Comparative microcomputed tomography and histological analysis of the effects of a horizontal platelet-rich fibrin bone block on maxillary sinus augmentation: A preclinical in vivo study.

BACKGROUND: While suggested to be effective in tissue regeneration, the effects of horizontal platelet-rich fibrin (H-PRF) bone block in sinus augmentation have not been verified in an animal model. METHODS: A total of 12 male New Zealand white rabbits that underwent sinus augmentation were divided into two groups: deproteinized bovine bone mineral (DBBM) only and H-PRF bone block. H-PRF was prepared at 700 × g for 8 min using a horizontal centrifuge. The H-PRF bone block was prepared by mixing 0.1 g DBBM with H-PRF fragments and then adding liquid H-PRF. Samples were collected after 4 and 8 weeks and analyzed using microcomputed tomography (micro-CT) for vertical bone gain of the sinus, bone volume/total volume (BV/TV) percentage, trabecular number (Tb.N), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp). Then, histological analyses were performed to investigate new blood vessels, material residue, bone formation and osteoclasts. RESULTS: Higher vertical bone gain of the sinus floor, BV/TV percentage, Tb.Th, and Tb.N and lower Tb.Sp were found in the H-PRF bone block group at both time points compared with the DBBM group. Higher amounts of new blood vessels and more osteoclasts were found in the H-PRF bone block group than in the DBBM group at both time points, especially in the regions close to the bone plate. More new bone formation and less material residue were observed in the H-PRF bone block group at 8 weeks. CONCLUSIONS: H-PRF bone block showed greater potential for sinus augmentation by promoting angiogenesis, bone formation and bone remodeling in a rabbit model.

Effect of relative centrifugal force on the biological properties of liquid platelet-rich fibrin produced via horizontal centrifugation.

OBJECTIVES: Platelet-rich fibrin (PRF) in liquid form has shown advantages in tissue engineering including acting as injectable fillers and drug carriers. However, few studies have investigated the best relative centrifugal force (RCF) for preparing liquid PRF. The aim of the present study was to find out optimal centrifugation force for preparing liquid PRF. MATERIALS AND METHODS: Liquid PRF was prepared using horizontal centrifugation (liquid H-PRF) with RCF ranging from 100 g, 300 g, 500 g, to 700 g for 8 min. The volume, weight, solidification time, and tensile properties were subsequently investigated. Scanning electron microscopy (SEM) and rheologic tests were carried out to investigate the microstructure and rheologic properties of liquid H-PRF after natural polymerization. The total number, concentration, and distribution of cells within each liquid H-PRF was evaluated by complete blood count (CBC) analysis and hematoxylin-eosin staining. RESULTS: As RCF values increased, the volume and weight of liquid H-PRF both increased accordingly. SEM images revealed that as the centrifugal force increased, the fibrin bundles became thinner with a denser fibrin network, and rheologic tests revealed improved mechanical properties. CBC analysis demonstrated that 500 g group had the highest number of leukocytes and neutrophils, whereas 100 g group yielded the highest concentration of leukocytes and platelets. Furthermore, histological analysis suggests that cells obtained by 500 g for 8 min were most evenly distributed in liquid H-PRF. CONCLUSIONS: In summary, the present study provided insights into the contents of liquid H-PRF prepared at different centrifugation forces, enabling clinicians to choose proper centrifugation forces based on their needs. CLINICAL RELEVANCE: The present findings provide theoretical basis for clinical choice of liquid H-PRF protocol from mechanical, cell contents, and histological aspects.

Comparison of platelet-rich fibrin (PRF) produced using 3 commercially available centrifuges at both high (~ 700 g) and low (~ 200 g) relative centrifugation forces.

