Collagen Fiber Alignment Research Articles

Natural Type II Collagen Hydrogel, Fibrin Sealant, and Adipose-Derived Stem Cells as a Promising Combination for Articular Cartilage Repair

Objective Articular cartilage is an avascular tissue with limited ability of self-regeneration and the current clinical treatments have restricted capacity to restore damages induced by trauma or diseases. Therefore, new techniques are being tested for cartilage repair, using scaffolds and/or stem cells. Although type II collagen hydrogel, fibrin sealant, and adipose-derived stem cells (ASCs) represent suitable alternatives for cartilage formation, their combination has not yet been investigated in vivo for focal articular cartilage defects. We performed a simple experimental procedure using the combination of these 3 compounds on cartilage lesions of rabbit knees. Design The hydrogel was developed in house and was first tested in vitro for chondrogenic differentiation. Next, implants were performed in chondral defects with or without ASCs and the degree of regeneration was macroscopically and microscopically evaluated. Results Production of proteoglycans and the increased expression of collagen type II (COL2α1), aggrecan (ACAN), and sex-determining region Y-box 9 (SOX9) confirmed the chondrogenic character of ASCs in the hydrogel in vitro. Importantly, the addition of ASC induced a higher overall repair of the chondral lesions and a better cellular organization and collagen fiber alignment compared with the same treatment without ASCs. This regenerating tissue also presented the expression of cartilage glycosaminoglycan and type II collagen. Conclusions Our results indicate that the combination of the 3 compounds is effective for articular cartilage repair and may be of future clinical interest.

Myosin phosphorylation on stress fibers predicts contact guidance behavior across diverse breast cancer cells.

During cancer progression the extracellular matrix is remodeled, forming aligned collagen fibers that proceed radially from the tumor, resulting in invasion. We have recently shown that different invasive breast cancer cells respond to epitaxially grown, aligned collagen fibrils differently. This article develops insight into why these cells differ in their contact guidance fidelity. Small changes in contractility or adhesion dramatically alter directional persistence on aligned collagen fibrils, while migration speed remains constant. The directionality of highly contractile and adhesive MDA-MB-231cells can be diminished by inhibiting Rho kinase or β1 integrin binding. Inversely, the directionality of less contractile and adhesive MTLn3 cells can be enhanced by activating contractility or integrins. Subtle, but quantifiable alterations in myosin II regulatory light chain phosphorylation on stress fibers explain the tuning of contact guidance fidelity, separate from migration per se indicating that the contractile and adhesive state of the cell in combination with collagen organization in the tumor microenvironment determine the efficiency of migration. Understanding how distinct cells respond to contact guidance cues will not only illuminate mechanisms for cancer invasion, but will also allow for the design of environments to separate specific subpopulations of cells from patient-derived tissues by leveraging differences in responses to directional migration cues.

Osteogenesis requires FAK-dependent collagen synthesis by fibroblasts and osteoblasts.

Focal adhesion kinase (FAK) is critical in adhesion-dependent signaling, but its role in osteogenesis in vivo is ill defined. We deleted Fak in fibroblasts and osteoblasts in Floxed-Fak mice bred with those expressing Cre-recombinase driven by 3.6-kb α1(I)-collagen promoter. Compared with wild-type (WT), conditional FAK-knockout (CFKO) mice were shorter (2-fold; P < 0.0001) and had crooked, shorter tails (50%; P < 0.0001). Microcomputed tomography analysis showed reduced bone volume (4-fold in tails; P < 0.0001; 2-fold in mandibles; P < 0.0001), whereas bone surface area/bone volume increased (3-fold in tails; P < 0.0001; 2.5-fold in mandibles; P < 0.001). Collagen density and fiber alignment in periodontal ligament were reduced by 4-fold (P < 0.0001) and 30% (P < 0.05), respectively, in CFKO mice. In cultured CFKO osteoblasts, mineralization at d 7 and mineralizing colony-forming units at d 21 were 30% (P < 0.0001) and >3-fold less than WT, respectively. Disruptions of FAK function in osteoblasts by conditional knockout, siRNA-knockdown, or FAK inhibitor reduced mRNA and protein expression of Runx2 (>30%), Osterix (>25%), and collagen-1 (2-fold). Collagen synthesis was abrogated in WT osteoblasts with Runx2 knockdown and in Fak-null fibroblasts transfected with an FAK kinase domain mutant or a kinase-impaired mutant (Y397F). These data indicate that FAK regulates osteogenesis through transcription factors that regulate collagen synthesis.-Rajshankar, D., Wang, Y., McCulloch, C. A. Osteogenesis requires FAK-dependent collagen synthesis by fibroblasts and osteoblasts.

