OBJECTIVE: The effects of the staurosporine on contraction of self-assembled constructs and extracellular matrix syntheses of goat temporomandibular joint discs were investigated. METHODS: Goat temporomandibular joint disc cells were isolated and cultured to P3, and 5.5×10⁶ cells were combined with different concentrations of staurosporine (0, 0.1, 1, 10, 100 nmol·L⁻¹) in agarose wells and cultured for one week. The samples were frozen and sectioned. Safranin-O,  Picro-sirius red and immunohistochemical staining were performed to observe the distributions of the extracellular matrix and the expression of alpha-smooth muscle actin (α-SMA). Enzyme linked immunosorbent assay (ELISA) and Blyscan kits were utilized to quan--titatively detect the contents of type Ⅰ collagen (ColⅠ) and glycosaminoglycans (GAGs). RESULTS: Each group of goat temporo-mandibular joint disc cells in the agarose wells were gathered to self-assemble into a disc-shaped base for 4 hours and then to gradually contract into a round shape. The Picro-sirius red staining was strong and indicated collagen distribution. The Safranin-O staining observed GAGs throughout the entire construct. The expression of ColⅠ was strongly posi-tive in the staurosporine groups; however, the expression of α-SMA was weak. ColⅠ and GAGs contents in the stau-rosporine groups were greater than that of the control group, especially in the 10 nmol·L⁻¹ group (P<0.01). CONCLUSIONS Staurosporine has a certain effect on the shrinkage of self-assembled constructs; however, such effect is not prominent. Staurosporine contributes to the construction synthesis of extracellular matrix.
Animals ; Collagen Type I ; Glycosaminoglycans ; Goats ; Staurosporine ; pharmacology ; Temporomandibular Joint ; Temporomandibular Joint Disc ; cytology ; drug effects

OBJECTIVES: This study aims to investigate the effects and mechanisms of chondroitin sulfate (CS), dermatan sulfate (DS), and heparin (HEP) on chondrogenesis of murine chondrogenic cell line (ATDC5) cells and the maintenance of murine articular cartilage in vitro. METHODS: ATDC5 and articular cartilage tissue explant were cultured in the medium containing different sulfated glycosaminoglycans. Cell proliferation, differentiation, cartilage formation, and mechanism were observed using cell proliferation assay, Alcian blue staining, real-time quantitative polymerase chain reaction (RT-qPCR), and Western blot, respectively. RESULTS: Results showed that HEP and DS primarily activated the bone morphogenetic protein (BMP) signal pathway, while CS primarily activated the protein kinase B (AKT) signal pathway, further promoted ATDC5 cell proliferation and matrix production, and increased Sox9, Col2a1, and Aggrecan expression. CONCLUSIONS This study investigated the differences and mechanisms of different sulfated glycosaminoglycans in chondrogenesis and cartilage homeostasis maintenance. HEP promotes cartilage formation and maintains the normal state of cartilage tissue in vitro, while CS plays a more effective role in the regeneration of damaged cartilage tissue.
Animals ; Mice ; Cartilage/metabolism* ; Cell Differentiation ; Cells, Cultured ; Chondrocytes/metabolism* ; Chondrogenesis/physiology* ; Glycosaminoglycans/pharmacology*

OBJECTIVETo investigate the effect of Coriolus versicolor polysaccharide B (CVP-B) on increased membrane glycosaminoglycans (GAG) expression and intracellular glutathione (GSH) of RAW264.7 macrophages exposed to angiotensin II (Ang II).

METHODSThe plasma membrane of RAW264.7 macrophages exposed to Ang II treatment was isolated by ultracentrifugation, and the membrane GAG expression was analyzed using 1, 9-dimethylmethylene blue (DMMB) spectrophotometric assay for sulfated GAG. The intracellular reduced GSH was determined using fluorophotometry.

RESULTSThe GAG content in the macrophage membranes increased by up to 54% following cell exposure to 1.0 micromol/L Ang II, whereas in presence of 1.0 micromol;/L Ang II, CVP-B at 1, 10, and 50 microg/ml decreased the GAG content by 13%, 43% (P<0.01), and 52% (P<0.01), respectively. The macrophage GSH activity decreased by 69% following incubation with 1.0 micromol;/L Ang II for 24 h, and CVP-B treatment at 1, 10, and 50 microg/ml in presence of 1.0 micromol;/L Ang II resulted in significant increment of GSH activity by 31%(P<0.05), 104% (P<0.01), and 168% (P<0.01), respectively.

