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Collagen barrier membranes are frequently used in guided tissue and bone regeneration. The aim of this study was to analyze the signature of human serum proteins adsorbed onto collagen membranes using a novel protein extraction method combined with mass spectrometry. Native porcine-derived collagen membranes (Geistlich Bio-Gide®, Wolhusen, Switzerland) were exposed to pooled human serum in vitro and, after thorough washing, subjected to protein extraction either in conjunction with protein enrichment or via a conventional surfactant-based method. The extracted proteins were analyzed via liquid chromatography with tandem mass spectrometry. Bioinformatic analysis of global profiling, gene ontology, and functional enrichment of the identified proteins was performed. Overall, a total of 326 adsorbed serum proteins were identified. The enrichment and conventional methods yielded similar numbers of total (315 vs. 309), exclusive (17 vs. 11), and major bone-related proteins (18 vs. 14). Most of the adsorbed proteins (n = 298) were common to both extraction groups and included several growth factors, extracellular matrix (ECM) proteins, cell adhesion molecules, and angiogenesis mediators involved in bone regeneration. Functional analyses revealed significant enrichment of ECM, exosomes, immune response, and cell growth components. Key proteins [transforming growth factor-beta 1 (TGFβ1), insulin-like growth factor binding proteins (IGFBP-5, -6, -7)] were exclusively detected with the enrichment-based method. In summary, native collagen membranes exhibited a high protein adsorption capacity in vitro. While both extraction methods were effective, the enrichment-based method showed distinct advantages in detecting specific bone-related proteins. Therefore, the use of multiple extraction methods is advisable in studies investigating protein adsorption on biomaterials. [ABSTRACT FROM AUTHOR]

Dimethyl fumarate (DMF), originally proposed to treat multiple sclerosis, is considered to have a spectrum of anti-inflammatory effects that effectively control periodontitis, mainly when applied with a hydrogel delivery system. Chemokine expression by gingival fibroblasts is a significant driver of periodontitis; thus, hydrogel-based strategies to deliver DMF, which in turn dampen chemokine expression, are of potential clinical relevance. To test this approach, we have established a bioassay where chemokine expression is induced by exposing gingival fibroblast to IL1β and TNFα, or with saliva. We show herein that DMF effectively reduced the expression of CXCL8, CXCL1, CXCL2, and CCL2—and lowered the phosphorylation of ERK and JNK—without affecting cell viability. This observation was confirmed by immunoassays with CXCL8. Consistently, the forced chemokine expression in HSC2 oral squamous epithelial cells was greatly diminished by DMF. To implement our hydrogel-based delivery system, gingival fibroblasts were cocultured with gellan gum hydrogels enriched for DMF. In support of our strategy, DMF-enriched gellan gum hydrogels significantly reduced the forced chemokine expression in gingival fibroblasts. Our data suggest that DMF exerts its anti-inflammatory activity in periodontal cells when released from gellan gum hydrogels, suggesting a potential clinical relevance to control overshooting chemokine expression under chronic inflammatory conditions. [ABSTRACT FROM AUTHOR]

Purpose: To compare different socket sealing approaches for alveolar ridge preservation and assess the dimensional changes and histologic characteristics of soft and hard tissues in a 4- to 6-month period. Material and Methods: A total of 22 patients with indicated single-tooth extraction in the maxillary nonmolar region were eligible for this study. After CBCT scanning and minimally traumatic tooth extraction, the alveolar sockets were filled with demineralized bovine bone mineral with collagen (DBBM-C) in patients from all groups except for those in the control group. Patients were divided into groups for socket sealing as follows: unsealed/spontaneous healing (control; n = 6), collagen matrix (n = 5), collagen membrane (n = 5), and autogenous graft (n = 6). A second CBCT scan was taken 4 to 6 months after extraction, and a trephine biopsy of soft and hard tissues was collected during implant placement. Tomographic dimensional changes were compared between groups. Intragroup tomographic evaluation and histological analysis were also performed. Results: Analysis of dimensional changes did not detect differences between the socket sealing groups (P > .05). In an intragroup evaluation, the height of the buccal bone and cross-sectional area of the alveolar ridge were significantly lower 4 to 6 months after extraction for the control group (P = .031). Histological analysis revealed that the socket sealing approach had no impact on hard and soft tissue formation. Conclusion: The data from the present study suggest that socket sealing with a collagen matrix, a collagen membrane exposed to the oral cavity, or an autogenous punch graft had no difference in the effects on volumetric maintenance and tissue formation in a period of 4 to 6 months. [ABSTRACT FROM AUTHOR]

