Search

Despite progress in use of decellularized lung scaffolds in ex vivo lung bioengineering schemes, including use of gels and other materials derived from the scaffolds, the detailed composition and functional role of extracellular matrix (ECM) proteoglycans (PGs) and their glycosaminoglycan (GAG) chains remaining in decellularized lungs, is poorly understood. Using a commonly utilized detergent-based decellularization approach in human autopsy lungs resulted in disproportionate losses of GAGs with depletion of chondroitin sulfate/dermatan sulfate (CS/DS) > heparan sulfate (HS) > hyaluronic acid (HA). Specific changes in disaccharide composition of remaining GAGs were observed with disproportionate loss of NS and NS2S for HS groups and of 4S for CS/DS groups. No significant influence of smoking history, sex, time to autopsy, or age was observed in native vs. decellularized lungs. Notably, surface plasmon resonance demonstrated that GAGs remaining in decellularized lungs were unable to bind key matrix-associated growth factors FGF2, HGF, and TGFβ1. Growth of lung epithelial, pulmonary vascular, and stromal cells cultured on the surface of or embedded within gels derived from decellularized human lungs was differentially and combinatorially enhanced by replenishing specific GAGs and FGF2, HGF, and TGFβ1. In summary, lung decellularization results in loss and/or dysfunction of specific GAGs or side chains significantly affecting matrix-associated growth factor binding and lung cell metabolism. GAG and matrix-associated growth factor replenishment thus needs to be incorporated into schemes for investigations utilizing gels and other materials produced from decellularized human lungs. STATEMENT OF SIGNIFICANCE: Despite progress in use of decellularized lung scaffolds in ex vivo lung bioengineering schemes, including use of gels and other materials derived from the scaffolds, the detailed composition and functional role of extracellular matrix (ECM) proteoglycans (PGs) and their glycosaminoglycan (GAG) chains remaining in decellularized lungs, is poorly understood. In the current studies, we demonstrate that glycosaminoglycans (GAGs) are significantly depleted during decellularization and those that remain are dysfunctional and unable to bind matrix-associated growth factors critical for cell growth and differentiation. Systematically repleting GAGs and matrix-associated growth factors to gels derived from decellularized human lung significantly and differentially affects cell growth. These studies highlight the importance of considering GAGs in decellularized lungs and their derivatives.

Summary: Heart failure (HF) and subarachnoid hemorrhage (SAH) chronically reduce cerebral perfusion, which negatively affects clinical outcome. This work demonstrates a strong relationship between cerebral artery cystic fibrosis transmembrane conductance regulator (CFTR) expression and altered cerebrovascular reactivity in HF and SAH. In HF and SAH, CFTR corrector compounds (C18 or lumacaftor) normalize pathological alterations in cerebral artery CFTR expression, vascular reactivity, and cerebral perfusion, without affecting systemic hemodynamic parameters. This normalization correlates with reduced neuronal injury. Therefore, CFTR therapeutics have emerged as valuable clinical tools to manage cerebrovascular dysfunction, impaired cerebral perfusion, and neuronal injury. Key Words: cognitive impairment, corrector compounds, cystic fibrosis transmembrane conductance regulator (CFTR), myogenic vasoconstriction, sphingosine-1-phosphate, tumor necrosis factor, vascular smooth muscle cells

The development of reliable tissue engineering methods using decellularized cadaveric or donor lungs could potentially provide a new source of lung tissue. The vast majority of current lung decellularization protocols are detergent based and incompletely removed residual detergents may have a deleterious impact on subsequent scaffold recellularization. Detergent removal and quality control measures that rigorously and reliably confirm removal, ideally utilizing nondestructive methods, are thus critical for generating optimal acellular scaffolds suitable for potential clinical translation. Using a modified and optimized version of a methylene blue-based detergent assay, we developed a straightforward, noninvasive method for easily and reliably detecting two of the most commonly utilized anionic detergents, sodium deoxycholate (SDC) and sodium dodecyl sulfate (SDS), in lung decellularization effluents. In parallel studies, we sought to determine the threshold of detergent concentration that was cytotoxic using four different representative human cell types utilized in the study of lung recellularization: human bronchial epithelial cells, human pulmonary vascular endothelial cells (CBF12), human lung fibroblasts, and human mesenchymal stem cells. Notably, different cells have varying thresholds for either SDC or SDS-based detergent-induced cytotoxicity. These studies demonstrate the importance of reliably removing residual detergents and argue that multiple cell lines should be tested in cytocompatibility-based assessments of acellular scaffolds. The detergent detection assay presented here is a useful nondestructive tool for assessing detergent removal in potential decellularization schemes or for use as a potential endpoint in future clinical schemes, generating acellular lungs using anionic detergent-based decellularization protocols.

