How to explain the beneficial effects of platelet-rich plasma.

Platelet-rich plasma (PRP) is the platelet and leukocyte-containing plasmatic fraction of anticoagulated autologous blood. While evidence supporting the clinical use of PRP in dentistry is low, PRP is widely used in sports medicine, orthopedics, and dermatology. Its beneficial activity is commonly attributed to the growth factors released from platelets accumulating in PRP; however, evidence is indirect and not comprehensive. There is thus a demand to revisit PRP with respect to basic and translational science. This review is to (i) recapitulate protocols and tools to prepare PRP; (ii) to discuss the cellular and molecular composition of PRP with a focus on platelets, leukocytes, and the fibrin-rich extracellular matrix of coagulated plasma; and finally (iii) to discuss potential beneficial effects of PRP on a cellular and molecular level with an outlook on its current use in dentistry and other medical fields.

Regeneration of alveolar bone defects in the experimental pig model: A systematic review and meta-analysis.

OBJECTIVE: Pigs are emerging as a preferred experimental in vivo model for bone regeneration. The study objective was to answer the focused PEO question: in the pig model (P), what is the capacity of experimental alveolar bone defects (E) for spontaneous regeneration in terms of new bone formation (O)? METHODS: Following PRISMA guidelines, electronic databases were searched for studies reporting experimental bone defects or extraction socket healing in the maxillae or mandibles of pigs. The main inclusion criteria were the presence of a control group of untreated defects/sockets and the assessment of regeneration via 3D tomography [radiographic defect fill (RDF)] or 2D histomorphometry [new bone formation (NBF)]. Random effects meta-analyses were performed for the outcomes RDF and NBF. RESULTS: Overall, 45 studies were included reporting on alveolar bone defects or extraction sockets, most frequently in the mandibles of minipigs. Based on morphology, defects were broadly classified as 'box-defects' (BD) or 'cylinder-defects' (CD) with a wide range of healing times (10 days to 52 weeks). Meta-analyses revealed pooled estimates (with 95% confidence intervals) of 50% RDF (36.87%-63.15%) and 43.74% NBF (30.47%-57%) in BD, and 44% RDF (16.48%-71.61%) and 39.67% NBF (31.53%-47.81%) in CD, which were similar to estimates of socket-healing [48.74% RDF (40.35%-57.13%) and 38.73% NBF (28.57%-48.89%)]. Heterogeneity in the meta-analysis was high (I2 > 90%). CONCLUSION: A substantial body of literature revealed a high capacity for spontaneous regeneration in experimental alveolar bone defects of (mini)pigs, which should be considered in future studies of bone regeneration in this animal model.

The use of mesenchymal stromal cell secretome to enhance guided bone regeneration in comparison with leukocyte and platelet-rich fibrin.

OBJECTIVES: Secretomes of mesenchymal stromal cells (MSC) represent a novel strategy for growth-factor delivery for tissue regeneration. The objective of this study was to compare the efficacy of adjunctive use of conditioned media of bone-marrow MSC (MSC-CM) with collagen barrier membranes vs. adjunctive use of conditioned media of leukocyte- and platelet-rich fibrin (PRF-CM), a current growth-factor therapy, for guided bone regeneration (GBR). METHODS: MSC-CM and PRF-CM prepared from healthy human donors were subjected to proteomic analysis using mass spectrometry and multiplex immunoassay. Collagen membranes functionalized with MSC-CM or PRF-CM were applied on critical-size rat calvaria defects and new bone formation was assessed via three-dimensional (3D) micro-CT analysis of total defect volume (2 and 4 weeks) and 2D histomorphometric analysis of central defect regions (4 weeks). RESULTS: While both MSC-CM and PRF-CM revealed several bone-related proteins, differentially expressed proteins, especially extracellular matrix components, were increased in MSC-CM. In rat calvaria defects, micro-CT revealed greater total bone coverage in the MSC-CM group after 2 and 4 weeks. Histologically, both groups showed a combination of regular new bone and 'hybrid' new bone, which was formed within the membrane compartment and characterized by incorporation of mineralized collagen fibers. Histomorphometry in central defect sections revealed greater hybrid bone area in the MSC-CM group, while the total new bone area was similar between groups. CONCLUSION: Based on the in vitro and in vivo investigations herein, functionalization of membranes with MSC-CM represents a promising strategy to enhance GBR.

