Establishing a through-puncture model for assessing post-injection leakage in the intervertebral disc.

PURPOSE: Needle injection through the outer annulus fibrosus of the intervertebral disc (IVD) is the most practical approach for delivery of therapeutic agents, which have been shown to potentially leak following needle retraction. The goal of this work was to establish a protocol for quantifying post-injection leakage and test its sensitivity to factors believed to affect needle track geometry. METHODS: A through-puncture defect procedure, followed by controlled injection, was performed on bovine caudal IVDs. Sensitivity to needle size was tested by injection of saline into unconstrained discs with either a 30G, 26G, or 21G hypodermic needle. Sensitivity to axial load was tested by repeated injection via a 26G needle with either no constraint, fixed height, or 10% axial compressive strain. Sensitivity to flexion was tested by applying combined 0.2 MPa compression and 15° of flexion following injection of 5% of disc volume. RESULTS: Needle diameter significantly affected maximum volume prior to leakage, ranging from 34.6 ± 31.9 µL when using 21G to 115.6 ± 23.6 µL when using 30G. While all unloaded discs leaked, axial compression decreased the incidence of leakage events by 50-100% depending on load history. Forward flexion resulted in a 22% incidence of leakage. CONCLUSION: Fluid injected into IVDs is at significant risk of leakage following needle retraction. This risk depends on factors which alter the geometry of the needle track, including needle size, pinching due to axial compression, and stretching as a result of forward flexion.

Radial trend in murine annulus fibrosus fiber orientation is best explained by vertebral growth.

PURPOSE: The intervertebral disc (IVD) annulus fibrosus (AF) is composed of concentric lamellae with alternating right- and left-handed helically oriented collagen fiber bundles. This arrangement results in anisotropic material properties, which depend on local fiber orientations. Prior measurements of fiber inclination angles in human lumbar and bovine caudal IVDs found a significantly higher inclination angle in the inner AF than outer, though it is currently unknown if this pattern is conserved in smaller mammalian species. Additionally, the physical mechanism behind this pattern remains un-determined. METHODS: In this study, AF fiber angles were measured histologically in murine caudal IVDs and compared to previously published values from bovine caudal IVDs. Fiber angles were also predicted using three theoretical models, including two based on adaptation to internal swelling pressure and one based on vertebral body growth. RESULTS: Fiber angle was found to significantly decrease from 49.5 ± 3.8° in the inner AF to 34.5 ± 6.6° in the outer AF. While steeper than in bovine discs at all locations, the trend with radial position was comparable between species. This trend was best fit by growth-based model and opposite of that predicted by the pressure vessel models. CONCLUSION: Trends in AF fiber orientation are conserved between mammalian species. Modeling results suggest that the AF tissue microstructure is more likely to be driven by adjacent vertebral body growth than adapted for optimal mechanical performance.

Level-wise differences in in vivo lateral bending moment are associated with microstructural alterations in bovine caudal intervertebral discs.

Despite its common use as a laboratory model, little is known about the in vivo forces and moments applied to the bovine caudal intervertebral disc. Such aspects are crucial, as intervertebral disc tissue is known to remodel in response to repeated loading. We hypothesized that the magnitude of loading from muscle contraction during a typical lateral bending motion varies between caudal levels and is accompanied by variations in tissue microstructure. This hypothesis was tested by estimating level-wise forces and bending moments using two independent approaches: a dynamic analytical model of the motion and analysis of muscle cross-sections obtained via computed tomography. Microstructure was assessed by measuring the collagen fiber crimp period in the annulus fibrosus, and composition was assessed via quantitative histology. Both the analytical model and muscle cross-sections indicated peak bending moments of over 3 N m and peak compressive force of over 125 N at the c1c2 level, decreasing distally. There was a significant downward trend from proximal to distal in the outer annulus fibrosus collagen crimp period in the anterior and posterior regions only, suggesting remodeling in response to the highest lateral bending moments. There were no observed trends in composition. Our results suggest that although the proximal discs in the bovine tail are subjected to forces and moments from muscle contraction that are comparable (relative to disc size) to those acting on human lumbar discs, the distal discs are not. The resulting pattern of microstructural alterations suggests that level-wise differences should be considered when using bovine discs as a research model.

