a job at a new biomaterials startup company. Your first project is to scale up the technologies from an academic project on engineering craniofacial bone using 3D printed ceramic scaffolds and stem cells in a small animal. What are the considerations for scaleup that you should consider?

There are two components to scaleup: from small animal to human and from a small number of samples to a large number of samples.

In scaling from animal to human, the dimensions of your system needed to engineer larger volumes of tissue (size of scaffold, increased number of cells), does scaffold design need to change or just scale proportionally? Will transport considerations and ability to induce vasculature change? Will Good Laboratory Practice manufacturing be required and, if so, will any changes to the processes be needed?

In scaling from lab experiment to mass production, some considerations include: the cost of raw materials, space, and time requirements for manufacturing a much larger number of devices and cells, potential need to redesign bioreactors.

5. Recombinant growth factors are a means of stimulating oral tissue regeneration, and several have advanced to in-human studies and commercial products. What do you think are the advantages and disadvantages of using recombinant factors? What do you think are the key issues that must be thought through/experimentally tested if this application is to achieve greater clinical reality?

Advantages: Potentially unlimited supply; avoidance of transplanting cells/cell culture issues; avoidance of issues of transplant survival; no rejection.

Potential disadvantages/critical issues: safety; efficacy; means of delivery (carrier, implantation); retention of activity upon delivery; specificity of activity to targeted need; off-target effects; off-target effects; functionality/stability of new tissue; cost.

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Chapter Exercises 1. Based on developments in wound dressings and skin substitutes during the past 30 years, what kinds of new products would you expect to see in the next 30 years?

Write a one-page summary.

2. Perform one or more literature reviews to extend your perspective beyond this chapter’s references. Consider presentation of the review(s) in a journal club or study group.

3. Perform lab exercises (e.g., antimicrobial testing, cell growth, and tissue morphogenesis) to gain experience with basic procedures related to the topics presented in this chapter.

4. Consider arranging a clinical shadowing assignment, independent study course, or internship to gain firstperson experience with the subject matter of this chapter.

5. Register for an elective course related to the subject matter of this chapter that is outside of, but complementary to, your major field of study.

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Exercises 1. The antigen included in a vaccine for Virus A is a protein present on the virus’ surface. What “type” of vaccine is it?

List the advantages and disadvantages of this kind of vaccine, and list several reasons why other types of vaccines may be unsuitable for immunizing against Virus A. • The vaccine is likely a subunit vaccine, which only include a component of the original pathogen (protein, polysaccharide) as the antigen. Other types of vaccines mentioned in the text are live/attenuated and inactivated whole pathogen, and toxoid vaccines, which include either the whole pathogen or a toxic byproduct as antigen. ◦ These other vaccine subtypes may be unsuitable if Virus A is too virulent to be included in liveattenuated form, or if it does not produce a toxic byproduct that can be immunized against.

• Advantages: Subunit vaccines are very safe, meaning they may be administered to the elderly and immunocompromised versus other vaccine types. Additionally, they immunize against a specific epitope, meaning that the resulting immune response can be more carefully controlled.

• Disadvantages: Immune responses with subunit vaccines tend to be shorter-lived and can require additional boosters.

2. List several of the ways in which Alum improves the efficacy of currently approved vaccines as an adjuvant, and suggest an alternate biomaterial strategy for each.

• Extended release of adsorbed antigen: Strategies may include conjugation or adsorption of antigen to the surface of or encapsulation within a biomaterial particle or surface, such as a nano/microparticle, or to/within a depot-forming biomaterial substrate.

Additional strategies could include degradable biomaterial formulations, linkages, etc.

• Protection of antigen: Strategies listed above and below could accomplish protection of antigen, particularly encapsulation of antigen in biomaterial particle or depot.

• Formation of depot (controversial, though is a valid answer): Injectable gels or polymeric matrices are strategies that would allow formation of a depot at the injection site.

• Attraction of immune cells to site of injection: Use of immunogenic biomaterials such as alginate or hydroxyapatite (mentioned in the text) to induce a local immune response and attract additional immune cells.

3. Below are several components of a currently approved vaccine. For each of these components, list a biomaterial design that could enhance the safety or efficacy of the vaccine. Please use information from the chapter, or cite if obtained from an outside source. a. Whole inactivated virus (antigen) b. Adjuvant

c. Stabilizing agents and preservatives • Answers may vary, this question is designed to be open-ended.

• Antigen: More control could be exerted over an inactivated vaccine in terms of antigen presentation by incorporating a subunit from the pathogen into a biomaterial nanoparticle, provided that the pathogen has an immunogenic characteristic peptide. Additionally, this antigen could be conjugated to a biomaterial for enhanced delivery to APCs, or encapsulated within for added protection from proteases. Use of materials that degrade in the endosomal environment (low pH, ROS, reductive) could allow for enhanced antigen presentation and cross-presentation. Additionally, antigen could be incorporated within a depot-forming material for slow release from the injection site.

• Adjuvant: Similarly, adjuvant could be incorporated into the same biomaterial nanocarrier as the antigen for co-delivery to the same APC in order to further enhance maturation and presentation/ cross-presentation, or incorporated into the depotforming material for extended release.

• Stabilizing agents and preservatives: Typical vaccines are comprised of several excipients that extend the life of the antigen. Incorporating a biomaterial into the vaccine that protects or encapsulates the antigen could allow for stabilization such that fewer preservatives are needed, improving the safety of the vaccine as well as the efficacy as the antigen or pathogen is protected until it reaches its cellular target.

4. Your lab aims to design a vaccine that eliminates the need for multiple doses. List several broad strategies that could be utilized to accomplish this.

• Stronger activation of immune system by targeted delivery to APCs. This can be accomplished using depot-forming materials, or nano/micro-particulate carriers to protect and co-deliver antigen and adjuvant.

• Use of biomaterial that adjuvants the immune system itself.

• Extended release formulation. This can be accomplished with depot-forming materials, slow-degrading polymers that allow protection of antigen for long periods of time.

• Other answers may be acceptable provided they are reasonable or discussed in the chapter.

5. An existing vaccine is needed in an underdeveloped nation without adequate refrigeration or access to medical professionals, and thus needs to be reformulated in order ensure stability in a new environment. a. List three engineering constraints of this new vaccine, and how biomaterials could provide a solution. b. Design a formulation using specific biomaterials listed in this Chapter or in the literature (please cite if obtained from an outside source).

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Exercises

• 1) The vaccine needs to be stable without refrigeration, meaning it must be formulated to allow the antigen to be stable in a range of temperatures.

Biomaterials could be used to stabilize the antigen by encapsulating it and thus protecting it from its environment, potentially allowing a lyophilized version to be utilized until delivering to patients.

A microneedle strategy could be employed here for the above reasons.

• 2) The vaccine should be formulated in a manner that avoids the use of medical professionals.

Biomaterials can help with this by potentially allowing for oral administration in pill form, which is easy and highly portable. It also allows breaking of the cold chain.

• 3) The vaccine should be long-lasting so that patients do not have to travel long distances for additional boosters. This can be accomplished by extended release formulations, biomaterials that protect antigen from degradation, or materials that allow for direct delivery to APCs. • Design a formulation using specific biomaterials listed in the Chapter: Answers will vary, this question is designed to be open-ended.

6. Say you are looking to engineer a biomaterial therapy that eliminates adoptively transferred T cells in a patient’s body after they are cured. Using strategies outlined in the chapter or in literature, hypothesize a biomaterial strategy that could accomplish this.

