The Non-neuronal Cholinergic System

An increasing body of knowledge indicates that the cholinergic system is not confined to the nervous system, but is practically ubiquitous. The present paper will address the question of the non-neuronal cholinergic system in vascular endothelial cells (EC). In tissue sections of human skin, immunohistochemical studies using confocal laser scanning microscopy showed ChAT (choline acetyltransferase) activity in the EC of dermal blood vessels. Positive ChAT immunoreactivity was also demonstrated in monolayer cultures of human umbilical vein EC (HUVEC) and a human angiosarcoma EC line (HAEND). That the synthesizing enzyme is not only present in EC, but also active was shown by measuring ChAT a…

Modulation of endotoxin-induced neutrophil transendothelial migration by alveolar epithelium in a defined bilayer model.

Within the alveolus, epithelial cells, due to their close association with endothelial cells, can potentially influence endothelial cell responsiveness during inflammation and their interaction with leukocytes. To investigate this, three lung epithelial cell lines (A549, Calu-3, or NCI-H441) were grown with endothelium on opposing surfaces of Transwell filters and the formation and stability of bilayers was rigorously evaluated. All epithelial lines disrupted endothelial monolayer formation on filters with 3- or 5-microm pores by breaching the filter, and this occurred regardless of seeding density, matrix composition, or duration of culture. Endothelial disruption was not detectable by ele…

Hyperbranched PEG by Random Copolymerization of Ethylene Oxide and Glycidol

The synthesis of hyperbranched poly(ethylene glycol) (hbPEG) in one step was realized by random copolymerization of ethylene oxide and glycidol, leading to a biocompatible, amorphous material with multiple hydroxyl functionalities. A series of copolymers with moderate polydispersity ($\overline {M} _{{\rm w}} /\overline {M} _{{\rm n}} $ < 1.8) was obtained with varying glycidol content (3-40 mol-%) and molecular weights up to 49 800 g mol(-1) . The randomly branched structure of the copolymers was confirmed by (1) H and (13) C NMR spectroscopy and thermal analysis (differential scanning calorimetry). MTS assay demonstrated low cell toxicity of the hyperbranched PEG, comparable to the highly…

Intraperitoneal adhesions-an ongoing challenge between biomedical engineering and the life sciences

Peritoneal adhesions remain a relevant clinical problem despite the currently available prophylactic barrier materials. So far, the physical separation of traumatized serosa areas using barriers represents the most important clinical strategy for adhesion prevention. However, the optimal material has not yet been found. Further optimization or pharmacological functionalization of these barriers could give an innovative input for peritoneal adhesion prevention. Therefore, a more complete understanding of pathogenesis is required. On the basis of the pathophysiology of adhesion formation the main barriers currently in clinical practice as well as new innovations are discussed in the present r…

The pre-vascularisation of a collagen-chondroitin sulphate scaffold using human amniotic fluid-derived stem cells to enhance and stabilise endothelial cell-mediated vessel formation

Abstract A major problem in tissue engineering (TE) is graft failure in vivo due to core degradation in in vitro engineered constructs designed to regenerate thick tissues such as bone. The integration of constructs post-implantation relies on the rapid formation of functional vasculature. A recent approach to overcome core degradation focuses on the creation of cell-based, pre-engineered vasculature formed within the TE construct in vitro , prior to implantation in vivo . The primary objective of this study was to investigate whether an amniotic fluid-derived stem cell (AFSC)–human umbilical vein endothelial cell (HUVEC) co-culture could be used to engineer in vitro vasculature in a collag…

Size- and coating-dependent uptake of polymer-coated gold nanoparticles in primary human dermal microvascular endothelial cells.

A library-orientated approach is used to gain understanding of the interactions of well-defined nanoparticles with primary human endothelial cells, which are a key component of the vasculature. Fifteen sequentially modified gold nanoparticles (AuNPs) based on three different core sizes (18, 35, 65 nm) and five polymeric coatings were prepared. The synthetic methodology ensured homogeneity across each series of particles to allow sequential investigation of the chemical features on cellular interactions. The toxicity of these nanoparticles, their uptake behavior in primary human dermal microvascular endothelial cells (HDMECs), and quantification of uptake were all investigated. The results o…

Recurrence of pleomorphic adenoma of the parotid gland-predictive value of cadherin-11 and fascin

The predictive value of cadherin-11, tenascin, fascin, and mucin-1 as markers for the likelihood of recurrence in pleomorphic adenoma of the parotid gland was examined. In this retrospective study we analysed 20 tumours from16 patients by immunohistochemistry. Staining intensities were measured using a semiquantitative scoring approach; localisation (tumour centre vs border) as well as clinical data were analysed and correlated with follow-up. Cadherin-11 was increased in recurrent tumours. However, no changes of fascin, tenascin or mucin-1 were observed. Cadherin-11 and fascin were increased in primary tumours of patients with later recurrence, with fascin upregulation restricted to the tu…

New aspects in the histological examination of polyethylene wear particles in failed total joint replacements.

The most important long-term complication in total joint replacements is aseptic osteolysis. Wear particles such as polyethylene (PE) debris are considered to be one of the causes that play a central role. Several studies indicated that PE can be visualised in paraffin-embedded tissue sections not only by polarised light, but also after oil red staining. To determine whether oil red staining enables sensitive detection of PE, we examined staining of mechanically-produced PE particles by oil red. Furthermore, we studied oil red staining of paraffin-embedded tissue specimens of patients with failed uncemented and cemented total knee and hip prostheses. We applied double labelling of sections …

Heterogeneous expression of cell adhesion molecules by endothelial cells in ARDS

ARDS (acute respiratory distress syndrome) can be associated with septic shock and multiple organ failure caused by an uncontrolled systemic inflammatory response to Gram-negative bacterial infection. While in animal models the key role of the endothelial adhesion molecules ICAM-1, E-selectin, and VCAM in ARDS has been extensively studied, there are scarcely any corresponding pathomorphological studies of human lung tissue. Hence, little is known about whether there is a comparable, or even heterogeneous, expression pattern of these molecules in the human pulmonary vasculature. This study was therefore undertaken to investigate the immunohistochemical expression of the constitutively expres…

Impact of polymer-modified gold nanoparticles on brain endothelial cells: exclusion of endoplasmic reticulum stress as a potential risk factor

A library of polymer-coated gold nanoparticles (AuNPs) differing in size and surface modifications was examined for uptake and induction of cellular stress responses in the endoplasmic reticulum (ER stress) in human brain endothelial cells (hCMEC/D3). ER stress is known to affect the physiology of endothelial cells (ECs) and may lead to inflammation or apoptosis. Thus, even if applied at non-cytotoxic concentrations ER stress caused by nanoparticles should be prevented to reduce the risk of vascular diseases and negative effects on the integrity of barriers (e.g. blood-brain barrier). We exposed hCMEC/D3 to twelve different AuNPs (three sizes: 18, 35, and 65 nm, each with four surface-modif…

Establishment and Characterization of an Angiosarcoma-Derived Cell Line, AS-M

A novel human endothelial cell line, AS-M, has been established from a cutaneous angiosarcoma on the scalp. The cells expressing platelet endothelial cell adhesion molecule-1 (CD31) were isolated using magnetic beads and subsequently cultured for a year. To date, the cells have undergone more than 100 population doublings (PDs). The AS-M cells manifested endothelial characteristics, such as active uptake of acetylated low-density lipoprotein labeled with 1,1'-dioctadecyl 3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil-Ac-LDL), capacity to bind the Ulex europeaus agglutin-I (UEA-I), and expression of von Willebrand factor (vWF) and CD31. The single cell-derived clone, AS-M.5, showed a…

Expression of apoptosis-related proteins, p53, and DNA fragmentation in sarcomas of the pulmonary artery

Department of Molecular Cell Biology, Max-Planck Institute for Physiology and Clinical Re-search, Bad Nauheim, Germany.The results of this article were presented at theannual Meeting of the German Society of Pathol-ogy, May 26–29, 1999, Jena, Germany.The authors acknowledge the excellent technicalsupport of Sandra Gerecht and Antonietta Ras-tiello, the photographic support of Peter Pulkowskiand Thomas Bo¨hm, and the helpful comments andcritical reading of the manuscript by Dr. NorbertSpeich (Imogen, Bonn, Germany) and Dr. Ron Un-ger.Address for correspondence: Andreas Gaumann,M.D., Department of Molecular Cell Biology, Max-Planck Institute for Physiology and Clincial Re-search, W. G. Kerckh…

Going beyond histology. Synchrotron micro-computed tomography as a methodology for biological tissue characterization: from tissue morphology to individual cells.

