Aligned Collagen Fibrils Research Articles

Matrix stiffening from collagen fibril density and alignment modulates YAP-mediated T-cell immune suppression

T-cells are essential components of the immune system, adapting their behavior in response to the mechanical environments they encounter within the body. In pathological conditions like cancer, the extracellular matrix (ECM) often becomes stiffer due to increased density and alignment of collagen fibrils, which can have a significant impact on T-cell function. In this study, we explored how these ECM properties—density and fibrillar alignment—affect T-cell behavior using three-dimensional (3D) collagen matrices that mimic these conditions. Our results show that increased matrix stiffness, whether due to higher density or alignment, significantly suppresses T-cell activation, reduces cytokine production, and limits proliferation, largely through enhanced YAP signaling. Individually, matrix alignment appears to lower actin levels in activated T-cells and changes migration behavior in both resting and activated T-cells, an effect not observed in matrices with randomly oriented fibrils. Notably, inhibiting YAP signaling was able to restore T-cell activation and improve immune responses, suggesting a potential strategy to boost the effectiveness of immunotherapy in stiff ECM environments. Overall, this study provides new insights into how ECM characteristics influence T-cell function, offering potential avenues for overcoming ECM-induced immunosuppression in diseases such as cancer.

Fibrotic Fibro-Adipogenic Progenitors Produce an Altered Extracellular Matrix that Impairs Myogenesis

Introduction: Fibrosis is the pathological accumulation of extracellular matrix (ECM) components, impairing tissue function. In skeletal muscle, the main source of ECM deposition are fibro-adipogenic progenitors (FAPs). FAPs can signal to muscle satellite cells (MuSCs) through soluble factors and ECM deposition. FAPs are more highly activated into myofibroblasts in fibrotic conditions, but how this affects their signaling to MuSCs is unknown. Cell-derived matrices (CDMs) can be synthesized from FAPs to investigate their ECM deposition. The goal of this study was to understand how FAPs are signaling to MuSCs through their ECM deposition. We hypothesized that FAPs isolated from mdx mice, a model for skeletal muscle fibrosis, would have increased collagen deposition compared to wt FAPs and would produce a matrix that reduces myogenesis. Methods: FAPs were isolated from the lower limb muscles of wt and mdx mice. FAPs were plated in media containing 50μM ascorbic acid to promote collagen deposition for 2 weeks in order to synthesize CDMs. CDMs were imaged using second harmonic generation (SHG) imaging to visualize collagen organization. Some CDMs were solubilized and replated to create a flat protein coating of matrix. MuSCs were then plated on the CDMs, solubilized matrix, in a transwell co-culture with FAPs, and on plastic in differentiation media for 5 days. Results: There was a significant negative correlation between the level of FAP activation into myofibroblasts and the MuSC differentiation index, independent of if the FAPs were isolated from wt or mdx mice. SHG images revealed CDMs derived from mdx FAPs had less aligned collagen fibrils compared to wt CDMs. Conclusions: High activation into myofibroblasts is often an indicator of a fibrotic FAP phenotype. The negative correlation between myofibroblasts and MuSC differentiation found here indicates this phenotype impairs myogenesis. FAPs in transwell co-culture did not have a significant effect on MuSC differentiation, highlighting the importance of ECM deposition rather than soluble signaling is important in FAP signaling to MuSCs. Targeting FAPs activation into myofibroblasts provides a therapeutic target to attenuate fibrosis and improve regeneration in skeletal muscle. Research reported in this publication was supported by the National Institute Of Arthritis And Musculoskeletal And Skin Diseases of the National Institutes of Health under Award Number F31AR082700. The content is solely the responsibility of the authors and does not necessarily represent the offcial views of the National Institutes of Health. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Glycosylation Modulates the Structure and Functions of Collagen: A Review.