• Similar to providing growth/survival factors to adoptively transferred cells ex vivo, the cells could be altered to express a unique marker that is targetable. At the desired time, one could administer a biomaterial containing 1) factors that would deplete the cells and 2) a targeting domain that is specific for the unique marker. However, there may be a variety of answers.

7. You are developing a new prophylactic vaccine in a mouse model and performing tests to assess its efficacy. Experiments show that your vaccine is not very effective in its current formulation. You check antibody titers in the blood and discover that insufficient quantities of antibody that can bind your pathogen are being produced. a. Why are antibody levels in the blood of interest when investigating prophylactic immunity? b. How might you modify your vaccine formulation to increase its potential to induce antibody production?

Prophylactic immunity is intended to prevent damage from a pathogen before it even begins, which can be achieved if the pathogen is neutralized before it has the ability to harm cells and is still within the extracellular space. The best way to achieve effective prophylactic immunity against a pathogen is to

have B cells that have already been exposed to it producing antibodies that bind to specific epitopes on that pathogen so that it can be neutralized. These antibodies circulate through the body and initiate neutralization of the pathogen very quickly upon encountering it.

Your vaccine can be designed to increase its potential to activate B cells by building antigenic components of you target pathogen into a biomaterial with multivalency of B cell epitopes and incorporation of T cellspecific antigens also associated with that pathogen to engage helper T cells in B cell activation. Including an adjuvant or using a different type of adjuvant if already using one may also be valuable.

8. Describe how biomaterials may be engineered to bias immune activation toward a humoral response or toward a cytotoxic T cell (cell-mediated) response.

Biomaterial vaccines designed with antigen multivalency and antigens associated with linked recognition may help stimulate B cell activation and increase humoral response. Designs with biomaterial carriers that degrade at endosomal pH or in the presence of endolysosomal lipases can increase the incidence of endosomal escape, resulting in more cross presentation by APCs and enhanced activation of cytotoxic T cells.

9. List the methods of tolerance induction discussed in this chapter. Would any of these treatments be compatible in combination with each other? Why or why not?

• Incomplete dendritic cell activation causing T cell anergy or deletion • Elevation of Treg activity • Suppression of B cell activation • Yes, some of these treatments could be used in combination with each other because they act upon different stages of the adaptive immune response and on different cell types. For example, biomaterials administered to suppress full activation of dendritic cells could reduce the priming of new self-antigen specific effector T cells, while simultaneous treatment with biomaterials releasing IL-2, TGF-beta, and rapamycin could further direct T cells in the lymph node toward Treg function.

10. Look up an autoimmune disease of your choice and identify the mechanism of action for this disease (what is the biological cause of the disease or its symptoms?).

What current treatment options are available? How might you design a biomaterial to induce tolerance in a way that improves upon these treatments?

Answers will vary – this is meant to be open ended to stimulate independent study of ways biomaterials can improve the current state of medical treatment.

Chapter Exercises With (Guided) Solutions Question 1: List four important biological design considerations for engineered tumor models.

Answer: 1. Tissue dimensionality 2. Mass and fluid transport/transport phenomena 3. ECM physicochemical properties/changes to stiffness, structure, chemical composition in malignancy, e.g., remodeling by stromal cells 4. Immunological considerations such as incorporation of immune cell types or immunocompetent animal models Question 2: Describe three model systems that serve to replicate changes in tissue dimensionality during tumorigenesis. Explain how they are manufactured and which specific aspects of the tumor microenvironment they can recapitulate.

Answer: 1. Spheroids (i) Formed via hanging drop, agarose-coated plates, or specialized plates (pyramidal microwells or plates with vertical channels) (ii) Promote cell–cell contacts in three dimensions, provide mass transport dynamics seen in tissues, and tissue-like structural features 2. Scaffolds (i) Encapsulation in polymeric scaffolds, hydrogels (both natural and synthetic) (ii) Cells get cues from their surroundings, adopt morphology different from 2D, changes to signaling and gene expression due to a variety of mechanisms including differential integrin engagement, cell–cell contact, as well as responses to tumor-intrinsic microenvironmental cues including hypoxia 3. Patterned hydrogels (i) Covalently couple adhesion molecules normally expressed on a cell’s membrane onto a PDMS stamped micropatterned PEG hydrogel; examples include N-cadherin, VE-cadherin, I-CAM, V-CAM, P-Sel, and Jag-1 (ii) This system allows for isolation and direct study of cell–cell contacts via interaction of specific protein binding pairs. These are also mechanically tunable allowing to compare effects of cell–cell adhesion in the presence and absence of varied substrate mechanical properties. This has identified how single cell division kinetics are impacted by intercellular adhesion proteins and how these correlate to in vivo function Question 3: Describe how changes in ECM dynamics can be recapitulated with hydrogel systems using examples from the text and explain the benefit of such systems to cancer research.

Answer: The tumor microenvironment is not static, but constantly changing. For example, spatial and temporal changes of ECM mechanical properties can be modulated by cell-mediated degradation. Studying the impact of

such changes can be achieved by incorporating specifically engineered cross-links into hydrogel systems. For example, covalent coupling of MMP degradable sequences enables cell-mediated degradation of the material and consequential changes in cell behavior. Additional chemistry can be incorporated to cause a secondary cross-linking reaction later during the culture period that inhibits additional enzymatic degradation. Mechanical properties can also be changed in a reversible manner through light-dependent stiffening or softening of materials. For example, reversible stiffening/softening of hyaluronic acid-based hydrogels can be achieved though incorporation of supramolecular cross-links formed via azobenzene bound to β-cyclodextrin where exposure to 365 nm light will soften and 400–500 nm light will stiffen the gels. PEG gels can also undergo delayed secondary cross-link reactions via thiol-ene polymerization. Such dynamic systems allow for cell-mediated remodeling, isolation of initial stiffness from another value, and separating the effect of morphology changes from those mediated by varied substrate mechanical properties.

Question 4: Which experimental technique is routinely used to measure the adhesive forces of cells on materials-based ECM mimics? Explain how this technique is performed.

Answer: Traction force microscopy. Fluorescent beads are embedded into polyacrylamide hydrogels. Cells are seeded on top and images are acquired of the cells as they exert a force on the underlying substrate. An enzyme called trypsin is then added to detach the cells from the gel. More images are taken in the same field of view of the gel in the relaxed state. The difference in the positions of the fluorescent beads is used to computationally determine the forces the cells were exerting.

Question 5: What are some immune-related changes that happen in cancer? To create a tumor model with functional immune response, what types of immune cells would you incorporate?

Answer: A. Changes: (1) Hijack inflammatory and immunomodulatory signals to enhance tumorigenesis (2) Suppressed cytotoxic activity of other immune cells (3) Increased remodeling due to upregulation of enzymes to degrade matrix (4) Changes to matrix mechanics and structure (intratumoral pressure, ECM density, fiber alignment).

B. Cell types: Dendritic cells, T cells (CD4+ and/or CD8+), macrophages (M1,M2), natural killer cells, lymphocytes. Other stromal cells such as endothelial cells or fibroblasts could also be useful.

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would differ from bone and how these would affect your choice of materials.

Answer: Differences include mechanical properties, cell types, and ECM proteins present. Brain tissue is much softer than bone and thus, the materials used to model brain must have a lower Young’s modulus than those used for bone.

