Increased Expression of Sox9 during Balance of BMSCs/Chondrocyte Bricks in Platelet-Rich Plasma Promotes Construction of a Stable 3-D Chondrogenesis Microenvironment for BMSCs

Sox9 is an intrinsic transcription factor related to the determination and maintenance of chondrogenic lineage of bone marrow mesenchymal stem cells (BMSCs). In recent research, we have proved that fragmented chondrocyte aggregates (cell bricks) could promote chondrogenesis of BMSCs in vivo. However, it is still unknown whether the ratio of BMSCs/chondrocyte bricks has a significant influence on 3-D cartilage regeneration and related molecular mechanism. To address this issue, the current study subcutaneously injected three groups of cell complex with different rabbit BMSCs/chondrocyte bricks’ ratios (1 : 2, 1 : 1, and 2 : 1) into nude mice. Gross morphology observation, histological and immunohistochemical assays, biochemical analysis, gene expression analysis, and western blot were used to compare the influence of different BMSCs/chondrocyte bricks’ ratios on the properties of tissue-engineered cartilage and explore the related molecular mechanism. The constructs of 1 : 1 BMSCs/chondrocyte bricks, (B1CB1) group resulted in persistent chondrogenesis with appropriate morphology and adequate central nutritional perfusion without ossification. The related mechanism is that increased expression of Sox9 in the B1C1 group promoted chondrogenesis and inhibited the osteogenesis of BMSCs through upregulating Col-II as well as downregulating RUNX2 and downstream of Col-X and Col-I by upregulating Nkx3.2. This study demonstrated that BMSCs/chondrocyte bricks 1:1 should be a suitable ratio and the Sox9-Nkx3.2-RUNX2 pathway was a related mechanism which played an important role in the niche for stable chondrogenesis of BMSCs constructed by chondrocyte bricks and PRP.

The Exaggerated Collagen Expression in Gvhd-Fibroblasts Is Effectively Inhibited By Therapeutic Concentration of Nilotinib

INTRODUCTION Dermal fibrosis and sclerosis are pathologic features shared by Scleroderma-like chronic graft-versus-host disease (Scl-cGVHD) and Systemic Scleroderma (SSc). Moreover, in both diseases stimulating anti-PDGF-R antibodies were found, leading to abnormal collagen production by fibroblasts, eventually contributing to organ damage. Targeted therapy with tyrosine kinase inhibitors (TKI) like Imatinib and Nilotinib demonstrated clinical efficacy in Scl-cGVHD; however, the molecular basis underpinning the clinical effects are not fully elucidated. We investigated here a potential terapeutical target of the dermal cGVHD pathophysiology: the cellular and molecular features of pathological skin fibroblasts (GVHD-Fbs) and the efficacy of Nilotinib on fibrosis modulation. MATERIALS AND METHODS Fibroblast cultures (GVHD-Fbs) were obtained from skin biopsies of affected skin from 6 patients with active cGVHD, control fibroblasts are Human Dermal Fibroblasts adult (n-FBS). Fibroblasts were characterized by flow cytometry (FACS CANTO II) for the detection of molecules: CD10, CD14, CD29, CD34, CD44, CD45, CD73, CD90, CD105, CD106, CD117, CD146. In order to evaluate the adipogenic, osteogenic or chondrogenic differentiation cGVHD-Fbs and n-Fbs (n = 3) were cultured in differentiation medium (respectively NH AdipoDiff, NH OsteoDiff, NH ChondroDiff) after four passages. Intracellular lipid droplets indicated adipogenic lineage differentiation. The differentiation potential in the osteogenic lineage was evaluated by calcium accumulation, as assessed by Alizarin Red. The pellet obtained from chondrogenic lineage differentiation was embedded in paraffin, cut in the microtome and the sections placed on a glass slide were stained with Alcian Blue [Junker JP, Cells Tissues Organs, 2010]. For incubation with Nilotinib (Santa Cruz Biotechnology) the 10 mM stock solution was diluted to the final concentration in DMEM supplemented with 0,2% FBS (starvation), added to cell cultures at a concentration of 1 μM or 2 μM for 48h, which covered the mean plasma levels in cGVHD patients after standard doses. In subsets of experiments, after starvation, fibroblasts were stimulated with recombinant TGFβ at 10 ng/ml (GIBCO, Invitrogen). After incubation, total RNA was isolated and reverse transcribed. Gene expression was quantified by real-time PCR using the Sybr Green Mix for qPCR. Specific primer pairs for COL1α1 and COL1α2 were designed with the Primer 3 software. The transcript levels were normalized for the expression of GAPDH constitutive gene. Differences were calculated with the threshold cycle (Ct) and the comparative Ct method for relative quantification. RESULTS GVHD-Fbs are morphologically and phenotypically similar to normal fibroblasts (n-FBS). GVHD-FBS did not show a different immunophenotype from n-Fbs, both in early and late culture passages. Also, no differences were noted between GVHD-Fbs and n-FBS in terms of multilineage differentiation capacity towards the adipogenic, osteogenic and chondrogenic lineage. Gene expression of COL1α1 and COL1α2 in GVHD-Fbs was respectively 4 and 1,6 times higher compared to n-FBS (p = 0.02). However, the increased collagen expression was exclusive of early-passage GVHD-Fbs; in late-passage (>4) GVHD-Fbs, collagen mRNA levels were similar to n-FBS (p=0.6 for COL1α1; p=0.4 for COL1α2). As expected, TGFβ boosted collagen expression in n-FBS, but it did not increase COL1α1 and COL1α2 mRNA levels in GVHD-Fbs. Therapeutic doses of Nilotinib (1μM) were able to reduce expression of COL1α1 and COL1α2 mRNA by 86,5% and 49%, respectively (p <0.01). CONCLUSIONS Early-passage GVHD-Fbs are a valuable cellular model to study the molecular mechanisms of cGVHD fibrosis in vitro, as they show increased collagen production, which is a strong hallmark of fibrosis. The failure to increase collagen expression in GVHD-Fbs upon TGFβ stimulation indirectly supports a TGFβ-dependent mechanism underpinning the fibrogenesis. Finally Nilotinib inhibits in vitro collagen expression in GVHD-Fbs confirming that the activity of TKI in Scl-cGVHD is mediated, at least in part, by direct antifibrotic effects on the fibroblasts. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Applied Osmotic Loading for Promoting Development of Engineered Cartilage

The avascular nature of cartilage and the harsh joint loading environment lead to a poor intrinsic healing capacity after injury, motivating the development of cell-based therapies for repair. Synovium-derived stem cells (SDSCs) have the potential for differentiating down a chondrogenic lineage and are thought to aid in articular cartilage repair after damage in vivo1. In the present study, we adopt a two-pronged strategy for growing clinically relevant cartilage grafts. Firstly, we compare the potential of SDSCs versus chondrocytes for engineering functional constructs. Secondly, we investigate the effect of extracellular osmolarity on mechanical and biochemical properties of SDSCs and similarly passaged chondrocytes in 3D culture. This approach is motivated by the fact that the in situ osmotic environment of chondrocytes varies with proteoglycan content and tissue deformation, altering the regulation of chondrocyte activity through mechanotransduction pathways2. We test the hypothesis that application of a hypertonic, more physiologic osmotic environment (created by addition of NaCl and KCl) relative to hypotonic media (300 mOsm), during 3D culture of SDSCs or chondrocytes in agarose hydrogels, improves the biochemical composition and mechanical properties of engineered tissue constructs.