gms | German Medical Science

Deutscher Kongress für Orthopädie und Unfallchirurgie (DKOU 2015)

20.10. - 23.10.2015, Berlin

rAAV-based genetic modification of human bone marrow aspirates seeded in 3D woven poly(epsilon-caprolactone) scaffolds

Meeting Abstract

  • presenting/speaker Magali Cucchiarini Madry - Zentrum für Experimentelle Orthopädie, Lehrstuhl für Exp. Orthopädie und Arthroseforschung, Universitätsklinikum des Saarlandes, Homburg, Germany
  • 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
  • Franklin Moutos - Department of Orthopaedic Surgery, Duke University Medical Center, Durham, United States
  • Farshid Guilak - Department of Orthopaedic Surgery, Duke University Medical Center, Durham, United States
  • Henning 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. DocGR16-81

doi: 10.3205/15dkou483, urn:nbn:de:0183-15dkou4838

Veröffentlicht: 5. Oktober 2015

© 2015 Cucchiarini Madry 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: Bone marrow aspirates are attractive platforms to deliver candidate genes to enhance the repair of cartilage defects. Yet, aspirates may not have a sufficient integrity to support joint loading in a defect repair site. Here, we tested the ability of rAAV vectors to modify human bone marrow aspirates seeded in biomechanically functional 3D woven poly(epsilon-caprolactone) (PCL) scaffolds.

Methods: rAAV-lacZ carries the E. coli beta-galactosidase gene (lacZ) and rAAV-RFP the Discosoma sp. red fluorescent protein gene (RFP). Bone marrow aspirates were obtained from the distal femurs of donors undergoing total knee arthroplasty and mixed with the vectors for application of the samples to woven PCL scaffolds using growth cultivation medium for 75 days. Transgene expression was monitored by X-Gal staining and detection of live fluorescence. Paraffin-embedded histological sections of the constructs were prepared for toluidine blue staining and detection of type-II collagen immunoreactivity. The DNA, proteoglycan, and type-II collagen contents were monitored by Hoechst 22358 assay, binding to DMMB, and ELISA, respectively. Total RNA was extracted and reverse transcription was carried out for cDNA amplification via SYBR Green real-time RT-PCR. Ct values were obtained for each target gene using GAPDH as a control for normalization and fold inductions (relative to untreated samples) were measured using the 2-DeltaDeltaCt method. Each condition was performed in duplicate in three independent experiments. The t-test was employed with P below 0.05 considered statistically significant.

Results and Conclusion: Intense lacZ expression was noted in the rAAV-lacZ constructs versus rAAV-RFP, already 4 days after vector application with a uniform seeding of the aspirates in the scaffolds, and for a prolonged period of time (at least 75 days). Efficient, sustained RFP expression was observed in the rAAV-RFP constructs compared with rAAV-lacZ for also at least 75 days. The constructs showed a high stability over time. Marked toluidine blue staining and type-II collagen deposition were noted on day 75, as signs of effective and stable chondrogenic differentiation, without visible differences with the untreated samples. There was also no significant difference in the DNA, proteoglycan, and type-II collagen contents and gene expression profiles at this late time point between rAAV-transduced and untreated samples.

rAAV can successfully and persistently modify human bone marrow aspirates in woven PCL scaffolds, providing a functional biomechanical environment for stable maintenance in 3D without impairing the chondrogenic differentiation processes. Direct therapeutic gene-based modification of bone marrow aspirates via rAAV in woven PCL scaffolds may have value in developing convenient, stable treatments for articular cartilage lesions.