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SCIENTIFIC ARTICLE

Tissue Engineering of Flexor Tendons: The Effect of a Tissue Bioreactor on Adipoderived Stem Cell–Seeded and Fibroblast-Seeded Tendon Constructs Ioannis K. Angelidis, MD, Johan Thorfinn, MD, PhD, Ian D. Connolly, Derek Lindsey, MS, Hung M. Pham, BS, James Chang, MD

Purpose Tissue-engineered flexor tendons could eventually be used for reconstruction of large tendon defects. The goal of this project was to examine the effect of a tissue bioreactor on the biomechanical properties of tendon constructs seeded with adipoderived stem cells (ASCs) and fibroblasts (Fs). Methods Rabbit rear paw flexor tendons were acellularized and seeded with ASCs or Fs. A custom bioreactor applied a cyclic mechanical load of 1.25 N at 1 cycle/minute for 5 days onto the tendon constructs. Three additional groups were used as controls: fresh tendons and tendons reseeded with either ASCs or Fs that were not exposed to the bioreactor treatment and were left in stationary incubation for 5 days. We compared the ultimate tensile stress (UTS) and elastic modulus (EM) of bioreactor-treated tendons with the unloaded control tendons and fresh tendons. Comparison across groups was assessed using one-way analysis of variance with the significance level set at p⬍.05. Pairwise comparison between the samples was determined by using the Tukey test. Results The UTS and EM values of bioreactor-treated tendons that were exposed to cyclic load were significantly higher than those of unloaded control tendons. Acellularized tendon constructs that were reseeded with ASCs and exposed to a cyclic load had a UTS of 66.76 MPa and an EM of 906.68 MPa; their unloaded equivalents had a UTS of 47.90 MPa and an EM of 715.57 MPa. Similar trends were found in the fibroblast-seeded tendon constructs that were exposed to the bioreactor treatment. The bioreactor-treated tendons approached the UTS and EM values of fresh tendons. Histologically, we found that cells reoriented themselves parallel to the direction of strain in response to cyclic strain. Conclusions The application of cyclic strain on seeded tendon constructs that were treated with the bioreactor helped achieve a UTS and an EM comparable with those of fresh tendons. Bioreactor pretreatment and alternative cell lines, such as ASCs and Fs, might therefore contribute to the in vitro production of strong tendon material. (J Hand Surg 2010;35A:1466–1472. Copyright © 2010 by the American Society for Surgery of the Hand. All rights reserved.) Key words Adipoderived stem cells (ASCs), bioreactor, fibroblasts (Fs), flexor tendon. FromtheSectionofPlasticSurgery,DepartmentofVeteransAffairs,andDivisionofPlasticSurgery,Stanford University Medical Center, Stanford, CA; Department of Plastic Surgery, Hand Surgery and Burns, University Hospital, Linköping, Sweden. Received for publication May 15, 2009; accepted in revised form June 17, 2010. J.C. received funding from a Veteran Affairs Medical Merit Review Award and a Veteran Affairs Rehabilitation Research and Development Merit Review Award. J.T. received funding from the Swedish Fulbright Commission, the Country Council of Östergötland (Sweden), Börje Gabrielsson’s Memorial Fund, the Swedish Society of Medicine, the Johan & Jacob Söderberg Foundation, and the Linköping Society of Medicine.

1466 䉬 ©  ASSH 䉬 Published by Elsevier, Inc. All rights reserved.

No benefits in any form have been received or will be received related directly or indirectly to the subject of this article. Correspondingauthor:IoannisAngelidis,MD,DivisionofPlasticandReconstructiveSurgery,Stanford University, 770 Welch Road, Palo Alto, CA 94304; e-mail: ianangelidis@hotmail.com. 0363-5023/10/35A09-0012$36.00/0 doi:10.1016/j.jhsa.2010.06.020


