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UBC Materials Engineering MTRL 466/467 Final Design Project Report

Design of Composite Porous Scaffolds Gabriel Hung, Calvina Martin, Hans Saputra, Ricky Tam Prof. Rizhi Wang November 28, 2011

EMAIL 309-6350 Stores Road, Vancouver, BC V6T 1Z4


Design Report Executive Summary In this project, an extensive literature review on the synthesis of a composite scaffold was done. Possible processing methods were considered including freeze-drying and electro-spinning. Our team decided to apply freeze drying method due to the simplicity of the method. Incorporation of CaP (Hydroxyapatite) was also considered to produce composite porous scaffold. Two methods of preparation/treatment were also considered such as addition of NaOH solution and Gas diffusion. Also, basic chemistry involved in this project including distribution diagram of tri-phosphoric acid, Chitosan solubility, and precipitation of Hydroxyapatite was studied. Aside from the chemistry aspect, mechanical properties relating to the structure of the scaffold was also studied. The purpose of combining Chitosan and Hydroxyapatite is to improve the mechanical properties of the structure. Thus, our group decided to synthesize the scaffolds with different wt% of HAp (0%, 10%, and 20%) incorporated into the chitosan. Scaffolds were successfully manufactured through two different approaches of increasing pH such as addition of NaOH (1M) and gas diffusion (NH3). The results and detail of the experiment will be discussed in the design section in this report. After the scaffolds were obtained, SEM and EDS analysis were performed. This is to analyze the overall structure, pores size, and also the chemical composition of the samples.

Design of Composite Porous Scaffolds

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Design Report Need and Constraint Background Chitosan has been a very influential material for the biomedical community. This is due to its biocompatibility and porous properties which makes it very flexible. It is derived from chitin, a biopolymer found from crustacean shells and fungi, where chitin undergoes deacetylation. This process removes the acetyl functional group from the polymer chain to bear chitosan as a result. Chitosan can be applied to multiple applications including wound dressings, porous scaffolds and as a drug releasing system. In our design project, we are concerned with the use of chitosan in the pursuit of a porous scaffold for bone regeneration. Potential customers will include biomedical engineers and researchers. They will provide a criterion for the design which will be detailed in the following section.

Design Needs and Constraints Over the recent years, many devices were invented to replace or support bone structures in the human body. This is due to the increasing demand for viable ways to increase bone regeneration. Presently, bone injuries can occur resulting from aging, sports or traffic accidents. Therefore, the focus of this project will be on porous scaffolds which are used to support bone regeneration. The scaffold has 3 major design criteria which must be met. Firstly, the chosen material must be biocompatible within the human body. Secondly, the scaffold must be strong enough to support any stresses experienced in skeletal frame in which it resides. Finally, a minimum porosity and pore size must be met to permit the in growth of cells into the scaffold.

Design of Composite Porous Scaffolds

Series 1

6

Series 2

Series 3

5

4 3

2 1

0 Category 1

Category 2

Category 3

Category 4

Figure 1. Cell Generation – 3 weeks after scaffold implantation

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Design Report Problem Specification As mentioned in the Background and Design Needs and Constraints, our ultimate goal is to economically produce a bone scaffold upon which the human body can regenerate lost tissue and bone. The bone scaffold will serve as a temporary bone replacement until the body has fully repaired the lost or damaged tissue. As such, the bone scaffold will need to mimic the structural properties of the bone itself and disintegrate after its job is done, thereby eliminating the need for surgical removal. An issue in the creation of such a scaffold is achieving the required strengths required to support the bone structure. A pure chitosan scaffold is much too weak to survive the rigours in the human body. To deal with this lack of strength, the team has chosen to incorporate a second phase into the Chitosan scaffold to produce a composite scaffold. The second material, Hydroxyapatite, was chosen for its biocompatibility, biodegradability, non-toxic nature, and most importantly, its high modulus. Hydroxyapatite is the mineral from which teeth and bone are made from, and this promotes osteoconductivity. The reasons for not using a purely Hydroxyapatite scaffold and ignoring chitosan completely are that pure Hydroxyapatite is brittle, has low crack resistance, and is difficult to form and shape. The parameters vital to the success of the bone scaffold are as follows. Porosity needs to be greater than 50% and pores need to be approximately 100Âľm wide and interconnected to allow for cell transplantation, blood flow and osteoconductivity. Next, the compressive strength and modulus need to mimic the cancellous bone found in a natural human body. If the modulus and strength are too low, the scaffold may fail during handling of the scaffold. It may also fail under initial usage, before the ingrowth of bone has occurred and can only support a limited load. Cancellous bone has Elastic Modulus of around 0.1 to 1 GPa. 100 MPa is therefore the target for our composite scaffold. However, different areas of the skeleton require different properties, and a model will be developed to predict the final mechanical strengths required by customer's needs. The team will also need to evaluate and choose a proper processing method to put theory into practice and physically produce the Chitosan/Hydroxyapatite composite scaffold to predetermined specifications.