Current light microscopic methods such as serial sectioning, confocal microscopy or multiphoton microscopy are severely limited in their ability to analyse rather opaque biological structures in three dimensions, while electron optical methods offer either a good three-dimensional topographic visualization (scanning electron microscopy) or high-resolution imaging of very thin samples (transmission electron microscopy). However, sample preparation commonly results in a significant alteration and the destruction of the three-dimensional integrity of the specimen. Depending on the selected photon energy, the interaction between X-rays and biological matter provides semi-transparency of the spe…

Neutrophil‐mediated enhancement of angiogenesis and osteogenesis in a novel triple cell co‐culture model with endothelial cells and osteoblasts

Repair and regeneration of critical‐sized bone defects remain a major challenge in orthopaedic and craniomaxillofacial surgery. Until now, attempts to bioengineer bone tissue have been hindered by the inability to establish proper angiogenesis and osteogenesis in the tissue construct. In the present study, we established a novel triple cell co‐culture model consisting of osteoblasts, endothelial cells, and neutrophils and conducted a systematic investigation of the effects of neutrophils on angiogenesis and osteogenesis. Neutrophils significantly increased angiogenesis in the tissue construct, evidenced by the formation of microvessel‐like structures with an extensive lattice‐like, stable t…

Development of a human co-culture model of a bronchial unit in vitro to investigate toxic pathomechanisms

Actinomyces in infected osteoradionecrosis--underestimated?

Infected osteoradionecrosis (IORN) is a severe complication of radiation therapy for head and neck cancer. Infected osteoradionecrosis can lead to fracture and often requires subsequent jaw resection. It is known that irradiated bone is highly susceptible to infections, mainly with Candida species and cariogenic bacteria. Only very few data exist on Actinomyces in IORN. The study population consisted of 31 patients (7 female, 24 male; median age, 58.3 years). All patients exhibited clinical and radiological signs of IORN (infection, mucosa or skin fistula, and sequestrated bone). To detect Actinomyces colonies, histological examination was performed using several staining procedures (hemato…

Development of a triple-culture model of the alveolar-capillary barrier

Myocarditis: from bench to bedside. LT Cooper Jr (Ed.). Humana Press, Totowa, NJ, 2002. ISBN: 1 58829 112 X/03

Endothelial cell colonization and angiogenic potential of combined nano- and micro-fibrous scaffolds for bone tissue engineering

Presently the majority of tissue engineering approaches aimed at regenerating bone relies only on postimplantation vascularization. Strategies that include seeding endothelial cells (ECs) on biomaterials and promoting their adhesion, migration and functionality might be a solution for the formation of vascularized bone. Nano/micro-fiber-combined scaffolds have an innovative structure, inspired by extracellular matrix (ECM) that combines a nano-network, aimed to promote cell adhesion, with a micro-fiber mesh that provides the mechanical support. In this work we addressed the influence of this nano-network on growth pattern, morphology, inflammatory expression profile, expression of structura…

Isolation and molecular characterization of brain microvascular endothelial cells from human brain tumors.

Brain tumor formation and growth is accompanied by the proliferation and infiltration of blood capillaries. The phenotypes of endothelial cells that make up capillaries are known to differ not only in the tissues in which endothelial cells are located but also as a result of the microenvironment to which they are exposed. For this reason, primary cultures of brain endothelial cells were isolated from human brain tumors removed by surgery and compared with cells from normal tissue. The primary confluent monolayers that grew out of isolated capillary fragments consisted of closely associated, elongated, fusiform-shaped cells. But brain tumor-derived endothelial cells in culture exhibited sign…

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 artific…

Effects of nano-scaled particles on endothelial cell function in vitro: studies on viability, proliferation and inflammation.

Recent studies give support for a connection between the presence of inorganic particles (of microm and nm size) in different organs and tissues and the development of inflammatory foci, called granulomas. As the potential source of particles (e.g. porcelain dental bridges) and the location of particle detection were topographically far apart, a distribution via the blood stream appears highly probable. Thus, endothelial cells, which line the inner surface of blood vessels, would come into direct contact with these particles, making particle-endothelial interactions potentially pathogenically relevant. The objective of this study was to evaluate the effects that five different nano-scaled p…

Crosstalk between osteoblasts and endothelial cells co-cultured on a polycaprolactone-starch scaffold and the in vitro development of vascularization.

The reconstruction of bone defects based on cell-seeded constructs requires a functional microvasculature that meets the metabolic demands of the engineered tissue. Therefore, strategies that augment neovascularization need to be identified. We propose an in vitro strategy consisting of the simultaneous culture of osteoblasts and endothelial cells on a starch-based scaffold for the formation of pre-vascular structures, with the final aim of accelerating the establishment of a vascular bed in the implanted construct. Human dermal microvascular endothelial cells (HDMECs) were co-cultured with human osteoblasts (hOBs) on a 3D starch-based scaffold and after 21 days of culture HDMEC aligned and…

Particulate Matter Contamination of Intravenous Antibiotics Aggravates Loss of Functional Capillary Density in Postischemic Striated Muscle

Through the increased use of less expensive and counterfeit medicines, the contamination of parenteral fluids and drugs by particulate matter poses an increasing health hazard worldwide. However, the mechanism of action of such contamination has never been conclusively demonstrated. We have systemically injected the particles contained in three different 1-g preparations of the antibiotic cefotaxime into hamsters and visualized the functional capillary density in striated skin muscle, using intravital fluorescence microscopy. Injection of particles from either of the three preparations did not affect capillary perfusion in normal muscle (n = 3 hamsters, each). However, injection of particle…

The role of oxidative stress in pro-inflammatory activation of human endothelial cells on Ti6Al4V alloy

Inflammation is an important step in the early phase of tissue regeneration around an implanted metallic orthopaedic device. However, prolonged inflammation, which can be induced by metallic corrosion products, can lead to aseptic loosening and implant failure. Cells in peri-implant tissue as well as metal corrosion can induce reactive oxygen species (ROS) formation, thus contributing to an oxidative microenvironment around an implant. Understanding cellular reactions to implant-induced oxidative stress and inflammatory activation is important to help prevent an adverse response to metallic materials. In an earlier study we have shown that endothelial cells grown on Ti6Al4V alloy are subjec…

Dynamic In Vivo Biocompatibility of Angiogenic Peptide Amphiphile Nanofibers

Biomaterials that promote angiogenesis have great potential in regenerative medicine for rapid revascularization of damaged tissue, survival of transplanted cells, and healing of chronic wounds. Supramolecular nanofibers formed by self-assembly of a heparin-binding peptide amphiphile and heparan sulfate-like glycosaminoglycans were evaluated here using a dorsal skinfold chamber model to dynamically monitor the interaction between the nanofiber gel and the microcirculation, representing a novel application of this model. We paired this model with a conventional subcutaneous implantation model for static histological assessment of the interactions between the gel and host tissue. In the stati…

A Quantitative In Vitro Approach to Study the Intracellular Fate of Gold Nanoparticles: From Synthesis to Cytotoxicity

Due to their physico-chemical characteristics, gold nanoparticles (AuNPs) seem to be suitable for biomedical and therapeutic applications even if conflicting data on their toxicological profiles are present in literature. In order to better understand if AuNPs could be safe we must consider different biological endpoints such as cytotoxicity, genotoxicity, inflammation and biopersistence. Starting from these considerations, one of the first issues to be assessed is to better understand if AuNPs can be internalized by cells. In this work, we propose a methodological approach to radioactivate AuNPs by neutron activation and the quantification of their internalization by two in vitro cell syst…

Mesenchymal stem cell proliferation and differentiation on load-bearing trabecular Nitinol scaffolds.

