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BACKGROUND: Capparis spinosa total alkaloids (CSTA) have certain effects on cell growth and extracellular matrix synthesis. The aging and apoptosis of nucleus pulposus cells are one of the main pathologies of intervertebral disc degeneration. Therefore, it is assumed that CSTA may have certain effect on the degeneration of the intervertebral disc. OBJECTIVE: To study the effect of CSTA on intervertebral disc degeneration rat model and nucleus pulposus cells. METHODS: Thirty-two Sprague-Dawley rats were randomly divided into sham, model, CSTA-H, and CSTA-L groups with eight in each group. Rat models of intervertebral disc degeneration were made in the model group, CSTA-L group and CSTA-H group. The CSTA-L and CSTA-H groups were given intragastric administration of CSTA 225 mg/kg/d and 450 mg/kg/d for 4 weeks respectively. Hematoxylin-eosin staining was used to observe the pathological changes of the intervertebral disc. Immunohistochemistry and western blot were used to detect the expression of type II collagen and aggrecan. Nucleus pulposus cells from the intervertebral disc of another two Sprague-Dawley rats were separated, cultured and divided into a control group, an oxygen-glucose deprivation (OGD) group and an administration group (OGD+CSTA 10 mg/L). After being cultured for 24 hours, the morphology of nucleus pulposus cells was observed, the cell proliferation ability was detected by cell counting kit-8, the cell apoptosis was detected by flow cytometry, and the expression of type II collagen and aggrecan were detected by western blot. RESULTS AND CONCLUSION: (1) Compared with the sham group, the intervertebral disc tissue of the model group showed fiber ring fissures, aggregation and shrinking of nucleus pulposus cells, and different improvements were found in the CSTA-L and CSTA-H groups. (2) The expression levels of type II collagen and aggrecan in the model group were significantly lower than those in the sham, CSTA-L and CSTA-H groups (P < 0.05). (3) Compared with the control group, the cells in the OGD group showed irregular morphology and death status, whereas the cell morphology in the administration group was improved. (4) Compared with the control group, nucleus pulposus cells in the OGD group showed lower proliferation, higher apoptotic rate, and lower levels of type II collagen and aggrecan (P < 0.05). Compared with the OGD group, nucleus pulposus cells in the administration group showed faster proliferation, lower apoptotic rate, and higher levels of type II collagen and aggrecan. To conclude, CSTA can improve intervertebral disc degeneration by promoting proliferation and inhibiting apoptosis of nucleus pulposus cells as well as inhibiting the degradation of extracellular matrix.
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ABSTRACT: In healthy cartilage, chondrocytes maintain an expression of collagens and proteoglycans and are sensitive to growth factors and cytokines that either enhance or reduce type II collagen synthesis. In osteoarthritis, pro-inflammatory cytokines, such as IL-6, induce overexpression of metalloproteinases (MMP) and decreasing synthesis of aggrecan. Use of chondroprotectors agents, such as Platelet-Rich Plasma (PRP) and triamcinolone (TA) are alternatives to reduce the progression of joint damage. In this study, we used chondrocytes extracted from metacarpophalangeal joints of healthy horses as the experimental model. Cells were treated in vitro with PRP or TA. No differences were observed between these treatments in comparison to the control group when the expressions of MMP9, MMP13, IL-6 and ACAN genes were evaluated (P<0.05). With these results, we can suggest that the treatments were not deleterious to equine cultured chondrocyte, once they did not stimulate MMPs and IL-6 synthesis or caused changes in ACAN.
