Dicalcium Silicate Induced Proinflammatory Responses through TLR2-Mediated NF-κB and JNK Pathways in the Murine RAW 264.7 Macrophage Cell Line.

Proinflammatory responses are important aspects of the immune response to biomaterials, which may cause peri-implantitis and implant shedding. The purpose of this study was to test the cytotoxicity and proinflammatory effects of dicalcium silicate particles on RAW 264.7 macrophages and to investigate the proinflammatory response mechanism induced by C2S and tricalcium phosphate (TCP). C2S and TCP particles were characterized using scanning electron microscopy (SEM), energy spectrum analysis (EDS) and X-ray diffraction (XRD). Cytotoxicity and apoptosis assays with C2S and TCP in the murine RAW 264.7 cell line were tested using the cell counting kit-8 (CCK-8) assay and flow cytometry (FCM). The detection results showed that C2S and TCP particles had no obvious toxicity in RAW 264.7 cells and did not cause obvious apoptosis, although they both caused an oxidative stress response by producing ROS when the concentrations were at 100 µg/mL. C2S particles are likely to induce a proinflammatory response by inducing high TLR2, TNF-α mRNA, TNF-α proinflammatory cytokine, p-IκB, and p-JNK1 + JNK2 + JNK3 expression levels. When we added siRNA-TLR2-1, a significant reduction was observed. These findings support the theory that C2S particles induce proinflammatory responses through the TLR2-mediated NF-κB and JNK pathways in the murine RAW 264.7 macrophage cell line.

The intricate mechanism of PLS3 in bone homeostasis and disease.

Since our discovery in 2013 that genetic defects in PLS3 lead to bone fragility, the mechanistic details of this process have remained obscure. It has been established that PLS3 variants cause syndromic and nonsyndromic osteoporosis as well as osteoarthritis. PLS3 codes for an actin-bundling protein with a broad pattern of expression. As such, it is puzzling how PLS3 specifically leads to bone-related disease presentation. Our review aims to summarize the current state of knowledge regarding the function of PLS3 in the predominant cell types in the bone tissue, the osteocytes, osteoblasts and osteoclasts. This is related to the role of PLS3 in regulating mechanotransduction, calcium regulation, vesicle trafficking, cell differentiation and mineralization as part of the complex bone pathology presented by PLS3 defects. Considering the consequences of PLS3 defects on multiple aspects of bone tissue metabolism, our review motivates the study of its mechanism in bone diseases which can potentially help in the design of suitable therapy.

The complete chloroplast genome of Eastern Asian fern Bolbitis laxireticulata (Dryopteridaceae).

Bolbitis laxireticulata is a potential ornamental plant, which is restricted to eastern Asia. Here, we sequenced the complete chloroplast (cp) genome of B. laxireticulata and constructed a phylogenetic cp tree of Dryopteridaceae to study their relationships. The cp genome of B. laxireticulata is 153,093 bp in length, being made up of large single-copy (LSC, 83,169 bp), small single-copy (SSC, 21,538 bp), and a pair of region inverted repeats (IRs, 24,193 bp). It has 124 genes including 83 protein-coding genes, 33 tRNA genes, and eight rRNA genes. With the maximum-likelihood tree indicating, B. laxireticulata is more closely related to B. subcordata.

Human Salivary Histatin-1 Attenuates Osteoarthritis through Promoting M1/M2 Macrophage Transition.

