Characterization of the cellular heterogeneity and bone regenerative potential of cultured human periosteal cells.

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 heterogeneity 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.

Non-destructive, spectrophotometric analysis of the thickness of the cell-multilayered periosteal sheet.

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.

Osteoclastogenic Potential of Tissue-Engineered Periosteal Sheet: Effects of Culture Media on the Ability to Recruit Osteoclast Precursors.

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.

Platelet-Rich Fibrin Extract: A Promising Fetal Bovine Serum Alternative in Explant Cultures of Human Periosteal Sheets for Regenerative Therapy.

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.

Loss of Stemness, EMT, and Supernumerary Tooth Formation in Cebpb-/-Runx2+/- Murine Incisors.

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.

Factors contributing to salivary human immunodeficiency virus type-1 levels measured by a Poisson distribution-based PCR method.

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.

Synergistic effects of the combined use of human-cultured periosteal sheets and platelet-rich fibrin on bone regeneration: An animal study.

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 × 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 µCT. 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.

Non-invasive, quantitative assessment of the morphology of γ-irradiated human mesenchymal stem cells and periosteal cells using digital holographic microscopy.

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.

Evaluating the Safety of Somatic Periosteal Cells by Flow-Cytometric Analysis Monitoring the History of DNA Damage.

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.

High-Resolution Three-Dimensional Computed Tomography Analysis of the Clinical Efficacy of Cultured Autogenous Periosteal Cells in Sinus Lift Bone Grafting.

BACKGROUND AND PURPOSE: Sinus 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 METHODS: SL 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. RESULTS: The 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. CONCLUSION: By 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.

Diagnostic value of cyclin-dependent kinase/cyclin-dependent kinase inhibitor expression ratios as biomarkers of locoregional and hematogenous dissemination risks in oral squamous cell carcinoma.

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.

X-ray and ultraviolet C irradiation-induced γ-H2AX and p53 formation in normal human periosteal cells in vitro: markers for quality control in cell therapy.

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.

Intraoperative assessment of surgical margins of oral squamous cell carcinoma using frozen sections: a practical clinicopathological management for recurrences.

BACKGROUND: Local recurrence remains a challenging clinical issue for the treatment of oral squamous cell carcinoma (SCC). We analyzed retrospectively how effective the frozen section technique (FS) was against recurrences of oral SCC. METHODS: We screened 343 surgical samples from 236 patients who had oral SCC, carcinoma in situ (CIS), or epithelial dysplasia, and we followed up their clinical outcomes for at least 5 years. Histopathological states of surgical margins were compared between FS and surgical materials in relapse and relapse-free groups, respectively. RESULTS: Among the 236 patients, 191 were classified into the relapse-free group, and 45 into the relapse group. FS was more frequently performed in the relapse-free group (128/191) than in the relapse group (83/152). Histopathologically, moderate dysplasia or CIS (borderline malignancies) and SCC were recognized in 55 samples of the relapse-free group and in 57 of the relapse group. For those surgical margins with borderline malignancies, additional incisions were performed in 38 of the 55 relapse-free cases, which reduced to 20 from the 38 margins with borderline malignancies (47.4% reduction), and in 39 of the 57 relapse cases, which reduced to only 3 of 39 (7.7% reduction). CONCLUSIONS: The intraoperative assessment of surgical margins by FS is essential in preventing recurrences of oral mucosal malignancies.

Real-time quantitative polymerase chain reaction and flow cytometric analyses of cell adhesion molecules expressed in human cell-multilayered periosteal sheets in vitro.

