Evaluation of the effect of injectable platelet-rich fibrin (i-PRF) in wound healing and growth factor release in rats: a split-mouth study.

This experimental study aimed to evaluate the effects of injectable platelet-rich fibrin (i-PRF) on mucosal healing and the release of growth factors in rats. 40 rats were used; i-PRF was administered in the right buccal area while saline was injected in the left. Cytokeratin, FGF, PDGF, TGF, and VEGF expressions were determined with immunohistochemistry. Gene expressions of EGF, TGF-ß, and VEGF were analysed. Epithelialization started on the 3rd day, and connective tissue maturation was more prominent in the i-PRF-applied group. Also, the releases of VEGF, EGF, TGF-ß, PDGF, and FGF were higher in the i-PRF group during the 14 days. Gene expression analysis showed that changes in TGF-ß at 14 days after i-PRF injection and VEGF after 21 days were statistically significant. The results of this study suggested that autologous i-PRF application enhanced the healing of oral mucosal wounds by increasing the release of growth factors for 21 days.

Lyophilized platelet rich fibrin and gelatin incorporated bioadhesive bone cement composite for repair of mandibular continuity defects.

Mandibular continuity defects stem from conditions such as malignancies, trauma, cysts, osteomyelitis and osteoradionecrosis, presenting significant challenges. If mandibular reconstruction fails, it can result in facial collapse, causing significant aesthetic and functional concerns for the patient. In the present study we developed a bio-adhesive Bone Cement (BC) enriched with lyophilised PRF and gelatin to enhance bone repair and induce regeneration. The developed BC consisted of a mixture of Tetracalcium Phosphate (TTCP) and O-Phospho-l-serine (OPLS) in addition to lyophilised Platelet Rich Fibrin (PRF) for sustained growth factor release and gelatin (GE) for improved cement resorption. It is primarily designed for in-situ application, conforming to the shape and size of the defect for effective bone repair and regeneration. The study evaluated four groups: (i) BC (control), (ii) BC-GE (control), (iii) BC-PRF, and (iv) BC-GE-PRF. All the four groups were characterised using FTIR, SEM and XRD. The mechanical studies of the prepared beads exhibited a significant increase in the compressive strength of the PRF loaded bone cement composites. In vitro degradation study of the beads over a 60-day period revealed a significantly higher percentage of bone cement resorption in the gelatin-incorporated groups, BC-GE (44 ± 0.5 %), and BC-GE-PRF (45 ± 2 %). The assessment of growth factor release (TGF-ß and VEGF) using ELISA revealed a prolonged and sustained release of both growth factors over a 28-day period. In vitro studies were performed on human Dental Follicle Stem Cells (DFSCs) to assess cell attachment, proliferation, mineralisation and osteogenic differentiation. These studies clearly depicted that BC-PRF and BC-GE-PRF showed significantly greater proliferation of DFSCs. Furthermore, BC-PRF and BC-GE-PRF samples exhibited notably elevated expression of Runx2 and OPN (osteogenic markers), as well as a higher intensity of alizarin red stain (mineralisation). Therefore, it was concluded that PRF incorporated bioadhesive bone cement composites greatly enhance the cell attachment, proliferation, mineralisation and osteogenic differentiation of the DFSCs. Thus, the PRF and gelatin incorporated bone cement composites is expected to facilitate effective and faster bone regeneration and healing in a wide range of dental and maxillofacial defects.

Improving properties of platelet-rich fibrin scaffold with tannic acid for wound healing.

Platelet-rich fibrin (PRF), which is the rich source of growth factors, has been used as an efficient scaffold in tissue engineering and wound healing. In this study, tannic acid as a green cross-linker with different concentrations (0.5%, 1%, 5% and 10%) was used to improve the properties of PRF. The cross-linked gel scaffolds were evaluated by analyses such as scanning electron microscopy, Fourier transform infrared spectroscopy, swelling and degradation, mechanical strength, cell toxicity, cell adhesion and antibacterial test. The results showed that the scaffold structure changes by increasing cross-linker concentration. The swelling rate decreased from 49% to 5% for the samples without the cross-linker and with tannic acid (10%), respectively. The degradation percentage for the cross-linked samples was 8%, which showed a lower degradation rate than the non-cross-linked samples (63%). The mechanical strength of the scaffold with the cross-linker increased up to three times (Young's modulus for the non-cross linked and the cross-linked samples: 0.01 and 0.6 MPa, respectively). Cytotoxicity was not observed up to 10% cross-linker concentration. The cells proliferated well on the cross-linked scaffolds and also showed a good antibacterial effect. In general, tannic acid can improve the physical and mechanical properties of PRF without negatively affecting its biological properties.

[Effects of advanced platelet-rich fibrin on deep partial-thickness burn wounds in nude mice].

