Calpain-1 Potentiates Periodontal Regeneration via PHLPP1-ERK-Driven Osteogenesis in Periodontal Ligament Stem Cells.

OBJECTIVES

In recent years, human periodontal ligament stem cells (hPDLSCs) are considered a promising seed cell source for bone tissue engineering, offering a prospective treatment for periodontal bone defects. The osteogenic differentiation of hPDLSCs is a critical process in bone tissue engineering. Calcium signalling regulates a myriad of cellular processes, including osteogenic differentiation; however, the function of calpain-1 (CAPN1), a calcium-dependent protein, remains incompletely elucidated.

METHODS

Intracellular Ca2+ levels were evaluated using Fluo-4 AM assay. CAPN1 was overexpressed by transfection of lentivirus. Knockdown of CAPN1 was performed using siRNA transfection. mRNA and protein expression were detected through Real-time PCR and Western blot, respectively. Immunofluorescence assay was performed using anti-CAPN1 and anti-phosphatase PH domain and leucine-rich repeat protein phosphatase 1 (PHLPP1) antibodies. Hydroxyapatite/beta-tricalcium phosphate loaded with CAPN1-overexpressing hPDLSCs were implanted into the dorsal region of nude mice. Periodontal bone defects were prepared using a trephine drill, and GelMA loaded with CAPN1-overexpressing hPDLSCs were implanted into the defect area.

RESULTS

Fluo-4 AM assay showed a continuous upregulation of intracellular Ca2+ levels, CAPN1 expression, and calpain activity during osteogenic induction of hPDLSCs. Overexpression of CAPN1 enhanced osteogenic differentiation of hPDLSCs and involved activation of the ERK1/2 signalling pathway. Knockdown CAPN1 or inhibiting CAPN1 by chemical inhibitor decreased osteogenic differentiation of hPDLSCs through ERK1/2 signalling pathway. Immunofluorescence staining and molecular docking analysis suggested that CAPN1 was colocalized and bound with PHLPP1. Furthermore, the combination of CAPN1-overexpressing hPDLSCs with hydroxyapatite/beta-tricalcium phosphate or GelMA hydrogel significantly enhanced bone regeneration in both subcutaneous implantation and periodontal bone defect models.

CONCLUSION

Our study reveals the role and mechanism of CAPN1 in the osteogenic differentiation of hPDLSCs, which provides a theoretical and technical foundation for targeting CAPN1 in bone regeneration.