An Experimental Study Of Adipose Derived Stem Cells With Articular Cartilage Acellular Matrices Scaffold Repairing Full-thickness Defects Of Knee Articular Cartilage In Rabbit

Background: The treatment of articular cartilage defects has been a problem in orthopedic surgery up to now .The defects may be caused by trauma or evolved during the course of diseases. Cartilage is a highly differentiated tissue and therefore has a limited capacity for self-repair. The large defects at articular cartilage are often repaired by fibrocartilage rather than normal hyaline cartilage. The repairing fibrocartilage tissues are different from hyaline cartilage in either biochemically or biomechanically and it ultimately deteriorates. Numerous strategies currently in clinical treatment are lavage and debridement, microfracture techniques, subcondral drilling, periosteal or perichondrial grafts, transplantation of osteochondral autografts or allografts. So many methods that mean no effective clinical treatment that can restore a damaged cartilage surface and prevent the outcome of degenerative joint disease except joint replacement. Cell-based therapies have also been developed in the treatment of this condition. The transplantation of tissue-engineered cartilage is an ideal method to repair the articular cartilage defects, and a important method to restoration the cartilage than replacement. Cartilage tissue engineering by means of cell amplification, which can be obtained and isolated from small biopsy specimens, and seeded onto biodegradable scaffold as cell-scaffold constructs to reconstruct the function of damaged tissue. Autogenous chondrocyte transplantation is promising but suffers from the same weaknesses as osteochondral autograft in incurring significant donor site morbidity. Stem cell therapy is an emerging field. Adult-derived stem cells, specifically mesenchymal stem cells, are more promising in this regard. At present the most common sources of adult-derived stem cells is the bone marrow. This source results in a low yield ofmesenchymal stem cells that necessitates ex vivo expansion. In addition, the heterogeneous nature of bone marrow with both hemopoietic and mesenchymal stem cells. On the basis of available therapeutic need, the ideal cell source would cause insignificant donor morbidity, be readily available, have no issues of immunogenicity, and good cell yield would obviate the need for ex vivo expansion. Adioise tissue is the ideal source. It causes insignificant donor morbidity. Its availability and good yield obviate the need for ex vivo. It exhibits wide differentiation potential with demonstrable cartilage, bone, fat and cardiomyocyte differentiation. The goal of present study was to investigate the feasibility of adipose derived stem cells(ADSCs) as the seed-cell of cartilage tissue engineering. The study was divided into three main parts. The first part was aimed at developing the method of isolating , expansion and chondrogenic differentiation of adipose derived stem cells(ADSCs) in vitro. The second part was aimed at constructing the tissue engineered cartilage with ADSCs-seeded human articular cartilage acellular matrix(ACAM) in vitro and in athymic mice. The third part was aimed at implantation of the cells/scaffold compounds for reconstituting osteochondral defects and assessing the reconstituted defects grossly, histologically, biochemically and biomechanically.Methods: (1) ADSCs isolated from adipose tissue, which was harvested under sterile condition from subcutaneous tissue of New Zealand White rabbit and culture-expanded in DMEM supplemented with 10% FBS at 37℃, 5%CO₂ atmosphere incubator. The 3^th passages ADSCs were treated according the groups: (A) common mediea (DMEM with 10%FBS). (B) DMEM with 10%FBS containing TGF-β₁ at 10ng/ml, bFGF at 25ng/ml and dexamethasone at 10^7-M, as induced ADSCs. (C) bone marrow-derived steml cells(MSCs) served as control. To determine the chondrogenic Phenotype of stem cells by staining with Safranin"O", Alcian blue and immunohistochemistry of collagen type II. Quantitative secreation of GAG was tested by dimethyl methylene blue and collagen II by ELISA. Cell proliferation capacity was tested by CCK-8. (2) ADSCs cultured in RCCS(rotarycell culture system), which contained the Cytodex-3 microcarriers in