A rat model of rheumatoid arthritis: TDZD-8 is associated with the protection against the induction of the synovium knee joint IL-17A/GSK3beta/ROS/alpha-SMA axis of fibrosis / Un modelo de rata de artritis reumatoide: TDZD-8 está asociado con la protección contra la inducción del eje de fibrosis de la articulación sinovial de la rodilla IL-17A/GSK3ß/ROS/α-SMA

SUMMARY: Rheumatoid arthritis (RA) that affects the synovial knee joint causes swelling of the synovial membrane and tissue damage. Interleukin-17A (IL-17A) and the enzyme glycogen synthase kinase-3β (GSK3β) are involved in the pathogenesis of RA. The link between IL-17A, GSK3β, the oxidative stress, and the profibrogenic marker alpha-smooth muscle actin (α-SMA) with and without TDZD-8, GSK3β inhibitor has not been studied before. Consequently, active immunization of rats was performed to induce RA after three weeks using collagen type II (COII) injections. The treated group received daily injection of 1 mg/kg TDZD-8 for 21 days following the immunization protocol (COII+TDZD-8). Blood and synovium tissue samples were harvested at the end of the experiment. RA development was confirmed as corroborated by a substantial increase in blood levels of the highly specific autoantibody for RA, anti-citrullinated protein antibody as well as augmentation of reactive oxidative species (ROS) levels measured as lipid peroxidation. RA induction also increased synovium tissue levels of IL-17A and the profibrogenic marker, α-SMA. All these parameters seemed to be significantly (p<0.0001) ameliorated by TDZD-8. Additionally, a significant correlation between IL-17A, ROS, and α-SMA and biomarkers of RA was observed. Thus, knee joint synovium RA induction augmented IL-17A/GSK3β/ROS/α-SMA axis mediated arthritis in a rat model of RA, which was inhibited by TDZD-8.

La artritis reumatoide (AR) que afecta la articulación sinovial de la rodilla provoca inflamación de la membrana sinovial y daño tisular. La interleucina-17A (IL-17A) y la enzima glucógeno sintasa quinasa-3β (GSK3β) están involucradas en la patogenia de la AR. No se ha estudiadol vínculo entre IL-17A, GSK3β, el estrés oxidativo y el marcador profibrogénico actina de músculo liso alfa (α-SMA) con y sin inhibidor de TDZD-8, GSK3β. En consecuencia, se realizó una inmunización activa de ratas para inducir la AR después de tres semanas usando inyecciones de colágeno tipo II (COII). El grupo tratado recibió una inyección diaria de 1 µg/ kg de TDZD-8 durante 21 días siguiendo el protocolo de inmunización (COII+TDZD-8). Se recogieron muestras de sangre y tejido sinovial al final del experimento. El desarrollo de AR se confirmó como lo corroboró el aumento sustancial en los niveles sanguíneos del autoanticuerpo altamente específico para AR, el anticuerpo antiproteína citrulinada, así como el aumento de los niveles de especies oxidativas reactivas (ROS) medidos como peroxidación lipídica. La inducción de AR también aumentó los niveles de tejido sinovial de IL-17A y el marcador profibrogénico, α-SMA. Todos estos parámetros parecían mejorar significativamente (p<0,0001) con TDZD-8. Además, se observó una correlación significativa entre IL- 17A, ROS y α-SMA y biomarcadores de AR. Por lo tanto, la inducción de AR en la sinovial de la articulación de la rodilla aumentó la artritis mediada por el eje IL-17A/GSK3β/ROS/α-SMA en un modelo de rata de AR, que fue inhibida por TDZD-8.