There are also compositional differences between the bone and brain ECM. Whereas the bone is composed predominantly of mineralized collagen, the brain contains fibronectin, vitronectin, tenascin C, laminin, and HA. The cellular composition of the brain and bone also vary and cocultures of brain cells with cancer cells would be very different from those used in bone. Finally, the interstitial pressure developing within the brain is significantly different given the space constraints mediated by the skull.

Question 7: What role does vasculature play in cancer?

List four materials and methods that could be used to model vasculature. What could be improved on existing models to more accurately model tumor vasculature?

Answer: Vasculature transports oxygen, nutrients, metabolic waste products, cells (e.g., metastatic cancer cells, immune cells), and drugs. Tumors must develop new vasculature as they grow to support the increased number of cells and the oxygen and nutrients they require. Often tumors outgrow the blood supply and develop a hypoxic necrotic core. When new vessels are created, they are often dysfunctional and leaky leading to impaired mass transport kinetics and the development of interstitial pressure.

A. Example model systems include: • Laser ablation of channels into PEGDA gels • Patterning vascular structures into hydrogel scaffolds using photolithography-based coupling of cell-adhesion peptides • Creating microfluidic networks within natural hydrogel matrices using soft lithography approaches • Using sacrificial materials to pattern channels into hydrogels • Performing vasculogenesis (i.e., individual endothelial cells forming vascular networks within hydrogel) within a microfluidic device • 3D printing in GelMA B. Improvements: Answers may vary, examples include: simulating leaky vessels, incorporating spheroid models with vascular models etc.

Question 8: A drug company has developed a new candidate drug that appears to target cellular adhesion mechanisms of cancer cells in solid tumors. When they tested the drug on prostate cancer cells seeded on a Petri dish, it was remarkably effective, but did not harm normal cells. However, investors are concerned that this experiment is not enough to justify the cost of preclinical animal testing. Outline and justify a series of experimental systems to test the drug’s efficacy, first in a precisely controlled environment, then in a more biologically complex system. Consider the advantages of natural and synthetic materials as well as the clarity of the data produced by different systems. Consider also additional factors that

may impact or confound your data.

Answer: 1. Controlled environments could utilize a synthetic hydro- gel material like PEG conjugated with one or more spe- cific adhesion sequences that are thought to play a role in the drug’s mechanism of action (e.g., RGD from fibro- nectin). The lack of biological complexity will help focus the experiment on adhesion while still enabling 3D cul- ture that is more faithful to tumor characteristics.

2. A more complex system could be a synthetic material that contains multiple peptide sequences, a composite material containing collagen or fibronectin, or a natural material. An experiment utilizing multiple cell types would be a closer approximation of a tumor.

Embed- ding individual cells in a 3D gel or matrix would make the system less cumbersome to use, while embedding a spheroid into a hydrogel adds more complexity and cost.

3. Confounding factors: material porosity, pH, stiffness, viscoelasticity, fiber alignment, and/or size. For gels: mass transfer and diffusion from culture medium.

Question 9: Researchers are studying cancer progres- sion in two groups of mice. One group of mice forms large aggressive tumors while the other group does not. The researchers hypothesize that it may have something to do with differences in the ECM properties between the two groups. a. Describe how this hypothesis could be studied using a cell-derived matrix model. b. Additional testing shows that the group of mice with larger tumors has stiffer ECM to start with.

Design a model using synthetic biomaterials to test whether dif- ferences in ECM mechanics could be responsible for differences in tumor growth between the two groups of mice. Explain why you chose the material you did. Answer: 1. Stromal cells can be isolated from each group of mice and cultured to produce matrices. The cultures can be decellularized to produce CDMs with unique properties to each group of mice. Cancer cells can be seeded on top of the matrices and analyzed for proliferation, morphol- ogy, migration, cluster formation, etc.

2. Many answers are possible. The main focus is on a material that has tunable mechanical properties.

Other considerations include 2D versus 3D ability (PA versus PEG), the inclusion of cell-adhesive ligands (RGD), and degradable properties of the material Question 10: Several immune cell types have previously been grown in coculture systems with cancer cells, but model systems that accurately model the role of the immune system in systemic regulation of cancer progression are still needed. Define the design criteria for a body-on-a- chip system that is comprised of three or more organoid systems relevant to cancer development, control, or metas- tasis. Your system should contain multiple cell types and consider physiological conditions necessary for cells to func- tion normally (as in the lung-

on-a-chip and endothelialized microvessel devices from the text).

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Answer: Possible design features—a primary tumor compartment with stromal cells of the primary site connected to other compartments by microfluidics or vascularized microvessels. Possible other compartments include common metastatic sites (liver, lung, brain, bone) as well as a lymphoid

compartment (lymph node, lymph vessels, skin, bone marrow, thymus, spleen, and liver). Physical design features will depend on the organs modeled, but mechanics, diffusion, and flow as well as multiple cell types relevant to the model designed should be considered.

Chapter Review Questions 1. Biomaterial DDSs are also applicable for use with vaccines. Vaccines are biological preparations typically containing a pathogen-mimicking agent, known as an antigen (e.g., weakened or killed whole pathogen, a surface protein or sugar, or a toxin), that improve immunity to disease. You have been hired by a biotechnology company to help develop a new vaccine against Epstein– Barr virus (EBV), the herpes virus that causes infectious mononucleosis. To date, the company has identified and purified key polypeptides from EBV glycoprotein gp350.

As the most abundant EBV glycoprotein, gp350 is a key virulence factor involved in the attachment of virus to B cells, so gp350 is hypothesized to be a good EBV vaccine candidate. Note: EBV is a ∼120–200 nm spherical virus with an outer viral envelope that contains almost a dozen glycoproteins, and is often spread through saliva.

Your manager informs you that the company is striving hard to implement alternative vaccine delivery systems to reduce the number of needle-based injections (e.g., booster shots) for patients, as well as overhead costs associated with the aseptic manufacturing processes necessary for parenterally delivered vaccines. In an attempt to reduce the number of booster shots needed, you suggest implementing a controlled-release delivery system that can gradually release antigen to the patient over time, thus reducing the need for additional booster shots.

Name and briefly describe two different biomaterial technologies that could be considered as a novel delivery platform for this vaccine.

Potential answers: a. Nanoparticles and microneedles. b. Nanoparticle vaccines can be used to conjugate, encapsulate, or adsorb antigens using a system that closely resembles the initial pathogen in size and shape. Additionally, they can be administered via sublingual or buccal delivery routes. Controlled release via degradation of the nanoparticle and release of antigen, degradation of a tether between the antigen and the nanoparticle, or

diffusive release of an encapsulated antigen are potential delivery platform options.

Microneedles can be coated or filled with antigens and then inserted into the upper layers of the skin to deliver or slowly release the antigen into the body.

These two approaches can be used alone or in combination to deliver controlled amounts of vaccine antigen over time that could have booster-like effects on the immune response. c. Hydrogel DDSs in the form of patches or oral films as well as nasal DDSs with appropriate supporting rationale.

2. You recently developed a new proton pump inhibitor for the treatment of heartburn and acid reflux. Unfortunately, the acid secretions of the stomach hydrolyze the drug, and the degradation products cause further irritation to the gastric mucosa. Your coworker suggested that you need to coat the drug to protect it from the stomach acid. Do you agree? If not, what other solution(s) can you offer to this problem? If so, which polymer (A, B, or C) would you use and why (i.e., how would it work)?

