Nanomechanical mapping of the osteochondral interface with contact resonance force microscopy and nanoindentation

The bone–cartilage, or osteochondral, interface resists remarkably high shear stresses and rarely fails, yet its mechanical characteristics are largely unknown. A complete understanding of this hierarchical system requires mechanical-property information at the length scales of both the interface and the connecting tissues. Here, we combined nanoindentation and atomic force microscopy (AFM) methods to investigate the multiscale mechanical properties across the osteochondral region. The nanoindentation modulus M ranged from that of the subchondral bone (M = 22.8 ± 1.8 GPa) to that of hyaline articular cartilage embedded in PMMA (M = 5.7 ± 1.0 GPa) across a narrow transition region <5 μm wide. Contact resonance force microscopy (CR-FM), which measures the frequency and quality factor of the AFM cantilever’s vibrational resonance in contact mode, was used to determine the relative storage modulus and loss tangent of the osteochondral interface. With better spatial resolution than nanoindentation, CR-FM measurements indicated an even narrower interface width of 2.3 ± 1.2 μm. Furthermore, CR-FM revealed a 24% increase in the viscoelastic loss tangent from the articular calcified cartilage into the PMMA-embedded hyaline articular cartilage. Quantitative backscattered electron imaging provided complementary measurement of mineral content. Our results provide insight into the multiscale functionality of the osteochondral interface that will advance understanding of disease states such as osteoarthritis and aid in the development of biomimetic interfaces.     

Micromechanics and ultrastructure of pyrolysed softwood cell walls

Pyrolytic conversion causes severe changes in the microstructure of the wood cell wall. Pine wood pyrolysed up to 325 °C was investigated by transmission electron microscopy, atomic force microscopy and nanoindentation measurements to monitor changes in structure and mechanical properties. Latewood cell walls were tested in the axial, radial and tangential directions at different temperatures of pyrolysis. A strong anisotropy of elastic properties in the native cell wall was found. Loss of the hierarchical structure of the cell wall due to pyrolysis resulted in elastic isotropy at 300 °C. The development of the mechanical properties with increasing temperature can be explained by alterations in the structure and it was found that the elastic properties were clearly related to length and orientation of the microfibrils.     

Mechanical properties and structure of the biological multilayered material system,Atractosteus spatula scales

During recent decades, research on biological systems such as abalone shell and fish armor has revealed that these biological systems employ carefully arranged hierarchical multilayered structures to achieve properties of high strength, high ductility and light weight. Knowledge of such structures may enable pathways to design bio-inspired materials for various applications. This study was conducted to investigate the spatial distribution of structure, chemical composition and mechanical properties in mineralized fish scales of the species Atractosteus spatula. Microindentation tests were conducted, and cracking patterns and damage sites in the scales were examined to investigate the underlying protective mechanisms of fish scales under impact and penetration loads. A difference in nanomechanical properties was observed, with a thinner, stiffer and harder outer layer (indentation modulus ～69 GPa and hardness ～3.3 GPa) on a more compliant and thicker inner layer (indentation modulus ～14.3 GPa and hardness ～0.5 GPa). High-resolution scanning electron microscopy imaging of a fracture surface revealed that the outer layer contained oriented nanorods embedded in a matrix, and that the nanostructure of the inner layer contained fiber-like structures organized in a complex layered pattern. Damage patterns formed during microindentation show complex deformation mechanisms. Images of cracks identify growth through the outer layer, then deflection along the interface before growing and arresting in the inner layer. High-magnification images of the crack tip in the inner layer show void-linking and fiber-bridging exhibiting inelastic behavior. The observed difference in mechanical properties and unique nanostructures of different layers may have contributed to the resistance of fish scales to failure by impact and penetration loading.     

Characterization of dielectrophoresis-aligned nanofibrous silk fibroin–chitosan scaffold and its interactions with endothelial cells for tissue engineering applications

