Fluorescent Cytochemical Detection of Polyphosphates Associated with Human Platelets

Polyphosphate (polyP) is released from activated platelets and activates the intrinsic coagulation pathway. However, polyP may also be involved in various pathophysiological functions related to platelets. To clarify these functions, we established a cytochemical method to reproducibly visualize polyP in platelets. Platelets obtained from healthy non-smoking donors were suspended in phosphate-buffered saline and quickly immobilized on glass slides using a Cytospin. After fixation and membrane permeabilization, platelets were treated with 4′,6- diamidino-2-phenylindole (DAPI) and examined using a fluorescence microscope with a blue-violet excitation filter block (BV-2A). Fixed platelets were also subjected to immunocytochemical examination to visualize serotonin distribution. Under the optimized conditions for polyP visualization, immobilized platelets were fixed with 10% neutral-buffered formalin for 4 h or longer and treated with DAPI at a concentration of 10 µg/mL in 0.02% saponin- or 0.1% Tween-20-containing Hanks balanced salt solution as a permeabilization buffer for 30 min at room temperature (22–25 °C). Based on the results obtained by using activated platelets, treatment with alkaline phosphatases, and serotonin release, the DAPI+ targets were identified as polyP. Therefore, this cytochemical method is useful for determining the amount and distribution of polyP in platelets

An intraductal papilloma of Blandin-Nuhn's gland and its consideration from the literature

本論文の要旨は平成5年10月の第38回日本口腔外科学会総会において発表した

An intraductal papilloma of Blandin-Nuhn's gland and its consideration from the literature

本論文の要旨は平成5年10月の第38回日本口腔外科学会総会において発表した

Platelet Adhesion on Commercially Pure Titanium Plates in Vitro II. Immunofluorescence Visualization of PDGF-B, TGFβ1, and PPARγ Released from Activated Adherent Platelets

Recent progress in the industrial development of dental implants has improved their surface bio-affinity, while clinical implantologists attempt to improve it through coating with various compounds, including platelet-rich plasma (PRP) in clinical settings. However, it is poorly understood how PRP acts on titanium surfaces. To validate this surface modification method and demonstrate how platelet-derived soluble biomolecules released from the activated adherent platelets act on plain, commercially pure-titanium (cp-Ti) plates, we evaluated the distribution of biomolecules by immunofluorescence. PPARγ, PDGF-B, and TGFβ1 were similarly released at immunofluorescence levels from activated adherent platelets, retained in the surrounding extra-platelet spaces for a while, and did not immediately diffuse away to distant spaces. Exogenously added CaCl2 augmented release and retention of those biomolecules along with activation and aggregation. Taken together with our previous data regarding platelet adhesion, these findings suggest that especially when treated with CaCl2, platelets immediately adhere on cp-Ti plates to release their stored biomolecules in the absence of plasma proteins and that these biomolecules do not diffuse away, but stay longer in extra-platelet spaces around the platelets by newly formed, immature fibrin fiber fragments. Consequently, these retained biomolecules are anticipated to cooperatively stabilize implants by stimulating alveolar bone regeneration and integration

Clinical Investigations of Two Advanced Aged Patients Demanding General Control during Dental Anaesthesia

本論文の要旨は平成4年7月の第7回中国四国歯科麻酔科研究会において発表した

Platelet adhesion on commercially pure titanium plates in vitro I: effects of plasma components and involvement of the von Willebrand factor and fibronectin

Abstract Background Platelet-rich plasma (PRP) is widely used in regenerative dentistry. Furthermore, it is often applied in the pretreatment of titanium implants to improve their surface bioaffinity and initial stability. However, effects of PRP application on implant surface at cellular and molecular levels remain poorly understood. Therefore, we examined platelet adhesion on commercially pure titanium (cp-Ti) plates, with a particular focus on fibrinogen (FGN), von Willebrand factor (vWF), and fibronectin (FN), in the presence or absence of plasma components. Methods Citrated blood samples were obtained from six healthy male volunteers, and pure-PRP (P-PRP) and pure platelet suspensions in phosphate-buffered saline (PBS) were prepared. Platelet adhesion on cp-Ti plate surface was evaluated by phalloidin staining and tetrazolium dye assay. Distribution of FGN, vWF, FN, albumin, CD62P, and CD63 was examined by immunocytochemical analysis. Results Platelets in PBS suspensions rapidly and time-dependently adhered to cp-Ti plate surface, but this adhesion was substantially disturbed by the presence of plasma components. FGN was most preferably adsorbed regardless of the presence or absence of plasma components, while vWF and FN showed greater accumulation on platelet adhesion area. Conclusions Although FGN is rapidly and abundantly adsorbed on cp-Ti plate surface, vWF and FN function as major platelet adhesion molecules in citrated blood samples. After pretreatment with P-PRP, however, platelets adhered to cp-Ti much less efficiently. Therefore, P-PRP pretreatment might not directly contribute to surface functionalization, initial stabilization, and osseointegration of machined or similar types of implants

