Development and in vivo response of hydroxyapatite/whitlockite from chicken bones as bone substitute using a chitosan membrane for guided bone regeneration

Biomaterials that meet the requirements to stimulate bone tissue formation play a vital role in orthopedics and dentistry. In this work, chitosan and a biphasic, non-cytotoxic material hydroxyapatite/whitlockite were obtained from natural sources, which are available as organic waste. The osteogenic activity was assessed using a rabbit model animal with a chitosan barrier membrane in combination with a bone-filling graft substitute composed of hydroxyapatite/whitlockite. FT-IR results showed the typical absorption bands of the chitosan and hydroxyapatite. Moreover, the X-ray diffraction pattern revealed a typical hexagonal phase of hydroxyapatite and rhombohedral structures related to whitlockite. Masson's trichrome stain showed an early formation of extracellular matrix mineralized, in accord with the surface morphology of a cortical mature bond observed by Scanning Electron Microscopy. The immunocytochemistry results showed a significant increase of positive immunoreactive cells to osteonectin in the treated defects in comparison with the control defects 6 and 8 weeks postoperatively. Overall, the results confirm that the use of this low-cost and versatile biomaterial as a barrier membrane and a bone substitute graft are useful for bone tissue engineering.