Cyclic stretch induced epigenetic activation of periodontal ligament cells.

Periodontal ligament (PDL) cells play a crucial role in maintaining periodontal integrity and function by providing cell sources for ligament regeneration. While biophysical stimulation is known to regulate cell behaviors and functions, its impact on epigenetics of PDL cells has not yet been elucidated. Here, we aimed to investigate the cytoskeletal changes, epigenetic modifications, and lineage commitment of PDL cells following the application of stretch stimuli to PDL. PDL cells were subjected to stretching (0.1 Hz, 10 %). Subsequently, changes in focal adhesion, tubulin, and histone modification were observed. The survival ability in inflammatory conditions was also evaluated. Furthermore, using a rat hypo-occlusion model, we verified whether these phenomena are observed in vivo. Stretched PDL cells showed maximal histone 3 acetylation (H3Ace) at 2 h, aligning perpendicularly to the stretch direction. RNA sequencing revealed stretching altered gene sets related to mechanotransduction, histone modification, reactive oxygen species (ROS) metabolism, and differentiation. We further found that anchorage, cell elongation, and actin/microtubule acetylation were highly upregulated with mechanosensitive chromatin remodelers such as H3Ace and histone H3 trimethyl lysine 9 (H3K9me3) adopting euchromatin status. Inhibitor studies showed mechanotransduction-mediated chromatin modification alters PDL cells behaviors. Stretched PDL cells displayed enhanced survival against bacterial toxin (C12-HSL) or ROS (H2O2) attack. Furthermore, cyclic stretch priming enhanced the osteoclast and osteoblast differentiation potential of PDL cells, as evidenced by upregulation of lineage-specific genes. In vivo, PDL cells from normally loaded teeth displayed an elongated morphology and higher levels of H3Ace compared to PDL cells with hypo-occlusion, where mechanical stimulus is removed. Overall, these data strongly link external physical forces to subsequent mechanotransduction and epigenetic changes, impacting gene expression and multiple cellular behaviors, providing important implications in cell biology and tissue regeneration.

Materials and extracellular matrix rigidity highlighted in tissue damages and diseases: Implication for biomaterials design and therapeutic targets.

Rigidity (or stiffness) of materials and extracellular matrix has proven to be one of the most significant extracellular physicochemical cues that can control diverse cell behaviors, such as contractility, motility, and spreading, and the resultant pathophysiological phenomena. Many 2D materials engineered with tunable rigidity have enabled researchers to elucidate the roles of matrix biophysical cues in diverse cellular events, including migration, lineage specification, and mechanical memory. Moreover, the recent findings accumulated under 3D environments with viscoelastic and remodeling properties pointed to the importance of dynamically changing rigidity in cell fate control, tissue repair, and disease progression. Thus, here we aim to highlight the works related with material/matrix-rigidity-mediated cell and tissue behaviors, with a brief outlook into the studies on the effects of material/matrix rigidity on cell behaviors in 2D systems, further discussion of the events and considerations in tissue-mimicking 3D conditions, and then examination of the in vivo findings that concern material/matrix rigidity. The current discussion will help understand the material/matrix-rigidity-mediated biological phenomena and further leverage the concepts to find therapeutic targets and to design implantable materials for the treatment of damaged and diseased tissues.

A Vitronectin-Derived Peptide Restores Ovariectomy-Induced Bone Loss by Dual Regulation of Bone Remodeling.

