Walter Bonani

Compressive Elasticity of Three-Dimensional Nanofiber Matrix Directs Mesenchymal Stem Cell Differentiation to Vascular Cells with Endothelial or Smooth Muscle Cell Markers

The importance of mesenchymal stem cells (MSC) in vascular regeneration is becoming increasingly recognized. However, few in vitro studies have been performed to identify the effects of environmental elasticity on the differentiation of MSC into vascular cell types. Electrospinning and photopolymerization techniques were used to fabricate a three-dimensional (3-D) polyethylene glycol dimethacrylate nanofiber hydrogel matrix with tunable elasticity for use as a cellular substrate. Compression testing demonstrated that the elastic modulus of the hydrated 3-D matrices ranged from 2 to 15 kPa, similar to the in vivo elasticity of the intima basement membrane and media layer. MSC seeded on rigid matrices (8-15 kPa) showed an increase in cell area compared with those seeded on soft matrices (2-5 kPa). Furthermore, the matrix elasticity guided the cells to express different vascular-specific phenotypes with high differentiation efficiency. Around 95% of MSC seeded on the 3-D matrices with an elasticity of 3 kPa showed Flk-1 endothelial markers within 24h, while only 20% of MSC seeded on the matrices with elasticity >8 kPa demonstrated Flk-1 marker. In contrast, ∼80% of MSC seeded on 3-D matrices with elasticity >8 kPa demonstrated smooth muscle α-actin marker within 24h, while fewer than 10% of MSC seeded on 3-D matrices with elasticity <5 kPa showed α-actin markers. The ability to control MSC differentiation into either endothelial or smooth muscle-like cells based purely on the local elasticity of the substrate could be a powerful tool for vascular tissue regeneration.

Biohybrid nanofiber constructs with anisotropic biomechanical properties.

Synthetic implant materials often lack of the anisotropic mechanical properties and cell-interactive surface which are shown by natural tissues. For example, engineered vascular grafts need to be developed to address the mechanical and biological problems associated with the graft materials. This study has demonstrated a double-electrospinning fabrication process to produce a poly(ε-caprolactone)-fibroin multilayer composite which shows well-integrated nanofibrous structure, endothelial-conducive surface and anisotropic mechanical property, suitable as engineered vascular constructs. Electrospinning parameters such as voltage, solution concentration, feed rate, and relative humidity were optimized to obtain defect-free, uniform nanofibers. To mimic the different mechanical properties of natural vessels in the circumferential and longitudinal directions, a rotating cylinder was used as collector, resulting in the production of constructs with anisotropic properties. The combination of the collector shape and the collector rotation allows us to produce a tubular structure with tunable anisotropic mechanical properties. Fourier transform infrared spectroscopy, differential scanning calorimetry, and uniaxial tensile tests were used to characterize the electrospun constructs. Cell cultures with primary endothelial cells demonstrated that cells showed spread morphology and strong adhesion on fibroin richer surfaces. The platform for producing robust multilayer scaffolds with intermixing nanofiber structure, tunable anisotropy ratio, and surface with specific compositions may hold great potential in tissue engineering applications.

Human mesenchymal stem cells cultured on silk hydrogels with variable stiffness and growth factor differentiate into mature smooth muscle cell phenotype.

