Method to Quantify Nanoscale Surface Charge in Liquid with Atomic Force Microscopy.

A theory is presented to obtain surface charge density on nanoscale objects from data in the snap-to-contact portion of an atomic force microscope force-separation curve. The mathematical model takes into account the tip's dielectric constant using the Self-Consistent Sum of Dipoles theory which includes the charge-charge interaction and the charge-dipole interaction with electrolyte-induced exponentially decaying screening, Debye and London dipolar force, and fluid viscosity including confined fluid layers to account for energy dissipation. Using previously published experimental data, the mathematical model is applied to measure the surface charge density on an individual nanoscale amine-modified polystyrene bead immobilized on the basal plane of highly oriented pyrolytic graphite in buffered aqueous solution. Within the experimental uncertainty, the magnitude of the charge density on a single bead obtained using the new method falls within the distribution of values determined by the manufacturer using titration and electron microscopy.

Charge Calibration Standard for Atomic Force Microscope Tips in Liquids.

An electric charge standard with nanoscale resolution is created using the known charge distribution of a single tobacco mosaic virus coat protein combined with the known packing of these proteins in the virus capsid. This advances the ability to measure charge on nanometric samples. Experimental atomic force microscope (AFM) force-distance curves are collected under aqueous conditions with controlled pH and ion concentration. A mathematical model that considers a polarizable dielectric tip immersed in an electrolyte is used to obtain charge density from the AFM measurements. Interactions between the tip and the sample are modeled using theory that includes monopolar electrostatic interactions, dipolar interactions, screening from both the dielectric nature of ambient water and solvated ions as described by the linear Poisson-Boltzmann equation, and hard-core repulsion. It is found that the tip charge density changes on a timescale of hours requiring recalibration of the tip for experiments lasting more than an hour. As an example of how a charge-calibrated tip may be used, the surface charge densities on 20 individual carboxylate-modified polystyrene (PS) beads are measured. The average of these AFM-measured bead charge densities is compared with the value obtained from conventional titration combined with electron microscopy. The two values are found to agree within 20%. While the comparison demonstrates similarity of the two charge measurements, hypotheses are put forward as to why the two techniques might be expected not to provide identical mean charge densities. The considerations used to build these hypotheses thus underscore the relevance of the method performed here if charge information is required on individual nanoparticles.

Euler-Bernoulli theory accurately predicts atomic force microscope cantilever shape during non-equilibrium snap-to-contact motion.

We prove that the Euler-Bernoulli elastic beam theory can be reliably used to describe the dynamics of an atomic force microscope cantilever during the far from equilibrium snap-to-contact event. In conventional atomic force microscope operation, force-separation curves are obtained by post-processing voltage versus time traces produced by measuring one point on the cantilever close to the hanging end. In this article, we assess the validity of the Euler-Bernoulli equation during the snap-to-contact event. The assessment is based on a direct comparison between experiment and theory. The experiment uses Doppler vibrometry to measure displacement versus time for many points along the long axis of the cantilever. The theoretical algorithm is based on a solution of the Euler-Bernoulli equation to obtain the full shape of the cantilever as a function of time. The algorithm uses as boundary conditions, experimentally obtained information only near the hanging end of the cantilever. The solution is obtained in a manner that takes into account non-equilibrium motion. Within experimental error, the theory agrees with experiment indicating that the Euler-Bernoulli theory is appropriate to predict the cantilever kinematics during snap-to-contact. Since forces on the tip can be obtained from the instantaneous shape of the cantilever, this work should allow for computation of tip-sample forces during the snap-to-contact event from a conventional force-distance measured input.

Effect of Drilling Speed on Dental Implant Insertion Torque.