BACKGROUND: Bone remodeling is tightly regulated through bone resorption and bone formation; imbalances in bone remodeling can cause various pathological conditions such as osteoporosis. Antiresorptive agents commonly used for treating osteoporosis do not substantially reverse osteoporotic bone loss. METHODS: We evaluated the effects of the RVYFFKGKQYWE motif (residues 270-281; VnP-16) of human vitronectin on the osteogenic differentiation of human mesenchymal stem cells (hMSCs) and osteoclastogenesis of bone marrow-derived macrophages. The effects of VnP-16 were also assessed in a mouse model of estrogen deficiency-induced osteoporosis (ovariectomized female C57BL/6 mice). To assay whether VnP-16 can reverse ovariectomy-induced bone loss, synthetic peptides or vehicle were subcutaneously injected into ovariectomized mice once a week for 4 weeks (n = 10/group). To evaluate the bone restorative effects of VnP-16, in-vivo micro-computed tomography analysis and histological staining were performed. RESULTS: VnP-16 induced osteogenic differentiation of hMSCs and inhibited the RANKL-RANK-TRAF6 axis in the osteoclastogenesis signaling pathway. Furthermore, systemic administration of VnP-16 reversed ovariectomy-induced bone loss in the femoral neck, distal femur and lumbar spine by increasing osteoblast differentiation and promoting bone formation, and concomitantly decreasing osteoclastogenesis and inhibiting bone resorption. The bone restorative effect of VnP-16 was observed one week after subcutaneous administration, and although the timing of the effect differed according to bone location, it persisted for at least 3 weeks. CONCLUSION: Our findings suggest that VnP-16 is a potential therapeutic agent for treating osteoporosis that mediates its effects through dual regulation of bone remodeling.

Erosion-Driven Enamel Crystallite Growth Phenomenon at the Tooth Surface In Vitro.

The erosion of tooth enamel is a common oral disease. The erosion pattern and location and the effects of nanoscale chemical composition on the erosion susceptibility of enamel have been well documented. However, the enamel remineralization accompanied by erosion and its underlying physicochemical mechanisms still remain poorly understood. Here, using rat molars selected for its good relevancy to human teeth, we investigated the remineralization behavior of the outermost enamel surface at the nanoscale level during erosion in diluted hydrochloric acid solutions. While particles on the outermost enamel surface that represent the termination of crystallites protruding to the surface from the near-surface core eroded by acid-attack, the lateral-growth of the particles (i.e., the main remineralization picture of the surface enamel) occurred concurrently. Ionic analyses indicate that the particle growth is driven by the local increase in pH near the eroding enamel surface as a result of the combination of the PO43- and CO32- released from the enamel surface with H+. As the pH increases eventually to the equilibrium pH level (∼5.5), a local supersaturation of solute ions is induced, resulting in particle growth. A simple growth model based on the experimental results together with an assumption that the particle growth is a diffusional process suggests that the particle growth rate is controlled by the degree of supersaturation and accommodation site for solute ions, which are affected by the pH of solution eroding the enamel surface. The remineralization mechanism presented by our study can explain how the enamel on being acid-exposed or tooth decay progress by beverage or food can naturally remineralize in the oral cavity and how remineralization can foster different surface topology at the nanoscale, depending on the pH value of etchant before the dental filling material is applied.

Dual actions of osteoclastic-inhibition and osteogenic-stimulation through strontium-releasing bioactive nanoscale cement imply biomaterial-enabled osteoporosis therapy.

Repair of defective hard-tissues in osteoporotic patients faces significantly challenges with limited therapeutic options. Although biomedical cements are considered promising materials for healthy bone repair, their uses for healing osteoporotic fracture are clinically limited. Herein, strontium-releasing-nanoscale cement was introduced to provide dual therapeutic-actions (pro-osteogenesis and anti-osteoclastogenesis), eventually for the regeneration of osteoporotic bone defect. The Sr-nanocement hardened from the Sr-doped nanoscale-glass particles was shown to release multiple ions including silicate, calcium and strontium at doses therapeutically relevant over time. When the Sr-nanocement was treated to pre-osteoblastic cells, the osteogenic mRNA level (Runx2, Opn, Bsp, Ocn), alkaline phosphatase activity, calcium deposition, and target luciferase reporter were stimulated with respect to the case with Sr-free-nanocement. When treated to pre-osteoclastic cells, the Sr-nanocement substantially reduced the osteoclastogenesis, such as osteoclastic mRNA level (Casr, Nfatc1, c-fos, Acp, Ctsk, Mmp-9), tartrate-resistant acid trap activity, and bone resorption capacity. In particular, the osteoclastic inhibition resulted in part from the interactive effect of osteoblasts which were activated by the Sr-nanocement, i.e., blockage of RANKL (receptor activator of nuclear factor-κB ligand) binding by enhanced osteoprotegerin and the deactivated Nfatc1. The Sr-nanocement, administered to an ovariectomized tibia defect (osteoporotic model) in rats, exhibited profound bone regenerative potential in cortical and surrounding trabecular area, including increased bone volume and density, enhanced production of osteopromotive proteins, and more populated osteoblasts, together with reduced signs of osteoclastic bone resorption. These results demonstrate that Sr-nanocement, with its dual effects of osteoclastic inhibition and osteogenic-stimulation, can be considered an effective nanotherapeutic implantable biomaterial platform for the treatment of osteoporotic bone defects.