Longitudinal Stretching for Maturation of Vascular Tissues Using Magnetic Forces.

Cellular spheroids were studied to determine their use as “bioinks” in the biofabrication of tissue engineered constructs. Specifically, magnetic forces were used to mediate the cyclic longitudinal stretching of tissues composed of Janus magnetic cellular spheroids (JMCSs), as part of a post-processing method for enhancing the deposition and mechanical properties of an extracellular matrix (ECM). The purpose was to accelerate the conventional tissue maturation process via novel post-processing techniques that accelerate the functional, structural, and mechanical mimicking of native tissues. The results of a forty-day study of JMCSs indicated an expression of collagen I, collagen IV, elastin, and fibronectin, which are important vascular ECM proteins. Most notably, the subsequent exposure of fused tissue sheets composed of JMCSs to magnetic forces did not hinder the production of these key proteins. Quantitative results demonstrate that cyclic longitudinal stretching of the tissue sheets mediated by these magnetic forces increased the Young’s modulus and induced collagen fiber alignment over a seven day period, when compared to statically conditioned controls. Specifically, the elastin and collagen content of these dynamically-conditioned sheets were 35- and three-fold greater, respectively, at seven days compared to the statically-conditioned controls at three days. These findings indicate the potential of using magnetic forces in tissue maturation, specifically through the cyclic longitudinal stretching of tissues.

Quantification of collagen fiber orientation in human tendons with the coefficient of variation of echogenicity

The grayscale distribution on the ultrasonic images of tendons may be reduced with alignment of collagen fibers, because ultrasound signal intensity changes with alterations in tendon collagen fiber orientation due to acoustic anisotropy in the tendons. The purpose of this study was to investigate changes in the coefficient of variation (CV) of echogenicity in the Achilles tendon during passive dorsiflexion (the angle task) and isometric plantar flexion (the contraction task). Achilles tendon transverse ultrasonic images were collected from 14 healthy individuals every 10° from 20° to −20° (positive values for plantar flexion) in the angle task and every 10% maximum voluntary contraction (MVC) from 0% to 70% MVC in the contraction task. The CV of echogenicity was measured in each image. In addition, relative changes in the measured variables between the former half (20–0° in the angle task, 0% to 30% MVC in the contraction task) and the latter half (0° to −20° in the angle task, 40% to 70% MVC in the contraction task) of each task were compared. The CV of echogenicity decreased with increases in the dorsiflexion angle and intensity of isometric contractions. Furthermore, relative changes in the CV of echogenicity were greater at more dorsiflexed positions in the angle task and at lower torque levels in the contraction task. These results suggested that decreases in the CV of echogenicity were partially related to the alignment of the tendon collagen fibers with tendon stretching.

Improvement of In Vitro Three-Dimensional Cartilage Regeneration by a Novel Hydrostatic Pressure Bioreactor.