Suppression of nitrosative stress and inflammation of the knee joint synovium in collagen type ii-induced rheumatoid arthritis by the inhibition of glycogen synthase kinase-3ß / Supresión del estrés nitrosativo e inflamación de la sinovial de la articulación de la rodilla en la artritis reumatoide inducida por colágeno tipo ii mediante la inhibición de la glucógeno sintasa quinasa-3 ß

SUMMARY: Rheumatoid arthritis (RA), an inflammatory autoimmune disease that causes cartilage degradation and tissue destruction, can affect synovial joints such as the knee joint. The link between the nitrosative stress enzyme inducible nitric oxide synthase (iNOS) and the cytokine interleukin-1 (IL-1β) in RA-induced knee joint synovial membrane damage with and without the incorporation of the GSK3β inhibitor TDZD-8 has never been studied. As a result, we used active immunization method with collagen type II (COII) for twenty one days to induce RA in rats. TDZD-8 (1 mg/kg; i.p.) was given daily into matched immunized rats for three weeks after day 21 (COII+TDZD-8). Blood and tissue samples were taken 42 days after immunization. A dramatic increase in rheumatoid factor (RF) blood levels, as well as considerable synovial tissue damage and inflammatory cell infiltration of the synovial membrane, were used to validate the onset of RA following COII immunization. COII immunization increased tissue levels of iNOS protein and IL- 1β mRNA and protein expression, which TDZD-8 suppressed considerably (p<0.0001). Furthermore, there was a significantly (p<0.001) positive correlation between iNOS, inflammatory biomarkers, and RF. We concluded that TDZD-8 reduced RA-induced IL-1β -iNOS axis-mediated arthritis in the rat knee joint synovium.

RESUMEN: La artritis reumatoide (AR), es una enfermedad autoinmune inflamatoria que causa la degradación del cartílago y la destrucción del tejido, pudiendo afectar las articulaciones sinoviales, como la articulación de la rodilla. No se ha estudiado el vínculo entre la óxido nítrico sintasa inducible por la enzima del estrés nitrosativo (iNOS) y la citocina interleucina-1 (IL-1β) en el daño de la membrana sinovial de la articulación de la rodilla provocado por AR con y sin la incorporación del inhibidor de GSK3β TDZD-8. Utilizamos el método de inmunización activa con colágeno tipo II (COII) durante veintiún días para inducir AR en ratas. Se administró TDZD-8 (1 mg/kg; i.p.) diariamente a ratas inmunizadas emparejadas durante tres semanas después del día 21 (COII+TDZD- 8). Se tomaron muestras de sangre y tejido 42 días después de la inmunización. Se observó un gran aumento de los niveles sanguíneos del factor reumatoideo (FR), así como un daño considerable del tejido sinovial e infiltración de células inflamatorias en la membrana sinovial, para validar la aparición de la AR después de la inmunización con COII. La inmunización con COII aumentó los niveles tisulares de la proteína iNOS y la expresión de proteína y ARNm de IL-1β, que TDZD-8 suprimió considerablemente (p<0,0001). Además, hubo una correlación positiva significativa (p<0,001) entre iNOS, biomarcadores inflamatorios y FR. Concluimos que TDZD- 8 redujo la artritis mediada por el eje IL-1β-iNOS inducida por la AR en la sinovial de la articulación de la rodilla de rata.

TDZD-8 suppresses rheumatoid arthritis induced by collagen type II in rats: potential role of apoptosis and inflammation / TDZD-8 suprime la artritis reumatoide inducida por colágeno tipo II en ratas: papel potencial de la apoptosis y la inflamación

SUMMARY: Rheumatoid arthritis (RA) is considered an autoimmune disease distinguished by chronic synovial membrane inflammation, degraded cartilage, as well as bone destruction, which lead to joints pain and stiffness. The pathogenesis of RA involved at least two mechanisms: Cellular proliferation and activation of glycogen synthase kinase-3β (GSK3β) enzyme. Thus, we tested the hypothesis that the GSK3binhibitor, TDZD-8, can treat the synovial tissue toward collagen type II (COII)-mediated RA linked to apoptosis induction and biomarker suppression of inflammation. Wistar rats were immunized with COII (the model group) for 21 days. Matched immunized rats were daily injected with TDZD-8 (1 mg/kg; i.p) for three additional weeks (COII+TDZD- 8).After 42 days of post-immunization, blood and tissues were collected. Histology (H&E) and immunohistochemistry (CD45; leukocyte common antigen) images showed that COII induced RA was demonstrated by profound damage to the synovial tissue and infiltration of the inflammatory cells, which were substantially ameliorated with TDZD-8. In addition, COII immunization caused the induction of rheumatoid factor (RF), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin 1 beta (IL-1β) that were substantially (p<0.05) suppressed by TDZD-8. Whereas, TDZD-8 augmented the apoptotic biomarker, Bcl-2-associated X protein (Bax), which was significantly (p<0.05) ameliorated by RA. We also showed a substantial relationship (p<0.001) between the blood levels of RF and the synovial tissue levels of TNF-α (r = 0.759), IL-1b (r = 0.969), IL-6 (r = 0.749), and Bax (r = - 0.914). These results indicate effective treatment of the injured synovial tissue by TDZD-8 against COII-induced RA in rats, which also decreases inflammatory biomarkers and augmentation of apoptosis.

