Objective To investigate the feasibility of cartilaginous implantscontaining bone marrow stromal cells(MSCs) derived from chondrocytes in biological resurfacing procedures for repairing articular cartilage defect. Methods MSCs derived from chondrocytes were obtained with high initial cell density subculture. An implant was constructed by dispersing the chondrocytes in a acid soluble type Ⅰ collagen gel(5×106cells/ml, final cell concentration). A fullthickness defect 3 mm×5 mm was created in the trochlear groove of femur in 36 rabbits. A piece of cotton soaked in 0.5% trypsin was laid into the defect for 5 minutes, then the defect was filled with MSC/collagen gel implant on one side(n=36), filledwith a plain collagen gel on the other side(n=18),and left empty as controls on the other side(n=18). The animals were sacrificed at 4, 8, 12, 24, 32,and 48 weeks. The repaired tissue was examined and evaluated with Pineda gradingscale. Results In MSCs group, the implanted cells resembled well differentiated chondrocytes and were surrounded by metachromatic matrix and the reparative tissue resembled hyaline cartilage after 4 weeks; bone was formed at the base of the defects, the thickness of new cartilage was larger than tht of normal one after 8 weeks; the thickness was reduced proximally, approximating to that of normal cartilage, and chondrocyte columns was formed and subchondral bone and tidemark reappeared after 12 weeks; the thickness of the new tissue was about 55% of the normal tissue, with smooth surface and there were hypertrophic chondrocytes near the tidemark after 24 weeks; no hypertrophic chondrocytes were observed, indicating cessation of endochondral ossification after 32 weeks; the tissue architecture was the same as that at 32 weeks, hyaline-like cartilage persisting, with subchondral bone and tidemark in continuity after 48 weeks. The four layer cell orientation was not as clear as that of normal cartilage. The defects were partially filled with fibrous tissue in controls. At 32 weeks, erosive cartilage, naked subchondralbone and proliferative synovial membrane indicated the presence of osteoarthrosis. There were no statistical difference according to Pineda tissue scales in the specimens from the MSCs group between 24, 32, and 48 weeks, but there was significant difference between 4 weeks and 24, 32 and 48 weeks (Plt;0.05). The joint function recovered after 2 weeks in MSCs group, while it deteriorated progressively incontrols. Conclusion MSCs derived from chondrocytes improve repair of largefullthickness defect in articular cartilage. The reparative hyaline-like cartilage is stable differentiation after 24 weeks, maintains good joint function after 48 weeks.
Superficial cartilage defect is an important factor that causes osteoarthritis. Therefore, it is very important to investigate the influence of superficial cartilage defects on its surface morphology and mechanical properties. In this study, the knee joint cartilage samples of adult pig were prepared, which were treated by enzymolysis with chymotrypsin and physical removal with electric friction pen, respectively. Normal cartilage and surface treated cartilage were divided into five groups: control group (normal cartilage group), chymotrypsin immersion group, chymotrypsin wiping group, removal 10% group with electric friction pen, and removal 20% group with electric friction pen. The surface morphology and structure of five groups of samples were characterized by laser spectrum confocal microscopy and environmental field scanning electron microscopy, and the mechanical properties of each group of samples were evaluated by tensile tests. The results show that the surface arithmetic mean height and fracture strength of the control group were the smallest, and the fracture strain was the largest. The surface arithmetic mean height and fracture strength of the removal 20% group with electric friction pen were the largest, and the fracture strain was the smallest. The surface arithmetic mean height, fracture strength and fracture strain values of the other three groups were all between the above two groups, but the surface arithmetic mean height and fracture strength of the removal 10% group with electric friction pen, the chymotrypsin wiping group and the chymotrypsin soaking group decreased successively, and the fracture strain increased successively. In addition, we carried out a study on the elastic modulus of different groups, and the results showed that the elastic modulus of the control group was the smallest, and the elastic modulus of the removal 20% group with electric friction pen was the largest. The above study revealed that the defect of the superficial area of cartilage changed its surface morphology and structure, and reduced its mechanical properties. The research results are of great significance for the prevention and repair of cartilage injury.