The eggshell membrane: A potential biomaterial for corneal wound healing.

The eggshell membrane (ESM) is an abundant resource with innate complex structure and composition provided by nature. With at least 60 million tonnes of hen eggs produced globally per annum, utilisation of this waste resource is highly attractive in positively impacting sustainability worldwide. Given the morphology and mechanical properties of this membrane, it has great potential as a biomaterials for wound dressing. However, to date, no studies have demonstrated nor reported this application. As such, the objective of this investigation was to identify and optimise a reproducible extraction protocol of the ESM and to assess the physical, chemical, mechanical and biological properties of the substrate with a view to use as a wound dressing. ESM samples were isolated by either manual peeling (ESM-strip) or via extraction using acetic acid [ESM-A0.5] or ethylenediaminetetraacetic acid, EDTA [ESM-E0.9]. Energy dispersive X-ray spectroscopy (EDS) confirmed that there were no traces of calcium residues from the extraction process. Fourier transform infrared (FTIR) spectroscopy revealed that the extraction method (acetic acid and EDTA) did not alter the chemical structures of the ESM and also clarified the composition of the fibrous proteins of the ESM. Scanning electron microscopy (SEM) analyses revealed a three-layer composite structure of the ESM: an inner layer as continuous, dense and non-fibrous (limiting membrane), a middle layer with a network of fibres (inner shell membrane) and the outer layer (outer shell membrane) of larger fibres. Material properties including optical transparency, porosity, fluid absorption/uptake, thermal stability, mechanical profiling of the ESM samples were performed and demonstrated suitable profiles for translational applications. Biological in vitro studies using SV40 immortalised corneal epithelial cells (ihCEC) and corneal mesenchymal stromal cells (C-MSC) demonstrated excellent biocompatibility. Taken together, these results document the development of a novel sustainable biomaterial that may be used for ophthalmic wounds and/or other biomedical therapies.

Therapeutic tissue regenerative nanohybrids self-assembled from bioactive inorganic core / chitosan shell nanounits.

Natural inorganic/organic nanohybrids are a fascinating model in biomaterials design due to their ultra-microstructure and extraordinary properties. Here, we report unique-structured nanohybrids through self-assembly of biomedical inorganic/organic nanounits, composed of bioactive inorganic nanoparticle core (hydroxyapatite, bioactive glass, or mesoporous silica) and chitosan shell - namely Chit@IOC. The inorganic core thin-shelled with chitosan could constitute as high as 90%, strikingly contrasted with the conventional composites. The Chit@IOC nanohybrids were highly resilient under cyclic load and resisted external stress almost an order of magnitude effectively than the conventional composites. The nanohybrids, with the nano-roughened surface topography, could accelerate the cellular responses through stimulated integrin-mediated focal adhesions. The nanohybrids were also able to load multiple therapeutic molecules in the core and shell compartment and then release sequentially, demonstrating controlled delivery systems. The nanohybrids compartmentally-loaded with therapeutic molecules (dexamethasone, fibroblast growth factor 2, and phenamil) were shown to stimulate the anti-inflammatory, pro-angiogenic and osteogenic events of relevant cells. When implanted in the in vivo calvarium defect model with 3D-printed scaffold forms, the therapeutic nanohybrids were proven to accelerate new bone formation. Overall, the nanohybrids self-assembled from Chit@IOC nanounits, with their unique properties (ultrahigh inorganic content, nano-topography, high resilience, multiple-therapeutics delivery, and cellular activation), can be considered as promising 3D tissue regenerative platforms.

