OBJECTIVETo explore and identify the method for IL-1beta induced New Zealand rabbit knee chondrocyte degeneration, thus providing experimental bases for Chinese medical research on osteoarthritis from in vitro cultured chondrocytes.

METHODSUnder aseptic conditions, bilateral knee joint cartilage was collected from 4-week old New Zealand rabbits. Chondrocytes were separated by type II collagenase digestion and mechanical blowing method. They were randomly divided into two groups when passaged to the 2nd generation, the normal control group (group Z) and the IL-1beta induced model group (group M). No intervention was given to those in group Z. 10% FBS culture media containing 10 ng/mL IL-1beta was added to group M. All cells were passaged to the 3rd generation. They were compared using morphological observation, toluidine blue staining, type II collagen immunohistochemical staining, and flow cytometry.

RESULTSUnder inverted microscope, the second and the 3rd generation chondrocytes' phenotype of group Z was stable with good proliferation. Most cells turned into fusiform and slabstone shaped. In group M, most cells turned into long spindle shape or irregular shape. Results of toluidine blue staining and immunohistochemistry showed that the positive expression of chondrocytes after staining in group Z was superior to that in group M. Results of flow cytometry showed that there was statistical difference in the apoptosis rate of the second generation chondrocytes between group M and group Z (P < 0.01).

CONCLUSIONIt was obviously seen that chondrocytes in IL-1beta induced New Zealand rabbit knee chondrocyte model obviously degenerated, which could be used in related experimental researches on osteoarthritis.

Animals ; Cartilage ; cytology ; drug effects ; Cells, Cultured ; Chondrocytes ; cytology ; drug effects ; Interleukin-1beta ; pharmacology ; Knee Joint ; cytology ; drug effects ; Rabbits

This study determined the effects of selenium on the growth of Fusarium strains and the effects of products extracted from the fungal cultures on relevant indicators of chondrocytes injury. The results showed that selenium supplementation resulted in differential effects on the mycelial growth of the strains. Levels of the chondrocyte injury indicators, including cell viability, proteoglycan and type II collagen contents and their mRNA expressions, were all reduced to varying degrees when the chondrocytes were incubated with fermentation extracts, the inhibitory effect varied depending on selenium content supplemented to fungal culture media. The results indicated that certain chain relations existed between the content of selenium in the environment, the production of some metabolites by fungi, and the occurrence of chondrocyte damage. The extent of this relationship and the role it plays in Kaschin-Beck disease pathogenesis merit further study.
Animals ; Cell Survival ; Cells, Cultured ; Chondrocytes ; pathology ; Fermentation ; Fusarium ; drug effects ; physiology ; Rabbits ; Selenium ; pharmacology

OBJECTIVE: To investigate the eff ect of icaritin on proliferation and apoptosis in rat chondrocytes and to provide new theory for osteochondropathy treatment. METHODS: Icaritin (with a purity of 99%) at different concentrations (0, 4, 8, 16, 32, 64 μmol/L) was incubated with rat chondrocytes for different time. The cell proliferation and apoptosis was assayed by MTT and fl ow cytometry, respectively. RESULTS: Compared with the control group, the cell proliferation was increased in the groups with icaritin at 4 or 8 μmol/L (P<0.05), whereas the proliferation was decreased in the groups with icaritin at 16, 32 or 64 μmol/L groups compared to the control group (P<0.05); the cell apoptosis ratio in the group with icaritin at 4 μmol/L was obviously lower than that in the control group aft er incubation of icaritin for 24 h and 48 h. Beyond 4 μmol/L, the higher concentration of icaritin, the higher apoptosis ratio of cell. However, it did not show a time-dependent manner at a same concentration of icaritin. CONCLUSION The icaritin at low concentration (4 or 8 μmol/L) can promote rat chondrocyte proliferation and inhibit cell apoptosis, while the effect of icaritin on rat chondrocyte at high concentration was reversed.
Animals ; Apoptosis ; Cell Proliferation ; Cells, Cultured ; Chondrocytes ; cytology ; drug effects ; Flavonoids ; chemistry ; Rats