RESUMO: Na cartilagem saudável, os condrócitos mantêm a expressão de colágenos e proteoglicanos, sendo sensíveis a fatores de crescimento e citocinas que aumentam ou reduzem a síntese de colágeno tipo II. Na osteoartrite, citocinas pró-inflamatórias, como a IL-6, estimulam a expressão de metaloproteinases (MMP) e reduzem a síntese de agrecano. O uso de condroprotetores, como o Plasma Rico em Plaquetas (PRP) e triancinolona (TA) é uma alternativa para se reduzir a progressão do dano articular. Neste estudo foram usados condrócitos extraídos das articulações metacarpofalangeanas de equinos saudáveis. As células foram tratadas in vitro com TA ou PRP. Não foram observadas diferenças entre os tratamentos comparando-se com o grupo controle quanto à expressão genética de MMP-9, MMP-13, IL-6 e ACAN (p<0,05). Assim, pode-se sugerir que os tratamentos não foram deletérios ao cultivo de condrócitos, uma vez que não estimularam a síntese de MMP e IL-6 e nem causaram alterações no ACAN.
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Objective: To investigate clinical efficacy of Duhuo Xuduan Tang in treatment of lumbar disc herniation with different syndrome types and its effect on serum pain factors, bone morphogenetic protein-7 (BMP-7) and Aggrecan. Method: A total of 121 patients with non-emergency lumbar disc herniation admitted to the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine from January 2017 to June 2018 were randomly divided into observation group (62 cases) and control group (59 cases). The two groups of patients were treated by repositioning maneuver before absolute bed rest. observation group was given Duhuo Xuduan Tang, 150 mL·time-1, 3 times·d-1 orally, while control group was orally given ibuprofen, 300 mg·time-1, vitamin B1, 10 mg·time-1, 2 times·d-1, drugs were taken for 5 days a week, and then stopped for 2 days. Both groups were treated continuously for 4 weeks. changes in visual analogue score (VAS), modified Oswestry dysfunction index (MODI), serum substance P (SP), dopamine (DA), serotonin (5-HT), BMP-7 and Aggrecan were observed before and after treatment with enzyme-linked immunosandwich assay (ELISA). Result: Compared with before treatment, VAS and modified Oswestry dysfunction index scores were lower in both groups (PPPPPConclusion: Duhuo Xuduan Tang has a certain efficacy on different types of lumbar disc herniation, with best efficacy for patients with liver and kidney yin deficiency.
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Objective: To study the effect of Bushen Zhuangjintang and glucosamine hydrochloride on the repairing of rat knee cartilage defect, the combined application of traditional Chinese and western medicine can promote the repairing of knee cartilage defect more effectively. To provide a new theoretical basis and method for the treatment of knee cartilage injury. Method: SPF rats (64 rats) were randomly divided into 5 groups, sham group,model group, Bushen Zhuangjintang group (7.5 g·kg-1·d-1), and glucosamine hydrochloride group(7.5 g·kg-1·d-1), Bushen Zhuangjin Decoction combined with glucosamine hydrochloride group[(7.5+7.5) g·kg-1·d-1] was administered to rats by drug gavage. The knee cartilage defects were repaired by gross observation and scanning electron microscopy at 4, 8 and 12 weeks. Real-time PCR was used to detect the expression of Collagen Ⅱ and Aggrecan mRNA in each group. Western blot was used to detect the expression of Collagen Ⅱ. Result: 4,8,12 weeks compared with group, Bushen Zhuangjintang combined with hyaluronic acid in the glucosamine hydrochloride group was filled with hyaline cartilage. The cartilage in the drilled area was smooth and smooth, and integrated with the surrounding cartilage tissue, which was superior to other groups. Real\|time PCR and Western blot analysis showed that the expression of Aggrecan protein and mRNA were significantly increased at 4, 8 and 12 weeks compared with the blank group (PPConclusion: The combined application of traditional Chinese and western medicine can obviously promote the repair of cartilage defects in the knee of rats. The possible mechanism of the treatment of cartilage injury by the combination of traditional Chinese and western medicine is analyzed from the level of gene protein expression and microstructure.
