Matrix Metalloproteinases as Regulators of Periodontal Inflammation

Periodontitis are infectious diseases characterized by immune-mediated destruction of periodontal supporting tissues and tooth loss. Matrix metalloproteinases (MMPs) are key proteases involved in destructive periodontal diseases. The study and interest in MMP has been fuelled by emerging evidence demonstrating the broad spectrum of molecules that can be cleaved by them and the myriad of biological processes that they can potentially regulate. The huge complexity of MMP functions within the protease web' is crucial for many physiologic and pathologic processes, including immunity, inflammation, bone resorption, and wound healing. Evidence points out that MMPs assemble in activation cascades and besides their classical extracellular matrix substrates, they cleave several signalling moleculessuch as cytokines, chemokines, and growth factors, among othersregulating their biological functions and/or bioavailability during periodontal diseases. In this review, we provide an overview of emerging evidence of MMPs as regulators of periodontal inflammation.Peer reviewe

Oral implant osseointegration model in C57Bl/6 mice: microtomographic, histological, histomorphometric and molecular characterization

Despite the successful clinical application of titanium (Ti) as a biomaterial, the exact cellular and molecular mechanisms responsible for Ti osseointegration remains unclear, especially because of the limited methodological tools available in this field. Objective: In this study, we present a microscopic and molecular characterization of an oral implant osseointegration model using C57Bl/6 mice. Material and Methods: Forty-eight male wild-type mice received a Ti implant on the edentulous alveolar crest and the peri-implant sites were evaluated through microscopic (μCT, histological and birefringence) and molecular (RealTimePCRarray) analysis in different points in time after surgery (3, 7, 14 and 21 days). Results: The early stages of osseointegration were marked by an increased expression of growth factors and MSC markers. Subsequently, a provisional granulation tissue was formed, with high expression of VEGFb and earlier osteogenic markers (BMPs, ALP and Runx2). The immune/inflammatory phase was evidenced by an increased density of inflammatory cells, and high expression of cytokines (TNF, IL6, IL1) chemokines (CXCL3, CCL2, CCL5 and CXC3CL1) and chemokine receptors (CCR2 and CCR5). Also, iNOS expression remained low, while ARG1 was upregulated, indicating predominance of a M2-type response. At later points in time, the bone matrix density and volume were increased, in agreement with a high expression of Col1a1 and Col21a2. The remodelling process was marked by peaks of MMPs, RANKL and OPG expression at 14 days, and an increased density of osteoclasts. At 21 days, intimate Ti/bone contact was observed, with expression of final osteoblast differentiation markers (PHEX, SOST), as well as red spectrum collagen fibers. Conclusions: This study demonstrated a unique molecular view of oral osseointegration kinetics in C57Bl/6 mice, evidencing potential elements responsible for orchestrating cell migration, proliferation, ECM deposition and maturation, angiogenesis, bone formation and remodeling at the bone-implant interface in parallel with a novel microscopic analysis

New Surgical Model for Bone–Muscle Injury Reveals Age and Gender-Related Healing Patterns in the 5 Lipoxygenase (5LO) Knockout Mouse

Signaling lipid mediators released from 5 lipoxygenase (5LO) pathways influence both bone and muscle cells, interfering in their proliferation and differentiation capacities. A major limitation to studying inflammatory signaling pathways in bone and muscle healing is the inadequacy of available animal models. We developed a surgical injury model in the vastus lateralis (VL) muscle and femur in 129/SvEv littermates mice to study simultaneous musculoskeletal (MSK) healing in male and female, young (3 months) and aged (18 months) WT mice compared to mice lacking 5LO (5LOKO). MSK defects were surgically created using a 1-mm punch device in the VA muscle followed by a 0.5-mm round defect in the femur. After days 7 and 14 post-surgery, the specimens were removed for microtomography (microCT), histopathology, and immunohistochemistry analyses. In addition, non-injured control skeletal muscles along with femur and L5 vertebrae were analyzed. Bones were microCT phenotyped, revealing that aged female WT mice presented reduced BV/TV and trabecular parameters compared to aged males and aged female 5LOKO mice. Skeletal muscles underwent a customized targeted lipidomics investigation for profiling and quantification of lipid signaling mediators (LMs), evidencing age, and gender related-differences in aged female 5LOKO mice compared to matched WT. Histological analysis revealed a suitable bone-healing process with osteoid deposition at day 7 post-surgery, followed by woven bone at day 14 post-surgery, observed in all young mice. Aged WT females displayed increased inflammatory response at day 7 post-surgery, delayed bone matrix maturation, and increased TRAP immunolabeling at day 14 post-surgery compared to 5LOKO females. Skeletal muscles of aged animals showed higher levels of inflammation in comparison to young controls at day 14 post-surgery; however, inflammatory process was attenuated in aged 5LOKO mice compared to aged WT. In conclusion, this new model shows that MSK healing is influenced by age, gender, and the 5LO pathway, which might serve as a potential target to investigate therapeutic interventions and age-related MSK diseases. Our new model is suitable for bone–muscle crosstalk studies

