Submandibular Gland Pathogenesis Following SARS-CoV-2 Infection and Implications for Xerostomia.

Although SARS-CoV-2 induces mucin hypersecretion in the respiratory tract, hyposalivation/xerostomia has been reported by COVID-19 patients. We evaluate the submandibular gland (SMGs) pathogenesis in SARS-CoV-2-infected K18-hACE2 mice, focusing on the impact of infection on the mucin production and structural integrity of acini, ductal system, myoepithelial cells (MECs) and telocytes. The spike protein, the nucleocapsid protein, hACE2, actin, EGF, TNF-α and IL-1ß were detected by immunofluorescence, and the Egfr and Muc5b expression was evaluated. In the infected animals, significant acinar hypertrophy was observed in contrast to ductal atrophy. Nucleocapsid proteins and/or viral particles were detected in the SMG cells, mainly in the nuclear membrane-derived vesicles, confirming the nuclear role in the viral formation. The acinar cells showed intense TNF-α and IL-1ß immunoexpression, and the EGF-EGFR signaling increased, together with Muc5b upregulation. This finding explains mucin hypersecretion and acinar hypertrophy, which compress the ducts. Dying MECs and actin reduction were also observed, indicating failure of contraction and acinar support, favoring acinar hypertrophy. Viral assembly was found in the dying telocytes, pointing to these intercommunicating cells as viral transmitters in SMGs. Therefore, EGF-EGFR-induced mucin hypersecretion was triggered by SARS-CoV-2 in acinar cells, likely mediated by cytokines. The damage to telocytes and MECs may have favored the acinar hypertrophy, leading to ductal obstruction, explaining xerostomia in COVID-19 patients. Thus, acinar cells, telocytes and MECs may be viral targets, which favor replication and cell-to-cell viral transmission in the SMG, corroborating the high viral load in saliva of infected individuals.

Reduction of osteoclast formation and survival following suppression of cytokines by diacerein in periodontitis.

Periodontitis causes an increase in several bioactive agents such as interleukins (IL), tumor necrosis factor (TNF)-α and receptor activator of NF-kB ligand (RANKL), which induce the osteoclast formation and activity. Since diacerein exerts anti-TNF-α and anti-IL-1 effects, alleviating bone destruction in osteoarthritis, we investigated whether this drug inhibits the formation and survival of osteoclast in the periodontitis. Rats were distributed into 3 groups: 1) group with periodontitis treated with 100 mg/kg diacerein (PDG), 2) group with periodontitis treated with saline (PSG) and group control (CG) without any treatment. After 7, 15 and 30 days, the maxillae were collected for light and transmission electron microscopy analyses. Gingiva samples were collected to evaluate the mRNA levels for Tnf, Il1b, Tnfsf11 and Tnfrsf11b by RT-qPCR. In PDG, the expression of Tnf and Il1b genes reduced significantly compared to PSG, except for Tnf expression at 7 days. The number of osteoclasts reduced significantly in the PDG in comparison with PSG at 7 and 15 days. In all periods, the IL-6 immunoexpression, RANKL/OPG immunoexpression and mRNA levels of Tnfsf11/Tnfrsf11b ratio were significantly lower in PDG than in PSG. PDG exhibited significantly higher frequency of TUNEL-positive osteoclasts than in PSG and CG at all time points. Osteoclasts with caspase-3-immunolabelled cytoplasm and nuclei with masses of condensed chromatin were observed in PDG, confirming osteoclast apoptosis. Diacerein inhibits osteoclastogenesis by decreasing Tnf and Il1b mRNA levels, resulting in decreased RANKL/OPG ratio, and induces apoptosis in osteoclasts of alveolar process of rat molars with periodontitis.

A new hydroxyapatite-alginate-gelatin biocomposite favor bone regeneration in a critical-sized calvarial defect model.

This study aimed to evaluate the osteogenic potential of hydroxyapatite (HA), Alginate (Alg), and Gelatine (Gel) composite in a critical-size defect model in rats. Twenty-four male rats were divided into three groups: a negative control with no treatment (Control group), a positive control treated with deproteinized bovine bone mineral (DBBM group), and the experimental group treated with the new HA-Alg-Gel composite (HA-Alg-Gel group). A critical size defect (8.5mm) was made in the rat's calvaria, and the bone formation was evaluated by in vivo microcomputed tomography analysis (µCT) after 1, 15, 45, and 90 days. After 90 days, the animals were euthanized and histological and histomorphometric analyses were performed. A higher proportion of mineralized tissue/biomaterial was observed in the DBBM group when compared to the HA-Alg-Gel and Control groups in the µCT analysis during all analysis periods. However, no differences were observed in the mineralized tissue/biomaterial proportion observed on day 1 (immediate postoperative) in comparison to later periods of analysis in all groups. In the histomorphometric analysis, the HA-Alg-Gel and Control groups showed higher bone formation than the DBBM group. Moreover, in histological analysis, five samples of the HA-Alg-Gal group exhibited formed bone spicules adjacent to the graft granules against only two of eight samples in the DBBM group. Both graft materials ensured the maintenance of defect bone thickness, while a tissue thickness reduction was observed in the control group. In conclusion, this study demonstrated the osteoconductive potential of HA-Alg-Gel bone graft by supporting new bone formation around its particles.

