Eva Dohle

Dynamic processes involved in the pre-vascularization of silk fibroin constructs for bone regeneration using outgrowth endothelial cells

For successful bone regeneration tissue engineered bone constructs combining both aspects, namely a high osteogenic potential and a rapid connection to the vascular network are needed. In this study we assessed the formation of pre-vascular structures by human outgrowth endothelial cells (OEC) from progenitors in the peripheral blood and the osteogenic differentiation of primary human osteoblasts (pOB) on micrometric silk fibroin scaffolds. The rational was to gain more insight into the dynamic processes involved in the differentiation and functionality of both cell types depending on culture time in vitro. Vascular tube formation by OEC was assessed quantitatively at one and 4 weeks of culture. In parallel, we assessed the temporal changes in cell ratios by flow cytometry and in the marker profiles of endothelial and osteogenic markers by quantitative real-time PCR. In terms of OEC, we observed an increase in tube length, tube area, number of nodes and number of vascular meshes within a culture period of 4 weeks, but a decrease in endothelial markers in real-time PCR. At the same time early osteogenic markers were downregulated, while marker expression associated with progressing mineralized matrix was upregulated in later stages of the culture. In addition, deposition of matrix components, such as collagen type I, known as a pro-angiogenic substrate for endothelial cells, appeared to increase with time indicated by immunohistochemistry. In summary, the study suggests a progressing maturation of the tissue construct with culture time which seems to be not effected by culture conditions mainly designed for outgrowth endothelial cells.

Improving vascularization of engineered bone through the generation of pro-angiogenic effects in co-culture systems.

One of the major problems with bone tissue engineering is the development of a rapid vascularization after implantation to supply the growing osteoblast cells with the nutrients to grow and survive as well as to remove waste products. It has been demonstrated that capillary-like structures produced in vitro will anastomose rapidly after implantation and become functioning blood vessels. For this reason, in recent years many studies have examined a variety of human osteoblast and endothelial cell co-culture systems in order to distribute osteoblasts on all parts of the bone scaffold and at the same time provide conditions for the endothelial cells to migrate to form a network of capillary-like structures throughout the osteoblast-colonized scaffold. The movement and proliferation of endothelial cells to form capillary-like structures is known as angiogenesis and is dependent on a variety of pro-angiogenic factors. This review summarizes human 2- and 3-D co-culture models to date, the types and origins of cells used in the co-cultures and the proangiogenic factors that have been identified in the co-culture models.

Outgrowth endothelial cells: sources, characteristics and potential applications in tissue engineering and regenerative medicine.

Endothelial progenitor cells from peripheral blood or cord blood are attracting increasing interest as a potential cell source for cellular therapies aiming to enhance the neovascularization of tissue engineered constructs or ischemic tissues. The present review focus on a specific population contained in endothelial progenitor cell cultures designated as outgrowth endothelial cells (OEC) or endothelial colony forming cells from peripheral blood or cord blood. Special attention will be paid to what is currently known in terms of the origin and the cell biological or functional characteristics of OEC. Furthermore, we will discuss current concepts, how OEC might be integrated in complex tissue engineered constructs based on biomaterial or co-cultures, with special emphasis on their potential application in bone tissue engineering and related vascularization strategies.

Cell Communication in a Coculture System Consisting of Outgrowth Endothelial Cells and Primary Osteoblasts

Bone tissue is a highly vascularized and dynamic system with a complex construction. In order to develop a construct for implant purposes in bone tissue engineering, a proper understanding of the complex dependencies between different cells and cell types would provide further insight into the highly regulated processes during bone repair, namely, angiogenesis and osteogenesis, and might result in sufficiently equipped constructs to be beneficial to patients and thereby accomplish their task. This study is based on an in vitro coculture model consisting of outgrowth endothelial cells and primary osteoblasts and is currently being used in different studies of bone repair processes with special regard to angiogenesis and osteogenesis. Coculture systems of OECs and pOBs positively influence the angiogenic potential of endothelial cells by inducing the formation of angiogenic structures in long-term cultures. Although many studies have focused on cell communication, there are still numerous aspects which remain poorly understood. Therefore, the aim of this study is to investigate certain growth factors and cell communication molecules that are important during bone repair processes. Selected growth factors like VEGF, angiopoietins, BMPs, and IGFs were investigated during angiogenesis and osteogenesis and their expression in the cultures was observed and compared after one and four weeks of cultivation. In addition, to gain a better understanding on the origin of different growth factors, both direct and indirect coculture strategies were employed. Another important focus of this study was to investigate the role of “gap junctions,” small protein pores which connect adjacent cells. With these bridges cells are able to exchange signal molecules, growth factors, and other important mediators. It could be shown that connexins, the gap junction proteins, were located around cell nuclei, where they await their transport to the cell membrane. In addition, areas in which two cells formed gap junctions were found.

Sonic Hedgehog-mediated synergistic effects guiding angiogenesis and osteogenesis.

