Strain inhibition of bacterial collagenase is consistent with a collagen fibril uncrimping mechanism in rat tail tendons.

Mechanical strain inhibits bacterial collagenase from cleaving collagen. Additionally, the toe region of a soft tissue's force-elongation curve arises from sequentially engaging collagen fibrils as the tissue lengthens. Together, these phenomena suggest that mechanical strain may gradually inhibit collagenase activity through a soft tissue's toe region. Therefore, this investigation sought to test this hypothesis. 92 rat tail tendon fascicles from 3 female sentinel animals underwent preliminary stiffness tests, and their force-elongation curves were fit to a collagen distribution model. This distribution-based model calculated the force magnitude corresponding to p% of collagen fibril engagement. Specimens were separated into one of five levels of p, and that level of force was maintained for two hours while being exposed to 0.054 U/mL of bacterial collagenase from C. histolyticum. The specimens were strained to failure following the creep test, and the relative reduction in stiffness was quantified to estimate the fraction of digested fibrils. Every 10% additional collagen engagement corresponded to a 6.3% (97% highest density interval: 4.3 - 8.4%) retention of stiffness, which indicated collagenase inhibition. The results of this investigation were consistent with a strain-inhibition hypothesis along with the established uncrimping mechanism in the toe region. These results support an interaction between mechanical strain and collagenolysis, which may be valuable for disease prevention or treatment.

Correlation between Mechanical Properties and Collagen Degeneration in Fibrous Tissue.

Fibrosis is a disease that causes abnormal accumulation of collagen and other extracellular matrix components. It can lead to organ failure and is responsible for one-third of all deaths worldwide. However, there is no cure for this disease, and the development of minimally invasive therapies is urgently needed. We have previously reported techniques for adjusting the shape and flexibility of fibrous tissue by traction while denaturing it with heat. However, studies comparing heat and traction on fibrous tissue are limited, so this paper examined that. Applying heat and traction to bovine Achilles tendon tissue has been shown to cause the denaturation of collagen molecules to accumulate in the tissue in response to these loads. Heat-induced collagen denaturation was nondirectional and omnidirectional, whereas mechanical stress-induced collagen denaturation was concentrated in the direction of traction. When both heat and traction were applied, collagen denaturation increased more than under a single load, indicating a synergistic effect.

A multiscale investigation into the role of collagen-hyaluronan interface shear on the mechanical behaviour of collagen fibers in annulus fibrosus - Molecular dynamics-cohesive finite element-based study.

Multi-directional deformation exhibited by annulus fibrosus (AF) is contributed by chemo-mechanical interactions among its biomolecular constituents' collagen type I (COL-I), collagen type II (COL-II), proteoglycans (aggrecan and hyaluronan) and water. However, the nature and role of such interactions on AF mechanics are unclear. This work employs a molecular dynamics-cohesive finite element-based multiscale approach to investigate role of COL-I-COL-II interchanging distribution and water concentration (WC) variations from outer annulus (OA) to inner annulus (IA) on collagen-hyaluronan (COL-HYL) interface shear, and the mechanisms by which interface shear impacts fibril sliding during collagen fiber deformation. At first, COL-HYL interface atomistic models are constructed by interchanging COL-I with COL-II and increasing COL-II and WC from 0 to 75%, and 65%-75% respectively. Thereafter, a multiscale approach is employed to develop representative volume elements (RVEs) of collagen fibers by incorporating COL-HYL shear as traction-separation behaviour at fibril-hyaluronan contact. Results show that increasing COL-II and WC increases interface stiffness from 0.6 GPa/nm to 1.2 GPa/nm and reduces interface strength from 155 MPa to 58 MPa from OA to IA, contributed by local hydration alterations. A stiffer and weaker interface enhances fibril sliding with increased straining at the contact - thereby contributing to reduction in modulus from 298 MPa to 198 MPa from OA to IA. Such reduction further contributes to softer mechanical response towards IA, as reported by earlier studies. Presented multiscale analysis provides deeper understanding of hierarchical structure-mechanics relationships in AF and can further aid in developing better substitutes for AF repair.

Characterization of the L4/L5 rat facet capsular ligament macromechanical and microstructural responses to tensile failure loading.

