Col V siRNA Engineered Tenocytes for Tendon Tissue Engineering

The presence of uniformly small collagen fibrils in tendon repair is believed to play a major role in suboptimal tendon healing. Collagen V is significantly elevated in healing tendons and plays an important role in fibrillogenesis. The objective of this study was to investigate the effect of a particular chain of collagen V on the fibrillogenesis of Sprague-Dawley rat tenocytes, as well as the efficacy of Col V siRNA engineered tenocytes for tendon tissue engineering. RNA interference gene therapy and a scaffold free tissue engineered tendon model were employed. The results showed that scaffold free tissue engineered tendon had tissue-specific tendon structure. Down regulation of collagen V α1 or α2 chains by siRNAs (Col5α1 siRNA, Col5α2 siRNA) had different effects on collagen I and decorin gene expressions. Col5α1 siRNA treated tenocytes had smaller collagen fibrils with abnormal morphology; while those Col5α2 siRNA treated tenocytes had the same morphology as normal tenocytes. Furthermore, it was found that tendons formed by coculture of Col5α1 siRNA treated tenocytes with normal tenocytes at a proper ratio had larger collagen fibrils and relative normal contour. Conclusively, it was demonstrated that Col V siRNA engineered tenocytes improved tendon tissue regeneration. And an optimal level of collagen V is vital in regulating collagen fibrillogenesis. This may provide a basis for future development of novel cellular- and molecular biology-based therapeutics for tendon diseases

In vitro suppression of the MMP-3 gene in normal and cytokine-treated human chondrosarcoma using small interfering RNA

<p>Abstract</p> <p>Background</p> <p>Matrix metalloproteinase (MMPs) synthesized and secreted from connective tissue cells have been thought to participate in degradation of the extracellular matrix. Increased MMPs activities that degrade proteoglycans have been measured in osteoarthritis cartilage. This study aims to suppress the expression of the <it>MMP-3 </it>gene in <it>in vitro </it>human chondrosarcoma using siRNA.</p> <p>Methods</p> <p>Cells were categorized into four groups: control (G.1); transfection solution treated (G.2); negative control siRNA treated (G.3); and <it>MMP-3 </it>siRNA treated (G.4). All four groups were further subdivided into two groups - treated and non-treated with IL-1β- following culture for 48 and 72 h. We observed the effects of gene suppression according to cell morphology, glycosaminoglycan (GAG) and hyaluronan (HA) production, and gene expression by using real-time polymerase chain reaction (PCR).</p> <p>Results</p> <p>In IL-1β treated cells the apoptosis rate in G.4 was found to be lower than in all other groups, while viability and mitotic rate were higher than in all other groups (<it>p </it>< 0.05). The production of GAG and HA in G.4 was significantly higher than the control group (<it>p </it>< 0.05). <it>MMP-3 </it>gene expression was downregulated significantly (<it>p </it>< 0.05).</p> <p>Conclusion</p> <p><it>MMP-3 </it>specific siRNA can inhibit the expression of <it>MMP-3 </it>in chondrosarcoma. This suggests that <it>MMP-3 </it>siRNA has the potential to be a useful preventive and therapeutic agent for osteoarthritis.</p

Decreasing NF-κB Expression Enhances Odontoblastic Differentiation and Collagen Expression in Dental Pulp Stem Cells Exposed to Inflammatory Cytokines

Inflammatory response in the dental pulp can alter the collagen matrix formation by dental pulp stem cells and lead to a delay or poor healing of the pulp. This inflammatory response is mediated by cytokines, including interleukin-1β and tumor necrosis factor-α. In this study, it is hypothesized that suppressing the actions of these inflammatory cytokines by knocking down the activity of transcription factor Nuclear Factor–κB will lead to dental pulp stem cell differentiation into odontoblasts and the production of collagen. Here, the role of Nuclear Factor–κB signaling and its reduction was examined during odontogenic behavior in the presence of these cytokines. The results showed a significant increase in Nuclear Factor–κB gene expression and p65 protein expression by interleukin-1β and tumor necrosis factor-α. Nuclear Factor–κB activation in the presence of these cytokines decreased significantly in a dose-dependent manner by a Nuclear Factor–κB inhibitor (MG132) and p65 siRNA. Down-regulation of Nuclear Factor–κB activity also enhanced the gene expression of the odontoblastic markers (dentin sialophosphoprotein, Nestin, and alkaline phosphatase) and displayed an odontoblastic cell morphology indicating the promotion of odontogenic differentiation of dental pulp stem cells. Finally, dental pulp stem cells exposed to reduced Nuclear Factor–κB activity resulted in a significant increase in collagen (I)-α1 expression in the presence of these cytokines. In conclusion, a decrease in Nuclear Factor-κB in dental pulp stem cells in the presence of inflammatory cytokines enhanced odontoblastic differentiation and collagen matrix formation.The open access fee for this work was funded through the Texas A&M University Open Access to Knowledge (OAK) Fund