Potential answer: Polymer B is the right polymer to use for enteric coating of the PPI drug. Polymer B will be protonated and hydrophobic in the stomach, forming an impermeable coating. It will switch to the ionized and soluble form in the intestine, releasing the drug in a favorable pH environment.

If you do not agree with your colleague, other possible approaches could be to deliver the drug from a twocompartment osmotic device, where each compartment is designed to release it only in the small intestines.

3. Delivery of leuprolide, a polypeptide drug, is used as a prostate cancer treatment. The amino acid sequence is: Glu-His-Trp-Ser-Tyr-DLeu-Leu-Arg-Pro-NHEt Leuprolide acetate a. Two strategies to achieve zero-order release are to use a reservoir/membrane device or to encapsulate the drug in a surface-degrading/eroding degradable polymer.

Cite a principal advantage of each of these strategies for the given application and a principal limitation.

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Potential answers: Reservoir/ membrane

Surfacedegrading/eroding polymer

Advantages

Disadvantages

well-controlled environment for a drug formulation, allowing increased drug stability and prolonged delivery times.

Increased drug stability.

No retrieval necessary, simple formulation (e.g., mix of drug and polymers)

Nondegradable— need for surgery for retrieval.

Underlying premise requires rapid polymer degradation, thus drug release may be limited to weeks rather than months; Zeroorder release may not be sustained especially at end of degradation due to surface area-to-volume changes over degradation time; Degradation products may lead to side effects

Potential answer: DDS PK follows the order of Liberation, Absorption, Distribution, Metabolism, and Elimination (LADME). Liberation is the release of drug from the DDS, which can occur by either diffusion, dissolution, mechanical release, or ion exchange.

This release typically occurs in the delivery site for local DDS or in the small intestine for oral delivery routes. Liberation is not a factor for administration directly to blood. Absorption is the next step, where the liberated drug is taken up, typically into systemic circulation. Uptake can occur through mucosa or capillary diffusion. Distribution is the subsequent localization of the drug into various tissues, typically through systemic circulation. Liver, lung, spleen, and kidney are common sites of distribution for most drugs, while the brain is less likely to uptake drug. Metabolism occurs when enzymes act on the drug to modify it, which can occur during delivery via enzymes in the skin, mucosa, and GI tract as well as during systemic circulation in the liver or target tissues. Metabolism can be exploited for prodrug strategies and certain DDS depot chemistries, but it is typically responsible for drug inactivation and clearance.

Elimination is the final step and typically occurs in the kidneys.

Drugs in systemic circulation are secreted into urine, and the rate of this elimination controls drug AUC. Answers for formulation changes can focus on any of the LADME steps; enteric coatings to protect drug from enzymatic degradation in the stomach, ion exchange resins to modify liberation rate, excipients to increase absorption, targeted DDSs to change distribution, and nanocarrier reservoir DDSs to modify elimination are all valid options.

5. a. What is the major barrier to drug permeation via the transdermal route of administration? b. For the following chemical structures being considered for the transdermal route, categorize each according to Lipinski’s rule of 5. c. Predict if transdermal permeation is possible and justify your answer based on (b). d. For the impermeable drugs, list and describe two methods that could be used to enhance drug permeation via the transdermal route of delivery.

OH

Cl O

OH

b. Your research group decides to first test encapsulation of leuprolide in PLGA matrices formed as implantable wafers. Explain what your biggest concern would be with the use of PLGA encapsulation for delivery of the polypeptide in a biological environment.

Potential answer: PLGA degradation products (lactic acid and glycolic acid) can build up in the matrix, causing locally acidic microenvironments that may expediate peptide chain scission and loss of potency due to degradation and increased drug release rates.

4. Name and describe the five parts or steps involved in DDS pharmacokinetics. Be sure to highlight the important anatomical structures and physiology of these struc-

HO

O

Cl

OH

O N O

P

NH

O

O

OH

O

NH2 O

OH

tures involved in the steps. For one of the steps, suggest a formulation strategy to improve drug delivery.

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Drug A

Drug D

Drug B

Solutions Manual for Biomaterials Science - An Introduction to Materials in Medicine 4th Edition by Wagner, William R.; Zhang, Guigen; Sakiyama-Elbert, Shelly E.; Yaszemski, Michael J.

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Chapter Review Questions

Drug A—Cyclophosphamide Drug B—Doxorubicin Drug C—Buprenorphine Drug D—Itraconazole Potential answers: a. Stratum corneum. b. Lipinsky’s rule of 5 states that a molecule with molecular weight >500, H-bond donors >5, H-bond acceptors >10, and log P > 5 will not penetrate cell membranes and is unlikely to be drug-like.

Compound hydrophobicity determined based on meeting Lipinski’s rule of 5: (A) MW < 500 g/mol, H-donor = 1, H-acceptor = 4, log P < 8 (B) MW = 542.53 g/mol, H-donor = 6, H-acceptor = 12, log P = 1.3 (C) MW < 500 g/mol, H-donor = 3, H-acceptor = 5, log P > 3—passively diffuses through stratum corneum per Table 2.5.12.4 (D) MW > 500 g/mol, H-donor = 0, H-acceptor = 12, log P>5 The drugs that are able to permeate the epidermis will need to be small (<500 g/mol) and lipophilic/hydrophobic (log P > 1). Drug C meets these criteria and can be expected to penetrate lipids well. Drug A is not currently intended for transdermal delivery; however, it has been shown to penetrate the stratum corneum through handling by nurses.

Drug B is too hydrophilic for these criteria, while drug D is too large and hydrophobic. Per Table 2.5.12.4, drug C has been formulated to be used for transdermal delivery as well, likely due to MW < 500 g/mol and log P > 3.

Approaches that can be used to enhance transdermal drug delivery of the remaining drugs could be iontophoresis, electrophoresis, microneedles, transdermal microjet, etc.

(Table 2.5.12.4).

6. Several key scientific findings in the 19th and early 20th centuries (e.g., Ehrlich’s magic bullet and Banting, Best, and Macleod’s discovery that insulin treated diabetes) laid the groundwork for DDS development in the late 20th century. What recent scientific findings have occurred since the start of the 21st century and how might they impact the future of DDS design and development? What challenges will need to be overcome to take full advantage of these scientific findings in DDS design and development?

Answers could include discoveries such as mapping of the human genome, siRNA, and CRISPR (as well as others). Certainly, these technologies will be useful for future design of targeted DDSs. For example, the human genome map will likely highlight genes or proteins that enable targeted cancer therapies to only destroy cancerous and potentially cancerous cells while leaving healthy cells unaffected. Technologies such as siRNA or CRISPR will likely provide specific molecular tools that facilitate this targeting process, but suitable DDS designs using these technologies will need to overcome multiple challenges, including biocompatibility, preliminary drug degradation, biological barriers to enable targeted drug delivery, etc.

Chapter Exercises

Chapter Exercise Answers

1. You and your team are designing a material that can offer three different degradation profiles both in vitro and in vivo. The tissue engineering scaffolds will be composed of blends of PLGA and PCL and will be porous.

1. Part 1:

Given the following scaffold formulations, which would you recommend for (a) the fastest degradation profile, (b) intermediate degradation, and (c) slowest degradation, and why?

• 25% PCL, 75% 50:50 PLGA • 50% PCL, 50% 50:50 PLGA • 75% PCL, 25% 50:50 PLGA

The greater the content of PCL, the slower the degradation profile as PCL is more hydrophobic and highly crystalline.