Bone tissue regeneration in load-bearing regions of the body requires high-strength porous scaffolds capable of supporting angiogenesis and osteogenesis. 70% porous Nitinol (NiTi) scaffolds with a regular 3-D architecture resembling trabecular bone were produced from Ni foams using an original reactive vapor infiltration technique. The "trabecular Nitinol" scaffolds possessed a high compressive strength of 79 MPa and high permeability of 6.9×10(-6) cm2. The scaffolds were further modified to produce a near Ni-free surface layer and evaluated in terms of Ni ion release and human mesenchymal stem cell (hMSC) proliferation (AlamarBlue), differentiation (alkaline phosphatase activity, ALP) and …

Microvessel-like structures from outgrowth endothelial cells from human peripheral blood in 2-dimensional and 3-dimensional co-cultures with osteoblastic lineage cells.

Tissue regeneration involves complex processes in the interaction between different cell types that control the process of neo-vascularization. In bone, osteoblasts and bone marrow stem cells provide cue elements for the proliferation of endothelial cells, differentiation of endothelial precursors, and the maturation of a vascular network. In this study, we investigated outgrowth endothelial cells (OECs), a potential source of autologous endothelial cells derived from human peripheral blood, in direct 2-dimensional (2-D) and 3-D co-culture systems with cells relevant for the regeneration of bone tissue, such as osteoblasts. In the co-cultures, OECs were evaluated in terms of their stability…

High-Temperature Sintering of Xenogeneic Bone Substitutes Leads to Increased Multinucleated Giant Cell Formation: In Vivo and Preliminary Clinical Results.

The present preclinical and clinical study assessed the inflammatory response to a high-temperature–treated xenogeneic material (Bego-Oss) and the effects of this material on the occurrence of multinucleated giant cells, implantation bed vascularization, and regenerative potential. After evaluation of the material characteristics via scanning electron microscopy, subcutaneous implantation in CD-1 mice was used to assess the inflammatory response to the material for up to 60 days. The clinical aspects of this study involved the use of human bone specimens 6 months after sinus augmentation. Established histologic and histomorphometric analysis methods were applied. After implantation, the mat…

Molecular basis of endothelial dysfunction in sepsis.

Sepsis is one of the major causes of mortality in critically ill patients and develops as a result of the host response to infection. A complex network of events is set into motion in the body by the infection and results in the pathogenesis of sepsis. This review article focuses on the molecular mechanisms and components involved in the pathogenesis of sepsis with a major emphasis on the endothelium. This includes sepsis-inducing bacterial components (e.g. endotoxins), cellular targets of these molecules and their responses, host reactions, intracellular and cytokine networks, individual susceptibility and new therapeutic targets in sepsis treatment.

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 p…

Scaffold vascularization in vivo driven by primary human osteoblasts in concert with host inflammatory cells.

Successful cell-based tissue engineering requires a rapid and thorough vascularization in order to ensure long-term implant survival and tissue integration. The vascularization of a scaffold is a complex process, and is modulated by the presence of transplanted cells, exogenous and endogenous signaling proteins, and the host tissue reaction, among other influencing factors. This paper presents evidence for the significance of pre-seeded osteoblasts for the in vivo vascularization of a biodegradable scaffold. Human osteoblasts, cultured on silk fibroin micronets in vitro, migrated throughout the interconnected pores of the scaffold and produced extensive bone matrix. When these constructs we…

Generation of human pulmonary microvascular endothelial cell lines.

The limited lifespan of human microvascular endothelial cells in cell culture represents a major obstacle for the study of microvascular pathobiology. To date, no endothelial cell line is available that demonstrates all of the fundamental characteristics of microvascular endothelial cells. We have generated endothelial cell lines from human pulmonary microvascular endothelial cells (HPMEC) isolated from adult donors. HPMEC were cotransfected with a plasmid encoding the catalytic component of telomerase (hTERT) and a plasmid encoding the simian virus 40 (SV40) large T antigen. Cells transfected with either plasmid alone had an extended lifespan, but the cultures eventually entered crisis aft…

Evaluation of the tissue reaction to a new bilayered collagen matrix in vivo and its translation to the clinic.

This study evaluates a new collagen matrix that is designed with a bilayered structure in order to promote guided tissue regeneration and integration within the host tissue. This material induced a mild tissue reaction when assessed in a murine model and was well integrated within the host tissue, persisting in the implantation bed throughout the in vivo study. A more porous layer was rapidly infiltrated by host mesenchymal cells, while a layer designed to be a barrier allowed cell attachment and host tissue integration, but at the same time remained impermeable to invading cells for the first 30 days of the study. The tissue reaction was favorable, and unlike a typical foreign body respons…

Signalling molecules and growth factors for tissue engineering of cartilage-what can we learn from the growth plate?

Modern tissue engineering concepts integrate cells, scaffolds, signalling molecules and growth factors. For the purposes of regenerative medicine, fetal development is of great interest because it is widely accepted that regeneration recapitulates in part developmental processes. In tissue engineering of cartilage the growth plate of the long bone represents an interesting, well-organized developmental structure with a spatial distribution of chondrocytes in different proliferation and differentiation stages, embedded in a scaffold of extracellular matrix components. The proliferation and differentiation of these chondrocytes is regulated by various hormonal and paracrine factors. Thus, mem…

Rapid vascularization of starchâ poly(caprolactone) in vivo by outgrowth endothelial cells in co-culture with primary osteoblasts

The successful integration of in vitro-generated tissues is dependent on adequate vascularization in vivo. Human outgrowth endothelial cells (OECs) isolated from the mononuclear cell fraction of peripheral blood represent a potent population of circulating endothelial progenitors that could provide a cell source for rapid anastomosis and scaffold vascularization. Our previous work with these cells in co-culture with primary human osteoblasts has demonstrated their potential to form perfused vascular structures within a starch–poly(caprolactone) biomaterial in vivo. In the present study, we demonstrate the ability of OECs to form perfused vascular structures as early as 48 h following subcut…

Impact of Ceramic and Metallic Nano-scaled Particles on Endothelial Cell Functionsin vitro

The sections in this article are Introduction Origin of Particles in the Human Environment Evidence for Size-dependent Toxicity of Particles Dissemination and Interferences of Nanoparticles within the Body Endothelial Cells and Nanoparticle Exposure Testing of Nanoparticle-induced Effects on Human Endothelial Cells In Vitro Materials and Methods Cell Culture Particles Transmission Electron Microscopy (TEM) Cytotoxicity Assay Detection of Ki67 Expression Quantification of IL-8 Release in Cell Culture Supernatant Quantification of E-selectin Cell Surface Protein Expression Fluorescence Staining Statistical Analysis Results Discussion Particle Internalization Particle Cytotoxicity Pro-inflamma…

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-li…

Microstructure, mechanical characteristics and cell compatibility of β-tricalcium phosphate reinforced with biodegradable Fe–Mg metal phase

The use of beta-tricalcium phosphate (β-TCP) ceramic as a bioresorbable bone substitute is limited to non-load-bearing sites by the material׳s brittleness and low bending strength. In the present work, new biocompatible β-TCP-based composites with improved mechanical properties were developed via reinforcing the ceramic matrix with 30 vol% of a biodegradable iron-magnesium metallic phase. β-TCP-15Fe15Mg and β-TCP-24Fe6Mg (vol%) composites were fabricated using a combination of high energy attrition milling, cold sintering/high pressure consolidation of powders at room temperature and annealing at 400 °C. The materials synthesized had a hierarchical nanocomposite structure with a nanocrystal…

