Z Orthop Unfall 2020; 158(S 01): S206-S207
DOI: 10.1055/s-0040-1717523
Vortrag
DKOU20-974 Grundlagenforschung->29. Biomaterialien und Implantate

Overexpression of bone morphogenetic protein 3 (BMP-3) via rAAV-mediated gene transfer on the osteochondrogenic fate of human bone marrow-derived mesenchymal stem cells

JK Venkatesan
*   präsentierender Autor
1   Zentrum für Experimentelle Orthopädie, Lehrstuhl für Exp. Orthopädie und Arthroseforschung, Universitätsklinikum des Saarlandes, Homburg
,
G Schmitt
1   Zentrum für Experimentelle Orthopädie, Lehrstuhl für Exp. Orthopädie und Arthroseforschung, Universitätsklinikum des Saarlandes, Homburg
,
H Madry
1   Zentrum für Experimentelle Orthopädie, Lehrstuhl für Exp. Orthopädie und Arthroseforschung, Universitätsklinikum des Saarlandes, Homburg
,
M Cucchiarini
1   Zentrum für Experimentelle Orthopädie, Lehrstuhl für Exp. Orthopädie und Arthroseforschung, Universitätsklinikum des Saarlandes, Homburg
› Institutsangaben
 

Objectives Injured adult articular cartilage is not capable of reliably regenerating an original hyaline structure. To improve the intrinsic healing activities of damaged cartilage, administration of genetically modified bone marrow-derived mesenchymal stem cells (MSCs) in cartilage lesions is a potent approach to improve cartilage repair. Here, we explored the potential benefits of overexpressing the bone morphogenetic protein 3 (BMP-3) via clinically relevant recombinant adeno-associated virus (rAAV) vectors upon the commitment of human MSCs (hMSCs) as future therapeutic platforms for cartilage repair.

Methods rAAV-lacZ carries the E. coli β -galactosidase (lacZ) reporter gene and rAAV-hBMP-3 a human BMP-3 sequence (OriGene), both controlled by the CMV-IE promoter/enhancer. Bone marrow drived hMSCS cells were transduced with rAAV (rAAV-hBMP-3 or rAAV-lacZ: 40 μ l each vector) or let untreated and kept either in monolayer cultures in DMEM, 10% FBS or as high-density aggregate cultures (2 x 105 cells) in chondrogenic medium (DMEM high glucose, ITS+ Premix, 1 mM pyruvate, 37.5 μ g/ml ascorbate 2-phosphate, 10-7 M dexamethasone, 10 ng/ml TGF- β) for up to 21 days. Expression of BMP-3 was monitored by immunohistochemistry or by ELISA (R&D Systems). Histological analyses were performed on fixed monolayer cultures (ALP staining) or on paraffin-embedded sections of the aggregate cultures (5 μ m) (toluidine blue staining). Total RNA was extracted using the RNeasy Protect Mini kit (Qiagen) 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

(SOX9, COL10A1) and GAPDH as a control for normalization, and fold inductions (relative to control 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 BMP-3 overexpression was observed both in hMSC monolayer and aggregate cultures by application of rAAV-hBMP-3 relative to the control conditions (lack of vector delivery). Application of rAAV-hBMP-3 significantly increased the levels of cell proliferation in hMSC monolayer and aggregate cultures relative to the control conditions.

Chondrogenic differentiation was evidenced by intense toluidine blue staining in hMSC aggregate cultures after 21 days, especially when the cells were treated with rAAV-hBMP-3. Similar results were noted regarding the osteogenic differentiation of the cells over time. A real-time RT-PCR analysis revealed a reduced hypertrophic differentiation of the cells following rAAV-hBMP-3 transduction relative to the control treatments, probably due to increased chondrogenic SOX9 gene expression levels. These results shows modifying hMSCs by therapeutic BMP-3 gene transfer as a potential tool to enhance the repair processes in sites of osteochondral lesions.

Stichwörter BMP-3, rAAV, hMSCs



Publikationsverlauf

Artikel online veröffentlicht:
15. Oktober 2020

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