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Microtissue engineering root canal dentin with biopolymeric nanoparticles has the potential to improve mechanical properties of iatrogenically compromised root dentin. This study aims to characterize the surface mechanical property, bulk biomechanical response, and fatigue resistance of microtissue-engineered root dentin using photodynamically (photodynamic-activated [PDA]) cross-linked chitosan nanoparticles (CSnps).Experiments were conducted in 3 parts: part 1, root canal dentin sections were subjected to nanoindentations before/after treatment with CSnps and chemically (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide [EDC] cross-linked CSnps) and photodynamically cross-linked CSnps to determine the properties of treated surfaces (n = 84 points/group); part 2, root canal dentin specimens treated with PDA cross-linked CSnps were subjected to strain analysis using customized moiré interferometry (n = 5/group); and part 3, root canal dentin specimens treated with EDC cross-linked CSnps, PDA cross-linked CSnps, and instrumented controls were tested using an accelerated fatigue loading protocol to evaluate the sustained loads and cycles at failure (n = 15/group). Data were analyzed using the paired sample t test, trend analysis, and Kaplan-Meier with log-rank tests at a significance of .05 in each experiment.Root dentin microtissue engineered with PDA cross-linked CSnps showed a 16.8% increase in elastic modulus and a conspicuous decrease in strain distribution in cervical root dentin (P < .01). There was a significant reduction in the tensile strain formed at the apical region of the instrumented root dentin after treatment (P < .05). Survival analysis showed a statistically significant difference (P < .05) among evaluated conditions in fatigue resistance (ie, PDA cross-linked CSnps > EDC cross-linked CSnps > control).This study highlighted the potential of root canal dentin microtissue engineering with PDA cross-linked CSnps to diminish radicular strain distribution and improve resistance to fatigue loads in endodontically treated teeth.