The influence of the stable expression of BMP2 in fibrin clots on the remodelling and repair of osteochondral defects.
ABSTRACT 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.
- SourceAvailable from: Magali Cucchiarini[Show abstract] [Hide abstract]
ABSTRACT: Direct gene transfer strategies are of promising value to treat articular cartilage defects. Here, we tested the ability of a recombinant adeno-associated virus (rAAV) SOX9 vector to enhance the repair of cartilage lesions in vivo. The candidate construct was provided to osteochondral defects in rabbit knee joints vis-à-vis control (lacZ) vector treatment and to cells relevant of the repair tissue (mesenchymal stem cells, chondrocytes). Efficient, long-term transgene expression was noted within the lesions (up to 16 weeks) and in cells in vitro (21 days). Administration of the SOX9 vector was capable of stimulating the biological activities in vitro and over time in vivo. SOX9 treatment in vivo was well tolerated, leading to improved cartilage repair processes with enhanced production of major matrix components. Remarkably, application of rAAV SOX9 delayed premature terminal differentiation and hypertrophy in the newly formed cartilage, possible due to contrasting effects of SOX9 on RUNX2 and β-catenin osteogenic expression in this area. Most strikingly, SOX9 treatment improved the reconstitution of the subchondral bone in the defects, possibly due to an increase in RUNX2 expression in this location. These findings show the potential of direct rAAV gene delivery as an efficient tool to treat cartilage lesions.Journal of Molecular Medicine 11/2012; · 4.77 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: A better understanding of osteogenesis at genetic and biochemical levels is yielding new molecular entities that can modulate bone regeneration and potentially act as novel therapies in a clinical setting. These new entities are motivating alternative approaches for bone repair by utilizing DNA-derived expression systems, as well as RNA-based regulatory molecules controlling the fate of cells involved in osteogenesis. These sophisticated mediators of osteogenesis, however, pose unique delivery challenges that are not obvious in deployment of conventional therapeutic agents. Viral and non-viral delivery systems are actively pursued in preclinical animal models to realize the potential of the gene-based medicines. This article will summarize promising bone-inducing molecular agents in the horizon as well as providing a critical review of delivery systems employed for their administration. Special attention was paid to synthetic (non-viral) delivery systems since they are more likely to be adopted for clinical testing due to safety considerations. We present a comparative analysis of dose-response relationships, as well as pharmacokinetics and pharmacodynamics features of various approaches, with the purpose of clearly defining the current frontier in the field. We conclude with the authors' perspective on the future of gene-based therapy of bone defects, articulating promising research avenues to advance the field to clinical bone repair. © 2013 American Society for Bone and Mineral Research.Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 04/2013; · 6.04 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Matrix-assisted chondrocyte transplantation (m-ACI) still lacks any standardization in its execution in terms of cell passage (P), cell yield (C) and in vitro membrane-holding time (T). It was the goal of this study to analyze the effect of shifting cell culture parameters (P, C, T) on the in vitro as well as in vivo effort of a regulated animal m-ACI. Autologous rabbit knee articular chondrocytes were seeded within bilayer collagen I/III 3-D matrices in variation of P, C and T. Each time, 2 PCT-identical by 2 PCT-identical cell-matrix-constructs (CMC)/animal were created. Simultaneously 2 (PCT-distinct) were re-implanted (CMC-e) autologous into artificial trochlear pristine chondral defects in vivo to remain for 12 weeks while the remaining 2 were harvested (CMC-i) for immediate in vitro analysis at the time of transplantation of their identical twins. mRNA of both, CMC-e regenerates and CMC-i membranes, was analyzed for Collagen-1,-2,-10, COMP, Aggrecan, Sox9 expression by use of a mixed linear model, multiple regression analysis. Generally, CMC-i values were higher than CMC-e values for differentiation targets; the opposite was true for dedifferentiation targets. Regarding individual gene expression, in vivo regenerate cell-matrix properties were significantly dependent on initial cell-matrix in vitro values as a sign of linearity. The parameter membrane-holding time (T) had strongest effects on the resulting mRNA expression with slightly less impact of the parameter passage (P), whereas cell yield (C) had clearly less effects. Noting differences between in vitro and in vivo data, in general, optimal expression patterns concerning chondrogenic differentiation were achieved by few passages, medium cellular yield, short membrane-holding time. Clinical m-ACI may benefit from optimal orchestration of the cell culture parameters passage, yield and time.Biomaterials 09/2011; 32(25):5810-8. · 8.31 Impact Factor