Ten years of injectable platelet-rich fibrin.

The use of platelet-rich fibrin (PRF) has seen widespread advantages over platelet-rich plasma (PRP) in many fields of medicine. However, until 2014, PRF remained clinically available only in its solid clotted form. Modifications to centrifugation protocols and tube technology have led to the development of a liquid injectable version of PRF (i-PRF). This narrative review takes a look back at the technological developments made throughout the past decade and further elaborates on their future clinical applications. Topics covered include improvements in isolation techniques and protocols, ways to further concentrate i-PRF, and the clinical impact and relevance of cooling i-PRF. Next, various uses of i-PRF are discussed, including its use in regenerative periodontology, implantology, endodontics, temporomandibular joint injections, and orthodontic tooth movement. Furthermore, various indications in medicine are also covered, including its use in sports injuries and osteoarthritis of various joints, treatment of diabetic ulcers/wound care, and facial esthetics and hair regrowth. Finally, future applications are discussed, mainly its use as a drug delivery vehicle for small biomolecules, such as growth factors, antibiotics, exosomes, and other medications that may benefit from the controlled and gradual release of biomolecules over time.

Platelet-rich fibrin combined with a particulate bone substitute versus guided bone regeneration in the damaged extraction socket: An in vivo study.

AIM: It has been proposed that platelet-rich fibrin (PRF) can be used to support bone regeneration during alveolar ridge augmentation. The aim of this study was to determine whether an approach utilizing PRF provides similar performance to the established guided bone regeneration (GBR) procedure. MATERIALS AND METHODS: Two-wall defects were surgically created in beagle dogs and treated in three experimental groups: (i) a sticky bone (SB) substitute prepared using liquid PRF and deproteinized porcine bone mineral (DPBM); (ii) SB covered with solid PRF compressed into a membrane; and (iii) GBR performed using DPBM covered by a collagen membrane. Quantitative reverse-transcription polymerase chain reaction was applied to the specimen after 1 week of healing, and microcomputed tomography (micro-CT) and histological outcomes were analysed after 8 weeks of healing. RESULTS: Compared with GBR, PRF resulted in a moderate increase in the expression levels of osteoblast and osteoclast markers, osteocalcin, and calcitonin receptor. Moreover, PRF modestly increased angiogenesis and the inflammation markers vascular endothelial growth factor (VEGF) and IL-6. Micro-CT and histological analyses confirmed the expected increased alveolar ridge area, with no significant differences between the three groups. Consistently, graft consolidation, as indicated by new bone formation at the defect site, did not differ significantly between groups. CONCLUSIONS: The present results demonstrate that PRF-based approaches perform comparably to the established GBR procedure in terms of the consolidation of DPBM in two-wall alveolar defects.

FasL is a catabolic factor in alveolar bone homeostasis.

AIM: Fas ligand (FasL) belongs to the tumour necrosis factor superfamily regulating bone turnover, inflammation, and apoptosis. The appendicular and axial skeleton phenotype of mature Faslgld mice has been reported. The impact of FasL on the alveolar bone providing support for the teeth at mature stages under healthy and induced inflammatory conditions remains unknown. MATERIALS AND METHODS: We performed a phenotypical analysis of mice carrying the homozygous Faslgld mutation and wild-type (WT) mice (C57BL/6) under healthy conditions and upon ligature-induced periodontitis. After 12 days, micro-computed tomography analysis revealed the distance between the cement enamel junction and the alveolar bone crest. Additional structural parameters, such as the bone volume fraction (BV/TV) and the periodontal ligament space volume, were measured. Histological analyses were performed to visualize the catabolic changes at the defect site. RESULTS: Healthy Faslgld mice were found to have more periodontal bone than their WT littermates. Faslgld had no significant effect on inflammatory osteolysis compared to WT controls with ligatures. Histology revealed eroded surfaces at the root and in the inter-proximal bone in both strains. CONCLUSIONS: Our findings suggest that FasL is a catabolic factor in alveolar bone homeostasis but it does not affect the inflammatory osteolysis.

The effect of osteocyte-derived RANKL on bone graft remodeling: An in vivo experimental study.

