OBJECTIVE: To observe the effects of silks on attachment, shape and function of chondrocytes cultured in vitro. METHODS: The silks from silk worm cocoons were digested by trypsin and coated with polylactic acid to from three dimensional scaffolds for rabbit rib chondrocyte culture. The growth and shape of chondrocytes were observed with phase contrast microscopy, scanning electron microscopy. RESULTS: The chondrocytes were adhered to silks slowly after chondrocytes were seeded into silk scaffolds and cells fixed on silks well 1 or 2 days later. Cells began to proliferate after 3 days and multiplicative growth was observed on the 6th day. Microholes of silk scaffolds were filled with chondrocytes 2 weeks later. Scanning electron microscopy showed that there was a lot of extracellular matrix surrounding cells. CONCLUSION: Silks are ideal for attachment, growth and function maintenance of chondrocytes, and silks can be used as scaffolds for chondrocytes in three dimensional culture.
OBJECTIVE This paper aims to investigate the suitable cell density and the best formation time of tissue engineered autologous cartilage and to provide theoretical basis and parameters for clinical application. METHODS The chondrocytes isolated from mini swines’ ears were mixed with injectable biocompatible matrix (Pluronic), and the density of cell suspensions were 10, 20, 30, 40, 50, 60, 70 x 10(4)/ml. The chondrocyte-polymer constructs were subcutaneously injected into the abdomen of autologous swine. The specimens were observed grossly and histologically after 6 weeks, and investigated the suitable cell density. Then the chondrocyte-polymer constructs with suitable cell density were transplanted into the abdomen of autologous swine and evaluated grossly and histologically in 1, 3, 6, 9, 15 weeks after transplantation to investigate the best formation time of tissue engineered cartilage. RESULTS The experiments demonstrated that the tissue engineered autologous cartilage was similar to the natural cartilage on animals with normal immune system in histological characteristics. The optimal chondrocyte density is 50 x 10(6)/ml, and the proper harvest time is the sixth week. CONCLUSION With tissue engineering skills, we have identified the optimal chondrocyte density and the proper harvest time.
Objective To study the effect of two cytokines, basic fibroblast growth factor(bFGF) and insulin-like growth factor-I(IGF-I), on cell proliferation in chondrocytes of adult rabbits. Methods The primary chondrocytes of adult rabbits were harvested and cultured with bFGF and IGF-I at different concentrations,respectively, as well as with the mixture of the two cytokines; the quantity of cultured chondrocytes was detected by MTT assay at the 24th, 48th and 72th hours; and the final fold increase of different groups was measured by cell count for the 3rd passage; and the proliferation index of the groups was recorded by flowing cytometer on the 14th day. Results ① The cultured chondrocytes with either bFGF, IGF-I or their mixture were significantly more than that of control group at the 24th, 48th and 72th hours (P＜0.01). ② After the 3rd passage, the final folds of proliferation were significantly higher in the groups with cytokinesthan in the control group (P＜0.01); and the final fold with the mixture ofcytokines was significantly higher than that of both IGF-I and bFGF (P＜0.01). ③ Theproliferation index was significantly higher in the groups with cytokines than in the control group (P＜0.01); the proliferation index with the mixture of cytokines was significantly higher than that of both IGF-I and bFGF (P＜0.05); besides, proliferation index was higher when cytokine was applied twice than once (P＜0.05). Conclusion bFGF and IGF-I could promote chondrocytes proliferation of adult rabbits obviously and they are synergistic in cell proliferation.
Objective To observe the main biological characteristics and chondrogenesis potency of bone marrow -derived stromal cells(MSCs) after cytokinesinduction or gene modification in vitro. Methods MSCs from an adult New Zealand white rabbit were isolated and cultivated, and then MSCs were divided into the common medium group(Group A, 15%FBS in DMEM), the induced group by cytokines (Group B), the transfected group(Group C)with adenovirus-hepatocyte growth factor transgene (adHGF). The medium of group B consisted of transforming growth factor-β1(TGF-β1,10 ng/ml), basic fibroblast growth factor(bFGF,25 ng/ml) addexamethasone (DEX,10-7mol/L) with 15%FBS in DMEM. Cartilage slices wereobtained from femoral condyles and patellar grove in the same rabbit. The minced cartilage was digested in Ⅱ collagenase (3 mg/ml) to obtain chondrocytes(Group D). The change of cell appearance, proliferation capacity, glycosaminoglycans(GAG), immunohistochemical staining for type Ⅰ, Ⅱ collagen were observed during the 5th passage MSCs and MSCs after induction or gene modification. Expression of mRNA for type Ⅰ and Ⅱ collagen was detected by RT-PCR. Results Primary MSCs proliferated as shortspindle shape, while the 5th MSCs showed longspindle shape. Positive stain of type Ⅰ collagen could be found in groups A, B and C, while positivestain of type Ⅱ collagen was shown in groups B and D. The content of GAG in group B was higher than that in group A, but there was no significant difference between them（Pgt;0.05）, and there was significant difference between groups A and D（Plt;0.05）. No significant difference was noted in groups A,B and C on proliferation by MTT（Pgt;0.05）,except that of at the fourth day after transfection between groups A and C（Plt;0.05）. RT-PCR demonstrated that MSCs always had higher levelsof mRNA type Ⅰ collagen in groups A, B and C. The expression of mRNA type Ⅱ collagen was identified in groups B and D, and only low levels of mRNA type Ⅱ collagen in group C. Conclusion The above results indicate MSCs have a natural tendency of osteogenic differentiation in vitro culture, and also demonstrate the chondrogenic potency with the technique of cytokines induction or gene modification after passage. MSCs can be transfected efficiently being seed cells in tissue engineered bone or cartilage to accept target genes such as adHGF, and have a higher levels of expression in vitro, which lasted 4 weeks at least.
