Nanoscale osseointegration of zirconia evaluated from the interfacial structure between ceria-stabilized tetragonal zirconia and cell-induced hydroxyapatite.

BACKGROUND: The osseointegration of zirconia implants has been evaluated based on their implant fixture bonding with the alveolar bone at the optical microscopic level. Achieving nano-level bonding between zirconia and bone apatite is crucial for superior osseointegration; however, only a few studies have investigated nanoscale bonding. This review outlines zirconia osseointegration, including surface modification, and presents an evaluation of nanoscale zirconia-apatite bonding and its structure. HIGHLIGHT: Assuming osseointegration, the cells produced calcium salts on a ceria-stabilized zirconia substrate. We analyzed the interface between calcium salts and zirconia substrates using transmission electron microscopy and found that 1) the cell-induced calcium salts were bone-like apatite and 2) direct nanoscale bonding was observed between the bone-like apatite and zirconia crystals without any special modifications of the zirconia surface. CONCLUSION: Structural affinity exists between bone apatite and zirconia crystals. Apatite formation can be induced by the zirconia surface. Zirconia bonds directly with apatite, indicating superior osseointegration in vivo.

Hyaluronan and chondroitin sulfate in chicken-vegetable bone broth delay osteoporosis progression.

Bone broth has recently gained worldwide recognition as a superfood that supplements several nutrients lacking in modern human diets; however, little is known of its efficacy on osteoporosis. Therefore, we aimed to identify the components of chicken-vegetable bone broth (CVBB) that are associated with osteoporosis prevention and verified the efficacy of these components using in vivo studies. In biochemical and cell biological experiments, CVBB was fractionated using ion exchange chromatography (IEC), and the effect of each IEC fraction on osteoclast differentiation was evaluated based on tartrate-resistant acid phosphatase (TRAP) activity, TRAP staining, and quantitative polymerase chain reaction analysis using mouse macrophage-like cells (RAW264 cell). In animal experiments, an ovariectomized (OVX) rat model was generated, followed by whole bone broth (OVX/CVBB) or IEC fraction (OVX/CVBB-Ext) administration and bone structural parameter characterization of OVX rat tibia based on micro-CT. Four CVBB fractions were obtained using IEC, and the fraction containing both hyaluronan and chondroitin sulfate (CVBB-Ext) led to the maximum inhibition of RAW264 cell differentiation. CVBB-Ext downregulated the expression of osteoclast differentiation marker genes. In animal experiments, the OVX group showed a clear decrease in bone density compared to that in the Sham operation group. The OVX/CVBB and OVX/CVBB-Ext groups showed increased bone mineral density and bone volume/tissue volume values compared to those in the OVX/control group. These results suggested that CVBB and CVBB-Ext slowed osteoporosis progression. Therefore, we conclude that hyaluronan and chondroitin sulfate in CVBB are key substances that impede osteoporosis progression. PRACTICAL APPLICATION: This study provides practical information on the effects of bone broth ingredients on osteoporosis to expand the current knowledge on the efficacy of bone broth, which is a widely consumed food. These results may help in the future development of bone broth as a dietary supplement for managing osteoporosis.

Synergistic effect of FGF-2 and TGF-ß1 on the mineralization of human umbilical cord perivascular cells.

OBJECTIVE: Human umbilical cord perivascular cells (HUCPVCs) are derived from the human umbilical cord perivascular tissue and are expected to replace mesenchymal stromal cells in the future. We investigated the synergistic effects of fibroblast growth factor 2 (FGF-2) and transforming growth factor-beta 1 (TGF-ß1) on HUCPVC mineralization. DESIGN: We prepared HUCPVCs with (FGF(+)HUCPVCs) or without FGF-2 (FGF(-)HUCPVCs) in the presence of activated vitamin D3, a bone morphogenic protein inhibitor, and TGF-ß1. We examined the cell proliferative capacity, expression of various hard tissue-forming cell gene markers, and mineralization induction ability and identified the crystalline phases of the mineralized nodules. RESULTS: FGF(+)HUCPVCs exhibited higher intracellular alkaline phosphatase (ALP) gene expression and ALP activity, and their cell proliferation rate was higher than that of FGF(-)HUCPVCs. The expression levels of osteoblast marker genes increased in FGF(+)HUCPVCs, whereas those of elastic fiber and muscle cell markers increased in FGF(-)HUCPVCs. The expression of genes related to matrix vesicle-mediated mineralization was increased in FGF(+)HUCPVCs. While FGF(-)HUCPVCs displayed myofibroblast-like properties and could not induce mineralization, FGF(+)HUCPVCs demonstrated the ability to produce mineralized nodules. The resulting mineralized nodules consisted of hydroxyapatite as the major phase and minor amounts of octacalcium phosphate. The mineralized nodules exhibited the morphological characteristics of bone hydroxyapatite, composed of fibrous hydroxyapatite nanorods and polycrystalline sheets. CONCLUSION: We found that FGF-2 synergizes with TGF-ß1 and is a key factor in the differentiation of HUCPVCs into osteoblast-like cells. Thus, HUCPVCs can potentially serve as a new stem cell source for future bone regeneration and dental treatments.

