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.

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.

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.

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.

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.

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.

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.

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.

Signal distortion in atomic force microscopy photodetector.

The frequency-dependent complex impedance of an atomic force microscope photodetector is measured. The inverse problem is solved obtaining the voltage that would have been collected with a hypothetical, perfectly flat-frequency-response photodetector from the experimentally available voltage. This information is used to study the distortion that the true input signal undergoes as it passes through the photodetector on the way to becoming the experimentally measured output signal. It is found that signals with features of interest shorter than 10 µs render noticeable differences between the true and measured raw voltages and forces. Signals with features shorter than 1 µs produce experimentally measured force curves that deviate substantially from the true force curves. A method is proposed for correcting the measured raw voltage signal.

Tension tests on mammalian collagen fibrils.

A brief overview of isolated collagen fibril mechanics testing is followed by presentation of the first results testing fibrils isolated from load-bearing mammalian tendons using a microelectromechanical systems platform. The in vitro modulus (326 ± 112 MPa) and fracture stress (71 ± 23 MPa) are shown to be lower than previously measured on fibrils extracted from sea cucumber dermis and tested with the same technique. Scanning electron microscope images show the fibrils can fail with a mechanism that involves circumferential rupture, whereas the core of the fibril stays at least partially intact.

Relative Contribution of Trabecular and Cortical Bone to Primary Implant Stability: An In Vitro Model Study.

The specific aim of this study was to examine the relative contributions to the implant insertion torque value (ITV) by cortical and trabecular components of an in vitro bone model. Simulated bone blocks of polyurethane were used with 2 densities of foam (0.08 g/cm(3) to mimic trabecular bone and 0.64 g/cm(3) to mimic cortical bone). We have developed a new platform technology to collect data that enables quantitative evaluation of ITV at different implant locations. Seven groups were used to model varying thicknesses of cortical bone over a lower-quality trabecular bone that have clinical significance: a solid 0.08 g/cm(3) block; 1 mm, 2 mm, and 3 mm thick 0.64 g/cm(3) sheets with no underlayer; and 1 mm, 2 mm, and 3 mm thick 0.64 g/cm(3) sheets laminated on top of a 4 cm thick 0.08 g/cm(3) block. The ITVs were recorded as a function of insertion displacement distance. Relative contributions of ITV ranged from 3% to 18% from trabecular bone, and 62% to 74% from cortical bone depending on the thickness of the cortical layer. Inserting an implant into 2-mm and 3-mm cortical layers laminated atop trabecular blocks had a synergistic effect on ITVs. Finally, an implant with a reverse bevel design near the abutment showed final average torque values that were 14% to 34% less than their maximum torque values. This work provides basic quantitative information for clinicians to understand the influence of composite layers of bone in relation to mechanical torque resistances during implant insertion in order to obtain desired primary implant stability.

Method to extract minimally damaged collagen fibrils from tendon.

A new method is presented to extract collagen fibrils from mammalian tendon tissue. Mammalian tendons are treated with a trypsin-based extraction medium and gently separated with tweezers in an aqueous solution. Collagen fibrils released in the solution are imaged using both dark-field light microscopy and scanning electron microscopy. The method successfully extracts isolated fibrils from rat tail and patellar tendons. To examine whether the method is likely to damage fibrils during extraction, sea cucumber dermis fibril lengths are compared against those obtained using only distilled water. The two methods produce fibrils of similar lengths. This is contrasted with fibrils being shortened when extracted using a tissue homogenizer. Scanning electron microscopy shows the new method preserves D-banding features on fibril surfaces and that fibril diameter does not vary substantially compared with water extracted fibrils.

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.

A selected review of the recent advances in craniomaxillofacial bone tissue engineering.

PURPOSE OF REVIEW: Craniofacial surgeons must continually make decisions about how to best reconstruct the craniomaxillofacial skeleton (CFS). A high priority has been placed on the search for bone substitute materials (BSMs) that are both mechanically and biologically optimized for these reconstructions. This review is intended to present the complexity of this undertaking to physicians and scientists by reviewing the technological advances published in the last 2 years. RECENT FINDINGS: Advances in bone tissue engineering took place in the areas of scaffolds, bioactive factors (e.g. growth factors, cytokines, and pharmaceuticals), and cellular components. Recent literature highlighted the complex interplay between these elements. Researchers also made great strides in merging high-resolution imaging with computer-aided tissue engineering. SUMMARY: Developing BSMs that fulfill the many needs in the CFS is difficult and there are multiple barriers to clinical translation. However, based on the progress in the last 2 years in the individual elements of BSM development as well as integration of those elements into implantable constructs, it appears that a product with specific CFS applications is on the horizon.

Nanophase bone substitute in vivo response to subcutaneous implantation.

A collagen-apatite composite designed as a load-bearing bone substitute implant is used to characterize the relationship between implant morphology and in vivo behavior. This nanophase bone substitute (NBS) is studied morphologically using a nondestructive imaging technique and biologically using the rodent subcutaneous model. Porosity and pore interconnectivity are correlated with histological outcomes showing cellular invasion occurs with average pore sizes below 100 µm. Crosslinking with D-ribose is shown to affect cellular infiltration in a dose-response manner. These data suggest that collagen-apatite bone substitutes can support cellular infiltration with pore size significantly smaller than 100 µm, an encouraging result regarding development of the NBS into a platform of biomaterials with enhanced mechanical properties. The data also indicate that increasing crosslinking density decreases cellular infiltration of NBS. Thus, modulating mechanical properties of the material by altering crosslink density is likely to produce decreased biological response within the material.

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.

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.

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.

Deformation micromechanisms of collagen fibrils under uniaxial tension.

Collagen, an essential building block of connective tissues, possesses useful mechanical properties due to its hierarchical structure. However, little is known about the mechanical properties of collagen fibril, an intermediate structure between the collagen molecule and connective tissue. Here, we report the results of systematic molecular dynamics simulations to probe the mechanical response of initially unflawed finite size collagen fibrils subjected to uniaxial tension. The observed deformation mechanisms, associated with rupture and sliding of tropocollagen molecules, are strongly influenced by fibril length, width and cross-linking density. Fibrils containing more than approximately 10 molecules along their length and across their width behave as representative volume elements and exhibit brittle fracture. Shorter fibrils experience a more graceful ductile-like failure. An analytical model is constructed and the results of the molecular modelling are used to find curve-fitted expressions for yield stress, yield strain and fracture strain as functions of fibril structural parameters. Our results for the first time elucidate the size dependence of mechanical failure properties of collagen fibrils. The associated molecular deformation mechanisms allow the full power of traditional material and structural engineering theory to be applied to our understanding of the normal and pathological mechanical behaviours of collagenous tissues under load.