Effects of cigarette smoke inhalation on the immune-inflammatory profile of experimental apical periodontitis in rats.

OBJECTIVE: To evaluate the effects of cigarette smoke inhalation on the immune-inflammatory profile of experimental apical periodontitis in rats. METHODOLOGY: In total, 32 male Wistar rats were divided into four groups (n = 8): AP-induced apical periodontitis; S-cigarette smoke inhalation; APS-induced AP and cigarette smoke inhalation; and C (control)-neither AP nor cigarette smoke inhalation. To induce cigarette smoke inhalation, the animals were kept in a chamber filled with tobacco smoke for 8 min thrice a day for 50 days. AP was induced 20 days after inhalation initiation by exposing their coronary pulp to their oral environment for 30 days. After animals were euthanized, their right hemimaxillae were removed for histopathological, semi-quantitative and immunohistochemical (F4/80, CD206 and iNOS) analyses. RESULTS: Quantitative data showed a moderate number of inflammatory infiltrates in AP and an intense number in APS (p < .05). Comparing F4/80+ cells showed no statistically significant differences among groups, but we found more CD206+ cells in AP than in C and S (p > .05). INOS+ immunostaining showed a significant increase in AP and APS, when compared with C and S (p < .05). APS had more iNOS+ cells than AP (p < .05). CONCLUSION: Cigarette smoke inhalation worsened AP, leading to a predominantly pro- inflammatory profile in our experimental model.

Influence of age and gender on alveolar bone healing post tooth extraction in 129 Sv mice: a microtomographic, histological, and biochemical characterization.

OBJECTIVES: To analyze the effect of biological sex and aging on craniofacial bone features in 129 Sv mice and their influence on dental socket healing post tooth extraction. MATERIALS AND METHODS: A total of 52 129 Sv mice were used, of which 28 were young (3-4 months) and 24 were aged (17-18 months), equally distributed according to biological sex. After an upper right incisor extraction, mice specimens were collected at 7, 14, and 21-days post-surgery for microtomographic (microCT) and comprehensive histological analysis. Mandible, skull bones, and maxillae at 21 days were analyzed by microCT, while blood plasma samples were collected for the detection of key bone turnover markers (P1NP and CTX-1) by enzyme-linked immunosorbent (ELISA) assay. RESULTS: Aged females depicted significantly decreased mineralized bone content in alveolar sockets in comparison to young females and aged males at day 7, and aged males at day 14. Mandible RCA and Ma.AR of aged females were also significantly decreased in comparison with young females. Histological evaluation revealed that all alveolar sockets healed at 21 days with inflammation resolution and deposition of new bone. Immunohistochemistry for TRAP revealed increased area density for osteoclasts in alveolar sockets of aged females when compared to young females at 21 days. While a significant increase in CTX-1 levels was detected in blood plasma of aged females when compared to young females, P1NP levels did not significantly change between young and older females. No significant changes were observed for males. CONCLUSIONS: Age and gender can significantly affect craniofacial bones of 129 Sv mice, especially maxilla and mandible in females. Considering the altered bone resorption parameters and delayed alveolar bone healing in older females, careful deliberation is necessary during development of pre-clinical models for craniofacial research. CLINICAL RELEVANCE: Aging can be a contributing factor to slower bone healing in craniofacial bones. However, there are no sufficient experimental studies that have addressed this phenomenon along with biological sex taken into consideration.

Dose-response effect of Montelukast on post-extraction dental socket repair and skeletal phenotype of mice.