Objective: To explore the effects of advanced platelet-rich fibrin (A-PRF) on deep partial-thickness burn wounds in nude mice and its mechanism. Methods: The experimental study method was adopted. Forty healthy volunteers in Subei People's Hospital were recruited, including 32 females and 8 males, aged 60 to 72 years. Leukocyte platelet-rich fibrin (L-PRF) and A-PRF membranes were prepared after venous blood was extracted from them. The microstructure of two kinds of platelet-rich fibrin (PRF) membranes was observed by field emission scanning electron microscope. The number of samples was 3 in the following experiments. The L-PRF and A-PRF membranes were divided into L-PRF group and A-PRF group and cultured, and then the release concentrations of platelet-derived growth factor-AB (PDGF-AB) and vascular endothelial growth factor (VEGF) in culture supernatant were determined by enzyme-linked immunosorbent assay on culture day 1, 3, 7, and 14. Mice L929 fibroblasts (Fbs) were divided into L-PRF group and A-PRF group, and cultured with L-PRF or A-PRF conditioned medium, respectively. On culture day 1, 3, and 7, the cell proliferation activity was detected by thiazole blue method. The cell migration rate was detected and calculated at 24 h after scratching by scratch test. Thirty-six male BALB/c nude mice aged 6-8 weeks were selected to make a deep partial-thickness burn wound on one hind leg, and then divided into normal saline group, L-PRF group, and A-PRF group, according to the random number table, with 12 mice in each group. The wounds of nude mice in normal saline group were only washed by normal saline, while the wounds of nude mice in L-PRF group and A-PRF group were covered with the corresponding membranes in addition. The wounds of nude mice in the 3 groups were all bandaged and fixed with dressings. On treatment day 4, 7, and 14, the wound healing was observed and the wound healing rate was calculated. Masson staining was used to observe the new collagen in wound tissue, and immunohistochemical staining was used to detect the percentage of CD31 positive cells in the wound. Data were statistically analyzed with independent sample t test, analysis of variance for repeated measurement, analysis of variance for factorial design, one-way analysis of variance, and least significant difference test. Results: L-PRF membrane's dense network structure was composed of coarse fibrin bundles, with scattered white blood cells and platelets with complete morphology. A-PRF membrane's loose network structure was composed of fine fibrin bundles, with scattered small amount of deformed white blood cells and platelets. On culture day 1, the release concentration of PDGF-AB in PRF culture supernatant in A-PRF group was significantly higher than that in L-PRF group (t=5.73, P<0.05), while the release concentrations of VEGF in PRF culture supernatant in the two groups were similar (P>0.05). On culture day 3, 7, and 14, the release concentrations of PDGF-AB and VEGF in PRF culture supernatant in A-PRF group were significantly higher than those in L-PRF group (with t values of 6.93, 7.45, 5.49, 6.97, 8.97, and 13.64, respectively, P<0.05). On culture day 3, 7, and 14, the release concentrations of PDGF-AB and VEGF in PRF culture supernatant in the two groups were all significantly higher than those in the previous time points within the group (P<0.05). On culture day 1, 3, and 7, the proliferation activity of mice Fbs in A-PRF group was 0.293±0.034, 0.582±0.054, and 0.775±0.040, respectively, which were significantly stronger than 0.117±0.013, 0.390±0.036, and 0.581±0.037 in L-PRF group (with t values of 8.38, 5.14, and 6.16, respectively, P<0.05). At 24 h after scratching, the migration rate of mice Fbs in A-PRF group was (60.9±2.2)%, which was significantly higher than (39.1±2.3)% in L-PRF group (t=11.74, P<0.05). On treatment day 4, the wound exudates of nude mice in L-PRF group and A-PRF group were less with no obvious signs of infection, while the wounds of nude mice in normal saline group showed more exudation. On treatment day 7, the wounds of nude mice in L-PRF group and A-PRF group were dry and crusted, while there was still a small amount of exudate in the wounds of nude mice in normal saline group. On treatment day 14, the wounds of nude mice in A-PRF group tended to heal; a small portion of wounds remained in nude mice in L-PRF group; the wound of nude mice was still covered with eschar in normal saline group. On treatment day 4, 7, and 14, the wound healing rate and percentage of CD31 positive cells of nude mice in L-PRF group were all significantly higher than those in normal saline group (P<0.05); compared with those in normal saline group and L-PRF group, the wound healing rate of nude mice in A-PRF group was significantly increased (P<0.05), the newborn collagen was orderly and evenly distributed, with no excessive deposition, and the percentage of CD31 positive cells was significantly increased (P<0.05). Conclusions: The stable fibrin network structure of A-PRF can maintain the sustained release of growth factors, accelerate cell proliferation, and promote cell migration, so as to shorten the healing time and improve the healing quality of deep partial-thickness burn wounds in nude mice.