Design of Composite Porous Scaffolds

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Design Report Technical Review Solubility of Chitosan Solubility of Chitosan is one of the many important aspects to be considered upon making Chitosan composite scaffold.

Figure 2. Comparison of Degree of Protonation of Chitosan as a Function of the Acid Concentration of HCl

The degree of protonation α, can be determined and plotted as a function of initial concentration of the Acetic Acid. The amount of acid needed depends on the quantity of Chitosan to be dissolved. In this project, 100 mL of 0.2 M Acetic Acid is used to dissolve pure Chitosan (95%). 0.2 M Acetic Acid corresponds to pH ≈ 2.7 which corresponds to α ≈0.2. The concentration needed to dissolve the Chitosan is at least equal to the concentration of –NH2 units involved (Rinaudo.M, Pavlov.G). As Chitosan is added to the system, the pH will increase and α will decrease. When α reaches zero, Chitosan powder cannot be dissolved in the solution anymore. The equilibrium reaction is as follow:

This reaction will help us approximate the maximum amount of Chitosan can be dissolved in 100 mL of 0.2M Acetic Acid. Since both the concentration of CH3COOH and the degree of protonation is approximately 0.2, the concentration on Chitosan in the system should not exceed 4%. In other words, 100 mL of 0.2M Acetic Acid can only dissolve approximately 4 grams of Chitosan.

Chemical Aspects of HAp Precipitation Precipitation of Calcium Phosphate (CaP) depends on the precipitation conditions such as temperature, degree of super saturation, pH and initial concentration of reagents. In this project, the study of precipitation of two stable forms of Calcium Phosphate at low temperature, mainly Hydroxyapatite (HAp) and Brushite (DCPD), will be studied. The precipitation is achieved by mixing equimolar quantities of Calcium Hydroxide in suspension with Phosphoric Acid solution at room temperature. Design of Composite Porous Scaffolds

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Design Report Formation of Brushite does not occur at the beginning of the reaction. On the other hand, Hydroxyapatite will precipitate at the beginning and will eventually evolve to Brushite. The precipitation process of Brushite can be simplified into five stages as follows: 1. 2. 3. 4.

Initiation of nucleation of Hydroxyapatite and pH stabilization Complete dissolution of Calcium Hydroxide and decrease in pH due to HAp growth Nucleation of Brushite Co-existance of two phases: Brushite and HAp

Another important aspect to understand is the involvement of ions in the formation of HAp.

Figure 3. Ions necessary for the growth of HAp: A, Hydryoxapatite; B, required ions for HAp growth; C, Present ions in solution; D, Hydrogen Ions

At low pH when the Phosphoric Acid and Calcium Hydroxide are initially mixed, some Brushite formed initially is dissolved while at the same time the Calcium Hydroxide is continuously dissolved in the solution. This may result in the constant Calcium concentration in the system. The consumption of Hydroxide ions should cause a decrease in pH, however, the dissolution of Calcium Hydroxide maintains the same pH level. At low pH, the Phosphate ions do not exist in significant amount. The most stable ions will be Dihydrogenphosphate (H2PO 42-). The consumption of Phosphate ion in the formation of HAp may result in the decrease in pH due to the release of [H +] from (H2PO 42-) dissociation. However, this does not happen at the beginning of the stage due to the continuous dissolution of Ca(OH)2. Once the Ca(OH)2 is completely dissolved, a decrease in Calcium concentration and a sudden decrease in pH is expected. In order to understand the last stage of Brushite formation, the solubility and ratio of Calcium concentration of the species present in the solution (HAp and Brushite) needs to be analyzed. At pH ranging from about 4.2 and above, the most stable form of Calcium Phosphate is Hydroxyapatite.

