Ectopic Cartilage Formation Induced by Mesenchymal Stem Cells on Porous Gelatin-Chondroitin-Hyaluronate Scaffold Containing Microspheres Loaded with TGF-β1

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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.

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