Aim: To fabricate a keratin hydrogel, characterize its functionality as a biomaterial and investigate the effects of keratin on growth and differentiation of odontoblast‐like cells.

Methodology: Keratins were extracted from sheep wool using a well‐established technique. The extracted proteins were purified by dialysis, quantified by gel electrophoresis, mass spectrometry, amino acid analysis and inductively coupled mass spectrometry. The microstructure of the fabricated keratin hydrogels was studied by scanning electron microscopy, flow characteristics by rheometer, hydrolytic stability and cytocompatibility by Live/Dead® cell assay. Furthermore, the influence of keratin on odontoblast‐like cells (MDPC‐23) was assessed to confirm their bioactivity at different dilutions. Cell proliferation was studied using alamarBlue® assay and differentiation by alkaline phosphatase enzyme activity, alizarin red staining and calcium quantification, reverse transcription polymerase chain reaction (rt‐PCR) and immunocytochemical staining for dentine matrix protein‐ 1 (DMP‐1) expression. anova with Tukey's tests was performed for statistical comparison.

Results: The characterized hydrogel was injectable with a highly porous architecture that underwent slow degradation, and its cytocompatibility was statistically equivalent to collagen hydrogel (P > 0.05). Cell proliferation and differentiation were enhanced at the optimal keratin concentration of 0.1 mg mL−1. At this concentration, the influence of keratin on cell differentiation was demonstrated by marked elevation in alkaline phosphatase activity (P < 0.05), calcium deposition (P < 0.01), gene expression (P < 0.01) and positive immunostaining for DMP‐1.

Conclusion: The presence of keratin enhanced odontoblast cell behaviour. Keratin hydrogels may be a potential scaffold for pulp–dentine regen‐eration.