Cell autonomous TGFß signaling is essential for stem/progenitor cell recruitment into degenerative tendons.

Understanding cell recruitment in damaged tendons is critical for improvements in regenerative therapy. We recently reported that targeted disruption of transforming growth factor beta (TGFß) type II receptor in the tendon cell lineage (Tgfbr2ScxCre) resulted in resident tenocyte dedifferentiation and tendon deterioration in postnatal stages. Here we extend the analysis and identify direct recruitment of stem/progenitor cells into the degenerative mutant tendons. Cre-mediated lineage tracing indicates that these cells are not derived from tendon-ensheathing tissues or from a Scleraxis-expressing lineage, and they turned on tendon markers only upon entering the mutant tendons. Through immunohistochemistry and inducible gene deletion, we further find that the recruited cells originated from a Sox9-expressing lineage and their recruitment was dependent on cell autonomous TGFß signaling. The cells identified in this study thus differ from previous reports of cell recruitment into injured tendons and suggest a critical role for TGFß signaling in cell recruitment, providing insights that may support improvements in tendon repair.

Aquatic Training after Joint Immobilization in Rats Promotes Adaptations in Myotendinous Junctions.

The myotendinous junction (MTJ) is the muscle-tendon interface and constitutes an integrated mechanical unit to force transmission. Joint immobilization promotes muscle atrophy via disuse, while physical exercise can be used as an adaptative stimulus. In this study, we aimed to investigate the components of the MTJ and their adaptations and the associated elements triggered with aquatic training after joint immobilization. Forty-four male Wistar rats were divided into sedentary (SD), aquatic training (AT), immobilization (IM), and immobilization/aquatic training (IMAT) groups. The samples were processed to measure fiber area, nuclear fractal dimension, MTJ nuclear density, identification of telocytes, sarcomeres, and MTJ perimeter length. In the AT group, the maintenance of ultrastructure and elements in the MTJ region were observed; the IM group presented muscle atrophy effects with reduced MTJ perimeter; the IMAT group demonstrated that aquatic training after joint immobilization promotes benefits in the muscle fiber area and fractal dimension, in the MTJ region shows longer sarcomeres and MTJ perimeter. We identified the presence of telocytes in the MTJ region in all experimental groups. We concluded that aquatic training is an effective rehabilitation method after joint immobilization due to reduced muscle atrophy and regeneration effects on MTJ in rats.

Histological and ultrastructural degenerative findings in the gluteus medius tendon after hip arthroplasty.

BACKGROUND: Despite gluteus medius (GMED) tendinosis being relatively common, its presence in association with hip osteoarthritis (OA) or total hip arthroplasty (THA) is not well studied. It was hypothesized that more tendon degeneration would be found in patients with OA of the hip and in those that had undergone THA than that in a control group. METHODS: One hundred patients were included between 2016 and 2019 and were included into 4 groups; the patients were undergoing revision surgery in two groups and primary THA in the other two groups; 22 patients had previously undergone primary THA through a direct lateral approach (involving sectioning of the GMED tendon), 24 patients had previously undergone primary THA through a posterior approach (leaving the GMED tendon intact), 29 patients had primary hip OA, and 25 patients who suffered a femoral neck fracture served as controls. Biopsies from the GMED tendon were obtained at the time of the primary THA or the hip revision surgery. The tendon biopsies were examined ultrastructurally and histologically. RESULTS: Ultrastructurally, the direct lateral and posterior revision groups had statistically significantly more collagen fibrils with smaller diameters compared with the fracture and primary THA groups. Moreover, the direct lateral revision group had more collagen fibrils with smaller diameters compared with the posterior revision group. Histologically, the direct lateral revision group had a higher total degeneration score (TDS) compared with the primary hip OA group. CONCLUSIONS: The GMED tendon shows more ultrastructural degeneration in patients who undergo hip revision arthroplasty than in patients with primary OA of the hip and control patients, who had suffered a femoral neck fracture. Furthermore, patients who had previously undergone primary THA through a direct lateral approach revealed more histological GMED tendon degeneration than patients who suffer primary hip OA.

Preparation and biological characteristics of a bovine acellular tendon fiber material.

