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Objective To find changes of urinary biomarkers of hydroxyproline (HYP) and C-terminal telopeptide of collagen type Ⅱ (CTX-Ⅱ) after celecoxib and anti-osteogenic tablets treatment among adult Kaschin-Beck disease (KBD) patients in Qinghai Province,and provide the scientific data for treatment effect of adult KBD.Methods According to the "Diagnosis of Kaschin-Beck Disease" (WS/T 207-2010),120 cases KBD patients and 89 cases healthy control were divided into three groups,including drug treatment group,non-drug therapy group and control group.The drug treatment group was taken celecoxib (200 mg/pill,1 pill/day,1 time/day) and anti-osteogenic tablets (0.4 g/pill,4 pills/time,2 times/day) for 6 month.The urine samples of these persons were detected with the method of enzyme linked immunosorbent assay (ELISA) for analyzing HYP and CTX-Ⅱ.The results were corrected by creatinine (Cr).Results There were 54 cases of KBD patients (24 males,30 females) in drug treatment group with average age of (47.51 ± 12.30) years old;there were 66 cases of KBD patients (31 males,35 females) in non-drug therapy group with average age of (46.85 ± 13.57) years old;and there were 89 healthy people (41 males,48 females) in control group with average age of (48.75 ± 13.92) years old.By comparing the three groups,there were no statistical significant differences in gender (x2 =0.820,P ＞ 0.05) and ages (F =0.379,P ＞ 0.05).Medians of urinary HYP contents among drug treatment group,non-drug therapy group and control group were 62.47,106.04,65.80 μg/μmol·Cr,respectively,and the difference was statistically significant (Z =12.114,P ＜ 0.01);medians of urinary CTX-Ⅱ contents among drug treatment group,non-drug therapy group and control group were 555.23,702.92,495.54 ng/μ mol· Cr,respectively,and the difference was statistically significant (Z =20.454,P ＜ 0.01).Conclusion Levels of HYP and CTX-Ⅱ among adult KBD patients have changed after treatment with celecoxib and anti-osteogenic tablets,and can be used to determine the effect of treatment.
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Objective To detect urinary bio-markers of hydroxyproline (HYP) and c-terminal telopeptide of collagen type Ⅱ (CTX-Ⅱ) among population from Kashin-Beck disease (KBD) regions in Qinghai Province,and to provide the scientific data for prevention and control of adult KBD.Methods According to the "Diagnosis of Kashin-Beck Disease" (WS/T 207-2010),using case-control study,120 KBD patients (males 55,females 65) and 89 healthy controls (males 41,females 48) in Qinghai KBD regions were divided into case group and control group.Morning urine samples were collected.HYP and CTX-Ⅱ contents were analyzed by enzyme-linked immunosorbent assay (ELISA),then these results were corrected with creatinine.All the data were analyzed by SPSS 17.0 software.Results There was no significant difference in the age of male and female between case group and control group (t =1.813,1.131,P ＞ 0.05).The medians of urinary HYP and CTX-Ⅱ contents among male patients were 74.91 μg/μmol Cr and 630.77 ng/μmol Cr,respectively,which were higher than those of control groups (51.38 μg/μ mol Cr,401.32 ng/μmol Cr,Z =3.068,3.246,P ＜ 0.01).The medians of urinary HYP and CTX-Ⅱ contents among female patients were 91.07 μg/μmol Cr and 637.17 ng/μmol Cr,respectively,compared with those of control groups (88.37μg/μmol Cr,546.47 ng/μmol Cr),there was no significant difference in HYP content (Z =0.273,P ＞ 0.05),however,the difference in CTX-Ⅱ content was statistically significant (Z =2.002,P ＜ 0.05).Conclusion The urinary HYP contents of male patients with KBD change significantly,while the degradation of type Ⅱ collagen in male and female patients increases,and CTX-Ⅱ could reflect the metabolic changes of collagen in KBD.
