Final irrigation with bioglass solution in regenerative endodontic procedure induces tissue formation inside the root canals, collagen maturation, proliferation cell and presence of osteocalcin.

AIM: To evaluate the influence of an experimental solution of cobalt-doped F18 bioactive glass (F18Co) on tissue repair following regenerative endodontic procedure (REP) in rat molars. METHODOLOGY: The F18Co solution was prepared at a ratio of 1:5 F18Co powder to distilled water. The right or left upper first molars of 12 Wistar rats were used, where the pulps were exposed, removed, and irrigated with 2.5% sodium hypochlorite (NaOCl), followed by 17% ethylenediaminetetraacetic acid (EDTA) (5 min each). Subsequently, the molars were divided into two groups (n = 6): REP-SS and REP-F18Co, where they received a final irrigation (5 min) with saline solution (SS) or F18Co solution, respectively. Then, intracanal bleeding was induced, and the tooth was sealed. Untreated molars were used as controls (n = 3). At 21 days, the rats were euthanized, and the specimens were processed for analysis of mineralized tissue and soft tissue formation inside the root canal using haematoxylin-eosin. The presence and maturation of collagen were evaluated by Masson's trichrome and picrosirius red staining. Immunolabelling analyses of proliferating cell nuclear antigen (PCNA) and osteocalcin (OCN) were performed. The data were submitted to the Mann-Whitney U-test (p < .05). RESULTS: There was a similar formation of mineralized tissue in thickness and length in REP-SS and REP-F18Co groups (p > .05). Regarding the presence of newly formed soft tissue, most specimens of the REP-F18Co had tissue formation up to the cervical third of the canal, whilst the REP-SS specimens showed formation up to the middle third (p < .05), and there was higher maturation of collagen in REP-F18Co (p < .05). The number of PCNA-positive cells found in the apical third of the root canal was significantly higher in the F18Co group, as well as the OCN immunolabelling, which was severe in most specimens of REP-F18Co, and low in most specimens of REP-SS. CONCLUSION: The final irrigation with F18Co bioactive glass solution in REP did not influence mineralized tissue formation but induced soft tissue formation inside the root canals, with higher collagen maturation, and an increase in PCNA-positive cells and OCN immunolabelling.

Bioactive glass-ceramic for bone tissue engineering: an in vitro and in vivo study focusing on osteoclasts.

Despite the crucial role of osteoclasts in the physiological process of bone repair, most bone tissue engineering strategies have focused on osteoblast-biomaterial interactions. Although Biosilicate® with two crystalline phases (BioS-2P) exhibits osteogenic properties and significant bone formation, its effects on osteoclasts are unknown. This study aimed to investigate the in vitro and in vivo effects of BioS-2P on osteoclast differentiation and activity. RAW 264.7 cells were cultured in osteoclastogenic medium (OCM) or OCM conditioned with BioS-2P (OCM-BioS-2P), and the cell morphology, viability, and osteoclast differentiation were evaluated. BioS-2P scaffolds were implanted into rat calvarial defects, and the bone tissue was evaluated using tartrate-resistant acid phosphatase (TRAP) staining and RT-polymerase chain reaction (PCR) after 2 and 4 weeks to determine the gene expressions of osteoclast markers and compare them with those of the bone grown in empty defects (Control). OCM-BioS-2P favored osteoclast viability and activity, as evidenced by an increase in the TRAP-positive cells and matrix resorption. The bone tissue grown on BioS-2P scaffolds exhibited higher expression of the osteoclast marker genes (Ctsk, Mmp 9, Rank) after 2 and 4 weeks and the RankL/Opg ratio after 2 weeks. Trap gene expression was lower at 2 weeks, and a higher number of TRAP-stained areas were observed in the newly formed bone on BioS-2P scaffolds at both 2 and 4 weeks compared to the Controls. These results enhanced our understanding of the role of bioactive glass-ceramics in bone repair, and highlighted their role in the modulation of osteoclastic activities and promotion of interactions between bone tissues and biomaterials.

