Biomedical subjects

Magali Cucchiarini

Publications and source records attributed to Magali Cucchiarini.

7 recordsLinked to original sources

PubMed2007

Restoration of the extracellular matrix in human osteoarthritic articular cartilage by overexpression of the transcription factor SOX9.

OBJECTIVE: Human osteoarthritis (OA) is characterized by a pathologic shift in articular cartilage homeostasis toward the progressive loss of extracellular matrix (ECM). The purpose of this study was to investigate the ability of rAAV-mediated SOX9 overexpression to restore major ECM components in human OA articular cartilage. METHODS: We monitored the synthesis and content of proteoglycans and type II collagen in 3-dimensional cultures of human normal and OA articular chondrocytes and in explant cultures of human normal and OA articular cartilage following direct application of a recombinant adeno-associated virus (rAAV) SOX9 vector in vitro and in situ. We also analyzed the effects of this treatment on cell proliferation in these systems. RESULTS: Following SOX9 gene transfer, expression levels of proteoglycans and type II collagen increased over time in normal and OA articular chondrocytes in vitro. In situ, overexpression of SOX9 in normal and OA articular cartilage stimulated proteoglycan and type II collagen synthesis in a dose-dependent manner. These effects were not associated with changes in chondrocyte proliferation. Notably, expression of the 2 principal matrix components could be restored in OA articular cartilage to levels similar to those in normal cartilage. CONCLUSION: These data support the concept of using direct, rAAV-mediated transfer of chondrogenic genes to articular cartilage for the treatment of OA in humans.

Adenoviridae↗

PubMed2006

Local stimulation of articular cartilage repair by transplantation of encapsulated chondrocytes overexpressing human fibroblast growth factor 2 (FGF-2) in vivo.

BACKGROUND: Defects of articular cartilage are an unsolved problem in orthopaedics. In the present study, we tested the hypothesis that gene transfer of human fibroblast growth factor 2 (FGF-2) via transplantation of encapsulated genetically modified articular chondrocytes stimulates chondrogenesis in cartilage defects in vivo. METHODS: Lapine articular chondrocytes overexpressing a lacZ or a human FGF-2 gene sequence were encapsulated in alginate and further characterized. The resulting lacZ or FGF-2 spheres were applied to cartilage defects in the knee joints of rabbits. In vivo, cartilage repair was assessed qualitatively and quantitatively at 3 and 14 weeks after implantation. RESULTS: In vitro, bioactive FGF-2 was secreted, leading to a significant increase in the cell numbers in FGF-2 spheres. In vivo, FGF-2 continued to be expressed for at least 3 weeks without leading to differences in FGF-2 concentrations in the synovial fluid between treatment groups. Histological analysis revealed no adverse pathologic effects on the synovial membrane at any time point. FGF-2 gene transfer enhanced type II collagen expression and individual parameters of chondrogenesis, such as the cell morphology and architecture of the new tissue. Overall articular cartilage repair was significantly improved at both time points in vivo. CONCLUSIONS: The data suggest that localized overexpression of FGF-2 enhances the repair of cartilage defects via stimulation of chondrogenesis, without adverse effects on the synovial membrane. These results may lead to the development of safe gene-based therapies for human articular cartilage defects.

Alginates↗

PubMed2005

Gene therapy for cartilage defects.

Focal defects of articular cartilage are an unsolved problem in clinical orthopaedics. These lesions do not heal spontaneously and no treatment leads to complete and durable cartilage regeneration. Although the concept of gene therapy for cartilage damage appears elegant and straightforward, current research indicates that an adaptation of gene transfer techniques to the problem of a circumscribed cartilage defect is required in order to successfully implement this approach. In particular, the localised delivery into the defect of therapeutic gene constructs is desirable. Current strategies aim at inducing chondrogenic pathways in the repair tissue that fills such defects. These include the stimulation of chondrocyte proliferation, maturation, and matrix synthesis via direct or cell transplantation-mediated approaches. Among the most studied candidates, polypeptide growth factors have shown promise to enhance the structural quality of the repair tissue. A better understanding of the basic scientific aspects of cartilage defect repair, together with the identification of additional molecular targets and the development of improved gene-delivery techniques, may allow a clinical translation of gene therapy for cartilage defects. The first experimental steps provide reason for cautious optimism.

Cartilage, Articular↗

PubMed2005

Improved tissue repair in articular cartilage defects in vivo by rAAV-mediated overexpression of human fibroblast growth factor 2.

Therapeutic gene transfer into articular cartilage is a potential means to stimulate reparative activities in tissue lesions. We previously demonstrated that direct application of recombinant adeno-associated virus (rAAV) vectors to articular chondrocytes in their native matrix in situ as well as sites of tissue damage allowed for efficient and sustained reporter gene expression. Here we test the hypothesis that rAAV-mediated overexpression of fibroblast growth factor 2 (FGF-2), one candidate for enhancing the repair of cartilage lesions, would lead to the production of a biologically active factor that would facilitate the healing of articular cartilage defects. In vitro, FGF-2 production from an rAAV-delivered transgene was sufficient to stimulate chondrocyte proliferation over a prolonged period of time. In vivo, application of the therapeutic vector significantly improved the overall repair, filling, architecture, and cell morphology of osteochondral defects in rabbit knee joints. Differences in matrix synthesis were also observed, although not to the point of statistical significance. This process may further benefit from cosupplementation with other factors. These results provide a basis for rAAV application to sites of articular cartilage damage to deliver agents that promote tissue repair.