Acellular tendon matrix is an ideal substitute for constructing tissue engineering ligaments, but using detergents causes damage to collagen and fibrin during the process of decellularization. In this study, fresh tendons were lyophilized and separated into fresh tendon fiber (FTF) bundles, and then the cellular components in FTF were removed to prepare acellular tendon fiber (ATF) without adding chemical detergent. H&E staining and DAPI fluorescence microscopy showed no nucleus and DNA residue. Compared with FTFs, the DNA content of ATFs was significantly lower without the collagen content change before and after decellularization. The microstructure of collagen fibrils in ATFs was intact under scanning electron microscopy (SEM), and the maximum tensile load and elastic modulus between FTFs and ATFs were not statistically different. The ATF bundles were cultured with SD rat tenocytes for 72 hr and cells attachment to fiber surfaces were observed under SEM. ATF bundles were then implanted into paraspinal muscles, and histological analysis showed fibroblast-like cells within the ATFs and was similar to the control group (fresh tendon autograft) in morphology. H&E staining showed that the number of lymphocytes and plasma cells in ATF was less than that in fresh tendon autograft. ATF bundles were twisted into linear fiber materials by hand, of which the maximum breaking strength was similar to silk with same diameter. These findings demonstrated that ATFs retain their original fibril structure and mechanical properties after decellularization by trypsin and pancreatic deoxyribonuclease without detergent. Lyophilized ATFs linear fiber material provides the possibility of preparing personalized ligament and other tissue engineering scaffolds.

Three-dimensional ultrastructure reconstruction of tendinous components at the bifurcation of the bovine superficial digital flexor tendon using array and STEM tomographies.

Tendons transmit force from muscle to bone for joint movement. Tenocytes are a specialized type of fibroblast that produces collagen fibrils in tendons. Their cytoplasmic processes form a network surrounding collagen fibrils to define a collagen fibre. Glycosaminoglycan (GAG) chains link collagen fibrils and adhere at the D-band of the collagen fibril. In this study, we used array and scanning transmission electron microscope (STEM) tomographies to reconstruct the three-dimensional ultrastructure of tenocytes, collagen fibres, collagen fibrils and GAG chains at the bifurcation of the bovine hindlimb superficial digital flexor tendon (SDFT). Collagen fibrils comprising a collagen fibre were not aligned uniformly and had at least two running directions. Spindle-shaped tenocytes were arranged along the long axis of a plurality of collagen fibres, where two groups of collagen fibrils with oblique directions to each other exhibited an oblique overlap of the two collagen fibril layers. Collagen fibrils with different running directions were observed in separating layers of about 300 nm in thickness and had diameters of 0-200 nm. About 40% of all collagen fibrils had a peak in the range of 20-40 nm. STEM analysis of the same site where the crossing of collagen fibres was observed by transmission electron microscopy demonstrated the outline of collagen fibrils with a clear D-banding pattern at a regular interval. Collagen fibrils were reconstructed three-dimensionally using continuous images acquired by STEM tomography, which confirmed that the collagen fibrils at the crossing sites did not orientate in layers, but were woven one by one. Higher magnification observation of GAG chains attached between the crossing collagen fibrils revealed numerous GAG chains arranged either vertically or obliquely on collagen fibrils. Furthermore, GAG chains at the cross of collagen fibrils connected the closest D-bands. GAG chains are thought to be universally present between collagen fibrils of the tendon. These observations by array and STEM tomographies increase our knowledge of the anatomy in the bifurcation of the bovine hindlimb SDFT and demonstrate the utility of these new imaging technologies.

Ultrastructural evidence for telocytes in equine tendon.

The three-dimensional ultrastructure of the tendon is complex. Two main cell types are classically supported: elongated tenocytes and ovoid tenoblasts. The existence of resident stem/progenitor cells in human and equine tendons has been demonstrated, but their location and relationship to tenoblasts and tenocytes remain unclear. Hence, in this work, we carried out an ultrastructural study of the equine superficial digital flexor tendon. Although the fine structure of tendons has been previously studied using electron microscopy, the presence of telocytes, a specific type of interstitial cell, has not been described thus far. We show the presence of telocytes in the equine inter-fascicular tendon matrix near blood vessels. These telocytes have characteristic telopodes, which are composed of alternating dilated portions (podoms) and thin segments (podomers). Additionally, we demonstrate the presence of the primary cilium in telocytes and its ability to release exosomes. The location of telocytes is similar to that of tendon stem cells. The telocyte-blood vessel proximity, the presence of primary immotile cilia and the release of exosomes could have special significance for tendon homeostasis.

