Avaliação dos princípios de fotobiomodulação e bioatividade na otimização do reparo ósseo na fixação de fraturas em ratas ovariectomizadas / Biomodulation and bioactivity principles evaluation in the optimization of bone repair in fixation of fractures in ovariectomized rats

Este trabalho teve como objetivo avaliar o efeito da biomodulação e bioatividade no reparo ósseo de ratas ovariectomizadas submetidas à simulação de fraturas femurais. Sessenta e quatro ratas Wistar (Rattus novergicus), fêmeas, com 6 meses de idade, em que metade dos animais foram submetidos à ovariectomia bilateral (OVX) e a outra metade à cirurgia fictícia de ovariectomia (SHAM), e um período de 3 meses foi acompanhado para a verificar se a osteoporose estava presente. As ratas foram submetidas à simulação de fratura em ambos os fêmures e a fratura foi fixada com miniplaca e parafusos do sistema 1,5 mm. A metade das amostrastiveram miniplacas com texturização de superfície tratadas por oxidação com plasma eletrolítico (PEO), que ficou em contato com o "gap" reparacional e, a outra metade com miniplacas convencionais, com superfície (CONV). Estes grupos experimentais não foram submetidos à biomodulação (CO). Para o mesmo desenho experimental dos grupos, no ato operatório, após a fixação das fraturas, os demais grupos experimentais foram submetidos à fotobiomodulação por meio da irradiação do laser de baixa intensidade (LASER), durante 5 minutos. Aos 14 e 42 dias de pós-operatório, foi administrada calceína e alizarina, respectivamente, pela via intramuscular, na dose de 20 mg/kg de peso. A eutanásia de todos os animais foi realizada aos 60 dias de pós-operatório. Os grupos experimentais (SHAM/LASER/CONV; SHAM/CO/CONV; SHAM/LASER/PEO; SHAM/CO/PEO; OVX/LASER/CONV; OVX/CO/CONV; OVX/LASER/PEO; OVX/CO/PEO), após a eutanásia, tiveram as regiões de interesse, inicialmente escaneadas em microtomografia computadorizada para avaliação dos parâmetros volumétricos do osso (BV/TV) e qualidade óssea PO(tot) (Tb.Th, Tb.Sp e Tb.N), em seguida as peças continuaram em processamento para inclusão em resina fotopolimerizável. As lâminas foram escaneadas em microscopia confocal a laser para análise da área em pixels2 de precipitação mineral óssea (APMO). Após este processo, as lâminas foram coradas em vermelho de alizarina e azul de Stevenel para histometria de área de osso neoformado (NBF) no "gap" reparacional e análise do padrão reparacional. Os resultados foram inferiores e estatisticamente significantes (p< 0,05) na análise de MicroCT na comparação do grupo OVX/CO/CONV com os demais grupos nos parâmetros (BV.TV, Tb.N e Tb.Th); APMO apresentou menor área para o fluorocromo vermelho de alizarina nos grupos OVX/CO/CONV e OVX/CO/PEO (p<0,05). No grupo OVX, o laser mostrou maior turnover ósseo (p< 0,05) para NBF e pior padrão histológico. Conclusão: A fotobiomodulação e bioatividade otimizaram o reparo ósseo de fraturas em fêmures de ratas ovariectomizadas(AU)