Cementum is key to periodontal tissue regeneration: A review on apatite microstructures for creation of novel cementum-based dental implants.

The cementum is the outermost layer of hard tissue covering the dentin within the root portion of the teeth. It is the only hard tissue with a specialized structure and function that forms a part of both the teeth and periodontal tissue. As such, cementum is believed to be critical for periodontal tissue regeneration. In this review, we discuss the function and histological structure of the cementum to promote crystal engineering with a biochemical approach in cementum regenerative medicine. We review the microstructure of enamel and bone while discussing the mechanism underlying apatite crystal formation to infer the morphology of cementum apatite crystals and their complex structure with collagen fibers. Finally, the limitations of the current dental implant treatments in clinical practice are explored from the perspective of periodontal tissue regeneration. We anticipate the possibility of advancing periodontal tissue regenerative medicine via cementum regeneration using a combination of material science and biochemical methods.

Characterization of bioactive substances involved in the induction of bone augmentation using demineralized bone sheets.

PURPOSE: To investigate the bone augmentation ability of demineralized bone sheets mixed with allogeneic bone with protein fractions containing bioactive substances and the interaction between coexisting bioactive substances and proteins. METHODS: Four types of demineralized bone sheets mixed with allogeneic bone in the presence or absence of bone proteins were created. Transplantation experiments using each demineralized bone sheet were performed in rats, and their ability to induce bone augmentation was analysed by microcomputed tomography images. Bioactive substances in bone proteins were isolated by heparin affinity chromatography and detected by the measurement of alkaline phosphatase activity in human periodontal ligament cells and dual luciferase assays. Noncollagenous proteins (NCPs) coexisting with the bioactive substances were identified by mass spectrometry, and their interaction with bioactive substances was investigated by in vitro binding experiments. RESULTS: Demineralized bone sheets containing bone proteins possessed the ability to induce bone augmentation. Bone proteins were isolated into five fractions by heparin affinity chromatography, and transforming growth factor-beta (TGF-ß) was detected in the third fraction (Hep-c). Dentin matrix protein 1 (DMP1), matrix extracellular phosphoglycoprotein (MEPE), and biglycan (BGN) also coexisted in Hep-c, and the binding of these proteins to TGF-ß increased TGF-ß activity by approximately 14.7% to 32.7%. CONCLUSIONS: Demineralized bone sheets are capable of inducing bone augmentation, and this ability is mainly due to TGF-ß in the bone protein mixed with the sheets. The activity of TGF-ß is maintained when binding to bone NCPs such as DMP1, MEPE, and BGN in the sheets.

Response of TGF-ß isoforms in epithelial-mesenchymal transition of enamel epithelial cells.

