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The purpose of this study was to assess the occurrence and prognosis of dental implant proximity or direct contact with the adjacent tooth and to evaluate the symptoms and complications in both the implant and the adjacent tooth. We then elaborate on treatment modalities and discuss the prevention of this phenomenon.This retrospective study was conducted based on the dental clinical and radiographic records of 43 patients with implant-tooth proximity of1.0 mm or direct implant-tooth contact. Multivariate Bayesian logistic regression was performed to examine the influence of individual variables on correcting major clinical variables.In the multivariate regression analysis, the rate of occurrence of tooth symptom decreased by about 95% with every increase of 1.0 mm distance between implant and tooth (odds ratio [OR] = 0.1, 95% confidence interval [CI]: 0.004-0.680, p = 0.024). In the case of implant-tooth root proximity in the anterior area, the OR of peri-implantitis occurrence was 30.4-fold greater than in posterior sites (OR = 30.4, 95% CI: 1.189-785.914, p = 0.040).Implant-tooth root proximity or direct implant-tooth contact is an iatrogenic factor that causes various complications, including devitalization of the adjacent tooth and peri-implantitis. Proactive prevention with surgical stents and intra-operative periapical radiographs is needed. If proximity or contact is found during surgery, repositioning of the fixture to the correct location is recommended in order to maintain peri-implant health and prevent complications.

This study aimed to investigate whether newly emerged periodontitis or apical periodontitis on the adjacent teeth affects osseointegrated dental implants in a beagle dog model. The mandibular second and fourth premolars on both sides of three beagles were extracted. Two months after extraction, four bone-level implant fixtures, two on both sides of each mandible, were placed in each beagle. Six weeks after implant surgery, healing abutments were connected. After sufficient osseointegration, plaque control was performed in the control group, while periodontitis and apical periodontitis were induced in the experimental groups. The beagles were euthanized for histological analyses five months after induction of experimental periodontitis. The implants in the control and apical periodontitis groups were well-maintained, while those in the periodontitis group showed clinical signs of inflammation with bone resorption. The bone-to-implant contact (BIC) and bone area (BA) values in the periodontitis group were lower than those in the other groups. The distance between the implant shoulder and the first BIC was significantly greater in the periodontitis group than in the control group. Unlike apical periodontitis, periodontitis of the teeth surrounding dental implants can induce peri-implantitis. Periodontal healthcare is essential for long-term dental implant survival.

Though the normal physiological level of oxidative stress is beneficial for maintaining bone homeostasis, however, the imbalance between reactive oxygen species (ROS) production and antioxidant defense can cause various bone diseases. The purpose of this study was to see whether nicotinamide (NAM), an NAD+ precursor, could support the maintenance of bone homeostasis via regulating osteoblasts. Here, we demonstrate that NAM enhanced osteoblast differentiation and mitochondrial metabolism. NAM increased the expression of antioxidant enzymes, which was due to increased FOXO3a transcriptional activity via SIRT3 activation. NAM has not only a preventive effect to a weak and chronic oxidative stress but also a therapeutic effect to a strong and acute exposure to H2O2 in osteoblasts differentiation. Collectively, NAM increased mitochondrial biogenesis and antioxidant enzyme expression through the activation of SIRT3-FOXO3a axis that consequently enhanced osteoblast differentiation. These results suggest NAM could be a potential preventive or therapeutic agent for the bone diseases caused by ROS.

This study aimed to histologically and histomorphometrically evaluate osseointegration following simultaneous implant placement and maxillary sinus augmentation. Three retrospective human cases are described in which implants were placed at the maxillary sinus site augmented with deproteinized bovine bone mineral (DBBM) and later retrieved due to implant fracture after 5 to 8 years of occlusal loading. The removed implants with bone were processed for histologic evaluation, and bone-to-implant contact (BIC), bone area (BA), and mirror-image bone area (MIBA) were measured. Mature lamella bone was mainly observed, and some unabsorbed grafted bone particles remained in all cases. The measured values of BIC, BA, and MIBA in the three consecutive threads with the highest values were 86.0% to 91.2%, 65.8% to 91.9%, and 73.0% to 90.4%, respectively, and there were no signs of inflammation. Within the limits of this study, these cases demonstrate successful bone formation after maxillary sinus bone augmentation with DBBM and simultaneous implant placement.

In an aging society, quality of life improvement is emerging as an important issue, and as implants are accepted as the core of oral rehabilitation treatment, competition for leadership in developing related technologies is intensifying. In this trend, unlike what is evident in the literature, the patent landscape shows the status of industrial-based technology development. A database analysis of a total of 32,237 dental implant patents shows improvements in technology, diverse geographical characteristics, and new advances toward technological convergence in this field. Technologically, dental implant technology has shown a tendency to develop from conventional implant materials and surface treatment technologies to new material technologies making use of substances such as pure zirconium and tantalum or software technologies related to diagnosis and prognosis. Regionally, dental implant technology, which was developed mainly in Europe and the Unites States in the past, is growing explosively in East Asian countries accompanied by the recent growth of the Asian market. In summary, dental implant technology seems to be developing while trying to converge with various technological areas based on the local market environment. Therefore, it is necessary to develop a new dental implant material technology that is highly applicable to the development of hybrid information/communication technology and is suitable for a new manufacturing method. Our study may provide important information to help basic and translational researchers and their financial supporters set their research directions in advancing the development of dental implants.

Aging causes dysfunction of innate immunity, although hematopoietic stem cells of aged bone marrow (BM) show an increased differentiation potential to myeloid lineage cells. The alteration of cellular heterogeneity and intercellular communications between BM immune cells may provide important clues to understanding age-dependent immune dysfunction. Here, we provide a deep single-cell transcriptomic analysis of total immune cell populations of young and old BM. We identified the well-organized differentiation status of 11 myeloid/lymphoid lineage cell populations and age-dependent alterations in the proportions of cells. The neutrophil lineages showed the most prominent alteration by aging, and subclustering of neutrophils indicated that the specific immature neutrophils are increased in old BM. In addition, we identified age-dependent alterations in secretory phenotypes associated with a decline in innate immunity and immune cell differentiation. Among these secretory phenotypes, SPP1 could be suggested as a representative signal that triggers myeloid skewing and immature neutrophil deposition in aged BM. Collectively, these results provide a novel link between the altered immune cell proportions in BM and age-dependent dysregulation of innate immunity.