ObjectiveTo investigate the mechanism of Semaphorin 3A (Sema3A) in fracture healing after nerve injury by observing the expression of Sema3A in the tibia fracture healing after traumatic brain injury (TBI). MethodsA total of 192 Wistar female rats, 8-10 weeks old and weighing 220-250 g, were randomly divided into tibia fracture group (group A, n=48), TBI group (group B, n=48), TBI with tibia fracture group (group C, n=48), and control group (group D, n=48). The tibia fracture model was established at the right side of group A; TBI model was made in group B by the improved Feeney method; the TBI and tibia fracture model was made in group C; no treatment was given in group D. The tissue samples were respectively collected at 3, 5, 7, 14, 21, and 28 days after operation; HE staining, immunohistochemistry staining, and Western blot method were used for the location and quantitative detection of Sema3A in callus tissue. ResultsHE staining showed that no obvious changes were observed at each time point in groups B and D. At 3 and 5 days, there was no obvious callus growth at fracture site with inflammatory cells and fibrous tissue filling in groups A and C. At 7 and 14 days, fibrous tissue grew from periosteum to fracture site in groups A and C; the proliferation of chondrocytes in exterior periosteum gradually formed osteoid callus at fracture site in groups A and C. The chondrocyte had bigger size, looser arrangement, and more osteoid in group C than group A. Group B had disorder periosteum, slight subperiosteal bone hyperplasia, and no obvious change of bone trabecula in group B when compared with group D. At 21 and 28 days, cartilage callus was gradually replaced by new bone trabecula in groups A and C. Group C had loose arrange, disorder structure, and low density of bone trabecula, big callus area and few chondrocyte and osteoid when compared with group A; group B was similar to Group D. Immunohistochemistry staining showed that Sema3A expression in chondrocytes in group C was higher than that in group A, particularly at 7, 14, and 21 day. Sema3A was significantly higher in osteoblasts of new bone trabecula in group A than group C, especially at 14 and 21 days (P<0.05). Western blot results showed that the Sema3A had the same expression trend during fracture healing in groups A and C. However, the expression of Sema3A protein was significantly higher in group C than group A (P<0.05) and in group B than group D (P<0.05) at 7, 14, 21, and 28 days. ConclusionAbnormal expression of Sema3A may play a role in fracture healing after nerve injury by promoting the chondrocytes proliferation and reducing the distribution of sensory nerve fibers and osteoblast differentiation.
【Abstract】 Objective To investigate the effect of retinoic acid (RA) on cell apoptosis and gene regulation of IGF-2in chondrocyte. Methods One 1-month-old Chinese rabbit weighted 500 g was used in this experiment. The chondrocyte from rabbit knee were cultured by enzyme digestion. Twenty-five μL all-trans-retinoic acid (ATRA) (1×10-6 mol/L) were added in the media of cultured chondrocyte for 24 hours as experimental group, while 25 μL DMEM were added as control group. The secretion of collagen Ⅱ was observed by immunohistochemistry method, cell apoptosis was detected by flow cytometry, IGF-2 mRNA and protein expression in chondrocyte were detected by RT-PCR and Western blot analysis. Results The expression of collagen Ⅱ was down-regulated by ATRA in the experimental group. The cell apoptosis in chondrocyte exposed to ATRA at 1 ×10-6 mol/L was 21% ± 2%, which increased 5 times compared with the control group（5% ± 1%）. The IGF-2 mRNA and protein level in the experimental group were decreased 75% and 57%, respectively, compared to the control group. There weresignificant difference between the experimental group and control group in each index (P lt; 0.05). Conclusion RA may down-regulate the secretion and cell prol iferation, but up-regulate the cell apoptosis in chondrocyte. The apoptotic effect may carry out through inhibiting the IGF-2 expression of chondrocyte.