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Objective To investigate the feasibility of bone marrow mesenchymal stem cells (BMSCs) combined with type Ⅱ collagen-hyaluronic acid-oxidized chondroitin sulfate (Col Ⅱ-HA-OCS) biomimetic hydrogel to repair articular cartilage defect in porcine and the role of the transplanted cells played in the process of cartilage repair.Methods A articular cartilage defect model which remaining cartilage calcified zone was created in the knee of Bama minipigs,the autologous BMSCs was used as seeds for transplantation and was labeled by the 5,6-carboxyfluorescein diacetate succinimidyl ester (CFDA SE).Animals were randomly divided into three groups:Group A (blank group) was left untreated,group B (cell-free biomimetic hydrogel group) was filled with biomimetic hydrogel and group C (BMSCs combined with biomimetic hydrogel group) was filled with the CFDA SE labeled autologous BMSCs combined with biomimetic hydrogel.One month after the operation,BrdU labeled liquid was injected intravenously into the animals 24 h and 48 h before specimens were taken from the executed animals.Partial cartilage repaired tissue in Group C was taken,cryosectioned and stained with DAPI and BrdU immunofluorescence.Confocal laser scanning microscope was used to observe and count the cells.Specimens of the three groups were analyzed through gross observation and histological staining,and scored according to the international cartilage repair society (ICRS) gross morphological score and ICRS histological score.Results Laser scanning confocal microscopy showed (97.3 ± 2.6) ％ of the cells were derived from the implanted BMSCs in repaired tissue and that the ratio of these cells with proliferative capacity was (76.6 ± 2.5) ％.Gross observation suggested most of the cartilage defect areas in Group C were filled with ivory tissue,but those in Group A and B were still obvious depression.Histological staining showed the cartilage defect areas in Group C were filled with cartilage like tissue,which was well integrated with the surrounding normal cartilage,presented a few cartilage lacunas could be seen,and had contents of Col Ⅱ and glycosaminoglycan similar with the adjacent normal cartilage.There was almost no filler in the defect area in Group A.There was little fibrous tissue in the defect area in Group B.ICRS gross score was (8.3 ± 1.0) points in Group C,higher than that in Group A [(0.5 ± 0.6) points] and Group B [(2.3 ± 0.5) points] (P ＜ 0.05).ICRS histological score was (10.3 ± 2.4) points in Group C,higher than that in Group A [(0.5 ± 0.6) points] and Group B [(4.5 ± 1.0) points] (P ＜ 0.05).Conclusions BMSCs combined with Col Ⅱ-HA-OCS biomimetic hydrogel for repairing porcine articular cartilage defects can achieve satisfactory results.Implanted BMSCs are the main component of the cell composition in the repaired tissue and gradually differentiated into chondrocytes.
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<p><b>OBJECTIVE</b>To observe the effects of close-to-bone needing combined with electroacupuncture (EA) on cartilage collagen type Ⅱ/discoidin domain receptor/matrix metalloproteinase 13 (collagen type Ⅱ/DDR2/MMP 13) signaling pathway in rabbits with knee osteoarthritis (KOA), and to explore the possible action mechanism of this method on repair of extracellular matrix of knee cartilage.</p><p><b>METHODS</b>Forty New Zealand white rabbits were randomly assigned into a normal group (10 rabbits) and a model establishing group (30 rabbits). The Hulth-Telhag technique was applied to establish the model of KOA, and X-ray was used for outcome assessment. The rabbits with successful modeling were randomly assigned into a model group, a close-to-bone needing group, a regular acupuncture group, 10 rabbits in each one. The rabbits in the close-to-bone needing group were treated with close-to-bone needing and EA; the rabbits in the regular acupuncture group were treated with regular acupuncture and EA. "Neixiyan" (EX-LE 4), "Dubi" (ST 35), "Yinlingquan" (SP 9), "Zusanli" (ST 36) and "Liangqiu" (ST 34) were selected in the two groups. The intervention was given for 20 min, once a day; the intervention of 5 days made 1 session, 2 days as the interval and totally 4 sessions were given. Rabbits in normal and model group were immobilized without any treatment. After the treatment, western blotting method was applied to evaluate the expression of DDR2 and collagen type Ⅱ; the activity of collagen type Ⅱ, DDR2 and MMP 13 was assessed by immunohistochemistry method; the mRNA expression of DDR2 and MMP 13 was determined by reverse transcription-polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>Compared with the normal group, the activity expression of collagen type Ⅱ were significantly reduced in the other groups (all<0.01),while the activity and mRNA expression of DDR2 and MMP 13 were notably increased (all<0.01). Compared with the model group, the activity expression of collagen type Ⅱ in the close-to-bone needing group and regular acupuncture group were increased (all<0.01), while the activity and mRNA expression of DDR2 and MMP 13 were reduced (all<0.01). Compared with the regular acupuncture group, the activity and mRNA expression of MMP 13 and DDR2 in the close-to-bone needing group were reduced (all<0.01), while the activity expression of collagen type Ⅱ were increased (<0.01).</p><p><b>CONCLUSIONS</b>The close-to-bone needing combined with EA and regular EA could both promote the repair of knee cartilage, where closing-to-bone needing combined with EA shows a superior efficacy. The mechanism may be associated with the blocking effect of collagen type Ⅱ/DDR2/MMP13 signaling pathway and the inhibiting effect of degradation in extracellular matrix of cartilage.</p>
