Flexor Tendon Tissue Engineering: a Comparison of Tenocytes Versus Stem Cells

Flexor Tendon Tissue Engineering: a Comparison of Tenocytes Versus Stem Cells

Title: Flexor Tendon Tissue Engineering: A Comparison of Tenocytes Versus Stem Cells

Authors: Gil S. Kryger MD, Hung Pham BS, Steven J. Bates MD, Cindy Wu BS, and James Chang MD

Introduction: When a divided flexor tendon cannot be repaired primarily because of tendon loss, tendon grafting must be performed to restore function to the digit. As the availability of donor tendons is limited, researchers are attempting to produce bioartificial tendons (BAT) by seeding an acellular scaffold with different types of cells. Most laboratories use a synthetic scaffold such as PGA, silk or Dacron. Our approach is to use acellular tendon. The options for cells include fibroblasts and mesenchymal stem cells (MSC). The goal of this study was to compare the properties of bone marrow derived MSC and fat derived MSC with sheath fibroblasts and tenocytes in an effort to determine the optimal cell type for tendon engineering.

Methods: New Zealand White Rabbit flexor tendons were harvested and treated to prepare cell cultures of sheath fibroblasts and tenocytes using previously described techniques. Rabbit bone marrow and fat were then harvested to prepare cell cultures of MSC. The four cell types were then compared morphologically. Next, the cells were stained with antibodies against collagens I, II and III. Immunocytochemistry was used to compare collagen synthesis. Growth characteristics, specifically proliferation curves and senescence, were studied. Cell counts were obtained by hemacytometry and MTT optical assay. Growth curves were plotted for early passage cells at passage 4 and later passage cells at passage 8. Doubling times were compared. To assess senescence, the different cell types were stained with beta galactosidase at passage 21.

Multiple combinations of growth factors in varying concentrations were then added and proliferation rates of the four cell types were measured. The optimal factors and concentrations were calculated.

Rabbit flexor tendons were harvested and acellularized using previously described techniques. These collagen scaffolds were seeded by suspension in each of the different cell types. The constructs were incubated for one week in vitro and compared by histology.

The constructs were then implanted into zone II flexor tendon defects that had been surgically created in rabbits. Five rabbits were grafted with each cell type. Three types of controls were used (three animals in each control group) to assess the inflammatory reaction to the constructs: acellular tendon not reseeded with any cell type; tendon allograft; and tendon autograft. The specimens from each group were harvested at 4 weeks (n=3) and at 8 weeks (n=2) and compared histologically. Controls were harvested after 4 weeks, 6 weeks and 8 weeks. Harvested grafts were compared histologically.

Results: All four cell types had a fibroblast-like morphology with spindle shaped cell bodies and elongated nuclei. All four cell types stained strongly positive for collagen I, weakly for collagen II, and strongly for collagen III.

All cell types showed exponential cell growth, reaching confluence by day six. Doubling times were similar for all cell types. Proliferation rates did not slow down from early to later passages.

The addition of IGF-1 100 ng/ml, basic FGF 1 ng/ ml, and PDGF-BB 50 ng/ml significantly increased the rate of tenocyte cell growth by a factor of 9x and sheath fibroblasts by a factor of 5x. Both fat and bone marrow MSC rates increased by factors of 3x and 4x respectively.

Beta galactosidase staining demonstrated roughly 1% of all cells were senescent at passage 21. There were no differences between the four cell types.

After one week in vitro, constructs seeded with all four cell types demonstrated clumping of cells on the surface of the scaffold without any penetration into the core. In comparison, acellular controls showed no cells on either the surface or the within the core of the acellular scaffold.

After eight weeks in vivo, constructs seeded with all four cell types demonstrated an intrasynovial tendon-like morphology. There was a single epitenon-like layer of cells on the surface of the construct and elongated endotenon-like cells throughout the core. In contrast, acellular controls showed only inflammatory cells on the surface of the scaffold without any penetration of cells into the core, even after 8 weeks. The inflammatory response was not different between any of the experimental or control animals.

Conclusion: As expected, tenocytes and sheath fibroblasts demonstrated a fibroblast-like morphology. The MSC’s were differentiated with media, and also appeared morphologically indistinguishable from the other cell types. As the heterogeneity of the MSC cultures decreased, we feel that it was fibroblasts that were proliferating in vitro and outgrowing the other cell morphologies.

Tenocytes synthesize collagens I and III. The finding that all four cell types stained strongly for these types of collagen (and not for collage II), is consistent with our expectations, as tendons are comprised mostly of collagens I and III. When these findings are taken in conjunction with the morphologies of the cells, it is reasonable to conclude that all four cell types adopted tenocyte-like features.

For a tissue engineering project to succeed, it is important that the cells that are used continue to proliferate readily. From this standpoint, all of the cell types studied were similar. They continued to proliferate readily as late as passage 21. Further studies of the proliferation rates are needed at even later passages. The addition of certain growth factors can enhance proliferation even further, as can the use of a tissue bioreactor.

The growth factors IGF-1, basic FGF and PDGF-BB are known to stimulate proliferation of fibroblasts. The addition of these factors significantly increased the proliferation rates of all cell types. The use of growth factors to optimize cellular proliferation is an integral part of tissue engineering experiments. Another component of tissue engineering is to mimic physiologic conditions. As tendons constantly experience tensile and shear forces, a bioreactor that can emulate these forces will presumably enhance tendon tissue engineering. We are currently studying such a device.

The artificial tendons continue to mature after implantation into flexor tendon zone II defects. Even though there is essentially no appreciable infiltration of cells into the core of the scaffold after one week in vitro, the constructs possess the morphology of intrasynovial tendons after 6 weeks in vivo. Because the acellular controls show no infiltration of cells into their core, we believe that elongated cells within the core of the experimental groups are in fact tenocytes and not inflammatory cells. However, whether these cells are from the host or the transplanted graft is unclear and further research, such as labeling stains or sex mismatched studies, is needed.

We lack long term studies of the collagen scaffold and do not yet know whether it will completely resorb and after how long. The ideal scaffold would be replaced with autogenous tissue without significant weakening.

In summary, bioengineered tendon grafts are a viable source of graft material for use in the reconstruction of flexor tendon defects. All four cell types studied in this experiment: tenocytes, sheath fibroblasts, bone marrow derived MSC’s and adipoderived MSC’s are similar with respect to the parameters we studied. No appreciable differences were found between their morphologies, collagen synthesis, growth characteristics and senescence, and their viability both in vitro and in vivo. Further studies are needed to determine the optimal cell type of these four, and to elucidate physiologic parameters of the constructs after implantation, such as adhesion formation, range of motion and tensile strength.


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