Characterization of Leukocyte- and Platelet-Rich Plasma Derived from Female Collage Athletes: A Cross-Sectional Cohort Study Focusing on Growth Factor, Inflammatory Cytokines, and Anti-Inflammatory Cytokine Levels.

Platelet-rich plasma (PRP) has been increasingly used in sports medicine owing to its various advantages. The purpose of our project was to standardize the parameters before performing large-scale clinical trials in the near future to precisely evaluate individual PRP quality. To examine the effects of regular exercise on PRP quality, this study focused on young female athletes, who have been relatively less studied. Blood samples were obtained from female college athletes (n = 35) and ordinary healthy adults (n = 30), which were considered as controls, and leukocyte-rich PRP (L-PRP) was prepared manually. Body composition indices were determined using a bathroom weight scale equipped with an impedance meter. Growth factors and cytokines were quantified using ELISA kits. Platelet-derived growth factor-BB (PDGF-BB) and Transforming-growth factors ß1 (TGFß1) levels (per platelet) in L-PRP were significantly lower in female athletes than in controls. In contrast, Interleukin-1ß and Interleukin 1 receptor antagonist (IL-1RA) levels (per platelet and L-PRP) in L-PRP were significantly higher in athletes, and this difference was more prominent in IL-1RA. These findings suggest that L-PRP from athletes may facilitate the inflammatory phase of the healing process by regulating the pro-inflammatory and anti-inflammatory balance. These chemical compositions can be adopted as "must-check" parameters to characterize individual PRP preparations prior to clinical trials.

Elevated IL-1ß and Comparable IL-1 Receptor Antagonist Levels Are Characteristic Features of L-PRP in Female College Athletes Compared to Male Professional Soccer Players.

Autologous platelet-rich plasma (PRP) therapy has been becoming popular for the treatment of musculotendinous injuries among athletes. However, for individual and practical variations, clinical success is hardly predictable. To overcome this difficulty, we have been exploring possible criterion candidates for monitoring its clinical effectiveness. In this study, we focused on sex-based differences in young elite athletes and compared the biochemical compositions of their PRP. Leukocyte-rich PRP (L-PRP) was manually prepared from blood samples collected from male professional soccer players (mPSPs) (n = 25) and female college athletes (fCAs) (n = 36). Platelet-derived growth factor-BB (PDGF-BB), transforming-growth factor-ß1 (TGFß1), platelet factor-4 (PF4), interleukin-1ß (IL-1ß), and IL-1 receptor antagonist (IL-1RA) were quantified using an enzyme-linked immunosorbent assay. The levels of PDGF-BB, TGFß1, and PF4 in L-PRP were significantly higher in mPSPs than in fCAs. Conversely, IL-1ß and IL-1RA were detected at significantly and slightly higher levels, respectively, in fCAs than in mPSPs. Our findings suggest that, even though L-PRP from fCAs may have lower potential to induce cell growth and differentiation than that of mPSPs, due to the latter's higher capacity to control inflammation, it does not necessarily imply that PRP treatment in fCAs is less effective. Thus, these cytokine levels should be checked before PRP therapy.

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.

A Strategic and Worldwide Cooperative Challenge Required for the Next Generation of Platelet Concentrates.

Blood-derived biomaterials, which are represented by platelet-rich plasma (PRP) or more recently by platelet-rich fibrin (PRF), have been used in regenerative therapy for almost 30 years [...].

Modulation of ATP Production Influences Inorganic Polyphosphate Levels in Non-Athletes' Platelets at the Resting State.

Platelets produce inorganic polyphosphate (polyP) upon activation to stimulate blood coagulation. Some researchers have linked polyP metabolism to ATP production, although the metabolic linkage is yet to be elucidated. We found evidence for this possibility in our previous study on professional athletes (versus non-athletes), and proposed that the regulatory mechanism might be different for these two groups. To explore this aspect further, we investigated the effects of modulated ATP production on polyP levels. Blood samples were obtained from Japanese healthy, non-athletes in the presence of acid-citrate-dextrose. The platelets in the plasma were treated with oligomycin, rotenone, and GlutaMAX to modulate ATP production. PolyP level was quantified fluorometrically and visualized using 4',6-diamidino-2-phenylindole. Correlations between polyP and ATP or NADH were then calculated. Contrary to the hypothesis, inhibitors of ATP production increased polyP levels, whereas amino acid supplementation produced the opposite effect. In general, however, polyP levels were positively correlated with ATP levels and negatively correlated with NADH levels. Since platelets are metabolically active, they exhibit high levels of ATP turnover rate. Therefore, these findings suggest that ATP may be involved in polyP production in the resting platelets of non-athletes.

