Development of a three-dimensional unit cell to model the micromechanical response of a collagen-based extracellular matrix

The three-dimensional microstructure and mechanical properties of the collagen fibrils within the extracellular matrix (ECM) is now being recognized as a primary factor in regulating cell proliferation and differentiation. Therefore, an appreciation of the mechanical aspects by which a cell interacts with its ECM is required for the development of engineered tissues. Ultimately, using these interactions to design tissue equivalents requires mathematical models with three-dimensional architecture. In this study, a three-dimensional model of a collagen fibril matrix undergoing uniaxial tensile stress was developed by making use of cellular solids. A structure consisting of thin struts was chosen to represent the arrangement of collagen fibrils within an engineered ECM. To account for the large deformation of tissues, the collagen fibrils were modeled as hyperelastic neo-Hookean or Mooney–Rivlin materials. The use of cellular solids allowed the fibril properties to be related to the ECM properties in closed form, which, in turn, allowed the estimation of fibril properties using ECM experimental data. A set of previously obtained experimental data consisting of simultaneous measures of the fibril microstructure and mechanical tests was used to evaluate the model’s capability to estimate collagen fibril mechanical property when given tissue-scale data and to predict the tissue-scale mechanical properties when given estimated fibril stiffness. The fibril tangent modulus was found to be 1.26 ± 0.70 and 1.62 ± 0.88 MPa when the fibril was modeled as neo-Hookean and Mooney–Rivlin material, respectively. There was no statistical significance of the estimated fibril tangent modulus among the different groups. Sensitivity analysis showed that the fibril mechanical properties and volume fraction were the two input parameters which required accurate values. While the volume fraction was easily obtained from the initial image of the gel, the fibril mechanical properties were not readily available. Therefore the fibril mechanical properties were estimated in the leave-one-out cross-validation (LOOCV) analysis. The LOOCV analysis showed that the model was able to predict the ECM stress–stretch curve with an average mean squared error of 9.71 kPa². The three-dimensional architecture expands on previous continuum models and two-dimensional representations to provide a useful model for studying the hierarchical effects of ECM microstructure on cell function. This model can be used as a design tool to engineer the optimum microstructure for cells to function.     

Topologically defined composites of collagen types I and V as in vitro cell culture scaffolds

Cell fate is known to be triggered by cues from the extracellular matrix, including its chemical, biological and physical characteristics. Specifically, mechanical and topological properties are increasingly recognized as important signals. The aim of this work was to provide an easily accessible biomimetic in vitro platform of topologically defined collagen I matrices to dissect cell behaviour under various conditions in vitro. We reconstituted covalently bound layers of three-dimensional (3-D) networks of collagen type I and collagen type V with a defined network topology. A new erosion algorithm enabled us to analyse the mean pore diameter and fibril content, while the mean fibril diameter was examined by an autocorrelation method. Different concentrations and ratios of collagen I and V resulted in pore diameters from 2.4 to 4.5 μm and fibril diameters from 0.6 to 0.8 μm. A comparison of telopeptide intact collagen I to telopeptide deficient collagen I revealed obvious differences in network structure. The good correlation of the topological data to measurements of network stiffness as well as invasion of human dermal fibroblasts proves that the topological analysis provides meaningful measures of the functional characteristics of the reconstituted 3-D collagen matrices.     

Heterogeneous micromechanical properties of the extracellular matrix in healthy and infarcted hearts

Infarcted hearts are macroscopically stiffer than healthy organs. Nevertheless, although cell behavior is mediated by the physical features of the cell niche, the intrinsic micromechanical properties of healthy and infarcted heart extracellular matrix (ECM) remain poorly characterized. Using atomic force microscopy, we studied ECM micromechanics of different histological regions of the left ventricle wall of healthy and infarcted mice. Hearts excised from healthy (n = 8) and infarcted mice (n = 8) were decellularized with sodium dodecyl sulfate and cut into 12 μm thick slices. Healthy ventricular ECM revealed marked mechanical heterogeneity across histological regions of the ventricular wall with the effective Young’s modulus ranging from 30.2 ± 2.8 to 74.5 ± 8.7 kPa in collagen- and elastin-rich regions of the myocardium, respectively. Infarcted ECM showed a predominant collagen composition and was 3-fold stiffer than collagen-rich regions of the healthy myocardium. ECM of both healthy and infarcted hearts exhibited a solid-like viscoelastic behavior that conforms to two power-law rheology. Knowledge of intrinsic micromechanical properties of the ECM at the length scale at which cells sense their environment will provide further insight into the cell–scaffold interplay in healthy and infarcted hearts.     