Collagens are fundamental constituents of the extracellular matrix and are the most abundant proteins in mammals. Collagens belong to the family of fibrous or fiber-forming proteins that self-assemble into fibrils that define their mechanical properties and biological functions. Up to now, 28 members of the collagen superfamily have been recognized. Collagen biosynthesis occurs in the endoplasmic reticulum, where specific post-translational modification-glycosylation-is also carried out. The glycosylation of collagens is very specific and adds β-d-galactopyranose and β-d-Glcp-(1→2)-d-Galp disaccharide through β-O-linkage to hydroxylysine. Several glycosyltransferases, namely COLGALT1, COLGALT2, LH3, and PGGHG glucosidase, were associated the with glycosylation of collagens, and recently, the crystal structure of LH3 has been solved. Although not fully understood, it is clear that the glycosylation of collagens influences collagen secretion and the alignment of collagen fibrils. A growing body of evidence also associates the glycosylation of collagen with its functions and various human diseases. Recent progress in understanding collagen glycosylation allows for the exploitation of its therapeutic potential and the discovery of new agents. This review will discuss the relevant contributions to understanding the glycosylation of collagens. Then, glycosyltransferases involved in collagen glycosylation, their structure, and catalytic mechanism will be surveyed. Furthermore, the involvement of glycosylation in collagen functions and collagen glycosylation-related diseases will be discussed.

Braided biomimetic PCL grafts for anterior cruciate ligament repair and regeneration

Anterior cruciate ligament (ACL) is a knee joint stabilizer with a limited regeneration capacity mainly because of low cellular content. State-of-the-art procedures are unable to restore the functions of the tissue as demonstrated by limited success rates. Regenerative engineering can offer a solution for restoring the functions of torn/ruptured ligaments provided that biomimetic grafts are available as grafts/scaffolds. However, a model construct to test behavior of cells to better understand the healing mechanism of ACL is still missing. This study, firstly, aimed at creating an injured rabbit ACL model. Then, the injured and healthy ACL tissues were characterized in terms of alignment and diameter distributions of collagen fibrils. Next, polycaprolactone (PCL) grafts were prepared from braided electrospun meshes and were characterized in terms of alignment and diameter distributions of fibers. Finally, biomechanical properties of ACL tissue and mechanical properties of PCL grafts were determined and compared. Findings demonstrated that distributions of the fiber diameters of PCL electrospun grafts were similar to diameter distribution of collagens of healthy and injured rabbit ACL. The novelty of this study relies on the determination of the diameter distribution of collagens of healthy and injured rabbit ACL tissues, and fabrication of PCL grafts with diameter distributions similar to that seen in healthy and injured ACLs. This study is significant because it addresses a worldwide clinical problem associated with millions of patients. The fibrous biomimetic graft designed in this study is different from the traditional grafts that exhibit unimodal distribution, and it is expected to have a significant contribution to ACL regeneration efforts.

Fabrication of Micropatterns of Aligned Collagen Fibrils.

Many tissues in vivo contain aligned structures such as filaments, fibrils, and fibers, which expose cells to anisotropic structural and topographical cues that range from the nanometer to micrometer scales. Understanding how cell behavior is regulated by these cues during physiological and pathological processes (e.g., wound healing, cancer invasion) requires substrates that can expose cells to anisotropic cues over several length scales. In this study, we developed a novel method of fabricating micropatterns of aligned collagen fibrils of different geometry onto PDMS-coated glass coverslips that allowed us to investigate the roles of topography and confinement on corneal cell behavior. When corneal cells were cultured on micropatterns of aligned collagen fibrils in the absence of confinement, the degree of cell alignment increased from 40 ± 14 to 82 ± 5% as the size of the micropattern width decreased from 750 to 50 μm. Although the cell area (∼2500 μm2), cell length (∼160 μm), and projected nuclear area (∼175 μm2) were relatively constant on the different micropattern widths, cells displayed an increased aspect ratio as the width of the aligned collagen fibril micropatterns decreased. We also observed that the morphology of cells adhering to the surrounding uncoated PDMS was dependent upon both the size of the aligned collagen fibril micropattern and the distance from the micropatterns. When corneal cells were confined to the micropatterns of aligned collagen fibrils by a Pluronic coating to passivate the surrounding area, a similar trend in increasing cell alignment was observed (35 ± 10 to 89 ± 2%). However, the projected nuclear area decreased significantly (∼210 to 130 μm2) as the micropattern width decreased from 750 to 50 μm. The development of this method allows for the deposition of aligned collagen fibril micropatterns of different geometries on a transparent and elastic substrate and provides an excellent model system to investigate the role of anisotropic cues in cell behavior.