Bone metabolism and repair are directly regulated by arachidonic acid metabolites. At present, we analyzed the dose-response effects of a selective cysteinyl leukotriene receptor type-1 antagonist during bone repair after tooth extraction and on non-injured skeleton. Sixty-three 129 Sv/Ev male mice composed the groups: C-Control (saline solution); MTK2-2 mg/Kg of Montelukast (MTK) and MTK4-4 mg/Kg of MTK, daily administered by mouth throughout all experimental periods set at 7, 14, and 21 days post-operative. Dental sockets were analyzed by computed microtomography (microCT), histopathology, and immunohistochemistry. Femurs, L5 vertebra and organs were also removed for observation. Blood was collected for plasma bone and liver markers. Histopathology and microCT analysis revealed early socket repair of MTK2 and MTK4 animals, with significant increased BV/TV at days 14 and 21 compared to C. Higher plasma calcium was detected at days 7 and 21 in MTK4 in comparison to C, while phosphate was significantly increased in MTK2 in the same periods in comparison to C and MTK4. No significant differences were found regarding plasma ALP and TRAP, neither for local TRAP and Runx2 immunolabeling at the healing sockets. Organs did not present histological abnormalities. Increased AST levels have been detected in distinct groups and periods. In general, femur phenotype was improved in MTK treated animals. Collectively, MTK promoted early bone formation after tooth extraction and increased bone quality of femurs and vertebra in a time-dose-dependent manner, and should be considered as an alternative therapy when improved post-extraction socket repair or skeleton preservation is required.

A Model Study to Evaluate Osseointegration and Fracture Healing Following Open Reduction and Internal Fixation (ORIF) in Diabetic Lewis Rats.

There is a higher risk of implant osseointegration failure after open reduction and internal fixation (ORIF) in patients with diabetes due to increased inflammatory conditions, associated metallic corrosion and infection. While it is possible to avoid elective osseous surgery in patients with diabetes, it may not be the case in nonelective cases, such as ORIF ankle fractures. A total of 30 male Lewis rats (12-15 weeks old) were distributed into diabetic (D) and nondiabetic (ND) groups. Fracture healing and osseointegration were evaluated at 2-, 10-, and 21-day time points. Microtomographic and histological analysis depicted distinct differences in fracture healing and osseointegration between D and ND animals. Immunohistochemical analysis exhibited elevated proliferation (PCNA) and osteogenic (Runx2) cells for ND animals, while HMGB1 (inflammatory marker) was elevated for D animals during healing. Bone resorption marker CTX-1 was elevated in the plasma of D animals at 2 days, while bone formation marker P1NP was higher for ND animals at 10 days. Overall, this model resulted in delayed implant osseointegration and fracture healing in diabetic animals, highlighting the importance of developing new biomaterials or implant coatings that can improve bone healing outcomes in this patient population.

Bioactive glass-ceramic bone repair associated or not with autogenous bone: a study of organic bone matrix organization in a rabbit critical-sized calvarial model.

OBJECTIVE: The aim of the study was to analyze bone matrix (BMX) organization after bone grafting and repair using a new bioactive glass-ceramic (Biosilicate®) associated or not with particulate autogenous bone graft. MATERIAL AND METHODS: Thirty rabbits underwent surgical bilateral parietal defects and divided into groups according to the materials used: (C) control-blood clot, (BG) particulate autogenous bone, (BS) bioactive glass-ceramic, and BG + BS. After 7, 14, and 30 days post-surgery, a fragment of each specimen was fixed in - 80 °C liquid nitrogen for zymographic evaluation, while the remaining was fixed in 10% formalin for histological birefringence analysis. RESULTS: The results of this study demonstrated that matrix organization in experimental groups was significantly improved compared to C considering collagenous organization. Zymographic analysis revealed pro-MMP-2, pro-MMP-9, and active (a)-MMP-2 in all groups, showing gradual decrease of total gelatinolytic activity during the periods. At day 7, BG presented more prominent gelatinolytic activity for pro-MMP-2 and 9 and a-MMP-2, when compared to the other groups. In addition, at day 7, a 53% activation ratio (active form/[active form + latent form]) was evident in C group, 33% in BS group, and 31% in BG group. CONCLUSION: In general, BS allowed the production of a BMX similar to BG, with organized collagen deposition and MMP-2 and MMP-9 disponibility, permitting satisfactory bone remodeling at the late period. CLINICAL RELEVANCE: The evaluation of new bone substitute, with favorable biological properties, opens the possibility for its use as a viable and efficient alternative to autologous bone graft.

Influence of rapid- and slow-rate resorption collagen membrane in maxillary sinus augmentation.