Flotillin-involved uptake of silica nanoparticles and responses of an alveolar-capillary barrier in vitro

AbstractDrug and gene delivery via nanoparticles across biological barriers such as the alveolar-capillary barrier of the lung constitutes an interesting and increasingly relevant field in nanomedicine. Nevertheless, potential hazardous effects of nanoparticles (NPs) as well as their cellular and systemic fate should be thoroughly examined. Hence, this study was designed to evaluate the effects of amorphous silica NPs (Sicastar) and (poly)organosiloxane NPs (AmOrSil) on the viability and the inflammatory response as well as on the cellular uptake mechanisms and fate in cells of the alveolar barrier. For this purpose, the alveolar epithelial cell line (NCI H441) and microvascular endothelial…

Expression of Matrix-Degrading Cysteine Proteinase Cathepsin K in Cholesteatoma

Cholesteatoma is a nonneoplastic lesion of the middle ear space or mastoid that is histologically characterized by a progressive bone erosion of the ossicles and surrounding bone. Several matrix-degrading enzymes have been implicated as mediators of this bone erosion. Because the novel cysteine proteinase cathepsin K has been shown to play a central role in bone resorption, we examined the expression of this enzyme in tissue specimens of cholesteatoma. Tissue specimens of 9 patients with cholesteatoma were obtained during middle-ear surgery. Expression of cathepsin K mRNA was determined by RT-PCR using specific primers. Immunohistochemical analysis of cathepsin K protein expression in tissu…

Osteonecrosis of the jaws in patients treated with bisphosphonates - histomorphologic analysis in comparison with infected osteoradionecrosis.

Background:  Patients treated with bisphosphonates because of bone metastases have been shown to develop osteonecrosis of the jaws. In the present study, we examined the histologic findings of these cases. As similarities between this disorder and infected osteoradionecrosis (IORN) are described, both lesions were compared. Methods:  We investigated eight patients with bisphosphonate treatment and osteonecrosis (four female, four male; median age: 65.6 years; cancer: multiple myeloma in five patients, breast cancer in three patients; mandibular involvement in five patients, maxillar involvement in three cases), and 10 patients suffering from IORN (all male; median age: 61.3 years; cancer: s…

The Role of Oxidative Stress in the Response of Endothelial Cells to Metals

The involvement of endothelial cells in inflammation and blood vessel formation (angiogenesis) makes them important for the integration of metal implants. Metal degradation products can, however, influence these processes, possibly leading to ineffective wound healing, prolonged inflammation and eventually aseptic loosening of the implant. Different metal degradation processes have been shown to lead to ROS formation. Oxidative stress, therefore, can mediate the reactions of the human body to the implant. While the response of endothelial cells to oxidative stress has been well studied, the effects of ROS produced as the result of metal degradation have not been addressed as yet. Therefore,…

Polyester vascular prostheses coated with a cyclodextrin polymer and activated with antibiotics: Cytotoxicity and microbiological evaluation

Abstract Polyester (PET) vascular grafts are used to replace or bypass damaged arteries. To minimize the risk of infection during and after surgical interventions, a PET vascular prosthesis (Polythese®) was functionalized with cyclodextrin polymers (PolyCDs) in order to obtain the controlled release of antibiotics (ABs: ciprofloxacin, vancomcyin and rifampicin). An epithelial cell line (L132) was used to determine the viability of the antibiotics, and human pulmonary microvascular endothelial cells (HPMEC) were used for cell proliferation by cell counting and cell vitality with Alamar Blue fluorescent dye. Staphylococcus aureus, Escherichia coli and Enteroccocus sp. were used to determine t…

The effects of metal implants on inflammatory and healing processes

Abstract Metal implants are known for their superior mechanical properties. However, cases of implant failure mainly due to aseptic loosening do occur. The formation of particulate wear debris and corrosion products, such as metal ions and reactive oxygen species, are considered to be crucial factors leading to the failure of metal implants. These metal degradation and corrosion products can induce inflammatory responses, mediated among others by neutrophils, macrophages and endothelial cells. Furthermore, these degradation products may affect blood vessel formation, one of the central processes in wound healing after implantation. Such events can lead to the aseptic loosening of implants c…

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…

Uptake and cytotoxicity of citrate-coated gold nanospheres : comparative studies on human endothelial and epithelial cells

Abstract Background The use of gold nanoparticles (AuNPs) for diagnostic applications and for drug and gene-delivery is currently under intensive investigation. For such applications, biocompatibility and the absence of cytotoxicity of AuNPs is essential. Although generally considered as highly biocompatible, previous in vitro studies have shown that cytotoxicity of AuNPs in certain human epithelial cells was observed. In particular, the degree of purification of AuNPs (presence of sodium citrate residues on the particles) was shown to affect the proliferation and induce cytotoxicity in these cells. To expand these studies, we have examined if the effects are related to nanoparticle size (1…

Tumorigenic conversion of endothelial cells.

Tumors of endothelial origin develop rarely. Until now, only two angiosarcoma (AS)-derived endothelial cell lines have been be isolated, ISO-HAS and AS-M. Both AS-derived endothelial cell lines presented the typical endothelial characteristics, such as the expression of CD31 and von Willebrand factor, but differed from normal endothelial cells in a nuclear expression of p53, in a delayed angiogenic reaction, and a reduced expression of caveolin. In addition, differences in the expression of cytokines and cell adhesion molecules responsive to proinflammatory stimuli were observed. While AS-M showed an expression pattern similar to that of human umbilical vein endothelial cells (HUVEC), ISO-H…

Immunogenic properties of renal cell carcinoma and the pathogenesis of osteolytic bone metastases.

The immunogenic properties of renal cell carcinoma (RCC) on bone osteolysis were investigated. mRNA expression of three proinflammatory cytokines, monocyte chemotactic protein-1 (MCP-1), interleukin-6 (IL-6) and interleukin-8 (IL-8), were determined in a panel of RCC lines (CRBM 1990, ACHN and Caki-1). Moreover proinflammatory cytokine mRNA expression and protein levels of adhesion molecules, intercellular adhesion molecule-1 (ICAM-1) and E-selectin, on human umbilical vein endothelial cells (HUVEC) incubated with the conditioned media from RCC lines were evaluated. RCC express mRNA of MCP-1, IL-6 and IL-8 that may induce a proinflammatory phenotype in endothelial cells. mRNA expression of …

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

AbstractEndotoxin, 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)…

The development of a tissue-engineered tracheobronchial epithelial model using a bilayered collagen-hyaluronate scaffold

Today, chronic respiratory disease is one of the leading causes of mortality globally. Epithelial dysfunction can play a central role in its pathophysiology. The development of physiologically-representative in vitro model systems using tissue-engineered constructs might improve our understanding of epithelial tissue and disease. This study sought to engineer a bilayered collagen-hyaluronate (CHyA-B) scaffold for the development of a physiologically-representative 3D in vitro tracheobronchial epithelial co-culture model. CHyA-B scaffolds were fabricated by integrating a thin film top-layer into a porous sub-layer with lyophilisation. The film layer firmly connected to the sub-layer with del…

Implantation of silicon dioxide-based nanocrystalline hydroxyapatite and pure phase beta-tricalciumphosphate bone substitute granules in caprine muscle tissue does not induce new bone formation

Abstract Background Osteoinductive bone substitutes are defined by their ability to induce new bone formation even at heterotopic implantation sites. The present study was designed to analyze the potential osteoinductivity of two different bone substitute materials in caprine muscle tissue. Materials and methods One gram each of either a porous beta-tricalcium phosphate (β-TCP) or an hydroxyapatite/silicon dioxide (HA/SiO2)-based nanocrystalline bone substitute material was implanted in several muscle pouches of goats. The biomaterials were explanted at 29, 91 and 181 days after implantation. Conventional histology and special histochemical stains were performed to detect osteoblast precurs…