Low back pain is a prevalent condition that affects the global population. The lumbar facet capsular ligament is a source of pain since the collagenous tissue of the ligament is innervated with sensory neurons that deform with the capsule's stretch. Regional differences in the microstructural and macrostructural anatomy of the spinal facets affect its capsule's mechanical behavior. Although there are many studies of the cervical facet in human and rodent models, the lumbar capsular ligament's multiscale behavior is less well-defined. This study characterizes the macroscale and fiber-scale changes of the rat lumbar facet capsule during tensile failure loading. An integrated polarized light imaging setup captured local fiber alignment during 0.08 mm/s distraction of 7 lumbar facets. Force, displacement, strain, and circular variance were measured at several points along the failure curve: the first instance when the local collagen fiber network realigns differentially (anomalous realignment), yield, the first peak in force corresponding to the capsule's first failure, and peak force, defined as ultimate rupture. Those outcomes were compared across events. While each of force, displacement, and average maximum principal strain increased with applied tension, so did the circular variance of the collagen, suggesting that the fibers were becoming more disorganized. From the fiber alignment maps collected at each mechanical event, the number of anomalous realignment events were counted and found to increase dramatically with loading. The increased collagen disorganization and increasing regions of such disorganization in the facet capsule during loading can provide insights about how loading to the ligament afferent nerves may be activated and thereby produce pain.

A biochemomechanical model of collagen turnover in arterial adaptations to hemodynamic loading.

The production, removal, and remodeling of fibrillar collagen is fundamental to mechanical homeostasis in arteries, including dynamic morphological and microstructural changes that occur in response to sustained changes in blood flow and pressure under physiological conditions. These dynamic processes involve complex, coupled biological, chemical, and mechanical mechanisms that are not completely understood. Nevertheless, recent simulations using constrained mixture models with phenomenologically motivated constitutive relations have proven able to predict salient features of the progression of certain vascular adaptations as well as disease processes. Collagen turnover is modeled, in part, via stress-dependent changes in collagen half-life, typically within the range of 10-70 days. By contrast, in this work we introduce a biochemomechanical approach to model the cellular synthesis of procollagen as well as its transition from an intermediate state of assembled microfibrils to mature cross-linked fibers, with mechano-regulated removal. The resulting model can simulate temporal changes in geometry, composition, and stress during early vascular adaptation (weeks to months) for modest changes in blood flow or pressure. It is shown that these simulations capture salient features from data presented in the literature from different animal models.

Structure-Function relationships in the skeletal muscle extracellular matrix.

The vast majority of skeletal muscle biomechanical studies have rightly focused on its active contractile properties. However, skeletal muscle passive biomechanical properties have significant clinical impact in aging and disease and are yet incompletely understood. This review focuses on the passive biomechanical properties of the skeletal muscle extracellular matrix (ECM) and suggests aspects of its structural basis. Structural features of the muscle ECM such as perimysial cables, collagen cross-links and endomysial structures have been described, but the way in which these structures combine to create passive biomechanical properties is not completely known. We highlight the presence and organization of perimysial cables. We also demonstrate that the analytical approaches that define passive biomechanical properties are not necessarily straight forward. For example, multiple equations, such as linear, exponential, and polynomial are commonly used to fit raw stress-strain data. Similarly, multiple definitions of zero strain exist that affect muscle biomechanical property calculations. Finally, the appropriate length range over which to measure the mechanical properties is not clear. Overall, this review summarizes our current state of knowledge in these areas and suggests experimental approaches to measuring the structural and functional properties of skeletal muscle.

Mouse Achilles tendons exhibit collagen disorganization but minimal collagen denaturation during cyclic loading to failure.

