Effect of Hydroxyapatite/ß-Tricalcium Phosphate on Osseointegration after Implantation into Mouse Maxilla.

In our previous study we established an animal model for immediately placed implants using mice and clarified that there were no significant differences in the chronological healing process at the bone-implant interface between immediately and delayed placed implants blasted with hydroxyapatite (HA)/ß-tricalcium phosphate (ß-TCP) (ratio 1:4). This study aimed to analyze the effects of HA/ß-TCP on osseointegration at the bone-implant interface after immediately placed implants in the maxillae of 4-week-old mice. Right maxillary first molars were extracted and cavities were prepared with a drill and titanium implants, blasted with or without HA/ß-TCP, were placed. The fixation was followed-up at 1, 5, 7, 14, and 28 days after implantation, and the decalcified samples were embedded in paraffin and prepared sections were processed for immunohistochemistry using anti-osteopontin (OPN) and Ki67 antibodies, and tartrate-resistant acid phosphatase histochemistry. The undecalcified sample elements were quantitatively analyzed by an electron probe microanalyzer. Bone formation occurred on the preexisting bone surface (indirect osteogenesis) and on the implant surface (direct osteogenesis), indicating that osseointegration was achieved until 4 weeks post-operation in both of the groups. In the non-blasted group, the OPN immunoreactivity at the bone-implant interface was significantly decreased compared with the blasted group at week 2 and 4, as well as the rate of direct osteogenesis at week 4. These results suggest that the lack of HA/ß-TCP on the implant surface affects the OPN immunoreactivity on the bone-implant interface, resulting in decreased direct osteogenesis following immediately placed titanium implants.

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.

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.

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.

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.

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.

N-Glycosylation with synthetic undecaprenyl pyrophosphate-linked oligosaccharide to oligopeptides by PglB oligosaccharyltransferase from Campylobacter jejuni.

The oligosaccharyltransferase PglB from Campylobacter jejuni catalyses the N-glycosylation reaction with undecaprenyl-pyrophosphate-linked Glc1 GalNAc5 Bac1 (Und-PP-Glc1 GalNAc5 Bac1 ). Experiments using chemically synthesized donors coupled to fluorescently tagged peptides confirmed that biosynthetic intermediate Und-PP-Bac1 and Und-PP-GalNAc2 Bac1 are transferred efficiently to the Asn residue in the consensus sequence (D/E-X'-N-X-T/S, X',X≠P). The products were analyzed in detail by tandem MS to confirm their chemical structures.

Identification and characterization of thermotolerant acetic acid bacteria strains isolated from coconut water vinegar in Sri Lanka.

From the pellicle formed on top of brewing coconut water vinegar in Sri Lanka, three Acetobacter strains (SL13E-2, SL13E-3, and SL13E-4) that grow at 42 °C and four Gluconobacter strains (SL13-5, SL13-6, SL13-7, and SL13-8) grow at 37 °C were identified as Acetobacter pasteurianus and Gluconobacter frateurii, respectively. Acetic acid production by the isolated Acetobacter strains was examined. All three strains gave 4% acetic acid from 6% initial ethanol at 37 °C, and 2.5% acetic acid from 4% initial ethanol at 40 °C. Compared with the two other strains, SL13E-4 showed both slower growth and slower acetic acid production. As well as the thermotolerant SKU1108 strain, the activities of the alcohol dehydrogenase and the aldehyde dehydrogenase of SL13E-2 and SL13E-4 were more stable than those of the mesophilic strain. The isolated strains were used to produce coconut water vinegar at higher temperatures than typically used for vinegar production.

Effect of leukocyte and platelet-rich plasma on osseointegration after implant placement in mouse maxilla.

