INTRODUCTION Intact articular cartilage is very important for providing the joint with an extraordinarily efficient lubrication. However, due to the low mitotic activity of chondrocytes it has limited capacity to restore defects (1, 2). For diffuse cartilage lesions in old people, total joint arthroplasty is one of the most common indications. But for focal cartilage defects in young adults the majority of the current therapies, such as microfracture, multiple drilling and so on can’t restore the normal cartilage and even induce further damage (3). Recently, tissue engineering has Regenerative Medicine The International Journal of Artificial Organs / Vol. 29 / no. 6, 2006 / pp. 602-611 Ectopic cartilage formation induced by mesenchymal stem cells on porous gelatin-chondroitin-hyaluronate scaffold containing microspheres loaded with TGF-β1 H. FAN 1,2 , Y. HU 1 , X. LI 1 , H. WU 3 , R. LV 1 , J. BAI 1 , J. WANG 1 , L. QIN 2 1 Department of Orthopedics and Traumatology, Xijing Hospital, the Fourth Military Medical University, Xi’an - People’s Republic of China 2 Musculoskeletal Research Laboratory, Department of Orthopaedics and Traumatology, the Chinese University of Hong Kong, Hong Kong SAR - People’s Republic of China 3 Department of Chemistry, the Fourth Military Medical University, Xi’an - People’s Republic of China © Wichtig Editore, 2006 0391-3988/602-10 $15.00/0 ABSTRACT: The study aimed to produce a novel porous gelatin-chondroitin-hyaluronate scaffold in combination with a controlled release of TGF-β1 and to evaluate its potentials in ectopic cartilage formation. The gelatin-chondroitin-hyaluronate scaffold was developed to mimic the natural extra cellular matrix of cartilage. Gelatin microspheres loaded with TGF-β1 (MS-TGFβ1) showed a fast cytokine release at initial phase (37.4%) and the ultimate accumulated release was 83.1% by day 18. Then MS-TGFβ1 were incorporated into scaffold. The MSCs seeded on scaffold with or without MS- TGFβ1 were incubated in vitro or implanted subcutaneously in nude mice. In vitro study showed that, compared to the scaffold, the scaffold/MS-TGFβ1 significantly augmented the proliferation of MSCs and GAG synthesis. Three weeks postoperatively histology observation showed that in MSCs/scaffold/MS-TGFβ1 implantation group, cells of newly formed ectopic cartilage were located within typical lacunae and demonstrated morphological characteristics of chondrocytes. Six weeks later the ectopic cartilage grew more and islands of cartilage were observed. The matrix was extensively metachromatic by safranin-O/Fast green staining. Immunohistochemical staining also indicated ectopic cartilage was intensely stained for type II collagen. Instead, in the MSCs/scaffold implantation group, no cartilage-like tissue formed and matrix showed negative or weak positive staining. The percentage of positive staining area was significantly larger in MSCs/scaffold/MS- TGFβ1 group (p<0.05) at each time point. The results indicated that the novel gelatin-chondroitin- hyaluronate scaffold with MS-TGFβ1 could induce the chondral differentiation of MSCs to form cartilage and might serve as a new way to repair cartilage defects. (Int J Artif Organs 2006; 29: 602-11) KEY WORDS: MSC (mesenchymal stem cell), Scaffold, TGF (transforming growth factor), Microsphere