Effects of Platelet-rich Plasma and Cell Coculture on Angiogenesis in Human Dental Pulp Stem Cells and Endothelial Progenitor Cells

doi:10.1016/j.joen.2014.07.022

Journal of Endodontics

Volume 40, Issue 11, November 2014, Pages 1810-1814

https://doi.org/10.1016/j.joen.2014.07.022 Get rights and content

Highlights

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We culture 2 kinds of cells (human dental pulp stem cells and endothelial progenitor cells) and prepare platelet-rich plasma successfully.
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We coculture human dental pulp stem cells and endothelial progenitor cells to investigate the potential effects on vasculogenic differentiation.
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We confirm that platelet-rich plasma not only maintains the growth and proliferation of cells but also promotes vasculogenesis.
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We confirm that coculture can promote vasculogenesis.
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Thus, platelet-rich plasma can be used in tissue engineering and might even be applied in tooth regeneration in the future.

Abstract

Introduction

Platelet-rich plasma (PRP) has been described as platelet concentrate. Growth factors released by activated platelets can improve wound vasculogenesis and enhance wound healing. In this study, we used PRP instead of serum to culture human dental pulp stem cells (hDPSCs) and endothelial progenitor cells (EPCs) and investigated revascularization ability. The effect of hDPSC and EPC coculture on vasculogenesis was also studied.

Methods

PRP was prepared by secondary centrifugation. Real-time polymerase chain reaction and Western blotting were used to determine the expression of vasculogenesis-related factors vascular endothelial growth factor, platelet-derived growth factor, fetal liver kinase 1 (Flk-1), and stromal cell-derived factor 1 (SDF-1) in cultured hDPSCs and EPCs. The cells were divided into 4 groups: EPCs + 10% fetal bovine serum (FBS), EPCs + 10% PRP, EPCs + hDPSCs + 10% FBS, and EPCs + hDPSCs + 10% PRP. Then, the formation of vessel-like structures was tested by the tube formation assay.

Results

On day 3, the expression levels of all the markers in the coculture groups were much higher than in the single-culture groups and were also higher in the PRP groups compared with the FBS groups (P < .05), except for SDF-1. Expression levels were significantly higher in the experimental groups (EPCs + 10% PRP, EPCs + hDPSCs + 10% FBS, and EPCs + hDPSCs + 10% PRP) than in the control group (EPCs + 10% FBS) and in the PRP groups/coculture groups compared with the FBS groups/single-culture groups (P < .01). The tube formation assay showed the area of vessel-like structures formed by the PRP group to be larger than in the FBS group (P < .05).

Conclusions

PRP and coculture can both promote vasculogenesis, and PRP can promote EPCs to form vessel-like structures.

Section snippets

PRP Preparation and Activation

The study was approved by the institutional research ethics committee of Southern Medical University, Guangzhou, China. Human umbilical cord blood was collected from healthy volunteers with informed consent in sterile tubes with anticoagulant. Platelets were separated from umbilical cord blood by secondary centrifugation; 14 mL blood was centrifuged at 360g for 20 minutes at room temperature. After the upper layer was collected, the sample was further centrifuged at 500g for 10 minutes. The

PRP Characterization

The platelet count range in whole blood was 116–230 × 10⁹/L, whereas PRP averaged 1306–1949 × 10⁹/L, which was significantly higher compared with the umbilical cord blood (P < .01).

Effect of PRP on mRNA Levels of Vasculogenesis-related Factors

The expression levels of VEGF, PDGF, Flk-1, and SDF-1 mRNA were detected by qRT-PCR on days 3, 7, and 14. The results of qRT-PCR are shown in Figure 1. On day 3, the expression of all the markers in the coculture groups (EDF and EDP) were higher than in the single-culture groups (EF and EP); similarly, all the

Discussion

PRP contains a large number of autologous growth factors and high levels of platelets. The platelet count in PRP has been found to be approximately 5 times that in peripheral human blood (16). Here, platelet counts in PRP averaged 1306–1949 × 10⁹/L, which is significantly higher compared with whole blood (P < .01). PRP activated by repetitive freeze thawing can release many active growth factors (data not shown). These PDGFs play an important role in tissue remodeling, including

Acknowledgments

Xinzhu Li and Jin Hou contributed equally to this work.

The authors thank Ms Jiao Hu and Dr Jun Wen for their assistance with this study.

Supported by a grant from National Natural Science Foundation of China (grant no. 81371137).

The authors deny any conflicts of interest related to this study.

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Basic ResearchEffects of Platelet-rich Plasma and Cell Coculture on Angiogenesis in Human Dental Pulp Stem Cells and Endothelial Progenitor Cells

Highlights

Abstract

Introduction

Methods

Results

Conclusions

Section snippets

PRP Preparation and Activation

PRP Characterization

Effect of PRP on mRNA Levels of Vasculogenesis-related Factors

Discussion

Acknowledgments

Cytotherapy

Arthroscopy

J Endod

Trends Biotechnol

J Endod

J Endod

J Endod

J Endod

J Oral Maxillofac Surg

Platelet quantification and growth factor analysis from platelet-rich plasma: implications for wound healing

Plast Reconstr Surg

Freeze-dried platelet-rich plasma shows beneficial healing properties in chronic wounds

Wound Repair Regen

Recombinant human platelet-derived growth factor-BB for the treatment of chronic pressure ulcers

Ann Plast Surg

Mechanism of action and in vivo role of platelet-derived growth factor

Physiol Rev

Basic Research
Effects of Platelet-rich Plasma and Cell Coculture on Angiogenesis in Human Dental Pulp Stem Cells and Endothelial Progenitor Cells