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.

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.

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.

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.

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.

Bioactivity of freeze-dried platelet-rich plasma in an adsorbed form on a biodegradable polymer material.

Owing to the necessity for the immediate preparation from patients' blood, autologous platelet-rich plasma (PRP) limits its clinical applicability. To address this concern and respond to emergency care and other unpredictable uses, we have developed a freeze-dried PRP in an adsorbed form on a biodegradable polymer material (Polyglactin 910). On the polymer filaments of PRP mesh, which was prepared by coating the polymer mesh with human fresh PRP and subsequent freeze-drying, platelets were incorporated, and related growth factors were preserved at high levels. This new PRP mesh preparation significantly and reproducibly stimulated the proliferation of human periodontal ligament cells in vitro and neovascularization in a chorioallantoic membrane assay. A full-thickness skin defect model in a diabetic mouse demonstrated the PRP mesh, although prepared from human blood, substantially facilitated angiogenesis, granulation tissue formation, and re-epithelialization without inducing severe inflammation in vivo. These data demonstrate that our new PRP mesh preparation functions as a bioactive material to facilitate tissue repair/regeneration. Therefore, we suggest that this bioactive material, composed of allogeneic PRP, could be clinically used as a promising alternative in emergency care or at times when autologous PRP is not prepared immediately before application.

A proposed protocol for the standardized preparation of PRF membranes for clinical use.

Upon clinical application, thick platelet-rich fibrin (PRF) is usually compressed to fit the implantation site. However, it is speculated that the preservation of platelets and plasma content depends on the compression methods used. To accurately evaluate the clinical outcome of PRF, the preparation protocol should be standardized. Freshly prepared PRF clots were compressed into a thin membrane by our novel PRF compression device. The localization of platelets was examined by SEM and immunostaining. Growth factor levels were evaluated by bioassays and cytokine-antibody array techniques. The angiogenic activity was examined by the chick chorioallantoic membrane assay and the scratch assay using HUVEC cultures. Platelets were concentrated on the surface of the region adjacent to the red thrombus and this region was subjected to the experiments. Compared to the PRF membrane compressed by dry gauze (G-PRF), the preservation of the plasma content, 3D-fibrin meshwork, and platelets was more intact in the compressor-prepared PRF membrane (C-PRF). Among the growth factors tested, C-PRF contained PDGF isoforms at higher levels, and significantly stimulated cell proliferation and neovascularization. C-PRF may be useful for grafting while minimizing the loss of bioactive factors. This C-PRF preparation protocol is proposed as a standardized protocol for PRF membrane preparation.

Manual cryopreservation of human alveolar periosteal tissue segments: Effects of pre-culture on recovery rate.

Cultured human periosteal sheets constitute a promising grafting material for periodontal tissue regenerative therapy. However, preparation of these sheets usually requires six weeks or longer, and this lengthy commitment and delay limits both clinical applicability and availability. The aim of this study is to develop an efficient, practical, cost-effective cryopreservation method for periosteal tissue segments (PTSs). Human PTSs were aseptically excised from alveolar bone and pre-cultured in Medium 199+10% fetal bovine serum (FBS) for the indicated number of days before they were slowly frozen down to -75°C in a commercial freezing vessel using medium containing 10% dimethyl sulfoxide (Me(2)SO) and various concentrations of FBS. After fast-thawing at 37°C, PTSs were again cultured, and their growth and responses to standard osteogenic induction were evaluated (vs. freshly excised PTSs). Proliferating cells were obtained at the highest levels from cryopreserved PTSs that were pre-cultured for 14 days before freezing. When a concentration of 50% or more FBS was included in the cryopreservation solution, cells migrated out more actively and grew faster. Importantly, osteoinduction up-regulated alkaline phosphatase (ALP) activity and osteoblastic marker mRNAs in cryopreserved PTS-derived sheets just as effectively as it did in native PTS-derived ones. These data suggest that pre-conditioned PTSs can be efficiently cryopreserved in a freezing solution containing high FBS by traditional manual cryopreservation methods without aid of a program freezer or more elaborate equipment.

In-vivo near-infrared optical imaging of growing osteosarcoma cell lesions xenografted in mice: dual-channel quantitative evaluation of volume and mineralization.

