Cost-effective 3D lung tissue spheroid as a model for SARS-CoV-2 infection and drug screening.

BACKGROUND: The SARS-CoV-2 pandemic has spurred an unparalleled scientific endeavor to elucidate the virus' structure, infection mechanisms, and pathogenesis. Two-dimensional culture systems have been instrumental in shedding light on numerous aspects of COVID-19. However, these in vitro systems lack the physiological complexity to comprehend the infection process and explore treatment options. Three-dimensional (3D) models have been proposed to fill the gap between 2D cultures and in vivo studies. Specifically, spheroids, composed of lung cell types, have been suggested for studying SARS-CoV-2 infection and serving as a drug screening platform. METHODS: 3D lung spheroids were prepared by coculturing human alveolar or bronchial epithelial cells with human lung stromal cells. The morphology, size, and ultrastructure of spheroids before and after SARS-CoV-2 infection were analyzed using optical and electron microscopy. Immunohistochemistry was used to detect spike protein and, thus, the virus presence in the spheroids. Multiplex analysis elucidated the cytokine release after virus infection. RESULTS: The spheroids were stable and kept their size and morphology after SARS-CoV-2 infection despite the presence of multivesicular bodies, endoplasmic reticulum rearrangement, tubular compartment-enclosed vesicles, and the accumulation of viral particles. The spheroid responded to the infection releasing IL-6 and IL-8 cytokines. CONCLUSION: This study demonstrates that coculture spheroids of epithelial and stromal cells can serve as a cost-effective infection model for the SARS-CoV-2 virus. We suggest using this 3D spheroid as a drug screening platform to explore new treatments related to the cytokines released during virus infection, especially for long COVID treatment.

A modified tunnel technique to treat multiple gingival recessions: Case Series

Abstract This case series reports a modified tunnel technique with connective tissue graft for the root coverage of multiple Miller Class I, II, and III gingival recessions. The modified approach presents an innovative suture technique to improve the stability and position of the graft. Ten patients with multiple gingival recessions (n=85 teeth) received surgical root coverage treatment. The gingival recession height and width were measured and presented as median, minimum, and maximum values. The percentage of the root coverage after at least 12 months expressed the treatment effectiveness. The Shapiro-Wilk test evaluated the normality; pared Wilcoxon test determined the exact P-value for the differences in the height of the gingival recession before and after surgical treatment (α = 0.05). An average of 97.9% (± 5.6%, p < 0.0001) root coverage after treatment occurred, and 73 out of 85 recessions presented complete root coverage after 12 months. Treatment of Miller class I and II gingival recessions resulted in root coverage higher than 99 and class III higher than 95% (p < 0.0001). The presented case series report the efficacy of a modified surgical technique promoting more than 95% of root coverage after 12 months in multiple Miller Class I, II, and III gingival recessions. Well-designed blind randomized controlled trials are needed to validate the proposed technique.

Resumo Esta série de casos relata uma técnica de túnel modificada com enxerto de tecido conjuntivo para o recobrimento radicular de múltiplas recessões gengivais Classe I, II e III de Miller. A abordagem modificada apresenta uma técnica de sutura inovadora para melhorar a estabilidade e a posição do enxerto. Dez pacientes com múltiplas recessões gengivais (n=85 dentes) receberam tratamento cirúrgico de recobrimento radicular. A altura e a largura da recessão gengival foram mensuradas e apresentadas como valores medianos, mínimos e máximos. A eficácia do tratamento foi expressa como uma porcentagem da cobertura radicular após pelo menos 12 meses. O teste de Shapiro-Wilk avaliou a normalidade; o teste de Wilcoxon pared determinou o valor P exato para as diferenças entre a altura da recessão gengival antes e após o tratamento cirúrgico (α = 0,05). Uma média de 97,9% (± 5,6%, p < 0,0001) de cobertura radicular após o tratamento ocorreu, e 73 das 85 recessões apresentaram cobertura radicular completa após 12 meses. O tratamento das recessões gengivais classe I e II de Miller resultou em recobrimento radicular superior a 99 e classe III superior a 95% (p < 0,0001). A série de casos apresentada relata a eficácia de uma técnica cirúrgica modificada promovendo mais de 95% de cobertura radicular após 12 meses em múltiplas recessões gengivais Classe I, II e III de Miller. Ensaios controlados randomizados cegos bem desenhados são necessários para validar a técnica proposta.

Does Melatonin Associated with Nanostructured Calcium Phosphate Improve Alveolar Bone Repair?

