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Optimized Repopulation of Tendon Hydrogel: Synergistic Effects of Growth Factor Combinations and Adipoderived Stem Cells
Simon Farnebo, MD, PhD1; Lovisa Farnebo, MD, PhD2; Maxwell Kim, BS1; Hung Pham, BS1; Colin Y. Woon, MD1; James Chang, MD1
1Division of Plastic Surgery, Stanford University Medical Center, Palo Alto, CA; 2Otolaryngology - Head and Neck Surgery, Stanford University, Palo Alto, CA

Background: Tendon-derived extracellular matrix (ECM) hydrogel has been shown to augment tendon healing in vivo. We hypothesized that reseeding of the gel with adipose-derived stem cells (ASCís) could further assist repopulation of the gel and that combinations of growth factors (GF) would improve the survival of these cells after reseeding.

Methods: Lyophilized decellularized tendons were milled and enzymatically digested. The resulting ECM solution was then supplemented with or without fetal calf serum (FCS) and varying concentrations of bFGF, IGF-1, and PDGF-BB, both individually and in combinations. The different gel conditions were then seeded with ASCís transfected with a GFP/luciferin construct. After 3 and 5 days in vitro, cell proliferation was determined using the MTT assay and histology. When the optimal condition for cell proliferation was established, gels were supplemented with the selected combination of GF, or no GF and injected into the back of immune competent Sprague Dawley rats. Bioluminescence of seeded gels was continuously followed up to 14 days after re-seeding in vivo. Histology and cell counts were performed after the gels were explanted at 14 days.

Results: There was enhanced proliferation of ASCís in gels supplemented with all individual growth factors in vitro. Among single growth factors, PDGF-BB at 100 ng/ml was the most efficient stimulator of proliferation. With multiple growth factors (combinations), the optimal concentration was determined to be 10 ng/ml bFGF, 100 ng/ml IGF-1, and 100 ng/ml PDGF-BB (increase 2.8-fold; p < 0.05). In vivo, bioluminesence showed an improved initial survival of cells in gels supplemented with the optimal concentration of GF compared with the control group (increase 10.6-fold at 8 days; p < 0.05). After 8 days a decline in cells was seen, and most replanted cells were not detectable by day 14. Cell counts of explants, however, showed a dramatic endogenous re-population of gels supplemented by GF+ASCís compared to both gels with GF but no ASCís (7.6-fold increase) and gels with ASCís but no GF (1.6-fold increase).

Conclusion: Synergistic effects of bFGF, IGF-1, and PDGF-BB can be used to improve cellular proliferation and repopulation of ASCís seeded to a tendon ECM gel. Reseeding with ASCís, with or without GF drastically stimulates endogenous repopulation of the gel in vivo and may be used to further augment tendon healing through this system.

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