Comparison of Osteoconductive Ability of Two Types of Cholesterol-Bearing Pullulan (CHP) Nanogel-Hydrogels Impregnated with BMP-2 and RANKL-Binding Peptide: Bone Histomorphometric Study in a Murine Calvarial Defect Model.

The receptor activator of NF-κB ligand (RANKL)-binding peptide is known to accelerate bone morphogenetic protein (BMP)-2-induced bone formation. Cholesterol-bearing pullulan (CHP)-OA nanogel-crosslinked PEG gel (CHP-OA nanogel-hydrogel) was shown to release the RANKL-binding peptide sustainably; however, an appropriate scaffold for peptide-accelerated bone formation is not determined yet. This study compares the osteoconductivity of CHP-OA hydrogel and another CHP nanogel, CHP-A nanogel-crosslinked PEG gel (CHP-A nanogel-hydrogel), in the bone formation induced by BMP-2 and the peptide. A calvarial defect model was performed in 5-week-old male mice, and scaffolds were placed in the defect. In vivo µCT was performed every week. Radiological and histological analyses after 4 weeks of scaffold placement revealed that the calcified bone area and the bone formation activity at the defect site in the CHP-OA hydrogel were significantly lower than those in the CHP-A hydrogel when the scaffolds were impregnated with both BMP-2 and the RANKL-binding peptide. The amount of induced bone was similar in both CHP-A and CHP-OA hydrogels when impregnated with BMP-2 alone. In conclusion, CHP-A hydrogel could be an appropriate scaffold compared to the CHP-OA hydrogel when the local bone formation was induced by the combination of RANKL-binding peptide and BMP-2, but not by BMP-2 alone.

Amelogenin as a regenerative endodontic molecule for immature teeth with apical periodontitis. An experimental study.

Vitality of the dentin-pulp complex depends on cell activity and signalling processes. Amelogenin protein regulates cell signalling pathways during tooth development by activating the Wnt/ß-catenin intercellular signalling pathway. This study aimed to regenerate a vascularized pulp using recombinant amelogenin protein, in necrotic root canals by cell homing. Access opening was performed for a total of 120 root canals and were left open to become contaminated with oral microbes for 14 days then cleaned. Canals were divided into 2 groups; in the First group, the canals were filled with amelogenin, while in the 2nd group the canals were left empty. Samples were evaluated histologically and with immunodetection of Sox2, Oct4, Vascular endothelial growth factor (VEGF), Wnt1a, Wnt 3a, Wnt 10b, and Glial Fibrillary Acidic Proteins (GFAP). IC50 was used to determine the cytotoxicity of amelogenin. Regenerated dense cellular tissue was seen in the apical part of amelogenin-treated root canals, and regenerated delicate vascularized tissue was observed in the radicular and pulp chamber. Cells found in the regenerated soft tissue expressed Wnt family members that regulate stem cell pluripotency. Also, Sox2 and Oct4, Pluripotency markers, could be identified in the newly formed apical papilla and dental follicle. Furthermore, VEGF in the regenerated pulps indicated neovascularization. While the GFAP immune reactivity demonstrated that the neuro-sensory organ was being replicated in the regenerated dental pulps. Finally, IC50 test showed that recombinant amelogenin protein has a safe dose at high-level concentrations. Recombinant amelogenin protein induces pulp regeneration most likely from the Sox2 identified stem cells within the apical papilla and can enhance apex formation in non-vital immature teeth.

Perforated Hydrogels Consisting of Cholesterol-Bearing Pullulan (CHP) Nanogels: A Newly Designed Scaffold for Bone Regeneration Induced by RANKL-Binding Peptides and BMP-2.

The receptor activator of NF-κB ligand (RANKL)-binding peptide, OP3-4, is known to stimulate bone morphogenetic protein (BMP)-2-induced bone formation, but peptides tend to aggregate and lose their bioactivity. Cholesterol-bearing pullulan (CHP) nanogel scaffold has been shown to prevent aggregation of peptides and to allow their sustained release and activity; however, the appropriate design of CHP nanogels to conduct local bone formation needs to be developed. In the present study, we investigated the osteoconductive capacity of a newly synthesized CHP nanogel, CHPA using OP3-4 and BMP-2. We also clarified the difference between perforated and nonperforated CHPA impregnated with the two signaling molecules. Thirty-six, five-week-old male BALB/c mice were used for the calvarial defect model. The mice were euthanized at 6 weeks postoperatively. A higher cortical bone mineral content and bone formation rate were observed in the perforated scaffold in comparison to the nonperforated scaffold, especially in the OP3-4/BMP-2 combination group. The degradation rate of scaffold material in the perforated OP3-4/BMP-2 combination group was lower than that in the nonperforated group. These data suggest that perforated CHPA nanogel could lead to local bone formation induced by OP3-4 and BMP-2 and clarified the appropriate degradation rate for inducing local bone formation when CHPA nanogels are designed to be perforated.

Tetracycline, an Appropriate Reagent for Measuring Bone-Formation Activity in the Murine Model of the Streptococcus mutans-Induced Bone Loss.

