Tissue Engineering of Flexor Tendons: Straining for Better ...



Tissue Engineering of Flexor Tendons: Straining for Better Results

Jonathan C. Riboh, BS, Alphonsus K. Chong, MD, Hung Pham, BS, Michael T. Longaker, MD, MBA, Christopher Jacobs, PhD, and James Chang, MD.

PURPOSE

Flexor tendon injuries requiring tendon grafting are a common clinical problem. Intrasynovial grafts are known to be clinically superior to extrasynovial grafts in the repair of these defects1, 2. However, there is a lack of good donor sites for intrasynovial tendons, and intrasynovial grafting hasn’t made it into surgical practice3. Tissue engineering of flexor tendons promises to address this challenge4-6. This technology will not be clinically useful however until large amounts of tendon can be grown quickly in the lab. To overcome the bottleneck of in vitro expansion of cells, we hypothesized that application of cyclic uniaxial strain to cultures could improve cell proliferation, collagen I production, and cell morphology.

METHODS

Four candidate cell lines were tested: epitenon tenocytes (E), tendon sheath fibroblasts (S), bone marrow-derived stem cells (bMSC), and adipoderived stem cells (ASC). These cells were first tested for their ability to adhere to extracellular matrix coated membranes (fibronectin). Cells were then subjected to 3 different strain regimens: Continuous Cyclic Strain (CCS: 8% elongation, 1 Hz, 100% duty cycle), Intermittent Cyclic Strain 1:2 (ICS 1h on : 2h off: 4% elongation, 0.1 Hz, 33% duty cycle) and ICS 1h on : 5h off (4% elongation, 0.1 Hz, 17% duty cycle). Cell adhesion and cell proliferation were measured using the colorimetric Alamar Blue assay. Simultaneously, collagen I levels in the conditioned media were measured by ELISA. Cell morphology was assessed by confocal microscopy after green fluorescent actin staining and red fluorescent nuclear staining. Experiments were performed in triplicate and analyzed with ANOVA and pair-wise t-tests corrected for multiple comparisons.

RESULTS

ASC (33% adhesion) and S (29% adhesion) adhered better to the fibronectin coated membranes than the other cell types (p < 10-10). Based on these results, only ASC and S were chosen for further analysis. CCS was shown to inhibit cell proliferation (p < 0.01), while ICS 1h on : 5h off caused a significant increase in cell proliferation (p = 0.05), Figure 1.

Figure 1: Effects of cyclic strain on cell proliferation

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CCS caused a large increase in per-cell collagen I production (p < 0.01), while ICS caused only moderate increases in collagen I production, Figure 2 A and B. However, when both collagen I levels and cell number are factored into an assessment of total collagen production, ICS 1h on : 2 h off is shown to be optimal, Figure 2 C and D.

Figure 2. Cyclic Strain Increases Collagen I Production

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Cyclic strain caused formation of parallel actin stress fibers, as well as nuclear elongation, Figure 3.

Figure 3. Cyclic Strain Alters Cell Morphology

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Control Cyclic Strain

CONCLUSIONS

This study describes a comprehensive review of the effect of cyclic uniaxial strain on candidate cell lines for flexor tendon tissue engineering. As mechanically active culture techniques become the norm in the engineering of musculoskeletal tissues, our results can serve as guidelines for the selection of strain regimes and cell sources. For flexor tendon engineering, we recommend the use of ASC, as they are easily isolated, grow quickly, adhere strongly to ECM coated membranes and respond well to mechanical challenge. We also recommend strain regimes that include rest periods, with a duty cycle of 33% being optimal in this study. This combination of informed cell selection and cautious application of cyclic uniaxial strain allowed us to optimize in vitro cell proliferation, collagen I production, and cell morphology.

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2. Seiler JG, 3rd, Chu CR, Amiel D, Woo SL, Gelberman RH. The Marshall R. Urist Young Investigator Award. Autogenous flexor tendon grafts. Biologic mechanisms for incorporation. Clin Orthop Relat Res. (345):239-247;1997

3. Chong AK, Chang J. Tissue engineering for the hand surgeon: a clinical perspective. J Hand Surg [Am], 31(3):349-358; 2006

4. Cao D, Liu W, Wei X, Xu F, Cui L, Cao Y. In vitro tendon engineering with avian tenocytes and polyglycolic acids: a preliminary report. Tissue Eng, 12(5):1369-1377; 2006

5. 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., 110(5):1280-1289; 2002

6. Zhang AY, Chang J. Tissue engineering of flexor tendons. Clin Plast Surg., 30(4):565-572; 2003

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