OBJECTIVE: To evaluate the effects of low- and rapid-resorption-rate bioabsorbable collagen membranes in maxillary sinus augmentation procedure in rabbits considering Schneiderian membrane (SM) reaction and bone tissue formation. MATERIALS AND METHODS: Eighteen male adult rabbits underwent bilateral maxillary sinus augmentation with particulate bovine hydroxyapatite to be divided into three groups, as follows: Group C - control, no membrane; Group RR - rapid resorbable collagen membrane; and Group SR - slow-resorbable collagen membrane. The animals were euthanized after 30 and 120 days for specimen's removal to be prepared and analyzed under light microscopy, histomorphometry, and immunohistochemistry for Runx2 and VEGF labeling. RESULTS: Histopathology evaluation presented similar healing pattern among the groups with a satisfactory response of SM, both at day 30 and day 120. Bone histomorphometry did not reveal significant differences among the groups, as well as immunohistochemistry analysis, which presented intense immunolabeling for both proteins in all groups. CONCLUSIONS: The presence of both membranes did not negatively interfere in bone formation and remodeling, and the focal mild inflammatory reaction caused by their degrading process did not impair the reconstructive procedure.

Experimental maxillary sinus augmentation using a highly bioactive glass ceramic.

Physicochemical characteristics of a biomaterial directly influence its biological behavior and fate. However, anatomical and physiological particularities of the recipient site also seem to contribute with this process. The present study aimed to evaluate bone healing of maxillary sinus augmentation using a novel bioactive glass ceramic in comparison with a bovine hydroxyapatite. Bilateral sinus augmentation was performed in adult male rabbits, divided into 4 groups according to the biomaterial used: BO-particulate bovine HA Bio-Oss(®) (BO), BO+G-particulate bovine HA + particulate autogenous bone graft (G), BS-particulate glass ceramic (180-212 µm) Biosilicate(®) (BS), and BS+G-particulate glass ceramic + G. After 45 and 90 days, animals were euthanized and the specimens prepared to be analyzed under light and polarized microscopy, immunohistochemistry, scanning electron microscopy (SEM), and micro-computed tomography (µCT). Results revealed different degradation pattern between both biomaterials, despite the association with bone graft. BS caused a more intense chronic inflammation with foreign body reaction, which led to a difficulty in bone formation. Besides this evidence, SEM and µCT confirmed direct contact between newly formed bone and biomaterial, along with osteopontin and osteocalcin immunolabeling. Bone matrix mineralization was late in BS group but became similar to BO at day 90. These results clearly indicate that further studies about Biosilicate(®) are necessary to identify the factors that resulted in an unfavorable healing response when used in maxillary sinus augmentation.

Effects of angiotensin II type I receptor blocker losartan on orthodontic tooth movement.

INTRODUCTION: Drugs that block the renin-angiotensin system (RAS) are widely used for treating hypertension, heart and kidney failure, and the harmful effects of diabetes. Components of the RAS have been identified in various organs, but little is known of their effects on bone remodeling. The aim of this study was to evaluate whether the blockage of the RAS influences strain-induced bone remodeling in a model of orthodontic tooth movement. METHODS: An orthodontic appliance was placed in C57BL6/J mice that were randomly divided into 2 groups: vehicle-treated mice (VH) and mice treated with losartan (an angiotensin II receptor blocker). Orthodontic tooth movement and the number of tartrate-resistant acid phosphatase-positive cells were determined by histopathologic analysis. The expression of mediators involved in bone remodeling was evaluated by quantitative real-time polymerase chain reaction. Blood pressure was measured before and during the experimental period. RESULTS: Orthodontic tooth movement and tartrate-resistant acid phosphatase-positive cells were significantly reduced in the losartan group compared with the VH group. mRNA levels of osteoclast markers (RANK, RANKL, cathepsin K, and metalloproteinase 13) were lower in the losartan mice than in the VH group, whereas the expressions of osteoblast markers and negative regulators of bone resorption (periostin, dentin matrix protein, alkaline phosphatase, collagen 1A1, semaphorin 3A3, metalloproteinase 2, and osteoprotegerin) were higher in the VH group. CONCLUSIONS: Blockage of the RAS system decreases osteoclast differentiation and activity and, consequently, results in decreased strain-induced bone remodeling in orthodontic tooth movement.

