Objectives <p>This study aimed to evaluate the efficacy of a Wnt pathway activator, lithium chloride (LiCl), and LiCl-stimulated human primary periodontal ligament cells (hPDLCs) cell sheets integrated with medical-grade melt-electrowritten polycaprolactone (mPCL) scaffolds for in vivo periodontal regeneration.</p> Methods <p>This study followed a three-stage experimental design. The first stage consisted of local administration of various concentrations of LiCl (10, 100, and 1000 mM) in rat periodontal defects for 3 weeks to evaluate its dosage effects on in vivo periodontal regeneration. Next, the impact of LiCl on cementogenic and osteogenic differentiation of human primary periodontal ligament cells (hPDLCs) was examined in vitro. Finally, LiCl-pretreated hPDLCs cell sheets, combined with mPCL scaffolds, were transplanted into rat periodontal defects for 4 weeks. Micro-CT, histology, and immunohistochemistry analyses were conducted to assess in vivo periodontal tissue formation.</p> Results <p>Local administration of LiCl (10 mM and 100 mM) promoted the regeneration of alveolar bone, cementum, and obliquely oriented PDL Sharpey fibres in the rat defect model. In vitro, 10 mM LiCl enhanced cementogenic and osteogenic differentiation in hPDLCs. Moreover, when implanting a 10 mM LiCl-treated hPDLCs cell sheet combined with a PCL scaffold into experimentally induced rat periodontal defects, significant regeneration occurred, including the formation of alveolar bone and cementum, along with Sharpey’s fibres inserted into the new cementum and alveolar bone.</p> Significance <p>This study demonstrates the potential of combining LiCl-stimulated hPDLCs cell sheets with mPCL scaffolds to enhance periodontal regeneration, highlighting the significance of LiCl as a promising therapeutic agent for periodontal regenerative treatment and tissue engineering strategies.</p> Graphical Abstract <p></p>