Toshimitsu Okudera

Growth factor and pro-inflammatory cytokine contents in platelet-rich plasma (PRP), plasma rich in growth factors (PRGF), advanced platelet-rich fibrin (A-PRF), and concentrated growth factors (CGF)

BackgroundThe development of platelet-rich fibrin (PRF) drastically simplified the preparation procedure of platelet-concentrated biomaterials, such as platelet-rich plasma (PRP), and facilitated their clinical application. PRF’s clinical effectiveness has often been demonstrated in pre-clinical and clinical studies; however, it is still controversial whether growth factors are significantly concentrated in PRF preparations to facilitate wound healing and tissue regeneration. To address this matter, we performed a comparative study of growth factor contents in PRP and its derivatives, such as advanced PRF (A-PRF) and concentrated growth factors (CGF).MethodsPRP and its derivatives were prepared from the same peripheral blood samples collected from healthy donors. A-PRF and CGF preparations were homogenized and centrifuged to produce extracts. Platelet and white blood cell counts in A-PRF and CGF preparations were determined by subtracting those counts in red blood cell fractions, supernatant acellular serum fractions, and A-PRF/CGF exudate fractions from those counts of whole blood samples. Concentrations of growth factors (TGF-β1, PDGF-BB, VEGF) and pro-inflammatory cytokines (IL-1β, IL-6) were determined using ELISA kits.ResultsCompared to PRP preparations, both A-PRF and CGF extracts contained compatible or higher levels of platelets and platelet-derived growth factors. In a cell proliferation assay, both A-PRF and CGF extracts significantly stimulated the proliferation of human periosteal cells without significant reduction at higher doses.ConclusionsThese data clearly demonstrate that both A-PRF and CGF preparations contain significant amounts of growth factors capable of stimulating periosteal cell proliferation, suggesting that A-PRF and CGF preparations function not only as a scaffolding material but also as a reservoir to deliver certain growth factors at the site of application.

Absorption and plasma kinetics of collagen tripeptide after peroral or intraperitoneal administration in rats

Collagen tripeptide (CTP) is a collagen-derived compound containing a high concentration of tripeptides with a Gly-X-Y sequence. In this study, the concentrations and metabolites of CTP were monitored in rat plasma after its administration. We performed a quantitative analysis using high-performance liquid chromatography tandem mass spectrometry according to the isotopic dilution method with stable isotopes. We confirmed that the tripeptides Gly-Pro-Hyp, Gly-Pro-Ala, and Gly-Ala-Hyp were transported into the plasma. Dipeptides, which are generated by degradation of the N- or C-terminus of the tripeptides Gly-Pro-Hyp, Gly-Pro-Ala, and Gly-Ala-Hyp, were also present in plasma. The plasma kinetics for peroral and intraperitoneal administration was similar. In addition, tripeptides and dipeptides were detected in no-administration rat blood. The pharmacokinetics were monitored in rats perorally administered with Gly-[3H]Pro-Hyp. Furthermore, CTP was incorporated into tissues including skin, bone, and joint tissue. Thus, administering collagen as tripeptides enables efficient absorption of tripeptides and dipeptides. Graphical abstract Plasma kinetics of peptides in the two routes of administration, and autoradiographs after peroral administration of Gly-[3H]Pro-Hyp.

Part II: Crystalline Fluorapatite-Coated Hydroxyapatite Implant Material: A Dog Study With Histologic Comparison of Osteogenesis Seen With FA-Coated HA Grafting Material Versus HA Controls: Potential Bacteriostatic Effect of Fluoridated HA

Success of osteogenesis in bone graft procedures can be enhanced by inhibiting oral bacterial infections through the use of prophylactic bacteriostatic fluoride within the grafting environment. Ideally, the fluoride ion should be chemically sequestered and thus unavailable unless needed at times during the process of early infection. As fluoride within fluorapatite is tightly bound at neutral pH and becomes available only during acidic conditions, fluorapatite is an ideal store for the fluoride ion which becomes released for bacteriostasis only during an acidic environment found with incipient bacterial infection. The purpose of this investigation was to compare the histologic properties of new bone formed surrounding fluorapatite (FA)-coated microcrystalline hydroxyapatite (HA) grafting material with comparable bone formed following the use of control HA material (OsteoGen, Impladent, Ltd, Holliswood, NY). The results of histologic analysis within dog studies here showed no detectable difference in new bone following therapeutic grafting procedures using each of the above 2 mineral coatings.