OBJECTIVE: During enamel formation, transforming growth factor-beta (TGF-ß) isoforms exhibit different activities for gene expression, apoptosis, and endocytosis. This study aimed to investigate the differential response of TGF-ß isoforms to epithelial-mesenchymal transition (EMT) in enamel epithelial cells. DESIGN: Using a mouse enamel epithelial cell line (mHAT9d) cultured in the presence of each TGF-ß isoform, (1) the morphological changes in EMT were explored, (2) EMT-related genes were analyzed by next-generation sequencing (NGS), (3) TGF-ß pathway for EMT was identified by inhibition experiments, and (4) the expression of the TGF-ß receptor gene in response to the binding affinity of the TGF-ß isoform were analyzed. RESULTS: EMT was observed in mHAT9d cultured in the presence of TGF-ß1 and ß3 but not TGF-ß2. The expression of both epithelial and mesenchymal marker genes was observed in mHAT9d exhibiting EMT. NGS analysis suggested extracellular signal-regulated kinase (ERK) and Rho pathways as TGF-ß signaling pathways associated with EMT. However, EMT in mHAT9d cultured in the presence of TGF-ß1 or ß3 occurred even in presence of an ERK1/2 inhibitor and was suppressed by Rho-kinase inhibitor. The expression of co-receptors for TGF-ß signaling in mHAT9d cells reduced following stimulation with each TGF-ß isoform. In contrast, endoglin levels increased following TGF-ß1 or ß3 stimulation, but no change was noted in response to TGF-ß2. CONCLUSIONS: We propose that in TGF-ß-stimulated enamel epithelial cells, EMT mainly occurred via the Rho signaling pathway, and the differences in response across TGF-ß isoforms were due to their endoglin-mediated binding affinity for the TGF-ß receptor.

Repurposing MDZ as a tool for tissue regeneration in dental cells.

BACKGROUND: Several recent studies have focused on the utility of drug repurposing to expand clinical application of approved therapeutics. Here, we investigate the efficacy of midazolam (MDZ) and cytokines for regenerating calcified tissue, using immortalized porcine dental pulp (PPU7) and mouse skeletal muscle derived myoblast (C2C12) cells, with the goal of repurposing MDZ as a new treatment to facilitate calcified tissue regeneration. HIGHLIGHTS: We noted that PPU7 and C2C12 cells cultured with various MDZ regimens displayed increased bone morphogenic protein (BMP-2), transforming growth factor beta (TGF-ß), and alkaline phosphatase activity. These increases were highest in PPU7 cells cultured with MDZ alone, and in C2C12 cells cultured with MDZ and BMP-2. PPU7 cells cultured under these conditions demonstrated markedly elevated expression of odontoblastic gene markers, indicating their likely differentiation into odontoblasts. Expression levels of osteoblastic gene markers also increased in C2C12 cells, suggesting that MDZ potentiates the effect of BMP-2, inducing osteoblast differentiation in these cells. Newly formed calcified deposits in both PPU7 and C2C12 cells were identified as hydroxyapatite via crystallographic and crystal engineering analyses. CONCLUSION: MDZ increases ALP activity, inducing expression of specific marker genes for both odontoblasts and osteoblasts while promoting hydroxyapatite production in both PPU7 and C2C12 cells. These responses were cell type specific. MDZ treatment alone could induce these changes in PPU7 cells, but C2C12 cell differentiation required BMP-2 addition.

Development and Characterization of Alkaline Phosphatase-Positive Human Umbilical Cord Perivascular Cells.

Human umbilical cord perivascular cells (HUCPVCs), harvested from human umbilical cord perivascular tissue, show potential for future use as an alternative to mesenchymal stromal cells. Here, we present the results for the characterization of the properties alkaline phosphatase-positive HUCPVCs (ALP(+)-HUCPVCs). These ALP(+)-HUCPVCs were created from HUCPVCs in this study by culturing in the presence of activated vitamin D3, an inhibitor of bone morphogenetic protein signaling and transforming growth factor-beta1 (TGF-ß1). The morphological characteristics, cell proliferation, gene expression, and mineralization-inducing ability of ALP(+)-HUCPVCs were investigated at the morphological, biological, and genetic levels. ALP(+)-HUCPVCs possess high ALP gene expression and activity in cells and a slow rate of cell growth. The morphology of ALP(+)-HUCPVCs is fibroblast-like, with an increase in actin filaments containing alpha-smooth muscle actin. In addition to ALP expression, the gene expression levels of type I collagen, osteopontin, elastin, fibrillin-1, and cluster of differentiation 90 are increased in ALP(+)-HUCPVCs. ALP(+)-HUCPVCs do not have the ability to induce mineralization nodules, which may be due to the restriction of phosphate uptake into matrix vesicles. Moreover, ALP(+)-HUCPVCs may produce anti-mineralization substances. We conclude that ALP(+)-HUCPVCs induced from HUCPVCs by a TGF-ß1 stimulation possess myofibroblast-like properties that have little mineralization-inducing ability.

