An Approach to Quantify Anisotropic Multiaxial Failure of the Annulus Fibrosus.

Tears in the annulus fibrosus (AF) of the intervertebral disk (IVD) occur due to multiaxial loading on the spine. However, most existing AF failure studies measure uniaxial stress, not the multiaxial stress at failure. Delamination theory, which requires advanced structural knowledge and knowledge about the interactions between the AF fibers and matrix, has historically been used to understand and predict AF failure. Alternatively, a simple method, the Tsai-Hill yield criteria, could describe multiaxial failure of the AF. This yield criteria uses the known tissue fiber orientation and an equation to establish the multiaxial failure stresses that cause failure. This paper presents a method to test the multiaxial failure stress of the AF experimentally and evaluate the potential for the Tsai-Hill model to predict these failure stresses. Porcine AF was cut into a dogbone shape at three distinct angles relative to the primary lamella direction (parallel, transverse, and oblique). Then, each dogbone was pulled to complete rupture. The Cauchy stress in the material's fiber coordinates was calculated. These multiaxial stress parameters were used to optimize the coefficients of the Tsai-Hill yield. The coefficients obtained for the Tsai-Hill model vary by an order of magnitude between the fiber and transverse directions, and these coefficients provide a good description of the AF multiaxial failure stress. These results establish both an experimental approach and the use of the Tsai-Hill model to explain the anisotropic failure behavior of the tissue.

MRI-based degeneration grades for lumbar facet joints do not correlate with cartilage mechanics.

Background: Lumbar facet joint arthritis is characterized by degeneration of articular cartilage, loss of joint spacing, and increased boney spur formation. These signs of facet joint degeneration have been previously measured using destructive biochemical and mechanical analysis. Nondestructive clinical evaluation of the facet joint has also been performed using MRI scoring, which ranks the health of the facet joint using the Fujiwara scale. However, nondestructive clinical evaluation of facet joint arthritis using standard MRI scoring provides low resolution images which result in high interobserver variability. Therefore, to assess the accuracy of nondestructive MRI analysis with regard to the health of the facet joint, this study determined whether any correlations existed between lumbar facet joint articular cartilage mechanics, facet articular cartilage biochemical signatures, and Fujiwara scores. Materials and Method: To accomplish this aim, human cadaveric lumbar spines were obtained and imaged using T1 MRI, then independently scored by three spine researchers. An osteochondral plug from each of the L2 thru L5 facet joints was obtained and loaded under unconfined compression. Results: The experiments showed no trends between histological images and changes in the Fujiwara score. The mechanical properties of articular cartilage (thickness, Young's modulus, instantaneous modulus, and permeability) also had no correlations with the Fujiwara score. Conclusions: These results show that the current Fujiwara score cannot accurately describe the biomechanics or biochemical composition of facet joint articular cartilage.

The Lumbar Facet Capsular Ligament Becomes More Anisotropic and the Fibers Become Stiffer With Intervertebral Disc and Facet Joint Degeneration.

Degeneration of the lumbar spine, and especially how that degeneration may lead to pain, remains poorly understood. In particular, the mechanics of the facet capsular ligament may contribute to low back pain, but the mechanical changes that occur in this ligament with spinal degeneration are unknown. Additionally, the highly nonlinear, heterogeneous, and anisotropic nature of the facet capsular ligament makes understanding mechanical changes more difficult. Clinically, magnetic resonance imaging (MRI)-based signs of degeneration in the facet joint and the intervertebral disc (IVD) correlate. Therefore, this study examined how the nonlinear, heterogeneous mechanics of the facet capsular ligament change with degeneration of the lumbar spine as characterized using MRI. Cadaveric human spines were imaged via MRI, and the L2-L5 facet joints and IVDs were scored using the Fujiwara and Pfirrmann grading systems. Then, the facet capsular ligament was isolated and biaxially loaded. The nonlinear mechanical properties of the ligament were obtained using a nonlinear generalized anisotropic inverse mechanics analysis (nGAIM). Then a Holzapfel-Gasser-Ogden (HGO) model was fit to the stress-strain data obtained from nGAIM. The facet capsular ligament is stiffer and more anisotropic at larger Pfirrmann grades and higher Fujiwara scores than at lower grades and scores. Analysis of ligament heterogeneity showed all tissues are highly heterogeneous, but no distinct spatial patterns of heterogeneity were found. These results show that degeneration of the lumbar spine including the facet capsular ligament appears to be occurring as a whole joint phenomenon and advance our understanding of lumbar spine degeneration.

