Periodontitis, a chronic inflammatory disease caused by microbial communities carrying pathogens, leads to the loss of tooth-supporting tissues and is a significant contributor to tooth loss. This study aims to develop a novel injectable cell-laden hydrogel consisted of collagen (COL), riboflavin, and a dental light-emitting diode (LED) photo-cross-linking process for periodontal regeneration. Utilizing α-SMA and ALP immunofluorescence markers, we confirmed the differentiation of human periodontal ligament fibroblasts (HPLFs) into myofibroblasts and preosteoblasts within collagen scaffolds in vitro. Twenty-four rats with three-wall artificial periodontal defects were divided into four groups, Blank, COL_LED, COL_HPLF, and COL_HPLF_LED, and histomorphometrically assessed after 6 weeks. Notably, the COL_HPLF_LED group showed less relative epithelial downgrowth (p< 0.01 for Blank,p< 0.05 for COL_LED and COL_HPLF), and the relative residual bone defect was significantly reduced in the COL_HPLF_LED group compared to the Blank and the COL_LED group (p< 0.05). The results indicated that LED photo-cross-linking collagen scaffolds possess sufficient strength to withstand the forces of surgical process and biting, providing support for HPLF cells embedded within them. The secretion of cells is suggested to promote the repair of adjacent tissues, including well-oriented periodontal ligament and alveolar bone regeneration. The approach developed in this study demonstrates clinical feasibility and holds promise for achieving both functional and structural regeneration of periodontal defects.