Influence of anti-sclerostin monoclonal antibody in the repair of post-extraction sockets of ovariectomized rats.

OBJECTIVE: This study assessed the impact of an anti-sclerostin monoclonal antibody (Scl-Ab)-based osteoporosis drug on the post-extraction alveolar repair of ovariectomized rats. DESIGN: Fifteen female rats were randomly distributed into three groups: CTR (healthy animals), OST (osteoporosis induced by ovariectomy), and OST+Scl-Ab (osteoporosis induction followed by Scl-Ab treatment). Ovariectomy or sham surgery was performed 30 days before baseline, and Scl-Ab or a vehicle was administered accordingly in the groups. After seven days, all rats underwent the first lower molar extraction and were euthanized 15 days later. Computed microtomography, histological analysis, and collagen content measurement were performed on post-extraction sockets and intact mandibular and maxillary bone areas. RESULTS: Microtomographic analyses of the sockets and mandibles did not reveal significant differences between groups on bone morphometric parameters (p > 0.05), while maxillary bone analyses resulted in better maintenance of bone architecture in OST+Scl-Ab, compared to OST (p < 0.05). Descriptive histological analysis and polarization microscopy indicated better post-extraction socket repair characteristics and collagen content in OST+Scl-Ab compared to OST (p < 0.05). CONCLUSIONS: Scl-Ab-based medication did not accelerate alveolar bone formation but exhibited better post-extraction repair characteristics, and collagen content compared to ovariectomized animals only.

Biocompatibility and bioactive potential of NeoPUTTY calcium silicate-based cement: An in vivo study in rats.

AIM: To evaluate the inflammatory reaction and the ability to induce mineralization activity of a new repair material, NeoPUTTY (NPutty; NuSmile, USA), in comparison with Bio-C Repair (BC; Angelus, Brazil) and MTA Repair HP (MTA HP; Angelus, Brazil). METHODOLOGY: Polyethylene tubes were filled with materials or kept empty (control group, CG) and implanted in subcutaneous tissue of rats for 7, 15, 30, and 60 days (n = 6/group). Capsule thickness, number of inflammatory cells (ICs), fibroblasts, collagen content, and von Kossa analysis were performed. Unstained sections were evaluated under polarized light and by immunohistochemistry for osteocalcin (OCN). Data were submitted to two-way anova followed by Tukey's test (p ≤ .05), except for OCN. OCN data were submitted to Kruskal-Wallis and Dunn and Friedman post hoc tests followed by the Nemenyi test at a significance level of 5%. RESULTS: At 7, 15, and 30 days, thick capsules containing numerous ICs were seen around the materials. At 60 days, a moderate inflammatory reaction was observed for NPutty, BC while MTA HP presented thin capsules with moderate inflammatory cells. In all periods, NPutty specimens contained the highest values of ICs (p < .05). From 7 to 60 days, the number of ICs reduced significantly while an increase in the number of fibroblasts and birefringent collagen content was observed. At 7 and 15 days, no significant difference was observed in the immunoexpression of OCN (p > .05). At 30 and 60 days, NPutty showed the lowest values of OCN (p < .05). At 60 days, a similar immunoexpression was observed for BC and MTA HP (p > .05). In all time intervals, capsules around NPutty, BC, and MTA HP showed von Kossa-positive and birefringent structures. CONCLUSIONS: Despite the greater inflammatory reaction promoted by NeoPutty than BC and MTA HP, the reduction in the thickness of capsules, the increase in the number of fibroblasts, and the reduction in the number of ICs indicate that this bioceramic material is biocompatible Furthermore, NeoPutty presents the ability to induce mineralization activity.

Bioactive potential of Bio-C Temp demonstrated by systemic mineralization markers and immunoexpression of bone proteins in the rat connective tissue.

