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

The transforming growth factor-beta 1 was known as having the most important influence on chondrocytes among various growth factors, being abundant in articular chondrocytes and osteocytes. We performed in vitro monolayer cultures of human articular chondrocytes from normal and osteoarthritic patients and studied the transforming growth factor-beta 1 responsiveness of those chondrocytes. The cell-growth curve indicated that the primary osteoarthritic chondrocyte culture with transforming growth factor-beta 1 showed a more rapid growth pattern than normal chondrocytes with or without TGF-beta 1 and osteoarthritic chondrocytes without TGF-beta 1. The osteoarthritic group showed a sharp decline in growth pattern with subsequent culture. The shape of osteoarthritic chondrocytes was bigger and more bizarre compared to those of normal chondrocytes. With subsequent culture, this change became prominent. The transforming growth factor-beta 1 increased the [3H]-TdR uptake in each group. The phenotypes of chondrocytes were more clearly expressed in the normal group. The chondrocytes lost their phenotype (production of collagen type II) following subculture in each group. The transforming growth factor-beta 1 could not inhibit or delay the dedifferentiation process (loss of phenotype).
Cartilage, Articular/drug effects* ; Cartilage, Articular/cytology ; Cell Division/drug effects ; Cells, Cultured ; Human ; Osteoarthritis/pathology ; Reference Values ; Transforming Growth Factor beta/pharmacology*

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 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 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

There is evidence from other studies that some degree of cartilage healing may take place after the initiation of an inflammatory response. It is postulated that the induction of the platelet-cartilage interaction may eventuate in cartilage repair. The treatment of fresh articular cartilage with proteolytic enzymes rendered the tissue active as a platelet aggregant. During platelet aggregation a host of active substances are released which are known to play a role in the inflammatory response (Thompson 1975). This study was undertaken to evaluate the effects of trypsin on the surface injury of rabbit hyaline cartilage. The results were as follows: 1) Hyaline cell regeneration was observed only in the group treated with trypsin and blood; 2) Hyaline cartilage regeneration did not occur in the group treated with a single injection of trypsin or blood; 3) There was no significant damage to the healthy articular cartilage by the single injection of trypsin or blood, or both; and 4) Platelets do not adhere to cartilage and superficial damaged cartilage does not induce platelet aggregation.
Animal ; Cartilage, Articular/*drug effects/physiology/ultrastructure ; Cell Division ; Mitosis/physiology ; Platelet Aggregation/drug effects ; Rabbits ; Regeneration ; Trypsin/*pharmacology ; Wound Healing/drug effects/physiology

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

Due to the poor repair ability of cartilage tissue, regenerative medicine still faces great challenges in the repair of large articular cartilage defects. Quercetin is widely applied as a traditional Chinese medicine in tissue regeneration including liver, bone and skin tissues. However, the evidence for its effects and internal mechanisms for cartilage regeneration are limited. In the present study, the effects of quercetin on chondrocyte function were systematically evaluated by CCK8 assay, PCR assay, cartilaginous matrix staining assays, immunofluorescence assay, and western blotting. The results showed that quercetin significantly up-regulated the expression of chondrogenesis genes and stimulated the secretion of GAG (glycosaminoglycan) through activating the ERK, P38 and AKT signalling pathways in a dose-dependent manner. Furthermore, in vivo experiments revealed that quercetin-loaded silk protein scaffolds dramatically stimulated the formation of new cartilage-like tissue with higher histological scores in rat femoral cartilage defects. These data suggest that quercetin can effectively stimulate chondrogenesis in vitro and in vivo, demonstrating the potential application of quercetin in the regeneration of cartilage defects.
Animals ; Cartilage/cytology* ; Chondrocytes/drug effects* ; Chondrogenesis/drug effects* ; Extracellular Matrix/metabolism* ; Quercetin/pharmacology* ; Rats ; Signal Transduction/drug effects* ; Tissue Scaffolds

A type of water-soluble carboxymethyl chitosan (O--CMC), with 76% degree of substitution determined by conductivity method, was prepared by using chloroacetic acid to react with C6-OH of chitosan. The solubility of O--CMC was characterized also. Animal experiment in rabbits showed that O--CMC could lubricate arthron, inhibit proliferation of fibroblast cells on rabbits' knee joints, benefit the process of repairing pathologic articular cartilage, and produce good therapeutic effect on rheumatoid arthritis.
Animals ; Arthritis, Experimental ; drug therapy ; pathology ; Cartilage, Articular ; drug effects ; Cell Proliferation ; drug effects ; Chitosan ; administration & dosage ; analogs & derivatives ; therapeutic use ; Female ; Fibroblasts ; drug effects ; Injections, Intra-Articular ; Knee Joint ; drug effects ; Male ; Rabbits

OBJECTIVETo investigate the effect of the basic fibroblast growth factor (b-FGF) to regenerate an autologous tissue-engineered cartilage in vitro.

METHODSThe Cells were harvested from the elastic auricular cartilage of swine,and were plated at the concentration of 1 x 10(4) cells/cm2 , studied in vitro at two different media enviroments: Group I contained Ham's F-12 with supplements and b-FGF, Group II contained Ham's F-12 only with supplements. The passage 2 cells (after 12.75 +/- 1.26 days) were harvested and mixed with 30% pluronic F-127/Ham's F-12 at the concentration of 50 x 10(6) cells/ml. It was injected subcutaneously at 0.5 ml per implant. The implants were harvested 8 weeks after the vivo culture and examined with the histological stains.

RESULTSThe chondrocytes displayed morphologically similar to the fibroblasts in the media containing basic-FGF. The number of cell doublings (after 12.75 +/- 1.26 days) in vitro culture was as the following: Group I, 70; Group II, 5.4. Eight 8 weeks after the vivo autologous implantation, the average weight (g) and volume (cm3) in each group was as the following: Group I, 0.371 g/0.370 cm3 Group II, 0.179 g/0.173 cm3 (P < 0.01). With the b-FGF in vitro culture, the cells were expanded by 70 times after 2 weeks. Histologically, all of the engineered cartilage in the two groups were similar to the native elastic cartilage.

CONCLUSIONThese results indicate that the basic-FGF could be used positively to enhance the quality and quantity of the seeding cells for the generation of the well-engineered cartilage.

Animals ; Cartilage ; cytology ; drug effects ; physiology ; Cell Division ; drug effects ; Cells, Cultured ; Chondrocytes ; cytology ; drug effects ; Female ; Fibroblast Growth Factors ; pharmacology ; physiology ; Male ; Regeneration ; drug effects ; Swine ; Tissue Engineering ; methods ; Transplantation, Autologous