Interactive effects of LPS and dentine matrix proteins on human dental pulp stem cells.

AIM: To investigate the combinatorial effects of lipopolysaccharide (LPS) and extracted dentine matrix proteins (eDMP) on regenerative and inflammatory responses in human dental pulp stem cells (DPSCs). METHODOLOGY: Culture media were supplemented with several concentrations of LPS, eDMP and combinations of both. Cell viability was assessed over 1 week by MTT assay; cell survival was evaluated after 24 h and 7 days by flow cytometry. The expression of mineralization-associated marker genes was determined by real-time quantitative polymerase chain reaction (RT-qPCR). To analyse the inflammatory response, secretion of interleukin 6 (IL-6) was quantified in the initial and the late phase of cell culture by enzyme-linked immunosorbent assay (ELISA). Data were treated nonparametrically and Mann-Whitney U-tests were performed to compare all experimental groups (α = 0.05). RESULTS: Whereas LPS had no impact on viability, eDMP led to a concentration-dependent decrease, which was significant after 7 days (P ≤ 0.024). A moderate decline of cell survival induced by LPS was detected after 48 h (P ≤ 0.026), whereas eDMP was able to reverse this effect. eDMP alone caused increased expression of tested marker genes, LPS had no regulatory effect. Combined eDMP and LPS induced an upregulation of collagen type I and osteocalcin, whereas expression levels of dentine matrix acidic phosphoprotein and dentine sialophosphoprotein were similar to the control. IL-6-secretion was increased by LPS over time. eDMP markedly elevated initial production of IL-6 (P ≤ 0.002), but suppressed LPS-induced cytokine production in the later phase. CONCLUSIONS: Lipopolysaccharide did not affect cell viability but interfered with odontoblast-like cell differentiation of DPSCs. Proteins from the dentine matrix may have a protective effect, attenuate the detrimental impact of LPS and thus play an important role during pulp repair.

Relationship between matrilin-1 gene polymorphisms and mandibular retrognathism.

INTRODUCTION: Mandibular retrognathism is a type of malocclusion that refers to an abnormal posterior position of the mandible as a result of a developmental abnormality. From the literature, it is evident that the mandibular growth pattern is determined by the intramembranous ossification of the mandibular body and endochondral ossification of the condyle. Matrilin-1 is a cartilage extracellular matrix protein, and matrilin-1 gene (MATN1) polymorphisms have been found to be involved in dental malocclusions of humans. In this study, we aimed to examine the association between MATN1 polymorphisms and the risk of mandibular retrognathism, in a case-control study with a South Indian population. METHODS: Eighty-one patients with mandibular retrognathism (SNB, <78°) and 71 controls having an orthognathic mandible (SNB, 80° ± 2°) were recruited. In both the patient and control groups, subjects with an orthognathic maxilla (SNA, 82° ± 2°) were included. Three single nucleotide polymorphisms of the MATN1 gene (rs1149048, rs1149042, and rs1065755) were genotyped using polymerase chain reaction-restriction fragment length polymorphism. The statistical association analysis was performed using the chi-square test. Pair-wise linkage disequilibrium was computed, and haplotypes were compared between subjects and controls. Nonparametric tests were used to compare cephalometric measurements between groups. RESULTS: No polymorphic site deviated from Hardy-Weinberg equilibrium in the controls. The rs1149042 genotypes and alleles were found to be associated with reduced risk of mandibular retrognathism. Furthermore, rs1149042 genotypes were associated with mandibular measurements (SNB and ANB). There was no strong and consistent linkage disequilibrium linkage disequilibrium across two different single nucleotide polymorphisms and haplotypes were not associated with mandibular retrognathism. CONCLUSIONS: The results of our study suggest an association between the MATN1 gene polymorphisms and mandibular retrognathism.

Investigations of Cartilage Matrix Degeneration in Patients with Early-Stage Femoral Head Necrosis.