Programming fibril alignment and mechanical response in reconstituted collagen fibers using reagent-free biomimetic energetic electron crosslinking

High energy electron beam treatment is shown to permanently imprint prestrain-induced alignment of collagen fibrils within macroscopic collagen fibers by introduction of biomimetic crosslinks, bridging orders of magnitude in Young's moduli.

Design matters: A comparison of natural versus synthetic skin substitutes across benchtop and porcine wound healing metrics: An experimental study.

Skin substitutes, essential tools for helping close full thickness wounds with minimal scarring, are available in both collagen-based and synthetic polyurethane constructions. Here we explore fundamental differences between two frequently used skin substitutes and discuss how these differences may impact in vivo performance. Polyurethane- and collagen-based matrices were characterized in vitro for pore size via scanning electron microscopy, hydrophobicity via liquid contact angle, conformability via bending angle, and biocompatibility via fibroblast and keratinocyte adhesion and proliferation. These matrices were then evaluated in a full-thickness excisional pig wound study followed by histological analysis. Statistical analysis was performed using t-tests or one-way analysis of variances with Tukey's multiple post hoc comparisons, where appropriate. Average pore diameter in the tested polyurethane matrix was over four times larger than that of the collagen matrix (589 ± 297 µm vs. 132 ± 91 µm). Through liquid contact angle measurement, the collagen matrix (not measurable) was found to be hydrophilic compared to the hydrophobic polyurethane matrix (>90°). The collagen matrix was significantly more conformable than the polyurethane matrix (9 ± 2° vs. 84 ± 5° bending angle, respectively). Fibroblast and keratinocyte adhesion and proliferation assays elucidated a significantly greater ability of both cell types to attach and proliferate on collagen versus polyurethane. While the porcine study showed minimal contraction of either matrix material, histological findings between the two treatments were markedly different. Collagen matrices were associated with early fibroblast infiltration and fibroplasia, whereas polyurethane matrices elicited a strong multinucleated giant cell response and produced a network of comparatively aligned collagen fibrils. The more favorable in vitro properties of the collagen matrix led to less inflammation and better overall tissue response in vivo. Overall, our findings demonstrate how the choice of biomaterial and its design directly translate to differing in vivo mechanisms of action and overall tissue quality.

New Insights on the Biomechanics of the Fetal Membrane.

During pregnancy, the Fetal Membrane (FM) is subjected to mechanical stretching that may result in preterm labor. The structural integrity of the FM is maintained by its collagenous layer. Disconnection and reconnection of molecular bonds between collagen fibrils is the fundamental process that governs the irreversible mechanical and supramolecular changes in the FM. At a critical threshold strain, bundling and alignment of collagen fibrils alter the super-molecular structure of the collagenous layer. Recent studies indicate that these changes are associated with inflammation and/or expression of specific proteins that are known to be related to uterine contractions and labor. The potential healing of stretching-induced damages in the FM by mediators involved in mechano-transduction is discussed.

Effects of Topography and PDGF on the Response of Corneal Keratocytes to Fibronectin-Coated Surfaces.

During corneal wound healing, corneal keratocytes are exposed to both biophysical and soluble cues that cause them to transform from a quiescent state to a repair phenotype. How keratocytes integrate these multiple cues simultaneously is not well understood. To investigate this process, primary rabbit corneal keratocytes were cultured on substrates patterned with aligned collagen fibrils and coated with adsorbed fibronectin. After 2 or 5 days of culture, keratocytes were fixed and stained to assess changes in cell morphology and markers of myofibroblastic activation by fluorescence microscopy. Initially, adsorbed fibronectin had an activating effect on the keratocytes as evidenced by changes in cell shape, stress fiber formation, and expression of alpha-smooth muscle actin (α-SMA). The magnitude of these effects depended upon substrate topography (i.e., flat substrate vs aligned collagen fibrils) and decreased with culture time. When keratocytes were simultaneously exposed to adsorbed fibronectin and soluble platelet-derived growth factor-BB (PDGF-BB), the cells elongated and had reduced expression of stress fibers and α-SMA. In the presence of PDGF-BB, keratocytes plated on the aligned collagen fibrils elongated in the direction of the fibrils. These results provide new information on how keratocytes respond to multiple simultaneous cues and how the anisotropic topography of aligned collagen fibrils influences keratocyte behavior.

Temporal application of lysyl oxidase during hierarchical collagen fiber formation differentially effects tissue mechanics.