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<p><b>OBJECTIVE</b>To observe the effects of close-to-bone needling (CBN) on the expressions of type-Ⅱcollagen, pro-collagen type Ⅱ C-terminal propeptide (PⅡCP) and C-telopeptide of type Ⅱ collagen (CTX-Ⅱ) in rabbits with knee osteoarthritis (KOA).</p><p><b>METHODS</b>Among 40 New Zealand rabbits, 10 rabbits were selected into a normal group; the remaining 30 rabbits were made into KOA model, and X-ray was used to evaluate the results of model establishment. After the model was successfully made, the rabbits were randomly divided into a model group, a CBN group and a regular acupuncture group, ten rabbits in each one.Rabbits in the CBN group and the regular acupuncture group were treated at "Neixiyan" (EX-LE 4), "Dubi" (ST 35), "Yinlingquan" (SP 9), "Zusanli" (ST 36) and "Liangqiu" (ST 34). The CBN group applied CBN, and the depth of needling was appropriate with needles reaching bone; the regular acupuncture group applied regular acupuncture. The electroacupuncture(EA) device was used in the two groups, 20 min per treatment, once a day.Five days of treatment were taken as one course, and totally 4 courses were given with an interval of 2 days between courses. The normal group received identical fixation as model group. After treatment, magnetic resonance imaging (MRI) was used to perform imaging observation on knee; transmission electron microscopy (TEM) was used to observe the cell structure of knee joint cartilage;HE staining was used to observe the pathological change of knee; TUNEL was used to observe the apoptotic index; the expressions of type-Ⅱ collagen proteins and mRNA were measured by Western-blot and reverse transcription-polymerase chain reaction (RT-PCR); the serum PⅡCP and CTX-Ⅱ levels were measured using enzyme-linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>After treatment, compared with the model group, in the CBN group and regular acupuncture group the articular cavity effusion was reduced without the subchondral bone edema; the cell structure of knee joint cartilage was regular with less apoptosis; the expressions of type-Ⅱ collagen proteins and mRNA were significantly increased (all<0.05), the PⅡCP levels were significantly increased (both<0.05), but the CTX-Ⅱ levels were significantly decreased (both<0.05).The differences of the expressions of type-Ⅱ collagen proteins and mRNA, the levels of PⅡCP and CTX-Ⅱ between the CBN group and the regular acupuncture group were significant (all<0.05); the differences between the CBN group and the normal group were non-significant (all>0.05).</p><p><b>CONCLUSIONS</b>CBN can significantly improve the pathological status of cartilage of KOA, reduce apoptosis, and is likely to regulate the expressions of PⅡCP and CTX-Ⅱ to promote the type-Ⅱ collagen, which is superior to regular acupuncture.</p>
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Objective:To study the influence of bone morphogenetic protein-7 ( BMP-7 ) on chondro-cyte secretion and expression of type Ⅱ collagen ( Col-Ⅱ) , aggrecan ( AGG ) and SRY-related high mobility group-box gene 9 ( Sox9 ) mRNA in porous tantalum-chondrocyte composites.Methods: The articular chondrocytes were isolated from 3-week-old New Zealand immature rabbits and identified.The 2nd generation of chondrocytes with 1 ×106/mL inoculate concentration was seeded in porous tantalum and divided into 4 groups, and control group ( tantalum/chondrocyte) , 50μg/L BMP-7 group (50μg/L BMP-7/tantalum/chondrocyte) , 100 μg/L BMP-7 group ( 100 μg/L BMP-7/tantalum/chondrocyte ) , and 200 μg/L BMP-7 group ( 200 μg/L BMP-7/tantalum/chondrocyte ) .The proliferation of chondro-cytes was measured by CCK-8 assay.The chondrocyte growth and morphology were observed by scanning electron microscopy ( SEM) .The synthesis of glycosaminoglycan ( GAG) in chondrocytes was tested by dimethyl methylene blue ( DMMB) colorimetric quantification method.Col-Ⅱ, AGG and Sox9 mRNA in chondrocytes were detected by real-time PCR.Results: The chondrocytes were spindle-shaped in 24 hours of primary cell culture and most cells became polygonal shaped in 4 days.The chondrocytes were affirmed by alcian blue, safranin O and Col-Ⅱimmunocytochemistry staining.The result of CCK-8 assay showed that the level of cell proliferation in 100 μg/L BMP-7 groups were higher than those in the other groups ( P<0 .05 ) .The chondrocytes implanted into porous tantalum scaffolds with BMP-7 had better functions, by which cytoplasmic processes developed and extended to the surface and inner of porous tan-talum by SEM observation.DMMB quantitative determination of GAG showed that GAG amount of chon-drocytes in 100 μg/L BMP-7 groups was significantly higher than those in the other groups ( P<0 .05 ) . The expressions of Col-Ⅱ, AGG and Sox9 mRNA in chondrocytes were up-regulated in the experimental groups, compared with the control group and the best effect appeared when concentration of BMP-7 was 200μg/L.(P<0.05).Conclusion:BMP-7/tantalum/chondrocytes composites enhanced in vitro chon-drocyte proliferation and extracellular matrix greatly, and can promote chondrogenic gene expression.
