| [Backgrounds]Cartilage is a kind of connective tissue with a single cell component,which is composed of a single chondrocyte,fiber and matrix.In vivo,cartilage mainly plays the physiological functions of connection,support,stress dispersion and vibration reduction.However,the inevitable occurrence of pathological phenomena such as trauma,inflammation and degeneration can easily lead to different degrees of injury or defect of cartilage tissue,and even lead to the occurrence of arthritis in serious cases,which has a great impact on the normal function of the joint and also brings inconvenience to the normal life of patients.Because the regeneration ability of chondrocytes is very limited,when cartilage defects need to be repaired by other ways,the research on the repair and treatment of cartilage damage is also the most important in orthopedics and clinical research.The repair process of cartilage defect is very complex,but the nuclear regulatory mechanism has not been revealed.?-tricalcium phosphate bio ceramic scaffold loaded by allogeneic chondroblasts plays an irreplaceable role in the repair of cartilage defects.The research of allogeneic chondrocyte osteoblasts is still in the exploratory stage,and it still has a long way to be widely used in clinical practice,which needs the comprehensive cooperation of a variety of detection methods.As for?-tricalcium phosphate bioceramic scaffolds,the choice of materials is the central part of the treatment of cartilage defects.Therefore,researchers need to weigh the risks and benefits of treatment,in order to provide a more secure guarantee for future clinical application.We believe that with the further combination and promotion of the technology,it will provide practical clinical guidance for the treatment of cartilage defects with allogeneic chondroblasts loaded with ?-tricalcium phosphate bioceramic scaffolds.The application of this comprehensive technology can provide scientific theoretical guidance for the patients with cartilage defects to formulate the corresponding treatment plan,so that the patients can recover as soon as possible,which lays a solid theoretical foundation for further research on cartilage defects,and can provide new treatment ideas.[Objects]In this study,firstly,the histocompatibility of allogeneic chondrocytes/osteoblasts loaded with ?-tricalcium phosphate(?-TCP)bioceramic scaffolds with cartilage was studied.Secondly,to explore the application value of 3D Artificial micro/nano scaffold in joint tissue repair.Finally,the effects of ?-tricalcium phosphate bioceramic scaffold on the energy metabolism and proliferation of chondrocytes were studied.[Methods]Part ?The bone marrow mesenchymal stem cells from beagle dogs were extracted and differentiated into chondrocytes and osteoblasts.The experimental cells were divided into three groups:control group,chondrocyte group and osteoblast group;experimental dogs were divided into three groups:?-TCP group(blank ?-TCP scaffold was placed in cartilage defect as experimental control),chondrocyte ?-TCP group(the cultured chondrocytes were fused with ?-TCP scaffold and then put into cartilage defect model dogs),chondrocyte/osteoblast ?-TCP group(the cultured chondrocytes were fused with ?-TCP scaffold)Three groups of experimental beagles were fed under the same conditions and killed 12 weeks later for experimental observation.Cell proliferation was detected by MTT.The apoptosis and viability of cells were detected by flow cytometry.The content of glycosaminoglycan(GAG)was determined by alcian blue.Western blotting was used to analyze the protein expression of col ? and BMP.Part ?The chondrocytes/osteoblasts loaded ?-TCP calcium phosphate bioceramic scaffolds and chondrocytes loaded ?-TCP bioceramic scaffolds were prepared through the culture of micro aggregate stem cells and the cell-loaded scaffolds based on bioreactor to treat the articular cartilage defects.The cartilage defects were repaired by the ?-TCP calcium phosphate bioceramic scaffolds loaded with allogeneic chondrocyte s/osteoblasts ?-TCP bioceramic scaffolds loaded with bone cells were set as positive control.Part ?Twenty four healthy beagles aged 4-10 months and weighing 7.5-10.0 kg were selected.The femoral trochlear of beagles was used as the research model of articular cartilage defect.The chondrocytes of costal cartilage and osteoblasts of periosteum were extracted from beagle dogs to ensure the homology.The experiment was divided into two groups:control group(cartilage defect model,implanted with ?-TCP bioceramic stent,n=12);complex group(cartilage defect model,implanted with allogeneic chondrocytes/osteoblasts loaded with ?-TCP bioceramic stent,n=12).The proliferation of cells on scaffolds was observed by tissue sections and microscopes.The levels of IL-1,IL-6 and TNF-? in chondrocytes were measured by ELISA.The content of type ?collagen and bone morphogenetic protein in chondrocytes of different treatment groups were analyzed by immunohistochemistry.The expression of transforming growth factor beta(TGF-?),insulin-like growth factor 2(IGF-2)and GLUT protein were analyzed by Western blot.The expression of hypoxia inducible factor-1?