Design of Composite Porous Scaffolds

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Design Report The speciation diagram of Phosphoric Acid can be obtained from the three dissociation equations as follow:

[ ] [ ]

[ ]

[ ] [ ]

[ ]

[ ]

[ ] [ ]

[ ]

[ ]

[ ] [ ]

[ ]

[ ]

[ ]

[Ca+2] vs. pH

Speciation Diagram

0

100%

-1

H 2 PO4 -

PO4 3-

HPO4 2-

80%

-2

-3

Fraction

log [Ca]

H 3 PO4

-4

-5 -6

60% 40% 20%

-7

-8 3

5

7

9

11

0%

13

0

pH HAp

5

[H3PO4]rel

DCPD (Brushite)

Figure 4. Calcium concentration of HAP and Brushite in the solution

[H2PO4-]rel

pH

10 [HPO42-]rel

15 [PO 43-]

Figure 5. Speciation Diagram of Triphosphoric Acid System

The incorporation of a second phase (CaP/Hydroxyapatite) into the Chitosan polymer can be challenging. In this project, our team is inspired by a basic idea of precipitation of Hydroxyapatite from aqueous solution. The equation involved is as follow: (

)

(

)

(

) (

)

According to the solubility diagram, at pH above 12, the Orthophosphate ions are predominant and thus the precipitation of Hydroxyapatite can be maximized. More detail explanation will be discussed in the design section.

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Design Report Preparation of Chitosan/HAp Composite Scaffold Various methods have been used to prepare Chitosan/HAp composite such as precipitation method, electro-spinning, simple in-situ hybridization, solvent casting and evaporation method, in-situ chemical method, freeze drying method, and simple mixing and membrane diffusion methods. In this project we are focusing on the combination of freeze drying and precipitation methods. The details of the preparation will be discussed in the “Design Section”.

Structure & Chemical Interaction Chitosan/HAp Composites Chitosan is a co-polymer consisting of β-(1-4)-2-acetamido-d-glucose and β-(1-4)-2-2amino-D-glucose unit linkages as shown figure 6.

Figure 6. Structure of Fully Deacetylated Chitosan

This structure allows the possibility of making Chitosan composite materials. In this project, Chitosan and HAp composite materials is considered. From the figure 7 below, Ca2+ ions appear on the terminated surface of HAp crystals, which have coordination number of seven strongly attached to the structure. Thus, there is a possibility to form coordination bonds between the –NH2 of Chitosan and Ca2+ of HAp (Vankatesan.J , Se-Kwon Kim).

Figure 7. Chemical Interaction between Chitosan and HAp

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Design Report Mechanical Properties of Chitosan/HAp Composite The mechanical properties of the Chitosan and HAp composites play a significant role in bone tissue engineering. The intermolecular Hydrogen bond and interaction between Chitosan and HAp contributes to good mechanical properties. As mentioned above, there is the possibility of the Ca+ ions to interact with the –NH2 and secondary OH group of Chitosan to form a strong bond which may contributes to the higher mechanical strength of the structure as compared to the Chitosan alone.

Chitosan and Carbon Nanotubes Composite Aside from Hydroxyapatite, Carbon nanotubes (CNTs) can also be used as a composite materials. CNTs are allotropes of Carbon with cylindrical nanostructure and length to diameter ratio of 28000000:1. CNTs have a high Young’s Modulus of about 1.5 TPa, high tensile strength of about 100 GPa as well as high ductility. This material has been used for many biomaterials engineering application to improve the mechanical strength of scaffold materials.

Predicting Mechanical Properties of the Composite Scaffold Through the analysis of previous experiments and literature, it has been found that the general rule of mixtures models the expected values of our desired mechanical properties really well. Although this rule of mixtures may not exactly predict the mechanical properties of the product, it will provide a good estimate of how the mechanical properties would be affected with respect to the amount of each constituent. This rule of mixtures is only valid for the Young's Modulus, yield strength, and hardness. It is these three properties that we are most interested in for the final product of our chitosan scaffold.

