Objective To review the latest progress of seeding cells for articular cartilage tissue engineering. Methods The recent original l iteratures on seeding cells for articular cartilage tissue engineering were extensively reviewed. Results The chondrocytes derived from BMSCs’ differentiation would be a main source of seeding cells articular cartilage for tissue engineering. Three-dimensional scaffolds and cultivation surroundings played important roles in the field of articular cartilage tissue engineering. Conclusion The util ization of cytokine and transgenic technology as well as improvements of three-dimensional scaffolds and cultivation surroundings will promote the development of articular cartilage tissue engineering.
OBJECTIVE To evaluate the results of free auto-periosteal graft in primary repair of cartilage defect accompanying severe comminuted fractured of patella. METHODS From January 1992 to August 1998, seventeen cases with extensive cartilage defect due to severe comminuted fracture of patella were primarily repaired with free auto-periosteal graft. In these cases, there were whole patellar fracture in 9 patients, upper two third patellar fracture in 3 patients and lower two third patellar fracture in 5 patients. During operation, "S"-shaped incision along medial side of knee through intra-cavity pathway were used. After fixation of the patellar fracture and clearance of the residual cartilage in the fracture area, the cancellous bone was exposed and trimmed. The free periosteum was incised from the anterior medial side of upper tibia and then transplanted to the region of cartilage defect. The size of grafted periosteum ranged from 3 cm x 4 cm 5 cm 6 x cm. The knee joint was received passive motion at 7 days after operation. RESULTS All cases were followed up 8 to 74 months. There were excellent recovery in 12 patients and the function of knee joint was normal, better recovery in 4 patients and the function of knee joint was nearly normal, and moderate recovery in 1 patient and the function of knee joint was limited mildly. CONCLUSION Free auto-periosteal graft is a simple and effective treatment in primary repair of cartilage defect accompanying patellar fracture. It is valuable to apply in clinical practice.
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.
OBJECTIVE To investigate the feasibility of repairing the whole layer defects of tibial plateau by implanting tissue-engineering cartilage. METHODS: The chondrocytes of 2-week-old rabbits were cultured and transferred to the 3rd generation, and mixed with human placenta collagen-sponge. The whole layer defects of tibial plateau in adult rabbits were repaired by the tissue-engineering cartilage in the experimental group; the defects were left un-repaired in control group. The repair results of defects were observed after 4, 12 and 24 weeks. RESULTS: In experimental group, no obvious new cartilage formation was seen 4 weeks after operation; some new cartilage formation was found after 12 weeks. Histological observation showed that chondrocytes had irregular edge, honeycombing structure and that cartilage cavities formed around the chondrocytes. After 24 weeks, obvious new cartilage formation was found with smooth surface, and linked with the tissues around it, but the defect was not repaired completely; histological results showed that cartilage cavities formed and that cartilage matrix was stained positively for toluidine blue. In control group, the defect was not repaired. CONCLUSION: The tissue-engineering cartilage can repair the defects of the whole layer cartilage of tibial plateau in rabbits, it is feasible to repair the whole layer cartilage defects of tibial plateau by this method.
To investigate the feasibility of using the pedicled patella for repaire of the superior articular surface of the medial tibial condyle, 37 lower limbs were studied by perfusion. In this series, there were 34 obsolete specimens and 3 fresh specimens of lower legs. Firstly, the vessels which supply to patella were observed by the methods of anatomy, section and casting mould. Then, the form and area of the patellar and tibial medial conylar articular surface were measured in 30 cases. The results showed: (1) the arteries supplied to patella formed a prepatellar arterial ring around patella, and the ring gave branches to patella; (2) medial inferior genicular artery and inferior patellar branches of the descending genicular arterial articular branch merge and acceed++ to prepatellar ring at inferior medial part of patella; (3) the articular surface of patella is similar to the superior articular surface of the tibial medial condyle on shape and area. It was concluded that the pedicled patella can be transposed to medial tibial condyle for repaire of the defect of the superior articular surface. The function of the knee can be reserved by this method.
