Phenotypic maintenance of chondrocytes in vitro under tensile stress enhanced by the extract of clematis chinensis / 中国组织工程研究

BACKGROUND: Mechanical load is crucial for the degeneration of chondrocytes and the development of osteoarthritis. Clematis chinensis can improve the inflammatory microenvironment of osteoarthritis, but its effect on mechanical load-induced degeneration of chondrocytes has not been elucidated. OBJECTIVE: To study the effect and mechanism of the extracts of Clematis chinensis on the degenerative changes of chondrocytes induced by intermittent cyclic mechanical tension (ICMT) in vitro. METHODS: Chondrocytes of the rabbit knee joint were isolated by type II collagenase digestion method and identified by Alcian blue staining. There were five groups in the experiment: Blank group, ICMT group, high-, medium- and low-dose Clematis chinensis groups. There was no intervention in the blank group, and the other groups were subjected to ICMT (10% tensile strength, 0.5 Hz, 8 hours per day, for a total of 2 days) for inducing chondrocyte degeneration. Three Clematis chinensis groups were concurrently given 0.5, 1, 2 g/L extracts of Clematis chinensis, respectively. The intervention time was 48 hours. Cell counting kit-8 assay was used for detection of chondrocyte proliferation. FITC-phalloidin staining was used for observation of cytoskeleton morphology. Real-time quantitative PCR and western blot assay were used for determination of collagen type II, matrix metalloproteinase 13, and transforming growth factor β at protein and gene levels, respectively. RESULTS AND CONCLUSION: (1) Compared with the blank group, the cytoskeleton of chondrocytes stimulated by ICMT was long- stretched, the proliferation activity of chondrocytes decreased, and the expressions of collagen type II and transforming growth factor β were down-regulated, while the expression of matrix metalloproteinase 13 was up-regulated. (2) Compared with the ICMT group, the extract of Clematis chinensis could promote the proliferation of chondrocytes, up-regulate the expressions of transforming growth factor β and collagen II, and down-regulate the expression of matrix metalloproteinase 13 in a concentration-dependent manner. To conclude, the extract of Clematis chinensis can inhibit the catabolism of chondrocyte induced by ICMT through regulating the expression of transforming growth factor β, promote the synthesis of extracellular matrix of chondrocytes, and maintain the phenotypic stability of chondrocytes.

Changes of subchondral bone in rat models of knee osteoarthritis treated by elcatonin / 中国组织工程研究

BACKGROUND: Imbalance between subchondral bone resorption and bone formation occurs in osteoarthritis. Our preliminary study has found that the subchondral bone changes precede the articular cartilage changes in knee osteoarthritis rats. OBJECTIVE: To observe the changes of subchondral bone in knee osteoarthritis rats, and to explore the effect of elcatonin on the expression of p38 MAPK and absorption of subchondral bone. METHODS: Female Sprague-Dawley rats, 3 months of age, were randomly divided into three groups: Sham operation group (n=6, the adipose tissue of the same size as the ovary was removed without ovariectomy, the bilateral knee joints were opened, but without cruciate ligament injury); model group (n=6, bilateral ovariectomy plus bilateral cruciate ligament injury, no treatment); elcatonin group (n=6, ovariectomy plus cruciate ligament injury, intramuscular injection of 5 lU/kg elcatonin, twice weekly). The bone mineral density and p38 protein expression levels in subchondral bone were detected at 12 weeks. The serum levels of C-terminal crosslinking telopeptides of type I collagen, C-terminal crosslinking telopeptides of type II collagen, interleukin-1, interleukin- 6 and bone-specific alkaline phosphatase, tartrate resistant acid phosphatase 5b were detected. RESULTS AND CONCLUSION: (1) The bone volume fraction and trabecular bone number in the elcatonin group were significantly higher than those in the model group (P < 0. 05), and the trabecular separation in the elcatonin group was lower than that in the model group (P < 0. 05). (2) The bone volume fraction, trabecular bone number and trabecular thickness in the model group were lower than those in the sham operation group (P < 0. 01 or P < 0. 05), and the trabecular separation was higher than that in the sham operation group (P < 0. 01). (3) The serum levels of C-terminal crosslinking telopeptides of type I collagen, C-terminal crosslinking telopeptides of type II collagen, interleukin-1, interleukin- 6 and tartrate resistant acid phosphatase 5b in the elcatonin group was significantly lower than those the model group (P < 0. 05), and the above levels in the model group were significantly higher than those in the sham operation group (P < 0. 05). (4) The p38 expression level in subchondral bone in the elcatonin group was significantly lower than that in the model group (P < 0. 01), and the level in the model group was significantly higher than that in the sham operation group (P < 0. 01). (5) These results indicate that elcatonin may inhibit the secretion of osteoarthritis pro-inflammatory factors and subchondral bone resorption by down-regulating the expression level of p38 MAPK.