The generalized rule of mixtures states that the final value of a mechanical property (Ec) is proportional to the mechanical property of each constituent (E1,E2,E3) and its respective volume fraction (V 1,V2,V3). J is a scaling factor which is based on the geometrical shape, spatial arrangement, orientation, and size distribution of the pores. In our specific case, we have 3 total phases in our composite scaffold. The two constituents of the scaffold are the chitosan and hydroxyapatite, while the third constituent, the porosity, or void space, must be considered as well. The void space has a value of zero for E, and its volume fraction is therefore the porosity of our scaffold. From this simple equation, several implications are quite clear. If we want to maximize the modulus and yield strength, then the volume fraction of the phase with the highest modulus or compressive strength should be increased. Hydroxyapatite has a modulus of approximately 95 GPa, which is significantly greater than the modulus of c hitosan. Therefore, we would want a higher amount of hydroxyapatite. Porosity, or void space, has a modulus of zero of course. Therefore, if we want a higher strength scaffold, we would want to maximize the hydroxyapatite content and to minimize porosity. However, one must not lose sight of the final goal in the pursuit of higher modulus. This is a bone scaffold after all, and there is a lower limit to the amount of porosity required. To develop a chitosan scaffold to the design requirements for a specific location and usage in the human body, we can use adjust and alter 4 variables; porosity, HAp content, chitosan content, and pore morphology. Of course, we are subject to the question of, "Is it actually possible?" Depending on the processing method, there are limits to how far the bounds of adjustability lie and that would require further research to define. The downside of using the generalized rule of mixtures in our quest to predict the mechanical properties is that we cannot explicitly estimate the value of J as well as the exact values to use for the properties of our constituents. In order to fully realize Design of Composite Porous Scaffolds

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Design Report the potential of this equation, the results of real world testing must be fit against the expectations of the equation. The v alue of J must be experimentally verified for each type of pore morphology. Only after this is done can a reliable prediction be made with that particular morphology of pores. If we vary the pore morphology, then a set of experiments must be conducted to verify the behaviour and reaction of the scaffold to varying parameters. Unfortunately, while several specimens have been produced, we were unable to test the mechanical properties to either prove or disprove the validity of our rule of mixtures.

Freeze Drying Freeze drying process can be simply understood as a dehydration process. Freeze drying works by freezing the Chitosan/Hydroxyapati te solution. This freeze solution is then placed in a vacuum where the pressure is decreased which allows the water to sublimate directly from solid phase to the gas phase. The sublimation process will ensure that the final scaffold product will have the desired porous structure without compromising the shape of the composite.

Figure 8. Freeze Solution in Vacuum Drying Machine

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Design Report Assess and Select Design Options Based on the technical review, freeze drying is the processing method chosen for this project. Also, Hydroxyapatite is chosen as our composite materials. Freeze drying method is relatively simpler than other methods mentioned in the technical review. This method is very reliable in terms of forming a porous structure. For example, the mold used prior to freeze drying determines the shape of the final desired structure. Thus, flexibility in forming the scaffold as well as a uniform porous structure can be achieved using this technique. Hydroxyapatite is a good candidate for the composite because Calcium Phosphate materials are osteoconductive, providing an ability to mimic the inorganic portion of natural bone. Combining Chitosan and Hydroxyapatite to form composite materials ( ) ( ) ] allows possibility to imitate both the organic and inorganic portion as of the natural bone. Hydroxyapatite [ is one of the most stable forms of Calcium Phosphate and is a major component of the bone (60% to 65%). Thus, HAp is the most attractive material to imitate some properties of natural bone. Figure 9 shows a thinking process for a specific freeze-drying processing method to meet the design requirement. Although it is not easy, we try to assess the contribution semi-quantitatively by providing a detail engineering design in the later section. It is clear that excellent biocompatibility, bioactivity, and sufficient porosity can be achieved. However, it is shown that the challenge is mainly on improving the mechanical properties. Two types of process preparations were considered in this project. First is the liquid-mixing method which involves the addition of 1M NaOH solution into the Chitosan/Acetic Acid + Calcium Nitrate + Ammonium Phosphate salts solution and second is the gas-diffusion method which involves the diffusion of Ammonia gas into the solution. The purpose of adding basic elements into the system is to increase the pH of the system. By doing so, the gelation and the formation of the Chitosan and the Hydroxyapatite are facilitated. The detail of these preparations method will be discussed in detail in the “Design Section�.