A genetic model for the secretory stage of dental enamel formation.

The revolution in genetics has rapidly increased our knowledge of human and mouse genes that are critical for the formation of dental enamel and helps us understand how enamel evolved. In this graphical review we focus on the roles of 41 genes that are essential for the secretory stage of amelogenesis when characteristic enamel mineral ribbons initiate on dentin and elongate to expand the enamel layer to the future surface of the tooth. Based upon ultrastructural analyses of genetically modified mice, we propose a molecular model explaining how a cell attachment apparatus including collagen 17, α6ß4 and αvß6 integrins, laminin 332, and secreted enamel proteins could attach to individual enamel mineral ribbons and mold their cross-sectional dimensions as they simultaneously elongate and orient them in the direction of the retrograde movement of the ameloblast membrane.

Coherent surface structure induces unique epitaxial overgrowth of metastable octacalcium phosphate on stable hydroxyapatite at critical fluoride concentration.

The phase transformation from soluble calcium phosphates to less-soluble hydroxyapatite (HAP) is a thermodynamically natural route. This process is irreversible, and effective use of poorly reactive HAP to repair teeth that have no cellular metabolism remains challenging. However, this thermodynamically controlled transformation may apparently be reversed through the fast nucleation and growth of metastable phases, leading to a reactive HAP surface. Here, the assembled HAP-nanorod phase is demonstrated to change into the metastable octacalcium phosphate (OCP) phase in a calcium phosphate solution containing 0.8 ppm fluoride. Grown OCPs display parallel surface streaks and their 11¯0 and 00l (l: odd) electron-diffraction spots are often not visible. The streaked, elongated OCP gradually grows into large plates with flat surfaces that exhibit an intense11¯0 spot. Crystal-structure models reveal that the unique epitaxial overgrowth of OCP on HAP occurs since both materials share coherent {100} faces, resulting in the distinctive disappearance of 11¯0 and 00l OCP spots. A polysynthetic twin model that reliably explains this disappearance is proposed for the growth of OCP. This apparent reverse phase transformation produces hybrid calcium phosphates consisting of HAP cores and highly reactive outer OCP layers that are promising for the repair of dentin caries. STATEMENT OF SIGNIFICANCE: This paper demonstrates important and interesting finding regarding formation of calcium phosphates in relation to their crystal structures. We first show that hydroxyapatite (HAP), the major constituent of human teeth and bone, can reversely change to its precursor, octacalcium phosphate (OCP), contrary to thermodynamic-stability rule. This apparent reverse phase transformation occurs through sharing the coherent {100} faces of both materials under controlled fluoride concentration. Nanoscale similarity of two crystal surfaces enables structurally shared epitaxial overgrowth of OCP on HAP aided by faster growth rate of OCP than that of HAP. This reaction produces hybrid crystal consisting of outer OCP and core HAP, that has not been known before and is able to be applied to dentin caries repair.

New insights into bioactivity of ceria-stabilized zirconia: Direct bonding to bone-like hydroxyapatite at nanoscale.

Osseointegration resulting from biomineralization means tight bone-implant attachment, which is clinically essential for successful dental implant treatment. The osseointegration ability of ceria-stabilized zirconia, a promising implant material, has been questionable and is unclear despite its clinical use due to zirconia's bioinert nature. The purpose of this research was to investigate the osseointegration ability of ceria-stabilized zirconia by clarifying its bioactivity. Here we show that ceria-stabilized zirconia is highly bioactive, contrary to the general consensus. Transmission electron microscopy observation revealed that the zirconia nanocrystals of a ceria-stabilized zirconia substrate directly bonded to osteoblastic cell-precipitated hydroxyapatite crystals at lattice fringe scale. This bonding was achieved without chemical treatment of the substrate surface before use. Hydroxyapatite crystals exhibited a morphology of flexible nanofibers less than 10 nm wide with nanometer-thick plates filling the spaces between nanofibers. Elemental analysis of the hydroxyapatites showed that they contained alkaline metal cations (Na, Mg, and K) as minor elements and that their average Ca/P atomic % ratio was ~1.40, similar to those of bone apatite. High bioactivity of ceria-stabilized zirconia resulted in direct bonding to bone-like hydroxyapatite, suggesting nanoscale direct osseointegration with bone in vivo that contributes to improving the success rate of dental implant treatment.