Objective To observe the biological characters of chondrocytes in articular loose body and to find out seeding cells for cartilage tissue engineering. Methods Samples from 5 loose body cartilages, 2 normal articular cartilages and 6 osteoarthritis articular cartilages were collected. Part of each sample’s cartilage was histologically studied to observe the chondrocytes distribution the morphologic changes by toluidine-blue staining, chondrocytes’ apoptosis by terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL). The rest of each cartilage was digested and isolated by 0.25% trypsin and 0.2% collagenase Ⅱ, and then were cultivated in 10%DMEM. Their morphologic changes were observed 24h later.Comparison was made btween three cartilages. Results Compared with normal cartilage and osteoarthritis articular cartilage, the cells density was higher, their lacunars were larger, cells distribution was irregular, and apoptosis was more apparent in loose body cartilage. Conclusion The characters of chondrocytes from loose body is more like fibroblasts so they can not serve as seeding cells directly for cartilage tissue engineering.
Objective To study the effects of the periosteum，synovium andcartilage tissues on the gene expressions of proteoglycan, collagen Ⅱ, andnuclear factor kappa B (NF-κB) and to investigate the different effects of these tissues on cartilage regeneration. Methods In 20 New Zealand white rabbits, 20 cartilage explants were taken from the knee joints in each rabbit, the sizeof which was 4 mm×4 mm×4 mm. All the cartilages were divided into the following 4 groups and cultured for 7 days: Group A, with 5 pieces (2 mm×2 mm) of the synovium of theknee joints in each dish; Group B, with 5 pieces (2 mm×2 mm) of the periosteum ineach dish; Group C, with 5 pieces (2 mm×2 mm×2 mm) of the cartilage in each dish; and Group D, with no addition of other tissues (control group). RNA was extracted from the cells of the cartilage explants (4 mm×4 mm×4 mm) in all the dishes. Thegene expressions of proteoglycan, collagen Ⅱ and NF-κB were defected by a reversetranscription-polymerase chain reaction (RT-PCR).Results In group A, the gene expression of proteoglycan was significantly decreased. The relative density of this gene expression had a significant difference when compared with that in group D (1.09±0.21 vs. 1.25±0.25, Plt;0.05); the gene expressions of collagen Ⅱ and NF-κB were also decreased, but they had no significant differences when compared with those in group D (Pgt;0.05). In groupB, the gene expressions of proteoglycan, collagen Ⅱ, and NF-κB were significantly increased. The relative densities of these gene expressions were 1.60±0.26, 1.57±0.24, and 4.20±2.22, respectively, which had significant differences when compared with those in group D (Plt;0.05). In group C, the relative density of the gene expression of collagen Ⅱ was 1.43±0.28, which had a significant difference when compared with that in group D (Plt;0.05), but therelative densities of the gene expressions of proteoglycan and NF-κB had no significant differences when compared with those in group D (Pgt;0.05). Conclusion The results indicate that the periosteum can up-regulate the gene expressions of proteoglycan, collagen Ⅱ and NF-κB. The NF-κB is likely to be an important nuclear transcription factor related to cartilage regeneration. The results also suggest that the periosteum maybe better in facilitating the cartilage repair and regeneration in clinical practice.
Objective To study the biological characteristic and potential of chondrocytes grafting cultured on fascia in repairing large defect of articular cartilage in rabbits. Methods Chondrocytes of young rabbits were isolated and subcultured on fascia. The large defect of articular cartilage was repaired by grafts of freeze-preserved and fresh chondrocytes cultured on fascia, and free chondrocytes respectively; the biological characteristic and metabolism were evaluated bymacroscopic, histological and immunohistochemical observations, autoradiography method and the measurement of nitric oxide content 6, 12, 24 weeks after grafting. Results The chondrocytes cultured on fascia maintained normal growth feature and metabolism, and there was no damage to chondrocytes after cryopreservation; the repaired cartilage was similar to the normal cartilage in cellular morphology and biological characteristics. Conclusion Chondrocytes could be cultured normally on fascia, which could be used as an ideal carrier of chondrocytes.
Objective To explore the ability of insulin-like growth factor-Ⅰ (IGF-Ⅰ) and hyaluracan acid in prompting chondrogenesis of engineering cartilage tissue.Methods Human articular chondrocytes were isolatedand cultured in DMEM plus 10% fetal bovine serum. They were divided into three groups：hyaluracan acid+chondrocytes + IGF-Ⅰ group(IGF-Ⅰ group), hyaluracan acid+chondrocytes group(cell group), hyaluracan acid group(control group). The ability of chondrogenesis was investigated by HE and toluidine blue staining, human collagen Ⅱ immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR).Results Both cell group and IGF-Ⅰ group could develop into cartilage tissue in the sixth week while control group could not. The number of cartilage lacuna in IGF-Ⅰ group were more than that in cell group. Human collagen Ⅱ immunohistochemistry showed that there were ber positive cell in IGF-Ⅰ group than in cell group, collagen Ⅱ mRNA expression was more higher and collagen Ⅰ mRNA expression was lower in IGF-Ⅰ group than in cell group. Conclusion Insulin growth factorⅠ can prompt chondrogenesis of engineering cartilage tissue and ameliorate the quality of engineering cartilage tissue in vitro.