BlpC-mediated selfish program leads to rapid loss of Streptococcus pneumoniae clonal diversity during infection.

Successful colonization of a host requires bacterial adaptation through genetic and population changes that are incompletely defined. Using chromosomal barcoding and high-throughput sequencing, we investigate the population dynamics of Streptococcus pneumoniae during infant mouse colonization. Within 1 day post inoculation, diversity was reduced >35-fold with expansion of a single clonal lineage. This loss of diversity was not due to immune factors, microbiota, or exclusive genetic drift. Rather, bacteriocins induced by the BlpC-quorum sensing pheromone resulted in predation of kin cells. In this intra-strain competition, the subpopulation reaching a quorum likely eliminates others that have yet to activate the blp locus. Additionally, this reduced diversity restricts the number of unique clones that establish colonization during transmission between hosts. Genetic variation in the blp locus was also associated with altered transmissibility in a human population, further underscoring the importance of BlpC in clonal selection and its role as a selfish element.

Mammalian Orthoreovirus Reassortment Proceeds with Little Constraint on Segment Mixing.

Segmentation of viral genomes provides the potential for genetic exchange within coinfected cells. However, for this potential to be realized, coinfecting genomes must mix during the viral life cycle. The efficiency of reassortment, in turn, dictates its potential to drive evolution. The opportunity for mixing within coinfected cells may vary greatly across virus families, such that the evolutionary implications of genome segmentation differ as a result of core features of the viral life cycle. To investigate the relationship between viral replication compartments and genetic exchange, we quantified reassortment in mammalian orthoreovirus (reovirus). Reoviruses carry a 10-segmented, double-stranded RNA genome, which is replicated within proteinaceous structures termed inclusion bodies. We hypothesized that inclusions impose a barrier to reassortment. We quantified reassortment between wild-type (wt) and variant (var) reoviruses that differ by one nucleotide per segment. Studies of wt/var systems in both T1L and T3D backgrounds revealed frequent reassortment without bias toward particular genotypes. However, reassortment was more efficient in the T3D serotype. Since T1L and T3D viruses exhibit different inclusion body morphologies, we tested the impact of this phenotype on reassortment. In both serotypes, reassortment levels did not differ by inclusion morphology. Reasoning that the merging of viral inclusions may be critical for genome mixing, we then tested the effect of blocking merging. Reassortment proceeded efficiently even under these conditions. Our findings indicate that reovirus reassortment is highly efficient despite the localization of many viral processes to inclusion bodies, and that the robustness of this genetic exchange is independent of inclusion body structure and fusion. IMPORTANCE Quantification of reassortment in diverse viral systems is critical to elucidate the implications of genome segmentation for viral evolution. In principle, genome segmentation offers a facile means of genetic exchange between coinfecting viruses. In practice, there may be physical barriers within the cell that limit the mixing of viral genomes. Here, we tested the hypothesis that localization of the various stages of the mammalian orthoreovirus life cycle within cytoplasmic inclusion bodies compartmentalizes viral replication and limits genetic exchange. Contrary to this hypothesis, our data indicate that reovirus reassortment occurs readily within coinfected cells and is not strongly affected by the structure or dynamics of viral inclusion bodies. We conclude that the potential for reassortment to contribute to reovirus evolution is high.

Collective interactions augment influenza A virus replication in a host-dependent manner.

Infection with a single influenza A virus (IAV) is only rarely sufficient to initiate productive infection. Instead, multiple viral genomes are often required in a given cell. Here, we show that the reliance of IAV on multiple infection can form an important species barrier. Namely, we find that avian H9N2 viruses representative of those circulating widely at the poultry-human interface exhibit acute dependence on collective interactions in mammalian systems. This need for multiple infection is greatly reduced in the natural host. Quantification of incomplete viral genomes showed that their complementation accounts for the moderate reliance on multiple infection seen in avian cells but not the added reliance seen in mammalian cells. An additional form of virus-virus interaction is needed in mammals. We find that the PA gene segment is a major driver of this phenotype and that both viral replication and transcription are affected. These data indicate that multiple distinct mechanisms underlie the reliance of IAV on multiple infection and underscore the importance of virus-virus interactions in IAV infection, evolution and emergence.