OBJECTIVES: Platelet-rich fibrin (PRF) has gained tremendous momentum in recent years as a natural autologous growth factor derived from blood capable of stimulating tissue regeneration. Owing to its widespread use, many companies have commercialized various centrifugation devices with various proposed protocols. The aim of the present study was to compare 3 different commercially available centrifuges at both high and low g-force protocols. MATERIALS AND METHODS: PRF was produced on three commercially available centrifuges including the IntraSpin Device (IntraLock), the Duo Quattro (Process for PRF), and Salvin (Salvin Dental). Two separate protocols were tested on each machine including the original leukocyte and platelet-rich fibrin (L-PRF) protocol (~ 700 RCF max (~ 400 RCF clot) for 12 min) as well as the advanced platelet-rich fibrin (A-PRF+) protocol (~ 200 g RCF max (~ 130 g RCF clot) for 8 min). Each of the tested groups was compared for cell numbers, growth factor release, scanning electron microscopy (SEM) for morphological differences, and clot size (both weight and length/width). RESULTS: The present study found that PRF clots produced utilizing the low-speed centrifugation speeds (~ 200 g for 8 min) produce clots that (1) contained a higher concentration of evenly distributed platelets, (2) secreted higher concentrations of growth factors over a 10 day period, and (3) were smaller in size. This was irrespective of the centrifugation device utilized and consistently observed on all 3 devices. The greatest impact was found between the protocols utilized (up to a 200%). Interestingly, it was further revealed that the centrifugation tubes used had a much greater impact on the final size outcome of PRF clots when compared to centrifugation devices. It was found that, in general, the Process for PRF tubes produced significantly greater-sized clots when compared to other commercially available tubes. The Salvin Dental tubes also produced significantly greater PRF clots when compared to the IntraLock tubes on each of the tested centrifugation devices. CONCLUSIONS: The present study demonstrated the reproducibility of a scientific concept (reduction in RCF produces PRF clots with more evenly distributed cells and growth factors) utilizing different devices. Furthermore, (and until now overlooked), it was revealed for the first time that the centrifugation tubes are central to the quality production of PRF. Future research investigating tube characteristics thus becomes critically important for the future optimization of PRF. CLINICAL RELEVANCE: This is the first study to reveal the marked impact of centrifugation tubes on the final production of PRF. Future study thus becomes markedly important to further optimize the quality of PRF-based matrices. It was further found that little variability existed between the centrifugation devices if optimized centrifugation protocols (lower centrifugation speeds) were utilized.

Drug delivery nanoparticles for preventing implant bacterial infections based on the bacteria and immunity mechanisms.

Implant dysfunction and failure during medical treatment can be attributed to bacterial infection with Staphylococcus aureus and Enterococcus faecalis, which are the prevalent strains responsible for implant infections. Currently, antibiotics are primarily used either locally or systemically to prevent and treat bacterial infections in implants. However, the effectiveness of this approach is unsatisfactory. Therefore, the development of new antimicrobial medications is crucial to address the clinical challenges associated with implant infections. In this study, a nanoparticle (ICG+RSG) composed of indocyanine green (ICG) and rosiglitazone (RSG), and delivered using 1,2-dipalmitoyl-snglycero-3-phosphocholine (DPPC) was prepared. ICG+RSG has photothermal and photodynamic properties to eliminate bacteria at the infection site by releasing reactive oxygen species and increasing the temperature. Additionally, it regulates phagocytosis and macrophage polarization to modulate the immune response in the body. ICG+RSG kills bacteria and reduces tissue inflammation, showing potential for preventing implant infections.

Characterization of Chitosan-Gallic Acid Graft Copolymer for Periodontal Dressing Hydrogel Application.

The postoperative periodontal wound is in a complex physiological environment; the bacteria accumulation, the saliva stimulation, and the food residues retention will aggravate the wound deterioration. Commercial periodontal dressings have been widely used for postoperative periodontal treatment, and there still exists some problems, such as poor biocompatibility, weak adhesion, insufficient antibacterial, and anti-inflammatory properties. In this study, a chitosan-gallic acid graft copolymer (CS-GA) is synthesized as a potential periodontal dressing hydrogel. CS-GA possesses high swelling rate, adjustable degradability, self-healing ability, biocompatibility, strong adhesion ability, high mechanical properties and toughness. Furthermore, CS-GA has good scavenging ability for ·OH, O2 - , and 1 O2. And CS-GA has good inhibition effect on different bacterial through bacterial membranes damage. CS-GA can stop bleeding in a short time and adsorb erythrocytes to form physical blood clots to enhance the hemostatic performance. In addition, CS-GA can reduce inflammatory factors expressions, increase collagen fibers deposition, and neovascularization to promote wounds healing, which makes it as a potential periodontal dressing for postoperative tissue restoration.

Microenvironment-Regulating Drug Delivery Nanoparticles for Treating and Preventing Typical Biofilm-Induced Oral Diseases.