BACKGROUND The purpose of this study was to explore changes in cartilage matrix in early-stage femoral head necrosis (FHN). MATERIAL AND METHODS Femoral head samples of patients with early FHN were collected during total hip arthroplasty (THA), high-field 7.0T MRI scans were performed in vitro, and the average T2 values were calculated. Cartilage samples were obtained from the weight-bearing area (FHN group) and non-weight-bearing area (Control group), divided into 3 equal parts and used for biochemical analysis, histopathological staining, and gene expression analysis. RESULTS T2 mapping of the femoral head specimens showed that the density distribution of cartilage surface was not uniform, and the average T2 value increased unevenly. Histological staining demonstrated that the number of chondrocytes was significantly decreased and they were irregularly arranged, SO staining was lost, and collagen fiber arrangement was slightly more irregular on the cartilage surface in the FHN group. The biochemical results in the FHN group showed that the water content increased significantly and the DNA content decreased significantly, while no significant changes in GAG and total collagen contents were detected. Gene expression analysis in the FHN group showed that SOX9 expression was significantly down-regulated, while COL10A1 and RUNX2 expressions were significantly up-regulated. The expression of ACAN and COL2A1 were decreased and COL1A1 was increased, but there was no significant difference compared with the Control group. CONCLUSIONS Taken together, the results of this study suggest that patients with early-stage FHN tend to have cartilage matrix degeneration, which provides new ideas for studying the pathogenesis of FHN and selecting treatment strategies.

Matrilin-3 Role in Cartilage Development and Osteoarthritis.

The extracellular matrix (ECM) of cartilage performs essential functions in differentiation and chondroprogenitor cell maintenance during development and regeneration. Here, we discuss the vital role of matrilin-3, an ECM protein involved in cartilage development and potential osteoarthritis pathomechanisms. As an adaptor protein, matrilin-3 binds to collagen IX to form a filamentous network around cells. Matrilin-3 is an essential component during cartilage development and ossification. In addition, it interacts directly or indirectly with transforming growth factor ß (TGF-ß), and bone morphogenetic protein 2 (BMP2) eventually regulates chondrocyte proliferation and hypertrophic differentiation. Interestingly, matrilin-3 increases interleukin receptor antagonists (IL-Ra) in chondrocytes, suggesting its role in the suppression of IL-1ß-mediated inflammatory action. Matrilin-3 downregulates the expression of matrix-degrading enzymes, such as a disintegrin metalloproteinase with thrombospondin motifs 4 (ADAMTS4) and ADAMTS5, matrix metalloproteinase 13 (MMP13), and collagen X, a hypertrophy marker during development and inflammatory conditions. Matrilin-3 essentially enhances collagen II and aggrecan expression, which are required to maintain the tensile strength and elasticity of cartilage, respectively. Interestingly, despite these attributes, matrilin-3 induces osteoarthritis-associated markers in chondrocytes in a concentration-dependent manner. Existing data provide insights into the critical role of matrilin-3 in inflammation, matrix degradation, and matrix formation in cartilage development and osteoarthritis.

MiR-202-5p/MATN2 are associated with regulatory T-cells differentiation and function in allergic rhinitis.

Regulatory T cells (Tregs) play a crucial role in allergic rhinitis (AR). However, the mechanism of how Tregs are regulated in AR is poorly understood. Here, we aimed to explore the role of Tregs in AR and how Tregs were regulated by miR-202-5P, which was demonstrated to be important in AR. Peripheral blood mononuclear cells (PBMC) were isolated from collected blood samples. Tregs were purified using Regulatory T Cell Isolation Kit, and differentiated from isolated CD4 T cells using recombinant human interleukin-2 (rhIL-2) and transforming growth factor beta (TGF-ß). mRNA expression levels of miR-202-5p, matrilin-2 (MATN2), TGF-ß1 and interleukin-10 (IL-10) were detected by real-time PCR. The concentrations of IL-4, interleukin-17 (IL-17), IL-10, interferon gamma (IFN-γ) and TGF-ß1 were detected by enzyme-linked immunosorbent assay (ELISA). MATN2 protein level was detected by Western blot. MiR-202-5p expression dramatically elevated in PBMCs, CD4+ T cells and Tregs of AR patients. In vitro, miR-202-5p promoted Tregs differentiation via targeting MATN2. MiR-202-5p/MATN2 axis mediated Tregs proliferation and functions. MiR-202-5p/MATN2 are associated with regulatory T-cells differentiation and function in allergic rhinitis.

