Ralph M. Albrecht

Three-dimensional extracellular matrix textured biomaterials

Clinical and experimental investigations have reported that manufactured surface topographies have significant effects on cell adhesion and tissue integration. However, essentially all previously examined topographies bear little relation to cell adhesion substrates found in biological tissues. In vivo, many cells are adherent to extracellular matrices (ECM), which have an extremely complex 3-D topography in the micrometre to nanometre range. In addition, many studies indicate that micro- and nano-scale mechanical stresses generated by cell-matrix adhesion have significant effects on cellular phenotypic behaviour. In this report we describe methodology for the fabrication of topographic replicas of the subendothelial ECM topography with a biomedical polyurethane. Using three-dimensional high resolution scanning electron microscopy, accurate replication of subendothelial ECM topography from the macroscopic to the macromolecular scale is demonstrated. Bovine aortic endothelial cells cultured on the ECM replicas spread more rapidly and had a three-dimensional appearance and spread areas at confluence which appeared more like endothelial cells in native arteries, compared with cells cultured on untextured control surfaces. Since the fabrication process may be used with many different types of materials, including polymers of synthetic and biological origin, these biomimetic ECM-textured surfaces may find both research and clinical applications.

Leukocyte Migration Is Regulated by L-Selectin Endoproteolytic Release

L-selectin mediates lymphocyte migration to peripheral lymph nodes and leukocyte rolling on vascular endothelium during inflammation. One unique feature that distinguishes L-selectin from other adhesion molecules is that it is rapidly cleaved from the cell surface after cellular activation. The biological significance of L-selectin endoproteolytic release was determined by generating gene-targeted mice expressing a modified receptor that was not cleaved from the cell surface. Blocking L-selectin cleavage on antigen-stimulated lymphocytes allowed their continued migration to peripheral lymph nodes and inhibited their short-term redirection to the spleen. Blocking homeostatic L-selectin cleavage also resulted in a constitutive 2-fold increase in overall L-selectin expression by leukocytes. As a result, neutrophils entered the inflamed peritoneum in greater numbers or for a longer duration. Thus, endoproteolytic cleavage regulates both homeostatic and activation-induced changes in cell surface L-selectin density, which directs the migration patterns of activated lymphocytes and neutrophils in vivo.

Platelet interaction with pyrolytic carbon heart-valve leaflets

Although the newest generation of mechanical heart-valve prosthetics constructed either partially or wholly of lowtemperature isotropic pyrolytic carbon (LTIC) have significantly reduced thromboembolic complications compared with early-generation mechanical valves (e.g., Starr-Edwards), thromboembolism remains an important clinical complication. In the present study, high-resolution, lowvoltage scanning electron microscopy (HR-LV-SEM) was used to examine the structure and platelet interaction properties of LTIC valve leaflets manufactured by both Carbo Medics, Inc. and by St. Jude Medical, Inc. Valve leaflets from both manufacturers, prepared and polished exactly as used in clinical heart valves, had similar surface energetics and elemental composition. Examination with LV-SEM revealed a rough and complex three-dimensional surface structure with nanometer- to micron-size features. In vitro adhesion of human platelets on the LTIC materials and Formvar were evaluated in the presence of 1 mg/mL albumin. Platelet-surface activation, as evaluated by shape change, spread area, and deposition, was extremely extensive on the LTIC materials compared with the Formvar positive control material. LTIC-adherent platelets were extremely thin, and closely followed the rough LTIC contours, greatly limiting their visibility with conventional SEM. These observations demonstrate that LTIC surfaces can extensively activate platelets even in the presence of albumin, thereby suggesting that platelet interactions with pyrolytic carbon may have a significant role in mechanical-valve thromboembolism.

The "active" rosette test in immunodeficiency diseases.

Abstract The “active” rosette test is a clinically useful test for assessing T cell function. When there is disparity between the result of the “active” and “total” rosette test, the clinical status and in vitro studies of the patient correlate better with active rosette formation. These studies also suggest that active rosette formation may serve as a marker of T cell differentiation.