While overuse is a prominent risk factor for tendinopathy, the fatigue-induced structural damage responsible for initiating tendon degeneration remains unclear. Denaturation of collagen molecules and collagen fiber disorganization have been observed within certain tendons in response to fatigue loading. However, no studies have investigated whether these forms of tissue damage occur in Achilles tendons, which commonly exhibit tendinopathy. Therefore, the objective of this study was to determine whether mouse Achilles tendons undergo collagen denaturation and collagen fiber disorganization when cyclically loaded to failure. Consistent with previous testing of other energy-storing tendons, we found that cyclic loading of mouse Achilles tendons produced collagen disorganization but minimal collagen denaturation. To determine whether the lack of collagen denaturation is unique to mouse Achilles tendons, we monotonically loaded the Achilles and other mouse tendons to failure. We found that the patellar tendon was also resistant to collagen denaturation, but the flexor digitorum longus (FDL) tendon and tail tendon fascicles were not. Furthermore, the Achilles and patellar tendons had a lower tensile strength and modulus. While this may be due to differences in tissue structure, it is likely that the lack of collagen denaturation during monotonic loading in both the Achilles and patellar tendons was due to failure near their bony insertions, which were absent in the FDL and tail tendons. These findings suggest that mouse Achilles tendons are resistant to collagen denaturation in situ and that Achilles tendon degeneration may not be initiated by mechanically-induced damage to collagen molecules.

Nonlinear time-dependent mechanical behavior of mammalian collagen fibrils.

The viscoelastic mechanical behavior of collagenous tissues has been studied extensively at the macroscale, yet a thorough quantitative understanding of the time-dependent mechanics of the basic building blocks of tissues, the collagen fibrils, is still missing. In order to address this knowledge gap, stress relaxation and creep tests at various stress (5-35 MPa) and strain (5-20%) levels were performed with individual collagen fibrils (average diameter of fully hydrated fibrils: 253 ± 21 nm) in phosphate buffered saline (PBS). The experimental results showed that the time-dependent mechanical behavior of fully hydrated individual collagen fibrils reconstituted from Type I calf skin collagen, is described by strain-dependent stress relaxation and stress-dependent creep functions in both the heel-toe and the linear regimes of deformation in monotonic stress-strain curves. The adaptive quasilinear viscoelastic (QLV) model, originally developed to capture the nonlinear viscoelastic response of collagenous tissues, provided a very good description of the nonlinear stress relaxation and creep behavior of the collagen fibrils. On the other hand, the nonlinear superposition (NSP) model fitted well the creep but not the stress relaxation data. The time constants and rates extracted from the adaptive QLV and the NSP models, respectively, pointed to a faster rate for stress relaxation than creep. This nonlinear viscoelastic behavior of individual collagen fibrils agrees with prior studies of macroscale collagenous tissues, thus demonstrating consistent time-dependent behavior across length scales and tissue hierarchies. STATEMENT OF SIGNIFICANCE: Pure stress relaxation and creep experiments were conducted for the first time with fully hydrated individual collagen fibrils. It is shown that collagen nanofibrils have a nonlinear time-dependent behavior which agrees with prior studies on macroscale collagenous tissues, thus demonstrating consistent time-dependent behavior across length scales and tissue hierarchies. This new insight into the non-linear viscoelastic behavior of the building blocks of mammalian collagenous tissues may serve as the foundation for improved macroscale tissue models that capture the mechanical behavior across length scales.

Larger interface area at the human myotendinous junction in type 1 compared with type 2 muscle fibers.

The myotendinous junction (MTJ) is structurally specialized to transmit force. The highly folded muscle membrane at the MTJ increases the contact area between muscle and tendon and potentially the load tolerance of the MTJ. Muscles with a high content of type II fibers are more often subject to strain injury compared with muscles with type I fibers. It is hypothesized that this is explained by a smaller interface area of MTJ in type II compared with type I muscle fibers. The aim was to investigate by confocal microscopy whether there is difference in the surface area at the MTJ between type I and II muscle fibers. Individual muscle fibers with an intact MTJ were isolated by microscopic dissection in samples from human semitendinosus, and they were labeled with antibodies against collagen XXII (indicating MTJ) and type I myosin (MHCI). Using a spinning disc confocal microscope, the MTJ from each fiber was scanned and subsequently reconstructed to a 3D-model. The interface area between muscle and tendon was calculated in type I and II fibers from these reconstructions. The MTJ was analyzed in 314 muscle fibers. Type I muscle fibers had a 22% larger MTJ interface area compared with type II fibers (p < 0.05), also when the area was normalized to fiber diameter. By the new method, it was possible to analyze the structure of the MTJ from a large number of human muscle fibers. The finding that the interface area between muscle and tendon is higher in type I compared with type II fibers suggests that type II fibers are less resistant to strain and therefore more susceptible to injury.