Sonic hedgehog (Shh) is a morphogen controlling the skeletal and vascular development in the embryo but is also reactivated during adult repair processes. Thus, this molecule holds great therapeutic potential for biotechnological and biomedical approaches aiming to enhance tissue regeneration or to replace damaged tissues. According to present knowledge, Shh signaling controls the expression of several families of growth factors involved in neovascularization and vessel maturation and acts upstream of the most prominent angiogenic growth factor, vascular endothelial growth factor. In this context, a very interesting feature of Shh is that it controls both angiogenic activity and vessel stabilization by mural cells. In parallel, Shh seems to have a direct effect on endothelial cell tube formation and seems to trigger the differentiation process of mesenchymal stem cells toward the osteogenic lineage. In this chapter, we will therefore shortly summarize the multifaceted potential of Shh for bone repair and vascularization according to the current literature. In addition, we will show how coculture models based on outgrowth endothelial cells and primary osteoblasts can be used to reveal some of the relevant mechanisms by which Shh drives and connects bone regeneration and vascularization.

Release of acetylcholine from murine embryonic stem cells: effect of nicotinic and muscarinic receptors and blockade of organic cation transporter.

AIMS The non-neuronal cholinergic system is widely expressed in nature. The present experiments were performed to characterize the non-neuronal cholinergic system in murine embryonic stem cells (CGR8 cell line). MAIN METHODS CGR8 cells were cultured in gelatinized flasks with Glasgows buffered minimal essential medium (Gibco, Germany). Acetylcholine was measured by HPLC combined with bioreactor and electrochemical detection. KEY FINDINGS CGR8 cells contained 1.08±0.12 pmol acetylcholine/10(6) cells (n=7) which was reduced to 0.50±0.06 pmol/10(6) cells (n=6; p<0.05) in the presence (4h) of 30μM bromoacetylcholine to block choline acetyltransferase. A time-dependent release of acetylcholine into the incubation medium was demonstrated, when cholinesterase activity was blocked by 10 μM physostigmine, with 97±13, 180±15 and 216±14 pmol being released from 65×10(6) cells after incubation periods of 2, 4 and 6h, respectively. The cumulative release corresponds to a fractional release rate of 2%/min. Blockade of nicotine or muscarine receptors did not significantly modulate the release of acetylcholine which was substantially reduced by 300 μM quinine (inhibitor of organic cation transporters). This inhibition showed considerable fading over the incubation period, indicating additional release mechanisms activated upon inhibition of organic cation transporters. SIGNIFICANCE Murine embryonic stem cells contain and release significant amounts of acetylcholine. The high fractional release rate and the compensation for blocked organic cation transporters indicate that non-neuronal acetylcholine may play a functional role in the homeostasis of murine embryonic stem cells.

TLR4 stimulation by LPS enhances angiogenesis in a co‐culture system consisting of primary human osteoblasts and outgrowth endothelial cells

The development of new approaches leading to fast and successful vascularization of tissue‐engineered constructs is one of the most intensively studied subjects in tissue engineering and regenerative medicine. Recently, TLR4 activation and LPS stimulation of endothelial cells have been reported to promote angiogenesis in a variety of settings. In this study, we demonstrate that TLR4 activation by Ultrapure LPS Escherichia coli 0111:B4 (LPS‐EB) significantly enhances microvessel formation in a co‐culture system consisting of outgrowth endothelial cells (OECs) and primary human osteoblasts (pOBs). The precise modes of TLR4 action on the process of angiogenesis have also been investigated in this study. Using quantitative fluorescence microscopy in monocultures of OECs and pOBs, it was found that TLR4 activation through LPS‐EB upregulates the expression level of TLR4/MYD88 and enhances both angiogenesis and osteogenesis. Furthermore, ELISA and qRT–PCR have shown that the level of two adhesion molecules (ICAM‐1 and E‐selectin), two cytokines (IL‐6 and IL‐8) and two growth factors (VEGF and PDGF‐BB) related to angiogenesis increase significantly after LPS‐EB treatment. This increased understanding of the role of TLR4 in angiogenesis could be of value in various settings related to tissue repair and tissue engineering. Moreover, since LPS and TLR4 agonists improve angiogenesis and osteogenesis, TLR4 agonists (endogenous or synthetic) could be used for angiogenesis intervention in vivo and therefore could be tested for their potential clinical applications in promoting angiogenesis in bone tissue engineering. Copyright

Angiogenesis control in spine regeneration

Abstract: The intervertebral disc (IVD) has a complex vascularisation pattern. While the nucleus pulposus is avascular, the annulus fibrosus as well as the endplates are vascularised. IVD degeneration is often accompanied, on the one hand, by blood vessel ingrowth into the nucleus pulposus and, on the other hand, by diminished vascularisation of the endplates. Tissue engineering of IVD, therefore, has to address the differences in the vascularisation of IVD compartments. This chapter summarises current knowledge about the mechanisms of angiogenesis and its physiological and pathological role in IVD biology. Different strategies to control angiogenesis are discussed in the chapter with examples from bone tissue engineering, ophthalmological and oncological disorders.