Uniform design-comprehensive scoring method was used to investigate the effects of ethanol dosage, ethanol concentration and extraction time, based on the evaluation index from transfer rates of epimedin A, epimedin B, epimedin C, icariin and baohuoside Ⅰ, which are the main active components in Epimedii Folium. Furthermore, the optimum conditions for the ethanol extraction process were determined by multiple linear stepwise regression and empirical test. Then, the ethanol extract of Epimedii Folium prepared according to the optimized technological conditions was used to intervene the injury model of chondrocyte induced by interleukin-1 beta(IL-1β). Annexin V-FITC/PI staining flow cytometry was used to detect the apoptotic rate of chondrocyte and analyze the effect of ethanol extract of Epimedii Folium on chondrocyte injury model. The optimum conditions of ethanol extraction were as follows. Crude powder of Epimedii Folium was added with 18 times of 70% ethanol solution, and extracted for twice in the refluxing process, for 60 minutes each time. Under the conditions, the extraction rates of the above five active components were 94.21%, 94.76%, 93.85%, 96.17% and 96.85%, respectively. The optimized ethanol extraction process of Epimedii Folium was reasonable, feasible and reproducible. This ethanol extract could significantly reduce the early apoptotic rate, late apoptotic and necrotic rate, total apoptotic rate(P<0.05 or P<0.01) of chondrocyte injury model induced by IL-1β, suggesting that the ethanol extract of Epimedii Folium can inhibit the apoptosis of chondrocytes induced by IL-1β to a certain extent, which lays a foundation for further study on its prevention and treatment of osteoarthritis.
Chondrocytes/drug effects* ; Epimedium/chemistry* ; Ethanol ; Humans ; Interleukin-1beta ; Plant Extracts/pharmacology* ; Plant Leaves/chemistry*

OBJECTIVETo investigate the effects of different concentrations of Gubishu containing serum on the proliferation of rabbit articular chondrocytes cultured in vitro.

METHODSArticular chondrocytes were obtained from the cartilage of 1-month rabbit and cultured in vitro. They were randomly divided into 8 groups,blank and Gubishu groups in different concentrations (5%, 10%,15%, 20%), MTT assay method was adopted to observe the influence of Gubishu containing serum with different concentrations to the proliferation of chondrocytes after incubated 1, 3, 5, 7 and 9 days.

RESULTSThe proliferation of chondrocytes was dependent on the concentration in Gubishu groups. At same time point,there was significant value between every groups, 20% concentration was greatest (P<0.05); There was significant differences between 5%, 10% and 20% concentration of the blank groups at same time point (P<0.05), and was not between 15% and 20% concentration at the 1, 3, 5 and 7 days (P>0.05), 20% concentration of the blank group was greatest. 20% concentrations of Gubishu containing serum was significantly greater than 20% concentrations of blank group at the 1, 3, 5 and 7 days (P<0.05).

CONCLUSION20% concentrations of Gubishu containing serum can significantly increase the proliferation of chondrocytes, and bring the logarithmic growth period forward to the 3 day.

Animals ; Cartilage, Articular ; cytology ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Chondrocytes ; drug effects ; physiology ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal ; pharmacology ; Female ; Male ; Rabbits ; Serum

OBJECTIVETo study the effects of Geniposide on SNP(sodium nitroprusside)-induced apoptosis of chondrocyte in vitro and cell cycle.

METHODSThe chondrocyte of three-week-old SD rats were separated and cultivated. The second generation of chondrocyte cells were involved in experiment. Chondrocyte proliferation was measured by assay; flow cytometer were adopted to observe cell cycle and apoptosis rate; NO examination adopted nitrate reductase method.

RESULTSGeniposide could significantly decrease the percentage of SNP-induced chondrocytes in G0/G1 phase and increased percentage in S phase and G2/M phase. The apoptosis of chondrocyte and the concentration of NO in the culture supernatants was reduced significantly (r=0.917, P<0.01).

CONCLUSIONGeniposide could impact SNP-induced apoptosis of chondrocyte by reducing the concentration of NO in the culture supernatants, promoting proliferation of chondrocytes, which is a probable and important mechanism of Geniposide preventing osteoarthritis.

Animals ; Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Chondrocytes ; drug effects ; physiology ; Female ; Iridoids ; pharmacology ; therapeutic use ; Male ; Nitroprusside ; pharmacology ; Osteoarthritis ; drug therapy ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo explore Poloxamer 188, a non-ionic surfactant as a potential tool for early intervention into the chondrocyte damaged by blunt mechanical trauma in vitro.

METHODSThree groups were control group (n = 6), no treatment group (n = 12) and Poloxamer 188 treatment group (n = 12). Two groups are then loaded to 20 MPa in unconfined compression. At 1 and 24 h the percentages of live and dead cells of superficial zone in compressed and control groups were determined with a cell viability stain.

RESULTSAt 1 h post-trauma, specimens of Poloxamer 188 treatment group (76%) had a significantly increased percentage of live cells in the superficial zone versus the no treatment group (55%). In 24 h the percentages of live cells in the superficial zone of the Poloxamer 188 treatment group (57%) were significantly greater than in the no treatment group (38%).

