BACKGROUNDChondrocytes' phenotype and biosynthesis of matrix are dependent on having an intact cytoskeletal structure. Microfilaments, microtubules, and intermediate filaments are three important components of the cytoskeletal structure of chondrocytes. The aims of this study were to determine and compare the effects of the disruption of these three cytoskeletal elements on the apoptosis and matrix synthesis by rabbit knee chondrocytes in vitro.

METHODSChondrocytes were isolated from full-thickness knee cartilage of two-month-old rabbits using enzymatic methods (n = 24). The isolated cells were stabilized for three days and then exposed to low, medium, and high doses of chemical agents that disrupt the three principal cytoskeletal elements of interest: colchicine for microtubules, acrylamide for intermediate filaments, and cytochalasin D for actin microfilaments. A group of control cells were treated with carrier. Early apoptosis was assessed using the Annexin-FITC binding assay by flow cytometry on days 1 and 2 after exposure to the disrupting chemical agents. The components and distribution of the cytoskeleton within the cells were analyzed by laser scanning confocal microscopy (LSCM) with immunofluorescence staining on day 3. The mRNA levels of aggrecan (AGG) and type II collagen (Col-2) and their levels in culture medium were analyzed using real-time PCR and enzyme-linked immunosorbent serologic assay (ELISA) on days 3, 6, and 9.

RESULTSIn the initial drug-dose-response study, there was no significant difference in the vitality of cells treated with 0.1 µmol/L colchicine, 2.5 mmol/L acrylamide, and 10 µg/L cytochalasin D for two days when compared with the control group of cells. The concentrations of colchicine and acrylamide treatment selected above significantly decreased the number of viable cells over the nine-day culture and disrupted significantly more cell nuclei. Real-time PCR and ELISA results showed that the mRNA levels and medium concentrations of AGG and Col-2 were significantly decreased for cultures treated with colchicine and acrylamide when compared with untreated cells at three, six, and nine days, and this inhibition was correlated with higher matrix metalloprotease-13 expression in these cells. Cellular proliferation, monolayer morphology, and matrix metabolism were unaffected in cytochalasin D-treated cells when compared with control cells over the nine-day culture period.

CONCLUSIONSThe disruption of the microtubulin and intermediate filaments induced chondrocyte apoptosis, increased matrix metalloprotease expression, and decreased AGG and Col-2 expression in rabbit knee chondrocyte cultures. Our findings suggest that microtubulin and intermediate filaments play a critical role in the synthesis of cartilage matrix by rabbit knee chondrocytes.

Animals ; Cartilage, Articular ; cytology ; metabolism ; Chondrocytes ; cytology ; Collagen ; metabolism ; Cytoskeleton ; metabolism ; Knee Joint ; cytology ; metabolism ; Microscopy, Confocal ; Rabbits

OBJECTIVETo investigate the effects in vitro of 17 beta-estradiol (E2) on the proliferation and metabolism of rabbit mandibular condylar cartilage cells.

METHODSChondrocytes were derived from neonatal rabbit mandibular condylar cartilage using a modified enzyme method. 17 beta-estradiol was added to the culture medium in a variety of concentrations. Cell growth and DNA, collagen, and proteoglycan synthesis were used as indicators of proliferation and differentiation of condylar chondrocytes. These were measured by cell number, 3H-proline and 35S-incorporation, respectively.

RESULTSE2 increased cell number and 3H-thymidine incorporation at 10(-8) to 10(-10) mol/L, and 10(-8) to 10(-11) mol/L in a dose-dependent manner, peaking at 10(-8) mol/L and 10(-9) mol/L, respectively. However, further increase in the concentration of estradiol caused inhibition of both cell number and 3H-thymidine incorporation, and this was significant at 10(-6) mol/L. The effect of E2 on proteoglycan synthesis was similar; the maximum stimulating effect was at 10(-8) mol/L, and inhibition was significant at 10(-6) mol/L. There was no obvious stimulatory effect of E2 on 3H-thymidine incorporation observed.

CONCLUSIONSEstradiol affects condylar chondrocyte cell growth, DNA, and proteoglycan synthesis in a biphasic manner depending on its concentration. This indicates that estrogen may be important in the proliferation and differentiation of mandibular condylar chondrocytes, and could be relevant to some aspects of certain temporomandibular joint diseases by modulating the function of the chondrocytes.