Bridging the Gap: Science and Technology Policy in the (Bio)Engineering Classroom.

Engineers and scientists have a key role to play in the creation and implementation of government policy. Policymakers need access to the technical expertise that is critical to our national progress and security; however, this need is often overlooked by engineering students, faculty, and professionals. Even though a substantial fraction of scientists and engineers end up pursuing jobs in government, engineering curricula do not usually provide any background in policy and for many, the policy-making process remains a black box. The good news is that there are some simple ways to make it more accessible and to encourage increased involvement. In this paper, we provide a brief overview of the federal policy-making process and present a collection of classroom learning activities that link policy-making and implementation to science and engineering. These can easily be added to existing courses without wholesale curricular changes. We also suggest professional development activities for engineers at all stages of their careers and discuss ways for engineers to become involved in the policy process. Introducing learning and career development activities focused on science and engineering policy will better prepare engineers to provide needed technical expertise to policymakers. It may also encourage engineers to consider careers in local, state, and federal government.

Mapping of Intervertebral Disk Annulus Fibrosus Compressive Properties Is Sensitive to Specimen Boundary Conditions.

Predicting the mechanical behavior of the intervertebral disk (IVD) in health and in disease requires accurate spatial mapping of its compressive mechanical properties. Previous studies confirmed that residual strains in the annulus fibrosus (AF) of the IVD, which result from nonuniform extracellular matrix deposition in response to in vivo loads, vary by anatomical regions (anterior, posterior, and lateral) and zones (inner, middle, and outer). We hypothesized that as the AF is composed of a nonlinear, anisotropic, viscoelastic material, the state of residual strain in the transverse plane would influence the apparent values of axial compressive properties. To test this hypothesis, axial creep indentation tests were performed, using a 1.6 mm spherical probe, at nine different anatomical locations on bovine caudal AFs in both the intact (residual strain present) and strain relieved states. The results showed a shift toward increased spatial homogeneity in all measured parameters, particularly instantaneous strain. This shift was not observed in control AFs, which were tested twice in the intact state. Our results confirm that time-dependent axial compressive properties of the AF are sensitive to the state of residual strain in the transverse plane, to a degree that is likely to affect whole disk behavior.

Phosphorylation and calcium antagonistically tune myosin-binding protein C's structure and function.

During each heartbeat, cardiac contractility results from calcium-activated sliding of actin thin filaments toward the centers of myosin thick filaments to shorten cellular length. Cardiac myosin-binding protein C (cMyBP-C) is a component of the thick filament that appears to tune these mechanochemical interactions by its N-terminal domains transiently interacting with actin and/or the myosin S2 domain, sensitizing thin filaments to calcium and governing maximal sliding velocity. Both functional mechanisms are potentially further tunable by phosphorylation of an intrinsically disordered, extensible region of cMyBP-C's N terminus, the M-domain. Using atomic force spectroscopy, electron microscopy, and mutant protein expression, we demonstrate that phosphorylation reduced the M-domain's extensibility and shifted the conformation of the N-terminal domain from an extended structure to a compact configuration. In combination with motility assay data, these structural effects of M-domain phosphorylation suggest a mechanism for diminishing the functional potency of individual cMyBP-C molecules. Interestingly, we found that calcium levels necessary to maximally activate the thin filament mitigated the structural effects of phosphorylation by increasing M-domain extensibility and shifting the phosphorylated N-terminal fragments back to the extended state, as if unphosphorylated. Functionally, the addition of calcium to the motility assays ablated the impact of phosphorylation on maximal sliding velocities, fully restoring cMyBP-C's inhibitory capacity. We conclude that M-domain phosphorylation may have its greatest effect on tuning cMyBP-C's calcium-sensitization of thin filaments at the low calcium levels between contractions. Importantly, calcium levels at the peak of contraction would allow cMyBP-C to remain a potent contractile modulator, regardless of cMyBP-C's phosphorylation state.

Full-length myosin Va exhibits altered gating during processive movement on actin.

