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.
The study aims to explore the effect of mesenchymal stem cells-derived exosomes (MSCs-Exo) on staurosporine (STS)-induced chondrocyte apoptosis before and after exposure to pulsed electromagnetic field (PEMF) at different frequencies. The AMSCs were extracted from the epididymal fat of healthy rats before and after exposure to the PEMF at 1 mT amplitude and a frequency of 15, 45, and 75 Hz, respectively, in an incubator. MSCs-Exo was extracted and identified. Exosomes were labeled with DiO fluorescent dye, and then co-cultured with STS-induced chondrocytes for 24 h. Cellular uptake of MSC-Exo, apoptosis, and the protein and mRNA expression of aggrecan, caspase-3 and collagenⅡA in chondrocytes were observed. The study demonstrated that the exposure of 75 Hz PEMF was superior to 15 and 45 Hz PEMF in enhancing the effect of exosomes in alleviating chondrocyte apoptosis and promoting cell matrix synthesis. This study lays a foundation for the regulatory mechanism of PEMF stimulation on MSCs-Exo in inhibiting chondrocyte apoptosis, and opens up a new direction for the prevention and treatment of osteoarthritis.
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 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 study the variation of CD105+/CD166+ cells and its multilineage potential in early osteoarthritis (OA) cartilage so as to lay a foundation for cartilage repair and pathologic cartilage remodeling in arthritis. Methods The knee OA model was established in the right knee of 30 adult New Zealand rabbits (8-12 months old). The chondrocytes were harvested from normal cartilage of the left knee (group A), OA cartilage of the right knee at 2 weeks (group B), at 4 weeks (group C), and at 8 weeks (group D) after modeling, and BMSCs were used in group E for the expression of CD105 and CD166. The percentage of CD105+/CD166+ cells in each group was counted by flow cytometry, and CD105+/CD166+ cells were isolated and purified by magnetic-activated cell sorting. The expressions of CD105 and CD166 were observed in 5 groups by laser scanning confocal microscope. Chondrogenesis, osteogenesis, and adipogenesis were evaluated with Alcian blue cytochemistry and collagen type II immunohistochemistry, by detecting the deposition of calcium, and with oil red O staining, respectively. Results The percentage of CD105+/CD166+ cells in group A, B, C, and D was significantly lower than that in group E (P lt; 0.05); it was significantly higher in groups B, C, and D than in group A (P lt; 0.05), and in group D than in groups B and C (P lt; 0.05), but no significant difference was found between groups B and C (P gt; 0.05). Laser scanning confocal microscope results confirmed the expressions of CD105+ and CD166+ cells in groups A, B, C, D, and E, no obvious difference in expression was shown among 5 groups. At 1 week after chondrogenic induction, positive expressions of proteoglycan and collagen type II were observed in 5 groups, no obvious difference was noticed among 5 groups. At 2 weeks after osteogenic induction, calcium level in group E was significantly higher than that in groups A, B, C, and D (P lt; 0.05), but no significant different was found among groups A, B, C, and D (P gt; 0.05). At 4 weeks after adipogenic induction, there were more red lipid droplets in group E than in groups A, B, C, and D. Conclusion CD105+/CD166+ cells in early OA cartilage increase, which show chondrogenic differentiation potential.
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.
ObjectiveTo summarize the research progress of pathological manifestations and mechanism of endochondral ossification in osteoarthritis (OA). MethodsThe literature about endochondral ossification, bone-cartilage remodeling in OA, and joints development was reviewed, analyzed, and summarized. ResultsChondrocyte hypertrophy and apoptosis, vascular invasion, replication of the tidemark, thickening calcified cartilage, and thinning superficial cartilage are the characteristics of cartilage degeneration in OA. Articular cartilage and growth plate are similar in structure, and cartilage degeneration in OA is similar to a process of endochondral ossification of the growth plate. ConclusionLoss of stability characterization from resting metabolic balance to a high conversion state of temporary cartilage in stimulation of abnormal mechanical stresses and cytokines would subsequently contributed to continual calcification and remodeling of articular cartilage, which may be the key link of the initiation and development of OA.
