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Hypertrophy is induced during the in vitro chondrogenic differentiation of human mesenchymal stem cells by bone morphogenetic protein-2 and bone morphogenetic protein-4 gene transfer

Andre F Steinert12, Benedikt Proffen1, Manuela Kunz1, Christian Hendrich1, Steven C Ghivizzani23, Ulrich Nöth1, Axel Rethwilm4, Jochen Eulert1 and Christopher H Evans2*

Author Affiliations

1 Orthopaedic Center for Musculoskeletal Research, Orthopaedic Clinic, König-Ludwig-Haus, Julius-Maximilians-University, Brettreichstrasse 11, 97074 Würzburg, Germany

2 Center for Molecular Orthopaedics, Harvard Medical School, 221 Longwood Avenue, BLI 152, Boston, MA 02115, USA

3 Department of Orthopaedics and Rehabilitation, University of Florida, 3450 Hull Road, Gainesville, FL 32607, USA

4 Institut für Virologie und Immunbiologie, Julius-Maximilians-University, Versbacherstrasse 7, 97078 Würzburg, Germany

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Arthritis Research & Therapy 2009, 11:R148  doi:10.1186/ar2822

Published: 2 October 2009



The present study compares bone morphogenetic protein (BMP)-4 and BMP-2 gene transfer as agents of chondrogenesis and hypertrophy in human primary mesenchymal stem cells (MSCs) maintained as pellet cultures.


Adenoviral vectors carrying cDNA encoding human BMP-4 (Ad.BMP-4) were constructed by cre-lox combination and compared to previously generated adenoviral vectors for BMP-2 (Ad.BMP-2), green fluorescent protein (Ad.GFP), or firefly luciferase (Ad.Luc). Cultures of human bone-marrow derived MSCs were infected with 5 × 102 viral particles/cell of Ad.BMP-2, or Ad.BMP-4, seeded into aggregates and cultured for three weeks in a defined, serum-free medium. Untransduced cells or cultures transduced with marker genes served as controls. Expression of BMP-2 and BMP-4 was determined by ELISA, and aggregates were analyzed histologically, immunohistochemically, biochemically and by RT-PCR for chondrogenesis and hypertrophy.


Levels of BMP-2 and BMP-4 in the media were initially 30 to 60 ng/mL and declined thereafter. BMP-4 and BMP-2 genes were equipotent inducers of chondrogenesis in primary MSCs as judged by lacuna formation, strong staining for proteoglycans and collagen type II, increased levels of GAG synthesis, and expression of mRNAs associated with the chondrocyte phenotype. However, BMP-4 modified aggregates showed a lower tendency to progress towards hypertrophy, as judged by expression of alkaline phosphatase, annexin 5, immunohistochemical staining for type X collagen protein, and lacunar size.


BMP-2 and BMP-4 were equally effective in provoking chondrogenesis by primary human MSCs in pellet culture. However, chondrogenesis triggered by BMP-2 and BMP-4 gene transfer showed considerable evidence of hypertrophic differentiation, with, the cells resembling growth plate chondrocytes both morphologically and functionally. This suggests caution when using these candidate genes in cartilage repair.