179 www.ecmjournal.org G-S Huang et al. Solid freeform-fabricated scaffolds European Cells and Materials Vol. 26 2013 (pages 179-194) DOI: 10.22203/eCM.v026a13 ISSN 1473-2262 Abstract Three-dimensional (3D) cellular spheroids have recently emerged as a new trend to replace suspended single cells in modern cell-based therapies because of their greater regeneration capacities in vitro. They may lose the 3D structure during a change of microenvironment, which poses challenges to their translation in vivo. Besides, the conventional microporous scaffolds may have diffculty in accommodating these relatively large spheroids. Here we revealed a novel design of microenvironment for delivering and sustaining the 3D spheroids. Biodegradable scaffolds with macroporosity to accommodate mesenchymal stem cell (MSC) spheroids were made by solid freeform fabrication (SFF) from the solution of poly(D,L-lactide-co-glycolide). Their internal surface was modifed with chitosan following air plasma treatment in order to preserve the morphology of the spheroids. It was demonstrated that human MSC spheroids loaded in SFF scaffolds produced a signifcantly larger amount of cartilage-associated extracellular matrix in vitro and in NOD/SCID mice compared to single cells in the same scaffolds. Implantation of MSC spheroid-loaded scaffolds into the chondral defects of rabbit knees showed superior cartilage regeneration. This study establishes new perspectives in designing the spheroid-sustaining microenvironment within a tissue engineering scaffold for in vivo applications. Keywords : Solid freeform fabrication; scaffolds; mesenchymal stem cell spheroids; chitosan; cartilage regeneration. *Address for correspondence: Shan-hui Hsu Institute of Polymer Science and Engineering National Taiwan University No. 1, Sec. 4 Roosevelt Road, Taipei 10617, Taiwan, R.O.C. Telephone Number: 886-2-3366-5313 FAX Number: 886-2-3366-5237 E-mail: shhsu@ntu.edu.tw Introduction Stem cells are multipotent cells that can differentiate into different lineages and expand while maintaining their undifferentiated state (self-renewal). Mesenchymal stem cells (MSCs) show adhesion to tissue culture polystyrene (TCPS) dish with fbroblast-like cell morphology and are normally characterised with their specifc lack of haematopoietic and endothelial markers but with variable expressions of several other surface antigens (e.g. CD73, CD90, and CD105). A three-dimensional (3D) culture environment is generally considered more favourable than 2D monolayer culture. Various culture systems have emerged lately to generate 3D multicellular spheroids from MSCs, such as suspension, hanging drop, micropatterned substrates, and non-adherent surface (Potapova et al., 2007; Wang et al. , 2009; Bhang et al., 2011; Su et al., 2013). MSC spheroids formed on micropatterned substrates had higher efficiency of osteogenic and adipogenic differentiations (Wang et al., 2009). Those generated from hanging drop or suspension had better anti-infammatory property (Bartosh et al., 2010; Ylostalo et al., 2012) and angiogenesis capacity (Bhang et al., 2011). A novel method has been established recently to generate MSC spheroids on chitosan-based substrates such as chitosan membranes (CS) (Hsu et al., 2012c) or hyaluronan-modifed chitosan membranes (CS-HA) (Huang et al., 2011). MSCs grown on these substrates were self-assembled into 3D cellular spheroids that kept migrating on the substrates. The self-renewal property and chondrogenic differentiation potential were signifcantly enhanced in these substrate-derived MSC spheroids (Huang et al., 2011; Hsu et al., 2012a). Although MSC spheroids are considered superior to single suspended cells in general, the delivery of relatively bulky spheroids without losing their 3D structure is a challenging task. Moreover, no study has ever followed if these MSC spheroids can truly maintain their 3D morphology upon a change of microenvironment. Scaffolds are a key component in cartilage tissue engineering, which provide a suitable microenvironment for cell development. Biodegradable scaffolds have been fabricated by a variety of methods, e.g., solvent casting/ particulate leaching, phase separation, freeze-drying, and fbre bonding (O’Shea and Miao, 2008). However, the pore size and porosity of these scaffolds are diffcult to control. Solid freeform fabrication (SFF) methods are a category SOLID FREEFORM-FABRICATED SCAFFOLDS DESIGNED TO CARRY MULTICELLULAR MESENCHYMAL STEM CELL SPHEROIDS FOR CARTILAGE REGENERATION G.-S. Huang 1 , C.-S. Tseng 2 , B. Linju Yen 3,4 , L.-G. Dai 5 , P.-S. Hsieh 1 and S.-h. Hsu 1,6 1 Institute of Polymer Science and Engineering, National Taiwan University, Taipei, Taiwan 2 Department of Mechanical Engineering, National Central University, Taoyuan, Taiwan 3 Institute of Cellular and System Medicine, National Health Research Institutes, Miaoli, Taiwan 4 Department of Obstetrics/Gynecology, Cathay General Hospital Shiji, New Taipei City, Taiwan 5 Department of Orthopedics, Kuang Tien General Hospital, Taichung, Taiwan 6 Research Center for Developmental Biology and Regenerative Medicine, National Taiwan University, Taipei, Taiwan