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Browsing by Author "Brown, James Augustus"

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    Comparison of bone marrow mesenchymal stem cells and tendon progenitor cells cultured on collagen surfaces
    Brown, James Augustus (Virginia Tech, 2010-04-29)
    Tendon injuries are a significant cause of morbidity in performance horses with superficial digital flexor tendon injury reported to represent up to 43% of overall Thoroughbred racehorse injuries. Natural repair is slow and results in inferior structural organization and biomechanical properties and, therefore, reinjury is common. The inability of tendon to regenerate after injury, or to heal with mechanical properties comparable to the original tissue, is likely attributable to low vascularity and cellularity of the tissue, low number of resident progenitor cells, and healing under weight-bearing conditions. Strategies to improve tendon healing have focused on enhancing the metabolic response of tenocytes, modulating the organization of the newly synthesized extracellular matrix, or administering progenitor cells to enhance repair. Significant research effort has been directed at the use of adult mesenchymal stem cells as a source of progenitor cells for equine tendon repair and recent clinical applications have utilized adult autologous stem cells derived either from adipose tissue or bone marrow aspirates. Isolation of a homogenous population of stem cells from bone marrow is time-consuming, and there is much variation in cell numbers, cell viability and growth rates among samples. Recently, a population of progenitor cells has been isolated from equine flexor tendons, thus providing an alternative source of progenitor cells from the target tissue for therapeutic intervention. The interaction between cells and the extracellular matrix (ECM) is an important factor in regulation of cell function. Proliferation, migration, differentiation and gene expression of many cell types are altered by adhesion to and interaction with matrix proteins and the extracellular environment. Tendon progenitor cells reside within a niche that comprises primarily parallel collagen fibers, and this niche plays an important role in regulating their function and differentiation. Culture conditions replicating this environment could be beneficial for both cell growth and matrix gene expression. The objectives of the study were to compare cell growth kinetics and biosynthetic capabilities of bone marrow mesenchymal stem cells (BMMSCs) and tendon derived progenitor cells (TPCs) cultured on commercially available bovine, highly purified bovine, porcine, and rattus collagen sources and standard tissue culture surfaces. We hypothesized that collagen type I matrix would preferentially support TPC proliferation and up regulate gene expression for collagens and organizational components of tendon and therefore provide a culture system and progenitor cell type with advantages over the current practice of BMMSC expansion on standard cell culture plastic surfaces. Cells were isolated from 6 young adult horses, expanded, and cultured on collagen-coated tissue culture plates, and no collagen control for 7 days. Samples were analyzed for cell number on days 4 and 7, and for mRNA expression of collagen type I, collagen type III, cartilage oligomeric matrix protein (COMP), and decorin on day 7. Glycosaminoglycan (GAG) synthesis was analyzed on day 7. Differences of cell number between collagen groups and cell type, and in gene expression and GAG synthesis between collagen groups and cell types, were evaluated by use of mixed-model repeated measures ANOVA. Pair-wise comparisons were made on significant differences identified with ANOVA using Tukey's post hoc test. Statistical significance was set at P<0.05. A statistical significant (P=0.05) increase in cell number for TPCs grown on rattus collagen versus control on day 4 was observed. No difference in GAG synthesis or expression of collagen type I, collagen type III, COMP or decorin mRNA was observed between collagen groups and non-collagen controls for either cell type on day 7. TPCs cultured on all collagen types yielded more cells than similarly cultured BMMSCs on day 4, but only porcine collagen was superior on day 7. TPCs synthesized more GAG than BMMSCs when cultured on control surfaces only. BMMSCs expressed more collagen type I mRNA when cultured on control, porcine and highly-purified collagen, and more collagen type III when cultured on control, porcine, highly-purified collagen, and rattus collagen, than TPCs. Tendon-progenitor cells expressed significantly more COMP when cultured on control and all collagen types, and decorin when cultured on porcine, highly purified bovine and bovine collagen when compared to BMMSCs. The results of this study revealed an advantage to culturing TPCs on randomly organized rattus collagen during the early growth phase. The beneficial effects of collagen-coated surfaces on cell proliferation is likely related to increased surface area for attachment and expansion provided by the random collagen matrix, and/or collagen-cell interactions. Tendon progenitor cells showed superior growth kinetics and expression of the matrix organizational components, COMP and decorin, than similarly cultured BMMSCs that expressed more collagen types III and I. TPCs synthesize more GAG compared to BMMSCs when cultured on plastic surfaces and there was no induction by collagen. Tendon progenitor cells should be considered as an alternative source of progenitor cells for injured equine tendons. Further in vitro studies characterizing factors that influence gene expression of both cell types is warranted.
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    Porcine urinary bladder matrix in an in vitro equine model of tenogenesis
    Khatibzadeh, Sarah M. (Virginia Tech, 2019-08-22)
    Extracellular matrix (ECM) is responsible for tendon strength and elasticity. Healed tendon ECM lacks structural integrity, leading to reinjury. Porcine urinary bladder matrix (UBM) provides a scaffold and source of bioactive proteins to improve tissue healing, but has received limited attention for treating tendon injuries. The objective of this study was to evaluate the ability of UBM to induce matrix organization and tenogenesis using a novel in vitro model. We hypothesized that addition of UBM to tendon ECM hydrogels would improve matrix organization and cell differentiation. Hydrogels seeded with bone marrow cells (n = 6 adult horses) were cast using rat tail tendon ECM ± UBM, fixed under static tension and harvested at 7 and 21 days for construct contraction, cell viability, histology, biochemistry, and gene expression. By day 7, UBM constructs contracted significantly from baseline, whereas control constructs did not. Both control and UBM constructs contracted significantly by day 21. In both groups, cells remained viable over time and changed from round and randomly oriented to elongated along lines of tension with visible compaction of the ECM. There were no differences over time or between treatments for nuclear aspect ratio, DNA, or glycosaminoglycan content. Decorin, matrix metalloproteinase 13, and scleraxis expression increased significantly over time, but not in response to UBM treatment. Mohawk expression was constant over time. Cartilage oligomeric matrix protein expression decreased over time in both groups. Using a novel ECM hydrogel model, substantial matrix organization and cell differentiation occurred; however, the addition of UBM failed to induce greater matrix organization than tendon ECM alone.