The specific aim of this study was to examine whether slow drilling speeds (15 rpm) produce pilot holes that result in different implant insertion torques than pilot holes made with higher speed drilling (1500 rpm). To accomplish this, a new method is presented for transferring samples from a drilling machine onto an implant insertion torque measuring apparatus while maintaining the same center of rotation. Simulated bone blocks of polyurethane were used with 2 densities of foam to mimic trabecular and cortical bone. Pilot holes drilled using both drilling methods were morphologically characterized at macro and micro scales. Nobel Biocare Nobel Active implants were then placed. Profilometer and optical imaging were used to determine changes in the pilot hole morphology. Recorded insertion torque measurements were used to quantitatively contrast implants inserted into holes drilled using the 2 speeds. Although there were slight qualitative and quantitative differences between the low- and high-speed drilled pilot holes, the differences were insufficient to cause a statistically significant change in insertion torque.

Electrostatic force curves in finite-size-ion electrolytes.

We obtain analytical expressions for electrostatic forces between an atomic force microscope tip and a sample immersed in an electrolyte. These simple expressions relate force to tip-sample separation explicitly incorporating tip size, solvent ion size, and solvent ion concentration. If the ions are much smaller than the tip-sample gap, the force decays monotonically, a consequence of the corresponding monotonic decays of the correlation function in the Debye-Hückel context. If the ions are of size comparable to the tip-sample gap, then oscillations appear superimposed on the overall decay, a consequence of the geometric mismatch between ion cluster size and the gap size.

Collagen fibrils from both positional and energy-storing tendons exhibit increased amounts of denatured collagen when stretched beyond the yield point.

Collagen molecules are the base structural unit of tendons, which become denatured during mechanical overload. We recently demonstrated that during tendon stretch, collagen denaturation occurs at the yield point of the stress-strain curve in both positional and energy-storing tendons. We were interested in investigating how this load is transferred throughout the collagen hierarchy, and sought to determine the onset of collagen denaturation when collagen fibrils are stretched. Fibrils are one level above the collagen molecule in the collagen hierarchy, allowing more direct probing of the effect of strain on collagen molecules. We isolated collagen fibrils from both positional and energy-storing tendon types and stretched them using a microelectromechanical system device to various levels of strain. We stained the fibrils with fluorescently labeled collagen hybridizing peptides that specifically bind to denatured collagen, and examined whether samples stretched beyond the yield point of the stress-strain curve exhibited increased amounts of denatured collagen. We found that collagen denaturation in collagen fibrils from both tendon types occurs at the yield point. Greater amounts of denatured collagen were found in post-yield positional fibrils than in energy-storing fibrils. This is despite a greater yield strain and yield stress in fibrils from energy-storing tendons compared to positional tendons. Interestingly, the peak modulus of collagen fibrils from both tendon types was the same. These results are likely explained by the greater crosslink density found in energy-storing tendons compared to positional tendons. The insights gained from this study could help management of tendon and other musculoskeletal injuries by targeting collagen molecular damage at the fibril level. STATEMENT OF SIGNIFICANCE: When tendons are stretched or torn, this can lead to collagen denaturation (damage). Depending on their biomechanical function, tendons are considered positional or energy-storing with different crosslink profiles. By stretching collagen fibrils instead of fascicles from both tendon types, we can more directly examine the effect of tensile stretch on the collagen molecule in tendons. We found that regardless of tendon type, collagen denaturation in fibrils occurs when they are stretched beyond the yield point of the stress-strain curve. This provides insight into how load affects different tendon sub-structures during tendon injuries and failure, which will help clinicians and researchers understand mechanisms of injuries and potentially target collagen molecular damage as a treatment strategy, leading to improved clinical outcomes following injury.

Platelet-mimicking procoagulant nanoparticles augment hemostasis in animal models of bleeding.