Background and objectives: Calcium phosphates have been widely used as bone substitutes, but their properties are limited to osteoconduction. The association of calcium phosphates with osteoinductive bioactive molecules has been used as a strategy in regenerative medicine. Melatonin has been studied due to its cell protection and antioxidant functions, reducing osteoclastic activity and stimulating newly formed bone. This study aimed to evaluate the effect of topical application of melatonin associated with nanostructured carbonated hydroxyapatite microspheres in the alveolar bone repair of Wistar rats through histological and histomorphometric analysis. Materials and Methods: Thirty female Wistar rats (300 g) were used, divided randomly into three experimental groups (n = 10), G1: nanostructured carbonated hydroxyapatite microspheres associated with melatonin gel (CHA-M); G2: nanostructured carbonated hydroxyapatite (CHA); G3: blood clot (without alveolar filling). The animals were euthanized after 7 and 42 days of the postoperative period and processed for histological and histomorphometric evaluation. Kruskal-Wallis and Dunn's post-test were applied to investigate statistical differences between the groups at the same time point for new bone and connective tissue variables. Mann-Whitney was used to assess statistical differences between different time points and in the biomaterial variable. Results: Results showed a greater volume of residual biomaterial in the CHA-M than the CHA group (p = 0.007), and there were no significant differences in terms of newly formed bone and connective tissue between CHA and CHA-M after 42 days. Conclusions: This study concluded that both biomaterials improved alveolar bone repair from 7 to 42 days after surgery, and the association of CHA with melatonin gel reduced the biomaterial's biodegradation at the implanted site but did not improve the alveolar bone repair.

The Use of Hydroxyapatite Loaded with Doxycycline (HADOX) in Dentoalveolar Surgery as a Risk-Reduction Therapeutic Protocol in Subjects Treated with Different Bisphosphonate Dosages.

Medication-related osteonecrosis of the jaw (MRONJ) is considered as a severe adverse side effect of specific drugs such as anti-resorptive and anti-angiogenic medications. Evidence suggests that MRONJ is linked to invasive dental procedures, mainly dentoalveolar surgery. Several preventive strategies to minimize the risk of developing MRONJ have been investigated. However, no investigation has been attempted to evaluate the therapeutic effect of local drug-delivery technology as a preventive strategy protocol. The aim of this study is to evaluate the efficacy of hydroxyapatite-containing doxycycline (HADOX) in rats with high-risk MRONJ development. All the rats used in this study were divided into seven groups. Six groups of rats out of seven were exposed to two different doses of antiresorptive drug therapy for four weeks before undergoing an upper incisor extraction. After 28 days, all the animals were euthanized, and the bone blocks were processed for histological and histomorphometrical evaluation. The histomorphometric analysis confirmed that newly formed bone (NFB) was present in all groups, with significant differences. NFB in the HADOX group treated with zoledronic acid at 4% showed (28.38; C.I. 22.29-34.48), which represents a significant increase compared to HA (15.69; C.I. 4.89-26.48) (p = 0.02). A similar pattern was observed in the HADOX group treated with zoledronic acid 8% ZA treatment (p = 0.001). Conclusions: HADOX did not inhibit any bone repair and reduced early inflammatory response. Hence, HADOX could promote bone healing in patients undergoing antiresorptive drug therapy.

Ionic concentration in periradicular medium after dissolution of endodontic file fragments: an in vitro study

Abstract The aim of this study was to obtain ionic quantification in periradicular medium after diffusion tests of the solution used inside root canals during the electrochemical dissolution of endodontic file fragments and the NiTi-containing dissolution product via an apical foramen. Thirty single-rooted extracted human teeth had root canals prepared and were attached to Eppendorf tubes filled with sterile saline. The samples were divided into 3 groups (n = 10) according to the solution used inside the root canal during the diffusion tests: Group 1: [NaF 12 g/L + NaCl 1 g/L]; Group 2: [NaF 12 g/L + NaCl 1 g/L + NiTi 0.50 g/L]; Group 3: [NaF 6 g/L + NaCl 0.5 g/L + NiTi 0.25 g/L]. The sample in each Eppendorf tube was then analyzed to assay the ionic quantification in periradicular medium. The groups were compared in relation to ionic quantifications (Kruskal-Wallis and Dunn's tests, p ≤ 0.05). Group 2 showed significantly higher F-, Ni and Ti quantities than groups 1 and 3 (p < 0.05). Group 3 showed significantly higher Ti and Ni quantities than group 1, where no measurable quantities of Ti and Ni were observed (p < 0.05). The conclusions were that a 50% dilution of the NiTi-containing dissolution product resulted in significantly lower F-, Ni and Ti quantities compared to the undiluted product. The quantifications observed here suggest that irrigation is recommendable during the electrochemical dissolution process to reduce the resultant ion concentrations in both the root canal and the periradicular medium.