RESUMEN: La artritis reumatoide (AR) es una enfermedad autoinmune que se distingue por la inflamación crónica de la membrana sinovial, el cartílago degradado y la destrucción de los huesos, lo que provoca dolor y rigidez en las articulaciones. La patogenia de la AR involucra al menos dos mecanismos: la proliferación celular y la activación de la enzima glucógeno sintasa quinasa-3b (GSK3β) Por lo tanto, probamos la hipótesis de que el inhibidor de GSK3β, TDZD-8, puede tratar el tejido sinovial hacia el colágeno tipo II (COII) - AR mediada por inducción de apoptosis y supresión de biomarcadores de inflamación. Se inmunizaron ratas Wistar con COII (el grupo modelo) durante 21 días. Se inyectaron diariamente ratas emparejadas inmunizadas con TDZD-8 (1 mg / kg; i.p) durante tres semanas adicionales (COII + TDZD-8). Después de 42 días de post-inmunización, se recolectó sangre y tejidos. Las imágenes de histología (H&E) e inmunohistoquímica (CD45; antígeno común de leucocitos) mostraron que la AR inducida por COII presentaba un daño profundo en el tejido sinovial e infiltración de las células inflamatorias, las que mejoraron con TDZD-8. Además, la inmunización con COII provocó la inducción de factor reumatoide (FR), factor de necrosis tumoral alfa (TNF-α), interleucina-6 (IL-6) e interleucina 1 beta (IL-1β) que fueron suprimidos por TDZD-8 de manera significativa (p < 0.05). Considerando que TDZD-8 aumentó el biomarcador apoptótico, la proteína X asociada a Bcl-2 (Bax), que fue mejorado (p <0,05) por RA. También se observó una relación sustancial (p <0,001) entre los niveles sanguíneos de RF y los niveles de tejido sinovial de TNF-α (r = 0,759), IL-1β (r = 0,969), IL-6 (r = 0,749), y Bax (r = -0,914). Estos resultados indicaron un tratamiento eficaz del tejido sinovial lesionado por TDZD-8 contra la AR inducida por COII en ratas, que también disminuye los biomarcadores inflamatorios y el aumento de la apoptosis.

Repair of Osteochondral Defects in Rabbit Knee Using Menstrual Blood Stem Cells Encapsulated in Fibrin Glue: A Good Stem Cell Candidate for the Treatment of Osteochondral Defects

BACKGROUND: In recent years, researchers discovered that menstrual blood-derived stem cells (MenSCs) have the potential to differentiate into a wide range of tissues including the chondrogenic lineage. In this study, we aimed to investigate the effect of MenSCs encapsulated in fibrin glue (FG) on healing of osteochondral defect in rabbit model. METHODS: We examined the effectiveness of MenSCs encapsulated in FG in comparison with FG alone in the repair of osteochondral defect (OCD) lesions of rabbit knees after 12 and 24 weeks. RESULTS: Macroscopical evaluation revealed that the effectiveness of MenSCs incorporation with FG is much higher than FG alone in repair of OCD defects. Indeed, histopathological evaluation of FG + MenSCs group at 12 weeks post-transplantation demonstrated that defects were filled with hyaline cartilage-like tissue with proper integration, high content of glycosaminoglycan and the existence of collagen fibers especially collagen type II, as well as by passing time (24 weeks post-transplantation), the most regenerated tissue in FG + MenSCs group was similar to hyaline cartilage with relatively good infill and integration. As the same with the result of 12 weeks post-implantation, the total point of microscopical examination in FG + MenSCs group was higher than other experimental groups, however, no significant difference was detected between groups at 24 weeks (p>0.05). CONCLUSION: In summary, MenSCs as unique stem cell population, is suitable for in vivo repair of OCD defects and promising for the future clinical application.

