gms | German Medical Science

Deutscher Kongress für Orthopädie und Unfallchirurgie (DKOU 2017)

24.10. - 27.10.2017, Berlin

Effective genetic modification of human articular chondrocytes via rAAV vectors in cartilage tissue analogs for cartilage engineering

Meeting Abstract

  • presenting/speaker Oliver Müller - Zentrum für Experimentelle Orthopädie, Universität des Saarlandes, Medizinische Fakultät Homburg, Homburg, Germany
  • Janina Frisch - Zentrum für Experimentelle Orthopädie, Universität des Saarlandes, Medizinische Fakultät Homburg, Homburg, Germany
  • Ana Rey Rico - Zentrum für Experimentelle Orthopädie, Universität des Saarlandes, Medizinische Fakultät Homburg, Homburg, Germany
  • Jagadeesh Venkatesan - Zentrum für Experimentelle Orthopädie, Universität des Saarlandes, Medizinische Fakultät Homburg, Homburg, Germany
  • Gertrud Schmitt - Zentrum für Experimentelle Orthopädie, Universität des Saarlandes, Medizinische Fakultät Homburg, Homburg, Germany
  • Henning Madry - Zentrum für Experimentelle Orthopädie, Universität des Saarlandes, Medizinische Fakultät Homburg, Homburg, Germany
  • George R. Dodge - Perelman School of Medicine, University of Pennsylvania, Translational Musculoskeletal Research Center, Philadelphia PA, United States
  • Magali Cucchiarini - Zentrum für Experimentelle Orthopädie, Universität des Saarlandes, Medizinische Fakultät Homburg, Homburg, Germany

Deutscher Kongress für Orthopädie und Unfallchirurgie (DKOU 2017). Berlin, 24.-27.10.2017. Düsseldorf: German Medical Science GMS Publishing House; 2017. DocPO29-331

doi: 10.3205/17dkou872, urn:nbn:de:0183-17dkou8720

Veröffentlicht: 23. Oktober 2017

© 2017 Müller 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: Repairing articular cartilage remain an illusive goal and is the subject of many investigations using tissue engineering platforms and biologics. Cartilage tissue analogs (CTAs) provide attractive high cell density, scaffold-free system to engineer articular cartilage with unique features of phenotype maintenance and cartilage like functional properties. Here, we examined the feasibility of generating CTAs using genetically modifying primary human chondrocytes with recombinant adeno-associated viral vectors (rAAV) and characterized the cartilage biomass formed.

Methods: Human articular chondrocytes were isolated the femoral head of the knee joint, washed in basal medium (DMEM), and kept in DMEM, 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin (growth medium). Cells were isolated by digestion in basal medium using collagenase for 36 h at 37°C. A 70µm cell strainer was used to isolate chondrocytes alongside with washing and centrifugation at 1,500 rpm (3x5 min each). rAAV vectors were packaged, purified, and titrated as describedin previous publications. rAAV-lacZ carries the E. coli beta-galactosidase gene (lacZ) and rAAV-RFP the Discosoma sp. red fluorescent protein gene (RFP), both controlled by the CMV-IE promoter/enhancer. Freshly isolated chondrocytes were transduced with rAAV-lacZ or rAAV-RFP (100 µl) and placed in growth medium at 37°C for 90 min. To create the CTAs, transduced cells were resuspended in complete medium (growth medium, 2.5 µg/ml fungizone, 1% MEM vitamin solution, 25 mM HEPES buffer, 50 µg/ml ascorbic acid) and plated at 106 cells/well in polyHEMA-coated 96-well plates (Ultra-Low Adhesion, Corning).After the formation of CTAs medium changes were performed once per week. Transgene expression was monitored by X-Gal staining (lacZ) and live fluorescence microscopy (RFP).

Results and Conclusion: Formation CTAs resulting from chondrocyte self-aggregation was evidenced after 12 h when cells began to coalesce, with a steady layer forming between days 5 and 7 and lasting over the whole course of the experiment (Figure 1 [Fig. 1]).

Effective, homogeneous, and sustained rAAV-mediated transgene expression was observed in rAAV-lacZ-transduced human CTAs compared with the control condition for at least 30 days.

Similar results were noted when applying rAAV-RFP, with an intense, prolonged fluorescent signal at least 60 days post-transduction relative to the control conditions. Using cell based cartilage repair approaches implantation of therapeutic rAAV-treated CTAs may offer workable, effective tool to treat articular cartilage lesions in patients and may offer a less complicated approach over those that employ foreign biocompatible materials.