Osseodensification enables bone healing chambers with improved low-density bone site primary stability: an in vivo study.

Primary implant stability is a prerequisite for successful implant osseointegration. The osseodensification technique (OD) is a non-subtractive drilling technique that preserves the bone tissue, increases osteotomy wall density, and improves the primary stability. This study aimed to investigate the hypothesis that OD, through a wider osteotomy, produces healing chambers (HCs) at the implant-bone interface without impacting low-density bone primary stability. Twenty implants (3.5 × 10 mm) with a nanohydroxyapatite (nHA) surface were inserted in the ilium of ten sheep. Implant beds were prepared as follows: (i) 2.7-mm-wide using subtractive conventional drilling (SCD) technique (n = 10); (ii) 3.8-mm-wide using an OD bur system (n = 10). The sheep were randomized to two groups, with samples collected at either 14-(n = 5) or 28-days (n = 5) post-surgery and processed for histological and histomorphometric evaluation of bone-implant contact (BIC) and bone area fraction occupancy (BAFO). No significant group differences were found with respect to final insertion torque and implant stability quotient (p > 0.050). BIC values were higher for SCD after 14 and 28 days (p < 0.050); however, BAFO values were similar (p > 0.050). It was possible to conclude that the OD technique allowed a wider implant bed preparation without prejudice on primary stability and bone remodeling.

Clinical efficacy of simvastatin gel combined with polypropylene membrane on the healing of extraction sockets: A triple-blind, randomized clinical trial.

OBJECTIVES: This study aimed to evaluate dimensional changes, level of soft tissue healing, and pain/discomfort perception in post-extraction sockets filling with 1.2% simvastatin (SIM) gel covered with polypropylene membranes (PPPM). MATERIAL AND METHODS: Twenty-six post-extraction sockets of posterior teeth were randomly allocated in two groups: (a) socket filling with 1.2% SIM gel and covered with PPPM (n = 13) and (b) socket filling with placebo gel and covered with PPPM (n = 13). Cone-beam computed tomography (CBCT) images before and 90 days after the extraction enabled alveolar bone dimensional changes calculation using horizontal and vertical measurements. The measurements occurred at three different levels for thickness located 1, 3, and 5 mm from the top of the bone crest. The vertical (depth) measure was assessed from the most apical portion of the socket to the bone crest's most coronal portion. Seven days after the extractions, the level of soft tissue healing and pain perception were also analyzed. RESULTS: After 90 days of extractions, the dimensional changes in thickness in the test group were significantly smaller in sections A (p = .044), B (p = .036) and C (p = .048) when compared to the control group. The test group showed a significantly lower height-dimensional change than the control group (p < .0001). Soft tissue healing index (p = .63), perception of pain (p = .23), and number of analgesics consumed (p = .25) were similar between groups. CONCLUSIONS: Simvastatin at 1.2% compared with placebo effectively reduced the dimensional changes in post-extraction sockets covered with PPPM. There was no significant difference in the level of soft tissue healing and postoperative pain between the test and control groups.

Cone-beam computed tomography cinematic rendering: clinical, teaching and research applications

Abstract Cone-beam computed tomography (CBCT) is an essential imaging method that increases the accuracy of diagnoses, planning and follow-up of endodontic complex cases. Image postprocessing and subsequent visualization relies on software for three-dimensional navigation, and application of indexation tools to provide clinically useful information according to a set of volumetric data. Image postprocessing has a crucial impact on diagnostic quality and various techniques have been employed on computed tomography (CT) and magnetic resonance imaging (MRI) data sets. These include: multiplanar reformations (MPR), maximum intensity projection (MIP) and volume rendering (VR). A recent advance in 3D data visualization is the new cinematic rendering reconstruction method, a technique that generates photorealistic 3D images from conventional CT and MRI data. This review discusses the importance of CBCT cinematic rendering for clinical decision-making, teaching, and research in Endodontics, and a presents series of cases that illustrate the diagnostic value of 3D cinematic rendering in clinical care.

Scaffold- and serum-free hypertrophic cartilage tissue engineering as an alternative approach for bone repair.