Reinforcement of colon anastomosis healing with leukocyte platelet-rich fibrin in rabbit model.

AIM: This study investigated the regenerative efficacy of leukocyte platelet-rich fibrin (L-PRF) on colon anastomotic healing in rabbits. MAIN METHODS: Thirty-six healthy male white New Zealand rabbits were subjected to complete transactions of the ascending colon. The rabbits were equally divided into two groups: the control group, where the transected colon ends were anastomosed by a simple interrupted suture pattern, and the L-PRF-treated group, in which L-PRF was wrapped entirely around the anastomotic line. The postoperative acute pain scale was assessed using the Bristol Rabbit Pain Scale before surgery and at each four-hour interval post-operatively. After euthanizing the rabbits, the adhesion degree score, anastomotic bursting pressure, and stenosis degree of the anastomotic colon were assessed, and histopathological examination at the 7th, 14th, and 28th days postoperatively. KEY FINDINGS: Rabbits in both groups showed a significant increase in pain scores compared to baseline. Postoperatively, the L-PRF group exhibited significantly lower pain scores, adhesion scores, and stenosis degrees than the control group. However, the anastomotic bursting pressure was significantly higher in the L-PRF group. Re-epithelialization, polymorphonuclear neutrophil infiltration, granulation tissue formation, and collagen deposition scores were improved considerably in the L-PRF group compared to the control group. Immunostaining of growth factor expression was significantly lower in the control than in the L-PRF group. SIGNIFICANCE: The L-PRF can augment collagen deposition, re-epithelialize the mucosa, promote angiogenesis, reduce adhesions, and diminish the stenosis degree scores. Therefore, it can be considered a promising aid in healing bowel anastomoses.

The Effects of Injectable Platelet-Rich Fibrin and Advanced-Platelet Rich Fibrin on Gingival Fibroblast Cell Vitality, Proliferation, Differentiation.

BACKGROUND: Injectable Platelet Rich Fibrin (I-PRF) and Advanced-Platelet Rich Fibrin (A-PRF) are autologous materials derived from patients' blood and employed in periodontal regenerative surgery. Although I-PRF and A-PRF have different characteristics, their biological effects on gingival tissue fibroblasts remain unclear. This research aims to compare the in vitro capacity in inducing gene expression and proliferation of human gingival fibroblasts between A-PRF and I-PRF. METHODS: Human donors undergoing dental implant surgery were sampled for normal human gingival fibroblasts (NHGFCs), followed by preparing A-PRF and I-PRF membranes. Enzyme-linked immunosorbent assay (ELISA) kit was used to assess the release of platelet-derived growth factor-AA (PDGF-AA), transforming growth factor-beta1 (TGF- ß1), and insulin growth factor-1 (IGF-1) at different periods. Cell viability and proliferation of A-PRF and I-PRF were compared using CCK-8 assay. The impacts of platelet concentration on human gingival fibroblast cells (HGFCs) were evaluated by quantifying the level or amount of phosphorylated extracellular signal-regulated protein kinase (p-ERK), and Matrix metalloproteinases (MMPs), MMP-1 and MMP-3. The effects of PRF on aged human gingival fibroblast cells were examined retrospectively. RESULTS: Overall, A-PRF demonstrated a higher release of TGF-B1 and PDGF-AA, while I-PRF reflected higher levels of IGF-1. A significantly higher level of cell proliferation was induced by higher cell proliferation by A-PRF and I-PRF. Additionally, in comparison to I-PRF, the expression of ERK phosphorylation and MMP-1 &MMP-3 in HGFCs was demonstrated by I-PRF and A-PRF. The increase in A-PRF was time-dependent (p < 0.05). CONCLUSION: Both I-PRF and A-PRF induced a stimulatory biological impact on the proliferation of human gingiva fibroblasts, with the latter demonstrating better capacity in facilitating the release of different growth factors. A-PRF also induced higher gene expression of p-ERK, MMP-1 &MMP-3, and the proliferation of fibroblasts.

Study of platelet-rich fibrin promoting endothelial cell differentiation and angiogenesis induced by transplantation of adipose-derived stem cells.