Tendon Multiscale Structure, Mechanics, and Damage Are Affected by Osmolarity of Bath Solution.

One of the most common bath solutions used in musculoskeletal mechanical testing is phosphate buffered saline (PBS). In tendon, swelling induced by physiological PBS results in decreased tendon modulus and induces microstructural changes. It is critical to evaluate the multiscale mechanical behavior of tendon under swelling to interpret prior work and provide information to design future studies. We compared the effects of physiological PBS and 8% polyethylene glycol and saline bathing solutions on tendon multiscale tendon mechanics and damage as well as microstructure with TEM in order to understand the effect of swelling on tendon. At the tissue level, tendons in PBS had a lower modulus than SPEG samples. PBS samples also showed an increased amount of non-recoverable sliding, which is an analog for microscale damage. SPEG had a higher microscale to tissue-scale strain ratio, showing the fibrils experienced less strain attenuation. From the TEM data, we showed the fibril spacing of SPEG samples was more similar to fresh control than PBS. We concluded that swelling alters multiscale mechanics and damage in addition to tendon microstructure. Future mechanical testing should consider using SPEG as a bath solution with an osmotic pressure which preserves fresh tissue water content.

Collagen XI regulates the acquisition of collagen fibril structure, organization and functional properties in tendon.

Collagen XI is a fibril-forming collagen that regulates collagen fibrillogenesis. Collagen XI is normally associated with collagen II-containing tissues such as cartilage, but it also is expressed broadly during development in collagen I-containing tissues, including tendons. The goals of this study are to define the roles of collagen XI in regulation of tendon fibrillar structure and the relationship to function. A conditional Col11a1-null mouse model was created to permit the spatial and temporal manipulation of Col11a1 expression. We hypothesize that collagen XI functions to regulate fibril assembly, organization and, therefore, tendon function. Previous work using cho mice with ablated Col11a1 alleles supported roles for collagen XI in tendon fibril assembly. Homozygous cho/cho mice have a perinatal lethal phenotype that limited the studies. To circumvent this, a conditional Col11a1flox/flox mouse model was created where exon 3 was flanked with loxP sites. Breeding with Scleraxis-Cre (Scx-Cre) mice yielded a tendon-specific Col11a1-null mouse line, Col11a1Δten/Δten. Col11a1flox/flox mice had no phenotype compared to wild type C57BL/6 mice and other control mice, e.g., Col11a1flox/flox and Scx-Cre. Col11a1flox/flox mice expressed Col11a1 mRNA at levels comparable to wild type and Scx-Cre mice. In contrast, in Col11a1Δten/Δten mice, Col11a1 mRNA expression decreased to baseline in flexor digitorum longus tendons (FDL). Collagen XI protein expression was absent in Col11a1Δten/Δten FDLs, and at ~50% in Col11a1+/Δten compared to controls. Phenotypically, Col11a1Δten/Δten mice had significantly decreased body weights (p < 0.001), grip strengths (p < 0.001), and with age developed gait impairment becoming hypomobile. In the absence of Col11a1, the tendon collagen fibrillar matrix was abnormal when analyzed using transmission electron microscopy. Reducing Col11a1 and, therefore collagen XI content, resulted in abnormal fibril structure, loss of normal fibril diameter control with a significant shift to small diameters and disrupted parallel alignment of fibrils. These alterations in matrix structure were observed in developing (day 4), maturing (day 30) and mature (day 60) mice. Altering the time of knockdown using inducible I-Col11a1-/- mice indicated that the primary regulatory foci for collagen XI was in development. In mature Col11a1Δten/Δten FDLs a significant decrease in the biomechanical properties was observed. The decrease in maximum stress and modulus suggest that fundamental differences in the material properties in the absence of Col11a1 expression underlie the mechanical deficiencies. These data demonstrate an essential role for collagen XI in regulation of tendon fibril assembly and organization occurring primarily during development.

Microstructural and Mechanical Properties of Grafts Commonly Used for Cruciate Ligament Reconstruction.

