Objective To explore the effect of rolling compression loading bioreactor on chondrogenesis of rabbit bone marrow mesenchymal stem cells (BMSCs) with different loading parameters. Methods BMSCs were isolated from New Zealand rabbits, aged 2.5 months. BMSCs at passage 3 were used to prepare BMSCs-agarose gels (4 mm in diameter and height, respectively). Samples were divided into 8 groups: 10% (group A1), 20% (group A2), and 30% (group A3) compression groups (0.4 Hz, 3 h/ d) and 20 minutes (group B1), 3 hours (group B2), and 12 hours (group B3) rolling time groups and static culture (control groups). The living cell rate, the collagen type II and Aggrecan gene expressions, and glycosaminoglycan (GAG) content were determined, and histological staining was done at 24 hours, 7 days, 14 days, and 21 days after culture. Results At 14 and 21 days, the living cell rates of groups A1 and A2 were significantly higher than that of group A3 (P lt; 0.05), groups B1 and B2 were significantly higher than group B3 (P lt; 0.05). Collagen type II and Aggrecan gene expressions of the experimental groups at each time point were significantly higher than those of the control groups (P lt; 0.05); at 14 and 21 days, collagen type II and Aggrecan gene expressions of groups A1 and A2 were significantly higher than those of group A3, and groups B1 and B2 were also significantly higher than group B3 (P lt; 0.05). At 14 and 21 days, the GAG contents of groups A1 and A2 were significantly higher than those of group A3 (P lt; 0.05); groups B1 and B2 were also significantly higher than group B3 (P lt; 0.05). At 21 days, toluidine blue staining showed that obvious blue-staining and even cartilage lacunae were seen in groups A2 and B2, but light and quite rare blue-staining in groups A1, A3, B1, and B3. Conclusion The rolling compression loading bioreactor has great promotion effect on chondrogenesis of rabbit BMSCs with rolling parameters of 0.4 Hz, 3 hours, and 20% compression.
Objective To evaluate the synergistic effect of bone morphogenetic protein 14 (BMP-14) and chondrocytes co-culture on chondrogenesis of adipose-derived stem cells (ADSCs) so as to optimize the source of seed cells for cartilage tissue engineering. Methods ADSCs and chondrocytes were isolated and cultured respectively from articular cartilage and subcutaneous fat of 2 male New Zealand white rabbits (weighing, 1.5 kg and 2.0 kg). The cells at passage 3 were harvested for experiment. ADSCs were identified by osteogenic induction (alizarin red staining), chondrogenic induction (alcian blue staining), and adipogenic induction (oil red O staining). The optimum multiplicity of infection (MOI) of transfection of adenovirus-cytomegalovirus (CMV)-BMP-14-internal ribosome entry site (IRES)-human renilla reniformis green fluorescent protein 1 (hrGFP-1) was determined and then ADSCs were transfected by the optimum MOI. The experiment was divided into 5 groups: group A, co-culture of ADSCs transfected by BMP-14 and chondrocytes (1 ∶ 1 in Transwell chambers); group B, co-culture of ADSCs and chondrocytes (1 ∶ 1 in Transwell chambers); group C, culture of ADSCs transfected by BMP-14; group D, simple chondrocytes culture; and group E, simple ADSCs culture. After 3 weeks, the glycosaminoglycan (GAG) content was detected by alcian blue staining; the expressions of collagen type II and BMP-14 protein were detected by Western blot; expression of Sox-9 gene was detected by RT-PCR. Results The cultured cells were proved to be ADSCs by identification. Inverted fluorescence microscope showed optimum transfection effect when MOI was 150. GAG content, expressions of collagen type II and BMP-14 protein, expression of Sox-9 gene were significantly higher in groups A and C than in the other 3 groups, in group A than in group C (P lt; 0.05), and groups B and D were significantly higher than group E (P lt; 0.05), but no significant difference was found between groups B and D (P gt; 0.05). Conclusion It can promote differentiation of ADSCs into chondrocytes by BMP-14 co-culture with chondrocytes, and they have a synergistic effect.