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OBJECTIVE: We have demonstrated that needle knife (acupotomy) treatment can improve knee osteoarthritis (KOA) in rabbits. The present study was designed to examine its effect on expression of phosphorylated focal adhesion kinase (p-FAK), phosphinositides 3 kinase (p-PI 3 K) and Aggrecan genes and proteins in the knee-joint cartilage tissues of KOA rabbits, so as to explore its partial molecular mechanism underlying improvement of KOA. METHODS: Forty-nine New Zealand male rabbits were randomly divided into normal control, model, model +inhibitor, needle knife, needle knife+ inhibitor, electroacupuncture (EA), EA +inhibitor groups (n=7 in each). The KOA model was established by modified Videman method (left hindlimb extension immobilization and ankle dorsal flexion 60°). Acupotomy relaxing manipulation was applied to the lateral collateral ligament and patellar ligament of the left knee-joint, two times a week for 4 weeks, and EA (2 Hz/100 Hz, 3 mA) was applied to the left "Liangmen" (ST 21), "Xuehai" (SP 10), "Neixiyan " (EX-LE 4) and "Dubi" (ST 35) for 20 min, three times a week, for 4 weeks. About 2 h before every needle-knife or EA treatment or at the corresponding time-point, intra-articular cavity injection of PF-562271(a specific antagonist of FAK, 200 μmol/L, 0.5 mL)was performed in the three inhibitor groups. The expression levels of p-FAK, p-PI 3 K, Aggrecan genes and proteins in the cartilage tissues were measured with quantitative Real-time PCR and Western blot, separately. RESULTS: After modeling, the expression levels of p-FAK and p-PI 3 K genes and proteins were significantly up-regulated (P<0.05, P<0.01), while those of Aggrecan protein and mRNA considerably down-regulated in the model group in comparison with the normal group (P<0.01). Following 4 weeks' needle-knife or EA treatment, the expression levels of p-FAK and p-PI 3 K and Aggrecan proteins in both EA and needle-knife groups, and Aggrecan mRNA in the needle knife group were significantly up-regulated (P<0.05, P<0.01). After administration of p-FAK antagonist, modeling-induced upregulation of expression of p-FAK mRNA and protein and p-PI 3 K protein, as well as modeling-induced down-regulation of Aggrecan mRNA and protein were significantly suppressed in the model+inhibitor group (P<0.05, P<0.01), and needle knife-induced and EA-induced up-regulation of expression of p-FAK, p-PI 3 K and Aggrecan mRNAs and proteins was notably suppressed respectively in comparison with the needle knife and EA groups (P<0.05, P<0.01). The effect of needle knife was significantly superior to that of EA in up-regulating p-PI 3 K and Aggrecan mRNAs as well as p-FAK, p-PI 3 K and Aggrecan proteins (P<0.05, P<0.01). CONCLUSION: Needle knife intervention can up-regulate the expression levels of p-FAK, p-PI 3 K and Aggrecan proteins and mRNAs in the cartilage tissue of the knee-joint in KOA rabbits, suggesting an involvement of FAK-PI 3 K signaling in the needle knife-induced improvement of KOA.
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Objective To investigate the role of vitamin D in the synthesis and degradation of aggrecan in rat articular chondrocytes at cellular level. Methods Rat articular chondrocytes were stimulated by IL-1α, IL-1β and TNF-α, respectively. Normal and inflammatory chondrocytes were treated with different doses of vitamin D, respectively. CCK8, Flow cytometry, real time-PCR and western blot analysis were used to examine the proliferation activity and apoptosis level of chondrocytes, and the expression of aggrecan, ADAMTS-4 and ADAMTS-5 at both mRNA and protein levels. Results IL-1α,IL-1β and TNF-α significantly decreased the proliferation activity and increased the apoptosis level of the chondrocytes. Furthermore, IL-1α, IL-1β and TNF-α significantly decreased the expression of aggrecan, and increased the expressions of ADAMTS-4 and ADAMTS-5 at both mRNA and protein levels in the chondrocytes. 1α,25 (OH)2D3supplementation significantly increased the proliferation activity and decreased the apoptosis level of chondrocytes stimulated by IL-1α, IL-1β and TNF-α in a dose-dependent manner, but not affected the normal chondrocytes. Meanwhile, 1α,25(OH)2D3also significantly increased the expression of aggrecan, and decreased the expressions of ADAMTS-4 and ADAMTS-5 at both mRNA and protein levels in the chondrocytes under inflammatory conditions. Conclusions Vitamin D may promote the anabolism of aggrecan and inhibit aggrecanase activity in chondrocytes under inflammatory conditions, which may impact overall protection for articular cartilage.
