Alkaline phosphatase dual-binding sites for collagen dictate cell migration and microvessel assembly in vitro.

Interactions between cell types, growth factors, and extracellular matrix components involved in angiogenesis are crucial for new vessel formation leading to tissue regeneration. This study investigated whether cocultures of fibroblasts and endothelial cells (ECs; from macro- or microvasculature) play a role in the formation of microvessel-like structures by ECs, as well as modulate fibroblast differentiation and growth factors production (vascular endothelial cell growth factor, basic fibroblast growth factor, active transforming growth factor-ß1, and interleukin-8), which are important for vessel sprouting and maturation. Data obtained revealed that in vitro coculture systems of fibroblasts and human ECs stimulate collagen synthesis and growth factors production by fibroblasts that ultimately affect the formation and distribution of microvessel-like structures in cell cultures. In this study, areas with activated fibroblasts and high alkaline phosphatase (ALP) activity were also observed in cocultures. Molecular docking assays revealed that ALP has two binding positions for collagen, suggesting its impact in collagen proteins' aggregation, cell migration, and microvessel assembly. These findings indicate that bioinformatics and coculture systems are complementary tools for investigating the participation of proteins, like collagen and ALP in angiogenesis.

Human endothelial cell-based assay for endotoxin as sensitive as the conventional Limulus Amebocyte Lysate assay.

Endotoxin, also known as lipopolysaccharide (LPS) produced by bacteria can be present in any liquid or on any biomaterial even if the material is sterile. Endotoxin in mammals can cause fever, inflammation, cell and tissue damage and irreversible septic shock and death. In the body, endothelial cells making up the blood vasculature and endothelial cells in vitro rapidly react to minute amounts of endotoxin resulting in a rapid induction of the cell adhesion molecule E-selectin. In this study we have used immunofluorescent staining to evaluate the expression of E-selectin on human microvascular endothelial cells from the skin (HDMEC) and human umbilical vein endothelial cells (HUVEC) exposed to various concentrations of LPS. In addition, the sensitivity of detection was compared with the most widely used assay for the presence of endotoxin, the Limulus Amebocyte Lysate assay (LAL). The detection of E-selectin on endothelial cells in the presence of LPS for 4 h was found to be at least as sensitive in detecting the same concentration using the LAL assay. A cell adhesion molecule-enzyme immunosorbent assay was also developed and used to quantify LPS using the endothelial cell model. A comparison of LAL and the immunofluorescent staining method was carried out with solutions, nanoparticles, biomaterial extracts and endothelial cells grown directly on biomaterials. Under all conditions, the endothelial/E-selectin model system was positive for the test samples that were positive by LAL. Thus, we propose the use of this highly sensitive, rapid, reproducible assay for the routine testing of endotoxin in all steps in the manufacturing process of materials destined for use in humans. This can give a rapid feedback and localization of bacterial contamination sources with the LAL being reserved for the testing of the final product.

Photoactivatable caged cyclic RGD peptide for triggering integrin binding and cell adhesion to surfaces.

We report the synthesis and properties of a photoactivatable caged RGD peptide and its application for phototriggering integrin- and cell-binding to surfaces. We analysed in detail 1) the differences in the integrin-binding affinity of the caged and uncaged forms by quartz crystal microbalance (QCM) studies, 2) the efficiency and yield of the photolytic uncaging reaction, 3) the biocompatibility of the photolysis by-products and irradiation conditions, 4) the possibility of site, temporal and density control of integrin-binding and therefore human cell attachment, and 5) the possibility of in situ generation of cell patterns and cell gradients by controlling the UV exposure. These studies provide a clear picture of the potential and limitations of caged RGD for integrin-mediated cell adhesion and demonstrate the application of this approach to the control and study of cell interactions and responses.

Human endothelial and osteoblast co-cultures on 3D biomaterials.

Increasingly, in vitro experiments are being used to evaluate the cell compatibility of novel biomaterials. Single cell cultures have been used to determine how well cells attach, grow, and exhibit characteristic functions on these materials and the outcome of such tests is generally accepted as an indicator of biocompatibility. However, organs and tissues are not made up of one cell type and the interaction of cells is known to be an essential factor for physiological cell function. To more accurately examine biomaterials for bone regeneration, we have developed methods to coculture osteoblasts, which are the primary cell type making up bone, and endothelial cells, which form the vasculature supplying cells in the bone with oxygen and nutrients to survive on 2- and 3-D biomaterials.