Effects of glass-ceramic produced by the sol-gel route in macrophages recruitment and polarization into bone tissue regeneration.

Effective bone substitute biomaterials remain an important challenge in patients with large bone defects. Glass ceramics produced by different synthesis routes may result in changes in the material physicochemical properties and consequently affect the success or failure of the bone healing response. To investigate the differences in the orchestration of the inflammatory and healing process in bone grafting and repair using different glass-ceramic routes production. Thirty male Wistar rats underwent surgical unilateral parietal defects filled with silicate glass-ceramic produced by distinct routes: BS - particulate glass-ceramic produced via the fusion/solidification route, and BG - particulate glass-ceramic produced via the sol-gel route. After 7, 14, and 21 days from biomaterial grafting, parietal bones were removed to be analyzed under H&E and Massons' Trichome staining, and immunohistochemistry for CD206, iNOS, and TGF-ß. Our findings demonstrated that the density of lymphocytes and plasma cells was significantly higher in the BS group at 45, and 7 days compared to the BG group, respectively. Furthermore, a significant increase of foreign body giant cells (FBGCs) in the BG group at day 7, compared to BS was found, demonstrating early efficient recruitment of FBGCs against sol-gel-derived glass-ceramic particulate (BS group). According to macrophage profiles, CD206+ macrophages enhanced at the final periods of both groups, being significantly higher at 45 days of BS compared to the BG group. On the other hand, the density of transformation growth factor beta (TGF-ß) positive cells on 21 days were the highest in BG, and the lowest in the BS group, demonstrating a differential synergy among groups. Noteworthy, TGF-ß+ cells were significantly higher at 21 days of BG compared to the BS group. Glass-ceramic biomaterials can act differently in the biological process of bone remodeling due to their route production, being the sol-gel route more efficient to activate M2 macrophages and specific FBGCs compared to the traditional route. Altogether, these features lead to a better understanding of the effectiveness of inflammatory response for biomaterial degradation and provide new insights for further preclinical and clinical studies involved in bone healing.

Revolutionizing fracture fixation in diabetic and non-diabetic rats: High mobility group box 1-based coating for enhanced osseointegration.

Chronic inflammation and hyperglycemia in diabetic patients increase the risk of implant failure and impaired fracture healing. We previously developed and characterized a titanium (Ti) coating strategy using an imidazolium-based ionic liquid (IonL) with a fully reduced, non-oxidizable High Mobility Group Box 1 (HMGB1) isoform (Ti-IonL-HMGB1) to immunomodulate tissue healing. In this study, we used an open reduction fracture fixation (ORIF) model in non-diabetic (ND) and diabetic (D) rats to further investigate the effectiveness of this Ti-IonL-HMGB1 coating on orthopedic applications. Ninety male Lewis rats (12-15 weeks) were divided into D (n = 45) and ND (n = 45) groups that were distributed into three subgroups based on the type of local treatment received: Ti (uncoated Ti), Ti-IonL, and Ti-IonL-HMGB1 implants. Fracture healing and osseointegration were evaluated using microtomographic, histological, and immunohistochemical analysis of proliferating cell nuclear antigen (PCNA), Runt-related transcription factor 2 (RUNX2), and HMGB1 markers at 2, 10, and 21 days post-ORIF. Scanning Electron Microscopy verified the coating stability after placement. Microtomographic and histological analysis demonstrated increased fracture healing and osseointegration for ND rats in all treatment groups at 10 days, with impaired healing for D rats. Immunohistochemical analysis exhibited elevated PCNA+ and RUNX2+ cells for D animals treated with Ti-IonL-HMGB1 at 21 days compared to all other groups. The immunohistochemical marker HMGB1 was elevated at all time points for D animals in comparison to ND animals, yet was lowered for D tissues near the Ti-IonL-HMGB1 treated implant. Improved osseous healing was demonstrated in D animals with Ti-IonL-HMGB1 treatment by 21 days, compared to D animals with other treatments. To the best of our knowledge, this is the first study analyzing Ti-IonL-HMGB1 implantation in an injury site through ORIF procedures in ND and D rats. This surface approach has potential for improving implanted biomaterials in diabetic environments.