Ti3C2Tx MXene Potentiates PSAT1-Mediated Osteogenesis Through miR-665/GSK-3β/β-Catenin Axis to Counteract Inflammation-Induced Bone Loss

Jinlong Zhang,1,2,* Xinyang Wang,3,4,* Jingwen Xiao,5,* Hongxiang Hong,1,2 Jiajia Chen,1,2 Chunshuai Wu,1,2 Guanhua Xu,1,2 Zhiming Cui1,2 1Department of Spine Surgery, Nantong City No.1 People’s Hospital and Second Affiliated Hospital of Nantong University, Nantong, Jiangsu Province, 226001, People’s Republic of China; 2Research Institute for Spine and Spinal Cord Disease of Nantong University, Nantong, Jiangsu, 226014, People’s Republic of China; 3Department of Stomatology, Affiliated Hospital of Nantong University, Medical School of Nantong University, Nantong, Jiangsu Province, 226001, People’s Republic of China; 4Department of Stomatology, Zhenjiang 359 Hospital, Zhenjiang, Jiangsu Province, 212000, People’s Republic of China; 5Department of Stomatology, Haimen District People’s Hospital, Nantong, Jiangsu Province, 226100, People’s Republic of China*These authors contributed equally to this workCorrespondence: Zhiming Cui, Department of Spine Surgery, Nantong City No.1 People’s Hospital and Second Affiliated Hospital of Nantong, Nantong, Jiangsu Province, 226001, People’s Republic of China, Email [email protected] Guanhua Xu, Department of Spine Surgery, Nantong City No.1 People’s Hospital and Second Affiliated Hospital of Nantong, Nantong, Jiangsu Province, 226001, People’s Republic of China, Email [email protected]: Inflammatory signaling-induced stem cell dysfunction severely impairs bone regeneration. This study aimed to develop a combinatorial strategy using Ti3C2Tx MXene scaffolds and PSAT1-engineered dental pulp stem cells (oe-PSAT1 DPSCs) to counteract inflammation-mediated osteogenic suppression.Methods: Dental pulp stem cells (DPSCs) were treated with TNF-α to simulate an inflammatory microenvironment. miR-665 expression and its targeting relationship with PSAT1 were analyzed via qRT-PCR, dual-luciferase reporter assay, and Western blot. The role of the miR-665/PSAT1/GSK-3β/β-catenin axis in osteogenic differentiation was evaluated using ALP activity, alizarin red staining, and immunofluorescence. Ti3C2Tx MXene was synthesized and characterized, and its effects on ROS scavenging and osteogenesis were assessed in vitro. In vivo efficacy was validated using a rat calvarial defect model with micro-CT, histological staining, and immunohistochemistry.Results: TNF-α stimulation upregulated miR-665, which directly targeted PSAT1 and inhibited the GSK-3β/β-catenin pathway, suppressing DPSCs osteogenic differentiation. PSAT1 overexpression rescued this suppression. Ti3C2Tx MXene scavenged ROS, enhanced calcium-dependent mineralization, and synergized with oe-PSAT1 DPSCs to amplify β-catenin activation. In rat models, the Ti3C2Tx MXene /oe-PSAT1 DPSCs combination achieved superior bone defect closure (higher BV/TV, Tb. Th, and mature collagen deposition) compared to Ti3C2Tx MXene alone.Discussion: This study identifies the miR-665/PSAT1/GSK-3β/β-catenin axis as a key regulator of inflammatory osteogenesis. The Ti3C2Tx MXene/oe-PSAT1 DPSCs strategy concurrently neutralizes oxidative stress and activates osteogenic signaling, providing a translatable platform for inflammatory bone regeneration. Keywords: DPSCs, osteogenic differentiation, miR-665, PSAT1, Ti3C2Tx