Engineering bone-like tissue in vitro using human bone marrow stem cells and silk scaffolds Lorenz Meinel, 1,2,3 Vassilis Karageorgiou, 3 Sandra Hofmann, 3,4 Robert Fajardo, 5 Brian Snyder, 5 Chunmei Li, 3 Ludwig Zichner, 2 Robert Langer, 1 Gordana Vunjak-Novakovic, 1 David L. Kaplan 3 1 Division of Health Sciences and Technology, Massachusetts Institute of Technology, E25-330, 45 Carleton Street, Cambridge, Massachusetts 02139 2 University Hospital for Orthopaedic Surgery Friedrichsheim, Marienburgstrasse 2, 60528 Frankfurt, Germany 3 Department of Biomedical Engineering, Tufts University, 4 Colby Street, Medford, Massachusetts 02155 4 Department of Chemistry and Applied Biosciences, ETH Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland 5 Orthopaedic Biomechanics Laboratory, Beth Israel Deaconess Medical Center, Harvard University, Boston, Massachusetts 02215 Received 24 February 2004; revised 21 May 2004; accepted 21 May 2004 Published online 12 August 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.30117 Abstract: Porous biodegradable silk scaffolds and human bone marrow derived mesenchymal stem cells (hMSCs) were used to engineer bone-like tissue in vitro. Two different scaffolds with the same microstructure were studied: colla- gen (to assess the effects of fast degradation) and silk with covalently bound RGD sequences (to assess the effects of enhanced cell attachment and slow degradation). The hM- SCs were isolated, expanded in culture, characterized with respect to the expression of surface markers and ability for chondrogenic and osteogenic differentiation, seeded on scaf- folds, and cultured for up to 4 weeks. Histological analysis and microcomputer tomography showed the development of up to 1.2-mm-long interconnected and organized bonelike trabeculae with cuboid cells on the silk-RGD scaffolds, fea- tures still present but to a lesser extent on silk scaffolds and absent on the collagen scaffolds. The X-ray diffraction pat- tern of the deposited bone corresponded to hydroxyapatite present in the native bone. Biochemical analysis showed increased mineralization on silk-RGD scaffolds compared with either silk or collagen scaffolds after 4 weeks. Expres- sion of bone sialoprotein, osteopontin, and bone morphoge- netic protein 2 was significantly higher for hMSCs cultured in osteogenic than control medium both after 2 and 4 weeks in culture. The results suggest that RGD-silk scaffolds are particularly suitable for autologous bone tissue engineering, presumably because of their stable macroporous structure, tailorable mechanical properties matching those of native bone, and slow degradation. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res 71A: 25–34, 2004 Key words: silk; stem cells; osteogenic; hydroxyapatite; tis- sue engineering INTRODUCTION In the United States, 2.5 million orthopedic and plastic reconstructions, including bone, cartilage, ten- don, ligament, and breast, are performed annually. 1 Most bone repair procedures require a replacement structure to restore tissue function, including total substitutes (artificial joints), or tissue harvested from a second anatomic location of the same patient or from other patients and transplanted to the compromised area. Tissue engineering can provide an alternative to traditional treatment protocols by replacing living tis- sue with tissue grown in vitro that is designed and engineered to meet the needs of each individual pa- tient and repair site. 2 In particular, tissue engineering of autologous bone using bone marrow derived hu- man mesenchymal stem cells (hMSCs) can potentially avoid autologous grafting techniques. The hMSCs can proliferate in an undifferentiated state and with the appropriate extrinsic signals, differentiate into cells of various mesenchymal lineages, including cartilage and bone. 3–6 To meet mechanical and functional requirements at the implant site, a mechanically stable slowly degrad- Correspondence to: D. L. Kaplan; e-mail: david.Kaplan@ tufts.edu Contract grant sponsor: German Alexander Von Hum- boldt Foundation Contract grant sponsor: National Institutes of Health; con- tract numbers: NIH R01DE13405-04, R01EB003210-01 Contract grant sponsor: National Science Foundation; contract grant number: NST DMR-0090384 Contract grant sponsor: NASA; contract grant number: NCC8-174 © 2004 Wiley Periodicals, Inc.