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injuries, large amounts of tendon grafts might be needed for reconstruction. In some extreme cases, the tendon losses are too large to be replaced solely by autologous grafts.1 Furthermore, intrasynovial tendons are superior to extrasynovial tendons because they might decrease the risk of adhesions and improve function.2 Unfortunately, the flexor digitorum longus tendon of the foot is the only source of intrasynovial grafting,3 which makes it imperative to use tendon grafts of extrasynovial origin, such as the palmaris longus tendon, the plantaris, the flexor digitorum superficialis, and the extensor digitorum longus tendons to the toes. However, extrasynovial tendons might not be an ideal option because adhesions forming between the tendon and its synovial sheath can lead to limited digital function and range of motion.4 – 6 Tissue engineering might address this need for production of additional intrasynovial tendon material. Previous work has focused on identifying an ideal cellscaffold framework that could be implanted into the body to replace the lost tendon. Different scaffold materials, such as fibroblast-seeded chitosan constructs,7 polyglycolic acid fiber construct seeded with avian tenocytes,8 or gel-based collagen scaffold-containing fibers,9,10 have had mixed results and limited applicability, mostly due to poor strength. Bioreactors have been used in tissue engineering of other musculoskeletal tissues. A bioreactor is a device that provides a controlled, sterile environment for the development of engineered tissue.11 It provides in vitro mechanical stimulation, aiming to replicate physiologic conditions.12 Recently, Laganà et al. introduced a bioreactor system to investigate the mechanobiology of cartilage tissue engineering.13 This system was able to support long-term cultures of chondrocyte-seeded, porous 3-dimensional constructs in a controlled environment, while exposing the cells to various combinations of hydrodynamic shear and hydrostatic pressure. Furthermore, Niklason et al. have shown that pulsatile stimulation applied by a bioreactor in vitro substantially improves the biomechanical properties of tissue-engineered blood vessels.14 Our laboratory has acquired a custom tissue bioreactor to apply oscillatory, uniaxial tensile stimulation to tissue-engineered flexor tendon constructs (Fig. 1). In previous experiments, we showed that acellularized tendon constructs reseeded with rabbit tenocytes improved their biomechanical properties when treated with the bioreactor system.15 We hypothesized that the application of cyclic strain on tendon constructs reseeded with 2 other candidate cell lines, adipoderived stem cells (ASCs) and fibroblasts (Fs) would also increase their

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FIGURE 1: The custom bioreactor system (LigaGen model L30-4C; DynaGen series; Tissue Growth Technologies). A chamber that can be loaded with up to 4 tendon constructs is connected with a stimulator that consists of a load cell that records the force generated by the motor. The whole system is supplied by a laptop, which shows the variation of the values (force, displacement, temperature) in the motor during each running.

ultimate tensile stress (UTS) and elastic modulus (EM) when compared with their reseeded unloaded equivalents. Finally, we also predicted that it would improve cell alignment, providing further validation for the implantation into rabbits. MATERIALS AND METHODS All rabbit experiments were performed in an animal facility that is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, and all animal protocols were approved by our Institutional Animal Care and Use Committee. Harvest of rabbit rear paw tendons Rear paw flexor digitorum profundus (FDP) tendons and cells were extracted from adult male New Zealand

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rabbits (about 3.5 kg). The rabbits were injected with lethal doses of Euthasol, and the rear paw FDP equivalents were dissected free. The tendons were then divided distally at the bony insertion and proximally at the common tendon origin. The harvested rear paw tendons were approximately 5 cm in length. Harvested tendons not immediately used were immersed in phosphate-buffered saline and stored at – 80°C. Isolation and in vitro expansion of adipoderived stem cells and sheath fibroblasts Adipoderived stem cells were obtained from inguinal fat pads, using a technique published by Zuk et al.16 The fat was cut with scissors and treated with 0.075% collagenase II in a shaking water bath at 37°C for 10 minutes. Collagenase was then inactivated using fresh media. The cell suspensions were centrifuged at 1,000 rpm for 5 minutes at 4°C. The supernatant was aspirated, and the pellet was resuspended with medium. The solution was filtered through a 100-␮im cell filter into a new tube and centrifuged at 1,000 rpm for 5 minutes. The cell pellet was resuspended with Ham’s F12 medium containing 10% rabbit serum. Finally, the cells were plated on a tissue culture dish. Sheath Fs were obtained using a previously established protocol.17,18 The tendons were digested with 0.5% collagenase type I (Sigma, St. Louis, MO) in 20 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer for 10 minutes at 37°C. The tendon sheath Fs were plated and cultured in Ham’s F12 medium supplemented with 10% rabbit serum. The cells were grown at 37°C in a humidified, standard incubator with 5% CO2. Culture media was changed every other day. For these experiments, only low-passage cultures (⬍6) were used. Acellularization of tendon Flexor digitorum profundus tendons were washed with phosphate-buffered saline and then immersed in phosphate-buffered saline and frozen at 80°C. They were then thawed to room temperature and placed into 0.05% trypsin and 0.53 mmol/L ethylenediaminetetraacetic acid (EDTA) for 24 hours at 37°C followed by Triton X-100 0.5% (Sigma, St. Louis, MO) for 24 hours at room temperature. Histological assessment using hematoxylin-eosin revealed that complete acellularization was achieved. A total of 17 rabbits were used for this study, and 112 FDP rear paw tendons were harvested, 78 of which were used for this experiment. To ensure that no viable cells remained after the process, acellularized tendons were treated with trypsin and collagenase, and no viable cells could be obtained in culture.