The aim of this study was to evaluate the effect of biomodulation and bioactivity on the bone repair of ovariectomized rats submitted to simulation of femoral fractures. 64 female adults Wistar rats (Rattus novergicus), which half of the animals was submitted to bilateral ovariectomy (OVX) and the other half to the dummy ovariectomy surgery (SHAM), and after 3-month the osteoporosis was induced. The rats were submitted to fracture simulation in both of the femurs and the fracture was fixed with miniplate and screws of the system 1.5 mm. Half of the samples had surface-textured miniplatestreated by with plasma electrolytic oxidation (PEO), which was in contact with the repair gap, and the other half with conventional miniplates with surface (CONV). These experimental groups were not submitted to biomodulation (CO). For the same experimental design of the groups, after fracture fixation, the other experimental groups were submitted to photobiomodulation through low level laser (laser), for 5 minutes. At 14 and 42 postoperative days, calcein and alizarin, respectively, were administered intramuscularly at a dose of 20 mg / kg body weight. The animals were euthanized at 60 postoperative days. The experimental groups (SHAM/LASER/CONV, SHAM/CO/CONV, SHAM/LASER/PEO, SHAM/CO/PEO, OVX/LASER/CONV, OVX/CO/CONV, OVX/LASER/PEO), after euthanasia, had the regions of interest, initially scanned in computerized microtomography to evaluate the bone volume parameters [BV / TV] and bone quality PO (tot) and (Tb.Th, Tb.Sp and Tb.N), then the samples were processed for inclusion in photopolymerizable resin.. The slides were scanned in confocal laser microscopy for analysis of the area in pixels2 of bone mineral precipitation (APMO). After this process, the slides were stained in alizarin red and Stevenel blue for neoformed bone area (NBF) in the reparative gap and analysis of the histology pattern. The results showed lower and statistically significant results (p <0.05) for MicroCT parameters (BV.TV, Tb.N and Tb.Th) in the comparison between OVX / CO / CONV and the other groups; APMO showed lower area for the alizarin red fluorochrome in the OVX / CO / CONV and OVX / CO / PEO groups (p <0.05). In the OVX, the laser showed greater bone turnover (P<0,05); The OVX / CO / CONV showed lower NBF (p <0.05) and worse histological pattern. Conclusion: The photobiomodulation and bioactivity optimized the bone repair of femur fractures in ovariectomized rats(AU)

Daily melatonin administration improves osseointegration in pinealectomized rats

Abstract The hypothesis of this study was that the peri-implant bone healing of the group of pinealectomized rats would differ from the control group. The samples were subjected to immunohistochemical, microtomographic (total porosity and connectivity density), and fluorochrome (mineralized surface) analyses. Objectives The goal of this study was to investigate the cellular changes and bone remodeling dynamics along the bone/implant interface in pinealectomized rats. Material and Methods The total of 18 adult male rats (Rattus norvegicus albinus, Wistar) was divided into three groups (n=6): control (CO), pinealectomized without melatonin (PNX) and pinealectomized with melatonin (PNXm). All animals were submitted to the first surgery (pinealectomy), except the CO group. Thirty days after the pinealectomy without melatonin, the second surgery was conducted, in which all animals received an implant in each tibia (36 titanium implants with surface treatment were installed - Implalife® São Paulo, SP, Brazil). By gavage, the rats of the PNX group received the vehicle solution, and the procedure. Results Immunohistochemical analysis for runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osteopontin (OP) and osteocalcin (OC) showed that the bone repair process in the PNXm group was similar to that of the CO group, whereas the PNX group showed a delay. The microtomographic parameters of total porosity [Po(tot)] and bone surface (BS) showed no statistically significant differences, whereas for the connective density (Conn.Dn) a statistical difference was found between the CO and PNXm groups. Fluorochrome analysis of the active mineralized surface showed statistically significant difference between the CO and PNX and between the CO and PNXm groups. Conclusion The absence of the pineal gland impaired the bone repair process during osseointegration, however the daily melatonin replacement was able to restore this response.

