The effect of mechanical loading on the metabolism of growth plate chondrocytes.

It is well known that mechanical loading influences the endochondral bone formation essential for the growth and development of longitudinal bones. The question was, however, asked whether the effect of mechanical loading on the chondrocyte metabolism is dependent on the loading frequency. This study was aimed at evaluating the effect of tensile loadings with various frequencies on the proliferation of growth plate chondrocytes and extracellular matrix synthesis. The chondrocytes obtained from rib growth plate cartilage of 4-week-old male Wistar strain rats were cultured by day 4 and day 11 and used as proliferating and matrix-forming chondrocytes, respectively. Intermittent tensile stresses with different frequencies were applied to each stage chondrocyte. DNA syntheses were examined by measuring the incorporation of [(3)H]thymidine into the cells. Furthermore, the rates of collagen and proteoglycan syntheses were determined by measuring the incorporation of [2,3-(3)H]proline and [(35)S]sulfate into the cells, respectively. At the proliferating stage, intermittent tensions with the frequencies of 30 cycles/min and 150 cycles/min significantly (p < 0.05) upregulated the syntheses of DNA, which indicates the promotion of chondrocyte proliferation. At the matrix-forming stage, collagen, and proteoglycan syntheses also enhanced with increase of the loading frequency. In particular, the intermittent tension with the frequencies of 30 cycles/min and 150 cycles/min increased significantly (p < 0.05 or p < 0.01) both the collagen and proteoglycan syntheses. These results suggest that the proliferation and differentiation of growth plate chondrocytes are regulated by the mechanical loading and that the chondrocyte metabolism enhanced with increase of loading frequency. These may give more insight into the possible mechanism leading to endochondral bone formation.

Multidifferentiation potential of mesenchymal stem cells in three-dimensional collagen gel cultures.

Mesenchymal stem cells (MSCs) have a great therapeutic potential resulting from their ability to differentiate into multiple tissues when cultured under specific conditions. However, it has not been clearly demonstrated whether or not MSCs exhibit a multidifferentiation potential in three-dimensional collagen gel cultures. This study was conducted to explore the multidifferentiation potential of MSCs cultured in three-dimensional collagen gels. Human MSCs were cultured in 0.3% collagen gel for 20 days in chondrogenic differentiation medium (CDM), and for 14 days in osteogenic differentiation medium (ODM). Increases in GAG deposits, intensity of toluidine blue staining, and mRNA expressions of chondrogenic markers (type II collagen and type X collagen) were found in human MSCs cultured in the collagen gel maintained in CDM. Positive staining for alkaline phosphatase (ALP) activity and alizarin red, and increases in mRNA expressions of osteogenic markers (type I collagen, bone sialoprotein and ALP) were noted in the MSCs maintained in ODM. These findings emphasize that human MSCs have an ability to differentiate into both bone and cartilaginous tissues in three-dimensional collagen gel cultures, indicating potential clinical applications of MSC transplant therapy with collagen gel as a scaffold for bone or cartilage regeneration in complicated tissue defects.

Mechanical regulation of terminal chondrocyte differentiation via RGD-CAP/beta ig-h3 induced by TGF-beta.

RGD-CAP (beta ig-h3), initially cloned as a transforming growth factor (TGF)-beta inducible gene in human lung adenocarcinoma cells, was demonstrated to have a negative regulatory function in mineralization in hypertrophic chondrocytes, and the expression was shown to be associated with mechanical stimulation. We hypothesized that mechanical stimulation may regulate the terminal chondrocyte differentiation through the TGF-beta pathway by enhancing the RGD-CAP expression. To test this hypothesis, we investigated the effects of mechanical strain on the terminal differentiation and mineralization of growth-plate chondrocytes and assessed the mechanical regulation of TGF-ss and RGD-CAP expression. A cyclic mechanical strain of 12% elongation was applied to the cultured pre-hypertrophic chondrocytes isolated from the rib cartilage of 4-week-old male rats at 30 cycles/min (loading and relaxation on every alternate second). The terminal differentiation and mineralization of chondrocytes were assessed by alkaline phosphatase (ALP) activity assay and alizarin red staining. The gene expressions of TGF-ss and RGD-CAP, as well as chondrocytic terminal differentiation markers such as type X collagen and ALP, were examined with real-time RT-PCR. Cyclic mechanical strain decreased the ALP activity and intensity of alizarin red staining in pre-hypertrophic chondrocytes, as well as the gene expressions of type X collagen and ALP. TGF-ss and RGD-CAP were upregulated in the pre-hypertrophic chondrocytes subjected to mechanical strain, whereas the level of PTHrP receptor mRNA was not affected by the mechanical strain. The neutralizing antibody for TGF-ss suppressed the reduction of the mineralization of chondrocyte cultures with the downregulation of RGD-CAP. These results suggest that mechanical strain negatively regulates the terminal differentiation of chondrocytes through the signal pathway of TGF-ss with the induction of RGD-CAP.

Induction of CD44 and MMP expression by hyaluronidase treatment of articular chondrocytes.

In this study, the effects of fragmentation of the glycosoaminoglycans of the cell-associated matrix by hyaluronidase (HAase) on the expression of CD44 receptor and matrix metalloproteinase (MMP) mRNAs in cultured articular chondrocytes were examined. Chondrocytes, isolated from rabbit and bovine articular cartilage, were treated with bovine testicular HAase (0-200 units/ml) in the presence or absence of an antibody for CD44. The mRNA levels of CD44, CD44 variant (CD44v), MMPs (MMP-1, -3 and -9), and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) were determined by RT-PCR. The treatment of cultured chondrocytes with HAase resulted in the production of low molecular weight fragments of hyaluronan (HA). The expression of CD44, CD44v and MMP (MMP-1, -3 and -9) mRNAs, but not TIMP-1 or TIMP-2 mRNA, was up-regulated in the cultures treated with HAase, whereas this expression was not affected by treatment with purified HA of 1.0 x 10(5) Da. Furthermore, the induction of CD44 and MMPs on treatment with HAase was suppressed by an anti-CD44 antibody. The results suggest that the fragmentation of HA may lead to cartilage destruction in terms of the enhanced expression of MMPs as well as the upregulation of CD44.