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PeerJ. 2016 Aug 23;4:e2229. doi: 10.7717/peerj.2229. eCollection 2016.

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

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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.
Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia.



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.


Bone; Chitosan; Normal human osteoblast; Normal human periodontal ligament fibroblast; Osteoproteogerin

Conflict of interest statement

The authors declare there are no competing interests.

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