The primary source of strength in menisci, tendons, and ligaments are hierarchical collagen fibers; however, these fibers are not regenerated after injury nor in engineered replacements, resulting in limited repair options. Collagen strength is reliant on fiber alignment, density, diameter, and crosslinking. Recently, we developed a culture system which guides cells in high-density collagen gels to develop native-like hierarchically organized collagen fibers, which match native fiber alignment and diameters by 6 weeks. However, tensile moduli plateau at 1MPa, suggesting crosslinking may be lacking. Collagen crosslinking is regulated by lysyl oxidase (LOX) which forms immature crosslinks that condense into mature trivalent crosslinks. Trivalent crosslinks are thought to be the primarily source of strength in fibers, but it's not well understood how they form. The objective of this study was to evaluate the effect of exogenous LOX in our culture system at different stages of hierarchical fiber formation to produce stronger replacements and to better understand factors affecting crosslink maturation. We found treatment with LOX isoform LOXL2 did not restrict hierarchical fiber formation, with constructs still forming aligned collagen fibrils by 2 weeks, larger fibers by 4 weeks, and early fascicles by 6 weeks. However, LOXL2 treatment did significantly increase mature pyridinium (PYD) crosslink accumulation and tissue mechanics, with timing of LOXL2 supplementation during fiber formation having a significant effect. Overall, we found one week of LOXL2 supplementation at 4 weeks produced constructs with native-like fiber organization, increased PYD accumulation, and increased mechanics, ultimately matching the tensile modulus of immature bovine menisci. STATEMENT OF SIGNIFICANCE: Collagen fibers are the primary source of strength and function in connective tissues throughout the body, however it remains a challenge to develop these fibers in engineered replacements, greatly reducing treatment options. Here we demonstrate lysyl oxidase like 2 (LOXL2) can be used to significantly improve the mechanics of tissue engineered constructs, but timing of application is important and will most likely depend on degree of collagen organization or maturation. Currently there is limited understanding of how collagen crosslinking is regulated, and this system is a promising platform to further investigate cellular regulation of LOX crosslinking. Understanding the mechanism that regulates LOX production and activity is needed to ultimately regenerate functional repair or replacements for connective tissues throughout the body.

Anisotropic dense collagen hydrogels with two ranges of porosity to mimic the skeletal muscle extracellular matrix

Despite the crucial role of the extracellular matrix (ECM) in the organotypic organization and function of skeletal muscles, most 3D models do not mimic its specific characteristics, namely its biochemical composition, stiffness, anisotropy, and porosity. Here, a novel 3D in vitro model of muscle ECM was developed reproducing these four crucial characteristics of the native ECM. An anisotropic hydrogel mimicking the muscle fascia was obtained thanks to unidirectional 3D printing of dense collagen with aligned collagen fibrils. The space between the different layers was tuned to generate an intrinsic network of pores (100 μm) suitable for nutrient and oxygen diffusion. By modulating the gelling conditions, the mechanical properties of the construct reached those measured in the physiological muscle ECM. This artificial matrix was thus evaluated for myoblast differentiation. The addition of large channels (600 μm) by molding permitted to create a second range of porosity suitable for cell colonization without altering the physical properties of the hydrogel. Skeletal myoblasts embedded in Matrigel®, seeded within the channels, organized in 3D, and differentiated into multinucleated myotubes. These results show that porous and anisotropic dense collagen hydrogels are promising biomaterials to model skeletal muscle ECM.

Bioprinted living tissue constructs with layer-specific, growth factor-loaded microspheres for improved enthesis healing of a rotator cuff