NF-kappa Bp65-specific siRNA inhibits expression of genes of COX-2, NOS-2 and MMP-9 in rat IL-1 beta-induced and TNF-alpha-induced chondrocytes

Objective: Small interfering RNA (siRNA) triggers RNA interference in mammalian somatic cells. Nuclear factor kappa B (NF-kappa B) is a transcription factor that is implicated in inflammation and immune activation. This study was to use NF-kappa Bp65-specific siRNA to inhibit the expression of genes of cyclooxygenase-2 (COX-2), nitric oxide synthase-2 (NOS-2) and matrix metalloproteinase-9 (MMP-9), which is paralleled with the initiation and progression of cartilage lesions in osteoarthritis (OA) model, in induced chondrocytes, and therefore to explore a new gene therapy for OA. Methods: Western blot and reverse transcriptase polymerase chain reaction (RT-PCR) were performed to optimize the silencing effects of NF-kappa Bp65-specific siRNA in cultured rat chondrocytes, and then to determine the expression of COX-2, NOS-2 and MMP-9 in induced chondrocytes. The activation of NF-kappa B was determined by electrophoretic mobility shift assay (EMSA). Western blot and RT-PCR were subjected to densitometric analysis and then band intensities were also determined. Results: The NF-kappa Bp65-specific siRNA inhibited the expression of NF-kappa Bp65 and activation of NF-kappa B, reducing significantly the expression of COX-2, NOS-2 and MMP-9 induced by interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) in cultured chondrocytes. Conclusions: NF-kappa Bp65-specific siRNA can inhibit the expression of COX-2, NOS-2 and MMP-9 in IL-1 beta-induced and TNF-alpha-induced chondrocytes. This suggests that NF-kappa Bp65-specific siRNA has potential to be a useful, preventive and therapeutic agent for OA at early stages. (c) 2005 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved.OrthopedicsRheumatologySCI(E)PubMed58ARTICLE4367-3761

Distinct Polysaccharide Utilization Determines Interspecies Competition between Intestinal Prevotella spp.

CD81 plays a role in a variety of physiological and pathological processes. Recent structural analysis of CD81 indicates that it contains an intramembrane cholesterol-binding pocket and that interaction with cholesterol may regulate a conformational switch in the extracellular domain of CD81. Therefore, CD81 possesses a potential cholesterol sensing mechanism; however, its relevance for protein function is thus far unknown. In this study we investigate CD81 cholesterol sensing in the context of its activity as a receptor for hepatitis C virus. Structure-led mutagenesis of the cholesterol-binding pocket reduced CD81-cholesterol association, but had disparate effects on HCV, both reducing and enhancing CD81 receptor activity. We reasoned that this could be explained by alterations in the consequences of cholesterol binding. To investigate this further we performed molecular dynamic simulations of CD81 with and without cholesterol; this identified an allosteric mechanism by which cholesterol binding regulates the conformation of CD81. To test this, we designed further mutations to force CD81 into either the open (cholesterol unbound) or closed (cholesterol bound) conformation. The open mutant of CD81 exhibited reduced receptor activity whereas the closed mutant was enhanced. These data are consistent with cholesterol switching CD81 between a receptor active and inactive state. CD81 interactome analysis also suggests that conformational switching may modulate the assembly of CD81-partner networks. This work furthers our understanding of the molecular mechanism of CD81 cholesterol sensing, how this relates to HCV entry and CD81's function as a molecular scaffold; these insights are relevant to CD81's varied roles in health and disease