A novel keratin-chitosan-tricalcium phosphate biocomposite as a potential scaffold for regenerative endodontics : an in vitro study

Abstract

The present study is the ‘first look’ at a novel biocomposite which may be used as a 3D implantable intracanal scaffold for regenerating dental pulp and periapical tissues. The main ingredient of the biocomposite was low molecular weight keratin protein extracted from sheep wool. Merino sheep wool offers a grand storehouse of keratin proteins which can extracted by many methods. This study used a novel chemical-free method using high temperature and pressure. The extracted proteins were of low molecular weight (3.5-15 kDa) and was water soluble. These proteins were used for the fabrication of the biocomposite along with other ingredients namely chitosan, tricalcium phosphate,barium sulphate and glycerol. This is, perhaps the first study that has explored the use of low molecular weight keratin in biomedical applications. Other constituents of the biocomposite were selected in order to provide specific properties to the composite. Keratin-chitosan formed a mechanically stable homogenous matrix. Chitosan also imparted an antimicrobial potential to the scaffold. Tricalcium phosphate acted as the filler and also a supplier of calcium ions. Barium sulphate provided radiopacity to the scaffold while glycerol was the plasticizer. The scaffold demonstrated many key characteristics relevant to tissue regeneration applications such as adequate porosity and degradation, as well as to endodontic applications such as moderate swelling and radiopacity.

Assessment of cytocompatibility yielded promising results. The scaffold promoted proliferation of MDPC 23 (odontoblast like cells) and OCCM 30 cells (cementoblast like cells). The cells were able to grow and achieve functional differentiation when cultured after exposure to scaffold extracts as evidenced by ALP assay which detected elevated ALP levels in culture. AR-S staining detected calcium deposits which further confirmed cell differentiation. Furthermore, immunocytochemical analysis revealed expression of DSPP by MDPC 23 cells which was indicative of odontogenic differentiation. These cell reactions demonstrated the regenerative potential of the biocomposite scaffold. The population density of viable stem cells and their successful differentiation is an absolute prerequisite for successful regenerative pulp therapy, so is the effective disinfection of the root canal system. The antimicrobial potential of the scaffold was tested against S.mutans which was a representative organism for primary infection and E faecalis which represented secondary or re-infection of the root canal system. The scaffold was able to successfully inhibit growth of both the species. This potent antibacterial action of the scaffold eliminated the need for using highly potent antibiotics and other antimicrobials detrimental to host cell survival during endodontic regenerative procedures.