Fine-tuning scaffolds for tissue regeneration: effects of formic acid processing on tissue reaction to silk fibroin

Formic acid (FA) plays a key role in the preparation of silk fibroin (SF) scaffolds from cocoons of Bombyx mori and is used for fibre distribution. In this study, we used a subcutaneous implantation model in Wistar rats to examine SF scaffolds prepared by treating the degummed cocoon with FA for either 30 or 60 min. The tissue reaction and inflammatory response to SF was assessed by qualitative histology at intervals from 3 to 180 days. Additionally, dynamic biomaterial-induced vascularization and biomaterial degradation were quantified using a technique for analysing an image of the entire implanted biomaterial. Varying the FA treatment time led to different scaffold morphologies and resul…

Tissue response to five commercially available peritoneal adhesion barriers-A systematic histological evaluation

Separating wounded serosa by physical barriers is the only clinically approved adjunct for postoperative adhesion prevention. Since the optimal adhesion barrier has not been found, it is essential to improve our pathogenic understanding of adhesion formation and to compare the effects of different barrier materials on tissue and cells. Wistar rats underwent standardized peritoneal damage and were treated either with Seprafilm, Adept, Intercoat, Spraygel, SupraSeal or remained untreated as a control. 14 days postoperatively, the lesions were explanted and histomorphologically analyzed using the European ISO score to evaluate material implants. Striking differences between the material groups…

Software-supported image quantification of angiogenesis in an in vitro culture system: application to studies of biocompatibility

Healing of soft tissue trauma and bone discontinuities following implantation involves acute inflammatory reactions and the formation of blood vessels (angiogenesis). During angiogenesis new capillary vessels arise from the existing vasculature. Endothelial cells (EC) are the major cell type involved in angiogenesis. Corrosion of orthopaedic metallic implant materials (e.g. CoCr alloys) can cause locally high concentrations of heavy metal ions in the peri-implant tissues. Some divalent metal ions (Co2+, Ni2+, Zn2+) lead to the activation of EC in vitro. Upon exposure to these ions. EC release cytokines and chemokines and increase the expression of cell surface adhesion molecules, which repr…

Modelling the regenerative niche: a major challenge in biomaterials research

By definition, biomaterials are developed for clinical application. In the field of regenerative medicine their principal function is to play a significant, and, if possible, an instructive role in tissue healing. In the last analysis the latter involves targeting the 'regenerative niche'. The present paper will address the problem of simulating this niche in the laboratory and adopts a life science approach involving the harnessing of heterotypic cellular communication to achieve this, that is, the ability of cells of different types to mutually influence cellular functions. Thus, co-culture systems using human cells are the methodological focus and will concern four exemplary fields of re…

Material‐Induced Cellular Interactions

Biological tolerance of different materials in bulk and nanoparticulate form in a rat model: sarcoma development by nanoparticles

In order to study the pathobiological impact of the nanometre-scale of materials, we evaluated the effects of five different materials as nanoparticulate biomaterials in comparison with bulk samples in contact with living tissues. Five groups out of 10 rats were implanted bilaterally for up to 12 months with materials of the same type, namely TiO 2 , SiO 2 , Ni, Co and polyvinyl chloride (PVC), subcutaneously with bulk material on one side of the vertebral column and intramuscularly with nanoparticulate material on the contralateral side. At the end of each implantation time, the site was macroscopically examined, followed by histological processing according to standard techniques. Malign…

In vitro methodologies to evaluate biocompatibility: status quo and perspective

Abstract The increasing use of biomaterials in clinical medicine to augment or replace failing organ function has heightened the need to apply relevant test systems to study the safety and efficacy of new medical devices. This becomes all the more important as the field of "tissue engineering" develops, in which the aim is to reconstruct tissue and organ function, for example, by using the patient's own cells seeded on to a three-dimensional (3-D) scaffold structure. In the biomaterial research field, there has been a necessary expansion of the concept of biocompatibility to address not only the biosafety issue, that is, the exclusion of cytotoxic and other deleterious effects of biomateria…

LATE-BREAKING ABSTRACT: Comparative studies on bronchuswall-thickness by histologic and computed tomographic measurements of porcine lungs

Aims: Histologic slides are commonly used as template in the evaluation and development of medical imaging methods.Diseases like Asthma and COPD show characteristic changes in airway morphology and airway measurement by computed tomography is a promising diagnostic approach.However,shrinkage caused by fixation and histological processing is known in lung tissue.In this study,the thickness of bronchus walls in paraffin and frozen sections as well as in CT and MicroCT were compared. Methods: Airway measurements of swine lungs were performed after freezing in ventilated condition in liquid nitrogen by measuring the wall thickness of 7 bronchi via CT and MicroCT as well as in frozen and paraffi…

Remodeling of peritoneal-like structures by mesothelial cells: its role in peritoneal healing.

Abstract Background. Intraabdominal adhesions are a common complication following laparotomy. Since the exact mechanisms involved in this processes are unknown we have analyzed in vitro the role of mesothelial cells in peritoneal healing. Material and methods. Human mesothelial cells from omental tissue were cultivated for 2 weeks in a three-dimensional culture either on or in a collagen type I matrix. The effects of blood and collagen matrix were analyzed by exposing mesothelial cells to an overlying blood clot, simulating intraperitoneal bleeding, or a second collagen layer. The production of collagen types III and IV, fibronectin, and laminin was analyzed with immunohistochemical methods…

Ovine Carotid Artery-Derived Cells as an Optimized Supportive Cell Layer in 2-D Capillary Network Assays

PLoS one 9(3), e91664 (2014). doi:10.1371/journal.pone.0091664

Co-culture systems for vascularization — Learning from nature

The endothelial cell (EC) is practically ubiquitous in the human body and forms the inner cellular lining of the entire cardiovascular system. Following tissue injury, the microcirculation becomes the stage for both the inflammatory response and the subsequent healing reaction to restore physiological function to the damaged tissue. The advent of the multidisciplinary field of Regenerative Medicine (RegMed), of which Tissue Engineering (TE) and drug delivery using modern stimuli-responsive or interactive biomaterials are important components, has opened up new approaches to the acceleration of the healing response. A central and rate-limiting role in the latter is played by the process of v…

NanoBioInterface: a multidisciplinary challenge

Nanotechnologies are being applied to many fields of science and engineering and represent a broad spectrum of methods, some of which have been known for a long time. However, the public interest as well as the promotion of this topic by funding agencies has led in some cases to a simple renaming of

Growth of human cells on a non-woven silk fibroin net: a potential for use in tissue engineering.

