gms | German Medical Science

Deutscher Kongress für Orthopädie und Unfallchirurgie (DKOU 2019)

22. - 25.10.2019, Berlin

Genetic modification of human bone marrow aspirates via rAAV-mediated overexpression of SOX9 and TGF-β upon vector delivery via pNaSS-coated poly(ε-caprolactone) scaffolds

Meeting Abstract

  • presenting/speaker Jagadeesh K. Venkatesan - Zentrum für Experimentelle Orthopädie, Lehrstuhl für Exp. Orthopädie und Arthroseforschung, Universitätsklinikum des Saarlandes, Homburg, Germany
  • Céline Falentin-Daudré - Université Paris 13-UMR CNRS 7244-CSPBAT-LBPS-UFR SMBH, Bobigny, France
  • Amélie Leroux - Université Paris 13-UMR CNRS 7244-CSPBAT-LBPS-UFR SMBH, Bobigny, France
  • Jean-Sébastien Baumann - Université Paris 13-UMR CNRS 7244-CSPBAT-LBPS-UFR SMBH, Bobigny, France
  • Veronique Migonney - Université Paris 13-UMR CNRS 7244-CSPBAT-LBPS-UFR SMBH, Bobigny, France
  • Magali Cucchiarini - Zentrum für Experimentelle Orthopädie, Lehrstuhl für Exp. Orthopädie und Arthroseforschung, Universitätsklinikum des Saarlandes, Homburg, Germany

Deutscher Kongress für Orthopädie und Unfallchirurgie (DKOU 2019). Berlin, 22.-25.10.2019. Düsseldorf: German Medical Science GMS Publishing House; 2019. DocAB34-743

doi: 10.3205/19dkou230, urn:nbn:de:0183-19dkou2307

Veröffentlicht: 22. Oktober 2019

© 2019 Venkatesan 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: Articular cartilage supports joint loading and motion but has a limited ability for self-healing. Gene transfer using the clinically relevant recombinant adeno-associated viral (rAAV) vectors is a powerful tool to enhance cartilage repair. Here, we tested the ability of poly(sodium styrene sulfonate) coated poly(Ε-caprolactone) scaffolds to deliver chondrogenic rAAV SOX9 and TFG-β vectors to human bone marrow aspirates, a potential source of reparative cells for cartilage repair.

Methods: The scaffolds were fabricated using a spin-coating method. The following conditions were tested: no coating and high coating (1.30 x 10-5 mol/g pNaSS).rAAV-FLAG-hsox9 carries a human SOX9 sequence and rAAV-hTGF-β human transforming growth factor-beta 1, both controlled by the CMV-IE promoter/enhancer. Immobilization of rAAV on the scaffolds was performed by 0.002% poly-L-lysine. Bone marrow aspirates were obtained from the distal femurs of donors undergoing total knee arthroplasty (n = 10). Aspirates (150 μ l) were placed on rAAV-coated scaffolds with a mixture of fibrinogen (17 mg/ml)/thrombin (5 U/ml) (Baxter, Volketswil, Switzerland). The constructs were maintained in DMEM, 10% fetal bovine serum, 100 U/ml penicillin, 100 μ g/ml streptomycin at 37ºC for 21 days. Histological and immunohistochemical analyses were performed on paraffin-embedded sections of the constructs (5 μ m) (toluidine blue staining; anti-TFG-β, anti-SOX9 and anti-type-II/-I/-X collagen immunostaining). Total RNA (Qiagen) was extracted and reverse transcription was carried out with the 1st Strand cDNA Synthesis kit (Qiagen). cDNA amplification was performed via SYBR Green real-time RT-PCR. Ct values were obtained for each target gene and GAPDH as a control for normalization, and fold inductions (relative to untreated samples) were measured using the 2- Δ Δ Ct method. 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: Successful transgene (SOX9 and TGF- β ) expression was specifically noted in human bone marrow aspirates incubated with rAAV SOX9- and TGF-β-treated pNaSS-coated PCL scaffolds over time relative to control condition (no vector treatment), regardless of the level of pNaSS coating. All systems underwent effective chondrogenic differentiation as seen by intense toluidine blue staining, while scaffold-mediated overexpression of SOX9 and of TGF-β further led to increased levels of type-II collagen immunostaining compared with control conditions. Interestingly, type-I and -X collagen deposition was less intense when rAAV SOX9 and rAAV TGF-β were provided to the aspirates via the scaffolds. These findings were corroborated by the results of a real-time RT-PCR analysis: Genetic delivery of therapeutic SOX9 and TGF-β rAAV vectors coated on pNaSS-grafted PCL scaffolds enhances the chondrogenic differentiation activities in human bone marrow aspirates versus control conditions.