Bioactive glass-ceramic for bone tissue engineering: an in vitro and in vivo study focusing on osteoclasts

Abstract: Despite the crucial role of osteoclasts in the physiological process of bone repair, most bone tissue engineering strategies have focused on osteoblast-biomaterial interactions. Although Biosilicate® with two crystalline phases (BioS-2P) exhibits osteogenic properties and significant bone formation, its effects on osteoclasts are unknown. This study aimed to investigate the in vitro and in vivo effects of BioS-2P on osteoclast differentiation and activity. RAW 264.7 cells were cultured in osteoclastogenic medium (OCM) or OCM conditioned with BioS-2P (OCM-BioS-2P), and the cell morphology, viability, and osteoclast differentiation were evaluated. BioS-2P scaffolds were implanted into rat calvarial defects, and the bone tissue was evaluated using tartrate-resistant acid phosphatase (TRAP) staining and RT-polymerase chain reaction (PCR) after 2 and 4 weeks to determine the gene expressions of osteoclast markers and compare them with those of the bone grown in empty defects (Control). OCM-BioS-2P favored osteoclast viability and activity, as evidenced by an increase in the TRAP-positive cells and matrix resorption. The bone tissue grown on BioS-2P scaffolds exhibited higher expression of the osteoclast marker genes (Ctsk, Mmp 9, Rank) after 2 and 4 weeks and the RankL/Opg ratio after 2 weeks. Trap gene expression was lower at 2 weeks, and a higher number of TRAP-stained areas were observed in the newly formed bone on BioS-2P scaffolds at both 2 and 4 weeks compared to the Controls. These results enhanced our understanding of the role of bioactive glass-ceramics in bone repair, and highlighted their role in the modulation of osteoclastic activities and promotion of interactions between bone tissues and biomaterials.

Scaffolds of bioactive glass-ceramic (Biosilicate®) and bone healing: A biological evaluation in an experimental model of tibial bone defect in rats.

This study aimed to investigate the in vivo tissue response of the Biosilicate® scaffolds in a model of tibial bone defect. Sixty male Wistar rats were distributed into bone defect control group (CG) and Biosilicate® scaffold group (BG).  Animals were euthanized 15, 30 and 45 days post-surgery. Stereomicroscopy, scanning electron microscopy, histopathological, immunohistochemistry and biomechanical analysis were used. Scaffolds had a total porosity of 44%, macroporosity of 15% with pore diameter of 230 µm. Higher amount of newly formed bone was observed on days 30 and 45 in BG. Immunohistochemistry analysis showed that the COX-2 expression was significantly higher on days 15 and 30 in BG compared with the CG. RUNX-2 immunoexpression was significantly higher in BG on days 15 and 45. No statistically significant difference was observed in RANKL immunoexpression in all experimental groups. BMP-9 immunoexpression was significantly upregulated in the BG on day 45. Biomechanical analysis showed a decrease in the biomechanical properties of the bone callus on days 30 and 45. The implantation of the Biosilicate® scaffolds was effective in stimulating newly bone formation and produced an increased immunoexpression of markers related to the bone repair.

Porous bioactive scaffolds: characterization and biological performance in a model of tibial bone defect in rats.

The aim of this study was to evaluate the effects of highly porous Biosilicate(®) scaffolds on bone healing in a tibial bone defect model in rats by means of histological evaluation (histopathological and immunohistochemistry analysis) of the bone callus and the systemic inflammatory response (immunoenzymatic assay). Eighty Wistar rats (12 weeks-old, weighing±300 g) were randomly divided into 2 groups (n=10 per experimental group, per time point): control group and Biosilicate® group (BG). Each group was euthanized 3, 7, 14 and 21 days post-surgery. Histological findings revealed a similar inflammatory response in both experimental groups, 3 and 7 days post-surgery. During the experimental periods (3-21 days post-surgery), it was observed that the biomaterial degradation, mainly in the periphery region, provided the development of the newly formed bone into the scaffolds. Immunohistochemistry analysis demonstrated that the Biosilicate® scaffolds stimulated cyclooxygenase-2, vascular endothelial growth factor and runt-related transcription factor 2 expression. Furthermore, in the immunoenzymatic assay, BG presented no difference in the level of tumor necrosis factor alpha in all experimental periods. Still, BG showed a higher level of interleukin 4 after 14 days post-implantation and a lower level of interleukin 10 in 21 days post-surgery. Our results demonstrated that Biosilicate® scaffolds can contribute for bone formation through a suitable architecture and by stimulating the synthesis of markers related to the bone repair.