Protective effect of glucosamine and risedronate (alone or in combination) against osteoarthritic changes in rat experimental model of immobilized knee / 대한해부학회지

Effect of Fengshi Qutong Capsules on synovial angiogenesis in rats with type Ⅱ collagen induced arthritis / 中国中药杂志

To observe the effect of Fengshi Qutong Capsules(FSQTC) on angiogenesis of rat aortarings and in knee joint synovium of type Ⅱ collagen-induced arthritis(CIA) rats. The blood vessel of aorta rings of normal SD rats were induced by vascular endothelial growth factor(VEGF) 20 μg·L~(-1 )in vitro, and were treated with FSQTC(0.02, 0.1 and 0.5 μg·L~(-1)) continuously for 9 days. The number, length and area of neovascularization of the vascular ring were measured. SD rats were immunized to establish collagen-induced arthritis. CIA rats were treated with FSQTC(0.25, 0.5, 1 g·kg~(-1)·d~(-1)) and methotrexate(0.2 mg·kg~(-1)·d~(-1)) daily for 19 days. Histopathological examination(HE) was performed to observe the vascular morphology and vascular density in the synovial membrane of the inflamed joint. Immunohistochemistry was performed to observe the expression of platelets-endothelial cell adhesion molecule(CD31), VEGF and VEGF receptor 2(VEGFR_2)in the synovium. Immunofluorescence was performed to observe the expression of CD31 and α smooth muscle actin(αSMA) in synovial membrane.TGF-β, PDGF and VEGFR_2 in serum were detected by enzyme-linked immunosorbent assay. The number, branch length and area of blood vessels of aorta rings were significantly increased induced by VEGF, and FSQTC could significantly reduce the number, branch length and area of blood vessels. Compared with the normal group, the vascular density, CD31 positive expression, CD31~+/αSMA~- immature and total vascular positive expression in the synovial membrane of the model group were significantly increased, and so as VEGF and VEGFR_2 in the synovium. The VEGFR_2, TGF-β and PDGF in sera were also significantly increased in model group. FSQTC reduced the synovial vascular density and inhibited the positive expression of CD31, CD31~+/αSMA~- immature blood vessels and total vascular. FSQTC has no significant effect on CD31~+/αSMA~+mature blood vessels. FSQTC also negatively inhibited the expression of VEGF, VEGFR_2, TGF-β and PDGF in synovial membrane and/or sera. The effect of methotrexate is similar with to the high dose group. Our results demonstrated that FSQTC could inhibit the angiogenesis of synovial tissue in CIA rats and of aortaring in rats, which is related to the reduction of angiogenesis regulatory factor.

Identification of a novel COL2A1 variant in a pedigree affected with spondyloepiphyseal dysplasia congenita / 中华医学遗传学杂志

OBJECTIVE@#To explore the molecular basis for a pedigree affected with spondyloepiphyseal dysplasia congenita (SEDC).@*METHODS@#The proband was subjected to whole exome sequencing. Suspected variant was verified by Sanger sequencing.@*RESULTS@#All patients from the pedigree were found to carry a novel missense variant c.1394G>C (p.Gly465Ala) of the COL2A1 gene. The variant was not reported previously. Provean, Polyphen-2 and Mutation Taster software predicted that the variant is highly likely to be pathogenic.@*CONCLUSION@#The c.1394G>C (p.Gly465Ala) variant of the COL2A1 gene probably underlies the SEDC in this pedigree.