Use of platelet-rich fibrin for the treatment of periodontal intrabony defects: a systematic review and meta-analysis.

OBJECTIVES: This study aims to compare the treatment outcomes of periodontal intrabony defects by using platelet-rich fibrin (PRF) with other commonly utilized modalities. MATERIALS AND METHODS: The eligibility criteria comprised randomized controlled trials (RCTs) comparing the clinical outcomes of PRF with that of other modalities. Studies were classified into 10 categories as follows: (1) open flap debridement (OFD) alone versus OFD/PRF; (2) OFD/bone graft (OFD/BG) versus OFD/PRF; (3) OFD/BG versus OFD/BG/PRF; (4-6) OFD/barrier membrane (BM), OFD/PRP, or OFD/enamel matrix derivative (EMD) versus OFD/PRF; (7) OFD/EMD versus OFD/EMD/PRF; (8-10) OFD/PRF versus OFD/PRF/metformin, OFD/PRF/bisphosphonates, or OFD/PRF/statins. Weighted means and forest plots were calculated for probing depth (PD), clinical attachment level (CAL), and radiographic bone fill (RBF). RESULTS: From 551 articles identified, 27 RCTs were included. The use of OFD/PRF statistically significantly reduced PD and improved CAL and RBF when compared to OFD. No clinically significant differences were reported when OFD/BG was compared to OFD/PRF. The addition of PRF to OFD/BG led to significant improvements in CAL and RBF. No differences were reported between any of the following groups (OFD/BM, OFD/PRP, and OFD/EMD) when compared to OFD/PRF. No improvements were also reported when PRF was added to OFD/EMD. The addition of all three of the following biomolecules (metformin, bisphosphonates, and statins) to OFD/PRF led to statistically significant improvements of PD, CAL, and RBF. CONCLUSIONS: The use of PRF significantly improved clinical outcomes in intrabony defects when compared to OFD alone with similar levels being observed between OFD/BG and OFD/PRF. Future research geared toward better understanding potential ways to enhance the regenerative properties of PRF with various small biomolecules may prove valuable for future clinical applications. Future research investigating PRF at histological level is also needed. CLINICAL RELEVANCE: The use of PRF in conjunction with OFD statistically significantly improved PD, CAL, and RBF values, yielding to comparable outcomes to OFD/BG. The combination of PRF with bone grafts or small biomolecules may offer certain clinical advantages, thus warranting further investigations.

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.

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

Cell culture media influence the characteristics of human osteogenic periosteal sheets. We have previously found that a stem cell medium facilitates growth and collagen matrix formation in vitro and osteogenesis in vivo. However, it has not yet been demonstrated which culture medium is superior for osteoclastogenesis, a prerequisite for reconstruction of normal bone metabolic basis. To address this question, we compared chemotaxis and osteoclastogenesis in tissue-engineered periosteal sheets (TPSs) prepared with two types of culture media. Periosteal tissues obtained from adult volunteers were expanded with the conventional Medium 199 or with the stem cell medium, MesenPRO. Hematopoietic enhanced-green-fluorescent-protein (EGFP)-nude mice were prepared by γ-irradiation of Balb/c nu/nu mice and subsequent transplantation of bone marrow cells from CAG-EGFP C57BL/6 mice. TPSs were implanted subcutaneously into the chimeric mice and retrieved after intervals for immunohistopathological examination. EGFP+ cells were similarly recruited to the implantation site in both the TPSs prepared, whereas the distribution of CD11b+ cells was significantly lower in the TPS prepared with the stem cell medium. Instead, osteoclastogenesis was higher in the TPS prepared with the stem cell medium than in the one prepared with the conventional medium. These findings suggest that the stem cell medium is preferable for the preparation of more functional TPSs.

A technical note on contamination from PRF tubes containing silica and silicone.

