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

Abstract

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.

Keywords:

• CORM-3
• hPDLSC
• osteo-specific marker
• micro-computed tomography
• osteopontin
• Runx2

Acknowledgments

This study was supported by Shandong Provincial Natural Science Foundation (ZR2020MH186), Shandong Provincial Science and Technology Development Plan (2010GSF10270), Jinan Science and Technology Innovation Program in Clinical Medicine (201805045).

Abbreviations

hPDLSCs, human periodontal ligament stem cells; ALP, alkaline phosphatase; qRT-PCR, quantitative real-time polymerase chain reaction; FBS, foetal bovine serum; PBS, phosphate buffered saline; CPC, cetylpyridinium chloride; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; OPN, osteopontin; Runx2, runt related transcription factor 2; PD, Periodontitis; PMSF, phenylmethanesulfonyl fluoride; SDS-PAGE, sulfate-polyacrylamide gel electrophoresis; PVDF, polyvinylidene fluoride; SD, standard deviation; MSCs, mesenchymal stem cells.

Ethics Approval and Consent to Participate

All guidelines on animal care and use applicable to international, national and/or institutions have been complied with.

Consent for Publication

All the participants in the study obtained informed consent.

Disclosure

The authors declare no conflicts of interest in this work.