Evidence for Contamination of Silica Microparticles in Advanced Platelet-Rich Fibrin Matrices Prepared Using Silica-Coated Plastic Tubes

Platelet-rich fibrin (PRF) therapy has been widely applied in regenerative dentistry, and PRF preparation has been optimized to efficiently form fibrin clots using plain glass tubes. Currently, a shortage of commercially available glass tubes has forced PRF users to utilize silica-coated plastic tubes. However, most plastic tubes are approved by regulatory authorities only for diagnostic use and remain to be approved for PRF therapy. To clarify this issue, we quantified silica microparticles incorporated into the PRF matrix. Blood samples were collected into three different brands of silica-containing plastic tubes and were immediately centrifuged following the protocol for advanced-PRF (A-PRF). Advanced-PRF-like matrices were examined using a scanning electron microscope (SEM), and silica microparticles were quantified using a spectrophotometer. Each brand used silica microparticles of specific size and appearance. Regardless of tube brands and individual donors, significant, but not accidental, levels of silica microparticles were found to be incorporated into the A-PRF-like matrix, which will be consequently incorporated into the implantation sites. Presently, from the increasing data for cytotoxicity of amorphous silica, we cannot exclude the possibility that such A-PRF-like matrices negatively influence tissue regeneration through induction of inflammation. Further investigation should be performed to clarify such potential risks

Distribution and quantification of activated platelets in platelet-rich fibrin matrices.

Platelet-rich fibrin (PRF) has been widely applied in regenerative therapy owing to its simple preparation protocol. To date, the original protocol for preparing leukocyte-rich (L)-PRF has been modified to produce derivatives such as advanced (A)-PRF, concentrated growth factors (CGF), and horizontal (H)-PRF. However, these derivatives have not been rigorously compared to explore possible differences. We previously developed and validated a nondestructive near-infrared (NIR) imaging method to quantitatively examine the platelet distribution in PRF matrices. To further evaluate the characteristics of platelets in PRF, we herein examined the distribution of activated platelets. Four types of PRF matrices were prepared under different centrifugal conditions from blood samples obtained from the same healthy donors. After fixation and compression, the matrices were stained immunohistochemically without sectioning and visualized using an NIR imager. Qualitative morphological analysis revealed that whole platelets were distributed widely and homogeneously in H-PRF and A-PRF, but localized along the distal tube surface in L-PRF and CGF. Activated platelets were distributed as were whole platelets in A-PRF, L-PRF, and CGF, but localized mainly in the "buffy coat" region in H-PRF. Quantitative analysis revealed that there was no significant difference in the ratio of activated to whole platelets between PRF derivatives. These findings suggest that platelet activation is similarly induced in fibrin matrices regardless of centrifugal speed or rotor angulation. However, only the H-PRF group was distinguishable from the other PRF derivatives in terms of activated platelet distribution

Fluorometric Quantification of Human Platelet Polyphosphate Using 4′,6-Diamidine-2-phenylindole Dihydrochloride: Applications in the Japanese Population

Polyphosphate (polyP), a biopolymer of inorganic phosphate, is widely distributed in living organisms. In platelets, polyP is released upon activation and plays important roles in coagulation and tissue regeneration. However, the lack of a specific quantification method has delayed the in-depth study of polyP. The fluorescent dye 4′,6-diamidine-2-phenylindole dihydrochloride (DAPI) has recently received attention as a promising probe for the visualization and quantification of cellular polyP levels. In this study, we further optimized quantification conditions and applied this protocol in quantification of platelet polyP levels in a Japanese population. Blood samples were collected from non-smoking, healthy Japanese subjects (23 males, 23 females). Washed platelets were fixed and probed with DAPI for fluorometric determination. PolyP levels per platelet count were significantly higher in women than that in men. A moderate negative correlation between age and polyP levels was found in women. Responsiveness to CaCl2 stimulation was also significantly higher in women than that in men. Overall, our optimized protocol requires neither purification nor degradation steps, reducing both the time and bias for reproducible quantification. Thus, we suggest that despite its low specificity, this DAPI-based protocol would be useful in routine laboratory testing to quantify platelet polyP levels efficiently and economically