Uncovering mechanisms of complex tissue assembly using dental organoids

The tooth is a complex organ comprised of molecularly and structurally diverse soft and mineralized tissues. Challenges in regenerating the tooth root lie in simultaneously rebuilding these unique tissues in a spatially- organized manner. Furthermore, sensory innervation and proper attachment to surrounding healthy alveolar bone is critical for regenerated dental tissue function. Carefully orchestrated spatial and temporal cues regulate tissue patterning of the tooth during development and ensure that the tooth and its supporting tissues are integrated into the jaw. Understanding these processes during repair will advance the field of regenerative dentistry. Using scaffold-free tissue engineering approaches, we have generated tooth root organoids using dental pulp stem/progenitor cells (DPSCs) and periodontal ligament (PDL) stem/progenitor cells (PDLSCs). These organoids comprise a striated structure of mineralized and unmineralized tissues, with a central pulp- like tissue enclosed within a mineralized dentino/cementogenic tissue surrounded by a soft periodontal ligament (PDL)-like tissue. Building this type of complex construct containing multiple, anatomically organized tissues with hugely varying structural, biochemical, and mechanical properties is a significant challenge in field of tissue engineering. Here, we propose utilizing tooth root organoids as novel 3D experimental models to define stages of DPSC and PDLSC fate that are critical for reparative tissue assembly and integration. In Aim 1, first, a time course evaluation will be performed to histologically map stages of cell fate and the progression of tissue assembly in tooth root organoids. Then, single cell transcriptomic analysis will be conducted to track the trajectories of cell differentiation and comprehensively define the cell populations comprising the organoids. The goal of Aim 2 is to uncover mechanisms regulating innervation of dental organoids. During natural tooth development, innervation is a tightly regulated spatiotemporal process. Here, we will investigate if dental stem/progenitor cells recapitulate these developmental phenomena while assembling dental organoids. In Aim 3, the ability of tooth root organoids to form physiological attachments to bone will be evaluated. Proper attachment of repaired or regenerated dental tissues to the alveolar bone remains a major clinical challenge. Tooth root organoids will be orthotopically implanted into healed tooth extraction sites in rats. Histological and microcomputed tomography analyses will be used to determine if the tooth root organoids are able to maintain their patterning and form Sharpey’s fibers to attach to the bone. In addition, the ability of these engineered dental tissues to become vascular and innervated in vivo will be assessed. The completion of this proposal will result in new knowledge on mechanisms governing tissue patterning in engineered tissues. This study will also provide important information on factors driving functional integration of regenerated tissue with the body. The outcomes of this work can be translated into developing new effective dental therapies and applied to regenerating organs throughout the body.