Open Access

Besides their extracellular activity crucial for several pathophysiological conditions, human cysteine cathepsins, in particular cathepsins K and S, represent important intracellular targets for drug development. In the present study, a prototypic dipeptide nitrile inhibitor structure was equipped with a coumarin moiety to function as a fluorescent reporter group. In a second inhibitor, a PEG linker was introduced between the dipeptide nitrile and the fluorophore. These tool compounds 6 and 7 were characterized by kinetic investigations as covalent reversible inhibitors of human cathepsins L, S, K and B. Probe 6 showed a pronounced inhibitory activity against cathepsins K and S, which was corroborated by modeling of inhibition modes. Probe 7 was highly potent (Ki = 93 nM) and selective for cathepsin S. To examine the ability of both probes to enter living cells, human embryonic kidney 293 cells were targeted. At a concentration of 10 µM, cellular uptake of probe 6 was demonstrated by fluorescence measurement after an incubation time of 30 min and 3 h, respectively. The probe’s concentration in cell lysates was ascertained on the basis of the emission at 492 nm upon excitation at 450 nm, and the results were expressed as concentrations of probe 6 relative to the protein concentration originating from the lysate. After incubation of 10 µM of probe 6 for 3 h, the cellular uptake was confirmed by fluorescence microscopy. HPLC was used to assess the probes’ lipophilicity, and the obtained log D7.4 value of 2.65 for probe 6 was in agreement with the demonstrated cellular uptake.

Three-dimensional scaffolds are known to directly influence proliferation and differentiation of mesenchymal stem cells into bone tissue due to their properties such as stiffness and topography. While conventional methods for chemical induction of differentiation processes are based on incorporation of growth factors and/or cytokines via blending or adhesion onto the scaffold surfaces, novel approaches use template-mediated biomineralization to mimic the stimuli stem cells receive in their natural niche. This chapter summarizes recent progresses in guided bone tissue engineering with particular focus on design and functionality of three dimensional scaffolds, chemical templates and promising approaches for the corresponding cellbased approaches for future therapies.

Background and Objectives: In advanced β-cell dysfunction, proinsulin is increasingly replacing insulin as major component of the secretion product. It has been speculated that proinsulin has at least the same adipogenic potency than insulin, leading to an increased tendency of lipid tissue formation in patients with late stage β-cell dysfunction. Methods and Results: Mesenchymal stem cells obtained from liposuction material were grown in differentiation media containing insulin (0.01 μmol), proinsulin (0.01 μmol) or insulin+proinsulin (each 0.005 μmol). Cell culture supernatants were taken from these experiments and an untreated control at weeks 1, 2, and 3, and were stored at -80°C until analysis. Cell differentiation was microscopically supervised and adiponectin concentrations were measured as marker for differentiation into mature lipid cells. This experiment was repeated three times. No growth of lipid cells and no change in adiponectin values was observed in the negative control group (after 7/14/12 days: 3.2±0.5/3.3±0.1/4.4±0.5 ng/ml/12 h). A continuous differentiation into mature adipocytes (also confirmed by Red-Oil-staining) and a corresponding increase in adiponectin values was observed in the experiments with insulin (3.6±1.9/5.1±1.4/13.3±1.5 ng/ml/12 h; p<0.05 week 1 vs. week 3) and proinsulin (3.3±1.2/3.5±0.3/12.2±1.2 ng/ml/12 h; p<0.05). Comparable effects were seen with the insulin/proinsulin combination. Conclusions: Proinsulin has the same adipogenic potential than insulin in vitro. Proinsulin has only 10∼20% of the glucose-lowering effect of insulin. It can be speculated that the adipogenic potential of proinsulin may be a large contributor to the increased body weight problems in patients with type 2 diabetes and advanced β-cell dysfunction.

Purpose – To describe the development of a novel polyether(meth)acrylate-based resin material class for stereolithography with alterable material characteristics. Design/methodology/approach – A complete overview of details to composition parameters, the optimization and bandwidth of mechanical and processing parameters is given. Initial biological characterization experiments and future application fields are depicted. Process parameters are studied in a commercial 3D systems Viper stereolithography system, and a new method to determine these parameters is described herein. Findings – Initial biological characterizations show the non-toxic behavior in a biological environment, caused mainly by the (meth)acrylate-based core components. These photolithographic resins combine an adjustable low Young’s modulus with the advantages of a non-toxic (meth)acrylate-based process material. In contrast to the mostly rigid process materials used today in the rapid prototyping industry, these polymeric formulations are able to fulfill the extended need for a soft engineering material. A short overview of sample applications is given. Practical implications – These polymeric formulations are able to meet the growing demand for a resin class for rapid manufacturing that covers a bandwidth from softer to stiffer materials. Originality/value – This paper gives an overview about the novel developed material class for stereolithography and should be therefore of high interest to people with interest in novel rapid manufacturing materials and technology.