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Objectives To analyze a rare autosomal recessive disease, aggrecan type spondylometaphyseal dysplasia (SEMD), which was caused by ACAN gene mutations. Methods A 7 years old girl was diagnosed with short stature after excluding growth hormone deficiency, idiopathic short stature, and hypothyroidism. Combining family history and clinical features, SEMD were suspected and genetic tests were performed. Results The patient was found with homozygous mutations of c.512C > T inACAN gene, and was diagnosed with aggrecan type SEMD. Her parents were found to be heterozygous mutation carrier. Conclusions In patients with high suspection of a special type of short stature, early genetic tests should be carried out for a clear diagnosis.
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Objective To detect the expression of a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4),Versican,and Aggrecan in hearts of myocardial infarction rats.Methods Male Wistar rats were chosen to make myocardial infarction model.Fifteen rats were randomly assigned into the myocardial infarction group and five rats into the sham-operation group.Rats were killed 3 d after surgery and the left ventricles were taken to produce frozen section.ADAMTS4,Versican,and Aggrecan protein expressions were detected by imnunohistochemistry method.Results ADAMTS4 expressed in myocardial cells at marginal zone of the infarcted area.ADAMTS4 expressed weakly while Versican expressed strongly in capillary endothelial cells of marginal zone of the infarcted area.Aggrecan showed no expression in the cardiac tissue and vascular endothelium of both myocardial infarction group and sham-operation group.Conclusions ADAMTS4 is involved in the inflammation after myocardial infarction.Degradation of ADAMTS4 to Versican exists in the vessels after myocardial infarction.Aggrecan is not involved in the pathophysiological process of myocardial infarction.
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Objective To detect the COMP and aggrecan content in serum of patients with KOA and explore its correlative factors.Methods COMP and aggrecan in serum of 100 patients with knee-osteoarthritis(KOA group)and 30 healthy volunteers (control group)were measured by ELISA.Correlative factors for COMP and aggrecan levels were tested by linear regression analy-sis.Results COMP and aggrecan levels in KOA group were significantly higher than that in control group (P < 0.05 ).Serum COMP and aggrecan levels were positively correlated with age,body mass index(BMI),WOMAC scores and X-ray K-L grade(P <0.05).Conclusion Serum COMP and aggrecan are useful markers in the diagnosis,which their level in KOA are higher than the normal people.Moreover,these rise of levels positively correlated with clinical disease parameters and radiological joint damage.
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Objective To explore the mechanism of therapeutic action of needle knife “regulating sinews and treating bones” on knee osteoarthritis (KOA) by observing the effect of needle knife intervention on KOA rabbit behaviors, mechanical characteristics of patellar ligament (PL), and expressions of interleukin-4 (IL-4), matrix metalloproteinase-3 (MMP-3) and aggrecan in knee cartilages.Methods Forty New Zealand rabbits were randomly allocated to blank, model, needle knife and electroacupuncture groups, 10 rabbits each. A rabbit model of KOA was made by the modified Videman method of immobilization in extension position. After model making, the needle knife and electroacupuncture groups received needle knife and electroacupuncture treatments, respectively. A behavioral assessment was made using the modified Lequesne MG knee grade evaluation method in every group after model making and treatment. The samples were taken after treatment. PL tension, stress relaxation and creep state were tested using a Bose Electro Force 3300 protracted test machine. Cartilage cell IL-4 expression was examined by ELISA. MMP-3 mRNA and aggrecan mRNA expressions were detected by real-time PCR.Results After model making, there was a statistically significant difference in the Lequesne MG score between the model and blank groups (P0.05). There was a statistically significant post-treatment difference in the Lequesne MG score between the needle knife or electroacupuncture group and the model group (P0.05).Conclusion The mechanism of action of needle knife treatment on KOA may be that it improves ligament mechanical characteristics, regulates intra-articular stress environment, and modulates aggrecan mRNA and MMP-3 mRNA expressions and inhibits cartilage degeneration through IL-4 mechanical signal pathway, to produce the therapeutic effect of “regulating sinews and treating bones”.