Intramuscular connective tissue content and mechanical properties: Influence of aging and physical activity in mice.

Aging is accompanied by morphological and mechanical changes to the intramuscular connective tissue (IMCT) of skeletal muscles, but whether physical exercise can influence these changes is debated. We investigated the effects of aging and exercise with high or low resistance on composition and mechanical properties of the IMCT, including direct measurements on isolated IMCT which has rarely been reported. Middle-aged (11 months, n = 24) and old (22 months, n = 18) C57BL/6 mice completed either high (HR) or low (LR) resistance voluntary wheel running or were sedentary (SED) for 10 weeks. Passive mechanical properties of the intact soleus and plantaris muscles and the isolated IMCT of the plantaris muscle were measured in vitro. IMCT thickness was measured on picrosirius red stained cross sections of the gastrocnemius and soleus muscle and for the gastrocnemius hydroxyproline content was quantified biochemically and advanced glycation end-products (AGEs) estimated by fluorometry. Mechanical stiffness, IMCT content and total AGEs were all elevated with aging in agreement with previous findings but were largely unaffected by training. Conclusion: IMCT accumulated with aging with a proportional increase in mechanical stiffness, but even the relatively high exercise volume achieved with voluntary wheel-running with or without resistance did not significantly influence these changes.

Editorial Commentary: Allogenic Dermal Fibroblasts in Collagen Matrix Scaffold Enhance Rotator Cuff Repair in an Animal Model.

There has been a recent surge of interest on the use of biologic supplements to facilitate rotator cuff repair healing. Experimental evidence appears to support use of allogenic dermal fibroblasts (ADFs), either in the form of local injection or tenocytes embedded in collagen matrix scaffold, to enhance healing of a repaired rotator cuff tendon tear in an animal model. When compared with the ADFs, the platelet-rich plasma (PRP)-induced response seems to be limited in terms of the specific increases in local collagen 1 concentration, thus resulting in a bone-tendon healing response that is inferior in both biology and biomechanical behavior under the same laboratory conditions. While on the one hand, there is pilot data supporting use of dermal fibroblast in the clinical setting, thus reinforcing the animal study findings, on the other hand, we are also aware of the encouraging biologic changes that occurred in the retrieved acellular dermal matrix (ADM) allograft that was used for superior capsular reconstruction as a treatment of irreparable rotator cuff tears. In theory, ADFs locally instilled as an injection should further enhance the healing response compared to the ADM. However, this needs to be further studied to be able to be widely applicable clinically.

Autophagy guards tendon homeostasis.

Tendons are vital collagen-dense specialized connective tissues transducing the force from skeletal muscle to the bone, thus enabling movement of the human body. Tendon cells adjust matrix turnover in response to physiological tissue loading and pathological overloading (tendinopathy). Nevertheless, the regulation of tendon matrix quality control is still poorly understood and the pathogenesis of tendinopathy is presently unsolved. Autophagy, the major mechanism of degradation and recycling of cellular components, plays a fundamental role in the homeostasis of several tissues. Here, we investigate the contribution of autophagy to human tendons' physiology, and we provide in vivo evidence that it is an active process in human tendon tissue. We show that selective autophagy of the endoplasmic reticulum (ER-phagy), regulates the secretion of type I procollagen (PC1), the major component of tendon extracellular matrix. Pharmacological activation of autophagy by inhibition of mTOR pathway alters the ultrastructural morphology of three-dimensional tissue-engineered tendons, shifting collagen fibrils size distribution. Moreover, autophagy induction negatively affects the biomechanical properties of the tissue-engineered tendons, causing a reduction in mechanical strength under tensile force. Overall, our results provide the first evidence that autophagy regulates tendon homeostasis by controlling PC1 quality control, thus potentially playing a role in the development of injured tendons.

Control of stem cell differentiation by using extrinsic photobiomodulation in conjunction with cell adhesion pattern.