Six tendons were treated this way to assess cell viability. Seeding of acellularized tendon Acellular tendon scaffolds to be reseeded were placed in media for 24 hours. Each tendon was placed into a test tube and immersed in media with either ASCs or Fs at a density of 2 ⫻ 106 cells/mL. The tubes with cell-scaffold constructs were mounted onto a rotator running with the lowest applicable speed (8 rpm). Low speed is important to have an equal distribution of cells on the surface of the constructs. The rotator was then placed in the incubator at 37°C in a humidified tissue culture chamber with 5% CO2 for 24 hours to allow attachment of the cells to the surface of the tendon scaffolds. Bioreactor optimization, biomechanical testing, and statistical analysis A custom tissue bioreactor providing uniaxial tendon strain (Ligagen L30 – 4C; DynaGen systems, Tissue Growth Technologies, Minnetonka, MN) was used to apply cyclic strain to the reseeded constructs. The cell– seeded constructs were placed in the bioreactor system in groups of 4 tendons per running. They were clamped between the bioreactor grips in a way that the exposed area was kept constant at 3 cm. The bioreactor system can accommodate up to 4 tendons. To ensure that the cyclic load was equally distributed to the tendon constructs, only an even number of constructs was placed in the bioreactor chamber. Experiments were conducted by comparing 4 bioreactor tendons with 4 nonbioreactor tendons or 2 bioreactor tendons with 2 nonbioreactor tendons. Tendons that were either contaminated during their bioreactor treatment or slipped during their testing were excluded. A total of 8 tendons were excluded. These tendons were replaced by performing additional experiments. Four tendons were used for histology to determine cell orientation after a 5-day bioreactor treatment or a 5-day stationary incubation. The cell–seeded constructs were subjected to a total cyclic load of 5 N, exposing each tendon to 1.25 N (5 N/4) over 5 days (n⫽4). The frequency used was 1 cycle/minute in alternating 1-hour periods of mechanical loading and rest. These settings had been optimized in previous studies. The experimental groups consisted of freshly harvested rear paw flexor FDP tendons that served as normal controls (n⫽15); acellularized FDP tendons that were reseeded with ASCs that were either exposed to bioreactor loading (ASC⫹,

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n⫽16) or no loading (ASC-, n⫽23) for 5 days; and FDP tendons that were reseeded with Fs and were either exposed to loading (F⫹, n⫽11) or no loading (F-, n⫽13 ) for 5 days. At the end of each 5-day period, tendons were kept moist in phosphate-buffered saline for biomechanical testing. The ends of the tendons were clamped between the grips of a materials testing system (MTS 858; MTS Inc., Minneapolis, MN). To prevent slippage of the tendon, the clamps were lined with fine sandpaper, and a cyanoacrylate glue was applied to the interface between the tendon and sandpaper. A 7-mm section of each end was clamped so that the intrasynovial portion of the constructs being tested between the clamps was kept constant at 2.5 cm until failure in this study. The UTS of each sample was calculated by dividing the force at which the construct broke by its cross-sectional area at the breaking point. The breaking point was usually located halfway between the middle of the tendon and the bottom of the grip. Slippage occurred in the pilot studies; however, it was prevented in the actual experiment with the proper amount of glue and sandpaper around the edges of the tendon constructs. The EM represents the construct’s tendency to be deformed elastically when a force is applied to it (reversible elasticity). The EM (Young modulus) was calculated from the slope of the stress-strain graph. To calculate the graph, we used values of stress and strain that were achieved during the last cycle of the construct’s running in the MTS machine. The biomechanical testing protocol applied 10 preconditioning load-unload cycles, and a final, 11th load cycle applied tensile force until tendon failure occurred. Long displacement data were recorded at a frequency of 100 Hz by the test system (Testware SX; MTS Inc.) and were analyzed using Microsoft Excel (Excel 10.0; Microsoft Corp., Redmond, WA). Comparison of group means was made with one-way analysis of variance and Tukey’s post hoc test using statistical software (SPSS 17.0; SPSS Inc., Chicago, IL). The significance level was set to p⬍.05. The cross–sectional area of the constructs was determined by obtaining 2 orthogonal photographs of each clamped tendon, using a tripod-mounted, high-resolution digital camera (Nikon D3; Nikon Inc., Melville, NY) at a fixed distance together with a known scale. The 2 orthogonal diameters were measured at the breaking point of the tendon construct, and the cross– sectional area was calculated on the basis of an ellipse