Human adipose stem/stromal cell (ASC) spheroids were used as a serum-free in vitro model to recapitulate the molecular events and extracellular matrix organization that orchestrate a hypertrophic cartilage phenotype. Induced-ASC spheroids (ø = 450 µm) showed high cell viability throughout the period of culture. The expression of collagen type X alpha 1 chain (COLXA1) and matrix metallopeptidase 13 (MMP-13) was upregulated at week 2 in induced-ASC spheroids compared with week 5 (P < .001) evaluated by quantitative real-time PCR. In accordance, secreted levels of IL-6 (P < .0001), IL-8 (P < .0001), IL-10 (P < .0001), bFGF (P < .001), VEGF (P < .0001), and RANTES (P < .0001) were the highest at week 2. Strong in situ staining for collagen type X and low staining for TSP-1 was associated with the increase of hypertrophic genes expression at week 2 in induced-ASC spheroids. Collagen type I, osteocalcin, biglycan, and tenascin C were detected at week 5 by in situ staining, in accordance with the highest expression of alkaline phosphatase (ALPL) gene and the presence of calcium deposits as evaluated by Alizarin Red O staining. Induced-ASC spheroids showed a higher force required to compression at week 2 (P < .0001). The human ASC spheroids under serum-free inducer medium and normoxic culture conditions were induced to a hypertrophic cartilage phenotype, opening a new perspective to recapitulate endochondral ossification in vivo.

Recent Patents on Impact of Lipopeptide on the Biofilm Formation onto Titanium and Stainless Steel Surfaces.

BACKGROUND: Numerous causes of infection in arthroplasties are related to biofilm formation on implant surfaces. In order to circumvent this problem, new alternatives to prevent bacterial adhesion biosurfactants-based are emerging due to low toxicity, biodegradability and antimicrobial activity of several biosurfactants. We revised all patents relating to biosurfactants of applicability in orthopedic implants. METHODS: This work aims to evaluate the capability of a lipopeptide produced by Bacillus subtilis ATCC 19659 isolates acting as inhibitors of the adhesion of Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213 onto titanium and stainless steel surfaces and its antimicrobial activity. RESULTS: The adhesion of the strains to the stainless-steel surface was higher than that of titanium. Preconditioning of titanium and stainless-steel surfaces with 10 mg mL-1 lipopeptide reduced the adhesion of E. coli by up to 93% and the adhesion of S. aureus by up to 99.9%, suggesting the strong potential of lipopeptides in the control of orthopedic infections. The minimal inhibitory concentration and minimum bactericidal concentration were 10 and 240 µg mL-1 for E. coli and S. aureus, respectively. CONCLUSION: The lipopeptide produced by Bacillus subtilis ATCC 19659 presented high biotechnological application in human health against orthopedic implants infections.

Randomized Controlled Clinical Trial of Nanostructured Carbonated Hydroxyapatite for Alveolar Bone Repair.

The properties of the biodegradation of bone substitutes in the dental socket after extraction is one of the goals of regenerative medicine. This double-blind, randomized, controlled clinical trial aimed to compare the effects of a new bioabsorbable nanostructured carbonated hydroxyapatite (CHA) with a commercially available bovine xenograft (Bio-Oss®) and clot (control group) in alveolar preservation. Thirty participants who required tooth extraction and implant placement were enrolled in this study. After 90 days, a sample of the grafted area was obtained for histological and histomorphometric evaluation and an implant was installed at the site. All surgical procedures were successfully carried out without complications and none of the patients were excluded. The samples revealed a statistically significant increase of new bone formation (NFB) in the CHA group compared with Bio-Oss® after 90 days from surgery (p < 0.05). However, the clot group presented no differences of NFB compared to CHA and Bio-Oss®. The CHA group presented less amount of reminiscent biomaterial compared to Bio-Oss®. Both biomaterials were considered osteoconductors, easy to handle, biocompatible, and suitable for alveolar filling. Nanostructured carbonated hydroxyapatite spheres promoted a higher biodegradation rate and is a promising biomaterial for alveolar socket preservation before implant treatment.

Method to determine the root canal anatomic dimension by using a new cone-beam computed tomography software

Abstract This study discusses a method to determine the root canal anatomic dimension by using e-Vol DX software. The methodology consists in initially establishes the correct positions which will be measured, define the point on the edge of the anatomical structure, and next adjust the intermediate position in the grayscale of CBCT image. Afterward, thin sections (0.10 mm) are obtained from 3D reconstructed slices in the filter for the measurements, in order to determine the edge of the anatomical surface in the axial plane. A replication of positions in 3D mode is done in multiplanar reconstruction (MPR) of CBCT images, where the correct position is established with the aid of a positioning guide. The 3D density is adjusted so that it is in the same dimension as the 2D image, and a dimension calibration occurs to the point where there is a coincidence between 3D and 2D. This calibration is done only at the beginning of the measurement. Next, the intermediate position of the division between the grayscale is verified in the CBCT scan. Once one side has been completed, it is moved to the other side and follows the same guidelines described above. When setting the position of the courses in the other margin, being that 2D mode is used as reference. Thus, one obtains the required measure, being checked in the two points. The creation of this filter in the e-Vol DX software for measurement, and its appropriate management, allows more effective applications when it is desired to obtain diameters of anatomical structures.