Structure and fracture resistance of alligator gar (Atractosteus spatula) armored fish scales

The alligator gar is a large fish with flexible armor consisting of ganoid scales. These scales contain a thin layer of ganoine (microhardness ～2.5 GPa) and a bony body (microhardness ～400 MPa), with jagged edges that provide effective protection against predators. We describe here the structure of both ganoine and bony foundation and characterize the mechanical properties and fracture mechanisms. The bony foundation is characterized by two components: a mineralized matrix and parallel arrays of tubules, most of which contain collagen fibers. The spacing of the empty tubules is ～60 μm; the spacing of those filled with collagen fibers is ～7 μm. Using micromechanical testing of such scales in a variable-pressure scanning electron microscope, we identify interactions between propagating cracks and the microstructure, and show that the toughness of the scales increases with crack extension in a classical resistance-curve response from the activation of extrinsic toughening mechanisms. We demonstrate how mechanical damage evolves in these structures, and further identify that the reinforcement of the mineral by the network of collagen fibers is the principal toughening mechanism resisting such damage. Additionally, we define the anisotropy of the toughness of the scales and relate this to the collagen fiber orientation.     

Electrospun nanostructured scaffolds for bone tissue engineering

The current challenge in bone tissue engineering is to fabricate a bioartificial bone graft mimicking the extracellular matrix (ECM) with effective bone mineralization, resulting in the regeneration of fractured or diseased bones. Biocomposite polymeric nanofibers containing nanohydroxyapatite (HA) fabricated by electrospinning could be promising scaffolds for bone tissue engineering. Nanofibrous scaffolds of poly-l-lactide (PLLA, 860 ± 110 nm), PLLA/HA (845 ± 140 nm) and PLLA/collagen/HA (310 ± 125 nm) were fabricated, and the morphology, chemical and mechanical characterization of the nanofibers were evaluated using scanning electron microscopy, Fourier transform infrared spectroscopy and tensile testing, respectively. The in vitro biocompatibility of different nanofibrous scaffolds was also assessed by growing human fetal osteoblasts (hFOB), and investigating the proliferation, alkaline phosphatase activity (ALP) and mineralization of cells on different nanofibrous scaffolds. Osteoblasts were found to adhere and grow actively on PLLA/collagen/HA nanofibers with enhanced mineral deposition of 57% higher than the PLLA/HA nanofibers. The synergistic effect of the presence of an ECM protein, collagen and HA in PLLA/collagen/HA nanofibers provided cell recognition sites together with apatite for cell proliferation and osteoconduction necessary for mineralization and bone formation. The results of our study showed that the biocomposite PLLA/collagen/HA nanofibrous scaffold could be a potential substrate for the proliferation and mineralization of osteoblasts, enhancing bone regeneration.     

Predicting bulk mechanical properties of cellularized collagen gels using multiphoton microscopy

Cellularized collagen gels are a common model in tissue engineering, but the relationship between the microstructure and bulk mechanical properties is only partially understood. Multiphoton microscopy (MPM) is an ideal non-invasive tool for examining collagen microstructure, cellularity and crosslink content in these gels. In order to identify robust image parameters that characterize microstructural determinants of the bulk elastic modulus, we performed serial MPM and mechanical tests on acellular and cellularized (normal human lung fibroblasts) collagen hydrogels, before and after glutaraldehyde crosslinking. Following gel contraction over 16 days, cellularized collagen gel content approached that of native connective tissues (～200 mg ml^–1). Young’s modulus (E) measurements from acellular collagen gels (range 0.5–12 kPa) exhibited a power-law concentration dependence (range 3–9 mg ml^–1) with exponents from 2.1 to 2.2, similar to other semiflexible biopolymer networks such as fibrin and actin. In contrast, cellularized collagen gel stiffness (range 0.5–27 kPa) produced concentration-dependent exponents of 0.7 uncrosslinked and 1.1 crosslinked (range ～5–200 mg ml^–1). The variation in E of cellularized collagen hydrogels can be explained by a power-law dependence on robust image parameters: either the second harmonic generation (SHG) and two-photon fluorescence (TPF) (matrix component) skewness (R² = 0.75, exponents of -1.0 and -0.6, respectively); or alternatively the SHG and TPF (matrix component) speckle contrast (R² = 0.83, exponents of ?0.7 and ?1.8, respectively). Image parameters based on the cellular component of TPF signal did not improve the fits. The concentration dependence of E suggests enhanced stress relaxation in cellularized vs. acellular gels. SHG and TPF image skewness and speckle contrast from cellularized collagen gels can predict E by capturing mechanically relevant information on collagen fiber, cell and crosslink density.     

