Cell-induced collagen alignment in a 3D in vitro culture during extracellular matrix production.

The bone extracellular matrix consists of a highly organized collagen matrix that is mineralized with carbonated hydroxyapatite. Even though the structure and composition of bone have been studied extensively, the mechanisms underlying collagen matrix organization remain elusive. In this study, we used a 3D cell culture system in which osteogenic cells deposit and orient the collagen matrix that is subsequently mineralized. Using live fluorescence imaging combined with volume electron microscopy, we visualize the organization of the cells and collagen in the cell culture. We show that the osteogenically induced cells are organizing the collagen matrix during development. Based on the observation of tunnel-like structures surrounded by aligned collagen in the center of the culture, we propose that osteoblasts organize the deposited collagen during migration through the culture. Overall, we show that cell-matrix interactions are involved in collagen alignment during early-stage osteogenic differentiation and that the matrix is organized by the osteoblasts in the absence of osteoclast activity.

Collagen architecture and biomechanics of gracilis and adductor longus muscles from children with cerebral palsy.

Cerebral palsy (CP) describes some upper motoneuron disorders due to non-progressive disturbances occurring in the developing brain that cause progressive changes to muscle. While longer sarcomeres increase muscle stiffness in patients with CP compared to typically developing (TD) patients, changes in extracellular matrix (ECM) architecture can increase stiffness. Our goal was to investigate how changes in muscle and ECM architecture impact muscle stiffness, gait and joint function in CP. Gracilis and adductor longus biopsies were collected from children with CP undergoing tendon lengthening surgery for hamstring and hip adduction contractures, respectively. Gracilis biopsies were collected from TD patients undergoing anterior cruciate ligament reconstruction surgery with hamstring autograft. Muscle mechanical testing, two-photon imaging and hydroxyproline assay were performed on biopsies. Corresponding data were compared to radiographic hip displacement in CP adductors (CPA), gait kinematics in CP hamstrings (CPH), and joint range of motion in CPA and CPH. We found at matched sarcomere lengths muscle stiffness and collagen architecture were similar between TD and CP hamstrings. However, CPH stiffness (R2 = 0.1973), collagen content (R2 = 0.5099) and cross-linking (R2 = 0.3233) were correlated to decreased knee range of motion. Additionally, we observed collagen fibres within the muscle ECM increase alignment during muscular stretching. These data demonstrate that while ECM architecture is similar between TD and CP hamstrings, collagen fibres biomechanics are sensitive to muscle strain and may be altered at longer in vivo sarcomere lengths in CP muscle. Future studies could evaluate the impact of ECM architecture on TD and CP muscle stiffness across in vivo operating ranges. KEY POINTS: At matched sarcomere lengths, gracilis muscle mechanics and collagen architecture are similar in TD patients and patients with CP. In both TD and CP muscles, collagen fibres dynamically increase their alignment during muscle stretching. Aspects of muscle mechanics and collagen architecture are predictive of in vivo knee joint motion and radiographic hip displacement in patients with CP. Longer sarcomere lengths in CP muscle in vivo may alter collagen architecture and biomechanics to drive deficits in joint mobility and gait function.

Ameloblastoma modifies tumor microenvironment for enhancing invasiveness by altering collagen alignment.

Tumor progression is profoundly affected by crosstalk between cancer cells and their stroma. In the past decades, the development of bioinformatics and the establishment of organoid model systems have allowed extensive investigation of the relationship between tumor cells and the tumor microenvironment (TME). However, the interaction between tumor cells and the extracellular matrix (ECM) in odontogenic epithelial neoplasms and the ECM remodeling mechanism remain unclear. In the present study, transcriptomic comparison and histopathologic analysis revealed that TME-related genes were upregulated in ameloblastoma compared to in odontogenic keratocysts. Tumoroid analysis indicated that type I collagen is required for ameloblastoma progression. Furthermore, ameloblastoma shows the capacity to remodel the ECM independently of cancer-associated fibroblasts. In conclusion, ameloblastoma-mediated ECM remodeling contributes to the formation of an invasive collagen architecture during tumor progression.