Objective To observe the chondrogenic differentiation of adipose-derived stem cells (ADSCs) by co-culturing chondrocytes and ADSCs. Methods ADSCs and chondrocytes were isolated and cultured from 8 healthy 4-month-old New Zealand rabbits (male or female, weighing 2.2-2.7 kg). ADSCs and chondrocytes at passage 2 were used. The 1 mL chondrocytes at concentration 2 × 104/mL and 1 mL ADSCs at concentration 2 × 104/mL were seeded on the upper layer and lower layer of Transwell 6-well plates separately in the experimental group, while ADSCs were cultured alone in the control group. The morphology changes of the induced ADSCs were observed by inverted phase contrast microscope. The glycosaminoglycan and collagen type II synthesized by the induced ADSCs were detected with toluidine blue staining and immunohistochemistry staining. The mRNA expressions of collagen type II, aggrecan, and SOX9 were detected with real-time fluorescent quantitative PCR. Results ADSCs in the experimental group gradually became chondrocytes-like in morphology and manifested as round; while ADSCs in the control group manifested as long spindle in morphology with whirlool growth pattern. At 14 days after co-culturing, the results of toluidine blue staining and immunohistochemistry staining were positive in the experimental group, while the results were negative in the control group. The results of real-time fluorescent quantitative PCR indicated that the expression levels of collagen type II, aggrecan, and SOX9 mRNA in the experimental group (1.43 ± 0.07, 2.13 ± 0.08, and 1.08 ± 0.08) were significantly higher than those in the control group (0.04 ± 0.03, 0.13 ± 0.04, and 0.10 ± 0.02) (P lt; 0.05). Conclusion ADSCs can differentiate into chondrocytes-like after co-culturing with chondrocytes.
Objective To investigate the effect of allogeneic chondrocytes-calcium alginate gel composite under the intervention of low intensive pulsed ultrasound (LIPUS) for repairing rabbit articular cartilage defects. Methods Bilateral knee articular cartilage were harvested from 8 2-week-old New Zealand white rabbits to separate the chondrocytes by mechanical-collagen type II enzyme digestion. The 3rd passage chondrocytes were diluted by 1.2% sodium alginate to 5 × 106 cells/mL, then mixed with CaCl2 solution to prepare chondrocytes-calcium alginate gel composite, which was treated with LIPUS for 3 days (F0: 1 MHz; PRF: 1 kHz; Amp: 60 mW/cm2; Cycle: 50; Time: 20 minutes). An articular cartilage defect of 3 mm in diameter and 3 mm in thickness was established in both knees of 18 New Zealand white rabbits (aged 28-35 weeks; weighing, 2.1-2.8 kg), and divided into 3 groups randomly, 6 rabbits in each group: LIPUS group, common group, and model group. Defect was repaired with LIPUS-intervention gel composite, non LIPUS-intervention gel composite in LIPUS group and common group, respectively; defect was not treated in the model group. The general condition of rabbits was observed after operation. The repair effect was evaluated by gross and histological observations, immunohistochemical staining, and Wakitani score at 8 and 12 weeks after operation. Results Defect was filled with hyaline chondroid tissue and white chondroid tissue in LIPUS and common groups, respectively. LIPUS group was better than common group in the surface smooth degree and the degree of integration with surrounding tissue. Defect was repaired slowly, and the new tissue had poor elasticity in model group. Histological observation and Wakitani score showed that LIPUS group had better repair than common group at 8 and 12 weeks after operation; the repair effect of the 2 groups was significantly better than that of model group (P lt; 0.05); and significant differences in repair effect were found between at 8 and 12 weeks in LIPUS and common groups (P lt; 0.05). The collagen type II positive expression area and absorbance (A) value of LIPUS and common groups were significantly higher than those of model group (P lt; 0.05) at 8 and 12 weeks after operation, and the expression of LIPUS group was superior to that of common group at 12 weeks (P lt; 0.05); and significant differences were found between at 8 and 12 weeks in LIPUS group (P lt; 0.05), but no significant difference between 2 time points in common and model groups (P gt; 0.05). Conclusion Allogeneic chondrocytes-calcium alginate gel composite can effectively repair articular cartilage defect. The effect of LIPUS optimized allogeneic chondrocytes-calcium alginate gel composite is better.
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.
Objective To investigate the feasibil ity of alendronate (ALN) in treating osteoarthritis (OA) by observing the effects of ALN on interleukin 1β (IL-1β) induced chondrocytes of rat in vitro. Methods The chondrocytes of knee articular surface from 15 SD rats (1-month-old, female or male, weighing 100-150 g) were cultured. The chondrocytes were observed by inverted phase contrast microscope and identified by toluidine blue staining and HE staining. The third passage chondrocytes were divided into 3 groups: the chondrocytes were cultured with DMEM for 5 days in group A, with 10 ng/mL IL-1β for 2 days and with DMEM for 3 days in group B, and with 10 ng/mL IL-1β for 2 days and with 1 × 10-6 mol/L ALN for 3 days in group C. Immunocytochemistry and real-time PCR were performed to determine the expression levels of collagen type II (Col II), matrix metalloproteinase 13 (MMP-13), and β-catenin. Results Toluidine blue staining proved that the cultured cells were chondrocytes. The integrated absorbency (IA) value of Col II in group C (10.290 7 ± 0.499 2) was lower than that in group A (15.377 0 ± 0.571 8) and higher than that in group B (5.463 2 ± 0.450 4), showing significant differences (P lt; 0.05). The IA value of MMP-13 in group C (3.068 6 ± 0.205 6) was significantly lower than that in group B (6.998 1 ± 0.329 7, P lt; 0.05), but there was no significant differenc when compared with group A (2.777 5 ± 0.199 6, P gt; 0.05). The IA value of β-catenin in group C (6.611 7 ± 0.381 8) was lower than that in group B (11.799 9 ± 0.348 7) and higher than that in group A (4.390 3 ± 0.551 9), showing significant differences (P lt; 0.05). The mRNA expression of Col II in group C was significantly higher than those in groups A and B (P lt; 0.05), the mRNA expression of MMP-13 in group C was significantly lower than that in group B (P lt; 0.05) but there was no significant difference when compared with group A (P gt; 0.05). The mRNA expression of β-catenin in group C was significantly lower than that in group B (P lt; 0.05) and higher than that in group A (P lt; 0.05). Conclusion ALN can protect rat chondrocyte from OA induced by IL-1β in vitro possibly by upregulating Col II and inhibiting the expression of MMP-13 and β-catenin in the chondrocytes.
