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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.

Since efficient transfer of foreign genes into primary articular chondrocytes (CC) is difficult, a VSV.G pseudotyped retroviral vector (Bullet) was developed for marker and growth factor gene transfer. Transduction efficiency was analysed by FACS. BMP2 production was determined by specific hBMP2-ELISA. BMP2 effect on cells regarding proteoglycan production was measured by alcian blue staining and dye quantification. Alkaline phosphatase activity was determined by enzymatic reaction with p-nitrophenyl phosphate at OD 405nm and proliferation rate was analysed by MTT-assay. ATDC5 cells (98.3+/-0.6%SD) were transduced to express the reporter gene eGFP. After 52 weeks 94.7+/-0.6%SD of cells were positive. Retroviral transduction efficiency for nlslacZ exceeded 92.3+/-6.1%SD in rabbit CC and expression remained high after 15 weeks (75.7+/-14.2%SD). ATDC5 cells and CC expressed the growth factor gene hBMP2 after retroviral transduction at different time-points. BMP2 led to an increase in proteoglycan and alkaline phosphatase production. Initially, the proliferation rate detected by MTT-assay increased in both the cell types; afterwards the proliferation rate was similar to controls. The described retroviral vector system achieved high initial transduction rates in ATDC5 cells and CC. Gene transfer was very stable over the time period analysed, rendering it a useful tool for future in vitro and in vivo studies on cartilage remodelling.

To examine a retroviral gene transfer to chondrocytes in vitro and in vivo in tissue-engineered cell-collagen constructs articular chondrocytes from rabbits and humans were isolated and transduced with VSV.G pseudotyped murine leukemia virus-derived retroviral vectors. Viral supernatants were generated by transient transfection of 293T cells using the pBullet retroviral vector carrying the nlslacZ gene, a Moloney murine leukemia virus gag/pol plasmid and a VSV.G coding plasmid. Transduction efficiency was analyzed by fluorescence-activated-cell-sorter analysis and transduced autologous chondrocytes from rabbits were seeded on collagen-scaffolds and implanted into osteochondral defects in the patellar groove of the rabbit's femur (n=10). LacZ-expression was analyzed by X-gal staining on total knee explants and histological sections. Retroviral transduction efficiency exceeded 92.3% (SEM+/-3.5%) in rabbit articular chondrocytes, 74.7% (SEM+/-1.8%) in human articular chondrocytes and 52.7% (SEM+/-5.8%) in osteoarthritic human chondrocytes. Reporter gene expression remained high after 15 weeks in 75.7% (SEM+/-8.2%) of transduced rabbit articular chondrocytes. In vivo, intraarticular beta-galactosidase activity could be determined in the majority of implanted chondrocytes in the osteochondral defects after 4 weeks.

Objective: The repair of osteochondral defects with chondrocytes genetically modified to express desired growth factors promises great potential in orthopaedic therapy. Controlled expression of the transgenes is required in many instances. The objective of the present study was to demonstrate the inducibility of tetracycline-responsive transgene expression in osteochondral defects in the knee joint filled with genetically modified chondrocyte implants., Methods: An expression plasmid containing the lacZ gene under the control of the minimal CMV promoter fused to the Tet-responsible element (TRE) as well as the reverse transactivator (rtTA2s-M2) was constructed and used to transfect isolated articular chondrocytes from New Zealand white rabbits. rtTA2s-M2 binds to the TRE in the presence of tetracycline and leads to the transcription of the transgene. Different concentrations of DNA and various DNA:lipid ratios were tested to determine best transfection conditions. Transfection efficiency and inducibility were analysed by histochemical analysis and flow-cytometry. To evaluate the system in vivo, collagen-sponges were seeded with transfected autologous chondrocytes and implanted in osteochondral defects in the knees of NZW-rabbits. Gene expression was induced by doxycycline and 3 weeks later, LacZ-expression in isolated knee joints was evaluated in histological sections by X-gal staining., Results: In vitro 13.5% (+/-1.32) of induced primary chondrocytes were LacZ-positive, while non-induced controls showed a background-staining in 0.6% (+/-0.2). In vivo, upon doxycycline treatment, induction of lacZ-gene-expression could be demonstrated in chondrocytes in 3-week-old, well-integrated implants., Conclusion: For the first time, tetracycline-inducible gene expression is demonstrated to work in the treatment of osteochondral defects. This demonstrates the feasibility for a gene therapy-assisted approach using controlled expression of therapeutic growth factors from transplanted genetically modified chondrocytes.