Matrilins.

Marilins mediate interactions between macromolecular components of the extracellular matrix, e.g., collagens and proteoglycans. They are composed of von Willebrand factor type A and epidermal growth factor-like domains and the subunits oligomerize via coiled-coil domains. Matrilin-1 and -3 are abundant in hyaline cartilage, whereas matrilin-2 and -4 are widespread but less abundant. Mutations in matrilin genes have been linked to chondrodysplasias and osteoarthritis and recently characterization of matrilin-deficient mice revealed novel functions in mechanotransduction, regeneration, or inflammation. Due to their intrinsic adhesiveness and partially also low abundance, the study of matrilins is cumbersome. In this chapter, we describe methods for purification of matrilins from tissue, analysis of matrilins in tissue extracts, recombinant expression, and generation of matrilin-specific antibodies.

Inhibitory effects of EGb761 on the expression of matrix metalloproteinases (MMPs) and cartilage matrix destruction.

Cytokines such as tumor necrosis factor alpha (TNF-α)-induced expression of matrix metalloproteinase (MMP) play a pivotal role in the destruction of articular cartilage in patients who are suffering from osteoarthritis (OA). Collagen type II, the basis for articular cartilage, can be degraded by MMP-1, MMP-3, and 13. EGb761, the standardized extract of Ginkgo biloba produced by Dr. Willar Schwabe Pharmaceuticals, has shown its anti-inflammatory capacity. This study aimed to determine a mechanism whereby EGb761 may inhibit cartilage degradation. Our results indicated that pretreatment with EGb761 abolishes MMP-1, MMP-3, and MMP-13 gene expression and protein expression induced by TNF-α in human chondrocyte monolayer. In addition, the reduction of the tissue inhibitor of metalloproteinase-1(TIMP-1) and metalloproteinase-2 gene expression induced by TNF-α was rescued by pretreatment with EGb761. Importantly, TNF-α-induced degradation of collagen type II was ameliorated by EGb761 in a dose-dependent manner. Mechanistically, our results indicated that EGb761 treatment attenuated TNF-α-induced NF-κB activation. These actions of EGb761 suggest a mechanism by which EGb761 may act to prevent cartilage breakdown in arthritis.

Axonally derived matrilin-2 induces proinflammatory responses that exacerbate autoimmune neuroinflammation.

In patients with multiple sclerosis (MS) and mice with experimental autoimmune encephalomyelitis (EAE), inflammatory axonal injury is a major determinant of disability; however, the drivers of this injury are incompletely understood. Here, we used the EAE model and determined that the extracellular matrix protein matrilin-2 (MATN2) is an endogenous neuronal molecule that is regulated in association with inflammatory axonal injury. Compared with WT mice, mice harboring a deletion of Matn2 exhibited reduced disease severity and axon damage following induction of EAE. Evaluation of neuron-macrophage cocultures revealed that exogenous MATN2 specifically signals through TLR4 and directly induces expression of proinflammatory genes in macrophages, promoting axonal damage. Moreover, the MATN2-induced proinflammatory response was attenuated greatly in macrophages from Myd88 KO mice. Examination of brain sections from patients with MS revealed that MATN2 is expressed in lesions but not in normal-appearing white matter. Together, our results indicate that MATN2 is a deleterious endogenous neuroaxonal injury response signal that activates innate immune cells and could contribute to early axonal damage in CNS inflammatory diseases like MS.