Platelet-rich fibrin (PRF) is prepared by spontaneous coagulation of fractionated blood. When squeezed between two plates, PRF is separated into solid PRF membranes and a liquid exudate, the PRF serum. The question arises regarding how much the overall activity remains in the PRF membranes and what is discarded into the PRF serum. To this end, we have exposed gingival fibroblasts to lysates prepared from PRF membranes and PRF serum, followed by bulk RNA sequencing. A total of 268 up- and 136 down-regulated genes in gingival fibroblasts exposed to PRF membrane lysates were significantly regulated under the premise of a minimum log2 with 2.5-fold change and a minus log10 significance level of two, respectively. PRF serum only caused 62 up- and 32 down-regulated genes under these conditions. Among the 46 commonly up-regulated genes were CXCL1, CXCL5, CXCL6, CXCL8, IL33, IL6, and PTGS2/COX2, stanniocalcin-1—all linked to an inflammatory response. PRF membrane lysates further increased chemokines CCL2, CCL7, CXCL2, CXCL3, and IL1R1, IL1RL1, and IL1RN, as well as the paracrine factors IL11, LIF, IGF1, BMP2, BMP6, FGF2, and CCN2/CTGF, and all hyaluronan synthases. On the other hand, PRF serum increased DKK1. The genes commonly down-regulated by PRF membrane lysates and PRF serum included interferon-induced protein with tetratricopeptide repeats (IFIT1, IFIT2, IFIT3) and odd-skipped-related transcription factors (OSR1 and OSR2), as well as FGF18 and GDF15, respectively. Taken together, PRF membrane lysates, compared to PRF serum, cause a more complex response in gingival fibroblasts, but each increased chemokine expression in gingival fibroblasts. [ABSTRACT FROM AUTHOR]

Platelet-rich fibrin (PRF), originally used to support soft tissue healing, is also considered a therapeutic option for treating oral lichen planus and leukoplakia. The progression from the two premalignant lesions to the aggressive malignant oral squamous cell carcinoma involves an inflammatory process linked to chemokine expression. Thus, there is a rationale for studying how PRF modulates the expression of chemokines in oral squamous carcinoma cells. To this aim, we expose the oral squamous carcinoma cell line HSC2 to IL1β and TNFα either alone or in the presence of lysates obtained from solid PRF membranes. We report here that in HSC2 cells, PRF lysates significantly reduce the forced transcription of chemokines, e.g., CXCL1, CXCL2, CXCL8, CXCL10, and CCL5. Moreover, PRF lysates attenuate the nuclear translocation of p65 in HSC2 oral epithelial cells when exposed to IL1β and TNFα. PRF lysates further reduce chemokine expression provoked by poly:IC HMW. Even though less pronounced, PRF lysates reduce IL1β- and TNFα-induced chemokine expression in TR146 cells. In primary oral epithelial cells, however, PRF lysates increase the basal expression of CXCL1, CXCL2 and CXCL8. Thus, PRF can exert a biphasic effect on chemokine expression in oral squamous cell carcinoma cell lines and primary oral epithelial cells. These findings suggest that PRF may reduce inflammation in a malignant environment while provoking an immunological response in healthy oral epithelium. [ABSTRACT FROM AUTHOR]

Gingival fibroblasts are a significant source of paracrine signals required to maintain periodontal homeostasis and to mediate pathological events linked to periodontitis and oral squamous cell carcinomas. Among the potential paracrine signals are stanniocalcin-1 (STC1), involved in oxidative stress and cellular survival; amphiregulin (AREG), a growth factor that mediates the cross-talk between immune cells and epithelial cells; chromosome 11 open reading frame 96 (C11orf96) with an unclear biologic function; and the inflammation-associated prostaglandin E synthase (PTGES). Gingival fibroblasts increasingly express these genes in response to bone allografts containing remnants of injured cells. Thus, the gene expression might be caused by the local release of damage-associated molecular patterns arising from injured cells. The aim of this study is consequently to use the established gene panel as a bioassay to measure the damage-associated activity of oral cell lysates. To this aim, we have exposed gingival fibroblasts to lysates prepared from the squamous carcinoma cell lines TR146 and HSC2, oral epithelial cells, and gingival fibroblasts. We report here that all lysates significantly increased the transcription of the entire gene panel, supported for STC1 at the protein level. Blocking TGF-β receptor 1 kinase with SB431542 only partially reduced the forced expression of STC1, AREG, and C11orf96. SB431542 even increased the PTGES expression. Together, these findings suggest that the damage signals originating from oral cells can change the paracrine activity of gingival fibroblasts. Moreover, the expression panel of genes can serve as a bioassay for testing the biocompatibility of materials for oral application. [ABSTRACT FROM AUTHOR]