Osteoarthritis (OA) is an inflammation-driven degenerative joint disease. Human salivary peptide histatin-1 (Hst1) shows pro-healing and immunomodulatory properties. but its role in OA treatment is not fully understood. In this study, we investigated the efficacy of Hst1 in the inflammation modulation-mediated attenuation of bone and cartilage damage in OA. Hst1 was intra-articularly injected into a rat knee joint in a monosodium iodoacetate (MIA)-induced OA model. Micro-CT, histological, and immunohistochemical analyses showed that Hst1 significantly attenuates cartilage and bone deconstruction as well as macrophage infiltration. In the lipopolysaccharide-induced air pouch model, Hst1 significantly reduced inflammatory cell infiltration and inflammation. Enzyme-linked immunosorbent assay (ELISA), RT-qPCR, Western blot, immunofluorescence staining, flow cytometry (FCM), metabolic energy analysis, and high-throughput gene sequencing showed that Hst1 significantly triggers M1-to-M2 macrophage phenotype switching, during which it significantly downregulated nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinases (MAPK) signaling pathways. Furthermore, cell migration assay, Alcian blue, Safranin O staining, RT-qPCR, Western blot, and FCM showed that Hst1 not only attenuates M1-macrophage-CM-induced apoptosis and matrix metalloproteinase expression in chondrogenic cells, but it also restores their metabolic activity, migration, and chondrogenic differentiation. These findings show the promising potential of Hst1 in treating OA.

Chondroinductive/chondroconductive peptides and their-functionalized biomaterials for cartilage tissue engineering.

The repair of articular cartilage defects is still challenging in the fields of orthopedics and maxillofacial surgery due to the avascular structure of articular cartilage and the limited regenerative capacity of mature chondrocytes. To provide viable treatment options, tremendous efforts have been made to develop various chondrogenically-functionalized biomaterials for cartilage tissue engineering. Peptides that are derived from and mimic the functions of chondroconductive cartilage extracellular matrix and chondroinductive growth factors, represent a unique group of bioactive agents for chondrogenic functionalization. Since they can be chemically synthesized, peptides bear better reproducibility, more stable efficacy, higher modifiability and yielding efficiency in comparison with naturally derived biomaterials and recombinant growth factors. In this review, we summarize the current knowledge in the designs of the chondroinductive/chondroconductive peptides, the underlying molecular mechanisms and their-functionalized biomaterials for cartilage tissue engineering. We also systematically compare their in-vitro and in-vivo efficacies in inducing chondrogenesis. Our vision is to stimulate the development of novel peptides and their-functionalized biomaterials for cartilage tissue engineering.

Incorporation of NGR1 promotes bone regeneration of injectable HA/nHAp hydrogels by anti-inflammation regulation via a MAPK/ERK signaling pathway.

Suitable bone grafts are commonly required to achieve successful bone regeneration, wherein much effort has been spent to optimize their osteogenesis. Increasing evidence has demonstrated that reducing the levels of TNF-α can enhance bone regeneration at the injury site. Notoginsenoside R1 (NGR1) has been extensively studied in the field of anti-inflammation and regenerative medicine. Nanosized hydroxyapatite (nHAp) possesses excellent biocompatibility and osteoconductivity. In this study, we fabricated a thermoresponsive, injectable hyaluronic acid/nHAp (HA/nHAp) composite hydrogel incorporated with NGR1 to promote bone regeneration. Furthermore, NGR1-HA/nHAp hydrogel could enhance bone regeneration than those of HA and HA/nHAp hydrogels, profited by the underlying osteoblastic mechanism that NGR1 could facilitate activation of the MAPK/ERK signaling pathway and down-regulate the expression of TNF-α, ultimately upregulated expression of osteogenic genes. In summary, the NGR1-HA/nHAp composite hydrogel with controlled inflammation, and excellent osteogenic effect, will have great potential for use in bone regeneration applications.

Dicalcium silicate-induced mitochondrial dysfunction and autophagy-mediated macrophagic inflammation promotes osteogenic differentiation of BMSCs.