Introduction: Osseointegration, the direct contact between an implant and bone, can be achieved by direct and/or indirect osteogenesis. Platelet-rich plasma accelerates tissue regeneration, wound healing, and osseointegration. This study aimed to analyze the effects of leukocyte and platelet-rich plasma (L-PRP) on direct and indirect osteogenesis after implant placement in a mouse maxilla. Methods: Blood was collected from the tail vein of 4-8-week-old male ICR mice and L-PRP was obtained after double-spin cycle centrifugation. After the right upper first molars of 4-week-old ICR mice were extracted while under deep anesthesia, the alveolar sockets were prepared with a drill, and titanium implants blasted with hydroxyapatite/ß-tricalcium phosphate were placed into the cavity filled with 1.5 µL of L-PRP. Samples were collected from the animals 3-28 days after implantation, and immunohistochemistry for osteopontin, Ki67 (cell proliferation marker), cathepsin-K (osteoclast marker), and osteonectin (osteoblast marker) was performed. Results: Cell proliferation was significantly higher in the L-PRP group than in the control group on postoperative days 3 and 5. The activities of osteoclast-lineage cells and osteoblasts increased significantly on day 5 in the L-PRP group, indicating that L-PRP evoked an active cellular response. Indirect osteogenesis was significantly higher on days 7, 14, and 28, and the osseointegration rate was significantly higher on day 28 in the L-PRP group compared with the control group. Conclusions: L-PRP enhances osseointegration by promoting mesenchymal cell proliferation, osteoclastic and osteoblastic activities, and indirect osteogenesis.

Plasma Gel Matrix as a Promising Carrier of Epigallocatechin Gallate for Regenerative Medicine.

Plasma gel (PG) is a protein matrix prepared from platelet-poor plasma and can be utilized as a drug carrier for controlled release. We previously demonstrated its applicability as a carrier of polyphosphate. Epigallocatechin-3-gallate (EGCG) is the main flavonoid found in green tea and functions as a strong antioxidant. To explore the applicability of PG as an EGCG carrier, we examined the release of EGCG from the PG matrix using an in vitro system. Pooled platelet-poor plasma (PPP) was prepared from four healthy adult male donors, mixed with EGCG, and heated at 75 °C for 10 or 20 min to prepare the PG matrix. The PG-EGCG matrix was incubated in PBS at 37 °C, and the EGCG released into PBS was determined using spectrophotometry. The antioxidant capacity was determined based on the principle of the iodine decolorization reaction. EGCG precipitated and incorporated into the PG matrix during thermal preparation. Trypsin, used to simulate the in vivo degradation of PG, released EGCG from the PG matrix over time. The released EGCG maintained its antioxidant capacity during incubation. These results indicate that thermally prepared PG matrices can be utilized as a promising EGCG carrier in the fields of tissue engineering and regenerative medicine.

Inhibitory effects of NaF on mitochondrial energy generation in human platelets in vitro.

Background: fluoride is a beneficial ion that has been used in various fields, from industrial products to therapeutics. However, due to its narrow therapeutic index, fluoride sometimes acts as a toxic agent at relatively higher concentrations in the human body. Based on the interest in genetic stability, its cytotoxic effects have been investigated mainly in nucleated, adherent cells, such as fibroblasts. However, the sensitivity of blood cells, especially anucleate platelets, to fluoride is poorly understood. To fill this gap in the literature, we investigated the effects of relatively low levels of fluoride on platelet energy metabolism, function, and viability. Methods: Platelet-rich plasma (PRP) was prepared from 15 non-smoking healthy male adults (age: 28-63) and treated with NaF (0.5 or 1.0 mM) in microtubes for up to 3 days. Platelet function was evaluated based on aggregation and adhesion activities. Platelet energy metabolism was evaluated based on intracellular ATP levels, extracellular lactate levels, and respiration activities. The mitochondrial membrane potential (Em) and localization of reactive oxygen species (ROS) were visualized using cytochemical methods. Platelet viability was evaluated by cell counting and tetrazolium reduction. Result: NaF (1 mM) significantly reduced platelet viability and inhibited functions. Behind these phenomena, NaF substantially decreased mitochondrial Em and increased ROS production along with significant decreases in oxygen consumption and ATP levels. Simultaneously, NaF increased the lactate levels. Although not statistically significant, similar effects were observed at 0.5 mM NaF. Conclusion: At relatively low levels, NaF has the potential to attenuate platelet function probably primarily through the inhibition of mitochondrial energy generation. Cytotoxicity may be directly related to ROS production. These findings suggest that when used topically, for example, for caries prevention in the oral cavity, NaF could interfere with wound healing and tissue regeneration by endogenous and exogenously added platelets in the form of PRP.

Metformin-suppressed platelet's function in vitro: Possible relation to delayed or failure of platelet-rich fibrin preparation.