Incomplete influenza A virus genomes occur frequently but are readily complemented during localized viral spread.

Segmentation of viral genomes into multiple RNAs creates the potential for replication of incomplete viral genomes (IVGs). Here we use a single-cell approach to quantify influenza A virus IVGs and examine their fitness implications. We find that each segment of influenza A/Panama/2007/99 (H3N2) virus has a 58% probability of being replicated in a cell infected with a single virion. Theoretical methods predict that IVGs carry high costs in a well-mixed system, as 3.6 virions are required for replication of a full genome. Spatial structure is predicted to mitigate these costs, however, and experimental manipulations of spatial structure indicate that local spread facilitates complementation. A virus entirely dependent on co-infection was used to assess relevance of IVGs in vivo. This virus grows robustly in guinea pigs, but is less infectious and does not transmit. Thus, co-infection allows IVGs to contribute to within-host spread, but complete genomes may be critical for transmission.

Dysregulation of M segment gene expression contributes to influenza A virus host restriction.

The M segment of the 2009 pandemic influenza A virus (IAV) has been implicated in its emergence into human populations. To elucidate the genetic contributions of the M segment to host adaptation, and the underlying mechanisms, we examined a panel of isogenic viruses that carry avian- or human-derived M segments. Avian, but not human, M segments restricted viral growth and transmission in mammalian model systems, and the restricted growth correlated with increased expression of M2 relative to M1. M2 overexpression was associated with intracellular accumulation of autophagosomes, which was alleviated by interference of the viral proton channel activity by amantadine treatment. As M1 and M2 are expressed from the M mRNA through alternative splicing, we separated synonymous and non-synonymous changes that differentiate human and avian M segments and found that dysregulation of gene expression leading to M2 overexpression diminished replication, irrespective of amino acid composition of M1 or M2. Moreover, in spite of efficient replication, virus possessing a human M segment that expressed avian M2 protein at low level did not transmit efficiently. We conclude that (i) determinants of transmission reside in the IAV M2 protein, and that (ii) control of M segment gene expression is a critical aspect of IAV host adaptation needed to prevent M2-mediated dysregulation of vesicular homeostasis.

Influenza A Virus Reassortment Is Limited by Anatomical Compartmentalization following Coinfection via Distinct Routes.

Exchange of gene segments through reassortment is a major feature of influenza A virus evolution and frequently contributes to the emergence of novel epidemic, pandemic, and zoonotic strains. It has long been evident that viral diversification through reassortment is constrained by genetic incompatibility between divergent parental viruses. In contrast, the role of virus-extrinsic factors in determining the likelihood of reassortment has remained unclear. To evaluate the impact of such factors in the absence of confounding effects of segment mismatch, we previously reported an approach in which reassortment between wild-type (wt) and genetically tagged variant (var) viruses of the same strain is measured. Here, using wt/var systems in the A/Netherlands/602/2009 (pH1N1) and A/Panama/2007/99 (H3N2) strain backgrounds, we tested whether inoculation of parental viruses into distinct sites within the respiratory tract limits their reassortment. Using a ferret (Mustella putorius furo) model, either matched parental viruses were coinoculated intranasally or one virus was instilled intranasally whereas the second was instilled intratracheally. Dual intranasal inoculation resulted in robust reassortment for wt/var viruses of both strain backgrounds. In contrast, when infections were initiated simultaneously at distinct sites, strong compartmentalization of viral replication was observed and minimal reassortment was detected. The observed lack of viral spread between upper and lower respiratory tract tissues may be attributable to localized exclusion of superinfection within the host, mediated by innate immune responses. Our findings indicate that dual infections in nature are more likely to result in reassortment if viruses are seeded into similar anatomical locations and have matched tissue tropisms.IMPORTANCE Genetic exchange between influenza A viruses (IAVs) through reassortment can facilitate the emergence of antigenically drifted seasonal strains and plays a prominent role in the development of pandemics. Typical human influenza infections are concentrated in the upper respiratory tract; however, lower respiratory tract (LRT) infection is an important feature of severe cases, which are more common in the very young, the elderly, and individuals with underlying conditions. In addition to host factors, viral characteristics and mode of transmission can also increase the likelihood of LRT infection: certain zoonotic IAVs are thought to favor the LRT, and transmission via small droplets allows direct seeding into lower respiratory tract tissues. To gauge the likelihood of reassortment in coinfected hosts, we assessed the extent to which initiation of infection at distinct respiratory tract sites impacts reassortment frequency. Our results reveal that spatially distinct inoculations result in anatomical compartmentalization of infection, which in turn strongly limits reassortment.

