Fibroblast cytoskeletal remodeling contributes to connective tissue tension

The visco-elastic behavior of connective tissue is generally attributed to the material properties of the extracellular matrix rather than cellular activity. We have previously shown that fibroblasts within areolar connective tissue exhibit dynamic cytoskeletal remodeling within minutes in response to tissue stretch ex vivo and in vivo. Here, we tested the hypothesis that fibroblasts, through this cytoskeletal remodeling, actively contribute to the visco-elastic behavior of the whole tissue. We measured significantly increased tissue tension when cellular function was broadly inhibited by sodium azide and when cytoskeletal dynamics were compromised by disrupting microtubules (with colchicine) or actomyosin contractility (via Rho kinase inhibition). These treatments led to a decrease in cell body cross-sectional area and cell field perimeter (obtained by joining the end of all of a fibroblast's processes). Suppressing lamellipodia formation by inhibiting Rac-1 decreased cell body cross-sectional area but did not affect cell field perimeter or tissue tension. Thus, by changing shape, fibroblasts can dynamically modulate the visco-elastic behavior of areolar connective tissue through Rho-dependent cytoskeletal mechanisms. These results have broad implications for our understanding of the dynamic interplay of forces between fibroblasts and their surrounding matrix, as well as for the neural, vascular, and immune cell populations residing within connective tissue.     

Biomaterial surface chemistry dictates adherent monocyte/macrophage cytokine expression in vitro

An in vitro human monocyte culture system was used to determine whether adherent monocyte/macrophage cytokine production was influenced by material surface chemistry. A polyethylene terephthalate (PET) base surface was modified by photograft copolymerization to yield hydrophobic, hydrophilic, anionic and cationic surfaces. Freshly isolated human monocytes were cultured onto the surfaces for periods up to 10 days in the presence or absence of interleukin-4 (IL-4). Semi-quantitative RT-PCR analysis on days 3, 7 and 10 of cell culture revealed that interleukin-10 (IL-10) expression significantly increased in cells adherent to the hydrophilic and anionic surfaces but significantly decreased in the cationic surface adherent monocytes/macrophages. Conversely, interleukin-8 (IL-8) expression was significantly decreased in cells adherent to the hydrophilic and anionic surfaces. Further analysis revealed that the hydrophilic and anionic surfaces inhibited monocyte adhesion and IL-4-mediated macrophage fusion into foreign body giant cells (FBGCs). Therefore, hydrophilic and anionic surfaces promote an anti-inflammatory type of response by dictating selective cytokine production by biomaterial adherent monocytes and macrophages. These studies contribute information necessary to enhance our understanding of biocompatibility to be used to improve the in vivo lifetime of implanted medical devices and prostheses.     

Tissue reactions to breast implants coated with polyurethane.

The histological features noted in the capsules from 7 polyurethane coated silicone breast prostheses are described. The polyurethane provoked a definite foreign body reaction and was slowly degraded, with some particles ejected from the capsule into the surrounding tissues. Separation of the polyurethane coating from the silicone prosthesis and the degradation of the polyurethane took about two years. Another much more resistant foreign material was found to occur in conjunction with the polyurethane in the capsules. It may be an adhesive or flakes off the silicone shell. Vacuolated spaces were noted in the inner layers of 3 capsules; it was assumed that they contained liquid silicone.     

Steric hindrance effects on surface reactions: applications to BIAcore

Because surface-volume reactions occur in many biological and industrial processes, understanding the rate of such reactions is important. The BIAcore surface plasmon resonance (SPR) biosensor for measuring rate constants has such a geometry. Though several models of the BIAcore have been presented, few take into account that large ligand molecules can block multiple receptor sites, thus skewing the sensogram data. In this paper some general mathematical principles are stated for handling this phenomenon, and a surface-reaction model is presented explicitly. An integro-partial differential equation results, which can be simplified greatly using perturbation techniques, yielding linear and nonlinear integrodifferential equations. Explicit and asymptotic solutions are constructed for cases motivated by experimental design. The general analysis can provide insight into surface-volume reactions occurring in various contexts. In particular, the steric hindrance effect can be quantified with a single dimensionless parameter. This work was supported in part by NIGMS Grant 1R01GM067244-01.     

Orchestrating cell/material interactions for tissue engineering of surgical implants

Research groups are currently recognising a critical clinical need for innovative approaches to organ failure and agenesis. Allografting, autologous reconstruction and prosthetics are hampered with severe limitations. Pertinently, readily available 'laboratory-grown' organs and implants are becoming a reality. Tissue engineering constructs vary in their design complexity depending on the specific structural and functional demands. Expeditious methods on integrating autologous stem cells onto nanoarchitectured 3D nanocomposites, are being transferred from lab to patients with a number of successful first-in-man experiences. Despite the need for a complete understanding of cell/material interactions tissue engineering is offering a plethora of exciting possibilities in regenerative medicine.     