Animals ; Cartilage, Articular ; cytology ; metabolism ; Cell Differentiation ; Cell Division ; drug effects ; Cells, Cultured ; Estradiol ; pharmacology ; Mandibular Condyle ; cytology ; metabolism ; Rabbits

OBJECTIVETo study proteins correlated with the mechanical properties of engineered cartilage by screening significantly changed proteins during cartilage formation by comparative proteomic analysis.

METHODSHuman chondrocyte, cultured and expanded, were seeded onto a polyglycolic acid/polylactic acid (PGA/PLA) scaffolds. After 4 weeks of culture in vitro, the constructs were divided into three groups. There were 6 specimens in each group. For the regular in vitro culture group (A), the constructs were kept in culture at the original condition for an additional 6 weeks. For in vivo groups, the constructs were implanted subcutaneously into nude mice for either 6 weeks (B) or 12 weeks (C). All specimens were harvested for gross observation, average wet weight and volume measurement, histology, immunohistochemistry and biomechanics to evaluate the results. Meanwhile, comparative proteomic analysis was performed for each group, and those proteins involved in extracellular matrix with at least 2 folds up-regulation were chosen for further exploration. The correlations between Young's modulus and the relative content of the selected proteins were analyzed by Pearson correlation coefficient.

RESULTSAll these samples in the three groups eventually formed hyaline-like cartilage structure. Specimens in C and B groups were similar with adult articular cartilage in appearance, and had multiple mature lacuna in histology. However, those specimens in A group had loose texture with irregular hypertrophy lacuna. Specimens implanted for 12 weeks in vivo had better wet weight (372.5 +/- 35.4) mg and Young's modulus (8.68 +/- 2.65) MPa than those cultured in vivo for 6 weeks (346 +/- 34.5) mg, (3.25 +/- 1.24) MPa (P < 0.01). In group A, they were (184.4 +/- 12.28) mg and (0.7 +/- 0.23) MPa. This study had detected 44 proteins in ECM by comparative proteomic analysis, then chosing the greatest ratio of 6 up-regulation proteins compared between C and A groups. The correlation results indicated the content of Decorin, Chondroadherin and Fibromodulin were linear correlation with the mechanical properties of engineered cartilage (P < 0.05).

CONCLUSIONSComparative proteomic analysis could provide large scale information of associated proteins, making it profit for advanced research on the relationship between extracellular matrix and mechanical properties of engineered cartilage by combination with tissue reconstruction techniques.

Animals ; Cartilage ; cytology ; metabolism ; physiology ; Cells, Cultured ; Chondrocytes ; cytology ; metabolism ; Fetus ; cytology ; Humans ; Mice, Nude ; Proteome ; metabolism ; Proteomics ; Tissue Engineering ; methods ; Tissue Scaffolds

To investigate the estrogen receptor (ER) expression in cartilage cell in the development of osteoarthritis induced by bilateral ovariectomy in guinea pig and to find their relationship. 30 two-month-old female guinea pigs were randomly divided into two groups (n = 15 each): sham operation (control) group and ovariectomized group (OVX); Scanning electorne microscope (SEM) and transmission electron microscope (TEM) were obtained to analysis the cartilage degeneration of the hind limb knee joint after 6 and 12 weeks of ovariectomy. Dextran-Coated-Charcoal (DCC) was taken to quantitively detect the expression of ER. The serum levels of estrogen and gestone were detected by immune contest assay. The results showed that ER do exist in the cartilages of the guinea pigs, with higher expression in the control group than in OVX group at the same time point (P < 0.05). It was increased also at 12 th week after operation than that of preoperation. The blood serum levels of estrogen and gestone showed a similar tendency to the expression of ER. Joint cartilage degeneration detected by SEM and TEM could be found at 6 th week, but severe degenerative lesions at 12 th week in the OVX group compared with the control group (P < 0.01). The data suggested that bilateral ovariectomy in guinea pig lead to severe osteoarthritis which mighgt be related to the lower serum level of estrogen and the downregulation of the expression of ER in the cartilage also.
Cartilage, Articular/cytology ; Cartilage, Articular/*metabolism ; Chondrocytes/metabolism ; Estrogens/*blood ; Osteoarthritis/*etiology ; Osteoarthritis/metabolism ; Ovariectomy ; Random Allocation ; Receptors, Estrogen/*biosynthesis ; Receptors, Estrogen/genetics

OBJECTIVETo observe the effect of single herb pilose antler (PA) on the expression of Smad2 and Smad3 in the cartilage of osteoarthritis (OA) rats.

