Aim: This study examined the effectiveness of human demineralized bone matrix (DBM) as a scaffold material in bone regenerative medicine using cultured periosteal cells (CPCs). Methods: A bone defect created in the parietal region of eight-week-old nude rats served as the graft bed. A polylactic acid resin dish shell was used as a covering material, and DBM (with (+) or without (-) CPC) was grafted at the site. Deproteinized bovine bone (DBB) was used in the control group. Four weeks later, the grafted site was sampled for micro-computed tomography and histological observations. The osteoblastic induction effect of DBM was evaluated by co-culturing with CPCs. Results: The DBM-CPC (+) group demonstrated a significantly larger new bone volume than the DBM-CPC (-) group. New bone formation with a distinct lining of alkaline phosphatase (ALP)-positive cells was observed in the DBM graft area, regardless of cell administration. However, the staining intensity and thickness tended to be higher in the DBM-CPC (+) group. Cells positive for tartrate-resistant acid phosphatase were more concentrated in areas close to the existing bone in the DBM-CPC (-) group and throughout the newly formed bone area in the DBM-CPC (+) group. Only slight bone conduction was observed adjacent to existing bone in the DBB graft group, with no new bone formation. The emergence of ALP-positive cells was confirmed in CPC co-cultured with DBM. Conclusions: These findings demonstrate the bone inductive effect of DBM and indicate the effectiveness of DBM as a cell transplantation carrier in bone regeneration cell therapy.

Background: Human periosteal cells (hPCs) possess high osteogenic potential and are considered promising candidates for regenerative therapy. Recombinant human collagen peptide (RCP), a xeno-free biomaterial enriched with arginine-glycine-aspartic acid (RGD) sequences, provides a stable and biocompatible scaffold that may enhance cellular functions. Objectives: This study aimed to evaluate the biocompatibility of RCP with hPCs and its effects on osteogenic functions in vitro and in vivo. Materials and methods: Cell viability was assessed by MTT assay at various RCP concentrations. Cell adhesion and migration were examined using fluorescence-based adhesion and Oris™ migration assays. Gene expression profiles were analyzed by RNA-sequencing and quantitative PCR. Alkaline phosphatase assay (ALP) and Alizarin Red staining (ARS) were performed to evaluate osteogenic differentiation. For in vivo analysis, a rat calvarial critical-size defect model was used to evaluate bone regeneration following transplantation of hPCs with or without RCP. Results: RCP exhibited no cytotoxic effects and enhanced the adhesion and migration of hPCs. RNA-seq and qPCR analyses revealed upregulation of extracellular matrix- and osteogenesis-related genes, including COL1A1, SERPINH1, ALP, RUNX2, and OCN. Moreover, RCP enhanced osteogenic differentiation, as demonstrated by increased ALP activity and greater calcium deposition in ARS. In vivo, micro-CT analysis showed no significant difference in new bone volume among groups, whereas immunohistochemical analysis revealed increased numbers of OCN- and BMP-2-positive cells in RCP-treated defects, indicating enhanced osteogenic activity. Conclusion: RCP supports the osteogenic potential of hPCs by promoting adhesion, migration, and osteogenesis-related gene expression, and enhancing osteogenic activity in vitro and in vivo. These findings suggest that RCP is a promising biomaterial for periosteal cell-based regenerative therapies.