Surface-mediated enzymatic reactions: simulations of tissue factor activation of factor X on a lipid surface.

Blood coagulation proceeds via reactions in which zymogen coagulation factors are activated to proteases. An essential step is the activation of factor X by a complex of tissue factor and factor VIIa. This complex usually is studied using phospholipid vesicles into which tissue factor is inserted. Because factor X exists free in solution and bound to the lipid-surface, it is difficult to establish experimentally the kinetic contribution of surfaces. We therefore developed a stochastic model to simulate such reactions and generate initial velocity data from which Michaelis-Menten parameters are estimated. Simulated Km values decrease slightly when substrate binding to lipid is increased and by a factor of four when the rates of surface diffusion are increased to that of fluid phase-diffusion. Simulations with various size planar surfaces established an enzyme capture radius of 32-64 nm. Simulations with different modes of enzyme-substrate complex assembly show that if the true substrate is lipid-bound, under certain conditions, the true Kcat is not measured; rather, the product "leaving rate" from the complex is the rate-limiting step that is measured as substrate is taken to infinity. This model is applicable to any surface-bound enzyme reaction.     

Polyelectrolyte/amphiphile interaction studied by surface tension measurements

The interaction of κ-carrageenan with three positively charged drug molecules with amphiphile character has been examined using surface tension measurements. The surface tension was measured by the pendant drop method which makes possible the determination at an apparent steady state which is important for polymeric systems. The results are compared with adsorption isotherms from dialysis equilibrium. The surface tension data, show that the presence of κ-carrageenan in the amphiphile solutions leads to an increased and pronounced lowering of the surface tension in a low concentration range of amphiphile. It is also shown that not only the hydrophobicity of the amphiphile but also the structure of the polyelectrolyte (charge density and helix-coil structure) largely determine the extent of interaction.     

Fluid structure interaction with contact surface methodology for evaluation of endovascular carotid implants for drug-resistant hypertension treatment

Drug-resistant hypertensive patients may be treated by mechanical stimulation of stretch-sensitive baroreceptors located in the sinus of carotid arteries. To evaluate the efficacy of endovascular devices to stretch the carotid sinus such that the induced strain might trigger baroreceptors to increase action potential firing rate and thereby reduce systemic blood pressure, numerical simulations were conducted of devices deployed in subject-specific carotid models. Two models were chosen--a typical physiologic carotid and a diminutive atypical physiologic model representing a clinically worst case scenario--to evaluate the effects of device deployment in normal and extreme cases, respectively. Based on the anatomical dimensions of the carotids, two different device sizes were chosen out of five total device sizes available. A fluid structure interaction (FSI) simulation methodology with contact surface between the device and the arterial wall was implemented for resolving the stresses and strains induced by device deployment. Results indicate that device deployment in the carotid sinus of the physiologic model induces an increase of 2.5% and 7.5% in circumferential and longitudinal wall stretch, respectively, and a maximum of 54% increase in von Mises arterial stress at the sinus wall baroreceptor region. The second device, deployed in the diminutive carotid model, induces an increase of 6% in both circumferential and longitudinal stretch and a 50% maximum increase in von Mises stress at the sinus wall baroreceptor region. Device deployment has a minimal effect on blood-flow patterns, indicating that it does not adversely affect carotid bifurcation hemodynamics in the physiologic model. In the smaller carotid model, deployment of the device lowers wall shear stress at sinus by 16% while accelerating flow entering the external carotid artery branch. Our FSI simulations of carotid arteries with deployed device show that the device induces localized increase in wall stretch at the sinus, suggesting that this will activate baroreceptors and subsequently may control hypertension in drug-resistant hypertensive patients, with no consequential deleterious effects on the carotid sinus hemodynamics.     

Dynamic Fibroblast Cultures: Response to Mechanical Stretching

Mechanical forces play an important role in the organization, growth and function of tissues. Dynamic extracellular environment affects cellular behavior modifying their orientation and their cytoskeleton. In this work, human fibroblasts have been subjected for three hours to increasing substrate deformations (1–25%) applied as cyclic uniaxial stretching at different frequencies (from 0.25 Hz to 3 Hz). Our objective was to identify whether and in which ranges the different deformations magnitude and rate were the factors responsible of the cell alignment and if actin cytoskeleton modification was involved in these responses. After three hours of cyclically stretched substrate, results evidenced that fibroblasts aligned perpendicularly to the stretch direction at 1% substrate deformation and reached statistically higher orientation at 2% substrate deformation with unmodified values at 5–20%, while 25% substrate deformation induced cellular death. It was also shown that a percentage of cells oriented perpendicularly to the deformation were not influenced by increased frequency of cyclical three hours deformations (0.25–3 Hz). Cyclic substrate deformation was shown also to involve actin fibers which orient perpendicularly to the stress direction as well. Thus, we argue that a substrate deformation induces a dynamic change in cytoskeleton able to modify the entire morphology of the cells.Key Words: mechanical stretching, cell orientation, stress fibers     