CONCLUSIONThese data provide the first evidence that CVP-B inhibits elevated GAG expression in RAW264.7 macrophage membrane induced by Ang II.

Agaricales ; chemistry ; Angiotensin II ; pharmacology ; Animals ; Cell Line ; Cell Membrane ; metabolism ; Glutathione ; analysis ; Glycosaminoglycans ; analysis ; Macrophages ; metabolism ; Mice ; Polysaccharides ; pharmacology

OBJECTIVETo evaluate the feasibility of growing tissue-engineered cartilage using chondrocytes seeded onto a biodegradable porous bioceramic, the beta-tricalcium phosphate (beta-TCP).

METHODSA porous bioceramic template of beta-TCP was created in the shape of a disc. Chondrocytes isolated from rabbit articular cartilage were seeded on the beta-TCP template and then kept in rotatory cell culture system (RCCS) for 1 week prior to subcutaneous transplantation into athymic mice. The three-dimensional structure was well-maintained 16 weeks after implantation. After 4, 8, 16 weeks, the specimens were harvested and examined macroscopically, histologically and immunohistochemically.

RESULTSGross morphological and histological analysis of the specimens from the chondrocyte-beta-TCP complex demonstrated new cartilage construction. The overall configuration of the experimental specimens closely resembled the structure of beta-TCP template.

CONCLUSIONThese findings suggest that porous bioceramic (beta-TCP) is a good "matrix" for chondrocyte, and can be used for cartilage engineering.

Animals ; Calcium Phosphates ; pharmacology ; Cartilage ; growth & development ; transplantation ; DNA ; analysis ; Female ; Glycosaminoglycans ; analysis ; Immunohistochemistry ; Mice ; Mice, Nude ; Tissue Engineering

OBJECTIVETo observe the influence of Pilose antler polypeptide on the glycosaminoglycan and type II collagen in the articular cartilage in experimental knee osteoarthritis.

METHODSTotally 64 New Zealand white rabbits of 6 months old were randomly divided into 2 groups:normal group (n = 8) and model group (n = 56). Model group was surgically induced into osteoarthritis model by method of Hulth. After successful modeling, the rabbits of model group were further divided into 2 groups: Pilose antler polypeptide-treatment group and control group, 24 rabbits in each group. Pilose antler polypeptide-treatment group received 0.5 ml intra-articular injection of Pilose antler polypeptide dilution liquid once in per 2 days for 30 days, while control group received 0.5 ml intra-articular injection of physiological saline. On days 7, 15 and 30 after intervention, articular cartilage samples were collected respectively. The content of glycosaminoglycan in articular cartilage was observed by toluidine blue staining and the expression of type II collagen in cartilage matrix was detected by immunohistochemical staining.

RESULTSAlong with the prolonging of time, the content of glycosaminoglycan and type II collagen in cartilage matrix of the Pilose antler polypeptide-treatment group and control group decreased gradually. On days 7, 15 and 30 after intervention, integrated optical density of the type II collagen positive area in cartilage matrix of the Pilose antler polypeptide-treatment group were (312.06 +/- 14.12), (273.31 +/- 12.42) and (248.34 +/- 10.41), which had statistically significant differences. Integrated optical density of the type II collagen positive area in cartilage matrix of the control group were (253.47 +/- 15.53), (215.67 +/- 9.72) and (160.01 +/- 13.23), which had statistically significant differences. At the same period, integrated optical density of the type II collagen positive area in cartilage matrix of the Pilose antler polypeptide-treatment group was higher than that of control group, which had statistically significant difference.

CONCLUSIONPilose antler polypeptide can inhibit reduction of the glycosaminoglycan and type II collagen in cartilage matrix and delay the degeneration of articular cartilage.

Animals ; Antlers ; chemistry ; metabolism ; Collagen Type II ; metabolism ; Disease Models, Animal ; Female ; Glycosaminoglycans ; metabolism ; Humans ; Male ; Osteoarthritis, Knee ; drug therapy ; metabolism ; Peptides ; metabolism ; pharmacology ; Rabbits

OBJECTIVETo investigate the antithrombotic mechanisms of holothurian glycosaminoglycan (GAG) extracted from sea cucumber.