Objective Melatonin (MLT) can increase the expression of cartilage-derived growth factor and stimulate the synthesis of cartilage matrix. To investigate the prevention and treatment effects of MLT on damaged cartilage through observing the expressions of bone morphogenetic protein 2 (BMP-2) and interleukin 1β (IL-1β) in articular cartilage of the rats with osteoarthritis (OA). Methods Forty SPF 4-week-old male SD rats (weighing 120-150 g) were randomly divided into 4 groups (n=10): normal control group (group A), OA group (group B), OA/pinealectomy group (group C), and OA/ pinealectomy/MLT group (group D). The rats of group A served as a control without treatment. The rats of groups B, C, andD underwent left knee joint injection of 0.2 mL 4% papain solution 1 time every other day for 2 weeks for establ ishing OAmodel. Two weeks after papain injection, the rats of groups C and D were exposed to continuous l ight for 24 hours (intensity of illumination: 500 lx) for creating pinealectomy models. And at the next day after pinealectomy model establ ishing, the rats of group D were treated with intra-articular injections of 0.2 mL 20 mg/mL MLT solution 4 times a week for 4 weeks. At 1 week after last MLT injection, the venous blood samples were taken in groups A, B, and C to test the level of serum MLT by ELISA, respectively, and then the specimens of left cartilage of femoral condyle were harvested for macroscopic, histological, and immunohistochemical examinations in 4 groups. Results The OA and pinealectomy models of rats were successfully establ ished, and all rats survived. There were significant differences in the serum MLT level among groups A, B, and C, and among different time points at the same group (P lt; 0.05). In group A, articular cartilage surface was smooth and elastic, and chondrocytes arranged regularly. In groups B and C, articular cartilage surface was rough, cartilage defects and subchondral bone exposure were observed in some areas, and chondrocytes arranged irregularly. In group D, cartilage surface was more smooth than that in groups B and C, and the degrees of cartilage defect and subchondral bone exposure decreased with regular arrangment of chondrocytes. There were significant differences in Mankin scores and integral absorbance values among 4 groups (P lt; 0.05). Conclusion Exposure to continuous l ight can accelerate degeneration process of articular cartilage of OA rats. Injections of 0.2 mL MLT solution (20 mg/mL) by intra-articular for 4 weeks can inhibit the progress of cartilage defects. Upregulationof anabol ic factor of BMP-2 as well as down-regulation of catabol ic factors of IL-1β is associated with cartilage repairin the pathological features of OA.
Objective To study the effect of adenovirus bone morphogenetic protein 2 gene(Ad-BMP-2) transfer inducing mesenchymal stem cells (MSCs) compounded with fibrin gel on repair of rabbit cartilage defect. Methods ①BMP-2 and collagen type Ⅱ in MSCs transferred by Ad-BMP-2 were examined by RT-PCR, aniline dyeing and immunohistochemical analysis in vitro. ②MSCs were cultured in fibrin gel for 9 days, and were examined with electron microscope. ③Fortytwo rabbits suffering from cartilage defect were divided into 3 groups:the defects were treated with Ad-BMP-2 transfer inducing MSCs compounded with fibrin in group A, with MSCs compounded with fibringel in group B and with no implants in group C as control. HE and aniline dyeing, immunohistochemical analysis and biomechanics study were carried out in the 4th, 8thand 12th weeks. Results ①The positive results were observed for BMP-2 and collagen type Ⅱ with RT-PCR on the 3rd day and 5th day respectively, being statisticallysignificant difference when compared with control group(P＜0.05). ②Ad-BMP-2 transfer inducing MSCs cultured in fibrin gel were positively stained by aniline dyeing and immunohistochemstry. ③The therapy effect of group A was better than that of the other two groups in histology, biochemistry and biomechanics, and the biomechanic and histological features of repaired cartilage were similar to those of the natural cartilage. Conclusion Ad-BMP-2 can induce the expressionof collagen type Ⅱ and mucopolysaccharide in MSCs by secreting BMP-2, and can reconstruct articular cartilage defects better when compounded with fibrin gel.
