Development of Periodontal Organoid-like Constructs Using Human Periodontal Ligament and Gingival Epithelial Cells: Comparison of Two Assembly Strategies

Periodontal organoid research remains underdeveloped, largely due to the lack of standardized fabrication protocols and multicellular constructs capable of reproducing epithelial–mesenchymal interactions relevant to periodontal biology. In particular, it remains unclear whether different spatial assembly strategies influence construct stability, multicellular organization, or early molecular behavior in periodontal three-dimensional (3D) systems. Therefore, this study aimed to develop and compare two different methods for generating dual-lineage 3D periodontal organoid-like constructs using human periodontal ligament cells (hPDL) and human gingival epithelial cells (hGEP). These strategies were selected to compare two biologically and technically distinct spatial configurations: bilayer assembly to partially mimic epithelial–connective tissue compartmentalization, and surface seeding as a simplified fabrication approach with potential advantages for reproducibility and workflow standardization. Both cell types were cultured in different media conditions (CnT-57, DMEM, and a 1:1 CnT-57/DMEM mixture) to determine compatibility for co-culture applications. Cell viability was assessed on days 1, 3, and 7 using the MTS assay. Constructs were produced in hyaluronic acid-based hydrogels using two strategies: Group 1–sequential photopolymerization of hPDL and hGEP layers to generate a bilayer construct; and Group 2—encapsulation of hPDL followed by direct seeding of hGEP onto the construct surface. Viability within constructs was evaluated on days 3 and 7 using the Live/Dead assay. Morphology was monitored using stereomicroscopy on days 0, 1, 3, and 7. Exploratory RNA sequencing was performed on day 7 to characterize transcriptomic profiles. All tested culture media maintained cellular viabilities above 70%, with no statistically significant differences among conditions (p > 0.05), indicating biocompatibility for both cell types. Group 1 exhibited viabilities of 86.54% ± 8.55% and 90.69% ± 7.88% on days 3 and 7, respectively, while Group 2 showed viabilities of 87.00% ± 9.58% and 88.08% ± 9.12%, with no significant intergroup differences (p > 0.05). Morphological analyses demonstrated preservation of construct integrity and progressive interaction between epithelial and mesenchymal compartments. Exploratory RNA sequencing revealed only subtle transcriptomic differences between assembly strategies. In conclusion, both methodologies successfully generated viable and structurally stable dual-lineage periodontal organoid-like constructs within a hyaluronic acid-based matrix. Comparison of these two assembly strategies demonstrates the feasibility of generating reproducible multicellular periodontal 3D models using distinct spatial configurations, establishing a proof-of-concept platform for future optimization toward periodontal disease modeling, regenerative studies, and advanced biofabrication applications.