Background and Objectives: Autologous bone grafting is the first choice for reconstructive surgery in bone defects due to trauma or malignant tumors. However, there is an increasing demand for minimally invasive alternatives involving bone regeneration using artificial materials. Biomimetic materials that replicate the body’s microscopic structure, such as Cellnest®, are gaining attention. Cellnest is a xeno-free recombinant peptide based on human type I collagen, containing a rich Arg-Gly-Asp (RGD) motif related to cell adhesion. The aim of this study was to compare the effects of Cellnest with existing collagen materials (Pelnac®, Integra®, Terudermis®) on bone regeneration and elucidate the underlying mechanisms. Materials and Methods: In vivo experiments involved a rat model of calvarial bone defects, in which Cellnest and other collagen materials were implanted into the defect area. Bone formation was assessed after 4 weeks using micro-computed tomography (micro-CT) and histological analysis. In vitro experiments included the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), adhesion, and migration assays, and a real-time polymerase chain reaction using rapidly expanding cells (RECs) to explore the mechanisms of Cellnest’s bone regenerative capacity. Results: The micro-CT analysis showed that the regenerated bone area was significantly greater in the Cellnest group (72.3%) than in the Pelnac® (25.5%), Integra® (31.6%), and Terudermis® (38.3%) groups. The histological analysis confirmed similar trends, with Cellnest showing 42.2% bone regeneration, outperforming the other materials. The in vitro assays revealed that Cellnest promoted cell proliferation, adhesion, and migration. Gene expression analysis demonstrated that Cellnest significantly increased the levels of the bone formation markers ALP and COL1. Conclusions: Cellnest, a human type I collagen-like peptide rich in RGD motifs, enhances bone regeneration by promoting MSC adhesion and migration, and bone formation-related gene expression. The findings suggest its potential as an effective material for bone defect reconstruction.

ObjectiveTo develop a clinically effective method to evaluate the bone formation process during gnathic bone regeneration therapy.Materials and MethodsTwenty patients who underwent sinus floor augmentation were divided into two groups: those who received graft materials with cultured autogenous periosteal cells [CAP (+)] and those who did not [CAP (-)]. Computed tomography (CT) images were obtained and examined before and 5 months, 1 year, and 3 years after the surgery using a three-dimensional (3D) image analysis system (SYNAPSE VINCENT, Fujifilm, Tokyo). The regenerated bone areas in the 3D images were stratified by their CT values according to Misch's classification as D1, D2, D3, D4, and D5; the distributions of the layers were monitored over the time points.ResultsAt 5 months after surgery, the proportion of D3, corresponding to the cancellous bone, was significantly higher, whereas that of D5, corresponding to the soft tissue, was significantly lower in the CAP (+) group compared to those in the CAP (-) group. The CT value-stratified 3D images demonstrated the promoting effects of CAP in the anabolic resorption of the graft-derived cortical bone granules and the formation of the bone structure in the CAP (+) group.ConclusionA time-course analysis of the distribution of the stratified CT values may help detect changes in the composition of the bone, thereby enabling the evaluation of the characteristics of the regenerated bone. This method could be widely applied to evaluate the bone quality in various treatment fields.

Introduction: Cell-based bone regenerative therapy exhibits considerable potential in the treatment of bone defects caused by trauma, disease, and congenital anomalies. The periosteum, a fibrous membrane covering the outer surface of bone, plays a crucial role in bone formation and regeneration by sourcing osteoprogenitor cells. The remarkable osteogenic potential of periosteal cells (PCs) has led to the effective clinical implementation of PC-based regenerative therapies and tissue engineering. The abundance of progenitor cells in cultured PCs is well established; however, the heterogeneity of the cell population and its impact on bone regeneration remain uncertain. In this study, we aimed to characterize the hetero-geneity of cultured PCs via single-cell RNA-sequencing (scRNA-seq) and to examine their osteogenic potential in vivo.Methods: Human PCs cultivated using the tissue explant method were utilized in this study. scRNA-seq and real-time PCR were performed to examine the cellular heterogeneity and osteogenic capacity of the cultured PCs. Experimental bone formation by the cultured PCs was examined using the rat model of subcutaneous implantation.Results: ScRNA-seq analysis showed that the cultured PCs were categorized into three cell types (osteoprogenitor cells, mesenchymal stem cells, and fibroblasts) with specific gene expression patterns. In addition, the cellular population and osteogenic capacity differed between the central and peripheral regions in the culture dish. The PCs in the central region showed higher osteogenic potential than those in the peripheral region. Conclusions: This study revealed the diversity of the composition of the PCs and their distinct osteogenic capabilities in different regions in the culture dish. The findings may provide promising prospects for the development of more efficacious regenerative therapeutic applications using cultured PCs in the future.(c) 2023, The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/ 4.0/).