Hyaluronic acid (HA) affects angiogenesis and promotes the migration and differentiation of mesenchymal cells, thereby activating the osteogenic ability of osteoblasts. Although studies on the action of HA during bone regeneration are being actively conducted, the optimal dose of HA required for bone regeneration remains unclear. Therefore, the purpose of this study was to elucidate the most effective HA dose for bone formation using a rat critical-size defect model.Thirty rats were randomly divided into 5 groups, with 6 rats in each group. An absorbable collagen sponge soaked with HA or saline was used to fill an 8-mm defect, which was then covered with a collagen membrane. Different treatments were performed for each group as follows: (1) saline control, (2) 1 mg/mL HA, (3) 25 mg/mL HA, (4) 50 mg/mL HA, or (5) 75 mg/mL HA. After a healing period of 4 weeks, micro-computed tomography and histological analysis were performed. The obtained values were analyzed using analysis of variance and the Tukey test (At week 4, the 75 mg/mL HA group had the highest bone volume/total volume ratio, new bone, and bone fill among the 5 groups, and these values were significantly different from those observed in the control group (The 75 mg/mL HA group showed better bone formation than the other groups (1, 25, and 50 mg/mL HA and control).

Patients with cleidocranial dysplasia (CCD) caused by mutations in RUNX2 have severe dental anomalies, including delayed or absent eruption of permanent teeth. This requires painful and expensive surgical/orthodontic intervention because of the absence of medicine for this condition. Here, we demonstrate that nicotinamide, a vitamin B3 and class III histone deacetylase inhibitor, significantly improves delayed tooth eruption in Runx2 +/− mice, a well-known CCD animal model, through the restoration of decreased osteoclastogenesis. We also found that Csf1 mRNA and protein levels were significantly reduced in Runx2 +/− osteoblasts as compared with wild type whereas RANKL and OPG levels had no significant difference between wild type and Runx2 +/− osteoblasts. The nicotinamide-induced restoration of osteoclastogenesis of bone marrow–derived macrophages in Runx2 +/− mice was due to the increased expression of RUNX2 and CSF1 and increased RANKL/OPG ratio. RUNX2 directly regulated Csf1 mRNA expression via binding to the promoter region of the Csf1 gene. In addition, nicotinamide enhanced the RUNX2 protein level and transacting activity posttranslationally with Sirt2 inhibition. Taken together, our study shows the potential and underlying molecular mechanism of nicotinamide for the treatment of delayed tooth eruption by using the Runx2 +/− murine model, suggesting nicotinamide as a candidate therapeutic drug for dental abnormalities in patients with CCD.

Male infertility is a multifactorial condition. Unexplained male infertility is often caused by spermatogenesis dysfunction. Knockout of Pin1, an important regulator of cell proliferation and differentiation, produces male infertility phenotypes such as testicular immaturity and azoospermia with spermatogonia depletion and blood-testis barrier (BTB) dysfunction. Gene therapy has been clinically considered for the treatment of male infertility, but it is not preferred because of the risks of adverse effects in germ cells. Direct intracellular protein delivery using nanoparticles is considered an effective alternative to gene therapy; however, in vivo testicular protein delivery remains a pressing challenge. Here, we investigated the direct intracellular protein delivery strategy using a fibroin nanoparticle-encapsulated cationic lipid complex (Fibroplex) to restore intratesticular PIN1. Local intratesticular delivery of PIN1 via Fibroplex in Pin1 knockout testes produced fertile mice, achieving recovery from the infertile phenotypes. Mechanistically, PIN1-loaded Fibroplex was successfully delivered into testicular cells, including spermatogonial cells and Sertoli cells, and the sustained release of PIN1 restored the gene expression required for the proliferation of spermatogonial cells and BTB integrity in Pin1 knockout testes. Collectively, testicular PIN1 protein delivery using Fibroplex might be an effective strategy for treating male infertility.

Cellular senescence is closely related to tissue aging including bone. Bone homeostasis is maintained by the tight balance between bone-forming osteoblasts and bone-resorbing osteoclasts, but it undergoes deregulation with age, causing age-associated osteoporosis, a main cause of which is osteoblast dysfunction. Oxidative stress caused by the accumulation of reactive oxygen species (ROS) in bone tissues with aging can accelerate osteoblast senescence and dysfunction. However, the regulatory mechanism that controls the ROS-induced senescence of osteoblasts is poorly understood. Here, we identified Peptidyl arginine deiminase 2 (PADI2), a post-translational modifying enzyme, as a regulator of ROS-accelerated senescence of osteoblasts via RNA-sequencing and further functional validations. PADI2 downregulation by treatment with H2O2 or its siRNA promoted cellular senescence and suppressed osteoblast differentiation. CCL2, 5, and 7 known as the elements of the senescence-associated secretory phenotype (SASP) which is a secretome including proinflammatory cytokines and chemokines emitted by senescent cells and a representative feature of senescence, were upregulated by H2O2 treatment or Padi2 knockdown. Furthermore, blocking these SASP factors with neutralizing antibodies or siRNAs alleviated the senescence and dysfunction of osteoblasts induced by H2O2 treatment or Padi2 knockdown. The elevated production of these SASP factors was mediated by the activation of NFκB signaling pathway. The inhibition of NFκB using the pharmacological inhibitor or siRNA effectively relieved H2O2 treatment- or Padi2 knockdown-induced senescence and osteoblast dysfunction. Together, our study for the first time uncover the role of PADI2 in ROS-accelerated cellular senescence of osteoblasts and provide new mechanistic and therapeutic insights into excessive ROS-promoted cellular senescence and aging-related bone diseases.

Significant research and development (RD) has been conducted to make the best dental implants while developing various patent applications and registrations. In this study, we evaluated the current status of patents on dental implants and identified the future direction of RD progress.A total of 29 711 patents related to dental implants were reviewed. These were published between 1909 and 2020 and retrieved from the Derwent Innovation patent database. The patents were grouped into three categories depending on the implant components: fixture, abutment, and artificial teeth.The category with most patents was "abutment," and the most cited patent was "screw-type dental implant anchor." Global patenting trends over the past 20 years showed that both applicants and applications increased in the early 2010s; however, these have since been on the decline. Currently, the United States holds the largest number of patents, and Nobel Biocare Holding AG is the top assignee. Technic maturation prediction analysis showed that the current dental implant technology is in the "decline stage."Trend analysis of the dental implant patent indicates the main contributors of development are profit-oriented companies. Recent reduction in the number of new patent applications suggests the technology is in the mature declining stage. The emergence of new materials or technologies that may close the gap in clinical unmet needs would reverse the trend.

