An Experimental Study Of Uninduced Adipose-Derived Stem Cells Repairing Defects Of Knee Articular Cartilage In Rabbit

Objective:Because adipose-derived stem cells (ADSCs) can be easily obtained in large quantities and with minimal discomfort, there has been increased interest in ADSCs for cartilage tissue engineering. But during chondrogenesis from ADSCs by traditional methods using growth factors, inadequate differentiation and hypertrophic differentiation are two important limitations. Furthermore, these methods are expensive. Recently, microenvironment has been proven to direct the progression of ADSCs into differentiated cells in vivo. And in addition to multilineage differentiation , ADSCs could secrete cytokines to repair damaged tissue . So the transplantion of uninduced ADSCs directly to repair defects of articular cartilage is possibly better. But it is unclear whether ADSCs can be induced into chondrocyte in articular microenvironment in pathologic state. Although chondrocytes can provide chondrogenic microenvironment to induce chondrogenic differentiation of BMSCs, it is unknown whether chondrocytes in pathologic state can induce chondrogenic differentiation of ADSCs. The purpose is to investigate the feasibility of ADSCs induced to differentiate into chondrocytes in pathologic microenvironment to repair defects of articular cartilage in vivo by co-culture with chondrocytes from osteoarthritis model in adult New Zealand white rabbits and animal test , and to provide a novel way for chondrogenesis of adipose-derived stem cells in vitro to construct engineered cartilage-like tissue.Materials and methods:1. Isolation and expansion of ADSCs were manipulated. 2. The appearance of ADSCs was observed by Oil Red O staining.3. The proliferation of ADSCs was detected by their growth curves before and after cryopreservation. 4. ADSCs were induced into osteogenic and chondrogenic differentiation in vitro and detected by von Kossa staining and toluidine blue staining, repectively. 5. ADSCs and articular chondrocytes from osteoarthritis model in adult New Zealand white rabbits were expanded and then seeded on plate and plug-in type millicell dish respectively. The co-culture cells were cultured in different conditions including different serum concentration (10% FBS and 2% FBS) and dimensions (fibrin scaffold and monolayer). The culture medium was changed every 3 days.7. The appearance of ADSCs before and after co-culture was observed by inverted microscope and transmission electron microscope.8. The expression of aggrecan and type II collagen genes in ADSCs were studied by toluidine blue staining and immunohistochemistry after co-culture for 14 days in vitro.9. The transcription of aggrecan , type II collagen, typeⅠcollagen and typeⅩcollagen genes in ADSCs were studied by RT-PCR.10. Fresh and old osteochondral defects were created in the femoral condyle of rabbits .These rabbits were divided into two groups : ADSCs with fibrin scaffold group and fibrin scaffold alone group. The test defects were filled with ADSCs with fibrin scaffold and the control defects were filled with fibrin scaffold alone. Specimens were harvested at 6 and 12 weeks postoperatively for gross observation and histological testing.Results:1. ADSCs were successfully obtained from adult New Zealand white rabbits and appeared fibrolast-like in the progress of culture. Their high proliferation rate and multilineage differentiation capacity were comfired in vitro. 2. The ADSCs showed no significant decrease in their proliferation after cryopreservation. 3. ADSCs became round at 7～9 days after in vitro co-culture. At 14 days after co-culture with chondrocytes, ADSCs was changed to chondrocyte-like cells morphologically and increased immunostaining particles of type II collagen and enhanced toluidine blue staining. And the transcription of type II collagen and aggrecan genes was also increased ,especially in the ADSC cultured in fibrin scaffold with 10% FBS. The transcription of typeⅠcollagen was showed in all groups, but the transcription of typeⅩcollagen genes never been found. 4. Gross osteochondrol defect reconstitution and histological grading in ADSCs with fibrin scaffold group was superior to fibrin alone repair both in fresh and old injure of articular cartilage.Conculsion:1. The ADSCs obtained could proliferate rapidly and be capable of multilineage differentiation. Moreover, the ADSCs showed no significant decrease in their proliferation after cryopreservation. So as seed-cell , ADSCs maybe well-suited for cartilage tissue engineering. 2. Chondrocytes in pathologic state can secrete some soluble cytokines to provide a chondrogenic microenvironment for chondrogenic differentiation of ADSC in vitro, which support the hypothesis that ADSCs could be induced to differentiate into chondrocytes in pathologic microenvironment of articular cartilage in vivo. 3. The co-culture of ADSCs with chondrocytes could be promoted to differentiate into chondrocytes by high serum and 3D culture., which can improve to construct engineered cartilage-like tissue in vitro. 4. The transplantation of uninduced ADSCs directly could repair damaged articular cartilage by induced to differentiate into chondrocytes in vivo.