Background Autologous tissue-engineered periosteal sheets, which have been clinically applied for periodontal regeneration, sinus lift, and alveolar ridge augmentation, are enriched with osteoblast precursor cells and the abundant deposition of collagen type I in the extracellular spaces. Their quality is inspected prior to clinical use; however, most criteria cannot be evaluated without sacrificing samples. To reduce such losses, we developed a non-destructive optical method that can quantitatively evaluate the thickness of the periosteal sheet. Methods Dispersed periosteal cells were inoculated into small pieces of collagen sponge (Terudermis®) and plated into 60-mm dishes for further explant culture using a conventional medium and a stem-cell culture medium. The thickness of periosteal sheets was evaluated using inverted microscopic, histological, labeling (CellVue®)-based imaging and spectrophotometric (Spectro-1®) methods. Results The three-dimensional growth of periosteal sheets did not necessarily correlate with two-dimensional growth. The periosteal sheet prepared with the stem-cell medium formed cell multilayers, a phenomenon that could be observed qualitatively by inverted microscopy. The spectrophotometric analysis enabled the quantitative evaluation of the thickness of the cell multilayer without sacrificing the samples processed for scheduled cell therapy. Conclusions The growth of periosteal sheets is influenced by several major factors, including the basic quality of the individual original periosteal tissue segments, the technical expertise of doctors and operators involved in tissue harvesting and processing, and culture conditions. This newly developed spectrophotometric analysis can quantify the thickness of cell-multilayered periosteal sheets for quality assurance in a non-destructive manner, thereby contributing to better bone augmentation prior to implant therapy.

Cell culture media influence the characteristics of human osteogenic periosteal sheets. We have previously found that a stem cell medium facilitates growth and collagen matrix formation in vitro and osteogenesis in vivo. However, it has not yet been demonstrated which culture medium is superior for osteoclastogenesis, a prerequisite for reconstruction of normal bone metabolic basis. To address this question, we compared chemotaxis and osteoclastogenesis in tissue-engineered periosteal sheets (TPSs) prepared with two types of culture media. Periosteal tissues obtained from adult volunteers were expanded with the conventional Medium 199 or with the stem cell medium, MesenPRO. Hematopoietic enhanced-green-fluorescent-protein (EGFP)-nude mice were prepared by γ-irradiation of Balb/c nu/nu mice and subsequent transplantation of bone marrow cells from CAG-EGFP C57BL/6 mice. TPSs were implanted subcutaneously into the chimeric mice and retrieved after intervals for immunohistopathological examination. EGFP+ cells were similarly recruited to the implantation site in both the TPSs prepared, whereas the distribution of CD11b+ cells was significantly lower in the TPS prepared with the stem cell medium. Instead, osteoclastogenesis was higher in the TPS prepared with the stem cell medium than in the one prepared with the conventional medium. These findings suggest that the stem cell medium is preferable for the preparation of more functional TPSs.

In 2004, we developed autologous periosteal sheets for the treatment of periodontal bone defects. This regenerative therapy has successfully regenerated periodontal bone and augmented alveolar ridge for implant placement. However, the necessity for 6-week culture is a limitation. Here, we examined the applicability of a human platelet-rich fibrin extract (PRFext) as an alternative to fetal bovine serum (FBS) for the explant culture of periosteal sheets in a novel culture medium (MSC-PCM) originally developed for maintaining mesenchymal stem cells. Small periosteum tissue segments were expanded in MSC-PCM + 2% PRFext for 4 weeks, and the resulting periosteal sheets were compared with those prepared by the conventional method using Medium199 + 10% FBS for their growth rate, cell multilayer formation, alkaline phosphatase (ALP) activity, and surface antigen expression (CD73, CD90, and CD105). Periosteal sheets grew faster in the novel culture medium than in the conventional medium. However, assessment of cell shape and ALP activity revealed that the periosteal cells growing in the novel medium were relatively immature. These findings suggest that the novel culture medium featuring PRFext offers advantages by shortening the culture period and excluding possible risks associated with xeno-factors without negatively altering the activity of periosteal sheets.

