Characterization of nano-hydroxyapatite incorporated carboxymethyl chitosan composite on human dental pulp stem cells.

AIM: To compare the odontogenic differentiation potential of a composite scaffold (CSHA) comprising of nano-hydroxyapatite (nHAp) and carboxymethyl chitosan (CMC) with Biodentine on human dental pulp stem cells (hDPSCs). METHODOLOGY: A CSHA scaffold was prepared through an ultrasonication route by adding nHAp and CMC (1:5 w/w) in water medium followed by freeze-drying. Physicochemical characterization was achieved using scanning electron microscopy with energy-dispersive X-ray spectroscopy, X-ray diffraction and Fourier transform infrared spectroscopy. In-vitro bioactivity and pH assessments were done by soaking in simulated body fluid (SBF) for 28 days. The angiogenic and odontogenic differentiation abilities were assessed by expression of vascular endothelial growth factor (VEGF) and Dentine sialophosphoprotein (DSPP) markers on cultured hDPSCs by flow cytometry and RT-qPCR at 7, 14 and 21 days. Cell viability/proliferation and biomineralization abilities of CSHA were compared with Biodentine by MTT assay, alkaline phosphatase (ALP) activity, Alizarin Red Staining (ARS) and osteopontin (OPN) expression on hDPSCs following 7 and 14 days. Data were statistically analysed with Kruskal Wallis and Friedman tests as well as one way anova followed by appropriate post hoc tests (p < .05). RESULTS: Characterization experiments revealed a porous microstructure of CSHA with pore diameter ranging between 60 and 200 µm and 1.67 Ca/P molar ratio along with the characteristic functional groups of both HAp and CMC. CSHA displayed bioactivity in SBF by forming apatite-like crystals and maintained a consistent pH value of 7.70 during 28 days' in vitro studies. CSHA significantly upregulated VEGF and DSPP levels on hDPSCs on day 21 compared with day 7 (p < .05). Further, CSHA supported cell viability/proliferation over 14 days like Biodentine with no statistical differences (p > .05). However, CSHA exhibited increased ALP and ARS activity with an intense OPN staining compared with Biodentine after 14 days (p < .05). CONCLUSION: The results highlighted the odontogenic differentiation and biomineralization abilities of CSHA on hDPSCs with significant VEGF and DSPP gene upregulations. Further, CSHA exhibited enhanced mineralization activity than Biodentine, as evidenced by increased ALP, ARS and OPN activity on day 14. The nHAp-CMC scaffold has the potential to act as an effective pulp capping agent; however, this needs to be further validated through in-vivo animal studies.

Eggshell derived nano-hydroxyapatite incorporated carboxymethyl chitosan scaffold for dentine regeneration: A laboratory investigation.

AIM: To assess odontogenic differentiation abilities of porous biomineralizable composite scaffolds comprising eggshell derived nano-hydroxyapatite (HAnp) and carboxymethyl chitosan (CMC) on cultured human dental pulp stem cells (hDPSCs). METHODOLOGY: Nano-hydroxyapatite was derived from eggshells using a simple combustion method and CMC was prepared from chitosan through a chemical route. Several compositions of HAnp-CMC (0:5, 5:0, 1:5, 2:5, 3:5, 4:5 and 1:1 w/w%) scaffolds were prepared by magnetic stirring and freeze-drying methods. HAnp-CMC scaffolds were characterized using high-resolution scanning electron microscopy combined with energy-dispersive X-ray spectroscopy, Fourier transform infrared spectroscopy and X-ray diffraction methods. In vitro bioactivity was determined following the interaction in simulated body fluid for 21 days. The optimized composite was then loaded onto hDPSCs to assess cell viability/proliferation, dentine sialophosphoprotein (DSPP) and vascular endothelial growth factor (VEGF) expressions using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, real-time quantitative polymerase chain reaction and flow cytometry methods, respectively, following 7, 14 and 21 days. For intergroup and intragroup comparisons, Kruskal-Wallis and Friedman tests were employed, respectively, followed by appropriate post hoc test (Dunn). Significant levels were set at *p < .05 and *p < .01. RESULTS: Synthesized hydroxyapatite (HAp) comprised crystals ranging from 20 to 50 nm (HAnp) with spherulite morphology and calcium/phosphorus (Ca/P) molar ratio of 1.67. The ultrastructure of all the scaffolds revealed a highly interconnected porous microstructure, whilst the chemical characterization displayed specific functional groups of both HAnp and CMC. In vitro bioactivity assessment confirmed the biomineralization potential of all scaffolds with an apatite-like crystal formation on the surface. The 1:5 HAnp-CMC revealed a favourable pore size (60-180 µm) that was suitable for cell seeding and was chosen for further experiments. Cell viability/proliferation rates of hDPSCs loaded 1:5 HAnp-CMC at 21st day was significantly greater than that at 7th day (p < .05). The mean relative quantification of DSPP expression by the scaffold was significantly higher (p < .05) on day 21 (3.16) than on day 7 (1.67). Mean fluorescence intensity of the VEGF expression at day 21 (32.5) was also significantly higher (p < .01) than at day 7 (12.54). CONCLUSION: hDPSCs on 1:5 HAnp-CMC scaffolds displayed increased cell viability/proliferation and enhanced DSPP as well as VEGF expressions. The 1:5 HAnp-CMC composite has the potential to serve as a promising scaffold for dentine regeneration.