Cartilage Regeneration By Selected Chondrogenic Clonal Mesenchymal Stem Cells In Cynomolgus Monkey Model

BackgroundArticular cartilage lesions can result from a variety of causes such as many joint diseases or trauma including work-or sports-related injuries. They are frequently associated with joint pain, dysfunction and disability, and are believed to progress to severe forms of osteoarthritis. Articular cartilage does not usually regenerate by forming original-quality tissue because of poor regenerative capacity of chondrocytes, particularly, in elderly patients. Autologous chondrocyte transplantation is a suitable technique for cartilage repair. However, there are several limitations to this application, including the limited availability of harvested cartilage, degeneration of the harvest site, dedifferentiation of chondrocytes in vitro.MSCs have been known as non-hematopoietic progenitor cells found in various adult tissues. They are characterized by their ease of isolation and their extensive proliferative ability while retaining the potential to differentiate along various lineages of mesenchymal origin, including chondrocytes, osteoblasts, and adipocytes. Additionally, MSCs possess potent immuno-modulatory and anti-inflammatory effects through either direct cell-cell interaction or secretion of various factors. They exert a tremendous effect on local tissue repair through modulating local environment and activation of endogenous progenitor cells. These features make MSC-based cell therapy as a hotly pursued subject of investigation in cartilage regeneration. However, a crucial requirement for MSC-based cartilage repair therapy is the delivery of the cells to the defect site.To improve the quality of the treatment, different studies have reported the application of various synthetic scaffolds in tissue engineering, which enable MSC penetration, help in maintaining the MSCs, provide chondroinductive matrix, allow nutrient delivery and gas exchange, and mimic the natural tissue geometry. However, many investigators are still concerned about the long-term safety and feasibility of synthetic scaffolds in clinic. In this study, a novel strategy was explored via developing a three-dimensional MSC-loaded acellular dermal matrix(ADM) in vitro, and replacing damaged cartilage by the transplantation of MSC-ADM grafts in an experimental, genetically close to human model of knee joint cartilage defect in Cynomolgus monkeys.To transfer the basic regenerative medicine knowledge to clinic, it is essential to use models which are genetically close to humans. In this background, aim of our study was to compare the differentiation capacity, cell yield, phenotype and proliferation of polyclonal MSCs (P-MSCs) and clonal MSCs (C-MSCs) in vitro, to evaluate the ability of transplanted cells to survive in cartilage and to investigate the capacity of autologous selected chondrogenic clonal MSCs (sC-MSCs) to repair damaged cartilage in a collagenase-induced Cynomolgus monkey model. Our aim was to investigate whether transplantation of selected chondrogenic clonal MSC-loaded ADM grafts could improve cartilage lesions in Cynomolgus monkeys, so that feasibility and acceptability of MSC-based cell therapy could be applied precisely for clinical research in humans.Part Ⅰ Bone Marrow Mesenchymal Stem Cells from Cynomolgus Monkeys: Isolation, Culture and IdentificationObjectives Isolated and cultivated Cynomolgus monkey MSCs. We compared the characteristics of monkey MSCs and evaluated their chondrogenic abilities in vitro.Materials and methods Harvest MSCs from Cynomolgus monkey to carry out chondrogenic culture and identification. MSCs were characterized for cell size, cell yield, phenotypes, proliferation and chondrogenic differentiation capacity. Chondrogenic differentiation assays were performed at different MSC passages by sulphated glycosaminoglycans (sGAG), collagen, and fluorescence activated cell sorter (FACS) analysis. Monkey clonal MSCs (C-MSCs) and polyclonal MSCs (P-MSCs) were characterized by determining cell size, cell yield, phenotypes, proliferation and chondrogenic differentiation capacity in vitro.Results MSCs expressed mesenchymal markers and differentiated into the chondrogenic lineage after stimulation with chondrogenic growth factors. Polyclonal and clonal MSCs could differentiate into the chondrogenic lineage after stimulation with suitable chondrogenic factors. They expressed mesenchymal markers and were negative for hematopoietic markers.Conclusions Monkey bone marrow-derived MSCs were found to differentiate into the chondrogenic lineage in this study. After positive selection, sC-MSCs retained better chondrogenic