An Evaluation of the Accuracy of the Subtraction Method Used for Determining Platelet Counts in Advanced Platelet-Rich Fibrin and Concentrated Growth Factor Preparations

Platelet concentrates should be quality-assured of purity and identity prior to clinical use. Unlike for the liquid form of platelet-rich plasma, platelet counts cannot be directly determined in solid fibrin clots and are instead calculated by subtracting the counts in other liquid or semi-clotted fractions from those in whole blood samples. Having long suspected the validity of this method, we herein examined the possible loss of platelets in the preparation process. Blood samples collected from healthy male donors were immediately centrifuged for advanced platelet-rich fibrin (A-PRF) and concentrated growth factors (CGF) according to recommended centrifugal protocols. Blood cells in liquid and semi-clotted fractions were directly counted. Platelets aggregated on clot surfaces were observed by scanning electron microscopy. A higher centrifugal force increased the numbers of platelets and platelet aggregates in the liquid red blood cell fraction and the semi-clotted red thrombus in the presence and absence of the anticoagulant, respectively. Nevertheless, the calculated platelet counts in A-PRF/CGF preparations were much higher than expected, rendering the currently accepted subtraction method inaccurate for determining platelet counts in fibrin clots. To ensure the quality of solid types of platelet concentrates chairside in a timely manner, a simple and accurate platelet-counting method should be developed immediately.

Part I: crystalline fluorapatite-coated hydroxyapatite, physical properties.

Crystalline fluorapatite-coated hydroxyapatite (FA-HA) is studied using scanning electron microscopy (SEM), X-ray diffraction (XD), energy dispersive X-ray analysis (EDX), and EDX analysis mapping (EDXM). Fluoridated HA (fluorapatite) was prepared by reacting resorbable synthetic HA (OsteoGen, Impladent, Ltd, Holliswood, NY) with 4.3% sodium fluoride (NaF) for 2 minutes. After washing and drying, the resultant powder was subjected to physical property analysis using the methods listed above. SEM showed little evidence of surface change. Changes, if any, consisted of a slightly more distinct crystalline clarity on the surface of the FA sample. XD patterns showed significant random noise dispersion of the untreated HA sample compared with the lack of noise patterns in the treated FA sample. Characteristic monetite peaks were noted in analysis of the nontreated HA control sample, whereas there was no evidence of monetite in XD analysis of the treated FA material. It was determined that the fluoridation reaction, as described, served as a purification procedure of the initial HA reagent to eliminate a more soluble monetite contaminant. Also, the reaction of fluoride ion with surface HA (whether it be from or a combination of dissolution-reapposition or isomorphic substitution) produces a more purified, crystalline FA sample that was characterized by a more characteristic and sharp XD pattern. EDX analysis of the FA sample revealed a fluoride peak at 0.70 KeV that was not seen in the nonfluoridated control. EDX mapping showed an evenly distributed needle-like crystalline-shaped particulate pattern over the entire surface of the FA sample, which was lacking in the HA control. From a variety of analytic methods (as described), it was concluded that reaction of synthetic resorbable HA with 4.3% NaF solution at neutral pH produces FA-coated HA.