METHODSOne hundred 3-month old female healthy SD rats, (200 +/- 20) g, were recruited and routinely fed for 1 week. They were randomly divided into 5 groups, i.e., the low dose PA group, the high dose PA group, the normal saline control group, the model group, and the normal control group, 20 in each group. The model was prepared using classic Hulth method except the normal control group. After 6-week modeling, the model was confirmed successful by pathologic observation. PA at 0.021 g/100 g and 0.084 g/1 00 g was given by gastrogavage to rats in the low dose PA group and the high dose PA group respectively. Normal saline was administered to those in the normal saline control group. No treatment was given to rats in the normal control group and the model group. Bilateral knee cartilages were harvested at week 2,4, and 6. mRNA and protein expressions of Smad2 and Smad3 were detected by immunohistochemical assay, fluorescent quantitative PCR, and Western blot.

RESULTSOA model was successfully prepared by pathological observation. Results of immunohistochemical assay showed that Smad2 and Smad3 expressed extensively in the cartilage, and located inside the chondrocyte membrane. Compared with the model group, mRNA expression of Smad2 and Smad3 obviously increased in the low dose PA group and the high dose PA group at week 2, 4, and 6, showing statistical difference (P < 0.05). Compared with the same group at week 4 after gastrogavage, mRNA expression of Smad2 and Smad3 obviously decreased in the low dose PA group and the high dose PA group at week 6, showing statistical difference (P < 0.05). Compared with the model group, protein expression of Smad2 and Smad3 obviously increased in the chondrocytes of the low dose PA group and the high dose PA group at week 2 and 4, showing statistical difference (P < 0.01). Compared with the same group at week 2 after gastrogavage, protein expression of Smad2 and Smad3 obviously increased in the low dose PA group and the high dose PA group at week 4, showing statistical difference (P < 0.01). Compared with the same group at week 4 after gastrogavage, protein expression of Smad2 and Smad3 obviously decreased in the low dose PA group and the high dose PA group at week 6, showing statistical difference (P < 0.01).

CONCLUSIONS(1) The pilose antler could repair cartilages by regulating mRNA and protein expressions of Smad2 and Smad3. (2) Up-regulating mRNA and protein expressions of Smad2 and Smad3 might be one of important mechanisms for the pathogenesis of OA.

Animals ; Antlers ; chemistry ; Cartilage ; cytology ; metabolism ; Chondrocytes ; drug effects ; metabolism ; Female ; Medicine, Chinese Traditional ; Osteoarthritis ; drug therapy ; metabolism ; Rats ; Rats, Sprague-Dawley ; Smad2 Protein ; metabolism ; Smad3 Protein ; metabolism

OBJECTIVE: To explore the effect of insulin-like growth factor (IGF-1) on the concentration of NO and PGE(2) in the supernatant of rabbit articular chondrocytes induced by IL-1, and to explore the mechanism of IGF-1 in the development of osteoarthritis (OA). METHODS: The samples were divided into 7 groups: IL-1beta 10 microg/L group, IL-1beta 10 microg/L+IGF-1 1 microg/L group, IL-1beta 10 microg/L+IGF-1 10 microg/L group, IL-1beta 10 microg/L+IGF-1 50 microg/L group, IL-1beta 10 microg/L+IGF-1 100 microg/L group, IGF-1 50 microg/L group, and a blank control group. The chondrocytes from the articular cartilage of 2 month old rabbits were cultivated and identified, and then co-cultured in the second filial generation chondrocytes on plates with or without recombinant human IGF-1 or IL-1. The concentration of NO was detected by nitrate reductase kit, and that of PGE(2) by enzyme-linked immunosorbent assay (ELISA). The results were analyzed by statistical method. RESULTS: The average value of NO and PGE(2) was (89.971+/-10.224) micromol/L and (22.028+/-8.731) micromol/L in the IL-1beta 10 microg/L group, and (12.404+/-8.809) micromol/L and (1.900+/-0.227) ng/L in the blank control group. The concentration of NO and PGE(2) in IL-1beta 10 microg/L group was significantly higher than that in the blank control group (P<0.05). At the same concentration of 10 microg/L, IGF-1 could dose-dependently decrease the increase of NO and PGE(2) concentration induced by IL-1beta in the chondrocytes supernatant in vitro, and the optimum concentration of IGF-1 was 50 microg/L. CONCLUSION IL-1 can significantly increase the concentration of NO and PGE(2), and IGF-1 can dose-dependently decrease the concentration of NO and PGE(2) in the chondrocytes supernatant in vitro. The optimum concentration of IGF-1 was 50 microg/L.
Animals ; Cartilage, Articular ; cytology ; metabolism ; Cells, Cultured ; Chondrocytes ; drug effects ; metabolism ; Dinoprostone ; metabolism ; Insulin-Like Growth Factor I ; pharmacology ; Interleukin-1 ; pharmacology ; Nitric Oxide ; metabolism ; Osteoarthritis ; metabolism ; Rabbits