Midface hypoplasia is a major manifestation of Apert syndrome. However, the tissue component responsible for midface hypoplasia has not been elucidated. We studied mice with a chondrocyte-specific Fgfr2S252W mutation (Col2a1-cre; Fgfr2S252W/+) to investigate the effect of cartilaginous components in midface hypoplasia of Apert syndrome. In Col2a1-cre; Fgfr2S252W/+ mice, skull shape was normal at birth, but hypoplastic phenotypes became evident with age. General dimensional changes of mutant mice were comparable with those of mice with mutations in EIIa-cre; Fgfr2S252W/+, a classic model of Apert syndrome in mice. Col2a1-cre; Fgfr2S252W/+ mice showed some unique facial phenotypes, such as elevated nasion, abnormal fusion of the suture between the premaxilla and the vomer, and decreased perpendicular plate of the ethmoid bone volume, which are related to the development of the nasal septal cartilage. Morphological and histological examination revealed that the presence of increased septal chondrocyte hypertrophy and abnormal thickening of nasal septum is causally related to midface deformities in nasal septum-associated structures. Our results suggest that careful examination and surgical correction of the nasal septal cartilage may improve the prognosis in the surgical treatment of midface hypoplasia and respiratory problems in patients with Apert syndrome.

For individuals who have experienced tooth loss, dental implants are an important treatment option for oral reconstruction. For these patients, alveolar bone augmentation and acceleration of osseointegration optimize implant stability. Traditional oral surgery often requires invasive procedures, which can result in prolonged treatment time and associated morbidity. It has been previously shown that chemical vapor deposition (CVD) polymerization of functionalized [2.2]paracyclophanes can be used to anchor gene encoding vectors onto biomaterial surfaces and local delivery of a bone morphogenetic protein (BMP)-encoding vector can increase alveolar bone volume and density in vivo. This study is the first to combine the use of CVD technology and BMP gene delivery on titanium for the promotion of bone regeneration and bone to implant contact in vivo. BMP-7 tethered to titanium surface enhances osteoblast cell differentiation and alkaline phosphatase activity in vitro and increases alveolar bone regeneration and % bone to implant contact similar to using high doses of exogenously applied BMP-7 in vivo. The use of this innovative gene delivery strategy on implant surfaces offers an alternative treatment option for targeted alveolar bone reconstruction.

Background The Encyclopedia of DNA Elements (ENCODE) project has advanced our knowledge of the functional elements in the genome and epigenome. The aim of this article was to provide the comprehension about current research trends from ENCODE project and establish the link between epigenetics and periodontal diseases based on epigenome studies and seek the future direction. Main body Global epigenome research projects have emphasized the importance of epigenetic research for understanding human health and disease, and current international consortia show an improved interest in the importance of oral health with systemic health. The epigenetic studies in dental field have been mainly conducted in periodontology and have focused on DNA methylation analysis. Advances in sequencing technology have broadened the target for epigenetic studies from specific genes to genome-wide analyses. Conclusions In line with global research trends, further extended and advanced epigenetic studies would provide crucial information for the realization of comprehensive dental medicine and expand the scope of ongoing large-scale research projects.

Nanofibrous engineered matrices have significant potential in cellular differentiation and tissue regeneration. Stem cells require specific extracellular signals that lead to the induction of different lineages. However, the mechanisms by which the nanofibrous matrix promotes mesenchymal stem cell (MSC) differentiation are largely unknown. Here, we investigated the mechanisms that underlie nanofibrous matrix-induced odontoblastic differentiation of human dental pulp MSCs (DP-MSCs). An electrospun polystyrene nanofibrous (PSF) matrix was prepared, and the cell responses to the PSF matrix were assessed in comparison with those on conventional tissue culture dishes. The PSF matrix promoted the expression of Wnt3a, Wnt5a, Wnt10a, BMP2, BMP4, and BMP7 in the DP-MSCs, concomitant with the induction of odontoblast/osteoblast differentiation markers, dentin sialophosphoprotein (DSPP), osteocalcin, and bone sialoprotein, whose levels were further enhanced by treatment with recombinant Wnt3a. The DP-MSCs cultured on the PSF matrix also exhibited a high alkaline phosphatase activity and intense Alizarin Red staining, indicating that the PSF matrix promotes odontoblast differentiation. Besides inducing the expression of Wnt3a, the PSF matrix maintained high levels of β-catenin protein and enhanced its translocation to the nucleus, leading to its transcriptional activity. Forced expression of LEF1 or treatments with LiCl further enhanced the DSPP expression. Blocking the Wnt3a-initiated signaling abrogated the PSF-induced DSPP expression. Furthermore, the cells on the PSF matrix increased the DSPP promoter activity. The β-catenin complex was bound to the conserved motifs on the DSPP promoter dictating its transcription. Transplantations of the preodontoblast-seeded PSF matrix to the subcutaneous tissues of nude mice confirmed the association of the PSF matrix with the Wnt3a and DSPP expressions in vivo. Taken together, these results demonstrate the nanofibrous engineered matrix strongly supports odontoblastic differentiation of DP-MSCs by enhancing Wnt/β-catenin signaling.

Embryonic stem cells have been a popular research topic in regenerative medicine owing to their pluripotency and applicability. However, due to the difficulty in harvesting them and their low yield efficiency, advanced cell reprogramming technology has been introduced as an alternative. Dental stem cells have entered the spotlight due to their regenerative potential and their ability to be obtained from biological waste generated after dental treatment. Cell reprogramming, a process of reverting mature somatic cells into stem cells, and transdifferentiation, a direct conversion between different cell types without induction of a pluripotent state, have helped overcome the shortcomings of stem cells and raised interest in their regenerative potential. Furthermore, the potential of these cells to return to their original cell types due to their epigenetic memory has reinforced the need to control the epigenetic background for successful management of cellular differentiation. Herein, we discuss all available sources of dental stem cells, the procedures used to obtain these cells, and their ability to differentiate into the desired cells. We also introduce the concepts of cell reprogramming and transdifferentiation in terms of genetics and epigenetics, including DNA methylation, histone modification, and non-coding RNA. Finally, we discuss a novel therapeutic avenue for using dental-derived cells as stem cells, and explain cell reprogramming and transdifferentiation, which are used in regenerative medicine and tissue engineering.