Characterization of Living Dental Pulp Cells in Direct Contact with Mineral Trioxide Aggregate.

Mineral trioxide aggregate (MTA) was introduced as a material for dental endodontic regenerative therapy. Here, we show the dynamics of living dental pulp cells in direct contact with an MTA disk. A red fluorescence protein (DsRed) was introduced into immortalized porcine dental pulp cells (PPU7) and cloned. DsRed-PPU7 cells were cultured on the MTA disk and cell proliferation, chemotaxis, the effects of growth factors and the gene expression of cells were investigated at the biological, histomorphological and genetic cell levels. Mineralized precipitates formed in the DsRed-PPU7 cells were characterized with crystal structural analysis. DsRed-PPU7 cells proliferated in the central part of the MTA disk until Day 6 and displayed a tendency to move to the outer circumference. Both transforming growth factor beta and bone morphogenetic protein promoted the proliferation and movement of DsRed-PPU7 cells and also enhanced the expression levels of odontoblastic gene differentiation markers. Mineralized precipitates formed in DsRed-PPU7 were composed of calcium and phosphate but its crystals were different in each position. Our investigation showed that DsRed-PPU7 cells in direct contact with the MTA disk could differentiate into odontoblasts by controlling cell-cell and cell-substrate interactions depending on cell adhesion and the surrounding environment of the MTA.

Combined Effect of Midazolam and Bone Morphogenetic Protein-2 for Differentiation Induction from C2C12 Myoblast Cells to Osteoblasts.

In drug repositioning research, a new concept in drug discovery and new therapeutic opportunities have been identified for existing drugs. Midazolam (MDZ) is an anesthetic inducer used for general anesthesia. Here, we demonstrate the combined effects of bone morphogenetic protein-2 (BMP-2) and MDZ on osteogenic differentiation. An immortalized mouse myoblast cell line (C2C12 cell) was cultured in the combination of BMP-2 and MDZ (BMP-2+MDZ). The differentiation and signal transduction of C2C12 cells into osteoblasts were investigated at biological, immunohistochemical, and genetic cell levels. Mineralized nodules formed in C2C12 cells were characterized at the crystal engineering level. BMP-2+MDZ treatment decreased the myotube cell formation of C2C12 cells, and enhanced alkaline phosphatase activity and expression levels of osteoblastic differentiation marker genes. The precipitated nodules consisted of randomly oriented hydroxyapatite nanorods and nanoparticles. BMP-2+MDZ treatment reduced the immunostaining for both α1 and γ2 subunits antigens on the gamma-aminobutyric acid type A (GABAA) receptor in C2C12 cells, but enhanced that for BMP signal transducers. Our investigation showed that BMP-2+MDZ has a strong ability to induce the differentiation of C2C12 cells into osteoblasts and has the potential for drug repositioning in bone regeneration.

Trafficking and secretion of keratin 75 by ameloblasts in vivo.

A highly specialized cytoskeletal protein, keratin 75 (K75), expressed primarily in hair follicles, nail beds, and lingual papillae, was recently discovered in dental enamel, the most highly mineralized hard tissue in the human body. Among many questions this discovery poses, the fundamental question regarding the trafficking and secretion of this protein, which lacks a signal peptide, is of an utmost importance. Here, we present evidence that K75 is expressed during the secretory stage of enamel formation and is present in the forming enamel matrix. We further show that K75 is secreted together with major enamel matrix proteins amelogenin and ameloblastin, and it was detected in Golgi and the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) but not in rough ER (rER). Inhibition of ER-Golgi transport by brefeldin A did not affect the association of K75 with Golgi, whereas ameloblastin accumulated in rER, and its transport from rER into Golgi was disrupted. Together, these results indicate that K75, a cytosolic protein lacking a signal sequence, is secreted into the forming enamel matrix utilizing portions of the conventional ER-Golgi secretory pathway. To the best of our knowledge, this is the first study providing insights into mechanisms of keratin secretion.

Porcine keratin 75 in developing enamel.