Comparison of pathological characteristics of Wistar and Lewis rat models of collagen-induced rheumatoid arthritis / 中国比较医学杂志

Objective To study the differences of symptoms and pathological features of Wistar and Lewis rat models of collagen-induced rheumatoid arthritis. Methods Wistar and Lewis rats were injected with intermixture of bovine TypeⅡ collage and complete Freund's adjuvant(CFA)for first immunization, then strengthen it after 14 days and observed the incidence of Wistar-RA group and Lewis-RA group. The degree of paws swelling and the titer of serum anti CII antibody were determined. The pathological changes in toe and joint tissues were examined at 12 weeks, and the expressions of VEGF, IL-6, IL-10 and IL-17A in the synovial membrane of ankle joint were detected. Results After collagen induction,the Wistar and Lewis rats showed paw swelling after 10 d and 14 d,and peaked at 21 d and 24 d,the titer of serum anti CII antibody was significantly increased at third week(P< 0.01), and arthritis index(AI)was also significantly increased(P< 0.01). In the Wistar-RA rat group, the rate of molding was 80%, and at fifth weeks, the swelling of the paws subsided and went into a flat level. The molding rate of the Lewis-RA group was 100%,at the seventh week,the swelling of paws subsided and went into a flat level. At 12 weeks,the two model groups showed severe articular cartilage erosion, synovial hyperplasia and inflammatory cell infiltration, neovascularization and pannus formation in the joint synovium,and the bone mineral density of the femur and tibia of the hind limbs was significantly decreased(P<0.01). The expression of VEGF,IL-6 and IL-17A in synovium was significantly increased(P< 0.05,P< 0.01). The expression of IL-10 was obviously decreased(P< 0.01). Compared with the Wistar-RA group,the paw volume and paw thickness were increased for a longer time in the Lewis-RA group,AI was higher than that of the Wistar-RA group,synovial angiogenesis and pannus formation were more distinct, the expression of VEGF in synovium was significantly higher than that of Wistar-RA group(P< 0.05), while the expression of IL-17A was significantly lower than that in the Wistar-RA group(P< 0.05). Conclusions Both the Wistar-RA rat model and Lewis-RA rat model show joint swelling,deformation and decreased activity. AI is increased,the expression of VEGF,IL-6 and IL-17A increased,and the expression of IL-10 decreased,which are consistent with the clinical manifestation. The Wistar-RA rat model has a short duration of swelling, while the Lewis-RA rat model has a longer swelling duration and more severe joint damages. The neovascularization and pannus formation are more obvious. The expression of IL-17A in the Wistar-RA rat model is higher, while the Lewis-RA model has a highly expressed VEGF,which may be related to its pathological characteristics.

A Case of Platyspondylic Lethal Skeletal Dysplasia Torrance Type

Platyspondylic lethal skeletal dysplasia, Torrance type (PLSD-T), is one of the phenotypes of type II collagenopathy and is characteristic of severe bone growth disorder. This phenotype may limit the growth and expansion of the lungs, which is known to cause death from respiratory failure during or shortly after birth, but in few less severe cases, patients have been reported to have survived to adulthood. We have experienced a case of PLSD-T in a preterm infant who was delivered via cesarean section at the gestational age of 29 weeks 3 days, with a birth weight of 1.15 kg. Physical examination of the infant revealed characteristic findings of short arms and legs, small thorax, distended abdomen, and cleft palate. On the basis of the subsequent genetic testing, the patient had a heterozygous mutation in the encoded c-propeptide region of collagen, type II, alpha 1 (COL2A1), c.4335G>A (p.Trp1445*) in exon 52. This is the first case of PLSD-T diagnosed in Korea, and we hereby report the case.

