Formulation and in vitro and in vivo evaluation of a new osteoprotegerin-chitosan gel for bone tissue regeneration

J Biomed Mater Res A. 2017 Feb;105(2):398-407. doi: 10.1002/jbm.a.35919. Epub 2016 Oct 18.


The osteoprotegerin (OPG) system plays a critical role in bone remodelling by regulating osteoclast formation and activity. The study aimed to determine the physicochemical properties and biocompatibility of a newly formulated OPG-chitosan gel. The OPG-chitosan gel was formulated using human OPG protein and water-soluble chitosan. The physicochemical properties were determined using Fourier transform infrared (FTIR) spectroscopy, thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). Gel morphology was determined using scanning electron microscopy (SEM) and then it was subjected to a protein release assay and biodegradability test. An in vitro cytotoxicity test on normal human periodontal ligament (NHPL) fibroblasts and normal human (NH) osteoblasts was carried out using the AlamarBlue assay. In vivo evaluation in a rabbit model involved creating critical-sized defects in calvarial bone, filling with the OPG-chitosan gel and sacrificing at 12 weeks. In vitro results demonstrated that the 25 kDa OPG-chitosan gel had the highest rate of protein release and achieved 90% degradation in 28 days. At 12 weeks, the defects filled with 25 kDa OPG-chitosan gel showed significant (p < 0.05) new bone formation and the highest expression of osteocalcin and osteopontin compared to controls. Thus, the 25 kDa OPG-chitosan gel could be a promising new biomaterial for tissue engineering. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 398-407, 2017.

Keywords: chitosan; normal human osteoblast cells; normal human periodontal ligament cells; osteoprotegerin; rabbit.

Publication types

  • Evaluation Study

MeSH terms

  • Bone Regeneration*
  • Cell Line
  • Chitosan* / chemistry
  • Chitosan* / pharmacology
  • Clergy
  • Female
  • Fibroblasts / metabolism
  • Fibroblasts / pathology
  • Humans
  • Hydrogels / chemistry
  • Hydrogels / pharmacology
  • Osteoblasts / metabolism*
  • Osteoblasts / pathology
  • Osteoprotegerin* / chemistry
  • Osteoprotegerin* / pharmacology
  • Periodontal Ligament / metabolism
  • Periodontal Ligament / pathology
  • Skull* / injuries
  • Skull* / metabolism
  • Skull* / pathology


  • Hydrogels
  • Osteoprotegerin
  • TNFRSF11B protein, human
  • Chitosan