Myosin Va (myoV) is a processive molecular motor that transports intracellular cargo along actin tracks with each head taking multiple 72-nm hand-over-hand steps. This stepping behavior was observed with a constitutively active, truncated myoV, in which the autoinhibitory interactions between the globular tail and motor domains (i.e., heads) that regulate the full-length molecule no longer exist. Without cargo at near physiologic ionic strength (100 mM KCl), full-length myoV adopts a folded (approximately 15 S), enzymatically-inhibited state that unfolds to an extended (approximately 11 S), active conformation at higher salt (250 mM). Under conditions favoring the folded, inhibited state, we show that Quantum-dot-labeled myoV exhibits two types of interaction with actin in the presence of MgATP. Most motors bind to actin and remain stationary, but surprisingly, approximately 20% are processive. The moving motors transition between a strictly gated and hand-over-hand stepping pattern typical of a constitutively active motor, and a new mode with a highly variable stepping pattern suggestive of altered gating. Each head of this partially inhibited motor takes longer-lived, short forward (35 nm) and backward (28 nm) steps, presumably due to globular tail-head interactions that modify the gating of the individual heads. This unique mechanical state may be an intermediate in the pathway between the inhibited and active states of the motor.

Molecular modulation of actomyosin function by cardiac myosin-binding protein C.

Cardiac myosin-binding protein C is a key regulator of cardiac contractility and is capable of both activating the thin filament to initiate actomyosin motion generation and governing maximal sliding velocities. While MyBP-C's C terminus localizes the molecule within the sarcomere, the N terminus appears to confer regulatory function by binding to the myosin motor domain and/or actin. Literature pertaining to how MyBP-C binding to the myosin motor domain and or actin leads to MyBP-C's dual modulatory roles that can impact actomyosin interactions are discussed.

Phosphorylation modulates the mechanical stability of the cardiac myosin-binding protein C motif.

Cardiac myosin-binding protein C (cMyBP-C) is a thick-filament-associated protein that modulates cardiac contractility through interactions of its N-terminal immunoglobulin (Ig)-like C0-C2 domains with actin and/or myosin. These interactions are modified by the phosphorylation of at least four serines located within the motif linker between domains C1 and C2. We investigated whether motif phosphorylation alters its mechanical properties by characterizing force-extension relations using atomic force spectroscopy of expressed mouse N-terminal cMyBP-C fragments (i.e., C0-C3). Protein kinase A phosphorylation or serine replacement with aspartic acids did not affect persistence length (0.43 ± 0.04 nm), individual Ig-like domain unfolding forces (118 ± 3 pN), or Ig extension due to unfolding (30 ± 0.38 nm). However, phosphorylation did significantly decrease the C0-C3 mean contour length by 24 ± 2 nm. These results suggest that upon phosphorylation, the motif, which is freely extensible in the nonphosphorylated state, adopts a more stable and/or different structure. Circular dichroism and dynamic light scattering data for shorter expressed C1-C2 fragments with all four serines replaced by aspartic acids confirmed that the motif did adopt a more stable structure that was not apparent in the nonphosphorylated motif. These biophysical data provide both a mechanical and structural basis for cMyBP-C regulation by motif phosphorylation.

Analysis of quantitative magnetic resonance imaging and biomechanical parameters on human discs with different grades of degeneration.

PURPOSE: To establish relationships between quantitative MRI (qMRI) and biomechanical parameters in order to help inform and interpret alterations of human intervertebral discs (IVD) with different grades of degeneration. MATERIALS AND METHODS: The properties of the nucleus pulposus (NP) and annulus fibrosus (AF) of each IVD of 10 lumbar spines (range, 32-77 years) were analyzed by qMRI (relaxation times T1 and T2, magnetization transfer ratio [MTR], and apparent diffusion coefficient [ADC]), and tested in confined compression and dynamic shear. RESULTS: T1 and T2 significantly decreased in both the NP and AF with increasing degeneration grades while the MTR increased significantly with grade 4. In contrast to the other qMRI parameters, the ADC had a tendency to decrease with increasing grade. Disc degeneration caused a decrease in the aggregate modulus, hydraulic permeability and shear modulus magnitude along with an increase in phase angle in the AF. In contrast, disc degeneration of NPs demonstrated decreases in shear modulus and phase angle. CONCLUSION: Our studies indicate that qMRI can be used as a noninvasive diagnostic tool in the detection of IVD properties with the potential to help interpret and detect early, middle, and late stages of degeneration. QMRI of human IVD can therefore become a very important diagnostic assessment tool in determining the functional state of the disc.