Introduction Although human immunodeficiency virus type 1 (HIV-1) infection through blood, breast milk, semen, and vaginal secretions has been established, no report has clearly demonstrated HIV-1 infection through saliva. We postulated that a low or absent infectivity of HIV-1 in saliva is due to the damage of viral RNA in virions. To test this hypothesis, we developed an RNA cleavage quantification method. Materials and methods A part of the gag gene was reverse transcribed from HIV-1 RNA extracted from saliva-treated virus, and the cDNA product was analyzed by quantitative real-time PCR at three positions that were located at various distances from the reverse transcription (RT) start site. The slope of plots of cycle threshold value vs distance between the RT start site and the primer-binding position was used to evaluate the degree of RNA damage. Results When free HIV-1 RNA was incubated in saliva, the RNA became undetectable within 5 minutes; however, when virions were incubated, cleavage of the viral RNA occurred at a much slower rate. Although heat treatment at 70 degrees C for 20 minutes did not influence the ability of saliva to cleave free HIV-1 RNA, it completely suppressed the RNA cleavage activity within the viral envelope, suggesting that viral structure was loosen or destroyed by thermolabile factors and then intravirion RNA was digested by salivary ribonucleases. Conclusions We detected thermolabile intravirion RNA cleavage activity in saliva. This activity may be related to a poor infectivity of HIV-1 in saliva.

Adult Cebpb KO mice incisors present amelogenin-positive epithelium pearls, enamel and dentin allopathic hyperplasia, fewer Sox2-positive cells in labial cervical loop epitheliums, and reduced Sox2 expression in enamel epithelial stem cells. Thus, Cebpb acts upstream of Sox2 to regulate stemness. In this study, Cebpb KO mice demonstrated cementum-like hard tissue in dental pulp, loss of polarity by ameloblasts, enamel matrix in ameloblastic layer, and increased expression of epithelial-mesenchymal transition(EMT) markers in a Cebpb knockdown mouse enamel epithelial stem cell line. Runx2 knockdown in the cell line presented a similar expression pattern. Therefore, the EMT enabled disengaged odontogenic epithelial stem cells to develop supernumerary teeth. Cebpb and Runx2 knockdown in the cell line revealed higher Biglycan and Decorin expression, and Decorin-positive staining in the periapical region, indicating their involvement in supernumerary tooth formation. Cebpb and Runx2 acted synergistically and played an important role in the formation of supernumerary teeth in adult incisors.

Objective To elucidate the mechanism underlying secretion of human immunodeficiency virus type 1 (HIV-1) into the oral cavity, by examining the relationships between various oral and systemic factors and the viral load in saliva. Methods Plasma and saliva samples from HIV-1 infected patients were assayed using the COBAS® AmpliPrep/COBAS® TaqMan® HIV-1 Test, version 1.0 and a Poisson distribution-based polymerase chain reaction (PCR) method for quantifying HIV-1 RNA and DNA. Results Forty-four pairs of samples were obtained from 18 patients. Salivary viral load was approximately 10% of the plasma viral load, but higher than the plasma load in two patients. The salivary viral DNA load was < 1% of the total HIV-1 nucleic acid load except in one patient who had more viral DNA than RNA. Multiple regression analysis showed that salivary viral load was significantly correlated with plasma viral load (partial correlation coefficient, 0.90) and the community periodontal index (–0.63). Conclusions The present results suggest that excretion through salivary glands, but not occult bleeding, may be a major pathway of HIV-1 into the oral cavity.