Figure 9. Contribution Diagram

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Design Report Detailed Design In order to determine whether the design is viable, a few samples with two different methods containing different Chitosan and HAp composition were manufactured. The microstructures of these samples were then analyzed with Scanning Electron Microscope (SEM) and Energy Dispersive Spectroscopy (EDS).

Sample Preparation In order to prepare the samples, a base solution of Chitosan was made by dissolving 2.5wt% Chitosan into 25 mL Acetic Acid. The solution was stirred with a magnetic stirrer until homogenously mixed. While still in agitation, 0.1 M Ca(NO 3)2 is added, followed by a drop-by-drop addition of 0.06 M (NH 4)2HPO 4. This Chitosan solution was then treated with two different methods to increase the pH which will promote the gelation process as well as the precipitation of Calcium Phosphate in the form of Hydroxyapatite.

Figure 10. Base Chitosan Solution

Liquid-Mixing Method The liquid-mixing method involves mixing 1 M NaOH into the solution while in agitation until precipitation was formed. This solution was then transferred into vials and freeze-dried to obtain the final composite scaffold sample. A unique observation was made during the NaOH addition. From figure 11, we are able to observe that a gel-like precipitation was formed during the first few hours of NaOH addition. This is because by adding a highly concentrated NaOH, immediate gelation of Chitosan occurred with the co-precipitation of Calcium Phosphate. However, this gel-like precipitation dissolved and mixed homogenously after left for 24 hours.

Design of Composite Porous Scaffolds

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Design Report

Figure 11. Co-precipitation of Hydroxyapatite

Gas-Diffusion Method The gas-diffusion method can be achieved through the diffusion of NH 3 gas through the Chitosan solution. The base Chitosan solution was transferred into the vials and placed, caps open, in the desiccator alongside with a sufficient amount of NH 3 placed in a beaker.

Samples

NH3 Figure 12. Gas-Diffusion Method Set-Up

The samples and NH3 solution were left in the desiccator for 24 hours. This allows the NH3 gas to diffuse into the samples in the samples which in turn will increase the pH, resulting in the gelation of Chitosan solution and the precipitation of Hydroxyapatite. These samples were then freeze-dried to obtain the final scaffold samples. For the gas-diffusion method, two different types of samples were produced: 10% HAp and 20% HAp. We chose these ratios in preparation to test the effects of the varying amounts of each constituent. However, we were unable to physically test our Design of Composite Porous Scaffolds

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Design Report samples. Through simple chemical calculation and the assumption that 100% precipitation of Calcium Phosphate, we were able to determine the amount of Ca(NO 3)2 and (NH4)2HPO 4 to be added in the base Chitosan solution. The compositions are as follows: Table 1. Amount of Solution Added According to HAp wt%

Input Chitosan (wt%) HAp (wt%) 80 20 90 10 100 0

Ca(NO3)2 (mL) 3.193 1.419 0

(NH4)2HPO4 (mL) 3.193 1.419 0

Final Scaffold Samples

Figure 13. Sample with 10wt% HAp + NaOH(aq)

Figure 14. Sample with 0wt% HAp + NH3(g)

Figure 15. Sample with 10wt% HAp + NH3(g)

Figure 16. Sample with 20wt% HAp + NH3(g)

As observed from the figures above, the samples treated with gas-diffusion method (figure 14 and 16) displayed more shrinkage comparing to the samples treated with liquid-mixing method (figure 13). This is due to the fact that the Design of Composite Porous Scaffolds

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Design Report precipitation of Calcium Phosphate was ‘crushed’ by the pressure from water expansion during the freeze-drying process, causing the overall scaffold structures to shrink. It is also observed that the samples with more wt% HAp have a better structure; this is due to the strong mechanical properties contributed from presence of Calcium Phosphate.

Figure 17. Freeze-Drying Process Diagram

In order to determine the properties of these samples, microstructure analysis was done. Each of the samples was prepared by cutting a small cross-section. Since these samples are not conductive, Au-Pd coatings were applied using the Sputter Coater. After the samples were ready, the microstructures were analyzed using SEM and EDS.