OBJECTIVES: Autologous bone is considered the gold standard for grafting, yet it suffers from a tendency to undergo resorption over time. While the exact mechanisms of this resorption remain elusive, osteocytes have been shown to play an important role in stimulating osteoclastic activity through their expression of receptor activator of NF-κB (RANK) ligand (RANKL). The aim of this study was to assess the function of osteocyte-derived RANKL in bone graft remodeling. MATERIALS AND METHODS: In Tnfsf11fl/fl ;Dmp1-Cre mice without osteocyte-specific RANKL as well as in Dmp1-Cre control mice, 2.6 mm calvarial bone disks were harvested and transplanted into mice with matching genetic backgrounds either subcutaneously or subperiosteally, creating 4 groups in total. Histology and micro-computed tomography of the grafts and the donor regions were performed 28 days after grafting. RESULTS: Histology revealed marked resorption of subcutaneous control Dmp1-Cre grafts and new bone formation around subperiosteal Dmp1-Cre grafts. In contrast, Tnfsf11fl/fl ;Dmp1-Cre grafts showed effectively neither signs of bone resorption nor formation. Quantitative micro-computed tomography revealed a significant difference in residual graft area between subcutaneous and subperiosteal Dmp1-Cre grafts (p < .01). This difference was not observed between subcutaneous and subperiosteal Tnfsf11fl/fl ;Dmp1-Cre grafts (p = .17). Residual graft volume (p = .08) and thickness (p = .13) did not differ significantly among the groups. Donor area regeneration was comparable between Tnfsf11fl/fl ;Dmp1-Cre and Dmp1-Cre mice and restricted to the defect margins. CONCLUSIONS: The results suggest an active function of osteocyte-derived RANKL in bone graft remodeling.

Oral squamous carcinoma cell lysates provoke exacerbated inflammatory response in gingival fibroblasts.

OBJECTIVES: To study whether damaged epithelial cells and gingival fibroblast could affect the expression of inflammatory cytokines in healthy cells. MATERIALS AND METHODS: Cell suspensions were submitted to different treatments to obtain the lysates: no treatment (supernatant control), sonication, and freeze/thawing. All treatments were centrifuged, and the supernatants of the lysates were used for experimentation. Cell viability assays, RT-qPCR of IL1, IL6 and IL8, IL6 immunoassay, and immunofluorescence of NF-kB p65 were applied to verify the inflammatory crosstalk of damaged cells over healthy plated cells. Furthermore, titanium discs and collagen membranes were treated with lysates and checked for IL8 expression by RT-qPCR. RESULTS: Lysates obtained upon sonication or freeze/thawing of oral squamous carcinoma cell lines provoked a robust increase in the expression of IL1, IL6, and IL8 by gingival fibroblasts, which was confirmed by IL6 immunoassays. Lysates obtained from the gingival fibroblasts failed to increase the expression of inflammatory cytokines in oral squamous carcinoma cells. Additionally, oral squamous carcinoma cell lysates caused the activation of the NF-kB signalling cascade in gingival fibroblasts as indicated by the phosphorylation and nuclear translocation of p65. Finally, oral squamous carcinoma cell lysates adhered to the titanium and collagen membrane surfaces and increased IL8 expression by gingival fibroblasts growing in these materials. CONCLUSIONS: Injured oral epithelial cells can release factors that incite gingival fibroblasts to become pro-inflammatory. CLINICAL RELEVANCE: Injuries affecting the oral mucosa generate epithelial fragments that may reach the underlying connective tissue and provoke inflammation. These injuries are routinely caused by mastication, sonication for teeth cleaning, teeth preparation, prostheses maladaptation, and implant drilling.

Bone Allograft Acid Lysates Change the Genetic Signature of Gingival Fibroblasts.

Bone allografts are widely used as osteoconductive support to guide bone regrowth. Bone allografts are more than a scaffold for the immigrating cells as they maintain some bioactivity of the original bone matrix. Yet, it remains unclear how immigrating cells respond to bone allografts. To this end, we have evaluated the response of mesenchymal cells exposed to acid lysates of bone allografts (ALBA). RNAseq revealed that ALBA has a strong impact on the genetic signature of gingival fibroblasts, indicated by the increased expression of IL11, AREG, C11orf96, STC1, and GK-as confirmed by RT-PCR, and for IL11 and STC1 by immunoassays. Considering that transforming growth factor-ß (TGF-ß) is stored in the bone matrix and may have caused the expression changes, we performed a proteomics analysis, TGF-ß immunoassay, and smad2/3 nuclear translocation. ALBA neither showed detectable TGF-ß nor was the lysate able to induce smad2/3 translocation. Nevertheless, the TGF-ß receptor type I kinase inhibitor SB431542 significantly decreased the expression of IL11, AREG, and C11orf96, suggesting that other agonists than TGF-ß are responsible for the robust cell response. The findings suggest that IL11, AREG, and C11orf96 expression in mesenchymal cells can serve as a bioassay reflecting the bioactivity of the bone allografts.