Dicalcium silicate (Ca2SiO4, C2S) has osteogenic potential but induces macrophagic inflammation. Mitochondrial function plays a vital role in macrophage polarization and macrophagic inflammation. The mitochondrial function of C2S-treated macrophages is still unclear. This study hypothesized: (i) the C2S modulates mitochondrial function and autophagy in macrophages to regulate macrophagic inflammation, and (ii) C2S-induced macrophagic inflammation regulates osteogenesis. We used RAW264.7 cells as a model of macrophage. The C2S (75-150 µg/ml) extract was used to analyze the macrophagic mitochondrial function and macrophage-mediated effect on osteogenic differentiation of mouse bone marrow-derived mesenchymal stem cells (BMSCs). The results showed that C2S extract (150 µg/ml) induced TNF-α, IL-1ß and IL-6 production in macrophages. C2S extract (150 µg/ml) enhanced reactive oxygen species level and intracellular calcium level but reduced mitochondrial membrane potential and ATP production. TEM images showed reduced mitochondrial abundance and altered the mitochondrial morphology in C2S (150 µg/ml)-treated macrophages. Protein level expression of PINK1, Parkin, Beclin1 and LC3 was upregulated but TOMM20 was downregulated. mRNA sequencing and KEGG analysis showed that C2S-induced differentially expressed mRNAs in macrophages were mainly distributed in the essential signaling pathways involved in mitochondrial function and autophagy. The conditioned medium from C2S-treated macrophage robustly promoted osteogenic differentiation in BMSCs. In conclusion, our results indicate mitochondrial dysfunction and autophagy as the possible mechanism of C2S-induced macrophagic inflammation. The promotion of osteogenic differentiation of BMSCs by the C2S-induced macrophagic inflammation suggests the potential application of C2S in developing immunomodulatory bone grafts.

Downregulation of Macrophage-Specific Act-1 Intensifies Periodontitis and Alveolar Bone Loss Possibly via TNF/NF-κB Signaling.

Periodontitis is a chronic inflammatory oral disease that affects almost half of the adult population. NF-κB activator 1 (Act1) is mainly expressed in immune cells, including macrophages, and modulates immune cells' function to regulate inflammation in inflammatory diseases. Macrophages play a vital role in the pathophysiology of periodontitis. However, the effect of macrophage-specific Act1 on periodontitis has not been investigated yet. This study aims to unravel the role of macrophage-specific Act1 on the pathophysiology of periodontitis. The expression of Act1 in healthy and periodontitis periodontal tissue was confirmed by immunohistochemistry. Macrophage-specific Act1 expression downregulated (anti-Act1) mice were developed by inserting anti-Act1 antisense oligonucleotides after the CD68 promoter of C57BL/6 mice. Ligature-induced periodontitis (LIP) was induced in anti-Act1 mice and wildtype mice. Micro-CT, histology, and TRAP staining analyzed the periodontal tissue status, alveolar bone loss, and osteoclast numbers. Immunohistochemistry, RT-qPCR, and ELISA analyzed the inflammatory cells infiltration, expression of inflammatory cytokines, and M1/M2 macrophage polarization. mRNA sequencing of in vitro bacterial lipopolysaccharide (LPS)-treated peritoneal macrophages analyzed the differentially expressed genes in anti-Act1 mice during inflammation. Anti-Act1 mice showed aggravated periodontitis and alveolar bone loss compared to wildtype. Periodontitis-affected periodontal tissue (PAPT) of anti-Act1 mice showed a higher degree of macrophage infiltration, and M1 macrophage polarization compared to wildtype. Levels of pro-inflammatory cytokines (IL-1ß, IL-6, and TNFα), and macrophage activity-related factors (CCL2, CCL3, and CCL4) were robustly high in PAPT of anti-Act1 mice compared to wildtype. mRNA sequencing and KEGG analysis showed activated TNF/NF-κB signaling in LPS-treated macrophages from anti-Act1 mice. In vitro studies on LPS-treated peritoneal macrophages from anti-act1 mice showed a higher degree of cell migration and expression of inflammatory cytokines, macrophage activity-related factors, M1 macrophage-related factors, and TNF/NF-κB signaling related P-p65 protein. In conclusion, downregulation of macrophage-specific Act1 aggravated periodontitis, alveolar bone loss, macrophage infiltration, inflammation, and M1 macrophage polarization. Furthermore, LPS-treated macrophages from anti-Act1 mice activated TNF/NF-κB signaling. These results indicate the distinct role of macrophage-specific Act1 on the pathophysiology of periodontitis possibly via TNF/NF-κB signaling.