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Objective To investigate the relationship between aggrecan and YKL-40 in knee articular cartilage of Sprague-Daw-lay(SD) rats with osteoarthritis (OA) .Methods Fifty-six healthy SD rats were randomly divided into 7 groups ,8 cases per group . The one side of knee joint was randomly selected for performing the anterior cruciate ligment transection (ACLT) and establishing the OA model .The rats in one group were randomly killed on the day of operation and at postoperative 0 ,2 ,4 ,8 ,12 ,16 ,20 weeks . The femoral condyle cartilage samples at different time periods in the operated side were collected for conducting safranin O /fast green staining and HE staining .Meanwhile ,the OA pathological grade was made out according to the modified Mankin scale .The expression of aggrecan and YKL-40 in the cartilage with different stages of OA were analyzed by the immunohistochemistry meth-od ,and the status of expression were measured by average optical density (AOD) .The correlation between aggrecan and YKL-40 was analyzed .Results With the aggravation of OA ,the expression of aggrecan was gradually reduced and the expression of YKL-40 was gradually increased .The differences during the early ,middle and late phases of OA had statistical significance (P<0 .05) . The expression of aggrecan was negatively correlated with the expression of YKL-40(P<0 .05) .Conclusion The level of aggrecan is gradually reduced with the aggravation of OA .Aggrecan is negatively correlated with the YKL-40 level ,which may reflect the dedifferentiation degree of joint chondrocyte to some extent .
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OBJECTIVE: Recurrent lumbar disc herniation has been reported to occur in 5% to 15% of surgically treated primary lumbar disc herniation cases. We investigated the molecular biologic characteristics of primary herniated discs and recurrent discs to see whether the recurrent discs has the similar biological features with primary herniated discs. METHODS: Primary herniated disc and recurrent disc cells were obtained by discectomy of lumbar disc patients and cells were isolated and then taken through monolayer cultures. We compared chondrogenic and osteogenic mRNA gene expression, and western blot between the two groups. RESULTS: The mRNA gene expression of recurrent disc cells were increased 1.47* times for aggrecan, 1.38 times for type I collagen, 2.04 times for type II collagen, 1.22 times for both Sox-9 and osteocalcin, and 1.31 times for alkaline phosphatase, respectively, compared with the primary herniated lumbar disc cells (*indicates p < 0.05). Western blot results for each aggrecan, type I collagen, type II collagen, Sox-9, osteocalcin, and alkaline phosphatase were similar between the primary herniated disc cells and recurrent disc cells. CONCLUSION: These results indicate that the recurrent disc cells have similar chondrogenic and osteogenic gene expression compared to primary herniated disc cells. Therefore, we assumed that the regeneration of remaining discs could fill the previous discectomy space and also it could be one of the factors for disc recurrence especially in the molecular biologic field.