Induction of Chondrogenic Differentiation in Human Mesenchymal Stem Cells Cultured on Human Demineralized Bone Matrix Scaffold under Hydrostatic Pressure

BACKGROUND: Articular cartilage damage is still a troublesome problem. Hence, several researches have been performed for cartilage repair. The aim of this study was to evaluate the chondrogenicity of demineralized bone matrix (DBM) scaffolds under cyclic hydrostatic pressure (CHP) in vitro. METHODS: In this study, CHP was applied to human bone marrow mesenchymal stem cells (hBMSCs) seeded on DBM scaffolds at a pressure of 5 MPa with a frequency of 0.5 Hz and 4 h per day for 1 week. Changes in chondrogenic and osteogenic gene expressions were analyzed by quantifying mRNA signal level of Sox9, collagen type I, collagen type II, aggrecan (ACAN), Osteocalcin, and Runx2. Histological analysis was carried out by hematoxylin and eosin, and Alcian blue staining. Moreover, DMMB and immunofluorescence staining were used for glycosaminoglycan (GAG) and collagen type II detection, respectively. RESULTS: Real-time PCR demonstrated that applying CHP to hBMSCs in DBM scaffolds increased mRNA levels by 1.3-fold, 1.2-fold, and 1.7-fold (p < 0.005) for Sox9, Col2, and ACAN, respectively by day 21, whereas it decreased mRNA levels by 0.7-fold and 0.8-fold (p < 0.05) for Runx2 and osteocalcin, respectively. Additionally, in the presence of TGF-β1 growth factor (10 ng/ml), CHP further increased mRNA levels for the mentioned genes (Sox9, Col2, and ACAN) by 1.4-fold, 1.3-fold and 2.5-fold (p < 0.005), respectively. Furthermore, in histological assessment, it was observed that the extracellular matrix contained GAG and type II collagen in scaffolds under CHP and CHP with TGF-β1, respectively. CONCLUSION: The osteo-inductive DBM scaffolds showed chondrogenic characteristics under hydrostatic pressure. Our study can be a fundamental study for the use of DBM in articular cartilage defects in vivo and lead to production of novel scaffolds with two different characteristics to regenerate both bone and cartilage simultaneously.

Combination therapy of curcumin and alendronate modulates bone turnover markers and enhances bone mineral density in postmenopausal women with osteoporosis

ABSTRACT Objective: This study evaluated the effects of combination therapy of curcumin and alendronate on BMD and bone turnover markers in postmenopausal women with osteoporosis. Subjects and methods: In a randomized, double-blind trial study, 60 postmenopausal women were divided into three groups: control, alendronate, and alendronate + curcumin. Each group included 20 patients. Total body, total hip, lumbar spine and femoral neck BMDs were measured by dual-energy X-ray absorptiometry (DXA) at baseline and after 12 months of therapy. Bone turnover markers such as bone-specific alkaline phosphatase (BALP), osteocalcin and C-terminal cross-linking telopeptide of type I collagen (CTx) were measured at the outset and 6 months later. Results: Patients in the control group suffered a significant decrease in BMD and increased bone turnover markers at the end of study. The group treated with only alendronate showed significantly decreased levels of BALP and CTx and increased levels of osteocalcin compared to the control group. The alendronate group also showed significant increases in the total body, total hip, lumbar spine and femoral neck BMDs at the end of study compared to the control group. In the curcumin + alendronate group, BALP and CTx levels decreased and osteocalcin levels increased significantly at the end of study compared to the control and alendronate groups. BMD indexes also increased in four areas significantly at the end of study compared to the control and alendronate groups. Conclusion: The combination of curcumin and alendronate has beneficial effects on BMD and bone turnover markers among postmenopausal women with osteoporosis. Arch Endocrinol Metab. 2018;62(4):438-45

Collagen Scaffolds in Cartilage Tissue Engineering and Relevant Approaches for Future Development