Competitive Dominance within Biofilm Consortia Regulates the Relative Distribution of Pneumococcal Nasopharyngeal Density.

Streptococcus pneumoniae is a main cause of child mortality worldwide, but strains also asymptomatically colonize the upper airways of most children and form biofilms. Recent studies have demonstrated that ∼50% of colonized children carry at least two different serotypes (i.e., strains) in the nasopharynx; however, studies of how strains coexist are limited. In this work, we investigated the physiological, genetic, and ecological requirements for the relative distribution of densities, and spatial localization, of pneumococcal strains within biofilm consortia. Biofilm consortia were prepared with vaccine type strains (i.e., serotype 6B [S6B], S19F, or S23F) and strain TIGR4 (S4). Experiments first revealed that the relative densities of S6B and S23F were similar in biofilm consortia. The density of S19F strains, however, was reduced to ∼10% in biofilm consortia, including either S6B, S23F, or TIGR4, in comparison to S19F monostrain biofilms. Reduction of S19F density within biofilm consortia was also observed in a simulated nasopharyngeal environment. Reduction of relative density was not related to growth rates, since the Malthusian parameter demonstrated similar rates of change of density for most strains. To investigate whether quorum sensing (QS) regulates relative densities in biofilm consortia, two different mutants were prepared: a TIGR4ΔluxS mutant and a TIGR4ΔcomC mutant. The density of S19F strains, however, was similarly reduced when consortia included TIGR4, TIGR4ΔluxS, or TIGR4ΔcomC Moreover, production of a different competence-stimulating peptide (CSP), CSP1 or CSP2, was not a factor that affected dominance. Finally, a mathematical model, confocal experiments, and experiments using Transwell devices demonstrated physical contact-mediated control of pneumococcal density within biofilm consortia.IMPORTANCEStreptococcus pneumoniae kills nearly half a million children every year, but it also produces nasopharyngeal biofilm consortia in a proportion of asymptomatic children, and these biofilms often contain two strains (i.e., serotypes). In our study, we investigated how strains coexist within pneumococcal consortia produced by vaccine serotypes S4, S6B, S19F, and S23F. Whereas S6B and S23F shared the biofilm consortium, our studies demonstrated reduction of the relative density of S19F strains, to ∼10% of what it would otherwise be if alone, in consortial biofilms formed with S4, S6B, or S23F. This dominance was not related to increased fitness when competing for nutrients, nor was it regulated by quorum-sensing LuxS/AI-2 or Com systems. It was demonstrated, however, to be enhanced by physical contact rather than by a product(s) secreted into the supernatant, as would naturally occur in the semidry nasopharyngeal environment. Competitive interactions within pneumococcal biofilm consortia regulate nasopharyngeal density, a risk factor for pneumococcal disease.

Human cartilage endplate permeability varies with degeneration and intervertebral disc site.