Therapeutic effects of Hedyotis diffusa Willd. on type II collagen-induced rheumatoid arthritis in rats / 中国应用生理学杂志

OBJECTIVE@#To investigate the therapeutic effects of Hedyotis diffusa Willd.on type Ⅱ collagen-induced rheumatoid arthritis in rats.@*METHODS@#According to the random number table, 60 SD rats were divided into the normal control group (=10, normal saline) and model group (=50).The collagen-induced arthritis model was established with the injection of type Ⅱ collagen into the back in rats other than the normal group and evaluated by arthritis score, then the model rats were randomly divided into model group (normal saline), tripterygium wilfordii polyglycoside (GTW) 6 mg/kg group (daily dose:0.4 mg/kg), HD 3, 6, 12 g/kg groups (daily dose:3, 6 and 12 g/kg, respectively), with 10 rats in each group. The rats were treated with corresponding agents by intragastric administration.The arthritis index and the pain threshold of all rats at different time points were observed and measured weekly.After treated by intragastric administration for 28 days, all rats were killed to measure the changes of serum cytokine levels including interleukin 1β (IL-lβ), tumor necrosis factor a (TNF-a), prostaglandin (PGE), receptor activator for nuclear factor-kappa B ligand (RANKL) and osteoprotegerin (OPG).@*RESULTS@#Compared with the control group, the arthritis index and the serum levels of IL-lβ, TNF-a, PGE, RANKL, OPG and RANKL/OPG of the model group were increased significantly (<0.05), the pain threshold of the model group was decreased significantly (<0.05); compared with the model group, the arthritis index and the serum levels of IL-lβ, TNF-a, PGE, RANKL, OPG and RANKL/OPG of the GTW group, HD low-dose, medium-dose, high dose groups were decreased significantly (<0.05), the pain threshold of the model group was increased significantly (<0.05).@*CONCLUSIONS@#Hedyotis diffusa Willd.can significantly reduce arthritis index and increase pain threshold, reduce the level of IL-lβ, TNF-a, PGE, RANKL, OPG, and RANKL/OPG, then can prevent CIA effectively.

Comparative study of degenerated versus non-degenerated nucleus pulposus cells in inducing mesenchymal stem cells differentiation / 医学研究生学报

Objective Normal nucleus pulposus cells (nNPCs) can induce the differentiation of mesenchymal stem cells (MSCs).However, the inductive effect of degeneration nucleus pul-posus cells ( dNPCs ) on MSCs has not been determined .This study aimed to compare nNPCs with dNPCs in inducing MSCs into nucleus pulposuslike cells. Methods MSCs (cell line) were co-cultured with NPCs (cell line/isolated from patients with intervertebral disc degeneration) in 3D in vitro.The cells were divided into 5 groups:nNPCs control group, dNPCs control group, MSCs control group, nNPCs-MSCs group (MSCs induced by nNPCs), dNPCs-MSCs group (MSCs induced by dNPCs).After co-cultured for 7 days, the expression level of aggrecan (ACAN), type II collagen (COL-2), SOX-9, matrix metalloproteinase-1 (MMP-1) and MMP-3 in all the 5 groups'cellswere detected by RT-PCR and Western-blot. Results The RT-PCR results showed the expression of ACAN , COL-2 and SOX-9 in nNPCs-MSCs group was higher than that in dNPCs control group and dNPCs -MSCs group (all P<0.05).The expression of MMP-1 and MMP-3 in nNPCs-MSCs group and dNPCs-MSCs group was lower than that in dNPCs control group ( all P<0.01) , but there was no difference when compared with nNPCs control group and MSCs control group (all P>0.05).Western-blot analysis showed that the expression of ACAN , COL-2 and SOX-9 in nNPCs-MSCs group was higher than that in dNPCs control group and dNPCs-MSCs group (all P<0.05).The expression of MMP-1 and MMP-3 in nNPCs-MSCs group and dNPCs-MSCs group was lower than that in nNPCs control group and dNPCs control group ( all P<0.01) , but there was no difference compared with MSCs control group ( all P>0.05) . Conclusion Both nNPCs and dNPCs could induce the differentiation of MSCs into nucleus pulposus cells .After co-cultured with nNPCs, the expression level of ACAN, COL-2 and SOX-9 of MSCs was similar to that of nNPCs, which was superior to the induc-tion of MSCs by dNPCs under the same culture conditions .