Aligned three-dimensional nanofibrous silk fibroin–chitosan (eSFCS) scaffolds were fabricated using dielectrophoresis (DEP) by investigating the effects of alternating current frequency, the presence of ions, the SF:CS ratio and the post-DEP freezing temperature. Scaffolds were characterized with polarized light microscopy to analyze SF polymer chain alignment, atomic force microscopy (AFM) to measure the apparent elastic modulus, and scanning electron microscopy and AFM to analyze scaffold topography. The interaction of human umbilical vein endothelial cells (HUVECs) with eSFCS scaffolds was assessed using immunostaining to assess cell patterning and AFM to measure the apparent elastic modulus of the cells. The eSFCS (50:50) samples prepared at 10 MHz with NaCl had the highest percentage of aligned area as compared to other conditions. As DEP frequency increased from 100 kHz to 10 MHz, fibril sizes decreased significantly. eSFCS (50:50) scaffolds fabricated at 10 MHz in the presence of 5 mM NaCl had a fibril size of 77.96 ± 4.69 nm and an apparent elastic modulus of 39.9 ± 22.4 kPa. HUVECs on eSFCS scaffolds formed aligned and branched capillary-like vascular structures. The elastic modulus of HUVEC cultured on eSFCS was 6.36 ± 2.37 kPa. DEP is a potential tool for fabrication of SFCS scaffolds with aligned nanofibrous structures that can guide vasculature in tissue engineering and repair.     

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.     

Elastic discontinuity due to ectopic calcification in a human fibrous joint

Disease can alter natural ramp-like elastic gradients to steeper step-like profiles at soft–hard tissue interfaces. Prolonged function can further mediate mechanochemical events that alter biomechanical response within diseased organs. In this study, a human bone–tooth fibrous joint was chosen as a model system, in which the effects of bacterial-induced disease, i.e. periodontitis, on natural elastic gradients were investigated. Specifically, the effects of ectopic biomineral, i.e. calculus, on innate chemical and elastic gradients within the cementum–dentin complex, both of which are fundamental parameters to load-bearing tissues, are investigated through comparisons with a healthy complex. Complementary techniques for mapping changes in physicochemical properties as a result of disease included micro X-ray computed tomography, microprobe micro X-ray fluorescence imaging, transmission electron and atomic force microscopy (AFM) techniques, and AFM-based nanoindentation. Results demonstrated primary effects as derivatives of ectopic mineralization within the diseased fibrous joint. Ectopic mineralization with no cementum resorption, but altered cementum physicochemical properties with increasing X-ray attenuation, exhibited stratified concretion with increasing X-ray fluorescence counts of calcium and phosphorus elements in the extracellular matrix in correlation with decreased hygroscopicity, indenter displacement, and apparent strain-relieving characteristics. Disease progression, identified as concretion through the periodontal ligament (PDL)–cementum enthesis, and sometimes the originally hygroscopic cementum–dentin junction, resulted in a significantly increased indentation elastic modulus (3.16 ± 1.19 GPa) and a shift towards a discontinuous interface compared with healthy conditions (1.54 ± 0.83 GPa) (Student’s t-test, P < 0.05). The observed primary effects could result in secondary downstream effects, such as compromised mechanobiology at the mechanically active PDL–cementum enthesis that can catalyze progression of disease.     

Micromechanical properties of single crystal hydroxyapatite by nanoindentation

Knowledge of the intrinsic properties of hydroxyapatite (HAp) single crystals is important for the design of natural systems and will assist further improvements of manufactured biomaterials. Nanoindentation provides a useful tool for determining mechanical properties such as the hardness, elastic modulus and fracture toughness of small samples. A Berkovich indenter was placed on the side and basal planes of a natural single crystal of Durango HAp. The hardness and elastic modulus values obtained revealed higher values for the base (7.1 and 150.4 GPa) compared to the side (6.4 and 143.6 GPa). The cracking threshold, i.e., the load at which cracking initiates, revealed earlier crack formation on the base (at 8 mN) compared to the side (at 11 mN). Fracture toughness was measured as 0.45 ± 0.09 and 0.35 ± 0.06 MPa m^1/2 for the side and basal plane, respectively. These results suggest that crystals are less prone to cracking and resist microcrack events better on the side, which is useful in bone, while exposing the base, the hardest face, to minimize mass loss from abrasion with teeth.     

Synchrotron X-ray diffraction study of load partitioning during elastic deformation of bovine dentin

The elastic properties of dentin, a biological composite consisting of stiff hydroxyapatite (HAP) nano-platelets within a compliant collagen matrix, are determined by the volume fraction of these two phases and the load transfer between them. We have measured the elastic strains in situ within the HAP phase of bovine dentine by high energy X-ray diffraction for a series of static compressive stresses at ambient temperature. The apparent HAP elastic modulus (ratio of applied stress to elastic HAP strain) was found to be 18 ± 2 GPa. This value is significantly lower than the value of 44 GPa predicted by the lower bound load transfer Voigt model, using HAP and collagen volume fractions determined by thermo-gravimetric analysis. This discrepancy is explained by (i) a reduction in the intrinsic Young’s modulus of the nano-size HAP platelets due to the high fraction of interfacial volume and (ii) an increase in local stresses due to stress concentration around the dentin tubules.     