Tetracycline is used as a fluorescent reagent to measure bone formation activity in bone histomorphometric analyses. However, there is a possibility to lead a different conclusion when it is used in a bacteria-infected murine model since the tetracycline is considered to work as an antibiotic reagent. There are non-antibiotic fluorescent reagents such as alizarin and calcein for measuring bone formation activity. The purpose of this study was to clarify whether tetracycline could be an appropriate reagent to measure bone formation activity in a murine bacterial model in the same way as a non-antibiotic fluorescent reagent. We used Streptococcus mutans (S. mutans), a normal inhabitant in the oral cavity and tetracycline-sensitive bacteria, for inducing the bacterial model. The murine bacterial model was generated by intravenously inoculating S. mutans to the tail vein, followed immediately by the injection of the first fluorescent reagent, and the second one was injected 2 days prior to euthanization. After one day of inoculation with S. mutans, the subcutaneously injected alizarin had a similar colony count derived from the liver and the bone marrow tissue compared to the phosphate buffered saline (PBS)-injected control group. On the other hand, subcutaneous injection of tetracycline led to a significantly lower colony count from the liver compared to alizarin- or calcein-injected group. However, on day seven, after S. mutans intravenous injections, bone mineral density of distal femurs was significantly reduced by the bacteria inoculation regardless of which fluorescent reagents were injected subcutaneously. Finally, S. mutans inoculation reduced bone-formation-activity indices in both the tetracycline-alizarin double-injected mice and the calcein-alizarin double-injected mice. These results suggested that a one-time injection of tetracycline did not affect bone formation indices in the S. mutans-induced bone loss model. Tetracycline could be used for measuring bone formation activity in the same way as non-antibiotic fluorescent reagent such as calcein and alizarin, even in a tetracycline-sensitive bacterium-infected model.

The Effects of Receptor Activator of NF-κB Ligand-Binding Peptides on Bone Resorption and Bone Formation.

Receptor activator of NF-κB ligand (RANKL)-binding peptides inhibit bone resorption and were recently shown to activate bone formation. The stimulatory mechanism underlying bone formation associated with these peptides was explained as RANKL-reverse signaling, wherein RANKL molecules on osteoblasts work as receptors to stimulate osteoblast differentiation. However, why RANKL-binding peptides stimulate osteoblast differentiation while osteoprotegerin (OPG), which is well known to bind to RANKL, cannot activate osteoblast differentiation has remained unclear. In this mini-review, we introduce three main issues: (1) The inhibitory effects of two RANKL-binding peptides (W9 and OP3-4) on bone resorption; (2) The stimulatory effects of the RANKL-binding peptides on osteoblast differentiation; and (3) The accumulation and membrane clustering of RANKL molecules at the cell surface of osteoblasts as a potential molecular switch stimulating osteoblast differentiation by RANKL-binding peptides.

Regeneration of Neural Networks in Immature Teeth with Non-Vital Pulp Following a Novel Regenerative Procedure.

BACKGROUND AND OBJECTIVES: Recombinant amelogenin protein (RAP) was reported to induce soft-tissue regeneration in canine infected endodontically treated permanent teeth with open apices. To characterize identities of the cells found in the RAP regenerated tissues compared to authentic pulp by identifying: 1) stem cells by their expression of Sox2; 2) nerve fibers by distribution of the axonal marker peripherin; 3) axons by their expression of calcitonin gene-related peptide (CGRP); 4) the presence of astrocytes expressing glial fibrillary acidic proteins (GFAP). METHODS: A total of 240 open-apex root canals in dogs were used. After establishment of oral contamination to the pulp, the canals were cleaned, irrigated, and 120 canals filled with RAP, and the other 120 with calcium hydroxide. RESULTS: After 1, 3, and 6 months, teeth were recovered for immune-detection of protein markers associated with native pulp tissues. Regenerated pulp and apical papilla of RAP group revealed an abundance of stem cells showing intense immunoreactivity to Sox2 antibody, immunoreactivity of peripherin mainly in the A-fibers of the odontoblast layer and immunoreactivity to CGRP fibers in the central pulp region indicative of C-fibres. GFAP immunoreactivity was observed near the odontoblastic, cell-rich regions and throughout the regenerated pulp. CONCLUSIONS: RAP induces pulp regeneration following regenerative endodontic procedures with cells identity by gene expression demonstrating a distribution pattern similar to the authentic pulp innervation. A- and C-fibers, as well as GFAP specific to astrocytic differentiation, are recognized. The origin of the regenerated neural networks may be derived from the Sox2 identified stem cells within the apical papilla.

The Effect of Using Bone Marrow Mesenchymal Stem Cells Versus Platelet Rich Plasma on the Healing of Induced Oral Ulcer in Albino Rats.

BACKGROUND AND OBJECTIVES: Oral ulceration is one of the most common debilitating condition that affects the oral cavity. In this study, the effect of locally injected platelet rich plasma (PRP) and bone marrow-derived mesenchymal stem cells (BMSCs) on the healing of oral ulcer was investigated. METHODS AND RESULTS: An ulcer was induced in buccal mucosa of rats by using 5mm biopsy punch followed by application of cotton swab soaked with formocresol for 60sec. The ulcer was left untreated in the control group, treated with intralesional injection of PRP, or isolated cultured BMSCs. Data were analyzed clinically, histologically and immunohistologically on day 3, 5, 7 and 10. BMSCs group showed smaller ulcer area throughout the whole experimental period than the other groups with complete resolution of the ulcer on day 10, unlike the control group. However, there was no significant difference with PRP, on day 5, 7 and 10, regarding clinical ulcer size. BMSCs group showed better histological results regarding the rate of epithelial cell migration, the number of inflammatory cells, thickness and organization of collagen fibres and the number of blood vessels, with complete re-epithelization on day 10. BMSCs group showed a greater number of anti-PCNA positive nuclei throughout the whole experimental period than the other groups except on day 5, PRP had higher mean numbers of anti-PCNA positive nuclei in both tissues. CONCLUSIONS: Both PRP and BMSCs accelerate wound healing and enhance the quality of the healing tissue with the latter being slightly more effective and faster.