Enamel Matrix Derivative Suppresses Chemokine Expression in Oral Epithelial Cells.

Epithelial cells in periodontitis patients increasingly express chemokines, suggesting their active involvement in the inflammatory process. Enamel matrix derivative (EMD) is an extract of porcine fetal tooth germs clinically applied to support the regrowth of periodontal tissues. Periodontal regeneration might benefit from the potential anti-inflammatory activity of EMD for epithelial cells. Our aim was, therefore, to set up a bioassay where chemokine expression is initiated in the HSC2 oral squamous carcinoma cell line and then test EMD for its capacity to lower the inflammatory response. To establish the bioassay, HSC2 cells being exposed to TNFα and LPS from E. coli (Escherichia coli) or P. gingivalis (Porphyromonas gingivalis) were subjected to RNAseq. Here, TNFα but not LPS caused a robust increase of chemokines, including CXCL1, CXCL2, CXCL8, CCL5, and CCL20 in HSC2 cells. Polymerase chain reaction confirmed the increased expression of the respective chemokines in cells exposed to TNFα and IL-1ß. Under these conditions, EMD reduced the expression of all chemokines at the transcriptional level and CXCL8 by immunoassay. The TGF-ß receptor type I kinase-inhibitor SB431542 reversed the anti-inflammatory activity. Moreover, EMD-activated TGF-ß-canonical signaling was visualized by phosphorylation of smad3 and nuclear translocation of smad2/3 in HSC2 cells and blocked by SB431542. This observation was confirmed with primary oral epithelial cells where EMD significantly lowered the SB431542-dependent expression of CXCL8. In summary, our findings suggest that TGF-ß signaling mediates the effects of EMD to lower the forced expression of chemokines in oral epithelial cells.

Proteomic Analysis of Mesenchymal Stromal Cells Secretome in Comparison to Leukocyte- and Platelet-Rich Fibrin.

Secretomes of mesenchymal stromal cells (MSCs) are emerging as a novel growth factor (GF)-based strategy for periodontal and bone regeneration. The objective of this study was to compare the secretome of human bone marrow MSC (BMSC) to that of leukocyte- and platelet-rich fibrin (L-PRF), an established GF-based therapy, in the context of wound healing and regeneration. Conditioned media from human BMSCs (BMSC-CM) and L-PRF (LPRF-CM) were subjected to quantitative proteomic analysis using liquid chromatography with tandem mass spectrometry. Global profiles, gene ontology (GO) categories, differentially expressed proteins (DEPs), and gene set enrichment (GSEA) were identified using bioinformatic methods. Concentrations of selected proteins were determined using a multiplex immunoassay. Among the proteins identified in BMSC-CM (2157 proteins) and LPRF-CM (1420 proteins), 1283 proteins were common. GO analysis revealed similarities between the groups in terms of biological processes (cellular organization, protein metabolism) and molecular functions (cellular/protein-binding). Notably, more DEPs were identified in BMSC-CM (n = 550) compared to LPRF-CM (n = 118); these included several key GF, cytokines, and extracellular matrix (ECM) proteins involved in wound healing. GSEA revealed enrichment of ECM (especially bone ECM)-related processes in BMSC-CM and immune-related processes in LPRF-CM. Similar trends for intergroup differences in protein detection were observed in the multiplex analysis. Thus, the secretome of BMSC is enriched for proteins/processes relevant for periodontal and bone regeneration. The in vivo efficacy of this therapy should be evaluated in future studies.

Transglutaminase Activity Is Conserved in Stratified Epithelia and Skin Appendages of Mammals and Birds.