Engineered dermal equivalent tissue in vitro by assembly of microtissue precursors

Tissue-engineered constructs can be fabricated by the assembly of smaller building blocks in order to mimic much of the native biology that is often made from repeating functional units. Our aim was to realize a three-dimensional (3-D) tissue-like construct in vitro by inducing the assembly of functional micrometric tissue precursors (μTPs). μTPs were obtained by dynamic cell seeding of bovine fibroblasts on porous gelatine microcarriers using a spinner flask bioreactor. During the dynamic seeding, cells adhered, proliferated and synthesized a thin layer of extracellular matrix (ECM) in and around the macroporous beads, generating the μTPs. The analysis showed that the ECM produced was rich in type I collagen. The cells and ECM layer around the μTPs allowed their biological sintering via cell–cell and cell–matrix interaction after only a few days of dynamic seeding. The assembling ability of μTPs was exploited by placing them in a maturation chamber. After 1 week of culture disc-shaped constructs (1 cm in diameter, 1 mm in thickness) of completely assembled μTPs were collected. The biohybrid obtained presented both a homogeneous and compact aspect. Moreover, histological and immunohistochemical analyses revealed an abundant ECM, rich in type I collagen, interconnecting the μTPs. The results obtained in this survey pave the way to realizing a 3-D dermal tissue equivalent by means of a bottom-up tissue engineering approach.     

Density–property relationships in collagen–glycosaminoglycan scaffolds

Collagen–glycosaminoglycan scaffolds for the regeneration of skin have previously been fabricated by freeze-drying a slurry containing a co-precipitate of collagen and glycosaminoglycan. The mechanical properties of the scaffold are low (e.g. the dry compressive Young’s modulus is roughly 30 kPa and the dry compressive strength is roughly 5 kPa). There is interest in using these scaffolds for tendon and ligament regeneration where there is a need for improved mechanical properties. Previous attempts to increase the mechanical properties of the scaffold by increasing the solid volume fraction of the scaffolds were limited by the increasing viscosity of the slurry, making it more difficult to mix and giving inhomogeneous scaffolds. Our recent work on mineralized collagen–glycosaminoglycan scaffolds used a vacuum filtration technique to increase the volume fraction of solids in the slurry, thereby increasing the density and mechanical properties of the scaffolds. In this work, we used this technique to fabricate collagen–glycosaminoglycan scaffolds with dry densities between 0.0076 and 0.0311 g cm^?3 and pore sizes between 250 and 350 μm, values appropriate for soft tissue growth. The compressive Young’s modulus and strength in the dry state increased from 32 to 127 kPa and from 5 to 19 kPa, respectively, with increasing density. The tensile Young’s modulus in the dry state increased from 295 to 3.1 MPa with increasing density. Finally, we showed that the attachment of cells onto the scaffold was directly proportional to the specific surface area of the scaffold, which defines the total internal surface area per volume of scaffold.     

Quercetin-crosslinked porcine heart valve matrix: Mechanical properties,stability, anticalcification and cytocompatibility