The feasibility of using gene therapy to treat full-thickness articular cartilage defects was investigated with respect to the transfection and expression of exogenous transforming growth factor (TGF)-beta 1 genes in bone marrow-derived mesenchymal stem cells (MSCs) in vitro. The full-length rat TGF-beta 1 cDNA was transfected to MSCs mediated by lipofectamine and then selected with G418, a synthetic neomycin analog. The transient and stable expression of TGF-beta 1 by MSCs was detected by using immunohistochemical staining. The lipofectamine-mediated gene therapy efficiently transfected MSCs in vitro with the TGF-beta 1 gene causing a marked up-regulation in TGF-beta 1 expression as compared with the vector-transfected control groups, and the increased expression persisted for at least 4 weeks after selected with G418. It was suggested that bone marrow-derived MSCs were susceptible to in vitro lipofectamine mediated TGF-beta 1 gene transfer and that transgene expression persisted for at least 4 weeks. Having successfully combined the existing techniques of tissue engineering with the novel possibilities offered by modern gene transfer technology, an innovative concept, i.e. molecular tissue engineering, are put forward for the first time. As a new branch of tissue engineering, it represents both a new area and an important trend in research. Using this technique, we have a new powerful tool with which: (1) to modify the functional biology of articular tissue repair along defined pathways of growth and differentiation and (2) to affect a better repair of full-thickness articular cartilage defects that occur as a result of injury and osteoarthritis.
Animals ; Bone Marrow Cells ; cytology ; metabolism ; Cartilage, Articular ; cytology ; Cells, Cultured ; Chondrocytes ; cytology ; Gene Transfer Techniques ; Rabbits ; Recombinant Proteins ; biosynthesis ; genetics ; Stem Cells ; cytology ; metabolism ; Tissue Engineering ; Transfection ; Transforming Growth Factor beta ; biosynthesis ; genetics

OBJECTIVETo investigate the effect of co-expression of bone morphogenetic protein 2 (BMP2) and Sox9 on chondrogenic differentiation of mesenchymal stem cells (MSCs) in vitro and provide experimental evidence for tissue engineering of cartilage.

METHODSMouse embryonic bone marrow MSC C3H10T1/2 cells were infected with recombinant adenovirus expressing BMP2, Sox9 and green fluorescent protein (GFP) for 3-14 days, with cells infected with the adenovirus carrying GFP gene as the control. The mRNA expression of the markers of chondrogenic differentiation, including collagen type II (Col2a1), aggrecan (ACAN), and collagen type X (Col10a1), were determined by real-time PCR. Alcian blue staining was used for quantitative analysis of sulfated glycosaminoglycan in the cellular matrix. The expression of Col2a1 protein was assayed by immunohistochemical staining and Western blot analysis.

RESULTSAdenovirus-mediated BMP2 expression induced chondrogenic differentiation of C3H10T1/2 cells. Overexpression of Sox9 effectively enhanced BMP2-induced expression of the chondrogenic markers Col2a1, aggrecan and Col10a1 mRNAs, and promoted the synthesis of sulfated glycosaminoglycan and Col2a1 protein in C3H10T1/2 cells.

CONCLUSIONCo-expression of BMP2 and Sox9 can promote chondrogenic differentiation of MSCs in vitro, which provides a new strategy for tissue engineering of cartilage.

