| Background:Bone marrow mesenchymal stem cells, BMSCs (BMSCs) are taken as a promising cell source for cartilage tissue engineering. Much effort has been focused on BMSCs which possess great research significance and extensive application prospects. However, cartilage graft derived from BMSCs shows a series of phenotypic alternation, such as losing the capability to secrete collagen II, during long term in vivo test, which leads to a serious influence on the long-term effect of cartilage defect repair using BMSCs. This series of phenomena occur similar to the process of endochondral bone formation. The engineering cartilage becomes markedly hypertrophic and calcified, which initiates the vascular invasion. As the transplanted time continues, augment of vascularization and mineralization are gradually proceeded and the new bone formed. The vascular invasion is a pivotal event, which accommodate the switch from chondrogenesis to osteogenesis in this process. Cartilage, however, is normally avascular even in the microenvironment of promotion of angiogenesis. Many studies have revealed that chondromodulin-I (ChM-â… ), specificly expressed in cartilage, is identified as an angiogenesis inhibitor. We presumed that ChM-â… was not or rarely expressed in BMSCs during the chondrogenesis, which might result in the vascular invasion and loss of cartilage phenotype. At present, there are no evidence showing whether ChM-â… is expressed in BMSCs and newly formed cartlage graft from BMSCs. Thus we began with whether expression difference of ChM-â… gene between BMSCs and chondrocytes, and then evaluated the function of inhibition of vascularization by ChM-â… in cartilage graft engineered from BMSCs in vivo, in order to investigate the contribution of ChM-â… during the process of loss of cartilage phenotype.Objective:This study was designed to assess the effects of chondrocytes on mineralization and vascularization of cartilage graft derived from BMSCs, compare the different expressions of ChM-I gene in chondrocytes and BMSCs, and then investigate the function of ChM-I gene inhibition of vascularization of ectopic cartilage engineered from BMSCs in vivo, which may offer a novel approach to enhance the phenotypic stability of BMSCs engineered cartilage.Methods and results:1. BMSCs and chondrocytes were co-seeded into natural coral scaffolds in different ratio of 3:1,1:1 and 1:3. Meanwhile, BMSCs and chondrocytes alone were also seeded as controls. The cell-coral composites were implanted subcutaneously into nude mice for 1 and 2 months. After harvesting, the specimens were observed and processed for histological examination to investigate the vascularization in newly formed tissue and the distribution of ChM-I. The gross observation and histological results showed that vascularization could be observed in BMSCs alone seeded scaffold, and mature bone could be detected in the graft. However, no obvious vascularization could be detected in the experimental groups'specimens seeded with chondrocytes, even though the percentage of which was just 25% in the cartilage graft. ChM-â… was markedly expressed in all experimental specimens by Immunofluorescence, as well as the positive control, but rarely in the BMSCs alone graft.2. The different expressions of ChM-â… gene in chondrocytes and BMSCs were compared by RT-PCR which revealed that ChM-â… was not expressed in primary BMSCs, as well as the gene of collagenâ…¡and aggrecan showed an obvious expression difference in both cells.3. ChM-â… gene was cloned by RT-PCR from rabbit articular cartilage, identified by DNA sequencing and then used to construct the recombinant adenovirus. We successfully obtained the gene and produced high titer adenovirus vector named Ad5-ChM-â… , containing the full-length cDNA of ChM-â… .4. The transfected BMSCs with Ad5-ChM-â… were seeded into natural coral and transplanted subcutaneously into nude mice for 1 and 2 months to investigate the alterations of phenotypes in vivo. The gross observation and histological results demonstrated that low vascularization and the expression of ChM-â… could be monitored in the cartilage graft derived from transfected BMSCs.Conclusions: Expressions of ChM-â… gene were significantly different in chondrocytes and BMSCs. The latter scarcely expressed ChM-â… . Chondrocytes and ChM-â… , a novel glycoprotein that specifically expressed in chondrocytes, inhibited the vascularization in cartilage graft engineered from BMSCs in vivo and suppressed the switch to bone so as to reinforce the cartilage phenotypic stability in some extent. Accordingly, adenovirus-mediated ChM-â… gene modified BMSCs could be regarded as an promising cell source for tissue engineering cartilage or/and cartilage defect repair to improve the effect of long-term therapy.
|
PDF Full Text Request