Biomaterials (v.27, #5)
Secrets of the code: Do vascular endothelial cells use ion channels to decipher complex flow signals? by Abdul I. Barakat; Deborah K. Lieu; Andrea Gojova (671-678).
The ability of vascular endothelial cells (ECs) to respond to changes in blood flow is essential for both vasoregulation and arterial wall remodelling, while abnormalities in endothelial responsiveness to flow play an important role in the development of atherosclerosis. Endothelial flow responses also have important implications for the field of vascular tissue engineering. In response to changes in fluid dynamic shear stress, ECs exhibit humoral, metabolic, and structural responses. Significantly, ECs respond differently to different types of shear stress. For instance, steady shear stress elicits a profile of responses that differs drastically from oscillatory shear stress. Although our understanding of flow-induced signaling has advanced greatly over the past two decades, how ECs sense shear forces remains to be established. Furthermore, the mechanisms by which ECs discriminate among different flow waveforms are unknown. Activation of flow-sensitive ion channels is one of the most rapid known responses to flow in ECs. In this paper, we argue in favor of an important role for ion channels in shear stress sensing in ECs and propose that these channels may endow ECs with the ability to resolve components of a complex flow signal and hence distinguish among different types of flow.
Keywords: Ion channels; Endothelial cells; Shear stress; Mechanotransduction; Cytoskeleton; Membrane;
Single molecule fluorescence studies of surface-adsorbed fibronectin by Meher Antia; Leon D. Islas; David A. Boness; Gretchen Baneyx; Viola Vogel (679-690).
The conformation of the extracellular matrix protein fibronectin plays a critical role in regulating cell function, including cell adhesion and migration. While average conformations of large ensembles of adhesion proteins have been previously measured, cells may sensitively respond to conformational outliers. We therefore applied both single molecule imaging and spectroscopy techniques to map a range of conformational states of individual fibronectin molecules adsorbed to glass, as well as to measure their conformational fluctuations in time. Single-step photobleaching experiments confirmed single molecule sensitivity. Single molecule spectra showed fluctuations in the peak wavelength, both over a spatial ensemble of molecules and in a single molecule over time, most likely indicating the different conformational states fibronectin can assume upon surface adsorption. Single-pair fluorescence resonance energy transfer (FRET) revealed that a fraction of fibronectin molecules existed in conformations that allowed for energy transfer between the labeled cysteine residues of the two dimeric arms folded upon each other, and that fluctuations occurred in the FRET efficiency. Fluorescence polarization experiments identified two possible sources of FRET fluctuations: changes in fluorophore orientation and conformational fluctuations of fibronectin over a time scale of seconds.
Keywords: Single molecule fluorescence; Fibronectin; RGD; Protein adsorption; Structural fluctuations; Biomaterials; Cell adhesion.;
Fabrication, characterization, and biological assessment of multilayered DNA-coatings for biomaterial purposes by Jeroen J.J.P. van den Beucken; Matthijn R.J. Vos; Peter C. Thüne; Tohru Hayakawa; Tadao Fukushima; Yoshio Okahata; X. Frank Walboomers; Nico A.J.M. Sommerdijk; Roeland J.M. Nolte; John A. Jansen (691-701).
This study describes the fabrication of two types of multilayered coatings onto titanium by electrostatic self-assembly (ESA), using deoxyribosenucleic acid (DNA) as the anionic polyelectrolyte and poly-d-lysine (PDL) or poly(allylamine hydrochloride) (PAH) as the cationic polyelectrolyte. Both coatings were characterized using UV-vis spectrophotometry, atomic force microscopy (AFM), X-ray photospectroscopy (XPS), contact angle measurements, Fourier transform infrared spectroscopy (FTIR), and for the amount of DNA immobilized. The mutagenicity of the constituents of the coatings was assessed. Titanium substrates with or without multilayered DNA-coatings were used in cell culture experiments to study cell proliferation, viability, and morphology. Results of UV-vis spectrophotometry, AFM, and contact angle measurements clearly indicated the progressive build-up of the multilayered coatings. Furthermore, AFM and XPS data showed a more uniform build-up and morphology of [PDL/DNA]-coatings compared to [PAH/DNA]-coatings. DNA-immobilization into both coatings was linear, and approximated 3 μg/cm2 into each double-layer. The surface morphology of both types of multilayered DNA-coatings showed elevations in the nanoscale range. No mutagenic effects of DNA, PDL, or PAH were detected, and cell viability and morphology were not affected by the presence of either type of multilayered DNA-coating. Still, the results of the proliferation assay revealed an increased proliferation of primary rat dermal fibroblasts on both types of multilayered DNA-coatings compared to non-coated controls. The biocompatibility and functionalization of the coatings produced here, will be assessed in subsequent cell culture and animal-implantation studies.
