A Synthetic Peptide Induces Chondrogenic Differentiation in Murine and Human Mesenchymal Stem Cells and Enhances Cartilage Repair In Vivo

Introduction: Chondrocytes found in articular cartilage are differentiated cells with limited regenerative capacities, and thus limited reparative capability following injury. Current treatments of cartilage damage mostly target pain relief and do not repair damaged articular cartilage. To repair damaged cartilage, transplant of chondrocytes, bone marrow stem cells, and other pluripotent cells is being actively investigated. Also, recombinant growth factors such as, insulin-like growth factor, plateletderived growth factor, bone morphogenetic protein (BMP) and others have been shown in research settings to facilitate cartilage repair[1,2]. B2A is a synthetic peptide that augments BMP-2 activity in osteogenic differentiation [3,4]. Furthermore, B2A increases bone formation and regeneration in animal spine fusion models [5,6]. The aims of this study is to investigate whether B2A peptide could induce the proliferation and differentiation of chondrocytes or chondrocyte precursor cells in vitro, and whether B2A enhance the repair of cartilage damage in an experimental osteoarthritis model. Materials and Methods: Normal human articular chondrocytes and human bone marrow mesenchymal stem (HMS) cells were from Cambrex Bio Science. C3H10T1/2 cells were from ATCC. All cell lines were maintained as per provider’s specifications. To evaluate the effects of B2A on cell proliferation, 1x103 cells were seeded into wells of 96-well plates and allowed to attach. The medium was replaced by DMEM containing 1% serum and B2A. After 3 days in culture, the relative cell number was monitored by a commercially available kit CyQuant (Invitrogen, Carlsbad, CA). The micromass cultures were established by seed high density (1 X107 cells/mL) cell suspensions into polypropylene tissue culture vessels in medium containing FBS, ascorbate, dexamethasone, and 5 μg/mL B2A. The culture medium was change every 3 days. Twelve days after the induction, the cell mass was fixed, paraffin embedded, and sectioned. The sulfated glycosaminoglycans and collagens in the cell mass were stained using Alcian blue (pH 1.0) and Masson Trichrome, respectively. To monitor collagen type II, cell aggregates were lysed and the cell lysates were resolved by SDS-PAGE and transferred onto PVDF membranes. Blocked membranes were incubated with a mouse anti-collagen type II (Chemicon) and the immuno-complexes were detected using chromogenic techniques. A rat model that mimicked aspects of osteoarthritis was used this study. Studies were conducted under IACUC-approved protocols and using specific pathogen-free, skeletally-mature male Sprague-Dawley rats. At day 0, monosodium iodoacetate (MIA) was injected to right rat knee joint to induce cartilage damage[7]. At day 7 and 14, the treatment group (N = 6) were treated with B2A by intra-articular injection to the right knee. Saline injection was used in the control group (N = 6). Twenty-one days after MIA injection, animals were euthanized and the knee joints were dissected, fixed, decalcified, and stained with either hemotoxylin and eosin or safranin O. The sections were examined and scored using a scoring system described by Nishda et al [7]. The scale is composed of cell morphology (by H & E) and matrix staining (by safranin O staining). The samples were assigned a score range from 0 to 3, 0 being normal and 3 being markedly damaged. Results: In monolayer culture, B2A increased the proliferation of HMS cells at concentrations of 1-10 μg/ml, but B2A did not affect the proliferation of C3H10T1/2 cells and the terminal differentiated chondrocytes. In micromass culture, B2A significantly increased the sulfated glycosaminoglycan deposition indicated by Alcian Blue staining. Masson Trichrome staining revealed that collagen deposition was increased in all three cell types upon the treatment of B2A. Furthermore, we investigated whether B2A increase the expression of the hyaline articular cartilage specific collagen, collagen type II. After 12 days stimulation, B2A increased collagen type II expression in C3H10T1/2 cells by more than 3 folds. In the MIA-induced osteoarthritis model, B2A was evaluated for its ability to increase the repair of damaged cartilage. If left untreated (saline group), the MIA-induced animals resulted in severe damage in the tibial trochlea cartilage. H & E staining in these animals revealed marked reduction in chondrocytes in the articuolar cartilage indicative of cell death. In addition, safranin O staining was markedly reduced in the untreated knee compared to the normal contra-lateral knee. In contrast, animals received B2A treatment exhibited significant repair of the damaged cartilage (Table 1). Compared to the saline treated group, there was a markedly increase in the number of chondrocytes found in B2A group and the difference between the saline and B2A group was statistically significant. Safranin O staining also revealed that improvement of matrix staining. The Safranin O score was reduced from 2.25 to 1.92 in the B2A treated group, however, the change did not reach the level of statistically significant. Discussion: The number of stem cells or chondrocyte progenitor cells in the site of damaged cartilage and their ability to differentiate to chondrocyte are important factors in the process of cartilage repair. In this study we reported that synthetic peptide B2A was able to increase proliferation in human bone marrow mesenchymal stem cells. Additionally, B2A increased the production of glycosaminoglcan and collagen. These finding suggested that B2A might have a role in promoting cartilage repair. The notion of B2A enhancing cartilage repair is further strengthen by the result that B2A increased the repair in damaged cartilage in vivo. B2A might facilitate the cartilage repair in multiple aspects: 1) increase the number of chondrocyte progenitor cells, 2) guide stem cells into chondrogenic differentiation pathway, and 3) increase the production of extracellular matrix by chondrocyte. Findings in this report suggests that B2A may be a hopeful therapeutic for cartilage repair in osteoarthiritis and other cartilage damages, and hence, continued study of B2A in additional animal models is warranted to further evaluate the efficacy of this peptide in cartilage repair. References: [1] Kuo et al. Osteoarthritis Cartilage 14, 1126-35, 2006 [2] Schmidt et al. Osteoarthritis Cartilage 14, 403-12, 2006 [3] Lin et al. J Orthop Res 25, 531-9, 2006 [4] Lin et al. J Bone Miner Res 20, 693-703, 2005 [5] Smucker et al. Abstract in 2007 ORS. [6] Smucker et al. (Submitted). [7] Nishida et al. J Bone Miner Res 19, 1308-19, 2004 Table 1. Histological score of rat tibial trocheas cartilage following MIA insult and treatment.