This article presents data related to the research article "Fibroin particle-supported cationic lipid layers for highly efficient intracellular protein delivery" and focuses on the delivery efficiency aspects of the fibroin particle-cationic lipid complex (Fibroplex), including its fabrication and the intracellular delivery to the mouse skin tissue. We introduced a stable lipid-particle complex called "Fibroplex", formed by loading cargo protein onto a silk fibroin spherical particle core complexed with cationic liposomes to address the intracellular recombinant protein delivery. This system exhibits cationic charge, which is advantageous for cellular uptake. The particle core is loaded with the cargo protein with high efficiency and shows long-term release in serum environment. Fibroplex can be formed simply by mixing the particle core and cationic liposome, and this spontaneous interaction does not cause any detrimental effects on the function of cargo proteins. Lipid-particle complex structure is stable over 10 days in the serum at 37 °C. Fibroplex was delivered at high efficiency to a wide variety of cells, including cancer cells and primary cell-lines. Also, Fibroplex loaded with two types of cargo successfully introduced them into the cytoplasm. Furthermore, Fibroplex shows successful intracellular delivery when injected with various cargo proteins such as GFP, HRP and Tyrosinase into mouse skin tissue as well as in vitro. The highlights of this article include: (1) Data for fabrication procedure of Fibroplex, (2) loading capacity, surface charge changes of Fibroplex, and (3) Intracellular delivery aspects of Fibroin in vitro and vivo.

Pin1 is a peptidyl prolyl cis-trans isomerase that specifically binds to the phosphoserine-proline or phosphothreonine-proline motifs of several proteins. We reported that Pin1 plays a critical role in the fate determination of Smad1/5, Runx2, and β-catenin that are indispensable nuclear proteins for osteoblast differentiation. Though several chemical inhibitors has been discovered for Pin1, no activator has been reported as of yet. In this study, we directly introduced recombinant Pin1 protein successfully into the cytoplasm via fibroin nanoparticle encapsulated in cationic lipid. This nanoparticle-lipid complex delivered its cargo with a high efficiency and a low cytotoxicity. Direct delivery of Pin1 leads to increased Runx2 and Smad signaling and resulted in recovery of the osteogenic marker genes expression and the deposition of mineral in Pin1-deficient cells. These result indicated that a direct Pin1 protein delivery method could be a potential therapeutics for the osteopenic diseases. J. Cell. Physiol. 232: 2798-2805, 2017. © 2016 Wiley Periodicals, Inc.

Cleidocranial dysplasia (CCD) is an autosomal dominant skeletal disorder caused by mutations in RUNX2, coding a key transcription factor of early osteogenesis. CCD patients suffer from developmental defects in cranial bones. Despite numerous investigations and clinical approaches, no therapeutic strategy has been suggested to prevent CCD. Here, we show that fetal administration of Entinostat/MS-275, a class I histone deacetylase (HDAC)-specific inhibitor, partially prevents delayed closure of cranial sutures in Runx2+/- mice strain of C57BL/6J by two mechanisms: 1) posttranslational acetylation of Runx2 protein, which stabilized the protein and activated its transcriptional activity; and 2) epigenetic regulation of Runx2 and other bone marker genes. Moreover, we show that MS-275 stimulates osteoblast proliferation effectively both in vivo and in vitro, suggesting that delayed skeletal development in CCD is closely related to the decreased number of progenitor cells as well as the delayed osteogenic differentiation. These findings provide the potential benefits of the therapeutic strategy using MS-275 to prevent CCD. © 2017 American Society for Bone and Mineral Research.

At present, various tissue engineering strategies in the regenerative medicine have been developed for multiple tissue regeneration and integrative structure formations in musculoskeletal system. However, the regenerations of dental tissues or tooth-supportive structures are still challenging due to the micro-interfacial compartmentalization of multiple tissues, their integrations for systematic responses, and spatiotemporal organizations of engineered fibrous tissues. In particular, the biomaterial-based approaches are limitedly investigated for spatiotemporal controls of periodontal regenerations and challenging to promote micron-scaled interfacial tissue compartmentalization with their integrations for functioning restorations. This chapter demonstrates biomaterial applications for periodontal tissue engineering and the advanced 3D fabrication technologies for preclinical applications and the limited clinical trial using 3D scaffolding systems.

To fully regenerate the periodontal tissues remains a challenge in our daily practice. Periodontal regeneration is a complex process involving a series of cellular and molecular events. In the last two decades, significant advances have been made in applying proteins and peptides to treat periodontal osseous defects. Several products are currently available and a few others are under development. Stem cell therapy has also been vigorously investigated for regenerating craniofacial tissues including periodontia. In this chapter, we review the current status of protein- and cell-based therapies for periodontal regeneration, with an emphasis on products that have been tested in clinical studies.

Bone regeneration has been a challenge for both researchers and clinicians. In the field of tissue engineering, much effort has been made to identify cell sources including stem cells. The present study aimed to induce trans-differentiation from adipocytes to osteoblasts using epigenetic modifiers; 5-aza-dC and/or trichostatin-A (TSA). 3 T3-L1 preadipocytes were treated with TSA (100 nM) and then with Wnt3a (50 ng/ml). Microscopic observation showed trans-differentiated cell morphology. Methylation-specific PCR and immunoblotting were performed to analyze the DNA methylation and histone acetylation patterns. The gene expression was determined by real-time PCR. Based on these in vitro experiments, in vivo mouse experiments supplemented the possibility of trans-differentiation by epigenetic modification. TSA induced the acetylation of lysine9 on histone H3, and a sequential Wnt3a treatment stimulated the expression of bone marker genes in adipocytes, suppressing adipogenesis and stimulating osteogenesis. Furthermore, TSA induced DNA hypomethylation, and a combined treatment with TSA and 5-aza-dC showed a synergistic effect in epigenetic modifications. The number of adipocytes and DNA methylation patterns of old (15 months) and young (6 weeks) mice were significantly different, and TSA and sequential Wnt3a treatments increased bone formation in the old mice. Collectively, our results confirmed cell trans-differentiation via epigenetic modifications and osteogenic signaling from adipocytes to osteoblasts for the bone regeneration in vitro and in vivo, and indicated that histone acetylation could induce DNA hypomethylation, enhancing the chance of trans-differentiation.

