Jayash Soher Nagi, Hashim Najihah M, Misran Misni, Baharuddin N A
Department of Restorative Dentistry, Faculty of Dentistry, University of Malaya, Kuala Lumpur, Malaysia.
Department of Pharmacy, Faculty of Medicine, University of Malaya,Kuala Lumpur,Malaysia; Centre For Natural Products And Drug Discovery (CENAR), Department of Chemistry, Faculty of Science, University of Malaya,Kuala Lumpur,Malaysia.
PeerJ. 2016 Aug 23;4:e2229. doi: 10.7717/peerj.2229. eCollection 2016.
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.
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.
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.
核因子κB受体激活剂（RANK）/RANK配体/骨保护素（OPG）系统通过调节破骨细胞的形成和活性在骨重塑中起关键作用。OPG已被全身用于治疗骨疾病。为了寻找更有效、更安全的骨疾病治疗方法，我们研究了新配制的OPG-壳聚糖复合物，其作为局部应用制剂，具有修复骨缺损的成骨潜力。
我们检测了与OPG结合的高、中、低分子量壳聚糖。使用正常人牙周膜（NHPL）成纤维细胞在二维和三维细胞培养中评估OPG在壳聚糖中的细胞毒性及其体外增殖情况。通过四唑盐还原（MTT）法和AlamarBlue法测量细胞存活率来比较这些组合的细胞毒性。在倒置显微镜下观察细胞形态变化。使用碘化丙啶和吖啶橙双重染色法评估细胞形态并量化活细胞和死细胞。通过蛋白质印迹法评估处理后的正常人成骨细胞中骨桥蛋白和骨钙素蛋白的表达水平。
结果表明，OPG与壳聚糖联合使用无毒，且OPG与低分子量壳聚糖联合使用对NHPL成纤维细胞的影响最为显著，并在治疗期间刺激细胞增殖。
