ARTHRITIS & RHEUMATISM Vol. 62, No. 4, April 2010, pp 1088–1096 DOI 10.1002/art.27328 © 2010, American College of Rheumatology Estradiol Inhibits Chondrogenic Differentiation of Mesenchymal Stem Cells via Nonclassic Signaling Zsuzsa Jenei-Lanzl, 1 Rainer H. Straub, 1 Thomas Dienstknecht, 1 Marion Huber, 1 Markus Hager, 2 Susanne Gr¨ assel, 3 Richard Kujat, 1 Martin K. Angele, 4 Michael Nerlich, 1 and Peter Angele 1 Objective. We undertook this study to examine the effects of estradiol on chondrogenesis of human bone marrow–derived mesenchymal stem cells (MSCs), with consideration of sex-dependent differences in cartilage repair. Methods. Bone marrow was obtained from the iliac crest of young men. Density-gradient centrifugation–separated human MSCs proliferated as a monolayer in serum-containing medium. After conflu- ence was achieved, aggregates were created and cultured in a serum-free differentiation medium. We added dif- ferent concentrations of 17-estradiol (E2) with or without the specific estrogen receptor inhibitor ICI 182.780, membrane-impermeable E2–bovine serum al- bumin (E2-BSA), ICI 182.780 alone, G-1 (an agonist of G protein–coupled receptor 30 [GPR-30]), and G15 (a GPR-30 antagonist). After 21 days, the aggregates were analyzed histologically and immunohistochemically; we quantified synthesized type II collagen, DNA content, sulfated glycosaminoglycan (sGAG) concentrations, and type X collagen and matrix metalloproteinase 13 (MMP-13) expression. Results. The existence of intracellular and membrane-associated E2 receptors was shown at vari- ous stages of chondrogenesis. Smaller aggregates and significantly lower type II collagen and sGAG content were detected after treatment with E2 and E2-BSA in a dose-dependent manner. Furthermore, E2 enhanced type X collagen and MMP-13 expression. Compared with estradiol alone, the coincubation of ICI 182.780 with estradiol enhanced suppression of chondrogenesis. Treatment with specific GPR-30 agonists alone (G-1 and ICI 182.780) resulted in a considerable inhibition of chondrogenesis. In addition, we found an enhancement of hypertrophy by G-1. Furthermore, the specific GPR-30 antagonist G15 reversed the GPR-30–mediated inhibition of chondrogenesis and up-regulation of hy- pertrophic gene expression. Conclusion. The experiments revealed a suppres- sion of chondrogenesis by estradiol via membrane re- ceptors (GPR-30). The study opens new perspectives for influencing chondrogenesis on the basis of classic and nonclassic estradiol signaling. Due to its limited capacity for spontaneous re- generation, articular cartilage damage is irreversible. The loss of this tissue in the joint caused by disease or trauma leads to clinical consequences such as osteoar- thritis (OA), which induces pain, limits mobility, and contributes to high economic costs (1). Over the last several years, cell-based tissue engineering has turned out to be an attractive regenerative therapeutic ap- proach for the repair of chondral defects (2–5) and for the prevention of trauma-dependent OA. Articular car- tilage is a sex hormone–sensitive tissue. Estrogens espe- cially have been shown to affect cartilage under physio- logic and pathologic conditions. Several studies have examined the role of estrogen in the regulation of the growth plate. Estrogen is crucial for normal longitudinal Supported by the DFG (Research Unit FOR696, project AN 309/2-1). 1 Zsuzsa Jenei-Lanzl, MS, Rainer H. Straub, MD, Thomas Dienstknecht, MD, Marion Huber, Richard Kujat, PhD, Michael Nerlich, MD, Peter Angele, MD: University Medical Center Regens- burg, Regensburg, Germany; 2 Markus Hager, MS: University of Regensburg, Regensburg, Germany; 3 Susanne Gra ¨ssel, PhD: Univer- sity Hospital Regensburg, Bad Abbach, Germany; 4 Martin K. Angele, MD: Ludwig Maximilians University, Munich, Germany. Dr. P. Angele has received consulting fees from B. Braun Aesculap (less than $10,000) and holds two patents (US and German) for biomaterials for chondrogenic regeneration. Address correspondence and reprint requests to Peter An- gele, MD, Department of Trauma Surgery, University Medical Center Regensburg, Franz-Josef-Strauss Allee 11, 93042 Regensburg, Ger- many. E-mail: Peter.Angele@klinik.uni-regensburg.de. Submitted for publication March 27, 2009; accepted in revised form December 28, 2009. 1088