The oral cavity comprises an environment full of microorganisms. Dysregulation of this microbial-cellular microenvironment will lead to a series of oral diseases, such as implant-associated infection caused by Staphylococcus aureus (S. aureus) biofilms and periodontitis initiated by Streptococcus oralis (S. oralis). In this study, a liposome-encapsulated indocyanine green (ICG) and rapamycin drug-delivery nanoparticle (ICG-rapamycin) is designed to treat and prevent two typical biofilm-induced oral diseases by regulating the microbial-cellular microenvironment. ICG-rapamycin elevates the reactive oxygen species (ROS) and temperature levels to facilitate photodynamic and photothermal mechanisms under near-infrared (NIR) laser irradiation for anti-bacteria. In addition, it prevents biofilm formation by promoting bacterial motility with increasing the ATP levels. The nanoparticles modulate the microbial-cellular interaction to reduce cellular inflammation and enhance bacterial clearance, which includes promoting the M2 polarization of macrophages, upregulating the anti-inflammatory factor TGF-ß, and enhancing the bacterial phagocytosis of macrophages. Based on these findings, ICG-rapamycin is applied to implant-infected and periodontitis animal models to confirm the effects in vivo. This study demonstrates that ICG-rapamycin can treat and prevent biofilm-induced oral diseases by regulating the microbial-cellular microenvironment, thus providing a promising strategy for future clinical applications.

Exploration of proper heating protocol for injectable horizontal platelet-rich fibrin gel.

PURPOSE: Platelet-rich fibrin (PRF) has been proposed as promising biomaterials with the advantages of host accumulation of platelets and leukocytes with entrapment of growth factors and fibrin scaffold. However, limitations including fast resorption rate (~ 2 weeks) restricts its clinical application. Recent studies have demonstrated heating treatment can prolong PRF degradation. Current published articles used the method of 75 °C for 10 min to obtain longer degradation, while few studies investigated the most suitable temperature for heating horizontal PRF. Our present study was to discover and confirm the optimum temperature for heat treatment before obtaining H-PRF gels by investigating their structure, mechanical properties, and bioactivity of the H-PRF gels after heating treatment. METHODS: In the present study, 2-mL upper layer of horizontal PRF was collected and heated at 45 °C, 60 °C, 75 °C, and 90 °C to heat 2-mL upper layer of horizontal PRF for 10 min before mixing with the 2-mL lower layer horizontal PRF. The weight, solidification time and the degradation properties were subsequently recorded. Scanning electron microscopy (SEM) and rheologic tests were carried out to investigate the microstructure and rheologic properties of each H-PRF gel. The biological activity of each H-PRF gel was also evaluated using live/dead staining. RESULTS: H-PRF gel prepared at 75 °C for 10 min had the fast solidification period (over a tenfold increase than control) as well as the best resistance to degradation. The number of living cells in H-PRF gel is greater than 90%. SEM showed that H-PRF gel becomes denser as the heating temperature increases, and rheologic tests also revealed that the heat treatment improved the mechanical properties of H-PRF gels when compared to non-heated control group. Future clinical studies are needed to further support the clinical application of H-PRF gels in tissue regeneration procedures. CONCLUSIONS: Our results demonstrated that the H-PRF gel obtained at 75 °C for 10 min could produce a uniform, moldable gel with a short time for solidification time, great rheologic behavior and, high percent of live cells in PRF gel. A promising use of the commonly utilized PRF gel was achieved facilitating tissue regeneration and preventing degradation.

Preparation, characterization and biological properties of a novel bone block composed of platelet rich fibrin and a deproteinized bovine bone mineral.