Keywords: AFM; Cell culture; Cell morphology; Cell proliferation; Cell viability; Electrostatic self-assembly; Fibroblast; FTIR; MIT assay; Mutagenicity; Nanotopography; SEM; Surface modification; Titanium;
Biocompatibility and remodeling potential of pure arterial elastin and collagen scaffolds by Dan T. Simionescu; Qijin Lu; Ying Song; JeoungSoo Lee; Tabitha N. Rosenbalm; Catherine Kelley; Naren R. Vyavahare (702-713).
Surgical therapy of cardiovascular disorders frequently requires replacement of diseased tissues with prosthetic devices or grafts. In typical tissue engineering approaches, scaffolds are utilized to serve as templates to support cell growth and remodeling. Decellularized vascular matrices have been previously investigated as scaffolds for tissue engineering. However, cell migration into these scaffolds was inadequate due to the very tight matrix organization specific to the aortic structure. To address this problem, we prepared two types of decellularized scaffolds from porcine vascular tissues. Pure elastin scaffolds and pure collagen scaffolds were prepared by selectively removing the collagen component or elastin, respectively. In the current study, we use a subdermal implantation model to demonstrate that arterial elastin and collagen scaffolds exhibit enhanced potential for repopulation by host cells in vivo. Notably, numerous new collagen fibers and bundles were found within the remodeled elastin scaffolds and new elastin fibers within collagen scaffolds, respectively, clearly indicating their ability to support de novo extracellular matrix synthesis. We also show that biological cues such as growth factors are required for efficient repopulation of elastin and collagen scaffolds. Finally, we bring evidence that these scaffolds can be endothelialized in vitro for thrombosis resistance and thus can serve as promising candidates for cardiovascular tissue engineering.
Keywords: Arterial tissue engineering; Collagen; Elastin; Vascular grafts; Remodeling;
The in vitro regulation of ovarian follicle development using alginate-extracellular matrix gels by Pamela K. Kreeger; Jason W. Deck; Teresa K. Woodruff; Lonnie D. Shea (714-723).
The extracellular matrix (ECM) provides a three-dimensional structure that promotes and regulates cell adhesion and provides signals that direct the cellular processes leading to tissue development. In this report, synthetic matrices that present defined ECM components were employed to investigate these signaling effects on tissue formation using ovarian follicle maturation as a model system. In vitro systems for follicle culture are being developed to preserve fertility for women, and cultures were performed to test the hypothesis that the ECM regulates follicle maturation in a manner that is dependent on both the ECM identity and the stage of follicle development. Immature mouse follicles were cultured within alginate-based matrices that were modified with specific ECM components (e.g., laminin) or RGD peptides. The matrix maintains the in vivo like morphology of the follicle and provides an environment that supports follicle development. The ECM components signal the somatic cells of the follicle, affecting their growth and differentiation, and unexpectedly also affect the meiotic competence of the oocyte. These effects depend upon both the identity of the ECM components and the initial stage of the follicle, indicating that the ECM is a dynamic regulator of follicle development. The development of synthetic matrices that promote follicle maturation to produce meiotically competent oocytes may provide a mechanism to preserve fertility, or more generally, provide design principles for scaffold-based approaches to tissue engineering.
Keywords: Alginate; Extracellular matrix; Cell encapsulation; Follicle; Ovary;
Electrospinning of collagen and elastin for tissue engineering applications by L. Buttafoco; N.G. Kolkman; P. Engbers-Buijtenhuijs; A.A. Poot; P.J. Dijkstra; I. Vermes; J. Feijen (724-734).