It is widely accepted that sandblasted/large-grit/acid-etched (SLA) surfaces of titanium (Ti) have a higher osteogenic potential than machined ones. However, most studies focused on differential gene expression without elucidating the underlying mechanism for this difference. The aim of this study was to evaluate how the surface roughness of dental Ti implants affects their osteogenic potential. Mouse preosteoblast MC3T3-E1 cells were seeded on machined and SLA Ti discs. The cellular activities of the discs were analyzed using confocal laser scanning microscopy, proliferation assays, and real-time polymerase chain reaction (PCR). DNA methylation was evaluated using a methylation-specific PCR. The cell morphology was slightly different between the two types of surfaces. While cellular proliferation was slightly greater on the machined surfaces, the osteogenic response of the SLA surfaces was superior, and they showed increased alkaline phosphatase (Alp) activity and higher bone marker gene expression levels (Type I collagen, Alp, and osteocalcin). The degree of DNA methylation on the Alp gene was lower on the SLA surfaces than on the machined surfaces. DNA methyltransferase inhibitor stimulated the Alp gene expression on the machined surfaces, similar to the SLA surfaces. The superior osteogenic potential of the SLA surfaces can be attributed to a different epigenetic landscape, specifically, the DNA methylation of Alp genes. This finding offers novel insights into epigenetics to supplement genetics and raises the possibility of using epidrugs as potential therapeutic targets to enhance osteogenesis on implant surfaces.

The canonical Wnt signaling pathway, in which β-catenin nuclear localization is a crucial step, plays an important role in osteoblast differentiation. Pin1, a prolyl isomerase, is also known as a key enzyme in osteogenesis. However, the role of Pin1 in canonical Wnt signal-induced osteoblast differentiation is poorly understood. We found that Pin1 deficiency caused osteopenia and reduction of β-catenin in bone lining cells. Similarly, Pin1 knockdown or treatment with Pin1 inhibitors strongly decreased the nuclear β-catenin level, TOP flash activity, and expression of bone marker genes induced by canonical Wnt activation and vice versa in Pin1 overexpression. Pin1 interacts directly with and isomerizes β-catenin in the nucleus. The isomerized β-catenin could not bind to nuclear adenomatous polyposis coli, which drives β-catenin out of the nucleus for proteasomal degradation, which consequently increases the retention of β-catenin in the nucleus and might explain the decrease of β-catenin ubiquitination. These results indicate that Pin1 could be a critical target to modulate β-catenin-mediated osteogenesis.

Purpose This study aimed to evaluate the effectiveness of the partial split-flap technique with a K-incision on vertical guided bone regeneration (vGBR) and to retrospectively analyze the clinical and radiographic outcomes of dental implantation using this approach. Methods In total, 78 patients who received 104 dental implants with vGBR, categorized as (1) pre-GBR and post-implantation and (2) simultaneous GBR and implantation, were enrolled. Data analysis was based on periapical radiographs, clinical photos, and dental records. The 2-sample t-test was used to compare the 2 surgical procedures. Results The baseline vertical bone level, augmented bone height (ABH), and treatment duration were significantly higher in the pre-GBR procedure group. The survival rates of the implants were 96.1% and 94.8% in implant- and patient-based analyses, respectively. In Cox regression analysis, high rates of implant failure were found in the presence of ABH of ≥4 mm, smoking, and diabetes. Conclusions Within the limitations of this retrospective study, the partial split-flap technique using a K-incision for vGBR showed stable clinical outcomes and favorable dental implant survival., Graphical Abstract

Peri-implantitis is similar to periodontitis in both symptoms and treatment; however, their level of similarity remains controversial. Here, we compared multiple cases of periodontitis and peri-implantitis through transcriptome and methylome profiling, and analyzed the effects of smoking as a typical risk factor. Human gingival tissues were obtained from 20 patients with periodontitis or peri-implantitis via periodontal surgical procedures. Total RNA and genomic DNA were isolated, and transcriptome and methylome datasets were generated. Comprehensive analysis of differential gene expression, DNA methylation, and protein-protein interactions indicated that periodontitis and peri-implantitis share biological similarities; however, hierarchical clustering between the two disease groups revealed distinct molecular characteristics. These differences might be related to structural differences in natural tooth-bone and implant-bone. Additionally, smoking differentially affected periodontitis and peri-implantitis in terms of host-defense mechanism impairment. Within the limitations of this study, the results provide evidence that peri-implantitis is distinct from periodontitis and that smoking potentially affects disease progression. Our study provides a foundation for the rational design of a large-scale study in the future for a more comprehensive analysis that includes microbiome and clinical data.

Objectives This 12-week clinical trial evaluated efficacy and adverse events for two recombinant human bone morphogenetic protein-2 (rhBMP-2) delivery systems in alveolar ridge preservation. Materials and methods Sixty-four patients had a single tooth that required replacement with an implant, surrounded by > 50% alveolar bone height. Two cohorts (n = 32 patients each) were randomized to receive a rhBMP-2-soaked absorbable collagen sponge (test group), or β-tricalcium phosphate and hydroxyapatite particles (control group) immersed in rhBMP-2, at the implant site. Bone height and width changes at 25%, 50%, and 75% of extraction socket level (ESL) were compared. Adverse events were assessed in the same period. In addition to the randomized controlled clinical trial, histological analysis of 21 patients (test group [n = 12], control group [n = 9]) was conducted, 4 months after alveolar ridge preservation. A non-inferiority test was used to analyze changes in alveolar bone height between groups (p = 0.05). A Wilcoxon rank-sum test was used to analyze changes in alveolar bone width and histomorphometric results between groups (p = 0.05). Results All patients showed good healing without severe adverse events. The lower limit of the one-sided 97.5% confidence interval in the difference between the two groups was 0.0033 (non-inferiority margin: -0.185); thus, the test group showed non-inferiority to the control group. Wilcoxon rank-sum test analysis did not show statistically significant differences between groups with regard to changes in alveolar bone width and histomorphometric analysis. Conclusions The delivery systems showed similar efficacy for alveolar ridge preservation without severe adverse events.