We have examined a novel biomaterial consisting of a non-woven fibroin net produced from silk (Bombyx mori) cocoons for its ability to support the growth of human cells. Various human cells of different tissue and cell types (endothelial, epithelial, fibroblast, glial, keratinocyte, osteoblast) were examined for adherence and growth on the nets by confocal laser microscopy after staining of the cells with calcein-AM and by electron microscopy. All the cells readily adhered and spread over the individual fibers of the nets. Most of the cells were able to grow and survive on the nets for at least 7 weeks and growth not only covered the individual fibers of the net but generally bridged the ga…

In Vitro Expression of the Endothelial Phenotype: Comparative Study of Primary Isolated Cells and Cell Lines, Including the Novel Cell Line HPMEC-ST1.6R

Endothelial cell lines are commonly used in in vitro studies to avoid problems associated with the use of primary endothelial cells such as the presence of contaminating cells, the difficulty in obtaining larger numbers of cells, as well as the progressive loss of cell viability and expression of endothelial markers in the course of in vitro propagation. We have analyzed the characteristics defining distinctive endothelial phenotypes in the cell lines EA.hy926, ECV304, EVLC2, HAEND, HMEC-1, ISO-HAS-1 and a cell line recently generated in our laboratory, HPMEC-ST1.6R, and have compared these phenotypes with those found in primary human endothelial cells isolated from umbilical vein (HUVEC), …

Atherosklerose - Progression durch unspezifische Aktivierung des Immunsystems

Die Atherosklerose ist eine Erkrankung mittelgroser und groser Gefase. Fruhlasionen sind gekennzeichnet durch die Adhasion und subendotheliale Emigration zirkulierender Monozyten, die als Schaumzellen Wegbereiterfunktionen fur spatere komplexe Plaques ubernehmen. Solche Lasionen sind bereits im Kindes- und Jugendalter zu finden. Der nachste Schritt der Atherogenese ist die Proliferation glattmuskularer Zellen innerhalb der Intima und der Media und damit ein gradueller Verschluss des Gefaslumens. Fibroblastare Zellen tragen ferner zum Lasionswachstum bei, indem sie exzessive Mengen extrazellularer Matrix bilden. Derartige Lasionen sind meist klinisch inapparent. In einer nachsten Phase kommt…

Gold nanoparticles induce cytotoxicity in the alveolar type-II cell lines A549 and NCIH441.

Abstract Background During the last years engineered nanoparticles (NPs) have been extensively used in different technologies and consequently many questions have arisen about the risk and the impact on human health following exposure to nanoparticles. Nevertheless, at present knowledge about the cytotoxicity induced by NPs is still largely incomplete. In this context, we have investigated the cytotoxicity induced by gold nanoparticles (AuNPs), which differed in size and purification grade (presence or absence of sodium citrate residues on the particle surface) in vitro, in the human alveolar type-II (ATII)-like cell lines A549 and NCIH441. Results We found that the presence of sodium citra…

An impaired alveolar-capillary barrier in vitro : effect of proinflammatory cytokines and consequences on nanocarrier interaction.

The alveolar region of the lung is an important target for drug and gene delivery approaches. Treatment with drugs is often necessary under pathophysiological conditions, in which there is acute inflammation of the target organ. Therefore, in vitro models of the alveolar-capillary barrier, which mimic inflammatory conditions in the alveolar region, would be useful to analyse and predict effects of novel drugs on healthy or inflamed tissues. The epithelial cell line H441 was cultivated with primary isolated human pulmonary microvascular endothelial cells (HPMECs) or the endothelial cell line ISO-HAS-1 on opposite sides of a permeable filter support under physiological and inflammatory condi…

Collagen-embedded hydroxylapatite–beta-tricalcium phosphate–silicon dioxide bone substitute granules assist rapid vascularization and promote cell growth

In the present study we assessed the biocompatibility in vitro and in vivo of a low-temperature sol-gel-manufactured SiO(2)-based bone graft substitute. Human primary osteoblasts and the osteoblastic cell line, MG63, cultured on the SiO(2) biomatrix in monoculture retained their osteoblastic morphology and cellular functionality in vitro. The effect of the biomaterial in vivo and its vascularization potential was tested subcutaneously in Wistar rats and demonstrated both rapid vascularization and good integration within the peri-implant tissue. Scaffold degradation was progressive during the first month after implantation, with tartrate-resistant acid phosphatase-positive macrophages being …

Western blotting as a method for studying cell-biomaterial interactions: The role of protein collection

Research of cell-biomaterial interactions is building on knowledge and methods available in cell and molecular biology. Western blotting is one of the options to characterize protein expression in cell populations. Method transfer to biomaterial model systems is not trivial because of the structure that exists in many biomaterials, preventing the collection of cell lysate by mechanical means. In this technical report, we describe the influence of different protein collection methods in a model system for cell-biomaterial interactions, consisting of endothelial cells exposed to different stimuli. In particular, the influence of trypsinization before lysis, and handling complexity were determ…

Expression of the endothelial markers PECAM-1, vWf, and CD34 in vivo and in vitro.

EC culture models are essential to study pathological alterations of endothelial cells (ECs) in pulmonary vascular diseases under standardized conditions. Nevertheless, little is known about the spectrum of alterations of vessel-specific endothelial phenotypes in monolayer cultures. For the comparative study of endothelial markers in vivo and in vitro we investigated immunohistochemically the expression of PECAM-1, vWf, and CD34 by pulmonary ECs in vivo and in stimulated/unstimulated human umbilical vein endothelial cells (HU-VEC) and human pulmonary microvascular endothelial cells (HPMEC). In vivo, vessel type-specific expression patterns were found for vWf and CD34, while PECAM-1 was homo…

Biological performance of cell-encapsulated methacrylated gellan gum-based hydrogels for nucleus pulposus regeneration

Limitations of current treatments for intervertebral disc (IVD) degeneration have promoted interest in the development of tissue-engineering approaches. Injectable hydrogels loaded with cells can be used as a substitute material for the inner IVD part, the nucleus pulposus (NP), and provide an opportunity for minimally invasive treatment of IVD degeneration. The NP is populated by chondrocyte-like cells; therefore, chondrocytes and mesenchymal stem cells (MSCs), stimulated to differentiate along the chondrogenic lineage, could be used to promote NP regeneration. In this study, the in vitro and in vivo response of human bone marrow-derived MSCs and nasal chondrocytes (NCs) to modified gellan…

Tomographic and Topographic Investigation of Poly-D,L-Lactide-Co-Glycolide Microspheres Loaded with Prostaglandine E&lt;sub&gt;2&lt;/sub&gt; for Extended Drug Release Applications

Polymeric, biodegradable microspheres represent a good reliable system to investigate the release of bioactive substances in both in vitro and in vivo applications. Common biomaterials for the synthesis of these microspheres are aliphatic polyesters of the poly(α-hydroxy)acids, especially poly-L-lactides (PLA) and polyglycolides (PGA) or their copolymers poly-D,L-lactide-co-glycolides (PLGA). In our own previous studies we have developed PLGA microspheres with integrated PGE2 as model substance for a wide range of biomedical applications, especially in angiogenesis, fracture healing and cartilage repair. The synthesis is based on a binary solvent in water emulsion approach, where two differ…

Crossing frontiers in biomaterials and regenerative medicine

From 28 May–1 June 2008, the 8th World Biomaterials Congress (WBC) was held in Amsterdam, The Netherlands. The meeting attracted 3052 participants from 82 countries, which has made this congress the largest meeting ever held in the field of biomaterials and regenerative medicine. More than 650 oral and 1500 poster contributions [1] were presented in 108 scientific sessions, divided into nine parallel sessions, including 48 dedicated symposia that had been proposed by international leaders in the field. The symposia each contained an invited keynote, providing expert opinions on current developments in biomaterials research. Historically, research in the field of biomaterials has focused on …

Biomaterial-Induced Sarcoma

In the study of carcinogenesis most interest has focused on carcinomas, as they represent the majority of human cancers. The recognition of the adenoma-carcinoma sequence both in humans and in animal experimental models has given the field of basic oncology the opportunity to elucidate individual mechanisms in the multistep development of carcinoma. The relative scarcity of human sarcomas coupled with the lack of adequate animal models has hampered understanding of the molecular genetic steps involved. We present an experimental model in the rat in which a high incidence of malignant mesenchymal tumors arise around a subcutaneously implanted biomaterial. Nine commercially available biomater…

Mechanisms of cell activation by heavy metal ions

Heavy metal ions can be released by corroding metallic implants into the surrounding tissue. When they enter blood vessels some of them are carried by proteins like albumin and can be taken up by endothelial cells lining the vessels. To study their involvement in the inflammatory response we investigated heavy metal ion induced effects in cultured human vascular endothelial cells (HUVECs). NiCl2 and CoCl2 upregulate, especially in concentrations of 1 mM, the expression of adhesion molecules (e.g., E-selectin and intercellular adhesion molecule-1), as well as the cytokines IL-6 and IL-8, as shown by enzyme immunoassay and Northern blot analysis. In addition, possible signal transduction mech…

In vitro methodologies to evaluate biocompatibility: Status quo and perspectives

Visions for regenerative medicine: interface between scientific fact and science fiction.