Microfluidically Aligned Collagen to Maintain the Phenotype of Tenocytes In Vitro.

Tendon is a highly organized tissue that transmits forces between muscle and bone. The architecture of the extracellular matrix of tendon, predominantly from collagen type I, is important for maintaining tenocyte phenotype and function. Therefore, in repair and regeneration of damaged and diseased tendon tissue, it is crucial to restore the aligned arrangement of the collagen type I fibers of the original matrix. To this end, a novel, user-friendly microfluidic piggyback platform is developed allowing the controlled patterned formation and alignment of collagen fibers simply on the bottom of culture dishes. Rat tenocytes cultured on the micropatterns of aligned fibrous collagen exhibit a more elongated morphology. The cells also show an increased expression of tenogenic markers at the gene and protein level compared to tenocytes cultured on tissue culture plastic or non-fibrillar collagen coatings. Moreover, using imprinted polystyrene replicas of aligned collagen fibers, this work shows that the fibrillar structure of collagen per se affects the tenocyte morphology, whereas the biochemical nature of collagen plays a prominent role in the expression of tenogenic markers. Beyond the controlled provision of aligned collagen, the microfluidic platform can aid in developing more physiologically relevant in vitro models of tendon and its regeneration.

Twisted-plywood-like tissue formation in vitro. Does curvature do the twist?

Little is known about the contribution of 3D surface geometry to the development of multilayered tissues containing fibrous extracellular matrix components, such as those found in bone. In this study, we elucidate the role of curvature in the formation of chiral, twisted-plywood-like structures. Tissues consisting of murine preosteoblast cells (MC3T3-E1) were grown on 3D scaffolds with constant-mean curvature and negative Gaussian curvature for up to 32 days. Using 3D fluorescence microscopy, the influence of surface curvature on actin stress-fiber alignment and chirality was investigated. To gain mechanistic insights, we did experiments with MC3T3-E1 cells deficient in nuclear A-type lamins or treated with drugs targeting cytoskeleton proteins. We find that wild-type cells form a thick tissue with fibers predominantly aligned along directions of negative curvature, but exhibiting a twist in orientation with respect to older tissues. Fiber orientation is conserved below the tissue surface, thus creating a twisted-plywood-like material. We further show that this alignment pattern strongly depends on the structural components of the cells (A-type lamins, actin, and myosin), showing a role of mechanosensing on tissue organization. Our data indicate the importance of substrate curvature in the formation of 3D tissues and provide insights into the emergence of chirality.

The biomechanics of wounds at physiologically relevant levels: Understanding skin's stress-shielding effect for the quantitative assessment of healing.

Wounds are responsible for the decrease in quality of life of billions of people around the world. Their assessment relies on subjective parameters which often delays optimal treatments and results in increased healthcare costs. In this work, we sought to understand and quantify how wounds at different healing stages (days 1, 3, 7 and 14 post wounding) change the mechanical properties of the tissues that contain them, and how these could be measured at clinically relevant strain levels, as a step towards quantitative wound tracking technologies. To achieve this, we used digital image correlation and mechanical testing on a mouse model of wound healing to map the global and local tissue strains. We found no significant differences in the elastic and viscoelastic properties of wounded vs unwounded skin when samples were measured in bulk, presumably as these were masked by the protective mechanisms of skin, which redistributes the applied loads to mitigate high stresses and reduce tissue damage. By measuring local strain values and observing the distinct patterns they formed, it was possible to establish a connection between the healing phase of the tissue (determined by the time post-injury and the observed histological features) and the overall mechanical behaviour. Importantly, these parameters were measured from the surface of the tissue, using physiologically relevant strains without increasing the tissue's damage. Adaptations of these approaches for clinical use have the potential to aid in the identification of skin healing problems, such as excessive inflammation or lack of mechanical progression over time. An increase, decrease, or lack of change in the elasticity and viscoelasticity parameters, can be indicative of wound state, thus ultimately leading to improved diagnostic outcomes.

Dimensionality Matters: Exploiting UV-Photopatterned 2D and Two-Photon-Printed 2.5D Contact Guidance Cues to Control Corneal Fibroblast Behavior and Collagen Deposition.