Diabetic patients are characterized by long wound healing time, and adipose stem cells (ADSCs) can secrete growth factors to promote angiogenesis and improve diabetic wound healing. In this research, we attempted to interrogate the impact of platelet-rich fibrin (PRF) on ADSCs in diabetic wound healing. ADSCs were harvested from human adipose tissues and identified through flow cytometry. After pretreatment with cultured medium supplemented with different concentrations of PRF (2.5%, 5%, and 7.5%), proliferation and differentiation capacity of ADSCs were assessed by CCK-8 assay, qRT-PCR and immunofluorescence (IF), respectively. Tube formation assay measured angiogenesis. Western blot analysis analyzed expression of endothelial markers and the extracellular signal-regulated kinase (ERK) and serine/threonine kinase (Akt) pathways in PRF-induced ADSCs. The CCK-8 experiment indicated that PRF enhanced proliferation of ADSCs in dose-dependent manner, relative to normal control group. The expression of endothelial markers and the capacity of tube formation were significantly promoted by 7.5% PRF. The release of growth factors containing vascular endothelial grow factor (VEGF) and insulin-like growth factor-1 (IGF-1) from PRF was increased with the extension of detection time. When the receptors of VEGF or/and IGF-1 were neutralized, ADSCs differentiation into endothelial cells were obviously inhibited. Additionally, PRF stimulated ERK and Akt pathways, and the inhibitors of ERK and Akt attenuated PRF-induced differentiation of ADSCs into endothelial cells. In conclusion, PRF promoted endothelial cell differentiation and angiogenesis induced by ADSCs in diabetic wound healing, which appears to give guidance for treating patients.

Histological, Immunohistochemical, Biomechanical, and Wettability Evaluations of the Leukocyte- and Platelet-Rich Fibrin Membranes Derived from Canine Blood.

This study aimed to perform histological, immunohistochemical, biomechanical, and wettability assessments of leukocyte- and platelet-rich fibrin (L-PRF) membranes obtained from the blood of healthy dogs. Ten client-owned Labrador Retriever dogs were enrolled. Blood samples were obtained from the external jugular vein using a vacuum tube without anticoagulant, which was immediately centrifuged at 400g for 12 min in a dedicated centrifuge. The L-PRF clot was removed from the tube, and the red clot was released from the buffy coat using a spatula. The membrane was produced using a PRF box. Histological examination identified the three portions of the L-PRF membranes. The first portion was composed mainly of red blood cells with the presence of a low number of leukocytes among them. The second portion was composed of white blood cells, mainly neutrophils. The third portion was composed of the fibrin network which was characterized by acidophilic staining. The immunohistochemical analysis showed that vascular endothelial growth factor and platelet-derived growth factor were expressed in all samples at different intensities, both in cellular components and fibrin mesh. The tensile test and wettability assessments were measured in membranes 30 min and 3 h after production. The 30 min L-PRF membranes supported twice the ultimate tensile strength compared to 3 h L-PRF membranes. The wettability of the 30 min sample membranes was statistically higher than the 3 h sample membranes. In conclusion, the centrifugation protocol allowed production of the L-PRF membrane using canine blood and this was confirmed by histological and immunohistochemical analysis. The mechanical resistance and wettability of the L-PRF membrane were significantly reduced over time.

Does the Choice of Preparation Protocol for Platelet-Rich Fibrin Have Consequences for Healing and Alveolar Ridge Preservation After Tooth Extraction? A Meta-Analysis.

PURPOSE: Multiple preparation protocols for platelet-rich fibrin (PRF) are in use today, and clinical results are often heterogeneous. This study analyzes the impact of the chosen PRF preparation protocol on 1) wound healing and 2) alveolar ridge preservation. METHODS: For this systematic review and meta-analysis, eligible studies were identified in PubMed and Cochrane databases. Included were randomized controlled and controlled clinical trials with healthy patients treated with PRF after atraumatic tooth extraction compared to untreated socket(s), reporting at least one of the following outcome variables: pain, swelling, soft tissue healing, alveolar osteitis risk, horizontal and vertical bone loss, socket fill, and new bone formation. Main predictor variable was relative centrifugal force (RCF) comparing high RCF (high PRF), intermediate RCF (standard [S-PRF]), low RCF (advanced PRF), and various RCF settings (concentrated growth factor preparation [CGF]). The type of centrifugation tubes (silica-coated plastic and glass) was a secondary predictor. Weighted or standardized mean differences, risk ratio and corresponding 95% confidence intervals were calculated. RESULTS: Forty studies published between 2012 and 2022 were selected. The pooled effects of all outcomes were significant against untreated sockets. Within the subgroups high PRF or advanced PRF had the lowest efficacy for many outcome parameters. Pain reduction (in visual analog scale units) was highest for S-PRF (-1.18 [-1.48, -0.88], P < .00001) and CGF (-1.03 [-1.16, -0.90], P < .001). The risk ratio of alveolar osteitis (0.09 [0.01, 0.69], P < .02) and soft tissue healing (standardized mean difference = 2.55 [2.06, 3.03], P < .001) were best for CGF. No subgroup differences were found for bone-related outcomes. No meaningful analysis of the tube material effect was possible. CONCLUSION: This study confirms that PRF is associated with reduced postoperative complications but indicates that preparation protocol influences clinical outcomes. S-PRF and CGF protocols appear to be superior for several outcome parameters.

Lipids of Platelet-Rich Fibrin Reduce the Inflammatory Response in Mesenchymal Cells and Macrophages.