The chemical composition of calcium phosphate (CaP) materials for the regenerative therapy of large bone defects is similar to that of bone. Additionally, calcium phosphates show an excellent biocompatibility. Besides the support of defect healing calcium phosphate implants should be completely degraded within an adequate time period to be replaced by newly formed bone. Although degradation of CaP-implants occurs mainly by dissolution of the material, it is important to characterize the osteoclastic resorption as well, which is involved in native bone remodeling. The degradation of bone substitutes made of calcium phosphate ceramics is influenced by various parameters, such as defect size and localization, the general health situation, and age of the patient, but also material properties are important. Especially, the calcium phosphate composition is crucial for the degradation behavior of a calcium phosphate material. Additionally, at the cellular level the micro- and macroporosity, including interconnecting pores, influences both, the dissolution and the osteoclastic resorption. In our study, three different calcium phosphate materials (hydroxyapatite, tricalcium phosphate, and a biphasic calcium phosphate) and two different geometries (dense 2D samples and porous 3D scaffolds) are compared regarding their dissolution and resorption behavior. The results show, that the dissolution of CaP-ceramics, as examined by the incubation in a degradation solution, depends mainly on the calcium phosphate phase but also on the porosity of the implant. Regarding the resorption, cell proliferation and differentiation of a monocytic cell line as well as the formation of resorption lacunas are analyzed. Cell proliferation is comparable on all phase compositions. Cell differentiation and resorption, however, are influenced by the calcium phosphate phase composition and by the implant porosity as well. By understanding these two mechanisms of degradation, bone substitute materials and, as a result, the bone regeneration of large bone defects using CaP-ceramics can be improved.

Introduction: Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease of yet unknown etiology. Recently, (re)activation of developmental pathways, such as Wnt/β-catenin in the alveolar epithelium has been linked to disease development. Regulation of Wnt/β-catenin activation has been shown to be dependent on Heparan Sulfate Proteoglycans (HSPGs). The role of Wnt-HSPG interaction in IPF, however, remains to be elusive. Methods: Murine and human lung epithelial cells were treated with Heparin, Desulfated (DS) Heparins or Heparinase II and Chondroitinase ABC, respectively. A Heparin binding assay was used to assess binding of Wnt3a to Heparin and DS Heparins. Immunofluorescence staining of ex vivo lung tissue for Wnt proteins and HSPGs was conducted. Results: Immunofluorescence staining of ex vivo tissue lung slices revealed upregulated WNT3a and HSPG expression in bleomycin-treated mouse lungs compared to controls. Heparin treatment of murine lung epithelial cells resulted in inhibition of TOP/FOP-flash reporter, indicating decreased canonical WNT signaling. In contrast, Heparinase II and Chondroitinase ABC treatment resulted in upregulated β-catenin-dependent gene transcription. Interestingly, 6-O-DS Heparin treatment of A549 cells did not decrease WNT signaling, while binding of WNT3a to 6-O-DS Heparin was not altered, thereby suggesting less inhibition of WNT signaling upon binding to 6-O-DS Heparin. Conclusion: Taken together, our findings revealed that HS sulfation and degradation modulates WNT/β-catenin signal activity. Altered sulfation states during ECM remodeling in IPF pathology might contribute to (re)activation of developmental pathways like WNT/β-catenin.