In the event of disease or injury, restoration of the native organization of cells and extracellular matrix is crucial for regaining tissue functionality. In the cornea, a highly organized collagenous tissue, keratocytes can align along the anisotropy of the physical microenvironment, providing a blueprint for guiding the organization of the collagenous matrix. Inspired by this physiological process, anisotropic contact guidance cues have been employed to steer the alignment of keratocytes as a first step to engineer in vitro cornea-like tissues. Despite promising results, two major hurdles must still be overcome to advance the field. First, there is an enormous design space to be explored in optimizing cellular contact guidance in three dimensions. Second, the role of contact guidance cues in directing the long-term deposition and organization of extracellular matrix proteins remains unknown. To address these challenges, here we combined two microengineering strategies-UV-based protein patterning (2D) and two-photon polymerization of topographies (2.5D)-to create a library of anisotropic contact guidance cues with systematically varying height (H, 0 µm ≤ H ≤ 20 µm) and width (W, 5 µm ≤ W ≤ 100 µm). With this unique approach, we found that, in the short term (24 h), the orientation and morphology of primary human fibroblastic keratocytes were critically determined not only by the pattern width, but also by the height of the contact guidance cues. Upon extended 7-day cultures, keratocytes were shown to produce a dense, fibrous collagen network along the direction of the contact guidance cues. Moreover, increasing the heights also increased the aligned fraction of deposited collagen and the contact guidance response of cells, all whilst the cells maintained the fibroblastic keratocyte phenotype. Our study thus reveals the importance of dimensionality of the physical microenvironment in steering both cellular organization and the formation of aligned, collagenous tissues.

Matricellular protein CCN1 promotes collagen alignment and scar integrity after myocardial infarction.

BACKGROUND: Members of the cellular communication network family (CCN) of matricellular proteins, like CCN1, have long been implicated in the regulation of cellular processes underlying wound healing, tissue fibrogenesis, and collagen dynamics. While many studies suggest antifibrotic actions for CCN1 in the adult heart through the promotion of myofibroblast senescence, they largely relied on exogenous supplementation strategies in in vivo models of cardiac injury where its expression is already induced-which may confound interpretation of its function in this process. The objective of this study was to interrogate the role of the endogenous protein on fibroblast function, collagen structural dynamics, and its associated impact on cardiac fibrosis after myocardial infarction (MI). METHODS/RESULTS: Here, we employed CCN1 loss-of-function methodologies, including both in vitro siRNA-mediated depletion and in vivo fibroblast-specific knockout mice to assess the role of the endogenous protein on cardiac fibroblast fibrotic signaling, and its involvement in acute scar formation after MI. In vitro depletion of CCN1 reduced cardiac fibroblast senescence and proliferation. Although depletion of CCN1 decreased the expression of collagen processing and stabilization enzymes (i.e., P4HA1, PLOD1, and PLOD2), it did not inhibit myofibroblast induction or type I collagen synthesis. Alone, fibroblast-specific removal of CCN1 did not negatively impact ventricular performance or myocardial collagen content but did contribute to disorganization of collagen fibrils and increased matrix compliance. Similarly, Ccn1 ablated animals subjected to MI showed no discernible alterations in cardiac structure or function one week after permanent coronary artery ligation, but exhibited marked increases in incidence of mortality and cardiac rupture. Consistent with our findings that CCN1 depletion does not assuage myofibroblast conversion or type I collagen synthesis in vitro, Ccn1 knockout animals revealed no measurable differences in collagen scar width or mass compared to controls; however, detailed structural analyses via SHG and TEM of scar regions revealed marked alterations in their scar collagen topography-exhibiting changes in numerous macro- and micro-level collagen architectural attributes. Specifically, Ccn1 knockout mice displayed heightened ECM structural complexity in post-MI scar regions, including diminished local alignment and heightened tortuosity of collagen fibers, as well as reduced organizational coherency, packing, and size of collagen fibrils. Associated with these changes in ECM topography with the loss of CCN1 were reductions in fibroblast-matrix interactions, as evidenced by reduced fibroblast nuclear and cellular deformation in vivo and reduced focal-adhesion formation in vitro; findings that ultimately suggest CCN1's ability to influence fibroblast-led collagen alignment may in part be credited to its capacity to augment fibroblast-matrix interactions. CONCLUSIONS: These findings underscore the pivotal role of endogenous CCN1 in the scar formation process occurring after MI, directing the appropriate arrangement of the extracellular matrix's collagenous components in the maturing scar-shaping the mechanical properties that support its structural stability. While this suggests an adaptive role for CCN1 in regulating collagen structural attributes crucial for supporting scar integrity post MI, the long-term protracted expression of CCN1 holds maladaptive implications, potentially diminishing collagen structural complexity and compliance in non-infarct regions.