Bone repair access of BoneCeramic™ in 5-mm defects: study on rat calvaria

Abstract Objective: The aim of this study was to evaluate the osteoconductive potential of BoneCeramic™ on bone healing in rat calvaria 5-mm defects. Material and Methods: A 5-mm calvaria bone defect was induced in three groups and the defect was not filled with biomaterial [Clot Group (CG)], autogenous bone (AG), or Bone Ceramic Group (BCG). Animals were euthanized after 14 or 28 days and the bone tissue within the central area of the bone defect was evaluated. Results were compared using ANOVA and Tukey test (p<0.05). Immunohistochemistry was performed using primary antibodies against osteocalcin, RUNX-2, TRAP, VEGF proteins, and 3-dimensional images of the defects in μCT were obtained to calculate bone mineral density (BMD). Results: In BCG, the defect was completely filled with biomaterial and new bone formation, which was statistically superior to that in the GC group, at both time-points (p<0.001 for 14 days; p=0.002 for 28 days). TRAP protein showed weak, RUNX-2 showed a greater immunolabeling when compared with other groups, VEGF showed moderate immunostaining, while osteocalcin was present at all time-points analyzed. The μCT images showed filling defect by BCG (BMD= 1337 HU at 28 days). Conclusion: Therefore, the biomaterial tested was found to be favorable to fill bone defects for the reporting period analyzed.

A SERM increasing the expression of the osteoblastogenesis and mineralization-related proteins and improving quality of bone tissue in an experimental model of osteoporosis

Abstract Raloxifene is an antiresorptive drug, selective estrogen receptor modulator (SERM) used in the treatment of osteoporosis. Objective To evaluate proteins related to bone repair at the peri-implant bone in a rat model of osteoporosis treated with raloxifene. Material and Methods 72 rats were divided into three groups: SHAM (healthy animals), OVX (ovariectomized animals), and RLX (ovariectomized animals treated with raloxifene). Raloxifene was administered by gavage (1 mg/kg/day). Tibial implantation was performed 30 days after ovariectomy, and animals were euthanized at 14, 42, and 60 days postoperatively. Samples were collected and analyzed by immunohistochemical reactions, molecular analysis, and microtomographic parameters. Results RLX showed intense staining of all investigated proteins at both time points except for RUNX2. These results were similar to SHAM and opposite to OVX, showing mild staining. The PCR gene expression of OC and ALP values for RLX (P<0.05) followed by SHAM and OVX groups. For BSP data, the highest expression was observed in the RLX groups and the lowest expression was observed in the OVX groups (P<0.05). For RUNX2 data, RLX and SHAM groups showed greater values compared to OVX (P<0.05). At 60 days postoperatively, microtomography parameters, related to closed porosity, showed higher values for (Po.N), (Po.V), and (Po) in RLX and SHAM groups, whereas OVX groups showed lower results (P<0.05); (BV) values (P=0.009); regarding total porosity (Po.tot), RLX group had statistically significant lower values than OVX and SHAM groups (P=0.009). Regarding the open porosity (Po.V and Po), the SHAM group presented the highest values, followed by OVX and RLX groups (P<0.05). The Structural Model Index (SMI), RLX group showed a value closer to zero than SHAM group (P<0.05). Conclusions Raloxifene had a positive effect on the expression of osteoblastogenesis/mineralization-related proteins and on micro-CT parameters related to peri-implant bone healing.

Short term sodium alendronate administration improves the peri-implant bone quality in osteoporotic animals

Abstract Sodium alendronate is a bisphosphonate drug that exerts antiresorptive action and is used to treat osteoporosis. Objective The aim of this study was to evaluate the bone repair process at the bone/implant interface of osteoporotic rats treated with sodium alendronate through the analysis of microtomography, real time polymerase chain reactions and immunohistochemistry (RUNX2 protein, bone sialoprotein (BSP), alkaline phosphatase, osteopontin and osteocalcin). Material and Methods A total of 42 rats were used and divided in to the following experimental groups: CTL: control group (rats submitted to fictitious surgery and fed with a balanced diet), OST: osteoporosis group (rats submitted to a bilateral ovariectomy and fed with a low calcium diet) and ALE: alendronate group (rats submitted to a bilateral ovariectomy, fed with a low calcium diet and treated with sodium alendronate). A surface treated implant was installed in both tibial metaphyses of each rat. Euthanasia of the animals was conducted at 14 (immunhostochemistry) and 42 days (immunohistochemistry, micro CT and PCR). Data were subjected to statistical analysis with a 5% significance level. Results Bone volume (BV) and total pore volume were higher for ALE group (P<0.05). Molecular data for RUNX2 and BSP proteins were significantly expressed in the ALE group (P<0.05), in comparison with the other groups. ALP expression was higher in the CTL group (P<0.05). The immunostaining for RUNX2 and osteopontin was positive in the osteoblastic lineage cells of neoformed bone for the CTL and ALE groups in both periods (14 and 42 days). Alkaline phosphatase presented a lower staining area in the OST group compared to the CTL in both periods and the ALE at 42 days. Conclusion There was a decrease of osteocalcin precipitation at 42 days for the ALE and OST groups. Therefore, treatment with short-term sodium alendronate improved bone repair around the implants installed in the tibia of osteoporotic rats.

