gms | German Medical Science

German Congress of Orthopedic and Trauma Surgery (DKOU 2018)

23.10. - 26.10.2018, Berlin

rAAV SOX9 gene transfer stimulates the chondrogenic differentiation activities in human peripheral blood aspirates

Meeting Abstract

  • presenting/speaker Jagadeesh K. Venkatesan - Zentrum für Experimentelle Orthopädie, Lehrstuhl für Exp. Orthopädie und Arthroseforschung, Universitätsklinikum des Saarlandes, Homburg, Germany
  • Patrick Orth - 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
  • 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 - Zentrum für Experimentelle Orthopädie, Lehrstuhl für Exp. Orthopädie und Arthroseforschung, Universitätsklinikum des Saarlandes, Homburg, Germany

Deutscher Kongress für Orthopädie und Unfallchirurgie (DKOU 2018). Berlin, 23.-26.10.2018. Düsseldorf: German Medical Science GMS Publishing House; 2018. DocPT19-429

doi: 10.3205/18dkou692, urn:nbn:de:0183-18dkou6925

Published: November 6, 2018

© 2018 Venkatesan et al.
This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 License. See license information at http://creativecommons.org/licenses/by/4.0/.


Outline

Text

Objectives: Implantation of genetically modified peripheral blood aspirates may be a promising approach to treat focal cartilage lesions. Here, we explored the beneficial effects of rAAV- SOX9 in human peripheral blood aspirates, allowing to durably enhance the chondrogenic differentiation activities in the samples.

Methods: rAAV-lacZ carries the E. coli -galactosidase (lacZ) gene and rAAV-FLAG-hsox9 a human FLAG-tagged sox9 sequence, both controlled by the CMV-IE promoter/enhancer. Peripheral blood (~ 3 ml) was collected in the presence of hirudin from human donors. Aspirates were immediately transduced with rAAV (40 l) or let untreated and kept in chondrogenic medium for up to 21 days. Histological and immunohistochemical analyses were performed on paraffin-embedded sections of the constructs (5 μ m) (toluidine blue staining; anti-SOX9 and anti-type-II/-I/-X collagen immunostaining). The proteoglycan contents in the aspirates were monitored by binding to dimethylmethylene blue dye and the DNA contents by Hoechst 33258 assay. Total RNA was extracted and reverse transcription carried out for cDNA amplification via real-time RT-PCR with GAPDH as control for normalization and the 2-Ct method relative to untreated samples. Each condition was performed in duplicate in three independent experiments. A t-test was employed with p ≤ 0.05 considered statistically significant.

Results and conclusion: Transgene (SOX9) expression was observed in rAAV SOX9-treated aspirates relative to control conditions over the period of evaluation. Enhanced chondrogenic differentiation was achieved in the aspirates transduced with rAAV SOX9 after 21 days as noted by stronger toluidine blue staining and type-II collagen immunostaining. Application of rAAV SOX9 significantly increased the proteoglycan contents in the aspirates relative to control treatments (1.8-fold) while no significant effects were noted on the DNA contents. Of further interest, immunoreactivity to type-I and -X collagen was less intense when rAAV SOX9 was provided to the aspirates. These findings were corroborated by results of a real-time RT-PCR analysis showing enhanced chondrogenic differentiation with SOX9 relative to the control treatments (up to 2- and 1.7-fold higher COL2A and ACN expression, respectively; p ≤ 0.001) and reduced hypertrophic differentiation (up to 2.7-fold lower COL1A1 and COL10A1 expression, respectively; p ≤ 0.001), probably resulting from increased levels of SOX9 expression (up to 5-fold difference; p ≤ 0.001). Genetic modification via rAAV-mediated SOX9 gene transfer allows to enhance the chondrogenic differentiation activities in human peripheral blood aspirates relative control conditions in vitro over time.These results show the potential of targeting human peripheral blood aspirates via therapeutic rAAV transduction as a novel, convenient tool to treat articular cartilage defects.