Resumo Este estudo discute um método para determinar a dimensão anatômica do canal radicular usando o software e-Vol DX. A metodologia consiste em inicialmente estabelecer as posições corretas que serão medidas, definir o ponto na borda da estrutura anatômica e ajustar a posição intermediária na escala de cinza na imagem em tomografia computadorizada de feixe cônico (TCFC). A seguir, slices finos (0,10mm) são obtidos a partir de cortes 3D reconstruídos no filtro para as medidas, a fim de determinar a borda da superfície anatômica no plano axial. Uma replicação de posições no modo 3D é feita em reconstrução multiplanar (MPR) em imagens de TCFC, onde a posição correta é estabelecida com o auxílio de um guia de posicionamento. A densidade 3D é ajustada de modo a ficar na mesma dimensão da imagem 2D, e então realiza-se uma calibração de dimensão até o ponto em que há uma coincidência entre o modo 3D e 2D. Essa calibração é feita apenas no início da medição. Posteriormente, a posição intermediária da divisão entre a escala de cinza é verificada na TCFC. Uma vez que um lado tenha sido concluído, o guia é movido para o outro lado, e segue-se as mesmas diretrizes descritas. Define-se a posição do marcador na outra margem, sendo que o modo 2D usado como referência. Assim, obtém-se a medida necessária, sendo verificado nas duas margens do canal radicular. A criação deste filtro no software e-Vol DX para medição e seu uso apropriado permite aplicações eficazes quando se deseja obter diâmetros de estruturas anatômicas.

Spheroids of stem cells as endochondral templates for improved bone engineering.

Osteodegenerative disease and bone fractures lead to bone damage or loss, requiring new bone formation to replace the damaged tissues. Classical 'top-down' tissue engineering relies on seeding cell suspensions into biomaterial scaffolds, and then guiding cell fate by growth factors. However, complex tissue fabrication using this approach has important limitations. 'Bottom-up' tissue engineering has the potential to overcome the drawbacks of the top-down approach, by using 'building blocks' of cell spheroids for tissue biofabrication without a scaffold. Spheroids are 3D structures that resemble the physiological tissue microenvironment and can be produced in vitro by different methods. Spheroids of mesenchymal stem cells (MSC) and adipose stem cells (ASC) have regenerative properties. Here we review, the use of spheroids as 'building blocks' in the 3D bioprinting of large-scale bone tissue and as a promising alternative for the treatment of osteodegenerative diseases and in bone engineering, including endochondral ossification (or developmental engineering).

In vitro and in vivo biocompatibility of reoss® in powder and putty configurations

Article This study evaluated comparatively two configurations (powder and putty) of a composite biomaterial based on PLGA (Poly(lactide-co-glycolide)/nanoescale hydroxyapatite (ReOss®, Intra-Lock International) through microscopic morphology, in vitro cytotoxicity, biocompatibility and in vivo response as a bone substitute. SEM and EDS characterized the biomaterials before/after grafting. Cytocompatibility was assessed with murine pre-osteoblasts. Osteoconductivity and biocompatibility were evaluated in White New Zealand rabbits. Both configurations were implanted in the calvaria of eighteen animals in non-critical size defects, with blood clot as the control group. After 30, 60 and 90 days, the animals were euthanized and the fragments containing the biomaterials and controls were harvested. Bone blocks were embedded in paraffin (n=15) aiming at histological and histomorphometric analysis, and in resin (n=3) aiming at SEM and EDS. Before implantation, the putty configuration showed both a porous and a fibrous morphological phase. Powder revealed porous particles with variable granulometry. EDS showed calcium, carbon, and oxygen in putty configuration, while powder also showed phosphorus. After implantation EDS revealed calcium, carbon, and oxygen in both configurations. The materials were considered cytotoxic by the XTT test. Histological analysis showed new bone formation and no inflammatory reaction at implant sites. However, the histomorphometric analysis indicated that the amount of newly formed bone was not statistically different between experimental groups. Although both materials presented in vitro cytotoxicity, they were biocompatible and osteoconductive. The configuration of ReOss® affected morphological characteristics and the in vitro cytocompatibility but did not impact on the in vivo biological response, as measured by the present model.

