Biomaterials 28 (2007) 814–824 Combined marrow stromal cell-sheet techniques and high-strength biodegradable composite scaffolds for engineered functional bone grafts Yefang Zhou a , Fulin Chen b , Saey Tuan Ho b , Maria Ann Woodruff b , Tit Meng Lim a , Dietmar W. Hutmacher b,c,Ã a Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore 119260, Singapore b Division of Bioengineering, Faculty of Engineering, National University of Singapore, Singapore 119260, Singapore c Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119260, Singapore Received 21 June 2006; accepted 20 September 2006 Available online 11 October 2006 Abstract In this study, cell sheets comprising multilayered porcine bone marrow stromal cells (BMSC) were assembled with fully interconnected scaffolds made from medical-grade polycaprolactone–calcium phosphate (mPCL–CaP), for the engineering of structural and functional bone grafts. The BMSC sheets were harvested from culture flasks and wrapped around pre-seeded composite scaffolds. The layered cell sheets integrated well with the scaffold/cell construct and remained viable, with mineralized nodules visible both inside and outside the scaffold for up to 8 weeks culture. Cells within the constructs underwent classical in vitro osteogenic differentiation with the associated elevation of alkaline phosphatase activity and bone-related protein expression. In vivo, two sets of cell-sheet-scaffold/cell constructs were transplanted under the skin of nude rats. The first set of constructs (5  5  4 mm 3 ) were assembled with BMSC sheets and cultured for 8 weeks before implantation. The second set of constructs (10  10  4 mm 3 ) was implanted immediately after assembly with BMSC sheets, with no further in vitro culture. For both groups, neo cortical and well-vascularised cancellous bone were formed within the constructs with up to 40% bone volume. Histological and immunohistochemical examination revealed that neo bone tissue formed from the pool of seeded BMSC and the bone formation followed predominantly an endochondral pathway, with woven bone matrix subsequently maturing into fully mineralized compact bone; exhibiting the histological markers of native bone. These findings demonstrate that large bone tissues similar to native bone can be regenerated utilizing BMSC sheet techniques in conjunction with composite scaffolds whose structures are optimized from a mechanical, nutrient transport and vascularization perspective. r 2006 Elsevier Ltd. All rights reserved. Keywords: Bone engineering; Bone marrow stromal cells; Mesenchymal stem cell; Composites; Biodegradable polymers; Functional tissue engineering 1. Introduction Tissue formation within the body, as part of a develop- ment, healing and/or repair process, is a complex event in which cell populations in combination with extra cellular matrix, self-assemble into functional units and ultimately into tissues and organs. There is intense academic and commercial interest in finding methods to stimulate and control these events, and eventually to replicate these events outside the body as closely as possible [1]. This interest has resulted in tissue engineering (TE) emerging as a well- recognized research area, in the arena of regenerative medicine. The most common concept underlying TE is to combine living cells, biologically active molecules and a structural scaffold to form a ‘‘tissue-engineering construct’’ (TEC) to promote the repair and regeneration of tissues [2]. In the bone TE literature [3–5], an increasing trend is seen towards the fabrication of polymer-based composite materials to obtain matrices with better osteoconductive properties. The main approach is to develop composite and biomimetic scaffolds with nanocrystallites of inorganic ARTICLE IN PRESS www.elsevier.com/locate/biomaterials 0142-9612/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2006.09.032 Ã Corresponding author. Division of Bioengineering, Faculty of Engineering, National University of Singapore, Singapore 119260, Singapore. Tel.: +65 68741036; fax: +65 67773537. E-mail addresses: scip1129@nus.edu.sg (Y. Zhou), cflfx@tom.com (F. Chen), g01029965@nus.edu.sg (S.T. Ho), biewma@nus.edu.sg (M.A. Woodruff), dbsltm@nus.edu.sg (T.M. Lim), biedwh@nus.edu.sg (D.W. Hutmacher).