Bioprosthetic heart valves, prepared by glutaraldehyde (GA) crosslinking, have some limitations due to poor durability, calcification and immunogenic reactions. The aim of this study was to evaluate the crosslinking effect of a natural product, quercetin, on decellularized porcine heart valve extracellular matrix (ECM). After crosslinking, the mechanical properties, stability, anticalcification and cytocompatibility were examined. The results showed that the tensile strength of quercetin-crosslinked ECM was higher than that of GA-crosslinked ECM. After crosslinking with quercetin, the thermal denaturation temperature of ECM was clearly increased. Quercetin-crosslinked ECM could be stored in D-Hanks solution for at least 30 days without any loss of ultimate tensile strength and elasticity. After soaking in D-Hanks solution for 36 days, there was only 11.55% non-crosslinked excess quercetin released and no further release thereafter. Cell culture study shows that no inhibition on proliferation of vascular endothelial cells occurred when the quercetin concentration was lower than 1 μg ml^?1. This non-cytotoxic concentration was 100 times higher than that of GA. The resistibility of quercetin-crosslinked ECM to in vitro enzymatic hydrolysis was comparable to that of GA-crosslinked ECM. An in vitro anticalcification experiment showed that quercetin crosslinking could protect ECM from deposition of minerals in simulated body fluid. The present study demonstrated that quercetin can crosslink porcine heart valve ECM effectively, which suggests that quercetin might be a new crosslinking reagent for the preparation of bioprosthetic heart valve xenografts and scaffolds for heart valve tissue engineering.     

Effect of three decellularisation protocols on the mechanical behaviour and structural properties of sheep aortic valve conduits

PurposeTo determine the best method for decellularisation of aortic valve conduits (AVCs) that efficiently removes the cells while preserving the extracellular matrix (ECM) by examining the valvular and conduit sections separately.Material/methodsSheep AVCs were decellularised by using three different protocols: detergent-based (1% SDS + 1% SDC), detergent and enzyme-based (Triton + EDTA + RNase and DNase), and enzyme-based (Trypsin + RNase and DNase) methods. The efficacy of the decellularisation methods to completely remove the cells while preserving the ECM was evaluated by histological evaluation, scanning electron microscopy (SEM), hydroxyproline analysis, tensile test, and DAPI staining.ResultsThe detergent-based method completely removed the cells and left the ECM and collagen content in the valve and conduit sections relatively well preserved. The detergent and enzyme-based protocol did not completely remove the cells, but left the collagen content in both sections well preserved. ECM deterioration was observed in the aortic valves (AVs), but the ultrastructure of the conduits was well preserved, with no media distortion. The enzyme-based protocol removed the cells relatively well; however, mild structural distortion and poor collagen content was observed in the AVs. Incomplete cell removal (better than that observed with the detergent and enzyme-based protocol), poor collagen preservation, and mild structural distortion were observed in conduits treated with the enzyme-based method.ConclusionsThe results suggested that the detergent-based methods are the most effective protocols for cell removal and ECM preservation of AVCs. The AVCs treated with this detergent-based method may be excellent scaffolds for recellularisation.     

Ab initio elastic properties and tensile strength of crystalline hydroxyapatite

We report elastic constant calculation and a “theoretical” tensile experiment on stoichiometric hydroxyapatite (HAP) crystal using an ab initio technique. These results compare favorably with a variety of measured data. Theoretical tensile experiments are performed on the orthorhombic cell of HAP for both uniaxial and biaxial loading. The results show considerable anisotropy in the stress–strain behavior. It is shown that the failure behavior of the perfect HAP crystal is brittle for tension along the z-axis with a maximum stress of 9.6 GPa at 10% strain. Biaxial failure envelopes from six “theoretical” loading tests show a highly anisotropic pattern. Structural analysis of the crystal under various stages of tensile strain reveals that the deformation behavior manifests itself mainly in the rotation of the PO₄ tetrahedron with concomitant movements of both the columnar and axial Ca ions. These results are discussed in the context of mechanical properties of bioceramic composites relevant to mineralized tissues.     

In situ forming collagen–hyaluronic acid membrane structures: Mechanism of self-assembly and applications in regenerative medicine

Bioactive, in situ forming materials have the potential to complement minimally invasive surgical procedures and enhance tissue healing. For such biomaterials to be adopted in the clinic, they must be cost-effective, easily handled by the surgeon and have a history of biocompatibility. To this end, we report a novel and facile self-assembling strategy to create membranes and encapsulating structures using collagen and hyaluronic acid (HA). Unlike membranes built by layer-by-layer deposition of oppositely charged biomolecules, the collagen–HA membranes described here form a diffusion barrier upon electrostatic interaction of the oppositely charged biomolecules, which is further driven by osmotic pressure imbalances. The resulting membranes have a nanofibrous architecture, a thicknesses of 130 μm and a tensile modulus (0.59 ± 0.06 MPa) that can increase 7-fold using carbodiimide chemistry (4.42 ± 1.46 MPa). Collagen–HA membranes support mesenchymal stem cell proliferation and have a slow and steady protein release profile (7% at day 28), offering opportunities for targeted tissue regeneration. We demonstrate the capacity to encapsulate cells by injecting HA into the collagen solution, and enhance allograft and implant biocompatibility through a coating technique. This study describes a novel mechanism of collagen–HA membrane formation and provides the groundwork to apply these membranes in a variety of tissue engineering applications.     