In vitro three‐dimensional (3D) cartilage regeneration is a promising strategy for repair of cartilage defects. However, inferior mechanical strength and tissue homogeneity greatly restricted its clinical translation. Simulation of mechanical stress through a bioreactor is an important approach for improving in vitro cartilage regeneration. The current study developed a hydrostatic pressure (HP) bioreactor based on a novel pressure‐transmitting mode achieved by slight deformation of a flexible membrane in a completely sealed stainless steel device. The newly developed bioreactor efficiently avoided the potential risks of previously reported pressure‐transmitting modes and simultaneously addressed a series of important issues, such as pressure scopes, culture chamber sizes, sealability, contamination control, and CO2 balance. The whole bioreactor system realized stable long‐term (8 weeks) culture under high HP (5–10 MPa) without the problems of medium leakage and contamination. Furthermore, the results of in vitro 3D tissue culture based on a cartilage regeneration model revealed that HP provided by the newly developed bioreactor efficiently promoted in vitro 3D cartilage formation by improving its mechanical strength, thickness, and homogeneity. Detailed analysis in cell proliferation, cartilage matrix production, and cross‐linking level of collagen macromolecules, as well as density and alignment of collagen fibers, further revealed the possible mechanisms that HP regulated in vitro cartilage regeneration. The current study provided a highly efficient and stable bioreactor system for improving in vitro 3D cartilage regeneration and thus will help to accelerate its clinical translation. Stem Cells Translational Medicine 2017;6:982–991

Optical metrics of the extracellular matrix predict compositional and mechanical changes after myocardial infarction.

Understanding the organization and mechanical function of the extracellular matrix (ECM) is critical for the development of therapeutic strategies that regulate wound healing following disease or injury. However, these relationships are challenging to elucidate during remodeling following myocardial infarction (MI) due to rapid changes in cellularity and an inability to characterize both ECM microstructure and function non-destructively. In this study, we overcome those challenges through whole organ decellularization and non-linear optical microscopy to directly relate the microstructure and mechanical properties of myocardial ECM. We non-destructively quantify collagen organization, content, and cross-linking within decellularized healthy and infarcted myocardium using second harmonic generation (SHG) and two photon excited autofluorescence. Tensile mechanical testing and compositional analysis reveal that the cumulative SHG intensity within each image volume and the average collagen autofluorescence are significantly correlated with collagen content and elastic modulus of the ECM, respectively. Compared to healthy ECM, infarcted tissues demonstrate a significant increase in collagen content and fiber alignment, and a decrease in cross-linking and elastic modulus. These findings indicate that cross-linking plays a key role in stiffness at the collagen fiber level following infarction, and highlight how this non-destructive approach to assessing remodeling can be used to understand ECM structure-function relationships.

Human pancreatic stellate cells modulate 3D collagen alignment to promote the migration of pancreatic ductal adenocarcinoma cells.

A hallmark of pancreatic ductal adenocarcinoma (PDAC) is the ability for cancer cells to aggressively infiltrate and navigate through a dense stroma during the metastatic process. Key features of the PDAC stroma include an abundant population of activated pancreatic stellate cells (PSCs) and highly aligned collagen fibers; however, important questions remain regarding how collagen becomes aligned and what the biological manifestations are. To better understand how PSCs, aligned collagen, and PDAC cells might cooperate during the transition to invasion, we utilized a microchannel-based in vitro tumor model and advanced imaging technologies to recreate and examine in vivo-like heterotypic interactions. We found that PSCs participate in a collaborative process with cancer cells by orchestrating the alignment of collagen fibers that, in turn, are permissive to enhanced cell migration. Additionally, direct contact between PSCs, collagen, and PDAC cells is critical to invasion and co-migration of both cell types. This suggests PSCs may accompany and assist in navigating PDAC cells through the stromal terrain. Together, our data provides a new role for PSCs in stimulating the metastatic process and underscores the importance of collagen alignment in cancer progression.

A model system for developing a tissue engineered meniscal enthesis.

The meniscal enthesis is a complex structure that is essential to mechanical stability of the meniscus and the knee joint. Several studies document the development of anatomically shaped tissue engineered meniscus constructs, but none have focused on how to integrate such tissues with underlying bone. This study establishes a simplified construct to model the meniscal enthesis composed of a collagen gel seeded with meniscal fibrochondrocytes integrated with decellularized cancellous bone. Mechanical fixation at the bony ends induced tissue integration of fibers into the bony tissue, which is critical for mechanical performance and has yet to be shown in enthesis literature. Our test platform is amenable to targeted experiments investigating mineralization gradients, collagen fiber alignment, cell population phenotype, and media conditioning with experimental impact on enthesis studies for meniscus, tendon, and ligament.