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Humans , Aggrecans , Alkaline Phosphatase , Blotting, Western , Collagen , Collagen Type I , Collagen Type II , Diskectomy , Durapatite , Extracellular Matrix , Gene Expression , Intervertebral Disc Displacement , Osteocalcin , Population Characteristics , Recurrence , Regeneration , RNA, MessengerABSTRACT
Objective By exploring the effects of recombinant human bone morphogenetic protein (rhBMP)2 and rhBMP13 on chondrocytes proteoglycan production and phenotype expression to establish the theoritical mechanisms for the treatment of disc degeneration with chondrocytes transplantation plus BMPs.Methods The dose-dependent effects of rhBMP2 and rhBMP13 on PG protein synthesis and gene expression were detected under different concentrations (0,25,125,and 625 ng/ml).The sulfated-glycosaminoglycan (s-GAG) in the culture media and the pericellular matrix was measured with a 1,9-dimethyl-methylene blue (DMMB) colorimetric assay.Reverse transcriptase polymerase chain reaction (RT-PCR) was performed respectively to quantify the relative abundance of aggrecan Mrna.Cell proliferation was examined by Hoechst Dye assay.Results All rhBMP2 and rhBMP13 in different concentrations could significantly increase s-GAG synthesis and gene expression in chondrocytes (P＜0.05).And at the same concentration.rhBMP2 was more potent than rhBMP13 on s-GAG synthesis.Hoechst Dye assay showed neither rhBMP2 nor rhBMP13 had significant effect on cell proliferation.Conclusion rhBMP2 and rhBMP13 are able to upregulate s-GAG synthesis,in addition,rhBMP2 is more potent than rhBMP13 on aggrecan gene expression regulation,but rhBMP2 and rhBMP13 do not have significant effect on chondrocyte proliferation.
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Objective To study the expressions of aggrecan (Agc) Ⅰ and Ⅱ collagen and Sox9 by bone mar-row mesenchymal stem cells exposed to electromagnetic fields (EMFs) and it's mechanisms involved. Methods Bone marrow mesenchymal stem cells were isolated from Sprague-Dawley rats and cultured in vitro. The third passage cells were harvested and exposed to 15 Hz 1 mT EMFs for 8 h/d. The semi-quantitative reverse transcription-polyme-rase chain reaction (RT-PCR) technique was used to measure parathyroid hormon receptor related peptide (PTHrp) ,Agc Ⅰ and Ⅱ collagen and Sox9 mRNA. Western blotting was used to measure type Ⅱ collagen expression. After the inhibitor of protein kinase A (PKA) H-89 and the inhibitor of protein kinase C (PKC) Go-6976 ( 12 μm) were added, the effects of EMFs on Agc Ⅰ and Ⅱ collagen and Sox9 mRNA expressions were measured again by using RT-PCR, and Western blotting technique. Results The EMFs induced significant increase of mRNA expressions of PTHrp, Agc Ⅰ and Ⅱ collagen and Sox9 in comparison to the controls, and promoted type Ⅱ collagen protein expres-sion. The Agc Ⅰ and Ⅱ collagen expressions decreased after PKA pathway inhibitor H-89 and PKC inhibitor Go-6976 were added, but the mRNA expression of Sox9 was not affected. Conclusion This study shows 15Hz 1mT EMFs can promote mRNA expressions of Agc Ⅰ and Ⅱ collagan and Sox9 of cbondrogenesis differentiation markers in bone marrow mesenchymal stem cells. The effect is correlated with PKA and PKC pathways.