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BACKGROUND:Studies have shown that the changes of extracellular matrix in degenerative intervertebral disc tissues mainly present as the decrease of col agen type Ⅱ and proteoglycan contents and the increase of col agen type Ⅰ content. OBJECTIVE:To explore the effect of adeno-associated virus-mediated bone morphogenetic protein 2 gene on nucleus pulposus Ⅰ and Ⅱ col agen levels in rabbit degenerative intervertebral disc tissues. METHODS:L 2-3 , L 3-4 , L 4-5 and L 5-6 lumbar discs of 12 New Zealand white rabbits were punctured to establish interverbral disc degeneration model. Subsequently, 12 rabbits were randomly divided into three groups, four rabbits in each group. The intervertebral discs in the adeno-associated virus-mediated bone morphogenetic protein 2 group were injected with the adeno-associated virus-mediated bone morphogenetic protein 2 gene, the intervertebral discs in the adeno-associated virus group were injected with adeno-associated virus only, while the discs in the normal saline group were injected with normal saline. Al rabbits were sacrificed after injected for 8 weeks, and the L 2-3 , L 3-4 , L 4-5 and L 5-6 lumbar discs of each rabbit were col ected, paraffin-embedded and sliced. The histological changes of nucleus pulposus were observed with hematoxylin-eosin staining, and the immunohistochemistry was used to detect the col agen type Ⅰ and Ⅱ expressions in nucleus pulposus. Semi-quantitative analysis was performed. RESULTS AND CONCLUSION:Hematoxylin-eosin staining showed that the nucleus pulposus in the intervertebral disc tissues was less in the adeno-associated virus-mediated bone morphogenetic protein 2 group, the nucleus pulposus was in single or clustered distribution with clear nucleus structure and without fibrous tissue fil ing. The tissue structures of nucleus pulposus were the same in the adeno-associated virus group and normal saline group, the cellnumber in nucleus pulposus was smal , the nucleus pulposus was shrunken and shriveled, and the cells were fil ed with fibrous tissue and arranged disorderly. Immunohistochemistry staining showed the expression of col age type Ⅰ in the intervertebral disc nucleus pulposus of adeno-associated virus-mediated bone morphogenetic protein 2 group was higher than that of the adeno-associated virus group and normal saline group (P<0.05);the expression of col age typeⅠ in the intervertebral disc nucleus pulposus of adeno-associated virus-mediated bone morphogenetic protein 2 group was lower than that of the adeno-associated virus group and normal saline group (P<0.05). The results indicate that adeno-associated virus-mediated bone morphogenetic protein 2 can inhibit the expression of col agen type Ⅰ in the intervertebral disc nucleus pulposus, promote the expression of col agen type Ⅱ. Maintaining the content of col agen in intervertebral disc can keep the histological structure and morphology of intervertebral disc, stabilize the environment for nucleus pulposus cellgrowth, and delay the intervertebral disc degeneration.
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BACKGROUND:Cervical decompression and fusion internal fixation wil accelerate adjacent segment disc degeneration, and it is not clear whether single segment instaibility can increase the adjacent segment disc degeneration. OBJECTIVE:To study the changes of morphology, proteoglycan and col agen type Ⅱ in the adjacent intervertebral disc of the cervical instability models. METHODS:Sixteen New Zealand white rabbits were divided into two groups randomly, with eight rabbits in the control group and eight rabbits in the model group. The animal cervical instability models were made by destroyed partly annulus fibrosus and absorbed C 5/6 nucleus pulposus through anterior cervical puncture. After 12 weeks, the animal models were tested by X-ray film. Al rabbits were sacrificed and 10 mg nucleus pulposus of the intervertebral discs of C 4/5 cut from sagittal plane were harvested and stored under 0 ℃. The content of proteoglycan in nucleus pulposus was tested with phloroglucinol method. Then, the paraffin sections of intervertebral disc tissues were taken for hematoxylin-eosin staining and SABC immunohistochemical staining. RESULTS AND CONCLUSION:The notochord cells of C4/5 intervertebral discs in the experimental group was decreased, and being replaced by fibroblast-like cells. Round chondrocytes could be seen occasional y and intervertebral discs annulus fibrosus became rough and arranged disorderly, the hyaline degeneration and pigmentation were observed as wel as the fibrochondrocytes, and there was a gap between inner and outer annulus fibrosus. The content of proteoglycan was decreased in the nucleus pulposus, and there was significant difference between two groups. The col agen type Ⅱ in the degenerative disc nucleus pulposus and annulus fibrosus of the experimental group was lower than that of the control group. Cervical instability can lead to adjacent intervertebral disc degeneration with the morphological changes and decreased content of proteoglycan and col agen type Ⅱ.