Treatment of bleeding disorders using transfusion of donor-derived platelets faces logistical challenges due to their limited availability, high risk of contamination, and short (5 to 7 days) shelf life. These challenges could be potentially addressed by designing platelet mimetics that emulate the adhesion, aggregation, and procoagulant functions of platelets. To this end, we created liposome-based platelet-mimicking procoagulant nanoparticles (PPNs) that can expose the phospholipid phosphatidylserine on their surface in response to plasmin. First, we tested PPNs in vitro using human plasma and demonstrated plasmin-triggered exposure of phosphatidylserine and the resultant assembly of coagulation factors on the PPN surface. We also showed that this phosphatidylserine exposed on the PPN surface could restore and enhance thrombin generation and fibrin formation in human plasma depleted of platelets. In human plasma and whole blood in vitro, PPNs improved fibrin stability and clot robustness in a fibrinolytic environment. We then tested PPNs in vivo in a mouse model of thrombocytopenia where treatment with PPNs reduced blood loss in a manner comparable to treatment with syngeneic platelets. Furthermore, in rat and mouse models of traumatic hemorrhage, treatment with PPNs substantially reduced bleeding and improved survival. No sign of systemic or off-target thrombotic risks was observed in the animal studies. These findings demonstrate the potential of PPNs as a platelet surrogate that should be further investigated for the management of bleeding.

Viscoelastic properties of isolated collagen fibrils.

Understanding the viscoelastic behavior of collagenous tissues with complex hierarchical structures requires knowledge of the properties at each structural level. Whole tissues have been studied extensively, but less is known about the mechanical behavior at the submicron, fibrillar level. Using a microelectromechanical systems platform, in vitro coupled creep and stress relaxation tests were performed on collagen fibrils isolated from the sea cucumber dermis. Stress-strain-time data indicate that isolated fibrils exhibit viscoelastic behavior that could be fitted using the Maxwell-Weichert model. The fibrils showed an elastic modulus of 123 ± 46 MPa. The time-dependent behavior was well fit using the two-time-constant Maxwell-Weichert model with a fast time response of 7 ± 2 s and a slow time response of 102 ± 5 s. The fibrillar relaxation time was smaller than literature values for tissue-level relaxation time, suggesting that tissue relaxation is dominated by noncollagenous components (e.g., proteoglycans). Each specimen was tested three times, and the only statistically significant difference found was that the elastic modulus is larger in the first test than in the subsequent two tests, indicating that viscous properties of collagen fibrils are not sensitive to the history of previous tests.

Membrane microviscosity regulates endothelial cell motility.

Endothelial cell (EC) movement is an initiating and rate-limiting event in the neogenesis and repair of blood vessels. Here, we explore the hypothesis that microviscosity of the plasma membrane (PM) is a key physiological regulator of cell movement. Aortic ECs treated with membrane-active agents, such as alpha-tocopherol, cholesterol and lysophospholipids, exhibited a biphasic dependency on membrane microviscosity, in which moderate increases enhanced EC migration, but increases beyond a threshold markedly inhibited migration. Surprisingly, angiogenic growth factors, that is, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), also increased membrane microviscosity, as measured in live cells by fluorescence recovery after photobleaching (FRAP). The localization of Rac to the PM was modified in cells treated with membrane-active agents or growth factors, suggesting a molecular mechanism for how membrane microviscosity influences cell movement. Our data show that angiogenic growth factors, as well as certain lipophilic molecules, regulate cell motility through alterations in membrane properties and the consequent relocalization of critical signalling molecules to membranes.

Obtaining charge distributions on geometrically generic nanostructures using scanning force microscopy.

We develop the self-consistent sum of dipoles (SCSD) theory for the purpose of recovering charge densities present on nanostructures using scanning force microscope (SFM) force-separation experiments. The dielectric probe is discretized into volume elements characterized by their atomic polarizabilities. Magnitudes of the induced dipole in each element are calculated based on discrete charges placed on the surfaces, dipole-dipole interactions, and dielectric and ionic properties of the surrounding medium. We perform two model-model comparisons, one with a macroscopic dielectric sphere and one with a nanocluster of silicon atoms. In both cases, using a single adjustable parameter, our SCSD theory agrees with the accepted theories to better than 99%. Force-separation curves between a silicon nitride probe and the basal plane of highly oriented pyrolytic graphite in nine ionic concentration and pH combinations were fit with a root-mean-square error of 3.6 pN, an improvement over the 12 pN error obtained using the Derjaguin approximation. These results suggest that the SCSD will be useful in modeling SFM force-separation data to obtain spatially varying charge densities on surfaces with complex geometries.