The bone marrow of healthy individuals is primarily composed of osteoblasts and hematopoietic cells, while that of osteoporosis patients has a larger portion of adipocytes. There is evidence that the epigenetic landscape can strongly influence cell differentiation. We have shown that it is possible to direct the trans-differentiation of adipocytes to osteoblasts by modifying the epigenetic landscape with a DNA methyltransferase inhibitor (DNMTi), 5'-aza-dC, followed by Wnt3a treatment to signal osteogenesis. Treating 3T3-L1 adipocytes with 5'-aza-dC induced demethylation in the hypermethylated CpG regions of bone marker genes; subsequent Wnt3a treatment drove the cells to osteogenic differentiation. When old mice with predominantly adipose marrow were treated with both 5'-aza-dC and Wnt3a, decreased fatty tissue and increased bone volume were observed. Together, our results indicate that epigenetic modification permits direct programming of adipocytes into osteoblasts in a mouse model of osteoporosis, suggesting that this approach could be useful in bone tissue-engineering applications.

It was indicated that tetragonal zirconia polycrystal (TZP) containing yttria (Y2O3) and niobium oxide (Nb2O5) ((Y,Nb)-TZP) could be an adequate dental material to be used at esthetically important sites. The (Y,Nb)-TZP was also proved to possess its osteogenic potential comparable with those conventional dental implant material, titanium (Ti). The objective of the current study was to characterize cellular response of human gingival fibroblasts (HGFs) to smooth and rough surfaces of the (Y,Nb)-TZP disc, which were obtained by polishing and sandblasting, respectively. Various microscopic, biochemical, and molecular techniques were used to investigate the disc surfaces and cellular responses for the experimental (Y,Nb)-TZP and the comparing Ti groups. Sandblasted rough (Y,Nb)-TZP (Zir-R) discs had the highest surface roughness. HGFs cultured on polished (Y,Nb)-TZP (Zir) showed a rounded cell morphology and light spreading at 6 h after seeding and its proliferation rate significantly increased during seven days of culture compared to other surfaces. The mRNA expressions of type I collagen, integrin α2 and β1 were significantly stimulated for the Zir group at 24 h after seeding. The current findings, combined with the previous results, indicate that (Y,Nb)-TZP provides appropriate surface condition for osseointegration at the fixture level and for peri-implant mucosal sealing at the abutment level producing a suitable candidate for dental implantation with an expected favorable clinical outcome.

Gain-of-function mutations in fibroblast growth factor receptors (FGFRs) cause congenital skeletal anomalies, including craniosynostosis (CS), which is characterized by the premature closure of craniofacial sutures. Apert syndrome (AS) is one of the severest forms of CS, and the only treatment is surgical expansion of prematurely fused sutures in infants. Previously, we demonstrated that the prolyl isomerase peptidyl-prolyl cis–trans isomerase interacting 1 (PIN1) plays a critical role in mediating FGFR signaling and that Pin1+/− mice exhibit delayed closure of cranial sutures. In this study, using both genetic and pharmacological approaches, we tested whether PIN1 modulation could be used as a therapeutic regimen against AS. In the genetic approach, we crossbred Fgfr2S252W/+, a mouse model of AS, and Pin1+/− mice. Downregulation of Pin1 gene dosage attenuated premature cranial suture closure and other phenotypes of AS in Fgfr2S252W/+ mutant mice. In the pharmacological approach, we intraperitoneally administered juglone, a PIN1 enzyme inhibitor, to pregnant Fgfr2S252W/+ mutant mice and found that this treatment successfully interrupted fetal development of AS phenotypes. Primary cultured osteoblasts from Fgfr2S252W/+ mutant mice expressed high levels of FGFR2 downstream target genes, but this phenotype was attenuated by PIN1 inhibition. Post-translational stabilization and activation of Runt-related transcription factor 2 (RUNX2) in Fgfr2S252W/+ osteoblasts were also attenuated by PIN1 inhibition. Based on these observations, we conclude that PIN1 enzyme activity is important for FGFR2-induced RUNX2 activation and craniofacial suture morphogenesis. Moreover, these findings highlight that juglone or other PIN1 inhibitors represent viable alternatives to surgical intervention for treatment of CS and other hyperostotic diseases.

Pin1 is a peptidyl prolyl cis-trans isomerase that specifically binds to the phosphoserine-proline or phosphothreonine-proline motifs of numerous proteins. Previously, we reported that Pin1 deficiency resulted in defects in osteoblast differentiation during early bone development. In this study, we found that adult Pin1-deficient mice developed osteoporotic phenotypes compared to age-matched controls. Since BMP2 stored in the bone matrix plays a critical role in adult bone maintenance, we suspected that BMP R-Smads (Smad1 and Smad5) could be critical targets for Pin1 action. Pin1 specifically binds to the phosphorylated linker region of Smad1, which leads to structural modification and stabilization of the Smad1 protein. In this process, Pin1-mediated conformational modification of Smad1 directly suppresses the Smurf1 interaction with Smad1, thereby promoting sustained activation of the Smad1 molecule. Our data demonstrate that post-phosphorylational prolyl isomerization of Smad1 is a converging signal to stabilize the Smad1 molecule against the ubiquitination process mediated by Smurf1. Therefore, Pin1 is a critical molecular switch in the determination of Smad1 fate, opposing the death signal transmitted to the Smad1 linker region by phosphorylation cascades after its nuclear localization and transcriptional activation. Thus, Pin1 could be developed as a major therapeutic target in many skeletal diseases.

Runx2 is the master transcription factor for bone formation. Haploinsufficiency of RUNX2 is the genetic cause of cleidocranial dysplasia (CCD) that is characterized by hypoplastic clavicles and open fontanels. In this study, we found that Pin1, peptidyl prolyl cis-trans isomerase, is a critical regulator of Runx2 in vivo and in vitro. Pin1 mutant mice developed CCD-like phenotypes with hypoplastic clavicles and open fontanels as found in the Runx2+/− mice. In addition Runx2 protein level was significantly reduced in Pin1 mutant mice. Moreover Pin1 directly interacts with the Runx2 protein in a phosphorylation-dependent manner and subsequently stabilizes Runx2 protein. In the absence of Pin1, Runx2 is rapidly degraded by the ubiquitin-dependent protein degradation pathway. However, Pin1 overexpression strongly attenuated uniquitin-dependent Runx2 degradation. Collectively conformational change of Runx2 by Pin1 is essential for its protein stability and possibly enhances the level of active Runx2 in vivo.