Study on effects of Yishen Juanbi Pill on histopathologic changes in ankle joint of type II collagen induced arthritis in rats and its mechanism / 中草药

Objective: To observe the therapeutical effect of Yishen Juanbi Pill (YJP) on type II collagen induced arthritis (CIA) in rats and the histopathologic changes in ankle joint and to explore its possible mechanism on rheumatoid arthritis (RA). Methods: The therapeutical effect of YJP on CIA rat paw swelling and the histopathologic morphology of the local tissue of ankle joint were observed by the type II CIA rat model. The expression of VEGF of the synovial membrane was assayed by the immunohistochemical method. The changes in VEGF gene expression of peripheral lymphocytes and hepatic tissue were analyzed with gene chip after the treatment with YJP. Results: The decreased swelling degrees of foot of rats in low-, mid-, and high-dose YJP groups were increased obviously than that of the model group (P 1.5). Conclusion: YJP can significantly inhibit joint swelling. YJP can significantly down-regulate the VEGF-B gene expression, thereby inhibit the formation of synovial tissue pannus and reduce cartilage and bone damages.

Cotransfected human chondrocytes: over-expression of IGF-I and SOX9 enhances the synthesis of cartilage matrix components collagen-II and glycosaminoglycans

Damage to cartilage causes a loss of type II collagen (Col-II) and glycosaminoglycans (GAG). To restore the original cartilage architecture, cell factors that stimulate Col-II and GAG production are needed. Insulin-like growth factor I (IGF-I) and transcription factor SOX9are essential for the synthesis of cartilage matrix, chondrocyte proliferation, and phenotype maintenance. We evaluated the combined effect of IGF-I and SOX9 transgene expression on Col-II and GAG production by cultured human articular chondrocytes. Transient transfection and cotransfection were performed using two mammalian expression plasmids (pCMV-SPORT6), one for each transgene. At day 9 post-transfection, the chondrocytes that were over-expressing IGF-I/SOX9 showed 2-fold increased mRNA expression of the Col-II gene, as well as a 57% increase in Col-II protein, whereas type I collagen expression (Col-I) was decreased by 59.3% compared with controls. The production of GAG by these cells increased significantly compared with the controls at day 9 (3.3- vs 1.8-times, an increase of almost 83%). Thus, IGF-I/SOX9 cotransfected chondrocytes may be useful for cell-based articular cartilage therapies.

Hearing Loss in Patients with Spondyloepiphyseal Dysplasia Congenita and Tarda.

Aim: To describe hearing loss in patients with spondyloepiphyseal dysplasia congenita and tarda. Methodology: A literature review of the National Library of Medicine's online database on hearing loss in patients with spondyloepiphyseal dysplasia congenita and tarda was performed. Results: Four articles were identified that reported hearing loss in subjects with spondyloepiphyseal dysplasia congenita and tarda. Including this study, a total of fourteen patients with hearing loss are reported. Eight patients with sensorineural loss and two patients with mixed hearing loss were identified. The type of hearing loss is unknown in 4 cases. Conclusion: Serial audiograms are recommended early in life in individuals with spondyloepiphyseal dysplasia congenita and when clinically indicated in patients with spondyloepiphyseal dysplasia tarda.

Effects of Amygdalin on TNF-α and sICAM-1 of Rats with Type II Collagen-induced Arthritis / 中国中医药信息杂志

Objective To discuss effects of anti-inflammatory mechanism of amygdalin on rats with type II collagen-induced arthritis (CIA). Methods Wistar rats were randomized into normal group, model group, amygdalin group, and tripterygium group. Type II CIA rat models were established. From the 15th day after the modeling establishment, each administration group was given corresponding dose of medicine for continuous 28 days. Levels of TNF-αand sICAM-1 were detected by ELISA in serum of rats, and expression of TNF-α was detected by immuno-histochemical method. Results TNF-α positive expression in amygdalin group and tripterygium group was similar and significantly reduced compared with model group. Levels TNF-α and sICAM-1 in amygdalin group and tripterygium group significantly decreased compared with those in model group (P0.05). Conclusion Amygdalin can inhibit the expression of TNF-α and levels of TNF-α and sICAM-1, in order to treat rheumatoid arthritis.

