Macrophage-mediated angiogenic activation of outgrowth endothelial cells in co-culture with primary osteoblasts

Abstract

The successful vascularisation of complex tissue engineered constructs for bone regeneration is still a major challenge in the field of tissue engineering. In this context, co-culture systems of endothelial cells and osteoblasts represent a promising approach to advance the formation of a stable vasculature as well as an excellent in vitro model to identify factors that positively influence bone healing processes, including angiogenesis. Under physiological conditions, the activation phase of angiogenesis is mainly induced by hypoxia or inflammation. Inflammatory cells such as macrophages secrete proinflammatory cytokines and proangiogenic growth factors, finally leading to the formation of new blood vessels. The aim of this study was to investigate if macrophages might positively influence the formation of microvessel-like structures via inflammatory mechanisms in a co-culture system consisting of human outgrowth endothelial cells (OECs) and primary osteoblasts. Treatment of co-cultures with macrophages (induced from THP-1) resulted in a higher number of microvessel-like structures formed by OECs compared to the co-culture. This change correlated with a significantly higher concentration of the proangiogenic VEGF in cell culture supernatants of triple-cultures and was accompanied by an increase in the expression of different proinflammatory cytokines, such as IL-6, IL-8 and TNFα. In addition, the expression of E-selectin and ICAM-1, adhesion molecules which are strongly involved in the interaction between leukocytes and endothelial cells during the process of inflammation was also found to be higher in triple-cultures compared to the double co-cultures, documenting an ongoing proinflammatory stimulus. These results raise the possibility of actively using pro-inflammatory stimuli in a tissue engineering context to accelerate healing mechanisms.

Platelet‐rich fibrin‐based matrices to improve angiogenesis in an in vitro co‐culture model for bone tissue engineering

“…Notably, the co‐culture system of OECs and pOBs has already been described in numerous studies and has been established as a highly beneficial tool to study regeneration mechanisms in vitro similar to the in vivo situation (Fuchs, Dohle, Kolbe, & Kirkpatrick, ; Fuchs et al, ; Fuchs, Ghanaati, et al, ; Fuchs, Jiang, et al, ). Furthermore, this in vitro model allows the identification of factors that might have a positive effect on processes involved in wound repair, in particular angiogenesis (Dohle et al, ; Dohle et al, ; Dohle et al, ; Li et al, ; Ma, Dohle, Li, & Kirkpatrick, ). Although the detailed interaction of endothelial cells with osteoblasts in this established co‐culture system underlies complex regulatory mechanisms that are still under investigation, microvessel‐like structure formation can be documented in long‐term co‐cultures after 4 weeks of co‐cultivation (Herzog, Dohle, Bischoff, & Kirkpatrick, ).…”

“…However, these co‐cultures of OECs and pOBs still possess some limitations due to the fact that an additional proangiogenic stimulus is essential to achieve a timely induction of the neovascularization process in vitro . Previous studies have documented that rapid initiation of angiogenesis could only be achieved when co‐cultures were treated with proangiogenic growth factors like vascular endothelial growth factor (VEGF), morphogens or even with activated macrophages mimicking the natural responses of the human body (Dohle et al, ; Dohle et al, ; Dohle et al, ). Furthermore, OEC/pOB co‐cultures strongly require the co‐implantation of proangiogenic matrices like Matrigel® for cell growth and induction of fast microvessel‐like structure formation in vivo (Fuchs, Ghanaati, et al, ).…”

Boon and Bane of Inflammation in Bone Tissue Regeneration and Its Link with Angiogenesis

“…Tissue formation relies on the supply of oxygen, nutrients, signaling molecules, and cells through the vasculature, and the vasculature also represents the best way to deposit of unwanted material (5,6). However, upon injury vessels are disrupted, supply ceases.…”

“…Novel approach to look at all three systems together Following this thought, however, we cant stop at the interdependency of the immune and skeletal systems while considering healing, but have to go one step further and include angiogenesis. Angiogenesis and inflammation are closely linked, especially during the early stages of a healing process (6,17). In the following we will try to highlight the interactions/ interdependency and interconnectivity of angiogenesis, immune reaction, and regeneration which should be considered together where healing is discussed.…”

“…Approaches are being considered in which the pro-angiogenic effect in tissue engineering is driven by inflammatory cells (6). Vessels have been proposed to be a source of osteogenic cells during the regenerative healing process of bone, however the communication between endothelial cells and osteoblastic cells is as yet not well understood (84), but could very well include immune cells signaling.…”