Avaliação do reparo ósseo na interface osso/implante em ratos pinealectomizados: análise histológica, imunoistoquimica, histométrica, biomecânica e por microct / Bone repair evaluation in bone/implant interface in pinealectomized rats: histological, immunohistochemistry, histometric, biomechanics and microCT analysis

O presente estudo teve como objetivo investigar a interface osso / implante em ratos pinealectomizados tratados com melatonina. Métodos: 30 ratos, machos, adultos foram divididos em 3 grupos: controle (CO), pinealectomizados (PnX) e pinealectomizado com melatonina (PNXm). Após 30 dias da Pinealectomia, um implante foi instalado em cada tíbia e a eutanásia foi realizada aos 42 dias. As amostras foram submetidas a histomorfométrica (ELCOI e AON), biomecânica (de torque reverso), microtomografia e análise imunoistoquímica. Os dados quantitativos foram submetidos à análise estatística (P <0,05). Para os resultados histométricos, valores ELCOI para PNX foram menores quando comparados com PNXm (P = 0,0393), enquanto AON não mostrou diferença entre os grupos (P = 0,761). Os valores de torque reverso não mostraram diferença entre os grupos PNX e PNXm (P = 0,5000), e as interações CO vs PNXm, e CO vs PNX mostraram diferença significativa (P <0,05). Os parâmetros volumétricas BV/TV (P = 0,933); Tr.N (P = 0,933); Tb.Th (P = 0,933), Tb.Sp (P = 0,200) não apresentaram diferença estatística. A imunomarcação para OPG, RANKL, TRAP e osteocalcina mostrou similaridade entre os grupos, apesar da PNXm parece assemelhar-se às condições fisiológicas (CO). Portanto, a melatonina melhorou a remodelação óssea na região peri-implante em ratos pinealectomizados.

This study aimed to investigate the interface bone/implant in pinealectomized rats treated with melatonin. 30 rats, male, adults were divided into 3 groups: control (CO), pinealectomized (PNX) and pinealectomized with melatonin (PNXm). After 30 days of the pinealectomy, one implant was installed on each tibia and euthanasia was performed at 42 days. The samples were submitted to histometric (BIC and NB), biomechanical (reverse torque), microtomography and immunohistochemical analysis. The quantitative data were subjected to statistical analysis (P<0.05). For the histometric results, BIC values to PNX were lower when compared to PNXm (P=0.0393), whereas NB did not showed difference among the groups (P=0.761). Reverse torque did not show difference between the groups PNX and PNXm (P=0,5000), and the interactions CO vs PNXm, and CO vs PNX showed significantly difference (P<0.05). The volumetric parameters BV/TV (P=0.933); Tr.N (P=0.933); Tb.Th (P=0.933), Tb.Sp (P=0.200) did not present statistical difference. The immunolabeling for OPG, RANKL, TRAP and osteocalcin showed similarity among the groups, despite the PNXm it seems to resemble to physiological conditions (CO). Therefore, melatonin improved the bone turnover in peri-implant region in pinealectomized rats.