Meshes of collagen and/or elastin were successfully prepared by means of electrospinning from aqueous solutions. Flow rate, applied electric field, collecting distance and composition of the starting solutions determined the morphology of the obtained fibres. Addition of PEO ( M w = 8 × 1 0 6 ) and NaCl was always necessary to spin continuous and homogeneous fibres. Spinning a mixture of collagen and elastin resulted in fibres in which the single components could not be distinguished by SEM. Increasing the elastin content determined an increase in fibres diameters from 220 to 600 nm. The voltage necessary for a continuous production of fibres was dependent on the composition of the starting solution, but always between 10 and 25 kV. Under these conditions, non-woven meshes could be formed and a partial orientation of the fibres constituting the mesh was obtained by using a rotating tubular mandrel as collector. Collagen/elastin (1:1) meshes were stabilized by crosslinking with N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). This treatment afforded materials with a high thermal stability ( T d = 79 ° C ) without altering their original morphology. Upon crosslinking PEO and NaCl were fully leached out. Smooth muscle cells grew as a confluent layer on top of the crosslinked meshes after 14 d of culture.
Keywords: Collagen; Elastin; Vascular grafts; Crosslinking;
Microintegrating smooth muscle cells into a biodegradable, elastomeric fiber matrix by John J. Stankus; Jianjun Guan; Kazuro Fujimoto; William R. Wagner (735-744).
Electrospinning permits fabrication of biodegradable elastomers into matrices that can resemble the scale and mechanical behavior of the native extracellular matrix. However, achieving high-cellular density and infiltration with this technique remains challenging and time consuming. We have overcome this limitation by electrospraying vascular smooth muscle cells (SMCs) concurrently with electrospinning a biodegradable, elastomeric poly(ester urethane)urea (PEUU). Trypan blue staining revealed no significant decrease in cell viability from the fabrication process and electrosprayed SMCs spread and proliferated similar to control unprocessed SMCs. The resulting SMC microintegrated PEUU constructs were cultured under static conditions or transmural perfusion. Higher cell numbers resulted with perfusion culture with 131% and 98% more viable cells versus static culture at days 4 and 7 ( p < 0.05 ). Fluorescent imaging and hematoxylin and eosin staining further illustrated high cell densities integrated between the elastomeric fibers after perfusion culture. SMC microintegrated PEUU was strong, flexible and anisotropic with tensile strengths ranging from 2.0 to 6.5 MPa and breaking strains from 850 to 1700% dependent on the material axis. The ability to microintegrate smooth muscle or other cell types into a biodegradable elastomer fiber matrix embodies a novel tissue engineering approach that could be applied to fabricate high cell density elastic tissue mimetics, blood vessels or other cardiovascular tissues.
Keywords: Bioreactor; Smooth muscle cell; Elastomer; Electrospinning; Polyurethane; Scaffold;
Tannic acid mimicking dendrimers as small intestine submucosa stabilizing nanomordants by Vladimir Kasyanov; Jason Isenburg; Robert A. Draughn; Starr Hazard; Jason Hodde; Iveta Ozolanta; Modra Murovska; S. Bart Halkes; Ioannis Vrasidas; Rob M.J. Liskamp; Roland J. Pieters; Dan Simionescu; Roger R. Markwald; Vladimir Mironov (745-751).
Chemical stabilization resulting in increased resistance to proteolytic degradation is one of the approaches in prevention of post-implantational aneurysm development in decellularized natural vascular scaffolds. Recently, tannic acid (TA) and tannic acid mimicking dendrimers (TAMD) have been suggested as potential stabilization agents for collagen and elastin. The aim of this work was to determine the stabilizing effects of TAMD on decellularized natural scaffolds. Vascular scaffolds fabricated from small intestine submucosa (SIS) and SIS plane sheets (Cook Biotech Inc.) were used. The biomechanical properties of the SIS vascular graft segments treated with TA and TAMD were tested. The effect of TAMD treatment on resistance to proteolytic degradation was evaluated by measuring biomechanical properties of TAMD stabilized and non-stabilized SIS specimens after incubation in collagenase solution. It was shown that treatment with TA as well as with TAMD increased the strength of tubular SIS as well as their resistance to proteolytic biodegradation manifested by preservation of biomechanical properties after collagenase treatment. Transmission electron microscopy demonstrated that treatment with TAMD increased the periodical pattern typical of collagen fiber ultrastructure as a result of the “mordant” effect. The possible collagen cross-linking effect of TAMD on SIS was investigated by differential scanning calorimetry (DSC). The treatment with TAMD induced a small, but detectable cross-linking effect, suggesting that TAMD do not establish extensive covalent cross links within the extracellular matrix but rather interact with collagen, thus rendering SIS scaffolds more resistant to proteolytic degradation.