Despite the critical functions the human cartilage endplate (CEP) plays in the intervertebral disc, little is known about its structural and mechanical properties and their changes with degeneration. Quantifying these changes with degeneration is important for understanding how the CEP contributes to the function and pathology of the disc. Therefore the objectives of this study were to quantify the effect of disc degeneration on human CEP mechanical properties, determine the influence of superior and inferior disc site on mechanics and composition, and simulate the role of collagen fibers in CEP and disc mechanics using a validated finite element model. Confined compression data and biochemical composition data were used in a biphasic-swelling model to calculate compressive extrafibrillar elastic and permeability properties. Tensile properties were obtained by applying published tensile test data to an ellipsoidal fiber distribution. Results showed that with degeneration CEP permeability decreased 50-60% suggesting that transport is inhibited in the degenerate disc. CEP fibers are organized parallel to the vertebrae and nucleus pulposus and may contribute to large shear strains (0.1-0.2) and delamination failure of the CEP commonly seen in herniated disc tissue. Fiber-reinforcement also reduces CEP axial strains thereby enhancing fluid flux by a factor of 1.8. Collectively, these results suggest that the structure and mechanics of the CEP may play critical roles in the solute transport and disc mechanics.

Novel human intervertebral disc strain template to quantify regional three-dimensional strains in a population and compare to internal strains predicted by a finite element model.

Tissue strain is an important indicator of mechanical function, but is difficult to noninvasively measure in the intervertebral disc. The objective of this study was to generate a disc strain template, a 3D average of disc strain, of a group of human L4-L5 discs loaded in axial compression. To do so, magnetic resonance images of uncompressed discs were used to create an average disc shape. Next, the strain tensors were calculated pixel-wise by using a previously developed registration algorithm. Individual disc strain tensor components were then transformed to the template space and averaged to create the disc strain template. The strain template reduced individual variability while highlighting group trends. For example, higher axial and circumferential strains were present in the lateral and posterolateral regions of the disc, which may lead to annular tears. This quantification of group-level trends in local 3D strain is a significant step forward in the study of disc biomechanics. These trends were compared to a finite element model that had been previously validated against the disc-level mechanical response. Depending on the strain component, 81-99% of the regions within the finite element model had calculated strains within one standard deviation of the template strain results. The template creation technique provides a new measurement technique useful for a wide range of studies, including more complex loading conditions, the effect of disc pathologies and degeneration, damage mechanisms, and design and evaluation of treatments. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1264-1273, 2016.

Gammaherpesvirus Co-infection with Malaria Suppresses Anti-parasitic Humoral Immunity.

Immunity to non-cerebral severe malaria is estimated to occur within 1-2 infections in areas of endemic transmission for Plasmodium falciparum. Yet, nearly 20% of infected children die annually as a result of severe malaria. Multiple risk factors are postulated to exacerbate malarial disease, one being co-infections with other pathogens. Children living in Sub-Saharan Africa are seropositive for Epstein Barr Virus (EBV) by the age of 6 months. This timing overlaps with the waning of protective maternal antibodies and susceptibility to primary Plasmodium infection. However, the impact of acute EBV infection on the generation of anti-malarial immunity is unknown. Using well established mouse models of infection, we show here that acute, but not latent murine gammaherpesvirus 68 (MHV68) infection suppresses the anti-malarial humoral response to a secondary malaria infection. Importantly, this resulted in the transformation of a non-lethal P. yoelii XNL infection into a lethal one; an outcome that is correlated with a defect in the maintenance of germinal center B cells and T follicular helper (Tfh) cells in the spleen. Furthermore, we have identified the MHV68 M2 protein as an important virus encoded protein that can: (i) suppress anti-MHV68 humoral responses during acute MHV68 infection; and (ii) plays a critical role in the observed suppression of anti-malarial humoral responses in the setting of co-infection. Notably, co-infection with an M2-null mutant MHV68 eliminates lethality of P. yoelii XNL. Collectively, our data demonstrates that an acute gammaherpesvirus infection can negatively impact the development of an anti-malarial immune response. This suggests that acute infection with EBV should be investigated as a risk factor for non-cerebral severe malaria in young children living in areas endemic for Plasmodium transmission.