Mandible Biomechanics and Continuously Erupting Teeth: A New Defect Model for Studying Load-Bearing Biomaterials.

Animals with elodont dentition and unfused mandible symphyses are hypothesized to have symmetric incisor morphology. Since these animals maintain their teeth by gnawing, they may provide physiologic feedback on mechanical function when unilateral mandible defects are created that manifest as ipsilateral changes in tooth structure. This defect model would potentially generate important information on the functional/mechanical properties of implants. Rats' and rabbits' mandibles and teeth are analyzed with µCT at baseline and post-intervention (n = 8 for each). Baseline incisors were compared. In a unilateral mandible pilot study, defects-ranging from critical size defect to complete ramus osteotomies-were created to assess effect on dentition (rats, n = 7; rabbits, n = 6). Within 90% confidence intervals, animals showed no baseline left/right differences in their incisors. There are apparent dental changes associated with unilateral defect type and location. Thus, at baseline, animals exhibit statistically significant incisor symmetry and there is an apparent relationship between mandible defect and incisor growth. The baseline symmetry proven here sets the stage to study the degree to which hemi-mandible destabilizing procedures result in measurable & reproducible disruption of dental asymmetry. In a validated model, an implant designed to function under load that prevents incisor asymmetry would provide supporting evidence that the implant has clinically useful load-bearing function.

In vitro fracture testing of submicron diameter collagen fibril specimens.

Mechanical testing of collagenous tissues at different length scales will provide improved understanding of the mechanical behavior of structures such as skin, tendon, and bone, and also guide the development of multiscale mechanical models. Using a microelectromechanical-systems (MEMS) platform, stress-strain response curves up to failure of type I collagen fibril specimens isolated from the dermis of sea cucumbers were obtained in vitro. A majority of the fibril specimens showed brittle fracture. Some displayed linear behavior up to failure, while others displayed some nonlinearity. The fibril specimens showed an elastic modulus of 470 ± 410 MPa, a fracture strength of 230 ± 160 MPa, and a fracture strain of 80% ± 44%. The fibril specimens displayed significantly lower elastic modulus in vitro than previously measured in air. Fracture strength/strain obtained in vitro and in air are both significantly larger than those obtained in vacuo, indicating that the difference arises from the lack of intrafibrillar water molecules produced by vacuum drying. Furthermore, fracture strength/strain of fibril specimens were different from those reported for collagenous tissues of higher hierarchical levels, indicating the importance of obtaining these properties at the fibrillar level for multiscale modeling.

Effect of neutrophil adhesion on the mechanical properties of lung microvascular endothelial cells.

Neutrophil adhesion to pulmonary microvascular endothelial cells (ECs) initiates intracellular signaling, resulting in remodeling of F-actin cytoskeletal structure of ECs. The present study determined the mechanical properties of ECs and the changes induced by neutrophil adhesion by atomic force microscopy. The elastic moduli of ECs were compared before neutrophils were present, as soon as neutrophil adhesion was detected, and 1 minute later. ECs that were adjacent to those with adherent neutrophils were also evaluated. Neutrophil adhesion induced a decrease in the elastic moduli in the 6.25-µm rim of ECs surrounding adherent neutrophils as soon as firmly adherent neutrophils were detected, which was transient and lasted less than 1 minute. Adjacent ECs developed an increase in stiffness that was significant in the central regions of these cells. Intercellular adhesion molecule-1 crosslinking did not induce significant changes in the elastic modulus of ECs in either region, suggesting that crosslinking intercellular adhesion molecule-1 is not sufficient to induce the observed changes. Our results demonstrate that neutrophil adhesion induces regional changes in the stiffness of ECs.

A Bottom-Up Approach Grafts Collagen Fibrils Perpendicularly to Titanium Surfaces.