Intracanal medications are used in endodontic treatment due to their antibacterial activity and ability to induce the periapical repair. Among the intracanal medications, the Calen (CAL; SS. White, Brazil) is a calcium hydroxide-based medication that provides an alkaline pH and releases calcium, exerting an antimicrobial activity. Bio-C Temp (BIO; Angelus, Brazil), a ready-to-use bioceramic intracanal medication, was designed to stimulate the mineralized tissues formation. Here, we investigated the bioactive potential of BIO in comparison to the CAL in the rat subcutaneous. Polyethylene tubes filled with medications, and empty tubes (control group, CG) were implanted in the subcutaneous tissue of rats. After 7, 15, 30 and 60 days, the blood was collected for calcium (Ca+2) and alkaline phosphatase (ALP) measurement, and the capsules around the implants were processed for morphological analyses. The data were submitted to two-way ANOVA and Tukey test (p < 0.05). At 7, 15 and 30 days, the ALP level was grater in BIO and CAL than in CG (p < 0.0001). At 7 and 15 days, greater Ca+2 level was seen in the serum of CAL samples. From 7 to 60 days, an increase in the number of fibroblasts, osteocalcin- and osteopontin-immunolabelled cells was observed in BIO and CAL groups (p < 0.0001). In all periods, BIO and CAL specimens showed von Kossa-positive structures. Moreover, ultrastructural analysis revealed globules of mineralization in the capsules around the BIO and CAL specimens. Thus Bio-C Temp caused an increase in the ALP, osteocalcin and osteopontin, which may have allowed the formation of calcite, suggesting bioactive potential.

Role of serotonin, estrogen, and TNF-α in the paroxetine-impaired steroidogenesis and testicular macrophages polarization.

BACKGROUND: Paroxetine, a selective serotonin reuptake inhibitor (SSRI) antidepressant, has caused male sexual dysfunction; however, the paroxetine mechanisms of action in testes are still unclear. OBJECTIVES: Paroxetine serotonergic effects in testes were evaluated, focusing on steroidogenesis and the correlation between macrophages population and possible TNF-α-derived oxidative stress. We also verified whether the changes are reversible following treatment interruption. MATERIALS AND METHODS: Adult rats received paroxetine (PG35 and PG65) or tap water (CG) for 35 days. PG65 was maintained without treatment for 30 more days. Intratesticular testosterone (IT), nitrite, and malondialdehyde concentrations were measured. To confirm serotonergic and estrogenic effects, Htr1b and Esr1 expressions were analyzed. The daily sperm production (DSP), frequency of abnormal seminiferous tubules (ST), SC number, ST area, and Leydig cells nuclear area (LCnu) were evaluated. TUNEL+ germ cells, M1 (CD68+ ), and M2 (Perls+ ) macrophages were quantified. 17ß-HSD7, CYP19A1, NDRG2, oxytocin, TNF-α, and iNOS were evaluated by immunoreactions. Oxytocin and NDRG2 protein levels as well as Tnfa mRNA expression were also analyzed. RESULTS: The Htr1b downregulation in testes confirmed the paroxetine serotonergic effect. The testicular sections showed abnormal ST frequency, ST atrophy and reduction of DSP, LCnu, SC number and Perls+ macrophages. TUNEL+ germ cells and LC were associated with strong NDRG2 immunoexpression. Paroxetine reduced IT levels and 17ß-HSD7 immunoexpression in parallel to increased CYP19A1, oxytocin, TNF-α and iNOS. Esr1 and Tnfa overexpression and increased number of CD68+ macrophages were also observed together with high nitrite and malondialdehyde levels. Most parameters were not recovered in PG65. CONCLUSIONS: Paroxetine serotonergic effect impairs LC steroidogenesis, via aromatization, increasing estrogen/testosterone ratio, which in turn upregulate NDRG2, promoting apoptosis, and impairing sperm production. Serotonin-estrogen pathways may be responsible for M2/M1 polarization, Tnfa upregulation, and induction of oxidative stress. The unrecovered testicular changes after treatment discontinuation are due to persistent paroxetine serotonin/estrogen effects.

A new hydroxyapatite-alginate-gelatin biocomposite favor bone regeneration in a critical-sized calvarial defect model

Abstract This study aimed to evaluate the osteogenic potential of hydroxyapatite (HA), Alginate (Alg), and Gelatine (Gel) composite in a critical-size defect model in rats. Twenty-four male rats were divided into three groups: a negative control with no treatment (Control group), a positive control treated with deproteinized bovine bone mineral (DBBM group), and the experimental group treated with the new HA-Alg-Gel composite (HA-Alg-Gel group). A critical size defect (8.5mm) was made in the rat's calvaria, and the bone formation was evaluated by in vivo microcomputed tomography analysis (µCT) after 1, 15, 45, and 90 days. After 90 days, the animals were euthanized and histological and histomorphometric analyses were performed. A higher proportion of mineralized tissue/biomaterial was observed in the DBBM group when compared to the HA-Alg-Gel and Control groups in the µCT analysis during all analysis periods. However, no differences were observed in the mineralized tissue/biomaterial proportion observed on day 1 (immediate postoperative) in comparison to later periods of analysis in all groups. In the histomorphometric analysis, the HA-Alg-Gel and Control groups showed higher bone formation than the DBBM group. Moreover, in histological analysis, five samples of the HA-Alg-Gal group exhibited formed bone spicules adjacent to the graft granules against only two of eight samples in the DBBM group. Both graft materials ensured the maintenance of defect bone thickness, while a tissue thickness reduction was observed in the control group. In conclusion, this study demonstrated the osteoconductive potential of HA-Alg-Gel bone graft by supporting new bone formation around its particles.