The rapid anastomosis between prevascularized networks on silk fibroin scaffolds generated in vitro with cocultures of human microvascular endothelial and osteoblast cells and the host vasculature.

The survival and functioning of a bone biomaterial upon implantation requires a rapidly forming and stably functioning vascularization that connects the implant to the recipient. We have previously shown that human microcapillary endothelial cells (HDMEC) and primary human osteoblast cells (HOS) in coculture on various 3-D bone biomaterial scaffolds rapidly distribute and self-assemble into a morphological structure resembling bone tissue. Endothelial cells form microcapillary-like structures containing a lumen and these were intertwined between the osteoblast cells and the biomaterial. This tissue-like self-assembly occurred in the absence of exogenously added angiogenic stimuli or artificial matrices. The purpose of this study was to determine whether this in vitro pre-formed microvasculature persists and functions in vivo and to determine how the host responds to the cell-containing scaffolds. The scaffolds with cocultures were implanted into immune-deficient mice and compared to scaffolds without cells or with HDMEC alone. Histological evaluation and immunohistochemical staining with human-specific antibodies of materials removed 14 days after implantation demonstrated that the in vitro pre-formed microcapillary structures were present on the silk fibroin scaffolds and showed a perfused lumen that contained red blood cells. This proved anastomosis with the host vasculature. Chimeric vessels in which HDMEC were integrated with the host's ingrowing (murine) capillaries were also observed. No HDMEC-derived microvessel structures or chimeric vessels were observed on implanted silk fibroin when precultured with HDMEC alone. In addition, there was migration of the host (murine) vasculature into the silk fibroin scaffolds implanted with cocultures, whereas silk fibroin alone or silk fibroin precultured only with HDMEC were nearly devoid of ingrowing host microcapillaries. Therefore, not only do the in vitro pre-formed microcapillaries in a coculture survive and anastomose with the host vasculature to become functioning microcapillaries after implantation, the coculture also stimulates the host capillaries to rapidly grow into the scaffold to vascularize the implanted material. Thus, this coculture-based pre-vascularization of a biomaterial implant may have great potential in the clinical setting to treat large bone defects.

Tissue-like self-assembly in cocultures of endothelial cells and osteoblasts and the formation of microcapillary-like structures on three-dimensional porous biomaterials.

The survival and functioning of a bone biomaterial requires a rapid and stable vascularization after implantation. However, the mechanisms involved in the context of the complex healing microenvironment are poorly understood. To evaluate the vascularization potential of bone biomaterials, angiogenic stimuli were added to human dermal microvascular endothelial cells (HDMEC) growing on three-dimensional (3-D) bone biomaterials consisting of porous hydroxyapatite, porous calcium phosphate, porous nickel-titanium, successfully being used in humans, and also silk fibroin nets. HDMEC did not migrate to form microcapillary-like structures as they did on cell culture plastic. In cocultures of HDMEC and primary human osteoblast cells (HOS) or the human osteoblast-like cell line MG-63 on these biomaterials, a tissue-like self-assembly of cells occurred with time, with endothelial cells forming microcapillary-like structures containing a lumen and giving a strong PECAM-1 expression at cell interfaces. These microcapillary-like structures were intertwined between cell layers of osteoblasts and did not form when exogenous angiogenic stimuli were added to these cocultures. The life span of HDMEC was also significantly enhanced by coculture; with HDMEC being present for up to at least 42 days, compared to the monoculture where cells began to die rapidly after 1 week without passage. This coculture system may be applicable to a prevascularization strategy for biomaterials prior to implantation. Irrespective of this, the coculture model holds promise for studies to deepen our understanding of bone regeneration on 3-D substrates. Most importantly, these data raise important questions concerning the exact nature of pro-angiogenic drug- or gene-delivery systems to be incorporated into scaffolds. Our results underline the necessity to take into account the in situ production of growth factors by invading mesenchymal cells in the regenerative niche.