J. Venkatesan (Homburg, DE)
Saarland University Experimentelle OrthopädiePresenter Of 1 Presentation
P046 - Implantation of Genetically Modified Human Bone Marrow Aspirates by rAAV Vector via pNaSS-grafted PCL Film in Human Osteochondral Model
Abstract
Purpose
Gene transfer using recombinant adeno-associated viral (rAAV) vectors is a powerful tool to enhance cartilage repair. Here, we tested the delivering therapeutic (chondrogenic sox9 and TGF-β) rAAV vectors in reparative human bone marrow aspirates by using poly (sodium styrene sulfonate) (pNaSS)-grafted poly(ε-caprolactone) (PCL) films for implantation in a human osteochondral defect model as a potential scaffold-guided gene therapy option.
Methods and Materials
The PCL films were tested in the following conditions: absence of pNaSS grafting (PCL conditions) versus pNaSS grafting (1.3 x 10-5 mol/g pNaSS; pNaSS-PCL conditions). rAAV-lacZ carries the E. coli β-galactosidase (lacZ) reporter gene, rAAV-FLAG-hsox9 a human sox9 sequence, and rAAV-hTGF-β a human transforming growth factor beta 1 sequence, all controlled by the CMV-IE promoter/enhancer. Immobilization of rAAV on the films was performed by adding the vectors (40 µl) with 0.002% poly-L-lysine. 150 µl of human bone marrow aspirates (hBMAs) were placed on the films with fibrinogen/thrombin and implanted in 4-mm diameter of human osteochondral defects (hOCDs) and kept in chondrogenic medium for 21 days. The biochemical assays and real-time RT-PCR analysis were assessed after 21 days. T-test was employed with P ≤ 0.05 considered statistically significant.
Results
Treatment with rAAV sox9 and TGF-β enhanced type-II collagen deposition in hBMAs via PCL film-guided gene transfer by implantation in hOCDs (Fig. 1 and Tables 1 and 2). Similar results were noted when measuring the normalized type-II collagen contents and increased the normalized proteoglycan contents but only with pNaSS-grafted films ((Tables 1 and 2). These results were corroborated by real-time RT-PCR of the profiles for COL2A1 and ACAN expression (Fig. 2) and reduced premature expression of COL1A1 and COL10A1 (Fig. 2).
Conclusion
These results show the potential of pNaSS-grafted PCL film-guided therapeutic rAAV gene transfer in hBMAs as a novel healing platform for implantation in articular cartilage defects.
Presenter Of 1 Presentation
P046 - Implantation of Genetically Modified Human Bone Marrow Aspirates by rAAV Vector via pNaSS-grafted PCL Film in Human Osteochondral Model
Abstract
Purpose
Gene transfer using recombinant adeno-associated viral (rAAV) vectors is a powerful tool to enhance cartilage repair. Here, we tested the delivering therapeutic (chondrogenic sox9 and TGF-β) rAAV vectors in reparative human bone marrow aspirates by using poly (sodium styrene sulfonate) (pNaSS)-grafted poly(ε-caprolactone) (PCL) films for implantation in a human osteochondral defect model as a potential scaffold-guided gene therapy option.
Methods and Materials
The PCL films were tested in the following conditions: absence of pNaSS grafting (PCL conditions) versus pNaSS grafting (1.3 x 10-5 mol/g pNaSS; pNaSS-PCL conditions). rAAV-lacZ carries the E. coli β-galactosidase (lacZ) reporter gene, rAAV-FLAG-hsox9 a human sox9 sequence, and rAAV-hTGF-β a human transforming growth factor beta 1 sequence, all controlled by the CMV-IE promoter/enhancer. Immobilization of rAAV on the films was performed by adding the vectors (40 µl) with 0.002% poly-L-lysine. 150 µl of human bone marrow aspirates (hBMAs) were placed on the films with fibrinogen/thrombin and implanted in 4-mm diameter of human osteochondral defects (hOCDs) and kept in chondrogenic medium for 21 days. The biochemical assays and real-time RT-PCR analysis were assessed after 21 days. T-test was employed with P ≤ 0.05 considered statistically significant.
Results
Treatment with rAAV sox9 and TGF-β enhanced type-II collagen deposition in hBMAs via PCL film-guided gene transfer by implantation in hOCDs (Fig. 1 and Tables 1 and 2). Similar results were noted when measuring the normalized type-II collagen contents and increased the normalized proteoglycan contents but only with pNaSS-grafted films ((Tables 1 and 2). These results were corroborated by real-time RT-PCR of the profiles for COL2A1 and ACAN expression (Fig. 2) and reduced premature expression of COL1A1 and COL10A1 (Fig. 2).
Conclusion
These results show the potential of pNaSS-grafted PCL film-guided therapeutic rAAV gene transfer in hBMAs as a novel healing platform for implantation in articular cartilage defects.