The induction and direction of stem cell differentiation into needed cell phenotypes is the central pillar of tissue engineering for repairing damaged tissues or organs. Conventionally, a special recipe of chemical factors is formulated to achieve this purpose for each specific target cell type. In this work, it is demonstrated that the combination of extrinsic photobiomodulation and collagen-covered microislands could be used to induce differentiation of Wharton's jelly mesenchymal stem cells (WJ-MSCs) with the differentiation direction dictated by the specific island topography without use of chemical factors. Both neurogenic differentiation and adipogenic differentiation could be attained with a rate surpassing that using chemical factors. Application of this method to other cell types is possible by utilizing microislands with a pattern tailored particularly for each specific cell type, rendering it a versatile modality for initiating and guiding stem cell differentiation.

Pseudohypoxic HIF pathway activation dysregulates collagen structure-function in human lung fibrosis.

Extracellular matrix (ECM) stiffening with downstream activation of mechanosensitive pathways is strongly implicated in fibrosis. We previously reported that altered collagen nanoarchitecture is a key determinant of pathogenetic ECM structure-function in human fibrosis (Jones et al., 2018). Here, through human tissue, bioinformatic and ex vivo studies we provide evidence that hypoxia-inducible factor (HIF) pathway activation is a critical pathway for this process regardless of the oxygen status (pseudohypoxia). Whilst TGFß increased the rate of fibrillar collagen synthesis, HIF pathway activation was required to dysregulate post-translational modification of fibrillar collagen, promoting pyridinoline cross-linking, altering collagen nanostructure, and increasing tissue stiffness. In vitro, knockdown of Factor Inhibiting HIF (FIH), which modulates HIF activity, or oxidative stress caused pseudohypoxic HIF activation in the normal fibroblasts. By contrast, endogenous FIH activity was reduced in fibroblasts from patients with lung fibrosis in association with significantly increased normoxic HIF pathway activation. In human lung fibrosis tissue, HIF-mediated signalling was increased at sites of active fibrogenesis whilst subpopulations of human lung fibrosis mesenchymal cells had increases in both HIF and oxidative stress scores. Our data demonstrate that oxidative stress can drive pseudohypoxic HIF pathway activation which is a critical regulator of pathogenetic collagen structure-function in fibrosis.

Biomechanics of mitral valve leaflets: Second harmonic generation microscopy, biaxial mechanical tests and tissue modeling.

Collagen fibers are the main load carrier in the mitral valve (MV) leaflets. Their orientation and dispersion are an important factor for the mechanical behavior. Most recent studies of collagen fibers in MVs lack either entire thickness study or high transmural resolution. The present study uses second harmonic generation (SHG) microscopy in combination with planar biaxial mechanical tests to better model and examine collagen fibers and mechanical properties of MV leaflets. SHG in combination with tissue clearing enables the collagen fibers to be examined through the entire thickness of the MV leaflets. Planar biaxial mechanical tests, on the other hand, enable the characterization of the mechanical tissue behavior, which is represented by a structural tissue model. Twelve porcine MV leaflets are examined. The SHG recording shows that the mean fiber angle for all samples varies on average by ±12° over the entire thickness and the collagen fiber dispersion changes strongly over the thickness. A constitutive model based on the generalized structure tensor approach is used for the associated tissue characterization. The model represents the tissue with three mechanical parameters plus the mean fiber direction and the dispersion, and predicts the biomechanical response of the leaflets with a good agreement (average r2=0.94). It is found that the collagen structure can be represented by a mean direction and a dispersion with a single family of fibers despite the variation in the collagen fiber direction and the dispersion over the entire thickness of MV leaflets. STATEMENT OF SIGNIFICANCE: Despite its prominent role in the mechanical behavior of mitral valve (MV) leaflets, the collagen structure has not yet been investigated over the entire thickness with high transmural resolution. The present study quantifies the detailed through thickness collagen fiber structure and examines the effects of its variation on MV tissue modeling. This is important because the study evaluates the assumption that the collagen fibers can be modeled with a representative single fiber family despite the variation across the thickness. In addition, the current comprehensive data set paves the way for quantifying the disruption of collagen fibers in myxomatous MV leaflets associated with disrupted collagen fibers.

Collagen polarization promotes epithelial elongation by stimulating locoregional cell proliferation.