BACKGROUND: Platelet-rich fibrin (PRF) has been widely utilized in modern medicine and dentistry owing to its ability to rapidly stimulate neoangiogenesis, leading to faster tissue regeneration. While improvements over traditional platelet rich plasma therapies (which use chemical additives such as bovine thrombin and calcium chloride) have been observed, most clinicians are unaware that many tubes utilized for the production of 'natural' and '100% autologous' PRF may in fact contain chemical additives without appropriate or transparent knowledge provided to the treating clinician. The aim of this overview article is therefore to provide a technical note on recent discoveries related to PRF tubes and describe recent trends related to research on the topic from the authors laboratories. METHODS: Recommendations are provided to clinicians with the aim of further optimizing PRF clots/membranes by appropriate understanding of PRF tubes. The most common additives to PRF tubes reported in the literature are silica and/or silicone. A variety of studies have been performed on their topic described in this narrative review article. RESULTS: Typically, PRF production is best achieved with plain, chemical-free glass tubes. Unfortunately, a variety of other centrifugation tubes commonly used for lab testing/diagnostics and not necessarily manufactured for human use have been utilized in clinical practice for the production of PRF with unpredictable clinical outcomes. Many clinicians have noted an increased variability in PRF clot sizes, a decreased rate of clot formation (PRF remains liquid even after an adequate protocol is followed), or even an increased rate in the clinical signs of inflammation following the use of PRF. CONCLUSION: This technical note addresses these issues in detail and provides scientific background of recent research articles on the topic. Furthermore, the need to adequately select appropriate centrifugation tubes for the production of PRF is highlighted with quantitative data provided from in vitro and animal investigations emphasizing the negative impact of the addition of silica/silicone on clot formation, cell behavior and in vivo inflammation.

Use of platelet-rich fibrin for the treatment of gingival recessions: a systematic review and meta-analysis.

OBJECTIVES: The aim of this systematic review and meta-analysis was to compare the use of platelet-rich fibrin (PRF) with other commonly utilized treatment modalities for root coverage procedures. MATERIALS AND METHODS: The eligibility criteria comprised randomized controlled trials (RCTs) comparing the performance of PRF with that of other modalities in the treatment of Miller class I or II (Cairo RT I) gingival recessions. Studies were classified into 5 categories as follows: (1) coronally advanced flap (CAF) alone vs CAF/PRF, (2) CAF/connective tissue graft (CAF/CTG) vs CAF/PRF, (3) CAF/enamel matrix derivative (CAF/EMD) vs CAF/PRF, (4) CAF/amnion membrane (CAF/AM) vs CAF/PRF, and (5) CAF/CTG vs CAF/CTG/PRF. Studies were evaluated for percentage of relative root coverage (rRC; primary outcome), clinical attachment level (CAL), keratinized mucosa width (KMW), and probing depth (PD) (secondary outcomes). RESULTS: From 976 articles identified, 17 RCTs were included. The use of PRF statistically significantly increased rRC and CAL compared with CAF alone. No change in KMW or reduction in PD was reported. Compared with PRF, CTG resulted in statistically significantly better KMW and RC. No statistically significant differences were reported between the CAF/PRF and CAF/EMD groups or between the CAF/PRF and CAF/AM groups for any of the investigated parameters. CONCLUSIONS: The use of CAF/PRF improved rRC and CAL compared with the use of CAF alone. While similar outcomes were observed between CAF/PRF and CAF/CTG for CAL and PD change, the latter group led to statistically significantly better outcomes in terms of rRC and KTW. In summary, the use of PRF in conjunction with CAF may represent a valid treatment modality for gingival recessions exhibiting adequate baseline KMW. CLINICAL RELEVANCE: The data indicate that the use of PRF in conjunction with CAF statistically significantly improves rRC when compared with CAF alone but did not improve KMW. Therefore, in cases with limited baseline KMW, the use of CTG may be preferred over PRF.

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.

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.

Imaging of Metastatic Cancer Cells in Sentinel Lymph Nodes using Affibody Probes and Possibility of a Theranostic Approach.

The accurate detection of lymph node metastases is essential for treatment success in early-stage malignant cancer. Sentinel lymph node (SLN) biopsy is the most effective procedure for detecting small or micrometastases that are undetectable by conventional imaging modalities. To demonstrate a new approach for developing a more efficient SLN biopsy procedure, we reported a two-stage imaging method combining lymphoscintigraphy and near-infrared (NIR) fluorescence imaging to depict metastatic cancer cells in SLNs in vivo. Furthermore, the theranostic potential of the combined procedure was examined by cell culture and xenograft mouse model. Anti-HER2 and anti-epidermal growth factor receptor (EGFR) affibody probes were used for NIR fluorescence imaging. Strong NIR fluorescence signal intensity of the anti-EGFR affibody probe was observed in SAS cells (EGFR positive). Radioactivity in the SLNs was clearly observed in the in vivo studies. High anti-EGFR affibody NIR fluorescence intensity was observed in the metastatic lymph nodes in mice. The addition of the IR700-conjugated anti-EGFR affibody to the culture medium decreased the proliferation of SAS cells. Decreased proliferation was shown in Ki-67 immunohistochemistry in xenograft tumors. Our data suggest that a two-stage combined imaging method using lymphoscintigraphy and affibody probes may offer the direct visualization of metastatic lymph nodes as an easily applied technique in SLN biopsy. Although further animal studies are required to assess the effect of treating lymphatic metastasis in this approach, our study results provide a foundation for the further development of this promising imaging and treatment strategy for earlier lymph node metastasis detection and treatment.