Platelet-rich fibrin (PRF) has a potent anti-inflammatory activity but the components mediating this effect remain unknown. Blood lipids have anti-inflammatory properties. The question arises whether this is also true for the lipid fraction of PRF. To answer this question, lipid fractions of solid and liquid PRF were tested for their potential to lower the inflammatory response of ST2 bone marrow stromal cells and primary bone marrow macrophages exposed to IL1ß and TNFα, and LPS, respectively. Cytokine production and the underlying signalling pathway were analysed by RT-PCR, immunoassays, and Western blotting. We report here that lipids from solid and liquid PRF substantially lowered cytokine-induced expression of IL6, CCL2 and CCL5 in ST2 cells. Moreover, the inflammatory response induced by Pam3CSK4, the agonist of Toll-like receptor (TLR) TLR2, was partially reduced by the lipid extracts in ST2 cells. The PRF lipids further reduced the LPS-induced expression of IL1ß, IL6 and CCL5 in macrophages at the transcriptional level. This was confirmed by showing the ability of PRF lipids to diminish IL6 at the protein level in ST2 cells and macrophages. Likewise, PRF lipid extracts reduced the phosphorylation of p38 and JNK and moderately decreased the phosphorylation of NFκB-p65 in ST2 cells. These findings suggest that the lipid fraction is at least partially responsible for the anti-inflammatory activity of PRF in vitro.

Skin and Bone Regeneration of Solid Bone Marrow Aspirate Concentrate Versus Platelet-Rich Fibrin.

Solid bone marrow aspirate concentrate (sBMAC) is harvested from bone marrow aspirate without anticoagulants by a centrifugation protocol similar to that for platelet-rich fibrin (PRF) prepared from peripheral blood. It was hypothesized that sBMAC could accelerate not only wound healing but also bone regeneration because of the abundant growth factor (GF) releases from enriched bone marrow cells. The purpose of the present study was to investigate skin wound healing and bone regenerative potential of sBMAC compared with arterial blood-derived PRF (Ar-PRF) and venous blood-derived PRF (Ve-PRF) in a skin defect and calvarial bone defect model in rabbits. GF release assays revealed significantly higher release of transforming growth factor-ß (TGF-ß), alkaline phosphatase (ALP), and osteocalcin (OCN) from sBMAC compared with PRFs for 24 h. In the skin defect animal model, sBMAC and PRFs promoted wound bed angiogenesis and re-epithelization in skin defect sites with higher collagen 1 synthesis, cytokeratin AE1/AE3, vascular endothelial growth factor (VEGF) expressions on week 1. Furthermore, a calvarial defect assay revealed that sBMAC promoted new bone formation with a sufficient bone marrow structure similar to that of intact bone in the bone defects. Ar-PRF achieved the second highest bone closure and new bone volume but yielded new bone that was thinner than the intact bone. In conclusion, sBMAC treatment might be a good option instead of PRF as an adjuvant therapy for both skin and bone tissue regeneration therapies in certain clinical situations. Impact statement Solid bone marrow aspirate concentrate (sBMAC) is new type of clot material prepared from bone marrow aspirate. The present study for the first time showed that sBMAC significantly accelerated both skin wound healing and bone formation in the defects, compared with conventional platelet-rich fibrin in rabbit experiment models.

Plasma rich in growth factors (PRGF) and leukocyte-platelet rich fibrin (L-PRF): comparative release of growth factors and biological effect on osteoblasts.

PURPOSE: To compare the release of platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF-I) and interleukin 1ß (IL-1ß) of plasma rich in growth factors (PRGF) and leucocyte platelet-rich fibrin (L-PRF) and to evaluate their biological implication in osteoblasts. METHODS: Blood from 3 healthy volunteers was processed into PRGF, immediate L-PRF (L-PRF 0') and L-PRF 30 min after collection (L-PRF-30') and a control group. Growth factors release were analyzed at 7 times by ELISA. Cell proliferation, collagen-I synthesis and alkaline phosphatase activity were assessed in primary cultures of human osteoblasts. RESULTS: A slower controlled release of IGF-I, VEGF and PDGF was observed in the PRGF group at day 14. A higher synthesis of type I collagen was also quantified in PRGF. L-PRF released significantly higher amounts of IL-1ß, that was almost absent in the PRGF. CONCLUSIONS: The addition of leukocytes dramatically increases the secretion of proinflammatory cytokines, which are likely to negatively influence the synthesis of type I collagen and alkaline phosphatase (ALP) by osteoblasts.

Comparison of physical, mechanical and biological effects of leucocyte-PRF and advanced-PRF on polyacrylamide nanofiber wound dressings: In vitro and in vivo evaluations.

