gms | German Medical Science

Deutscher Kongress für Orthopädie und Unfallchirurgie (DKOU 2017)

24.10. - 27.10.2017, Berlin

Overexpression of human DKK1 via rAAV stimulates the chondrogenic differentiation processes

Meeting Abstract

  • presenting/speaker Jagadeesh K. Venkatesan - 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
  • Janina Frisch - 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 2017). Berlin, 24.-27.10.2017. Düsseldorf: German Medical Science GMS Publishing House; 2017. DocIN27-518

doi: 10.3205/17dkou064, urn:nbn:de:0183-17dkou0646

Veröffentlicht: 23. Oktober 2017

© 2017 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: Injured adult articular cartilage does not regenerate its original structure and functions. Genetic modification of mesenchymal stem cells (MSCs) is a potential strategy to enhance the chondroreparative activities. Here, we tested the delivery of a Dickkopf-related protein 1 (DKK1)via rAAV to human MSCs to stimulate cell chondrogenic differentiation processes.

Methods: rAAV-lacZ carries the E. coli beta-galactosidase ( β-gal) marker gene (lacZ) and rAAV-DKK1 a human DKK1 sequence, both controlled by the CMV-IE promoter/enhancer. Vectors were packaged, purified, and titrated as previously described. Bone marrow aspirates were obtained from the distal femurs of donors undergoing total knee arthroplasty (n = 8). Human mesenchymal stem cells (hMSCs) were prepared as previously described.Cells were transduced with rAAV vectors both in monolayer culture (20 μ l vector) and in high-density aggregate cultures (40  μ l) and maintained for up to 21 days in chondrogenic medium. Transgene (DKK1) expression was monitored by immunocyto-/histochemistry on fixed monolayer cultures and on paraffin-embedded sections of aggregates (5μm),respectively. Sections from aggregates were also stained with toluidine blue and to detect type-II, -I, and -X collagen.The DNA, proteoglycan, and type-II collagen contents were monitored by Hoechst 22358 assay, binding to DMMB, and ELISA, respectively. Total RNA was extracted using the RNeasy Protect Mini kit (Qiagen) and reverse transcription was carried out with the 1st Strand cDNA Synthesis kit (AMV) (Roche). cDNA was amplified by SYBR Green real-time PCR for SOX9,type-II collagen (COL2A1), type-I collagen (COL1A1), type-X collagen (COL10A1)and GAPDH in control. Data were calculated using the 2- Δ Ct method. Each condition was performed in two independent experiments.

Results and Conclusion: Sustained, effective DKK1 expression was noted in rAAV-DKK1-transduced hMSCs over time both in monolayer and aggregate cultures relative to control (rAAV-lacZ) treatment. Successful chondrogenic differentiation was achieved in rAAV-DKK1-transduced hMSC aggregates after day 21 as seen by more intense toluidine blue staining and type-II collagen immunostaining compared with control transduction. Concordant with that, the proteoglycan and type-II collagen contents were more elevated using the DKK1 vector. In contrast, reactivity to type-I and type-X collagen was less intense with rAAV-DKK1. These findings were corroborated by the results of a real-time RT-PCR analysis showing up to 2.8- and 2-fold increases in SOX9 and COL2A1 expression and up to 3.3- and 10-fold decreases in COL1A1 and COL10A1 expression with DKK1 relative to control conditions. Finally, the DNA contents were higher with the DKK1 vector. DKK1 can be successfully overexpressed in hMSCs via rAAV vectors, leading to enhance chondrogenic cell differentiation over time.Gene-based modification of hMSCs via rAAV-DKK1 may find value in developing novel approaches to treat cartilage lesions.