To construct the retroviral vector containing human interleukin 1 receptor antagonist (IL-1Ra)and to investigate the property of the transfected articular chondrocytes from osteoarthritic patients in vitro. Methods Retroviral vector PLXRN carrying IL-1Ra (PLXRN-IL-1Ra) gene was constructed by inserting IL-1Ra gene at the sites of Sal I and BamH I. The recombinant retroviral plasmid was homologously recombinated in bacterial cells. After screening and ampl ification, the recombinant retroviral plasmid was obtained and transfected into PT67 cells. The repl ication-defective retrovirus PLXRN-IL- 1Ra was packed and ampl ified in the PT67 cells. Viral titer was determined by infecting NIH/3T3 cells with serially diluted viral supernatants produced with a control vector. Experiments were divided into 3 groups: non-transducted group (group A), PLXRN transduction group (group B), PLXRN-IL-1Ra transduction group (group C). Primary articular chondrocytes from osteoarthritic patients were transduced with PLXRN and PLXRN-IL-1Ra.The positive chondrocytes clones, which were G418- resistant, were cultured for 3-4 weeks after being selected by G418. The expression of IL-1Ra mRNA in the chondrocytes was determined by RT-PCR. Levels of IL-1Ra protein synthesis in the supernatants were measured by ELISA. Results Restric tive endonuclease identification and gene sequencing confirmed that the recombinant contained IL-1Ra cDNA．Virus titer could reach 3 × 104 CFU/mL. Primary chondrocytes cultured in vitro were polygonal or spindle and were stained with purple particles by toluidine blue staining. After stable transduction into the chondrocytes the 311 bp fragment of IL-1Ra was detected in group C by semi-quantitative RT-PCR. ELISA showed that IL-1Ra in supernatants of the group A and group B were below the level of detection. The concentrations were（60.47 ± 15.13）ng/L in group C .There were significant differences between gene transduction group and control groups (P lt; 0.05). Conclusion The construction of recombinant retrovirus vector by homologous recombination in bacterial cells can be quickly and easily performed. Stable and effective expression of IL-1Ra can be achieved by transduction with retroviral vectors in osteoarthritic articular chondrocytes, indicating potential util ity in gene therapy for osteoarthritis．
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 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 investigate the role of transforming growth factor β(TGF-β)in the regulation of the gene expression of matrix metalloproteinase 13(MMP-13)in the human hyaline chondrocytes. Methods The human hyaline chondrocytes harvested enzymatically and cultured in DMEM supplemented with 20% fetus calf serum were divided into 7 groups. Group 1 was used as a contol, and 1 ng/ml TGF-β(group 2), 10 ng/ml TGF-β(group 3), 100 ng/ml TGF-β(group 4), 1 ng/ml TGF-β＋10 ng/ml IL-1β(group 5), 10 ng/ml TGF-β＋10 ng/ml IL-1β(group 6),and 100 ng/ml TGF-β＋10 ng/ml IL1β(group 7) were given for 12-hour coculture. The MMP-13 mRNA levels of passaged human hyaline chondrocytes were assessed by reverse transcriptionpolymerase chain reaction(RT-PCR) and real-time fluorescent quantitative PCR. Results TGF-β can increase the MMP-13 mRNA level respectively in the passagedhyaline chondrocytes. In the multifactor treated groups, TGF-β can decrease the MMP-13 mRNA level respectively and there was significant difference between groups (Plt;0.05).The level of MMP-13 mRNA expression had significant coherence withthe dosage of TGF-β. Conclusion The above results show that human chondrocytes express MMP-13 mRNA. TGF-β could cause a dosedependent stimulation on MMP-13 gene expression in human chondrocytes and have a potent effect of antagonizing IL-1β in osteoarthritis. TGF-β may play a crucial role in the occurrence anddevelopment of osteoarthritis through regulating MMP-13.