Serum‐free culture of rabbit meniscal fibrochondrocytes: Proliferative response

Abstract

We have formulated a serum-free medium capable of supporting DNA synthesis in rabbit meniscal fibrochondrocytes at a level equivalent to 10% fetal bovine serum (FBS). The medium consists of a 1:1 mixture of Dulbecco's Modified Eagle's Medium and Ham's F-12 medium supplemented with transferrin (1 microgram/ml), selenium (1 pg/ml), trace metal mix (1:100), dexamethasone (100 ng/ml), insulin-like growth factors I and II (50 ng/ml each), pituitary fibroblast growth factor (100 ng/ml), and lactalbumin hydrolysate (2 micrograms/ml). Endothelial cell growth supplement could be substituted for lactalbumin hydrolysate to obtain similar results. Ventrex PC-1, a commercially available, low-protein, serum-free medium, was found to support proliferation of fibrochondrocytes but not as well as 10% FBS or our medium formulation. Lipid supplements, which are known to support the serum-free growth of hyaline chondrocytes, were found to be either of no value or antagonistic for the culture of fibrochondrocytes. Likewise, vitamin E alone, progesterone, putrescine, and hydrocortisone were also without benefit in our culture system. The cells had a more chondrocytic morphology when grown in defined medium as opposed to 10% FBS. The results of this study should now make it possible to identify and quantitate those factors necessary to affect meniscal repair by utilizing further techniques in vitro.

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

“…The present data indicate that sustained and efficient rAAV-mediated FGF-2 secretion promoted cell proliferation and survival in a three-dimensional in vitro culture system and in meniscal explants, consistent with the established mitogenic properties of FGF-2. 18,19,46 The absolute concentrations of FGF-2 produced in the explants after transduction by rAAV-hFGF-2 were higher than those achieved in isolated meniscal fibrochondrocytes using similar vector doses, although the cell densities in situ are inferior to those in established monolayer cultures, most likely because of the higher amounts of cells present in the entirely transduced explants. The concentrations of FGF-2 obtained here in vitro were in the range of those measured when applying the same vector to rabbit chondrocytes (about 12 ng per 10 7 cells per 24 h when adding 300 ml rAAV to 10 6 cells, i.e.…”

“…The effects documented in situ were noted both in the peripheral zone of the intact meniscus and in the central, avascular part with low intrinsic repair ability as described earlier when applying a recombinant FGF-2 molecule. 18,19,46 Most importantly, the proliferative activities of meniscal fibrochondrocytes were also stimulated by the candidate treatment in areas surrounding the experimental meniscal lesions, an effect that was accompanied by a significant decrease in the amplitude of the artificial defects compared with a control gene administration.…”

“…This might have occurred at later time points or when applying superior vector doses to yield higher FGF-2 secretion levels, 18,19,46 and in which case presumably higher repopulating cell concentrations, 14,15 but it is more reasonable to think that healing of meniscal defects may necessitate the activation of additional processes. Interestingly, administration of the rAAV-hFGF-2 vector did not significantly enhance the synthesis of major extracellular matrix components in the systems evaluated, consistent with data using recombinant FGF-2 at approximate concentrations 19 and with our earlier observations in articular cartilage.…”

“…49,51 Relevant to the present approach, increased production of proteoglycan and type-II collagen has been documented in aggregate cultures of human meniscal cells treated by FGF-2 in the presence of transforming growth factor beta, 20 a factor well known for its metabolic properties in the meniscus. [24][25][26][27]31,37 Application of combinations of mitogenic and anabolic factors that also include insulin-like growth factors I and II, 18 platelet-derived growth factor, 19,21-23 bone morphogenic proteins 2 and 7, 22,23 and hepatocyte growth factor 22 may thus be of additonal value to improve the healing of meniscal lesions. Alternatively, factors such as the vascular endothelial growth factor, 52 hepatocyte growth factor, 22,33 and platelet-derived growth factor 22 might be considered with FGF-2 53 for their therapeutic effects on cell migration and vasculogenesis.…”

“…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.…”