A human-cultured alveolar bone-derived periosteal (hCP) sheet is an osteogenic grafting material used clinically in periodontal regenerative therapy, while platelet-rich fibrin (PRF), a platelet concentrate with fibrin clot, is considered to augment the wound healing process. Therefore, whether the combined use of hCP-PRF complex could facilitate bone regeneration synergistically was evaluated in animal models. Human periosteal segments (1 x 1 mm) were cultured initially on plastic dishes and formed an hCP sheet. The hCP sheet was implanted with freshly prepared human PRF into subcutaneous tissue (hCP: n = 4, hCP + PRF: n = 4) and 4 mm diameter calvarial bone defect models (hCP: n = 4, hCP + PRF: n = 4, control [defect-only]: n = 4) that prepared in nude mice. At 4 weeks postimplantation, new bone formation was evaluated by using mu CT. Cell growth and neovascularization were evaluated by histochemical and immunohistological methods. In the subcutaneous tissue, mineral deposit formation, collagen deposition, and number of vessels were higher in the hCP + PRF group than in the hCP alone group. In the calvarial defect models, new bone formation was significantly higher in the hCP + PRF group than in the hCP alone group and defect-only control group. The numbers of vessels and PCNA-positive cells in calvarial defects were also increased in the hCP + PRF group more than in the hCP alone group. Platelet-rich fibrin preparations support the proliferation and the growth of periosteal cells to form well-combined active biological materials. Platelet-rich fibrin also stimulates the local angiogenesis in the implantation site. Therefore, the combined use of hCP and PRF could be clinically applicable in bone regeneration therapy.

In preparing cell-based products for regenerative therapy, cell quality should be strictly controlled. Methodologies for evaluating cell viability, identity, and purity are established and used routinely, whereas current methodologies for evaluating cell safety, particularly genetic integrity or tumorigenicity, are time-consuming and relatively insensitive. As part of developing a more practical screening system, the authors previously demonstrated that γ-H2AX and p53 were useful markers for evaluating the history of DNA damage. To validate these markers further and develop a more quantitative methodology, single cell-based expression of these markers and two additional candidates have now been examined using flow cytometry (FCM). FCM analysis and immunofluorescent staining demonstrated that γ-ray-irradiation suppressed proliferation, enlarged cells, and cell nuclei, and immediately upregulated γ-H2AX and p21(waf1) in large numbers of cells for up to 12 days. Gamma-H2AX foci were formed in the nuclei of many affected cells. An initial sharp increase in p53 expression declined slowly over 12 days, while Rb expression increased linearly. The present findings suggest that this high-throughput, cell-based, combinational evaluation of protein markers and cell size enables a small number of cells with a history of DNA damage to be detected quickly and routinely from within a very large cell population. Using this screening methodology will improve the ability to verify the quality of cell-based products used in regenerative therapy.

Abstract Purpose: To assure the quality of cells to be used in cell therapy, we examined the applicability of digital holographic microscopy (DHM) for non-invasive, quantitative assessment of changes in cell morphology. Materials and methods: Mesenchymal stem cells derived from adipose tissue (MSC-AT) and bone marrow (MSC-BM), in addition to human alveolar periosteal cells (PC) as a reference, were γ-ray irradiated (1 and 4 Gy), and their morphological changes were quantified without fixation using holographic microscopy. After detachment and fixation with ethanol, cell number and surface antigen expression were determined using an automated cell counter kit and flow-cytometry, respectively. Results: Among various indexes, only indexes related to cell size were significantly changed after γ-irradiation. Both BMC-AT and BMC-BM were enlarged and more sensitive to a low dose of γ-irradiation than PC. In contrast to PC, proteins related to DNA damage repair (γ-H2AX, p21waf1, p53 and Rb) were not substantially upregulated or sustained for a week in either MSC-AT or MSC-BM. Conclusion: Instead of DNA damage markers, we suggest that cell morphological parameters (e.g. cell volume) that are monitored by DHM could be a useful and more stable marker of MSC quality.

AbstractBackground and PurposeSinus lift (SL) using cultured autogenous periosteal cells (CAPCs) combined with autogenous bone and platelet‐rich plasma (PRP) was performed to evaluate the effect of cell administration on bone regeneration, by using high‐resolution three‐dimensional computed tomography (CT).Materials and MethodsSL with autogenous bone and PRP plus CAPC [CAPC(+)SL] was performed in 23 patients. A piece of periosteum taken from the mandible was cultured in M199 medium with 10% fetal bovine serum (FBS) for 6 weeks. As control, 16 patients received SL with autogenous bone and PRP [CAPC(−)SL]. Three‐dimensional CT imaging was performed before and 4 months and 1 year after SL, and stratification was performed based on CT numbers (HUs) corresponding to soft tissue and cancellous or cortical bone.ResultsThe augmented bone in CAPC(+)SL revealed an increase in HUs corresponding to cancellous bone as well as a decrease in HUs corresponding to grafted cortical bone. In addition, HUs corresponding to cancellous bone in the graft bed were increased in CAPC(+)SL but were decreased in CAPC(−)SL. Insertion torque during implant placement was significantly higher in CAPC(+)SL.ConclusionBy promoting bone anabolic activity both in augmented bone and graft bed, CAPCs are expected to aid primary fixation and osseointegration of implants in clinical applications.