Platelet-rich fibrin (PRF) is extracted from the blood without biochemical interference and, also, with the ability of a long-term release of growth factors that can stimulate tissue repair and regerenation. Here, leucocyte- and platelet-rich fibrin (L-PRF) and advanced platelet-rich fibrin (A-PRF) were extracted and utilized for the creation of nanofibers containing polyacrylamide (PAAm), PAAm / L-PRF and PAAm / A-PRP through electrospinning processing technique. The effect of the type of PRF on the physical, mechanical and biological properties of the resultant nanofiberous wound dressings are thoroughly evaluated. The results presented in the current study reveals that the fiber diameter is grealtly reduced through the utilization of L-PRF. In addition, mechanical property is also positively affected by L-PRF and the degradation rate is found to be higher compared to A-PRF group. The L929 cells proliferation and adhesion, angiogenesis potential and wound healing ability was significantly higher in PAAm/A-PRF nanofibers compared to pure PAAm and PAAm/L-PRF nanofibers owed to the release of vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF). Overall, the utilization of L-PRF or A-PRF can improve the physical, mechanical and biological behavior of nanofiber making them an ideal candidate for wound dressings, with the emphasis on the skin tissue repair and regeneration applications.

Effectiveness of Bone Marrow-Derived Platelet-Rich Fibrin on Rotator Cuff Healing in a Rabbit Degenerative Model.

BACKGROUND: Platelet-rich fibrin (PRF) is a second-generation platelet concentrate. Although peripheral blood-derived PRF (P-PRF) is commonly applied in biological augmentation, there is no report about the therapeutic effect of bone marrow-derived PRF (BM-PRF) for degenerative rotator cuff tears (RCTs). PURPOSE/HYPOTHESIS: To examine the effects of platelet-rich plasma (PRP), P-PRF, and BM-PRF during rotator cuff repair (RCR) in degenerative RCTs in rabbits. We hypothesized that BM-PRF would accelerate the bone-tendon healing after RCR. STUDY DESIGN: Controlled laboratory study. METHODS: Degenerative RCT models were created 2 weeks before beginning the study, and 68 juvenile rabbits were divided into 4 groups: the control, PRP, P-PRF, and BM-PRF groups. RCR without augmentation was done in the control group. PRP was prepared by centrifuging peripheral blood twice using a plastic tube. P-PRF and BM-PRF were prepared by centrifuging peripheral blood and bone marrow, respectively, using a glass tube. Rabbits from PRP, P-PRF, and BM-PRF groups were administered the augmentation in a similar fashion for RCR, between the rotator cuff and the footprint of the humerus. At 4, 8, and 12 weeks, rabbits were euthanized and histologically assessed using hematoxylin and eosin staining, Alcian blue staining, and immunohistochemical staining for type I and III collagen. The sections were also evaluated with immunofluorescence staining of vascular endothelial growth factor (VEGF) at 4 weeks. RESULTS: The continuity was significantly better in the BM-PRF group at 4 weeks (P < .05). Immunofluorescence staining demonstrated that VEGF-positive stained cells were significantly greater in the BM-PRF group than in the control group (P < .01). The modified tendon maturing score was significantly greater in the BM-PRF group than in the control and PRP groups at 12 weeks (P < .05). There was no significant difference in the modified tendon maturing score of the P-PRF group compared with the control group. CONCLUSION: The rabbit model of degenerative RCTs demonstrated that RCR combined with BM-PRF enhanced tendon-bone continuity and increased the VEGF-positive cells at 4 weeks and obtained preferable tendon-bone maturation at 12 weeks. CLINICAL RELEVANCE: RCR augmented with BM-PRF has the potential to improve clinical outcomes for RCTs.

Platelet-Rich Fibrin Reduces IL-1ß Release from Macrophages Undergoing Pyroptosis.

BACKGROUND: Pyroptosis is a catabolic process relevant to periodontal disorders for which interleukin-1ß (IL-1ß) inflammation is central to the pathophysiology of the disease. Despite platelet-rich fibrin (PRF) anti-inflammatory properties and its application to support periodontal regeneration, the capacity of PRF to modulate pyroptosis, specifically the production and release of IL-1ß, remains unknown. The question arises whether PRF could regulate IL-1ß release from macrophages in vitro. METHODS: To answer this question, RAW 264.7 macrophages and primary macrophages obtained from murine bone marrow were primed with PRF before being challenged by lipopolysaccharide (LPS). Cells were then analysed for the pyroptosis signalling components by gene expression analyses and IL-1ß secretion at the protein level. The release of mitochondrial reactive oxygen species (ROS) was also detected. RESULTS: PRF lowered the LPS-induced expression of IL-1ß and NLRP3 inflammasome, caspase-11 and IL-18 in primary macrophages, and IL-1ß and caspase-11 in RAW 264.7 cells. Additionally, PRF diminished the secretion of IL-1ß at the protein level in LPS-induced RAW 264.7 cells. This was shown through immunoassays performed with the supernatant and further confirmed by analysing the lysates of permeabilised cells. Furthermore, PRF reduced the ROS release provoked by LPS in RAW 264.7 cells. Finally, to enhance IL-1ß release from the LPS-primed macrophages, we introduced a second signal with adenosine triphosphate (ATP). In this setting, PRF significantly reduced IL-1ß release in RAW 264.7 cells and a trend to diminish IL-1ß release in primary macrophages. CONCLUSION: These findings suggest that PRF can reduce IL-1ß release and, at least in part, inhibit pyroptosis-related factors in LPS-challenged macrophages.

