Multilayered Matrices Research Articles

Advantages and limitations of using cell viability assays for 3D bioprinted constructs

Bioprinting shows promise for bioengineered scaffolds and three-dimensional (3D) disease models, but assessing the viability of embedded cells is challenging. Conventional assays are limited by the technical problems that derive from using multi-layered bioink matrices dispersing cells in three dimensions. In this study, we tested bioprinted osteogenic bioinks as a model system. Alginate- or gelatin-based bioinks were loaded with/without ceramic microparticles and osteogenic cells (bone tumor cells, with or without normal bone cells). Despite demonstrating 80%–90% viability through manual counting and live/dead staining, this was time-consuming and operator-dependent. Moreover, for the alginate-bioprinted scaffold, cell spheroids could not be distinguished from single cells. The indirect assay (alamarBlue), was faster but less accurate than live/dead staining due to dependence on hydrogel permeability. Automated confocal microscope acquisition and cell counting of live/dead staining was more reproducible, reliable, faster, efficient, and avoided overestimates compared to manual cell counting by optical microscopy. Finally, for 1.2 mm thick 3D bioprints, dual-photon confocal scanning with vital staining greatly improved the precision of the evaluation of cell distribution and viability and cell–cell interactions through the z-axis. In summary, automated confocal microscopy and cell counting provided superior accuracy for the assessment of cell viability and interactions in 3D bioprinted models compared to most commonly and currently used techniques.

Local extensional flows promote long-range fiber alignment in 3D collagen hydrogels

Randomly oriented type I collagen (COL1) fibers in the extracellular matrix are reorganized by biophysical forces into aligned domains extending several millimeters and with varying degrees of fiber alignment. These aligned fibers can transmit traction forces, guide tumor cell migration, facilitate angiogenesis, and influence tissue morphogenesis. To create aligned COL1 domains in microfluidic cell culture models, shear flows have been used to align thin COL1 matrices (<50 µm in height) in a microchannel. However, there has been limited investigation into the role of shear flows in aligning 3D hydrogels (>130 µm). Here, we show that pure shear flows do not induce fiber alignment in 3D atelo COL1 hydrogels, but the simple addition of local extensional flow promotes alignment that is maintained across several millimeters, with a degree of alignment directly related to the extensional strain rate. We further advance experimental capabilities by addressing the practical challenge of accessing a 3D hydrogel formed within a microchannel by introducing a magnetically coupled modular platform that can be released to expose the microengineered hydrogel. We demonstrate the platform’s capability to pattern cells and fabricate multi-layered COL1 matrices using layer-by-layer fabrication and specialized modules. Our approach provides an easy-to-use fabrication method to achieve advanced hydrogel microengineering capabilities that combine fiber alignment with biofabrication capabilities.

Application of Ion Exchange and Adsorption Techniques for Separation of Whey Proteins from Bovine Milk

Background:The world production of whey was estimated to be more than 200 million tons per year. Although whey is an important source of proteins with high nutritional value and biotechnological importance, it is still considered as a by-product of the dairy industry with low economic value due to low industrial exploitation. There are several challenges in the separation of whey proteins: low concentration, the complexity of the material and similar properties (pI, molecular mass) of some proteins.Methods:A narrative review of all the relevant papers on the present methodologies based on ion-exchange and adsorption principles for isolation of whey proteins, known to the authors, was conducted.Results:Traditional ion-exchange techniques are widely used for the separation and purification of the bovine whey proteins. These methodologies, based on the anion or cation chromatographic procedures, as well as combination of aforementioned techniques are still preferential methods for the isolation of the whey proteins on the laboratory scale. However, more recent research on ion exchange membranes for this purpose has been introduced, with promising potential to be applied on the pilot industrial scale. Newly developed methodologies based either on the ion-exchange separation (for example: simulated moving bed chromatography, expanded bed adsorption, magnetic ion exchangers, etc.) or adsorption (for example: adsorption on hydroxyapatite or activated carbon, or molecular imprinting) are promising approaches for scaling up of the whey proteins’ purification processes.Conclusion:Many procedures based on ion exchange are successfully implemented for separation and purification of whey proteins, providing protein preparations of moderate-to-high yield and satisfactory purity. However, the authors anticipate further development of adsorption-based methodologies for separation of whey proteins by targeting the differences in proteins’ structures rather than targeting the differences in molecular masses and pI. The complex composite multilayered matrices, including also inorganic components, are promising materials for simultaneous exploiting of the differences in the masses, pI and structures of whey proteins for the separation.

