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Articular cartilage defects that do not repair spontaneously induce osteoarthritic changes in joints over a long period of observation. In this study, we examined the usefulness of transplanting culture-expanded bone marrow mesenchymal cells into osteochondral defects of joints with cartilage defects. First, we performed experiments on rabbits and up on obtaining good results proceeded to perform the experiments on humans. Macroscopic and histological repair with this method was good, and good clinical results were obtained although there was no significant difference with the control group. Recent reports have indicated that this procedure is comparable to autologous chondrocyte implantation, and concluded that it was a good procedure because it required one step less than that required by surgery, reduced costs for patients, and minimized donor site morbidity. Although some reports have previously shown that progenitor cells formed a tumor when implanted into immune-deficient mice after long term in vitro culture, the safety of the cell transplantation was confirmed by our clinical experience. Thus, this procedure is useful, effective, and safe, but the repaired tissues were not always hyaline cartilage. To obtain better repair with this procedure, treatment approaches using some growth factors during in vitro culture or gene transfection are being explored. J. Cell. Physiol. 225: 291–295, 2010. © 2010 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

A morphological parameter of polygonal index was defined as the ratio of cell adhesion area versus the square of the major cell axis, and cells that had an adhesion area larger than 4000 μm2 and a polygonal index larger than 0.3 were considered large polygonal cells. Cell morphology tended to change from fibroblast-like to polygonal and the percentage of the large polygonal cells increased almost in proportion to aggrecan mRNA expression level during the differentiation culture of mesenchymal stem cells (MSCs) to chondrocytes. Approximately 80% of the large polygonal cells were negative for MSC marker (CD90, CD166) expression and the aggrecan mRNA expression level of the large polygonal cells was markedly higher than that of cells with other morphologies. [ABSTRACT FROM AUTHOR]

Fibroin‐hydrogel sponge and collagen gel were used as scaffold for in vitro cartilage regeneration. Fibroin‐hydrogel sponge was formed by phase separation from freezed fibroin solution. Chondrocytes were harvested from proximal humerus, distal femur and proximal tibia of 4‐week‐old Japanese white rabbits and inoculated in the fibroin–hydrogel sponge and collagen gel. Those constructs were cultured in DMEM supplemented with 10% FCS and 50 ml L‐ascorbate at 37°C. Histological observation, measurement of sulfated glycosaminoglycan and cell density were carried out at 3, 7, and 14 days after the cultivation. Well‐defined cartilage tissue can be seen both in the fibroin–hydrogel sponge and in the collagen gel. The matrix was intensely stained by safranin‐O and showed a metachromatic reaction in both group. However, the quantity of sulfated glycosaminoglycan and cell density of the fibroin–hydrogel sponge group were increased more rapidly than these of the collagen gel group. Thus, the chondrocytes proliferated in the fibroin sponge without losing their differentiated phenotype. It is possible that culture environment in the fibroin sponge was suitable for chondrocytes regeneration. [ABSTRACT FROM AUTHOR]

The dynamic visco‐elastic properties of regenerated cartilage tissue were measured to evaluate its mechanical function during cultivation. Harvested chondrocytes from 4‐week‐old Japanese white rabbits were inoculated into fibroin sponge at a cell concentration of about 5×10[sup 7] cells/ml. Dynamic visco‐elasticity measurements were performed under compressive loading to evaluate the load bearing function of the articular cartilage. The dynamic modulus and the dynamic loss of the regenerated cartilage increased and the peak value of tanδ, as well as the frequency at the peak, decreased with increasing cultivation time. The pores of the fibroin sponge became filled with newly formed tissue as cultivation time increased. These changes in the visco‐elastic properties of the regenerated cartilage were compared with those of a model system, ethylene propylene diene monomer sponge with interstitial fluid, and appear to be a result of increased fluid flow resistance and internal loss. We conclude that the changes in the dynamic visco‐elastic properties of the regenerated cartilage were caused because of narrowing of the fluid path by synthesized extracellular matrix. [ABSTRACT FROM AUTHOR]

