The Molecular Mechanism and Targeted Reconstruction of Peritubular Dentin Mineralization - PROJECT SUMMARY/ABSTRACT Dentin contains dentinal tubules that house odontoblastic processes, sensory nerve endings, and dentinal fluids. These components are essential for the proper mineralization of dentin and for dental sensation of and response to external stimuli. Homeostasis within dentinal tubules is protected by a layer of hypermineralized peritubular dentin. Notably, in common dentin defects like deep caries into dentin, peritubular dentin is the frontline mineralized tissues to be impacted. To restore defective dentin, bioengineered approaches are being actively developed. A consensus of these approaches is to restore the physiological dentin with a tubular system; thus, peritubular dentin is an essential target for dentin regeneration. Currently, a strategy for this is lacking. Therefore, it is vital to reveal the molecular mechanism of peritubular dentin mineralization to develop strategies to physiologically reconstruct defective dentin with bioengineering dentin containing dentinal tubules capable of hypermineralization. Our preliminary data indicate that peritubular dentin mineralization is primarily regulated by chondroitin sulfate (CS), dentin sialoprotein (DSP), and dentin phosphoprotein (DPP), each with distinct roles. We hypothesize that negatively charged CS chains extending from DSP form an organic scaffold with the acidic DPP to promote peritubular dentin mineralization. We propose to use 6 mouse models with varied amounts of DSP, DPP, and CS in peritubular dentin to study the molecular mechanism of peritubular dentin mineralization. We further propose to reconstruct peritubular dentin with a targeted delivery strategy. In aim 1, we will reveal the dependent relationships of DSP, DPP, and CS in the organic molecular architecture of peritubular dentin matrix. We will first generate a mouse model, in which the CS attachment sites on DSP are ablated. Then, we will reveal CS species, dentin matrix proteins, and their molecular architectures in the peritubular dentin matrix using the 6 mouse models. In aim 2, we will determine the contributions of DSP, DPP, and CS to the mineralized ultrastructure and mechanical properties of peritubular dentin. Using the 6 modified mouse strains, we will define the contributions of DSP, DPP, and CS to overall dentin mineralization, peritubular dentin morphology, crystal structure, size, and orientation, and mechanical properties of peritubular dentin. In aim 3, we will reconstruct defective peritubular dentin with targeted delivery of DSP and DPP. We will use a mineral-targeting peptide and an inducible AAV receptor overexpression mouse model for the targeted delivery. We will generate rAAV8 constructs for DSP and DPP expression, deliver them to mice lacking DSP and/or DPP, and evaluate their peritubular dentin quality. Results from this project will resolve the mechanism of peritubular dentin mineralization (a longstanding mystery in the field of biomineralization) and improve methods for bioengineering dentin regeneration. Our investigations using the rAAV8 system will be a starting point for the development of targeted delivery strategies to odontoblasts to treat common dentin disorders like deep caries into dentin and dentinogenesis imperfecta.
