Intrinsic healing in the equine superficial digital flexor tendon in vitro : effects of hyaluronate and polysulfated glycosaminoglycans on matrix synthesis and cell proliferation
Riley, Christopher Bruce
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The use of representative in vitro models has been favoured for the study of biologic and pharmacologic responses of the equine superficial digital flexor tendon (SDFT). The objectives of this study were to determine the effects of exogenous sodium hyaluronate (NaHA) and polysulfated glycosaminoglycan (PSGAG) on extracellular matrix (ECM) synthesis and cell proliferation by explants of the equine SDFT, and to compare the effects of NaHA and PSGAG at equivalent concentrations. Explants of the tensile SDFT were cultured in RPMI 1640/10% DHS/100 μg/ml ascorbate in sealed bottles on rollers (14 rpm) at 36.5 ± 0.5°C. After a pre-treatment culture period of 18 days, cultures were randomized into treatment groups, and the drug being tested added to the medium. Cultures were treated for 6 days prior to pulse-chase radiolabeling with 35S-sulfate, L-(2,3,4,5-3H) proline and methyl-C14-thymidine to determine rates of glycosaminoglycans (GAGs) synthesis, protein synthesis and cell proliferation respectively. The explants and aliquots of medium were hydrolysed in 6 M HCl, scintillation counted, and explant and medium counts summed in order to determine molar incorporation rates per mg dry weight of tissue. Protein in aliquots of the medium was precipitated and hydrolysed. The rates of collagen and noncollagen protein synthesis were determined from the derivatized amino acids by RP-HPLC. Hyaluronate synthesis was determined by radiometric assay in one experiment. NaHA was evaluated at concentrations of 0 to 2000 μg/ml. The rates of proline incorporation into protein, collagen synthesis and GAGs synthesis were increased in a dose-dependent manner. Cell proliferation was not significantly affected by NaHA. PSGAG was added to the medium in concentrations of 0 to 5000 μg/ml. The rates of proline incorporation into protein, collagen and noncollagen protein synthesis, and GAGs synthesis were significantly increased in the presence at concentrations higher than 100 μg/ml. The degree of response to PSGAG is strongly dose-related, with optimal concentrations for increased synthesis occurring at 1000 to 2000 μg/ml PSGAG. The effects of NaHA were compared to those of PSGAG, both at 1000 μg/m. The exogenous administration of PSGAG results in a wider range and greater degree of cellular responses than NaHA. Exogenous PSGAG resulted in increased collagen, noncollagen protein, and sulfated GAG synthesis, increased cell proliferation, and decreased NaHA synthesis. However, exogenously administered NaHA only increased collagen and sulfated GAG synthesis and the degree of response was significantly less than the response to PSGAG. This data provides empirical evidence that substantiates and identifies a cellular basis for the beneficial effects of NaHA and PSGAG on equine tendon repair. The results suggest that PSGAG may be a more potent drug for the stimulation or modulation of cell proliferation and ECM in the injured equine SDFT.