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.

Effects of SARS-CoV-2 mRNA vaccines on platelet polyphosphate levels and inflammation: A pilot study.

Platelets function as immune cells in conjunction with white blood cells, targeting invading pathogens and inducing immune reactions. Intercellular communications among these immune cells are partly mediated by platelet polyphosphate (polyP), which was originally recognized as a thrombotic and hemostatic biomolecule. To determine the involvement of polyP in SARS-CoV-2-mRNA vaccine-induced immune responses, specifically in inflammatory responses, the effects of mRNA vaccines on platelet polyP levels were examined. Before and after vaccination with the COVID-19 vaccine (BNT162b2), blood samples were obtained from healthy, non-smoking individuals who did not have any systemic diseases. Test group demographics skewed somewhat towards either older males (first vaccination, n=6; second vaccination, n=8) or younger females (first vaccination, n=14; second vaccination, n=23). polyP levels in washed platelets from the blood samples were determined using the fluorometric method with DAPI. The side-effects of vaccination were recorded as scores. In the female group, platelet polyP levels decreased after the first vaccination, and the side-effect score increased after the second vaccination. Moderate correlation coefficients were observed between the reduction in polyP levels and the side-effect scores and pre-vaccination polyP levels. Despite the small sample size, this pilot study suggests that platelet polyP may suppress the side effects induced by the mRNA vaccines after the first vaccination, but not the second vaccination in younger female subjects, who generally have higher immune responsiveness than their male counterparts.

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.

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.

Platelet adhesion on commercially pure titanium plates in vitro III: effects of calcium phosphate-blasting on titanium plate biocompatibility.

BACKGROUND: Platelet-rich plasma (PRP) is often used to improve surface biocompatibility. We previously found that platelets rapidly adhere to plain commercially pure titanium (cp-Ti) plates in the absence, but not in the presence, of plasma proteins. To further expand on these findings, in the present study, we switched titanium plates from a plain surface to a rough surface that is blasted with calcium phosphate (CaP) powder and then examined platelet adhesion and activation. METHODS: Elemental distribution in CaP-blasted cp-Ti plates was analyzed using energy-dispersive X-ray spectroscopy. PRP samples prepared from anticoagulated blood samples of six healthy, non-smoking adult male donors were loaded on CaP-blasted cp-Ti plates for 1 h and fixed for examination of platelet morphology and visualization of PDGF-B and platelet surface markers (CD62P, CD63) using scanning electron microscopy and fluorescence microscopy. Plain SUS316L stainless steel plates used in injection needles were also examined for comparison. RESULTS: Significant amounts of calcium and phosphate were detected on the CaP-blasted cp-Ti surface. Platelets rapidly adhered to this surface, leading to higher activation. Platelets also adhered to the plain stainless surface; however, the levels of adhesion and activation were much lower than those observed on the CaP-blasted cp-Ti plate. CONCLUSIONS: The CaP-blasted cp-Ti surface efficiently entraps and activates platelets. Biomolecules released from the activated platelets could be retained by the fibrin matrix on the surface to facilitate regeneration of the surrounding tissues. Thus, PRP immersion could not only eliminate surface air bubbles but also improve the biocompatibility of the implant surface.

Concentrated Growth Factor Matrices Prepared Using Silica-Coated Plastic Tubes Are Distinguishable From Those Prepared Using Glass Tubes in Platelet Distribution: Application of a Novel Near-Infrared Imaging-Based, Quantitative Technique.