Directly delivering therapeutic proteins into cells has promise as an intervention without side effects for protein deficiencies caused by genetic defects. However, as negatively charged macromolecules, proteins require carriers for achieving cellular uptake and maintaining their activity in the cytoplasm. The biodegradable natural polymer silk fibroin has demonstrated outstanding advantages as a protein drug scaffold in vitro and in vivo, but its usage has been limited in the extracellular space because of its negatively charged character. Here, we present an intracellular protein delivery system based on fibroin particles coated with cationic lipid layers, denoted as Fibroplex, the surface charge of which can be modulated. Fibroplex showed higher delivery efficiency than conventional delivery methods as well as long-term cargo release in the cytoplasm without toxicity. Furthermore, in vivo experiments showed that Fibroplex efficiently delivered tyrosinase and horseradish peroxidase, which led to hyper-pigmentation and tumor regression, respectively, suggesting its potential for therapeutic protein applications in hereditary diseases or cancer.

Pin1 is a peptidyl prolyl cis-trans isomerase that specifically binds to the phosphoserine-proline or phosphothreonine-proline motifs of several proteins. We reported that Pin1 plays a critical role in the fate determination of Smad1/5, Runx2, and β-catenin that are indispensable nuclear proteins for osteoblast differentiation. Though several chemical inhibitors has been discovered for Pin1, no activator has been reported as of yet. In this study, we directly introduced recombinant Pin1 protein successfully into the cytoplasm via fibroin nanoparticle encapsulated in cationic lipid. This nanoparticle-lipid complex delivered its cargo with a high efficiency and a low cytotoxicity. Direct delivery of Pin1 leads to increased Runx2 and Smad signaling and resulted in recovery of the osteogenic marker genes expression and the deposition of mineral in Pin1-deficient cells. These result indicated that a direct Pin1 protein delivery method could be a potential therapeutics for the osteopenic diseases. J. Cell. Physiol. 232: 2798-2805, 2017. © 2016 Wiley Periodicals, Inc.

Cleidocranial dysplasia (CCD) is an autosomal dominant skeletal disorder caused by mutations in RUNX2, coding a key transcription factor of early osteogenesis. CCD patients suffer from developmental defects in cranial bones. Despite numerous investigations and clinical approaches, no therapeutic strategy has been suggested to prevent CCD. Here, we show that fetal administration of Entinostat/MS-275, a class I histone deacetylase (HDAC)-specific inhibitor, partially prevents delayed closure of cranial sutures in Runx2

In previous studies by the authors, it was demonstrated that a fibronectin (FN)-derived oligopeptide, termed F20, stimulates osteoblast differentiation in vitro and bone formation in vivo. However, the fundamental molecular mechanism by which F20 stimulates osteogenesis remains unknown. Therefore, in this study the molecular mechanism underlying the effect of F20 in osteoblast differentiation is investigated.The role of F20 in osteoblast differentiation was examined using mouse bone-marrow-derived ST2 cell line. The effect of Smad1/5 was determined following small interfering RNA knockdown. Runt-related transcription factor (Runx) 2, alkaline phosphatase (Alp), and osteocalcin (Oc) mRNA levels were determined by quantitative real-time polymerase chain reaction, and their transcriptional activation was assessed using luciferase reporter assays. Extracellular signal-regulated kinase (ERK) phosphorylation was visualized via immunoblotting.Synthetic oligopeptide F20 stimulated expression of bone marker genes Runx2, Alp, and Oc in ST2 cells via Smad and ERK or mitogen-activated protein kinase signaling pathways as did bone morphogenic protein 2 (BMP2). Furthermore, Runx2 acted as a transcription factor during F20-induced osteoblast differentiation.Collectively, these results indicate that F20 induces osteoblast differentiation with a pattern similar to that mediated by BMP2 signaling pathway. The authors' previous data also showed that FN-derived oligopeptide improved wound healing, and it is suggested that F20 might serve as a therapeutic biomolecule to facilitate periodontal tissue regeneration.

Introduction This article reports 3 representative cases of interdisciplinary management of a palatogingival groove in maxillary lateral incisors. The development, pathology, and effectiveness of management approaches in cases involving a combined periodontal-endodontic lesion with a palatogingival groove are discussed. Methods We describe 3 patients with a noncontributory medical history presenting with a chief complaint related to a maxillary incisor and diagnosed with a combined periodontal-endodontic lesion with a palatogingival groove at Seoul National University Dental Hospital, Seoul, Korea. Results Palatogingival grooves were mostly associated with deep periodontal pockets connected to a periapical lesion. Optional collaborative treatments were performed according to the condition as follows: case 1, root canal treatment (RCT), open flap debridement, odontoplasty, and guided tissue regeneration; case 2, RCT, apicoectomy, open flap debridement, and odontoplasty; and case 3, RCT, crown restoration, root planning, and odontoplasty. After clinical examination and radiographic assessments, the periapical lesion and periodontal deep pocket were successfully resolved with periodontal-endodontic collaborative treatment involving both periodontal surgical procedures (cases 1 and 2) and a nonsurgical procedure (case 3). Conclusions Within the limitations of this study, these case reports show that accurate diagnosis of developmental anomalies and elimination of inflammatory irritants are key factors for favorable long-term outcomes.

Dentin sialophosphoprotein (DSPP), a typical dentin-specific protein, is mainly expressed in the dentin extracellular matrix and plays a role in dentin mineralization. BMP-2 provides a strong signal for differentiation and mineralization of odontoblasts and osteoblasts. Previously, BMP-2 treatment is reported to stimulate Dspp expression in the MD10-F2 pre-odontoblast cells through activation of the heterotrimeric transcription factor Y (NF-Y). The canonical BMP signaling pathway is known to contribute greatly to biomineralization, however, it is not known whether it is involved in Dspp expression. Here, we investigated this question. Activation of the canonical BMP-2 signaling pathway in MDPC-23, preodontoblast cell, by overexpression of constitutively active Smad1/5 or downstream transcription factors Dlx5 and Runx2 stimulated Dspp expression. Conversely, knockdown of each element with siRNA significantly blocked the BMP-2-induced Dspp expression. To test whether these transcription factors downstream of BMP-2 are directly involved in regulating Dspp, we analyzed the mouse Dspp promoter. There are 5 well conserved homeodomain binding elements, H1 to H5, in Dspp proximal promoter regions (−791 to +54). A serial deletion of H1 and H2 greatly changed basal promoter activity and responsiveness to Dlx5 or Msx2. However, further deletions did not change the responsiveness to Dlx5 or Msx2. H1 and H2 sites can be suggested as specific response elements of Dlx5 and Msx2, respectively, based on their promoter activity modulation. Thus, the canonical BMP-2 signaling pathway plays a crucial part in the regulation of Dspp expression through the action of Smads, Dlx5, Runx2, and Msx2.