the chondrogenic medium. Static culture ADSCs with chondrgenic medium served as control. Growth of ADSCs on Cytodex3 microcarriers was observed dynamically under phase contrast microscope and SEM . The differentiation of ADSCs into chondrocyte was determined by histochemical stain including Safranin-O, tolidine blue and immunocytochemical analysis for type II collagen. (3) ADSCs was seeded into the ACAM scaffolds (diameter 4mm, thickness 2mm)at a final cell density of 5×10⁷ /mL. The cells/scaffold compounds were incubated with chondrogenic medium in the RCCS at 37℃, 5%CO₂ for 21 days. The parallel static culture was performed as control group. Gross appearance , histological analysis, histochemistry stain including Safranin-O and Alcian blue, immunohistochemistry stain were investigated after 3 week.. (4) The cells/scaffold compounds which constructed with induced ADSCS or common ADSCs were incubated for 1 week at 37℃, 5%CO₂ incubator, induced MSCs as control, and then implanted respectively into subcutaneous pockets on the backs of 5 athymic mice. The composites were harvested and examined with histology at 3 weeks after implantation. ( 5 ) A full-thickness articular-cartilage defect (4mm in diameter) was created in the patellar grove of distal femur of rabbits. The rabbits were divided into six groups:(A) the defects were left empty.(B) filled with ACAM. (C) ADSCs/scaffold. (D) induced ADSCs/scaffold. (E) induced MSCs/scaffold. (F) ADSCs induced in RCCS/scaffold. Specimens were harvested at 3, 6and 12 months postoperatively, and assessing the reconstituted defects grossly, histologically, biochemically and biomechanically.Results: ADSCs was havested after passaged three to two weeks while MSCs three weeks in vitro. Type I collagen was identified in all ADSCs and MSCs without induced. Type II collagen was identified in induced ADSCs and MSC. The proliferation of these cells was same. The quantity of secreting GAG and collagen II in induced ADSCs is higher than ADSCs while the same as induced MSCs cells. The ADSCs induced in RCCS attached rapidly to the surface of Cytodex3 microcarriers in 24h. Quick growth of these cells was observed after they fully spread onto themicrocarriers. The density of ADSCs increased about 20 times in later stage of culture as compared with the initial density. Histochemical and Immunocytochemical analysis revealed that Safranin-O, tolidine blue and II collagen stain were obviously positive. The cells/scaffold compounds were incubated with chondrogenic medium in the RCCS preserved a better mechanical strength than control group and the most part of scaffold had been absorbed with a significant cell proliferation. Histological stain showed a large mount extracellular matrix around the cells. Histochemical and immunohistochemical analysis revealed that Safranin-O, and II collagen stain were obviously positive. Histological examination showed that the composite constructed with cytokines induced ADSCs or MSCs qualified osteochondrogenic capacity in nude mice model. In animal experimental, results indicated that every group have been repaired by fibrocartilage or hyaline cartilage in different degree. By histological and histochemical grading scale evaluation of area of defect, the method filled with ADSCs induced in RCCS/scaffold was the best way to repair full-thicks cartilage defect. The induced ADSCs and MSCs which were no apparent difference between them but better than other groups. The same results can be seen in biochemical and biomechanical testing. Conclusions:(1) The methods to isolate, culture and induce ADSCs into chondrocytes have been established successfully.(2) Microcarrier culture of ADSCs in RCCS can yield a large quantity of cells within a short time and a successful differentiation into chondrocyte at the same time .(3) Bioreactor can efficient promote the cell proliferation and chondrogenic differentiation, and it is a preferable method for cartilage differentiation of three — dimension biomaterial in vitro.(4) This study demonstrated that construction of a tissue-engineered osteochondral graft with ADSCs and ACAM can repair articularcartilage and subchondral defects. (5) ADSCs is qualified as the seed-cell of cartilage tissue engineering. Thestudy indicates ADSCs shows the same chondrogenic potential asMSCs.