Platelet-rich fibrin (PRF) matrices were originally prepared using plain glass tubes without the aid of coagulation factors because coagulation factor XII is activated by glass surfaces. Recently, the use of silica-coated plastic tubes as a substitute of glass tubes has been recommended for PRF preparation. This recommendation is owing not only to the shortage of glass tubes for medical use in the market, but also the higher coagulation activity of silica-coated plastic tubes and equal quality of PRF. However, these matrices are not the same. To evaluate the differences, we compared glass- and silica-coated plastic tubes in terms of platelet distribution and quantity in concentrated growth factors (CGF). CGF matrices were immediately prepared from freshly collected blood samples, fixed after red thrombus removal, and divided into two equal pieces sagittally. One piece was used for CD41 detection and the other was applied as an isotype control. Platelet distribution in CGF matrices was examined, without embedding or sectioning, by a novel method using invisible near-infrared imaging. The dehydrated membranous CGF matrix was more transparent. Thus, the fluorescence signal was clearly detectable with less scattering. Platelets were distributed mainly in the distal side of the glass-prepared CGF matrix, but homogeneously in the silica-prepared CGF matrix. Platelet count was positively correlated with fluorescence intensity. Although not yet fully developed, this imaging technique enabled us to recognize the differences in platelet distribution and quantity in CGF matrices by excluding bias caused by the technical limitations of scanning electron microscopy and conventional immunohistochemical methods.

Quantitative Near-Infrared Imaging of Platelets in Platelet-Rich Fibrin (PRF) Matrices: Comparative Analysis of Bio-PRF, Leukocyte-Rich PRF, Advanced-PRF and Concentrated Growth Factors.

Platelet-rich fibrin (PRF) is a fibrin matrix enriched with platelets. The PRF matrix is thought to form a steep gradient of platelet density around the region corresponding to the buffy coat in anticoagulated blood samples. However, this phenomenon has not yet been proven. To visualize platelet distribution in PRF in a non-invasive manner, we utilized near-infrared (NIR) imaging technology. In this study, four types of PRF matrices, bio-PRF, advanced-PRF (A-PRF), leukocyte-rich PRF (L-PRF), and concentrated growth factors (CGF) were compared. Blood samples collected from healthy, non-smoking volunteers were immediately centrifuged using four different protocols in glass tubes. The fixed PRF matrices were sagittally divided into two equal parts, and subjected to modified immunohistochemical examination. After probing with NIR dye-conjugated secondary antibody, the CD41+ platelets were visualized using an NIR imager. In L-PRF and CGF, platelets were distributed mainly on and below the distal surface, while in bio-PRF and A-PRF, platelet distribution was widespread and homogenous. Among three regions of the PRF matrices (upper, middle, and lower), no significant differences were observed. These findings suggest that platelets aggregate on polymerizing fibrin fibers and float up as a PRF matrix into the plasma fraction, amending the current "gradient" theory of platelet distribution.

A Comparative Study of The Effects of Anticoagulants on Pure Platelet-Rich Plasma Quality and Potency.

It is generally accepted that citrate or the A-form of acid-citrate-dextrose (ACD-A) are suitable for preparing platelet-rich plasma (PRP) for regenerative therapy. However, this is based on evidence from blood transfusions and not from regenerative medicine. Thus, we examined the effects of anticoagulants, such as ACD-A, ethylenediaminetetraacetic acid (EDTA), and heparin, on the regenerative quality of PRP to address this gap. The blood samples were collected in the presence of anticoagulants and were processed to prepare pure-PRP. Platelet size, activation status, and intra-platelet free Ca2+ concentration were determined while using a hematology analyzer and flow cytometer. Platelet-derived growth factor-BB (PDGF-BB) was quantified while using an ELISA. In pure-PRP samples, EDTA caused platelet swelling and activation, but yielded the highest number of platelets. Heparin aggregated platelets and disturbed the overall counting of blood cells. However, no significant differences in PDGF-BB levels were observed among the anticoagulants tested. Moreover, when considering the easy preparation of platelet suspensions, without the need for high-level pipetting skills, these findings suggest the comparable potency of EDTA-derived pure-PRP in tissue regeneration and support the use of EDTA in the preparation of pure-PRP. Further in vivo studies are required in animal models to exclude the possible negative effects of including EDTA in pure-PRP preparations.

Acute cytotoxic effects of silica microparticles used for coating of plastic blood-collection tubes on human periosteal cells.