Platelet rich plasma associated with heterologous fresh and thawed chondrocytes on osteochondral lesions of rabbits / Associação de plasma rico em plaquetas e condrócitos heterólogos frescos e descongelados em lesão osteocondral de coelhos

Chondrocytes obtained from stifle joint of New Zealand White rabbits were cultivated. Half of cells were maintained in culture for later implantation and the others frozen during six months to evaluate viability. A circular osteochondral defect was created in the right stifle of other twenty seven rabbits. The control group (CG) received no treatment. The thawed (TH) and fresh (FH) heterologous groups received, respectively, an implant of cultivated thawed or fresh heterologous chondrocytes associated with platelet rich plasma (PRP). The CG group showed greatest pain and lameness compared to the other groups seven days after the implantation. Microscopically, at 45 and 90 days, the TH and FH groups showed filling with cartilaginous tissue containing chondrocytes surrounded by a dense matrix of glycosaminoglycans. In the CG group, healing occurred with vascularized fibrous connective tissue without integration to the subchondral bone. Cryopreserved heterologous chondrocytes were viable for implantation and healing of osteochondral lesions; the association with PRP allows the fixation of cells in the lesion and offers growth factors which accelerates repair with tissue similar to articular hyaline cartilage.

Cultivaram-se condrócitos obtidos da articulação do joelho de coelhos. Metade das células foi mantida em cultura para posterior implantação, e a outra metade foi congelada durante seis meses com a finalidade de avaliar a viabilidade. Criou-se um defeito circular osteocondral no joelho direito de outros vinte e sete coelhos. O grupo controle (GC) não recebeu tratamento. Os grupos descongelado (TH) e fresco (FH) receberam, respectivamente, implantes heterólogos de condrócitos cultivados descongelados e frescos, associados com PRP. O grupo GC apresentou maior dor e claudicação em comparação com os outros grupos aos sete dias após o implante. Microscopicamente, aos 45 e 90 dias, os grupos TH e FH mostraram preenchimento da falha com tecido cartilaginoso contendo condrócitos circundados por uma matriz densa de glicosaminoglicanos. Nesse período, no grupo CG, a cura ocorreu com tecido conjuntivo fibroso vascularizado e sem integração com o osso subcondral. Condrócitos heterólogos criopreservados foram viáveis para implantação e tratamento de lesões osteocondrais; a associação com o PRP permitiu a fixação de células na lesão e ofereceu fatores de crescimento que aceleraram a reparação com o tecido semelhante à cartilagem hialina articular.

In vitro blockade of SDF-1/CXCR4 signaling pathway by T140 to prevent degradation of type Ⅱ collagen in human articular cartilage / 实用医学杂志

Objective To explore the impact of blockade of SDF-1/CXCR4 signaling pathway by T140 on degradation of typeⅡ collagen in human articular cartilage and to define the mechanism of action of T140. Methods 144 pieces of cartilage (Mankin score of 0 or 1) were obtained from osteoarthritis patients undergoing total knee replacement ( OA cartilage group) and 144 pieces of cartilage (Mankin score of 0 or 1) were obtained from patients receiving traumatic amputation (normal cartilage group). Each group was treated with SDF-1 of 100 ng/mL, then divided into three subgroups A, B, and C. The cartilage tissue in each group was cultured in the nutrient solution containing of T140, MAB310, or SDF-1 (1 000 nmol/L) for 48 or 96 hours. RT-PCR was used to detect expression of typeⅡcollagen mRNA in the cartilage tissue. Results Level of type Ⅱcollagen mRNA was markedly higher in subgroup A than in subgroup B and subgroup C at the same group and the same time (P <0.05). The expression level of type Ⅱcollagen mRNA at the same time and in the same subgroup of OA cartilage group were lower than that in normal cartilage group (P < 0.05). Conclusions SDF-1 induces degradation of typeⅡcollagen in human articular cartilage thruogh the SDF-1/CXCR4 signaling pathway. T140 can block the SDF-1/CXCR4 signaling pathway and reduce the degradation of type II collagen.