A biomechanical model of wound contraction and scar formation.

We propose a biomechanical model for investigating wound contraction mechanism and resulting scarring. Extracellular matrix is modeled as fiber-reinforced anisotropic soft tissue, with its elastic properties dynamically changing with the density and orientation of collagen fibers. Collagen fibers are deposited by fibroblasts infiltrating the wound space, and are dynamically aligned with both migrating fibroblasts and tissue residing tension field. Our new 2D hybrid agent-based model provides a comprehensive platform for examining the mechanobiology in wound contraction and scar formation. Simulation results are consistent with experimental observations and are able to reveal the effects of wound morphology and mechanical environment on contraction patterns. Our model results support the hypothesis that scar formation is the product of collagen fiber synthesis and alignment in the presence of the tensile stress field generated by a wound contraction process.

3D printing of mechanically functional meniscal tissue equivalents using high concentration extracellular matrix inks.

Decellularized extracellular matrix (dECM) has emerged as a promising biomaterial in the fields of tissue engineering and regenerative medicine due to its ability to provide specific biochemical and biophysical cues supportive of the regeneration of diverse tissue types. Such biomaterials have also been used to produce tissue-specific inks and bioinks for 3D printing applications. However, a major limitation associated with the use of such dECM materials is their poor mechanical properties, which limits their use in load-bearing applications such as meniscus regeneration. In this study, native porcine menisci were solubilized and decellularized using different methods to produce highly concentrated dECM inks of differing biochemical content and printability. All dECM inks displayed shear thinning and thixotropic properties, with increased viscosity and improved printability observed at higher pH levels, enabling the 3D printing of anatomically defined meniscal implants. With additional crosslinking of the dECM inks following thermal gelation at pH 11, it was possible to fabricate highly elastic meniscal tissue equivalents with compressive mechanical properties similar to the native tissue. These improved mechanical properties at higher pH correlated with the development of a denser network of smaller diameter collagen fibers. These constructs also displayed repeatable loading and unloading curves when subjected to long-term cyclic compression tests. Moreover, the printing of dECM inks at the appropriate pH promoted a preferential alignment of the collagen fibers. Altogether, these findings demonstrate the potential of 3D printing of highly concentrated meniscus dECM inks to produce mechanically functional and biocompatible implants for meniscal tissue regeneration. This approach could be applied to a wide variety of different biological tissues, enabling the 3D printing of tissue mimics with diverse applications from tissue engineering to surgical planning.

Bioprinting of structurally organized meniscal tissue within anisotropic melt electrowritten scaffolds.