Validation and application of an intervertebral disc finite element model utilizing independently constructed tissue-level constitutive formulations that are nonlinear, anisotropic, and time-dependent.

Finite element (FE) models are advantageous in the study of intervertebral disc mechanics as the stress-strain distributions can be determined throughout the tissue and the applied loading and material properties can be controlled and modified. However, the complicated nature of the disc presents a challenge in developing an accurate and predictive disc model, which has led to limitations in FE geometry, material constitutive models and properties, and model validation. The objective of this study was to develop a new FE model of the intervertebral disc, to validate the model's nonlinear and time-dependent responses without tuning or calibration, and to evaluate the effect of changes in nucleus pulposus (NP), cartilaginous endplate (CEP), and annulus fibrosus (AF) material properties on the disc mechanical response. The new FE disc model utilized an analytically-based geometry. The model was created from the mean shape of human L4/L5 discs, measured from high-resolution 3D MR images and averaged using signed distance functions. Structural hyperelastic constitutive models were used in conjunction with biphasic-swelling theory to obtain material properties from recent tissue tests in confined compression and uniaxial tension. The FE disc model predictions fit within the experimental range (mean ± 95% confidence interval) of the disc's nonlinear response for compressive slow loading ramp, creep, and stress-relaxation simulations. Changes in NP and CEP properties affected the neutral-zone displacement but had little effect on the final stiffness during slow-ramp compression loading. These results highlight the need to validate FE models using the disc's full nonlinear response in multiple loading scenarios.

Human L3L4 intervertebral disc mean 3D shape, modes of variation, and their relationship to degeneration.

Intervertebral disc mechanics are affected by both disc shape and disc degeneration, which in turn each affect the other; disc mechanics additionally have a role in the etiology of disc degeneration. Finite element analysis (FEA) is a favored tool to investigate these relationships, but limited data for intervertebral disc 3D shape has forced the use of simplified or single-subject geometries, with the effect of inter-individual shape variation investigated only in specialized studies. Similarly, most data on disc shape variation with degeneration is based on 2D mid-sagittal images, which incompletely define 3D shape changes. Therefore, the objective of this study was to quantify inter-individual disc shape variation in 3D, classify this variation into independently-occurring modes using a statistical shape model, and identify correlations between disc shape and degeneration. Three-dimensional disc shapes were obtained from MRI of 13 human male cadaver L3L4 discs. An average disc shape and four major modes of shape variation (representing 90% of the variance) were identified. The first mode represented disc axial area and was significantly correlated to degeneration (R(2)=0.44), indicating larger axial area in degenerate discs. Disc height variation occurred in three distinct modes, each also involving non-height variation. The statistical shape model provides an average L3L4 disc shape for FEA that is fully defined in 3D, and makes it convenient to generate a set of shapes with which to represent aggregate inter-individual variation. Degeneration grade-specific shapes can also be generated. To facilitate application, the model is included in this paper׳s supplemental content.

Different effects of whole-cell and acellular vaccines on Bordetella transmission.

BACKGROUND: Vaccine development has largely focused on the ability of vaccines to reduce disease in individual hosts, with less attention to assessing the vaccine's effects on transmission between hosts. Current acellular vaccines against Bordetella pertussis are effective in preventing severe disease but have little effect on less severe coughing illness that can mediate transmission. METHODS: Using mice that are natural host's of Bordetella bronchiseptica, we determined the effects of vaccination on shedding and transmission of this pathogen. RESULTS: Vaccination with heat-killed whole-cell B. bronchiseptica or B. pertussis inhibited shedding of B. bronchiseptica. Differences in neutrophil and B-cell recruitment distinguished sham-vaccine from whole-cell-----vaccine responses and correlated with shedding output. Both B and T cells were essential for vaccine-induced control of shedding. Adoptive transfer of antibodies was able to limit shedding, while depletion of CD4(+) T cells led to increased shedding in vaccinated mice. Finally, whole-cell vaccination was able to prevent transmission, but an acellular vaccine that effectively controls disease failed to control shedding and transmission. CONCLUSIONS: Our results highlight discrepancies between whole-cell and acellular vaccination that could contribute to the increased incidence of B. pertussis infection since the transition to the use of acellular vaccination.

