Local application of osteoprotegerin-chitosan gel in critical-sized defects in a rabbit model

Background Osteoprotegerin (OPG) is used for the systemic treatment of bone diseases, although it has many side effects. The aim of this study was to investigate a newly formulated OPG-chitosan gel for local application to repair bone defects. Recent studies have reported that immunodetection of osteopontin (OPN) and osteocalcin (OC) can be used to characterise osteogenesis and new bone formation. Methods The osteogenic potential of the OPG-chitosan gel was evaluated in rabbits. Critical-sized defects were created in the calvarial bone, which were either left unfilled (control; group I), or filled with chitosan gel (group II) or OPG-chitosan gel (group III), with rabbits sacrificed at 6 and 12 weeks. Bone samples from the surgical area were decalcified and treated with routine histological and immunohistochemical protocols using OC, OPN, and cathepsin K (osteoclast marker) antibodies. The toxicity of the OPG-chitosan gel was evaluated by biochemical assays (liver and kidney function tests). Results The mean bone growth in defects filled with the OPG-chitosan gel was significantly higher than those filled with the chitosan gel or the unfilled group (p < 0.05). At 6 and 12 weeks, the highest levels of OC and OPN markers were found in the OPG-chitosan gel group, followed by the chitosan gel group. The number of osteoclasts in the OPG-chitosan gel group was lower than the other groups. The results of the liver and kidney functional tests indicated no signs of harmful systemic effects of treatment. In conclusion, the OPG-chitosan gel has many characteristics that make it suitable for bone repair and regeneration, highlighting its potential benefits for tissue engineering applications

Local application of osteoprotegerin-chitosan gel in critical-sized defects in a rabbit model

Background Osteoprotegerin (OPG) is used for the systemic treatment of bone diseases, although it has many side effects. The aim of this study was to investigate a newly formulated OPG-chitosan gel for local application to repair bone defects. Recent studies have reported that immunodetection of osteopontin (OPN) and osteocalcin (OC) can be used to characterise osteogenesis and new bone formation. Methods The osteogenic potential of the OPG-chitosan gel was evaluated in rabbits. Critical-sized defects were created in the calvarial bone, which were either left unfilled (control; group I), or filled with chitosan gel (group II) or OPG-chitosan gel (group III), with rabbits sacrificed at 6 and 12 weeks. Bone samples from the surgical area were decalcified and treated with routine histological and immunohistochemical protocols using OC, OPN, and cathepsin K (osteoclast marker) antibodies. The toxicity of the OPG-chitosan gel was evaluated by biochemical assays (liver and kidney function tests). Results The mean bone growth in defects filled with the OPG-chitosan gel was significantly higher than those filled with the chitosan gel or the unfilled group (p < 0.05). At 6 and 12 weeks, the highest levels of OC and OPN markers were found in the OPG-chitosan gel group, followed by the chitosan gel group. The number of osteoclasts in the OPG-chitosan gel group was lower than the other groups. The results of the liver and kidney functional tests indicated no signs of harmful systemic effects of treatment. In conclusion, the OPG-chitosan gel has many characteristics that make it suitable for bone repair and regeneration, highlighting its potential benefits for tissue engineering applications

In vitro evaluation of osteoprotegerin in chitosan for potential bone defect applications

Background The receptor activator of nuclear factor kappa-B (RANK)/RANK ligand/osteoprotegerin (OPG) system plays a critical role in bone remodelling by regulating osteoclast formation and activity. OPG has been used systemically in the treatment of bone diseases. In searching for more effective and safer treatment for bone diseases, we investigated newly formulated OPG-chitosan complexes, which is prepared as a local application for its osteogenic potential to remediate bone defects. Methods We examined high, medium and low molecular weights of chitosan combined with OPG. The cytotoxicity of OPG in chitosan and its proliferation in vitro was evaluated using normal, human periodontal ligament (NHPL) fibroblasts in 2D and 3D cell culture. The cytotoxicity of these combinations was compared by measuring cell survival with a tetrazolium salt reduction (MTT) assay and AlamarBlue assay. The cellular morphological changes were observed under an inverted microscope. A propidium iodide and acridine orange double-staining assay was used to evaluate the morphology and quantify the viable and nonviable cells. The expression level of osteopontin and osteocalcin protein in treated normal human osteoblast cells was evaluated by using Western blot. Results The results demonstrated that OPG in combination with chitosan was non-toxic, and OPG combined with low molecular weight chitosan has the most significant effect on NHPL fibroblasts and stimulates proliferation of cells over the period of treatment

Sexually dimorphic effects of prenatal alcohol exposure on the murine skeleton

BackgroundPrenatal alcohol exposure (PAE) can result in lifelong disabilities known as foetal alcohol spectrum disorder (FASD) and is associated with childhood growth deficiencies and increased bone fracture risk. However, the effects of PAE on the adult skeleton remain unclear and any potential sexual dimorphism is undetermined. Therefore, we utilised a murine model to examine sex differences with PAE on in vitro bone formation, and in the juvenile and adult skeleton.MethodsPregnant C57BL/6J female mice received 5% ethanol in their drinking water during gestation. Primary calvarial osteoblasts were isolated from neonatal offspring and mineralised bone nodule formation and gene expression assessed. Skeletal phenotyping of 4- and 12-week-old male and female offspring was conducted by micro-computed tomography (µCT), 3-point bending, growth plate analyses, and histology.ResultsOsteoblasts from male and female PAE mice displayed reduced bone formation, compared to control (≤30%). Bglap and Ahsg were upregulated with PAE in both sexes compared to control, whereas Vegfa, Bmp6, Tgfbr1 and Flt1 were downregulated in PAE male osteoblasts only. In 12-week-old mice, µCT analysis revealed a sex and exposure interaction across several trabecular bone parameters. PAE was detrimental to the trabecular compartment in male mice compared to control, yet PAE females were unaffected. Both male and female mice had significant reductions in cortical parameters with PAE. Whilst male mice were negatively affected along the tibia length, females were only distally affected. Posterior cortical porosity was increased in PAE females only. Mechanical testing revealed PAE males had significantly reduced bone stiffness compared to controls; maximum load and yield was reduced in both sexes. PAE had no effect on total body weight or tibial bone length in either sex. However, total growth plate width in male PAE mice compared to control was reduced, whilst female PAE mice were unaffected. 4-week-old mice did not display the altered skeletal phenotype with PAE observed in 12-week-old animals.ConclusionsEvidence herein suggests for the first time that PAE exerts divergent sex effects on the skeleton, possibly influenced by underlying sex specific transcriptional mechanisms of osteoblasts. Establishing these sex differences will support future policies and clinical management of FASD