The meniscus is characterised by an anisotropic collagen fibre network which is integral to its biomechanical functionality. The engineering of structurally organized meniscal grafts that mimic the anisotropy of the native tissue remains a significant challenge. In this study, inkjet bioprinting was used to deposit a cell-laden bioink into additively manufactured scaffolds of differing architectures to engineer fibrocartilage grafts with user defined collagen architectures. Polymeric scaffolds consisting of guiding fibre networks with varying aspect ratios (1:1; 1:4; 1:16) were produced using either fused deposition modelling (FDM) or melt electrowriting (MEW), resulting in scaffolds with different internal architectures and fibre diameters. Scaffold architecture was found to influence the spatial organization of the collagen network laid down by the jetted cells, with higher aspect ratios (1:4 and 1:16) supporting the formation of structurally anisotropic tissues. The MEW scaffolds supported the development of a fibrocartilaginous tissue with compressive mechanical properties similar to that of native meniscus, while the anisotropic tensile properties of these constructs could be tuned by altering the fibre network aspect ratio. This MEW framework was then used to generate scaffolds with spatially distinct fibre patterns, which in turn supported the development of heterogenous tissues consisting of isotropic and anisotropic collagen networks. Such bioprinted tissues could potentially form the basis of new treatment options for damaged and diseased meniscal tissue. STATEMENT OF SIGNIFICANCE: This study describes a multiple tool biofabrication strategy which enables the engineering of spatially organized fibrocartilage tissues. The architecture of MEW scaffolds can be tailored to not only modulate the directionality of the collagen fibres laid down by cells, but also to tune the anisotropic tensile mechanical properties of the resulting constructs, thereby enabling the engineering of biomimetic meniscal-like tissues. Furthermore, the inherent flexibility of MEW enables the development of zonally defined and potentially patient-specific implants.

Collagen Fibril Orientation Instructs Fibroblast Differentiation Via Cell Contractility.

Collagen alignment is one of the key microarchitectural signatures of many pathological conditions, including scarring and fibrosis. Investigating how collagen alignment modulates cellular functions will pave the way for understanding tissue scarring and regeneration and new therapeutic strategies. However, current approaches for the fabrication of three-dimensional (3D) aligned collagen matrices are low-throughput and require special devices. To overcome these limitations, a simple approach to reconstitute homogeneous 3D collagen matrices with adjustable degree of fibril alignment using 3D printed inclined surfaces is developed. By characterizing the mechanical properties of reconstituted matrices, it is found that the elastic modulus of collagen matrices is enhanced with an increase in the alignment degree. The reconstituted matrices are used to study fibroblast behavior to reveal the progression of scar formation where a gradual enhancement of collagen alignment can be observed. It is found that matrices with aligned fibrils trigger fibroblast differentiation into myofibroblasts via cell contractility, while collagen stiffening through a crosslinker does not. The results suggest the impact of collagen fibril organization on the regulation of fibroblast differentiation. Overall, this approach to reconstitute 3D collagen matrices with fibril alignment opens opportunities for biomimetic pathological-relevant tissue in vitro, which can be applied for other biomedical research.

A Review of the Collagen Orientation in the Articular Cartilage.

OBJECTIVE: There has been a debate as to the alignment of the collagen fibers. Using a hand lens, Sir William Hunter demonstrated that the collagen fibers ran perpendicular and later aspects were supported by Benninghoff. Despite these 2 historical studies, modern technology has conflicting data on the collagen alignment. DESIGN: Ten mature New Zealand rabbits were used to obtain 40 condyle specimens. The specimens were passed through ascending grades of alcohol, subjected to critical point drying (CPD), and viewed in the scanning electron microscope. Specimens revealed splits from the dehydration process. When observing the fibers exposed within the opening of the splits, parallel fibers were observed to run in a radial direction, normal to the surface of the articular cartilage, radiating from the deep zone and arcading as they approach the surface layer. After these observations, the same samples were mechanically fractured and damaged by scalpel. RESULTS: The splits in the articular surface created deep fissures, exposing parallel bundles of collagen fibers, radiating from the deep zone and arcading as they approach the surface layer. On higher magnification, individual fibers were observed to run parallel to one another, traversing radially toward the surface of the articular cartilage and arcading. Mechanical fracturing and scalpel damage induced on the same specimens with the splits showed randomly oriented fibers. CONCLUSION: Collagen fiber orientation corroborates aspects of Hunter's findings and compliments Benninghoff. Investigators must be aware of the limits of their processing and imaging techniques in order to interpret collagen fiber orientation in cartilage.

Directional cues in the tumor microenvironment due to cell contraction against aligned collagen fibers.

