Synergistic effects of the combined use of human-cultured periosteal sheets and platelet-rich fibrin on bone regeneration: An animal study.

A human-cultured alveolar bone-derived periosteal (hCP) sheet is an osteogenic grafting material used clinically in periodontal regenerative therapy, while platelet-rich fibrin (PRF), a platelet concentrate with fibrin clot, is considered to augment the wound healing process. Therefore, whether the combined use of hCP-PRF complex could facilitate bone regeneration synergistically was evaluated in animal models. Human periosteal segments (1 × 1 mm) were cultured initially on plastic dishes and formed an hCP sheet. The hCP sheet was implanted with freshly prepared human PRF into subcutaneous tissue (hCP: n = 4, hCP + PRF: n = 4) and 4 mm diameter calvarial bone defect models (hCP: n = 4, hCP + PRF: n = 4, control [defect-only]: n = 4) that prepared in nude mice. At 4 weeks postimplantation, new bone formation was evaluated by using µCT. Cell growth and neovascularization were evaluated by histochemical and immunohistological methods. In the subcutaneous tissue, mineral deposit formation, collagen deposition, and number of vessels were higher in the hCP + PRF group than in the hCP alone group. In the calvarial defect models, new bone formation was significantly higher in the hCP + PRF group than in the hCP alone group and defect-only control group. The numbers of vessels and PCNA-positive cells in calvarial defects were also increased in the hCP + PRF group more than in the hCP alone group. Platelet-rich fibrin preparations support the proliferation and the growth of periosteal cells to form well-combined active biological materials. Platelet-rich fibrin also stimulates the local angiogenesis in the implantation site. Therefore, the combined use of hCP and PRF could be clinically applicable in bone regeneration therapy.

In vitro immunological and biological evaluations of the angiogenic potential of platelet-rich fibrin preparations: a standardized comparison with PRP preparations.

BACKGROUND: Platelet-rich fibrin (PRF), a platelet-rich plasma (PRP) derivative mainly composed of fibrin networks, has been increasingly demonstrated to be effective in wound healing in clinical and pre-clinical animal studies. However, there has still been a concern that major growth factors may significantly be loss from PRF during its preparation through the slow clotting process. To address this concern, we compared the angiogenic potential of PRF and PRP by standardization of procedures based on volume ratios. METHODS: PRP, PRF, and platelet-poor plasma (PPP) were prepared from the peripheral blood of healthy donors. PRF preparations were squeezed or homogenized to produce exudate (PRFexu) or extract (PRFext), respectively. Concentrations of the angiogenic factors and their bioactivities were determined using ELISA kits, a scratch assay using endothelial cells and a chicken chorioallantoic membrane (CAM) assay. RESULTS: In PRP and PRF preparations, both VEGF and PDGF-BB were significantly more concentrated than PPP. In the scratch assay, PRFexu and PRFext were the most effective for wound closure. In the CAM assay, PRF membranes were the most effective for neovascularization. CONCLUSIONS: It is suggested that PRF preparations efficiently preserve the angiogenic factors and function not only as a scaffolding material but as a reservoir of angiogenic factors in wound healing.

The heat-compression technique for the conversion of platelet-rich fibrin preparation to a barrier membrane with a reduced rate of biodegradation.

Platelet-rich fibrin (PRF) was developed as an advanced form of platelet-rich plasma to eliminate xenofactors, such as bovine thrombin, and it is mainly used as a source of growth factor for tissue regeneration. Furthermore, although a minor application, PRF in a compressed membrane-like form has also been used as a substitute for commercially available barrier membranes in guided-tissue regeneration (GTR) treatment. However, the PRF membrane is resorbed within 2 weeks or less at implantation sites; therefore, it can barely maintain sufficient space for bone regeneration. In this study, we developed and optimized a heat-compression technique and tested the feasibility of the resulting PRF membrane. Freshly prepared human PRF was first compressed with dry gauze and subsequently with a hot iron. Biodegradability was microscopically examined in vitro by treatment with plasmin at 37°C or in vivo by subcutaneous implantation in nude mice. Compared with the control gauze-compressed PRF, the heat-compressed PRF appeared plasmin-resistant and remained stable for longer than 10 days in vitro. Additionally, in animal implantation studies, the heat-compressed PRF was observed at least for 3 weeks postimplantation in vivo whereas the control PRF was completely resorbed within 2 weeks. Therefore, these findings suggest that the heat-compression technique reduces the rate of biodegradation of the PRF membrane without sacrificing its biocompatibility and that the heat-compressed PRF membrane easily could be prepared at chair-side and applied as a barrier membrane in the GTR treatment.

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.

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 short-term preservation of human cultured periosteal sheets, osteogenic grafting materials, using a commercial preservation solution containing epigallocatechin-3-gallate (Theliokeep(®)) under hypothermic conditions.

In the past decade, it has increasingly been reported that epigallocatechin-3-gallate (EGCG), a major catechin derivative extracted from Green tea, has various bioactivities, including a cell-protective action on mammalian cells and tissues. In this study, we have tested a commercial preservation solution containing EGCG (Theliokeep(®)) in both two- and three-dimensional cultures of human periosteal sheets, which have been used as an osteogenic grafting material for periodontal regenerative therapy. When periosteal sheets were 3D-cultured on collagen mesh, cell viability was maintained for 2 days using the hypothermic EGCG preservation solution. Replenishment of EGCG solution with 2-day intervals prevented the time-dependent decline in cell viability at 3 days and later. As observed in nonpreserved control cultures, most cells were positive for proliferating cell-nuclear antigen (PCNA) in the cultures preserved at 4°C in the EGCG solution, whereas PCNA-negative cells were increased in the cultures preserved at 4°C in the MesenPRO medium. In periosteal sheets 2D-cultured in plastic dishes, the EGCG solution occasionally was associated with vacuole formation in the cytoplasm, but cells could again expand in the culture medium at 37°C. As observed in the nonpreserved periosteal sheets control, the osteogenic induction upregulated alkaline phosphatase in those cells and tissues preserved in the EGCG solution. The EGCG solution protected cells from the cold shock-induced membrane phospholipid peroxidation. Our data suggest that the EGCG solution acts as an antioxidant to protect periosteal cells from cold shock and preserves cells under chilled conditions. The limited period of preservation time could be expanded by repeating replenishment of the EGCG solution or by optimizing the formula to be more favorable for human periosteal sheets without sacrificing cell viability. This methodology of preserving human cultured periosteal sheets with EGCG would be expected to support and spread the clinical use of regenerative therapy with autologous periosteal sheets.