HGMB1 and RAGE as Essential Components of Ti Osseointegration Process in Mice

The release of the prototypic DAMP High Mobility Group Box 1 (HMGB1) into extracellular environment and its binding to the Receptor for Advanced Glycation End Products (RAGE) has been described to trigger sterile inflammation and regulate healing outcome. However, their role on host response to Ti-based biomaterials and in the subsequent osseointegration remains unexplored. In this study, HMGB1 and RAGE inhibition in the Ti-mediated osseointegration were investigated in C57Bl/6 mice. C57Bl/6 mice received a Ti-device implantation (Ti-screw in the edentulous alveolar crest and a Ti-disc in the subcutaneous tissue) and were evaluated by microscopic (microCT [bone] and histology [bone and subcutaneous]) and molecular methods (ELISA, PCR array) during 3, 7, 14, and 21 days. Mice were divided into 4 groups: Control (no treatment); GZA (IP injection of Glycyrrhizic Acid for HMGB1 inhibition, 4 mg/Kg/day); RAP (IP injection of RAGE Antagonistic Peptide, 4 mg/Kg/day), and vehicle controls (1.5% DMSO solution for GZA and 0.9% saline solution for RAP); treatments were given at all experimental time points, starting 1 day before surgeries. HMGB1 was detected in the Ti-implantation sites, adsorbed to the screws/discs. In Control and vehicle groups, osseointegration was characterized by a slight inflammatory response at early time points, followed by a gradual bone apposition and matrix maturation at late time points. The inhibition of HMGB1 or RAGE impaired the osseointegration, affecting the dynamics of mineralized and organic bone matrix, and resulting in a foreign body reaction, with persistence of macrophages, necrotic bone, and foreign body giant cells until later time points. While Control samples were characterized by a balance between M1 and M2-type response in bone and subcutaneous sites of implantation, and also MSC markers, the inhibition of HMGB1 or RAGE caused a higher expression M1 markers and pro-inflammatory cytokines, as well chemokines and receptors for macrophage migration until later time points. In conclusion, HMGB1 and RAGE have a marked role in the osseointegration, evidenced by their influence on host inflammatory immune response, which includes macrophages migration and M1/M2 response, MSC markers expression, which collectively modulate bone matrix deposition and osseointegration outcome

Primum non nocere – Are chloroquine and hydroxychloroquine safe prophylactic/treatment options for SARS-CoV-2 (covid-19)?

ABSTRACT Chloroquine (CQ) and its analog hydroxychloroquine (HCQ) were recently included in several clinical trials as potential prophylactic and therapeutic options for SARS-CoV-2 infection/covid-19. However, drug effectiveness in preventing, treating, or slowing the progression of the disease is still unknown. Despite some initial promising in vitro results, rigorous pre-clinical animal studies and randomized clinical trials have not been performed yet. On the other hand, while the potential effectiveness of CQ/HCQ is, at best, hypothetical, their side effects are factual and most worrisome, particularly when considering vulnerable groups of patients being treated with these drugs. In this comment, we briefly explain the possible mechanisms of action of CQ/HCQ for treating other diseases, possible actions against covid-19, and their potent side effects, in order to reinforce the necessity of evaluating the benefit-risk balance when widely prescribing these drugs for SARS-CoV-2 infection/covid-19. We conclude by strongly recommending against their indiscriminate use. DESCRIPTORS: Coronavirus Infections. Chloroquine, toxicity. Hydroxychloroquine, toxicity. Contraindications, Drug

Participação de DAMPs na modulação da resposta de macrófagos à implantação de um biomaterial clássico (Ti) e seu impacto no processo de reparo e osseointegração subsequentes