It is well established that collagen alignment in the breast tumor microenvironment provides biophysical cues to drive disease progression. Numerous mechanistic studies have demonstrated that tumor cell behavior is driven by the architecture and stiffness of the collagen matrix. However, the mechanical properties within a 3D collagen microenvironment, particularly at the scale of the cell, remain poorly defined. To investigate cell-scale mechanical cues with respect to local collagen architecture, we employed a combination of intravital imaging of the mammary tumor microenvironment and a 3D collagen gel system with both acellular pNIPAAm microspheres and MDA-MB-231 breast carcinoma cells. Within the in vivo tumor microenvironment, the displacement of collagen fiber was identified in response to tumor cells migrating through the stromal matrix. To further investigate cell-scale stiffness in aligned fiber architectures and the propagation of cell-induced fiber deformations, precise control of collagen architecture was coupled with innovative methodology to measure mechanical properties of the collagen fiber network. This method revealed up to a 35-fold difference in directional cell-scale stiffness resulting from contraction against aligned fibers. Furthermore, the local anisotropy of the matrix dramatically altered the rate at which contractility-induced fiber displacements decayed over distance. Together, our results reveal mechanical properties in aligned matrices that provide dramatically different cues to the cell in perpendicular directions. These findings are supported by the mechanosensing behavior of tumor cells and have important implications for cell-cell communication within the tissue microenvironment. STATEMENT OF SIGNIFICANCE: It is widely appreciated that the architecture of the extracellular matrix impacts cellular behavior in normal and disease states. Numerous studies have determined the fundamental role of collagen matrix architecture on cellular mechanosensing, but effectively quantifying anisotropic mechanical properties of the collagen matrix at the cell-scale remains challenging. Here, we developed innovative methodology to discover that collagen alignment results in a 35-fold difference in cell-scale stiffness and alters contractile force transmission through the fiber network. Furthermore, we identified bias in cell response along the axis of alignment, where local stiffness is highest. Overall, our results define cell-scale stiffness and fiber deformations due to collagen architecture that may instruct cell communication within a broad range of tissue microenvironments.

Geometrical confinement controls cell, ECM and vascular network alignment during the morphogenesis of 3D bioengineered human connective tissues.

Engineered tissues featuring aligned ECM possess superior regenerative capabilities for the healing of damaged aligned tissues. The morphofunctional integration in the host's injury site improves if the aligned ECM elicits the unidirectional growth of vascular network. In this work we used a bottom-up tissue engineering strategy to produce endogenous and highly aligned human connective tissues with the final aim to trigger the unidirectional growth of capillary-like structures. Engineered microtissues, previously developed by our group, were casted in molds featured by different aspect ratio (AR) to obtain final centimeter-sized macrotissues differently shaped. By varying the AR from 1 to 50 we were able to vary the final shape of the macrotissues, from square to wire. We demonstrated that by increasing the AR of the maturation space hosting the microtissues, it was possible to control the alignment of the neo-synthesized ECM. The geometrical confinement conditions at AR = 50, indeed, promoted the unidirectional growth and assembly of the collagen network. The wire-shaped tissues were characterized by parallel arrangement of the collagen fiber bundles, higher persistence length and speed of migrating cells and superior mechanical properties than the square-shaped macrotissues. Interestingly, the aligned collagen fibers elicited the unidirectional growth of capillary-like structures. STATEMENT OF SIGNIFICANCE: Alignment of preexisting extracellular matrices by using mechanical cues modulating cell traction, has been widely described. Here, we show a new method to align de novo synthesized extracellular matrix components in bioengineered connective tissues obtained by means of a bottom-up tissue engineering approach. Building blocks are cast in maturation chambers, having different aspect ratios, in which the in vitro morphogenesis process takes place. High aspect ratio chambers (corresponding to wire-shaped tissues) triggered spontaneous alignment of collagenous network affecting cell polarization, migration and tensile properties of the tissue as well. Aligned ECM provided a contact guidance for the formation of highly polarized capillary-like network suggesting an in vivo possible application to trigger fast angiogenesis and perfusion in damaged aligned tissues.

Fibroblasts treated with macrophage conditioned medium results in phenotypic shifts and changes in collagen organization.