Resumo Este estudo teve como objetivo avaliar o potencial osteogênico de um compósito de hidroxiapatita (HA), alginato (Alg) e gelatina (Gel) em um modelo de defeito de tamanho crítico em ratos. Vinte e quatro ratos machos foram divididos em três grupos: um controle negativo sem tratamento (grupo controle), um controle positivo tratado com osso bovino desproteinizado (grupo DBBM) e o grupo experimental tratado com o novo compósito HA-Alg-Gel (grupo HA-Alg-Gel). Um defeito de tamanho crítico (8,5mm) foi feito na calvária dos ratos, e a formação óssea foi avaliada por análise de microtomografia computadorizada in vivo (µCT) após 1, 15, 45 e 90 dias. Após 90 dias, os animais foram eutanasiados e análises histológicas e histomorfométricas foram realizadas. Uma maior proporção de tecido mineralizado/biomaterial foi observada no grupo DBBM quando comparado aos grupos HA-Alg-Gel e controle na análise de µCT durante todos os períodos de análise. Entretanto, não foram observadas diferenças na proporção tecido mineralizado/biomaterial no dia 1 (pós-operatório imediato) em relação aos períodos posteriores de análise em todos os grupos. Na análise histomorfométrica, os grupos HA-Alg-Gel e controle apresentaram maior formação óssea do que o grupo DBBM. Além disso, na análise histológica, cinco amostras do grupo HA-Alg-Gal exibiram espículas ósseas formadas adjacentes aos grânulos do enxerto contra apenas duas das oito amostras do grupo DBBM. Ambos os materiais de enxerto garantiram a manutenção da espessura óssea do defeito, enquanto uma redução da espessura do tecido foi observada no grupo controle. Em conclusão, este estudo demonstrou o potencial osteocondutor do enxerto ósseo de HA-Alg-Gel, promovendo a formação de osso novo ao redor das suas partículas.

Additional Insights Into the Role of Osteocalcin in Osteoblast Differentiation and in the Early Steps of Developing Alveolar Process of Rat Molars.

This study investigated whether osteocalcin (OCN) is present in osteoblast precursors and its relationship with initial phases of alveolar process formation. Samples of maxillae of 16-, 18-, and 20-day-old rat embryos (E16, E18, and E20, respectively), and 05-, 10-, and 15-day-old postnatal rats (P05, P10, and P15, respectively) were fixed and embedded in paraffin or araldite. Immunohistochemistry for osterix (Osx), alkaline phosphatase (ALP), and OCN detection was performed and the number of immunolabelled cells was computed. Non-decalcified sections were subjected to the von Kossa method combined with immunohistochemistry for Osx or OCN detection. For OCN immunolocalization, samples were fixed in 0.5% glutaraldehyde/2% formaldehyde and embedded in LR White resin. The highest number of ALP- and OCN-immunolabelled cells was observed in dental follicle of E16 specimens, mainly in basal portions of dental alveolus. In corresponding regions, osteoblasts in differentiation adjacent to von Kossa-positive bone matrix exhibited Osx and OCN immunoreactivity. Ultrastructural analysis revealed OCN immunoreactive particles inside osteoblast in differentiation, and in bone matrix associated with collagen fibrils and within matrix vesicles, at early stages of alveolar process formation. Our results indicate that OCN plays a role in osteoblast differentiation and may regulate calcium/phosphate precipitation during early mineralization of the alveolar process.

Possible role of annexin A1/FPR2 pathway in COX2/NLRP3 inflammasome regulation in alveolar bone cells of estrogen-deficient female rats with diabetes mellitus.