The mechanical stress–strain properties of single electrospun collagen type I nanofibers

Knowledge of the mechanical properties of electrospun fibers is important for their successful application in tissue engineering, material composites, filtration and drug delivery. In particular, electrospun collagen has great potential for biomedical applications due to its biocompatibility and promotion of cell growth and adhesion. Using a combined atomic force microscopy (AFM)/optical microscopy technique, the single fiber mechanical properties of dry, electrospun collagen type I were determined. The fibers were electrospun from a 80 mg ml^?1 collagen solution in 1,1,1,3,3,3-hexafluro-2-propanol and collected on a striated surface suitable for lateral force manipulation by AFM. The small strain modulus, calculated from three-point bending analysis, was 2.82 GPa. The modulus showed significant softening as the strain increased. The average extensibility of the fibers was 33% of their initial length, and the average maximum stress (rupture stress) was 25 MPa. The fibers displayed significant energy loss and permanent deformations above 2% strain.     

Elastic modulus and collagen organization of the rabbit cornea: Epithelium to endothelium

The rabbit is commonly used to evaluate new corneal prosthetics and study corneal wound healing. Knowledge of the stiffness of the rabbit cornea would better inform the design and fabrication of keratoprosthetics and substrates with relevant mechanical properties for in vitro investigations of corneal cellular behavior. This study determined the elastic modulus of the rabbit corneal epithelium, anterior basement membrane (ABM), anterior and posterior stroma, Descemet’s membrane (DM) and endothelium using atomic force microscopy (AFM). In addition, three-dimensional collagen fiber organization of the rabbit cornea was determined using nonlinear optical high-resolution macroscopy. The elastic modulus as determined by AFM for each corneal layer was: epithelium, 0.57 ± 0.29 kPa (mean ± SD); ABM, 4.5 ± 1.2 kPa, anterior stroma, 1.1 ± 0.6 kPa; posterior stroma, 0.38 ± 0.22 kPa; DM, 11.7 ± 7.4 kPa; and endothelium, 4.1 ± 1.7 kPa. The biophysical properties, including the elastic modulus, are unique for each layer of the rabbit cornea and are dramatically softer in comparison to the corresponding regions of the human cornea. Collagen fiber organization is also dramatically different between the two species, with markedly less intertwining observed in the rabbit vs. human cornea. Given that the substratum stiffness considerably alters the corneal cell behavior, keratoprosthetics that incorporate mechanical properties simulating the native human cornea may not elicit optimal cellular performance in rabbit corneas that have dramatically different elastic moduli. These data should allow for the design of substrates that better mimic the biomechanical properties of the corneal cellular environment.     

The mechanical properties of individual,electrospun fibrinogen fibers

We used a combined atomic force microscopic (AFM)/fluorescence microscopic technique to study the mechanical properties of individual, electrospun fibrinogen fibers in aqueous buffer. Fibers (average diameter 208 nm) were suspended over 12 μm-wide grooves in a striated, transparent substrate. The AFM, situated above the sample, was used to laterally stretch the fibers and to measure the applied force. The fluorescence microscope, situated below the sample, was used to visualize the stretching process. The fibers could be stretched to 2.3 times their original length before breaking; the breaking stress was 22 × 10⁶ Pa. We collected incremental stress–strain curves to determine the viscoelastic behavior of these fibers. The total stretch modulus was 17.5 × 10⁶ Pa and the relaxed elastic modulus was 7.2 × 10⁶ Pa. When held at constant strain, electrospun fibrinogen fibers showed a fast and slow stress relaxation time of 3 and 55 s.Our fibers were spun from the typically used 90% 1,1,1,3,3,3-hexafluoro-2-propanol (90-HFP) electrospinning solution and re-suspended in aqueous buffer. Circular dichroism spectra indicate that α-helical content of fibrinogen is ～70% higher in 90-HFP than in aqueous solution.These data are needed to understand the mechanical behavior of electrospun fibrinogen structures. Our technique is also applicable to study other nanoscopic fibers.     