The cross-linking of structural proteins is critical for establishing the mechanical stability of the epithelial compartments of the skin and skin appendages. The introduction of isopeptide bonds between glutamine and lysine residues depends on catalysis by transglutaminases and represents the main protein cross-linking mechanism besides the formation of disulfide bonds. Here, we used a fluorescent labeling protocol to localize the activity of transglutaminases on thin sections of the integument and its appendages in mammals and birds. In human tissues, transglutaminase activity was detected in the granular layer of the epidermis, suprabasal layers of the gingival epithelium, the duct of sweat glands, hair follicles and the nail matrix. In the skin appendages of chickens, transglutaminase activity was present in the claw matrix, the feather follicle sheath, the feather sheath and in differentiating keratinocytes of feather barb ridges. During chicken embryogenesis, active transglutaminase was found in the cornifying epidermis, the periderm and the subperiderm. Transglutaminase activity was also detected in the filiform papillae on the tongue of mice and in conical papillae on the tongue of chickens. In summary, our study reveals that transglutaminase activities are widely distributed in integumentary structures and suggests that transglutamination contributes to the cornification of hard skin appendages such as nails and feathers.

Preoperative buccal bone volume predicts long-term graft retention following augmentation in the esthetic zone: A retrospective case series.

OBJECTIVES: Buccal bone augmentation in the esthetic zone is routinely used to achieve optimal clinical outcomes. Nonetheless, long-term data are sparse, and it is unknown how baseline buccal bone volume affects the retention of the augmented volume over time. MATERIAL AND METHODS: This is a long-term follow-up retrospective case series. After a preoperative computed tomography scan, implants were placed in the anterior maxilla following guided bone regeneration, autogenous block grafting, or both. At the follow-up, patients received a computed tomography scan and a clinical examination. Buccal bone volume was the primary outcome. Buccal bone thickness, peri-implant, and esthetic parameters were secondary outcomes. RESULTS: After a median follow-up of 6.7 years (interquartile range: 4.9-9.4), 28 implants in 19 patients (median age at augmentation: 43.3 years, interquartile range: 34.4-56.7, 53% female) were followed up. Preoperative buccal bone volume at baseline (V0 ) showed a moderate correlation to final buccal bone volume (Vt , rs  = .43) but a strong correlation to the absolute volumetric change (ΔV = Vt -V0 , rs  = -.80). A linear mixed model for Vt had a large intercept of 91.39 (p < .001) and a rather small slope of .11 for V0 (p = .11). Observed differences between treatments were not statistically significant in the mixed model. V0 above 105 mm3 predicted a negative volume change (ΔV < 0) with a specificity of 100% and a sensitivity of 96%. CONCLUSIONS: The results suggest higher gains in sites with lower V0 and point to a cutoff V0 above which the augmented volume is not retained long-term.

Dalbergiones lower the inflammatory response in oral cells in vitro.

OBJECTIVES: Periodontitis is a global health burden that underlines the demand for anti-inflammatory treatment. Dalbergia melanoxylon being a rich source of flavonoids has been widely used in traditional medicine but the potential anti-inflammatory activity of its dalbergiones remains to be shown. MATERIAL AND METHODS: We have isolated 3'-hydroxy-4,4'-dimethoxydalbergione, 4-methoxydalbergione, and 4'-hydroxy-4-methoxydalbergione from Dalbergia melanoxylon and tested their potential anti-inflammatory activity. RESULTS: All dalbergiones are potent inhibitors of an LPS-induced inflammatory response of RAW 264.7 macrophages. This is specified by IL1ß and IL6 production, and the p65 nuclear translocation. Consistently, in primary macrophages, the dalbergiones caused an M1-to-M2 polarization switch indicated by the decreased ration of IL1ß and IL6 versus arginase 1 and YM1 expression. To implement oral cells, we have used gingival fibroblasts exposed to IL1ß and TNFα. Consistently, all dalbergiones reduced the expression of IL6 and IL8 as well as the nuclear translocation of p65. CONCLUSION: These findings increase the accumulating knowledge on dalbergiones and extend it towards its capacity to lower the inflammatory response of oral cells. CLINICAL RELEVANCE: These findings are another piece of evidence that supports the use of herbal medicine to potentially lower inflammatory events related to dentistry.

Enamel Matrix Derivative Decreases Pyroptosis-Related Genes in Macrophages.