In tissue regeneration, the goal is to regenerate tissue similar to what was damaged or missing while preventing fibrotic scarring, which may lead to decreased mechanical strength and dissimilar tissue characteristics compared to native tissue. We believe collagen orientation plays a critical role in wound contraction and scarring and that it is modulated by myofibroblasts. We used macrophage conditioned medium to simulate complex events that can influence the fibroblast phenotype during the wound healing process. In addition to examining the effect of macrophage phenotype on fibroblasts, we inhibited focal adhesion kinase (FAK), Rho-associated protein kinase (ROCK), and myosin II for fibroblasts cultured on both tissue culture plastic and methacrylated gellan gum to understand how different pathways and materials influence fibroblast responses. Collagen orientation, α-SMA expression, focal adhesion area, and cell migration were altered by inhibition of FAK, ROCK, or myosin II and macrophage phenotype, along with the substrate. An increase in either focal adhesion area or α-smooth muscle actin (α-SMA) expression correlated with an aligned collagen orientation. Gellan gum hydrogels upregulated α-SMA expression in ROCK inhibited conditioned media and downregulated the FAK area in FAK and ROCK inhibited conditioned media. Myosin II had no impact on the α-SMA expression on the substrate compared to coverslip except for M2 conditioned medium. Gellan gum hydrogel significantly increased cell migration under FAK and Myosin II mediated conditioned media and unconditioned media. Collectively, our study examined how macrophage phenotype influences fibroblast response, which would be beneficial in controlling scar tissue formation.

Mechanical Properties and Microstructural Collagen Alignment of the Ulnar Collateral Ligament During Dynamic Loading.

BACKGROUND: The ulnar collateral ligament (UCL) microstructural organization and collagen fiber realignment in response to load are unknown. PURPOSE/HYPOTHESIS: The purpose was to describe the real-time microstructural collagen changes in the anterior bundle (AB) and posterior bundle (PB) of the UCL with tensile load. It was hypothesized that the UCL AB is stronger and stiffer with more highly aligned collagen during loading when compared with the UCL PB. STUDY DESIGN: Descriptive laboratory study. METHODS: The AB and PB from 34 fresh cadaveric specimens were longitudinally sectioned to allow uniform light passage for quantitative polarized light imaging. Specimens were secured to a tensile test machine and underwent cyclic preconditioning, a ramp-and-hold stress-relaxation test, and a quasi-static ramp to failure. A division-of-focal-plane polarization camera captured real-time pixelwise microstructural data of each sample during stress-relaxation and at the zero, transition, and linear points of the stress-strain curve. The SD of the angle of polarization determined the deviation of the average direction of collagen fibers in the tissue, while the average degree of linear polarization evaluated the strength of collagen alignment in those directions. Since the data were nonnormally distributed, the median ± interquartile range are presented. RESULTS: The AB has larger elastic moduli than the PB ( P < .0001) in the toe region (median, 2.73 MPa [interquartile range, 1.1-5.6 MPa] vs 0.65 MPa [0.44-1.5 MPa]) and the linear region (13.77 MPa [4.8-40.7 MPa] vs 1.96 MPa [0.58-9.3 MPa]). The AB demonstrated larger stress values, stronger collagen alignment, and more uniform collagen organization during stress-relaxation. PB collagen fibers were more disorganized than the AB during the zero ( P = .046), transitional ( P = .011), and linear ( P = .007) regions of the stress-strain curve. Both UCL bundles exhibited very small changes in collagen alignment (SD of the angle of polarization) with load. CONCLUSION: The AB of the UCL is stiffer and stronger, with more strongly aligned and more uniformly oriented collagen fibers, than the PB. The small changes in collagen alignment indicate that the UCL response to load is due more to its static collagen organization than to dynamic changes in collagen alignment. CLINICAL RELEVANCE: The UCL collagen organization may explain its susceptibility to injury with repetitive valgus loads.

Achilles tendon structure differs between competitive distance runners and nonrunners despite no clinical signs or symptoms of midsubstance tendinopathy.

