gms | German Medical Science

Deutscher Kongress für Orthopädie und Unfallchirurgie (DKOU 2015)

20.10. - 23.10.2015, Berlin

Influence of IGF-I overexpression via rAAV gene transfer upon the chondrogenic differentiation potential of human bone marrow aspirates

Meeting Abstract

  • presenting/speaker Janina Frisch - Zentrum für Experimentelle Orthopädie, Lehrstuhl für Exp. Orthopädie und Arthroseforschung, Universitätsklinikum des Saarlandes, Homburg, Germany
  • Ana Rey Rico - Zentrum für Experimentelle Orthopädie, Lehrstuhl für Exp. Orthopädie und Arthroseforschung, Universitätsklinikum des Saarlandes, Homburg, Germany
  • Jagadeesh K. Venkatesan - Zentrum für Experimentelle Orthopädie, Lehrstuhl für Exp. Orthopädie und Arthroseforschung, Universitätsklinikum des Saarlandes, Homburg, Germany
  • Gertrud Schmitt - Zentrum für Experimentelle Orthopädie, Lehrstuhl für Exp. Orthopädie und Arthroseforschung, Universitätsklinikum des Saarlandes, Homburg, Germany
  • Henning Madry - Zentrum für Experimentelle Orthopädie, Lehrstuhl für Exp. Orthopädie und Arthroseforschung, Universitätsklinikum des Saarlandes, Homburg, Germany
  • Magali Cucchiarini Madry - Zentrum für Experimentelle Orthopädie, Lehrstuhl für Exp. Orthopädie und Arthroseforschung, Universitätsklinikum des Saarlandes, Homburg, Germany

Deutscher Kongress für Orthopädie und Unfallchirurgie (DKOU 2015). Berlin, 20.-23.10.2015. Düsseldorf: German Medical Science GMS Publishing House; 2015. DocGR22-82

doi: 10.3205/15dkou536, urn:nbn:de:0183-15dkou5368

Veröffentlicht: 5. Oktober 2015

© 2015 Frisch et al.
Dieser Artikel ist ein Open-Access-Artikel und steht unter den Lizenzbedingungen der Creative Commons Attribution 4.0 License (Namensnennung). Lizenz-Angaben siehe http://creativecommons.org/licenses/by/4.0/.


Gliederung

Text

Objectives: Implantation of bone marrow aspirates modified by recombinant adeno-associated viral (rAAV) vectors is a promising approach to improve the healing of articular cartilage defects. In the present study, we investigated the effects of prolonged overexpression of the mitogenic and pro-anabolic insulin-like growth factor I (IGF-I) via rAAV gene transfer on the biological activities of human bone marrow aspirates.

Methods: All transgenes were under the control of the CMV-IE promoter/enhancer: rAAV-lacZ carries the E. coli β-galactosidase gene (lacZ) and rAAV-hIGF-I a human insulin-like growth factor I (hIGF-I) cDNA fragment. Bone marrow aspirates were obtained from the distal femurs of patients undergoing total knee arthroplasty, aliquoted, and immediately transduced with rAAV vectors (40 µl) in chondrogenic medium for up to 21 days. Histological sections of aspirates were processed for IGF-I immunohistochemical analysis. The DNA and proteoglycan contents were determined with a fluorimetric assay using Hoechst 22358 and by binding to the dimethylmethylene blue dye, respectively. The chondrogenic differentiation processes in the aspirates were monitored by histological (toluidine blue staining) and immunohistochemical analyses (type-II collagen immunodetection). Real-time RT-PCR was performed to analyze the expression of chondrogenic (SOX9, COL2A1, ACAN) and hypertrophic (COL1A1, COL10A1) markers. Each condition was performed in duplicate in three independent experiments. The t-test was used where appropriate with P ≤ 0.05 considered statistically significant.

Results and Conclusion: Results:

Successful transgene (IGF-I) expression was achieved as noted by the higher IGF-I deposition in the IGF-I-treated samples compared with the control (lacZ) condition. Application of rAAV-hIGF-I enhanced the DNA and proteoglycan contents in the samples relative to lacZ. Enhanced chondrogenic differentiation was noted in the IGF-I-treated aspirates compared with lacZ, as noted by more robust toluidine blue staining and higher type-II collagen deposition on day 21 after gene delivery. These findings were confirmed by the results of a real-time RT-PCR analysis, showing an increase in the expression of chondrogenic markers upon rAAV-hIGF-I treatment (SOX9, COL2A1, ACAN). Of further note, the profiles of hypertrophic markers (COL1A1, COL10A1) were also increased upon IGF-I transduction.

Conclusion:

Overexpression of rAAV-hIGF-I via rAAV gene transfer improves the metabolic activities and chondrogenic differentiation processes in human bone marrow aspirates compared with control conditions. A tight regulation of the IGF-I production levels in these samples is further required to control premature terminal differentiation and hypertrophy. Direct, convenient gene-based modification of bone marrow aspirates via rAAV is a promising approach to develop novel treatments for articular cartilage lesions.