Optimization of Bone-Tissue Engineering in Goats Moyo C. Kruyt, 1 Wouter J. A. Dhert, 1 Cumhur Oner, 1 Clemens A. van Blitterswijk, 2,3 Abraham J. Verbout, 1 Joost D. de Bruijn 2 1 Department of Orthopedics, University Medical Center Utrecht, The Netherlands 2 IsoTis NV Bilthoven, The Netherlands 3 Twente University, The Netherlands Received 9 December 2002; revised 4 April 2003; accepted 23 April 2003 Published online 27 February 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.b.10073 Abstract: Successful bone-tissue engineering (TE) has been reported for various strategies to combine cells with a porous scaffold. In particular, the period after seeding until implantation of the constructs may vary between hours and several weeks. Differences between these strategies can be reduced to (a) the presence of extracellular matrix, (b) the differentiation status of the cells, and (c) the presence of residual potentially immunogenic serum proteins. These parameters are investigated in two types of calcium phosphate scaffolds in a goat model of ectopic bone formation. Culture-expanded bone-marrow stromal cells from eight goats were seeded onto two types of hydroxyapatite granules: HA60/400 (60% porosity, 400-m average pore size) and HA70/800. Scaffolds seeded with cells and control scaffolds were cultured for 6 days in medium containing autologous or semisynthetic serum, in the presence or absence of dexamethasone. Other scaffolds were seeded with cells just before implantation in medium with or without serum. All conditions were implanted autologously in the paraspi- nal muscles. After 12 weeks, bone had formed in 87% of all TE constructs, as demonstrated by histology. Histomorphometry indicated significantly more bone in the HA70/800 scaffolds. Furthermore, a significant advantage in bone formation was found when the constructs had been cultured for 6 days. In conclusion, both scaffold characteristics (porosity) and TE strategy (culturing of the constructs) were demonstrated to be important for bone TE. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 69B: 113–120, 2004 Keywords: tissue engineering, serum, extracellular matrix, bone, ectopic, goats, cells INTRODUCTION Tissue engineering of autologous bone is a promising alter- native for the surgically derived autologous bone graft. Since the early nineties, many investigators have demonstrated the concept of combining osteoprogenitor cells with an appropri- ate scaffold to be osteogenic ectopically in rodents and larger mammals. 1–4 Recently, ectopic tissue-engineered bone for- mation in a goat model has been demonstrated. 5,6 Although less informative with respect to clinical application, the ad- vantage of an ectopic model is that it allows fundamental research without the disturbing influence of host bone as present orthotopically. Furthermore, many experimental con- ditions can be evaluated in one animal for comparative stud- ies. The scaffolds most frequently selected for bone TE are porous structures of hydroxyapatite (HA), often as a compos- ite with -tricalcium phosphate (TCP). 2,7 The osteoconduc- tivity and biocompatibility of these materials are advanta- geous for future orthotopic application. The cells mostly used are bone marrow stromal cells (BMSCs). 2,8,9 These BMSCs can be administered by soaking the scaffold in fresh bone marrow, 10,11 or after culture expansion, 8,12,13 which was shown to be superior. 1,4,6,14,15 Although many scaffold characteristics and the selection and expansion of BMSCs is quite similar between various studies, the strategy for building the final construct is remark- ably different. For instance, some investigators seed undif- ferentiated “stem” cells under serum-free conditions on the scaffold, followed by implantation of the constructs within several hours after seeding. 4,15,16 Others culture the cells in the constructs 1 or more weeks prior to implantation, to allow extracellular-matrix formation and to stimulate osteogenic differentiation by addition of specific differentiation factors such as dexamethasone. 3,5,14,17,18 Differences between these strategies may be reduced to the following parameters: (a) the Correspondence to: Moyo C. Kruyt, Department of Orthopedics, G05.228, Uni- versity Medical Center, Utrecht, P.O. Box 85500, 3508 GA Utrecht, The Netherlands (e-mail: m.c.kruijt@chir.azu.nl) Contract grant sponsor: The Netherlands Technology Foundation (STW), contract grant number UGN.4966) © 2004 Wiley Periodicals, Inc. 113