Depth-dependent patterns in shear modulus of temporomandibular joint cartilage correspond to tissue structure and anatomic location.

To fully understand TMJ cartilage degeneration and appropriate repair mechanisms, it is critical to understand the native structure-mechanics relationships of TMJ cartilage and any local variation that may occur in the tissue. Here, we used confocal elastography and digital image correlation to measure the depth-dependent shear properties as well as the structural properties of TMJ cartilage at different anatomic locations on the condyle to identify depth-dependent changes in shear mechanics and structure. We found that samples at every anatomic location showed qualitatively similar shear modulus profiles as a function of depth. In every sample, four distinct zones of mechanical behavior were observed, with shear modulus values spanning 3-5 orders of magnitude across zones. However, quantitative characteristics of shear modulus profiles varied by anatomic location, particularly zone size and location, with the most significant variation in zonal width occurring in the fibrocartilage surface layer (zone 1). This anatomic variation suggests that different locations on the TMJ condyle may play unique mechanical roles in TMJ function. Furthermore, zones identified in the mechanical data corresponded on a sample-by-sample basis to zones identified in the structural data, indicating the known structural zones of TMJ cartilage may also play unique mechanical roles in TMJ function.

Local tissue heterogeneity may modulate neuronal responses via altered axon strain fields: insights about innervated joint capsules from a computational model.

In innervated collagenous tissues, tissue scale loading may contribute to joint pain by transmitting force through collagen fibers to the embedded mechanosensitive axons. However, the highly heterogeneous collagen structures of native tissues make understanding this relationship challenging. Recently, collagen gels with embedded axons were stretched and the resulting axon signals were measured, but these experiments were unable to measure the local axon strain fields. Computational discrete fiber network models can directly determine axon strain fields due to tissue scale loading. Therefore, this study used a discrete fiber network model to identify how heterogeneous collagen networks (networks with multiple collagen fiber densities) change axon strain due to tissue scale loading. In this model, a composite cylinder (axon) was embedded in a Delaunay network (collagen). Homogeneous networks with a single collagen volume fraction and two types of heterogeneous networks with either a sparse center or dense center were created. Measurements of fiber forces show higher magnitude forces in sparse regions of heterogeneous networks and uniform force distributions in homogeneous networks. The average axon strain in the sparse center networks decreases when compared to homogeneous networks with similar collagen volume fractions. In dense center networks, the average axon strain increases compared to homogeneous networks. The top 1% of axon strains are unaffected by network heterogeneity. Based on these results, the interaction of tissue scale loading, collagen network heterogeneity, and axon strains in native musculoskeletal tissues should be considered when investigating the source of joint pain.

The influence of chondrocyte source on the manufacturing reproducibility of human tissue engineered cartilage.

Multiple human tissue engineered cartilage constructs are showing promise in advanced clinical trials but identifying important measures of manufacturing reproducibility remains a challenge. FDA guidance suggests measuring multiple mechanical properties prior to implantation, because these properties could affect the long term success of the implant. Additionally, these engineered cartilage mechanics could be sensitive to the autologous chondrocyte source, an inherently irregular manufacturing starting material. If any mechanical properties are sensitive to changes in the autologous chondrocyte source, these properties may need to be measured prior to implantation to ensure manufacturing reproducibility and quality. Therefore, this study identified variability in the compressive, friction, and shear properties of a human tissue engineered cartilage constructs due to the chondrocyte source. Over 200 constructs were created from 7 different chondrocyte sources and tested using 3 distinct mechanical experiments. Under confined compression, the compressive properties (aggregate modulus and hydraulic permeability) varied by orders of magnitude due to the chondrocyte source. The friction coefficient changed by a factor of 5 due to the chondrocyte source and high intrapatient variability was noted. In contrast, the shear modulus was not affected by changes in the chondrocyte source. Finally, measurements on the local compressive and shear mechanics revealed variability in the depth dependent strain fields based on chondrocyte source. Since the chondrocyte source causes large amounts of variability in the compression and local mechanical properties of engineered cartilage, these mechanical properties may be important measures of manufacturing reproducibility. STATEMENT OF SIGNIFICANCE: Although the FDA recommends measuring mechanical properties of human tissue engineered cartilage constructs during manufacturing, the effect of manufacturing variability on construct mechanics is unknown. As one of the first studies to measure multiple mechanical properties on hundreds of human tissue engineered cartilage constructs, we found the compressive properties are most sensitive to changes in the autologous chondrocyte source, an inherently irregular manufacturing variable. This sensitivity to the autologous chondrocyte source reveals the compressive properties should be measured prior to implantation to assess manufacturing reproducibility.