BACKGROUND: Annexin A1 (ANXA1) and the NOD-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome play important roles in bone remodeling. However, expression profiles of these factors in bone cells under diabetes mellitus (DM) and estrogen-deficient conditions are poorly understood. This study investigated the immunoexpression of ANXA1 and its formyl peptide receptor 2 (FPR2), as well as NLRP3 inflammasome mediators, during remodeling of the alveolar process in diabetic and estrogen-deficient rats. METHODS: Twenty adult female Wistar rats were divided into four groups (n = 5): Sham-operated (SHAM) and ovariectomized (OVX) rats received a vehicle solution, and SHAM and OVX rats were intraperitoneally administered 60 mg/kg/body weight (BW) of streptozotocin (STZ) to induce DM (SHAM-Di and OVX-Di groups). After 7 weeks, the rats were euthanized and their maxillae were fixed in phosphate-buffered 4% formaldehyde and embedded in paraffin. Sections were stained with hematoxylin/eosin (H&E) and picrosirius red or subjected to immunohistochemical detection of ANXA1, FPR2, NLRP3, interleukin-1ß (IL-1ß), and cyclooxygenase-2 (COX2). RESULTS: Estrogen deficiency and DM were associated with deleterious effects in bone tissue, as evidenced by a lower number of osteocytes and higher number of empty lacunae in the SHAM-Di and OVX-Di groups compared to the nondiabetic groups. Both diabetic groups showed a smaller vascular area and weaker collagen fiber birefringence intensity in alveolar bone tissue. A significantly higher number of ANXA1/FPR2-positive osteoblasts, osteocytes, and osteoclasts was accompanied by a significantly higher number of these cells immunolabeled for COX2, NLRP3, and IL-1ß in the diabetic and OVX groups, especially in both estrogen-deficient and diabetic rats. CONCLUSION: These results indicate a possible role for the ANXA1/FPR2 pathway as a fine-tuning/anti-inflammatory regulator to counterbalance exacerbated COX2/NLRP3/IL-1ß activation in bone cells during bone remodeling under estrogen deficiency and DM.

Biocompatibility, bioactive potential, porosity, and interface analysis calcium silicate repair cements in a dentin tube model.

OBJECTIVES: This study is to evaluate biocompatibility, bioactive potential, porosity, and dentin/material interface of Bio-C Repair (BIOC-R), MTA Repair HP (MTAHP), and Intermediate Restorative Material (IRM). MATERIALS AND METHODS: Dentin tubes were implanted into subcutaneous of rats for 7, 15, 30, and 60 days. Thickness of capsules, number of inflammatory cells (ICs), interleukin-6 (IL-6), osteocalcin (OCN), and von Kossa were evaluated. Porosity and material/dentin interface voids were also analyzed. Data were submitted to ANOVA and Tukey's tests (p < 0.05). RESULTS: IRM capsules were thicker and contained greater ICs and IL-6-immunopositive cells at 7 and 15 days. BIOC-R capsules exhibited higher thickness and ICs at 7 days and greater IL-6 at 7 and 15 days than MTAHP (p < 0.05). At 30 and 60 days, no significant difference was observed among the groups. OCN-immunopositive cells, von Kossa-positive, and birefringent structures were observed in BIOC-R and MTAHP. MTAHP exhibited higher porosity and interface voids (p < 0.05). CONCLUSIONS: BIOC-R, MTAHP, and IRM are biocompatible. Bioceramics materials demonstrate bioactive potential. MTAHP presented the highest porosity and presence of voids. CLINICAL RELEVANCE: BIOC-R and MTAHP have adequate biological properties. BIOC-R demonstrated lower porosity and presence of voids, which may represent better sealing for its clinical applications.

Venlafaxine increases aromatization, reduces apical V-ATPase in clear cells and induces increased number of mast cells and smooth muscle cells death in rat cauda epididymis.

Depressive disorders (DD) have affected millions of people worldwide. Venlafaxine, antidepressant of the class of serotonin and norepinephrine reuptake inhibitors, has been prescribed for the treatment of DD. In rat testes, venlafaxine induces testosterone (T) aromatization and increases estrogen levels. Aromatase is a key enzyme for the formation of estrogen in the epididymis, an essential organ for male fertility. We investigated the impact of serotonergic/noradrenergic venlafaxine effect on the epididymal cauda region, focusing on aromatase, V-ATPase and EGF epithelial immunoexpression, smooth muscle (SM) integrity and mast cells number (MCN). Male rats were distributed into control (CG; n = 10) and venlafaxine (VFG, n = 10) groups. VFG received 30 mg/kg b.w. of venlafaxine for 35 days. The epididymal cauda was processed for light and transmission electron microscopy (TEM). The expression of connexin 43 (Cx43) and estrogen alpha (Esr1), adrenergic (Adra1a) and serotonergic (Htr1b) receptors were analyzed. Clear cells (CCs) area, SM thickness, viable spermatozoa (VS) and MCN were evaluated. Apoptosis was confirmed by TUNEL and TEM. The following immunoreactions were performed: T, aromatase, T/aromatase co-localization, V-ATPase, EGF, Cx43 and PCNA. The increased Adra1a and reduced Htr1b expressions confirmed the noradrenergic and serotonergic venlafaxine effects, respectively, corroborating the increased MCN, apoptosis and atrophy of SM. In VFG, the epithelial EGF increased, explaining Cx43 overexpression and basal cells mitotic activity. T aromatization and Esr1 downregulation indicate high estrogen levels, explaining CCs hypertrophy and changes in the V-ATPase localization, corroborating VS reduction. Thus, in addition to serotonergic/noradrenergic effects, T/estrogen imbalance, induced by venlafaxine, impairs epididymal structure and function.