Keywords: Dendrimer; Tannic acid; Acellular scaffold; Vascular prosthesis; Biomechanical properties; Nanomordant;
Correlating subjective and objective descriptors of ultra high molecular weight wear particles from total joint prostheses by Brian T. McMullin; Ming-Ying Leung; Arun S. Shanbhag; Donald McNulty; Jay D. Mabrey; C. Mauli Agrawal (752-757).
A total of 750 images of individual ultra-high molecular weight polyethylene (UHMWPE) particles isolated from periprosthetic failed hip, knee, and shoulder arthroplasties were extracted from archival scanning electron micrographs. Particle size and morphology was subsequently analyzed using computerized image analysis software utilizing five descriptors found in ASTM F1877-98, a standard for quantitative description of wear debris. An online survey application was developed to display particle images, and allowed ten respondents to classify particle morphologies according to commonly used terminology as fibers, flakes, or granules. Particles were categorized based on a simple majority of responses. All descriptors were evaluated using a one-way ANOVA and Tukey–Kramer test for all-pairs comparison among each class of particles. A logistic regression model using half of the particles included in the survey was then used to develop a mathematical scheme to predict whether a given particle should be classified as a fiber, flake, or granule based on its quantitative measurements. The validity of the model was then assessed using the other half of the survey particles and compared with human responses. Comparison of the quantitative measurements of isolated particles showed that the morphologies of each particle type classified by respondents were statistically different from one another ( p < 0.05 ). The average agreement between mathematical prediction and human respondents was 83.5% (standard error 0.16%). These data suggest that computerized descriptors can be feasibly correlated with subjective terminology, thus providing a basis for a common vocabulary for particle description which can be translated into quantitative dimensions.
Keywords: Wear debris; Arthroplasty; Polyethylene; Joint replacement;
Improved surgical mesh integration into the rat abdominal wall with arginine administration by M.A. Arbos; J.M. Ferrando; M.T. Quiles; J. Vidal; M. López-Cano; J. Gil; J.M. Manero; J. Peña; P. Huguet; S. Schwartz-Riera; J. Reventós; M. Armengol (758-768).
Prosthetic meshes are used as the standard of care in abdominal wall hernia repair. However, hernia recurrences and side effects remain unsolved problems. The demand by health care providers for increasingly efficient and cost-effective surgery encourages the development of newer strategies to improve devices and outcomes. Here, we evaluated whether l-arginine administration was able to ameliorate long-term polypropylene prostheses incorporation into the abdominal wall of Sprague–Dawley rats. Meshes were placed on-lay and continuous l-arginine was administered. In vivo biocompatibility was studied at 7, 25 and 30 days post-implantation. Effectively, l-arginine administration in combination with mesh triggered subtle changes in ECM composition that impinged on critical biochemical and structural features. Lastly, tensile strength augmented and stiffness decreased over the control condition. This could help to restructure the mechanical load transfer from the implant to the brittle surrounding tissues, i.e., impact load and fatigue load associated with mechanical tensions could be distributed between the mesh and the restored tissue in a more balanced manner, and ultimately help to reduce the incidence of loosening, recurrences, and local wound complications. Since the newly formed tissue is more mechanically stable, this approach could eventually be introduced to human hernia repair.
Keywords: Surgical mesh; l-arginine; ECM; Angiogenesis; Environmental scanning electron microscopy (ESEM); Soft tissue biomechanics;
A comparison of mineral affinity of bisphosphonate–protein conjugates constructed with disulfide and thioether linkages by Jennifer E.I. Wright; Sebastien A. Gittens; Geeti Bansal; Pavel I. Kitov; Dennis Sindrey; Cezary Kucharski; Hasan Uludağ (769-784).