Resumo Este estudo avaliou comparativamente duas configurações (pó e massa) de um biomaterial composto com base de PLGA (Poli(láctico-co-glicólico)/hidroxiapatita em nanoescala (ReOss®, Intra-Lock International) através da morfologia microscópica, citotoxicidade in vitro, biocompatibilidade e resposta in vivo como substituto ósseo. MEV e EDS caracterizaram os biomateriais antes/após o enxerto. A citocompatibilidade foi avaliada em pré-osteoblastos murinos. A osteocondutividade e a biocompatibilidade foram avaliadas em coelhos Branco da Nova Zelândia. Ambas as configurações foram implantadas na calvária de dezoito animais em defeitos não-críticos, com coágulo sanguíneo como grupo controle. Após 30, 60 e 90 dias, os animais foram eutanasiados e os fragmentos contendo os biomateriais e controles coletados. Blocos ósseos foram embebidos em parafina (n=15) destinados às análises histológica e histomorfométrica, e em resina (n=3) destinadas à MEV e EDS. Antes da implantação, a configuração massa mostrou ambas fases morfológicas porosa e fibrosa. O pó revelou partículas porosas com granulometria variável. EDS mostrou cálcio, carbono e oxigênio na configuração massa, enquanto o pó mostrou também fósforo. Após a implantação a EDS revelou cálcio, carbono e oxigênio em ambas configurações. Os materiais foram considerados citotóxicos pelo teste XTT. A análise histológica mostrou nova formação óssea e nenhuma reação inflamatória nos sítios de implante. Entretanto, a análise histomorfométrica indicou que a quantidade de osso neoformado não foi estatisticamente diferente entre os grupos experimentais. Embora ambos os materiais tenham apresentado citotoxicidade in vitro, foram biocompatíveis e osteocondutores. A configuração do ReOss® afetou as características morfológicas e a citocompatibilidade in vitro, porém não impactou a resposta biológica in vivo, como medido pelo presente modelo.

Chronic Periodontitis and RANKL/OPG Ratio in Peri-Implant Mucosae Inflammation

Abstract tHistory of chronic periodontitis (CP) is a risk factor for oseointegration failure. The osteoclastogenesis system (RANK, RANKL and OPG) is critical for bone homeostatic control. We investigated the levels of OPG and RANKL in peri-implant tissues from volunteers with and without a history of CP and their association with mucosae inflammation. This is a single-blind case-contro study. Diagnosis of a history of CP and peri-implant examination was performed on 46 volunteers, divided into control (without history of CP, n=26) and CP group (with history of CP, n=20). Gingival biopsies were harvested during implant exposure. Quantitative PCR evaluated OPG/RANKL mRNA expressions. OPG and RANKL proteins were analyzed by western blot and immunohistochemistry assay. The chi-square test analyzed the significance of nominal variables between groups while continuous variables were analyzed by T-test or Mann-Whitney test, after Shapiro-Wilk test evaluation. The 2-ΔΔCT Livak method calculation evaluated the gene expression. Values of p<0.05 were considered statistically significant. Volunteers with CP history had 23 times higher chance of developing mucosae inflammation. High mucosae levels of RANKL (p=0.04) and RANKL/OPG (p=0.001) mRNA expressions were observed in CP group. CP volunteers showed increased RANKL protein levels in opposition to decreased OPG expression. Even without active periodontitis, volunteers with a history of CP had elevated gingival levels of RANKL/OPG and higher correlation with peri-implant mucosae inflammation and implant loss.

Resumo A história de periodontite crônica (CP) é um fator de risco para falhas na osseointegração. O sistema de osteoclastogênese (RANK, RANKL e OPG) é crucial para o controle da homeostase óssea. O objetivo deste estudo foi investigar os níveis de OPG e RANKL no tecido peri-implantar de voluntários com e sem histórico de CP e sua associação com inflamação da mucosa. Este é um estudo tipo caso-controle. O exame para diagnóstico de CP e na região peri-implantar foi realizado em 46 voluntários, divididos em controle (sem história CP, n=26) e grupo CP (com histórico de CP, n=20). Descartes gengivais foram obtidos durante a exposição do implante. PCR quantitativo avaliou a expressão do RNAm de OPG/RANKL. As proteínas OPG e RANKL foram analisadas por western blot e imunohistoquímica. O teste do qui-quadrado analisou a significância entre as variáveis nominais enquanto as variáveis contínuas foram analisadas pelo teste-t e Mann-Whitney, após o teste de Shapiro-wilk. O método do Livak 2--ΔΔCT avaliou a expressão gênica. Os voluntários com CP apresentaram 23 vezes mais chances de desenvolver inflamação da mucosa. Expressão elevada no RNAm de RANKL (p=0.04) e RANKL/OPG (p=0.001) foram observadas no grupo CP. Voluntários com CP mostraram aumento dos níveis da proteína RANKL em contraste com diminuída expressão de OPG. Mesmo sem periodontite ativa, voluntários com histórico de CP apresentaram elevado nível gengival de RANKL/OPG e alta correlação com inflamação peri-implantar e perda do implante.