Currently, titanium dental implant apposition to bone is achieved via osseointegration leading to ankylosis. A biomimetic Sharpey's fiber-type interface could be constructed around collagen fibrils robustly attached and projecting perpendicularly from the titanium surface. We present a proof-of-concept for a method to create upright-standing collagen nanofibrils covalently bonded to a titanium surface. The method involves activation of the titanium surface using a plasma discharge treatment followed by functionalization with an oxyamine-terminated silane coupling molecule. Using Rapoport's salt, the N-termini of individual type I collagen monomers are converted to ketones. When presented to the functionalized titanium surface, these ketones form oxime linkages with the silanes thus immobilizing the collagen. In a two-step process, these covalently bonded monomers act as sites for the formation of fibrils. Many fibril-surface junctions were observed by scanning electron microscopy on three different surfaces. These findings set the stage for working toward a high surface density of such features which might act as a platform from which to build a synthetic ligament.

Polarization of plasma membrane microviscosity during endothelial cell migration.

Cell movement is characterized by anterior-posterior polarization of multiple cell structures. We show here that the plasma membrane is polarized in moving endothelial cells (EC); in particular, plasma membrane microviscosity (PMM) is increased at the cell leading edge. Our studies indicate that cholesterol has an important role in generation of this microviscosity gradient. In vitro studies using synthetic lipid vesicles show that membrane microviscosity has a substantial and biphasic influence on actin dynamics; a small amount of cholesterol increases actin-mediated vesicle deformation, whereas a large amount completely inhibits deformation. Experiments in migrating ECs confirm the important role of PMM on actin dynamics. Angiogenic growth factor-stimulated cells exhibit substantially increased membrane microviscosity at the cell front but, unexpectedly, show decreased rates of actin polymerization. Our results suggest that increased PMM in lamellipodia may permit more productive actin filament and meshwork formation, resulting in enhanced rates of cell movement.

Stress-strain experiments on individual collagen fibrils.

Collagen, a molecule consisting of three braided protein helices, is the primary building block of many biological tissues including bone, tendon, cartilage, and skin. Staggered arrays of collagen molecules form fibrils, which arrange into higher-ordered structures such as fibers and fascicles. Because collagen plays a crucial role in determining the mechanical properties of these tissues, significant theoretical research is directed toward developing models of the stiffness, strength, and toughness of collagen molecules and fibrils. Experimental data to guide the development of these models, however, are sparse and limited to small strain response. Using a microelectromechanical systems platform to test partially hydrated collagen fibrils under uniaxial tension, we obtained quantitative, reproducible mechanical measurements of the stress-strain curve of type I collagen fibrils, with diameters ranging from 150-470 nm. The fibrils showed a small strain (epsilon < 0.09) modulus of 0.86 +/- 0.45 GPa. Fibrils tested to strains as high as 100% demonstrated strain softening (sigma(yield) = 0.22 +/- 0.14 GPa; epsilon(yield) = 0.21 +/- 0.13) and strain hardening, time-dependent recoverable residual strain, dehydration-induced embrittlement, and susceptibility to cyclic fatigue. The results suggest that the stress-strain behavior of collagen fibrils is dictated by global characteristic dimensions as well as internal structure.

Changes in the hyperelastic properties of endothelial cells induced by tumor necrosis factor-alpha.

Mechanical properties of living cells can be determined using atomic force microscopy (AFM). In this study, a novel analysis was developed to determine the mechanical properties of adherent monolayers of pulmonary microvascular endothelial cells (ECs) using AFM and finite element modeling, which considers both the finite thickness of ECs and their nonlinear elastic properties, as well as the large strain induced by AFM. Comparison of this model with the more traditional Hertzian model, which assumes linear elastic behavior, small strains, and infinite cell thickness, suggests that the new analysis can predict the mechanical response of ECs during AFM indentation better than Hertz's model, especially when using force-displacement data obtained from large indentations (>100 nm). The shear moduli and distensibility of ECs were greater when using small indentations (<100 nm) compared to large indentations (>100 nm). Tumor necrosis factor-alpha induced changes in the mechanical properties of ECs, which included a decrease in the average shear moduli that occurred in all regions of the ECs and an increase in distensibility in the central regions when measured using small indentations. These changes can be modeled as changes in a chain network structure within the ECs.