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ObjectiveTo observe the preventive effect of type Ⅱ collagen on experimental rat articular cartilage damage induced by T-2 toxin,to explore molecular biomarkers of articular cartilage damage and repair,and to provide a theoretical basis for control of articular cartilage damage.MethodsEighty Wistar rats were randomly divided into 4 groups according to their body weights:negative control,positive control,high-dose intervention,and low-dose intervention groups,20 rats in each group.Animals in negative control group were fed with standard rat chow,and animals in other three groups were fed with T-2-toxin-contaminated chow( 100 ng/kgfeed).Animals in negative and positive control groups drank distilled water,animals in high-dose intervention and low-dose intervention groups drank water containing type Ⅱ collagen(0.5,5.0 g/L,respectively).These rats were sacrificed after 3 and 5 months,respectively,and bilateral knee joints were collected.Histopathologic changes in hyaline cartilage were examined by light microscope,serum levels of type Ⅱ collagen carboxyl terminal peptide (CTX-Ⅱ ),cartilage oligomeric matrix protein (COMP) and urinary deoxypyridinoline (DPD) were determined by enzyme-linked immunosorbent assay(ELISA).ResultsHE staining showed,that the positive control articular chondrocytes were disarranged,deformated,degenerated,with necrosis and extensive areas of chondrocyte loss;but the two intervention groups only showed fibril formation and swelling and surface cartilage cells became round,flat cartilage cells decreased in number,and cartilage cells clustered and so on early pathological changes of osteoarthritis.At the ends of 3 month and 5 month experiment,the levels of serum CTX- Ⅱ in different groups were,negative control[(18.77 ± 4.61),(25.07 ± 9.17)μg/L],high-dose intervention[ (21.11 ± 5.02),(33.20 ± 9.74)μg/L ],low-dose intervention [ ( 19.87 ± 4.53 ),( 29.73 ± 9.32 ) μg/L ] and positive control [ ( 24.43 ± 5.23 ),( 39.17 ±10.49 ) μg/L ] ; the levels of serum COMP were,negative control group [ (5.43 ± 2.75 ),( 6.38 ± 2.23 ) μg/L ],highdose intervention group[ (17.27 ± 4.77),(20.32 ± 4.74)μg/L],low-dose intervention group[(20.13 ± 5.07),(19.44 ± 4.92)μg/L] and positive control group[ (21.37 ± 4.72),(24.52 ± 4.26)μg/L].At the end of 3 month,compared with negative control group,the level of serum CTX- Ⅱ in other three groups increased,but only positive control group increased significantly(P ＜ 0.05) ; at the end of 5 month,compared with negative control group,the level of serum CTX-Ⅱ in other three groups increased significantly,and the difference was statistically significant (all P ＜ 0.05),and the level of CTX-Ⅱ in the two intervention groups was significantly lower compared with that of positive control group(all P ＜ 0.05).Compared with negative control group,the level of serum COMP in other groups increased significantly at the end of 3 month (all P ＜ 0.05) and only the level of serum COMP in high-dose intervention group was significantly lower compared with that of positive control group(P ＜ 0.05).At the end of 5 month,compared with negative control group,the level of serum COMP in other three groups increased significantly,the difference were statistically significant (all P ＜ 0.05) ; the levels of serum COMP in the two intervention groups were significantly lower than that of positive control group(all P ＜ 0.05).At the ends of 3 month and 5 month,the content of urinary DPD in negative control group were[ (3.47 ± 2.20),(4.14 ± 1.06)μg/L],positive control group[ (4.09 ± 2.48),(4.33 ± 3.43)μg/L],high-dose intervention group[ (3.86 ± 2.31 ),(5.72 ± 3.89)μg/L] and low-dose intervention group[ (3.58 ± 2.77),(4.23 ± 2.90)μg/L].The difference between the 4 groups were not statistically significant (F =2.608,2.436,all P ＞ 0.05).ConclusionsType Ⅱ collagen could effectively reduce the level of serum CTX-Ⅱ and COMP in experimental rats and delay the process of articular cartilage damage induced by T-2 toxin.
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Objective To study the relationship between the medial artery calcification and expression of core-binding factor alpha 1 (Cbf α-1) and collagen Ⅱ (Col Ⅱ) in chronic kidney disease (CKD) stage 5 patients.Methods Pieces of radial arteries were taken from 40 patients with CKD stage 5 during internal arteriovenous fistula operation.Ten patients with subtotal gastrectomy and normal renal function were chosen as control.The vessels were examined for calcification by von Kossa stain and for the presence of Cbfα-1 and Col Ⅱ by immunohistochemistry.According to von Kossa stain,CKD stage 5 patients were divided into no calcification group,mild-moderate calcification group and severe calcification group.Other related factors including serum calcium,phosphate,intact parathyroid hormone (iPTH),C-reactive protein (CRP),triglyceride(TG),cholesterol(TC) and lowdensity lipoproteins(LDL) were also detected.Results Seventeen (42.5％) of CKD Stage 5 patients showed vascular calcification,while calcification was not found in controls.Most calcification occurred in medial layer.Positive immunohistochemical staining of core-binding factor and Col Ⅱ was found in the smooth muscular cell plasma of medial layer in the vessels with calcification.However,above positive staining was also observed in 78.3％ of no calcification group.But there was little staining in control group.Positive staining score of Cbfα-1 and Col Ⅱ in severe calcification group was significantly higher than that in no calcification group.Same findings were obtained in mild-moderate calcification group,but the difference between them was not statistically significant.CRP and Ca × P were positively correlated with staining score of Cbfα-1 and Col Ⅱ.Serum phosphate was positively correlated with Cbfα-1 (r=0.786,P＜0.01) and Col Ⅱ (r=0.785,P＜0.01) respectively.Conclusions 42.5％ of CKD stage 5 patients in our group shows vascular calcification,which occurrs mainly in medial layer.High expression of Cbfα-1 and Col Ⅱ can be observed in vascular calcification of radial arteries,which is earlier than vascular histological changes.Cbfα-1 and Col Ⅱ may be involved in the development of vascular calcification.