Objective To investigate the influenceof insulin-like growth factor-I (IGF-I) on biological characteristics of articular chondrocytes cultured in vitro of rabbits. Methods Monolayer articular chondrocytes of 4week old rabbits were cultured in medium with IGF-I, at the concentrations of 3, 10, 30, 100, and300ng/ml. The DNA content in cells and glucuronic acid content in matrix were detected on the 2nd, 4th, 6th days after culture. Results The DNA content in cells and the glucuronic acid content in matrix in articular chondrocytes cultured in medium with IGF-I at concentrations of 3-300ng/ml were all significantly higher than those in control group (P＜0.01), which reached the peak at the concentrations of 30-100mg/ml on the 4th day. Conclusion IGF-I could obviously promote theproliferation of articular chondrocytes in vitro, and there exist time-dependent and dose-dependent effect.
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.
In order to observe the effects of different facing directions of the germinal layer of periosteum on the cartilage regeneration, the human fibrin adhesive agent was used to adhere autogenous periosteum to repair the articular cartilage defect of rabbits. Twentyfour rabbits with 48 knee joints were divided randomly into two groups. A 0.6cm×1.2cm articular cartilage defect was created on the femoral trochlea until there was bleeding from the subchondral bone. A piece of periosteum, sized 0.75cm×1.5cm, was removed from the medial aspect of upper tibia. The periosteum was adhered to the defect by human fibrin adhesive agent. In Group 1 the germinal layer faced the subchondral bone and in Group 2 the germinal layer faced the joint cavity. The cartilage regeneration in both groups was observed by naked eyes and light microscope in 2nd and 6th weeks and by electron microscope after Safronin Ostained in 12th and 20th weeks. The results showed that before the 6th week, the cartilage regeneration was faster in Group 2 than that in Group 1. After that there was no significant difference in regeneration between the two groups. This suggested that the facing direction of the germinal layer was not a critical factor on cartilage regeneration. It was also found that the strength of the adhesive agent was not enough. The regenerated cartilage was proved to be hyaline cartilage.
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.
ObjectiveTo investigate the effects of micro-fracture and insul in-l ike growth factor 1 (IGF-1) in treatment of articular cartilage defect in rabbits. MethodsTwenty-four New Zealand white rabbits (aged, 4-6 months; weighing, 2.5-3.5 kg) were randomly divided into 4 groups (n=6):micro-fractures and recombinant human IGF-1 (rhIGF-1) treatment group (group A), micro-fracture control group (group B), rhIGF-1 treatment control group (group C), and blank control group (group D). Full thickness articular cartilage defects of 8 mm×6 mm in size were created in the bilateral femoral condyles of all rabbits. The micro-fracture surgery was performed in groups A and B. The 0.1 mL rhIGF-1 (0.01 μg/μL) was injected into the knee cavity in groups A and C at 3 times a week for 4 weeks after operation, while 0.1 mL sal ine was injected in groups B and D at the same time points. At 4, 12, and 24 weeks, the gross, histological, and immunohistochemical observations were performed, and histological score also was processed according to Wakitani's score criteria. The collagen contents in the repair tissues and normal patellofemoral cartilage were detected by the improved hydroxyproline (HPR) method at 24 weeks. Electron microscope was used to observe repair tissues of groups A and B at 24 weeks. Results All animals were survival at the end of experiment. At 24 weeks after operation, defect was repaired with time, and the repair tissue was similar to normal cartilage in group A; the repair tissue was even without boundary with normal cartilage in group B; and the repair tissue was uneven with clear boundary with normal cartilage in groups C and D. Histological staining showed that the repair tissues had no difference with normal cartilage in group A; many oval chondrocytes-l ike cells and l ight-colored matrix were seen in the repair tissues of group B; only a few small spindle-shaped fibroblasts were seen in groups C and D. Moreover, histological scores of group A were significantly better than those of groups B, C, and D (P<0.05) at 4, 12, and 24 weeks. Electron microscope observation showed that a large number of lacuna were seen on the surface of repair tissue in group A, and chondrocytes contained glycogen granules were located in lacunae, and were surrounded with the collagen fibers, which was better than that in group B. Collagen content of the repair tissue in group A was significantly higher than that in groups B, C, and D (P<0.05), but it was significantly lower than that of normal cartilage (P<0.05). Conclusion Combination of micro-fracture and rhIGF-1 for the treatment of full thickness articular cartilage defects could promote the repair of defects by hyaline cartilage.