Similarly, for the following formulations, which would you recommend for (a) the fastest degradation profile, (b) intermediate degradation, and (c) slowest degradation, and why?

• 50% PCL, 50% 75:25 PLGA • 50% PCL, 50% 25:75 PLGA • 50% PCL, 50% 50:50 PLGA 2. A company wants to 3D print mesenchymal stem cells into a commercial tissue engineering scaffold system.

Which 3D printing method would you recommend for them to use and why?

3. You are designing a tissue engineering scaffold for pancreatic islet cell regeneration. Which cell type would you choose for your scaffold and why? For the tissue engineering scaffold, would you choose (a) a hydrogel, (b) a calcium phosphate cement, or (c) an electrospun PCL mesh? Why? 4. You have an electrospun tissue engineering scaffold that you intend to use as a cancer model for Ewing Sarcoma to study the efficacy of chemotherapeutics in vitro. Which cell seeding method would you use to seed your scaffold? Why? 5. You have synthesized a new polyaddition polymer and need to characterize the chemical structure and molecular weight.

Which characterization techniques would you use?

6. You and your team want to create a highly porous PCL scaffold with both nano- and microporosity, encapsulating growth factors that cannot be dissolved in an organic solvent. Which scaffold preparation method should you and your team choose and why?

(a) 25% PCL, 75% 50:50 PLGA (b) 50% PCL, 50% 50:50 PLGA (c) 75% PCL, 25% 50:50 PLGA

Part 2: (a) 50% PCL, 50% 25:75 PLGA (b) 50% PCL, 50% 50:50 PLGA (c) 50% PCL, 50% 75:25 PLGA

The greater the content of PLA, compared to PLGA, the slower the degradation rate given the increased hydrophobicity and crystallinity of PLA. 2. Extrusion based printing would be recommended as that system for 3D printing is compatible with many biological polymers, as well as crosslinking systems that do not require exposure to high temperatures and harsh initiators.

3. A variety of cell types could be chosen. Induced pluripotent stem cells (iPSCs) would be a good choice and a hydrogel would be the best material for the intended application given the material mechanical properties being the closest to native pancreatic tissue.

4. The best seeding method for the situation would be perfusion-based seeding of the Ewing Sarcoma cells. The system would also necessitate bioreactor culture in order to better recreate the in vivo environment when determining chemotherapeutic efficacy.

5. 1H-NMR can be used to characterize the polymer chemical structure and either APC/GPC can be used to characterize the molecular weight.

6. Gas foaming would be a very good option as CO2 is readily soluble in PCL and gas foaming does not require heating the mixture to high temperatures that would denature or disrupt the bioactivity of any growth factors incorporated into the system.

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Chapter Exercises

applications? Choose one type of polymer and provide ways to overcome the disadvantages listed.

Answer:

1. What are the advantages and disadvantages of using natural and synthetic polymers for soft tissue-engineering

Synthetic

Natural

Advantages

Disadvantages

Ways to Overcome

–Easily tunable properties (mechanics, degradation, modifications) –Highly reproducible –Bioactive –Generally biocompatible

–Lacks bioactivity –May use cytotoxic reagents or degradation by-products

–Combines with bioactive peptides, extracellular matrix components, or natural polymers to increase bioactivity

–Degrades rapidly –Lowers mechanical strength –Variability due to source and processing

–Crosslinks the materials or combines with synthetic polymers to increase mechanical strength

2. Strategies to engineer the soft tissues of the GI tract have been slow to translate to clinical significance. What are some possible reasons for this slow translation? a. The GI tract consists of multiple organs with dramatically different properties. While the system is continuous, efforts to regenerate individual organs are largely discrete. Strategies that show promise in the esophagus may not be feasible in the colon.

An analogy can be drawn to cancer: it is frequently referred to as if it were a single—albeit major— problem to be solved, yet it is unlikely that any single approach will be able to cure the many different cancer types. b. The soft tissues of the GI tract are complex and highly interrelated. The success to date has been in the regeneration of the epithelial layer, presumably because the epithelium does not contain the abundant vasculature of the underlying tissues. For this reason, its regeneration is easier. This is not the case for other tissues, such as the smooth muscle. Although regeneration of smooth muscle has been demonstrated, maintenance of phenotype over the long term is required. This will rely upon simultaneous regeneration not only of the smooth muscle, but also the enteric nervous system, vasculature, and lymphatics. c. There are still key gaps in our understanding of how the GI tract functions. We know, for example, that the ICC are vital to GI motility, but the precise mechanisms by which the ICC help to drive peristalsis are still active research areas. Without a clear picture of the processes that govern GI function, it may be difficult to engineer soft tissues capable of recapitulating those functions.

3. There are multiple biomaterial technologies on the market for soft tissue fillers; however, there is still much research in finding optimal materials. Explain the limitations of current soft tissue fillers.

The main limitation is that current fillers serve only to provide augmentation without inducing functional tissue regeneration. Also, most fillers on the market are degradable and without tissue formation, therefore it is necessary to have repeat injections to maintain tissue volume.

4. Design a biomaterial to stimulate adipogenesis. Explain the reasons behind the biomaterial you choose and any other components you include.

This is an open-ended question; however, the main factor to include will be to provide biochemical cues either through the biomaterial itself or by adding in bioactive components.

Other properties include appropriate mechanical properties similar to native adipose tissue, sufficient degradation as tissue is formed, and to include cells.

5. Using your biomaterial developed in the previous question, design an experiment to determine if it can stimulate adipogenesis.

Experiments can be either in vitro or in vivo. For in vitro tests, the type of cells used should be specified if the proposed material does not already include cells. For in vivo tests, the animal model and location of implantation/injection should be specified. For either setup, adipose tissue-related assays such as immunohistochemistry of adipocytes (Oil Red O) or new ECM productions (collagen type IV, laminin, fibronectin), gene expression of adipogenic and lipogenic factors (RT-PCR of PPARγ, GLUT4, FABP4, ACS, GPDH), lipid quantification (AdipoRed, triglyceride), or measuring metabolic/enzymatic activity (lipolysis, glucose update, GPDH enzyme activity) can be performed.

6. Discuss challenges to the development of skin equivalents.

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Exercises

tissue-engineering approaches, which include: recapitulation of human biology, biomaterials choice (mechanics, degradation, biocompatibility), or other issues pertaining to manufacturing, including production cost, manufacturing process, stability, etc.

7. For maintenance of systems, what are the main considerations or parameters?

Answers should include concepts such as nutrient transfer, cell culture composition, shelf life, and ability of cells to expand appropriately before transplantation.

8. For clinical substitutes, name three aspects that are important to consider in product development.

Can include: cell sourcing, scaleup, sterility, shelf life, storage.

9. Propose an experimental design to investigate a disease process in the skin. What components should be included to evaluate the process? Provide a process flow diagram of your design.

Answers should develop a skin equivalent that is capable of probing some aspect of the disease in a logical way—for example, if the student chose melanoma, it would follow that they should design a skin model that includes keratinocytes and melanocytes, and their experimental protocol should include some method to investigate that process such as inducing an ultraviolet event externally, or attempting to physically recapitulate a cancer-like environment. The cells could also be isolated from a patient with the disease to provide comparisons.