This article gives a brief overview of the authors' views on the future development of tissue engineering with respect to the challenges both to the materials and life sciences. Emphasis will be placed on the advantages of three-dimensional bioresorbable polymers in combination with relevant molecular cues and the application of autologous stem or progenitor cells. There is a requirement for much more diversity in the synthesis of so-called "intelligent" materials, which respond to external stimuli, as well as the development of novel drug and gene delivery systems. In addition, much more basic research is necessary in developmental biology and the application of modern cell and molecular b…

Collagen-low molecular weight hyaluronic acid semi-interpenetrating network loaded with gelatin microspheres for cell and growth factor delivery for nucleus pulposus regeneration.

Intervertebral disc (IVD) degeneration is one of the main causes of low back pain. Current surgical treatments are complex and generally do not fully restore spine mobility. Development of injectable extracellular matrix-based hydrogels offers an opportunity for minimally invasive treatment of IVD degeneration. Here we analyze a specific formulation of collagen-low molecular weight hyaluronic acid (LMW HA) semi-interpenetrating network (semi-IPN) loaded with gelatin microspheres as a potential material for tissue engineering of the inner part of the IVD, the nucleus pulposus (NP). The material displayed a gel-like behavior, it was easily injectable as demonstrated by suitable tests and did …

The effect of human osteoblasts on proliferation and neo-vessel formation of human umbilical vein endothelial cells in a long-term 3D co-culture on polyurethane scaffolds

Angiogenesis is a key element in early wound healing and is considered important for tissue regeneration and for directing inflammatory cells to the wound site. The improvement of vascularization by implementation of endothelial cells or angiogenic growth factors may represent a key solution for engineering bone constructs of large size. In this study, we describe a long-term culture environment that supports the survival, proliferation, and in vitro vasculogenesis of human umbilical vein endothelial cells (HUVEC). This condition can be achieved in a co-culture model of HUVEC and primary human osteoblasts (hOB) employing polyurethane scaffolds and platelet-rich plasma in a static microenvir…

Identification of neuronal and angiogenic growth factors in an in vitro blood-brain barrier model system: Relevance in barrier integrity and tight junction formation and complexity.

We previously demonstrated that the co-cultivation of endothelial cells with neural cells resulted in an improved integrity of the in vitro blood-brain barrier (BBB), and that this model could be useful to evaluate the transport properties of potential central nervous system disease drugs through the microvascular brain endothelial. In this study we have used real-time PCR, fluorescent microscopy, protein arrays and enzyme-linked immunosorbent assays to determine which neural- and endothelial cell-derived factors are produced in the co-culture and improve the integrity of the BBB. In addition, a further improvement of the BBB integrity was achieved by adjusting serum concentrations and grow…

Expression and function of the non-neuronal cholinergic system in endothelial cells

Increasing evidence has shown the expression of the non-neuronal cholinergic system in endothelial cells. In the present experiments the expression of choline acetyltransferase (ChAT) was investigated in human endothelial cells by anti-ChAT immunohistochemistry and anti-ChAT immunofluorescence. Positive ChAT immunoreactivity was found in cultures of human umbilical endothelial cells (HUVEC) and a human angiosarcoma cell line (HAEND). In HUVEC and HAEND choline acetyltransferase activity and small amounts of acetylcholine were also detected. Positive ChAT-immunoreactivity was demonstrated in situ in endothelial cells of the human umbilical cord. In addition, in experiments with confocal lase…

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 vasculatu…

Characterization of oriented protein-ceramic and protein-polymer-composites for cartilage tissue engineering using synchrotron μ-CT

Abstract In this paper we report on the synthesis of three different gelatine based scaffold materials for the reconstruction of articular cartilage defects. The first scaffold design is based on an unmodified, oriented gelatine network, while the second design further comprises an attached inorganic hydroxyapatite layer and the third design includes poly(l-lactide) microspheres as a model material for future drug-release applications. All three scaffold designs were characterized and imaged using synchrotron μ-CT, obtaining a complete volumetric reconstruction of a previously defined sample region. Furthermore, two unmodified scaffolds were cultivated for one week with porcine chondrocytes…

Analysis of the Biological Response of Endothelial and Fibroblast Cells Cultured on Synthetic Scaffolds with Various Hydrophilic/Hydrophobic Ratios: Influence of Fibronectin Adsorption and Conformation

In this study we developed polymer scaffolds intended as anchorage rings for cornea prostheses among other applications, and examined their cell compatibility. In particular, a series of interconnected porous polymer scaffolds with pore sizes from 80 to 110 microns were manufactured varying the ratio of hydrophobic to hydrophilic monomeric units along the polymer chains. Further, the effects of fibronectin precoating, a physiological adhesion molecule, were tested. The interactions between the normal human fibroblast cell line MRC-5 and primary human umbilical vein endothelial cells (HUVECs) with the scaffold surfaces were evaluated. Adhesion and growth of the cells was examined by confocal…

Biocompatibility studies of endothelial cells on a novel calcium phosphate/SiO 2 -xerogel composite for bone tissue engineering

The bone biomaterial BONITmatrix®, a nanoporous, granular scaffold composed of hydroxylapatite, calcium phosphate and SiO2, linked by a dense collagen mesh, was tested for its biocompatibility using endothelial cells (EC) in the form of macrovascular HUVEC, microvascular HDMEC and the endothelial cell line ISOHAS-1. Cells were examined for their adherence and growth on the biomaterial and this was followed by confocal laser scanning microscopy after vital staining or immunocytochemical reactions, as well as by scanning electron microscopy. Macro- and microvascular ECs predominantly spread on BONITmatrix®-collagen mesh-covered surfaces and fibres and maintained their typical morphology. As E…

Uptake of poly(2-hydroxypropylmethacrylamide)-coated gold nanoparticles in microvascular endothelial cells and transport across the blood-brain barrier.

The facile and modular functionalization of gold nanoparticles makes them versatile tools in nanomedicine, for instance, photothermal therapy, contrast agents or as model nanoparticles to probe drug-delivery mechanisms. Since endothelial cells from various locations in the body exhibit unique phenotypes we quantitatively examined the amount of different sized poly(2-hydroxypropylmethacrylamide)-coated gold nanoparticles internalized into primary human dermal endothelial cells or human brain endothelial cells (hCMEC/D3) by inductively coupled plasma atomic emission spectroscopy (ICP-AES) and visualized the nanoparticles using light and electron microscopy. Poly(2-hydroxypropylmethacrylamide)…

Comparative Study of Adhesion Molecule Expression in Cultured Human Macro- and Microvascular Endothelial Cells

Culture systems as models for disease are only valid as long as they are comparable to in vivo conditions. The phenotype of cultured endothelial cells (ECs) has only been sporadically compared to the corresponding phenotype in vivo. Thus, we compared by immunolocalization the endothelial expression of ICAM-1, VCAM, and E-selectin in vivo in stimulated/unstimulated human umbilical vein endothelial cells (HUVEC) as a model for macrovascular ECs and stimulated/unstimulated HPMEC (human pulmonary microvessel endothelial cells) as a model for pulmonary microvascular ECs with that in human lungs in vivo (normal and ARDS). Proinflammatory stimuli in vitro were used to stimulate conditions relevant…

Influence of β-tricalcium phosphate granule size and morphology on tissue reaction in vivo.

