Cell-based strategies to engineer a meniscus substitute has been suggested as an approach to the treatment of meniscal defects. However, attempts to expand human meniscus cells in monolayer culture have resulted in decreased gene expression of ECM components of importance in meniscus function, such as type II collagen [
7], which is located mostly in the inner region of the tissue and is thought to endow properties suitable for compressive load distribution [
13]. In this study we have investigated the combination of culture under conditions of low oxygen tension and FGF2-stimulated cell expansion as a strategy to augment the re-expression of type II collagen and a matrix-forming phenotype in human meniscus cells. Human meniscus cells showed a chondrogenic response (increased collagen II gene and protein expression) when cultured in cell aggregates regardless of FGF2 presence or absence during monolayer expansion (Figure
3). However, the response was much greater in cell aggregate cultures derived from FGF2-expanded cells (Figure
3). The type II collagen protein was notably more localized in the matrix at the periphery of the cell aggregates and more pericellularly at the central region of the cell aggregates (Figure
8). The chondrogenic response was further enhanced by low oxygen tension, which caused increased gene expression of SOX9. However, the expression of L-SOX5 and SOX6 remained unchanged and low. This was surprising because L-SOX5 and SOX6 interact cooperatively with SOX9 to promote the expression of cartilage-specific genes (such as those encoding COL2A1 and aggrecan) [
28]. The enhanced chondrogenic response at low oxygen tension may involve the transcriptional activity of HIF-1α, (hypoxia inducible factor) which modulates the expression of a variety of hypoxia-inducible genes under low oxygen tension [
32]. It has been reported that hypoxia promotes the differentiation of murine mesenchymal stroma cells along a chondrocyte pathway in part by activating SOX9 via a HIF-1α-dependent mechanism [
33]. Furthermore, HIF-1α has been shown to bind to cAMP-response element-binding protein (CREB)-binding protein (CBP)/p300 [
34], which SOX9 uses to exert its cartilage-specific type II collagen gene promoter activity [
35]. It was noticeable that in monolayer there was no significant chondrogenic response in changing from normal oxygen tension (20%) to low oxygen tension (5%) compared with changing from monolayer to aggregate (Figure
3). In the comparison between the expression of cells in aggregates and in monolayer, the 3D structure of a cell aggregate, together with oxygen consumption by the cells, would result in a lower oxygen tension within the aggregate than in cells in a monolayer. However, because cell aggregates showed a strong chondrogenic response at 5% and 20% oxygen, any small difference in oxygen tension was clearly not a major factor driving the chondrogenic response.
It was notable that the high gene expression of COL1A2 in cell aggregates formed from FGF2-expanded cells was not correlated with the matrix immunostaining, which was weak with anti-type I collagen. This suggested that there is a more complex control of type I collagen translation, fibrillogenesis and matrix deposition.
Further characterization of the chondrogenic response by human meniscus cells was by gene expression analysis of proteoglycan common to cartilage and meniscus. Aggrecan gene expression was low in meniscal cells and was not influenced by FGF2-mediated cell expansion, but its expression increased in cell aggregate cultures. FGF2-expanded cells expressed higher levels of biglycan and fibromodulin in cell aggregates, and this was unaffected by low oxygen. In non-FGF2-expanded cells, biglycan and fibromodulin expression was similar in monolayer and cell aggregates, but biglycan was increased by low oxygen tension in cell aggregates formed from FGF2-expanded cells. Histology showed an increase in safranin-O staining in cell aggregates formed from FGF2-expanded cells at low oxygen tension. Although this did not reflect a significant statistical increase in GAG/DNA ratio under low-oxygen conditions, the cell aggregates formed were of higher wet weight and this might correspond to a greater increase in cell number.
This study showed that P2 meniscus cells after growth stimulation with FGF2 were able to re-express type II collagen and proteoglycans at both the gene and protein levels. Furthermore, this ability was enhanced by 5% oxygen culture conditions and was higher than with meniscus cells expanded in the absence of FGF2. The cells used in this study were from all regions of the meniscus and thus include cells from the inner avascular region, which contains more collagen type II than the outer vascular region. FGF2 may favour the selective proliferation of the cells from this region and thus sustain the population of meniscus cells with chondrogenic potential. Expansion with FGF2 has been reported to increase the chondrogenic potential of human bone marrow stromal cells [
36]. Previous studies by Nakata and colleagues [
7] have reported three distinguishable cell types within the human meniscus tissue: small round chondrocyte-like cells, elongated fibroblast-like cells and polygonal cells; they related the loss of collagen II expression in meniscus cells during monolayer expansion with the gradual loss of both the chondrocyte-like and polygonal cell populations to leave predominantly fibroblast-like cells. The mechanism by which FGF2 conferred this ability to re-express type II collagen and proteoglycan in meniscus cells is therefore either by the selective proliferation of chondrogenic cells within the culture or by maintaining the cells in a more plastic and responsive state to chondrogenic stimuli [
16].