Chronic Periodontitis and RANKL/OPG Ratio in Peri-Implant Mucosae Inflammation.

tHistory of chronic periodontitis (CP) is a risk factor for oseointegration failure. The osteoclastogenesis system (RANK, RANKL and OPG) is critical for bone homeostatic control. We investigated the levels of OPG and RANKL in peri-implant tissues from volunteers with and without a history of CP and their association with mucosae inflammation. This is a single-blind case-contro study. Diagnosis of a history of CP and peri-implant examination was performed on 46 volunteers, divided into control (without history of CP, n=26) and CP group (with history of CP, n=20). Gingival biopsies were harvested during implant exposure. Quantitative PCR evaluated OPG/RANKL mRNA expressions. OPG and RANKL proteins were analyzed by western blot and immunohistochemistry assay. The chi-square test analyzed the significance of nominal variables between groups while continuous variables were analyzed by T-test or Mann-Whitney test, after Shapiro-Wilk test evaluation. The 2-ΔΔCT Livak method calculation evaluated the gene expression. Values of p<0.05 were considered statistically significant. Volunteers with CP history had 23 times higher chance of developing mucosae inflammation. High mucosae levels of RANKL (p=0.04) and RANKL/OPG (p=0.001) mRNA expressions were observed in CP group. CP volunteers showed increased RANKL protein levels in opposition to decreased OPG expression. Even without active periodontitis, volunteers with a history of CP had elevated gingival levels of RANKL/OPG and higher correlation with peri-implant mucosae inflammation and implant loss.

Association Between Polymorphisms in the Genes of Estrogen Receptors and the Presence of Temporomandibular Disorders and Chronic Arthralgia.

PURPOSE: The high prevalence of painful temporomandibular disorders (TMDs) in women suggests that estrogen and its receptors play a fundamental etiologic role in the development of this joint pathology through complex action mechanisms. The aim of this study was to evaluate the possible association between polymorphisms in the ESR1 (estrogen receptor-1) and ESRRB (estrogen-related receptor-ß) genes and the risk of simultaneous development of TMDs and pain in other joints in the body. MATERIALS AND METHODS: All participants were clinically evaluated for the presence of TMD (Research Diagnostic Criteria for TMD) and asked about the presence of chronic joint pain. The control group consisted of 72 patients without TMD and without pain. Participants with arthralgia were divided into 3 groups: with muscular TMD (n = 42), with articular TMD (n = 16), and without TMD and with systemic arthralgia (n = 82). Eight single-nucleotide polymorphisms in the ESR1 (rs12154178, rs1884051, rs2273206, rs7774230) and ESRRB (rs1676303, rs4903399, rs10132091, rs7151924) genes were investigated. The χ2 test and Student t and Mann-Whitney tests were used to assess the relevance of nominal and continuous variables, respectively. A P value less than .05 was considered significant. RESULTS: The TT (timin/timin) genotype for the ESR1 (rs2273206) gene was strongly associated with the risk of developing muscle TMDs and temporomandibular joint pain (P = .04). For the ESRRB (rs1676303) gene, an association was observed between the CC (cytosine/cytosine) genotype and the presence of articular TMDs associated with other chronic arthralgia (P = .02). These results were confirmed by the increased risk of developing articular TMDs associated with the C allele (P = .04). CONCLUSIONS: This study supports the hypothesis that changes in the ESR1 and ESRRB genes influence the presence of TMDs associated with chronic joint pain.

