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…

Development and in vitro Evaluation of Antigen-Loaded Poly(amidoamine) Nanoparticles for Respiratory Epithelium Applications

A poly(amidoamine) with disulfide linkages in the main chain and 4-hydroxybutyl and ω-carboxy-PEG groups (9:1 ratio) as side chains was prepared by Michael addition polymerization of cystamine bisacrylamide with 4-hydroxybutylamine and ω-carboxy-PEG-amine. To develop therapeutic protein formulations for improved delivery of antigen via the intranasal route, nanoparticles were prepared from this polymer by self-assembly with p24 or ovalbumin as the model proteins and CpG as the adjuvant. The nanoparticles incorporated the antigens and adjuvant from the feed solution with high efficiency (∼90 %) and have sizes of 112 and 169 nm, respectively, with low positive surface charge (∼+2 mV). Formula…

An injectable bone substitute composed of beta-tricalcium phosphate granules, methylcellulose and hyaluronic acid inhibits connective tissue influx into its implantation bed in vivo

In this study, the in vivo tissue reaction to a new triphasic and injectable paste-like bone-substitute material composed of beta-tricalcium phosphate (β-TCP), methylcellulose and hyaluronic acid was analyzed. Using a subcutaneous implantation model, the interaction of these materials and the peri-implant tissue reaction were tested in Wistar rats for up to 60 days by means of established histological methods, including histomorphometrical analysis. The study focused on tissue integration, classification of the cellular inflammatory response and the degradation of the material. Groups composed of animals injected only with β-TCP granules, sham-operated animals and animals injected with sali…

Tissue reaction to sealing materials: different view at biocompatibility

Abstract The biodegradability of root canal sealers in areas other than the root canal system is crucial to the overall success rate of endodontic treatment. The aim of the present study was to investigate, the cell and tissue reaction to GuttaFlow and AHPlus, both in vitro and in vivo. For the in vitro experiments the materials were incubated with Human Periodontal Ligament Fibroblasts and cell proliferation and cytotoxicity analyses were performed. Additional fluorescence-microscope stainings were carried out in order to visualize cell growth and morphology. For assessment of the tissue reaction to the materials a subcutaneous implantation model in Wistar rats was employed and the inflamm…

Tissue engineered pre-vascularized buccal mucosa equivalents utilizing a primary triculture of epithelial cells, endothelial cells and fibroblasts

Artificial generated buccal mucosa equivalents are a promising approach for the reconstruction of urethral defects. Limiting in this approach is a poor blood vessel supply after transplantation, resulting in increased morbidity and necrosis. We generated a pre-vascularized buccal mucosa equivalent in a tri-culture of primary buccal epithelial cells, fibroblasts and microvascular endothelial cells, using a native collagen membrane as a scaffold. A successful pre-vascularization and dense formation of capillary-like structures at superficial areas was demonstrated. The lumen size of pre-formed blood vessels corresponded to the capillary size in vivo (10-30 μm). Comparing native with a highly …

Nanostructured medical sutures with antibacterial properties

Bacterial repellence in suture materials is a desirable property that can potentially improve the healing process by preventing infection. We describe a method for generating nanostructures at the surface of commercial sutures of different composition, and their potential for preventing biofilm formation. We show how bacteria attachment is altered in the presence of nanosized topographies and identify optimum designs for preventing it without compromising biocompatibility and applicability in terms of nanostructure robustness or tissue friction. These studies open new possibilities for flexible and cost-effective realization of topography-based antibacterial coatings for absorbable biomedic…

Growth of human cells on polyethersulfone (PES) hollow fiber membranes.

A novel material of porous hollow fibers made of polyethersulfone (PES) was examined for its ability to support the growth of human cells. This material was made in the absence of solvents and had pore diameters smaller than 100 microm. Human cell lines of different tissue and cell types (endothelial, epithelial, fibroblast, glial, keratinocyte, osteoblast) were investigated for adherence, growth, spread and survival on PES by confocal laser microscopy after staining of the cells with Calcein-AM. Endothelial cell attachment and growth required pre-coating PES with either fibronectin or gelatin. The other cell types exhibited little difference in growth, spread or survival on coated or uncoa…

Improving cytocompatibility of Co28Cr6Mo by TiO 2 coating: gene expression study in human endothelial cells

Cobalt-based materials are widely used for coronary stents, as well as bone and joint implants. However, their use is associated with high corrosion incidence. Titanium alloys, by contrast, are more biocompatible owing to the formation of a relatively inactive titanium oxide (TiO 2 ) layer on their surface. This study was aimed at improving Co28Cr6Mo alloy cytocompatibility via sol–gel TiO 2 coating to reduce metal corrosion and metal ion release. Owing to their role in inflammation and tissue remodelling around an implant, endothelial cells present a suitable in vitro model for testing the biological response to metallic materials. Primary human endothelial cells seeded on Co28Cr6Mo showe…

Design and physicochemical characterization of poly(amidoamine) nanoparticles and the toxicological evaluation in human endothelial cells: applications to peptide delivery to the brain

In this study, we investigated nanoparticles formulated by self-assembly of a biodegradable poly(amidoamine) (PAA) and a fluorescently labeled peptide, in their capacity to internalize in endothelial cells and deliver the peptide, with possible applications for brain drug delivery. The nanoparticles were characterized in terms of size, surface charge, and loading efficiency, and were applied on human cerebral microvascular endothelial cells (hCMEC/D3) and human umbilical vein endothelial cells (Huvec) cells. Cell-internalization and cytotoxicity experiments showed that the PAA-based nanocomplexes were essentially nontoxic, and the peptide was successfully internalized into cells. The result…

