Michelle L. Oyen

Extracellular-matrix tethering regulates stem-cell fate

To investigate how substrate properties influence stem-cell fate, we cultured single human epidermal stem cells on polydimethylsiloxane (PDMS) and polyacrylamide (PAAm) hydrogel surfaces, 0.1 kPa-2.3 MPa in stiffness, with a covalently attached collagen coating. Cell spreading and differentiation were unaffected by polydimethylsiloxane stiffness. However, cells on polyacrylamide of low elastic modulus (0.5 kPa) could not form stable focal adhesions and differentiated as a result of decreased activation of the extracellular-signal-related kinase (ERK)/mitogen-activated protein kinase (MAPK) signalling pathway. The differentiation of human mesenchymal stem cells was also unaffected by PDMS stiffness but regulated by the elastic modulus of PAAm. Dextran penetration measurements indicated that polyacrylamide substrates of low elastic modulus were more porous than stiff substrates, suggesting that the collagen anchoring points would be further apart. We then changed collagen crosslink concentration and used hydrogel-nanoparticle substrates to vary anchoring distance at constant substrate stiffness. Lower collagen anchoring density resulted in increased differentiation. We conclude that stem cells exert a mechanical force on collagen fibres and gauge the feedback to make cell-fate decisions.

Biomimetic layer-by-layer assembly of artificial nacre

Nacre is a technologically remarkable organic-inorganic composite biomaterial. It consists of an ordered multilayer structure of crystalline calcium carbonate platelets separated by porous organic layers. This microstructure exhibits both optical iridescence and mechanical toughness, which transcend those of its constituent components. Replication of nacre is essential for understanding this complex biomineral, and paves the way for tough coatings fabricated from cheap abundant materials. Fabricating a calcitic nacre imitation with biologically similar optical and mechanical properties will likely require following all steps taken in biogenic nacre synthesis. Here we present a route to artificial nacre that mimics the natural layer-by-layer approach to fabricate a hierarchical crystalline multilayer material. Its structure-function relationship was confirmed by nacre-like mechanical properties and striking optical iridescence. Our biomimetic route uses the interplay of polymer-mediated mineral growth, combined with layer-by-layer deposition of porous organic films. This is the first successful attempt to replicate nacre, using CaCO(3).

Spherical Indentation Creep Following Ramp Loading

Elastic-viscoelastic correspondence, utilizing Boltzmann integral operators, was used to generate displacement–time solutions for spherical indentation testing of viscoelastic materials. Solutions were found for creep following loading at a constant loading rate and compared with step-loading solutions. Experimental creep tests were performed with different loading rate–peak load level combinations on glassy and rubbery polymeric materials. The experimental data were fit to the spherical indentation ramp–creep solutions to obtain values of shear modulus and time-constants; good agreement was found between the experimental results and known modulus values. A multiple ramp-and-hold protocol was examined for the measurement of creep responses at several loads (and depths) within the same test. Emphasis is given to the use of multiple experiments (or multiple levels within a single experiment) to test a priori assumptions made in the correspondence solutions regarding linear viscoelastic material behavior and the creep function.

Analytical techniques for indentation of viscoelastic materials

Indentation of linearly viscoelastic materials is explored using elastic–viscoelastic correspondence analysis for both conical-pyramidal and spherical indentation. Boltzmann hereditary integrals are used to generate displacement–time solutions for loading at constant rate and creep following ramp loading. Experimental data for triangle- and trapezoidal-loading are examined for commercially-available polymers and compared with analytical solutions. Emphasis is given to the use of multiple experiments to test the fidelity and predictive capability of the obtained material creep function. Plastic deformation occurs in sharp indentation of glassy polymers and is found to complicate the viscoelastic analysis. A new method is proposed for estimating a material time-constant from peak displacement or hardness data obtained in pyramidal indentation tests performed at different loading rates.

A practical guide for analysis of nanoindentation data

Mechanical properties of biological materials are increasingly explored via nanoindentation testing. This paper reviews the modes of deformation found during indentation: elastic, plastic, viscous and fracture. A scheme is provided for ascertaining which deformation modes are active during a particular indentation test based on the load-displacement trace. Two behavior maps for indentation are presented, one in the viscous-elastic-plastic space, concerning homogeneous deformation, and one in the plastic versus brittle space, concerning the transition to fracture behavior when the threshold for cracking is exceeded. Best-practice methods for characterizing materials are presented based on which deformation modes are active; the discussion includes both nanoindentation experimental test options and appropriate methods for analyzing the resulting data.