BACKGROUND AIMS: Cultured human periosteal sheets more effectively function as an osteogenic grafting material at implantation sites than do dispersed periosteal cells. Because adherent cell growth and differentiation are regulated by cell-cell and cell-extracellular matrix contacts, we hypothesized that this advantage is a result of the unique cell adhesion pattern formed by their multiple cell layers and abundant extracellular matrix. To test this hypothesis, we prepared three distinct forms of periosteal cell cultures: three-dimensional cell-multilayered periosteal sheets, two-dimensional dispersed cell cultures, and three-dimensional hybrid mock-ups of cells dispersed onto collagen sponges. METHODS: Periosteal cells were obtained from human alveolar bone. Cell adhesion and extracellular matrix molecules were quantitatively determined at the messenger RNA and protein levels by means of real-time quantitative polymerase chain reaction and flow cytometry, respectively. RESULTS: Real-time quantitative polymerase chain reaction analysis demonstrated that regardless of culture media α1 integrin, vascular cell adhesion molecule-1, fibronectin and collagen type 1 were substantially upregulated, whereas CD44 was strongly downregulated in periosteal sheets compared with dispersed cell monolayers. With increased thickness, stem cell medium upregulated several integrins (ß1, α1 and α4), CD146, vascular cell adhesion molecule-1, fibronectin and collagen type 1 in the periosteal sheets. Flow cytometric analysis revealed that the active configuration of ß1 integrin was substantially downregulated in the stem cell medium-expanded cell cultures. The cell adhesion pattern found in the mock-up cultures was almost identical to that of genuine periosteal sheets. CONCLUSIONS: Integrin α1ß1 and CD44 function as the main cell adhesion molecule in highly cell-multilayered periosteal sheets and dispersed cells, respectively. This difference may account for the more potent osteogenic activity shown by the thicker periosteal sheets.

ITGA3 and ITGB4 expression biomarkers estimate the risks of locoregional and hematogenous dissemination of oral squamous cell carcinoma.

BACKGROUND: Molecular biomarkers are essential for monitoring treatment effects, predicting prognosis, and improving survival rate in oral squamous cell carcinoma. This study sought to verify the effectiveness of two integrin gene expression ratios as biomarkers. METHODS: Gene expression analyses of integrin α3 (ITGA3), integrin ß4 (ITGB4), CD9 antigen (CD9), and plakoglobin (JUP) by quantitative real-time PCR were conducted on total RNA from 270 OSCC cases. The logrank test, Cox proportional hazards model, and Kaplan-Meier estimates were performed on the gene expression ratios of ITGA3/CD9 and ITGB4/JUP and on the clinicopathological parameters for major clinical events. RESULTS: A high rate (around 80%) of lymph node metastasis was found in cases with a high ITGA3/CD9 ratio (high-ITGA3/CD9) and invasive histopathology (YK4). Primary site recurrence (PSR) was associated with high-ITGA3/CD9, T3-4 (TNM class), and positive margin, indicating that PSR is synergistically influenced by treatment failure and biological malignancy. A high ITGB4/JUP ratio (high-ITGB4/JUP) was revealed to be a primary contributor to distant metastasis without the involvement of clinicopathological factors, suggesting intervention of a critical step dependent on the function of the integrin ß4 subunit. Kaplan-Meier curves revealed positive margin as a lethal treatment consequence in high-ITGA3/CD9 and YK4 double-positive cases. CONCLUSION: Two types of metastatic trait were found in OSCC: locoregional dissemination, which was reflected by high-ITGA3/CD9, and distant metastasis through hematogenous dissemination, uniquely distinguished by high-ITGB4/JUP. The clinical significance of the integrin biomarkers implies that biological mechanisms such as cancer cell motility and anchorage-independent survival are vital for OSCC recurrence and metastasis.

Tissue-engineered cultured periosteum sheet application to treat infrabony defects: case series and 5-year results.

One-year data after autologous grafting of infrabony periodontal defects with human cultured periosteum sheets in combination with platelet-rich plasma and hydroxyapatite granules have shown favorable clinical and radiographic results. A 5-year follow-up evaluation of 22 selected patients indicated that treated infrabony defects remained stable. Radiographically, there was an increase in osseous radiopacity and bone trabeculation suggesting further bone maturation. This novel tissue-engineered periodontal treatment approach has resulted in significant clinical improvement, and defects remained stable after 5 years.

Biomechanical evaluation by AFM of cultured human cell-multilayered periosteal sheets.

