Growth factors like BMP2 have been tested for osteochondral repair, but transfer methods used until now were insufficient. Therefore, the aim of this study was to analyse if stable BMP2 expression after retroviral vector (Bullet) transduction is able to regenerate osteochondral defects in rabbits. Fibrin clots colonized by control or BMP2-transduced chondrocytes were generated for in vitro experiments and implantation into standardized corresponding osteochondral defects (n=32) in the rabbit trochlea. After 4 and 12 weeks repair tissue was analysed by histology (HE, alcian-blue, toluidine-blue), immunohistochemistry (Col1, Col2, aggrecan, aggrecan-link protein), ELISA (BMP2), and quantitative RT-PCR (BMP2, Col1, Col2, Col10, Cbfa1, Sox9). In vitro clots were also analysed by BMP2-ELISA, histology (alcian-blue), quantitative RT-PCR and in addition by electron microscopy. BMP2 increased Col2 expression, proteoglycan production and cell size in vitro. BMP2 transduction by Bullet was efficient and gene expression was stable in vivo over at least 12 weeks. Proteoglycan content and ICRS-score of repair tissue were improved by BMP2 after 4 and 12 weeks and Col2 expression after 4 weeks compared to controls. However, in spite of stable BMP2 expression, a complete repair of osteochondral defects could not be demonstrated. Therefore, BMP2 is not suitable to regenerate osteochondral lesions completely.
"As shown in this study, articular cartilage repair was improved at 8 and 12 weeks in vivo. A variety of therapeutic genes like BMP-2, BMP-7, IGF-I, FGF-2, and TGF-β (7,9,10,76,108-113) has been studied so far, with significant improvements in articular cartilage repair (7,9,10,76,108,110-113) (Figure 4). The choice of therapeutic genes is based on the strategy selected for improving articular cartilage repair. "
[Show abstract][Hide abstract] ABSTRACT: Articular cartilage defects do not regenerate. Transplantation of autologous articular chondrocytes, which is clinically being performed since several decades, laid the foundation for the transplantation of genetically modified cells, which may serve the dual role of providing a cell population capable of chondrogenesis and an additional stimulus for targeted articular cartilage repair. Experimental data generated so far have shown that genetically modified articular chondrocytes and mesenchymal stem cells (MSC) allow for sustained transgene expression when transplanted into articular cartilage defects in vivo. Overexpression of therapeutic factors enhances the structural features of the cartilaginous repair tissue. Combined overexpression of genes with complementary mechanisms of action is also feasible, holding promises for further enhancement of articular cartilage repair. Significant benefits have been also observed in preclinical animal models that are, in principle, more appropriate to the clinical situation. Finally, there is convincing proof of concept based on a phase I clinical gene therapy study in which transduced fibroblasts were injected into the metacarpophalangeal joints of patients without adverse events. To realize the full clinical potential of this approach, issues that need to be addressed include its safety, the choice of the ideal gene vector system allowing for a long-term transgene expression, the identification of the optimal therapeutic gene(s), the transplantation without or with supportive biomaterials, and the establishment of the optimal dose of modified cells. As safe techniques for generating genetically engineered articular chondrocytes and MSCs are available, they may eventually represent new avenues for improved cell-based therapies for articular cartilage repair. This, in turn, may provide an important step toward the unanswered question of articular cartilage regeneration.
Croatian Medical Journal 06/2011; 52(3):245-61. DOI:10.3325/cmj.2011.52.245 · 1.31 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bone morphogenetic protein 2 (BMP2), a member of the transforming growth factor-β (TGF-β) superfamily, plays a key role in the induction of the differentiation of mesenchymal cells into chondrocytes to form cartilage tissue. However, it is not clear whether BMP2 regulates the proliferation of chondrocytes. In the present study, the effect of BMP2 on the proliferation of chondrocytes and its underlying mechanism was investigated. Chondrocytes isolated from the knee of SD rats were cultured and identified using toluidine blue staining. The second generation chondrocytes were collected and stimulated with or without BMP2 for 48 h. Cell viability was analyzed using the MTT assay. mRNA and protein expression levels of β-catenin, GSK-3β, Dvl1 and Cyclin D1 were detected using real-time RT-PCR and Western blotting, respectively. The cell cycle distribution of the chondrocytes was analyzed by flow cytometry. BMP2 stimulation was found to significantly increase cell viability. In addition, following BMP2 treatment, β-catenin, Cyclin D1 and Dvl1 expression was significantly increased, whereas GSK-3β expression was significantly decreased. Moreover, the percentage proportion of chondrocytes in the G0/G1 phase was significantly decreased, whereas that in the S phase was significantly increased. The results indicate that BMP2 promotes chondrocyte proliferation via the Wnt/β-catenin signaling pathway.
Molecular Medicine Reports 07/2011; 4(4):621-6. DOI:10.3892/mmr.2011.474 · 1.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Treatment of cartilage defects is still challenging, primarily because of the poor self-healing capacity of articular cartilage. Gene therapy approaches have gained considerable attention, but, depending on the vector system used, they can lead to either limited or unrestrained gene expression, and therefore regulation of gene expression is necessary. This study was undertaken to construct an efficient tetracycline (Tet)-regulated, lentivirally mediated system for the expression of growth factor bone morphogenetic protein 2 (BMP-2) in primary rabbit chondrocytes that will allow for the induction and termination of growth factor gene expression once cartilage regeneration is complete.
Chondrogenic ATDC5 cells and primary rabbit chondrocytes were lentivirally transduced with different tetracycline-on (Tet-On)-regulated, self-inactivating vectors for the induction of expression of enhanced green fluorescent protein (eGFP) or BMP-2, using either a 1-vector system or a 2-vector system.
Expression of eGFP was induced on ATDC5 cells and chondrocytes. The highest induction rate and highest level of gene expression were reached when the spleen focus-forming virus long terminal repeat promoter was used to drive the reverse transactivator expression, after the addition of doxycycline, in chondrocytes. An up to 20-fold induction of Tet-mediated BMP-2 expression was observed on ATDC5 cells. The extent of induction and expression level of BMP-2 in chondrocytes were similar between the 1-vector system- and 2-vector system-infected cells (mean +/- SD 15.5 +/- 1.1 ng/ml and 14.6 +/- 0.4 ng/ml, respectively). In addition, prolonged induction and switching-off of BMP-2 expression, as well as repeated induction, were demonstrated. Production of proteoglycans, as shown by Alcian blue staining, demonstrated the functionality of the lentivirally expressed BMP-2 under induced conditions.
The lentivirally mediated Tet-On system is an effective strategy for efficient, repeatedly inducible expression of BMP-2 in primary rabbit chondrocytes. Therefore, use of this system in in vivo experiments may be a promising approach as a treatment strategy for cartilage defects.
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