Simple Summary: This research investigated the impact of different surface modifications on the osseointegration of dental implants. The study conducted experiments on rabbit tibiae comparing conventional machined surfaces (MSs) with those modified using a laser beam (LSs) or a laser beam incorporating hydroxyapatite (HA) by means of biomimetic methods without thermic treatment (LHSs). Utilizing scanning electron microscopy coupled with energy-dispersive X-ray spectrometry (SEM/EDX) and fluorochrome labeling techniques, the study evaluated bone formation over a four-week period. The results demonstrated significantly improved bone contact and newly formed bone area with LSs and LHSs compared to MSs. These findings suggest that surface modifications, particularly LS modifications and LHS modifications, enhance osseointegration, potentially leading to an enhanced longevity and performance of dental implants in clinical settings. (1) Objective: The aim of this study was to assess the biological behavior of bone tissue on a machined surface (MS) and modifications made by a laser beam (LS) and by a laser beam incorporated with hydroxyapatite (HA) using a biomimetic method without thermic treatment (LHS). (2) Methods: Scanning electron microscopy coupled with energy-dispersive X-ray spectrometry (SEM/EDX) was performed before and after installation in the rabbit tibiae. A total of 20 Albinus rabbits randomly received 30 implants of 3.75 × 10 mm in the right and left tibias, with two implants on each surface in each tibia. In the animals belonging to the 4-week euthanasia period group, intramuscular application of the fluorochromes calcein and alizarin was performed. In implants placed mesially in the tibiofemoral joint, biomechanical analysis was performed by means of a removal torque (N/cm). The tibias with the implants located distally to the joint were submitted for analysis by confocal laser microscopy (mineral apposition rate) and for histometric analysis by bone contact implant (%BIC) and newly formed bone area (%NBA). (3) Results: The SEM showed differences between the surfaces. The biomechanical analysis revealed significant differences in removal torque values between the MSs and LHSs over a 2-week period. Over a 4-week period, both the LSs and LHSs demonstrated removal torque values statistically higher than the MSs. BIC of the LHS implants were statistically superior to MS at the 2-week period and LHS and LS surfaces were statistically superior to MS at the 4-week period. Statistical analysis of the NBA of the implants showed difference between the LHS and MS in the period of 2 weeks. (4) Conclusions: The modifications of the LSs and LHSs provided important physicochemical modifications that favored the deposition of bone tissue on the surface of the implants. [ABSTRACT FROM AUTHOR]

Platelet-rich fibrin (PRF), the coagulated plasma of fractionated blood, is widely used to support tissue regeneration in dentistry, and the underlying cellular and molecular mechanisms are increasingly being understood. Periodontal connective tissues steadily express CXCL8, a chemokine that attracts granulocytes and lymphocytes, supporting homeostatic immunity. Even though PRF is considered to dampen inflammation, it should not be ruled out that PRF increases the expression of CXCL8 in gingival fibroblasts. To test this hypothesis, we conducted a bioassay where gingival fibroblasts were exposed to PRF lysates and the respective serum. We show here that PRF lysates and, to a lesser extent, PRF serum increased the expression of CXCL8 by the gingival fibroblasts, as confirmed by immunoassay. SB203580, the inhibitor of p38 mitogen-activated protein kinase, reduced CXCL8 expression. Consistently, PRF lysates and, to a weaker range, the PRF serum also caused phosphorylation of p38 in gingival fibroblasts. Assuming that PRF is a rich source of growth factors, the TGF-β receptor type I kinase inhibitor SB431542 decreased the PRF-induced expression and translation of CXCL8. The findings suggest that PRF lysates and the respective serum drive CXCL8 expression by activating TGF-β and p38 signaling in gingival fibroblasts. [ABSTRACT FROM AUTHOR]