The tissue reactions of mice to infection with Heligmosmoides polygyrus

The intestines of normal and resistant LAF1 mice were subjected to histologic study to determine the timing and mechanisms of resistance to reinfection by Heligmosmoides polygyrus. During reinfection third-stage larvae are less able to penetrate the intestinal wall. Larvae which are able to encyst develop at a slower rate and provoke an increase in nonspecific inflammation around their cysts. After emergence from intestinal cysts, preadults are rapidly lost, but at no time were injured or destroyed larvae or adults noted. Exsheathed larvae were injected via tail vein into control, sensitized and resistant BALB/c mice. The inflammatory response around entrapped larvae in the lung was measured at 1, 2, 4, and 8 days. A heightened inflammatory response, consisting primarily of polymorphonuclear cells with some round cells which peaked in size on day 2, was observed in both sensitized and resistant mice. A similar heightened inflammatory response was also observed in both AKR (non-resistant) BALB/c (resistant) mice vaccinated subcutaneously with exsheathed larvae.     

Bioelectronics meets nanomedicine for cardiovascular implants: PEDOT-based nanocoatings for tissue regeneration

Background

An exciting direction in nanomedicine would be to analyze how living cells respond to conducting polymers. Their application for tissue regeneration may advance the performance of drug eluting stents by addressing the delayed stent re-endothelialization and late stent thrombosis.

Methods

The suitability of poly (3, 4-ethylenedioxythiophene) (PEDOT) thin films for stents to promote cell adhesion and proliferation is tested in correlation with doping and physicochemical properties. PEDOT doped either with poly (styrenesulfonate) (PSS) or tosylate anion (TOS) was used for films' fabrication by spin coating and vapor phase polymerization respectively. PEGylation of PEDOT: TOS for reduced immunogenicity and biofunctionalization of PEDOT: PSS with RGD peptides for induced cell proliferation was further applied. Atomic Force Microscopy and Spectroscopic Ellipsometry were implemented for nanotopographical, structural, optical and conductivity measurements in parallel with wettability and protein adsorption studies. Direct and extract testing of cell viability and proliferation of L929 fibroblasts on PEDOT samples by MTT assay in line with SEM studies follow.

Results

All PEDOT thin films are cytocompatible and promote human serum albumin adsorption. PEDOT:TOS films were found superior regarding cell adhesion as compared to controls. Their nanotopography and hydrophilicity are significant factors that influence cytocompatibility. PEGylation of PEDOT:TOS increases their conductivity and hydrophilicity with similar results on cell viability with bare PEDOT:TOS. The biofunctionalized PEDOT:PSS thin films show enhanced cell proliferation.

Conclusions

The application of PEDOT polymers has evolved as a new perspective to advance stents.

General significance

In this work, nanomedicine involving nanotools and novel nanomaterials merges with bioelectronics to stimulate tissue regeneration for cardiovascular implants. This article is part of a Special Issue entitled Organic Bioelectronics — Novel Applications in Biomedicine.     

Transplantation and cellular reactions to tissue antigens in Streptococcus-immunized rabbits]

The immunization of rabbits with the cells and the disintegration products of fractions of the cytoplasmic membranes of group A streptococcus (type 1) in incomplete Freund adjuvant, introduced in a single injection into the pads of the paws, caused lesions in autoplastic skin grafts and accelerated the rejection of alloplastic skin grafts. The rabbits showed positive delayed-type skin reactions to streptococcus and homologous skin antigens, and lymphocytes specifically reacting with FITC-labeled homologous skin antigen were found in their blood. Prolonged intravenous immunization with streptococcus, which induced the formation of complement fixing antibodies to homologous skin antigens, did not influence the taking of autoplastic and alloplastic skin grafts. The injection of hyperimmune streptococcus rabbit antiserum containing antibodies to skin antigens to intact rabbits produced no lesions in the autoplastic skin grafts and prolonged the lift of the alloplastic skin grafts.     

A method to estimate in vivo dynamic articular surface interaction

This paper describes a method to calculate and visualize the proximity of subchondral bone surfaces during dynamic movement. This method combines high-speed biplane radiographic image data and three-dimensional (3D) bone surface information derived from computed tomography to determine subchondral bone motion during dynamic activities. Knowledge of in vivo subchondral bone motion may be useful in the study of osteoarthritis, in biomechanical modeling, and in identifying normal and pathological joint mechanics. This method can be used to identify the regions of close contact during dynamic motion, to calculate the surface area of subchondral bone within close contact, and to determine the changing position of the close contact area during dynamic activities. None of this informations can be obtained using other currently available 3D motion analysis techniques. Example applications showing dynamic in vivo tibio-femoral bone surface motion during canine gait and human one-legged hopping are presented.