Regeneration processes of alveolar bone and microvascular changes after the application of platelet-rich fibrin.

OBJECTIVES: Platelet-rich fibrin (PRF) is a promising agent for bone regeneration (BR). Platelets contain several growth factors that promote angiogenesis and BR. In this study, we observed the morphology of alveolar BR. METHODS: PRF (Advanced PRF: A-PRF) was prepared by extracting 10 mL of blood from each dog in a collection tube before tooth extraction. The samples were centrifuged at 200 × g for 8 min and incubated for 10 min to allow clotting. The alveolar socket on the dentition's right side was densely filled with PRF. The opposite side, which did not receive PRF, served as the control group. Different methods were used for specimen preparation and observation. Sections stained with hematoxylin and eosin were observed under a light microscope. Bone specimens were observed using stereoscopic microscopy. The resin cast models were examined using a scanning electron microscope. Moreover, bone formation ratio and height were measured. RESULTS: Fourteen days postoperatively, angiogenesis and bone deposition were more advanced in the PRF group than in the control group. Thirty days postoperatively, both groups developed porous bone. In the PRF group, new bone trabeculae (BT) and a network of blood vessels were formed in the bone marrow. Ninety days postoperatively, the resin cast showed a normal bone structure with BT and bone marrow. Thick BT were observed in the PRF group. CONCLUSIONS: Growth factors in PRF stimulate microcirculation and promote angiogenesis and bone deposition. The benefits of PRF include safety and increased bone formation.

Advanced platelet-rich fibrin (A-PRF) has an impact on the initial healing of gingival regeneration after tooth extraction.

OBJECTIVES: Platelet-rich fibrin (PRF) is widely used in wound healing because it contains several growth factors, including vascular endothelial growth factor (VEGF). In this study, we investigated the effects of advanced PRF (A-PRF) in early-stage gingival regeneration after tooth extraction. METHODS: Blood sample was collected from females beagle dogs (age: 12 months) before tooth extraction for A-PRF preparation. All animals were sacrificed by perfusion-fixation on postoperative days 1, 3, and 7. The upper jaws were prepared for hematoxylin and eosin staining and immunostaining (for CD34 and VEGF). The lower jaw samples were prepared for scanning electron microscope observations. Blood flow in the gingiva before and after surgery was measured using laser Doppler flowmetry. RESULTS: In the A-PRF group, a large number of microvessels were observed in the gingival tissue on postoperative day 1. The microvessels in the control group were fewer and sparse. Regarding the vascular resin cast, a large number of new blood vessels were observed on postoperative day 1 in the A-PRF group. A stronger CD34-positive signal was obtained around the blood vessels in the A-PRF group than in the control group. Further, a strong VEGF-positive signal was observed in the perivascular tissue in the A-PRF group. Gingival blood flow was significantly higher in the A-PRF group after surgery. CONCLUSION: A-PRF had a positive impact on angiogenesis in the gingiva through the induction of VEGF expression. Thus, A-PRF may be beneficial for gingival tissue regeneration.

Application of deproteinized bovine bone mineral as proangiogenic scaffold for alveolar bone formation in beagle dogs.

Alveolar bone repair after tooth extraction is essential after oral surgeries. Various grafting materials are used to promote the regeneration of lost alveolar bone. This study analysed the morphological features of the tissue regeneration process using deproteinized bovine bone mineral (DBBM). DBBM was used to densely fill the extraction sockets in beagle dogs. Following resin casting of the vasculature, stereomicroscopy and scanning electron microscopy were used to observe blood vessels and hard tissues in haematoxylin and eosin-stained sections on postoperative days 14, 30 and 90 in conjunction with vascular endothelial growth factor (VEGF) immunostaining to evaluate alveolar bone vascularization. On day 14 post-operation, the DBBM granules tightly filled the extraction sockets, maintained alveolar margin height and formed a scaffold for aiding angiogenesis and new bone formation. On day 30, new bone formation was observed around the DBBM granules. By day 90, bone tissue regeneration progressed in both groups but was more pronounced in the DBBM group. Alveolar margin height was maintained in the DBBM group throughout the study. Furthermore, VEGF expression in the DBBM group was detected around newly formed bone. We conclude that DBBM acts as a suitable scaffold for new bone generation, as well as angiogenesis around healing alveolar bone, and that it has the potential to play a key role in vascularization and bone formation.