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ObjectiveTo investigate oral collagen type Ⅱ peptide-cholera toxin B subunit-liposome complex induce tolerance to collagen-induced arthritis (CIA).MethodsDBA/1 mice were divided into four groups,group Ⅰ:normal control,group Ⅱ:CIA control,group Ⅲ:oral collagen type Ⅱ peptidecholera toxin B subunit-liposome complex,and group Ⅳ:for testing IgG2a (on day 14 after primary immunization).Arthritis scores and histopathologic assessment were analyzed.The levels of serum IgG2a were examined by ELISA.ResultsThere was no arthritis development in group Ⅰ.The incidence of arthritis in group Ⅱ was higher than that in group Ⅲ ( 100％ vs 28.6％,P＜0.05).The arthritis score in group Ⅱ was higher than that in group Ⅲ (5.40 vs 0.4,3,P＜0.01).Histopathologic score was higher in group Ⅱ than that in group Ⅲ (16.00 vs 2.85,p＜0.05).Level of serum IgG2a of group Ⅰ was very 1ow(38 ng/ml).Mice of group Ⅱ produced significantly higher level of IgG2a than mice of group Ⅲ (3922 ng/ml vs 3219ng/ml,P＜0.05).IgG2a of group Ⅳ was 98 ng/ml which was significantly higher than that of group Ⅰ (P＜0.01 ).ConclusionOral collagen type Ⅱ peptide-cholera toxin B subunit-liposome complex could inhibit CIA progression through immune tolerance.
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Objective To observe the effect of the emodin thermosensitive hydrogel on collagenase in gingival crevicular fluid of patients with chronic periodontitis and to evaluate the clinical efficacy.Methods Forty patients with chronic periodontitis were selected.For each patient after scaring and root planning,the one teeth on one side in a person was assigned at the test group and treated with the emodin thermosensitive hydrogel in the pocket once a week for four weeks.The teeth on the other side were assigned as the control group,just with the primary periodontal treatment.The PLI、SBI、PD、AL、MD and content of collagenase in gingival crevicular fluid were examined at baseline,before administration and after administration four weeks.The COL-II level in GCF was detected using ELISA method.Results There were no significant differences in PLI、SBI、PD、AL、MD and content of collagenase in gingival crevicular fluid before the treatment(P ＞ 0.05).While there are some evidences that periodontal indexes dropped down and the type Ⅱ collagenase level in gingival crevicular fluid also declined after using the emodin thermosensitive hydrogel than the control group(t =3.46,4.02,4.18,3.03,2.79,4.29,all P ＜ 0.05).Conclusion Ultrasonic scaling using the emodin thermosensitive hydrogelas was more effective in reducing the type Ⅱ collagenase level in gingival crevicular fluid.And improving clinical parameters associated with periodontal health in patients with chronic periodontitis.
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ObjectiveThe aim of this study is to investigate whether oral administration of collagen Ⅱ peptide (250-270)[C Ⅱ (250-270)]-cholera toxin B subunit (CTB)complex could effectively set up oral immune tolerance to collagen-induced arthritis (CIA) in mice. MethodsDBA/1 mice were divided into Ⅰ, Ⅱ, Ⅲ and Ⅳgroups. Group Ⅰ was normal control group. Collagen type Ⅱ emulsified in Freund's complete adjuvant were injected to mice of groups Ⅱ , Ⅲ and Ⅳ twice from the base of the tail. Mice of group Ⅲ were fed with C Ⅱ (250-270)-CTB covalent complex twice after the arthritis was developed. Mice of group Ⅳ were fed with C Ⅱ(250-270) and CTB mix at the 14th day after primary immunization. Visual scores and histopathologic scores of arthritis were recorded. The frequencies of arthritis between the groups were compared usingFisher's exact test. The clinical and histological severity of arthritis were analyzed by ANOVA.Results The frequencies of arthritis in groups Ⅰ , Ⅱ , Ⅲ and Ⅳ were 0, 100％, 100％ and 25％ respectively. Average accumulative scores of arthritis were 0, 5.0±1.7, 10.8±2.8 and 1.0±2.0 respectively. Average accum-ulative histopathological scores of arthritis were 0, 16±8, 32±13 and 7±6 respectively. Conclusion Oral administration of C Ⅱ (250-270) and CTB mix in arthritis mice after C Ⅱ immunization can suppress the onset and severity of arthritis. Oral administration of C Ⅱ (250-270)-CTB covalent complex in the acute stage of arthritis can accelerate arthritis.