WP9QY (W9) is a receptor activator of nuclear factor-κB ligand (RANKL)-binding peptide that inhibits osteoclastogenesis by blunting the RANKL-RANK interaction, and also increases osteoblastogenesis via RANKL reverse signaling. W9 has dual effects on osteoclasts and osteoblasts; however, it is unknown whether the peptide has an effect on chondrocytes. Here, we report that W9 induces proliferation and differentiation of chondrocytes in vitro and repairs full-thickness articular cartilage defects in vivo. W9 stimulated chondrocyte differentiation in a two-dimensional (2D) culture of human mesenchymal stem cells (hMSCs), and transforming growth factor β3 (TGF-β3) showed synergistic effects with W9 on chondrogenesis. W9 enlarged the size of 3D pellet cultures of hMSCs and produced chondrocyte-specific matrices, especially in combined treatment with TGF-β3. The peptide also stimulated proliferation of hMSCs with induction of expression of chondrogenesis-related genes. Several RANKL inhibitors had no effect on chondrocytic differentiation. RANKL-knockdown experiments showed that W9 did not induce chondrogenesis through RANKL, but did induce osteoblastogenesis through RANKL. Intraarticular injection of W9 resulted in significant repair of full-thickness articular cartilage defects in rabbits. Taken together, these results suggest that W9 ameliorates the articular cartilage defects by increasing the volume of cartilaginous matrices with accompanying induction of proliferation and differentiation of chondrocytes via mechanisms independent of RANKL inhibition and RANKL reverse signaling. Since no pharmaceuticals are clinically available for treatment of cartilage damage such as osteoarthritis, our findings demonstrate the potential of W9 to address the unmet medical needs. • Accumulation of cartilaginous matrices in chondrocytes with WP9QY peptide. • Synergy between WP9QY and TGF-β3 in chondrogenesis. • Unknown mechanisms independent of RANKL inhibition and RANKL reverse signaling. • Potential to address unmet medical needs for cartilage damage such as osteoarthritis. • Important clues for understanding regulation of cartilage regeneration. [ABSTRACT FROM AUTHOR]

Abstract: The effect of the addition of monosaccharides composing glycosaminoglycans (GAGs) on the accumulation of type 2 collagen (COL(II)) in a three-dimensional (3D) culture of porcine chondrocyte cells was investigated for possible application to cartilage regenerative medicine. Primary chondrocytes from porcine cartilage were cultivated in three-dimension employing atelo collagen gel for 3 weeks with the addition of several saccharides. The addition of d-glucuronic acid (d-GlcA), N-acetyl-d-galactosamine (d-GalNAc), chondroitin sulfate C (CSC), d-galactose, N-acetyl-d-glucosamine, and l-iduronic acid increased markedly not aggrecan but COL(II) accumulation although the addition of d-fructose and d-mannose not composing GAGs did not show such an effect. The addition of d-GlcA and d-GalNAc had no synergistic effect. The addition of CSC, d-GlcA, and d-GalNAc also increased COL(II) mRNA expression while aggrecan mRNA expression was not increased by these compounds. In conclusion, the addition of monosaccharides composing GAGs might be valuable for increasing COL(II) accumulation in the 3D culture of chondrocytes. [Copyright &y& Elsevier]

The effect of glycosaminoglycan addition on a three-dimensional (3D) culture of porcine chondrocyte cells was investigated with a view to use in cartilage regenerative medicine. Chondroitin sulfate C increased the mRNA expression of type 2 collagen, while chondroitin sulfate A did not. Hyaluronic acid of high molecular weight markedly decreased the mRNA expression of both aggrecan and type 2 collagen, although hyaluronic acid of low molecular weight showed no apparent effect. [Copyright &y& Elsevier]

Abstract: Porcine articular cartilage tissue was crushed using Multi-Beads-Shocker™ and digested with trypsin and collagenase type II. Chondrocyte cell yield was maximized by automatic crushing for 10 s, which was 4 times that by manual dicing with a surgical blade and might be useful for the automation of the cell processing. [Copyright &y& Elsevier]