The importance of size-exclusion characteristics of type I collagen in bonding to dentin matrices

The mineral phase of dentin is located primarily within collagen fibrils. During development, bone or dentin collagen fibrils are formed first and then water within the fibril is replaced with apatite crystallites. Mineralized collagen contains very little water. During dentin bonding, acid-etching of mineralized dentin solubilizes the mineral crystallites and replaces them with water. During the infiltration phase of dentin bonding, adhesive comonomers are supposed to replace all of the collagen water with adhesive monomers that are then polymerized into copolymers. The authors of a recently published review suggested that dental monomers were too large to enter and displace water from collagen fibrils. If that were true, the endogenous proteases bound to dentin collagen could be responsible for unimpeded collagen degradation that is responsible for the poor durability of resin–dentin bonds. The current work studied the size–exclusion characteristics of dentin collagen, using a gel-filtration-like column chromatography technique, using dentin powder instead of Sephadex. The elution volumes of test molecules, including adhesive monomers, revealed that adhesive monomers smaller than ～1000 Da can freely diffuse into collagen water, while molecules of 10,000 Da begin to be excluded, and bovine serum albumin (66,000 Da) was fully excluded. These results validate the concept that dental monomers can permeate between collagen molecules during infiltration by etch-and-rinse adhesives in water-saturated matrices.     

Local micromechanical properties of decellularized lung scaffolds measured with atomic force microscopy

Bioartificial lungs re-engineered from decellularized organ scaffolds are a promising alternative to lung transplantation. Critical features for improving scaffold repopulation depend on the mechanical properties of the cell microenvironment. However, the mechanics of the lung extracellular matrix (ECM) is poorly defined. The local mechanical properties of the ECM were measured in different regions of decellularized rat lung scaffolds with atomic force microscopy. Lungs excised from rats (n = 11) were decellularized with sodium dodecyl sulfate (SDS) and cut into ～7 μm thick slices. The complex elastic modulus (G¹) of lung ECM was measured over a frequency band ranging from 0.1 to 11.45 Hz. Measurements were taken in alveolar wall segments, alveolar wall junctions and pleural regions. The storage modulus (G′, real part of G¹) of alveolar ECM was ～6 kPa, showing small changes between wall segments and junctions. Pleural regions were threefold stiffer than alveolar walls. G′ of alveolar walls and pleura increased with frequency as a weak power law with exponent 0.05. The loss modulus (G″, imaginary part of G¹) was 10-fold lower and showed a frequency dependence similar to that of G′ at low frequencies (0.1–1 Hz), but increased more markedly at higher frequencies. Local differences in mechanical properties and topology of the parenchymal site could be relevant mechanical cues for regulating the spatial distribution, differentiation and function of lung cells.     

Mechanical properties of bioactive glass (13-93) scaffolds fabricated by robotic deposition for structural bone repair