Biomaterials with permanent hydrophilic surfaces and low protein adsorption properties

Low protein adsorbing polymer films have been prepared with which to fabricate intravenous containers, designed for compatibility with low concentrations of protein drugs. The material is economically manufactured utilizing physical melt blending of water-soluble surface-modifying polymers (PEO, PEOX, PVA, and PNVP) with a base polymer (EVA, PP, PETG, PMMA, SB, and nylon). Permanency of the hydrophilic surfaces so generated was confirmed by surface contact angle experiments and total organic carbon leachables analysis of the aqueous contacting solutions. Binding of IgG, albumin and insulin was studied. A sixfold reduction of protein adsorption was obtained by adding 5% PVA13K to EVA, for IgG at a bulk concentration of 2.5 ppm. Surface bound protein measured by micro-BCA colorimetry, agreed with the solution protein lost, as determined by the Fluoraldehyde procedure. Imaging of the protein exposed plastic surfaces by silver enhanced protein conjugated gold staining agreed with the quantitative assay determinations.

The Potent Platelet Inhibitory Effects of S-Nitrosated Albumin Coating of Artificial Surfaces

OBJECTIVES We studied the antithrombotic effect of coating glass, collagen and metal stent surfaces with bovine serum albumin (BSA) covalently modified to carry S-NO functional groups denoted (pS-NO-BSA). METHODS Video-enhanced light microscopy was used to visualize canine blood platelet adhesion and aggregation in a parallel plate glass chamber. Platelet adhesion was observed for 60 min on glass, glass coated with BSA, glass coated with pS-NO-BSA, collagen I (CO) surface, CO coated with BSA and CO coated with pS-NO-BSA. We also coated Palmaz-Shatz (P-S) stents with pS-NO-BSA. Coated and uncoated stents were then immersed in porcine platelet-rich plasma for two min and the platelet cyclic GMP level was measured. In six anesthetized pigs, coated and uncoated stents were placed in the carotid arteries and [111In]-labeled platelets were circulated for 2 h. The stented arteries were then removed and placed in a gamma well counter. RESULTS There was significantly less platelet attachment, adhesion and aggregation on the pS-NO-BSA coated surfaces compared with the BSA coated and uncoated surfaces. The pS-NO-BSA coating increased the platelet cGMP levels to 5.9+/-0.7 pmoles/10(8) platelets compared with 2.7+/-0.9 pmoles/10(8) platelets for control (p < 0.01). The average gamma ray count from [111In]-labeled platelets that attached to the coated stents was 90,000+/-42,000/min and 435,000+/-290,000/min for the uncoated stents (p < 0.01). CONCLUSIONS The pS-NO-BSA coating of thrombogenic surfaces reduces platelet adhesion and aggregation, possibly by increasing the platelet cGMP. This inhibitory effect appears to be a consequence of the direct antiplatelet actions of NO combined with the antiadhesive properties of albumin.

Correlative Instrumental Neutron Activation Analysis, Light Microscopy, Transmission Electron Microscopy, and X-ray Microanalysis for Qualitative and Quantitative Detection of Colloidal Gold Spheres in Biological Specimens

: Colloidal gold, conjugated to ligands or antibodies, is routinely used as a label for the detection of cell structures by light (LM) and electron microscopy (EM). To date, several methods to count the number of colloidal gold labels have been employed with limited success. Instrumental neutron activation analysis (INAA), a physical method for the analysis of the elemental composition of materials, can be used to provide a quantitative index of gold accumulation in bulk specimens. Given that gold is not naturally found in biological specimens in any substantial amount and that colloidal gold and ligand conjugates can be prepared to yield uniform bead sizes, the amount of label can be calculated in bulk biological samples by INAA. Here we describe the use of INAA, LM, transmission EM, and X-ray microanalysis (EDX) in a model to determine both distribution (localization) and amount of colloidal gold at the organ, tissue, cellular, and ultrastructural levels in whole animal systems following administration. In addition, the sensitivity for gold in biological specimens by INAA is compared with that of inductively coupled plasma-mass spectrometry (ICP-MS). The correlative use of INAA, LM, TEM, and EDX can be useful, for example, in the quantitative and qualitative tracking of various labeled molecular species following administration in vivo.