Elastic, permeability and swelling properties of human intervertebral disc tissues: A benchmark for tissue engineering.

The aim of functional tissue engineering is to repair and replace tissues that have a biomechanical function, i.e., connective orthopaedic tissues. To do this, it is necessary to have accurate benchmarks for the elastic, permeability, and swelling (i.e., biphasic-swelling) properties of native tissues. However, in the case of the intervertebral disc, the biphasic-swelling properties of individual tissues reported in the literature exhibit great variation and even span several orders of magnitude. This variation is probably caused by differences in the testing protocols and the constitutive models used to analyze the data. Therefore, the objective of this study was to measure the human lumbar disc annulus fibrosus (AF), nucleus pulposus (NP), and cartilaginous endplates (CEP) biphasic-swelling properties using a consistent experimental protocol and analyses. The testing protocol was composed of a swelling period followed by multiple confined compression ramps. To analyze the confined compression data, the tissues were modeled using a biphasic-swelling model, which augments the standard biphasic model through the addition of a deformation-dependent osmotic pressure term. This model allows considering the swelling deformations and the contribution of osmotic pressure in the analysis of the experimental data. The swelling stretch was not different between the disc regions (AF: 1.28±0.16; NP: 1.73±0.74; CEP: 1.29±0.26), with a total average of 1.42. The aggregate modulus (Ha) of the extra-fibrillar matrix was higher in the CEP (390kPa) compared to the NP (100kPa) or AF (30kPa). The permeability was very different across tissue regions, with the AF permeability (64 E(-16)m(4)/Ns) higher than the NP and CEP (~5.5 E(-16)m(4)/Ns). Additionally, a normalized time-constant (3000s) for the stress relaxation was similar for all the disc tissues. The properties measured in this study are important as benchmarks for tissue engineering and for modeling the disc's mechanical behavior and transport.

Biaxial tension of fibrous tissue: using finite element methods to address experimental challenges arising from boundary conditions and anisotropy.

Planar biaxial tension remains a critical loading modality for fibrous soft tissue and is widely used to characterize tissue mechanical response, evaluate treatments, develop constitutive formulas, and obtain material properties for use in finite element studies. Although the application of tension on all edges of the test specimen represents the in situ environment, there remains a need to address the interpretation of experimental results. Unlike uniaxial tension, in biaxial tension the applied forces at the loading clamps do not transmit fully to the region of interest (ROI), which may lead to improper material characterization if not accounted for. In this study, we reviewed the tensile biaxial literature over the last ten years, noting experimental and analysis challenges. In response to these challenges, we used finite element simulations to quantify load transmission from the clamps to the ROI in biaxial tension and to formulate a correction factor that can be used to determine ROI stresses. Additionally, the impact of sample geometry, material anisotropy, and tissue orientation on the correction factor were determined. Large stress concentrations were evident in both square and cruciform geometries and for all levels of anisotropy. In general, stress concentrations were greater for the square geometry than the cruciform geometry. For both square and cruciform geometries, materials with fibers aligned parallel to the loading axes reduced stress concentrations compared to the isotropic tissue, resulting in more of the applied load being transferred to the ROI. In contrast, fiber-reinforced specimens oriented such that the fibers aligned at an angle to the loading axes produced very large stress concentrations across the clamps and shielding in the ROI. A correction factor technique was introduced that can be used to calculate the stresses in the ROI from the measured experimental loads at the clamps. Application of a correction factor to experimental biaxial results may lead to more accurate representation of the mechanical response of fibrous soft tissue.