10wt% HAp + NaOH

10wt% HAp + NH3(g)

20wt% HAp + NH3(g)

0wt% HAp + NH3(g)

Figure 18. Samples with Gold Coatings - Ready for SEM and EDS Analysis

Design of Composite Porous Scaffolds

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Design Report Microstructure Analysis Through SEM and EDS analysis, we are able to analyze the overall structure as well as the pore structure, surface morphology and chemical distribution of each samples. The overall structure of each sample can be observed through SEM analysis at 80x magnification. From figure 19 to figure 22, we are able conclude that different manufacturing method will results in different microstructure. By comparing the microstructures, we can see that the sample treated with liquid-mixing method (figure 19) produces microstructure with smoother surface morphology and thinner walls. However, the samples treated with gas-diffusion method (figure 20 to figure 22) produce microstructure with rougher surface and thicker walls.

10wt% CaP + NaOH

0wt% CaP +NH3

Figure 19. 80x Magnification of 10wt% HAp + NaOH

Figure 20. 80x Magnification of 0wt% HAp + NH3

+NH310wt% CaP +NH3

20wt% CaP +NH3

Figure 21. 80x Magnification of 10wt% HAp + NH3

Figure 22. 80x Magnification of 20wt% HAp + NH3

In addition, from the 80x magnifications of each sample, we can conclude that all the samples have similar pore sizes. However, the sample treated with liquid-mixing method has a more uniform pore size compared to the samples treated with gas-diffusion method.

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Design Report The surface morphologies of the samples treated with different methods can be further examined by comparing the 10wt% HAp samples treated with NaOH and NH3. By comparing figure 23 and figure 24, we can clearly observe the difference in the surface morphology as well as the pore structure. The liquid-mixing treated samples displays a much softer and smoother surface with thinner walls whereas the gas-diffusion treated samples displays rough surface – almost needle-like – with much thicker wall structures.

10wt% CaP + NaOH

10wt% CaP

Figure 23. 500x Magnification 10wt% HAp + NAOH

Figure 24. 700x Magnification 10wt% HAp + NH3

In order to analyze the thick wall, we did a spot analysis with EDS to determine the composition of the thick wall. The resul ts shows that the thick wall contains Calcium and Phosporus composition ratio of 1.69. This is a very satisfying result since the human bone has the theretetical ratio of 1.67 which is very close to the experimental value. Table 2. Composition at Point A

Composition wt%

C 54.01

O 37.02

P 3.33

Ca 5.63

A

Figure 25. 1000x Magnification 10wt% HAp + NH3 Spot Analysis at Point A

Figure 26. Graph of Composition at Point A Design of Composite Porous Scaffolds

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Design Report In order to confirm that our composite scaffold contains both Chitosan and Hydroxyapatite composition, particle distribution analysis is done. Using EDS, we are able to compare the particle distribution for samples undergone both the liquid-mixing method and the gas-diffusion method. From figure 27 and figure 28, we can observe that the Calcium Phosphate is evenly distributed throughout both of the sample. This result is very satisfying since an evenly distributed Calcium Phosphate increase the total mechanical properties throughout the whole scaffold body.

10wt% CaP + NaOH

Figure 27. Particle Distribution for samples with 10%wt CaP + NAOH

Design of Composite Porous Scaffolds

20wt% CaP + NH 3

Figure 28. Particle Distribution for samples with 10%wt CaP + NH3

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Design Report Socio-Economic Assessment of Design In order to describe the cost of this design, a simple scenario must be setup to help determine what contributes to the economic assessment. Before proceeding, a few assumptions must be made for simplicity. First, a lab workplace is available to use without any cost but no equipment has been provided. Second, the client for this operation is a distributor which provides the requirements needed for the scaffold design; therefore the need to obtain approval from the FDA is not necessary. Lastly, the lab is arranged to produce scaffolds specifically for the Greater Vancouver community. There are approximately 6.8 million bone fracture injuries reported in the States every year. This translates to 2 percent of the population which then estimates to around 1100 broken bone cases in Vancouver. Now, considering all of these cases use scaffolds as means of repair, the lab will average production of 3 scaffolds per day. This minute quantity is definitely feas ible to accomplish. The following tables will outline the costs of each section. As well, the scaffolds that will be produced are designed to be large enough to fit into an average adult's ulna bone in their arm. Table 3. Reagent Costs for a Single Scaffold