Alveolar bone repair with strontium- containing nanostructured carbonated hydroxyapatite

ABSTRACT Objective: This study aimed to evaluate bone repair in rat dental sockets after implanting nanostructured carbonated hydroxyapatite/sodium alginate (CHA) and nanostructured carbonated hydroxyapatite/sodium alginate containing 5% strontium microspheres (SrCHA) as bone substitute materials. Methods: Twenty male Wistar rats were randomly divided into two experimental groups: CHA and SrCHA (n=5/period/group). After one and 6 weeks of extraction of the right maxillary central incisor and biomaterial implantation, 5 μm bone blocks were obtained for histomorphometric evaluation. The parameters evaluated were remaining biomaterial, loose connective tissue and newly formed bone in a standard area. Statistical analysis was performed by Mann-Withney and and Wilcoxon tests at 95% level of significance. Results: The histomorphometric results showed that the microspheres showed similar fragmentation and bio-absorbation (p>0.05). We observed the formation of new bones in both groups during the same experimental periods; however, the new bone formation differed significantly between the weeks 1 and 6 (p=0.0039) in both groups. Conclusion: The CHA and SrCHA biomaterials were biocompatible, osteoconductive and bioabsorbable, indicating their great potential for clinical use as bone substitutes.

Assessing the association between hypoxia during craniofacial development and oral clefts

Abstract Objectives To evaluate the association between hypoxia during embryo development and oral clefts in an animal model, and to evaluate the association between polymorphisms in the HIF-1A gene with oral clefts in human families. Material and Methods The study with the animal model used zebrafish embryos at 8 hours post-fertilization submitted to 30% and 50% hypoxia for 24 hours. At 5 days post-fertilization, the larvae were fixed. The cartilage structures were stained to evaluate craniofacial phenotypes. The family-based association study included 148 Brazilian nuclear families with oral clefts. The association between the genetic polymorphisms rs2301113 and rs2057482 in HIF-1A with oral clefts was tested. We used real time PCR genotyping approach. ANOVA with Tukey's post-test was used to compare means. The transmission/disequilibrium test was used to analyze the distortion of the inheritance of alleles from parents to their affected offspring. Results For the hypoxic animal model, the anterior portion of the ethmoid plate presented a gap in the anterior edge, forming a cleft. The hypoxia level was associated with the severity of the phenotype (p<0.0001). For the families, there was no under-transmitted allele among the affected progeny (p>0.05). Conclusion Hypoxia is involved in the oral cleft etiology, however, polymorphisms in HIF-1A are not associated with oral clefts in humans.

Accelerated Healing Period with Hydrophilic Implant Placed in Sheep Tibia

Abstract The objective of this study was to evaluate the early osseointegration of two different implants surfaces, a sandblasted and acid-etched surface (TN) compared with same geometry and surface roughness modified to be hydrophilic/wettable by conditioning in an isotonic solution of 0.9% sodium-chloride (TA) through histological and histomorphometric analysis after sheep tibia implantation. Forty dental implants, divided in two groups (TN and TA) were placed in the left tibia of twenty healthy, skeletally mature Santa Ines sheep (n=5/experimental period). After 7, 14, 21 and 28 days post-implantation, the samples were removed and the sheep were kept alive. Analysis of resin sections (30 μm) allowed the quantification of bone area (BA) and bone-to-implant contact (BIC). TA group presented nearly 50% increase in BA at 14 days (p<0.001, ANOVA - Tukey's post test) compared with 7 days. The TA presented higher values than the TN for BA and BIC at 14, 21, and 28 days after placement, stabilizing bone healing. TA hydrophilic surface promoted early osseointegration at 14 and 21 days compared to TN, accelerating bone healing period post-implant placement in sheep tibia.

Resumo O objetivo deste estudo foi avaliar a osseointegração precoce de duas superficies diferentes de implantes, uma superfície tratada com jateamento e ataque ácido (TN) comparada com outra superfície com a mesma geometria e rugosidade e modificada com hidrofilicidade / molhabilidade através do condicionamento em solução isotônica de 0,9% de cloreto de sódio (TA) através de análises histológica e histomorfométrica após a implantação em tíbia de ovelhas. Quarenta implantes dentários, divididos em dois grupos (TN e TA), foram implantados na tíbia esquerda de vinte ovelhas Santa Inês (n=5/período experimental) saudáveis e esqueleticamente maduras. Após 7, 14, 21 e 28 dias pós-implantação, as amostras foram coletadas e as ovelhas foram mantidas vivas. A análise dos cortes em resina (30 μm) permitiu a quantificação da área de osso (BA) e do contato osso-implante (BIC). O grupo TA apresentou aumento de quase 50% na BA aos 14 dias (p <0,001, ANOVA - pós teste de Tukey) em comparação com 7 dias. O grupo TA apresentou valores maiores do que o TN para BA e BIC aos 14, 21 e 28 dias após a implantação, estabilizando a cicatrização óssea. A superfície hidrofílica TA promoveu uma osseointegração precoce aos 14 e 21 dias em relação à TN, acelerando o período de cicatrização óssea após a instalação dos implantes em tíbias de ovelha.