Experimental Studies On Repair Of Articular Cartilage Damage With ADSCs Induced By CDMP1

-6-Articular cartilage is a narrow layer of specialized connective tissue that permits smooth, frictionless movement of diarthrodial joints. It is comprised of a relatively small number of cells (chondrocytes) embedded in an abundant extracellular matrix. The latter consists predominantly of type-II collagen, proteoglycans andwater, along with smaller amounts of other collagen types and non-collagenous proteins.Damaged articular cartilage has a limited capacity for self-repair. Joint surface defects that exceed a critical size heal poorly and usually lead to osteoarthritis.Since cartilage is an avascular tissue, spontaneous repopulation of the defects with cells forming repair cartilage is commonly not observed. In the end, joint replacement is still the only therapeutic option for many patients to regain mobility and relieve chronic pain.Multiple joints can be affected in osteoarthritis, but mostly in hip and knee joints. Osteoarthritis is characterised by destruction of cartilage, subchondral bone eburnation, cyst formation, fibrilation of capsule, and eventually, joint deformation and function loss.It is well known that the capacity of articular cartilage for repair is limited. Injuries of the articular cartilage that do not penetrate the subchondral bone do not heal and usually progress onto the degeneration of the articular surface.Cartilage injuries are a common clinical problem that, if left untreated, could lead to osteoarthritis. Degeneration of articular cartilage is one of the great clinical challenges that still lack a convincing therapeutic solution.Recently, tissue engineering has emerged as a new method in which a combination of cells, scaffold, and bioactive agents is used to fabricate functional new tissue to replace damaged cartilage.The damage and loss of organs and tissues lead to metabolic and structural changes that can cause significant morbidity and decrease the quality of life. Currently employed therapies for the treatments of joint tissues loss or disease are unsatisfactory as they rely on metal joints prosthesis which offer structural replacement albeit limited functionality. Furthermore, artificial implants lack tissue's physiological activities and often do not provide the lifelong solution for the patient. The field of tissue engineering (TE) has emerged over the past decades to improve the treatments for tissue and organ failure.Adipose-derived mesenchymal cells (AMCs) are an attractive source of multipotent mesenchymal cells (MSCs) for use in tissue engineering and clinical applications .Their relative abundance and easy access in adult tissues make them ideal candidates for cell-based therapies, and much current research is devoted to further elucidate the pathways involved in these cells'differentiation to bone,cartilage, and other tissues. Cartilage-derived morphogenetic protein-1 (CDMP-1: also known as growth differentiation factor-5, GDF-5) is a key regulatory factor in regulation of the development of the appendicular skeleton, particularly at the early stages of chondrogenesis of limb Growth/differentiation factor 5 (GDF5) is a member of the bone morphogenetic protein (BMP) family, which has been implicated in several skeletogenic events including cartilage and bone formation. CDMP-1 promotes differentiation of chondrocytes into hypertrophy and enhances commitment of mesenchymal cells into the chondrocytic lineage.This research work was designed to investigate the feasibility of diferentiation of Sprague-Dawley rat adipose-derived stem cells(ADSCs) in vitro into chondrogenic phenotype with induction of cartilage-derived morphogenetic protein(CDMP1)growth factor at both in vitro and in vivo levels. Firstly, ADSCs was induced by different dose of CDMP1 and the proliferation and differentiation of ADSCs after induced by CDMP1 was analyzed with MTT ; Sencondly , to investigate the feasibility of using Sprague-Dawley rat adipose-derived stem cells which was induced by cartilage-derived morphogenetic protein(CDMP1)growth factor and combined with the scaffold that was made of spongy bone of cattle forming into cartilage in vivo of nude mouse ; Finaly , to investigate the feasibility of heterogeneity cartilage to be the seeds cell of cartilage tissue engineering by repairing knee joints defect in rabbits with the cell induced by CDMP1 of Sprague-Dawley rat. The contents and results of this work were as follows.1. Effects of the proliferation and differentiation of ADSCs after induced by CDMP1 