Effects of size scaling on cellular dynamics and tissue patterning in tooth root organoids

Organoids offer a novel platform to study developmental and reparative processes and hold promise as model systems for drug discovery and personalized medicine. To maximize their utility, we investigated the capacity of organoids, specifically tooth root organoids, to be scaled down in size. We previously established that dental pulp stem/progenitor cells (DPSCs) and periodontal ligament (PDL) stem/progenitor cells (PDLSCs) co-cultured using scaffold-free tissue engineering approaches, self-assemble into tooth root organoids with anatomically organized layers of pulp-, dentin-, cementum-, and PDL-like tissues. These organoids were originally formed in 6 well plates. To test the effects of scaling down construct size, DPSC-PDLSC constructs were now generated with fixed initial cell density in 6-, 12-, and 24-well plates. While construct diameter initially scaled linearly with culture surface area, over time samples generated in the larger wells condensed more than their smaller counterparts resulting in final construct sizes which deviated from the initial linear relationship. Furthermore, this differential size decrease coincided with altered mineralized tissue patterning, where samples generated in the smaller dishes had greater relative mineral deposition that lacked predictable patterning unlike the distinct anatomically patterned mineralized and soft tissue layers seen in constructs formed in the larger wells. Interestingly, samples formed in the smaller wells had increased apoptotic activity at the periphery, suggesting that cellular dynamics vary with initial construct size. These findings demonstrate that reducing initial cell number in scaffold-free DPSC-PDLSC constructs alters cellular behavior and tissue architecture, suggesting the existence of a critical cell mass required for consistent and proper tooth root organoid formation.