Chemical conjugation of bisphosphonates (BPs) to therapeutic proteins is an effective means to impart mineral affinity to proteins. Such conjugates can be implanted with mineral-based matrices to control the local delivery kinetics of the proteins. BPs linked to proteins with reversible (i.e., cleavable) linkages are desirable over conjugates with stable linkages to release the protein in free form. This study conducted a direct comparison of mineral affinity of BP–protein conjugates linked together with cleavable disulfide and non-cleavable thioether linkages. Bovine serum albumin (BSA) was used as a model protein and the desired conjugates were created with N-succinimidyl-3-(2-pyridyldithio)propionate (disulfide) and succinimidyl-4-(N-maleimido-methyl)cyclohexane-1-carboxylate (thioether) linkers. The disulfide-linked conjugates were cleaved in the presence of a major thiol constituent of serum, cysteine. The imparted mineral affinity, as assessed by hydroxyapatite binding in vitro, was lost upon the cleavage of the disulfide-linked aminoBP. The presence of the serum did not accelerate the cleavage of disulfide-linked conjugates. The aminoBP–BSA conjugates formed with disulfide and thioether linkages were subcutaneously implanted in rats with two different mineral-based matrices to assess protein loss from the matrices. All conjugates exhibited a higher retention in mineral matrices as compared to unmodified BSA. However, no significant differences in in situ pharmacokinetics of the disulfide- and thioether-linked conjugates were observed. We conclude that disulfide-linked BP conjugates were readily cleavable by the amino acid cysteine in vitro, but in vivo cleavage of the disulfide-linked conjugates was not evident when the proteins were implanted adsorbed to mineral-based matrices. BP–protein conjugates with faster-cleaving tethers might be required to significantly influence the release of the BP conjugates from the mineral matrices.
Keywords: Bone tissue engineering; Drug delivery; Hydroxyapatite; Protein adsorption; Coupling agent;
Mechanical properties of human stratum corneum: Effects of temperature, hydration, and chemical treatment by Kenneth S. Wu; William W. van Osdol; Reinhold H. Dauskardt (785-795).
An in vitro mechanics approach to quantify the intercellular delamination energy and mechanical behavior of isolated human stratum corneum (SC) in a direction perpendicular to the skin surface is presented. The effects of temperature, hydration, and a chloroform–methanol treatment to remove intercellular lipids were explored. The delamination energy for debonding of cells within the SC layer was found to be sensitive to the moisture content of the tissue and to the test temperature. Delamination energies for untreated stratum corneum were measured in the range of 1–8 J/m2 depending on test temperature. Fully hydrated specimen energies decreased with increasing temperature, while room-humidity-hydrated specimens exhibited more constant values of 2–4 J/m2. Lipid-extracted specimens exhibited higher delamination energies of ∼12 J/m2, with values decreasing to ∼4 J/m2 with increasing test temperature. The peak separation stress decreased with increasing temperature and hydration, but lipid-extracted specimens exhibited higher peak stresses than untreated controls. The delaminated surfaces revealed an intercellular failure path with no evidence of tearing or fracture of cells. The highly anisotropic mechanical behavior of the SC is discussed in relation to the underlying SC structure.
Keywords: Mechanical properties; Fracture toughness; Epithelial cell; Stratum corneum; Tissue treatment;
Mathematical modeling of material-induced blood plasma coagulation by Zhe Guo; Karen M. Bussard; Kaushik Chatterjee; Rachel Miller; Erwin A. Vogler; Christopher A. Siedlecki (796-806).
Contact activation of the intrinsic pathway of the blood coagulation cascade is initiated when a procoagulant material interacts with coagulation factor XII, (FXII) yielding a proteolytic enzyme FXIIa. Procoagulant surface properties are thought to play an important role in activation. To study the mechanism of interaction between procoagulant materials and blood plasma, a mathematical model that is similar in form and in derivation to Michaelis–Menten enzyme kinetics was developed in order to yield tractable relationships between dose (surface area and energy) and response (coagulation time (CT)). The application of this model to experimental data suggests that CT is dependent on the FXIIa concentration and that the amount of FXIIa generated can be analyzed using a model that is linearly dependent on contact time. It is concluded from these experiments and modeling analysis that the primary mechanism for activation of coagulation involves autoactivation of FXII by the procoagulant surface or kallikrein-mediated reciprocal activation of FXII. FXIIa-induced self-amplification of FXII is insignificant.
Keywords: Coagulation; Blood compatibility; Plasma proteins; Modeling;