Despite the successful clinical application of titanium (Ti) as a biomaterial, the exact cellular and molecular mechanisms responsible for Ti osseointegration remain unclear. Indeed, specific knowledge still lacks on what elements are present at biomaterial/host interface and how these factors can trigger inflammatory pathways involved in the subsequent osseointegration process. In this context, we hypothesize that the surgical trauma inherent to the biomaterial grafting results in the release of DAMPs (damage-associated molecular patterns), endogenous proteins that act as triggers of immune inflammatory response upon cellular/tissue stress and/or damage. HMGB1 comprises the prototypic DAMP, which triggers host response via its cognate receptor RAGE, present at leucocytes and somatic cells surfaces. In this context, the aim of this thesis is to study the influence of DAMPs on the biomaterial/host interface and its role in mediating a constructive inflammatory process along tissue repair and osseointegration outcome. Methods and Results: In the article 1, we first characterized an oral osseointegration model in C57Bl/6 mice. This model of oral osseointegration was performed by using Ti screws (6AL-4V, Ø0.6mm, length of 1.5 mm) implanted in the edentulous alveolar crest of mice maxilla. The peri-implant sites were evaluated by microCT, as well histological and molecular assessments. In the article 2, we confirm the presence of DAMPs (HMGB1, HSP60, HSP70, S100A, Byglican, and Fibronectin) at Ti/host interface, analyzing Ti discs (6AL-4V, Ø6mm, 2mm of thick) implanted in the subcutaneous tissue of C57Bl/6 mice. Subsequently, the impact of HMGB1 and RAGE on the tissue repair around Ti discs was investigated by using HMGB1 (GZA 200mg/Kg) or RAGE (RAP, 4m/Kg/day) pharmacological inhibitors. The HMGB1/RAGE axis actively influences the inflammatory response post biomaterial implantation and the blocking of both molecules can negatively affect the subcutaneous tissue repair surrounding Ti disc in mice. In the article 3, Ti screws were implanted in the maxillary edentulous alveolar crest of C57Bl/6 mice, treated or untreated with GZA and RAP and the osseointegration process was evaluated by microscopic and molecular analysis (such as characterized in the article 1). The failure of osseointegration process was observed in mice treated with RAP or GZA, which present a disruption of the inflammatory process followed by foreign body reaction. In conclusion, HMGB1 and RAGE actively influence the tissue repair and osseointegration process in response to Ti-devices grafting, influencing the genesis and regulation of inflammatory immune response, which include the modulation of macrophages polarization state, MSC migration and differentiation in bone cells and consequent bone deposition.Apesar do sucesso clínico do Titânio (Ti) como biomaterial, os exatos mecanismos celulares e moleculares que levam à sua osseointegração permanecem incertos. De fato, ainda há uma lacuna de conhecimento sobre quais elementos estão presentes na interface hospedeiro/biomaterial e como esses fatores poder deflagrar as vias inflamatórias envolvidas no subseqüente processo de osseointegração. Neste contexto, sugere-se que o trauma cirúrgico inerente à implantação do biomaterial resulta na liberação de DAMPs (do inglês damage-associated molecular patterns), os quais são proteínas endógenas que agem como ativadoras da resposta imune/inflamatória sob um estresse ou dano celular e tecidual. HMGB1 constitui um DAMP prototípico, o qual ativa a resposta do hospedeiro via seu receptor cognato RAGE, que por sua vez está presente na superfície de leucócitos e células somáticas. Neste contexto, o objetivo da presente tese é estudar a influencia de DAMPs na interface hospedeiro/biomaterial e seu papel na modulação de um processo inflamatório construtivo ao longo do reparo tecidual e da osseointegração. Material e Métodos: No artigo 1, caracterizou-se um modelo de osseointegração oral em camundongos C57Bl/6 . Tal modelo foi desenvolvido utilizando parafusos de Ti (6AL-4V, Ø0,6mm, 1.5 de comprimento) implantados no rebordo alveolar edentulo da maxila de camundongos, cujos tecidos peri-implatares foram avaliados por meio de microCT, bem como análises histológicas e moleculares. No artigo 2, inicialmente confirmou-se a presença de DAMPs (HMGB1, HSP60, HSP70, S100A, Biglicana e Fibronectina) na interface Ti/hospedeiro, analisando amostras com discos de Ti (6AL-4V, Ø6mm x 2mm de espessura) implantados no tecido subcutâneo de camundongos C57Bl/6. Posteriormente, o impacto de HMGB1 e RAGE no reparo tecidual ao redor dos discos de Ti foi analisado por meio de uso de inibidores farmacológicos de HMGB1 (GZA 200mg/Kg/dia) e RAGE (RAP, 4m/Kg/dia). O eixo HMGB1/RAGE influencia ativamente a resposta inflamatória pós implantação do biomaterial, e o bloqueio de ambas as moléculas pode afetar negativamente o reparo tecidual subcutâneo ao redor de discos de Ti em camundongos. No artigo 3, parafuso de Ti foram implantados no rebordo edentulo da maxila de camundongos C57Bl/6, tratados e não tratados com GZA e RAP; e o processo de osseointegração foi avaliado por meio de análises microscópicas e moleculares (tal como caracterizado no artigo 1). A falha da osseointegração foi observada em camundongos tratados com RAP ou GZA, os quais apresentaram alterações importantes no processo inflamatório seguidas por uma reação de corpo estranho nos perídos mais tardios. Em suma, conclui-se que HMGB1 e RAGE influenciam ativamente o processo de reparo tecidual e de osseointegração frente à implantação de dispositivos de Ti, influenciando a geração e a regulação da resposta imune inflamatória, a qual inclui a modulação da polarização de macrófagos, a migração de MSCs e a diferenciação de células ósseas para subsequente deposição óssea