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To investigate the influence of recombinant adenovirus carrying tissue inhibitor of metalloproteinase-3 (RAdTIMP-3) on the main compositions of rabbits intervertebral discs and to assess its potential in treatment for intervertebral disc degeneration.[Method]RadTIMP-3 and empty adenovims vector with Lac-Z gene (Rad66) was propagated in 293 Cells and was purified, identified and tittered. Thirty Japanese white rabbits were randomly divided into 5 groups. And 25 μl of various reagents were injected to the L4、5 and L5、6 intervertebral discs of the rabbits as follows:normal saline in group 1, 1.0×1010 OPU/ml of RAd66 in Group 2, and 1.0×1010 OPU/ml of RAdTIMP-3 in group 3, 4 and 5. The intervertebral discs of each group were collected after 2, 2, 1, 2 and 4 weeks after injection respectively.Then X-gal staining, And Group 1, RT-PCR for TIMP-3 and aggrecan core protein,TUNEL staining, immunohistochemical staining for TIMP-3 and type I! Collagen and Safranin O-Fast green staining was carried out to assess the effects of RadTIMP-3 transfection.[Result](1)concentration of RAdTIMP-3 reached 1.9×1012 OPU/ml after propagation and purification. (2)RT-PCR shows that the expression of TIMP-3 was significantly raised in group 3, 4, 5, as compared with group 1 or 2. And the expression of core protein gene in group 3, 4, 5 increased slightly than in group 1 and 2. (3) TUNEL staining revealed that there was not significant difference between the positive-staining rates of any two of the groups. (4)TIMP-3 staining exhibited an obvious increase of positive-staining rates in group 3, 4 and 5 as compared with groupi or 2. The staining density of Safranin O-Fast Green staining and immunohistochemical staining for type II collagen of group 5 was obviously higher than that of group 1 or 2.[Conclusion]RAdTIMP-3 can express widely and safely in rabbit intervertebral discs, and improve the quantity and quality of matrix. It has the potential to be used in treatment for intervertabral disc degeneration.
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PURPOSE: The aim of this study was to evaluate the expression of type II collagen, aggrecan, VEGF-A and PEDF mRNAs in the human chondrocytes derived from the articular cartilage of the femoral heads with avacular necrosis (AVN). MATERIALS AND METHODS: We cultured human chondrocytes that were primarily derived from the articular cartilage of femoral heads with AVN. We evaluated the mRNA expression of type II collagen, aggrecan, VEGF-A and PEDF. RESULTS: The chondrocytes of the AVN group showed decreased expressions of type II collagen mRNA and aggrecan mRNA (p0.05). CONCLUSION: The cartilage matrix's formation ability was found to be decreased in the chondrocytes of the femoral heads affected by AVN.
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Humans , Aggrecans , Cartilage , Cartilage, Articular , Chondrocytes , Collagen Type II , Extracellular Matrix , Head , Necrosis , RNA, Messenger , Vascular Endothelial Growth Factor AABSTRACT
[Objective] To examine the specific gene and protein expression of primary and passaged chondrocytes in normal oxygen tension and hypoxia.[Methods]Articular chondrocytes were isolated from limb joint cartilage of C57BL/6 mice(3~5 days).Primary chondrocytes(P0)and passaled chondrocytes(P1,P2)were cultured in normal oxygen tension and hypoxia respectively for two days.The mRNA levels of collagen II,aggrecan,sox9,Indian hedgehog(ihh),parathyroid hormone-related protein(PTHrP),bone morphogenetic protein(BMP)-4 and wnt5a were examined with RT-PCR,and protein levels of collagen II,aggrecan were examined with immunohistochemistry and toluidine blue staining.[Results]During the culture of primary chondrocyte,the expression of collagen II and aggrecan increased under hypoxia,mRNA levels of collagen II,wnt5a increased and ihh decreased at the same time.The mRNA levels of special genes and differentiation related genes of passaged chondrocytes were not altered under hypoxia.[Conclusion]Protein and mRNA level of Collagen II of primary chondrocyte under hypoxia increased.It may be regulated by chondrocyte differentiation gene ihh,and wnt5a.The chondrocyte phenotype could not be resumed under hypoxia through short-term monolayer culture in vitro.
