Production of three-dimensional tissue-engineered cartilage through mutual fusion of chondrocyte pellets.

In this study, the mutual fusion of chondrocyte pellets was promoted in order to produce large-sized tissue-engineered cartilage with a three-dimensional (3D) shape. Five pellets of human auricular chondrocytes were first prepared, which were then incubated in an agarose mold. After 3 weeks of culture in matrix production-promoting medium under 5.78g/cm(2) compression, the tissue-engineered cartilage showed a sufficient mechanical strength. To confirm the usefulness of these methods, a transplantation experiment was performed using beagles. Tissue-engineered cartilage prepared with 50 pellets of beagle chondrocytes was transplanted subcutaneously into the cell-donor dog for 2 months. The tissue-engineered cartilage of the beagles maintained a rod-like shape, even after harvest. Histology showed fair cartilage regeneration. Furthermore, 20 pellets were made and placed on a beta-tricalcium phosphate prism, and this was then incubated within the agarose mold for 3 weeks. The construct was transplanted into a bone/cartilage defect in the cell-donor beagle. After 2 months, bone and cartilage regeneration was identified on micro-computed tomography and magnetic resonance imaging. This approach involving the fusion of small pellets into a large structure enabled the production of 3D tissue-engineered cartilage that was close to physiological cartilage tissue in property, without conventional polyper scaffolds.

The application of atelocollagen gel in combination with porous scaffolds for cartilage tissue engineering and its suitable conditions.

For improving the quality of tissue-engineered cartilage, we examined the in vivo usefulness of porous bodies as scaffolds combined with an atelocollagen hydrogel, and investigated the suitable conditions for atelocollagen and seeding cells within the engineered tissues. We made tissue-engineered constructs using a collagen sponge (CS) or porous poly(L-lactide) (PLLA) with human chondrocytes and 1% hydrogel, the concentration of which maximized the accumulation of cartilage matrices. The CS was soft with a Young's modulus of less than 1 MPa, whereas the porous PLLA was very rigid with a Young's modulus of 10 MPa. Although the constructs with the CS shrank to 50% in size after a 2-month subcutaneous transplantation in nude mice, the PLLA constructs maintained their original sizes. Both of the porous scaffolds contained some cartilage regeneration in the presence of the chondrocytes and hydrogel, but the PLLA counterpart significantly accumulated abundant matrices in vivo. Regarding the conditions of the chondrocytes, the cartilage regeneration was improved in inverse proportion to the passage numbers among passages 3-8, and was linear with the cell densities (10(6) to 10(8) cells/mL). Thus, the rigid porous scaffold can maintain the size of the tissue-engineered cartilage and realize fair cartilage regeneration in vivo when combined with 1% atelocollagen and some conditioned chondrocytes.

Selection of highly osteogenic and chondrogenic cells from bone marrow stromal cells in biocompatible polymer-coated plates.

To enrich the subpopulation that preserves self-renewal and multipotentiality from conventionally prepared bone marrow stromal cells (MSCs), we attempted to use 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer-coated plates that selected the MSCs with strong adhesion ability and evaluated the proliferation ability or osteogenic/chondrogenic potential of the MPC polymer-selected MSCs. The number of MSCs that were attached to the MPC polymer-coated plates decreased with an increase in the density of MPC unit (0-10%), whereas no significant difference in the proliferation ability was seen among these cells. The surface epitopes of CD29, CD44, CD105, and CD166, and not CD34 or CD45, were detectable in the cells of all MPC polymer-coated plates, implying that they belong to the MSC category. In the osteogenic and chondrogenic induction, the MSCs selected by the 2-5% MPC unit composition showed higher expression levels of osteoblastic and chondrocytic markers (COL1A1/ALP, or COL2A1/COL10A1/Sox9) at passage 2, compared with those of 0-1% or even 10% MPC unit composition, while the enhanced effects continued by passage 5. The selection based on the adequate cell adhesiveness by the MPC polymer-coated plates could improve the osteogenic and chondrogenic potential of MSCs, which would provide cell sources that can be used to treat the more severe and various bone/cartilage diseases.

Involvement of fibroblast growth factor 18 in dedifferentiation of cultured human chondrocytes.