Role of CCR2+ cells in the alveolar bone repair process in mice: histomorphometric and molecular characterization

O processo de reparo ósseo depende de uma resposta inflamatória inicial e transitória, a qual envolve a participação de diversos leucócitos, como células da linhagem monócito/macrófago. O receptor CCR2 é importante para o recrutamento de macrófagos durante as respostas imunes, além de ter um papel na regulação da osteoclastogênese. Assim, o objetivo do presente estudo foi investigar papel de células CCR2+ no processo de reparo ósseo alveolar pós-exodontia em camundongos, por meio de análises microscópicas (MicroCT, histomorfometria, análise de birrefringência e imuno-histoquímica) e moleculares (PCRArray) comparativas entre as linhagens C57Bl/6 (WT) e CCR2KO, ao longo dos períodos de 0 hora, 7, 14 e 21 dias pós-exodontia do incisivo superior direito. Como resultado geral das análises microscópicas, constatamos que a ausência de células CCR2+ não afetou o resultado final do reparo ósseo alveolar em camundongos CCR2KO, mas levou a alterações transitórias e estatisticamente significantes (p<0,05) para quantificação de infiltrado inflamatório, vasos sanguíneos, fibroblastos, fibras colágenas, osteoblastos e osteoclastos. Além disso, a ausência de células CCR2+ resultou em diminuição (p<0,05) de células F4/80+ e CCR5+ no infiltrado inflamatório ao longo do processo de reparo ósseo alveolar de camundongos CCR2KO, demonstrando o papel do receptor CCR2 no recrutamento de macrófagos (células F4/80+), bem como sugerindo que as células F4/80+ apresentam dupla positividade para os receptores CCR2 e CCR5. Neste contexto, o receptor CCR5 seria o responsável pela migração remanescente, ainda que reduzida, de células F4/80+ nos animais CCR2KO. Considerando os resultados moleculares, a ausência de CCR2 resultou na alteração da expressão de diferentes marcadores em camundongos CCR2KO, tais como: o fator de crescimento TGF1, marcadores de matriz COL1, MMP1a, MMP2 e MMP9, marcadores ósseos RUNX2, DMP1, RANKL, RANK e CTSK, e marcadores de MSCs CD106, COT-4, NANOG, CD146 e CD105, bem como de marcadores imunológicos como as citocinas IL-6 e TNF-a, receptores de quimiocinas CCR1, CCR5 e CXCR1,e as quimiocinas CCL12, CCL20, CCL25 e CXCL12. Em conclusão, estes resultados indicam que células CCR2+ desempenham diferentes funções no reparo ósseo alveolar em camundongos, influenciando tanto a resposta inflamatória, como os eventos teciduais observados ao longo deste processo.The bone repair process depends of an initial and transitory inflammatory response, which involves the participation of various leukocytes subsets, as of the monocyte/macrophage lineage. The CCR2 receptor is important to macrophage recruitment during immune responses, and play an active role in the regulation of osteoclastogenesis. Thereby, the purpose of this study was to investigate the role of CCR2+ cells in the alveolar bone repair process in mice, by means of microscopic (MicroCT, histomorphometry, birefringence analysis and immunohistochemistry) and molecular (PCRArray) comparative analysis between C57BL / 6 (WT) and CCR2KO mice during periods of 0 hour, 7, 14 and 21 days post-extraction of the right upper incisor. As a result of the microscopic analysis, we noted that the absence of CCR2+ cells did not affect in the overall outcome of alveolar bone repair in CCR2KO mice, but resulted in transient and statistically significant (p<0.05) alterations of inflammatory infiltrate, blood vessels, fibroblasts, collagen fibers, osteoblasts and osteoclasts counts. Furthermore, the absence of CCR2+cells resulted in a decrease (p<0.05) of CCR5+ and F4/80+ cells in the inflammatory infiltrate along the alveolar bone repair process in CCR2KO mice, demonstrating the role of CCR2 receptor in macrophages migration (F4/80+ cells), as well as suggesting that the F4/80+ cells are double positive for CCR2 and CCR5. In this context, CCR5 receptor could be responsible for the remaining (but reduced) migration, of the F4/80 + cells in CCR2KO mice. According to molecular results, the absence of CCR2 resulted in an altered expression of different markers in CCR2KO mice, such as: growth factor TGF1, the matrix markers COL1, MMP1a, MMP2 and MMP9, the bone markers RUNX2, DMP1 RANKL, RANK and CTSK, and MSCs markers CD106, OCT-4, NANOG, CD146 and CD105, as well as immunological markers as IL-6 and TNF-, chemokine receptors CCR1, CXCR1 and CCR5, and the chemokines CCL12, CCL20, CCL25 and CXCL12. In conclusion, these results indicate that CCR2+ cells have different functions in alveolar bone repair in mice, influencing the inflammatory response and also tissue events observed throughout the process events