Biocompatibility of corrosion-resistant zeolite coatings for titanium alloy biomedical implants

Titanium alloy, Ti6Al4V, is widely used in dental and orthopedic implants. Despite its excellent biocompatibility, Ti6Al4V releases toxic Al and V ions into the surrounding tissue after implantation. In addition, the elastic modulus of Ti6Al4V (～110 GPa) is significantly higher than that of bone (10–40 GPa), leading to a modulus mismatch and consequently implant loosening and deosteointegration. Zeolite coatings are proposed to prevent the release of the toxic ions into human tissue and enhance osteointegration by matching the mechanical properties of bone. Zeolite MFI coatings are successfully synthesized on commercially pure titanium and Ti6Al4V for the first time. The coating shows excellent adhesion by incorporating titanium from the substrate within the zeolite framework. Higher corrosion resistance than the bare titanium alloy is observed in 0.856 M NaCl solution at pHs of 7.0 and 1.0. Zeolite coatings eliminate the release of cytotoxic Al and V ions over a 7 day period. Pluripotent mouse embryonic stem cells show higher adhesion and cell proliferation on the three-dimensional zeolite microstructure surface compared with a two-dimensional glass surface, indicating that the zeolite coatings are highly biocompatible.     

Effects of dehydration-induced structural and material changes on the apparent modulus of cancellous bone

Dehydration is known to cause an increase in the elastic modulus of bone tissue. However, it also causes structural changes (i.e. shrinkage) which can themselves significantly alter the mechanical properties, particularly in cancellous bone. The current study attempts to estimate the contribution of these two competing factors to the net change of dehydration on the apparent modulus of bovine cancellous bone. Cylindrical cores from the lumbar vertebrae were tested in tension, while hydrated and again after dehydration. The bone volume fractions (BV/TV) were measured in both conditions. The results indicate that the average overall increase in the apparent modulus after dehydration is 14 ± 14% (mean ± SD), which represents the net effect of a 27% increase in modulus due to increased tissue modulus offset by a modulus decrease of 13% due to reductions in bone volume fraction. These observations underscore the need to consider both structural and material changes when comparing hydrated and dehydrated mechanical behaviour.     

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.     

Surviving the surf: The tribomechanical properties of the periostracum of Mytilus sp.

We investigated the friction and wear behavior as well as the mechanical properties of the periostracum of Mytilus sp. Tribological properties were determined with a reciprocal sliding microtribometer, while mechanical characterization was performed using a nanoindenter. Measurements were performed in dry and wet conditions. On the dry periostracum we found a low friction coefficient of 0.078 ± 0.007 on the young parts and a higher one of 0.63 ± 0.02 on the old parts of the shell. Under wet, saline, conditions we only observed one average coefficient of friction of 0.37 ± 0.01. Microscopic ex situ analysis indicated that dry periostracum wore rather rapidly by plowing and fatigue, while it exhibited a high wear resistance when immersed in salt water. The Young’s modulus and hardness of the periostracum were also investigated in both dry and wet conditions. Under dry conditions the Young’s modulus of the periostracum was 8 ± 3 GPa, while under wet conditions it was 0.21 ± 0.05 GPa. The hardness of dry periostracum samples was 353 ± 127 MPa, whereas the hardness of wet samples was 5 ± 2 MPa. It was found that, in the wet state, viscous behavior plays a significant role in the mechanical response of the periostracum. Our results strongly indicate that the periostracum can provide an important contribution to the overall wear resistance of Mytilus sp. shell.     

Recombinant production and film properties of full-length hornet silk proteins

Full-length versions of the four main components of silk cocoons of Vespa simillima hornets, Vssilk1–4, were produced as recombinant proteins in Escherichia coli. In shake flasks, the recombinant Vssilk proteins yielded 160–330 mg recombinant protein l⁻¹. Films generated from solutions of single Vssilk proteins had a secondary structure similar to that of films generated from native hornet silk. The films made from individual recombinant hornet silk proteins had similar or enhanced mechanical performance compared with films generated from native hornet silk, possibly reflecting the homogeneity of the recombinant proteins. The pH-dependent changes in zeta (ζ) potential of each Vssilk film were measured, and isoelectric points (pI) of Vssilk1–4 were determined as 8.9, 9.1, 5.0 and 4.2, respectively. The pI of native hornet silk, a combination of the four Vssilk proteins, was 4.7, a value similar to that of Bombyx mori silkworm silk. Films generated from Vssilk1 and 2 had net positive charge under physiological conditions and showed significantly higher cell adhesion activity. It is proposed that recombinant hornet silk is a valuable new material with potential for cell culture applications.     