Minor Review: An Overview of a Synthetic Nanophase Bone Substitute.

Material is reviewed that consists of reconstituted collagen fibril gel mineralized in a manner that produces biomimetically sized nanoapatites intimately associated with the fibrils. This gel is formed into usable shapes with a modulus and strength that allow it to be surgically press fitted into bony defects. The design paradigm for the material is that the nanoapatites will dissolve into soluble Ca2+ as the collagen is degraded into RGD-containing peptide fragments due to osteoclastic action. This is intended to signal to the osteoclasts to continue removing the material in a biomimetic fashion similar to bony remodeling. Preliminary experiments in a subcutaneous rat model show that the material is biocompatible with respect to inflammatory and immunogenic responses, and that it supports cellular invasion. Preliminary experiments in a critical-sized mandibular defect in rats show that the material is resorbable and functions well as a bone morphogenetic 2 (BMP-2) carrier. We have produced a range of mechanical and biological responses by varying mechanical and chemical processing of the material.

Nanophase bone substitute for craniofacial load bearing application: Pilot study in the rodent.

An exploratory pilot study shows that a rodent mandibular defect model is useful in determining the biological response to a nanophase collagen/apatite composite designed as a biomimetic load-bearing bone substitute. Using a critical size defect, eight groups of rats (n = 3) were implanted with four renditions of the nanophase bone substitute (NBS) biomaterial. Each rendition was tested with and without recombinant human bone morphogenetic protein 2 (BMP2). NBS biomaterial renditions were: baseline, hyper-densified, d-ribose crosslinked, and d-ribose crosslinked and hyper-densified. Biological outcomes were assessed surgically, radiologically, and histologically. With the limited power available due to the small N's involved, some interesting hypotheses were generated that will be more fully investigated in future studies. BMP2 loaded NBS, when uncrosslinked, resulted in robust bone formation in the entire defect volume (regardless of porosity). Unloaded NBS were well tolerated but did not cause significant new bone formation in the defect volume. Densification alone had little effect on in vivo performance. Crosslinking thwarted implant uptake of BMP2 and resulted in fibrous encapsulation. It is concluded that the nanophase bone substitute is well tolerated in this bone defect model. When loaded with BMP2, implantation resulted in complete bony healing and defect closure with implant density (porosity) having little effect on bone healing or remodeling. Without BMP2 the biomaterial did not result in defect closure. Crosslinking, necessary to increase mechanical properties in an aqueous environment, disrupts osteointegration and BMP2 uptake. Alternate implant fabrication strategies will be necessary to achieve an improved balance between material strength and osteointegration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 520-532, 2018.

Multiscale mechanics of hierarchical structure/property relationships in calcified tissues and tissue/material interfaces.

This paper presents a review plus new data that describes the role hierarchical nanostructural properties play in developing an understanding of the effect of scale on the material properties (chemical, elastic and electrical) of calcified tissues as well as the interfaces that form between such tissues and biomaterials. Both nanostructural and microstructural properties will be considered starting with the size and shape of the apatitic mineralites in both young and mature bovine bone. Microstructural properties for human dentin and cortical and trabecular bone will be considered. These separate sets of data will be combined mathematically to advance the effects of scale on the modeling of these tissues and the tissue/biomaterial interfaces as hierarchical material/structural composites. Interfacial structure and properties to be considered in greatest detail will be that of the dentin/adhesive (d/a) interface, which presents a clear example of examining all three material properties, (chemical, elastic and electrical). In this case, finite element modeling (FEA) was based on the actual measured values of the structure and elastic properties of the materials comprising the d/a interface; this combination provides insight into factors and mechanisms that contribute to premature failure of dental composite fillings. At present, there are more elastic property data obtained by microstructural measurements, especially high frequency ultrasonic wave propagation (UWP) and scanning acoustic microscopy (SAM) techniques. However, atomic force microscopy (AFM) and nanoindentation (NI) of cortical and trabecular bone and the dentin-enamel junction (DEJ) among others have become available allowing correlation of the nanostructural level measurements with those made on the microstructural level.