Introduction: Keratin is the major structural fibrous protein providing outer covering such as hair, wool, feathers, nails, hooves and horns of mammals, reptiles and birds[1]. Wool consists of 95% keratin proteins (KPs). The potential of these KPs biomaterials and their effect on soft and hard tissue regeneration have also been demonstrated[2]-[5]. Several in vivo and clinical studies have shown their biocompatibility, non-toxicity, and tissue healing/repairing performances. But only a few reports are available on usage of keratin in dental applications[6]. We are currently studying KPs for dental applications, where KPs were reconstituted into gels, and their effects on growth and differentiation behaviour of odontoblasts-like cells. Furthermore, these gels were implanted in tooth pulp to evaluate the pulp-dentine regeneration, with the aim of translating these findings for clinical endodontic applications.
Materials and Methods: Clean New Zealand sheep wool was solubilized by a mixture of 7M urea, SDS (Sodium dodecyl sulphate) and β-mercaptoethanol, filtered and purified (dialysis using a 20kDa membrane), followed by freeze-drying to obtain KPs powder. KPs powder (10, 15 and 20% (w/v)) was reconstituted in deionized water and 3% glycerol to create a homogenous gels. Visco-elastic properties (elastic modulus (G') and loss modulus (G")) of gels were assessed using a rheometer. The 20% w/v KP gel was selected, gamma sterilised (25 kGy) and exposed to odontoblasts-like cells for evaluating cytocompatibility by live-dead assay. Gel was implanted into rat upper-molar teeth (n=7) following a partial pulpotomy to investigate the pulpal (repair/regenerative) response. Control pulps (n=7) were treated with hard setting Ca(OH)2. Rats were sacrificed (28 d), en bloc resected maxillae were processed for histological and immunohistochemically staining of dentine matrix protein (DMP-1), and the pulp-dentine regeneration response was evaluated in a quantitative manner.
Results and Discussion: Visco-elastic behaviour of gel showed a gradual increase in G' with increasing KPs concentration with the maximum recorded for 20%. This is due to the presence of α-keratins with higher disulphide bonds (cross-linked), yielding dimensionally stable gel at room temperature. In addition, 20% KPs gel also demonstrated best injectability performance through a 21 gauge needle and cytocompatibility with no significant difference compared to control collagen hydrogels. The in vivo pulpal implantation of 20% gel showed that the inflammatory response was similar to that of control Ca(OH)2. Furthermore, the implantation of KPs resulted in mineralized dentine-like deposits, positive for DMP-1 expression, which suggest reparative response of radicular pulp.
Conclusion: Reconstituted 20%KPs gel showed dimensionally stable with effective injectability and cytocompatibility performance from the study. The in vivo pilot study clearly exhibited a dentin-like deposits in the radicular pulp-tissue with no adverse reactions, which suggest KPs could be a potential biomaterial source for pulp-tissue engineering (regeneration) in clinical applications.
All animal procedures were done after prior approval from University of Otago Animal Ethics committee (AEC - 90/13).This research was supported by New Zealand Dental Association Research Fund and University of Otago postgraduate scholarhsip fund; This research was supported by New Zealand Dental Association Research Fund and the University of Otago Postgraduate Research Fund.
References:
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