BACKGROUND: Cartilage tissue engineering (CTE) aims to obtain a structure mimicking native cartilage tissue through the combination of relevant cells, three-dimensional scaffolds, and extraneous signals. Implantation of ‘matured’ constructs is thus expected to provide solution for treating large injury of articular cartilage. Type I collagen is widely used as scaffolds for CTE products undergoing clinical trial, owing to its ubiquitous biocompatibility and vast clinical approval. However, the long-term performance of pure type I collagen scaffolds would suffer from its limited chondrogenic capacity and inferior mechanical properties. This paper aims to provide insights necessary for advancing type I collagen scaffolds in the CTE applications. METHODS: Initially, the interactions of type I/II collagen with CTE-relevant cells [i.e., articular chondrocytes (ACs) and mesenchymal stem cells (MSCs)] are discussed. Next, the physical features and chemical composition of the scaffolds crucial to support chondrogenic activities of AC and MSC are highlighted. Attempts to optimize the collagen scaffolds by blending with natural/synthetic polymers are described. Hybrid strategy in which collagen and structural polymers are combined in non-blending manner is detailed. RESULTS: Type I collagen is sufficient to support cellular activities of ACs and MSCs; however it shows limited chondrogenic performance than type II collagen. Nonetheless, type I collagen is the clinically feasible option since type II collagen shows arthritogenic potency. Physical features of scaffolds such as internal structure, pore size, stiffness, etc. are shown to be crucial in influencing the differentiation fate and secreting extracellular matrixes from ACs and MSCs. Collagen can be blended with native or synthetic polymer to improve the mechanical and bioactivities of final composites. However, the versatility of blending strategy is limited due to denaturation of type I collagen at harsh processing condition. Hybrid strategy is successful in maximizing bioactivity of collagen scaffolds and mechanical robustness of structural polymer. CONCLUSION: Considering the previous improvements of physical and compositional properties of collagen scaffolds and recent manufacturing developments of structural polymer, it is concluded that hybrid strategy is a promising approach to advance further collagen-based scaffolds in CTE.

Inhibition of the Expression of Matrix Metalloproteinases in Articular Chondrocytes by Resveratrol through Affecting Nuclear Factor-Kappa B Signaling Pathway

In the present study, we tried to examine whether resveratrol regulates the expression of matrix metalloproteinases (MMPs) through affecting nuclear factor-kappa B (NF-κB) in articular chondrocytes. Rabbit articular chondrocytes were cultured in a monolayer, and reverse transcription-polymerase chain reaction (RT-PCR) was used to measure interleukin-1β (IL-1β)-induced gene expression of MMP-3, MMP-1, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), ADAMTS-5 and type II collagen. Effect of resveratrol on IL-1β-induced secretion of MMP-3 was investigated in rabbit articular chondrocytes using western blot analysis. To elucidate the action mechanism of resveratrol, effect of resveratrol on IL-1β-induced NF-κB signaling pathway was investigated in SW1353, a human chondrosarcoma cell line, by western blot analysis. The results were as follows: (1) resveratrol inhibited the gene expression of MMP-3, MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5, but increased the gene expression of type II collagen; (2) resveratrol reduced the secretion of MMP-3; (3) resveratrol inhibited IL-1β-induced activation (phosphorylation) of inhibitory kappa B kinase (IKK), and thus phosphorylation and degradation of inhibitory kappa Bα (IκBα); (4) resveratrol inhibited IL-1β-induced phosphorylation and nuclear translocation of NF-κB p65. This, in turn, led to the down-regulation of gene expression of MMPs in SW1353 cells. These results suggest that resveratrol can regulate the expression of MMPs through affecting NF-κB by directly acting on articular chondrocytes.

Micromass cultures are effective for differentiation of human amniotic fluid stem cells into chondrocytes

OBJECTIVES: Articular cartilage is vulnerable to injuries and undergoes an irreversible degenerative process. The use of amniotic fluid mesenchymal stromal stem cells for the reconstruction of articular cartilage is a promising therapeutic alternative. The aim of this study was to investigate the chondrogenic potential of amniotic fluid mesenchymal stromal stem cells from human amniotic fluid from second trimester pregnant women in a micromass system (high-density cell culture) with TGF-β3 for 21 days. METHODS: Micromass was performed using amniotic fluid mesenchymal stromal stem cells previously cultured in a monolayer. Chondrocytes from adult human normal cartilage were used as controls. After 21 days, chondrogenic potential was determined by measuring the expression of genes, such as SOX-9, type II collagen and aggrecan, in newly differentiated cells by real-time PCR (qRT-PCR). The production of type II collagen protein was observed by western blotting. Immunohistochemistry analysis was also performed to detect collagen type II and aggrecan. This study was approved by the local ethics committee. RESULTS: SOX-9, aggrecan and type II collagen were expressed in newly differentiated chondrocytes. The expression of SOX-9 was significantly higher in newly differentiated chondrocytes than in adult cartilage. Collagen type II protein was also detected. CONCLUSION: We demonstrate that stem cells from human amniotic fluid are a suitable source for chondrogenesis when cultured in a micromass system. amniotic fluid mesenchymal stromal stem cells are an extremely viable source for clinical applications, and our results suggest the possibility of using human amniotic fluid as a source of mesenchymal stem cells.