Reagent Costs (Single Scaffold) Chitosan

6

Acetic Acid

21.2

Calcium Nitrate Ammonium Diphosphate

$/5g $/200mL

0.476

$/2g

3.56

$/1g

Ammonia

0.979

$/0.25g

Total Cost

32.22

$/scaffold

Total Cost per year

35275.43

$/year

In table 3, it is clear that acetic acid makes up a large portion of the bulk cost to produce a single scaffold. Hydrochloric acid (HCl) is another possible acid that could be used to replace the high cost of acetic acid but in fact HCl is almost twice as expensive as acetic acid. On the other hand, chitosan and other reagents are relatively inexpensive. Table 4. Equipment Costs Used as Initial Investment

Equipment Costs Magnetic Stirrer Beakers

Design of Composite Porous Scaffolds

350 75

$ $/12 beakers

Dessicator

675

$

Pipettes

430

$/800

Lyophilizer

5000

$

Total Cost

6530

$/10 years

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Design Report Since the lab will not be provided with equipment, an initial investment must be made to start production. Table 4 lists the mandatory apparatus that will be used within the lab and the total cost that is needed. It should be noted that a scanning electron microscope (SEM) is not listed because the product is assumed to be thoroughly tested beforehand. Table 5. Maintenance and Operation Costs

Maintenance/Operation Costs Lab Technicians Lab Upkeep Total Cost

22000

$/year

5000

$/year

27000

$/year

All salaries, electricity and water are calculated in Table 5.

NPV vs. # of Year 15000 10000

5000 0

0

2

4

6

8

10

12

-5000 -10000

-15000

Years Figure 29. Net Present Value Graph

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Design Report Figure 29 illustrates the net present value of the production. It is shown that it will take around four years in order to breakeven and start earning a profit. These numbers are based on changing the amount each scaffold is sold for until the internal rate of return reaches 25%. The final result is shown in the table below: Table 6. Scaffold Selling Cost

Chitosan Scaffold Selling Cost

60 $/scaffold 65700 $/year

The luxury of this design project is that it barely impacts the environment. Every procedure occurs within the lab and all reagents are safe to obtain. Chitosan may be the only compound that can have any environmental impact due to its derivation from chitin. Fortunately, most chitin are taken from crustacean waste so as not to disturb the wildlife cycle. Chitosan can also be obtained from various forms of fungi. Furthermore, all safety hazards pertaining to this project will be involved with any lab facility. This means that all lab technicians must wear appropriate protection and handle machinery with care to prevent mishaps. Therefore potential endangerment is minimal if standard procedure is taken.

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Design Report Recommendations In conclusion, chitosan scaffolds have been proven to be a potentially useful medical device to aid in bone repair. The production phase is a very simple procedure that can be very flexible depending on what properties are needed. In this design project, co-precipitation is the preferred technique combined with gas immersion to increase the pH to form the precipitate. The resulting sample is a porous and biocompatible composite scaffold containing chitosan and hydroxyapatite. However, much more work is needed to verify the rule of mixtures is applicable. To produce a mathematical model to predict final mechanical properties by varying the amounts of each constituent and porosity, we need to do several things. First we need to produce more samples with varying ratios of Chitosan and HAp, porosity, and pore morphology. From here, we need to test these samples to find the modulus and yield strength of our samples, and compare the values to our expected values derived from the rule of mixtures as well as to determine the value of J, the scaling factor, to each type of pore morphology. In the future, it is to the best interest to promote the beneficial factors provided by chitosan scaffolds. Presently, this f orm of bone repair is a fairly new procedure and future work should assist in making scaffolds to be the primary method for bone healing. In order to proceed with this goal, further research must be done to answer all the uncertainties that may arise fro m this design. For example, what needs to be done to combat the degradation rate of chitosan when present in the body? How can one simulate whether the scaffold can be accepted by the body and how long does it take to regenerate bone cells compared with the conventional way?