Thick nanoporous matrices of polystyrene nanoparticles and their potential for electrochemical biosensing

Solid-state nanopores with diameter in units of nanometer can be formed by assembling spherical nanoparticles in a dense arrangement. In the current work, the properties of multi-layered highly ordered assemblies of polystyrene nanoparticles were studied, and their feasibility for electrochemical biosensing was probed. These thick matrices were built using a step-by-step deposition technique. Each individual layer of NPs exhibited distinct color which was caused by the thin film interference effect (a color of specific wavelength was characteristic for matrix of specific thickness). The electrochemical characteristics of matrices were investigated with impedance spectroscopy. The impedance spectra of multi-layered matrices exhibited formation of an additional semicircle (RC component additional to the one in a common Randles equivalent circuit). Further, model biosensing based on nanopore blocking was performed using human serum albumin as an antigen and the corresponding monoclonal antibody as an analyte (serology format). Resulting data disfavored the direct employment of the multi-layered matrices for biosensing purposes as the efficiency decreased with increasing thickness of matrices. However, the data revealed highly valuable information about the diffusion and redox processes in the thick nanoporous matrices.

3D Bioprinting of Human Adipose-Derived Stem Cells and Their Tenogenic Differentiation in Clinical-Grade Medium.

Defining the best combination of cells and biomaterials is a key challenge for the development of tendon tissue engineering (TE) strategies. Adipose-derived stem cells (ASCs) are ideal candidates for this purpose. In addition, controlled cell-based products adherent to good manufacturing practice (GMP) are required for their clinical scale-up. With this aim, in this study, ASC 3D bioprinting and GMP-compliant tenogenic differentiation were investigated. In detail, primary human ASCs were embedded within a nanofibrillar-cellulose/alginate bioink and 3D-bioprinted into multi-layered square-grid matrices. Bioink viscoelastic properties and scaffold ultrastructural morphology were analyzed by rheology and scanning electron microscopy (SEM). The optimal cell concentration for printing among 3, 6 and 9 × 106 ASC/mL was evaluated in terms of cell viability. ASC morphology was characterized by SEM and F-actin immunostaining. Tenogenic differentiation ability was then evaluated in terms of cell viability, morphology and expression of scleraxis and collagen type III by biochemical induction using BMP-12, TGF-β3, CTGF and ascorbic acid supplementation (TENO). Pro-inflammatory cytokine release was also assessed. Bioprinted ASCs showed high viability and survival and exhibited a tenocyte-like phenotype after biochemical induction, with no inflammatory response to the bioink. In conclusion, we report a first proof of concept for the clinical scale-up of ASC 3D bioprinting for tendon TE.

Engineering clinically-relevant human fibroblastic cell-derived extracellular matrices.

