gms | German Medical Science

German Congress of Orthopedic and Trauma Surgery (DKOU 2017)

24.10. - 27.10.2017, Berlin

Attenuation of human MSC hypertrophy in 3D culture via treatment with a retinoic acid receptor inverse agonist

Meeting Abstract

  • presenting/speaker Moritz Riedl - Universitätsklinikum Regensburg, Klinik und Poliklinik für Unfallchirurgie, Regensburg, Germany
  • Girish Pattappa - Universitätsklinikum Regensburg, Regensburg, Germany
  • Michael Nerlich - Universitätsklinikum Regensburg, Klinik und Poliklinik für Unfallchirurgie, Regensburg, Germany
  • Peter Angele - Universitätsklinikum Regensburg, Laboratory for Experimental trauma surgery, Department of Trauma surgery, Regensburg, Germany
  • Michael Müller - Universitätsklinikum Regensburg, Klinik und Poliklinik für Unfallchirurgie, Regensburg, Germany
  • Matthias Koch - Universitätsklinikum Regensburg, Klinik und Poliklinik für Unfallchirurgie, Regensburg, Germany
  • Denitsa Docheva - Universitätsklinikum Regensburg, Regensburg, Germany
  • Christian Pfeifer - Universitätsklinikum Regensburg, Klinik und Poliklinik für Unfallchirurgie, Regensburg, Germany

Deutscher Kongress für Orthopädie und Unfallchirurgie (DKOU 2017). Berlin, 24.-27.10.2017. Düsseldorf: German Medical Science GMS Publishing House; 2017. DocPO12-594

doi: 10.3205/17dkou625, urn:nbn:de:0183-17dkou6259

Published: October 23, 2017

© 2017 Riedl et al.
This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 License. See license information at http://creativecommons.org/licenses/by/4.0/.


Outline

Text

Objectives: Human Mesenchymal stem cells (hMSCs) are a promising source for articular cartilage repair. Unfortunately in vitro chondrogenically differentiated hMSCs tend to undergo hypertrophy such as the transient 'chondrocytes' in the growth plate. Retinoic acid (RA) signalling plays a key role in growth plate hypertrophy. Whilst RA agonists block chondrogenesis and foster hypertrophy during later stages, RAR inverse agonists (IA) enhance chondrogenesis when applied early in culture. Therefore, we hypothesized that treatment of chondrogenically differentiated hMSCs with RAR IA, will attenuate hypertrophy during the hypertrophic stage of chondrogenesis.

Methods: Pellets of passage 2 hMSCs were formed in V-bottom well plates by centrifugation and pre-differentiated in a chemically defined medium containing 10ng/mL TGF? (CM+) for 14 days. Thereafter, pellets were cultured for an additional 14 days under 6 conditions: CM+, CM- (w/out TGF?), and hypertrophic medium (CM- with 25 ng/ml BMP 4, w/out dexamethasone). Each of these first three conditions was additionally supplemented with the RA receptor (RAR) inverse agonist BMS493 (BMS) at 2 μM after 14 days of chondrogenic pre- differentiation. One additional BMP4 group was supplemented with BMS from the beginning of chondrogenic differentiation until day 14 (CM+BMS/BMP4). On 28, pellets were assessed for gene expression (Col 2, Col 10, Col 1 and MMP13). Cartilage matrix and phenotype was assessed histologically using dimethyl methylene blue (DMMB), alkaline phosphatase staining (ALP) and collagen II and X immunohistochemistry.

Results and Conclusion: CM+/BMP4 hMSCs showed a hypertrophic phenotype with increased cell volume, collagen X content and ALP activity compared to CM+/CM+. Hypertrophy was reduced by addition of BMS at day 14 (CM+/BMP4BMS) and further reduced by addition from the beginning (CM+BMS/BMP4). BMS treatment resulted in smaller cells under hypertrophic conditions, higher collagen II content in chondrogenic groups (CM+/CM+BMS) and reduction in collagen X production and ALP activity in every condition. Gene expression data for hypertrophic markers, collagen X and MMP13, were upregulated under the influence of BMP4 but MMP13 expression was down regulated upon addition of BMS during the late stage differentiation (CM+/BMP4BMS) and further reduced upon addition during early stage chondrogenesis (CM+BMS/BMP4). Furthermore, Collagen X expression was reduced by early BMS treatment.

Chondrogenically differentiated hMSCs show hypertrophic conversion but the inclusion of the RAR IA, BMS, attenuated these hypertrophic changes as demonstrated by histology, immunohistochemistry and PCR. These findings suggest an additional approach to attenuate hypertrophy in chondrogenically differentiated hMSCs. Current studies are exploring the timing and dose of BMS to most efficaciously prevent hypertrophy. Furthermore, we are investigating the pathways through which BMS493 exerts its effects and will translate these findings in vivo.