Cell culture of rabbit meniscal fibrochondrocytes: Proliferative and synthetic response to growth factors and ascorbate

Abstract

This study was undertaken to determine whether the cells of the fibrocartilaginous meniscal substance are capable of proliferation and matrix synthesis. Cells were isolated from the fibrocartilaginous menisci of young New Zealand white rabbits, and grown in two alternative culture regimens differing only in the basal nutrient medium used to initiate primary monolayer growth. Under each culture regimen, the cells attached and proliferated both initially and after passage into secondary (2 degrees) culture. Differences were noted in cell morphology and time to reach confluence in primary (1 degrees) culture. Upon passage into 2 degrees culture, the fibrochondrocytes assumed two distinct morphologies depending upon the type of medium used for 1 degree culture. These morphological changes were accompanied by differences in the population doubling time and incorporation of 35SO4 into sulfated proteoglycans. The proliferation of both fibrochondrocyte subtypes was stimulated by the addition of either pituitary fibroblast growth factor (FGF) or human platelet lysate in a dose-dependent manner. Both FGF (10 ng/ml) and ascorbate (40 micrograms/ml) decreased 35-sulfate incorporation, whereas only ascorbate was found to alter the amount of sulfated glycosaminoglycan in the pericellular coat. We conclude that the fibrochondrocytes of the meniscal substance are capable of replication and synthesis of matrix macromolecules if given the proper stimuli. Additionally, there may be two subpopulations of fibrochondrocytes that can be distinguished by their in vitro behavior.

In vitro cell proliferation and proteoglycan synthesis of rabbit meniscal fibrochondrocytes as a function of age and sex

“…Neither age nor sex appeared to make any difference in the time required for the majority of the cells to attach to the surface of the culture flask (<24 hours), the time requiired for the cells to flatten or "spreadout" once attached ( 2 4 days), or the time necessary to reach a confluent monolayer in primary culture (9-12 days) ( Table 1). Although there was some variation between culture characteristics of the cells from different rabbits, all values were within the limits previously found for 6-month-old females (22).…”

“…For reasons that are obscure at present, meniscal fibrochondrocytes do not, in most instances, initiate a reparative response. This might be due to the tissue's avascularity (43) and a consequent lack of contact between the cells and these serum factors (22). However, despite the several differences found between males and females and between animals of different ages, this study demonstrates that the fibrochondrocytes from both skeletally immature and skeletally mature rabbits retain their ability to proliferate and synthesize matrix macromolecules when stimulated with progression factors and competence factors.…”

“…The growth activity of the fibrochondrocytes, with and without 24 hours of exposure to pituitary FGF (10 ng/ml) in secondary culture, was measured by the incorporation of 3H-thymidine into DNA. This concentration of FGF was used because it has previously been shown to elicit a nearmaximal response with this cell type (22). Cells grown in multiwell culture dishes, as described above, were labeled for 1 hour with 'H-thymidine (5 pCi/ml, specific activity 64 CilmM).…”

rAAV-mediated overexpression of FGF-2 promotes cell proliferation, survival, and α-SMA expression in human meniscal lesions

“…14,15 Delivery of therapeutic genes in the meniscus is an attractive approach to enhance meniscal repair. 16,17 Candidate factors that stimulate the cellular activities of meniscal fibrochondrocytes include the insulin-like growth factors I and II, 18 fibroblast growth factor 2 (FGF-2), [18][19][20] platelet-derived growth factor, 19,[21][22][23] transforming growth factor beta, [23][24][25][26][27] hepatocyte growth factor, 22 and the bone morphogenic proteins 2 and 7. 22,23 Although several studies have been performed to evaluate the efficacy of nonviral and viral vectors to deliver reporter genes in meniscal cells in vitro, [28][29][30][31][32][33][34] in situ, 30,35 and in vivo, 30,34,36 little is known about the value of applying therapeutic gene sequences in the injured meniscus.…”

Growth factors and fibrochondrocytes in scaffolds

“…Although growth factors have been studied on monolayer cultures of fibrochondrocytes, meniscal explants, in alginate beads, as well as open defects and wounds [6,8,14,23,24,27], there are no studies that look at the effects of growth factors on fibrochondrocytes seeded in scaffolds. Growth factors have been previously shown to increase extracellular matrix (ECM) production, migration, and proliferation of meniscal cells [6,8,14,27]. Different growth factors at different concentrations can have varying effects on the type of stimulation they provide.…”