Three-dimensional (3D) culturing models, replicating in vivo tissue microenvironments that incorporate native extracellular matrix (ECM), have revolutionized the cell biology field. Fibroblastic cells generate lattices of interstitial ECM proteins. Cell interactions with ECMs and with molecules sequestered/stored within these are crucial for tissue development and homeostasis maintenance. Hence, ECMs provide cells with biochemical and biomechanical cues to support and locally control cell function. Further, dynamic changes in ECMs, and in cell-ECM interactions, partake in growth, development, and temporary occurrences such as acute wound healing. Notably, dysregulation in ECMs and fibroblasts could be important triggers and modulators of pathological events such as developmental defects, and diseases associated with fibrosis and chronic inflammation such as cancer. Studying the type of fibroblastic cells producing these matrices and how alterations to these cells enable changes in ECMs are of paramount importance. This chapter provides a step-by-step method for producing multilayered (e.g., 3D) fibroblastic cell-derived matrices (fCDM). Methods also include means to assess ECM topography and other cellular traits, indicative of fibroblastic functional statuses, like naïve/normal vs. inflammatory and/or myofibroblastic. For these, protocols include indications for isolating normal and diseased fibroblasts (i.e., cancer-associated fibroblasts known as CAFs). Protocols also include means for conducting microscopy assessments, querying whether fibroblasts present with fCDM-dependent normal or CAF phenotypes. These are supported by discrete semi-quantitative digital imaging analyses, providing some imaging processing advice. Additionally, protocols include descriptions for effective fCDM decellularization, which renders cellular debris-free patho/physiological in vivo-like scaffolds, suitable as 3D substrates for subsequent cell culturing.

Multi-Compartmental Hydrogel Microparticles Fabricated by Combination of Sequential Electrospinning and Photopatterning.

Multi-compartmental non-spherical hydrogel microparticles were fabricated by combining electrospinning and photopatterning. Sequential electrospinning produced multi-layered fiber matrices with different composition in which each layer became a compartment of the particle. Photopatterning of the hydrogel in the presence of the multi-layered fiber matrix generated multi-compartmental microparticles with different vertical functionalities. While the shapes of the hydrogel microparticles were determined by the design of the photomask, the chemical properties and size of each compartment were independently controlled by changing the molecules incorporated into each fiber matrix and the electrospinning times, respectively. The resultant multi-compartmental hydrogel microparticles could carry out not only the release of different growth factors with independent kinetics but also binding of multiple targets at different compartments.

A REVIEW ON BI-LAYER TABLETS - AN EMERGING TREND

Bi-layer tablets have been developed to achieve controlled delivery of different drugs with pre-defined release profiles. In the last decade, interest in developing a combination of two or more Active Pharmaceutical Ingredients (API) in a single dosage form (bilayer tablet) has increased in the pharmaceutical industry, promoting patient convenience and compliance. Bi-layer tablet is suitable for sequential release of two drugs in combination, separate two incompatible substances and also for sustained release tablet in which one Layer is immediate release as initial dose and second layer is maintenance dose. Bilayer tablets can be a primary option to avoid chemical incompatibilities between API by physical separation, and to enable the development of different drug release profiles (immediate release with extended release). There is various application of the bilayer tablet it consist of monolithic partially coated or multilayered matrices.

Combined single cell AFM manipulation and TIRFM for probing the molecular stability of multilayer fibrinogen matrices

Adsorption of fibrinogen on various surfaces produces a nanoscale multilayer matrix, which strongly reduces the adhesion of platelets and leukocytes with implications for hemostasis and blood compatibility of biomaterials. The nonadhesive properties of fibrinogen matrices are based on their extensibility, ensuing the inability to transduce strong mechanical forces via cellular integrins and resulting in weak intracellular signaling. In addition, reduced cell adhesion may arise from the weaker associations between fibrinogen molecules in the superficial layers of the matrix. Such reduced stability would allow integrins to pull fibrinogen molecules out of the matrix with comparable or smaller forces than required to break integrin-fibrinogen bonds. To examine this possibility, we developed a method based on the combination of total internal reflection fluorescence microscopy, single cell manipulation with an atomic force microscope and microcontact printing to study the transfer of fibrinogen molecules out of a matrix onto cells. We calculated the average fluorescence intensities per pixel for wild-type HEK 293 (HEK WT) and HEK 293 cells expressing leukocyte integrin Mac-1 (HEK Mac-1) before and after contact with multilayered matrices of fluorescently labeled fibrinogen. For contact times of 500 s, HEK Mac-1 cells show a median increase of 57% of the fluorescence intensity compared to 6% for HEK WT cells. The results suggest that the integrin Mac-1-fibrinogen interactions are stronger than the intermolecular fibrinogen interactions in the superficial layer of the matrix. The low mechanical stability of the multilayer fibrinogen surface may contribute to the reduced cell adhesive properties of fibrinogen-coated substrates. We anticipate that the described method can be applied to various cell types to examine their integrin-mediated adhesion to the extracellular matrices with a variable protein composition.

