PURPOSE
The adhesions and contractures that result from the use of extrasynovial grafts in flexor tendon repairs are a major challenge in hand surgery. Tissue engineering of intransynovial tendon grafts can best be accomplished by combining biological and mechanical stimuli in a bioreactor to simulate physiological conditions. We hypothesized that in candidate cell lines, proliferation, collagen production, and cell morphology could be optimized through application of continuous cyclic strain (CCS).
METHODS
Epitenon tenocytes (E), tendon sheath fibroblasts (S), bone marrow mesenchymal stem cells (bMSC) and adipose mesenchymal stem cells (ASC) were plated on pronectin-coated silicone membranes and subjected to 8% strain, 1 Hz for 4 days; or no strain. The number of adherent cells after a 12h incubation was assessed using the colorimetric Alamar Blue assay. Cell proliferation was measured every 2 days using the same assay. Simultaneously, collagen I levels in the conditioned media were measured by ELISA. Cell morphology was assessed by confocal microscopy. Experiments were performed in triplicate and analyzed with ANOVA and pair-wise t-tests corrected for multiple comparisons.
RESULTS
S and ASC adhered better to pronectin than E and bMSC (33% and 29% adhesion vs. 21% and 6% respectively, p <10-10). CCS caused a decrease in proliferation in all cell lines (p < 0.004 for all), with net cell death between baseline and day 2, and a slow recovery of growth thereafter. bMSC did not show growth recovery. CCS caused collagen I production to increase 2 fold in S, ASC and E (p < 0.03 for all). In bMSC, collagen I production increased nearly 10 fold (p = 0.0001). Strained cells from all cell lines became fusiform, with nuclear elongation and parallel alignment of actin filaments.
CONCLUSIONS
In order to achieve tendon healing and regeneration, the cells seeded on tissue engineered flexor tendons must proliferate, secrete collagen, and differentiate into tenocytes. This study demonstrates that CCS inhibits cell proliferation yet increases collagen I production in all candidate cell lines for flexor tendon engineering. The data suggests that S and ASC are the most appropriate of the 4 cell lines studied given their high adhesion to ECM, high proliferative rate and their response to mechanical strain. Finally, we have shown that CCS helps multipotent cells assume a morphology similar to that of tendon fibroblasts. Future studies will focus on varying strain patterns to achieve maximal cell proliferation and collagen production.