Fluorescent labeling with 5-DTAF reduces collagen fiber uncrimping in loaded tendons.

A fluorescent dye commonly used to image tissues under load (5-DTAF) has previously been shown to stiffen tendons. This study hypothesized that 5-DTAF staining stiffens tendons through reduced fiber sliding, altering the rate at which crimped collagen fibers straighten under load. This was tested by using reflected cross-polarized light microscopy to measure fiber crimp period of cervine extensor digitorum longus tendon specimens under axial load. Specimens were treated with either phosphate buffered saline (negative control), genipin (positive control), or 5-DTAF. In saline treated specimens, crimp period (relative to unstretched) increased at approximately 2.5 times the applied axial strain, indicating substantial fiber sliding. In both 5-DTAF and genipin treated specimens, this ratio was reduced to 1:1, indicating no fiber sliding. These results add further evidence that care should be taken when using 5-DTAF to stain tissue for studying microscale deformations in tissues.

Cargo properties play a critical role in myosin Va-driven cargo transport along actin filaments.

High-resolution experiments revealed that a single myosin-Va motor can transport micron-sized cargo on actin filaments in a stepwise manner. However, intracellular cargo transport is mediated through the dense actin meshwork by a team of myosin Va motors. The mechanism of how motors interact mechanically to bring about efficient cargo transport is still poorly understood. This study describes a stochastic model where a quantitative understanding of the collective behaviors of myosin Va motors is developed based on cargo stiffness. To understand how cargo properties affect the overall cargo transport, we have designed a model in which two myosin Va motors were coupled by wormlike chain tethers with persistence length ranging from 10 to 80 nm and contour length from 100 to 200 nm, and predicted distributions of velocity, run length, and tether force. Our analysis showed that these parameters are sensitive to both the contour and persistence length of cargo. While the velocity of two couple motors is decreased compared to a single motor (from 531 ± 251 nm/s to as low as 318 ± 287 nm/s), the run length (716 ± 563 nm for a single motor) decreased for short, rigid tethers (to as low as 377 ± 187 µm) and increased for long, flexible tethers (to as high as 1.74 ± 1.50 µm). The sensitivity of processive properties to tether rigidity (persistence length) was greatest for short tethers, which caused the motors to exhibit close, yet anti-cooperative coordination. Motors coupled by longer tethers stepped more independently regardless of tether rigidity. Therefore, the properties of the cargo or linkage must play an essential role in motor-motor communication and cargo transport.

Reflected cross-polarized light microscopy as a method for measuring collagen fiber crimp in musculoskeletal tissues.

Many musculoskeletal tissues are composed primarily of type I collagen, which takes on a periodic crimp morphology that allows large tensile strains in the tissue. The spatial period of collagen fiber crimp may be used to infer internal strains in a tissue and is typically measured using transmitted cross-polarized light imaging of thin slices. However, slicing may induce specimen distortion and precludes mechanical loading of the specimen during imaging. We hypothesized that reflected cross-polarized light imaging of thick tissue explants would yield crimp period measurements comparable to those obtained from transmitted light imaging of thin slices. We further hypothesized that these measurements would be sensitive to applied uniaxial strain in the fiber direction. These hypotheses were tested by imaging both intervertebral disc outer annulus fibrosus and medial collateral ligament tissue specimens. We found that both transmitted and reflected light yielded similar crimp period measurements for intervertebral disc tissue, with an overall average of 43.5 ± 11.5 µm. Reflected light yielded a significantly higher crimp period with lower variance than transmission through thin specimens (54.1 ± 10.6 µm versus 50.4 ± 16.0 µm) in the ligament. Upon application of axial tension, crimp periods in both fibers increased at a rate of approximately three times the applied strain (with 3.17% applied strain yielding a 9.64 ± 4.4% increase in crimp period in the disc and an 11.7 ± 3.7% increase in the ligament), indicating significant fibril sliding. In support of our hypotheses, these findings suggest that reflected cross-polarized light is a suitable method for measuring collagen fiber crimp in musculoskeletal tissues, both statically and under tension.