We previously demonstrated that thicker periosteal sheets with enhanced cell layering maintain their component cells at relatively immature stages of differentiation but express a high in vivo osteogenic potential. As it has been recently proposed that stiff scaffolds provide a mechanical cue to various cell types that promotes differentiation, we postulated that the maintenance of immature cells in our periosteal sheets is due to the mechanical stiffness of the multilayered-cell architecture. To demonstrate the biomechanical characteristics of our periosteal sheets, we have determined their stiffnesses with atomic force microscopy (AFM) and evaluated the expression of extracellular matrix (ECM) components specifically by both immunocytochemistry and a complementary DNA microarray technology. Compared to osteoblastic Saos2 cells, the cytoskeletal fibers were developed more in the periosteal cells, but the periosteal cells in monolayer culture developed before either the cells in the peripheral or central regions of the periosteal sheets developed. However, the nanoindentation by AFM distinguished the central region from the peripheral region. The peak stiffness values of cells were ordered as follows: tissue culture polystyrene (1.66GPa)≫dispersed (9.99kPa)>central region (5.20kPa)>peripheral regions (3.67kPa). Similarly, the degree of development of α-smooth muscle actin (αSMA) filaments within cells was dispersed>central region>peripheral region. In conjunction with the abundantly deposited ECM in the periosteal sheets, these findings suggest that the order of cell stiffness may depend on the integration of the stiffness of individual ECM components and the extent of cytoskeletal fiber formation. Because recently published data have demonstrated that the optimal stiffness for osteogenic differentiation is 25-40kPa, it is plausible that the periosteal cells residing in the less-stiff multilayer regions could be maintained at relatively immature stages under the control of the stem-cell medium in vitro but start differentiating when exposed to the proper stiffness upon release from the culture conditions at the implantation site.

[Clinical and microbiological analysis of community-acquired bacteremia admitted to a tertiary teaching hospital].

OBJECTIVES: To investigate clinical and microbiological characteristics of community-acquired bacteremia (CAB). METHODS: We retrospectively analyzed subjects with CAB hospitalized at Saga University Hospital between January 2009 and September 2011. We investigated causative organisms, primary infection sites, and subject summaries and complications, and analyzed the mortality factor. RESULTS: CAB incidence was 185 cases, with 192 organisms cultured. Causative organisms were gram-positive bacteria in 81 strains (42%), 9 (11%) of which were methicillin-resistant Staphylococcus aureus (MRSA). Gram-negative bacteria were identified in 111 strains (58%), with 80% Enterobacteriaceae. Five of the 111 (5%) were caused by extended-spectrum beta-lactamase (ESBL) producing bacteria. The most frequent bacteremia portal was intraabdominal infection (29%, 54/185). During hospitalization of 1-180 days, 20 subjects eventually died. Neutropenia on admission was associated with significantly higher mortality than without (30% vs. 3%, p < 0.001). Septic shock rates were higher in non-survivors than survivors (45% vs. 14%, p = 0.002), and more complications were documented in non-survivors than survivors (50% vs. 25%, p = 0.017). No specific pathogen or primary infection site was associated with higher mortality. CONCLUSIONS: Antimicrobial-resistant pathogens such as MRSA and ESBL producers should be considered even in CAB, especially in subjects with healthcare-associated infection, regardless of how small the number. The CAB treatment course should consider subjects summaries, severity, and complications.

An improved freeze-dried PRP-coated biodegradable material suitable for connective tissue regenerative therapy.

We previously published an investigation indicating freeze-dried platelet-rich plasma (PRP)-coated polyglactin mesh was a promising wound-dressing material. However, one of its disadvantages was the inflammatory nature due to degradation of the polyglactin. Therefore, in this study, we investigated the use of a collagen sponge as the carrier for PRP. When implanted subcutaneously in nude mice, the PRP-coated sponge alone rapidly induced angiogenesis and infiltration of surrounding connective tissue without inducing appreciable inflammation. Moreover, addition of periosteal fibroblastic cells substantially augmented the angiogenic response. With in vitro studies, the PRP-coated sponge provided various major growth factors at high levels to stimulate the proliferation of cells cultured on plastic dishes, but did not stimulate the proliferation of cells inoculated into the PRP-coated sponge. Cells were embedded in the fibrin mesh and maintained their spherical shape without stretching. The atomic force microscopic analysis demonstrated that the fibrin gel formed on the PRP-coated sponge was much softer (approx. 22 kPa) than the cross-linked collagen that formed the sponge base (appox. 1.9 MPa). Because insoluble matrices have recently and increasingly been considered important regulatory factors of cellular behavior, as are soluble growth factors, it is suggested that this soft fibrin mesh possibly suppresses cell survival. Overall, our investigation has successfully demonstrated improved wound-healing and regenerative potential of the PRP-coated mesh by combining it with the collagen sponge. In the clinical setting, this PRP-coated collagen sponge is a promising material for connective tissue regenerative therapy, such as periodontal therapy, burn victim treatment and in cosmetic or plastic surgery.