UNLABELLED Cell-matrix and cell-biomolecule interactions play critical roles in a diversity of biological events including cell adhesion, growth, differentiation, and apoptosis. Evidence suggests that a concise crosstalk of these environmental factors may be required to direct stem cell differentiation toward matured cell type and function. However, the culmination of these complex interactions to direct stem cells into highly specific phenotypes in vitro is still widely unknown, particularly in the context of implantable biomaterials. In this study, we utilized tunable hydrogels based on a simple high pressure CO2 method and silk fibroin (SF) the structural protein of Bombyx mori silk fibers. Modification of SF protein starting water solution concentration results in hydrogels of variable stiffness while retaining key structural parameters such as matrix pore size and β-sheet crystallinity. To further resolve the complex crosstalk of chemical signals with matrix properties, we chose to investigate the role of 3D hydrogel stiffness and transforming growth factor (TGF-β1), with the aim of correlating the effects on the vascular commitment of human mesenchymal stem cells. Our data revealed the potential to upregulate matured vascular smooth muscle cell phenotype (myosin heavy chain expression) of hMSCs by employing appropriate matrix stiffness and growth factor (within 72h). Overall, our observations suggest that chemical and physical stimuli within the cellular microenvironment are tightly coupled systems involved in the fate decisions of hMSCs. The production of tunable scaffold materials that are biocompatible and further specialized to mimic tissue-specific niche environments will be of considerable value to future tissue engineering platforms. STATEMENT OF SIGNIFICANCE This article investigates the role of silk fibroin hydrogel stiffness and transforming growth factor (TGF-β1), with the aim of correlating the effects on the vascular commitment of human mesenchymal stem cells. Specifically, we demonstrate the upregulation of mature vascular smooth muscle cell phenotype (myosin heavy chain expression) of hMSCs by employing appropriate matrix stiffness and growth factor (within 72h). Moreover, we demonstrate the potential to direct specialized hMSC differentiation by modulating stiffness and growth factor using silk fibroin, a well-tolerated and -defined biomaterial with an impressive portfolio of tissue engineering applications. Altogether, our study reinforce the fact that complex differentiation protocols may be simplified by engineering the cellular microenvironment on multiple scales, i.e. matrix stiffness with growth factor.

Electrodeposition of Silk Fibroin on Metal Substrates

Silk fibroin, one of the most promising natural materials for tissue engineering, has positive interactions with the biological environment, particularly in the field of bone and cartilage regeneration. A new approach was developed to create hydrogels from water-based fibroin solutions by applying an electric field to effect protein migration and coagulation at the anode (Aluminium or Ti6Al4V alloy) of an electrochemical cell. The process was easily controlled by the voltage applied to the electrodes (3, 10, and 30 V), solution concentration (1%, 2%, 2.6% w/v), time (up to 100 s) and electrode distance (1—6 mm). The hydrogel thickness can be increased up to 60 μm and, depending on processing conditions, porous coatings or compact films can be obtained. The ability of electrodeposited fibroin hydrogels to coat metal objects with complex shape and surface morphology, together with the acclaimed properties of fibroin, makes it a promising technique to enhance the osteointegration of dental or orthopedic prostheses.

Mechanical and biocompatible characterizations of a readily available multilayer vascular graft.

There is always a considerable clinical need for vascular grafts. Considering the availability, physical and mechanical properties, and regenerative potential, we have developed and characterized readily available, strong, and compliant multilayer grafts that support cell culture and ingrowth. The grafts were made from heterogeneous materials and structures, including a thin, dense, nanofibrous core composed of poly-ε-caprolactone (PCL), and a thick, porous, hydrogel sleeve composed of genipin-crosslinked collagen-chitosan (GCC). Because the difference in physicochemical properties between PCL and GCC caused layer separation, the layer adhesion was identified as a determinant to graft property and integrity under physiological conditions. Thus, strategies to modify the layer interface, including increasing porosity of the PCL surface, decreasing hydrophobicity, and increasing interlayer crosslinking, were developed. Results from microscopic images showed that increasing PCL porosity was characterized by improved layer adhesion. The resultant graft was characterized by high compliance (4.5%), and desired permeability (528 mL/cm(2)/min), burst strength (695 mmHg), and suture strength (2.38 N) for readily grafting. Results also showed that PCL mainly contributed to the graft mechanical properties, whereas GCC reduced the water permeability. In addition to their complementary contributions to physical and mechanical properties, the distinct graft layers also provided layer-specific structures for seeding and culture of vascular endothelial and smooth muscle cells in vitro. Acellular graft constructs were readily used to replace abdominal aorta of rabbits, resulting in rapid cell ingrowth and flow reperfusion. The multilayer constructs capable of sustaining physiological conditions and promoting cellular activities could serve as a platform for future development of regenerative vascular grafts.