Heterogeneous matrix deposition in human tissue engineered cartilage changes the local shear modulus and resistance to local construct buckling.

Human tissue engineered cartilage is a promising solution for focal cartilage defects, but these constructs do not have the same local mechanical properties as native tissue. Most clinically relevant engineered cartilage constructs seed human chondrocytes onto a collagen scaffold, which buckles at low loads and strains. This buckling creates local regions of high strain that could cause cell death and damage the engineered tissue. Since human tissue engineered cartilage is commonly grown in-vivo prior to implantation, new matrix deposition could improve the local implant mechanics and prevent local tissue buckling. However, the relationship between local biochemical composition and the local mechanics or local buckling probability has never been quantified. Therefore, this study correlated the local biochemical composition of human tissue engineered cartilage constructs using Fourier transform infrared spectroscopy (FTIR) with the local shear modulus and local buckling probability. The local shear modulus and local buckling probability were obtained using a confocal elastography technique. The local shear modulus increased with increases in local aggrecan content in the interior region (inside the scaffold). A minimum amount of aggrecan was required to prevent local construct buckling at physiologic strains. Since the original scaffold was primarily composed of collagen, increases in collagen content due to new matrix deposition was minimal and had little effect on the mechanical properties. Thus, we concluded that aggrecan deposition inside the scaffold pores is the most effective way to improve the mechanical function and prevent local tissue damage in human tissue engineered cartilage constructs.

Multiscale mechanics of tissue-engineered cartilage grown from human chondrocytes and human-induced pluripotent stem cells.

Tissue-engineered cartilage has shown promising results in the repair of focal cartilage defects. However, current clinical techniques rely on an extra surgical procedure to biopsy healthy cartilage to obtain human chondrocytes. Alternatively, induced pluripotent stem cells (iPSCs) have the ability to differentiate into chondrocytes and produce cartilaginous matrix without the need to biopsy healthy cartilage. However, the mechanical properties of tissue-engineered cartilage with iPSCs are unknown and might be critical to long-term tissue function and health. This study used confined compression, cartilage on glass tribology, and shear testing on a confocal microscope to assess the macroscale and microscale mechanical properties of two constructs seeded with either chondrocyte-derived iPSCs (Ch-iPSCs) or native human chondrocytes. Macroscale properties of Ch-iPSC constructs provided similar or better mechanical properties than chondrocyte constructs. Under compression, Ch-iPSC constructs had an aggregate modulus that was two times larger than chondrocyte constructs and was closer to native tissue. No differences in the shear modulus and friction coefficients were observed between Ch-iPSC and chondrocyte constructs. On the microscale, Ch-iPSC and chondrocyte constructs had different depth-dependent mechanical properties, neither of which matches native tissue. These observed depth-dependent differences may be important to the function of the implant. Overall, this comparison of multiple mechanical properties of Ch-iPSC and chondrocyte constructs shows that using Ch-iPSCs can produce equivalent or better global mechanical properties to chondrocytes. Therefore, iPSC-seeded cartilage constructs could be a promising solution to repair focal cartilage defects. The chondrocyte constructs used in this study have been implanted into humans for clinical trials. Therefore, Ch-iPSC constructs could also be used clinically in place of the current chondrocyte construct.

Stribeck Curve Analysis of Temporomandibular Joint Condylar Cartilage and Disc.

Temporomandibular joint (TMJ) diseases such as osteoarthritis and disc displacement have no permanent treatment options, but lubrication therapies, used in other joints, could be an effective alternative. However, the healthy TMJ contains fibrocartilage, not hyaline cartilage as is found in other joints. As such, the effect of lubrication therapies in the TMJ is unknown. Additionally, only a few studies have characterized the friction coefficient of the healthy TMJ. Like other cartilaginous tissues, the mandibular condyles and discs are subject to changes in friction coefficient due to fluid pressurization. In addition, the friction coefficients of the inferior joint space of the TMJ are affected by the sliding direction and anatomic location. However, these previous findings have not been able to identify how all three of these parameters (anatomic location, sliding direction, and fluid pressurization) influence changes in friction coefficient. This study used Stribeck curves to identify differences in the friction coefficients of mandibular condyles and discs based on anatomic location, sliding direction, and amount of fluid pressurization (friction mode). Friction coefficients were measured using a cartilage on glass tribometer. Both mandibular condyle and disc friction coefficients were well described by Stribeck curves (R2 range 0.87-0.97; p < 0.0001). These curves changed based on anatomic location (Δµ ∼ 0.05), but very few differences in friction coefficients were observed based on sliding direction. Mandibular condyles had similar boundary mode and elastoviscous mode friction coefficients to the TMJ disc (µmin ∼ 0.009 to 0.19) and both were lower than hyaline cartilage in other joints (e.g., knee, ankle, etc.). The observed differences here indicate that the surface characteristics of each anatomic region cause differences in friction coefficients.