BACKGROUND: Injuries to the anterior cruciate ligament and posterior cruciate ligament are common, and often are treated with reconstruction. Limited quantitative data are available describing material properties of grafts used for reconstructions such as the bone-patellar tendon-bone (BPTB), hamstring tendon (HS), and quadriceps tendon (QT). The purpose of this study was to quantify and compare microstructural and mechanical properties of BPTB, HS, and QT grafts. METHODS: Forty specimens (13 BPTB, 13 HS, and 14 QT grafts) from 24 donors were used. Specimens were subjected to preconditioning, stress relaxation, and ramp to failure. Mechanical parameters were calculated for each sample, and polarization imaging was used to evaluate the direction and strength of collagen fiber alignment during testing. RESULTS: QT had the largest modulus values, and HS had the smallest. BPTB exhibited the least disperse collagen organization, while HS were the least strongly aligned. Microstructural properties showed more strongly aligned collagen with increasing load for all grafts. All tissues showed stress relaxation and subtle microstructural changes during the hold period. CONCLUSIONS: The mechanical and microstructural properties differed significantly among BPTB, HS, and QT grafts. QT exhibited the largest moduli and greatest strength of collagen alignment, while HS had the smallest moduli and least strongly aligned collagen. CLINICAL RELEVANCE: This study identified mechanical and microstructural differences among common grafts and between these grafts and the cruciate ligaments they replace. Further research is needed to properly interpret the clinical relevance of these differences.

Three-dimensional structural interrelations between cells, extracellular matrix, and mineral in normally mineralizing avian leg tendon.

The spatial-temporal relationship between cells, extracellular matrices, and mineral deposits is fundamental for an improved understanding of mineralization mechanisms in vertebrate tissues. By utilizing focused ion beam-scanning electron microscopy with serial surface imaging, normally mineralizing avian tendons have been studied with nanometer resolution in three dimensions with volumes exceeding tens of micrometers in range. These parameters are necessary to yield sufficiently fine ultrastructural details while providing a comprehensive overview of the interrelationships between the tissue structural constituents. Investigation reveals a complex lacuno-canalicular network in highly mineralized tendon regions, where ∼100 nm diameter canaliculi emanating from cell (tenocyte) lacunae surround extracellular collagen fibril bundles. Canaliculi are linked to smaller channels of ∼40 nm diameter, occupying spaces between fibrils. Close to the tendon mineralization front, calcium-rich deposits appear between the fibrils and, with time, mineral propagates along and within them. These close associations between tenocytes, tenocyte lacunae, canaliculi, small channels, collagen, and mineral suggest a concept for the mineralization process, where ions and/or mineral precursors may be transported through spaces between fibrils before they crystallize along the surface of and within the fibrils.

Flexor tendon grafts for pulley reconstruction - Morphological aspects.

BACKGROUND: Pulleys are crucial to convert flexor tendon excursion into angular motion at the metacarpophalangeal and interphalangeal joints. Loss of pulley function can lead to significant impairment of hand function and may require surgical reconstruction. This reconstruction can be achieved using different flexor tendons grafts, such as the intrasynovial flexor digitorum superficialis (FDS) or the extrasynovial palmaris longus (PL). However, there is limited knowledge on the micromorphology of human pulleys and the suitability of flexor tendon grafts for their reconstruction remains elusive. METHODS: In the present cadaver study A2 and A4 pulleys were compared with FDS and PL tendons by means of scanning electron microscopy (SEM), histology and immunohistochemistry. Surface morphology, core structure and vascularization of the specimens were analyzed. RESULTS: SEM imaging of the gliding surfaces revealed morphological differences between tendons and pulleys. Moreover, the core structure of FDS samples was characterized by bundles of individual collagen fibrils whereas PL tendons exhibited a less hierarchical microstructure. In contrast, pulleys consisted of lamellar sheets of densely packed collagen fibrils. Finally, immunohistochemical analyses revealed that the flexor tendons and pulleys contain similar numbers of CD31+ microvessels, indicating a comparable tissue vascularization. CONCLUSION: This study provides novel SEM and immunohistochemical insights into the micromorphology of human pulleys and flexor tendon grafts. Intrasynovial flexor tendons may be particularly suitable for pulley reconstruction and preserving the paratenon may be crucial for graft revascularization.

Increased Collagen Turnover Impairs Tendon Microstructure and Stability in Integrin α2ß1-Deficient Mice.