Processing and characterization of diatom nanoparticles and microparticles as potential source of silicon for bone tissue engineering

Silicon plays an important role in bone formation and maintenance, improving osteoblast cell function and inducing mineralization. Often, bone deformation and long bone abnormalities have been associated with silica/silicon deficiency. Diatomite, a natural deposit of diatom skeleton, is a cheap and abundant source of biogenic silica. The aim of the present study is to validate the potential of diatom particles derived from diatom skeletons as silicon-donor materials for bone tissue engineering applications. Raw diatomite (RD) and calcined diatomite (CD) powders were purified by acid treatments, and diatom microparticles (MPs) and nanoparticles (NPs) were produced by fragmentation of purified diatoms under alkaline conditions. The influence of processing on the surface chemical composition of purified diatomites was evaluated by X-ray photoelectron spectroscopy (XPS). Diatoms NPs were also characterized in terms of morphology and size distribution by transmission electron microscopy (TEM) and Dynamic light scattering (DLS), while diatom MPs morphology was analyzed by scanning electron microscopy (SEM). Surface area and microporosity of the diatom particles were evaluated by nitrogen physisorption methods. Release of silicon ions from diatom-derived particles was demonstrated using inductively coupled plasma optical emission spectrometry (ICP/OES); furthermore, silicon release kinetic was found to be influenced by diatomite purification method and particle size. Diatom-derived microparticles (MPs) and nanoparticles (NPs) showed limited or no cytotoxic effect in vitro depending on the administration conditions.

Modulating the release of drugs from alginate matrices with the addition of gelatin microbeads

Injectable drug-loaded matrices and controlled release technology offer numerous advantages over conventional dosages. Cross-linkable alginate hydrogels have been proposed for in vivo injection, but their large initial burst release of encapsulated drugs represents a limitation for the transition to the clinical phase. To reduce this effect, a new drug delivery system was prepared by combining uncross-linked, drug-loaded gelatin microbeads with cross-linkable alginate solution. Gelatin microbeads ranging from 5 to 50 µm were obtained depending on gelatin concentration, stirring rate, and emulsifying time. The release behavior of drug-loaded gelatin microbeads encapsulated within cross-linked alginate gel was characterized both at room temperature and 37°C and compared with the release from gelatin microbeads and cross-linked alginate gel alone. Gelatin microbeads reduced the initial burst release of fluorescein from cross-linked alginate matrix, with a corresponding decrease in the release efficiency. Burst release in the first 2 h was reduced from 30% to about 5%, while cumulative release at 37°C declined from about 95% to 50% after 7 days. This system represents a promising approach for the development of novel and versatile injectable drug delivery systems.

Stress-sensitive nutrient consumption via steady and non-reversing dynamic shear in continuous-flow rotational bioreactors ☆

