| IntroductionArticular cartilage defect is a common clinical disease.As the articular cartilage lacks of blood vessels,nerves,and lymphatic system,and contains few chondrocytes with limited migration and proliferation ability,once it is injured,it would be difficult to realize self-healing.If not treated promptly and effectively,the injury would continue to intensify and cause osteoarthritis,resulting in joint pain and swelling,or movement disorders.The current common clinical treatments for articular cartilage defect are micro fractures and autologous chondrocyte transplantation,but there is no treatment method that has been proved to be able to successfully restore normal hyaline cartilage,and the cartilage repair tissue is mostly fibrocartilage.In the past few decades,with the deepening of research and the development of technology,tissue engineering technology has become a promising method for cartilage repair compared with other treatments.Bone marrow mesenchymal stem cells(BMSCs)has the advantages of convenient access,good proliferation and differentiation ability,no immune rejection;it can differentiate into cartilage cells with appropriate biochemical and physical stimuli,making it an ideal cell source for cartilage repair.So far,there are abundant researches using BMSCs as seeding cell for tissue engineered cartilage,but many studies mainly focus on the repair effect,the selection of scaffold materials and bioactive factors to promote and induce the differentiation of BMSCs into chondrocytes;it still lacks sufficient study on the survival and prognosis of BMSCs during the cartilage repair process.By enhancing these understanding,it would help us to objectively evaluate the result of cartilage tissue engineering based on BMSCs and optimize this technology,contributing to the future clinical application of BMSCs.The purpose of this study is to apply the type II collagen-hyaluronic acid-chondroitin sulfate oxidation(Col II-HA-OCS)biomimetic extracellular matrix gel developed by the research group to simulate the extracellular matrix of normal cartilage using carboxyfluorescein(5,6-carboxyfluorescein diacetate succinimidyl ester,from CFDA SE)labeled autologous BMSCs as seed cells to repair porcine articular cartilage defect and explore the survival,proliferation and differentiation of BMSCs transplantation in the repair tissue.Methods1.12 Ba Ma mini pigs were used in this research.The porcine BMSCs were isolated from bone marrow blood.After cultured P2,BMSCs were labeled by CFDA SE and the labeling rate was evaluated.2.One knee joint of each pig was chosen and used for experiment,totaling 12 knees.Surgical incision of the joint capsule was done to expose the surface of the trochlear joint of the femur.The defect in the weight-bearing area of the cartilage was drilled with 8mm hollow ring,the cartilage in the defect area was debrided and the model of articular cartilage defect was completed.The 12 knees were randomly divided into 3 groups,4 knees in each group.Group A was the control group with no treatment of defect;group B was acellular biomimetic gel group;group C was autologous BMSCs complex biomimetic gel group,with autologous BMSCs labeled by CFDA SE.3.One month after the operation,5-bromo-2-deoxyuridine(Brd U)labeling fluid was injected intravenously into the experimental animals follow 25mg/Kg,24 h and 48 h before specimens were taken from the executed animals.The gross morphological and histological observations were carried out and scored by ICRS gross morphological score and ICRS histological score.Partial cartilage repair tissue was taken in the autologous BMSC biomimetic composite gel group for frozen sections;DAPI staining and Brd U immunofluorescence staining was conducted;confocal laser scanning microscope was used to observe,count,and calculate the cell survival and cell ratio accounted for proliferative viable engraftment rate.Results1.The instant marking rate of the CFDA SE labeled mini pig BMSCs was(99.34 ± 0.43)%,followed by(98.72 ± 0.31)%,(98.51 ± 0.45)%,(98.33 ± 0.37)%,and(98.17 ± 0.34)% on 1,3,5 and 7 d.2.After one month,there was no filler in the defect area of the control group;there was a small amount of fibrous tissue at the bottom of the defect area of the acellular biomimetic gel group with good repairing effect and low content of cells near the edge of defect;the defect area of the autologous BMSCs biomimetic complex gel group was filled with cartilage like tissue,which was well integrated with the surrounding normal cartilage,the cell content was more and of irregular distribution,a small amount of cartilage lacuna could be seen,and the histological staining result was similar with the adjacent normal cartilage.3.The repaired tissue of autologous BMSCs complex biomimetic gel group was observed and calculated by laser scanning confocal microscopy,the ratio of implanted cells to viable cells was(97.33 ± 2.57)%,and the ratio of implanted viable cells with proliferative capacity was(76.57 ± 2.51)%.4.The ICRS score of gross morphology: the control group(0.50 ± 0.58),acellular bionic gel group(2.25 ± 0.50),autologous BMSCs complex biomimetic gel group(8.25 ± 0.96)points;ICRS histological score: the control group(0.50 ± 0.58),acellular bionic gel group(4.50 ± 1.00),autologous BMSCs complex biomimetic gel group(10.25 ± 2.36)points.The scores of the autologous BMSCs complex bionic gel group were significantly higher than those of the other two groups,the difference was statistically significant(P < 0.05).Conclusion1.CFDA SE labeled porcine BMSCs is an efficient and stable method of cell labeling,and the implanted cells can be clearly observed one month after implantation in animals.2.Autologous BMSCs complex biomimetic gel for repairing porcine articular cartilage defects can achieve satisfactory result close to normal cartilage tissue,which is significantly better than the control group and acellular bionic gel group.3.In the repaired tissue of autologous BMSCs complex biomimetic gel group,the viable cells were basically derived from the implanted BMSCs,the proliferation ability of these cells were decreased,these cells secreted Col II and glycosaminoglycans and gradually differentiated into chondrocytes. |
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