was analyzed with MTTThe second generation ADSCs were cultured with the density of 1X103/well in 96-well plate in high-glucose Dulbecco's modified Eagle's medium (DMEM) +10 ml/L neonatal bovine serum (NBS) and induced with CDMP1 50 ng/mL (group A),CDMP1 100 ng/mL (group B),CDMP1 150ng/mL (group C) and CDMP1 0 ng/mL (group D) after 12 h . To analyzed the proliferation and differentiation of ADSCs after induced by CDMP1 . As a result , CDMP1 had contribution to the proliferation and differentiation of ADSCs and the optimal concentration of CDMP1 should be 50ng/mL since there was no statistically significant difference in proliferation and differention of ADSCs between groups A,B,C.2. The fuction of the diferentiation of Sprague-Dawley rat adipose-derived stem cells(ADSCs) in vitro into chondrogenic phenotype with induction of cartilage-derived morphogenetic protein(CDMP1)growth factor The second generation ADSCs were cultured with the density of 1X104/well in 24-well plate in high-glucose Dulbecco's modified Eagle's medium (DMEM) +10 ml/L neonatal bovine serum (NBS) and induced with CDMP1 50 ng/mL (group A),CDMP1 100 ng/mL (group B),CDMP1 150ng/mL (group C) and CDMP1 0 ng/mL (group D) after 12 h .)The changes of the cell shape were observed with inverted phase contrast microscope, and the proliferation and differentiation of ADSCs after induced by CDMP1 was analyzed with MTT. Expression of collogenâ…¡was detected by immunohistochemistry and the expression of GAG was detected by toluidine blue O. ADSCs from SD rat, can differentiate into chondrogenic phenotype with CDMP1 induction in vitro. And it supplies us great hopes in sources of seed cell for tissue engineering.3. To observe the shape of the scaffold of spongy bone of cattle with scanning electron microscope (SEM)The SEM showed: cartilage and ADSCs induced by CDMP1 which both were combined into the scaffolds of spongy bone of cattle growed well .The interval porosity of the scaffold of spongy bone of cattle was modicus , the average aperture was 382 um . It gived a very good three-dimensional scaffolds for the growth of cells. 4. The fuction of rat adipose-derived stem cells forming into cartilage with induction of cartilage-derived morphogenetic protein (CDMP1) growth factor in vivo of nude mouseThe second generation cartilage cells of SD rat were cultured with elementary nutrient liquid which contains DMEM+10 mL/L NBCS only with the scaffold for another two weeks . There were twenty nude mice which were imbedded the composite of cells-scaffold of the experimental group in their left armpits and the composite of cells-scaffold of the control group in their right armpits. After eight weeks, toluidine blue staining showed the slices of the experimental group was positive and the shape of cells became globular , and there were apparent lacunas of cartilage while the control group was negative and there were no lacunas of cartilage. But the scaffolds of both the groups were degradation entirely. It's possible to form into cartilage in vivo of nude mouse by using ADSCs of SD rat which was induced by CDMP1 and combined with the scaffold of spongy bone of cattle.5. Study of reparing knee joints defection in rabbit with the ADSCs induced by CDMP1 which were combined with the scaffold made of the spongy bone of cattleTwenty adult New Zealand rabbits were included in this study. To make the models of knee joints defect of rabbits and embed the composites of ADSCs-scaffold into the defect of the left legs of the rabbits as the experimental group while embedding the scaffold only into the defect of the right legs of the rabbits as the control group. Every six rabbits were killed after eight, sixteen and twenty-four weeks and to make the slices of safranine o and toluidine blue staining .The repairing of the experimental group were satisfactory and the cells of the area of repairing and the normal circumjacent area were very similar in morphous .But the control group were still cavitates and the boundary between the repairing area and the normal circumjacent area was visible . Toluidine blue and safranine o staining showed the slices of the experimental group was positive and the shape of cells were globular , and there were apparent lacunas of cartilage while the control group was negative and there were no lacunas of cartilage. The knee joints defect of rabbits were repaired successfully by using cartilage of SD rat combining with the scaffold of spongy bone of cattle and it's possible that heterogeneity cartilage to be the seeds cell of cartilage tissue engineering .