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Recently, new centrifugation protocols for the preparation of platelet-rich fibrin (PRF) have been introduced in an attempt to further improve the beneficial impact of these 2nd generation platelet concentrate membranes. This in-vitro study aimed to compare the biological and physical characteristics of three types of PRF membranes using two different centrifuges with adapted relative centrifugal forces (RCF): leucocyte- and platelet-rich fibrin, advanced platelet-rich fibrin, and advanced platelet-rich fibrin+. Release of growth factors, macroscopic dimensions, cellular content and mechanical properties of the respective membranes, prepared from blood of the same individual were explored. Furthermore, the impact of timing (blood draw-centrifugation and centrifugation-membrane preparation) was assessed morphologically as well as by electron microscopy scanning. No statistically significant differences amongst the three PRF modifications could be observed, neither in their release of growth factors or the cellular content, nor in clot/membrane dimensions. The difference between both centrifuges were negligible when the same g-force was used. A lower g-force, however, reduced membrane tensile strength. Timing in the preparation process had a significant impact. Adaptation of RCF only had a minimal impact on the final characteristics of PRF membranes.

Various studies have described the biological properties of the Leucocyte- and Platelet Rich Fibrin (L-PRF) such as the antimicrobial effect against wound bacteria, but less is known about the effect against periodontal pathogens. The aim of this study was to evaluate the antibacterial properties of the L-PRF membrane and L-PRF exudate against the main periopathogens cultured on agar plates and in planktonic solution. This study demonstrated the antibacterial effect of the L-PRF membrane against P. intermedia, F. nucleatum, and A. actinomycetemcomitans, but especially against P. gingivalis. The L-PRF exudate also showed a strong inhibition against P. gingivalis on agar plates. No inhibition could be observed for the other bacterial strains. Moreover, L-PRF exudate decreased the number of viable P.gingivalis in a planktonic solution in a dose-dependent way. However, A. actinomycetemcomitans showed an increased growth in planktonic solution when in contact with the L-PRF exudate.

Previous research identified potential prebiotic substrates for oral health like the structural analogues N-acetyl-d-mannosamine (NADM) and N-acetyl-d-glucosamine (NADG). The main hypothesis of the current study was twofold. Firstly, it was hypothesized that the modulatory effects of NADM are not limited to changes in multi-species oral biofilm composition, but also include effects on metabolism, virulence, and inflammatory potential. Secondly, the presence and orientation of their N-acetyl group could play a role. Therefore, a comparison was made between the effects of NADM, NADG and d-(+)-mannose on multi-species oral biofilms. Besides a beneficial compositional shift, NADM-treated biofilms also showed an altered metabolism, a reduced virulence and a decreased inflammatory potential. At a substrate concentration of 1 M, these effects were pronounced for all biofilm aspects, whereas at ~ 0.05 M (1%(w/v)) only the effects on virulence were pronounced. When comparing between substrates, both the presence and orientation of the N-acetyl group played a role. However, this was generally only at 1 M and dependent on the biofilm aspect. Overall, NADM was found to have different effects at two concentrations that beneficially modulate in vitro multi-species oral biofilm composition, metabolism, virulence and inflammatory potential. The presence and orientation of the N-acetyl group influenced these effects.

The oral use of antimicrobial agents embedded in toothpastes and mouth rinses results in an oral microbial massacre with high amounts of dead bacteria in close proximity to few surviving bacteria. It was hypothesized that this provides the surviving pathogenic bacteria a large amount of dead microbial biomass as a nutritional source for growth (necrotrophy). This study demonstrated the necrotrophic growth of periodontal pathogens in the presence of different dead oral species. In addition, the presence of dead bacteria resulted in an outgrowth of several periodontal pathogens in complex multi-species biofilms. Additionally, upon contact with dead oral bacteria, virulence genes of P. intermedia and P. gingivalis were up-regulated (necrovirulence). This resulted in a more pronounced epithelial cytotoxicity (necrotoxicity). These findings indicate that presence of dead bacteria induce necrotrophy, necrovirulence and necrotoxicity in several oral pathogens.