Basic characteristics of plasma rich in growth factors (PRGF): blood cell components and biological effects

Platelet‐rich plasma (PRP) is widely used in regenerative medicine because of its high concentrations of various growth factors and platelets. However, the distribution of blood cell components has not been investigated in either PRP or other PRP derivatives. In this study, we focused on plasma rich in growth factors (PRGF), a PRP derivative, and analyzed the distributions of platelets and white blood cells (WBCs). Peripheral blood samples were collected from healthy volunteers (N = 14) and centrifuged to prepare PRGF and PRP. Blood cells were counted using an automated hematology analyzer. The effects of PRP and PRGF preparations on cell proliferation were determined using human periosteal cells. In the PRGF preparations, both red blood cells and WBCs were almost completely eliminated, and platelets were concentrated by 2.84‐fold, whereas in the PRP preparations, both platelets and WBCs were similarly concentrated by 8.79‐ and 5.51‐fold, respectively. Platelet counts in the PRGF preparations were positively correlated with platelet counts in the whole blood samples, while the platelet concentration rate was negatively correlated with red blood cell counts in the whole blood samples. In contrast, platelet counts and concentration rates in the PRP preparations were significantly influenced by WBC counts in whole blood samples. The PRP preparations, but not the PRGF preparations, significantly suppressed cell growth at higher doses in vitro. Therefore, these results suggest that PRGF preparations can clearly be distinguished from PRP preparations by both inclusion of WBCs and dose‐dependent stimulation of periosteal cell proliferation in vitro.

Quality Assessment of Platelet-Rich Fibrin-Like Matrix Prepared from Whole Blood Samples after Extended Storage

The platelet-rich fibrin–like matrix (PRFM) is usually prepared onsite and immediately used for regenerative therapy. Nonetheless, to meet the clinical necessity of preserving the PRFM without quality deterioration, we developed a method for preparation of PRFMs from short-term-stored whole blood (WB) samples. In this study, to evaluate the practical expiration date of storage, we extended the storage time of WB samples from 2 to 7 days and assessed the quality of the resulting PRFMs. WB samples collected with acid-citrate-dextrose were stored with gentle agitation at ambient temperature. To prepare PRFMs, the stored WB samples were mixed with CaCl2 in glass tubes and centrifuged. Fibrin fiber networks, CD41 and CD62P expression, and Platelet Derived Growth Factor-BB (PDGF-BB) levels were examined by scanning electron microscopy (SEM), flow cytometry, and an Enzyme-Linked ImmunoSorbent Assay (ELISA), respectively. Long-term storage had no significant effect on either blood cell counts or platelet functions tested. The resulting PRFMs were visually identical to freshly prepared ones. PDGF-BB levels did not markedly decrease in a time-dependent manner. However, fibrin fibers gradually became thinner after storage. Although the coagulation activity may diminish, we propose that PRFMs can be prepared—without evident loss of quality—from WB samples stored for up to 7 days by our previously developed method.

An on-site preparable, novel bone-grafting complex consisting of human platelet-rich fibrin and porous particles made of a recombinant collagen-like protein: BONE SUBSTITUTE CONSISTING OF SYNTHETIC RGD PROTEIN AND PRF

Abstract Platelet‐rich fibrin (PRF) is widely used in regenerative medicine. Nonetheless, major issues include its controversial effects on bone regeneration and a lack of quality‐assured glass tubes required for coagulation. We used porous particles (FBG) comprising a recombinant RGD motif‐enriched collagen I‐like protein to activate the coagulation pathway and examined the effects of the resulting PRF–FBG complex on bone regeneration. Human whole‐blood samples were mixed with FBG in plastic tubes and centrifuged to prepare a PRF–FBG complex. Platelet‐derived growth factor‐BB (PDGF‐BB) levels and cell growth activity were determined by ELISA and a bioassay using osteoblasts. Bone regenerative activity was assessed using a mouse model of calvarial bone defect. FBG facilitated PRF‐like matrix formation during centrifugation. In this PRF–FBG complex, the microstructure of fibrin fibers was similar to that of PRF prepared conventionally in glass tubes. PDGF‐BB levels and mitogenic action were not significantly influenced by FBG. In the bone defect model, although PRF did not exert any significant positive effects on its own, in combination with FBG, it synergistically stimulated new bone formation. This study demonstrated that incorporation of FBG into whole‐blood samples induces PRF formation without the aid of glass tubes. The resulting PRF–FBG complex could be a promising bone grafting material in clinical settings.