(HIF1 ?),amp activated kinase(AMPK)and ERK mRNA were analyzed by Western blot.Western blot analysis of glycolysis,oxidative phosphorylation,PPP and glycogen synthesis pathway key enzymes,3-phosphoglyceraldehyde dehydrogenase(GAPDH),nicotinamide adenine dinucleotide phosphate(NADPH)and glycogen synthase kinase 3? in different treatment groups 3.GSK3?)protein expression.LC3 and beclin-1 levels of chondrocytes in different treatment groups were detected by enzyme-linked immunosorbent assay.[Results]Part ?After 4 hours in culture,the chondrocyte group had higher cell proliferation than the control group(P<0.05),the proliferation of osteoblasts was higher than that of the control group(P<0.05).After 24 hours of culture,the cell proliferation of the chondrocyte group was higher than that of the control group(P<0.05),and that of osteoblasts was higher than that of the control group(P<0.05).Chondrocyte group and osteoblast group had lower apoptosis than control group(P<0.05),and osteoblast had lower apoptosis rate than chondrocytes(P<0.05).Compared with the control group,the chondrocyte group and osteoblast group had higher cell viability(P<0.05),and the osteoblasts had higher viability than chondrocytes(P<0.05).The content of GAG in chondrocyte group and osteoblast group was higher than that in control group(P<0.05).There was no difference in the content of GAG in chondrocyte group and osteoblast group(P>0.05).The expression of Col ? protein was higher in the chondrocyte group than in the control group(P<0.05),and the protein of Col ? in the osteoblast group was lower than that in the chondrocyte group(P<0.05).Elevated(P<0.05),there was no difference in BMP protein expression between the control group and the chondrocyte group(P>0.05).The cartilage/osteoblast ?-TCP group had a higher cell morphology score than the chondrocyte?-TCP group(P<0.05),and the chondrocyte ?-TCP group had a higher cell morphology score than the ?-TCP group(P<0.05).The cartilage/osteoblast ?-TCP group had a higher surface morphology score than the chondrocyte ?-TCP group and ?-TCP group(P<0.05).Compared with chondrocyte ?-TCP group and ?-TCP group,the expression of IL-1? in cartilage/osteoblast ?-TCP group was decreased(P<0.05).The expression of TNF-? mRNA in ?-TCP group was higher than that in chondrocyte ?-TCP group and cartilage/osteogenic ?-TCP group(P<0.05).The cartilage/osteoblast ?-TCP group had lower IL-6 mRNA expression than the chondrocyte ?-TCP group and ?-TCP group(P<0.05).Part ?Chondrocytes and osteoblasts can be successfully induced from allogeneic bone marrow stromal cells by culturing them in vitro.The?-TCP bioceramic scaffolds of chondrocytes/osteoblasts,?-TCP bioceramic scaffolds of chondrocytes,?-TCP bioceramic scaffolds of chondrocytes and ?-TCP bioceramic scaffolds were successfully prepared by cell-loaded scaffolds culture based on bioreactor.The scaffold was used in the treatment of articular cartilage defect in beagle dogs.The relative cartilage regeneration ability of beagle dogs' femoral trochlear is as follows:?-TCP bioceramic scaffolds of chondrocytes/osteoblasts,?-TCP bioceramic scaffolds of chondrocytes,?-TCP bioceramic scaffolds of chondrocytes,?-TCP bioceramic scaffolds.Part ?Compared with the control group,the cell proliferation rate of the complex group increased at 1 week,3 weeks,5 weeks and 8 weeks(P<0.05).Compared with the control group,the levels of IL-1,IL-6 and TNF-? in the complex group decreased(P<0.05).Compared with the control group,the content of type ? collagen and bone morphogenetic protein in the complex group increased(P<0.05).Compared with the control group,the expression of TGF-?,IGF-2 and GLUT protein in the complex group increased(P<0.05).Compared with the control group,the expression of HIF-1?,AMPK and ERK protein in the complex group increased(P<0.05).Compared with the control group,the expression of GAPDH,NADPH and GSK3 ? protein in the complex group decreased(P<0.05).Compared with the control group,the levels of LC3 and beclin-1 in the complex group decreased(P<0.05),indicating that the complex inhibited autophagy.[Conclusions]1.To explore the feasibility of cartilage defects with BMSCs-derived Allogeneic chondrocyte osteoblast-loaded?-tricalcium phosphate bioceramic scaffolds.A llochondrocyte/osteoblast-loaded ?-TCP bioceramics scaffolds can produce good biocompatibility with surrounding cartilage.2.Micro mass stem cell culture and bioreactor based cell loaded scaffold culture can be used to prepare chondrocyte/osteoblast loaded ?-TCP bioceramic scaffolds for the treatment of articular cartilage defects.This suggests that ?-TCP bioceramic scaffold loaded with allogeneic chondrocytes/osteoblasts can be potentially used in the treatment of cartilage defects.3.Allogeneic chondrocyte osteoblast-loaded ?-tricalcium phosphate bioceramic scaffolds increase chondrocyte energy metabolism and proliferation,and reduce cell autophagy. |
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