There is a need to develop synthetic scaffolds to repair large defects in load-bearing bones. Bioactive glasses have attractive properties as a scaffold material for bone repair, but data on their mechanical properties are limited. The objective of the present study was to comprehensively evaluate the mechanical properties of strong porous scaffolds of silicate 13-93 bioactive glass fabricated by robocasting. As-fabricated scaffolds with a grid-like microstructure (porosity 47%, filament diameter 330 μm, pore width 300 μm) were tested in compressive and flexural loading to determine their strength, elastic modulus, Weibull modulus, fatigue resistance, and fracture toughness. Scaffolds were also tested in compression after they were immersed in simulated body fluid (SBF) in vitro or implanted in a rat subcutaneous model in vivo. As fabricated, the scaffolds had a strength of 86 ± 9 MPa, elastic modulus of 13 ± 2 GPa, and a Weibull modulus of 12 when tested in compression. In flexural loading the strength, elastic modulus, and Weibull modulus were 11 ± 3 MPa, 13 ± 2 GPa, and 6, respectively. In compression, the as-fabricated scaffolds had a mean fatigue life of ～10⁶ cycles when tested in air at room temperature or in phosphate-buffered saline at 37 °C under cyclic stresses of 1–10 or 2–20 MPa. The compressive strength of the scaffolds decreased markedly during the first 2 weeks of immersion in SBF or implantation in vivo, but more slowly thereafter. The brittle mechanical response of the scaffolds in vitro changed to an elasto-plastic response after implantation for longer than 2–4 weeks in vivo. In addition to providing critically needed data for designing bioactive glass scaffolds, the results are promising for the application of these strong porous scaffolds in loaded bone repair.     

Cell–matrix interactions and dynamic mechanical loading influence chondrocyte gene expression and bioactivity in PEG-RGD hydrogels

The pericellular matrix (PCM) surrounding chondrocytes is thought to play an important role in transmitting biochemical and biomechanical signals to the cells, which regulates many cellular functions including tissue homeostasis. To better understand chondrocytes interactions with their PCM, three-dimensional poly(ethylene glycol) (PEG) hydrogels containing Arg–Gly–Asp (RGD), the cell-adhesion sequence found in fibronectin and which is present in the PCM of cartilage, were employed. RGD was incorporated into PEG hydrogels via tethers at 0.1, 0.4 and 0.8 mM concentrations. Bovine chondrocytes were encapsulated in the hydrogels and subjected to dynamic compressive strains (0.3 Hz, 18% amplitude strain) for 48 h, and their response assessed by cell morphology, ECM gene expression, cell proliferation and matrix synthesis. Incorporation of RGD did not influence cell morphology under free swelling conditions. However, the level of cell deformation upon an applied strain was greater in the presence of RGD. In the absence of dynamic loading, RGD appears to have a negative effect on chondrocyte phenotype, as seen by a 4.7-fold decrease in collagen II/collagen I expressions in 0.8 mM RGD constructs. However, RGD had little effect on early responses of chondrocytes (i.e. cell proliferation and matrix synthesis/deposition). When isolating RGD as a biomechanical cue, cellular response was very different. Chondrocyte phenotype (collagen II/collagen I ratio) and proteoglycan synthesis were enhanced with higher concentrations of RGD. Overall, our findings demonstrate that RGD ligands enhance cartilage-specific gene expression and matrix synthesis, but only when mechanically stimulated, suggesting that cell–matrix interactions mediate chondrocyte response to mechanical stimulation.     

Dynamic shear-influenced collagen self-assembly

The ability to influence the direction of polymerization of a self-assembling biomolecular system has the potential to generate materials with extremely high anisotropy. In biological systems where highly-oriented cellular populations give rise to aligned and often load-bearing tissue such organized molecular scaffolds could aid in the contact guidance of cells for engineered tissue constructs (e.g. cornea and tendon). In this investigation we examine the detailed dynamics of pepsin-extracted type I bovine collagen assembly on a glass surface under the influence of flow between two plates. Differential Interference Contrast (DIC) imaging (60×-1.4NA) with focal plane stabilization was used to resolve and track the growth of collagen aggregates on borosilicate glass for 4 different shear rates (500, 80, 20, and 9 s^?1). The detailed morphology of the collagen fibrils/aggregates was examined using Quick Freeze Deep Etch (QFDE) electron microscopy. Nucleation of fibrils on the glass was observed to occur rapidly (～2 min) followed by continued growth of the fibrils. The growth rates were dependent on flow in a complex manner with the highest rate of axial growth (0.1 μ/s) occurring at a shear rate of 9 s^?1. The lowest growth rate occurred at the highest shear. Fibrils were observed to both branch and join during the experiments. The best alignment of fibrils was observed at intermediate shear rates of 20 and 80 s^?1. However, the investigation revealed that fibril directional growth was not stable. At high shear rates, fibrils would often turn downstream forming what we term “hooks” which are likely the combined result of monomer interaction with the initial collagen layer or “mat” and the high shear rate. Further, QFDE examination of fibril morphology demonstrated that the assembled fibrillar structure did not possess native D-periodicity. Instead, fibrils comprised a collection of generally aligned, monomers which were self-assembled to form a fibril-like aggregate. In conclusion, though constant shear-rate clearly influences collagen fibrillar alignment, the formation of highly-organized collagenous arrays of native-like D-banded fibrils remains a challenge. Modulation of shear in combination with surface energy patterning to produce a highly-aligned initial mat may provide significant improvement of both the fibril morphology and alignment.     