Objective To compare biological characteristics between articular chondrocyte and meniscal fibrochondrocyte cultured in vitro andto investigate the possibility of using cultured cartilage as a substitute for meniscus.Methods Chondrocytes isolated from articular cartilage and meniscus of rabbits aged 3 weeks were respectively passaged in monolayer and cultured in centrifuge tube. Cartilages cultured in centrifuge tube and meniscus of rabbit aged 6 weeks were detected by histological examination and transmission electron microscopy. Growth curves of articular chondrocytes and meniscalfibrochondrocytes were compared; meanwhile, cell cycles of articular chondrocytes and meniscal fibrochondrocytes in passage 2and 4 were separately measured by flow cytometry.Results Articular chondrocytes in passage 4 were dedifferentiated. Articular chondrocytes formed cartilage 2 weeks after cultivation in centrifuge tube, but meniscal fibrochondrocytes could not generate cartilage. The differences in ultrastructure and histology obviously existed between cultured cartilage and meniscus; moreover, apoptosis of chondrocytes appeared in cultured cartilage. Proportion of subdiploid cells in articular chondrocytes passage 2 and 4 was markedly higher than that in passage 2 and 4 fibrochondrocytes(Plt;0.05). Conclusion Meniscal fibrochondrocytes can not form cartilage after cultivationin centrifuge tube, while cartilage cultured in centrifuge tube from articular chondrocytes can not be used as graft material for meniscus. Articular cartilage ismarkedly different from meniscus.
Objective To construct recombinant lentiviral expression vectors of porcine transforming growth factor β1 (TGF-β1) gene and transfect bone marrow mesenchymal stem cells (BMSCs) so as to provide TGF-β1 gene-modified BMSCs for bone and cartilage tissue engineering. Methods The TGF-β1 cDNA was extracted and packed into lentiviral vector, and positive clones were identified by PCR and gene sequencing, then the virus titer was determined. BMSCs were isolated frombone marrow of the 2-month-old Bama miniature pigs (weighing 15 kg), and the 2nd and 3rd generations of BMSCs wereharvested for experiments. BMSCs were then transfected by TGF-β1 recombinant lentiviral vectors (TGF-β1 vector group)respectively at multi pl icity of infection (MOI) of 10, 50, 70, 100, and 150; then the effects of transfection were detected bylaser confocal microscope and Western blot was used to determine the optimal value of MOI. BMSCs transfected by empty vector (empty vector group) and non-transfected BMSCs (non-transfection group) were used as control group. RT-PCR, immunocytochemistry, and ELISA were performed to detect the expressions of TGF-β1 mRNA, TGF-β1 protein, and collagen type II. Results Successful construction of recombinant lentiviral vectors of porcine TGF-β1 gene was identified by PCR and gene sequencing, and BMSCs were successfully transfected by TGF-β1 recombinant lentiviral vectors. Green fluorescence was observed by laser confocal microscope. Western blot showed the optimal value of MOI was 70. The expression of TGF-β1 mRNA was significantly higher in TGF-β1 vector group than in empty vector group and non-transfection group (P lt; 0.05). Immunocytochemistry results revealed positive expression of TGF-β1 protein and collagen type II in BMSCs of TGF-β1 vector group, but negative expression in empty vector group and non-transfection group. At 21 days after transfection, high expression of TGF-β1 protein still could be detected by ELISA in TGF-β1 vector group. Conclusion TGF-β1 gene can be successfully transfected into BMSCs via lentiviral vectors, and long-term stable expression of TGF-β1 protein can be observed, prompting BMSCs differentiation into chondrocytes.
In order to investigate the effect of motion on repairing articular cartilage defect following autogenous periosteal graft, sixty adult rabbits were divided randomly into three groups: out-cage motion (OCM), in-cage motion (ICM) and immobilization (IMM). A defect of the articular cartilage, 1 cm x 0.5 cm in size, was made in the patellar-groove of femur of each hind limb. Free autogenous periosteal graft from the proximal tibia was sutured on the base of the left defect, while the right limb was served as control. The animals were sacrificed at 4, 8 and 12 weeks, respectively, after operation. The regeneration of the cartilage implanted was observed through gross, histology, histochemical assay and electronic microscope. The influence of different amount of motion on the chondrogenesis from the periosteal implant was also compared. The result showed that the hyaline cartilage produced from periosteal implant could be capable to repair full-thickness of articular cartilage. From statistical study, there was significant difference between OCM and ICM groups (P lt; 0.05), ICM and IMM (P lt; 0.05) as well as OCM and IMM (P lt; 0.01). It was suggested that the periosteal graft was effective in repair of defect of articular cartilage and the amount of motion was important for chondrogenesis.
ObjectiveTo explore the relationship between subchondral bone reconstruction and articular cartilage regeneration in a rabbit model of spontaneous osteochondral repair.