Atherosclerosis is a chronic disease of the arteries and accounts for about 50 percent of all deaths in industrialized countries. For its treatment, patients primarily need to undergo lifestyle changes, concerning their diet or sportive behavior, while additional pharmaceutical approaches help to reduce major risk factors such as hypertension and hyperlipidemia. However, these two areas of treatment are only briefly mentioned here. Instead, this article focuses on literature and patents from the last decade focusing on invasive surgical procedures necessary for treatment of diseased blood vessels in severe cases of atherosclerosis. Described herein are synthetic grafts and so-called autografts, which are harvested from the patient for bypass surgery. In addition, implantable stents are discussed, which deal with different atherosclerotic aspects, such as restenosis, re-endothelialization, neointimal hyperplasia and thrombosis. And finally, publications and inventions are presented from the relatively new field of artificial tissue engineering incorporating stem cells or biomaterials to construct new vessels as substitutes for diseased veins and arteries.

This review is divided into two interconnected parts, namely a biological and a chemical one. The focus of the first part is on the biological background for constructing tissue-engineered vascular grafts to promote vascular healing. Various cell types, such as embryonic, mesenchymal and induced pluripotent stem cells, progenitor cells and endothelial- and smooth muscle cells will be discussed with respect to their specific markers. The in vitro and in vivo models and their potential to treat vascular diseases are also introduced. The chemical part focuses on strategies using either artificial or natural polymers for scaffold fabrication, including decellularized cardiovascular tissue. An overview will be given on scaffold fabrication including conventional methods and nanotechnologies. Special attention is given to 3D network formation via different chemical and physical cross-linking methods. In particular, electron beam treatment is introduced as a method to combine 3D network formation and surface modification. The review includes recently published scientific data and patents which have been registered within the last decade.

In search of novel gene signatures for osteo-differentiation of mesenchymal stem cells (MSCs), we submitted cDNA preparations from undifferentiated and differentiating MSCs to differential display- and semiquantitative-PCR and found down-regulation of COL5A1 in osteo-induced cultures at days 21 and 28, when the mineralized matrix accumulates. We also cultured osteo-differentiating MSCs onto type V collagen substrates and found a decrease in the accumulation of extracellular calcium compared to those grown in uncoated flasks. To our knowledge, this is first evidence that type V collagen might represent a stromal component that impairs osteogenesis.

The biological effects of bilirubin, still poorly understood, are concentration-dependent ranging from cell protection to toxicity. Here we present data that at high nontoxic physiological concentrations, bilirubin inhibits growth of proliferating human coronary artery smooth muscle cells by three events. It impairs the activation of Raf/ERK/MAPK pathway and the cellular Raf and cyclin D1 content that results in retinoblastoma protein hypophosphorylation on amino acids S608 and S780. These events impede the release of YY1 to the nuclei and its availability to regulate the expression of genes and to support cellular proliferation. Moreover, altered calcium influx and calpain II protease activation leads to proteolytical degradation of transcription factor YY1. We conclude that in the serum-stimulated human vascular smooth muscle primary cell cultures, bilirubin favors growth arrest, and we propose that this activity is regulated by its interaction with the Raf/ERK/MAPK pathway, effect on cyclin D1 and Raf content, altered retinoblastoma protein profile of hypophosphorylation, calcium influx, and YY1 proteolysis. We propose that these activities together culminate in diminished 5 S and 45 S ribosomal RNA synthesis and cell growth arrest. The observations provide important mechanistic insight into the molecular mechanisms underlying the transition of human vascular smooth muscle cells from proliferative to contractile phenotype and the role of bilirubin in this transition.

Medicine was revolutionized in the last two centuries and its advances have more than doubled life expectancy. Nevertheless, some problems are as old as mankind and although the underlying causes might have changed, the problems themselves have not. Musculoskeletal disorders and tooth loss are such problems; they are the major reasons for the ever-growing need for bone replacement, which cannot always be realized by autologous material. New, multidisciplinary strategies are needed for the development of novel materials to meet the demand. Stem-cell-based approaches combined with newly designed scaffold materials seem to be promising tools for constructing tissue replacements. Human mesenchymal stem cells and their remarkable differentiation potential are an interesting cell source for the development of bio-engineered tissues. Scaffolds based on natural and synthetic materials with or without the use of bioactive molecules are constructed to mimic the natural environment. They can improve proliferation and differentiation of the scaffold-seeded cells. Combined, they can provide specific remedies for hard tissue replacement, which will be discussed in this chapter.

Exposure to microgravity conditions causes cardiovascular deconditioning in astronauts during spaceflight. Until now, no specific drugs are available for countermeasure, since the underlying mechanism is largely unknown. Endothelial cells (ECs) and smooth muscle cells (SMCs) play key roles in various vascular functions, many of which are regulated by purinergic 2 (P2) receptors. However, their function in ECs and SMCs under microgravity conditions is still unclear. In this study, primary ECs and SMCs were isolated from bovine aorta and verified with specific markers. We show for the first time that the P2 receptor expression pattern is altered in ECs and SMCs after 24 h exposure to simulated microgravity using a clinostat. However, conditioned medium compensates this change in specific P2 receptors, for example, P2X7. Notably, P2 receptors such as P2X7 might be the important players during the paracrine interaction. Additionally, ECs and SMCs secreted different cytokines under simulated microgravity, leading into a pathogenic proliferation and migration. In conclusion, our data indicate P2 receptors might be important players responding to gravity changes in ECs and SMCs. Since some artificial P2 receptor ligands are applied as drugs, it is reasonable to assume that they might be promising candidates against cardiovascular deconditioning in the future.