Hydroxyapatite (HA) ceramic is a widely used synthetic bone substitute material for the regeneration of bone defects. We manufactured HA scaffolds with adjustable pore sizes and pore geometry by dispense-plotting. In addition, we attached peptides covalently onto the HA surface and are able to simultaneously quantify the amount of covalently attached and adsorbed peptide down to the picomolar range with a novel fluorescence-based detection method. In cell culture assays with stromal bone marrow cells, we observed a positive effect of biofunctionalization on cell differentiation after 21 days of culture when comparing the scaffold functionalized with the RGD motif containing adhesion peptide to an unmodified scaffold.

Accumulation of leukocytes, particularly of polymorphonuclear neutrophils (PMNLs), is considered as an important response in tissue injury or infection. Recent investigations in a variety of organs, such as bowel (Granger et al. 1989), skeletal muscle (Belkin et al. 1989), and brain (Bednar et al. 1991; Grogaard et al. 1989; Mori et al. 1992), suggest an involvement of leukocytes in ischemia mechanisms. Although recirculation after ischemia is a most important factor of functional recovery, interactions between circulating blood elements and the tissue injured by ischemia could lead to further damage (Harlan 1987). Owing to specific rheologic and adhesive properties, leukocytes may plug capillaries in ischemic tissue (Engler et al. 1983; del Zoppo et al. 1991). An increase in vascular resistance leading to an impaired tissue reperfusion could result in additional damage from secondary ischemia. Further, a release of toxic material of PMNLs such as superoxide anion radicals or elastase might be involved (Weiss 1989).

Palladium based Heusler alloys according to the general formula (Pd 1− x X x ) 2 MnSn ( X = Co, Ni; x = 0–1) were prepared. They were all single phased with the L 2 1 structure. Magnetic investigation was done in a temperature range of 80–1100 K at fields up to 1.3 T. Curie temperatures as well as paramagnetic Curie temperatures reveal a linear dependence of composition. In the alloys containing Ni, Mn only bears a magnetic moment of approximately 4 μ B ; the effective moment corresponds well. For the Co containing compounds an additional moment for Co, which was very sensitive to composition, was encountered. Ferromagnetic behaviour is mainly due to the localized character of the Mn moments.

The magnetic properties of TlFe3Te3 are studied on powdered samples and single crystals. Below 220 K TlFe3Te3, is a very anisotropic ferromagnet, the crystallographic c axis being the easy axis of magnetization. While in the easy direction saturation is achieved below 0.5 kOe, in the hard direction saturation is reached at 64 kOe. The angular dependence of the magnetization follows closely a cos ¦ϑ¦ law. The magnetic transition is very abrupt at low external fields, suggesting a first order phase transition. It is accompanied by a small anomaly in the thermal dilatation of the c axis. The magnetization shows an anomalous increase below 50 K suggesting a phase transition.

New mixed Heusler alloys of the system Cu 2 (Mn 1− x Ni x )Sn were prepared with x = 0–1. Magnetic measurements in the ferromagnetic region were undertaken. For manganese, the only atom displaying a magnetic moment in this row of Heusler alloys, 4μ B was found, deviations were due to the degree of order. Measurements in the paramagnetic region were not possible because of phase transitions on heating leading to polyphase samples. The critical concentration of magnetic ions was estimated to 13 at.% and compared with models given by Duff and Cannella [6]. With the same plot the Curie point for Cu 2 MnSn could be determined (630 K).

In the applications of thin film technology, the chemical nature of the substrate surface is known to affect the film/substrate interactions. Undesirable contaminants in the interfacial region may affect film nucleation, adhesion, and electrical or optical properties. Often, glow‐discharge sputter cleaning is used as a surface preparation technique prior to thin film deposition. Since this method of sputter cleaning is performed in a gaseous environment where the mean free path of the sputtered atoms is short, there is a possibility of surface recontamination by material sputtered from the substrate and from surrounding fixturing. In this investigation, we have used soft‐x‐ray appearance‐potential spectroscopy to monitor in situ the substrate surface composition after sputter cleaning with both dc and rf discharges. Data are presented which show that common contaminants such as carbon and oxide layers may be adequately removed by either dc or rf glow‐discharge sputter cleaning. Direct current sputter clea...