Platelet-rich fibrin (PRF) is a popular autologous blood-derived biomaterial that is used in regenerative therapy. Owing to its simple preparation without additional factors, the PRF quality directly reflects the characteristics of individual blood samples. Antiplatelet or anticoagulant drugs can hamper the successful preparation of PRF. We recently observed similar phenomena in metformin-taking type-2 diabetics (T2DM). Thus, we hypothesized that metformin interferes with platelet function, thereby suppressing coagulation. For practical reasons, leukocyte- and platelet-rich plasma was prepared from healthy male donors (n = 9-15, age: 26-80 years) and treated with metformin (1-10 mM) for 24-72 h. Intrinsic and extrinsic coagulation activities were evaluated using prothrombin time (PT) and activated partial thromboplastin time (ATPP). Platelet adhesion and aggregation assays were performed using ADP stimulation. Among the parameters tested, APTT was the most sensitive and was significantly prolonged in the concentration range of 1-10 mM in a time- and concentration-dependent manner. Although obtained from healthy platelets and relatively higher concentrations of metformin, these findings suggest that metformin may induce further dysfunction of platelets to suppress intrinsic coagulation activity in T2DM patients, leading to failure of PRF preparation. This phenomenon may not have a severe impact on clinical diabetology or hematology. However, clinicians using PRF are recommended to be more sensitive to such information to avoid unexpected events in clinical settings.

Plasma Gel Made of Platelet-Poor Plasma: In Vitro Verification as a Carrier of Polyphosphate.

Plasma gel (PG) is a blood-derived biomaterial that can be prepared by heating or chemical cross-linking without the aid of intrinsic coagulation activity and has gradually been applied in the field of esthetic surgery. To explore the applicability of PG in regenerative therapy or tissue engineering, in this study, we focused on the advantages of the heating method and verified the retention capacity of the resulting PG for polyphosphate (polyP), a polyanion that contributes to hemostasis and bone regeneration. Pooled platelet-poor plasma (PPP) was prepared from four healthy male adult donors, mixed with synthetic polyP, and heated at 75 °C for 10 or 30 min to prepare PG in microtubes. The PG was incubated in PBS at 37 °C, and polyP levels in the extra-matrix PBS were determined by the fluorometric method every 24 h. The microstructure of PG was examined using scanning electron microscopy. In the small PG matrices, almost all of the added polyP (~100%) was released within the initial 24 h. In contrast, in the large PG matrices, approximately 50% of the polyP was released within the initial 24 h and thereafter gradually released over time. Owing to its simple chemical structure, linear polyP cannot be theoretically retained in the gel matrices used in this study. However, these findings suggest that thermally prepared PG matrices can be applied as carriers of polyP in tissue engineering and regenerative medicine.

Responses of promyelocytic leukemia HL60 cells as an inflammatory cell lineage model to silica microparticles used to coat blood collection tubes.

BACKGROUND: The preparation of platelet-rich fibrin (PRF) requires glass blood collection tubes, and thus, the shortage or unavailability of such tubes has driven clinicians to search for suitable substitutes, such as silica-coated plastic tubes. However, we have previously demonstrated the cytotoxicity of silica microparticles (MPs) used in plastic tubes to cultured human periosteal cells. To further establish the effects of silica MPs on inflammation, we examined silica MP-induced changes in a human promyelocytic cell model in vitro. METHODS: Human promyelocytic HL60 cells were used either without chemical induction or after differentiation induced using phorbol myristate acetate (PMA) or dimethyl sulfoxide. HL60 cells, osteoblastic MG63, and Balb/c mouse cells were treated with silica MPs, and their surface ultrastructure and numbers were examined using a scanning electron microscope and an automated cell counter, respectively. Differentiation markers, such as acid phosphatase, non-specific esterase, and CD11b, were visualized by cytochemical and immunofluorescent staining, and superoxide dismutase (SOD) activity was quantified. RESULTS: Regardless of SOD activity, silica cytotoxicity was observed in MG63 and Balb/c cells. At sub-toxic doses, silica MPs slightly or moderately upregulated the differentiation markers of the control, PMA-induced monocytic, and dimethyl sulfoxide-induced granulocytic HL60 cells. Although SOD activity was the highest (P < 0.05) in PMA-induced cells, a silica-induced reduction in cell adhesion was observed only in those cells (P < 0.05). CONCLUSIONS: Silica MP contamination of PRF preparations can potentially exacerbate inflammation at implantation sites. Consequently, unless biomedical advantages can be identified, silica-coated plastic blood collection tubes should not be routinely used for PRF preparations.