In vitro culture increases mechanical stability of human tissue engineered cartilage constructs by prevention of microscale scaffold buckling.

Many studies have measured the global compressive properties of tissue engineered (TE) cartilage grown on porous scaffolds. Such scaffolds are known to exhibit strain softening due to local buckling under loading. As matrix is deposited onto these scaffolds, the global compressive properties increase. However the relationship between the amount and distribution of matrix in the scaffold and local buckling is unknown. To address this knowledge gap, we studied how local strain and construct buckling in human TE constructs changes over culture times and GAG content. Confocal elastography techniques and digital image correlation (DIC) were used to measure and record buckling modes and local strains. Receiver operating characteristic (ROC) curves were used to quantify construct buckling. The results from the ROC analysis were placed into Kaplan-Meier survival function curves to establish the probability that any point in a construct buckled. These analysis techniques revealed the presence of buckling at early time points, but bending at later time points. An inverse correlation was observed between the probability of buckling and the total GAG content of each construct. This data suggests that increased GAG content prevents the onset of construct buckling and improves the microscale compressive tissue properties. This increase in GAG deposition leads to enhanced global compressive properties by prevention of microscale buckling.

Mechanical properties and structure-function relationships of human chondrocyte-seeded cartilage constructs after in vitro culture.

Autologous Chondrocyte Implantation (ACI) is a widely recognized method for the repair of focal cartilage defects. Despite the accepted use, problems with this technique still exist, including graft hypertrophy, damage to surrounding tissue by sutures, uneven cell distribution, and delamination. Modified ACI techniques overcome these challenges by seeding autologous chondrocytes onto a 3D scaffold and securing the graft into the defect. Many studies on these tissue engineered grafts have identified the compressive properties, but few have examined frictional and shear properties as suggested by FDA guidance. This study is the first to perform three mechanical tests (compressive, frictional, and shear) on human tissue engineered cartilage. The objective was to understand the complex mechanical behavior, function, and changes that occur with time in these constructs grown in vitro using compression, friction, and shear tests. Safranin-O histology and a DMMB assay both revealed increased sulfated glycosaminoglycan (sGAG) content in the scaffolds with increased maturity. Similarly, immunohistochemistry revealed increased lubricin localization on the construct surface. Confined compression and friction tests both revealed improved properties with increased construct maturity. Compressive properties correlated with the sGAG content, while improved friction coefficients were attributed to increased lubricin localization on the construct surfaces. In contrast, shear properties did not improve with increased culture time. This study suggests the various mechanical and biological properties of tissue engineered cartilage improve at different rates, indicating thorough mechanical evaluation of tissue engineered cartilage is critical to understanding the performance of repaired cartilage. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2298-2306, 2017.

Minimization of treatment time for in vitro 1.1 MHz destruction of Pseudomonas aeruginosa biofilms by high-intensity focused ultrasound.

Medical implants are prone to colonization by bacterial biofilms. Normally, surgery is required to replace the infected implant. One promising noninvasive modality is to destroy biofilms with high-intensity focused ultrasound. In our study, Pseudomonas aeruginosa biofilms were grown on implant-mimicking graphite disks in a flow chamber for 3 days prior to exposing them to ultrasound pulses. Exposure time at each treatment location was varied between 5, 15 and 30s. Burst period was varied between 1, 3, 6 and 12 milliseconds (ms). The pulses were 20 cycles in duration at 1.1 MHz from a spherically focused transducer (f/1, 63 mm focal length), creating peak compressional and rarefactional pressures at the graphite disk surface of 30 and 13 MPa, respectively. P. aeruginosa were tagged with green fluorescent protein, and killed cells were visualized using propidium iodide before determining the extent of biofilm destruction. The exposure-induced temperature rise was measured to be less than 0.2°C at the focus, namely the interface between graphite disk and water. Then, the temperature rise was measured at the focus between the graphite disk and a tissue-mimicking phantom to evaluate therapy safety. Two thresholds, of bacteria destruction increase and of complete bacteria removal, respectively, were identified to divide our eight exposure conditions. Results indicated that 30-s exposure and 6-ms pulse period were sufficient to destroy the biofilms. However, the 15-s exposure and 3-ms pulse period were viewed as optimum when considering exposure time, efficacy, and safety.