capacity than that in P-and C-MSCs, the average amount of sGAG, type Ⅱ collagen protein and cell size were increased in later passages. Part Ⅱ Preparation and Characterization of Acellular Dermal MatrixObjectives To prepare a type of acellular dermal matrix (ADM) and analyze its properties.Materials and methods ADM was prepared by means of combined treatments with physical, chemical and biological methods, and its structure, composition and properties were evaluated. Selected chondrogenic clonal MSCs were seeded onto ADM scaffold and sC-MSCs-loaded ADM grafts were analyzed by confocal microscopy and scanning electron microscopy.Results The porosity of ADM reached to80.26%and the tensile strength was about4.52MPa. The selected clonal MSCs were seeded on ADM and cultured for21days, the average amount of sGAG and type II collagen, protein were increased. Thus, prolonged sC-MSCs-ADM scaffold interactions favored enhanced chondrogenic potential. The results of scanning electron microscopy and confocal fluorescence microscopy showed that sC-MSCs can survive in ADM grafts.Conclusions We used natural monkey acellular dermal matrix as scaffold that presents a more natural microenvironment to support growth and proliferation of sC-MSCs than synthetic scaffolds. Ultimately, it will be directed towards developing a clinically feasible strategy for use of sC-MSCs-ADM in the reconstruction of articular cartilage. Part III Cartilage Regeneration by Selected Chondrogenic Clonal Mesenchymal Stem Cells in the Collagenase-induced Monkey ModelObjectives Osteoarthritis (OA) is the most common form of degenerative arthritis.Current therapies do not regenerate damaged cartilage. Mesenchymal stem cell (MSC)-based therapy is a promising novel approach for repairing articular cartilage whose success depends upon high-quality MSCs and delivery mode. We evaluated the repair potential of selected chondrogenic clonal MSCs (sC-MSCs) via their delivery to the injured cartilage site in a collagenase-induced OA model of Cynomolgus monkeys.Materials and methods OA-like cartilage lesions were generated by intraarticular bacterial collagenase injections. The articular cartilage lesions were treated with normal saline (NS), autologous P-MSCs, and sC-MSCs, respectively, by direct delivery and adherence technology. The clinical parameters, radiographic images, histological (Hematoxylin and Safranin0/Fast green) and immunohistochemical examinations of Type I and II Collagen, MMP-1and MMP-3were analyzed at week8,16, and24post-treatment.Results MSCs expressed mesenchymal markers and differentiated into the chondrogenic lineage after stimulation with chondrogenic growth factors. Abrasion and necrosis of articular cartilage were significantly improved and repaired by MSC-based treatment, particularly, in sC-MSC-treated group, which displayed consistently higher histological scores than those of other groups.Conclusions Treatment with sC-MSCs could effectively improve the cartilage lesions in Cynomolgus monkey collagenase-induced OA model. Due to close genetic proximity of monkey and human, the applicability of this knowledge to repair human OA cartilage defects is worth-investigating. Part Ⅳ Reconstruction of Cartilage Defects with Selected Chondrogenic Clonal Mesenchymal Stem Cell loaded Acellular Dermal Matrix in Cartilage Defect Model in Monkey ModelObjectives Articular cartilage defects are commonly associated with trauma, inflammation and osteoarthritis. Mesenchymal stem cell (MSC)-based therapy is a promising novel approach for repairing articular cartilage. Direct intra-articular injection of uncommitted MSCs does not regenerate high-quality cartilage. This study explored utilization of a new three-dimensional,selected chondrogenic clonal MSC-loaded monkey acellular dermal matrix (MSC-ADM) scaffold to repair damaged cartilage in an experimental model of knee joint cartilage defect in Cynomolgus monkeys.Materials and methods Cartilage defects were treated with normal saline, clonal MSCs and clonal MSC-ADM grafts, respectively. The clinical parameters, histological and immunohistochemical examinations were evaluated at week8,16,24post-treatment, respectively.Results Articular cartilage defects were considerably improved and repaired by selected chondrogenic clonal MSC-based treatment, particularly, in MSC-ADM-treated group. The histological scores in MSC-ADM-treated group were consistently higher than that those of other groups.Conclusions Our results suggest that selected chondrogenic clonal MSC-loaded ADM grafts could improve the cartilage lesions in Cynomolgus monkey model, which may be applicable for repairing similar human cartilage defects.