Background: Pyroptosis is a caspase-dependent catabolic process relevant to periodontal disorders for which inflammation is central to the pathophysiology of the disease. Although enamel matrix derivative (EMD) has been applied to support periodontal regeneration, its capacity to modulate the expression of pyroptosis-related genes remains unknown. Considering EMD has anti-inflammatory properties and pyroptosis is linked to the activation of the inflammasome in chronic periodontitis, the question arises whether EMD could reduce pyroptosis signalling. Methods: To answer this question, primary macrophages obtained from murine bone marrow and RAW 264.7 macrophages were primed with EMD before being challenged by lipopolysaccharide (LPS). Cells were then analysed for pyroptosis-signalling components by gene expression analyses, interleukin-1ß (IL-1ß) immunoassay, and the detection of caspase-1 (CAS1). The release of mitochondrial reactive oxygen species (ROS) was also detected. Results: We report here that EMD, like the inflammasome (NLRP3) and CAS1 specific inhibitors-MCC950 and Ac-YVAD-cmk, respectively-lowered the LPS-induced expression of NLRP3 in primary macrophages (EMD: p = 0.0232; MCC950: p = 0.0426; Ac-YVAD-cmk: p = 0.0317). EMD further reduced the LPS-induced expression of NLRP3 in RAW 264.7 cells (p = 0.0043). There was also a reduction in CAS1 and IL-1ß in RAW 264.7 macrophages on the transcriptional level (p = 0.0598; p = 0.0283; respectively), in IL-1ß protein release (p = 0.0313), and CAS1 activity. Consistently, EMD, like MCC950 and Ac-YVAD-cmk, diminished the ROS release in activated RAW 264.7 cells. In ST2 murine mesenchymal cells, EMD could not be tested because LPS, saliva, and IL-1ß + TNF-α failed to provoke pyroptosis signalling. Conclusion: These findings suggest that EMD is capable of dampening the expression of pyroptosis-related genes in macrophages.

Blood Clots versus PRF: Activating TGF-ß Signaling and Inhibiting Inflammation In Vitro.

The preparation of platelet-rich fibrin (PRF) requires blood centrifugation to separate the yellow plasma from the red erythrocyte fraction. PRF membranes prepared from coagulated yellow plasma are then transferred to the defect sites to support tissue regeneration. During natural wound healing, however, it is the unfractionated blood clot (UBC) that fills the defect site. It is unclear whether centrifugation is necessary to prepare a blood-derived matrix that supports tissue regeneration. The aim of the present study was to compare lysates prepared from PRF and UBC based on bioassays and degradation of the respective membranes. We report here that lysates prepared from PRF and UBC membranes similarly activate TGF-ß signaling, as indicated by the expression of interleukin 11 (IL-11), NADPH oxidase 4 (NOX-4) and proteoglycan 4 (PRG4) in gingival fibroblasts. Consistently, PRF and UBC lysates stimulated the phosphorylation and nuclear translocation of Smad3 in gingival fibroblasts. We further observed that PRF and UBC lysates have comparable anti-inflammatory activity, as shown by the reduction in lipopolysaccharide (LPS)-induced IL-6, inducible nitric oxidase synthase (iNOS) and cyclooxygenase 2 (COX-2) expression in RAW264.7 cells. Moreover, inflammation induced by Poly (1:C) HMW and FSL-1, which are agonists of Toll-like receptor (TLR) 3 and 2/6, respectively, was reduced by both PRF and UBC. PRF and UBC lysates reduced the nuclear translocation of p65 in LPS-induced RAW264.7 cells. In contrast to the similar activity observed in the bioassays, UBC membranes lack the structural integrity of PRF membranes, as indicated by the rapid and spontaneous disintegration of UBC membranes. We show here that the lysates prepared from PRF and UBC possess robust TGF-ß and anti-inflammatory activity. However, visual inspection of the PRF and UBC membranes confirmed the negative impact of erythrocytes on the structural integrity of membranes prepared from whole blood. The data from the present study suggest that although both UBC and PRF have potent TGF-ß and anti-inflammatory activity, UBC does not have the strength properties required to be used clinically to prepare applicable membranes. Thus, centrifugation is necessary to generate durable and clinically applicable blood-derived membranes.

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.

TGF-ß Signalling Mediates the Anti-Inflammatory Activity of Enamel Matrix Derivative In Vitro.

