Remodeling of Human Osteochondral Defects via rAAV-Mediated Co-Overexpression of TGF-β and IGF-I from Implanted Human Bone Marrow-Derived Mesenchymal Stromal Cells
Abstract
:1. Introduction
2. Experimental Section
2.1. Study Design
2.2. Reagents
2.3. Cell and Osteochondral Defect Cultures
2.4. Plasmids and rAAV Vectors
2.5. rAAV-Mediated Gene Transfer
2.6. Transgene Expression
2.7. Histological and Immonohistochemical Analyses
2.8. Histomorphometric Analyses
2.9. Biochemical Analyses
2.10. Real-Time RT-PCR Analyses
2.11. Statistical Analysis
3. Results
3.1. Effective rAAV-Mediated Co-Overexpression of TGF-β and IGF-I in Human Osteochondral Defects Upon Implantation of Genetically Modified hMSC Aggregates
3.2. Biological and Chondrogenic Differentiation Activities in Human Osteochondral Defects Mediated by Co-Overexpression of TGF-β and IGF-I via rAAV in Implanted hMSC Aggregates
3.3. Influence of Co-Overexpression of TGF-β and IGF-I via rAAV Upon the Osteogenic and Hypertrophic Differentiation Activities in Human Osteochondral Defects Upon Implantation of Genetically Modified hMSC Aggregates
3.4. Real-Time RT-PCR Analyses in Human Osteochondral Defects Mediated by Co-Overexpression of TGF-β with IGF-I via rAAV in Implanted hMSC Aggregates
4. Discussion
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Assay | lacZ | TGF-β/lacZ | IGF-I/lacZ | TGF-β/IGF-I |
---|---|---|---|---|
TGF-β+ | 156 ± 74 | 333 ± 58 a,c | 158 ± 90 | 556 ± 70 a,b,c |
IGF-I+ | 53 ± 7 | 55 ± 8 | 71 ± 16 a,b | 73 ± 18 a,b |
Assay | lacZ | TGF-β/lacZ | IGF-I/lacZ | TGF-β/IGF-I | |
---|---|---|---|---|---|
TGF-β+ cells | OCD | 12 ± 2 | 77 ± 8 a,c | 12 ± 5 | 93 ± 1 a,b,c |
surrounding cartilage | 2 ± 1 | 62 ± 6 a,c | 3 ± 1 | 74 ± 6 a,b,c | |
IGF-I+ cells | OCD | 13 ± 3 | 18 ± 4 | 76 ± 3 a,b | 86 ± 2 a,b,c |
surrounding cartilage | 6 ± 2 | 15 ± 4 | 56 ± 3 a,b | 64 ± 5 a,b,c | |
Cell densities | OCD | 3467 ± 115 | 5767 ± 1156 a | 5608 ± 300 a | 11,308 ± 1556 a,b,c |
surrounding cartilage | 145 ± 21 | 238 ± 75 a,c | 168 ± 11 | 286 ± 80 a,b,c | |
Safranin O | OCD | 0.3 ± 0.4 | 2.1 ± 0.8a,c | 0.1 ± 0.4 | 2.4 ± 0.7 a,c |
surrounding cartilage | 1.3 ± 0.5 | 2.6 ± 1.0 a | 1.9 ± 0.6 a | 3.8 ± 0.5 a,b,c | |
Type-II collagen | OCD | 1.0 ± 0.8 | 3.0 ± 0.1 a,c | 2.3 ± 0.5 a | 3.8 ± 0.5 a,b,c |
surrounding cartilage | 0.5 ± 0.1 | 2.5 ± 0.6 a,c | 1.8 ± 0.5 a | 3.0 ± 0.8 a,c | |
Type-I collagen | OCD | 3.1 ± 0.8 | 1.5 ± 0.7 a,c | 3.0 ± 0.5 | 1.1 ± 0.4 a,c |
surrounding cartilage | 2.9 ± 0.6 | 2.0 ± 0.8 a | 2.6 ± 0.7 | 1.5 ± 0.5 a,c | |
Type-X collagen | OCD | 3.0 ± 0.8 | 1.9 ± 1.1 a | 2.3 ± 0.9 | 1.3 ± 0.5 a |
surrounding cartilage | 2.9 ± 0.6 | 2.6 ± 0.5 | 2.1 ± 1.0 | 0.8 ± 0.7 a,b,c |
Assay | lacZ | TGF-β/lacZ | IGF-I/lacZ | TGF-β/IGF-I |
---|---|---|---|---|
DNA | 0.39 ± 0.26 | 0.45 ± 0.30 | 0.39 ± 0.25 | 0.52 ± 0.32 a,b,c |
Proteoglycans | 12 ± 7 | 17 ± 12 a | 15 ± 8 | 22 ± 13 a,b,c |
Proteoglycans/DNA | 0.11 ± 0.05 | 0.13 ± 0.07 a | 0.12 ± 0.05 | 0.15 ± 0.07 a,b,c |
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Morscheid, S.; Venkatesan, J.K.; Rey-Rico, A.; Schmitt, G.; Cucchiarini, M. Remodeling of Human Osteochondral Defects via rAAV-Mediated Co-Overexpression of TGF-β and IGF-I from Implanted Human Bone Marrow-Derived Mesenchymal Stromal Cells. J. Clin. Med. 2019, 8, 1326. https://doi.org/10.3390/jcm8091326
Morscheid S, Venkatesan JK, Rey-Rico A, Schmitt G, Cucchiarini M. Remodeling of Human Osteochondral Defects via rAAV-Mediated Co-Overexpression of TGF-β and IGF-I from Implanted Human Bone Marrow-Derived Mesenchymal Stromal Cells. Journal of Clinical Medicine. 2019; 8(9):1326. https://doi.org/10.3390/jcm8091326
Chicago/Turabian StyleMorscheid, Stephanie, Jagadeesh Kumar Venkatesan, Ana Rey-Rico, Gertrud Schmitt, and Magali Cucchiarini. 2019. "Remodeling of Human Osteochondral Defects via rAAV-Mediated Co-Overexpression of TGF-β and IGF-I from Implanted Human Bone Marrow-Derived Mesenchymal Stromal Cells" Journal of Clinical Medicine 8, no. 9: 1326. https://doi.org/10.3390/jcm8091326