BACKGROUND: In a previous study using a rodent osteosarcoma-grafted rat model, in which cell-dependent mineralization was previously demonstrated to proportionally increase with growth, we performed a quantitative analysis of mineral deposit formation using (99m)Tc-HMDP and found some weaknesses, such as longer acquisition time and narrower dynamic ranges (i.e. images easily saturated). The recently developed near-infrared (NIR) optical imaging technique is expected to non-invasively evaluate changes in living small animals in a quantitative manner. PURPOSE: To test the feasibility of NIR imaging with a dual-channel system as a better alternative for bone scintigraphy by quantitatively evaluating mineralization along with the growth of osteosarcoma lesions in a mouse-xenograft model. MATERIAL AND METHODS: The gross volume and mineralization of osteosarcoma lesions were evaluated in living mice simultaneously with dual-channels by NIR dye-labeled probes, 2-deoxyglucose (DG) and pamidronate (OS), respectively. To verify these quantitative data, retrieved osteosarcoma lesions were then subjected to ex-vivo imaging, weighing under wet conditions, microfocus-computed tomography (µCT) analysis, and histopathological examination. RESULTS: Because of less scattering and no anatomical overlapping, as generally shown, specific fluorescence signals targeted to the osteosarcoma lesions could be determined clearly by ex-vivo imaging. These data were well positively correlated with the in-vivo imaging data (r > 0.8, P < 0.02). Other good to excellent correlations (r > 0.8, P < 0.02) were observed between DG accumulation and tumor gross volume and between OS accumulation and mineralization volume. CONCLUSION: This in-vivo NIR imaging technique using DG and OS is sensitive to the level to simultaneously detect and quantitatively evaluate the growth and mineralization occuring in this type of osteosarcoma lesions of living mice without either invasion or sacrifice. By possible mutual complementation, this dual imaging system might be useful for accurate diagnosis even in the presence of overlapping tissues.

Osteogenic activity of human periosteal sheets cultured on salmon collagen-coated ePTFE meshes.

Our animal implantation studies have demonstrated that, after osteogenic processing, cultured human periosteal sheets form osteoid tissue ectopically without the aid of conventional scaffolding materials. To improve the osteogenic activity of these periosteal sheets, we have tested the effects of including a scaffold made of salmon collagen-coated ePTFE mesh. Periosteal sheets were produced with minimal manipulation without enzymatic digestion. Outgrown cells penetrated into the coated mesh fiber networks to form complex multicellular layers and increased expression of alkaline phosphatase activity in response to the osteoinduction. In vitro mineralization was notably enhanced in the original tissue segment regions, but numerous micro-mineral deposits were also formed on the coated-fiber networks. When implanted subcutaneously into nude mice, periosteal sheets efficiently form osteoid around the mineral deposits. These findings suggest that the intricate three-dimensional mesh composed of collagen-coated fibers substantially augmented the osteogenic activity of human periosteal sheets both in vitro and in vivo.

Impact of Local Drug Delivery of Minocycline on the Subgingival Microbiota during Supportive Periodontal Therapy: A Randomized Controlled Pilot Study.

The aim of this study was to examine the effect of adjunct local minocycline administration on the microbiological parameters of subgingival plaque samples in the residual periodontal pockets. Ten chronic periodontitis patients under a supportive periodontal therapy regimen were recruited. After subgingival debridement, either 2% minocycline gel, Periocline™, (Test Group) or a placebo (Control Group) was administered to the selected sites once a week for three weeks. Subgingival plaque was collected at baseline, and at four weeks and eight weeks. The microbiological composition was analyzed by 16S ribosomal RNA sequencing. In the Test Group, α-diversity (evenness) decreased compared to the baseline (p = 0.005) and was lower compared to the control group at four weeks (p = 0.003). The microbial community composition between the two groups was significantly different at four weeks (p = 0.029). These changes were attributable to a decrease in the bacteria associated with periodontitis and an increase in the bacteria associated with periodontal health. Additionally, the improvement in bleeding on probing continued at eight weeks; however, there were little microbial effects of 2% minocycline gel observed at eight weeks. The control group demonstrated no change throughout the eight-week experimental period. Thus, local minocycline administration can change the subgingival microbial community of residual periodontal pockets.

Treatment of human infrabony periodontal defects by grafting human cultured periosteum sheets combined with platelet-rich plasma and porous hydroxyapatite granules: case series.