OBJECTIVE: To provide in vivo biochemical evidence for the isolation, identification, and characterization of porcine keratin 75 (K75) in developing enamel. METHODS: Immunolocalization of K75 was observed in mandibles from mice at postnatal days 5 and 11. K75 gene expression was analyzed by quantitative reverse transcription-polymerase chain reaction using enamel organ epithelium (EOE) of incisors from pigs at 5 months of age. Enamel protein was extracted and isolated from both immature and mature enamel of second molars from 5-month-old pigs, and the K75 antibody-positive fraction was analyzed by liquid chromatography-mass spectrometry (LC-MS/MS). In vitro protease digestion of K75-antibody-positive fraction was carried out using porcine kallikrein 4 (pKLK4) or recombinant human enamelysin (rhMMP20) and their degradation patterns were characterized by both SDS-PAGE and western blotting. RESULTS: Specific immunostaining for K75 was restricted to the layers of stratum intermedium and the enamel side of ameloblasts in mice at postnatal day 5, and to the papillary layer at postnatal day 11. Porcine K75 was expressed throughout enamel formation, but its transcript levels were significantly higher in the transition EOE than in the secretory- and maturation-stage EOE. Porcine K75 was extracted from the neutral soluble fraction from both immature and mature enamel. It was identified by LC-MS/MS analysis, and was found not to be degraded by either pKLK4 or rhMMP20. CONCLUSION: We propose that K75 is present in the developing enamel and undergoes different processing/degradation compared to other enamel proteins.

Potential function of TGF-ß isoforms in maturation-stage ameloblasts.

OBJECTIVES: To investigate potential functions of transforming growth factor-beta (TGF-ß) isoforms in maturation-stage ameloblasts during amelogenesis. METHODS: In vivo activation of TGF-ß was characterized by using matrix metalloproteinase 20 null (Mmp20-/-) and wild-type (Mmp20+/+) mice. Using mHAT9d cells cultured in the presence of each TGF-ß isoform, (1) cell proliferation was determined by MTS assay, (2) immunostaining with anti-cleaved caspase-3 monoclonal antibody was performed and apoptotic indices were measured, (3) gene expression was analyzed by RT-qPCR, and (4) the uptake of amelogenin into mHAT9d cells was directly observed using a fluorescence microscope. RESULTS: TGF-ß1 and TGF-ß3 were present in the enamel matrix of developing teeth which were activated by MMP20 in vivo. A genetic study revealed that the three TGF-ß isoforms upregulate kallikrein 4 (KLK4) mRNA levels but downregulate carbonic anhydrase II. Moreover, TGF-ß1 and TGF-ß2 significantly upregulated the mRNA level of amelotin, whereas TGF-ß3 dramatically downregulated the mRNA levels of odontogenic ameloblast-associated protein (ODAM), family with sequence similarity 83 member H (FAM83H), and alkaline phosphatase (ALP). Immunostaining analysis showed that the apoptosis of mHAT9d cells is induced by three TGF-ß isoforms, with TGF-ß3 being most effective. Both TGF-ß1 and TGF-ß3 induced endocytosis of amelogenin. CONCLUSIONS: We propose that TGF-ß is regulated in an isoform-specific manner to perform multiple biological functions such as gene expression related to the structure of basal lamina/ameloblasts, mineral ion transport, apoptosis, and endocytosis in maturation-stage ameloblasts.

Protocols for Studying Formation and Mineralization of Dental Tissues In Vivo: Extraction Protocol for Isolating Dentin Matrix Proteins from Developing Teeth.

The organic material in developing dentin is 90% type I collagen and 10% non-collagenous proteins. The key to understanding dentin biomineralization is to study how these proteins collectively precipitate and organize hydroxyapatite crystals. The first step in characterizing the proteins within a mineralizing matrix is to efficiently extract and isolate the essential molecular participants and elucidate their structural and biochemical properties. In this study, we expanded previous approaches to develop an improved strategy for the extraction of extracellular matrix proteins from the dentin of developing teeth. Proteins in dentin powder were sequentially extracted in the order Tris-guanidine buffer, HCl-formic acid solution, acetic acid-NaCl solution, Tris-NaCl buffer, and a second Tris-guanidine buffer. Individual fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), by gelatin or casein zymography, and by Western blot analysis using dentin sialoprotein (DSP)- or dentin glycoprotein (DGP)-specific antibodies. This approach was used to purify assorted porcine dentin non-collagenous proteins.

