Jyh-Horng Wang

Decoy receptor 3 (DcR3) induces osteoclast formation from monocyte/macrophage lineage precursor cells

AbstractRecent evidence indicates that the decoy receptor 3 (DcR3) of the TNF receptor superfamily, which initially though prevents cytokine responses of FasL, LIGHT and TL1A by binding and neutralization, can modulate monocyte function through reverse signaling. We show in this work that DcR3 can induce osteoclast formation from human monocytes, murine RAW264.7 macrophages, and bone marrow cells. DcR3-differentiated cells exhibit characteristics unique for osteoclasts, including polynuclear giant morphology, bone resorption, TRAP, CD51/61, and MMP-9 expression. Consistent with the abrogation of osteoclastogenic effect of DcR3 by TNFR-Fc, DcR3 treatment can induce osteoclastogenic cytokine TNF-α release through ERK and p38 MAPK signaling pathways. We conclude that DcR3 via coupling reverse signaling of ERK and p38 MAPK and stimulating TNF-α synthesis is a critical regulator of osteoclast formation. This action of DcR3 might play an important role in significant osteoclastic activity in osteolytic bone metastases.

Tissue engineering-based cartilage repair with mesenchymal stem cells in a porcine model†‡

This in vivo pilot study explored the use of mesenchymal stem cell (MSC) containing tissue engineering constructs in repair of osteochondral defects. Osteochondral defects were created in the medial condyles of both knees of 16 miniature pigs. One joint received a cell/collagen tissue engineering construct with or without pretreatment with transforming growth factor β (TGF‐β) and the other joint from the same pig received no treatment or the gel scaffold only. Six months after surgery, in knees with no treatment, all defects showed contracted craters; in those treated with the gel scaffold alone, six showed a smooth gross surface, one a hypertrophic surface, and one a contracted crater; in those with undifferentiated MSCs, five defects had smooth, fully repaired surfaces or partially repaired surfaces, and one defect poor repair; in those with TGF‐β‐induced differentiated MSCs, seven defects had smooth, fully repaired surfaces or partially repaired surfaces, and three defects showed poor repair. In Pineda score grading, the group with undifferentiated MSC, but not the group with TGF‐β‐induced differentiated MSCs, had significantly lower subchondral, cell morphology, and total scores than the groups with no or gel‐only treatment. The compressive stiffness was larger in cartilage without surgical treatment than the treated area within each group. In conclusion, this preliminary pilot study suggests that using undifferentiated MSCs might be a better approach than using TGF‐β‐induced differentiated MSCs for in vivo tissue engineered treatment of osteochondral defects.

Modulation of gene expression and collagen production of anterior cruciate ligament cells through cell shape changes on polycaprolactone/chitosan blends

Our previous study has illustrated that chitosan could enhance human anterior cruciate ligament (ACL) cells to exhibit a dramatic effect on increasing the gene expression of transforming growth factor beta1 (TGF-beta1), which is a specific gene for wound healing and collagen synthesis. However, human ACL cells could not adhere and proliferate well on chitosan. In order to overcome this drawback, we introduced polycaprolactone (PCL) into chitosan by the method of blending in this study. It was found that the morphology, viability and gene expression of human ACL cells on the chitosan/PCL blends could be effectively regulated. With the increase of PCL content in blends, human ACL cells presented more flatten shape, well-organized cytoskeleton, and higher proliferated ability. Compared to flatten shape, human ACL cells with round shape exhibited higher levels of mRNA expression of TGF-beta1 and collagen type III through 3-day culture period. Furthermore, these blended materials could upregulate protein synthesis of human ACL cells, which corresponded to their gene expressions. Therefore, it is possible to combine the advantages of chitosan and PCL to create a new blended material, which could control cellular morphologies specifically, and further to regulate the gene expression and protein production of cells for specific applications. We expected this concept, controlling the cell shape through biomaterial to modulate the behavior of cells, could provide a new vision for the material selection of ligament tissue engineering.

Nitric oxide induces osteoblast apoptosis through the de novo synthesis of Bax protein

Nitric oxide (NO) plays a crucial role in the physiological and pathophysiological regulations of osteoblast functions. This study is designed to evaluate the toxic effects of NO released by sodium nitroprusside (SNP), an NO donor, on neonatal Wistar rat calvarial osteoblasts from the analyses of cell viability, alkaline phosphatase (ALP) activity, cell morphology, apoptotic cells, terminal deoxynucleotidyl transferase‐mediated dUTP nick end‐label (TUNEL) assay, DNA ladder, and immunocytochemistry and Western blot for proapoptotic Bax protein. SNP increased the levels of nitrite, an oxidative product of NO, in the culture medium of osteoblasts in concentration‐ and time‐dependent manners, and altered cell morphologies to round and shrinkage shapes. Administration of osteoblasts with SNP resulted in concentration‐ and time‐dependent decreases of cell viability and ALP activity. Analysis of apoptotic cells revealed that SNP increased the percentages of osteoblasts processing apoptosis. Analyses of TUNEL and DNA ladder showed that SNP caused DNA fragmentation. Pretreatment with cycloheximide, an inhibitor of protein synthesis, partially blocked SNP‐induced osteoblast apoptosis. Imunocytochemical and immunoblotting analyses revealed that SNP increased Bax protein in osteoblasts. This study suggests that SNP could increase the levels of NO in osteoblasts, and cause osteoblast apoptosis possibly through the de novo synthesis of proapoptotic Bax protein.