Because of its simple operation, platelet-rich fibrin (PRF) is becoming more popular than the original form, platelet-rich plasma (PRP), in regenerative dentistry. PRF preparation requires plain glass blood-collection tubes, but not either anticoagulants or coagulation factors. However, such glass tubes designed for laboratory testing are no longer commercially available. Although several glass tubes specifically designed for PRF preparation are available, many clinicians prefer to obtain stably supplied substitutes, such as silica-coated plastic tubes produced by major medical device companies. The quality of PRF prepared by silica-coated tubes has not been assessed and we previously reported significant contamination of silica microparticles in the resulting PRF matrix and alerted clinicians against the use for PRF preparation. To further assess the biosafety of the silica microparticles, we presently examined their effects on human normal periosteal cells derived from alveolar bone. The periosteal cells were obtained from explant cultures of small periosteal tissues obtained from healthy donors. Silica microparticles were obtained from silica-coated tubes and added to cell cultures. Cellular responses were monitored using a tetrazolium assay, phase-contract inverted microscopy, an immunofluorescence method, and scanning electron microscopy. Silica microparticles adsorbed onto the cell surface with seemingly high affinity and induced apoptosis, resulting in significant reduction of cell proliferation and viability. These findings suggest that silica microparticles contained in plastic tubes for the purpose of blood coagulation are hazardous for various cell types around sites where silica-contaminated PRF matrices are implanted.

Striking Differences in Platelet Distribution between Advanced-Platelet-Rich Fibrin and Concentrated Growth Factors: Effects of Silica-Containing Plastic Tubes.

Compared with platelet-rich plasma, the preparation of platelet-rich fibrin (PRF) is simple and has not been overly modified. However, it was recently demonstrated that centrifugation conditions influence the composition of PRF and that silica microparticles from silica-coated plastic tubes can enter the PRF matrix. These factors may also modify platelet distribution. To examine these possibilities, we prepared PRF matrices using various types of blood-collection tubes (plain glass tubes and silica-containing plastic tubes) and different centrifugation speeds. The protocols of concentrated growth factors and advanced-PRF represented high- and low-speed centrifugation, respectively. Platelet distribution in the PRF matrix was examined immunohistochemically. Using low-speed centrifugation, platelets were distributed homogeneously within the PRF matrix regardless of tube types. In high-speed centrifugation, platelets were distributed mainly on one surface region of the PRF matrix in glass tubes, whereas in silica-coated tubes, platelet distribution was commonly more diffusive than in glass tubes. Therefore, both blood-collection tube types and centrifugal conditions appeared to influence platelet distribution in the PRF matrix. Platelets distributed in the deep regions of the PRF matrix may contribute to better growth factor retention and release. However, clinicians should be careful in using silica-coated tubes because their silica microparticles may be a health hazard.

Spectrophotometric Determination of the Aggregation Activity of Platelets in Platelet-Rich Plasma for Better Quality Control.

Although platelet-rich plasma (PRP) is now widely used in regenerative medicine and dentistry, contradictory clinical outcomes have often been obtained. To minimize such differences and to obtain high quality evidence from clinical studies, the PRP preparation protocol needs to be standardized. In addition, emphasis must be placed on quality control. Following our previous spectrophotometric method of platelet counting, in this study, another simple and convenient spectrophotometric method to determine platelet aggregation activity has been developed. Citrated blood samples were collected from healthy donors and used. After centrifugation twice, platelets were suspended in phosphate buffered saline (PBS) and adenosine diphosphate (ADP)-induced aggregation was determined using a spectrophotometer at 615 nm. For validation, platelets pretreated with aspirin, an antiplatelet agent, or hydrogen peroxide (H2O2), an oxidative stress-inducing agent, were also analyzed. Optimal platelet concentration, assay buffer solution, and representative time point for determination of aggregation were found to be 50-100 × 104/µL, PBS, and 3 min after stimulation, respectively. Suppressed or injured platelets showed a significantly lower aggregation response to ADP. Therefore, it suggests that this spectrophotometric method may be useful in quick chair-side evaluation of individual PRP quality.