Integrins are a family of transmembrane proteins, involved in substrate recognition and cell adhesion in cross-talk with the extra cellular matrix. In this study, we investigated the influence of integrin α2ß1 on tendons, another collagen type I-rich tissue of the musculoskeletal system. Morphological, as well as functional, parameters were analyzed in vivo and in vitro, comparing wild-type against integrin α2ß1 deficiency. Tenocytes lacking integrin α2ß1 produced more collagen in vitro, which is similar to the situation in osseous tissue. Fibril morphology and biomechanical strength proved to be altered, as integrin α2ß1 deficiency led to significantly smaller fibrils as well as changes in dynamic E-modulus in vivo. This discrepancy can be explained by a higher collagen turnover: integrin α2ß1-deficient cells produced more matrix, and tendons contained more residual C-terminal fragments of type I collagen, as well as an increased matrix metalloproteinase-2 activity. A greatly decreased percentage of non-collagenous proteins may be the cause of changes in fibril diameter regulation and increased the proteolytic degradation of collagen in the integrin-deficient tendons. The results reveal a significant impact of integrin α2ß1 on collagen modifications in tendons. Its role in tendon pathologies, like chronic degradation, will be the subject of future investigations.

Optical Microscopy and Electron Microscopy for the Morphological Evaluation of Tendons: A Mini Review.

The morphological characteristics of tendons have been thoroughly evaluated via microscopy. Optical microscopy and electron microscopy are the most commonly used techniques for tendon tissue observation. According to the principles of both microscopy types, preparation and evaluation methods vary. Simple optical microscopy is commonly used in the observation of cells and extracellular matrix, and many stains, including hematoxylin-eosin, Van Gieson, Prussian blue, Alcian blue, and toluidine blue, are used for evaluating cells, collagen fiber arrangement, and noncollagenous proteins. Histological scoring systems have been used in many studies for semi-quantification. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) are the most commonly used electron microscopy types, and special consideration is needed for the fixation and embedding protocols. Glutaraldehyde followed by osmium is most commonly used in the chemical fixation of tendon tissue, followed by epoxy resin embedment. Longitudinal sections captured in SEM images show the arrangement of collagen fibrils and the cells and lipid drops among them, while cross sections captured in TEM images show the diameter and distribution of collagen fibrils. SEM and TEM are used together for comprehensive evaluations. This mini review is focused on the preparation methodology and related evaluation indexes for the morphological evaluation of tendons.

Three-sectioning method: A procedure for studying hard tissues and large pieces under light and electron microscopy.

The histological study of hard pieces such as tendons and calcified lesions and tissues is a field that has been gaining increased attention owing to the rapid development of implantable prostheses, among other factors. In these studies, serial sectioning is utilized to detect areas of interest throughout the entire piece, as it enables the application of the appropriate light and electron microscopy techniques in these areas. We propose the "three-sectioning method" that subjects the pieces to three consecutive cycles of embedding and sectioning to localize and study the areas of interest, as an efficient technique for these histological studies. The pieces were first embedded in epoxy resin and then cut into thick sections (approximately 300 µm) for the first cycle. Next, areas of interest selected on these thick sections were re-embedded in epoxy resin to be sectioned again (second sectioning) to obtain a series of semithin sections (1-3 µm). These semithin sections are usually studied using the most relevant techniques for light microscopy. Smaller areas of interest are selected to be cut into ultrathin sections (60-90 nm) for transmission electron microscopy. If necessary, the selected areas of the semithin sections can be embedded again, and then cut into new ultrathin sections. The different kinds of sections we have described here may also be studied using scanning electron microscopy. This systematic method facilitates correlative microscopy from lower to higher magnifications along with the usage of a broad variety of histological techniques including electron microscopy.

Tendon Extracellular Matrix Remodeling and Defective Cell Polarization in the Presence of Collagen VI Mutations.