There is evidence that pathogenic bacteria can adapt to antiseptics upon repeated exposure. More alarming is the concomitant increase in antibiotic resistance that has been described for some pathogens. Unfortunately, effects of adaptation and cross-adaptation are hardly known for oral pathogens, which are very frequently exposed to antiseptics. Therefore, this study aimed to determine the in vitro increase in minimum inhibitory concentrations (MICs) in oral pathogens after repeated exposure to chlorhexidine or cetylpyridinium chloride, to examine if (cross-)adaptation to antiseptics/antibiotics occurs, if (cross-)adaptation is reversible and what the potential underlying mechanisms are. When the pathogens were exposed to antiseptics, their MICs significantly increased. This increase was in general at least partially conserved after regrowth without antiseptics. Some of the adapted species also showed cross-adaptation, as shown by increased MICs of antibiotics and the other antiseptic. In most antiseptic-adapted bacteria, cell-surface hydrophobicity was increased and mass-spectrometry analysis revealed changes in expression of proteins involved in a wide range of functional domains. These in vitro data shows the adaptation and cross-adaptation of oral pathogens to antiseptics and antibiotics. This was related to changes in cell surface hydrophobicity and in expression of proteins involved in membrane transport, virulence, oxidative stress protection and metabolism.

Leukocyte- and Platelet-Rich Fibrin (L-PRF) is an autologous platelet concentrate, consisting of a fbrin matrix enriched with platelets, leukocytes and a plethora of cytokines and growth factors. Since L-PRF is produced bedside from whole blood without the use of an anti-coagulant, it is becoming a popular adjuvant in regenerative medicine. While other types of platelet concentrates have been described to stimulate blood vessel formation, little is known about the angiogenic capacities of L-PRF. Therefore, this study aimed to fully characterize the angiogenic potential of L-PRF. With an antibody array, the growth factors released by L-PRF were determined and high levels of CXC chemokine receptor 2 (CXCR-2) ligands and epidermal growth factor (EGF) were found. L-PRF induced in vitro key steps of the angiogenic process: endothelial proliferation, migration and tube formation. In addition, we could clearly demonstrate that L-PRF is able to induce blood vessel formation in vivo, the chorioallantoic membrane assay. In conclusion, we could demonstrate the angiogenic capacity of L-PRF both in vitro and in vivo, underlying the clinical potential of this easy-to-use platelet concentrate. Tis research was supported by grants to I. Lambrichts and J. Ratajczak from the Research Foundation-Flanders (‘Fonds Wetenschappelijk onderzoek Vlaanderen- FWO’, grant n° G089213N). A. Bronckaers, P. Gervois and P. Hilkens are postdoctoral fellows of the FWO. G. Merckx is funded by ‘Bijzonder Onderzoeksfonds (BOF, BOF16DOC06). All authors wish to cordially thank blood donors involved in this study.

More insight into the biological fundamentals of leukocyte platelet-rich fibrin (L-PRF) guided healing is necessary to recommend its application, in particular in deficient bone sites that need to support implants. This study investigated the short-term bone healing effect of L-PRF treatment in cylindrical non-critical sized bone defects with 3 mm diameter and 6 mm depth in tibiae of 18 adult male New Zealand White rabbits. After a randomization process, 96 bone defects were prepared and half of them were filled with a L-PRF membrane, while untreated defects in the opposite tibia served as control group. The rabbits were euthanized after 7, 14 or 28 days of healing. The bone healing of the cortical and medullary areas was investigated by micro-CT, while the expression of molecular markers (RUNX2, VEGFA, COL1A2 and BMP2) was assessed by qRT-PCR. Treatment with L-PRF did not affect the micro-structural bone characteristics of the repaired bone tissue, except for a decrease in the trabecular connectivity at the cortical level after 14 days of healing. At this time, RUNX2 and VEGFA mRNA levels were significantly lower in the treated defects. L-PRF membranes thus had a temporary negative influence on the bone microarchitecture (Tb.Pf) and on the RUNX2 and VEGFA expression during early bone healing. Overall, L-PRF treatment did not enhance bone regeneration in these non-critical size defects after 28 days. ispartof: Scientific Reports vol:7 ispartof: location:England status: published