Achilles tendinopathy affects many running athletes and often leads to chronic pain and functional deficits. Although changes in tendon structure have been linked with tendinopathy, the effects of distance running on tendon structure are not well understood. Therefore, the purpose of this study was to characterize structural differences in the Achilles tendons in healthy young adults and competitive distance runners using quantitative ultrasound analyses. We hypothesized that competitive distance runners with no clinical signs or symptoms of tendinopathy would have quantitative signs of tendon damage, characterized by decreased collagen alignment and echogenicity, in addition to previous reports of thicker tendons. Longitudinal ultrasound images of the right Achilles tendon midsubstance were acquired in competitive distance runners and recreationally active adults. Collagen organization, mean echogenicity, and tendon thickness were quantified using image processing techniques. Clinical assessments confirmed that runners had no signs or symptoms of tendinopathy, and controls were only included if they had no history of Achilles tendon pain or injuries. Runner tendons were 40% less organized, 48% thicker, and 41% less echogenic compared with the control tendons ( P < 0.001). Young adults engaged in competitive distance running have structurally different tendons than recreationally active young adults. NEW & NOTEWORTHY In this study, we quantified the Achilles tendon substructure in distance runners, and a control group of young adults, to determine whether distance running elicits structural adaptations of the tendon. We found that competitive distance runners have structurally compromised Achilles tendons despite not showing any clinical signs or symptoms of tendon injury. These findings suggest that distance running may stimulate structural changes as a protective mechanism against tendon pain and dysfunction.

Collagen Fiber Orientation Regulates 3D Vascular Network Formation and Alignment.

Alignment of collagen type I fibers is a hallmark of both physiological and pathological tissue remodeling. However, the effects of collagen fiber orientation on endothelial cell behavior and vascular network formation are poorly understood because of a lack of model systems that allow studying these potential functional connections. By casting collagen type I into prestrained (0, 10, 25, 50% strain), poly(dimethylsiloxane) (PDMS)-based microwells and releasing the mold strain following polymerization, we have created collagen gels with varying fiber alignment as confirmed by structural analysis. Endothelial cells embedded within the different gels responded to increased collagen fiber orientation by assembling into 3D vascular networks that consisted of thicker, more aligned branches and featured elevated collagen IV deposition and lumen formation relative to control conditions. These substrate-dependent changes in microvascular network formation were associated with altered cell division and migration patterns and related to enhanced mechanosignaling. Our studies indicate that collagen fiber alignment can directly regulate vascular network formation and that culture models with aligned collagen may be used to investigate the underlying mechanisms ultimately advancing our understanding of tissue development, homeostasis, and disease.

Tendon structure quantified using ultrasound imaging differs based on location and training type.

Achilles tendinopathy is 10 times more common among running athletes compared with age-matched peers. Load-induced tendon remodeling and its progression in an at-risk population of developing symptomatic tendinopathy are not well understood. The purpose of this study was to prospectively characterize Achilles and patellar tendon structure in competitive collegiate distance runners over different competitive seasons using quantitative ultrasound imaging. Twenty-two collegiate cross-country runners and eleven controls were examined for this study. Ultrasound images of bilateral Achilles and patellar tendons were obtained near the start and end of the collegiate cross-country season and the conclusion outdoor track season. Collagen organization, mean echogenicity, tendon thickness, and neovascularity were determined using well-established image processing techniques. Achilles tendon collagen was less aligned in runners compared with controls (28% greater) but improved slightly (7% decrease) after the completion of the track season. Conversely, patellar tendons in runners were similar to control tendons throughout the cross-country season but underwent collagen alignment (17% decrease) and tendon hypertrophy (21% increase). Our findings indicate that Achilles tendon structure in trained runners differs structurally from control tendons but is stable throughout training while patellar tendon structure changes in response to the transition in training volume between cross-country and track seasons. These findings expand upon prior reports that some degree of tendon remodeling may act as a protective adaptation for sport specific loading.NEW & NOTEWORTHY In this study we prospectively examined the Achilles and patellar tendon structure of distance runners to determine if continued training through multiple seasons elicits tendon remodeling or pathology. We found that Achilles and patellar tendons respond uniquely to the changing loads required during each season. Achilles tendon collagen alignment is mostly stable throughout the competitive cycle, but the patellar tendon structurally remodels following the transition from cross-country to track season.