Matrix-driven formation of mesenchymal stem cell–extracellular matrix microtissues on soft alginate hydrogels

Mesenchymal stem cells (MSCs) can be made to rearrange into microtissues in response to specific matrix cues, a process that depends on a balance between cell–matrix and cell–cell interactions. The effect of such cues, and especially their interplay, is still not fully understood, particularly in three-dimensional (3-D) systems. Here, the behaviour of human MSCs cultured within hydrogel matrices with tailored stiffness and composition was evaluated. MSC aggregation occurred only in more compliant matrices (G′ ⩽ 120 Pa), when compared to stiffer ones, both in the presence and in the absence of matrix-bound arginine–glycine–aspartic acid cell–adhesion ligands (RGD; 0, 100 and 200 μM). Fibronectin assembly stabilized cell–cell contacts within aggregates, even in non-adhesive matrices. However, MSCs were able to substantially contract the artificial matrix only when RGD was present. Moreover, compliant matrices facilitated cell proliferation and provided an environment conducive for MSC osteogenic differentiation, even without RGD. Cell interactions with the original matrix became less important as time progressed, while the de novo-produced extracellular matrix became a more critical determinant of cell fate. These data provide further insights into the mechanisms by which MSCs sense their microenvironment to organize into tissues, and provide new clues to the design of cell-instructive 3-D matrices.     

Ultrastructural organization and micromechanical properties of shark tooth enameloid

The outer part of shark teeth is formed by the hard and mineral-rich enameloid that has excellent mechanical properties, which makes it a very interesting model system for the development of new bio-inspired dental materials. We characterized the microstructure, chemical composition and resulting local mechanical properties of the enameloid from teeth of Isurus oxyrinchus (shortfin mako shark) by performing an in-depth analysis using various high-resolution analytical techniques, including scanning electron microscopy, qualitative energy-dispersive X-ray spectroscopy and nanoindentation. Shark tooth enameloid reveals an intricate hierarchical arrangement of thin (50–80 nm) and long (>1 μm) crystallites of fluoroapatite with a high degree of structural anisotropy, which leads to exceptional mechanical properties. Both stiffness and hardness are surprisingly homogeneous in the shiny layer as well as in the enameloid: although both tooth phases differ in structure and composition, they show almost no orientation dependence with respect to the loading direction of the enameloid crystallites. The results were used to determine the structural hierarchy of shark teeth, which can be used as a base for establishing design criteria for synthetic bio-inspired and biomimetic dental composites.     

Compressive and tensile properties of articular cartilage in axial loading are modulated differently by osmotic environment

Aims of the present study were to test the hypotheses that (1) the compressive properties of articular cartilage are affected more by changes in the medium ionic concentration than the tensile properties, (2) collagen network controls the compression–tension nonlinearity of articular cartilage, and (3) proteoglycan (PG) and collagen contents are primary determinants of the compressive and tensile properties of cartilage, respectively. These hypotheses were experimentally tested by axial compressive and tensile tests (perpendicular to the cartilage surface) of bovine articular cartilage samples immersed in 0.005 M (n = 6), 0.15 M (n = 12) and 1.0 M (n = 6) saline solutions. Compressive and tensile behaviour was analyzed by a nonlinear fibril-reinforced poroelastic model. Tissue PG and collagen contents were measured using Fourier transform infrared imaging spectroscopy (FT-IRIS). The compressive modulus of cartilage varied significantly (n = 6, p < 0.05) as the medium concentration changed. The tensile modulus changed significantly only as the medium concentration was reduced from 0.15 to 0.005 M (n = 6, p < 0.05). The fibril-reinforced poroelastic model with stiff, nonlinear collagen fibrils predicted the experimentally measured compression–tension nonlinearity of cartilage. Tissue PG and collagen contents accounted for the compressive and tensile properties of cartilage.