A comparative study of nonoperative versus operative treatment of developmental dysplasia of the hip in patients of walking age

We retrospectively reviewed our results of treatment of two groups of children with developmental dysplasia of the hip (DDH) aged between 13 and 17 months. The nonoperative group consisted of 16 patients (17 hips) who were treated by closed reduction and casting with or without skin traction. There were four hips with mild degrees of avascular necrosis, and one hip with a failed reduction. Before further treatment, there were one hip in Severin class I, nine in class III, six in class IV, and one in class VI. Subsequent open reduction or pelvic osteotomy was required in those hips with failed reduction and residual dysplasia. The operative group consisted of 32 patients (32 hips). Open reduction and Salter osteotomy were performed without preoperative traction. There was one hip with redislocation due to improper use of a short spica and two hips with a mild degree of avascular necrosis. In > 2 years and 3 months of follow-up, 13 hips were in Severin class I, 18 in class II, and one in class III. The treatment time was significantly shortened in the operative group. We conclude that neglected DDH in patients of walking age can be safely and effectively treated by open reduction plus Salter osteotomy.

Simvastatin inhibits cysteine‐rich protein 61 expression in rheumatoid arthritis synovial fibroblasts through the regulation of sirtuin‐1/FoxO3a signaling

OBJECTIVE To examine the role of sirtuin-1 (SIRT-1)/FoxO3a in the expression of cysteine-rich protein 61 (CYR-61) in rheumatoid arthritis synovial fibroblasts (RASFs) and the influence of simvastatin on this pathway, and to determine the relationship between disease progression and FoxO3a/CYR-61 signaling in synovial fibroblasts in vivo using a rat model of collagen-induced arthritis (CIA). METHODS In RASFs, the expression of CYR-61 and SIRT-1, the localization of FoxO3a in the nucleus/cytoplasm, and the phosphorylation/acetylation of FoxO3a were examined by Western blotting. Secretion of CCL20 was assessed by enzyme-linked immunosorbent assay. Promoter activity of the Cyr61 gene was evaluated by luciferase assay, with or without forced expression of FoxO3a and SIRT-1 by lentiviral transduction. FoxO3a-Cyr61 promoter interaction was examined by chromatin immunoprecipitation. In rats with CIA, the expression of CYR-61 and phosphorylated FoxO3a in synovial fibroblasts was examined by immunohistochemistry. RESULTS In RASFs, simvastatin suppressed the tumor necrosis factor α (TNFα)-induced production of CYR-61 and CCL20. Nuclear levels of FoxO3a were decreased after TNFα stimulation of RASFs, and forced expression of FoxO3a reversed the inductive effects of TNFα on CYR-61. Simvastatin inhibited the nuclear export, phosphorylation, and acetylation of FoxO3a and maintained its binding to the Cyr61 promoter. Forced expression of SIRT-1 in RASFs led to decreased levels of CYR-61 and deacetylation of FoxO3a. Following treatment with simvastatin, the expression of SIRT-1 was up-regulated and SIRT-1/FoxO3a binding was enhanced in RASFs. In rats with CIA, intraarticular injection of simvastatin alleviated arthritis and suppressed CYR-61 expression and FoxO3a phosphorylation in synovial fibroblasts. CONCLUSION CYR-61 is important in the pathogenesis of RA, and SIRT-1/FoxO3a signaling is crucial to induction of CYR-61 in RASFs. Simvastatin plays a beneficial role in inflammatory arthritis through its up-regulation of SIRT-1/FoxO3a signaling in synovial fibroblasts. Continued study of the pathways linking sirtuins, FoxO proteins, and the inflammatory responses of RASFs may provide new insights into the pathophysiology of RA.

Denbinobin upregulates miR-146a expression and attenuates IL-1β-induced upregulation of ICAM-1 and VCAM-1 expressions in osteoarthritis fibroblast-like synoviocytes