Mutations in collagen VI genes cause two major clinical myopathies, Bethlem myopathy (BM) and Ullrich congenital muscular dystrophy (UCMD), and the rarer myosclerosis myopathy. In addition to congenital muscle weakness, patients affected by collagen VI-related myopathies show axial and proximal joint contractures, and distal joint hypermobility, which suggest the involvement of tendon function. To gain further insight into the role of collagen VI in human tendon structure and function, we performed ultrastructural, biochemical, and RT-PCR analysis on tendon biopsies and on cell cultures derived from two patients affected with BM and UCMD. In vitro studies revealed striking alterations in the collagen VI network, associated with disruption of the collagen VI-NG2 (Collagen VI-neural/glial antigen 2) axis and defects in cell polarization and migration. The organization of extracellular matrix (ECM) components, as regards collagens I and XII, was also affected, along with an increase in the active form of metalloproteinase 2 (MMP2). In agreement with the in vitro alterations, tendon biopsies from collagen VI-related myopathy patients displayed striking changes in collagen fibril morphology and cell death. These data point to a critical role of collagen VI in tendon matrix organization and cell behavior. The remodeling of the tendon matrix may contribute to the muscle dysfunction observed in BM and UCMD patients.

Independent, Controllable Stretch-Perfusion Bioreactor Chambers to Functionalize Cell-Seeded Decellularized Tendons.

Tissue-engineered decellularized matrices can progress clinical replacement of full-thickness ruptures or tendon defects. This study develops and validates a custom-made automated bioreactor, called oscillating stretch-perfusion bioreactor (OSPB), consisting of multiple, independent culture chambers able to combine a bidirectional perfusion with a programmable, uniaxial strain to functionalize cell-seeded decellularized tendons. Decellularized tendon matrices were seeded on their surfaces and within the tendon fibers with mesenchymal stem cells. Then, they were subjected to a bidirectional perfusion and programmed stretching cycles of 15-30-60 min on-off two times per day for 7 days of culture. In vitro analyses showed viable cells, homogenously distributed on the surface of the constructs. More importantly, cell-seeded decellularized tendon grafts undergoing cyclic load in our bioreactor had a superior production and organization of newly formed collagen matrix compared to static cultured constructs. The coherency and local alignment of the new collagen deposition within the inner injected channels quantitatively supported histological findings. The designed OSPB could be considered a unique, cost-effective system able to involve multiple independently controlled chambers in terms of biological and mechanical protocols. This system allows parallel processing of several customized tendon constructs to be used as grafts to enhance the surgical repair of large tendon defects.

Connective Tissue Ultrastructure: A Direct Comparison between Conventional Specimen Preparation and High-Pressure Freezing/Freeze-Substitution.

It is generally agreed within the microscopy community that the quality of ultrastructure within the connective tissue matrix resulting from high-pressure freezing followed by freeze-substitution (HPF/FS) far exceeds that gained following the "conventional" preparation method, which includes aqueous fixation, dehydration, and embedding. Exposure to cryogen at high pressure is the only cryopreservation method capable of vitrifying tissue structure to a depth exceeding 200 µm. Cells within connective tissues prepared by HPF/FS are universally larger, filling the commonly seen void at the juncture between cell and matrix. Without significant shrinkage of cells and the coincident extraction of the cytosolic components, well-resolved organelles are less clustered within an expanded cytosol. Much of the artifact from "conventional" methods occurs as large space filling and also smaller fibril-associated proteoglycans are extracted during fixation. However, the visualization of some matrix features by electron microscopy is actually dependent on the collapse or extraction of these "masking" components. Herein, we argue that an impression of ultrastructure within commonly studied matrices, in particular skin, is best gained following the evaluation of both conventional preparations and tissue prepared by HPF/FS. Anat Rec, 2019. © 2019 American Association for Anatomy.

Integration of polarized spatial frequency domain imaging (pSFDI) with a biaxial mechanical testing system for quantification of load-dependent collagen architecture in soft collagenous tissues.

