Chitosan in association with osteogenic factors as a cell-homing platform for dentin regeneration: Analysis in a pulp-in-a-chip model.

OBJECTIVE: In this paper we propose the association of ß-glycerophosphate (ßGP) and calcium-hydroxide with chitosan (CH) to formulate a porous bioactive scaffold suitable as a cell-homing platform for dentin regeneration. METHODS: Calcium hydroxide and ßGP solutions were incorporated into chitosan to modulate scaffold architecture and composition by a phase separation technique. Architecture, chemical composition, and degradability were evaluated, and biological characterizations were performed by the seeding of dental pulp cells (DPCs) onto scaffolds, or by cultivating them in contact with leachable components (extracts), to determine cytocompatibility and odontoblastic differentiation. Cell-free scaffolds were then positioned in intimate contact with a 3D culture of DPCs in a pulp-in-a-chip platform under simulated pulp pressure. Cell mobilization and odontoblastic marker expression were evaluated. Deposition of mineralized matrix was assessed in direct contact with dentin, in the absence of osteogenic factors. RESULTS: Incorporation of calcium hydroxide and ßGP generated a stable porous chitosan scaffold containing Ca-P nanoglobule topography (CH-Ca-ßGP), which favored cell viability, alkaline phosphatase activity, and mineralized matrix deposition by cells seeded onto the scaffold structure and at a distance. The pulp-in-a-chip assay denoted its chemotactic and bioactive potential, since dentin sialoprotein-positive DPCs from 3D culture adhered to CH-Ca-ßGP more than to plain chitosan. The higher deposition of mineralized matrix onto the scaffold and surrounding dentin was also observed. SIGNIFICANCE: A CH-Ca-ßGP scaffold creates a microenvironment capable of mobilizing DPC migration toward its structure, harnessing the odontogenic potential and culminating in the expression of a highly mineralizing phenotype, key factors for a cell-homing strategy.

Chitosan-Calcium-Simvastatin Scaffold as an Inductive Cell-Free Platform.

The development of biomaterials based on the combination of biopolymers with bioactive compounds to develop delivery systems capable of modulating dentin regeneration mediated by resident cells is the goal of current biology-based strategies for regenerative dentistry. In this article, the bioactive potential of a simvastatin (SV)-releasing chitosan-calcium-hydroxide (CH-Ca) scaffold was assessed. After the incorporation of SV into CH-Ca, characterization of the scaffold was performed. Dental pulp cells (DPCs) were seeded onto scaffolds for the assessment of cytocompatibility, and odontoblastic differentiation was evaluated in a microenvironment surrounded by dentin. Thereafter, the cell-free scaffold was adapted to dentin discs positioned in artificial pulp chambers in direct contact with a 3-dimensional (3D) culture of DPCs, and the system was sealed to simulate internal pressure at 20 cm/H2O. In vivo experiments with cell-free scaffolds were performed in rats' calvaria defects. Fourier-transform infrared spectroscopy spectra proved incorporation of Ca and SV into the scaffold structure. Ca and SV were released upon immersion in a neutral environment. Viable DPCs were able to spread and proliferate on the scaffold over 14 d. Odontoblastic differentiation occurred in the DPC/scaffold constructs in contact with dentin, in which SV supplementation promoted odontoblastic marker overexpression and enhanced mineralized matrix deposition. The chemoattractant potential of the CH-Ca scaffold was improved by SV, with numerous viable and dentin sialoprotein-positive cells from the 3D culture being observed on its surface. Cells at 3D culture featured increased gene expression of odontoblastic markers in contact with the SV-enriched CH-Ca scaffold. CH-Ca-SV led to intense mineralization in vivo, presenting mineralization foci inside its structure. In conclusion, the CH-Ca-SV scaffold induces differentiation of DPCs into a highly mineralizing phenotype in the presence of dentin, creating a microenvironment capable of attracting pulp cells to its surface and inducing the overexpression of odontoblastic markers in a cell-homing strategy.