Collagen-Induced Arthritis Analysis in Rhbdf2 Knockout Mouse

Rhomboid family member 2 gene (Rhbdf2) is an inactive homologue lacking essential catalytic residues of rhomboid intramembrane serine proteases. The protein is necessary for maturation of tumor necrosis factor-alpha (TNF-α) converting enzyme, which is the molecule responsible for the release of TNF-α. In this study, Rhbdf2 knockout (KO) mice were produced by CRISPR/CAS9. To see the effects of the failure of TNF-α release induced by Rhbdf2 gene KO, collagen-induced arthritis (CIA), which is the representative TNF-α related disease, was induced in the Rhbdf2 mutant mouse using chicken collagen type II. The severity of the CIA was measured by traditional clinical scores and histopathological analysis of hind limb joints. A rota-rod test and grip strength test were employed to evaluate the severity of CIA based on losses of physical functions. The results indicated that Rhbdf2 mutant mice showed clear alleviation of the clinical severity of CIA as demonstrated by the significantly lower severity indexes. Moreover, a grip strength test was shown to be useful for the evaluation of physical functional losses by CIA. Overall, the results showed that the Rhbdf2 gene has a significant effect on the induction of CIA, which is related to TNF-α.

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.

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.

Differentiation potential of canine mesenchymal stem cells on hydrogel scaffold-based three-dimensional environment / 대한수의학회지

Mesenchymal stem cells (MSCs) are useful candidates for tissue engineering and cell therapy. Physiological cell environment not only connects cells to each other, but also connects cells to the extracellular matrix that provide mechanical support, thus exposing the entire cell surface and activating signaling pathways. Hydrogel is a polymeric material that swells in water and maintains a distinct 3-dimensional (3D) network structure by cross linking. In this study, we investigated the optimized cellular function for canine adipose tissue-derived MSCs (cAD-MSCs) using hydrogel. We observed that the expression levels of Ki67 and proliferating cell nuclear antigen, which are involved in cell proliferation and stemness, were increased in transwell-hydrogel (3D-TN) compared to the transwell-normal (TN). Also, transforming growth factor-β1 and SOX9, which are typical bone morphogenesis-inducing factors, were increased in 3D-TN compared to the TN. Collagen type II alpha 1, which is a chondrocyte-specific marker, was increased in 3D-TN compared to the TN. Osteocalcin, which is a osteocyte-specific marker, was increased in 3DTN compared to the TN. Collectively, preconditioning cAD-MSCs via 3D culture systems can enhance inherent secretory properties that may improve the potency and efficacy of MSCs-based therapies for bone regeneration process.

In Vitro Anti-Inflammation and Chondrogenic Differentiation Effects of Inclusion Nanocomplexes of Hyaluronic Acid-Beta Cyclodextrin and Simvastatin

The aim of this study was to prepare inclusion nanocomplexes of hyaluronic acid-β-cyclodextrin and simvastatin (HA-β-CD/SIM) and evaluate in vitro anti-inflammation effects on lipopolysaccharide (LPS)-activated synoviocytes and chondrogenic differentiation effects on rat adipose-derived stem cells (rADSCs). The β-CD moieties in HA-β-CD could incorporate SIM to form HA-β-CD/SIM nanocomplexes with diameters of 297–350 nm. HA-β-CD/SIM resulted in long-term release of SIM from the nanocomplexes for up to 63 days in a sustained manner. In vitro studies revealed that HA-β-CD/SIM nanocomplexes were able to effectively and dose-dependently suppress the mRNA expression levels of proinflammatory markers such as matrix metallopeptidase-3 (MMP-3), MMP-13, cyclooxygenase-2 (COX-2), a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5), interleukin-6 (IL-6), and tumor necrosis factor (TNF-α) in LPS-stimulated synoviocytes. HA-β-CD/SIM-treated rADSCs significantly and dose-dependently enhanced mRNA expressions of aggrecan, collagen type II (COL2A1), and collagen type X (COL10A1), implying that HA-β-CD/SIM greatly induced the chondrogenic differentiation of rADSCs. Conclusively, HA-β-CD/SIM nanocomplexes will be a promising therapeutic material to alleviate inflammation as well as promote chondrogenesis.