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ObjectiveTo investigate the effect of vascular endothelial growth factor (VEGF) on collagen Ⅱ expression in rat articular chondrocytes in vitro.MethodsChondrocytes were isolated and cultured.Then rats were divided into 4 groups:group A(control):without any intervention; group B:10 ng/ml VEGF was added; group C:10 ng/ml IL-1β was added; group D:10 ng/ml VEGF and 10 ng/ml IL-1β were added.Messenger RNA (mRNA) expression of collagen Ⅱ was detected by using real time polymerase chain reaction (real time PCR),and the protein expression level of collagen Ⅱ was detected by Western blotting.Comparisons between groups were performed by one-way ANOVA.ResultsThe collagen Ⅱ mRNA expression levels of group B (0.78+0.07),group C (0.67+0.06) and group D (0.57+0.04) were significantly lower than those of the group A (1.00±0.08),and there was significant difference between B and D,C and D.Compared with group A(0.95+0.21),the expression of collagen Ⅱ protein in group B(0.71+0.14),group C(0.60±0.11) and group D(0.31 +0.09) was significantly suppressed.The expression of collagen Ⅱ protein in group D was significantly lower than those of group B and C.ConclusionVEGF can significantly suppress the expression of collagen II in rat articular chondrocytes.VEGF may play an important role in the development of osteoarthritis.
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ObjectiveTo establish rabbit knee joint cartilage injury models to evaluate effects of the type Ⅱ collagen sponge in repair of the articular cartilage.MethodsThe type Ⅱ collagen sponge was prepared according to previous method and the pore size of the sponges was measured based on the collagen autofluorescence characteristics.The type Ⅱ collagen sponge was transplanted into the injury lesions of the animal model for experimental study.The regeneration of the cartilage defects was observed by using MRI, histologic HE staining, Safranin O, sirius red polarized light staining, areas determination of the newly grown cartilage and immunohistochemistry of type Ⅱ collagen.ResultsAutofluorescent images of confocal microscope layer scanning showed that the pore size was (93.26 + 38.40) μm in diameter, suitable for chondrocyte growth.Comparison between MRI and H&E staining results showed quicker effusion absorption in the treatment groups than that in the control group, while the level of inflammatory response in the treatment group was lower than that in the control group.The sporadic cartilage signals first appeared at the 6th week.The newly formed cartilage with the expression of glycosaminoglycan and type Ⅱ collagen matrix was confirmed by Safranin O staining and immunohistochemical analysis.The sirius red polarized light staining showed that areas of the newly formed cartilage were significantly larger in the treatment group than that in the control group (P ＜ 0.01).Conclusion The type Ⅱ collagen sponge developed from purification can effectively repair the damaged cartilage tissues of the rabbit knee joints, as has been verified either by MRI or histology.
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AIM: To explore the effects of glucocorticoids on fracture healing in a rat model of tibial fracture. METHODS: Sixty three months old female SD rats were divided into control and glucocorticoid-treated group. A glucocorticoid-induced osteoporosis model was established by intramuscular injection of prednisolone acetate (5 mg·kg~(-1)·d~(-1) for 3 weeks), in which the tibial was osteotomized by a wire saw as fracture healing model and internal fixed with a Kirschner pin. The rats were scarified at different time points after operation. The callus formation was monitored over a period of 6 weeks by histological method, bone mineral density (BMD) detection and biomechanical examination. Western blotting was used to measure the expression of type II collagen. RESULTS: A glucocorticoid-induced osteoporosis model was successfully established and conformed by BMD measurement. The formation of primary callus was observed in both groups 3 days after fracture. At 2 weeks after injury, the glucocorticoid-treated group had a lower BMD and less cartilage matrix as compared to control group. An increase in bone callus and chondrogenesis was observed at 4 to 6 weeks after fracture in glucocorticoid-treated group as compared to control group. The expression of type II collagen was delayed in glucocorticoid-treated group. Biomechanical measurement showed that the actual maximum load was increased by 35.8% in control group as compared to glucocorticoid-treated group at 6th week. CONCLUSION: These results indicate that chondrogenesis and transformation from cartilage callus to bony callus are delayed by glucocorticoids. The retardation of collagen Ⅱ production may be the reason for the inhibition of fracture healing.
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Objective To study the effect of type Ⅱ collagen (C Ⅱ) from Zaoeys dummies (Cantor)on the immune functions of rats with collagen-induced arthritis and to understand the mechanisms of RA treated with Zaoeys dhumnades (Cantor).Methods The rats with collagen-induced arthritis were randomly divided into three group:C Ⅱ from Zaocys dhumnades,bovine C Ⅱ and arthritis control group,a normal control group was set up,too.Every group had 7 rats.The C Ⅱ from Zaocys dhumnades and the bovine C Ⅱ group were fed with C Ⅱ from Zaocys dhumnades (15 mg/kg) and bovine C Ⅱ (15 mg/kg) per day respectivelyfor 15 days.The CD4/CD8 subset ratio,serum levels of anti-C Ⅱ antibody,TNF-a,IL-10,IL-1 and IL-4 in.rats were measured.Results In the arthritis group,CD4/CD8 subset ratio (P＜0.05) and serum levels of type Ⅱ collagen antibody (P＜0.01) and TNF-a (P＜0.01) were significant increased and IL-10 (P＜0.01) was significantly decreased.In the C Ⅱ from Zaocys dhumnades and bovine C Ⅱ group,CD4/CD8 subset ratio (P＜0.05) and the level of TNF-a (P＜0.01) were significantly decreased compared with the arthritis group,and had no difference compared with the normal group.The level of anti-C Ⅱ antibody was declined significantly compared with the arthritis group (P＜0.05) and had statistical difference with the normal group (P＜0.01).The level of IL-10 was significantly increased(P＜0.01),but lower than the normal group(P＜0.05).There was no statistical difference in the level of IL-1 and IL--4 in.all four groups.Conclusion C Ⅱ from Zaoeys dhum-nades (Cantor) is as effective as bovine C Ⅱ in modifying the immune functions of collagen-induced arth-ritis in rats.They can decrease the level of anti-C Ⅱ antibody,the level of TNF-a,CD4/CD8 subset ratio and increase the level of IL-10 in the peripheral blood of rats with collagen-induced arthritis.C Ⅱ from Zaocys dhumnades may be one of the important pharmacological active components that have the potential in treating rheumatoid arthritis.