Solutions Manual for Biomaterials Science - An Introduction to Materials in Medicine 4th Edition by Wagner, William R.; Zhang, Guigen; Sakiyama-Elbert, Shelly E.; Yaszemski, Michael J.

Chapter Questions for the Student 1. When a product, for any reason, no longer meets customer expectations or is no longer a benefit compared to risks due to outmoded technology, should the manufacturer be encouraged to remove the device from the market?

2. If the methods for retiring a device at the end of its natural product lifecycle due to obsolescence were easier, might patients benefit from an orderly market retraction?

3. What might device retraction from the market mean for a patient who may still have the implanted device?

4. Does this suggest a need in medical device regulatory authority to provide for an orderly retraction of a device from the market without the risk that a manufacturer will face major liability because the device is no longer state of the art?

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Chapter Study Guide 1. Can a medical device manufacturer leverage prior use of a biomaterial in a previous device that has a different intended use and avoid having to do biological testing?

2. If a biomaterial-based medical device manufacturer changes the sterilization method for a slightly different device, using the same material and manufacturing process, can the manufacturer avoid additional biocompatibility testing?

3. When a manufacturer can show that a prior device used the same biomaterial, can the manufacturer avoid additional testing, and, if so, what would the manufacturer have to demonstrate in their own application?

4. After conducting testing in ISO 10993-1, can a manufacturer claim that the materials in the new medical device are biocompatible?

5. If a manufacturer’s new device fails cytotoxicity testing, does that mean that the materials in the device are toxic and not appropriate for use in the medical device?

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Solutions Manual for Biomaterials Science - An Introduction to Materials in Medicine 4th Edition by Wagner, William R.; Zhang, Guigen; Sakiyama-Elbert, Shelly E.; Yaszemski, Michael J.

Chapter Exercises 1. How is bioburden on a material or device typically determined? Is this different from “soil” that is observed in clinical settings? How do the two affect each other and the processing of the material or device for use or reuse?

2. What is the SAL? What is the generally accepted value of SAL? Are most medical devices sterilized at, above, or below the SAL and how is that choice rationalized?

3. What are the advantages of EO sterilization and conversely under what conditions may EO sterilization not be the appropriate choice?

4. Gamma irradiation may not be feasible with many polymeric biomaterials. Which polymers must special consideration be given to in this regard?

5. Processing of biological samples or materials containing encapsulated cells are limited in how they may be processed for sterilization. What are the considerations for each category of biological material and how can sterilization and safety needs be addressed alternatively?

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Chapter Study Questions 1. For each of the items listed here, determine if it is a design input or a design specification, and explain your choice.

The diameter of the catheter must be 4 ± 0.1 cm The catheter must be slippery enough to fit through small blood vessels of the brain The catheter must comply with ISO 10993 standards for biocompatibility

Answer: Item

Input or Specification?

The diameter of the catheter must be 4 ± 0.1 cm

Specification

The catheter must be slippery enough to fit through small blood vessels of the brain

Input

Item The catheter must comply with ISO 10993 standards for biocompatibility.

Input or Specification?

Input or specification

Explanation This one is tricky.

Input documents often give lists of what standards the device needs to observe. In that sense, this is an input. However, the specific tests within ISO 10993 are more detailed in regard to what constitutes passing or failing. In that sense, this is a specification.

A DSD may use the line just as is for the specification, or it may break each ISO 10993 test into a separate line to create multiple specifications out of this one.

Explanation This is a specification because it is specific about a size and has a tolerance range.

Inputs are generally not as specific as this.

This is an input because it talks about a user need without giving specifics on how slippery.

This input would likely translate into a specification on the coefficient of friction, with a tolerance range.

2. Consider a bone plate made from a novel polymeric biomaterial. One of the specifications for this device says that the “Bone plate shall be constructed from said novel polymer with a molecular weight between 100 and 150 kDa after a standard 12-hour EtO sterilization cycle.” Describe the setup of one or more verification tests that would address whether or not the prototypes are in spec.

Answer: The first thing to realize from this question is that it must use a sterilized prototype because it is asking for a device’s molecular weight after sterilization to be within a certain range. Any experiment created to verify this spec would need to start with a sterilization cycle called for in the spec, i.e., a “standard” 12-h EtO cycle.

Of course, sterilizing one sample is probably not a good idea. There will need to be a number of samples that show that the prototype manufacturing process can create these samples consistently in that molecular weight range. For tests like this, having 10 or more samples is a good idea.

Having those samples created on different days by different people on different equipment would be a better idea.

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Subjecting 10 prototype samples to the sterilization cycle and then using some method of molecular weight measurement, such as gel permeation chromatography, would be a good start. The results would look at whether all 10 samples met the spec, and if not how many failed. As a bonus, it would not be a bad idea to also include measurements of nonsterilized samples as a control to get a better clue on how much the sterilization process affects the molecular weight.

3. For each of the items listed here, determine if it is describing a verification test or a validation test and explain your choice.

Having people wear a brace for 10 h and then filling out a survey on its comfort Measuring 20 specimens of a device for their length, averaging the measurements, and calculating the standard deviation of the lengths Running a clinical trial on a novel polymeric bone screw to determine its Kaplan–Meier survivorship at 3 years

Answer:

Item Having people wear a brace for 10 h and then filling out a survey on its comfort

Verification or Validation?

Validation

Explanation This test is expressly looking at User Needs, probably based on some Design input that talked about comfort requirements

Item

Verification or Validation?

Explanation

Measuring 20 specimens of a device for their length, averaging the measurements, and calculating the standard deviation of the lengths

Verification

This test is looking at a Design specification for length, not a User Need. In general, User Needs are not concerned with tolerances and specific numerical length ranges. For a User Need, dimensions just need to be “enough to do the job.”

Running a clinical trial on a novel polymeric bone screw to determine its Kaplan– Meier survivorship at 3 years

Validation

This one is tricky. The item talks about Kaplan–Meier survivorship, which is a statistical measurement of how long the implant functions in patients before failure. That could be interpreted as a specification, but in fact this statistic is usually not a required specification for devices. Instead it is considered a user need: a device needs to be “State of The art,” meaning it needs to last as long as all the other modern devices in the industry that do the same thing.

Each implant has a typical expected lifetime in the industry according to technology and patient need.

Theoretically, this statistic could be listed as a device specification but then it would subject the device to a lengthy clinical trial to prove it, which would delay the device’s entry onto the market.

Chapter Study Questions

4. Consider polymer microspheres with a small amount of temporary radioactivity that are loaded into a syringe and injected precisely into a cancerous tumor. The microspheres are supposed to deliver high doses of intense radiation to a small area within the tumor for a limited amount of time, after which they rapidly become less radioactive and resorb to be eventually excreted through the kidneys and lungs. Contrast the verification and validation testing for such a device.

Answer: Since verification testing checks that inputs = outputs, the verification testing of these polymeric microspheres would check all the specifications and make sure they are functional and in range. This would involve looking at the size ranges of the microspheres. The amount of radioactivity and the pace of its dropoff would also be specifications of concern.

The other important component of this device is the syringe because it delivers the microspheres. Testing the volume of injection, rate of injection, and other aspects of the syringe function would be important. It would also be necessary to make sure that the syringe material can withstand the radioactivity under storage. It would not be good if the plastic syringe became brittle and shattered after storing the device on the shelf for a few weeks!

Meanwhile, validation testing would be concerned with patient needs. Does this device perform safely? Is it efficacious? A clinical trial or perhaps multiple clinical trials would be necessary to prove how well it functions in

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decreasing tumor size and/or increasing patient survival.