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FIGURE 2: The UTS (MPa) of fresh control tendons (black), ASC-seeded constructs either exposed to bioreactor treatment (ASC⫹, diagonal stripes) or not exposed to bioreactor treatment (ASC-, horizontal stripes), and F-seeded constructs either exposed to bioreactor treatment (F⫹, diamonds) or not exposed to bioreactor treatment (F-, squares). T-bars indicate 1 SD.

with image analysis software (ImageJ; National Institutes of Health, Bethesda, MD). Histological analysis Histologic appearances were examined for cell orientation for constructs that went through the bioreactor and those that were exposed to stationary incubation. SYTO green11 fluorescent nucleic acid stain (Invitrogen, Carlsbad, CA) was used The cryostat was used (Fig. 2). to prepare the histological slides. The thickness of the section was 10 ␮im. RESULTS Ultimate Tensile Stress Reseeded tendon constructs that were exposed to a 1 cycle/minute load in alternating 1-hour periods of mechanical loading and rest for 5 days were found to have a statistically significant increase (p ⬍ .001) in UTS compared with unloaded reseeded controls in both of our experimental groups (Fig. 2). The Tukey test demonstrated that there was no notable difference between the fresh, intact tendons and the bioreactor-treated tendons. The bioreactor–treated tendon constructs that were reseeded with fibroblasts presented the highest UTS value. Elastic modulus Reseeded tendon constructs that were exposed to a 1 cycle/minute load in alternating 1-hour periods of mechanical loading and rest for 5 days were also found to have a statistically significant increase (p ⬍ .001) in

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FIGURE 3: The EM (MPa) of fresh control tendons (black), ASC-seeded constructs either exposed to bioreactor treatment (ASC⫹, diagonal stripes) or not exposed to bioreactor treatment (ASC-, horizontal stripes), and F-seeded constructs either exposed to bioreactor treatment (F⫹, diamonds) or not exposed to bioreactor treatment (F-, squares). T-bars indicate 1 SD.

EM compared with unloaded reseeded groups in both of our experimental groups (Fig. 3). The Tukey test demonstrated that there was no notable difference between the fresh, intact tendons and the bioreactortreated tendons. The fresh, intact tendons still resulted in the highest EM values. Histology Cyclic strain caused the cells in both of our experimental groups (ASC⫹ and F⫹) to elongate parallel to the direction of strain. This alignment was in stark contrast to the random cell orientation of unloaded constructs (Fig. 4). Cyclic strain had similar effects on both the ASC-seeded and F-seeded tendon constructs. Cells were distributed equally throughout the tendon’s surface, with far fewer cells in the core of the tendon.

DISCUSSION Flexor tendons experience cyclic mechanical stress during normal daily hand movements. In our study, we demonstrated that acellularized tendons—seeded with either ASCs or Fs and treated with cyclic strain in a bioreactor—improved their biomechanical properties, achieving values that approached that of intact, fresh tendons. This provides evidence that simulation of in vivo conditions might be important in the in vitro phase of tendon tissue engineering. Bioreactor treatment has become important in other tissues and models of tissue engineering. Abousleiman and Sikavitsas have recently shown that cyclically tensioned human umbilical veins that were acellularized and reseeded with mesenchymal stem cells were stronger than their unstretched equivalents.11 Joshi and Webb have shown that F-seeded substrates cultured under the default of cyclic strain exhibited an increased EM.19 In addition, Webb et al. showed that application of cyclic strain on F–seeded polyurethane constructs improved their EM.20 Our data on tendons are consistent with these previous studies. Although the scaffold material that was used for seeding differed in all these models, the findings are similar. The application of cyclic strain on reseeded constructs improved their biomechanical properties. In our study, the bioreactor–treated seeded tendon constructs were considerably stronger than the unloaded ones. Our data also showed that, in unstressed conditions, both types of reseeded tendon constructs (ASC and F) exhibited almost the same values of UTS and EM, independent of the cell type with which they were reseeded. In prior studies in our laboratory, acellularized tendon constructs that were seeded with tenocytes and were then subjected to cyclic strain had improved biomechanical properties.21 These tenocyte

FIGURE 4: A, B The ASCs (top). C, D The Fs (bottom). A, C Loaded tendons demonstrated elongation of the nuclei and parallelization to the direction of force (left). B, D Unloaded tendons show random orientation of nuclei (right).

