OBJECTIVE: To study chondrogenesis of calcium alginate-chondrocytes predetermined shapes. METHODS: Chondrocytes isolated from ears of rabbit by type II collagenase digestion, and then were mixed with 1.5% solidium alginate solution. The suspension was gelled to create three spatial shapes as triangle, circle and quadrilateral by immersed into 2.5% CaCl2 for 90 minutes, and then was implanted into the subcutaneous pocket on the dorsum of the rabbit. Samples were harvested at 6 and 12 weeks after implantation. RESULTS: Gross examination of excised specimens at 6 and 12 weeks after implantation revealed the presence of new cartilage of approximately the same dimensions as the original construct. Histologic evaluation using hematoxylin and eosin stains confirmed the presence of cartilage nodules at 6 weeks after implantation. After 12 weeks, mature cartilage was observed and histologic analysis confirmed the presence of well formed cartilaginous matrix. CONCLUSION: Predetermined shapes neocartilage can be regenerated using calcium alginate as a carrier of chondrocytes in the bodies of immune animals.
Objective To investigate the effect of collagen type I concentration on the physical and chemical properties of the collagen hydrogel, and to analyze the effect of different concentrations of collagen type I hydrogel on the phenotype and gene expression of the chondrocytes in vitro. Methods Three kinds of collagen hydrogels with concentrations of 12, 8, and 6 mg/ mL (C12, C8, and C6) were prepared, respectively. The micro-structure, compressive modulus, and swelling ratio of the hydrogels were measured and analyzed. The chondrocytes at 2nd passage were cocultured with three kinds of collagen hydrogels in vitro, respectively. After 1-day culture, the samples were stained with fluorescein diacetate (FDA) / propidium iodide (PI) and the cell activity was observed under confocal laser microscope. After 14-day culture, HE staining and toluidine blue staining were carried out to observe the histological morphology, and mRNA expressions of chondrocytes related genes (collagen type II, Aggrecan, collagen type I, collagen type X, Sox9) were determined by real-time fluorescent quantitative PCR. Results With the increase of collagen type I concentration from 6 to 12 mg/mL, the physical and chemical properties of the collagen hydrogels changed significantly: the fiber network became dense; the swelling ratios of C6, C8, and C12 were 0.260 ± 0.055, 0.358 ± 0.072, and 0.539 ± 0.033 at 192 hours, respectively, showing significant differences among 3 groups (P lt; 0.05); and the compression modulus were (4.86 ± 0.96), (7.09 ± 2.33), and (11.08 ± 3.18) kPa, respectively, showing significant differences among 3 groups (P lt; 0.05). After stained with FDA/PI, most cells were stained green, and few were stained red. The histological observation results showed that the chondrocytes in C12 hydrogels aggregated obviously with b heterochromia, chondrocytes in C8 hydrogels aggregated partly with obvious heterochromia, and chondrcytes in C6 hydrogels uniformly distributed with weak heterochromia. Real-time fluorescent quantitative PCR results showed that the mRNA expressions of collagen type II and Aggrecan were at the same level in C12, C8, and C6; the expressions of collagen type I, Sox9, and collagen type X were up-regulated with the increase of collagen type I hydrogels concentration, and the expressions were the highest at 12 mg/mL and were the lowest at 6 mg/mL, showing significant differences among 3 groups (P lt; 0.05). Conclusion Increasing the concentration of collagen hydrogels leads to better mechanical properties and higher shrink-resistance, but it may induce the up-regulation of cartilage fibrosis and hypertrophy related gene expression.
ObjectiveTo assess the role and effect of Wharton's jelly of human umbilical cord oriented scaffold on chondrocytes co-cultured in vitro. MethodsChondrocytes from shoulder cartilage of adult New Zealand rabbits were isolated,cultured,amplified,and labelled using fluorescent dye PKH26.Cells were extracted from human umbilical cord tissue using wet-grinding chemical technology to prepare the Wharton's jelly of human umbilical cord oriented scaffold by freeze-drying and cross-linking technology.Second generation of chondrocytes were cultured with Wharton's jelly of human umbilical cord oriented scaffold.Inverted microscope and scanning electron microscope (SEM) were used to observe the cell distribution and adhesion on the scaffold; extracellular matrix secretion of the chondrocytes were observed by toluidine blue and safranin O staining.Cells distribution and proliferation on the scaffold were assessed by fluorescein diacetate-propidium iodide (FDA-PI) and Hoechst33258 staining.The viability of the in vitro cultured and PKH26 fluorescence labelled chondrocytes on the scaffold were assessed via fluorescence microscope. ResultsInverted microscope showed that the cells cultured on the scaffold for 3 days were round or oval shaped and evenly distributed into space of the scaffold.SEM observation showed that large number of cultured cells adhered to the pores between the scaffolds and were round or oval shape,which aggregated,proliferated,and arranged vertically on longitudinally oriented scaffold at 7 days after culture.Histological observation showed that cells distributed and proliferated on the scaffold,and secreted large amount of extracellular matrix at 7 days.Scaffold could guide cell migration and proliferation,and could effectively preserve and promote the secretion of extracellular matrix.Cell viability assessments at 3 days after culture showed most of the adhered cells were living and the viability was more than 90%.PKH26 labelled chondrocytes were seen,which distributed uniformly along the pore of oriented scaffold,and exuberantly proliferated. ConclusionWharton's jelly of human umbilical cord oriented scaffold favors adhesion,proliferation,and survival of chondrocytes.It possesses a favorable affinity and cell compatibility.Thus,it is an ideal scaffold for cartilage tissue engineering.