In a previous publication from our research group we reported that a combination of platelet-rich plasma (PRP) and hydroxyapatite (HA) granules resulted in a significantly favorable clinical result in treating osseous defects. The aim of this study is to evaluate the clinical response of human cultured periosteum (HCP) sheets in combination with autologous PRP and HA granules used as a tissue-engineered grafting material for the treatment of infrabony defects in three female patients. Periosteum cell specimens were harvested from the mandible of each patient and cultured for six weeks until HCP sheets were formed. The periodontal surgery fully exposed the osseous defects and debridement and root planing were carried out. The coagulated PRP and HA mixture were placed into the osseous defects, and then they were covered with autologous HCP sheets. Six months post-surgery there were gains in clinical attachment and radiographic evidence of infrabony osseous fill. It may be concluded that HCP sheets combined with PRP and HA granules showed significant promise for treating human periodontal infrabony defects. A factor contributing to these favorable clinical results would be the presence of osteogenic cells in the HCP sheets, which provided greater regeneration potential.

Platelet Adhesion on Commercially Pure Titanium Plates in Vitro II. Immunofluorescence Visualization of PDGF-B, TGFß1, 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 TGFß1 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.

Distribution of platelets, transforming growth factor-ß1, platelet-derived growth factor-BB, vascular endothelial growth factor and matrix metalloprotease-9 in advanced platelet-rich fibrin and concentrated growth factor matrices.

AIM: Platelet-rich fibrin (PRF) matrices are compared with regard to their ability to retain and release growth factors. Although this ability is thought to influence regenerative outcomes, it remains unclear how it is regulated. To address this question, we compared advanced PRF (A-PRF) and concentrated growth factor (CGF) matrices in terms of distribution of platelets, transforming growth factor-ß1, platelet-derived growth factor-BB, vascular endothelial growth factor and matrix metalloprotease-9 (MMP9). METHODS: Blood samples were obtained in glass tubes and immediately centrifuged to prepare A-PRF or CGF matrix according to their specific protocols. Both matrices were compressed, embedded in paraffin and subjected to immunohistochemical examination. RESULTS: Leukocytes and plasma proteins were localized on the proximal surface including the interface corresponding to buffy coat. In A-PRF, platelets were distributed homogenously, while growth factors and fibronectin were localized on the distal surface and MMP9 was mainly colocalized with leukocytes. In CGF, in contrast, platelets were localized on and below the proximal surface like leukocytes, growth factors were diffused homogenously and MMP9 was found in the plasma protein layers. CONCLUSION: Although these preparations do not allow accurate quantification, platelet counts and growth factor levels seemed higher and leukocytes were less activated in A-PRF. This may explain A-PRF's higher ability to release growth factors.

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.

Tissue-engineered cultured periosteum used with platelet-rich plasma and hydroxyapatite in treating human osseous defects.

BACKGROUND: The aim of the present controlled clinical study was to compare the clinical response of human cultured periosteum (HCP) sheets in combination with platelet-rich plasma (PRP) and porous hydroxyapatite (HA) granules to a mixture of PRP and HA in the treatment of human infrabony periodontal defects. METHODS: Thirty interproximal infrabony osseous defects in 30 healthy, non-smoking subjects diagnosed with chronic periodontitis were included in this study. The subjects were randomly assigned to the test group (HCP sheets combined with PRP and HA) or the control group (PRP with HA). Clinical and radiographic measurements were made at baseline and the 12-month post-surgical evaluation. RESULTS: Compared to baseline, the 12-month results indicated that both treatment modalities resulted in statistically significant changes (P <0.01) in the gingival index, bleeding on probing, probing depth, clinical attachment level, and radiographic infrabony defect depth. Compared to the control group, the test group exhibited a statistically significantly more favorable change in clinical attachment gain (3.9 +/- 1.6 mm versus 2.7 +/- 1.3 mm; P <0.05), vertical relative attachment gain (83.5% +/- 31.7% versus 55.0% +/- 21.9%; P <0.05), and radiographic infrabony defect fill (4.9 +/- 1.2 mm versus 3.2 +/- 1.1 mm; P <0.01). CONCLUSIONS: Compared to PRP with HA, treatment with a combination of HCP sheets, PRP, and HA led to a significantly more favorable clinical improvement in infrabony periodontal defects. A factor likely contributing to these favorable clinical results is the presence of osteogenic cells in the HCP sheets, which provided greater regeneration potential.