Microstructural and Mechanical Properties of the Posterior Cruciate Ligament: A Comparison of the Anterolateral and Posteromedial Bundles.

The microstructural organization (collagen fiber alignment) of the posterior cruciate ligament (PCL), which likely corresponds with its functional properties, has only been described qualitatively in the literature, to our knowledge. The goal of this study was to quantify the tensile mechanical and microstructural properties of the PCL and compare these qualities between the anterolateral and posteromedial bundles. Twenty-two knee specimens from 13 donors (8 male and 5 female; mean age [and standard deviation] at the time of death, 43.0 ± 4.1 years; mean body mass index, 30.0 ± 6.7 kg/m2) were dissected to isolate the PCL, and each bundle was split into 3 regions. Mechanical testing of each regional sample consisted of preconditioning followed by a ramp-and-hold stress-relaxation test and a quasi-static ramp-to-failure test. Microstructural analysis was performed with use of a high-resolution, division-of-focal-plane polarization camera to evaluate the average direction of collagen orientation and the degree to which the collagen fibers were aligned in that direction. Results were compared between the anterolateral and posteromedial bundles and across the regions of each bundle. The anterolateral and posteromedial bundles demonstrated largely equivalent mechanical and microstructural properties. Elastic moduli in the toe and linear regions were not different; however, the posteromedial bundle did show significantly more stress relaxation (p = 0.004). There were also few differences in microstructural properties between bundles, which again were seen only in stress relaxation. Comparing regions within each bundle, several mechanical and microstructural parameters showed significant relationships across the posteromedial bundle, following a gradient of decreasing strength and alignment from anterior to posterior. The PCL has relatively homogenous microstructural and mechanical properties, with few differences between the anterolateral and posteromedial bundles. This finding suggests that distinct functions of the PCL bundles result primarily from size and anatomical location rather than from differences in these properties. These properties of the PCL can be used to assess the utility of graft choices and operative techniques for PCL reconstruction and may partly explain limited differences in the outcomes of single-bundle compared with double-bundle reconstruction techniques for the PCL.

Experimental and Clinical Evidence Based Rationality of Incorporation of Deep Fascia in Tissue Transfer for Reconstructive Surgery

ABSTRACTIntense clinical research since 1984 through 93 parameters has unveiled the rationality of reconstructive procedures with several new concepts and innovative techniques of various compositions. Such extensive work has been done totally in our country popularizing the techniques amongst the plastic surgeons of the world. This presentation deals with the tissue constituents and vascular network of the deep fascia. This sustained research was conducted at inter-departmental, interfaculty and interinstitutional levels.Intense clinical research since 1984 through 93 parameters has unveiled the rationality of reconstructive procedures with several new concepts and innovative techniques of various compositions. Such extensive work has been done totally in our country popularizing the techniques amongst the plastic surgeons of the world. This presentation deals with the tissue constituents and vascular network of the deep fascia. This sustained research was conducted at inter-departmental, interfaculty and interinstitutional levels.Histology of deep fascia showed rich subfascial and suprafascial arterioles and capillaries. Intrafascial course of the perforating vessels from the subfascial plane to the suprafascial plane was visualized. Confocal microscopic analysis of fluorecenised deep fascia under 40X magnification showed longitudinally aligned collagen fibres and nuclei of multiple fibroblasts. Electron microscopic Y of deep fascia revealed (a) Elastic tissue and collagen fibres, (b) Lymphatic vessel, (c) Thin walled venule with single layer of muscle fiber, endothelial cell nucleus, Venules with multiple RBCs, mast cell with granules and capillary showing endothelium and endothelial cells.Angiography in experimental model and patients, demonstrated longitudinally oriented vascular network in deep fascia and fasciocutaneous flap. Live microcirculation and Lymphatic circulation in the human deep fascia was demonstrated first time in the world under 150 and 600 magnifications.

