Characterizing Mineralization Potential of Nano-hydroxyapatite-Carboxymethyl-Chitosan Scaffold for Dentin-Pulp Regeneration

Objectives: Minimal-Invasive-Dentistry has brought about paradigm shift in caries treatment, which includes remineralization of incipient enamel lesions to preservation and regeneration of affected dentin in dentinal lesions. Our objective was to develop and characterize a nano-hydroxyapatite(nHAp) incorporated carboxymethy-chitosan (CMC) scaffold (CSHA) to enhance cell proliferation and mineralization potential.
Methods: Experiments were conducted in two stages. Experiment-1: CMC solution was prepared by reacting monochloro-acetic acid with chitosan(carboxymethylation). This was added to nHAp to prepare hydrogel that was freeze-dried to obtain CSHA scaffold. CSHA was characterized using SEM-EDX, XRD and FTIR. Experiment-2: hDPSCs were isolated and cultured from extracted premolars. CSHA, Biodentine and osteogenic medium (control), previously sterilized were placed in 96 well-plates, over which hDPSCs were directly seeded to assess for cell viability (MTT assay), mineralization potential using alkaline phosphatase (ALP) expression (colorimetric assay), deposition of calcific nodules (alizarin-red-S staining-ARS) and detection of biomarker osteopontin (OPN) (indirect immunocytochemistry) after 7 and14 days interactions.
Statistical analysis using non-parametric Kruskal Wallis and Freidman test(MTT assay) and parametric one-way ANOVA and Independent-T test(ALP,ARS,cell count) with significance level at p<0.05.
Results: SEM-EDX revealed highly interconnected porous(60-200μ) scaffold with uniformly seeded nHAp crystals, and Ca/P molar-ratio of 1.6 suggesting HAp presence. XRD also revealed peaks characteristic of both HAp and CMC. CSHA and Biodentine showed similar cytotoxicity. There was significant cellular proliferation (cell count) with CSHA as compared to Biodentine. Mineralization potential determined using ALP activity was significantly higher with CSHA, with corresponding higher degrees of calcific nodule formation at 7 and14 days interval. CSHA exhibited intense, wider OPN staining
Conclusions: CSHA showed better cellular proliferation, ALP and OPN expressions and higher calcific nodule formation thus promising potential for application in dentine-pulp regeneration.