HER2-Targeted Multifunctional Silica Nanoparticles Specifically Enhance the Radiosensitivity of HER2-Overexpressing Breast Cancer Cells.

We investigated the effects of targeted functionalized silica nanoparticles on the radiosensitivity of cancer cells. Better control of the local concentration of silica nanoparticles may facilitate their use as an adjuvant in conjunction with ionizing radiation to target cancer cells while preventing damage to normal cells. Hyperbranched polyamidoamine (PAMAM) was grafted onto the surface of amorphous silica nanoparticles to functionalize them. The PAMAM-coated silica nanoparticles (PCSNs) were then conjugated with fluorescent dyes. Anti-HER2 antibodies were covalently attached to the labeled PCSNs. The HER2-overexpressing SK-BR3 breast cancer cell line was incubated in medium containing the PCSN probes. After incubation; the cells were exposed to X-ray radiation. Cells were counted in all samples using cell proliferation assays; and apoptotic cells were detected. The cell survival results showed that the combination of the targeted PCSN probes and radiation reduced the survival rate of SK-BR3 cells to a greater extent than when either PCSN probes, PCSNs or radiation were applied individually. The results also showed an increase in apoptosis in the SK-BR3 cells that internalized the PCSN probes and were then irradiated. Based on these data, PCSN probes act as specific radiosensitizing agents for HER2-overexpressing cells.

Dual-Labeled Near-Infrared/(99m)Tc Imaging Probes Using PAMAM-Coated Silica Nanoparticles for the Imaging of HER2-Expressing Cancer Cells.

We sought to develop dual-modality imaging probes using functionalized silica nanoparticles to target human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer cells and achieve efficient target imaging of HER2-expressing tumors. Polyamidoamine-based functionalized silica nanoparticles (PCSNs) for multimodal imaging were synthesized with near-infrared (NIR) fluorescence (indocyanine green (ICG)) and technetium-99m ((99m)Tc) radioactivity. Anti-HER2 antibodies were bound to the labeled PCSNs. These dual-imaging probes were tested to image HER2-overexpressing breast carcinoma cells. In vivo imaging was also examined in breast tumor xenograft models in mice. SK-BR3 (HER2 positive) cells were imaged with stronger NIR fluorescent signals than that in MDA-MB231 (HER2 negative) cells. The increased radioactivity of the SK-BR3 cells was also confirmed by phosphor imaging. NIR images showed strong fluorescent signals in the SK-BR3 tumor model compared to muscle tissues and the MDA-MB231 tumor model. Automatic well counting results showed increased radioactivity in the SK-BR3 xenograft tumors. We developed functionalized silica nanoparticles loaded with (99m)Tc and ICG for the targeting and imaging of HER2-expressing cells. The dual-imaging probes efficiently imaged HER2-overexpressing cells. Although further studies are needed to produce efficient isotope labeling, the results suggest that the multifunctional silica nanoparticles are a promising vehicle for imaging specific components of the cell membrane in a dual-modality manner.

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.

Platelet-rich plasma and its derivatives as promising bioactive materials for regenerative medicine: basic principles and concepts underlying recent advances.

Over the past decade, platelet-rich plasma (PRP), a platelet-concentrated plasma fraction, has been widely investigated and applied to regenerative medicine. The clinical utility of PRP is supported by evidence that PRP contains high concentrations of platelet-related growth factors and normal concentrations of plasma-derived fibrinogen, both of which contribute synergistically to the regenerative process. Additionally, its superior cost-efficacy versus conventional therapies is attractive to many clinicians. However, current disadvantages of PRP include a relatively complicated preparation procedure and variable operator-dependent efficacy. An additional disadvantage is the use of bovine thrombin, an animal-derived biological, as a coagulant. Many of these disadvantages are overcome by recent advances in preparation procedures and devices; for example, Joseph Choukroun simplified the platelet-rich fibrin preparation procedure and improved handling efficiency without the aid of animal-derived factors. With advancements in cell processing technology, there has been a general shift in cell therapy from autologous to allogeneic treatment; however, autologous PRP therapy will not easily be replaced by allogeneic treatment in the near future. Therefore, to provide more predictable regenerative therapy outcomes using autologous PRP, further investigations should address developing a standardized procedure for PRP preparation to augment its efficacy and potency, independent of donor variability. We would then propose that operators and clinicians prepare PRP according to the standardized protocol and to carefully evaluate the clinical scenario (i.e., recipient factors comprising skeletal defects) to determine which factor(s) should be added to PRP preparations. This careful approach will lead to improved clinical outcomes for patients.