Penetrating annulus fibrosus injuries affect dynamic compressive behaviors of the intervertebral disc via altered fluid flow: an analytical interpretation.

Extensive experimental work on the effects of penetrating annular injuries indicated that large injuries impact axial compressive properties of small animal intervertebral discs, yet there is some disagreement regarding the sensitivity of mechanical tests to small injury sizes. In order to understand the mechanism of injury size sensitivity, this study proposed a simple one dimensional model coupling elastic deformations in the annulus with fluid flow into and out of the nucleus through both porous boundaries and through a penetrating annular injury. The model was evaluated numerically in dynamic compression with parameters obtained by fitting the solution to experimental stress-relaxation data. The model predicted low sensitivity of mechanical changes to injury diameter at both small and large sizes (as measured by low and high ratios of injury diameter to annulus thickness), with a narrow range of high sensitivity in between. The size at which axial mechanics were most sensitive to injury size (i.e., critical injury size) increased with loading frequency. This study provides a quantitative hypothetical model of how penetrating annulus fibrosus injuries in discs with a gelatinous nucleus pulposus may alter disc mechanics by changing nucleus pulposus fluid pressurization through introduction of a new fluid transport pathway though the annulus. This model also explains how puncture-induced biomechanical changes depend on both injury size and test protocol.

Needle puncture injury of the rat intervertebral disc affects torsional and compressive biomechanics differently.

Needle puncture is a common method of inducing intervertebral disc (IVD) degeneration in small animal models and may have some similarities to IVD injury conditions such as herniation. Yet, the influence of puncture injuries on IVD biomechanics is not well understood. This study quantified the acute effects of anular injury on the biomechanics of rat caudal IVDs in compression and torsion following puncture with 30, 25 and 21 G needles. In compression, puncture injury reduced elastic stiffness by 20% for all needle sizes, but differences between control and punctured discs did not remain after compressive overload. In contrast, torsional parameters associated with anular fiber tension were affected proportionally with needle size. We conclude that IVD injuries that penetrate through the thickness of the annulus affect IVD biomechanics through different mechanisms for compression and torsion. Anular injuries affect torsional properties in a manner directly related to the amount of fiber disruption and compressive properties in a manner that affects pressurization.

A growth-based model for the prediction of fiber angle distribution in the intervertebral disc annulus fibrosus.

There is a growing interest in the development of patient-specific finite element models of the human lumbar spine for both the assessment of injury risk and the development of treatment strategies. A current challenge in implementing these models is that the outer annulus fibrosus of the disc is composed of concentric sheets of aligned collagen fibers, the helical angles of which vary spatially. In finite element models, fiber angle is typically assumed to be constant, based on average experimental measurements from a small number of locations. The present study hypothesized that the full spatial distribution of fiber angles in the annulus fibrosus may be predicted for any disc geometry by assuming growth from a thin cylinder with constant fiber angle. This hypothesis was tested by developing an analytical model of disc growth and calibrating it with fiber angle measurements of adult bovine caudal discs. The calibrated model was then run on a representative human lumbar disc geometry. The model was able to accurately predict fiber angle distributions in both the experimental bovine caudal disc measurements and literature-reported human lumbar disc measurements. Despite its theoretical basis in development, the model requires only mature state geometry, making it practical for implementation in patient-specific finite element analyses, in which disc geometry is obtained from clinical imaging.

Slow depressurization following intradiscal injection leads to injectate leakage in a large animal model.

Needle injection has been indicated as the most practical method of delivering therapeutic agents to the intervertebral disc due to the disc's largely avascular nature. As the disc is characterized by both high stiffness and low permeability, injection requires substantial pressure, which may not relax on practical time scales. Additionally, needle puncture results in a localized disruption to the annulus fibrosus that can provide a leakage pathway for pressurized injectate. We hypothesized that intradiscal injection would result in slow relaxation of injectate pressure, followed by leakage upon needle retraction. This hypothesis was tested via controlled injection of fluorescently labeled saline into bovine caudal discs via a 21 gauge needle. Injections were performed with 10% of total disc volume injected at 3%/s followed by a 4-minute dwell. An analytical poroelastic model was calibrated to the experimental data and used to estimate injectate delivery with time. Experimental results confirmed both pressurization (with a peak of 199 ± 45 kPa) and slow recovery (final pressure of 81 ± 23 kPa). Injectate leakage through the needle puncture was verified following needle retraction in all samples. Histological sections of the discs displayed a clear defect at each disc's injection site with strong fluorescent labeling indicating a leakage pathway. The modeling results suggest that less than one-fourth of the injected volume was absorbed by the tissue in 4 minutes. Taken together these results suggest that needle injection is a feasible, albeit inefficient method for delivery of therapeutic agents into the intervertebral disc. Particular care should be taken to aspirate un-absorbed injectate prior to needle retraction to prevent leakage and exposure of surrounding tissues.