Interleukin-1β (IL-1β) upregulates intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expressions in osteoarthritis fibroblast-like synoviocytes (OA-FLS) via nuclear factor (NF)-κB-mediated mechanism; enhancement of leukocyte infiltration and upregulation of proinflammatory mediators play a crucial role in OA pathophysiology. MicroRNA (miR)-146a suppresses inflammatory responses by inhibiting NF-κB activity and target gene expression, and epigenetic mechanisms are reportedly involved in miR expression regulation. Here, we aimed to verify the inhibition of ICAM-1/VCAM-1 expression in OA-FLS on denbinobin treatment and to determine whether this inhibition was due to the miR-146a-dependent pathway. We also assessed the epigenetic regulation caused by histone acetyltransferases involved in denbinobin action. Denbinobin attenuated the upregulation of IL-1β-induced ICAM-1/VCAM-1 expression and monocyte adhesion to OA-FLS. The mechanism underlying the inhibitory effects of denbinobin involved miR-146a induction, which in turn inhibited NF-κB signaling. This is because miR-146a inhibitor abrogated the inhibitory effects of denbinobin. Furthermore, histone acetyltransferase inhibitor attenuated the denbinobin-induced upregulation of miR-146a expression and inhibited the acetylation of NF-κB-binding sites located within the miR-146a promoter region. These data suggest that an epigenetic mechanism plays a crucial role in the upregulation of miR-146a expression in response to denbinobin treatment. Our overall findings suggest that denbinobin can be used as a potent anti-inflammatory agent.Key message• Denbinobin inhibited IL-1β-induced ICAM-1/VCAM-1 expression and monocyte adhesion to OA-FLS.• It was due to denbinobin increased miR-146a level, which in turn inhibited NF-κB signaling.• Our overall findings suggest that denbinobin can be used as a potent anti-inflammatory agent.

Development of biodegradable polyesterurethane membranes with different surface morphologies for the culture of osteoblasts

To evaluate the biocompatibility of biodegradable polyesterurethane membranes with different surface morphologies for their possible use as orthopedic biomaterials, rat osteoblasts were cultured on smooth, sunken, and particulate polyesterurethane membranes. A close interaction between cells and exposed particles on the particulate membranes was found. Cells on the particulate surfaces were well spread and flattened and had the greatest adhesion while cells on the smooth surfaces were more rounded, less spread, and less adhered. In addition, in order to investigate their in vivo degradation rates, the morphologic changes in retrieved membranes from 2, 4, and 8 weeks after subcutaneous implantation were observed by scanning electron microscopy and their average molecular weight changes were determined by gel permeation chromatography. These analyses showed that smooth membranes, compared with the two other surface membrane types, had the greatest rate and degree of molecular weight change. In contrast, the molecular weight of particulate membranes, which favor the osteoblast culture, had not changed significantly at 8 weeks postimplantation. Thus particulate polyesterurethane membrane surfaces may be of use as an orthopedic biomaterial, and polyesterurethane membranes certainly provide an ideal system for further study of the relative contributions to biocompatibility and degradation derived from surface morphology.

The phenotypic responses of human anterior cruciate ligament cells cultured on poly(ϵ‐caprolactone) and chitosan

The purpose of this study is to evaluate the phenotypic responses of human anterior cruciate ligament (ACL) cells on two biodegradable materials: poly(epsilon-caprolactone) (PCL) and chitosan. ACL cells cultured on PCL displayed phenotypes that were well spread with a developed cytoskeleton. In comparison, chitosan was not an appropriate substrate to support the attachment and spreading of ACL cells, which was attributed to the low fibronectin (FN) adsorption of chitosan. However, ACL cells cultured on chitosan exhibited a dramatic effect on increasing transcripts of transforming growth factor beta1 (TGF beta1) and collagen III. After coating FN on chitosan surface, cell morphology and the mRNA levels of all tested genes had the similar levels on PCL and FN-coated chitosan. This indicates the expression of TGF beta1 and collagen III mRNA of human ACL cells was seem to correlate closely with the adhesion behavior of human ACL cells and was influenced by the underlying substrate properties. Since an ideal scaffold used in ACL tissue engineering is not only for cell attachment but also for extracellular matrix deposition during ligament regeneration, chitosan may be considered as a scaffold for ACL tissue engineering, which can upregulate the expression of specific genes of matrix production and wound healing in human ACL cells to synthesize more quantity of FN and TGF beta1 proteins.

Characterization of the rheological behavior of UHMWPE gels using parallel plate rheometry

The effects of shear flow, temperature, and gel concentration on the rheological behavior of the ultrahigh-molecular-weight polyethylene (UHMWPE) gel in gel spinning process were investigated. The gel point was determined using parallel plate rheometry in rotation mode with controlled stress. Likewise, the flow curves at various temperatures were determined with controlled shear rate from 10−2 to 10 s−1. Whereas the shear storage modulus (G′) was obtained in oscillation mode with controlled strain from 1 to 100%. The result shows that the gel point of the UHMWPE gel increases with increasing gel concentration. The result from the strain sweep indicates that G′ of the gel is 1.5 × 103 Pa, and it exhibits a plateau at low strain, but it is reduced with increasing strain. At low shear rates, for temperatures above gel point, all flow curves exhibit a plateau, then go down with increasing shear rate. Studying contributions from UHMWPE gel concentration, temperature, and shear rate for rheological view, we found that spinning at 6% UHMWPE (MW : 1.4 × 106 g/mol) gel and 140°C gives the best effect on formation of fiber structure.