Elsevier

Journal of Endodontics

Volume 42, Issue 9, September 2016, Pages 1355-1361
Journal of Endodontics

Regenerative Endodontics
Calcium Hydroxide–induced Proliferation, Migration, Osteogenic Differentiation, and Mineralization via the Mitogen-activated Protein Kinase Pathway in Human Dental Pulp Stem Cells

https://doi.org/10.1016/j.joen.2016.04.025Get rights and content

Highlights

  • Calcium hydroxide activated mitogen-activated protein kinases in human DPSCs.

  • JNK and p38 are involved in calcium hydroxide–induced proliferation in DPSCs.

  • JNK, p38, and ERK involved in calcium hydroxide–induced migration, alkaline phosphatase expression, and mineralization in DPSCs.

Abstract

Introduction

Calcium hydroxide has been extensively used as the gold standard for direct pulp capping in clinical dentistry. It induces proliferation, migration, and mineralization in dental pulp stem cells (DPSCs), but the underlying mechanisms are still unclear. The aim of this study was to investigate the role of the mitogen-activated protein (MAP) kinase pathway in calcium hydroxide–induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs.

Methods

Human DPSCs between passages 3 and 6 were used. DPSCs were preincubated with inhibitors of MAP kinases and cultured with calcium hydroxide. The phosphorylated MAP kinases were detected by Western blot analysis. Cell viability was analyzed via the methylthiazol tetrazolium assay. Cell migration was estimated using the wound healing assay. Alkaline phosphatase (ALP) expression was analyzed using the ALP staining assay. Mineralization was studied by alizarin red staining analysis.

Results

Calcium hydroxide significantly promoted the phosphorylation of the c-Jun N-terminal kinase (JNK), p38, and extracellular signal–regulated kinase. The inhibition of JNK and p38 signaling abolished calcium hydroxide–induced proliferation of DPSCs. The inhibition of JNK, p38, and extracellular signal–regulated kinase signaling suppressed the migration, ALP expression, and mineralization of DPSCs.

Conclusions

Our study showed that the MAP kinase pathway was involved in calcium hydroxide–induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs.

Section snippets

Cell Isolation and Culture

Human dental pulp tissues were obtained from healthy young donors (14–22 years) who extracted the third molars for orthodontic reasons at Peking University People's Hospital, Beijing, China. This study conforms to the Declaration of Helsinki, and all processes were approved by the Beihang University Ethical Committee, Beijing, China. Informed consent from each patient was obtained. The pulp tissue was digested with 3 mg/mL collagenase type I (Sigma-Aldrich, St Louis, MO) and 4 mg/mL Dispase

Calcium Hydroxide–induced Phosphorylations of MAP Kinases in Human DPSCs

Calcium hydroxide, 10 μg/mL, was added into the medium, and the same volume of solution that was used to dissolve calcium hydroxide was added into the control group (3). The phosphorylations of MAP kinases were analyzed by Western blot analysis after 5, 10, 30, and 60 minutes, respectively. The results showed that calcium hydroxide induced MAP kinase phosphorylation rapidly. Phosphorylated-p38 (p-p38) and phosphorylated-JNK (p-JNK) increased after 5 minutes of incubation and lasted for 1 hour.

Discussion

One of the main goals of vital pulp therapy is to stimulate the pulp to initiate reparative tertiary dentin formation. This process integrates steps of migration, proliferation, and mineralization of pulp cells (20). As the gold standard of direct pulp capping materials in clinical vital pulp therapy, calcium hydroxide increases the proliferation of DPSCs (3). The phosphorylated levels of p38, JNK, and ERK are all appreciably increased in response to calcium hydroxide in 10 to 30 minutes. It

Conclusion

Our findings suggested that MAP kinases involved in calcium hydroxide induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs. Calcium hydroxide rapidly promoted the phosphorylation of JNK, p38, and ERK. JNK and p38 played roles in calcium hydroxide–induced proliferation. JNK, p38, and ERK were involved in calcium hydroxide–induced migration, osteogenic differentiation, and mineralization in human DPSCs. Our work revealed the mechanism of calcium

Acknowledgments

Supported by the National Natural Science Foundation of China (grant nos. 11572030, 11102015, 61227902, 11120101001, 11421202, 11302020, and 11402017), the Fundamental Research Funds for the Central Universities, Beijing Higher Education Young Elite Teacher Project, the 111 Project (B13003), grant YWF-15-YG-003 (Beihang University), Specialized Research Fund for the Doctoral Program of Higher Education (20131102130004), and National Basic Research Program of China (973 program, 2011CB710901).

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