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Optimized Repopulation Of Extracellular Matrix Hydrogel: Synergistic Effects Of Combinations Of Growth Factors And Adipoderived Stem Cells
Simon Farnebo, MD PHD, Maxwell Kim, MSC, Lovisa Farnebo, MD PHD, Hung Pham, MSC, James Chang, MD.
Stanford Medical University, Palo Alto, CA, USA.
Poor healing of acute and chronic tendon injuries is a common and unsolved problem in plastic and hand surgery. We have previously described how tendon healing can be augmented by the addition of a tendon-derived, extracellular matrix (ECM) hydrogel. In a rat tendon injury model, an accelerated healing with a more mature collagen composition, and improved biomechanical strength was observed. We now hypothesized that reseeding of the gel with adipose-derived stem cells (ASC’s) could further assist repopulation of the gel. Also, we aimed at investigating if combinations of growth factors (GF) - basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF)-1, and platelet-derived growth factor (PDGF)-BB would improve the growth survival of these cells.
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 ASC’s transfected with a GFP and 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 Wistar 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. Scanning electron microscopy was used to study difference in ultrastructure. Statistical analysis of biomechanical data was performed using a paired Student’s T-test.
There was enhanced proliferation of ASC’s 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 2.9-fold; p < 0.05) (Figure 1). 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 -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+ASC’s compared to both gels with GF but no ASC’s (7.6-fold increase) and gels with ASC’s but no GF (1.6-fold increase). (Figure 2)
Synergistic effects of bFGF, IGF-1, and PDGF-BB can be used to improve cellular proliferation and repopulation of ASC’s seeded to a tendon ECM gel. Reseeding with ASC’s, 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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