Enamel matrix derivative (EMD) prepared from extracted porcine fetal tooth material can support the regrow of periodontal tissues. Previous findings suggest that EMD has anti-inflammatory properties and TGF-ß activity in vitro. However, the anti-inflammatory activity of EMD is mediated via TGF-ß has not been considered. To this aim, we first established a bioassay to confirm the anti-inflammatory activity of EMD. The bioassay was based on the RAW 264.7 macrophage cell line and proven with primary macrophages where EMD significantly reduced the forced expression of IL-6. We then confirmed the presence of TGF-ß1 in EMD by immunoassay and by provoking the Smad2/3 nuclear translocation in RAW 264.7 macrophages. Next, we took advantage of the TGF-ß receptor type I kinase-inhibitor SB431542 to block the respective signalling pathway. SB431542 reversed the anti-inflammatory activity of EMD and TGF-ß in a bioassay when IL-6 and CXCL2 expression was driven by the LPS stimulation of RAW 264.7 macrophages. This central observation was supported by showing that SB431542 reversed the anti-inflammatory activity of EMD using IL-1ß and TNF-α-stimulated ST2 bone marrow stromal cells. Together, these findings implicate that the TGF-ß activity mediates at least part of the anti-inflammatory activity of EMD in vitro.

Proof-of-Principle Study Suggesting Potential Anti-Inflammatory Activity of Butyrate and Propionate in Periodontal Cells.

Short-chain fatty acids (SCFAs) are potent immune modulators present in the gingival crevicular fluid. It is therefore likely that SCFAs exert a role in periodontal health and disease. To better understand how SCFAs can module inflammation, we screened acetic acid, propionic acid, and butyric acid for their potential ability to lower the inflammatory response of macrophages, gingival fibroblasts, and oral epithelial cells in vitro. To this end, RAW 264.7 and primary macrophages were exposed to LPSs from Porphyromonas gingivalis (P. gingivalis) with and without the SCFAs. Moreover, gingival fibroblasts and HSC2 oral epithelial cells were exposed to IL1ß and TNFα with and without the SCFAs. We report here that butyrate was effective in reducing the lipopolysaccharide (LPS)-induced expression of IL6 and chemokine (C-X-C motif) ligand 2 (CXCL2) in the RAW 264.7 and primary macrophages. Butyrate also reduced the IL1ß and TNFα-induced expression of IL8, chemokine (C-X-C motif) ligand 1 (CXCL1), and CXCL2 in gingival fibroblasts. Likewise, butyrate lowered the induced expression of CXCL1 and CXCL2, but not IL8, in HSC2 cells. Butyrate further caused a reduction of p65 nuclear translocation in RAW 264.7 macrophages, gingival fibroblasts, and HSC2 cells. Propionate and acetate partially lowered the inflammatory response in vitro but did not reach the level of significance. These findings suggest that not only macrophages, but also gingival fibroblasts and oral epithelial cells are susceptive to the anti-inflammatory activity of butyrate.

Heartwood of Dalbergia cochinchinensis: 4,7,2'-Trihydroxy-4'-methoxyisoflavanol and 6,4'-Dihydroxy-7-methoxyflavane Reduce Cytokine and Chemokine Expression In Vitro.

Dalbergia cochinchinensis has been widely used in traditional medicine because of its flavonoids; however, the impact of the flavonoids to modulate the inflammatory response to oral cells remains to be described. For this aim, we isolated 4,7,2'-trihydroxy-4'-methoxyisoflavanol (472T4MIF) and 6,4'-dihydroxy-7-methoxyflavane (64D7MF) from the heartwood of D. cochinchinensis and confirmed the chemical structure by nuclear magnetic resonance. We show here that both flavonoids are inhibitors of an inflammatory response of murine RAW 264.7 inflammatory macrophages stimulated by LPS. This is indicated by interleukin (IL)1, IL6, and chemokine CCL2 production besides the phosphorylation of p65. Consistently, in primary murine macrophages, both flavonoids decreased the inflammatory response by lowering LPS-induced IL1 and IL6 expression. To introduce oral cells, we have used human gingival fibroblasts and provoked the inflammatory response by exposing them to IL1ß and TNFα. Under these conditions, 472T4MIF, but not 64D7MF, reduced the expression of chemokines CXCL1 and CXCL2. Taken together, we identified two flavonoids that can reduce the expression of cytokines and chemokines in macrophages and fibroblastic cells.