Root coverage with cultured gingival dermal substitute composed of gingival fibroblasts and matrix: a case series.

Cultured gingival dermal substitute (CGDS), composed of gingival fibroblasts and matrix and fabricated using tissue-engineering techniques, has been used for root coverage procedures. Fourteen sites from four patients with > or = 2 mm of Miller Class I or II facial gingival tissue recession were treated. The autologous CGDS sheet, prepared prior to surgical treatment, was grafted over the teeth with gingival recession and then covered with a coronally positioned flap. Vertical and horizontal recession was measured at baseline (prior to the surgical procedure) and 13 to 40 weeks (average: 30.7 +/- 9.6 weeks) after surgery. The average vertical and horizontal root coverage after surgery was 79.1% +/- 25.7% and 75.2% +/- 31.4%, respectively. Moreover, there was a significant increase of keratinized and attached gingival tissue at the final clinical evaluation compared with preoperative measurements (P < .05). These results demonstrate CGDS as a promising grafting material for use with root coverage procedures in periodontal therapy.

Comprehensive Quality Control of the Regenerative Therapy Using Platelet Concentrates: The Current Situation and Prospects in Japan.

Platelet concentrates (PCs), represented by platelet-rich plasma (PRP), have been widely applied in the fields of regenerative and aesthetic therapies. PCs' mechanisms of action, however, are too complicated, and it is not easy to present the whole picture; besides, clinical outcomes are hardly reproducible in many cases. Therefore, several medically advanced countries seemingly intend to regulate PC therapies weakly or strictly because of the increasing popularity. Japan established laws and regulations for PC therapy in the "Act on the Safety of Regenerative Medicine" along with the "Pharmaceuticals, Medical Devices and Other Therapeutic Products Act" in 2014, which, to our knowledge, represent the strictest regulatory framework for production and therapeutic use of PCs in the world. According to these laws and regulations, PCs produced for topical use should be prepared as cell-based medicinal products, essentially as should stem cells, in accordance with their registered ("licensed" under actual conditions) standard operating procedures. Nonetheless, criteria for their quality are not standardized. In this review, we discuss the quality of PC preparations by focusing on the basic concept and regulatory framework of regenerative medicine in Japan. Within the new framework, PC therapy is regulated by a specific notification and registration system, as is stem cell therapy. In comparison with the latter, however, risk factors that hamper successful PC therapy are much fewer. Via appropriate evaluation of patients' conditions and whole-blood samples by simple and sensitive but not yet fully standardized assays, it is theoretically possible that PC quality will be controlled nearly completely. In addition to or instead of standardization of preparation protocols, standardization of preoperative examination of individual PC preparations is an urgent task for improving and guaranteeing the safety and efficacy of PC therapy.

Platelet Counts in Insoluble Platelet-Rich Fibrin Clots: A Direct Method for Accurate Determination.

Platelet-rich fibrin (PRF) clots have been used in regenerative dentistry most often, with the assumption that growth factor levels are concentrated in proportion to the platelet concentration. Platelet counts in PRF are generally determined indirectly by platelet counting in other liquid fractions. This study shows a method for direct estimation of platelet counts in PRF. To validate this method by determination of the recovery rate, whole-blood samples were obtained with an anticoagulant from healthy donors, and platelet-rich plasma (PRP) fractions were clotted with CaCl2 by centrifugation and digested with tissue-plasminogen activator. Platelet counts were estimated before clotting and after digestion using an automatic hemocytometer. The method was then tested on PRF clots. The quality of platelets was examined by scanning electron microscopy and flow cytometry. In PRP-derived fibrin matrices, the recovery rate of platelets and white blood cells was 91.6 and 74.6%, respectively, after 24 h of digestion. In PRF clots associated with small and large red thrombi, platelet counts were 92.6 and 67.2% of the respective total platelet counts. These findings suggest that our direct method is sufficient for estimating the number of platelets trapped in an insoluble fibrin matrix and for determining that platelets are distributed in PRF clots and red thrombi roughly in proportion to their individual volumes. Therefore, we propose this direct digestion method for more accurate estimation of platelet counts in most types of platelet-enriched fibrin matrix.