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.

In vivo imaging of endoplasmic reticulum and distribution of mutant α-amylase in Aspergillus oryzae.

Properly folded proteins destined for secretion exit through a specific subdomain of the endoplasmic reticulum (ER) known as transitional ER (tER) sites or ER exit sites (ERES). While such proteins in filamentous fungi localize at the hyphal tips overlapping the Spitzenkörper, the distribution of misfolded proteins remains unknown. In the present study, we analyzed the distribution of mutant protein as well as ER and tER sites visualized by expression of AoClxA and AoSec13 fused with fluorescent protein, respectively, in the filamentous fungus Aspergillus oryzae. Discrete tER subdomains were visualized as the punctate dots of AoSec13 overlapping or associated with AoClxA distribution. Both ER and tER sites were concentrated near hyphal tips and formed apical gradients. Interestingly, while the expression of wild-type α-amylase fusion protein (AmyB-mDsRed) showed its localization coinciding with the Spitzenkörper, a disulfide bond-deletion in AmyB causing its misfolding resulted in its accumulation in the subapical and basal ER, creating a reciprocal gradient to the tER sites. Furthermore, the reciprocal gradient enabled a clear distinction between the tER sites and the mutant AmyB accumulation sites near the apex. Based on these findings, we conclude that A. oryzae accumulates aberrant proteins toward basal hyphae while maintaining polarized tER sites for secretion of properly folded proteins at the hyphal tip.

Aspergillus luchuensis fatty acid oxygenase ppoC is necessary for 1-octen-3-ol biosynthesis in rice koji.

Awamori is a distilled spirit produced in Okinawa Prefecture, in southern Japan. Awamori contains the volatile organic compound 1-octen-3-ol, an important flavor component. Here, using solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GCMS), we demonstrate that the black koji mold Aspergillus luchuensis produces 1-octen-3-ol in rice koji. To examine the role of the fatty acid oxygenase genes ppoA and ppoC in 1-octen-3-ol biosynthesis by A. luchuensis, we constructed ppoA and ppoC disruptants, ΔppoA and ΔppoC, respectively, via protoplast-PEG transformation. No clear differences in growth and conidiation were observed between the transformants and the parent strain. Volatile compounds in rice koji prepared using these gene disruptants were analyzed by SPME-GCMS. The amount of 1-octen-3-ol contained in koji produced by the ΔppoA strain was the same as that produced by the parental strain. In contrast, although the ΔppoC strain grew on the rice koji, 1-octen-3-ol was not detected. These results indicate that ppoC is involved in 1-octen-3-ol biosynthesis in A. luchuensis.

Awamori fermentation test and 1-octen-3-ol productivity analysis using fatty acid oxygenase disruptants of Aspergillus luchuensis.

1-Octen-3-ol is a major aroma component of awamori, a traditional distilled liquor produced in Okinawa Prefecture, Japan. As 1-octen-3-ol is thought to affect the sensory properties of awamori, it is important to fully characterize the compound's biosynthetic pathway and control mechanism. We previously reported that the fatty acid oxygenase ppoC (ppo: psi-produced oxygenase) of Aspergillus luchuensis is directly involved in the production of 1-octen-3-ol in rice koji (Kataoka et al., J. Biosci. Bioeng., 129, 192-198, 2020). In the present study, we constructed A. luchuensis ppoD disruptants to characterize the role of ppo genes in 1-octen-3-ol biosynthesis. A small-scale awamori fermentation test was performed using ppoA, ppoC, and ppoD single disruptants (ΔppoA, ΔppoC, and ΔppoD, respectively), along with the parent strain, ΔligD. 1-Octen-3-ol was not detected in the distillate prepared using the ΔppoC strain. We conclude that A. luchuensis ppoC is the only 1-octen-3-ol-producing factor in the awamori brewing process. Because ΔppoA and ΔppoD slightly enhanced 1-octen-3-ol productivity, these two genes may play a role in negatively controlling 1-octen-3-ol biosynthesis.

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.

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.