Jörg Bornemann

Gold Nanoparticles of Diameter 1.4 nm Trigger Necrosis by Oxidative Stress and Mitochondrial Damage

Gold nanoparticles (AuNPs) are generally considered nontoxic, similar to bulk gold, which is inert and biocompatible. AuNPs of diameter 1.4 nm capped with triphenylphosphine monosulfonate (TPPMS), Au1.4MS, are much more cytotoxic than 15-nm nanoparticles (Au15MS) of similar chemical composition. Here, major cell-death pathways are studied and it is determined that the cytotoxicity is caused by oxidative stress. Indicators of oxidative stress, reactive oxygen species (ROS), mitochondrial potential and integrity, and mitochondrial substrate reduction are all compromised. Genome-wide expression profiling using DNA gene arrays indicates robust upregulation of stress-related genes after 6 and 12 h of incubation with a 2 x IC50 concentration of Au1.4MS but not with Au15MS nanoparticles. The caspase inhibitor Z-VAD-fmk does not rescue the cells, which suggests that necrosis, not apoptosis, is the predominant pathway at this concentration. Pretreatment of the nanoparticles with reducing agents/antioxidants N-acetylcysteine, glutathione, and TPPMS reduces the toxicity of Au1.4MS. AuNPs of similar size but capped with glutathione (Au1.1GSH) likewise do not induce oxidative stress. Besides the size dependency of AuNP toxicity, ligand chemistry is a critical parameter determining the degree of cytotoxicity. AuNP exposure most likely causes oxidative stress that is amplified by mitochondrial damage. Au1.4MS nanoparticle cytotoxicity is associated with oxidative stress, endogenous ROS production, and depletion of the intracellular antioxidant pool.

The osteogenic differentiation of adult bone marrow and perinatal umbilical mesenchymal stem cells and matrix remodelling in three-dimensional collagen scaffolds.

Adult human mesenchymal stem cells from bone marrow (BM-MSC) represent a promising source for skeletal regeneration. Perinatal MSC from Whartons Jelly of the umbilical cord (UC-MSC) are expected to possess enhanced differentiation capacities due to partial expression of pluripotency markers. For bone tissue engineering, it is important to analyse in vitro behaviour of stem cell/biomaterial hybrids concerning in vivo integration into injured tissue via migration, matrix remodelling and differentiation. This study compares the cell-mediated remodelling of three-dimensional collagen I/III gels during osteogenic differentiation of both cell types. When activated through collagen contact and subjected to osteogenic differentiation, UC-MSC differ from BM-MSC in expression and synthesis of extracellular matrix (ECM) proteins as shown by histology, immunohistochemistry, Western Blot analysis and realtime-RT-PCR. The biosynthetic activity was accompanied in both cell types by the ultrastructural appearance of hydroxyapatite/calcium crystals and osteogenic gene induction. Following secretion of matrix metalloproteinases (MMP), both MSC types migrated into and colonised the collagenous matrix causing matrix strengthening and contraction. These results indicate that UC-MSC and BM-MSC display all features needed for effective bone fracture healing. The expression of ECM differs in both cell types considerably, suggesting different mechanisms for bone formation and significant impact for bone tissue engineering.

Proximal tubular cells contain a phenotypically distinct, scattered cell population involved in tubular regeneration.

Regeneration of injured tubular cells occurs after acute tubular necrosis primarily from intrinsic renal cells. This may occur from a pre‐existing intratubular stem/progenitor cell population or from any surviving proximal tubular cell. In this study, we characterize a CD24‐, CD133‐, and vimentin‐positive subpopulation of cells scattered throughout the proximal tubule in normal human kidney. Compared to adjacent ‘normal’ proximal tubular cells, these CD24‐positive cells contained less cytoplasm, fewer mitochondria, and no brush border. In addition, 49 marker proteins are described that are expressed within the proximal tubules in a similar scattered pattern. For eight of these markers, we confirmed co‐localization with CD24. In human biopsies of patients with acute tubular necrosis (ATN), the number of CD24‐positive tubular cells was increased. In both normal human kidneys and the ATN biopsies, around 85% of proliferating cells were CD24‐positive – indicating that this cell population participates in tubular regeneration. In healthy rat kidneys, the novel cell subpopulation was absent. However, upon unilateral ureteral obstruction (UUO), the novel cell population was detected in significant amounts in the injured kidney. In summary, in human renal biopsies, the CD24‐positive cells represent tubular cells with a deviant phenotype, characterized by a distinct morphology and marker expression. After acute tubular injury, these cells become more numerous. In healthy rat kidneys, these cells are not detectable, whereas after UUO, they appeared de novo – arguing against the notion that these cells represent a pre‐existing progenitor cell population. Our data indicate rather that these cells represent transiently dedifferentiated tubular cells involved in regeneration. Copyright

Clearance of Fetuin-A–Containing Calciprotein Particles Is Mediated by Scavenger Receptor-A