Proteoglycans contribute locally to swelling, but globally to compressive mechanics, in intact cervine medial meniscus.

Loss of charged proteoglycans in the knee meniscus, which aid in the support of compressive loads by entraining water, is an effect of degeneration and is often associated with osteoarthritis. In healthy menisci, proteoglycan content is highest in the inner white zone and decreases towards the peripheral red zone. We hypothesized that loss of proteoglycans would reduce both osmotic swelling and compressive stiffness, spatially localized to the avascular white zone of the meniscus. This hypothesis was tested by targeted enzymatic digestion of proteoglycans using hyaluronidase in intact cervine medial menisci. Mechanics were quantified by creep indentation on the femoral surface. Osmotic swelling changes were assessed by measuring collagen fiber crimp period in the radial-axial plane in the lamellar layer along both the tibial and femoral contacting surfaces. All measurements were made in the inner, middle, and outer zones of the anterior, central, and posterior regions. Mechanical measurements showed variation in creep behavior with anatomical location, along with spatially uniform decreases in viscosity (average of 21%) and creep stiffness (average of 15%) with hyaluronidase treatment. Lamellar collagen crimp period was significantly decreased (average of 27%) by hyaluronidase, indicating a decrease in osmotic swelling, with the largest decreases seen in locations with the highest proteoglycan content. Taken together, these results suggest that while proteoglycans have localized effects on meniscus swelling, the resulting effect on compressive properties is distributed throughout the tissue.

Increased lumbar spinal column laxity due to low-angle, low-load cyclic flexion may predispose to acute injury.

Lumbar spinal column laxity contributes to instability, increasing the risk of low back injury and pain. Until the laxity increase due to the cyclic loads of daily living can be quantified, the associated injury risk cannot be predicted clinically. This work cyclically loaded 5-vertebra lumbar motion segments (7 skeletally-mature cervine specimens, 5 osteoporotic human cadaver specimens) for 20 000 cycles of low-load low-angle (15°) flexion. The normalized neutral zone lengths and slopes of the load-displacement hysteresis loops showed a similar increase in spinal column laxity across species. The intervertebral kinematics also changes with cyclic loading. Differences in the location and magnitude of surface strain on the vertebral bodies (0.34% ± 0.11% in the cervine specimens, and 3.13% ± 1.69% in the human cadaver specimens) are consistent with expected fracture modes in these populations. Together, these results provide biomechanical evidence of spinal column damage during high-cycle low-load low-angle loading.

A numerical study to determine pericellular matrix modulus and evaluate its effects on the micromechanical environment of chondrocytes.

Chondrocyte biosynthesis is highly sensitive to mechanical strain. A thin pericellular matrix (PCM) surrounds the cell and plays an important role in mechanotransduction. PCM material properties are difficult to measure directly because of its size and connectivity to both cell and extracellular matrix (ECM). The purpose of this study was to develop a method of calculating linear elastic properties of the PCM using an inverse finite element approach with experimental properties of cell and chondron taken from the literature. Finite element models were constructed of both the equivalent chondron case and the chondrocyte-PCM structure, and a Fibonacci search obtained PCM moduli that matched the ECM strain field between the two cases. The most important result was that ECM strain adjacent to a chondron inclusion was sensitive to the chondron properties and may be used to calculate PCM mechanical properties, consistent with our strain field hypotheses. PCM moduli obtained through this method range from 43 to 240 kPa, were significantly higher than previously published but resulted in only a 0.5-21% decrease in relative effective cell strain. Similarities between effective strain ratios led to the conclusion that matching experimental techniques used to measure cell and PCM properties was more important than absolute values of the properties.