In this study, we report that using in vitro micromass culture models of chondrogenesis, the peptide B2A was able to up-regulate a number of genes associated with chondrogenesis. That up-regulation was reflected in an increased production of sulfated glycosaminoglycans and type II collagen. In vivo, using a chemically-induced model of osteoarthritis in rat knees, B2A increased cartilage repair as monitored histologically.
The micromass culture model has been used extensively to study chondrogenic differentiation(16–20)
. In this model, chondrocyte precursors, including C3H10T1/2 cells, undergo processes similar to cartilage development. Under high cell density conditions and in the presence of appropriate growth factors, cells differentiate and express collagen type II and cartilage-specific proteoglycans, and eventually form tissue masses that resemble cartilage. In the present study, C3H10T1/2 cells were used in concert with PCR to evaluate gene expression following B2A stimulation.
Twist1, a member of the basic helix-loop-helix transcription factors family, was up-regulated upon B2A treatment. A high level of Twist1 is required to promote and maintain chondrogenic differentiation in immature chondrocytes(15)
, and thus its up-regulation would be expected. As the chondrocytes turn into mature hypertrophic chondrocytes, Twist1 level decreases. The higher level of Twist1 found in the 7-day micromass culture suggests that the cells are in transition from stem cells to premature chondrocytes.
Additionally, Fgf1, Fgfr1, and Fgfr2 were up-regulated in the B2A treated micromass cultures. B2A stimulated the production of PDGF-AA. PDGF-AA has been found to be a specific PDGF isoform associated with chondrogenic phenotype(21)
. It is a potent mitogenic and chemotactic factor for mesenchymal stem cells and chondrocytes, and can also increase proteoglycan production in chondrocytes. The increased Pdgfa might serve as an autocrine loop to propel the chondrogenic differentiation. Interestingly, Bmp1, a member of the BMP1/TLD metalloproteinases (review see Hopkins et al (22)
), was found significantly up-regulated (~500 fold). In addition to playing critical roles in regulating the formation of various extracellular matrixes, BMP1 also regulates activity of TGF-β family members. BMP1 activates BMP2/4 by cleaving their antagonist, chordin (23, 24)
. In vivo
, BMP1 is able to induce ectopic cartilage (25, 26)
. The increase of Bmp1 expression might play a role in the B2A enhancement of cartilage repair. Intriguingly, Col3a1 and Col11a1 were found increased significantly at day 7. Col3a1 and Col11a1 are detected in articular cartilage as an intrinsic part of the type II collagen fibril. They consist of 10- and 3% of the total collagen found in the cartilage matrix, respectively (review see Eyre et al (27)
). The up-regulation of Col3a1 and Col11a1 by B2A might play a role in articular cartilage matrix repair and remodeling.
Productions of glycosaminoglycans and type II collagen are two hallmarks of chondrogenic differentiation. It has been reported that TGF-β, BMP-2, and GDF5, growth factors that play positive roles in chondrogenic differentiation during embryonic development and cartilage repair in adulthood, significantly increase the glycosaminoglycan and type II collagen in vitro. B2A also stimulated the production of glycosaminoglycans and type II collagen of both hBMSC and hNAC in micromass culture. These findings suggest that B2A might enhance chondrogenic differentiation and cartilage repair in vivo as well. It should be noted that in the experiments described here TGF-beta was not included in the medium as is normally done for most micromass cultures, and thus the effect of B2A was specific.
The ability of B2A in vitro
to influence gene expression and cartilage-like phenotypes suggests that B2A might have a role in promoting cartilage repair. In this report, we demonstrate that B2A increased the repair in damaged cartilage in vivo
. MIA inhibits glycolysis in the joint and causes the death of chondrocytes(28)
. MIA, as used in the present study, causes progressive cartilage degeneration and produces a pathologic manifestation similar to that found in arthritis(14)
. Guzman and colleagues reported that MIA causes chondrocyte degeneration at as early as day 1 post-injection and by day 5 and 7, marked loss of chondrocytes and moderate collapse of the cartilaginous matrix was observed(29)
. In the present study, at 7 days after MIA injection, B2A treatment was initiated (7- and 14 days). The separation of MIA injection and B2A treatment minimized the possibility that B2A could interact with MIA and compromise interpretation of results. B2A significantly improved the cartilage repair in the damaged knees. While this study indicates that B2A improves cartilage repair, additional studies focused on optimizing the B2A dose and carrier would be useful. Moreover, testing the B2A effects in models other than MIA and study of the B2A-enhanced repair kinetics will also provide further information to determine whether B2A applied in the synovial space is a viable therapeutic approach.
While it is clear that additional studies are needed to clarify the role that B2A might play in cartilage repair, the results reported here are encouraging. Repair of articular cartilage(30)
is a significant clinical challenge in part because cartilage has no vascularity and low cell density(31)
, and chondrocytes have a low proliferation capacity. Repopulation of cartilage defects does occur, at least to some degree in humans, and perhaps more so in experimental animals where the origin of the repopulating cells appears to be primitive mesenchymal cells of the marrow and synovial fat pad(32)
. B2A may also play a role in the therapeutic use of MSCs as an agent to help expand cell populations and to prime the cells towards the chondrogenic differentiation prior to implantation.
In conclusion, B2A might facilitate cartilage repair via multiple aspects: 1) increasing the expression of many genes associated with chondrocyte maturation in progenitor cells, 2) stimulating chondrogenesis in stem cells as evidenced by the induction of multiple genes associated with chondrocyte differentiation, and 3) increasing the production of extracellular matrix by chondrocytes. Continued study of B2A is warranted to further evaluate this peptide in cartilage repair.