Of course, these would all be set in the indications for use, which are not specified in this problem, nor is the type of cancer it would treat. A device like this would likely treat a specific type of cancer in a specific type of patient.

Aside from patient needs, validation would also look at physician needs, if the physician is the one injecting the treatment. How hard is it to push the plunger? How long does that take? How hard is it to attach a Luer lock to the end of the syringe if that is what the physician wants to do?

Is such a thing even recommended? These questions would be answered by consulting with physicians who would use the device and collecting the results.

5. Consider a design input document that has 23 design inputs listed. How many design specifications and design outputs will that yield?

Answer: It is not possible to tell from the number of design inputs how many specifications will wind up proliferating from them. Often, a single design input will yield multiple design specifications. A single design specification can sometimes yield multiple design outputs. A thoughtful answer to this question might be “at least 23 specifications and 23 outputs,” but that is also not always the case. Sometimes it is possible for multiple design inputs to feed into a single specification, so one specification covers more than one design input. In such a case, there could actually be fewer than 23 specifications and outputs stemming from the inputs, but it would be rare.

Solutions Manual for Biomaterials Science - An Introduction to Materials in Medicine 4th Edition by Wagner, William R.; Zhang, Guigen; Sakiyama-Elbert, Shelly E.; Yaszemski, Michael J.

Student Questions 1. What are some of the recent changes that affect the success of a new medical device?

2. What are some of the differences between linear development and agile product development?

3. Give an example of Class 1 and Class 2 data.

4. How do medical device companies fulfill different stakeholder needs?

5. What are some of the skills needed for a successful product development team?

6. Can you speculate how product development for emerging markets might differ from the product development process in developed markets?

7. Explain the differences between FDA approval and CMS approval and how it affects the success of a medical device.

8. During which stages of development does a company secure funding for their idea?

9. What are the four marketing claims a product can make?

Which do you think is the strongest?

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Chapter Questions: True or False 1. Medical device regulation is harmonized globally and therefore once a device is approved in the United States by the FDA it can be sold in any country.

2. A Class I device is not regulated in the United States.

3. Risk management standard ISO 14971 provides the requirements for determining how to conduct biocompatibility testing.

4. ISO 10993 says that prior human clinical use of the material reduces the risk of biomaterial toxicity and the

prior use makes the material so safe no other testing is necessary.

5. Japan recognizes US PMA determination, so a product approved in the United States does not require a separate application for sale in Japan or Korea.

6. ISO 13485 quality requirements are identical to the US 21 CFR 820, so an ISO certificate means a US company will not be inspected by the US FDA. 7. Submission of the Clinical Evaluation Report to the notified body for the CE mark exempts a foreign company from needing to file a 510(k) in the United States.

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Homework Questions 1. Which one below lists all the types of ASTM standards?

A. Classification and Test Methods.

B. Classification, Guide, Specification, Terminology and Test Methods.

C. Classification, Guide, Practice, Specification, and Test Methods.

D. Classification, Guide, Practice, Specification, Terminology, and Test Methods.

2. Describe the differences between an ASTM specification standard and a test method standard.

3. Who uses standards?

A. Manufacturers and Test laboratories B. Manufacturers, Consumers, Test laboratories, College professors and their students.

C. Manufacturers, Test laboratories, College professors and their students.

4. Which below best completes this sentence? Compliance with a standard …

A. is voluntary.

B. is nonvoluntary.

C. may be either voluntary or nonvoluntary.

5. What are the names of some standards development organizations (SDOs) that prepare consensus standards through voting (lists are not exhaustive)?

A. AAMI, ASTM, ISO, and ADA B. AAMI, ASTM, ISO, and ANSI 6. Briefly describe the ASTM standards development process including words like task groups, subcommittees, and ballots.

7. Both the subcommittees and main committees of ASTM must be balanced between what categories of stakeholder groups?

8. How can domination by any one stakeholder or interest group be prevented during development of a standard?

A. Only one vote per voting interest is allowed.

B. Only one member per voting interest may join the SDO. C. Through discussion at working groups or taskforce group meetings.

9. Should a standard be based on well-established technologies or on new areas of research? Circle one.

10. (A) Why are there so many different standards organizations, and (B) accreditation by what organization brings some measure of uniformity of process to the many SDOs in the USA? 11. Identify a standard within ASTM (www.astm.org) that covers the testing needed to establish the biocompatibility of a new material for a medical device.

12. Which type of testing below is an example of in vitro testing: A. in a cell culture plate B. implantation in an animal C. a simulation on a computer 13. Are materials proposed for medical use tested by direct contact, by using extracts obtained by soaking the

implant in liquids that simulate body fluids or by both methods (search www.astm.org)?

14. What animal is skin irritation typically tested in (search www.astm.org)?

A. Rabbit B. Dog C. Cat D. Mouse 15. Are guinea pigs used in testing for allergic reactions?

Search the www.astm.org website to find out.

16. Search the www.astm.org website to find three examples of standards for biomaterials, biomolecules, cells and tissue-engineered constructs that apply to tissueengineered medical products.

17. Standards development in the area of nanomaterials has been slow. Suggest possibilities for what has held up standards development in the field of nanotechnology.

Answer Key for Homework Questions 1. What are the types of ASTM standards?

Answer: D. Classification, Guide, Practice, Specification, Terminology, and Test Methods.

2. Describe the differences between an ASTM specification standard and an ASTM test method standard.

Answer: A specification describes chemical, mechanical, physical, and electrical properties of a material. A test method standard describes the test specimen to be used, the conditions under which it is to be tested, how many specimens and what controls are to be tested, and how the data are to be analyzed. Often, the reproducibility of test methods is assessed by “interlaboratory testing,” meaning that several laboratories have followed the method and the results are analyzed to determine the degree to which they agree.

3. Who uses standards?

Answer: B. Manufacturers, Consumers, Test laboratories, College professors and their students.

4. Which below best completes this sentence? Compliance with a standard …

C. may be either voluntary or nonvoluntary.

5. What are the names of standards development organizations (SDOs) that prepare consensus standards through voting?

Answer: A. AAMI, ASTM, ISO and ADA. Note: ANSI does not write standards but instead coordinates the activity of standards development organizations in the United States.

6. Briefly describe the ASTM standards development process including words like task groups, subcommittees, and ballots.

Answer: This is a brief description of the ASTM standards development process: A standard work item can be proposed by anyone and then approved by the executive committee of a subcommittee based on a rationale and scope provided by the person or group initiating the standard. A task group is created that includes 1484.e1

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5–20 stakeholders and they draft the standard. It is written using an ASTM template for one of the six defined types of standards. It is sent out for ballot at the subcommittee level first and then the main committee.

Persuasive negative votes will stop the process and may require revision of the standard. If no negative votes are received at either subcommittee or main committee, then the item becomes a published standard.

7. Both the subcommittees and main committees of ASTM must be balanced between what category of stakeholder groups?

Answer: Producers and nonproducers.

Note: Nonproducers include users, consumers, and general interest.

8. How is domination by any one stakeholder or interest group prevented during development of a standard?

Answer: A. Only one vote per voting interest is allowed.

9. Should a standard be based on well-established technologies or on new areas of research? Circle one.

Answer: Well-established technologies.