Epithelial networks are commonly generated by processes where multicellular aggregates elongate and branch. Here, we focus on understanding cellular mechanisms for elongation using an organotypic culture system as a model of mammary epithelial anlage. Isotropic cell aggregates broke symmetry and slowly elongated when transplanted into collagen 1 gels. The elongating regions of aggregates displayed enhanced cell proliferation that was necessary for elongation to occur. Strikingly, this locoregional increase in cell proliferation occurred where collagen 1 fibrils reorganized into bundles that were polarized with the elongating aggregates. Applying external stretch as a cell-independent way to reorganize the extracellular matrix, we found that collagen polarization stimulated regional cell proliferation to precipitate symmetry breaking and elongation. This required ß1-integrin and ERK signaling. We propose that collagen polarization supports epithelial anlagen elongation by stimulating locoregional cell proliferation. This could provide a long-lasting structural memory of the initial axis that is generated when anlage break symmetry.

Collagen XIV Is an Intrinsic Regulator of Corneal Stromal Structure and Function.

Collagen XIV is poorly characterized in the body, and the current knowledge of its function in the cornea is limited. The aim of the current study was to elucidate the role(s) of collagen XIV in regulating corneal stromal structure and function. Analysis of collagen XIV expression, temporal and spatial, was performed at different postnatal days (Ps) in wild-type C57BL/6 mouse corneal stromas and after injury. Conventional collagen XIV null mice were used to inquire the roles that collagen XIV plays in fibrillogenesis, fibril packing, and tissue mechanics. Fibril assembly and packing as well as stromal organization were evaluated using transmission electron microscopy and second harmonic generation microscopy. Atomic force microscopy was used to assess stromal stiffness. Col14a1 mRNA expression was present at P4 to P10 and decreased at P30. No immunoreactivity was noted at P150. Abnormal collagen fibril assembly with a shift toward larger-diameter fibrils and increased interfibrillar spacing in the absence of collagen XIV was found. Second harmonic generation microscopy showed impaired fibrillogenesis in the collagen XIV null stroma. Mechanical testing suggested that collagen XIV confers stiffness to stromal tissue. Expression of collagen XIV is up-regulated following injury. This study indicates that collagen XIV plays a regulatory role in corneal development and in the function of the adult cornea. The expression of collagen XIV is recapitulated during wound healing.

Mechanical plasticity of collagen directs branch elongation in human mammary gland organoids.

Epithelial branch elongation is a central developmental process during branching morphogenesis in diverse organs. This fundamental growth process into large arborized epithelial networks is accompanied by structural reorganization of the surrounding extracellular matrix (ECM), well beyond its mechanical linear response regime. Here, we report that epithelial ductal elongation within human mammary organoid branches relies on the non-linear and plastic mechanical response of the surrounding collagen. Specifically, we demonstrate that collective back-and-forth motion of cells within the branches generates tension that is strong enough to induce a plastic reorganization of the surrounding collagen network which results in the formation of mechanically stable collagen cages. Such matrix encasing in turn directs further tension generation, branch outgrowth and plastic deformation of the matrix. The identified mechanical tension equilibrium sets a framework to understand how mechanical cues can direct ductal branch elongation.

Clinical Importance of Bone Matrix Damage Mechanisms for Fracture Prevention.

PURPOSE OF REVIEW: Bone matrix exhibits great complexity in its composition, structure and mechanics. Here, we provide a review of recent research articles and appraise the evidence that bone matrix quality is clinically important and possibly targetable for fracture prevention. RECENT FINDINGS: Deformation of mineralised collagen fibrils determines bone fracture mechanics. Slipping and separation at the mineral-fibril and fibril-fibril interfaces, respectively, are the structural mechanisms for plastic deformation and microcrack nucleation. Existing technologies for assessing bone tissue in vivo cannot measure matrix structure or fracture mechanics but have shown limited use in clinical settings for identifying fragility or following treatment outcomes based on composition. Matrix is biomechanically and clinically important, but the knowledge has not translated into clinical practice. The structural mechanisms by which a load is transferred from mineralised collagen fibrils to the whole bone via microcracking have been proven too complex to measure in vivo. The mineral-fibril or fibril-fibril interfaces might be suitable targets for diagnosing fragility or delivering molecules that reduce fracture risk by strengthening the mineral bonds while maintaining flexibility in the fibrils.