The influence of preadsorbed canine von Willebrand factor, fibronectin and fibrinogen on ex vivo artificial surface-induced thrombosis

We have examined the effects of preadsorption of several canine plasma proteins on surface-induced thrombogenesis in a canine ex vivo model. Our technique allowed determination of initial deposition and subsequent embolization of 51Cr-labeled platelets and 125I-fibrinogen onto and from polymeric arterio-venous shunts in non-anticoagulated canines. Segments of the tubing were removed at various time points between 2 and 120 minutes of blood contact for examination of the morphology of the thrombus by scanning electron microscopy. Thrombus deposition was measured on uncoated plasticized poly(vinyl chloride) (PVC) and PVC precoated with canine von Willebrand factor (vWF), fibronectin, partially purified fibrinogen (fibrinogen which contained vWF and fibronectin as impurities), or purified fibrinogen (fibrinogen which had been further purified to remove fibronectin and vWF). Preadsorption of all proteins studied enhanced the thrombogenic response relative to that of the uncoated surface. Precoating with vWF or partially purified fibrinogen resulted in the deposition of the greatest number of thrombi, and embolization was slower than on shunts precoated with canine fibronectin or purified fibrinogen. The deposition-embolization profiles for the fibronectin and purified fibrinogen-coated surfaces were similar. The amount and time sequence of initial adhesion and spreading of platelets was related to the extent and time sequence of peak thrombus formation. The partially purified fibrinogen-coated and vWF-coated surfaces had more adhered and spread platelets at the earliest time points and a greater number of larger thrombi at the peak deposition times. The slowest rate of platelet adhesion and spreading was seen on the purified fibrinogen-coated surface. White blood cells were present very early on surfaces precoated with vWF and partially purified fibrinogen, and were present prior to embolization on all surfaces. Major conclusions from this work indicate that, although fibrinogen and fibronectin promote thrombogenesis when adsorbed to a surface, vWF is even more active in promoting platelet deposition and in anchoring thrombi to the surface of biomaterials. Thus, differences in vWF adsorption to biomaterials may be a determinant of surface-induced thrombogenesis.

Nano- and Microscale Holes Modulate Cell-Substrate Adhesion, Cytoskeletal Organization, and

Human corneal epithelial cells (HCECs) interface with a basement membrane in vivo that possesses complex nanoscale topographic features. We report that synthetic substrates patterned with nano- and microscale holes differentially modulate the proliferation, shape and adhesion of SV40 human corneal epithelial cells (SV40-HCECs) as a function of feature size: 1) Cell proliferation was inhibited on nanoscale features (features size less than 800 nm in pitch) compared to microscale features or planar substrates in identical culture conditions. 2)Cells on nanoscale holes had a stellate morphology compared to those on microscale features that were more evenly spread. 3) Cells adhered more to nanoscale features than to microscale features when exposed to shear stress in a laminar flow chamber. Transmission electron microscopy showed that cells cultured on the 400 nm pitch patterns had longer and more numerous filopodia and retraction fibers than cells cultured on the 1600 nm pitch patterns. Immunogold labeling of -beta1 integrins revealed that these receptors were localized at the cell periphery and in the aforementioned cytoskeletal elements. Our findings indicate that surface discontinuities and the activation of mechanochemical cell signaling mechanisms may contribute to the observed responses exhibited by SV40-HCECs cultured on nano- and microscale topography

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Diphosphoryl lipid A derived from the nontoxic LPS of Rhodobacter sphaeroides (RsDPLA) has been shown to be a powerful LPS antagonist in both human and murine cell lines. In addition, RsDPLA also can protect mice against the lethal effects of toxic LPS. In this study, we complexed both the deep rough LPS from Escherichia coli D31 m4 (ReLPS) and RsDPLA with 5- and 30-nm colloidal gold and compared their binding to the RAW 264.7 cell line by electron microscopy. Both ReLPS and RsDPLA bound to the cells with the following observations. First, binding studies revealed that pretreatment with RsDPLA completely blocked the binding and thus internalization of ReLPS-gold conjugates to these cells at both 37°C and 4°C. Second, ReLPS was internalized via micropinocytosis (noncoated plasma membrane invaginations) involving formation of caveolae-like structures and leading to the formation of micropinocytotic vesicles, macropinocytosis (or phagocytosis), formation of clathrin-coated pits (receptor mediated), and penetration through plasma membrane into cytoplasm. Third, in contrast, RsDPLA was internalized predominantly via macropinocytosis. These studies show for the first time that RsDPLA blocks the binding and thus internalization of LPS as observed by scanning and transmission electron microscopy.