Functional anatomy of the equine temporomandibular joint: Collagen fiber texture of the articular surfaces

In the last decade, the equine masticatory apparatus has received much attention. Numerous studies have emphasized the importance of the temporomandibular joint (TMJ) in the functional process of mastication. However, ultrastructural and histological data providing a basis for biomechanical and histopathological considerations are not available. The aim of the present study was to analyze the architecture of the collagen fiber apparatus in the articular surfaces of the equine TMJ to reveal typical morphological features indicating biomechanical adaptions. Therefore, the collagen fiber alignment was visualized using the split-line technique in 16 adult warmblood horses without any history of TMJ disorders.Within the central two-thirds of the articular surfaces of the articular tubercle, the articular disc and the mandibular head, split-lines ran in a correspondent rostrocaudal direction. In the lateral and medial aspects of these articular surfaces, the split-line pattern varied, displaying curved arrangements in the articular disc and punctual split-lines in the bony components. Mediolateral orientated split-lines were found in the rostral and caudal border of the articular disc and in the mandibular fossa. The complex movements during the equine chewing cycle are likely assigned to different areas of the TMJ. The split-line pattern of the equine TMJ is indicative of a relative movement of the joint components in a preferential rostrocaudal direction which is consigned to the central aspects of the TMJ. The lateral and medial aspects of the articular surfaces provide split-line patterns that indicate movements particularly around a dorsoventral axis.

Advanced quantitative imaging and biomechanical analyses of periosteal fibers in accelerated bone growth

PurposeThe accepted mechanism explaining the accelerated growth following periosteal resection is that the periosteum serves as a mechanical restraint to restrict physeal growth. To test the veracity of this mechanism we first utilized Second Harmonic Generation (SHG) imaging to measure differences of periosteal fiber alignment at various strains. Additionally, we measured changes in periosteal growth factor transcription. Next we utilized SHG imaging to assess the alignment of the periosteal fibers on the bone both before and after periosteal resection. Based on the currently accepted mechanism, we hypothesized that the periosteal fibers adjacent to the physis should be more aligned (under tension) during growth and become less aligned (more relaxed) following metaphyseal periosteal resection. In addition, we measured the changes in periosteal micro- and macro-scale mechanics. Methods30 seven-week old New Zealand White rabbits were sacrificed. The periosteum was imaged on the bone at five regions using SHG imaging. One centimeter periosteal resections were then performed at the proximal tibial metaphyses. The resected periosteal strips were stretched to different strains in a materials testing system (MTS), fixed, and imaged using SHG microscopy. Collagen fiber alignment at each strain was then determined computationally using CurveAlign. In addition, periosteal strips underwent biomechanical testing in both circumferential and axial directions to determine modulus, failure stress, and failure strain. Relative mRNA expression of growth factors: TGFβ-1, -2, -3, Ihh, PTHrP, Gli, and Patched were measured following loading of the periosteal strips at physiological strains in a bioreactor. The periosteum adjacent to the physis of six tibiae was imaged on the bone, before and after, metaphyseal periosteal resection, and fiber alignment was computed. One-way ANOVA statistics were performed on all data. ResultsImaging of the periosteum at different regions of the bone demonstrated complex regional differences in fiber orientation. Increasing periosteal strain on the resected strips increased periosteal fiber alignment (p<0.0001). The only exception to this pattern was the 10% strain on the tibial periosteum, which may indicate fiber rupture at this non-physiologic strain. Periosteal fiber alignment adjacent to the resection became less aligned while those adjacent to the physes remained relatively unchanged before and after periosteal resection. Increasing periosteal strain on the resected strips increased periosteal fiber alignment (p<0.0001). The only exception to this pattern was the 10% strain on the tibial periosteum, which may indicate fiber rupture (and consequent retraction) at this non-physiologic strain. Increasing periosteal strain revealed a significant increase in relative mRNA expression for Ihh, PTHrP, Gli, and Patched, respectively. ConclusionPeriosteal fibers adjacent to the growth plate do not appear under tension in the growing limb, and the alignments of these fibers remain unchanged following periosteal resection. SignificanceThe results of this study call into question the long-accepted role of the periosteum acting as a simple mechanical tether restricting growth at the physis.