Animals↗

PubMed2004

Menisci are efficiently transduced by recombinant adeno-associated virus vectors in vitro and in vivo.

BACKGROUND: Meniscal tears remain an unsolved problem in sports medicine. Gene transfer is a potential approach to enhancing meniscal repair. Recombinant adeno-associated virus is a method of gene transfer that has advantages over previously used approaches to this problem. HYPOTHESIS: Direct gene transfer to meniscal cells can be accomplished using recombinant adeno-associated virus in vitro and in vivo. STUDY DESIGN: Controlled laboratory study. METHODS: Recombinant adeno-associated viruses containing the reporter gene lacZ were tested for their ability to achieve gene transfer into lapine and human meniscal cells in vitro and into lapine meniscal defects in vivo. Results were assessed by detecting beta-galactosidase, the enzyme encoded by the lacZ gene. RESULTS: Maximal efficiency of gene transfer was 81.6% +/- 6.6% for lapine and 87.2% +/- 14.8% for human meniscal cells in vitro. Expression of the transferred gene continued for the 28-day duration of the study. When the recombinant adeno-associated virus vector was injected into meniscal tears in a lapine meniscal tear model, transgene expression continued in meniscal cells adjacent to the tear for at least 20 days in vivo. CONCLUSIONS: The data suggest that recombinant adeno-associated virus vectors can directly and efficiently transfer and stably express foreign genes in isolated lapine and human meniscal cells in vitro and in lapine meniscal defects in vivo. CLINICAL RELEVANCE: This direct gene transfer approach may form a basis for improved treatments of meniscal tears.

Aged↗

PubMed2003

Recombinant adeno-associated virus vectors efficiently and persistently transduce chondrocytes in normal and osteoarthritic human articular cartilage.

Successful gene transfer into articular cartilage is a prerequisite for gene therapy of articular joint disorders. In the present study we tested the hypothesis that recombinant adeno-associated virus (rAAV) vectors are capable of effecting gene transfer in isolated articular chondrocytes in vitro, articular cartilage tissue in vitro, and sites of articular damage in vivo. Using an rAAV vector carrying the Escherichia coli beta-galactosidase gene (lacZ) under the control of the cytomegalovirus (CMV) immediate-early promoter/enhancer (rAAV-lacZ), transduction efficiency exceeded 70% for isolated normal human adult articular chondrocytes, and osteoarthritic human articular chondrocytes. These were comparable to the transduction efficiency obtained with neonatal bovine articular chondrocytes. Transduction of explant cultures of articular cartilage resulted in reporter gene expression within the tissue of all three cartilage types to a depth exceeding 450 microm, which remained present until 150 days. When rAAV-lacZ vectors were applied to femoral chondral defects and osteochondral defects in vivo in a rat knee model, reporter gene expression was achieved for at least 10 days after transduction. These data suggest that AAV-based vectors can efficiently transduce and stably express foreign genes in articular chondrocytes, including chondrocytes of normal and osteoarthritic human articular cartilage. The data further suggest that the same rAAV vectors are capable of transducing chondrocytes in situ within their native matrix to a depth sufficient to be of potential clinical significance. Finally, the data demonstrate that these rAAV vectors are capable of effectively delivering recombinant genes to chondral and osteochondral defects in vivo.

Animals↗

PubMed2003

Sustained transgene expression in cartilage defects in vivo after transplantation of articular chondrocytes modified by lipid-mediated gene transfer in a gel suspension delivery system.

BACKGROUND: Genetically modified chondrocytes may be able to modulate articular cartilage repair. To date, transplantation of modified chondrocytes into cartilage defects has been restricted to viral vectors. We tested the hypothesis that a recombinant gene can be delivered to sites of cartilage damage in vivo using chondrocytes transfected by a lipid-mediated gene transfer method. METHODS: Isolated lapine articular chondrocytes were transfected with an expression plasmid vector carrying the P. pyralis luciferase gene using the reagent FuGENE 6. Transfected chondrocytes were encapsulated in alginate spheres and implanted into osteochondral defects in the knee joints of rabbits. RESULTS: In vitro, luciferase activity in pCMVLuc-transfected spheres showed an early peak at day 2 post-transfection and remained elevated at day 32, the longest time point evaluated. The number of viable chondrocytes in non-transfected and transfected spheres increased over the period of cultivation. In vivo, luciferase activity was maximal at day 5 post-transfection, declined by day 16, but was still present at day 32. On histological analysis, the alginate-chondrocyte spheres filled the cartilage defects and were surrounded by a fibrous repair tissue composed of spindle-shaped cells. CONCLUSIONS: These data demonstrate the successful introduction of articular chondrocytes modified by lipid-mediated gene transfer in a gel suspension delivery system into osteochondral defects and the sustained expression of the transgene in vivo. This method may be used to define the effects of genes involved in cartilage repair and may provide alternative treatments for articular cartilage defects.

Alginates↗