Nanotherapeutics for regeneration of degenerated tissue infected by bacteria through the multiple delivery of bioactive ions and growth factor with antibacterial/angiogenic and osteogenic/odontogenic capacity.

Therapeutic options are quite limited in clinics for the successful repair of infected/degenerated tissues. Although the prevalent treatment is the complete removal of the whole infected tissue, this leads to a loss of tissue function and serious complications. Herein the dental pulp infection, as one of the most common dental problems, was selected as a clinically relevant case to regenerate using a multifunctional nanotherapeutic approach. For this, a mesoporous bioactive glass nano-delivery system incorporating silicate, calcium, and copper as well as loading epidermal growth factor (EGF) was designed to provide antibacterial/pro-angiogenic and osteo/odontogenic multiple therapeutic effects. Amine-functionalized Cu-doped bioactive glass nanospheres (Cu-BGn) were prepared to be 50-60 nm in size, mesoporous, positive-charged and bone-bioactive. The Cu-BGn could release bioactive ions (copper, calcium and silicate ions) with therapeutically-effective doses. The Cu-BGn treatment to human umbilical vein endothelial cells (HUVEC) led to significant enhancement of the migration, tubule formation and expression of angiogenic gene (e.g. vascular endothelial growth factor, VEGF). Furthermore, the EGF-loaded Cu-BGn (EGF@Cu-BGn) showed pro-angiogenic effects with antibacterial activity against E. faecalis, a pathogen commonly involved in the pulp infection. Of note, under the co-culture condition of HUVEC with E. faecalis, the secretion of VEGF was up-regulated. In addition, the osteo/odontogenic stimulation of the EGF@Cu-BGn was evidenced with human dental pulp stem cells. The local administration of the EGF@Cu-BGn in a rat molar tooth defect infected with E. faecalis revealed significant in vivo regenerative capacity, highlighting the nanotherapeutic uses of the multifunctional nanoparticles for regenerating infected/damaged hard tissues.

Physical Properties and Biofunctionalities of Bioactive Root Canal Sealers In Vitro.

Calcium silicate-based bioactive glass has received significant attention for use in various biomedical applications due to its excellent bioactivity and biocompatibility. However, the bioactivity of calcium silicate nanoparticle-incorporated bioactive dental sealer is not much explored. Herein, three commercially available bioactive root canal sealers (Endoseal MTA (EDS), Well-Root ST (WST), and Nishika Canal Sealer BG (NBG)) were compared with a resin-based control sealer (AH Plus (AHP)) in terms of physical, chemical, and biological properties. EDS and NBG showed 200 to 400 nm and 100 to 200 nm nanoparticle incorporation in the SEM image, respectively, and WST and NBG showed mineral deposition in Hank's balanced salt solution after 28 days. The flowability and film thickness of all products met the ISO 3107 standard. Water contact angle, linear dimensional changes, and calcium and silicate ion release were significantly different among groups. All bioactive root canal sealers released calcium ions, while NBG released ~10 times more silicon ions than the other bioactive root canal sealers. Under the cytocompatible extraction range, NBG showed prominent cytocompatibility, osteogenecity, and angiogenecity compared to other sealers in vitro. These results indicate that calcium silicate nanoparticle incorporation in dental sealers could be a potential strategy for dental periapical tissue regeneration.

RNA interference in glial cells for nerve injury treatment.

Drivers of RNA interference are potent for manipulating gene and protein levels, which enable the restoration of dysregulated mRNA expression that is commonly associated with injuries and diseases. This review summarizes the potential of targeting neuroglial cells, using RNA interference, to treat nerve injuries sustained in the central nervous system. In addition, the various methods of delivering these RNA interference effectors will be discussed.

The laminin-211-derived PPFEGCIWN motif accelerates wound reepithelialization and increases phospho-FAK-Tyr397 and Rac1-GTP levels in a rat excisional wound splinting model.