Numerical modelling and experimental investigation of drug release from layered silicone matrix systems

Medical devices and polymeric matrix systems that release drugs or other bioactive compounds are of interest for a variety of applications. The release of the drug can be dependent on a number of factors such as the solubility, diffusivity, dissolution rate and distribution of the solid drug in the matrix. Achieving the goal of an optimal release profile can be challenging when relying solely on traditional experimental work. Accurate modelling complementing experimentation is therefore desirable. Numerical modelling is increasingly becoming an integral part of research and development due to the significant advances in computer simulation technology.This work focuses on numerical modelling and investigation of multi-layered silicone matrix systems. A numerical model that can be used to model multi-layered systems was constructed and validated by comparison with experimental data. The model could account for the limited dissolution rate and effect of the drug distribution on the release profiles. Parametric study showed how different factors affect the characteristics of drug release. Multi-layered medical silicone matrices were prepared in special moulds, where the quantity of drug in each layer could be varied, and release was investigated with Franz-diffusion cell setup. Data for long-term release was fitted to the model and the full depletion of the system predicted. The numerical model constructed for this study, whose input parameters are the diffusion, effective dissolution rate and dimensional solubility coefficients, does not require any type of steady-state approximation. These results indicate that numerical model can be used as a design tool for development of controlled release systems such as drug-loaded medical devices.

Electrospun matrices for localised controlled drug delivery: release of tetracycline hydrochloride from layers of polycaprolactone and poly(ethylene-co-vinyl acetate).

We report the controlled release of tetracycline (Tet) HCl from a three-layered electrospun matrix for the first time. Five formulations of electrospun poly-ε-caprolactone (PCL) and poly(ethylene-co-vinyl acetate) (PEVA) have been designed, prepared as micro/nanofibre layers, and assayed for the controlled release of the clinically useful antibiotic Tet HCl with potential applications in wound healing and especially in complicated skin and skin-structure infections. Tet HCl was also chosen as a model drug possessing a good ultraviolet (UV) chromophore and capable of fluorescence together with limited stability. Tet HCl was successfully incorporated (essentially quantitatively at 3%, w/w) and provided controlled release from multilayered electrospun matrices. The Tet HCl release test was carried out by a total immersion method on 2 × 2cm(2) electrospun fibrous mats in Tris or phosphate-buffered saline heated to 37°C. The formulation PCL/PEVA/PCL with Tet HCl in each layer gave a large initial (burst) release followed by a sustained release. Adding a third layer to the two-layered formulations led to release being sustained from 6days to more than 15days. There was no detectable loss of Tet chemical stability (as shown by UV and NMR) or bioactivity (as shown by a modified Kirby-Bauer disc assay). Using Tet HCl-sensitive bacteria, Staphylococcus aureus (ATCC 25923), the Tet HCl-loaded three-layered matrix formulations were still showing significantly higher antibacterial effects on days4 and 5 than commercially available Antimicrobial Susceptibility Test Discs of Tet HCl. Electrospinning provides good encapsulation efficiency of Tet HCl within PCL/PEVA/PCL polymers in micro/nanofibre layers which display sustained antibiotic release.