Background: The combination of a β-tricalcium phosphate (βTCP) block with a scaffold-free chondrocyte sheet formed by the centrifugation of chondrocytes in a well was investigated with the aim of constructing an osteochondral-like structure. Methods: Human and porcine articular cartilage chondrocytes were respectively centrifuged in a 96-well plate or cell culture insert (0.32 cm) that was set in a 24-well plate, cultivated in the respective vessel for 3 weeks, and the cell sheets were harvested. In some cases, a cylindrical βTCP block (diameter 5 mm, height 3 mm) was placed on the sheet on days 1-7. The sheet size, cell number, and sulfated glycosaminoglycan accumulation were determined. Results: The use of a 96-well plate for not suspension but adhesion culture and the initial centrifugation of a well containing cells were crucial to obtaining a uniformly thick cell sheet. The glycosaminoglycan density of the harvested cell sheet was comparable to that of the pellet culture. An inoculum cell number of more than 31 × 10 cells tended to result in a curved cell sheet. Culture involving 18.6 × 10 cells and the 96-well plate for adhesion culture showed no curving of the cell sheet (thickness of 0.85 mm), and these were found to be the best of the culture conditions tested. The timing of the addition of a βTCP block to the cell sheet (1-7 days) markedly affected the balance between the thickness of cell sheet parts on and in the βTCP block. Conclusion: Centrifugation and subsequent cultivation of chondrocytes (18.6 × 10 cells) in a 96-well plate for adhesion culture led to the production of a scaffold-free cartilage-like cell sheet with a thickness of 0.85 mm. A combined osteochondral-like structure was produced by putting a βTCP block on the cell sheet. The thickness of the cell sheet on the βTCP block and the binding strength between the cell sheet and the βTCP block could be optimized by adjusting the inoculum cell number and timing of βTCP block addition. [ABSTRACT FROM AUTHOR]

Abstract: Although tribological function is the most important mechanical property of articular cartilage, few studies have examined this function in tissue-engineered cartilage. We investigated changes in the frictional properties of cartilage regenerated from the inoculation of rabbit chondrocytes into fibroin sponge. A reciprocating friction-testing apparatus was used to measure the friction coefficient of the regenerated cartilage under a small load. The specimen was slid against a stainless steel plate in a water vessel filled with physiological saline. The applied load was 0.03N, the stroke length was 20mm, and the mean sliding velocity was 0.8mm/s. The friction coefficient of the regenerated cartilage decreased with increasing cultivation time, because a hydrophilic layer of synthesized extracellular matrix was formed on the fibroin sponge surface. The friction coefficient of the regenerated cartilage was as low as that of natural cartilage in the early stages of the sliding tests, but it increased with increasing duration of sliding owing to exudation of interstitial water from the surface layer. [Copyright &y& Elsevier]

Abstract: The cell-specific production rate of melanoma inhibitory activity was quantitatively proportional to the aggrecan gene expression level in the early and middle phase during not only the redifferentiation culture of human cartilage chondrocytes but also the differentiation culture of human bone marrow mesenchymal stem cells to chondrocytes. [Copyright &y& Elsevier]

The effects of the density of human mesenchymal stem cells (MSCs) on their differentioation to chondrocytes in a differentiation medium supplemented with dexamethasone, TGFβ3, and IGF-1 were investigated for the regenerative therapy of cartilage. The increase in the initial density of MSCs from 0.05×104 to 0.9×104 cells/cm2 accelerated the increase in the expression level of aggrecan mRNA during the differentiation culture for 7 d. The conditioned medium harvested at 7 d from the differentiation culture with an initial MSC density of 0.3×104 cells/cm2 accelerated the initial increase in the expression level for 3 d in the differentiation culture with an initial MSC density of 0.3×104 cells/cm2, whereas the conditioned medium harvested at 7 d in the differentiation culture with an initial MSC density of 0.05×104 cells/cm2 did not. The differentiation culture after 14 d with an initial MSC concentration of 0.3×104 cells/cm2 showed an expression level 1.7-fold that in the case of the culture with an initial MSC concentration of 0.05×104 cells/cm2. Thus, a high MSC inoculum density might be appropriate for the rapid differentiation of MSCs to chondrocytes. [Copyright &y& Elsevier]