We previously reported that the PPFEGCIWN motif (Ln2-LG3-P2-DN3), residues 2678-2686 of the human laminin α2 chain, promotes cell attachment of normal human epidermal keratinocytes (NHEKs) and dermal fibroblasts (NHDFs); however, its in vivo effects on cutaneous wound healing have not yet been examined. In this study, we sought to determine whether Ln2-LG3-P2-DN3 could promote full-thickness cutaneous wound healing by accelerating wound reepithelialization and wound closure in vivo. Ln2-LG3-P2-DN3 had significantly higher cell attachment and spreading activities than vehicle or scrambled peptide control in both NHEKs and NHDFs in vitro. The wound area was significantly smaller in rats treated with Ln2-LG3-P2-DN3 than in those treated with vehicle or scrambled peptide in the early phase of wound healing. Furthermore, Ln2-LG3-P2-DN3 significantly accelerated wound reepithelialization relative to vehicle or scrambled peptide and promoted FAK-Tyr397 phosphorylation and Rac1 activation. Collectively, our findings suggest that the PPFEGCIWN motif has potential as a therapeutic agent for cutaneous regeneration via the acceleration of wound reepithelization and wound closure.

Nano-graphene oxide/polyurethane nanofibers: mechanically flexible and myogenic stimulating matrix for skeletal tissue engineering.

For skeletal muscle engineering, scaffolds that can stimulate myogenic differentiation of cells while possessing suitable mechanical properties (e.g. flexibility) are required. In particular, the elastic property of scaffolds is of importance which helps to resist and support the dynamic conditions of muscle tissue environment. Here, we developed highly flexible nanocomposite nanofibrous scaffolds made of polycarbonate diol and isosorbide-based polyurethane and hydrophilic nano-graphene oxide added at concentrations up to 8%. The nano-graphene oxide incorporation increased the hydrophilicity, elasticity, and stress relaxation capacity of the polyurethane-derived nanofibrous scaffolds. When cultured with C2C12 cells, the polyurethane-nano-graphene oxide nanofibers enhanced the initial adhesion and spreading of cells and further the proliferation. Furthermore, the polyurethane-nano-graphene oxide scaffolds significantly up-regulated the myogenic mRNA levels and myosin heavy chain expression. Of note, the cells on the flexible polyurethane-nano-graphene oxide nanofibrous scaffolds could be mechanically stretched to experience dynamic tensional force. Under the dynamic force condition, the cells expressed significantly higher myogenic differentiation markers at both gene and protein levels and exhibited more aligned myotubular formation. The currently developed polyurethane-nano-graphene oxide nanofibrous scaffolds, due to their nanofibrous morphology and high mechanical flexibility, along with the stimulating capacity for myogenic differentiation, are considered to be a potential matrix for future skeletal muscle engineering.

A laminin-211-derived bioactive peptide promotes the osseointegration of a sandblasted, large-grit, acid-etched titanium implant.

Early implant loading is very important for reducing the duration of missing teeth in human patients. The laminin-derived peptide, DLTIDDSYWYRI motif (Ln2-P3), accelerates bone healing. Therefore, to investigate the hypothesis that Ln2-P3 increases the bone response to sandblasted, large-grit, acid-etched (SLA) titanium implants, the effect of the Ln2-P3 peptide on the osseointegration of SLA titanium implants was evaluated in vitro and in vivo. Human osteoblast-like cells were cultured on untreated, scrambled peptide (SP)-treated, and Ln2-P3-treated SLA titanium discs, and the cellular responses of these cells were evaluated. The Ln2-P3 treatment augmented osteoblast attachment and spreading, alkaline phosphatase activity, and the expression of osteogenic marker genes. Furthermore, the untreated and Ln2-P3-treated SLA titanium implants were inserted into the tibiae of rabbits for 9 and 11 days. Compared with the untreated implants, the Ln2-P3-treated implants showed a significantly higher bone-to-implant contact ratio at Day 9 after implantation and an increased bone area. The Ln2-P3 treatment of the SLA titanium implant surface augmented osteoblastic activity and accelerated peri-implant bone formation at the bone-implant interface. Overall, these results indicated that compared with the SLA titanium surface alone, the Ln2-P3 peptide-treated SLA titanium surface enhances initial osseointegration, thereby facilitating earlier implant loading.