Effect of advanced platelet-rich fibrin on accelerating alveolar bone formation in dogs: a histological and immunofluorescence evaluation.

Several methods have been developed to regenerate lost alveolar bone. Platelet-rich fibrin (PRF) is a useful adjunct for new bone formation in dentistry. To elucidate the effect of advanced PRF (A-PRF) on bone formation, we inserted A-PRF clots in sockets after tooth extraction. Premolars were extracted from beagle dogs, and A-PRF was applied to the socket. New bone formation was assessed using histological and immunofluorescence examinations, and the bone formation ratio was evaluated 14 and 30 days postoperatively. Histological examination revealed newly formed bone filling the sockets up to the center in the A-PRF group at 14 days postoperatively, while thick and regular bone trabeculae were arranged in porous bone after 30 days. Higher expressions of osteocalcin and osteopontin were observed in newly formed bone in the A-PRF group, compared to the control group. The bone formation ratio was also higher in the A-PRF group than in the control group. Thus, A-PRF application may result in enhanced new bone formation and may aid in accelerating bone formation. A-PRF was more rapid than a self-limiting process during induction of bone formation by enhancing osteoblast activity and may be useful for bone formation in clinical medicine.

An on-site preparable, novel bone-grafting complex consisting of human platelet-rich fibrin and porous particles made of a recombinant collagen-like protein.

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. © 2018 The Authors. Journal of Biomedical Materials Research Part B: Applied Biomaterials published by Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1420-1430, 2019.

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.

Mechanical and degradation properties of advanced platelet-rich fibrin (A-PRF), concentrated growth factors (CGF), and platelet-poor plasma-derived fibrin (PPTF).

BACKGROUND: Fibrin clot membranes prepared from advanced platelet-rich fibrin (A-PRF) or concentrated growth factors (CGF), despite their relatively rapid biodegradability, have been used as bioactive barrier membranes for alveolar bone tissue regeneration. As the membranes degrade, it is thought that the growth factors are gradually released. However, the mechanical and degradable properties of these membranes have not well been characterized. The purpose of this study was to mechanically and chemically characterize these membranes. METHODS: A-PRF and CGF clots were prepared from blood samples collected from non-smoking, healthy donors and were compressed to form 1-mm-thick membranes. Platelet-poor plasma-derived fibrin (PPTF) clots were prepared by adding bovine thrombin to platelet-poor plasma. A tensile test was performed at the speed of 1 mm/min. Morphology of the fibrin fibers was examined by SEM. A digestion test was performed in PBS containing trypsin and EDTA. RESULTS: In the tensile test, statistical difference was not observed in Young's modulus, strain at break, or maximum stress between A-PRF and CGF. In strain at break, PPTF was significantly weaker than CGF. Likewise, fibrin fiber thickness and crosslink density of PPTF were less than those of other membranes, and PPTF degraded faster than others. CONCLUSIONS: Although the centrifugal conditions are different, A-PRF and CGF are prepared by essentially identical mechanisms. Therefore, it is conceivable that both membranes have similar mechanical and chemical properties. Only PPTF, which was prepared by a different mechanism, was characterized as mechanically weaker and enzymatically more degradable.

Platelet-rich fibrin prepared from stored whole-blood samples.