Multi-scale structural and tensile mechanical response of annulus fibrosus to osmotic loading.

This study investigates differential multi-scale structure and function relationships of the outer and inner annulus fibrosus (AF) to osmotic swelling in different buffer solutions by quantifying tensile mechanics, glycoasamino-glycan(GAG) content, water content and tissue swelling, and collagen fibril ultrastructure. In the outer AF, the tensile modulus decreased by over 70% with 0.15 M PBS treatment but was unchanged with 2 M PBS treatment. Moreover, the modulus loss following 0.15 M PBS treatment was reversed when followed by 2 M PBS treatment, potentially from increased interfibrillar and interlamellar shearing associated with fibril swelling. In contrast, the inner AF tensile modulus was unchanged by 0.15 M PBS treatment and increased following 2 M treatment. Transmission electron microscopy revealed that the mean collagen fibril diameters of the untreated outer and inner AF were 87.8 ± 27.9 and 71.0 ± 26.9 nm, respectively. In the outer AF, collagen fibril swelling was observed with both 0.15 M and 2 M PBS treatments, but inherently low GAG content remained unchanged. In the inner AF, 2 M PBS treatment caused fibril swelling and GAG loss, suggesting that GAG plays a role in maintaining the structure of collagen fibrils leading to modulation of the native tissue mechanical properties. These results demonstrate important regional variations in structure and composition, and their influence on the heterogeneous mechanics of the AF. Moreover, because the composition and structure is altered as a consequence of progressive disk degeneration, quantification of these interactions is critical for study of the AF pathogenesis of degeneration and tissue engineering

Human Annulus Fibrosus Dynamic Tensile Modulus Increases with Degeneration.

The annulus fibrosus (AF) of the intervertebral disc experiences cyclic tensile loading in vivo at various states of mechanical equilibrium. Disc degeneration is associated with alterations in the biochemical composition of the AF including decreased water content, decreased proteoglycan concentration, and increased collagen deposition that affect mechanical function of the AF in compression and shear. Such changes may also affect the dynamic viscoelastic properties of the AF and thus alter the disc's ability to dissipate energy under physiologic loading. The objectives of this study were to quantify the dynamic viscoelastic properties of human AF in circumferential tension and to determine the effect of degeneration on these properties. Nondegenerated and degenerated human AF tensile samples were tested in uniaxial tension over a spectrum of loading frequencies spanning 0.01Hz to 2Hz at several states of equilibrium strain to determine the dynamic viscoelastic properties (dynamic modulus, phase angle) using a linear viscoelastic model. The AF dynamic modulus increased at higher equilibrium strain levels. The AF behaved more elastically at higher frequencies with a decreased phase angle. Degeneration resulted in a higher dynamic modulus at all strain levels but had no effect on phase angle. The findings from this study elucidate the effect of degeneration on the dynamic viscoelastic properties of human AF and lend insight into the mechanical role of the AF in cyclic loading conditions.

Effect of orientation and targeted extracellular matrix degradation on the shear mechanical properties of the annulus fibrosus.

The intervertebral disc experiences combinations of compression, torsion, and bending that subject the disc substructures, particularly the annulus fibrosus (AF), to multidirectional loads and deformations. Combined tensile and shear loading is a particularly important loading paradigm, as compressive loads place the AF in circumferential hoop tension, and spine torsion or bending induces AF shear. Yet the anisotropy of AF mechanical properties in shear, as well as important structure-function mechanisms governing this response, are not well-understood. The objective of this study, therefore, was to investigate the effects of tissue orientation and enzymatic degradation of glycosaminoglycan (GAG) and elastin on AF shear mechanical properties. Significant anisotropy was found: the circumferential shear modulus, Gθz, was an order of magnitude greater than the radial shear modulus, Grθ. In the circumferential direction, prestrain significantly increased the shear modulus, suggesting an important role for collagen fiber stretch in shear properties for this orientation. While not significant and highly variable, ChABC treatment to remove GAG increased the circumferential shear modulus compared to PBS control (p=0.15). Together with the established literature for tensile loading of fiber-reinforced GAG-rich tissues, the trends for changes in shear modulus with ChABC treatment reflect complex, structure-function relationships between GAG and collagen that potentially occur over several hierarchical scales. Elastase digestion did not significantly affect shear modulus with respect to PBS control; further contributing to the notion that circumferential shear modulus is dominated by collagen fiber stretch. The results of this study highlight the complexity of the structure-function relationships that govern the mechanical response of the AF in radial and circumferential shear, and provide new and more accurate data for the validation of material models and tissue-engineered disc replacements.