Planar biaxial extension of the lumbar facet capsular ligament reveals significant in-plane shear forces

The lumbar facet capsular ligament (FCL) articulates with six degrees of freedom during spinal motions of flexion/extension, lateral bending, and axial rotation. The lumbar FCL is composed of highly aligned collagen fiber bundles on the posterior surface (oriented primarily laterally between the rigid articular facets) and irregularly oriented elastin on the anterior surface. Because the FCL is a capsule, it has multiple insertion sites across the lumbar facet joint, which, along with its material structure, give rise to complicated deformations in vivo. We performed planar equibiaxial mechanical tests on excised healthy cadaveric lumbar FCLs (n=6) to extract normal and shear reaction forces, and fit sample-specific two-fiber-family finite element models to the experimental force data. An eight-parameter anisotropic, hyperelastic model was used. Shear forces at maximum extension (mean values of 1.68N and 3.01N in the two directions) were of comparable magnitude to the normal forces perpendicular to the aligned collagen fiber bundles (4.67N) but smaller than normal forces in the fiber direction (16.11N). Inclusion of the experimental shear forces in the model optimization yielded fits with highly aligned fibers oriented at a specific angle across all samples, typically with one fiber population aligned nearly horizontally and the other at an oblique angle. Conversely, models fit to only the normal force data resulted in a broad range of fiber angles with low specificity. We found that shear forces generated through planar equibiaxial extension aided the model fit in describing the anisotropic nature of the FCL surface.

Computer simulation of lumbar flexion shows shear of the facet capsular ligament

Background ContextThe lumbar facet capsular ligament (FCL) is a posterior spinal ligament with a complex structure and kinematic profile. The FCL has a curved geometry, multiple attachment sites, and preferentially aligned collagen fiber bundles on the posterior surface that are innervated with mechanoreceptive nerve endings. Spinal flexion induces three-dimensional (3D) deformations, requiring the FCL to maintain significant tensile and shear loads. Previous works aimed to study 3D facet joint kinematics during flexion, but to our knowledge none have reported localized FCL surface deformations likely created by this complex structure. PurposeThe purpose of this study was to elucidate local deformations of both the posterior and anterior surfaces of the lumbar FCL to understand the distribution and magnitude of in-plane and through-plane deformations, including the prevalence of shear. Study Design/SettingThe FCL anterior and posterior surface deformations were quantified through creation of a finite element model simulating facet joint flexion using a realistic geometry, physiological kinematics, and fitted constitutive material. MethodsGeometry was obtained from the micro-CT data of a healthy L3–L4 facet joint capsule (n=1); kinematics were extracted from sagittal plane fluoroscopic data of healthy volunteers (n=10) performing flexion; and average material properties were determined from planar biaxial extension tests of L4–L5 FCLs (n=6). All analyses were performed with the non-linear finite element solver, FEBio. A grid of equally spaced 3×3 nodes on the posterior surface identified regional differences within the strain fields and was used to create comparisons against previously published experimental data. This study was funded by the National Institutes of Health and the authors have no disclosures. ResultsInhomogeneous in-plane and through-plane shear deformations were prominent through the middle body of the FCL on both surfaces. Anterior surface deformations were more pronounced because of the small width of the joint space, whereas posterior surface deformations were more diffuse because the larger area increased deformability. We speculate these areas of large deformation may provide this proprioceptive system with an excellent measure of spinal motion. ConclusionsWe found that in-plane and through-plane shear deformations are widely present in finite element simulations of a lumbar FCL during flexion. Importantly, we conclude that future studies of the FCL must consider the effects of both shear and tensile deformations.

