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.
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.
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.
【Abstract】 Objective To investigate the protective effect of early motion on articular cartilage after joint allograft by performing a controlled trial between different post-operation strategies after joint allograft in an animal model. Methods Twenty hemi-knee joints were harvested from 10 6-month-old New Zealand white rabbits (male or female, weighing 2.5-3.0 kg); 10 hemi-knee joints by deep frozen treatment (donors) were transplanted to unilateral knee joints (recipients) of 10 6-month-old Chinchilla rabbits (male or female, weighing 2.5-3.0 kg), which were divided into early motion group (n=5) and sustained fixation group (n=5); and 10 hemi-knee joints were used as blank control (n=5) and frozen control (n=5). The articular cartilage of allogenic joints was detected by X-ray film, gross, and histology at 6 weeks after operation. Results Gross observation: no obvious limitation of joint movements was observed in early motion group, but obvious limitation in sustained fixation group. X-ray films: the bone ends between donor and recipient healed well with good paraposition and alignment on the operation day and 2 weeks after operation; at 6 weeks, angulation deformity was observed in early motion group of 3 rabbits, and paraposition and alignment were satisfactory in sustained fixation group. Histological observation: HE staining showed that the chondrocytes had normal quantity and morphology with few nuclear fragmentation and karyolysis in early motion group, but the quantity of chondrocytes sharply decreased with dissolved nuclei and numerous fibrous tissues in the cartilage matrix in sustained fixation group. The cell survival rate of the early motion group (49.66% ± 2.15%) was significantly higher than that of the sustained fixation group (20.68% ± 1.24%) (P lt; 0.05). Scanning electron microscopy observation: nuclear membrane was intact with chromatin condensation and edema of mitochondria and rough surfaced endoplasmic reticulum in early motion group, and that the membrane of chondrocyte vanished with blurring border between chondrocyte and matrix, rupture of nuclear membrane and the disappearance of chromatin and organelles could be found in sustained fixation group. Conclusion Early motion has protective effect on articular cartilage after joint allograft, but cannot completely prevent degeneration of the allogenic articular cartilage.
Objective To investigate the effect of “two-phase” tissue engineered cartilage constructed by autologous marrow mesenchymal stem cells(MSCs) and allogeneic bone matrix gelatin(BMG) in repairing articular cartilage defects. Methods Thirty-twoNew Zealand white rabbits were involved in the experiment. “Two-phase” allogeneic BMG scaffold (one side of porous cancellous bone and the other side of cortical bone; 3 mm both in diameter and in thickness) was prepared from iliac bone and limb bone of 5 rabbits by sequentially chemical method. The MSCs wereseparated from 18 New Zealand white rabbits and induced to express chondrocyticphenotype. The chondrocyte precursor cells were seeded onto “two-phase” allogeneic BMG to construct tissue engineering cartilage. Masson’s trichrome staining, PAS staining and scanning electronic microscopic observation were carried out at 1, 3 and 5 weeks. The defects of full thickness articular cartilage(3 mm both in diameter and in depth) were made at both sides of femoral medial condyles in 27 rabbits(including 18 of separated MSCs and the remaining 9). The defects were repaired with the tissue engineered cartilage at the right side (group A, n=18), with BMG at the left side(group B, n=18), and without any implant at both sides in the remaining 9 rabbits as a control( group C, n=18). After 1, 3 and6 months, the 6 specimens of femoral condyles were harvested in 3 groups, respectively. Gross observation, Masson’s trichrome and Alcian blue staining, modified Wakitani scoring and in situ hybridization of collagen type Ⅱ were carried out to assess the repair efficacy of tissue engineered cartilage. Results The “two-phase” BMG consisted of the dense cortical part and the loose cancellous part. In cancellous part, the pore size ranged 100-800 μm, in which the chondrocyte precursor cells being induced from MSCs proliferated and formed the cell-rich cartilaginous part of tissue engineered cartilage. In cortical part, the pore size ranged 10-40 μm, on which the cells arranged in a layer and formed the hard part of subchondral bone. After 1 month of transplantation, the cartilage and subchondral bone were regenerated in group A; during observation, the regenerated cartilage graduallythinned, but defect was repaired and the structure of the articular surface ansubchondral bone was in integrity. In groups B and C, defects were not repaired, the surrounding cartilage of defect was abrased. According to the modified Wakitani scoring, the indexes in group A were significantly higher than those in group B and C(Plt;0.01) except the thickness of cartilage at 6 months. The positive cell rate of in situ hybridization for collagen type Ⅱ in group A was also higher than those in groups B and C(Plt;0.01). Conclusion “Two-phase” allogeneic BMG is a prospective scaffold for tissue engineered cartilage,which combines with autologous chondrocyte precursor cells induced from MSCs toconstruct the tissue engineering cartilage. The tissue engineered cartilage can repair defects of articular cartilage and subchondral bone.