OBJECTIVE: Chondrocytes inevitably decrease production of cartilaginous matrices during long-term cultures with repeated passaging; this is termed dedifferentiation. To learn more concerning prevention of dedifferentiation, we have focused here on the fibroblast growth factor (FGF) family that influences chondrocyte proliferation or differentiation. MATERIALS AND METHODS: We have compared gene expression between differentiated cells in passage 3 (P3) and dedifferentiated ones in P8 of human cultured chondrocytes. We also performed ligand administration of the responsive factor or its gene silencing, using small interfering RNA (siRNA). RESULTS: FGFs 1, 5, 10, 13 and 18 were higher at P8 compared to P3, while FGFs 9 and 14 were lower. Especially, FGF18 showed a 10-fold increase by P8. Ligand administration of FGF18 in the P3 cells, or its gene silencing using siRNA in the P8 cells, revealed dose-dependent increase and decrease respectively in type II collagen/type I collagen ratio. Exogenous FGF18 also upregulated expression of transforming growth factor beta (TGF-beta), the anabolic factor of chondrocytes, in P3 chondrocytes, but P8 cells maintained a low level of TGF-beta expression, suggesting a decrease in responsiveness of TGF-beta to FGF18 stimulation in the dedifferentiated chondrocytes. CONCLUSION: FGF18 seems to play a role in maintenance of chondrocyte properties, although its expression was rather high in dedifferentiated chondrocytes. Upregulation of FGF18 in dedifferentiated chondrocytes implied that it may be a marker of dedifferentiation.

Histochemical evaluation for the biological effect of menatetrenone on metaphyseal trabeculae of ovariectomized rats.

To evaluate the biological effects of vitamin K2 (menatetrenone, MK-4) on ovariectomy (OVX)-induced bone loss, we have examined histological alterations of femoral metaphyses of sham-operated (sham group), ovariectomized (OVX group), and MK-4 dietary-supplemented OVX (MK-4 group; 50 mg/kg per day) female Fischer rats 1, 2, 5, and 8 weeks after OVX. In the first week, rats of the OVX and MK-4 groups showed discontinuous trabeculae compared with sham-operated rats. At 2 weeks after OVX, the OVX rats revealed many large tartrate resistant acid phosphatase (TRAP)-positive osteoclasts, while osteoclasts in the MK-4-treated rats were similar in size to those of the sham group. At 5 weeks, the OVX and MK-4 groups revealed fragmented trabeculae in femoral metaphyses. The cartilage matrix was partially exposed due to stimulated bone resorption in the OVX group, but not in the MK-4 group. After 8 weeks, the OVX rats had little metaphyseal trabeculae, whereas the MK-4-treated rats had maintained short trabeculae. Despite the presence of intense alkaline phosphatase-positive osteoblasts on trabeculae in the MK-4 group, TRAP-positive osteoclasts were flattened without developing ruffled borders. Therefore, MK-4 appeared to lessen the increase in osteoclastic bone resorption induced by OVX, as well as to maintain the accelerated osteoblastic activity. It is of importance to identify the target cells for MK-4 in bone. Autoradiography localized [3H]-labeled MK-4 mainly in osteoblasts and adjacent bone matrices, but not in osteoclasts, indicating that MK-4 targets osteoblasts. Thus, MK-4 appears to target osteoblasts, consequently inhibiting bone loss induced by ovariectomy.

Mouse epididymal sperm contain active P450 aromatase which decreases as sperm traverse the epididymis.

Recently we reported that mouse germ cells in the testis contain active P450 aromatase (P450arom), the enzyme that converts androgens to estrogens. This finding suggested that germ cells have the ability to produce estrogen. Further studies have shown that germ cells in the testis of several species contain P450arom. The goal of this study was to determine if epididymal sperm contain P450arom and if P450arom activity in sperm changes during traversion of the epididymis in the adult mouse. P450arom was localized in sperm present in the efferent ductules and epididymis by immunocytochemistry using an antiserum generated against purified human placental cytochrome P450arom. P450arom immunostaining in sperm was most prominent in sperm located in the proximal caput epididymis, decreased as sperm traversed the corpus epididymis, and was only slightly apparent in sperm in the cauda epididymis. The immunolocalization of P450arom in epididymal sperm was supported by the measurement of P450arom activity in sperm by the 3H2O assay. We found that P450arom activity in sperm significantly decreases as sperm traverse the epididymis. Based upon these observations, we conclude that sperm can synthesize estrogen and that the synthesis of estrogen by sperm present in the efferent ductules and caput epididymis could be important in the process of sperm maturation.