CONCLUSIONSPoloxamer 188 surfactant could help restore the integrity of cell membranes in cartilage damaged by blunt mechanical trauma. Models of mechanical cartilage injury in vitro may explain aspect of the interactions between mechanical forces and degradative pathways which lead to osteoarthritis progression.

Apoptosis ; drug effects ; Cartilage, Articular ; pathology ; Cell Survival ; drug effects ; Chondrocytes ; drug effects ; pathology ; Humans ; In Vitro Techniques ; Poloxamer ; pharmacology ; Random Allocation ; Weight-Bearing

OBJECTIVETo establish an immortalized marrow mesenchymal stem cell line to facilitate advances in cartilage engineering research.

METHODSHuman telomerase reverse transcriptase (hTERT) cDNA was transferred into primary human marrow mesenchymal stem cells (hMSC) by retroviral vector pLEGFP-C1-hTERT. Subsequently G418 resistant cell clone was screened and expanded for further studies. hMSC biomarkers and hTERT expression were confirmed by examination. Transfected hMSC was induced to differentiate into chondrocyte using TGF-P1 and dexamethasone.

RESULTSUp-regulated hTERT expression was detected in transfected hMSC. hMSC-hTERT cells could be induced to differentiate into chondrocyte. Higher telomerase activity in transfected cells was maintained for 50 population doublings so far. Collagen II could be detected in induced transfected hMSC by immunocytochemical and hybridization in situ.

CONCLUSIONSEctopic expression of hTERT can effectively immortalize hMSC in vitro. Immortalized hMSC can be induced to differentiate into chondrocyte under certain condition. It may be an ideal target of further studies in cartilage engineering.

Bone Marrow Cells ; cytology ; drug effects ; metabolism ; Cell Differentiation ; drug effects ; Cell Line, Transformed ; Chondrocytes ; cytology ; Humans ; Mesenchymal Stromal Cells ; cytology ; drug effects ; metabolism ; Telomerase ; genetics ; metabolism ; Transfection

OBJECTIVETo study the effect of growth hormone (GH) on the proliferation and type II collagen secretion of chondrocytes of mandibular condyle in rabbit in vitro.

METHODSFlow cytometry (FCM) and immunohistochemical technique were employed to observe the possible changes.

RESULTS(1) The exogenic GH can enhance the proliferation and synthesis of DNA of the chondrocytes of mandibular condyle in rabbit in vitro. The suitable concentration of GH is 10 microg/ml. The synthesis of DNA reaches the highest level after 12 hours, while the proliferation index (PI) hits the highest after 24 hours. (2) GH (10 microg/ml) can stimulate the secretion of type II collagen of the chondrocytes.

CONCLUSIONThe exogenic GH can enhance the proliferation, the synthesis of DNA and the secretion of type II collagen of the chondrocytes of mandibular condyle in rabbit in vitro.

Animals ; Cartilage, Articular ; cytology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Chondrocytes ; drug effects ; secretion ; Collagen Type II ; drug effects ; Growth Hormone ; pharmacology ; Mandibular Condyle ; cytology ; Rabbits

OBJECTIVETo study the effects of Bushen Zhuangjin Decoction (BZD) containing serum on the apoptosis of chondrocytes induced by mechanics stimulus.

METHODSThe BZD containing serum was extracted. The chondrocyte nutritive media was divided into 3 groups, i.e., the common nutritive medium group, the blank rabbit serum medium group, and the BZD nutritive medium group. The apoptosis of chondrocytes was induced by continuing mechanics stimulus in 24 h. Then the chondrocytes were collected. The apoptosis rate of chondrocytes was determined by flow cytometry. The contents of interleukin 1beta (IL-1beta) and nitric oxide (NO) in the corresponding media were determined.

RESULTSThe apoptosis of chondrocytes in the BZD nutritive medium group (19.55 +/- 7.98)% was lower than that of the common nutritive medium group (39.32 +/- 13.45)% and the blank rabbit serum medium group (37.87 +/- 9.67)%, showing statistical difference (P < 0.05). The contents of IL-1beta and NO were also lower in the BZD nutritive medium group with statistical difference when compared with those of the other two groups (P < 0.05).

CONCLUSIONBZD containing serum could protect mechanics stimulus induced apoptosis of chondrocytes.

Animals ; Apoptosis ; drug effects ; Cartilage, Articular ; drug effects ; Cells, Cultured ; Chondrocytes ; cytology ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Flow Cytometry ; Interleukin-1beta ; analysis ; Nitric Oxide ; analysis ; Rabbits ; Serum