Supportive angiogenic and osteogenic differentiation of mesenchymal stromal cells and endothelial cells in monolayer and co-cultures.

Florian Böhrnsen,Henning Schliephake

doi:10.1038/ijos.2016.39

Abstract

Sites of implantation with compromised biology may be unable to achieve the required level of angiogenic and osteogenic regeneration. The specific function and contribution of different cell types to the formation of prevascularized, osteogenic networks in co-culture remains unclear. To determine how bone marrow-derived mesenchymal stromal cells (BMSCs) and endothelial cells (ECs) contribute to cellular proangiogenic differentiation, we analysed the differentiation of BMSCs and ECs in standardized monolayer, Transwell and co-cultures. BMSCs were derived from the iliac bone marrow of five patients, characterized and differentiated in standardized monolayers, permeable Transwells and co-cultures with human umbilical vein ECs (HUVECs). The expression levels of CD31, von Willebrand factor, osteonectin (ON) and Runx2 were assessed by quantitative reverse transcriptase polymerase chain reaction. The protein expression of alkaline phosphatase, ON and CD31 was demonstrated via histochemical and immunofluorescence analysis. The results showed that BMSCs and HUVECs were able to retain their lineage-specific osteogenic and angiogenic differentiation in direct and indirect co-cultures. In addition, BMSCs demonstrated a supportive expression of angiogenic function in co-culture, while HUVEC was able to improve the expression of osteogenic marker molecules in BMSCs.

Highlights

Sites of implantation with compromised biology may be unable to achieve the required level of osteogenic activity, regeneration and osseointegration.[1,2] The successful healing of bone grafts in these sites of implantation is based on the supply of oxygen and nutrients to the grafted tissue
The results showed that bone marrow-derived mesenchymal stromal cells (BMSCs) and human umbilical vein endothelial cell (EC) (HUVECs) were able to retain their lineage-specific osteogenic and angiogenic differentiation in direct and indirect co-cultures
Angiogenetic differentiation of HUVEC and BMSC co-cultures Immunostaining revealed the expression of the angiogenic marker molecule CD31 in monolayer, Transwell and co-culture differentiation (Figure 2a and 2b)

Summary

Introduction

Sites of implantation with compromised biology may be unable to achieve the required level of osteogenic activity, regeneration and osseointegration.[1,2] The successful healing of bone grafts in these sites of implantation is based on the supply of oxygen and nutrients to the grafted tissue. In non-vascularized bone grafts, revascularization originating from the recipient site is the key process for bone graft survival and successful repair. This applies even more to the use of tissue-engineered bone grafts, the biological quality of which is still inferior to that of the native bone grafts. Monitoring of the integration of cancellous bone grafts using Technetium-99 (99Tc) bone scans has shown that rapid revascularization of large graft areas occurs during the first postoperative week.[3,4] As the velocity of this process is far beyond the growth rate of proliferating capillaries at the recipient site, it has been suggested that revascularization of grafted cancellous bone tissue is accomplished through the direct connection of proliferating vessels at the recipient site to existing capillary networks inside the grafts. For the use of tissue-engineered bone grafts, the generation of functional capillary networks inside the cultured constructs may be important to overcome the current limitations in the clinical application of bone tissue engineering.[5,6] The prevascularization of tissue-engineered bone grafts is considered to be an essential step towards a graft structure that may enhance the early revascularization of tissue-engineered grafts

Methods

Results

Conclusion