METHODSHuman endothelial cell line EA. hy926 cells were treated with 10 mg/L GAG or 10U/mL unfractionated heparin (UFH) by short-term (15 min - 2 h) and longer-time incubation (6 h - 48 h). Different doses of GAG were used to stimulate EA. hy926. Released free tissue factor pathway inhibitor(TFPI) was determined by ELISA assay. TFPI expression was investigated by immunofluorescent method and TFPI mRNA level by real-time PCR. In a 96-wells microtitre plate, pooled normal plasma containing different concentrations of GAG was allowed to clot by addition of thrombin and calcium chloride, fibrinolysis was induced by addition of t-PA. TRR (TAFI-related retardation of clot lysis) was used to assess thrombin-activatable fibrinolysis inhibitor(TAFI) functional activity.

RESULTSGAG increased TFPI synthesis, expression and secretion in a dose- and time dependent manner. GAG at low concentrations could lengthen while at intermediate concentrations could shorten clot lysis times significantly as compared to control values. TRR was dose-dependently decreased on addition of GAG.

CONCLUSIONSGAG increases TFPI synthesis, expression and secretion of endothelial cells. GAG at intermediate concentrations significantly affects clot stability of a developing clot by means of diminishing TAFI activation.

Animals ; Carboxypeptidase B2 ; antagonists & inhibitors ; Cell Line ; Dose-Response Relationship, Drug ; Endothelial Cells ; drug effects ; metabolism ; Glycosaminoglycans ; pharmacology ; Heparin ; pharmacology ; Holothuria ; Humans ; Lipoproteins ; biosynthesis ; genetics ; RNA, Messenger ; genetics ; Tissue Extracts ; pharmacology

OBJECTIVETo explore the effect of Ray cartilage glycosaminoglycans (RCG) on the expression of MMP-9 in Lewis lung carcinoma of mice.

METHODThe model of mice with Lewis lung carcinoma was induced. The experimental mice were randomly divided into normal saline group, RCG groups at varied concentrations and CTX group. Tumor growth state was observed, and tumor inhibitory rate of primary tumor and number of lung metastasis focus were measured. The expression of MMP-9 mRNA and protein in Lewis lung carcinoma was determined with RT-PCR and Western blot.

RESULTAs compared with normal saline group, tumor growth curves in RCG groups were smooth, there were significant differences of inhibitory rates of primary tumor and number of lung metastasis focus between RCG groups and normal saline group, and MMP-9 mRNA and protein expression levels in RCG groups were reduced significantly.

CONCLUSIONRCG can inhibit effectively the growth and metastasis of implanted Lewis lung carcinoma in C57BL/6 mice, which is probably attributed to reducing the expression of MMP-9 mRNA and protein.

Animals ; Antineoplastic Agents ; isolation & purification ; pharmacology ; Carcinoma, Lewis Lung ; enzymology ; pathology ; Cartilage ; chemistry ; Cell Line, Tumor ; Glycosaminoglycans ; isolation & purification ; pharmacology ; Matrix Metalloproteinase 9 ; biosynthesis ; genetics ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Skates (Fish)

OBJECTIVETo examine the effects of high and low concentrations of transforming growth factor (TGF) β(1) and insulin-like growth factor-I (IGF-I) on the extracelluar matrix synthesis of the self-assembled constructs of temporomandibular joint (TMJ) disc.

METHODSThe experimental groups of self-assembled constructs were exposed to IGF-I (10, 100 µg/L) and TGF-β(1) (5, 50 µg/L), the control groups were not added with any growth factors. All groups were examined at 3 and 6 weeks for gross morphological, histological, and biochemical changes. Safranin-O/fast green staining was used to examine glycosaminoglycan (GAG) distribution, picrosirius red and immunohistochemical staining to observe type I collagen distribution. Type I collagen contents were tested by ELISA assay kit, GAG contents were measured by Blyscan GAG assay kit, and the cell numbers were quantified with a Picogreen reagent kit.