Effects of prednisone on trabecular microstructure and biomechanical properties of femur in a rat model of type II collagen-induced arthritis / 中国药理学通报

Aim To investigate the effects of predni-sone on trabecular microstructure and biomechanical properties of femur in a rat model of type II collagen-induced arthritis (CIA ) using micro-CT and biome-chanics.Methods Forty 8-week-old male Lewis rats were randomly divided into 2 groups:control (CON ) group with 6 rats,and the remaining 34 rats were used to establish the CIA model.3 weeks after immunization screening CIA rats were randomly divided into CIA group,CIA plus prednisone 4.5 mg · kg-1 · d -1 group and CIA plus prednisone 9 mg · kg-1 · d -1 group.Rats in CON group were given vehicle as well as in CIA group.Rats in the other two groups were treated with prednisone at 4.5 mg·kg-1 ·d -1 or 9 mg ·kg-1 · d -1 .After 90 days treatment,all rats were euthanized,and the left femur was collected for biome-chanics,micro-CT scanning and three-dimensional re-construction.Results Micro-CT data showed that tra-becular thickness,trabecular number,bone volume/total volume,bone mineral density in CIA group were significantly lower than those in CON group.While tra-becular separation,structure model index were signifi-cantly higher than those in CON group.Compared with CON group,biomechanical properties (elastic load, maximum load,break load and stiffness)were signifi-cantly decreased in CIA group.Compared with CIA group,bone volume/total volume and trabecular num-ber were increased,while trabecular separation was significantly decreased in two prednisone groups.Com-pared with CIA group,there was no significant change in biomechanical properties in two prednisone groups. Conclusions Treatment with prednisone for 3 months can ameliorate the damage of trabecular microstructure of the femur in CIA rats,but it has no effect on biome-chanical properties and bone mineral density.

Efeitos do congelamento sobre condrócitos cultivados de coelhos / Effect of freezing on rabbit cultured chondrocytes

This work evaluated the effect of freezing on chondrocytes maintained in culture, aiming the establishment of a cell bank for future application as heterologous implant. Chondrocytes extracted from joint cartilage of nine healthy New Zealand White rabbits were cultivated and frozen with the cryoprotector 5 percent dimethylsulfoxide for six months. Phenotypic and scanning electron microscopy analyses were carried out to identify morphological and functional differences between fresh and thawed cells. After enzymatic digestion, a total of 4.8x10(5)cells per rabbit were obtained. Fresh chondrocytes showed a high mitotic rate and abundant matrix was present up to 60 days of culture. Loss of phenotypic stability was notable in the thawed chondrocytes, with a low labeling of proteoglycans and weak immunostaining of type II collagen. The present study showed important loss of chondrocyte viability under the freezing conditions. For future in vivo studies of heterologous implant, these results suggests that a high number of cells should be implanted in the host site in order to achieve an adequate number of viable cells. Furthermore, the chondrocytes should be implanted after two weeks of culture, when the highest viability rate is found.

Avaliaram-se os efeitos do congelamento sobre condrócitos mantidos em cultura, com o objetivo de se estabelecer um banco celular para futura aplicação como implante heterólogo. Condrócitos extraídos da cartilagem articular de nove coelhos saudáveis, da raça Nova Zelândia Branca, foram cultivados e submetidos ao congelamento, com o citoprotetor sulfóxido de dimetila a 5 por cento, por um período de seis meses. Análises fenotípicas e de microscopia eletrônica de varredura foram realizadas com o objetivo de identificar diferenças morfológicas e funcionais entre as células frescas e as descongeladas. Após a digestão enzimática, foram obtidas 4,8x10(5) células por coelho. Os condrócitos frescos apresentaram elevada taxa mitótica e abundante presença de matriz até os 60 dias de cultura. Nas culturas dos condrócitos descongelados, a perda de estabilidade fenotípica foi marcante, o que foi demonstrado pela baixa intensidade da coloração dos proteoglicanos e pela fraca imunomarcação do colágeno tipo II. Sob as condições de congelamento utilizadas, houve importante perda de viabilidade condrocítica. Para futuros estudos in vivo de implante heterólogo, estes resultados sugerem que o elevado número de células deve ser implantado no sítio hospedeiro, com o objetivo de se obter maior quantidade de células viáveis, e que os condrócitos deverão ser implantados com duas semanas de cultivo, período em que apresentam melhor taxa de viabilidade.

