International dental journal · 2025
Calpain-1 Potentiates Periodontal Regeneration via PHLPP1-ERK-Driven Osteogenesis in Periodontal Ligament Stem Cells.
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Li Yuejia, Yang Bin, Mei Ziyi, Deng Pingmeng, Fu Tiwei, Li Jie
Аннотация
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.
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.