The aim of the present study was to investigate the diagnostic value of cell cycle-related genes in oral squamous cell carcinoma (OSCC) by examining the expression of the following genes in 77 OSCC tissues by quantitative polymerase chain reaction: Cyclin genes (CCNA1, CCND1, CCND2 and CCNE1), cyclin-dependent kinase (CDK) genes (CDK1, CDK2 and CDK4), CDK inhibitor genes (CDKN2A, CDKN1A, CDKN1B and CDKN1C), and integrin and associated genes that we previously reported (ITGA3, ITGB4, CD9 and JUP). The expression ratios of 66 combinations of the 11 cell cycle-related genes were analyzed to examine their associations with major clinical events using Mann-Whitney U and log-rank tests. Three expression ratios (CDK1/CDKN1B, CDK2/CDKN1A and CCNE1/CDK2) showed associations on univariate analyses and their diagnostic value was re-analyzed with integrin gene expression biomarkers (ITGA3/CD9 and ITGB4/JUP) using the Cox proportional hazards model and Kaplan-Meier estimates. Lymph node metastasis occurred in >90% of double-positive cases (high-ITGA3/CD9 and high-CDK1/CDKN1B) irrespective of tumor size (P<0.0001). Primary site recurrence was found in >30% of double-positive cases (high-ITGA3/CD9 and high-CDK2/CDKN1A) with tumors >20 mm (P=0.003). Triple-positive (high-ITGB4/JUP, high-ITGA3/CD9 and high-CDK2/CDKN1A) was associated with distant metastasis (P<0.0001), but not with other clinical parameters. Disease-specific death occurred in 55% of double-positive cases (high-ITGA3/CD9 and high-CDK2/CDKN1A) (P<0.0001) and a positive surgical margin was a significant factor for fatality in these cases. Reliable prediction of locoregional and hematogenous dissemination risks in OSCC using the four CDK and integrin gene expression ratios is a promising biomarker system. Clinical use of these parameters may improve the control rate with the use of new therapeutic strategies.

Background aims For successful cell transplantation therapy, the quality of cells must be strictly controlled. Unfortunately, to exclude inappropriate cells that possess structurally abnormal chromosomes, currently only karyotyping functions as an assessment. Unfortunately, this methodology is time-consuming and only effective for metaphasic cells. To develop a more efficient, inclusive and sensitive methodology, we examined the phosphorylation of histone H2AX and the p53 levels in normal human periosteal cells exposed to x-rays or other oxidative stressors. Methods Periosteal cells were obtained from human alveolar bone before being exposed to x-rays, ultraviolet C or hydrogen peroxide. The cell cycle, electric nuclear volume and CD44 expression were evaluated using flow cytometry, and the phosphorylated H2AX (γ-H2AX), p53, p21 and proliferating cell nuclear antigen (PCNA) levels were evaluated by Western blot analyses. Results Each oxidative stress dose-dependently arrested cell growth and partially induced premature cellular senescence. In parallel, each oxidative stress rapidly phosphorylated H2AX and stabilized p53, and intense stress sustained these high levels for at least 8 days. Conclusions Intensive oxidative stress induces sustained high levels of γ-H2AX and p53, which force cells toward senescence or non-apoptotic cell death. Lower doses of oxidative stress induced more modest and transient increases in γ-H2AX and p53, and these cells eventually survive. However, because DNA is repaired without a template in the majority of these cells, G1 mutations accumulate. Therefore, we recommend that any cell population expressing elevated γ-H2AX and p53 levels be excluded from cell transplantation therapy.