Mild Heat Stress Enhances Angiogenesis in a Co-culture System Consisting of Primary Human Osteoblasts and Outgrowth Endothelial Cells

Abstract

The repair and regeneration of large bone defects, including the formation of functional vasculature, represents a highly challenging task for tissue engineering and regenerative medicine. Recent studies have shown that vascularization and ossification can be stimulated by mild heat stress (MHS), which would offer the option to enhance the bone regeneration process by relatively simple means. However, the mechanisms of MHS-enhanced angiogenesis and osteogenesis, as well as potential risks for the treated cells are unclear. We have investigated the direct effect of MHS on angiogenesis and osteogenesis in a co-culture system of human outgrowth endothelial cells (OECs) and primary osteoblasts (pOBs), and assessed cytotoxic effects, as well as the levels of various heat shock proteins (HSPs) synthesized under these conditions. Enhanced formation of microvessel-like structures was observed in co-cultures exposed to MHS (41°C, 1 h), twice per week, over a time period of 7–14 days. As shown by real-time polymerase chain reaction (PCR), the expression of vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), and tumor necrosis factor-alpha was up-regulated in MHS-treated co-cultures 24 h post-treatment. At the protein level, significantly elevated VEGF and Ang-1 concentrations were observed in MHS-treated co-cultures and pOB mono-cultures compared with controls, indicating paracrine effects associated with MHS-induced angiogenesis. MHS-stimulated co-cultures and OEC mono-cultures released higher levels of Ang-2 than untreated cultures. On the other hand MHS treatment of co-cultures did not result in a clear effect regarding osteogenesis. Nevertheless, real-time PCR demonstrated that MHS increased the expression of mitogen-activated protein kinase, interleukin-6, and bone morphogenetic protein 2, known as HSP-related molecules in angiogenic and osteogenic regulation pathways. In agreement with these observations, the expression of some selected HSPs also increased at both the mRNA and protein levels in MHS-treated co-cultures.