Layered chitosan-collagen hydrogel/aligned PLLA nanofiber construct for flexor tendon regeneration

The aim of our study was to develop a tendon construct of electrospun aligned poly (l-lactic acid) (PLLA) nanofibers, to mimic the aligned collagen fiber bundles and layering PLLA fibers with chitosan-collagen hydrogel, to mimic the glycosaminoglycans of sheath ECM for tendon regeneration. The hydrogel coated electrospun membrane was rolled and an outer coating of alginate gel was given to prevent peritendinous adhesion. The developed constructs were characterized by SEM, FT-IR and tensile testing. Protein adsorption studies showed lower protein adsorption on coated scaffolds compared to uncoated scaffolds. The samples were proven to be non-toxic to tenocytes. The chitosan-collagen/PLLA uncoated scaffolds and alginate gel coated chitosan-collagen/PLLA scaffolds showed good cell proliferation. The tenocytes showed good attachment and spreading on the scaffolds. This study indicated that the developed chitosan-collagen/PLLA/alginate scaffold would be suitable for flexor tendon regeneration.

Abstract IA24: Forcing through tumor metastasis: Regulation of epithelial-mesenchymal plasticity by matrix stiffness

Abstract In cancer, the presence of dense clusters of collagen fibrils indicates increased matrix stiffness and correlates with poor survival. Previous studies have identified a functional role for matrix stiffness in driving tumor progression, but it is unclear how mechanical inputs are transduced into transcriptional outputs to drive tumor metastasis. To investigate mechanisms of tumor cell mechanotransduction, we utilized a 3D Matrigel culture of murine (Eph4Ras) or human (MCF10A, MCF10DCIS, Bt-549) mammary epithelial cells coupled with a polyacrylamide hydrogel base with calibrated elastic moduli ranging between ~150Pa of normal mammary gland and ~5700Pa of breast tumor tissues. This approach allows for the interrogation of the effects of changes in matrix stiffness independent of other biochemical variables in extracellular matrix (ECM). Using this system, we show that increasing matrix stiffness induced morphological changes resembling the Epithelial-Mesenchymal Transition (EMT) program. Therefore, we hypothesized that increasing matrix stiffness could directly signal through EMT-inducing transcription factors to induce EMT and promote invasion. Using a combination of bioengineering, cell biollogy, proteomic, imaging, and murine tumor modeling approaches, we found that the EMT-inducing transcription factor TWIST1 is an essential mechanomediator that promotes EMT in response to increasing matrix stiffness. Loss of Twist1 prevents matrix stiffness-induced EMT and tumor cell invasion. TGF-beta signaling, a major EMT-inducing biochemical signal, acts synergistically with increasing matrix stiffness, the mechanical cure to induce a complete EMT program in a Twist1-dependent manner. At the compliant matrix stiffness, TWIST1 is sequestered in the cytoplasm by its cytoplasmic binding partner G3BP2, thus preventing TWIST1 from accessing DNA to drive EMT. High matrix stiffness promotes nuclear translocation of TWIST1 by releasing TWIST1 from G3BP2. Loss of G3BP2 leads to constitutive TWIST1 nuclear localization and synergizes with increasing matrix stiffness to induce EMT and invasion in a Twist1-dependent manner. In MCF10DCIS breast tumor xenografts, loss of G3BP2 promotes local invasion and distant metastasis in the lung. In human stage-3 breast tumors, collagen fiber alignment, a marker of increasing matrix stiffness, and reduced expression of G3BP2 together predict poor survival. Together, our findings reveal a TWIST1-G3BP2 mechanotransduction pathway that responds to biomechanical signals from the tumor microenvironment to drive EMT, invasion and metastasis. Citation Format: Jing Yang. Forcing through tumor metastasis: Regulation of epithelial-mesenchymal plasticity by matrix stiffness. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr IA24.