Objective To establish a kind of gene therapy method of rheumatoid arthritis, to construct the interleukin-18-PE38 fusion gene expression vectorand to explore the expression of the fusion gene in the chondrocytes and 3T3 cells. Methods Interleukin-18-PE38 fusion gene was cleaved from plasmid PRKL459k-IL-18-PE38 by restriction enzyme digestion,then linked with vectors PsecTag2B and transformed into competence bacteria, positive clones were selected and confimed by restrictive enzyme(EcoRI) digestion assay. The rearrangement plasmid PsecTag2B-IL-18-PE38 was transfected into 3T3 cells and mouse chondrocytes by liposome protocol(experimental group),null vector was used as negative control, and the transient expression was identified by fluorescence immunocytochemical assay. Results Restrictive enzymes digestion analysis revealed thatthe length of theinterleukin-18-PE38 fusion gene was 6 000 bp. Fluorescence immunocytochemical method showed that fluorescence intensity of the experimental group is b,whilefluorescence intensity of the control group is weak. Conclusion the eukaryoticexpression vector PsecTag2B-IL-18-PE38 is established successfully which canbeexpressed in the 3T3 cells and mouse chodrocytes. Our results lay a foundationfor the further investigation for rheumatoid arthritis therapy.
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 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.
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 observe the effect of engineered epiphyseal cartilage regenerated in vitro with 3-D scaffold by chondrocytes from epiphyseal plate in repairing the tibial epiphyseal defect, and to explore the methods to promote the confluence between engineered cartilage and epiphyseal plate. METHODS: Chondrocytes were isolated enzymatically from the epiphyseal plates of immature rabbits, and then planted into the tissue culture flasks and cultivated. The first passage chondrocytes were collected and mixed fully with the self-made liquid biological gel at approximately 2.5 x 10(7) cells/ml to form cell-gel fluid. The cell-gel fluid was dropped into the porous calcium polyphosphate fiber/poly-L-lactic acid(CPPf/PLLA)scaffold, and a cell-gel-scaffold complex formed after being solidified. The defect models of 40% upper tibial epiphyseal plate were made in 72 immature rabbits; they were divided into 4 groups: group A(the cell-gel-scaffold complex was transplanted into the defect and the gap filled with chondrocyte-gel fluid), group B (with noncell CPPf/PLLA scaffold), group C(with fat) and group D(with nothing). The changes of roentgenograph, gross and histology were investigated after 2, 4, 6, 8, 12 and 16 weeks of operation. RESULTS: In group A, the typical histological structure of epiphyseal plate derived from the engineered cartilage with a fine integration between host and donor tissues after 2 weeks. The repaired epiphyseal plate had normal histological structure without deformation of tibia after 4 weeks. The early histological change of epiphyseal closure appeared in the repaired area with varus and shortening deformation of the tibia after 8 weeks. The epiphyseal plate was closed in the repaired area with more evident deformation of tibia; the growth function of repaired epiphyseal plate was 43.6% of the normal one. In groups B, C and D, deformation of tibia occurred after 2 weeks; the defect area of epiphyseal plate was completely closed after 4 weeks. The deformation was very severe without growth of the injured epiphyseal plate after 16 weeks, and no significant difference was observed between the three groups. CONCLUSION: Engineered epiphyseal cartilage can repair the epiphyseal defect in the histological structure with partial recovery of the epiphyseal growth capability. Injecting the suspension of fluid chondrocyte-gel into the defects induces a fine integration of host and donor tissues.
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 investigate apoptosis of chondrocytes cultured in vitro and related expression of caspase-3. METHODS: Apoptosis of chondrocytes were detected by flow cytometry analysis and TUNEL staining. The expression of caspase-3 was determined by RT-PCR and Western blot, and caspase-3 protein activity was determined by ELISA. RESULTS: Apoptosis was observed in chondrocytes cultured in vitro from passage 1 to passage 4 at various degrees. The percentage of apoptosis of chondrocytes on day 7 was much higher than that on day 3 (15.7% +/- 0.3% vs 8.9% +/- 0.6%, P lt; 0.01). caspase-3 mRNA and protein expressed in chondrocytes during whole culture process. Along with the culture time extension in vitro, caspase-3 expression and protein activity up-regulated, coincident with apoptosis of chondrocyte. caspase-3 was activated and a fragment of 20 kDa was detected after 7 days of culture. CONCLUSION: caspase-3 is involved in apoptosis of chondrocytes cultured in vitro.