Biocompatibility and bioactive potential of an experimental tricalcium silicate-based cement in comparison with Bio-C repair and MTA Repair HP materials.

AIM: To evaluate the tissue reaction of a tricalcium silicate-based repair material associated with 30% calcium tungstate (TCS + CaWO4 ) in comparison to Bio-C Repair (Bio-C; Angelus) and to MTA Repair HP (MTA HP; Angelus). METHODOLOGY: Polyethylene tubes filled with one of the materials or left empty (control group, CG) were implanted into the subcutaneous tissues of rats for 7, 15, 30 and 60 days (n = 32/group). The capsule thickness, number of inflammatory cells, collagen content, interleukin-6 (IL-6), osteocalcin (OCN), von Kossa reaction and analysis under polarized light were evaluated. The data were subjected to generalized linear models for repeated measures, except the OCN. OCN data were submitted to Kruskal-Wallis and Dunn's post hoc test and Friedman followed by Nemenyi's test at significance level of 5%. RESULTS: At all time points, significant differences in the number of inflammatory cells were not observed between TCS + CaWO4 and Bio-C, whereas, at 15, 30 and 60 days, no significant difference was detected between TCS + CaWO4 and MTA HP. At all periods, significant differences were not detected in the number of fibroblasts in TCS + CaWO4 versus MTA HP, and, at 60 days, no significant difference was demonstrated between these groups and CG. Significant differences in the immunoexpression of IL-6 were not detected amongst bioceramic materials at all periods. From 7 to 60 days, significant reduction in the number of inflammatory cells, number of IL-6-immunopositive cells and in the capsule thickness was accompanied by significant increase in the collagen in all groups. OCN-immunolabelled cells, von Kossa-positive structures and amorphous calcite deposits were observed around all materials, whereas, in the CG, these structures were not seen. CONCLUSIONS: These findings indicate that the experimental material (TCS + CaWO4 ) is biocompatible and has a bioactive potential, similar to the MTA HP and Bio-C Repair, and suggest its use as a root repair material.

Participation of fibroblast growth factor-1 and interleukin-10 in connective tissue repair following subcutaneous implantation of bioceramic materials in rats.

AIM: To evaluate whether the bioceramic materials Bio-C Pulpo (Bio-C, Angelus) and mineral trioxide aggregate (MTA) Repair HP (MTA-HP, Angelus) induce fibroblast proliferation and release of interleukin-10 (IL-10), an anti-inflammatory cytokine, stimulating connective tissue remodelling. The tissue response of Bio-C and MTA-HP was compared with the White MTA (WMTA; Angelus) since studies have demonstrated that WMTA induces tissue repair. METHODOLOGY: Bio-C, MTA-HP and WMTA were inserted into polyethylene tubes and implanted in the subcutaneous tissue of Holtzman rats for 7, 15, 30 and 60 days. As a control group (CG), empty tubes were implanted subcutaneously. The number of fibroblasts (FB), Ki-67-, fibroblast growth factor-1- (FGF-1) and IL-10-immunolabelled cells and collagen content in the capsules was obtained. The data were subjected to two-way anova followed by Tukey's test (p ≤ .05). RESULTS: At 7 days, significant differences in the number of FB were not detected amongst Bio-C, MTA-HP and WMTA groups (p Ëƒ .05). The capsules of all groups exhibited a significant increase in the number of FB and content of collagen over time. From 7 to 60 days, a significant reduction in the number of FGF-1- and Ki-67-immunolabelled cells was seen in the capsules of all specimens. In all periods, no significant difference in the number of FGF-1-immunolabelled cells was detected between Bio-C and CG specimens. At 60 days, significant differences in the immunoexpression of FGF-1 were not observed amongst the groups. At 7 and 15 days, the highest immunoexpression for Ki-67 was present in Bio-C specimens whilst, after 30 and 60 days, no significant difference was observed amongst the bioceramic materials. At 7 days, few IL-10 immunolabelled cells were present in the capsules of all specimens whereas, at 60 days, a significant increase in the IL-10-immunostaining was present in all groups. At 60 days, the Bio-C, MTA-HP and WMTA groups showed a greater number of IL-10-immunolabelled cells than in the CG specimens (p < .0001). CONCLUSIONS: Bio-C, MTA-HP and WMTA stimulate fibroblast proliferation, leading to the formation of collagen-rich capsules. FGF-1 and IL-10 may mediate the remodelling of capsules around Bio-C, MTA-HP and WMTA bioceramic materials.