The Expression of Extracellular Matrix mRNA in the Chondrocytes of the Femoral Head Affected by Avascular Necrosis

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.

Bone and Cartilage Turnover Markers, Bone Mineral Density, and Radiographic Damage in Men with Ankylosing Spondylitis

PURPOSE: To determine the levels of bone and cartilage turnover markers in men with ankylosing spondylitis (AS) and to investigate their associations with disease activity, bone mineral density, and radiographic damage of the spine. PATIENTS AND METHODS: This cross-sectional study enrolled 35 men with newly diagnosed AS. The bone mineral densities (BMD) of their lumbar spines and proximal femurs, Bath AS Disease Activity Index (BASDAI), and Bath AS Radiographic Index (BASRI) were evaluated. Urinary C-terminal telopeptide fragments of type I collagen (CTX-I) and type II collagen (CTX-II) levels were determined by enzyme-linked immunosorbent assay, and serum levels of bone-specific alkaline phosphatase (BALP) and osteocalcin were determined by an enzyme immunoassay. Levels of biochemical markers were compared with those of 70 age-matched healthy men. RESULTS: Patients with AS had significantly higher mean urinary CTX-I and CTX-II levels than control subjects (p < 0.05). Elevated urinary CTX-I levels correlated well with BASDAI, femoral BMD, and femoral T score (p < 0.05), and elevated urinary CTX-II levels correlated well with spinal BASRI (p < 0.05) in patients with AS. Mean serum BALP and osteocalcin levels did not differ between patients and controls and did not show any significant correlations with BMD, BASDAI, or BASRI in men with AS. Conclusions: Elevated CTX-I reflects disease activity and loss of femoral BMD while elevated CTX-II levels correlate well with radiographic damage of the spine, suggesting the usefulness of these markers for monitoring disease activity, loss of BMD, and radiographic damage in men with AS.

Cross-talk between Integrin and SMAD Signal Pathway in Articular Chondrocyte / 대한정형외과연구학회지

PURPOSE: Identifying the signal cross-talk between integrin signaling cascade and TGF-beta 1 signaling cascade in articular chondrocytes. MATERIALS AND METHODS: To analyze integrin or TGF-beta 1 mediated signaling pathways from extracellular stimuli, type II collagen was coated on the cell culture plate and TGF-beta 1 was added to cell culture media. Chondrocytes were cultured in the conditioned media with each or both stimuli. Altered activation of signaling proteins detected with western blot technique. RESULTS: More rapid attachment of cells was observed in the type II collagen coated group than non-coated group. The phosphorylated SMAD 2 and 3 were expressed in the type II collagen coated group and synergistically up-regulated phosphorylation in the co-treated group. The phosphorylated FAK at tyrosine 925 was activated by TGF-beta 1 treatment and synergistically up-regulated by both stimuli. But there was no meaningfully changed phosphorylation of extracellular signal regulated protein kinase (ERK) 1/2 and p38, as known downstream molecules of FAK cascade. CONCLUSION: This result means that SMAD 2, SMAD 3 and tyrosine 925 of FAK are involved in this signal cross-talking in articular chondrocytes.

Therapeutic effects of recombinant human interleukin 1 receptor antagonist on type II collagen-induced arthritis rats / 中国药理学通报

Aim: To study the therapeutic effects of recombinant human interleukin 1 receptor antagonist (rhIL-1ra) on type II collagen-induced arthritis (CIA) rats. Methods: SD rats were divided randomly into six groups including normal, model, rhIL-1ra (7.5,30,120 mg·kg-1) and anakinra(120 mg· kg-1) groups. Collagen II emulsion was used to induce CIA model in rats. The body weight was observed once a week. Paw swelling of CIA rats was measured with volume meter. C II induced delayed-type hypersensitivity (DTH) was measured. Meanwhile, the level of anti-C II IgG antibody in serum was assayed by ELISA. Results: The onset of paw swelling was on dlO after injection of emulsion. The level of serum anti-C II IgG antibody was increased significantly in CIA. Pathological changes in joints of CIA rats showed hyperplastic synovium of CIA, inflammatory cells infiltration, pannus, destruction of cartilage and bone. rhIL-1ra(7.5,30,120 mg·kg-1·d-1 x 7 d) and anakinra (120 mg·kg-1·d-1 x 7 d) subcutaneous injection (sc) inhibited inflammatory swelling. rhIL-1ra(30, 120 mg·kg-1·d-1 x 7 d) significantly suppressed the DTH reaction induced with C II in CIA rats. Moreover, rhIL-1ra reduced the level of anti-C II IgG antibody in serum. Pathological examination showed rhIL-1ra(120 mg·kg-1·d-1 x 7 d) significantly improved subchondral inflammation, synovium hyperplasia, pannus and cartilage damage. Conclusion: rhIL-1ra has therapeutic effects on CIA rats.