RESULTSThe growth factor groups all upregulated the matrix synthesis of the self-assembled constructs compared with control groups. TGF-β(1) (5 µg/L) and IGF-I (10 µg/L) were the two most potent concentration in increasing type I collagen and GAG synthesis and cells proliferation. IGF-I group (10 µg/L) produced nearly 2 times (109.16 ± 5.12 µg) as much type I collagen as the control group (69.13 ± 5.94 µg) at 3 weeks. The matrix contents and the number of the proliferated cells in control group and all GF groups at 6 weeks were more than those at 3 weeks.

CONCLUSIONSIGF-I (10 µg/L) is the most beneficial growth factor and can be applied in tissue-engineering stratigies of the temporomandibular joint disc. At the same time, the exposure time of growth factors is another key factor that affects matrix synthesis of TMJ disc constructs.

Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; Collagen Type I ; biosynthesis ; Extracellular Matrix ; metabolism ; Glycosaminoglycans ; biosynthesis ; Goats ; Insulin-Like Growth Factor I ; pharmacology ; Temporomandibular Joint Disc ; cytology ; metabolism ; Tissue Engineering ; methods ; Transforming Growth Factor beta1 ; pharmacology

OBJECTIVETo investigate the potential effect of proteoglycan (PG) and glycosaminoglycan (GAG) on the bonding of etch and rinse adhesive to dentin, in order to improve the bonding effect of dentin.

METHODSForty-two extracted molars were used to obtain standard dentin bonding surface, and the specimens were etched for 15 s with 37% phosphoric acid and divided into three groups using a table of random numbers. Then the three groups undergone different incubating procedures as follow: specimens in chondroitinase ABC (C-ABC) group were incubated with C-ABC at 37 °C for 48 h in vibrator. Specimens in trypsin (TRY) group were incubated with trypsin, and specimens in the control group were incubated with deionized water for 48 h in the oscillators. Then specimens in each group were randomly assigned into two subgroups, A (Adper(TM) Single Bond 2) and B (Prime & Bond NT) (n = 7). The microtensile bond strength (µTBS), fracture mode and bonding interface morphology of the specimens were evaluated via microtensile testing, stereo microscope and field emission scanning electron microscopy (FE-SEM) respectively after specimens being incubated in 37 °C water for 24 h.

RESULTSThe immediate µTBS of C-ABC group bonding with adhesive A and B [(32.9±2.5) and (26.8±2.2) MPa] were significantly lower than that of the control group [(40.7±3.3) and (34.6±3.7) MPa] (P < 0.05). While the immediate µTBS of TRY group [(49.0 ± 3.6) and (44.5 ± 3.0) MPa] were significantly higher than that of the control group(P < 0.05).

CONCLUSIONSDentin PG participates in the dentin bonding process. Removal of PG increased the immediate µTBS of dentin and total etching adhesives, while removal of GAG decreased the immediate µTBS.

Acid Etching, Dental ; methods ; Bisphenol A-Glycidyl Methacrylate ; pharmacology ; Chondroitin ABC Lyase ; pharmacology ; Dental Bonding ; Dental Stress Analysis ; Dentin ; chemistry ; Dentin-Bonding Agents ; pharmacology ; Glycosaminoglycans ; pharmacology ; Hot Temperature ; Humans ; Phosphoric Acids ; Polymethacrylic Acids ; Proteoglycans ; pharmacology ; Random Allocation ; Resin Cements ; Tensile Strength ; drug effects ; Trypsin ; pharmacology

The present paper is aimed to observe the effects of basic fibroblast growth factor (bFGF) on bone marrow mesenchymal stem cell (BMSCs) differentiation. The bFGF was used to stimulate BMSCs and histology, immunohistochemistry and enzyme linked immunosorbent assay (ELISA) were used to examine the extracellular matrix produced by induced BMSCs, evaluated the feasibility of BMSCs being the seeding cells of temporomandibular joint (TMJ) disc tissue engineering. The results showed that having been induced with bFGF, the BMSCs could differentiate into fibroblast-like cells, which could synthesize GAG and collagen type I matrix. So it is feasible for BMSCs as seeding cells for engineered TMJ disc.
Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Collagen Type I ; biosynthesis ; Fibroblast Growth Factor 2 ; pharmacology ; Glycosaminoglycans ; biosynthesis ; Goats ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Temporomandibular Joint Disc ; cytology ; Tissue Engineering