Platelet-rich fibrin, the coagulated plasma fraction of blood, is commonly used to support natural healing in clinical applications. The rat calvaria defect is a standardized model to study bone regeneration. It remains, however, unclear if the rat calvaria defect is appropriate to investigate the impact of human PRF (Platelet-Rich Fibrin) on bone regeneration. To this end, we soaked Bio-Gide® collagen membranes in human or rat liquid concentrated PRF before placing them onto 5 mm calvarial defects in Sprague Dawley rats. Three weeks later, histology and micro-computed tomography (μCT) were performed. We observed that the collagen membranes soaked with rat PRF show the characteristic features of new bone and areas of mineralized collagen matrix, indicated by a median mineralized volume of 1.5 mm3 (range: 0.9; 5.3 mm3). Histology revealed new bone growing underneath the membrane and hybrid bone where collagen fibers are embedded in the new bone. Moreover, areas of passive mineralization were observed. The collagen membranes soaked with human PRF, however, were devoid of histological features of new bone formation in the center of the defect; only occasionally, new bone formed at the defect margins. Human PRF (h-PRF) caused a median bone volume of 0.9 mm3 (range: 0.3–3.3 mm3), which was significantly lower than what was observed with rat PRF (r-PRF), with a BV median of 1.2 mm3 (range: 0.3–5.9 mm3). Our findings indicate that the rat calvaria defect model is suitable for assessing the effects of rat PRF on bone formation, but caution is warranted when extrapolating conclusions regarding the efficacy of human PRF. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Bio-Gide® is a collagen membrane routinely used in guided bone regeneration. Recent studies have shown that this collagen membrane has osteoconductive properties, meaning that it can support the growth of new bone. However, it has also been observed that the collagen membrane has areas of mineralized fibers which can occur spontaneously and independently of osteoblasts. To better understand how this works, we established a model using minced collagen membranes to reduce the active mineralization of intact collagen membranes in favor of passive mineralization. We thus compared the original intact membrane with a minced collagen membrane in a 5 mm calvarial defect model in Sprague Dawley rats. After three weeks of healing, histology and microcomputed tomography (μCT) were performed. Histological analysis confirmed the osteoconductive properties, with new bone growing inside the intact collagen membrane. However, in minced collagen membranes, the osteoconductive properties were restricted to the defect margins. Interestingly, histology revealed large mineralized areas indicating passive mineralization with no signs of bone formation. In the μCT analysis, the intact collagen membranes caused a higher median mineralized volume (1.5 mm3) compared with the minced group (0.4 mm3), but this lacked significance (p = 0.09). The μCT analysis needs to be interpreted carefully, particularly in defects filled with minced membranes, considering that the mineralized tissue may not necessarily be bone but also the result of passive mineralization. Taken together, the findings suggest that Bio-Gide® collagen membranes support bone formation while also exhibiting potential for passive mineralization. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Based on the evidence of a significant communication and connection pathway between the bone and immune systems, a new science has emerged: osteoimmunology. Indeed, the immune system has a considerable impact on bone health and diseases, as well as on bone formation during grafts and its stability over time. Chronic inflammation induces the excessive production of oxidants. An imbalance between the levels of oxidants and antioxidants is called oxidative stress. This physio-pathological state causes both molecular and cellular damage, which leads to DNA alterations, genetic mutations and cell apoptosis, and thus, impaired immunity followed by delayed or compromised wound healing. Oxidative stress levels experienced by the body affect bone regeneration and maintenance around teeth and dental implants. As the immune system and bone remodeling are interconnected, bone loss is a consequence of immune dysregulation. Therefore, oral tissue deficiencies such as periodontitis and peri-implantitis should be regarded as immune diseases. Bone management strategies should include both biological and surgical solutions. These protocols tend to improve immunity through antioxidant production to enhance bone formation and prevent bone loss. This narrative review aims to highlight the relationship between inflammation, oxidation, immunity and bone health in the oral cavity. It intends to help clinicians to detect high-risk situations in oral surgery and to propose biological and clinical solutions that will enhance patients' immune responses and surgical treatment outcomes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The laser treatment and hydroxyapatite coating of dental implants are supposed to enhance osseointegration, but prior to preclinical testing, any negative impact on cell viability should be ruled out. This study aimed to evaluate the response of murine osteogenic cell lineage MC3T3-E1 and the bone marrow-derived stromal cells ST2 to surface modifications of machined titanium discs, e.g., laser treatment without and with hydroxyapatite coating, as well as sandblasting followed by acid etching. Scanning electron microscopy and the contact angle measurements revealed that laser treatment caused a honeycomb surface and higher wettability compared to a machined or sandblasting acid-etched surface. Hydroxyapatite coating, however, not only reduced the viability of MC3T3-E1 and ST2 cells but also provoked the expression and release of interleukin-6. These findings suggest that the laser treatment of titanium supports its hydrophilicity, but adding hydroxyapatite can reduce cell viability and induce the concomitant release of inflammatory cytokines. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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-β (TGF-β) is stored in the bone matrix and may have caused the expression changes, we performed a proteomics analysis, TGF-β immunoassay, and smad2/3 nuclear translocation. ALBA neither showed detectable TGF-β nor was the lysate able to induce smad2/3 translocation. Nevertheless, the TGF-β receptor type I kinase inhibitor SB431542 significantly decreased the expression of IL11, AREG, and C11orf96, suggesting that other agonists than TGF-β 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. [ABSTRACT FROM AUTHOR]