Fiber angle and aspect ratio influence the shear mechanics of oriented electrospun nanofibrous scaffolds.

Fibrocartilages, including the knee meniscus and the annulus fibrosus (AF) of the intervertebral disc, play critical mechanical roles in load transmission across joints and their function is dependent upon well-defined structural hierarchies, organization, and composition. All, however, are compromised in the pathologic transformations associated with tissue degeneration. Tissue engineering strategies that address these key features, for example, aligned nanofibrous scaffolds seeded with mesenchymal stem cells (MSCs), represent a promising approach for the regeneration of these fibrous structures. While such engineered constructs can replicate native tissue structure and uniaxial tensile properties, the multidirectional loading encountered by these tissues in vivo necessitates that they function adequately in other loading modalities as well, including shear. As previous findings have shown that native tissue tensile and shear properties are dependent on fiber angle and sample aspect ratio, respectively, the objective of the present study was to evaluate the effects of a changing fiber angle and sample aspect ratio on the shear properties of aligned electrospun poly(ε-caprolactone) (PCL) scaffolds, and to determine how extracellular matrix deposition by resident MSCs modulates the measured shear response. Results show that fiber orientation and sample aspect ratio significantly influence the response of scaffolds in shear, and that measured shear strains can be predicted by finite element models. Furthermore, acellular PCL scaffolds possessed a relatively high shear modulus, 2-4 fold greater than native tissue, independent of fiber angle and aspect ratio. It was further noted that under testing conditions that engendered significant fiber stretch, the aggregate resistance to shear was higher, indicating a role for fiber stretch in the overall shear response. Finally, with time in culture, the shear modulus of MSC laden constructs increased, suggesting that deposited ECM contributes to the construct shear properties. Collectively, these findings show that aligned electrospun PCL scaffolds are a promising tool for engineering fibrocartilage tissues, and that the shear properties of both acellular and cell-seeded formulations can match or exceed native tissue benchmarks.

Axial creep loading and unloaded recovery of the human intervertebral disc and the effect of degeneration.

The intervertebral disc maintains a balance between externally applied loads and internal osmotic pressure. Fluid flow plays a key role in this process, causing fluctuations in disc hydration and height. The objectives of this study were to quantify and model the axial creep and recovery responses of nondegenerate and degenerate human lumbar discs. Two experiments were performed. First, a slow compressive ramp was applied to 2000 N, unloaded to allow recovery for up to 24 h, and re-applied. The linear-region stiffness and disc height were within 5% of the initial condition for recovery times greater than 8 h. In the second experiment, a 1000 N creep load was applied for four hours, unloaded recovery monitored for 24 h, and the creep load repeated. A viscoelastic model comprised of a "fast" and "slow" exponential response was used to describe the creep and recovery, where the fast response is associated with flow in the nucleus pulposus (NP) and endplate, while the slow response is associated with the annulus fibrosus (AF). The study demonstrated that recovery is 3-4X slower than loading. The fast response was correlated with degeneration, suggesting larger changes in the NP with degeneration compared to the AF. However, the fast response comprised only 10%-15% of the total equilibrium displacement, with the AF-dominated slow response comprising 40%-70%. Finally, the physiological loads and deformations and their associated long equilibrium times confirm that diurnal loading does not represent "equilibrium" in the disc, but that over time the disc is in steady-state.