Viscoelastic properties of bone as a function of hydration state determined by nanoindentation

Spherical indentation creep testing was used to examine the effect of hydration state on bone mechanical properties. Analysis of creep data was based on the elastic–viscoelastic correspondence principle and utilized a direct solution for the finite loading-rate experimental conditions. The zero-time shear modulus was computed from the creep compliance function and compared to the indentation modulus obtained via conventional indentation analysis, based on an elastic unloading response. The method was validated using a well-known polymer material under three different loading conditions. The method was applied to bone samples prepared with different water content by partial exchange with ethanol, where 70% ethanol was considered as the baseline condition. A hydration increase was associated with a 43% decrease in stiffness, while a hydration decrease resulted in a 20% increase in bone tissue stiffness.

Poroelastic nanoindentation responses of hydrated bone

Indentation techniques are used for the measurement of mechanical properties of a wide range of materials. Typical elastic analysis for spherical indentation is applicable in the absence of time-dependent deformation, but is inappropriate for materials with time-dependent creep responses active in the experimental time frame. In the current work, a poroelastic analysis—a mechanical theory incorporating fluid motion through a porous elastic network—is used to examine spherical indentation creep responses of hydrated biological materials. Existing analytical and finite element solutions for the poroelastic Hertzian indentation problem are reviewed, and a poroelastic parameter identification scheme is developed. Experimental data from nanoindentation of hydrated bone immersed in water and polar solvents (ethanol, methanol, acetone) are examined within the poroelastic framework. Immersion of bone in polar solvents with decreasing polarity results in increased stiffness, decreased Poisson’s ratio, and decreased hydraulic permeability. Nanoindentation poroelastic analysis results are compared with existing literature for bone poroelasticity at larger length scales, and the effective pore size probed in indentation creep experiments was estimated to be 1.6 nm, consistent with the scale of fundamental collagen–apatite interactions. Results for water permeability in bone were compared with studies of water diffusion through fully dense bone.

Composite electrospun gelatin fiber-alginate gel scaffolds for mechanically robust tissue engineered cornea.

A severe shortage of good quality donor cornea is now an international crisis in public health. Alternatives for donor tissue need to be urgently developed to meet the increasing demand for corneal transplantation. Hydrogels have been widely used as scaffolds for corneal tissue regeneration due to their large water content, similar to that of native tissue. However, these hydrogel scaffolds lack the fibrous structure that functions as a load-bearing component in the native tissue, resulting in poor mechanical performance. This work shows that mechanical properties of compliant hydrogels can be substantially enhanced with electrospun nanofiber reinforcement. Electrospun gelatin nanofibers were infiltrated with alginate hydrogels, yielding transparent fiber-reinforced hydrogels. Without prior crosslinking, electrospun gelatin nanofibers improved the tensile elastic modulus of the hydrogels from 78±19 kPa to 450±100 kPa. Stiffer hydrogels, with elastic modulus of 820±210 kPa, were obtained by crosslinking the gelatin fibers with carbodiimide hydrochloride in ethanol before the infiltration process, but at the expense of transparency. The developed fiber-reinforced hydrogels show great promise as mechanically robust scaffolds for corneal tissue engineering applications.

Composite bounds on the elastic modulus of bone

Advances in diagnosis and treatment of some bone disorders can be made by understanding the linkage between mineral content and mechanical function. Bone is approximately half by volume a hydrated protein network, and the remainder is a biomineral analogue of hydroxyapatite. In the current work, paired measurements of mechanical properties, using nanoindentation, and of bone mineral volume fraction, computed from quantitative back-scattered electron imaging, were made on six different types of normal and outlier bone samples. Local elastic modulus was plotted against mineral fraction and compared with predictions of engineering bounds for a two-phase composite material. Experimental data spanning the composite bounds showed no one-to-one relationship between mechanical stiffness and bone composition, excluding the possibility of any single, simple composites model for bone at nanometer length-scales.

Nanofibrous hydrogel composites as mechanically robust tissue engineering scaffolds

Hydrogels closely resemble the extracellular matrix (ECM) and can support cell proliferation while new tissue is formed, making them materials of choice as tissue engineering scaffolds. However, their sometimes-poor mechanical properties can hinder their application. The addition of meshes of nanofibers embedded in their matrix forms a composite that draws from the advantages of both components. Given that these materials are still in the early stages of development, there is a lack of uniformity across methods for characterizing their mechanical properties. Here, we propose a simple metric to enable comparisons between materials. The fibrous constituent improves the mechanical properties of the hydrogel, while the biocompatibility and functionality of the gels are maintained or even improved.