Rationale: Fetuin-A is a liver-derived plasma protein involved in the regulation of calcified matrix metabolism. Biochemical studies showed that fetuin-A is essential for the formation of protein-mineral complexes, called calciprotein particles (CPPs). CPPs must be cleared from circulation to prevent local deposition and pathological calcification. Objective: We studied CPP clearance in mice and in cell culture to identify the tissues, cells, and receptors involved in the clearance. Methods and Results: In mice, fetuin-A–containing CPPs were rapidly cleared by the reticuloendothelial system, namely Kupffer cells of the liver and marginal zone macrophages of the spleen. Macrophages from scavenger receptor-AI/II (SR-A)-deficient mice cleared CPPs less efficiently than macrophages from wild-type mice, suggesting that SR-AI/II is involved in CPP binding and endocytosis. Accordingly, we found reduced clearance of CPPs in SR-A/MARCO–deficient mice. Conclusions: We could demonstrate that fetuin-A–containing CPPs facilitate the clearance of mineral debris by macrophages via SR-A. Since the same receptor also contributes to the uptake of modified low-density lipoprotein particles in atherosclerosis, defective endocytosis of both types of particle may impinge on lipid as well as mineral debris clearance in calcifying atherosclerosis.

Simultaneous dual-band ultra-high resolution optical coherence tomography

Ultra-high resolution optical coherence tomography (OCT) imaging is demonstrated simultaneously at 840 nm and 1230 nm central wavelength using an off-the-shelf turn-key supercontinuum light source. Spectral filtering of the light source emission results in a double peak spectrum with average powers exceeding 100 mW and bandwidths exceeding 200 nm for each wavelength band. A free-space OCT setup optimized to support both wavelengths in parallel is introduced. OCT imaging of biological tissue ex vivo and in vivo is demonstrated with axial resolutions measured to be < 2 mum and < 4 mum at 840 nm and 1230 nm, respectively. This measuring scheme is used to extract spectroscopic features with outstanding spatial resolution enabling enhanced image contrast.

Long-term survival and characterisation of human umbilical cord-derived mesenchymal stem cells on dermal equivalents.

During early embryogenesis, mesenchymal cells arise from the primitive epithelium and can revert to an epithelial phenotype by passing through mesenchymal-to-epithelial transition (MET). Mesenchymal stem cells (MSC) of the Whartons Jelly of the umbilical cord (UC-MSC) express pluripotency markers underlining their primitive developmental state. As mesenchymal stem cells from bone marrow (BM-MSC) possess a strong propensity to ameliorate mesenchymal tissue damage, UC-MSC might also be able to differentiate into cells apart from the mesoderm, allowing replacement of ectodermal and mesodermal tissues. In this study, we analysed the possible epidermal differentiation of UC-MSC on dermal equivalents (DEs) consisting of collagen I/III with dermal fibroblasts and subjected to the culture conditions for tissue engineering of skin with keratinocytes. The culture conditions were further modified by pre-treating the cells with 5-azacytidine or by supplementing the medium with all trans retinoic acid. Interestingly, a subpopulation of UC-MSC (29%) co-expressed pan-cytokeratin (epithelial marker; pan-CK) and vimentin (mesenchymal marker) after isolation. Under the three-dimensional conditions of skin, the number of pan-CK(+)-cells increased to >30% after 21 days of cultivation, while under osteogenic culture conditions the cells were pan-CK-negative, thus showing the influence of the artificial niche. Nevertheless, the pan-CK-expression was neither accompanied by typical epithelial morphology nor expression of other epidermal markers. The pan-CK-detection can be explained by the expression of cytokeratins in myofibroblasts. UC-MSC expressed alpha-smooth muscle actin after isolation and displayed all features of functional myofibroblasts like morphology, cell-mediated contraction of a collagen gel and production of components of the extracellular matrix (ECM). The treatment with all trans retinoic acid or 5-azacytidine could neither induce an epidermal differentiation nor enhance the myofibroblastic differentiation. Concluding, UC-MSC might be an interesting cell source to support the regeneration of wounds by their differentiation into myofibroblasts and their extensive synthesis of ECM components.

The role of biomaterials in the direction of mesenchymal stem cell properties and extracellular matrix remodelling in dermal tissue engineering

Recently, a new generation of dermal equivalents (DE) was presented which are solely generated on a human fibroblast-derived dermal matrix. Since human mesenchymal stem cells from bone marrow (BM-MSC) and Whartons Jelly of the umbilical cord (UC-MSC) are characterised by a distinct biosynthetic and paracrine activity, they are an appealing alternative approach for generating cell-based DE. This study compares the epithelial-mesenchymal interaction and extracellular matrix (ECM) remodelling of cell-based and collagen-based DE using fibroblasts, BM-MSC or UC-MSC, respectively, in co-culture with the keratinocyte cell line HaCaT. While fibroblast-based DE exhibit normal matrix synthesis, proliferation and differentiation of keratinocytes, mesenchymal stem cell-based DE resulted in excessive production of inhomogenous matrix aggregates, loss of polarisation of the epidermal cell layer and an inconstant paracrine activity. In contrast, collagen-embedded MSC revealed a homogenous growth pattern as well as a constant expression of growth factors and ECM proteins without a negative influence on the epidermal layer as shown by histology, electron microscopy, immunohistochemistry and realtime-RT-PCR. These results indicate the necessity of an instructive biomaterial-based scaffold to direct stem cell differentiation, proliferation, paracrine activity as well as regulation of ECM deposition.