The Mechanism of the Formation of Nonadhesive Fibrinogen Matrices Examined by Atomic Force Microscopy

The plasma protein fibrinogen prevents adhesion of blood cells such as platelets and leukocytes to various surfaces, including fibrin gels. We have previously reported that the decrease in cell adhesion results from fibrinogen aggregation and the formation of an extensible multilayered matrix incapable of transducing strong mechanical forces through cellular integrins1,2. Here, using the various capabilities of Atomic Force Microscopy (AFM), we have examined the physical properties of matrices produced from human plasma fibrinogen (hFg) and two recombinant fibrinogens: recombinant normal fibrinogen (rFg) and fibrinogen with truncated αC domains (FgAα251). We have determined the thickness of matrices using AFM lithography, and also performed the high-resolution topographical visualization of single molecules within the matrices. With force spectroscopy, we have measured the extensibility, adhesion forces and the energy of AFM tip-fibrinogen matrix interactions. Our results indicate that hFg and rFg adsorbed at high concentrations form an extensible matrix containing 8-9 molecular layers. In contrast, FgAα251 forms only 2-3 molecular layers. These data indicate that the αC domains are critically involved in the formation of a fibrinogen multilayer. Furthermore, the inability of FgAα251 to form a thick multilayered matrix results in sustained cell adhesion, unlike hFg which completely prevents cell adhesion. To evaluate the contribution of the αC domains to the formation of multilayered matrices, we developed a model based on intermolecular domain interactions to simulate the adsorption process for hFg and FgAα251. These findings have implications for processes where deposition of fibrinogen occurs, such as thrombus formation and adsorption of fibrinogen on implanted biomaterials.1. Podolnikova, N. P. et al., Biochemistry, 49, 68-77 (2010).2. Yermolenko, I. S. et al., Langmuir, 26, 17269-17277 (2010).

Analysis of crack deviation in ceramic matrix composites and multilayers based on the Cook and Gordon mechanism

In ceramic matrix composites, cracks that initiate in the matrix may be deflected in the fibre/matrix region, depending on interface characteristics. The most recent CMCs possess multilayered matrices and interphases. Cracks can be deflected within the layers or along interfaces depending on layer bonding. In this paper, the extension/deflection of a crack located in the vicinity of an interface is examined. All situations, including multilayers, interphases, etc. are covered by a single elementary cell made of two materials, one of which is cracked. A crack deflection condition is proposed. The model is based on the Cook and Gordon mechanism which considers debonding ahead of the crack tip as a result of the multiaxial stress state induced by the crack. Finally, the model is applied to various fibre/matrix combinations involving carbon or silicon carbide, and pyrocarbon or boron nitride based interphases.

Treatment of stress variables in advanced multilayered plate elements based upon Reissner’s mixed variational theorem

The subject of the present work consists of multilayered finite elements that are able to furnish an accurate description of strain/stress fields in multilayer flat structure analysis. The formulation of the finite elements is based upon Reissner’s mixed variational theorem, which allows one to assume two independent fields for displacements and transverse stress variables. The resulting advanced finite element can describe, a priori, the interlaminar continuous transverse shear and normal stress fields, and the so called C z 0 - requirements can be satisfied. This paper is mainly concerned about the treatment of stress variables in the mixed formulation. In particular, two layer-wise finite elements are compared. The first finite element examined, called LMN, uses a layer-wise mixed formulation and the displacements and transverse stresses are expanded along the thickness of a generic layer using a Legendre polynomial of N degree. In the assembling process, the transverse stress variables are eliminated at element level (static-condensation technique) after the generation of the element multilayered matrices. In the second finite element, called LMNF, the approach taken in the first finite element (LMN) is used, except that the static-condensation technique is not applied, and both displacement and stress variables appear as problem unknown. This last approach guarantees that the transverse stresses between two adjacent elements are continuous functions (this does not happen if the static-condensation method is used). Therefore, it can be concluded that LMNF is a powerful two-dimensional tool to analyze very thick multilayered plates.