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values were similar to those of seeded constructs with either ASCs or Fs that were also pretreated with the bioreactor. Therefore, the specific cell type tested might not affect the strength of the new tendon construct as much as the bioreactor treatment itself. In this same previous study, acellularized tendons (without any cells reseeded) that were exposed to the same loading parameters of the bioreactor were not found to have a marked increased UTS compared with nonloaded, acellularized controls. Therefore, this bioreactor effect is not related to the effect of mechanical strain on the tendon’s collagen structure itself. As far as acellularized tendon constructs are concerned, Saber et al.21 demonstrated that there is a significant difference between freshly harvested tendons and acellularized tendons (control, freshly harvested tendons, UTS ⫽ 72.97 ⫾ 14.53 N vs acellularized tendons, UTS ⫽ 40.92 ⫾ 9.31 N; p⫽.0023).15 On the other hand, Chong et al.21 demonstrated that there was no significant difference on the mechanical properties between freshly harvested tendons (UTS ⫽ 66.01 ⫾ 20.79 MPa vs acellularized tendons, UTS ⫽ 55.09 ⫾ 17.43 MPa p⫽.736).21 These controls were not repeated in this current study because the current work focuses directly on the effect of bioreactor treatment on tendons seeded with either ASCs or Fs. Tendon acellularization has resulted in mixed results in terms of strength preservation, as seen in our data. Baseline strength of the acellularized tendon scaffold material will be important for translation to clinical cases. Current work in the laboratory has focused on methods to better preserve biomechanical strength in acellularized tendons, specifically in a human tendon model. Loesberg et al. investigated cell orientation in response to cyclic stress in culture and found that cultured fibroblasts tend to align perpendicular to the direction of strain.22 This has also been confirmed by Lee et al. who seeded type 1 collagen– coated plates with ASCs and exposed them to mechanical stimulation.23 In that study, cyclic strain also caused alignment of the cells perpendicular to the direction of strain. However, in our current study and in previous work in our laboratory, after candidate cells are seeded onto a 3-dimensional scaffold, they change alignment from perpendicular 22 to parallel to the direction of strain. Our data confirm the findings of Wang et al., showing that cells seeded onto a collagen scaffold exposed to uniaxial cyclic strain will reorient parallel to the direction of force.24 Cells react