Osteonal lamellae elementary units: Lamellar microstructure,curvature and mechanical properties

The mechanical and structural properties of the sublayers of osteonal lamellae were studied. Young’s modulus (E) of adjacent individual lamellae was measured by nanoindentation of parallel slices every 1–3 μm, in planes parallel and perpendicular to the osteon axis (OA). In planes parallel to the OA, the modulus of a lamella could vary significantly between sequential slices. Significant modulus variations were also sometimes found on opposing sides of the osteonal canal for the same lamella. These results are rationalized by considerations involving the microstructural organization of the collagen fibrils in the lamellae. Scanning electron microscope imaging of freeze fractured surfaces revealed that the substructure of a single lamella can vary significantly on the opposing sides of the osteonal axis. Using a serial surface view method, parallel planes were exposed every 8–10 nm using a dual-beam microscope. Analysis of the orientations of fibrils revealed that the structure is rotated plywood like, consisting of unidirectional sublayers of fibrils of several orientations, with occasional randomly oriented sublayers. The dependence of the measured mechanical properties of the lamellae on the indentation location may be explained by the observed structure, as well as by the curvature of the osteonal lamellae through simple geometrical-structural considerations. Mechanical advantages arising from the curved laminate structure are discussed.     

人体主动脉瓣膜钙化组织的力学性质

目的采用纳米压痕测试方法,测量人体主动脉瓣取出物的钙化组织的材料力学性能。方法采集5名主动脉瓣狭窄患者的瓣叶取出物,选取钙化瓣叶进行纳米压痕测试,获得钙化组织弹性模量、硬度等材料力学参数。结果瓣叶钙化组织的弹性模量为(15.69±3.89) GPa,硬度为(0.59±0.15) GPa。结论通过纳米压痕测试方法得到瓣叶钙化组织的弹性模量和硬度,为瓣膜的生物力学研究提供实验数据参考。     

Mechanical properties,electronic structure and bonding of α- and β-tricalcium phosphates with surface characterization

The mechanical properties and electronic structure of α- and β-tricalcium phosphate (TCP) crystals are studied by using two ab initio density functional methods, the Vienna Ab initio Simulation Package (VASP) and the orthogonalized linear combination of atomic orbitals method. Based on the VASP optimized crystal structures, the elastic constants of α- and β-TCP are obtained using an effective stress–strain computational scheme. From the calculated elastic constants, the bulk modulus, shear modulus, Young’s modulus and Poisson’s ratios are obtained. The results show that the mechanical properties of the two crystals are comparable and that α-TCP is somewhat softer than β-TCP. Comparison with experimental extrapolations of the elastic constants shows significant differences, which attest to the difficulty of obtaining single crystal samples. The calculated electronic structure results show that both crystals are large gap insulators with a direct band gap of 4.89 eV for α-TCP and 5.25 eV for β-TCP. Effective charge calculations show that, on average, β-TCP has slightly less charge transfer per Ca than α-TCP. The (0 1 0) ((0 0 1)) surface model for α-TCP (β-TCP) is studied using a supercell slab geometry and fully relaxed to obtain the optimized structures. The estimated surface formation energies are 0.777 and 0.842 J m^?2 for α-TCP and β-TCP, respectively. The electronic structures of the two surface models are compared with the bulk models. Charge density analysis shows that the surfaces of both TCP crystals are positively charged overall owing to the presence of Ca ions near the surfaces.     

Shaping the micromechanical behavior of multi-phase composites for bone tissue engineering

Mechanical stiffness is a fundamental parameter in the rational design of composites for bone tissue engineering in that it affects both the mechanical stability and the osteo-regeneration process at the fracture site. A mathematical model is presented for predicting the effective Young’s modulus (E) and shear modulus (G) of a multi-phase biocomposite as a function of the geometry, material properties and volume concentration of each individual phase. It is demonstrated that the shape of the reinforcing particles may dramatically affect the mechanical stiffness: E and G can be maximized by employing particles with large geometrical anisotropy, such as thin platelet-like or long fibrillar-like particles. For a porous poly(propylene fumarate) (60% porosity) scaffold reinforced with silicon particles (10% volume concentration) the Young’s (shear) modulus could be increased by more than 10 times by just using thin platelet-like as opposed to classical spherical particles, achieving an effective modulus E ～ 8 GPa (G ～ 3.5 GPa). The mathematical model proposed provides results in good agreement with several experimental test cases and could help in identifying the proper formulation of bone scaffolds, reducing the development time and guiding the experimental testing.