Biodegradable scaffolds based on plant stems for application in regenerative medicine.

Several synthetic and natural materials have been studied for the confection of temporary grafts for application in regenerative medicine, however, the development of a material with adequate properties remains a challenge, mainly because its degradation kinetics in biological systems. Nature provides materials with noble properties that can be used as such for many applications, thus, taking advantage of the available morphology and assembled structures of plants, we propose to study the vegetable stems for use as temporary graft. Since thein vivodegradation is maybe one of the most important features of the temporary grafts, here we have implanted the plant stems from pumpkin, papaya, and castor into the subepithelial tissue of animals and followed their biodegradation process and the local inflammatory response. Mechanical tests, FTIR and contact angle with water were also analysed. The results indicated the mechanical properties and the contact angle were adequate for use in regenerative medicine. The results of thein vivostudies indicated a beneficial inflammatory process and a gradual disintegration of the materials within 60 days, suggesting the plants stems as new and potential materials for development of grafts for use in the field of regenerative medicine.

A Denture Use Model Associated with Candida spp. in Immunocompetent Male and Female Rats.

Denture stomatitis (DS) is a common infection in denture wearers, especially women. This study evaluated the induction of DS using acrylic devices attached to the palate of rats combined with inoculation of Candida spp. Immunocompetent male and female rats received a carbohydrate-rich diet. Impressions were taken from the rats' palate to individually fabricate acrylic devices. Mono- and multispecies biofilms of C. albicans, C. glabrata, and C. tropicalis were grown on the devices, which were then cemented on posterior teeth and kept in the rats' palate for four weeks. Microbial samples from the palate and the device were quantified. Oral microbiome of rats inoculated with C. albicans was analyzed by 16S rRNA gene sequencing. Log10(CFU/mL) were analyzed by mixed or two-way MANOVA (α = 0.05). Candida spp. and acrylic device did not induce palatal inflammation macroscopically nor microscopically. Although there was an increase (p < 0.001) of the total microbiota and female rats demonstrated higher (p = 0.007) recovery of Candida spp. from the palate, the gender differences were not biologically relevant. The microbiome results indicate an increase in inflammatory microbiota and reduction in health-associated micro-organisms. Although Candida spp. and acrylic device did not induce DS in immunocompetent rats, the shift in microbiota may precede manifestation of inflammation.

Bioactive potential of Bio-C Pulpo is evidenced by presence of birefringent calcite and osteocalcin immunoexpression in the rat subcutaneous tissue.

As the biocompatibility and bioactive potential of repair materials are desired characteristics in dentistry, the tissue response of Bio-C Pulpo, a bioceramic material launched on the marked by Angelus (Brazil), was compared with Biodentine (Septodont, France) and White MTA (WMTA; Angelus, Brazil). In 32 rats, 148 polyethylene tubes filled with Bio-C Pulpo, Biodentine or WMTA, and empty (CG, control group) were implanted into subcutaneous tissues for 7, 15, 30, and 60 days. The capsule thickness, numerical density of inflammatory cells (IC) and fibroblasts (Fb), amount of collagen, immunohistochemistry detection of interleukin-6 (IL-6) and osteocalcin (OCN), von Kossa and analysis under polarized light were performed. Data were subjected to two-way ANOVA followed by Tukey's test (p ≤ 0.05). At 7 and 15 days, the capsules around Bio-C Pulpo were thicker than in WMTA while, at 30 and 60 days, significant differences were not observed among the groups. Although at 7, 15, and 30 days, a greater number of IL-6-immunostained cells was found in Bio-C Pulpo and Biodentine than in WMTA, no significant difference was detected among the groups at 60 days. In all groups, the number of Fb and collagen content increased significantly over time. The capsules around materials exhibited von Kossa-positive and birefringent structures, and OCN-immunostained cells whereas, in the CG, these structures were not observed. Bio-C Pulpo, similarly to Biodentine and WMTA, is biocompatible, allows the connective tissue repair and presents bioactive potential in connective tissue of rats.

Impact of glycine and erythritol/chlorhexidine air-polishing powders on human gingival fibroblasts: An in vitro study.