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 The changes of the aquaporins 1 (AQP-1) expression may be related to the chondrocyte apoptosis. To explore the correlation between the expression of AQP-1 and chondrocyte apoptosis by observing the expression of the AQP-1 and the Caspase-3, so as to provide experimental evidence for the further study in the pathogenesis of osteoarthritis (OA). Methods Seventy-two 8-week-old clean grade male Sprague Dawley rats, weighing 286-320 g (mean, 300 g), were randomly divided into the operated group (n=24), the sham-operated group (n=24), and the control group (n=24).OA models were made by amputating the anterior cruciate l igament and medial collateral l igament, and partial excision of medial meniscus in operated group; the articular cavity was exposed only in sham-operated group; and no treatment was given in control group. The general condition of the rat was observed after model was establ ished. At 1, 2, 4, and 8 weeks, the specimens of knee joints were harvested to perform the gross and histological observations; the mRNA expressions of AQP-1 and Caspase-3 were determined by real-time fluorescent quantitative PCR; and the activity of the Caspase-3 protease was detected. The correlations between the expression of AQP-1 mRNA and the expressions of Caspase-3 mRNA and protease were analyzed. Results Totally 6 rats died after operation, and the rats were suppl ied immediately; the other rats survived to the end of experiment. The appearance and structure of knee articular cartilage were normal in control group and sham-operated group. While in operated group, the cartilage had a rough surface with fissure and vegetation, and fibrosis and irregular cell arrangement were seen on the surface of cartilage. There were significant differences in the Mankin score between the operated group and sham-operated group, control group at 2, 4, and 8 weeks (P lt; 0.05). There was no significant difference in expressions of the AQP-1 mRNA and Caspase-3 mRNA, and the activity of the Caspase-3 protease among 3 groups at 1 week after operation (P gt; 0.05); while the expressions of the AQP-1 mRNA, Caspase-3 mRNA, and the activity of the Caspase-3 protease in operated group were significantly higher than those in sham-operated group and control group at 2, 4, and 8 weeks after operation (P lt; 0.05), andthere was an increased trend over time. There was significantly positive correlation (r=0.817, P=0.000) between the expressions of AQP-1 mRNA and Caspase-3 mRNA, and the regression equation was y=0.426 7x2+0.051 5x; meanwhile, there was also significantly positive correlation (r=0.945, P=0.000) between the expression of AQP-1 mRNA and the activity of Caspase-3 protease, and the regression equation was y=15.423 0x+4.392 8. Conclusion The up-regulation of AQP-1 expression in OA cartilage may be related to the chondrocyte apoptosis, and the changes of AQP-1 expression may involve in the pathogenesis of OA.
Objective To investigate the feasibil ity and effect of inducing adi pose-derived stem cells (ADSCs) treated with growth differentiation factor 5 (GDF-5) to undergo chondrogenic differentiation in vitro. Methods Six healthy Japanese rabbits aged 3 months (2-3 kg) of clean grade were chosen, irrespective of sex. ADSCs were isolated and cultured with collagenase digestion, then were detected and identified by vimentin immunohistochemistry and CD44, CD49d, CD106immunofluorescence staining. ADSCs at passage 3 were used and the cell density was adjusted to 1 × 106/mL, then the ADSCs were treated with 0, 10, 100 ng/mL GDF-5 and common cultural medium, respectively. The morphology changes of the induced ADSCs were observed by inverted contrast phase microscope and their growth state were detected by MTT. The mRNA quantities of Col II and proteoglycan expressed by the induced ADSCs were detected with RT-PCR. The Col II proteoglycan synthesized by the induced ADSCs were detected with alcian blue staining, toluidine blue staining, immunohistochemistry staining, and Western blot method. Results ADSCs mostly presented small sphere, fusiform and polygon shape with positive expression of CD44 and CD49d and negative expression of CD106 and vimentin. The ADSCs treated with 100 ng/mL GDF-5 presented sphere or sphere-l ike change and vigorous prol iferation. The mRNA quantities of Col II and proteoglycan synthesized by the induced ADSCs treated with 0, 10, 100 ng/mL GDF-5 and common cultural medium increased in a dose-dependent manner at 7 days. There were significant differences among all the groups (P lt; 0.05), except that no significant difference was evident between the 0 ng/mL group and the 10 ng/mL group (P gt; 0.05). When ADSCs were treated with 100 ng/mL GDF-5 for 14 days, the Col II and the mRNA and protein quantities of ptoteoglycan reached the peak, and the results of alcian blue, toluidine blue and Col IIimmunohistochemistry staining were positive. Conclusion ADSCs treated with certain concentration of GDF-5 have higher expression of Col II and proteoglycan and possess partial biological function of chondrocyte.