Recent advances in the field of ceramic fibers and ceramic matrix composites

Progress achieved during the last decade in the field of ceramic fibers and related ceramic matrix composites is reviewed. Both SiC-based and alumina-based fine fibers have been improved in terms of thermal stability and creep resistance with temperature limit of about 1400 and 1200°C, respectively. Two concepts for achieving damage-tolerant ceramic matrix composites have been identified : (i) that of non-oxide composites with a dense matrix in which matrix cracks formed under load are deflected and arrested in a weak fiber coating referred to as the interphase and (ii) that of all-oxide composites with a highly porous matrix with no need of any fiber coating. The lifetime under load of non-oxide composites in oxidizing atmospheres, is improved through the use of multilayered self-healing interphases and matrices deposited from gaseous precursors by chemical vapor infiltration (CVI). Lifetime ranging from 1000 to 10,000 hours at 1200°C under cyclic loading in air are foreseen. Alumina-based composites although attractive for long term exposures in oxidizing atmospheres up to 1200°C. are still experimental materials.

High strain to failure matrices produced by multiple crack stopping interfaces: comparison of polymeric and CVD matrices

Fibre reinforced ceramic matrix composites are a promising class of structural materials. While tensile failure strains for composites often approach one percent, matrix microcracking frequently occurs at just a small fraction of the failure strain. Multiple crack-deflecting interfaces and whisker reinforcement were introduced within the matrix to increase the strain at which matrix microcracking occurred. Two processes, polymeric precursor and CVD infiltration, were evaluated for producing the multilayered matrices. Improvements in matrix strains of over 200%, and also composite strengths were observed.

A Tridimensional Self-Thinning Model for Multilayered Intertidal Mussels.

Intertidal mussels usually form complex multilayered matrices with density-dependent effects on survival and growth, and self-thinning scaling between biomass (B) and density (N) is expected. This article develops a tridimensional model of space-driven self-thinning that in addition to B-N explicitly includes the degree of packing of the mussels, measured as the number of layers (L). The structure of our model (B-N-L) encompasses previous bidimensional models (B-N) of self-thinning as special cases and enables comparisons between mono- and multilayered populations. We contrast the predictions of the bi- and tridimensional models using data obtained from Perumytilus purpuratus mussel beds on the rocky shores of central Chile monitored during a 28-mo period. The tridimensional model suggests that density dependence is much more frequent than hitherto indicated by bidimensional models. We propose that our space-driven tridimensional model may be applied not only to mussels but also to other species where spatial overlapping configurations occur.

Mathematical model for controlled diffusional release of dispersed solute drugs from monolithic implants.

New mathematical models are formulated and analytical solutions are presented for the diffusional release of a solute from both non-erodible and biodegradable multi-layered slab matrices in which the initial drug loading c0 is greater than the solubility limit cs. A Stefan problem with moving boundaries results from this formulation. An inward moving diffusional front separates the reservoir (unextracted region) containing the undissolved drug from the partially extracted region. The cumulative mass released is determined as a function of time. The ultimate goal of such an investigation is to provide a reliable design tool for the fabrication of specialized implantable capsule/drug combinations to deliver prespecified and reproducible dosages over a wide spectrum of conditions and required durations of therapeutic treatment. Such a mathematical/computational tool may also prove effective in the prediction of suitable dosages for other drugs of differing chemical or molecular properties without additional elaborate animal testing.

Multi-layered hydrophilic matrices as constant release devices (Geomatrix TM Systems)

In recent years many attempts have been made to modify drug release profiles from hydrophilic matrix tablets. Recently a new method based on the application of an inert impermeable coating on different sides of a matrix tablet was proposed. The coating delays the interaction of the matrix with the dissolution medium. In this way the hydration rate of the active core is slowed and its release surface is reduced. As a result the release extent is increased, while the dissolution profile tends to shift toward constant drug release compared with the uncoated matrix. In this article the design and technological development (up to the industrial production) of these types of devices is described: the film coatings are replaced by a polymeric barrier applied on the tablet bases during tableting. The effectiveness, the reproducibility and the technological characteristics of the new multi-layer systems are described.