BACKGROUND: Supra- and subgingival air-polishing has been used in periodontitis and gingivitis therapy for years. Low-abrasive types of powders have facilitated the application in subgingival areas. In this study, the cellular effects of a glycine powder and an erythritol/chlorhexidine (CHX) powder on human gingival fibroblasts (HGF) were investigated. METHODS: HGF were obtained from sound gingiva of three healthy donors. After 12 h and 24 h of incubation time, cell viability testing and, after 24 h and 48 h, a cell proliferation assay was conducted. Additionally, the individual components erythritol and CHX were investigated for cell viability. In vitro wound healing was monitored for 48 h and scanning electron microscopy (SEM) analysis was performed after 24 h. Statistical analysis was accomplished by ANOVA and post hoc Dunnett's and Tukey's tests (p < 0.05) were performed. RESULTS: Erythritol/CHX powder and in a lower extent, glycine powder decreased cell viability and cell proliferation. The negative effect of erythritol/CHX was mainly based on the CHX component. In vitro wound healing was negatively influenced in both types of powders compared to control. Cell size was altered in both test groups, whereas cell morphology was affected only in the erythritol/CHX group. CONCLUSIONS: The investigated powders for subgingival air-polishing can influence cell viability, morphology, and proliferation, as well as wound closure in vitro. These actions on fibroblasts are discernible, with the cytotoxic effect of erythritol/CHX powder being very clear and mainly due to the CHX component. Our results suggest that subgingivally applied powders can exert direct effects on gingival fibroblasts.

Obesity influences the proteome of periodontal ligament tissues following periodontitis induction in rats.

BACKGROUND AND OBJECTIVES: Many studies have been conducted to better understand the molecular mechanism involved with periodontitis progression. There has been growing interest in the potential impact of obesity on periodontitis onset and progression, but the mechanisms involved remain to be elucidated. The present study was designed to determine the impact of obesity on experimentally induced periodontitis in rats and identify novel pathways involved. METHODS: Sixteen Holtzman rats were distributed into two groups (n = 8): ligature-induced periodontitis (P) and obesity plus ligature-induced periodontitis (OP). Obesity was induced by a high-fat diet for 70 days, whereas periodontitis was induced for 20 days, with a cotton thread placed around the upper first molars bilaterally. Alveolar bone loss was measured by microtomographic analysis and histologically by histometry on the hemimaxillae. The protein composition of the periodontal ligament was evaluated by proteomic analysis. RESULTS: Data analysis (body weight, adipose tissue weight, and blood test) confirmed obesity induction, whereas bone loss was confirmed by micro-CT and histologic analyses. Proteome analysis from the periodontal ligament tissues (PDL) identified 819 proteins, 53 exclusive to the P group, 28 exclusive to the OP group, and 738 commonly expressed. Validation was performed by immunohistochemistry for selected proteins (spondin1, vinculin, and TRAP). CONCLUSION: Histologically, it was found that obesity did not significantly affect bone loss resulting from periodontitis. However, the present study's findings indicated that obesity affects the proteome of PDL submitted to experimental periodontitis, allowing for identifying potential targets for personalized approaches.

Bilateral asymmetry in bullfrog testes and fat bodies: correlations with steroidogenic activity, mast cells number and structural proteins.

In seasonal breeders, such as amphibians, testicular functions depend on complex processes that change according to seasonality, including Leydig cell (LC) differentiation and lipid-dependent steroidogenesis, extracellular proteins remodeling and actin-dependent cellular dynamics. Speculating that fat bodies (FB) could support some of these processes in L. catesbeianus, we evaluated bilaterally the FB weights, correlating them to testicular parameters such as weight, testosterone (T) immunoexpression, mast cells (MC) number, vascularization and structural proteins. In an attempt to better understand the testicular asymmetry in amphibians, correlations between these different testicular parameters were also established. Right testes (RT), left testes (LT) and associated FB of bullfrogs were weighed, and testes were processed for light and transmission electron microscopy. Collagen content (COL) and MC number were quantified. T and actin immunoexpressions and vascular areas were measured. Statistical analyses and Pearson's correlation were performed. The LT and its associated FB were heavier than the right ones, and showed intense T and actin immunoexpressions, numerous lipid-rich LC, and greater MC number, COL and vascularization than the RT. Positive correlations were detected between: a) FB and testis weights, b) T immunoexpression and testis and FB weights, c) T and actin immunoexpressions and COL. Otherwise, MC number was inversely correlated to T immunoexpression and COL. In right and left sides, the proportional correlation between T immunoexpression and FB weight suggests that FB-stored lipid amount depends on the steroidogenic demand of its associated testis. Thus, the asymmetry in the testes and FB may be associated, at least in part, to the LC steroidogenic activity, which tends to be more intense in LT than in RT. The results also point to a role of COL and mast cells in the LC differentiation and steroidogenesis. Actin was also greater in LT and correlated with T immunoexpression, indicating that the amount of this structural protein depends on androgenic control. Therefore, the testicular asymmetry in bullfrogs seems to be associated to different morphofunctional processes occurring, bilaterally, at different intensities. In this case, there is a tendency of LT, in association with its FB, to be more active than RT. The findings highlight the FB-testis interplay for the comprehension of reproduction in amphibians.