Visualization of the Basement Membrane Zone of the Bladder by Optical Coherence Tomography: Feasibility of Noninvasive Evaluation of Tumor Invasion

OBJECTIVES Imaging techniques with high resolution are evolving rapidly for medical applications and may substitute invasive diagnostic techniques. The use of ultrahigh resolution optical coherence tomography (UHR-OCT) to image healthy and morphologically altered bladder tissue with virtual histology is evaluated ex vivo to define parameters necessary for future, diagnostically relevant in vivo systems. Here, special focus is on the visualization of the basement membrane zone. METHODS Optical coherence tomography examinations were performed by using a modified commercial OCT system comprising a Ti:sapphire femtosecond laser to support an enhanced resolution of 3 microm axial x 10 microm lateral. Tomograms of 142 fresh human bladder tissue samples from cystectomies, radical prostatectomies, and transurethral tumor resections were recorded and referenced to histologic sections using standard hematoxylin and eosin staining. RESULTS OCT of normal bladder mucosa allows for a clear differentiation of urothelium and lamina propria. The basement membrane zone is identified as a narrow, low-scattering band between these layers. This allows for reliable exclusion of invasion. Healthy urothelial tissue, carcinoma in situ, and transitional cell carcinoma can be differentiated using this imaging technique. Sensitivity of UHR-OCT for malignant bladder tissue could be determined to be 83.8%, and specificity to be 78.1%. CONCLUSIONS UHR-OCT is considered promising in the attempt to strive for fluorescence cystoscopy-guided virtual histology as a means of supporting therapeutic decisions for bladder neoplasia.

Peptide-Functionalized Gold Nanorods Increase Liver Injury in Hepatitis

Targeted nanomedicine holds enormous potential for advanced diagnostics and therapy. Although it is known that nanoparticles accumulate in liver in vivo, the impact of cell-targeting particles on the liver, especially in disease conditions, is largely obscure. We had previously demonstrated that peptide-conjugated nanoparticles differentially impact macrophage activation in vitro. We thus comprehensively studied the distribution of gold nanorods (AuNR) in mice in vivo and assessed their hepatotoxicity and impact on systemic and hepatic immune cells in healthy animals and experimental liver disease models. Gold nanorods were stabilized with either cetyltrimethylammonium bromide or poly(ethylene glycol) and additional bioactive tripeptides RGD or GLF. Gold nanorods mostly accumulated in liver upon systemic injection in mice, as evidenced by inductively coupled plasma mass spectrometry from different organs and by non-invasive microcomputerized tomography whole-body imaging. In liver, AuNR were only found in macrophages by seedless deposition and electron microscopy. In healthy animals, AuNR did not cause significant hepatotoxicity as evidenced by biochemical and histological analyses, even at high AuNR doses. However, flow cytometry and gene expression studies revealed that AuNR polarized hepatic macrophages, even at low doses, dependent on the respective peptide sequence, toward M1 or M2 activation. While peptide-modified AuNR did not influence liver scarring, termed fibrosis, in chronic hepatic injury models, AuNR-induced preactivation of hepatic macrophages significantly exacerbated liver damage and disease activity in experimental immune-mediated hepatitis in mice. Bioactively targeted gold nanoparticles are thus potentially harmful in clinically relevant settings of liver injury, as they can aggravate hepatitis severity.

Exposure to Uremic Serum Induces a Procalcific Phenotype in Human Mesenchymal Stem Cells

Objective— Medial artery calcification in patients with chronic kidney disease proceeds through intramembranous ossification resulting from osteoblast-induced calcification of the collagen extracellular matrix. The current study is based on the hypothesis that mesenchymal stem cells (MSC) constitute critical cells for procalcific extracellular matrix remodeling in patients with chronic kidney disease. Methods and Results— Human MSC were cultured in media supplemented with pooled sera from either healthy or uremic patients (20%). Exposure to uremic serum enhanced the proliferation of MSC (cell counting, BrdU incorporation) whereas apoptosis and necrosis were not affected (annexin V and 7-amino-actinomycin staining). Uremic serum–exposed MSC recapitulated osteogenesis by matrix calcification and expression of bone-related genes (bone morphogenetic protein [BMP]-2 receptor, alkaline phosphatase, osteopontin, and Runx2) in 35 days. The uremic serum–induced osteogenesis was completely blocked by a BMP-2/4 neutralizing antibody or the natural antagonist NOGGIN. Calcification and matrix remodeling were further analyzed in a collagen-embedded osteogenesis model recapitulating the vascular collagen I/III environment. The uremic serum–induced calcification was shown to occur along collagen fibers as shown by scanning electron microscopy, energy-dispersive X-ray spectroscopy, and von Kossa staining and was accompanied by extensive matrix remodeling. Conclusion— Uremic serum induced in a BMP-2/4-dependent manner an osteoblast-like phenotype in MSC accompanied by matrix remodeling and calcification.