MethodsTwenty-four 6-month-old New Zealand white rabbits were included. The osteochondral defects (4 mm in diameter and 3 mm in depth) were created in the trochlear groove of the unilateral femur, which penetrated the subchondral bone without any treatment. The rabbits were sacrificed at 1, 4, 12, and 24 weeks after operation, respectively. The specimens were obtained for macroscopic, histological, and immunohistochemical observations. According to the International Cartilage Repair Society (ICRS) histological scoring, the effect of cartilage repair was assessed. The histomorphometrical parameters of subchondral bone were analyzed by micro-CT scan and reconstruction, and the relationship between cartilage repair and the histomorphometrical parameters of the subchondral bone were also analyzed.
ResultsOsteochondral defects could be repaired spontaneously in rabbit model. With time, defect was gradually filled with repaired tissue, subchondral bone plate under the defect region gradually migrated upward. Bone mineral density, bone volume fraction, tissue mineralized density, trabecula number, and trabecula thickness were increased, while trabecula spacing was decreased. Significant difference was found in the other parameters between different time points (P<0.05) except for trabecula thickness between at 4 and 12 weeks after operation (P>0.05). Histological examination showed that fibrous repair was predominant with rare hyaline cartilage. With time, ICRS scores increased gradually, showing significant differences between other time points (P<0.05) except for between at 4 and 12 weeks after operation (P>0.05). Among the histomorphometrical parameters of subchondral bone, the trabecula spacing was negatively correlated with ICRS score (r=-0.584, P=0.039), and the other histomorphometrical parameters were positively correlated with ICRS score (r=0.680-0.891).
ConclusionThere is relevant correlation as well as independent process between cartilage regeneration and subchondral bone reconstruction in the rabbit model of spontaneous osteochondral repair, and fast subchondral bone remodeling may adversely affect articular cartilage repair.
The repair of defects of articular cartilage has continued to be a difficult problem. This article provided a collective review from literature pertaining to the advances gained in the repair of cartilaginous defects. In the spontaneous repair, if the defect of the cartilage was less than 3 mm, might result in complete or partial repair, but in those the diameter was more than 3 mm, the defect could not be repaired by normal cartilage. Although the cartilaginous autograft could give good result, but it could not be widely applied because short of supply of the autogenous cartilage. Cartilagious allograft could not be taken to repair cartilaginous defect because of reaction from tissue rejection. The transplantation of periosteal or perichondral graft had been tried but was eventually abandoned because of poor long-term result. The transplantation of free chondrocytes might be a method of hope. In general, transplantation of free chondrocytes into the cartilaginous defect will be lost. The supply of autogenous chondrocytes was very limited, and the heterogenous chondrocytes would inflict immunoreaction after being transplanted. In late of 1980, a new concept of tissue engineering was proposed. The problem that a scaffold of appropriate material which could hold the free chondrocytes in place from being lost might undergo proliferation and differentiation into new cartilage was far from being solved. Although tissue engineering still had various problems needed further investigation, but it will probably be the main direction of development in this field.
OBJECTIVE To investigate the possibility of repairing the cartilage cartilage defect with homogeneous chondrocytes combined with Pluronic. METHODS: Homogeneous cartilage chondrocytes of adult New Zealand rabbits were harvested and cultured in vitro, which were marked by 3H-TdR and mixed with Pluronic. The medial or lateral condyle defects were made (phi 4 mm, extending down to the calcified zone) in 20 rabbits. In the experimental group, the right defects were repaired by homogeneous chondrocytes combined with Pluronic; in the control group, the left defects were repaired by Pluronic only or were left un-repaired. The animals were sacrificed in the 4th, 8th and 16th weeks after operation respectively. The repair results were observed and the cell source of repair tissue was distinguished. RESULTS: In the experimental group, the cartilage defects were repaired by the cartilage-like tissue after 8 weeks of operation; the defects were completely filled with mature cartilage tissue, which integrated smoothly with articular cartilage 16 weeks later. In the control group, only a small amount fibrous tissues were seen on the surface of defects. Autoradiographic assessment showed that the repair cells came from the implants, but not from self-chondrocytes. CONCLUSION: It is a good way to repair articular cartilage defects with homograft of tissue engineering cartilage. It is a convenient method to mark with 3H-TdR to discriminate the resource of the repair cells.