Detection of the Urinary Biomarkers PYD, CTX-II, and DPD in Patients with Kashin-Beck Disease in the Qinghai Province of China / 生物医学与环境科学（英文）

Kashin-Beck disease (KBD) is an endemic degenerative osteoarthropathy of uncertain etiology. The aim of our study was to identify changes in C-telopeptide of type II collagen (CTX-II), pyridinoline (PYD), and deoxypyridinoline (DPD) among KBD patients. 54 KBD patients and 78 healthy controls were included this study. Urinary samples were collected and measured by ELISA. The median quantities of PYD, CTX-II, and DPD of KBD patients were 1107.73 ng/μmol.cre, 695.11 ng/μmol.cre, and 1342.34 pml/μmol.cre, while the median quantities of healthy controls were 805.59 ng/μmol.cre, 546.47 ng/μmol.cre, and 718.15 pml/μmol.cre, respectively. The differences between KBD patients and healthy controls were statistically significant (Z = 4.405, 3.653, and 3.724; P < 0.001). The higher levels of PYD, CTX-II, and DPD detected in KBD patients indicate that they could be used as biomarkers of KBD.

Effects of Type I Collagen Concentration in Hydrogel on the Growth and Phenotypic Expression of Rat Chondrocytes

It is controversial whether type I collagen itself can maintain and improve chondrogenic phenotype of chondrocytes in a three-dimensional (3D) environment. In this study, we examined the effect of type I collagen concentration in hydrogel (0.5, 1, and 2 mg/ml) on the growth and phenotype expression of rat chondrocytes in vitro. All collagen hydrogels showed substantial contractions during culture, in a concentration-dependent manner, which was due to the cell proliferation. The cell viability was shown to be the highest in 2 mg/ml collagen gel. The mRNA expression of chondrogenic phenotypes, including SOX9, type II collagen, and aggrecan, was significantly up-regulated, particularly in 1 mg/ml collagen gel. Furthermore, the production of type II collagen and glycosaminoglycan (GAG) content was also enhanced. The results suggest that type I collagen hydrogel is not detrimental to, but may be useful for, the chondrocyte culture for cartilage tissue engineering.

Effect of oleanolic acid on the activity, secretion and gene expression of matrix metalloproteinase-3 in articular chondrocytes in vitro and the production of matrix metalloproteinase-3 in vivo

In the present study, we tried to examine whether oleanolic acid regulates the activity, secretion and gene expression of matrix metalloproteinase-3 (MMP-3) in primary cultured rabbit articular chondrocytes, as well as the production of MMP-3 in the knee joint of rat to evaluate the potential chondroprotective effect of oleanolic acid. Rabbit articular chondrocytes were cultured in a monolayer, and reverse transcription-polymerase chain reaction (RT-PCR) was used to measure interleukin-1β (IL-1β)-induced gene expression of MMP-3, MMP-1, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), ADAMTS-5 and type II collagen. In rabbit articular chondrocytes, the effects of oleanolic acid on IL-1β-induced secretion and proteolytic activity of MMP-3 were investigated using western blot analysis and casein zymography, respectively. The effect of oleanolic acid on in vivo MMP-3 protein production was also examined, after intra-articular injection to the knee joint of rat. The results were as follows: (1) oleanolic acid inhibited the gene expression of MMP-3, MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5, but increased the gene expression of type II collagen; (2) oleanolic acid reduced the secretion and proteolytic activity of MMP-3; (3) oleanolic acid suppressed the production of MMP-3 protein in vivo. These results suggest that oleanolic acid can regulate the activity, secretion and gene expression of MMP-3, by directly acting on articular chondrocytes.