HUVECs-conditioned medium has a better potential to stimulate differentiation of dental pulp stromal cells toward an osteoblastic lineage

Introduction
One of the biggest challenges in tissue engineering technique is adequate vascularization to prevent tissue necrosis. This problem often arises in large bone defect reconstruction. During osteogenesis, vascularization involves cross-talk regulation between endothelial and osteoblast cells. It was the foundation of this study.

Objectives
To evaluate the effect of co-culture and conditioned medium of human umbilical vein endothelial cells (HUVECs) on the expression of collagen I, fibronectin proteins and alkaline phosphates (ALP) activity released by dental pulp cells (DPSCs) on 3D scaffold composed of hydroxyapatite/tricalcium phosphate/chitosan (HA/TCP/chitosan).

Material and methods
The three main research group were (1) DPSCs co-cultured with various ratios of HUVECs, (2) DPSCs cultured with various concentrations of HUVECs-conditioned medium (CM), (3) DPSCs cultured with normal medium as the control. Culture media were collected at day 3, 5, 7 to examine the collagen type 1, fibronection protein and ALP activity levels.

Results
DPSCs-HUVECs indirect co-culture increased the protein expression of collagen I and fibronectin. However, the upregulation was higher when DPSCs were cultured with HUVECs CM. ALP was not changed in co-culture systems while it increased in the HUVECs CM group. The increase in three different markers tested in HUVECs CM compared to the co-culture system might be explained by the presence of signaling molecules in the HUVECs CM that stimulate DPSCs. Fibronectin expression was necessary for DPSCs’ attachment to the 3D scaffold, while collagen I and ALP expressions might indicate the differentiation of DPSCs to osteoblasts.

Conclusions
HUVECs-conditioned medium has a better potential to be used to stimulate the differentiation of dental pulp cells towards an osteoblastic lineage, the condition that is necessary for the success of bone tissue engineering.