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 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 establ ish a porcine model of articular full-thickness cartilage defect characterized byremaining cartilage calcified zone on femoral trochlea, so as to provide a considerable and comparative control group forinvestigating repair effects of tissue engineered scaffolds in articular cartilage defects with cartilage calcified zone remaining.Methods The full-thickness cartilage column defects (6 mm in diameter, 0.2-0.5 mm in depth) without damage on calcifiedcartilage zone were made on the femoral trochlea in 9 clean-grade 6-month-old Guizhou mini pigs by standard cartilage-defectmakingsuites. Microscopical observation was performed after modeling. Scanning were made by 3.0T MRI at 4 weeks. Thengeneral observation, stereomicroscope, and histological staining were used to observe cartilage repair. Results All animals wereal ive. No infection of incisions or patellar dislocations occurred; they were able to walk with partial weight-bearing immediatelyafter surgery and could move freely without limp at 1 week. Obvious signal discontinuity in trochlea and subchondral bone couldbe observed in MRI, without deep signal change in defects surrounding. Microscopical observation showed a few repair tissueand petechia at base of the defect with clear boundary. Nearly intact calcified zone of cartilage and zonal collapse of subchondralbone in defects could be observed with stereomicroscope. Under common microscope, no chondrocytes was found in defects,as well as negative staining of fast green-safranin O and alcian blue. Under polarized microscope, the bottom of defects werefilled with a l ittle of fibrous tissue presenting continuous and b l ight-refraction by sirius red staining. Conclusion Theanimal model of articular full-thickness cartilage defect on femoral trochlea by standard cartilage-defect-making suites can beapplied for the research of cartilage disease in early human osteoarthritis and function of calcified cartilage zone in pig.
Objective To investigate the possibility of sheep joint cartilage defect repair with tissue engineered cartilage constructed by using porous bioceramics as scaffold and TGF-β induced autologous bone marrow derived mesenchymal stem cells(MSCs) as seed cell. Methods In the experimental group(n=12), autologous MSCs were isolated and expanded in vitro and then implanted into the pre molded porous β-TCP; the cell β-TCP complex was implanted into sheep right humeral cartilage defect. The defects in β-TCP (n= 12) group were repaired by B-TCP only, while defects in the control group (n= 4) were left un-repaired. Samples were extracted 12 and 24 weeks after operation for histological, histochemical and immunohistochemical analysis. Results In the experimental group, cartilage-like tissue formation could be seen on the surface of the implants. Microscopic analysis demonstrated obvious degradation of B-TCP and extensive new cartilage formation 12 weeks after operation, containing rich extracellularmatrix. The cells were stained positively with type II collagen. The bioceramics had almo st completely been degraded and abundant cartilage formation could be seen in the whole defects 24 weeks later. In the B-TCP group, marginal cartilage ingrowth could be seen 12 weeks after operation and the number of chondrocytes increasedmarkedly after 24wee s. However, no cartilage can be found in the middle of the material. In the control group, only a small quantity of new cartilage formation could be seenalong the margin of defects. Conclusion It is feasible to generate tissue engineered cartilage with porous B-TCP and auto logousM SCs for cartilage defect repair.
It is very difficult to repair large articular cartilage defect of the hip. From May 1990 to April 1994, 47 hips in 42 patients of large articuler cartilage defects were repaired by allograft of skull periosteum. Among them, 14 cases, whose femoral heads were grade. IV necrosis, were given deep iliac circumflex artery pedicled iliac bone graft simultaneously. The skull periosteum had been treated by low tempreturel (-40 degrees C) before and kept in Nitrogen (-196 degrees C) till use. During the operation, the skull periosteum was sutured tightly to the femoral head and sticked to the accetabulum by medical ZT glue. Thirty eight hips in 34 patients were followed up for 2-6 years with an average of 3.4 years. According to the hip postoperative criteria of Wu Zhi-kang, 25 cases were excellent, 5 cases very good, 3 cases good and 1 case fair. The mean score increased from 6.4 before operation to 15.8 after operation. The results showed, in compare with autograft of periosteum for biological resurface of large articular defect, this method is free of donor-site morbidity. Skull periosteum allograft was effective for the treatment of large articular cartilage defects in hip.