Alveolar bone defects caused by tooth loss often lead to challenges in implant dentistry, with a need for development of optimal bone biomaterials to predictably rebuild these tissues. To address this problem, we fabricated a novel bone block using platelet-rich fibrin (PRF) and Deproteinized Bovine Bone Mineral (DBBM), and characterized their mechanical and biological properties. The bone block was prepared by mixing DBBM, Liquid-PRF, and Solid-PRF fragments in various combinations as follows: (1) BLOCK-1 made with Solid-PRF fragments + DBBM, (2) BLOCK-2 made with Liquid-PRF + DBBM, (3) BLOCK-3 made with Solid-PRF fragments + Liquid-PRF + DBBM. The time for solidification and the degradation properties were subsequently recorded. Scanning electron microscopy (SEM) and tensile tests were carried out to investigate the microstructure and mechanical properties of each block. The bioactivity of the three groups towards osteoblast differentiation was also evaluated by culturing cells with the conditioned medium from each of the three groups including cell proliferation assay, cell migration assay, alkaline phosphatase (ALP) staining, and alizarin red staining (ARS), as well as by real-time PCR for genes encoding runt-related transcription factor 2 (RUNX2), ALP, collagen type I alpha1(COL1A1) and osteocalcin (OCN). BLOCK-3 made with Solid-PRF fragments + Liquid-PRF + DBBM had by far the fastest solidification period (over a 10-fold increase) as well as the most resistance to degradation. SEM and tensile tests also revealed that the mechanical properties of BLOCK-3 were superior in strength when compared to all other groups and further induced the highest osteoblast migration and osteogenic differentiation confirmed by ALP, ARS and real-time PCR. PRF bone blocks made through the combination of Solid-PRF fragments + Liquid-PRF + DBBM had the greatest mechanical and biological properties when compared to either used alone. Future clinical studies are warranted to further support the clinical application of PRF bone blocks in bone regeneration procedures.

Antibacterial effects of platelet-rich fibrin produced by horizontal centrifugation.

Platelet-rich fibrin (PRF) has been widely used owing to its ability to stimulate tissue regeneration. To date, few studies have described the antibacterial properties of PRF. Previously, PRF prepared by horizontal centrifugation (H-PRF) was shown to contain more immune cells than leukocyte- and platelet-rich fibrin (L-PRF). This study aimed to compare the antimicrobial effects of PRFs against Staphylococcus aureus and Escherichia coli in vitro and to determine whether the antibacterial effects correlated with the number of immune cells. Blood samples were obtained from eight healthy donors to prepare L-PRF and H-PRF. The sizes and weights of L-PRF and H-PRF were first evaluated, and their antibacterial effects against S. aureus and E. coli were then tested in vitro using the inhibition ring and plate-counting test methods. Flow-cytometric analysis of the cell components of L-PRF and H-PRF was also performed. No significant differences in size or weight were observed between the L-PRF and H-PRF groups. The H-PRF group contained more leukocytes than the L-PRF group. While both PRFs had notable antimicrobial activity against S. aureus and E. coli, H-PRF demonstrated a significantly better antibacterial effect than L-PRF. Furthermore, the antimicrobial ability of the PRF solid was less efficient than that of wet PRF. In conclusion, H-PRF exhibited better antibacterial activity than L-PRF, which might have been attributed to having more immune cells.

A novel method for evaluating and quantifying cell types in platelet rich fibrin and an introduction to horizontal centrifugation.

Platelet rich fibrin (PRF) has been utilized clinically as a platelet concentrate capable of stimulating tissue regeneration. Interestingly, several protocols have been proposed with little data obtained regarding the final cell counts following centrifugation. The aim of the present study was to compare different commercially available centrifuges and their respective protocols utilizing a novel method to quantify cells. One millimeter blood layers following centrifugation were sequentially pipetted from the upper layer downward until all 10 mL were harvested in sequential samples. Thereafter, each sample was sent for CBC analysis to accurately quantify precisely cell numbers within each separate blood layer following centrifugation. The results from this study revealed that L-PRF protocols (2700 rpm × 12 min) produced a clot with the majority of platelets and leukocytes concentrated within the buffy coat with relatively no cells found within the first 4 mL of L-PRF. Slower centrifugation protocols produced using the A-PRF protocols (1300 rpm × 8 min) produced a more evenly distributed number of platelets throughout PRF. Injectable-PRF (i-PRF) protocols produced the highest concentration of leukocytes/platelets, however, the total number of leukocytes and platelets were significantly lower owing to the decreased total volume collected. Horizontal centrifugation produced a significant increase in both the number and concentration of platelets and leukocytes (up to 3.5× higher for either solid/liquid PRF). When compared to either fixed or angled centrifuge (InstraSpin, Process for PRF). In conclusion, the present study revealed a novel/accurate method to quantify cells following PRF protocols. Furthermore, PRF produced via horizontal centrifugation accumulated a higher number and concentration of platelets/leukocytes when compared to either fixed-angle centrifugation.