BACKGROUND: In regenerative therapy, self-clotted platelet concentrates, such as platelet-rich fibrin (PRF), are generally prepared on-site and are immediately used for treatment. If blood samples or prepared clots can be preserved for several days, their clinical applicability will expand. Here, we prepared PRF from stored whole-blood samples and examined their characteristics. METHODS: Blood samples were collected from non-smoking, healthy male donors (aged 27-67 years, N = 6), and PRF clots were prepared immediately or after storage for 1-2 days. Fibrin fiber was examined by scanning electron microscopy. Bioactivity was evaluated by means of a bioassay system involving human periosteal cells, whereas PDGF-BB concentrations were determined by an enzyme-linked immunosorbent assay. RESULTS: Addition of optimal amounts of a 10% CaCl2 solution restored the coagulative ability of whole-blood samples that contained an anticoagulant (acid citrate dextrose) and were stored for up to 2 days at ambient temperature. In PRF clots prepared from the stored whole-blood samples, the thickness and cross-links of fibrin fibers were almost identical to those of freshly prepared PRF clots. PDGF-BB concentrations in the PRF extract were significantly lower in stored whole-blood samples than in fresh samples; however, both extracts had similar stimulatory effects on periosteal-cell proliferation. CONCLUSIONS: Quality of PRF clots prepared from stored whole-blood samples is not reduced significantly and can be ensured for use in regenerative therapy. Therefore, the proposed method enables a more flexible treatment schedule and choice of a more suitable platelet concentrate immediately before treatment, not after blood collection.

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.

Microcirculation alterations in experimentally induced gingivitis in dogs.

The present study aimed to morphologically examine the gingival microvascular network using a microvascular resin cast (MRC) technique, and to investigate how inflammatory disease functionally affects gingival microcirculation using laser Doppler flowmetry (LDF). We used four beagle dogs with healthy periodontal tissue as experimental animals. To cause periodontal inflammation, dental floss was placed around the cervical neck portions of the right premolars. The unmanipulated left premolars served as controls, and received plaque control every 7 days. After 90 days, gingivitis was induced in the experimental side, while the control side maintained healthy gingiva. To perform morphological examinations, we used an MRC method involving the injection of low-viscosity synthetic resin into the blood vessels, leading to peripheral soft-tissue dissolution and permitting observation of the bone, teeth, and vascular cast. Gingival blood flow was estimated using an LDF meter. The control gingival vasculature showed hairpin-loop-like networks along the tooth surface. The blood vessels had diameters of 20-40 µm and were regularly arranged around the cervical portion. On the other hand, the vasculature in the experimental group was twisted and gathered into spiral forms, with blood vessels that had uneven surfaces and smaller diameters of 8-10 µm. LDF revealed reduced gingival blood flow in the group with experimentally induced gingivitis compared to controls. The actual measurements of gingival blood flow by LDF were in agreement with the alterations that would be expected based on the gingivitis-induced morphological alterations observed with the MRC technique.

Enhancement of mechanical strength and in vivo cytocompatibility of porous ß-tricalcium phosphate ceramics by gelatin coating.

BACKGROUND: In an attempt to prepare scaffolds with porosity and compressive strength as high as possible, we prepared porous ß-tricalcium phosphate (TCP) scaffolds and coated them with regenerative medicine-grade gelatin. The effects of the gelatin coating on the compressive strength and in vivo osteoblast compatibility were investigated. METHODS: Porous ß-TCP scaffolds were prepared and coated with up to 3 mass% gelatin, and then subjected to thermal cross-linking. The gelatin-coated and uncoated scaffolds were then subjected to compressive strength tests and implantation tests into bone defects of Wistar rats. RESULTS: The compressive strength increased by one order of magnitude from 0.45 MPa for uncoated to 5.1 MPa for gelatin-coated scaffolds. The osteoblast density in the internal space of the scaffold increased by 40 % through gelatin coating. CONCLUSIONS: Coating porous bone graft materials with gelatin is a promising measure to enhance both mechanical strength and biomedical efficacy at the same time.

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).

BACKGROUND: The 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). METHODS: PRP 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-ß1, PDGF-BB, VEGF) and pro-inflammatory cytokines (IL-1ß, IL-6) were determined using ELISA kits. RESULTS: Compared 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. CONCLUSIONS: These 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.

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.

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-[(3)H]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.

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.

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.