Regional Variation in the Mechanical and Microstructural Properties of the Human Anterior Cruciate Ligament

Background: The anteromedial (AM) bundle of the anterior cruciate ligament (ACL) has a higher modulus and failure stress than does the posterolateral (PL) bundle. However, it is unknown how these properties vary within each bundle. Purpose: To quantify mechanical and microstructural properties of samples within ACL bundles to elucidate any regional variation across the ligament. We hypothesized that there are no differences within each bundle in contrast to cross-bundle variation. Study Design: Descriptive laboratory study. Methods: Sixteen human ACLs were dissected into AM and PL bundles. Three samples were taken from each bundle in an ordered sequence from AM (region 1 AM bundle) to PL (region 6 PL bundle). Each sample was tested in uniaxial tension, using quantitative polarized light imaging (QPLI) to quantify collagen fiber alignment. After preconditioning, samples were subjected to a stress-relaxation (SR) test followed by quasistatic ramp-to-failure (RF). Peak and equilibrium stress values were computed from the SR test and modulus quantified in the toe- and linear-regions of the RF. QPLI values describing collagen orientation (angle of polarization [AoP]) and strength of alignment (degree of linear polarization [DoLP]) were computed for the SR test and at points corresponding to the zero, transition point, and linear region of the RF. Results: Toe- and linear-region modulus values decreased from region 1 to 6. Slopes of regression lines increased for the average DoLP during RF, with significance at higher strains. The standard deviation of AoP values decreased during RF, indicating tighter distribution of orientation angles, with significant correlations at all points of the RF. During SR, stress values uniformly decreased but did not show significant linear regression by region. DoLP and AoP values changed slightly during SR and demonstrated significant linear variation by region at both peak and equilibrium points. Conclusion: Most microstructural and material properties evaluated in this study appear to follow a linear gradient across the ACL, rather than varying by bundle. Clinical Relevance: This AM-to-PL variation provides a more accurate description of functional tissue anatomy and can be used to assess and guide techniques of ACL reconstruction.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

The effect of tendon stem/progenitor cell (TSC) sheet on the early tendon healing in a rat Achilles tendon injury model

Tissue-engineering approaches have a great potential to improve the treatment of tendon injuries that affect millions of people. The present study tested the hypothesis that introduction of a tendon derived stem/progenitor cell (TSC) sheet accelerates tendon healing and tendon regeneration in a rat model. TSC sheets were produced on temperature-responsive culture dishes. Then, they were grafted on unwounded Achilles tendons and at sites of a 3mm of Achilles tendon defect. At 2 and 4weeks after implantation tendons were examined by histology, immunohistochemistry, transmission electron microscopy (TEM) and mechanical testing. The results showed that the implanted TSC sheet remained stably attached on the tendon surface at 4 weeks after implantation. Moreover, in the tendon defect model, tendon defect area where TSC sheet was implanted was well regenerated and had better organized collagen fibers with elongated spindle shaped cells, compared to relatively disorganized collagen fibers and round shaped cells in the control group. TEM observations revealed longitudinally aligned collagen fibers and thick collagen fibrils in the TSC sheet implanted group. Finally, at 4weeks mechanical property of the TSC sheet implanted tendon had better ultimate load than the control. In conclusion, this study demonstrates the feasibility of implanting TSC sheets on tendons in vivo. Introduction of the cell sheets into a tendon defect significantly improved histological properties and collagen content at both 2 and 4 weeks after implantation, indicating that TSC sheets may effectively promote tendon remodeling in the early stages of tendon healing. Statement of SignificanceTendon injury is a highly prevalent clinical problem that debilitates millions of people worldwide in both occupational and athletic settings. It also costs billions of healthcare dollars in treatment every year. In this study, we showed the feasibility of using tendon derived stem cell sheet to deliver biologically active tenogenic-constructs and promote tendon regeneration. This work has the potential to impact the orthopaedic surgery and sports medicine fields in the treatment of tendon injury.