Surface-modified 3D starch-based scaffold for improved endothelialization for bone tissue engineering

Providing adequate vascularization is one of the main hurdles to the widespread clinical application of bone tissue engineering approaches. Due to their unique role in blood vessel formation, endothelial cells (EC) play a key role in the establishment of successful vascularization strategies. However, currently available polymeric materials do not generally support EC growth without coating with adhesive proteins. In this work we present argon plasma treatment as a suitable method to render the surface of a 3D starch-based scaffold compatible for ECs, this way obviating the need for protein pre-coating. To this end we studied the effect of plasma modification on surface properties, protein …

Tissue response and biomaterial integration: the efficacy of in vitro methods

Implantation involves tissue trauma, which evokes an inflammatory response, coupled to a wound healing reaction, involving angiogenesis, fibroblast activation and matrix remodelling. Until now the type and extent of such reactions to give optimal integration of various biomaterials are practically unknown. Three principal fields of research can yield useful data to understand these phenomena better: studies on explanted biomaterials, animal models and relevant in vitro techniques. This paper will present examples of the latter field and the application of endothelial cell (EC) culture systems to study the effects of important tissue (e.g. pro-inflammatory cytokines, chemokines) and material…

Monocyte preseeding leads to an increased implant bed vascularization of biphasic calcium phosphate bone substitutes via vessel maturation

The present study analyzes the influence of the addition of monocytes to a biphasic bone substitute with two granule sizes (400-700 μm and 500-1000 μm). The majority of the added monocytes was detectable as mononuclear cells, while also low amounts of (chimeric) multinucleated giant cells (MNGCs) were found. No increase in the total number of MNGCs was established, but a significantly increased percent vascularization. Altogether, the results show that the added monocytes become involved in the tissue response to a biomaterial without marked changes in the overall reaction. Monocyte addition enables an increased implant bed vascularization especially via induction of vessel maturation and, …

Endothelialization of a non-woven silk fibroin net for use in tissue engineering: growth and gene regulation of human endothelial cells

Abstract We have previously shown that a biomaterial consisting of a non-woven fibroin net produced from silk (Bombyx mori) cocoons is an excellent scaffolding material for a wide variety of human cells of different tissue types. Endothelialization must take place for a biomaterial to be successful after implantation. Therefore, primary human endothelial cells and the human endothelial cell lines, HPMEC-ST1.6R and ISO-HAS-1, were examined for adherence and growth patterns on the fibroin nets by confocal laser scanning microscopy after vital staining of the cells and by electron microscopy. Endothelial cells adhered and spread along individual fibers of the nets and did not fill the gaps bet…

Bioresponsive poly(amidoamine)s designed for intracellular protein delivery.

Poly(amidoamine)s with bioreducible disulfide linkages in the main chain (SS-PAAs) and pH-responsive, negatively charged citraconate groups in the sidechain have been designed for effective intracellular delivery and release of proteins with a net positive charge at neutral pH. Using lysozyme as a cationic model protein these water soluble polymers efficiently self-assemble into nanocomplexes by charge attraction. At pH 5 (the endosomal pH) the amide linkages connecting the citraconate groups in the sidechains of the SS-PAAs are hydrolyzed by intramolecular catalysis, resulting in expulsion of the negative citraconate groups and formation of protonated amine groups, resulting in charge reve…

Cell Culture Systems for Studying Biomaterial Interactions with Biological Barriers

The human body has numerous physical barriers that prevent most harmful or foreign compounds from entering the body. These barriers are formed by unique cell types, which through their location-specific biological cell characteristics prevent compounds from passing between or through them or selectively allow only specific compounds to move across the barrier that they form. Multiple cell types are involved that together form the functioning barrier in a particular organ or tissue. In many cases, in vitro human multicellular culture systems have been developed. These in vitro cell culture models have been extremely valuable in determining the toxic effects of novel compounds on cellular fun…

Porous Gelatin Hydrogels:  2. In Vitro Cell Interaction Study

We report on the feasibility of applying porous gelatin hydrogels, prepared by a novel and controlled cryogenic treatment, as cell-interactive scaffolds for tissue engineering applications. Despite the large number of publications on gelatin as a biomaterial, a detailed study of screening a limited number of gelatin scaffolds for their interaction with a panel of human cells has, to the best of our knowledge, not yet been published. In the present work, we have evaluated two types of porous gelatin scaffolds that differ in their pore geometry and pore size. Type I hydrogels contained top-to-bottom transverse channels (i.e. cones) with a decreasing diameter from the top (330 microm) to the b…

Phenotypic redifferentiation and cell cluster formation of cultured human articular chondrocytes in a three-dimensional oriented gelatin scaffold in the presence of PGE2- first results of a pilot study

Modern tissue engineering strategies comprise three elemental parameters: cells, scaffolds and growth factors. Articular cartilage represents a highly specialized tissue which allows frictionless gliding of corresponding articulating surfaces. As the regenerative potential of cartilage is low, tissue engineering-based strategies for cartilage regeneration represent a huge challenge. Prostaglandins function as regulators in cartilage development and metabolism, especially in growth plate chondrocytes. In this study, it was analyzed if prostaglandin E2 (PGE2) has an effect on the phenotypic differentiation of human chondrocytes cultured in a three-dimensional (3D) gelatin-based scaffold made …

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 examp…

Porous gelatin cryogels as cell delivery tool in tissue engineering.