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[Objective]Porous collagen type Ⅱ matrices crosslinked by 1-ethyl-3-(3-dimethyl aminopropyl)carbodⅡmide(EDC) and N-hydroxysucinimide(NHS) were developed as scaffolds for cartilage tissue engineering.[Method]Collagen type Ⅱ matrix was crosslinked by EDC/NHS,and then freeze-dried to achieve collagen scaffolds.The porous characteristics of the prepared scaffolds were tested by using diffenent methods,including laser scanning confocal microscopy (LSCM),scanning electron microscopy (SEM) and mechanical testing machine.The chondrocytes labeled by GFP were planted in crosslinked collagen type Ⅱ,and observed by LSCM.[Result](1) The mechanical properties of cross-linked matrices increased significantly,reaching 2.18?0.47MPa.(2)The degradation of cross-linked matrices decreased by 8.28% after 4 hours.The pore size was about 90?m,and their average porosity and water capacity reached 93.39% and 97.78%,respectively.3.Chondrocytes were in good condition in the crosslinked collagen.[Conclusion]The present work indicates that EDC/NHS-crosslinked collagen type Ⅱ could keep the properties and biocompatibility of collagen,besides,the mechanical strength increased and the degradation was decreased.It will be suitable for cartilage tissue engineering purposes.
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Objectives To establish a reliable method for isolating and purifying soluble collagen type Ⅱ(SCⅡ)by optimizing preparative procedure.Method The chicken sternal cartilage was selected as raw material.Guanidine hydrochloride was used to remove the proteoglycans.The digestion manners of pepsin,sodium chloride concentrations for salting,types of DEAE anion resin were studied for extracting SCⅡ.The SCⅡ identification was made by SDS-PAGE,absorption spectrum and amino acid analysis.Result It was convenient for pre-treatments of chicken sternal cartilage.The proteoglycans could be efficiently removed by 4 mol/L guanidine hydrochloride.The satisfied results were obtained by limited enzyme digestion of pepsin added by two steps.The optimizing concentration of sodium chloride for salting was 2.4mol/L.SDS-PAGE maps revealed that the bands of purified SCⅡand standard CⅡ were at the same location.The absorption peak of SCⅡ was at 230nm.The concentrations of Gly,Pro and Ala were the highest in 15 amino acids.Conclusion The improved method has significant advantages of simple working process,result reliability and convenient source of raw material.It is suitable for purifying the SCⅡ at variable scales in research works and clinic application.The SCⅡ product obtained has high purity and accords with the characteristics of collagen type Ⅱ.
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Objective To establish the type Ⅱ collagen specific T cell line of Wistar rat and observe its effect on transferring arthritis.Methods The Wistar rats were immunized with emulsified chicken type Ⅱ collagen (CCⅡ) in complete Freund′s adjuvant by intradermal injection to induce the rat model of collagen induced arthritis (CIA).The lymphocytes were obtained from mesenteric lymph nodes of CIA rats,and the type Ⅱ collagen reactive T cell line was selected and propagated by CCⅡ stimulating in vitro .The proliferation response and phenotype were analyzed by 3 H TdR incorporation and fluorescence activated cell sorter (FACS).The onset of arthritis and pathological characteristic in ankle joints of recipient rats were observed with naked eye and histochemical examination.Anti CCⅡ antibody in serum was assayed by enzyme linked immunosorbent assay (ELISA).Results A T cell line was successfully established.The results of FACS labeled with fluorescent antibodies showed that 98 2% of the line cells were T cells,of which 89 7% were CD4 + T cells.The results of adoptive transfer showed that the incidence of arthritis was 50% when the injected cell number was 5?10 7,meanwhile the level of anti CCⅡ antibody in serum was elevated more than that of the control.Conclusion A cell line has been successfully established.The result of arthritis transferring by T cell line shows that the T cell plays a great role in the pathogenesis of CIA and provides a research datum for rheumatoid arthritis therapy with T cell vaccine.