Tranexamic acid integrated into platelet-rich fibrin produces a robust and resilient antihemorrhagic biological agent: a human cohort study.

OBJECTIVE: To investigate the incorporation of the antifibrinolytic agent tranexamic acid (TA) during platelet-rich fibrin (PRF) formation to produce a robust fibrin agent with procoagulation properties. STUDY DESIGN: Blood from healthy volunteers was collected. Into 3 tubes, TA was immediately added in 1-mL, 0.4-mL, and 0.2-mL volumes, and the fourth tube was without additions. After PRF preparation, the clots were weighed in their raw (clot) and membrane forms. PRF physical properties were analyzed using a universal testing system (Instron). Protein and TA levels in the PRF were analyzed using a bicinchoninic acid assay and a ferric chloride assay, respectively. RESULTS: The addition of TA to PRF led to a robust weight compared with sham control. PRF weight was greater in females in all tested groups. The addition of TA also led to greater resilience to tears, especially at 1-mL TA addition to the blood. Furthermore, TA addition led to a greater value of total protein within the PRF and entrapment of TA in the PRF. CONCLUSIONS: Addition of TA to a PRF preparation leads to robust PRF with greater protein levels and the amalgamation of TA into the PRF. Such an agent may enhance the beneficial properties of PRF and attribute procoagulation properties to it.

Distinctive Roles of Wnt Signaling in Chondrogenic Differentiation of BMSCs under Coupling of Pressure and Platelet-Rich Fibrin.

BACKGROUND: Although newly formed constructs of feasible pressure-preadjusted bone marrow mesenchymal stem cells (BMSCs) and platelet-rich fibrin (PRF) showed biomechanical flexibility and superior capacity for cartilage regeneration, it is still not very clear how BMSCs and seed cells feel mechanical stimuli and convert them into biological signals, and the difference in signal transduction underlying mechanical and chemical cues is also unclear. METHODS: To determine whether mechanical stimulation (hydrostatic pressure) and chemical cues (platelet-rich fibrin, PRF) activate canonical or noncanonical Wnt signaling in BMSCs, BMSCs cocultured with PRF were subjected to hydrostatic pressure loading, and the activation of the Wnt signaling molecules and expression of cartilage-associated proteins and genes were determined by western blotting and polymerase chain reaction (PCR). Inhibitors of canonical or noncanonical Wnt signaling, XVX-939 or L690,330, were adopted to investigate the role of Wnt signaling molecules in mechanically promoted chondrogenic differentiation of BMSCs. RESULTS: Hydrostatic pressure of 120 kPa activated both Wnt/ß-catenin signaling and Wnt/Ca2+ signaling, with the the maximum promotion effect at 60 min. PRF exerted no synergistic effect on Wnt/ß-catenin signaling activation. However, the growth factors released by PRF might reverse the promotion effects of pressure on Wnt/Ca2+ signaling. Real-time PCR and Western blotting results showed that pressure could activate the expression of Col-II, Sox9, and aggrecan in BMSCs cocultured with PRF. Blocking experiment found a positive role of Wnt/ß-catenin signaling, and a negative role of Wnt/Ca2+ signaling in chondrogenic differentiation of the BMSCs. Mutual inhibition exists between canonical and noncanonical Wnt signaling in BMSCs under pressure. CONCLUSION: Wnt signaling participates in the pressure-promoted chondrogenesis of the BMSCs co-cultured with PRF, with canonical and noncanonical pathways playing distinct roles during the process.

Effects of advanced platelet-rich fibrin combined with xenogenic bone on human periodontal ligament stem cells.

