Synergistic Effects of Theobromine Derived Copper Nanoparticles on Periodontal Ligament Cell Proliferation and Migration

Regeneration of periodontal tissues relies on effective cell proliferation, migration, and matrix remodeling. Recent advances in nanomedicine have demonstrated that phytochemical-based nanoparticles may offer synergistic benefits in enhancing these cellular processes. This study explores the effects of Theobromine-Derived Copper Nanoparticles (Theobro-CuNPs) on human periodontal ligament fibroblasts (PDLFs), with a focus on proliferation, migration, and gene expression of key regenerative markers. Theobro-CuNPs were synthesized via a green chemistry method using theobromine as a reducing agent and copper sulfate as the metal precursor. Characterization of nanoparticles was conducted using SEM, UV-Vis spectroscopy and EDX analytical techniques. PDLFs were treated with Theobro-CuNPs using doses of 1,5 and 10 µg/ml, for durations of either 24 or 48 hours. The MTT assay was utilized to evaluate cell proliferation, meanwhile migratory behavior assessment employed scratch wound assays. Gene expression levels of VEGF, COL1A1, FN1, and MMP2 were quantified using qRT-PCR. Characterization confirmed the successful formation of spherical, nanoscale Theobro-CuNPs with a distinct surface plasmon resonance was detected near 575 nanometers, confirming nanoparticles formation. MTT assay revealed a concentration-dependent improvement in cellular viability was observed to be peaking at 10 µg/mL. Scratch assay results showed significantly enhanced wound closure in the Theo-CuNP-treated group compared to controls. qRT-PCR demonstrated significant upregulation of VEGF (3.2-fold), COL1A1 (2.8-fold), FN1 (2.5-fold), and MMP2 (3.6-fold), indicating strong pro-regenerative and matrix-remodeling effects. Theobro-CuNPs significantly enhance periodontal ligament cell proliferation, migration, and the upregulation of critical genes involved in tissue regeneration. These results imply that Theobro-CuNPs hold potential as a bioactive therapeutic platform for periodontal tissue engineering and regenerative dentistry.