Chondrogenic Differentiation Capacity of Human Mesenchymal Progenitor Cells Derived from Subchondral Cortico-Spongious Bone Katja Neumann, 1 Tilo Dehne, 2 Michaela Endres, 1,2 Christoph Erggelet, 3 Christian Kaps, 1,2 Jochen Ringe, 2,4 Michael Sittinger 2,4 1 TransTissue Technologies GmbH, Tucholskystrasse 2, 10117 Berlin, Germany, 2 Charite ´ –Universita ¨tsmedizin Berlin, Department of Rheumatology and Clinical Immunology, Laboratory for Tissue Engineering, Tucholskystrasse 2, 10117 Berlin, Germany, 3 Department of Orthopedic Surgery and Traumatology, University of Freiburg, Freiburg, Germany, 4 Berlin–Brandenburg Center for Regenerative Therapies, Charite ´ –Universita ¨tsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany Received 16 November 2007; accepted 21 December 2007 Published online 7 May 2008 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jor.20635 ABSTRACT: Microfracture is frequently used to repair articular cartilage defects and allows mesenchymal progenitors to migrate from subchondral bone into the defect and form cartilaginous repair tissue. The aim of our study was to analyze the cell surface antigen pattern and the differentiation capacity of cells derived from human subchondral bone. Human progenitor cells were derived from subchondral cortico- spongious bone and grown in the presence of human serum. Stem cell-related cell surface antigens were analyzed by flowcytometry. Cortico- spongious progenitor (CSP) cells showed presence of CD73, CD90, CD105, and STRO-1. Multilineage differentiation potential of CSP cells was documented by histological staining and by gene expression analysis of osteogenic, adipogenic, and chondrogenic marker genes. CSP cells formed a mineralized matrix as demonstrated by von Kossa staining and showed induction of osteocalcin, independent of osteogenic stimulation. During adipogenic differentiation, the adipogenic marker genes fatty acid binding protein 4 and peroxisome proliferative activated receptor g were induced. Immunohistochemical staining of cartilage-specific type II collagen and induction of the chondrocytic marker genes cartilage oligomeric matrix protein, aggrecan, and types II and IX collagen confirmed TGFb3-mediated chondrogenic lineage development. CSP cells from subchondral bone, as known from microfracture, are multipotent stem cell-like mesenchymal progenitors with a high chondrogenic differentiation potential. ß 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1449– 1456, 2008 Keywords: microfracture; stem cells; cartilage repair; chondrogenesis Injuries of the articular cartilage of the knee are common and were documented in about 60% of the patients subjected to knee arthroscopies. 1,2 These cartilage lesions are predominantly classified as focal chondral or osteochondral lesions (67%) and osteo- arthritic changes (29%). The majority of defects (70%) are nonisolated cartilage lesions. 3 Since injured arti- cular cartilage does not heal and has only a low regenerative capacity, reparative surgical therapy options aim at the reconstruction of the articular surface and formation of adequate cartilage repair tissue. In particular, techniques that stimulate the bone marrow like Pridie-drilling, abrasion, or the micro- fracture technique are frequently used for the treat- ment of focal cartilage defects. 4–6 These techniques have in common that mesenchymal progenitor cells from the subchondral spongious bone marrow are allowed to populate the defect and subsequently form a cartilaginous repair tissue. For instance, the mini- mally invasive microfracture technique induces the healing sequence by directly accessing the subchondral bone marrow within the defect site. Arthroscopically, the cartilage lesion is subjected to debridement, while damage to the subchondral bone plate is avoided and a firmly attached healthy cartilage rim is prepared. Access to the bone marrow is established by introducing multiple, evenly distributed perforations across the defect and adjacent to the cartilage rim. Bleeding is observed and blood-derived cells and bone marrow- derived mesenchymal stem and progenitor cells are flushed into the defect, forming a blood clot. It is suggested that growth factors from the subchondral bone or even from the synovial fluid may stimulate these progenitors to form a cartilaginous repair tissue of a nonhyaline appearance, filling the defect. 7–9 Clinically, the outcome after microfracture treatment of full- thickness cartilage defects showed good and satisfactory short-term results in up to 77% of patients. 10–12 Patients showed significant clinical improvement, 2 and 5 years after microfracture, compared to the preoperative situation as assessed by the Lysholm, Tegner and SF-36 physical component score. 10,11 How- ever, in a group of 85 patients with full-thickness cartilage defects, it has been shown that microfracture treatment significantly improved the clinical situation as assessed by the International Cartilage Repair Society (ICRS) score at 18 months. In the long-term, at 36 months, the ICRS score was significantly higher compared to the preoperative situation, but was significantly decreased compared to the 18 months follow-up scores. 12 Therefore, microfracture treatment shows good short-term results, but clinical results may become impaired in the long-term. From the cellular point of view, mesenchymal pro- genitor or stem cells from bone marrow, e.g. isolated from iliac crest, have a multipotential differentiation capacity that allows development along the chondrogenic, osteo- genic, and adipogenic lineage. 13 These cells can be isolated, extensively grown in vitro while maintaining JOURNAL OF ORTHOPAEDIC RESEARCH NOVEMBER 2008 1449 Correspondence to: Christian Kaps (T: þ49 30 450 513 293; F: þ49 30 450 513 957; E-mail: christian.kaps@transtissue.com) ß 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.