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Design Report Team Responsibilities Our group consists of 4 members: Gabriel Hung, Calvina Martin, Hans Saputra and Ricky Tam. The workload for this design project is equally allocated to each of the group members. However, each of us will provide help and assistance to everyone i n the team whenever a member faces difficulties. Gabriel is responsible for the background and introduction of both the presentation and the report. He also carries out numerous amounts of literature reviews as the basis of our project. In addition, he provides assistance to Hans on the chemistry aspects as well as to Calvina on the different processes evaluation to produce the Chitosan/Hydroxyapatite composite. For the final report, he is responsible for the socio-economic analysis as well as the recommendation. Calvina is responsible for the literature reviews on freeze drying and how the cooling rate will affect the final pore structures of the scaffold. She is also responsible to research on the methods of preparing Chitosan/Hydroxyapatite solution. This information will allow the initial planning of processing the scaffold product that will be produced at the end of this project as well as give an overall idea of the chemical aspects required to prepare the solutions. For the final report, she is responsible for the sample preparation as well as the microstructure analysis. Hans is responsible for the chemistry aspects of the design project. He has successfully modeled and generates the Speciation Diagram for the Phosphate ions concentration as well as constructs a Calcium concentration vs. pH graph. These two graphs will help us determine the pH level required to form and precipitate the Chitosan/Hydroxyapatite composite. The literature on the hydroxyapatite formation has also been reviewed. Also, he and Gabriel have planned the processing method using flowcharts. For the final report, he is responsible for the chemical aspects of the technical review. Ricky is responsible for the mechanical aspects of this design project. He will be focusing on the mechanical modeling of the scaffold. Some of the mechanical properties that will be assessed are the modulus and compressive strength of the final scaffold. He is also responsible to evaluate the weight ratio for the Chitosan/Hydroxyapatite composite that will provide the most optimum mechanical properties by varying the porosity of the final scaffold product. For the final report, he is responsible for reviewing the Need and Constraints and Problem Specification sections as well as updating the Mechanical Properties Analysis section. The whole team is involved in laboratory work which includes making the scaffold samples as well as performing the microstructure analysis with SEM and EDS.

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Design Report References [1] E. Landi et al. I Acta Biomaterialia 4 (2008): 1620-1626 [2] Fan, Min, Qiaoling Hu and Kai Shen. "Preparation and structure of chitosan sol uble in wide pH range." 2009. [3] Ferreira, A., C. Oliveira and F. Rocha. "The different phases in the precipitation of dicalcium phosphate dihydrate." Journal of Crystal Growth (2003): 599-611. [4] Ji, Shaocheng, Qi Gu and Bin Xia. "Porosity dependence of mechanical properties of solid materials." J Mater Science (2006): 1757-1768. [5] Kong, Lijun, et al. "Preparation and characterization of nano-hydroxyapatite/chitosan composite scaffolds." Wiley Periodicals (2005). [6] Landi, Elena, Federica Valentini and Anna Tampieri. "Porous hydroxyapatite/gelatine scaffolds with ice -designed channel-like porosity for biomedical applications." Acta Biomaterialia 4 (2008): 1620-1626. [7] Lu, Shaojie, et al. "Preparation of Water-Soluble Chitosan." Journal of Applied Polymer Science, Vol. 91 (2004): 34973503. [8] Yamaguchi, I., et al. "Preparation and microstructure analysis of chitosan/hydroxyapatite nanocomposites." John Wiley & Sons (2000). [9] Kashiwazaki, H., et al. " Fabrication of porouschitosan/hydroxyapatite nanocomposites:Their mechanical and biological properties." Bio-Medical Materials and Engineering 19 (2009) [10] Mubarez, H. " Mechanical and Chemical Characterization of Chitosan Hydroxyapatite Composites" (2011) [11] Nwe, N., Furuike, T., Tamura, H. "The Mechanical and Biological Properties of Chitosan Scaffoldsfor Tissue Regeneration Templates Are Significantly Enhancedby Chitosan from Gongronella butleri" Materials (2009) [12] Rinaudo.M, Pavlov.G. "Influence of acetic acid concentration on the solubilization of chitosan." Polymer (1999): 7029-7032. [13] Vankatesan.J , Se-Kwon Kim. "Chitosan Composites for Bone Tissue Engineering -An Overview." Marine Drugs (2010)

Design of Composite Porous Scaffolds

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Design Project - Composite Biomaterials scaffold