Selective Expansion of TCR V beta 3+CD4+ T Cells in Collagen-induced Arthritis in DBA/1 Mice

BACKGROUND: Collagen-induced arthritis (CIA) in mice is animal model of autoimmune disease known as rheumatic arthritis in human. We investigated CII-specific CD4+ T cell receptor usage in CIA mice. METHODS: In CIA model, draining lymph node (dLN) CD4+ T cells and splenocytes at 3rd, 5th, 8th week, we investigated CII-specific T cell proliferation, production of IL-17, IFN-gamma, TNF-alpha, IL-4 and IL-10. And we also performed anti-CII IgG Ab measurements in serum level, TCRVbeta usage and T cell clonality with RT-PCR-SSCP analysis. Also, we performed proliferative response against CII when CII-specific T cell subset is deleted. RESULTS: CIA mice showed more increase in the serum level of anti-CII IgG than normal mice after induction of arthritis. And the level of anti-CII IgG2a in CIA mice was increased after 3rd week after primary immunization, while anti-CII IgG1 was decreased. Draining LN CD4+T cells have proliferated against CII stimulation at 3rd week after 1st immunization. CD4+T cells derived from dLN of CIA mice produced proinflammatory cytokine IFN-gamma, IL-17 etc. Draining LN CD4 T cells of CIA presented higher proportion of CD4+Vbeta +subsets compared to those of normal mice at 3rd week after 1st immunization, and they were increased in proportion by CII stimulation. Draining LN CD4+ T cells without TCRVbeta +/Vbeta 8.1/8.2+/Vbeta 10b+cells were not responsive against CII stimulation. But, CII-reactive response of TCRVbeta 3-/Vbeta 8.1/8.2-/Vbeta 10b- T cells was recovered when Vbeta 3+ T cells were added in culture. CONCLUSION: Our results indicate that CD4+Vbeta 3+ T cells cells are selectively expanded in dLN of CIA mice, and their recovery upon CII re-stimulation in vitro, as well as the production Th1-type cytokines, may play pivotal role in CIA pathogenesis.

Reciprocal Activation of Fibroblast-like Synoviocyte and Type II Collagen-reactive T cell in Rheumatoid Arthritis / 대한류마티스학회지

OBJECTIVE: To investigate the interaction between type II collagen (CII)-reactive T cell and fibroblast-like synoviocyte in rheumatoid arthritis (RA). METHODS: Peripheral blood T cells from RA patients were cultured with bovine CII and analyzed by flow cytometry. After co-culture with CII-reactive T cells and fibroblast-like synoviocytes (FLS), the expression of cytokines (IL-15 and TNF-alpha from FLS, IFN-gamma and IL-17 from CII-reactive T cells) were determined by ELISA and RT-PCR. RESULTS: CII-reactive T cells expressed CD69, one of the early activation markers, and produced significant amount of IFN-gamma, and proliferated. IL-15 and TNF-alpha expression from FLS were significantly elevated when co-culture with CII-reactive T cells and inhibited by physical interruption of cell-to-cell contact or anti-CD40 antibody. IFN-gamma and IL-17 expression from CII-reactive T cells were also significantly elevated when co-culture with FLS and inhibited by anti-IL-15 monoclonal antibody. CONCLUSIONS: CII-reactive T cells can activate FLS to secret proinflammatoy cytokines and interactions between these two cells drive further activation of each other. These data suggest that CII-reactive T cell may play a important role in pathogenesis of RA.