Collagen fiber networks provide the structural strength of tissues, such as tendons, skin and arteries. Quantifying the fiber architecture in response to mechanical loads is essential towards a better understanding of the tissue-level mechanical behaviors, especially in assessing disease-driven functional changes. To enable novel investigations into these load-dependent fiber structures, a polarized spatial frequency domain imaging (pSFDI) device was developed and, for the first time, integrated with a biaxial mechanical testing system. The integrated instrument is capable of a wide-field quantification of the fiber orientation and the degree of optical anisotropy (DOA), representing the local degree of fiber alignment. The opto-mechanical instrument''s performance was assessed through uniaxial loading on tendon tissues with known collagen fiber microstructures. Our results revealed that the bulk fiber orientation angle of the tendon tissue changed minimally with loading (median ± 0.5*IQR of 52.7° ± 3.3° and 51.9° ± 3.3° under 0 and 3% longitudinal strains, respectively), whereas on a micro-scale, the fibers became better aligned with the direction of loading: the DOA (mean ± SD) increased from 0.149 ± 0.032 to 0.198 ± 0.056 under 0 and 3% longitudinal strains, respectively, p < 0.001. The integrated instrument was further applied to study two representative mitral valve anterior leaflet (MVAL) tissues subjected to various biaxial loads. The fiber orientations within these representative MVAL tissue specimens demonstrated noticeable heterogeneity, with the local fiber orientations dependent upon the sample, the spatial and transmural locations, and the applied loading. Our results also showed that fibers were generally better aligned under equibiaxial (DOA = 0.089 ± 0.036) and circumferentially-dominant loading (DOA = 0.086 ± 0.037) than under the radially-dominant loading (DOA = 0.077 ± 0.034), indicating circumferential predisposition. These novel findings exemplify a deeper understanding of the load-dependent collagen fiber microstructures obtained through the use of the integrated opto-mechanical instrument. STATEMENT OF SIGNIFICANCE: In this study, a novel quantitative opto-mechanical system was developed by combining a polarized Spatial Frequency Domain Imaging (pSFDI) device with a biaxial mechanical tester. The integrated system was used to quantify the load-dependent collagen fiber microstructures in representative tendon and mitral valve anterior leaflet (MVAL) tissues. Our results revealed that MVAL's fiber architectures exhibited load-dependent spatial and transmural heterogeneities, suggesting further microstructural complexity than previously reported in heart valve tissues. These novel findings were possible through the system's ability to, for the first time, capture the load-dependent collagen architecture in the mitral valve anterior leaflet tissue over a wide field of view (e.g., 10 × 10 mm for the MVAL tissue specimens). Such capabilities afford unique future opportunities to improve patient outcomes through concurrent mechanical and microstructural assessments of healthy and diseased tissues in conditions such as heart valve regurgitation and calcification.

Spin-lattice relaxation in liquid entrapped in a nanocavity.

We consider the spin lattice relaxation in bulk liquid and liquid entrapped in a nanocavity. The kinetic equation which describes the spin lattice relaxation is obtained by using the theory of the nonequilibrium state operator. A solution of the kinetic equation gives the quadrature expression for the relaxation time, T1. The calculated relaxation time agrees well with the experimental data. The spin-lattice relaxation time is calculated for nanocavities with a characteristic size much less than 700 nm, with the assumption that the spin-lattice relaxation mechanism is determined by nanocavity fluctuations. The resulting expression shows an explicit dependence of the relaxation time T1 on the volume, density of nuclear spins, and parameters of the cavity (shape and orientation relatively to the applied field). To compare with the experiment on the detection of the anisotropy of the relaxation time, we average the expression that describes the relaxation time over the orientation of the nanocavities relative to the applied magnetic field. The good agreement with the experimental data for fibril tissues was achieved by adjustment of few fitting parameters - the standard deviation, averaged fiber direction, and weight factors - which characterize the ordering of fibrils.

Helical fibrillar microstructure of tendon using serial block-face scanning electron microscopy and a mechanical model for interfibrillar load transfer.

Tendon's hierarchical structure allows for load transfer between its fibrillar elements at multiple length scales. Tendon microstructure is particularly important, because it includes the cells and their surrounding collagen fibrils, where mechanical interactions can have potentially important physiological and pathological contributions. However, the three-dimensional (3D) microstructure and the mechanisms of load transfer in that length scale are not known. It has been postulated that interfibrillar matrix shear or direct load transfer via the fusion/branching of small fibrils are responsible for load transfer, but the significance of these mechanisms is still unclear. Alternatively, the helical fibrils that occur at the microstructural scale in tendon may also mediate load transfer; however, these structures are not well studied due to the lack of a three-dimensional visualization of tendon microstructure. In this study, we used serial block-face scanning electron microscopy to investigate the 3D microstructure of fibrils in rat tail tendon. We found that tendon fibrils have a complex architecture with many helically wrapped fibrils. We studied the mechanical implications of these helical structures using finite-element modelling and found that frictional contact between helical fibrils can induce load transfer even in the absence of matrix bonding or fibril fusion/branching. This study is significant in that it provides a three-dimensional view of the tendon microstructure and suggests friction between helically wrapped fibrils as a mechanism for load transfer, which is an important aspect of tendon biomechanics.