Damaged cells that appear as a consequence of invasive dental procedures or in response to dental materials are supposed to release damage-associated signals. These damage-associated signals not only support tissue regeneration but might also contribute to unwanted fibrosis. The aim of this study was to identify a molecular target that reflects how fibroblasts respond to necrotic oral tissue cells. To simulate the cell damage, we prepared necrotic cell lysates by sonication of the osteocytic cell line IDG-SW3 and exposed them to gingival fibroblasts. RNAseq revealed a moderate increase in IL11 expression in the gingival fibroblasts, a pleiotropic cytokine involved in fibrosis and inflammation, and also in regeneration following trauma. Necrotic lysates of the human squamous carcinoma cell lines HSC2 and TR146, as well as of gingival fibroblasts, however, caused a robust increase in IL11 expression in the gingival fibroblasts. Consistently, immunoassay revealed significantly increased IL11 levels in the gingival fibroblasts when exposed to the respective lysates. Considering that IL11 is a TGF-β target gene, IL11 expression was partially blocked by SB431542, a TGF-β receptor type I kinase inhibitor. Moreover, lysates from the HSC2, TR146, and gingival fibroblasts caused a moderate smad2/3 nuclear translocation in the gingival fibroblasts. Taken together and based on IL11 expression, our findings show that fibroblasts are sensitive to damaged oral tissue cells. [ABSTRACT FROM AUTHOR]

Platelet-rich fibrin (PRF) is an autologous fibrin-rich matrix where activated platelets and leucocytes accumulate. PRF has a wide spectrum of clinical indications with the overall aim of supporting tissue regeneration which in dentistry includes the healing of healthy oral mucosa with epithelial cells. In oral squamous cell carcinoma lesions, however, epithelial cells undergo malignant transformation, indicated by their unrestricted proliferation and migration potential, which should not be further enhanced by a wound-healing formula. Yet, little is known about how oral squamous cell carcinomas respond to PRF lysates. The aim of the present study was, therefore, to test the capacity of PRF lysates to change the transcriptome of HSC2 oral squamous carcinoma cells and perform bioassays to support the findings. Based on the RNAseq analysis, PRF lysates caused an increase in the genes functionally linked to cell replication and migration. In support of this screening approach, PRF lysates enhanced the proliferation of HSC2 oral squamous carcinoma cells, as indicated by 3[H]-thymidine incorporation, cell counting, and the expression of proliferation-related genes. Moreover, PRF lysates sped up cell migration in a scratch assay requiring actin polymerization. Taken together, our data showing that PRF lysates are mitogenic and stimulate motility of oral squamous carcinoma cell lines could be an indication that treatment with PRF in cases of oral carcinoma should be carefully considered. [ABSTRACT FROM AUTHOR]

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-1β. Under these conditions, EMD reduced the expression of all chemokines at the transcriptional level and CXCL8 by immunoassay. The TGF-β receptor type I kinase-inhibitor SB431542 reversed the anti-inflammatory activity. Moreover, EMD-activated TGF-β-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-β signaling mediates the effects of EMD to lower the forced expression of chemokines in oral epithelial cells. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]