OBJECTIVES: In this study, we aimed to investigate the effects of a mixture of advanced platelet-rich fibrin (A-PRF) and xenogenic bone substitute material (XBSM) on the proliferation and migration of human periodontal ligament stem cells (hPDLSCs) based on the in vitro release of growth factors. MATERIAL AND METHODS: The concentrations of platelet-derived growth factor-AB (PDGF-AB) and vascular endothelial growth factor (VEGF) released by the A-PRF-XBSM mixture were estimated using enzyme-linked immunoassay for up to 7 d. The A-PRF-XBSM mixture exudate was incubated with hPDLSCs. At Days 1, 3, 5, and 7, cell proliferation and migration were investigated by cell counting and wound-healing assays. RESULTS: PDGD-AB and VEGF were released from the A-PRF-XBSM mixture exudate for up to 7 days. hPDLSCs were cultured in media with various concentrations of the A-PRF-XBSM mixture exudate and exhibited their proliferation and migration ability. Furthermore, the factors released from the 100% A-PRF-XBSM mixture exudate had a substantial effect on cell migration, whereas those released from 4% and 20% A-PRF-XBSM mixture exudates stimulated hPDLSC proliferation. CONCLUSIONS: A-PRF-XBSM mixture continuously released growth factors over 7 days and enhanced hPDLSC proliferation and migration. Therefore, A-PRF in combination with XBSM might provide potential advantages for periodontal tissue regeneration.

Impact of PRP and PRF on Viability of Zoledronic Acid Treated Osteoblasts in 2D and 3D Cell Culture.

BACKGROUND/AIM: Zoledronic acid (ZA) treatment of in vitro cultured osteoblasts (OB) results in reduction in viability, proliferation and differentiation. These effects are slightly attenuated when platelets-rich fibrin and plasma (PRF and PRP) are added. However, it is still unknown whether application of PRP/PRF on ZA-treated OB in a 3D-environment would influence the viability in relation to 2D-cultivation. MATERIALS AND METHODS: Non-treated and ZA-treated OB were cultivated in 2D conditions or seeded in a 3D collagen scaffold with and without PRP/PRF. MTT test was carried out after 5 days of colonization. 4,6-diamidino-2'-phenylindole, dihydrochloride (DAPI)-staining was performed in OB grown in 3D scaffolds to ensure spatial distribution of OB. RESULTS: ZA led to a significant reduction in cell viability compared to the control group. Addition of either PRF or PRP to the 3D colonized and ZA-treated OB significantly enhanced their survival and viability in relation to 2D monolayer cultivation. CONCLUSION: The use of 3D-scaffolds has a positive effect on OB viability, and stimulation by PRF and PRP may provide a therapeutic approach to transfer these results into clinical routine for the treatment of patients with bisphosphonate related osteonecrosis of the jaw (BR-ONJ).

A Standardized g-Force Allows the Preparation of Similar Platelet-Rich Fibrin Qualities Regardless of Rotor Angle.

Platelet-rich fibrin (PRF) is an autologous blood concentrate that supports tissue regeneration. The effect of the centrifuge rotor angle in the fabrication of PRF is still not fully elucidated. The hypothesis of this study is: When applying the same g-force (relative centrifugal force [RCF]) and centrifugation time, PRF components and bioactivity are not modified using either a swing-out rotor or a fixed angle rotor. For this purpose, peripheral blood samples (from five donors) were used to gain solid (710 ×g, 8 min) and liquid (44 ×g, 8 min) PRF matrices using three different centrifuges (one fixed angle as a control and two different swing-out rotor centrifuges). The physical characteristics of the solid PRF were measured to evaluate the clot formation and cellular distribution. The liquid PRF was used to evaluate the cell number, bioactivity, and influence on primary human osteoblasts (pOBs) and primary human fibroblasts (pHFs) in vitro. Solid PRF clots were significantly larger in the group of fixed rotor centrifuges compared with either of the two evaluated swing-out rotor centrifuges. No differences were observed when evaluating the cellular distribution within the solid PRF. No statistically significant differences were documented in the cell's density in liquid PRF samples (platelets, lymphocytes, neutrophils, eosinophils, and basophils) among the differently gained PRF samples. No statistically significant differences were documented for the released growth factors (vascular endothelial growth factor, epidermal growth factor, and transforming growth factor beta 1) over 7 days. pOBs and pHFs viability after treatment with PRF conditioned media showed no statistically significant differences between the evaluated groups. However, the number of adherent cells treated with PRF obtained with the use of the fixed angle rotor was significantly higher when compared with those treated with PRF obtained by using the swing-out rotors. The presented results confirm that regardless of the centrifuge rotor used, the components and bioactivity of solid and liquid PRF matrices are modified by the applied RCF and centrifugation time. These findings are of great importance for highlighting the essential role of adapting the centrifugation protocols when using different centrifuges and to correctly report the used centrifugation protocols in scientific research to allow for reproducible results. Impact statement Platelet-rich fibrin (PRF) is prepared from autologous peripheral blood and is widely applied in research and clinical treatments. The centrifugation parameters used during the preparation of PRF directly influence its components and bioactivity. By using a standardized protocol, the present study demonstrated that adapting various centrifuges to a standardized relative centrifugal force and centrifugation protocol resulted in reproducible PRF matrices with similar bioactivity, regardless of the centrifuge rotor angle. These findings underline the necessity to carefully adapt and correctly report the used centrifuge and centrifugation protocols in scientific research to allow reproducible results.