10. (A) Why are there so many different standards organizations, and (B) accreditation by what organization brings some measure of uniformity of process to the many SDOs in the USA? Answer: (A) Each SDO provides valuable standards for a particular area of technology and has the right stakeholders and experts to write these standards. (B) ANSI accreditation provides a measure of uniformity and consistency of process to the many SDOs. 11. Identify a standard within ASTM (www.astm.org) that covers the testing needed to establish the biocompatibility of a new material for a medical device.

Answer: There are many standards that could provide a correct answer. Some of them are listed here: F748 Standard Practice for Selecting Generic Biological Test Methods for Materials and Devices Medical Devices.

F3206 Standard Guide for Assessing Medical Device Cytocompatibility with Delivered Cellular Therapies.

F813 Standard Practice for Direct Contact Cell Culture Evaluation of Materials for Medical Devices.

F1027 Standard Practice for Assessment of Tissue and Cell Compatibility of Orofacial Prosthetic Materials and Devices.

12. Which type of testing below is an example of in vitro testing: Answer: A. In a cell culture plate.

13. Are materials proposed for medical use tested directly, by using extracts obtained by soaking the implant in liquids that simulate body fluids or both?

Answer: Both.

Note: Under ASTM Committee F04.16, see the following: ASTM F749 Practice for Evaluating Material Extracts

by Intracutaneous Injection in the Rabbit ASTM F750 Practice for Evaluating Material Extracts

by Systemic Injection in the Mouse ASTM F813 Practice for Direct Contact Cell Culture Evaluation of Materials for Medical Devices 14. What animal is skin irritation typically tested in?

Answer: A. Rabbit.

Note: See ASTM F719 Standard Practice for Testing Biomaterials in Rabbits for Primary Skin Irritation.

Humans are also used. See ASTM F2808 Standard Test Method for Performing Behind-the-Knee (BTK) Test for Evaluating Skin Irritation Response to Products and Materials That Come into Repeated or Extended Contact with Skin.

15. Are guinea pigs used in testing for allergic reactions?

Search the www.astm.org website to find out.

Answer: Yes. Note: See ASTM F720 Practice for Testing Guinea Pigs for Contact Allergens: Guinea Pig Maximization Test.

16. Search the www.astm.org website to find examples of standards for cells and tissue-engineered constructs that apply to tissue-engineered medical products.

Answer: There are many standards that could provide a correct answer. Some of them are listed here: ASTM F3259 Standard Guide for Micro-computed Tomography of Tissue Engineered Scaffolds ASTM F2211 Standard Classification for Tissue Engineered Medical Products (TEMPs) ASTM F2315 Standard Guide for Immobilization or Encapsulation of Living Cells or Tissue in Alginate Gels ASTM F2739 Standard Guide for Quantifying Cell Viability within Biomaterial Scaffolds 17. Standards development in the area of nanomaterials has been slow. Suggest possibilities for what has held up standards development in the field of nanotechnology.

Answer: Standards for nanoparticles have been delayed due to the lack of high-precision methods required to ensure reliability of the results. It is not possible to standardize something that is not reproducible or reliably measured. A lack of reproducibility has also delayed standards for cells in the field of tissue engineering and regenerative medicine. Uncertainty with test methods and high variability in the results, and in the cells themselves, has made it difficult to define appropriate quality measures.

Solutions Manual for Biomaterials Science - An Introduction to Materials in Medicine 4th Edition by Wagner, William R.; Zhang, Guigen; Sakiyama-Elbert, Shelly E.; Yaszemski, Michael J.

Chapter Exercises (With Answers) 1. Identify the characteristics that define an implant, as recognized by the National Institutes of Health.

Answer: An implant is defined as a type of medical device that is placed inside or on the surface of the human body, as having an intended lifespan spanning months or years, as being made of biomaterials and penetrating living tissues, as having a physiological interaction in the human body, and as being retrievable.

2. Define implant retrieval analysis and identify two general goals for it.

Answer: Implant retrieval and evaluation (commonly, implant retrieval analysis or explant analysis) describes a method of scientific analysis for determining the efficacy and safety or biocompatibility of medical implants in vivo.

General goals include: • Determine rates, modes, and mechanisms of implant failure • Identify effects of patient and implant factors on implant performance and overall clinical outcome • Establish clinical and design factors that promote implant success • Determine dynamics, temporal variations, and mechanisms of tissue–biomaterial and blood–biomaterial interactions • Develop design criteria for future implants • Determine adequacy and appropriateness of animal models • Identify biomarkers of implant reactivity.

3. Assume you are involved in characterizing biomaterials for the development of a new implant. Devise a preclinical testing plan that makes use of information gained through implant retrieval and evaluation.

Answer: This question is open-ended and has many possible correct answers.

A preclinical testing plan could include: (1) Inspection of unimplanted prototypes to evaluate changes induced by the fabrication process (2) In vitro testing of implant biomaterials followed by implant retrieval and evaluation to evaluate biocompatibility, durability of the biomaterials, or potential failure modes (3) In vivo animal models followed by implant retrieval and evaluation to evaluate biocompatibility, durability, implant–tissue interactions, or safety and effectiveness of the design.

4. Argue the advantages of integrating implant retrieval and evaluation programs into implant registries and other intentional surveillance activities.

Answer: This question is open-ended and has many possible correct answers.

Quantitative and systematic implant retrieval and evaluation …

• Plays a critical role in the evolution of medical devices throughout the development process and clinical use • Informs the medical device industry through their corrective and preventive action (CAPA) systems for meeting FDA quality system regulation requirements • Encourages technology development through identification of failure modes and mechanisms, providing a benchmark for assessing future improvements in implant performance and longevity • Provides an evidence-based approach for driving change in surgical techniques and implant biomaterials and designs • Is a suitable tool for evaluating the safety and effectiveness of high-risk devices, devices that have long in vivo durations, devices in which substantial design variations provide the potential for significant variations in outcomes, and those in which device failure has serious implication for the public health.

5. What failure mechanisms did implant retrieval and evaluation reveal about early Björk-Shiley prosthetic heart valve designs? Describe the designs of modern heart valves that help to minimize the future risk of similar failures.

Answer: The tilting disk mechanical prosthetic heart valve design was associated with an unacceptable late failure rate owing to thrombotic occlusion. The redesigned Björk-Shiley C–C heart valve had fatigue fracture of the metallic outlet strut initiated at or near intrinsic weld flaws and the point of maximum bending stress, leading to disk escape, pronounced wear at the tip of the outlet struts, and localized pyrolytic carbon wear deposits at sites where the closing disk contacted the strut.

Designs of modern heart valves achieve low peak stresses throughout the cardiac cycle to minimize the potential for creep deformation or fatigue.

6. In the early 1970s, what biomaterial problems were revealed by implant retrieval analysis that helped to prompt the need for FDA regulation of medical devices, as authorized through the Medical Device Amendments of 1976?

Answer: Explant analysis of the Dalkon Shield intrauterine device (IUD) revealed the multifilament tail string served as a wick for vaginal fluid and bacterial access inside the uterus. Deterioration of the outer nylon sheath, especially in regions of high stress adjacent to the knotted attachment side, provided a route for bacteria to exit the sheath into the sterile uterine cavity and contributed to string breakage during attempted removal.

The absence of heat-sealed ends on the multifilament tail strings and deterioration of the outer nylon sheath were identified as contributing factors in the high infection rates and the FDA identified the multifilament tail string as a hazard for its role in the infections.

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