Effects of biosilicate(®) scaffolds and low-level laser therapy on the process of bone healing.

OBJECTIVE: This study aimed to investigate the in vivo tissue performance of the association of Biosilicate(®) scaffolds and low-level laser therapy (LLLT) in a tibial bone defects model in rats. BACKGROUND DATA: Many studies have been demonstrating the osteogenic potential of Biosilicate and LLLT. However, there is a need to investigate the effects of both treatments for bone consolidation. METHODS: The animals were divided into control group (CG), Biosilicate scaffold group (BG), and Biosilicate scaffolds plus LLLT group (BLG). Animals were euthanized after 15, 30, and 45 days post-injury. RESULTS: The histological analysis revealed that all the experimental groups showed inflammatory infiltrate and granulation tissue, at the area of the defect at day 15. After 30 days, CG still showed granulation tissue and bone ingrowth. Both Biosilicate groups presented newly formed bone and interconected trabeculae. At 45 days, CG showed immature newly formed bone. A more mature newly formed bone was observed in BG and BLG. On day 15, BG demonstrated a statistically higher expression of cyclooxygenase (COX)-2 compared with CG and BLG. No statistically significant difference was observed in COX-2 immunoexpression among the groups at 30 and 45 days. Similar expression of bone morphogenetic protein (BMP)-9 was demonstrated for all experimental groups at 15 and 30 days. At 45 days, the BMP-9 immunoexpression was statistically upregulated in the BLG compared with the CG and BG. No statistically significant difference was observed in the receptor activator of nuclear factor kappa-B ligand (RANKL) immunoexpression among the groups in all periods evaluated. Biosilicate groups presented a decrease in biomechanical properties compared with CG at 30 and 45 days post-surgery. CONCLUSIONS: Our findings suggest that Biosilicate presented osteogenic activity, accelerating bone repair. However, laser therapy was not able to enhance the bioactive properties of the Biosilicate.

Effect of 830 nm laser phototherapy on osteoblasts grown in vitro on Biosilicate scaffolds.

OBJECTIVE: The purpose of this study was (i) to develop a method for successfully seeding osteoblasts onto a glass-ceramic scaffold designed for use in clinical settings, and (ii) to determine whether the application of laser phototherapy at 830 nm would result in osteoblast proliferation on the glass-ceramic scaffold. BACKGROUND: The use of bioscaffolds is considered a promising strategy for a number of clinical applications where tissue healing is sub-optimal. As in vitro osteoblast growth is a slow process, laser phototherapy could be used to stimulate osteoblast proliferation on bioscaffolds. METHODS: A methodology was developed to seed an osteoblastic (MC3T3) cell line onto a novel glass-ceramic scaffold. Seeded scaffolds were irradiated with a single exposure of 830 nm laser at 10 J/cm(2) (at diode). Non-irradiated seeded scaffolds acted as negative controls. Cell proliferation was assessed seven days after irradiation. RESULTS: Osteoblastic MC3T3 cells were successfully grown on discs composed of a glass-ceramic composite. Laser irradiation produced a 13% decrease in MC3T3 cell proliferation on glass-ceramic discs (mean +/- SD = 0.192 +/- 0.002) compared with control (non-irradiated) discs (mean +/-SD = 0.22 +/- 0.002). CONCLUSIONS: Despite successful seeding of bioscaffolds with osteoblasts, laser phototherapy resulted in a reduction in cell growth compared to non-irradiated controls. Future research combining laser phototherapy and glass-ceramic scaffolds should take into account possible interactions of the laser with matrix compounds.