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Objective To study aggrecan, collagen type Ⅰ and collagen type Ⅱ gene expression of intervertebral disk degeneration through destroying bilateral zygapophysial joints of New-Zealand rabbit. Methods Thirty male New-Zealand rabbits were randomly divided into operation groups on the bone and operation group on the soft tissue. In operation group of the bone, L4 and L5 inferior articular processes were en bloc excised, L5 and L6 superior articular processes were retained. In the operation group of soft-tissue, only L3 to L7 paravertebral muscles were stripped. In operation group of the bone, L4-5 and L5-6 intervertebral disks were acted as experimental group; L3-4 and L6-7 acted as self-control group. In the operation of soft tissue, L4-5 and L5-6 were acted as experimentalcontrol group. One、two、four and eight months post-operation, New-Zealand rabbits were killed. Aggrecan, collagen type Ⅰ and collagen type Ⅱ gene expression were performed with semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR). Results Aggrecan and collagen type Ⅱ mRNA levels decreased markedly, whereas type Ⅰ collagen mRNA gradually increased. At the same time, aggrecan, collagen type Ⅱ gene expression were the least in experiment group, whereas were most in the experiment-control group. Collagen type Ⅰ showed the contra-tendency. Conclusion Intervertebral disk degeneration can be induced through destroying L4-5 and L5-6 zygapophysial joints of New-Zealand rabbit.Aggrecan, collagen type Ⅰ and collagen type Ⅱ gene expression can reflect intervertebral disk degeneration.
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OBJECTIVES: To determine effect of transforming growth factor-beta1 and bone morphogenetic protein-2 in matrix synthesis and expression of chondrogenic phenotype in human intervertebral disc cells. MATERIALS AND METHODS: The intervertebral disc cells were harvested and cultured from the surgical patients for the degenerative disc disease. TGF-beta1 was purchased from R&D and BMP-2 was produced by transfection of pcDNA3.1/Hygro/BMP-2 to CHO cell using Lipofectamine 2000. rhBMP-2 was separated by Heparin-Sepharose A chromatography. TGF-bata1 and BMP-2 were administered to culture. Proteoglycan synthesis was assessed by 35S incorporation and expression of matrix mRNA was analyzed by RT-PCR for collagen I, collagen II, aggrecan, and osteocalcin. RESULTS: TGF-bata1 and BMP-2 showed increased proteoglycan synthesis and expression of collagen I, collagen II and aggrecan mRNA in dose dependent manner respectively. There was no recognizable synergistic effect in matrix synthesis and matrix mRNA expression. Throughout dosage, expression of osteogenic phenotype (osteocalcin mRNA) was not noted. CONCLUSION: TGF-beta1 and BMP-2 proved to be effective anabolic agent for maximizing matrix synthesis without evidence of osteogenesis.
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Animals , Cricetinae , Humans , Aggrecans , CHO Cells , Chromatography , Collagen , Intervertebral Disc , Osteocalcin , Osteogenesis , Phenotype , Proteoglycans , RNA, Messenger , Transfection , Transforming Growth Factor beta1ABSTRACT
PURPOSE: We intended to check the growth rates and phenotypic markers of chondrocytes in the dedifferentiated cells cultivated in various conditions in order to establish the ideal culture system for implantation. MATERIALS AND METHODS: Culturing rabbit chondrocytes from proximal tibia, we checked the phenotypes at first, second, and third week. Then we cultured the chondrocytes in different circumstances such as monolayer or three dimensional gel in the presence or abscence of TGF-B1, and checked the growth rates and phenotypic markers. RESULTS: There was no difference in growth rates and mRNA level of type I, type II collagen and aggrecan between the cells cultured in monolayer and three dimensional gel of collagen. However, the responses of the cells to TGF-B1, were quite different between these two groups. In monolayer culture, the expression of type I collagen was depressed by TGF-B1 while the growth rate was markedly increased. Oppositely in three dimensional culture, the mRNA level of type I collagen was markedly increased and the growth rate was completely suppressed by TGF-B1. The expression of type II collagen could be detected only in TGF-B1-treated cells cultured in three dimensional gel for 4 or more days. The mRNA level of aggrecan was also increased by TGF-B1, in the cells cultured in three dimensional gel. CONCLUSIONS: These results suggest that the number of chondrocytes can be efficiently expanded by culturing the cells in monolayer and the phenotypes of chondrocyte can be restored by culturing the cells in three dimensional gel containing TGF-B1. The application of semi-solid gel containing differentiated chondrocytes in physeal implantation should be further evaluated