In this study the tissue reaction to five different β-tricalcium phosphate (β-TCP)-based bone substitute materials differing only in size, shape and porosity was analyzed over 60 days, at 3, 10, 15, 30 and 60 days after implantation. Using the subcutaneous implantation model in Wistar rats both the inflammatory response within the implantation bed and the resulting vascularization of the biomaterials were qualitatively and quantitatively assessed by means of standard and special histological staining methods. The data from this study showed that all investigated β-TCP bone substitutes induced the formation of multinucleated giant cells. Changes in size, shape and porosity influenced the int…

Addition of blood to a phycogenic bone substitute leads to increased in vivo vascularization

The present study aimed to analyze the effects of the addition of blood to the phycogenic bone substitute Algipore(®) on the severity of in vivo tissue reaction. Initially, Fourier-transform infrared spectroscopy (FTIR) of the bone substitute was conducted to analyze its chemical composition. The subcutaneous implantation model in Balb/c mice was then applied for up to 30 d to analyze the tissue reactions on the basis of specialized histochemical, immunohistochemical, and histomorphometrical methods. The data of the FTIR analysis showed that the phycogenic bone substitute material is mainly composed of hydroxyapatite with some carbonate content. The in vivo analyses revealed that the additi…

Tissue expansion of lung bronchi due to tissue processing for histology – A comparative analysis of paraffin versus frozen sections in a pig model

Tissue shrinking due to fixation and processing is well known. However, the degree of shrinking varies significantly with the tissue type as well as the processing method and is not well studied in various tissues. In daily pathological routine workflow, histological specimens from frozen and paraffin sections are performed from the same tissue. In the present study we compared the thickness of bronchus walls obtained from paraffin and frozen sections.Pig lungs were frozen in ventilated condition in liquid nitrogen and 36 bronchi were isolated after dissection. Frozen sections of 5 μm thickness were performed and the remaining tissue was fixed and embedded in paraffin after fixation in 4% f…

Interactions of silica nanoparticles with lung epithelial cells and the association to flotillins

Amorphous silica nanoparticles (aSNPs) gain increasing popularity for industrial and therapeutic claims. The lung with its surface area of 100-140 m(2) displays an ideal target for therapeutic approaches, but it represents also a serious area of attack for harmful nanomaterials. The exact nature of the cytotoxic effects of NPs is still unknown. Furthermore, cellular pathways and the destiny of internalized NPs are still poorly understood. Therefore, we examined the cytotoxicity (MTS, LDH) and inflammatory responses (IL-8) for different-sized aSNPs (30, 70, 300 nm) on our lung epithelial cells line NCI H441 and endothelial cell line ISO-HAS-1. Additionally, colocalization studies have been c…

An in vitro and in vivo study of peptide-functionalized nanoparticles for brain targeting: The importance of selective blood-brain barrier uptake

Targeted delivery of drugs across endothelial barriers remains a formidable challenge, especially in the case of the brain, where the blood-brain barrier severely limits entry of drugs into the central nervous system. Nanoparticle-mediated transport of peptide/protein-based drugs across endothelial barriers shows great potential as a therapeutic strategy in a wide variety of diseases. Functionalizing nanoparticles with peptides allows for more efficient targeting to specific organs. We have evaluated the hemocompatibilty, cytotoxicity, endothelial uptake, efficacy of delivery and safety of liposome, hyperbranched polyester, poly(glycidol) and acrylamide-based nanoparticles functionalized wi…

Prostaglandin E2 (PGE2) plays an important role in bone development and metabolism. To interfere therapeutically in the PGE2 pathway, however, knowledge about the involved enzymes (cyclooxygenases) and receptors (PGE2 receptors) is essential. We therefore examined the production of PGE2 in cultured growth plate chondrocytes in vitro and the effects of exogenously added PGE2 on cell proliferation. Furthermore, we analysed the expression and spatial distribution of cyclooxygenase (COX)-1 and COX-2 and PGE2 receptor types EP1, EP2, EP3 and EP4 in the growth plate in situ and in vitro. PGE2 synthesis was determined by mass spectrometry, cell proliferation by DNA [3H]-thymidine incorporation, mR…

Endothelial cells stimulate osteogenic differentiation of mesenchymal stem cells on calcium phosphate scaffolds

The interaction of mesenchymal stem cells (MSCs) with endothelium in vivo is significant for regenerative processes in organisms. To design concepts for tissue engineering for bone regeneration based on this interaction, the osteogenic differentiation of human bone marrow-derived MSCs in a co-culture with human dermal microvascular endothelial cells (HDMECs) was studied. The experiments were focussed on the regulation of MSCs in a co-culture with HDMECs on different calcium phosphate scaffolds. Alkaline phosphatase (ALP) activity and mRNA expression of various osteogenic markers increased significantly when cells were co-cultured on materials with calcium phosphate scaffolds compared to tis…

Response of micro- and macrovascular endothelial cells to starch-based fiber meshes for bone tissue engineering.

The establishment of a functional vasculature is as yet an unrealized milestone in bone reconstruction therapy. For this study, fiber-mesh scaffolds obtained from a blend of starch and poly(caprolactone) (SPCL), that have previously been shown to be an excellent material for the proliferation and differentiation of bone marrow cells and thereby represent great potential as constructs for bone regeneration, were examined for endothelial cell (EC) compatibility. To be successfully applied in vivo, this tissue engineered construct should also be able to support the growth of ECs in order to facilitate vascularization and therefore assure the viability of the construct upon implantation. The ma…

Implanted neonatal human dermal fibroblasts influence the recruitment of endothelial cells in mice

The vascularization of new tissue within a reasonable time is a crucial prerequisite for the success of different cell- and material-based strategies. Considering that angiogenesis is a multi-step process involving humoral and cellular regulatory components, only in vivo assays provide the adequate information about vessel formation and the recruitment of endothelial cells. The present study aimed to investigate if neonatal human dermal fibroblasts could influence in vivo neovascularization. Results obtained showed that fibroblasts were able to recruit endothelial cells to vascularize the implanted matrix, which was further colonized by murine functional blood vessels after one week. The ve…

Response of human endothelial cells to oxidative stress on Ti6Al4V alloy.

Titanium and its alloys are amongst the most frequently used materials in bone and dental implantology. The good biocompatibility of titanium(-alloys) is attributed to the formation of a titanium oxide layer on the implant surface. However, implant failures do occur and this appears to be due to titanium corrosion. Thus, cells participating in the wound healing processes around an implanted material, among them endothelial cells, might be subjected to reactive oxygen species (ROS) formed by electrochemical processes during titanium corrosion. Therefore, we studied the response of endothelial cells grown on Ti6Al4V alloy to H(2)O(2) and compared this with the response of endothelial cells gr…

Emulsion-based synthesis of PLGA-microspheres for the in vitro expansion of porcine chondrocytes.

Abstract The in vitro cell expansion of autologous chondrocytes is of high interest in regenerative medicine since these cells can be used to treat joint cartilage defects. In order to preserve chondrocyte phenotype, while optimizing adhesion on microspheres, several processing parameters for the microsphere synthesis were varied. In this study three different polylactide-co-glycolides were used with differing lactide–glycolide ratios (85:15 and 50:50) and differing inherent viscosities. An emulsion route was established, where the polymer was dissolved in chloroform and then injected into a stirred polyvinyl alcohol–water solution at different polymer concentrations and different stirring …

Impact of polymer-modified gold nanoparticles on brain endothelial cells: exclusion of endoplasmic reticulum stress as a potential risk factor

A library of polymer-coated gold nanoparticles (AuNPs) differing in size and surface modifications was examined for uptake and induction of cellular stress responses in the endoplasmic reticulum (ER stress) in human brain endothelial cells (hCMEC/D3). ER stress is known to affect the physiology of endothelial cells (ECs) and may lead to inflammation or apoptosis. Thus, even if applied at non-cytotoxic concentrations ER stress caused by nanoparticles should be prevented to reduce the risk of vascular diseases and negative effects on the integrity of barriers (e.g. blood–brain barrier). We exposed hCMEC/D3 to twelve different AuNPs (three sizes: 18, 35, and 65 nm, each with four surface-modif…