Carbon Monoxide Releasing Molecule-3 Enhances Osteogenic Differentiation of Human Periodontal Ligament Stem Cells by Carbon Monoxide Release

Purpose: Limited intrinsic regeneration capacity following bone destruction remains a significant medical problem. Multiple regulatory effects of carbon monoxide releasing molecule-3 (CORM-3) have been reported. The aim of this study was to investigate the effect of CORM-3 on the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) during osteogenesis.

Patients and methods: hPDLSCs obtained from healthy periodontal ligament tissues were cultured and identified with specific surface antigens by flow cytometry. Effect of CORM-3 on the proliferation of hPDLSCs was determined by CCK-8 assay. Alizarin red staining and alkaline phosphatase (ALP) activity were used to assess the osteogenic differentiation of hPDLSCs. Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis were used to detect the expression of the indicated genes. Critical-sized skull defect was made in Balb/c-nude mice, microcomputed tomography (Micro-CT) and Masson trichrome staining were used to assess the new bone regeneration in mice.

Results: CORM-3 (400 μmol/l) significantly promoted the proliferation of hPDLSCs. CORM-3 pretreatment not only notably enhanced the mRNA and protein expression of osteo-specific marker OPN, Runx2 and ALP, but also increased mineral deposition and ALP activity by the release of CO on day 3, 7 and 14 (P<0.05). Degassed CORM-3 did not show the same effect as CORM-3. In animal model, application of CORM-3 with hPDLSCs transplantation highly increased new bone formation in skull defect region.

Conclusion: CORM-3 promoted osteogenic differentiation of hPDLSCs, and increased hPDLSCs-induced new bone formation in mice with critical-sized skull defect, which suggests an efficient and promising strategy in the treatment of disease with bone defect.