Animals ; Bone Morphogenetic Protein 2 ; genetics ; metabolism ; Cartilage ; cytology ; Cell Differentiation ; Cells, Cultured ; Chondrocytes ; cytology ; Humans ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Mice ; SOX9 Transcription Factor ; genetics ; metabolism ; Tissue Engineering

Normal articular cartilage is composed of chondrocytes embedded within an extracellular matrix (ECM). The patterns of integrin expression determine the adhesive properties of cells by modulating interactions with specific ECMs. Our hypothesis is that chondrocyte integrin expression changes in response to changes in their microenvironment. Porcine articular chondrocytes were encapsulated in alginate beads with several ECMs (collagen type I, collagen type II and fibronectin) for 7 days, subjected to RT-PCR, western blot analysis and immunofluorescence staining. It was found that chondrocytes in different ECMs showed different patterns of integrin expression. Integrin alpha5 and beta1 were strongly expressed in all groups, but integrin alpha1 was strongly expressed only in collagen type I and fibronectin conjugated alginate beads, and integrin alpha2 was strongly expressed only in collagen type II conjugated alginate beads. These findings suggest that the addition of different ECMs to chondrocytes can modulate the patterns and levels of integrin expression possibly through a feedback mechanism. These finding suggest that the modulation of ECM interactions may play a critical role in the pathogenesis of osteoarthritis.
Animals ; Cartilage, Articular/cytology/*metabolism ; Chondrocytes/*metabolism ; Extracellular Matrix/*physiology ; Fluorescent Antibody Technique ; Integrins/genetics/*metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Support, Non-U.S. Gov't ; Swine

OBJECTIVEThis study was to explore the cytotoxic effect and the related injury mechanism of deoxynivalenol (DON) on articular chondrocytes in human embryo.

METHODSArticular cartilage cells were isolated from knees of human embryo and cultured in DMEM/F12 medium. The cells of the 4th generation were divided into five groups and incubated with varying concentrations of DON as the followings: control group and group with DON of 0.1, 0.2, 0.4, 1.0 µg/ml. The effects of DON were observed 72 hours after incubation. Cell apoptosis was assayed by flow cytometry (FCM) with Annexin V-FITC/PI staining; MMP-13 and PGE2 were detected by ELISA kits; NO was measured by Griess assay with spectrophotometer. Inducible nitric oxide synthase (iNOS) and collagen II in cells were detected by FCM. The expression levels of iNOS, mRNA and collagen II mRNA were measured with RT-PCR.

RESULTSThe rates of cell apoptosis in DON groups were 6.78% - 19.05%, which were significantly higher than that in control (1.20%, F = 174.761, P < 0.05). The levels of NO in DON groups were 20.8 - 40.7 µmol/L, which were significantly higher than that in control (10.2 µmol/L, F = 91.966, P < 0.05). The levels of MMP-13 in DON groups were 0.25 - 0.56 µmol/L, which were significantly higher than that in control (0 µmol/L, F = 78.420, P < 0.05). The levels of PGE2 in DON groups were 3.2-20.6 µmol/L, which were significantly higher than that in control (11.6 µmol/L, F = 276.453, P < 0.05). The proportions of cells with positive iNOS in DON groups were 14.8% - 56.8% which were significantly higher than that in controls (7.1%, F = 214.614, P < 0.05). The proportions of cells with positive collagen II in groups with DON of 0.4 µg/ml and 1.0 µg/ml were 56.7% and 52.7%, which were significantly lower than that in control (62.2%, F = 5.134, P < 0.05). The relative absorbance values of iNOS mRNA in DON groups were 1.07 - 1.33, which were significantly higher than that in control (0.62, F = 8.358, P < 0.05). The levels of collagen II mRNA in groups with DON of 0.4 µg/ml and 1.0 µg/ml were 0.83 and 0.82, which were significantly lower than that in control (1.14, F = 7.887, P < 0.05).

CONCLUSIONDON could promote anabolism of NO in articular cartilage cells by which up-regulated the expression of PGE2 and MMP-13, which both promoted resolution of articular cartilage matrix such as collagen II. DON induced apoptosis in articular cartilage cells.

Cartilage, Articular ; cytology ; embryology ; Cells, Cultured ; Chondrocytes ; drug effects ; metabolism ; Dinoprostone ; metabolism ; Humans ; Matrix Metalloproteinase 13 ; metabolism ; Nitric Oxide ; biosynthesis ; Trichothecenes ; toxicity