Boston-type craniosynostosis is caused by a single amino acid substitution, P148H, in the transcription factor MSX2. The increased binding affinity of MSX2 (P148H) to the response element has led many to hypothesize that the substitution is a gain-of-function mutation. However, there have been conflicting reports on the function of MSX2, and by extension, the nature of the P148H mutation remains unclear. In this study, we have examined the molecular mechanism of MSX2 function and the nature of the P148H mutation. During cranial suture closure of rodent, Msx2 expression was detected in the suture space. Overexpression of wild type MSX2 in mesenchymal cells stimulated cell proliferation and cyclin D1 expression, whereas P148H mutant did not. These results indicated that MSX2 is involved in maintaining the suture space by stimulating suture mesenchymal cell proliferation and that P148H is defective in this process. The protein levels of P148H were lower than wild type Msx2 (Msx2-WT), and pulse-chase experiments indicated that the mutant protein has a shorter half-life than the Msx2-WT protein. The ubiquitylation level of P148H was greater than that of Msx2-WT. The degradation of Msx2 was mediated by Praja1, and the P148H mutant was degraded more effectively than WT. The ubiquitylation of Msx2-WT was higher in the presence of Msx2 (P148H), which indicated that P148H functions as a dominant-negative mutant. Collectively, the primary function of MSX2 in suture closure is the induction of cell proliferation and suture maintenance, and the mutation results in an increased susceptibility of both wild type and mutant MSX2 to proteasomal degradation.

Our previous studies demonstrated that a recombinant fibronectin (FN)-derived oligopeptide that we named F20 stimulated osteoblast adhesion, proliferation, and differentiation in vitro and in vivo. In the present study, we used a synthetic oligopeptide and investigated the osteogenic potential of F20 coating on titanium discs, to stimulate superior osseointegration for dental implant surface modification. Surface characteristic analysis of titanium was performed by confocal laser scanning microscopy (CLSM) observation. Synthetic F20 was coated onto the machined or SLA titanium discs by an adsorption procedure. ST2 cells were seeded on the titanium discs. We evaluated cell adhesion with SEM and CLSM observation, cell proliferation with picogreen assay, and osteoblast differentiation with real-time PCR, ALP activity assay, immunoblot assay and ALP staining. FITC-labeled F20 coating on the discs was detected by fluorescence, showing good F20 adsorption and different coating patterns according to the surface roughness. In the SEM and CLSM observations, cells were well attached on the machined surface and greater stress fiber formation was seen on discs coated with F20 than on other discs. F20 stimulated cellular proliferation, as well as osteoblast differentiation through the extracellular signalregulated kinase (Erk) signaling pathway. These cellular responses to F20 were slightly better on the machined titanium surface than the SLA surface. These results suggest that F20 promotes osteogenesis through the Erk pathway and is a suitable biomolecule for surface modification of dental implants for improved osseointegration.

The bone marrow of healthy individuals is primarily composed of osteoblasts and hematopoietic cells, while that of osteoporosis patients has a larger portion of adipocytes. There is evidence that the epigenetic landscape can strongly influence cell differentiation. We have shown that it is possible to direct the trans-differentiation of adipocytes to osteoblasts by modifying the epigenetic landscape with a DNA methyltransferase inhibitor (DNMTi), 5'-aza-dC, followed by Wnt3a treatment to signal osteogenesis. Treating 3T3-L1 adipocytes with 5'-aza-dC induced demethylation in the hypermethylated CpG regions of bone marker genes; subsequent Wnt3a treatment drove the cells to osteogenic differentiation. When old mice with predominantly adipose marrow were treated with both 5'-aza-dC and Wnt3a, decreased fatty tissue and increased bone volume were observed. Together, our results indicate that epigenetic modification permits direct programming of adipocytes into osteoblasts in a mouse model of osteoporosis, suggesting that this approach could be useful in bone tissue-engineering applications.

PURPOSE This study was performed to evaluate the osteogenic potential of 3mol% yttria-stabilized tetragonal zirconia polycrystals (3Y-TZP) and niobium oxide containing Y-TZPs with specific ratios, new (Y,Nb)-TZPs, namely YN4533 and YN4533/Al20 discs. MATERIALS AND METHODS 3Y-TZP, YN4533 and YN4533/Al20 discs (15 mm diameter and 1 mm thickness) were prepared and their average surface roughness (Ra) and surface topography were analyzed using 3-D confocal laser microscope (CLSM) and scanning electron microscope (SEM). Mouse pre-osteoblast MC3T3-E1 cells were seeded onto all zirconia discs and evaluated with regard to cell attachment and morphology by (CLSM), cell proliferation by PicoGreen assay, and cell differentiation by Reverse-Transcription PCR and Quantitative Real-Time PCR, and alkaline phosphatase (Alp) staining. RESULTS The cellular morphology of MC3T3-E1 pre-osteoblasts was more stretched on a smooth surface than on a rough surface, regardless of the material. Cellular proliferation was higher on smooth surfaces, but there were no significant differences between 3Y-TZP, YN4533, and YN4533/Al20. Osteoblast differentiation patterns on YN4533 and YN4533/Al20 were similar to or slightly higher than seen in 3Y-TZP. Although there were no significant differences in bone marker gene expression (alkaline phosphatase and osteocalcin), Alp staining indicated better osteoblast differentiation on YN4533 and YN4533/Al20 compared to 3Y-TZP. CONCLUSION Based on these results, niobium oxide containing Y-TZPs have comparable osteogenic potential to 3Y-TZP and are expected to be suitable alternative ceramics dental implant materials to titanium for aesthetically important areas.