Potential for Drug Repositioning of Midazolam for Dentin Regeneration.

Drug repositioning promises the advantages of reducing costs and expediting approvalschedules. An induction of the anesthetic and sedative drug; midazolam (MDZ), regulatesinhibitory neurotransmitters in the vertebrate nervous system. In this study we show the potentialfor drug repositioning of MDZ for dentin regeneration. A porcine dental pulp-derived cell line(PPU-7) that we established was cultured in MDZ-only, the combination of MDZ with bonemorphogenetic protein 2, and the combination of MDZ with transforming growth factor-beta 1. Thedifferentiation of PPU-7 into odontoblasts was investigated at the cell biological and genetic level.Mineralized nodules formed in PPU-7 were characterized at the protein and crystal engineeringlevels. The MDZ-only treatment enhanced the alkaline phosphatase activity and mRNA levels ofodontoblast differentiation marker genes, and precipitated nodule formation containing a dentinspecificprotein (dentin phosphoprotein). The nodules consisted of randomly orientedhydroxyapatite nanorods and nanoparticles. The morphology, orientation, and chemicalcomposition of the hydroxyapatite crystals were similar to those of hydroxyapatite that hadtransformed from amorphous calcium phosphate nanoparticles, as well as the hydroxyapatite inhuman molar dentin. Our investigation showed that a combination of MDZ and PPU-7 cellspossesses high potential of drug repositioning for dentin regeneration.

Structural features, processing mechanism and gene splice variants of dentin sialophosphoprotein.

Dentin sialophosphoprotein (DSPP) plays an important role in the formation of dentin. Understanding its structure and function would provide important insights into the regulation of dentin mineralization. For the past 15 years, we have been studying DSPP-derived proteins isolated from pig dentin. Porcine DSPP is synthesized and secreted by odontoblasts and processed into three proteins, i.e., dentin sialoprotein (DSP), dentin glycoprotein (DGP), and dentin phosphoprotein (DPP), by bone morphogenetic protein 1 and matrix metalloproteinase-20 and -2. DSP is a proteoglycan that forms covalent dimers, DGP is a phosphorylated glycoprotein, and DPP is a highly phosphorylated intrinsically disordered protein with genetic polymorphisms. Furthermore, DPP is not detected in dental pulp. This is possibly due to the existence of two mRNA variants of the DSPP gene: one that encodes the DSP region alone and another that encodes full-length DSPP. The mRNA variant encoding DSP alone is expressed in dental pulp and odontoblasts, but the variant encoding full-length DSPP is predominantly expressed in odontoblasts and barely in dental pulp.

Effects of Er:YAG and Diode Laser Irradiation on Dental Pulp Cells and Tissues.

Vital pulp therapy (VPT) is to preserve the nerve and maintain healthy dental pulp tissue. Laser irradiation (LI) is beneficial for VPT. Understanding how LI affects dental pulp cells and tissues is necessary to elucidate the mechanism of reparative dentin and dentin regeneration. Here, we show how Er:YAG-LI and diode-LI modulated cell proliferation, apoptosis, gene expression, protease activation, and mineralization induction in dental pulp cells and tissues using cell culture, immunohistochemical, genetic, and protein analysis techniques. Both LIs promoted proliferation in porcine dental pulp-derived cell lines (PPU-7), although the cell growth rate between the LIs was different. In addition to proliferation, both LIs also caused apoptosis; however, the apoptotic index for Er:YAG-LI was higher than that for diode-LI. The mRNA level of odontoblastic gene markers-two dentin sialophosphoprotein splicing variants and matrix metalloprotease (MMP)20 were enhanced by diode-LI, whereas MMP2 was increased by Er:YAG-LI. Both LIs enhanced alkaline phosphatase activity, suggesting that they may help induce PPU-7 differentiation into odontoblast-like cells. In terms of mineralization induction, the LIs were not significantly different, although their cell reactivity was likely different. Both LIs activated four MMPs in porcine dental pulp tissues. We helped elucidate how reparative dentin is formed during laser treatments.