Stress-sensitive biological response is simulated in a modified parallel-disk viscometer that implements steady and unidirectional dynamic shear under physiological conditions. Anchorage-dependent mammalian cells adhere to a protein coating on the surface of the rotating plate, receiving nutrients and oxygen from an aqueous medium that flows radially and tangentially, accompanied by transverse diffusion in the z-direction toward the active surface. This process is modeled as radial convection and axial diffusion with angular symmetry in cylindrical coordinates. The reaction/diffusion boundary condition on the surface of the rotating plate includes position-dependent stress-sensitive nutrient consumption via the zr- and zTheta-elements of the velocity gradient tensor at the cell/aqueous-medium interface. Linear transport laws in chemically reactive systems that obey Curies theorem predict the existence of cross-phenomena between scalar reaction rates and the magnitude of the second-rank velocity gradient tensor, selecting only those elements of nabla v experienced by anchorage-dependent cells that are bound to protein-active sites. Stress sensitivity via the formalism of irreversible thermodynamics introduces a zeroth-order contribution to heterogeneous reaction rates that must be quenched when nutrients, oxygen, chemically anchored cells, or vacant active protein sites are not present on the surface of the rotating plate. Computer simulations of nutrient consumption profiles via simple nth-order kinetics (i.e., n=1,2) suggest that rotational bioreactor designs should consider stress-sensitivity when the shear-rate-based Damköhler number (i.e., ratio of the stress-dependent zeroth-order rate of nutrient consumption relative to the rate of nutrient diffusion toward active cells adhered to the rotating plate) is greater than approximately 25% of the stress-free Damköhler number. Rotational bioreactor simulations are presented for simple 1st-order, simple 2nd-order, and complex stress-free kinetics, where the latter includes a 4th-order rate expression that considers adsorption/desorption equilibria via the Fowler-Guggenheim modification of the Langmuir isotherm for receptor-mediated cell-protein binding, accompanied by the formation of receptor complexes. Dimensionless parameters are identified to obtain equivalent stress-free nutrient consumption in the exit streams of 2-dimensional creeping-flow rotational bioreactors and 1-dimensional laminar-flow tubular bioreactors. Modulated rotation of the active plate at physiological frequencies mimics pulsatile cardiovascular flow and demonstrates that these rotational bioreactors must operate above the critical stress-sensitive Damköhler number, identified under steady shear conditions, before dynamic shear has a distinguishable effect on bioreactor performance.

Bioactivity and mineralization of natural hydroxyapatite from cuttlefish bone and Bioglass® co-sintered bioceramics.

In this study, bioactive hydroxyapatite (HAP)‐based bioceramics starting from cuttlefish bone powders have been prepared and characterized. In particular, fragmented cuttlefish bone was co‐sintered with 30 wt% of Bioglass®‐45S5 to synthesize HAP‐based powders with enhanced mechanical properties and bioactivity. Commercial synthetic HAP was treated following the same procedure and used as a reference. The structure and composition of the bioceramics formulations were characterized using Fourier transform infrared spectroscopy, X‐ray diffraction and scanning electron microscopy. After the thermal treatment of cuttlefish bone powder added with 30 wt% Bioglass, new phases with compositions of sodium calcium phosphate [Na3Ca6(PO4)5], β‐tricalcium phosphate [Ca3(PO4)] and amorphous silica were detected. In vitro cell culture studies were performed by evaluating proliferation, metabolic activity and differentiation of human osteoblast‐like cells (MG63). Scaffolds made with cuttlefish bone powder exhibited increased apatite deposition, alkaline phosphatase activity and cell proliferation compared with commercial synthetic HAP. In addition, the ceramic compositions obtained after the combination with Bioglass® further enhanced the metabolic activity of MG63 cell and promoted the formation of a well‐developed apatite layer after 7 days of incubation in Dulbeccos modified Eagles medium.

Evaluation of alternative sources of collagen fractions from Loligo vulgaris squid mantle.

Acid-Solubilized Collagen (ASC) and Pepsin-Solubilized Collagen (PSC) were extracted from the mantle of the common European squid, and were comparatively characterized. ASC and PSC were isolated with an extraction yield of 5.1 and 24.2% (on dry weight basis), respectively. SDS-PAGE showed that the ASC was mostly comprised of α1- and α2-chains; while the PSC presented relevant β- and γ-components. GPC analysis confirmed that both the ASC and the PSC consisted of fractions characterized by different molecular weight. Thermal denaturation behavior of ASC and PSC were followed by calorimetric and rheological analyses; denaturation temperature was estimated to be 22°C for ASC and 21°C for PSC. Amino acid composition and solubility of collagen were also investigated. Finally, the cytotoxicity of the isolated collagen was evaluated in vitro and no cytotoxic activity caused by the collagen extracts was observed. This study demonstrated that squid mantle has potential as an alternative source of collagen-derived materials.