Molecular-Level Understanding of the Influence of Ions and Water on HMGB1 Adsorption Induced by Surface Hydroxylation of Titanium Implants

Due to its excellent chemical and mechanical properties, titanium has become the material of choice for orthopedic and dental implants to promote rehabilitation via bone anchorage and osseointegration. Titanium osseointegration is partially related to its capability to form a TiO2 surface layer and its ability to interact with key endogenous proteins immediately upon implantation, establishing the first bone-biomaterial interface. Surgical trauma caused by implantation results in the release of High Mobility Group Box 1 (HMGB1) protein, which is a prototypic DAMP (Damage Associated Molecular Pattern) with multiple roles in inflammation and tissue healing. To develop different surface strategies that improve the clinical outcome of titanium-based implants by controlling their biological activity, a molecular-scale understanding of HMGB1-surface interactions is desired. Here, we use molecular dynamics (MD) computer simulations to provide direct insight into the HMGB1 interactions and the possible molecular arrangements of HMGB1 on fully hydroxylated and non-hydroxylated rutile (110) TiO2 surfaces. The results establish that HMGB1 is most likely to be adsorbed directly onto the surface regardless of surface hydroxylation, which is undesirable because it could affect its biological activity by causing structural changes to the protein. The hydroxylated TiO2 surface shows a greater affinity for HMGB1 than the non-hydroxylated surface. The water layer on the non-hydroxylated TiO2 surface prevents ions and the protein from directly contacting the surface. However, it was observed that if the ionic strength increases, the total number of ions adsorbed on the two surfaces increases, and the protein’s direct adsorption ability decreases. These findings will help to understand the HMGB1-TiO2 interactions upon implantation, as well as the development of different surface strategies by introducing ions or ionic materials to the titanium implant surface to modulate its interactions with HMGB1 to preserve biological function.</div

A novel bioactive vitroceramic presents similar biological responses as autogenous bone grafts

Bioactive glasses represent an interesting class of bone substitute's biomaterials. the present study investigated the repair of bone defects filled with a novel bioactive vitroceramic (Biosilicate(A (R))), alone or in association with particulate autogenous bone grafts in calvaria defects of rabbits. After 7, 14, and 30 days the specimens were retrieved for histological, histomorphometric and immunohistochemistry analysis. Satisfactory bone formation was observed in all groups, and direct bone-biomaterial surface was noted. Histomorphometric assessment did not show statistically significant differences in bone formation among the groups and periods, except for BG group at day 14. Immunoexpression of Runx-2 was similar among the groups containing the graft and the biomaterial, being more intense than in control group. Similar result was observed for VEGF expression, especially in the last experimental period. These results revealed that Biosilicate(A (R)) presented a favorable behavior, comparable to autogenous bone graft.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Univ Sagrado Coracao, Dept Hlth Sci, Discipline Oral & Maxillofacial Surg, BR-17013070 Bauru, SP, BrazilUniv Sagrado Coracao, Sch Dent, BR-17011160 Bauru, SP, BrazilUniv Sagrado Coracao, Dept Hlth Sci, Discipline Oral & Maxillofacial Surg, BR-17011160 Bauru, SP, BrazilUniv Sagrado Coracao, Dept Hlth Sci, Discipline Periodontol, BR-17011160 Bauru, SP, BrazilUniversidade Federal de São Paulo, Dept Biosci, BR-11060001 Santos, SP, BrazilUniversidade Federal de São Paulo, Dept Biosci, BR-11060001 Santos, SP, BrazilFAPESP: 2008/11485-8FAPESP: 2009/17294-1Web of Scienc