LncRNA H19 promotes odontoblastic differentiation of human dental pulp stem cells by regulating miR-140-5p and BMP-2/FGF9.

BACKGROUND: Increasing evidence has revealed that long non-coding RNAs (lncRNAs) exert critical roles in biological mineralization. As a critical process for dentin formation, odontoblastic differentiation is regulated by complex signaling networks. The present study aimed to investigate the biological role and regulatory mechanisms of lncRNA-H19 (H19) in regulating the odontoblastic differentiation of human dental pulp stem cells (hDPSCs). METHODS: We performed lncRNA microarray assay to reveal the expression patterns of lncRNAs involved in odontoblastic differentiation. H19 was identified and verified as a critical factor by qRT-PCR. The gain- and loss-of-function studies were performed to investigate the biological role of H19 in regulating odontoblastic differentiation of hDPSCs in vitro and in vivo. Odontoblastic differentiation was evaluated through qRT-PCR, Western blot, and Alizarin Red S staining. Bioinformatics analysis identified that H19 could directly interact with miR-140-5p, which was further verified by luciferase reporter assay. After overexpression of miR-140-5p in hDPSCs, odontoblastic differentiation was determined. Moreover, the potential target genes of miR-140-5p were investigated and the biological functions of BMP-2 and FGF9 in hDPSCs were verified. Co-transfection experiments were conducted to validate miR-140-5p was involved in H19-mediated odontoblastic differentiation in hDPSCs. RESULTS: The expression of H19 was significantly upregulated in hDPSCs undergoing odontoblastic differentiation. Overexpression of H19 stimulated odontoblastic differentiation in vitro and in vivo, whereas downregulation of H19 revealed the opposite effect. H19 binds directly to miR-140-5p and overexpression of miR-140-5p inhibited odontoblastic differentiation of hDPSCs. H19 acted as a miR-140-5p sponge, resulting in regulated the expression of BMP-2 and FGF9. Overexpression of H19 abrogated the inhibitory effect of miR-140-5p on odontoblastic differentiation. CONCLUSION: Our data revealed that H19 plays a positive regulatory role in odontoblastic differentiation of hDPSCs through miR-140-5p/BMP-2/FGF9 axis, suggesting that H19 may be a stimulatory regulator of odontogenesis.

Dicalcium silicate microparticles modulate the differential expression of circRNAs and mRNAs in BMSCs and promote osteogenesis via circ_1983-miR-6931-Gas7 interaction.

Dicalcium silicate microparticle (C2S)-based biomaterials have a potential for bone and dental tissue regenerative applications. The C2S-mediated transcriptome level mechanism in mesenchymal stem cells (MSCs) during bone-defect healing has not been investigated yet. In this study, we elucidated the differential expression pattern of messenger RNAs (mRNAs) and circular RNAs (circRNAs) in C2S-treated MSCs and their involvement in the osteogenesis process. C2S robustly enhanced the osteogenic differentiation of MSCs and cranial bone defect healing. C2S-treatment modulated the differential expression of mRNAs and circRNAs in MSCs. Differentially expressed circRNAs and mRNAs were involved in competing endogenous RNA (ceRNA-interaction networks). These ceRNA-interaction networks regulated the signaling pathways associated with osteogenesis, e.g., Wnt, PI3K-Akt, MAPK, and JAK/STAT signaling. C2S-treatment upregulated the expression of circ_1983, Gas7, and Runx2 in BMSCs. RNase R and luciferase activity assay confirmed the stability and miR-6931 sponging property of circ_1983, respectively. Knockdown of circ_1983 enhanced miR-6931 expression but inhibited Gas7 and Runx2 expression and osteogenic differentiation in C2S-treated MSCs. In conclusion, for the first time, we report the role of cicr_1983-miR-6931-Gas7 ceRNA-interaction in C2S-induced osteogenic differentiation of MSCs and bone defect healing. This study opens a new research stream "the role of circRNAs-mediated ceRNA-interaction in biomaterials and stem cell-based bone tissue engineering".