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differently to mechanical stimulation, depending on cell culture versus cell seeding onto scaffolds. Bioreactor application of cyclic stain improves strength during the in vitro phase of flexor tendon tissue engineering. Both ASCs and Fs are candidate cell lines for seeding, although Fs might be easier to harvest and culture. These optimized variables will be important in achieving the ultimate goal—production of strong, nonimmunogenic tendon material that could be used clinically in complex hand reconstruction. REFERENCES 1. Zhang AY, Chang J. Tissue Engineering of Flexor Tendons. Clin Plast Surg 2003;30:565–572. 2. Leversedge FJ, Zelouf D, Williams C, Gelberman RH, Seiler JG III. Flexor tendon grafting to the hand: an assessment of the intrasynovial donor tendon—a preliminary single-cohort study. J Hand Surg 2000;25A:721–730. 3. Seiler JG III, Reddy AS, Simpson LE, Williams CS, Hewan-Lowe K, Gelberman RH. The flexor digitorum longus: an anatomic and microscopic study for use as a tendon graft. J Hand Surg 1995;20A: 492– 495. 4. Amadio PC, Wood MB, Cooney WP III, Bogard SD. Staged flexor tendon reconstruction in the fingers and hand. J Hand Surg 1988; 13A:559 –562. 5. LaSalle WB, Strickland JW. An evaluation of the two-stage flexor tendon reconstruction technique. J Hand Surg 1983;8:263–267. 6. Wehbé MA, Mawr B, Hunter JM, Schneider LH, Goodwyn BL. Two-stage flexor-tendon reconstruction. Ten-year experience. J Bone Joint Surg 1986;68A:752–763. 7. Funakoshi T, Majima T, Iwasaki N, Suenaga N, Sawaguchi N, Shimode K, et al. Application of tissue engineering techniques for rotator cuff regeneration using a chitosan-based hyaluronan hybrid fiber scaffold. Am J Sports Med 2005;33:1193–1201. 8. Cao Y, Liu Y, Liu W, Shan Q, Buonocore SD, Cui L. Bridging tendon defects using autologous tenocyte engineered tendon in a hen model. Plast Reconstr Surg 2002;110:1280 –1289. 9. Juncosa-Melvin N, Boivin GP, Gooch C, Galloway MT, West JR, Dunn MG, et al. The effect of autologous mesenchymal stem cells on the biomechanics and histology of gel-collagen sponge constructs used for rabbit patellar tendon repair. Tissue Eng 2006; 12:369 –379. 10. Nirmalanandhan VS, Rao M, Shearn JT, Juncosa-Melvin N, Gooch C, Butler DL. Effect of scaffold material, construct length and mechanical stimulation on the in vitro stiffness of the engineered tendon construct. J Biomech 2008;41:822– 828. 11. Abousleiman RI, Sikavitsas VI. Bioreactors for tissues of the musculoskeletal system. Adv Exp Med Biol 2006;585:243–259. 12. Butler DL, Hunter SA, Chokalingam K, Cordray MJ, Shearn J, Juncosa-Melvin N, et al. Using functional tissue engineering and bioreactors to mechanically stimulate tissue-engineered constructs. Tissue Eng Part A 2009;4:741–749. 13. Laganà K, Moretti M, Dubini G, Raimondi MT. A new bioreactor for the controlled application of complex mechanical stimuli for cartilage tissue engineering. Proc Inst Mech Eng H 2008;222:705–715. 14. Niklason LE, Gao J, Abbott WM, Hirschi KK, Houser S, Marini R, et al. Functional arteries grown in vitro. Science 1999;284:489 – 493. 15. Saber S, Zhang AY, Ki SH, Lindsey DP, Smith RL, Riboh J, et al. Flexor tendon tissue engineering: bioreactor cyclic strain increases construct strength. Tissue Eng Part A 2010;16:2085–2090. 16. Kryger GS, Chong AK, Costa M, Pham H, Bates SJ, Chang J. A comparison of tenocytes and mesenchymal stem cells for use in flexor tendon tissue engineering. J Hand Surg 2007;32A:597– 605.

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17. Banes AJ, Horesovsky G, Larson C, Tsuzaki M, Judex S, Archambault J, et al. Mechanical load stimulates expression of novel genes in vivo and in vitro in avian flexor tendon cells. Osteoarthritis Cartilage 1999;7:141–153. 18. Klein MB, Pham H, Yalamanchi N, Chang J. Flexor tendon wound healing in vitro: the effect of lactate on tendon cell proliferation and collagen production. J Hand Surg 2001;26A:847– 854. 19. Joshi SD, Webb K. Variation of cyclic strain parameters regulates development of elastic modulus in fibroblast/substrate constructs. J Orthop Res 2008;26:1105–1113. 20. Webb K, Hitchcock RW, Smeal RM, Li W, Gray SD, Tresco PA. Cyclic strain increases fibroblast proliferation, matrix accumulation, and elastic modulus of fibroblast-seeded polyurethane constructs. J Biomech 2006;39:1136 –1144.

21. Chong AK, Riboh J, Smith RL, Lindsey DP, Pham HM, Chang J. Flexor tendon tissue engineering: acellularized and reseeded tendon constructs. Plast Reconstr Surg 2009;123:1759 –1766. 22. Loesberg WA, Walboomers XF, van Loon JJ, Jansen JA. The effect of combined cyclic mechanical stretching and microgrooved surface topography on the behavior of fibroblasts. J Biomed Mater Res A 2005;75:723–732. 23. Lee WC, Maul TM, Vorp DA, Rubin JP, Marra KG. Effects of uniaxial cyclic strain on adipose-derived stem cell morphology, proliferation, and differentiation. Biomech Model Mechanobiol 2007;6:265–273. 24. Wang B, Liu W, Zhang Y, Jiang Y, Zhang WJ, Zhou G, et al.. Engineering of extensor tendon complex by an ex vivo approach. Biomaterials 2008;29:2954 –2961.

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