Substantial challenges remain in constructing the native tendon-to-bone interface for rotator cuff healing owing to the enthesis tissues' highly organized structural and compositional gradients. Herein, we propose to bioprint living tissue constructs with layer-specific growth factors (GFs) to promote enthesis regeneration by guiding the zonal differentiation of the loaded stem cells in situ. The sustained release of tenogenic, chondrogenic, and osteogenic GFs was achieved via microsphere-based delivery carriers embedded in the bioprinted constructs. Compared to the basal construct without GFs, the layer-specific tissue analogs realized region-specific differentiation of stem cells in vitro. More importantly, bioprinted living tissue constructs with layer-specific GFs rapidly enhanced the enthesis regeneration in a rabbit rotator cuff tear model in terms of biomechanical restoration, collagen deposition, and alignment, showing gradient interface of fibrocartilage structures with aligned collagen fibrils and an ultimate load failure of 154.3 ± 9.5 N resembling those of native enthesis tissues in 12 weeks. This exploration provides a feasible strategy to engineer living tissue constructions with region-specific differentiation potentials for the functional repair of gradient enthesis tissues. Statement of significancePrevious studies that employed acellular layer-specific scaffolds or stem cells for the reconstruction of the rotator cuff faced challenges due to their insufficient capability to rebuild the anisotropic compositional and structural gradients of native enthesis tissues. This manuscript proposed a living tissue construct with layer-specific, GFs-loaded µS, which can direct in situ and region-specific differentiation of the embedded stem cells to tenogenic, chondrogenic, and osteogenic lineages for functional regeneration of the enthesis tissues. This bioprinted living tissue construct with the unique capability to reduce fibrovascular scar tissue formation and simultaneously facilitate enthesis tissue remodeling might provide a promising strategy to repair complex and gradient tissues in the future.

Distinctive structure, composition and biomechanics of collagen fibrils in vaginal wall connective tissues associated with pelvic organ prolapse

Collagen is the predominant structural protein within connective tissues. Pelvic organ prolapse (POP) is characterized by weakening of the pelvic floor connective tissues and loss of support for pelvic organs. In this study, we examined the multiscale structure, molecular composition and biomechanics of native collagen fibrils in connective tissues of the posterior vaginal fornix collected from healthy women and POP patients, and established the correlation of these properties with clinical POP quantification (POP-Q) scores. The collagen characteristics, including collagen amount, ratio of Collagen I and Collagen III, collagen fibril d-period, alignment and stiffness, were found to change progressively with the increase of the clinical measurement of Point C, a measure of uterine descent and apical prolapse. The results imply that a severe prolapse is associated with stiffer collagen fibrils, reduced collagen d-period, increased fibril alignment and imbalanced collagen synthesis, degradation and deposition. Additionally, prolapse progression appears to be synchronized with deterioration of the collagen matrix, suggesting that a POP-Q score obtained via a non-invasive clinical test can be potentially used to quantitatively assess collagen abnormality of a patient's local tissue. Statement of significanceAbnormal collagen metabolism and deposition are known to associate with connective tissue disorders, such as pelvic organ prolapse. Quantitative correlation of the biochemical and biophysical characteristics of collagen in a prolapse patient's tissue with the clinical diagnostic measurements is unexplored and unestablished. This study fills the knowledge gap between clinical prolapse quantification and the individual's cellular and molecular disorders leading to connective tissue failure, thus, provides the basis for clinicians to employ personalized treatment that can best manage the patient's condition and to alert pre-symptomatic patients for early management to avoid unwanted surgery.

A Biomimetic High Throughput Model of Cancer Cell Spheroid Dissemination onto Aligned Fibrillar Collagen.

Cell dissemination during tumor development is a characteristic of cancer metastasis. Dissemination from three-dimensional spheroid models on extracellular matrices designed to mimic tissue-specific physiological microenvironments may allow us to better elucidate the mechanism behind cancer metastasis and the response to therapeutic agents. The orientation of fibrillar collagen plays a key role in cellular processes and mediates metastasis through contact-guidance. Understanding how cells migrate on aligned collagen fibrils requires in vitro assays with reproducible and standardized orientation of collagen fibrils on the macro-to-nanoscale. Herein, we implement a spheroid-based migration assay, integrated with a fibrillar type I collagen matrix, in a manner compatible with high throughput image acquisition and quantitative analysis. The migration of highly proliferating U2OS osteosarcoma cell spheroids onto an aligned fibrillar type I collagen matrix was quantified. Cell dissemination from the spheroid was polarized with increased invasion in the direction of fibril alignment. The resulting area of cell dissemination had an aspect ratio of 1.2 ± 0.1 and an angle of maximum invasion distance of 5° ± 44° relative to the direction of collagen fibril alignment. The assay described here can be applied to a fully automated imaging and analysis pipeline for the assessment of tumor cell migration with high throughput screening.

Structure and Strength of Bovine and Equine Amniotic Membrane.

Simple SummaryThin, strong scaffold materials are needed for surgical applications. There is a limited selection of available materials and new materials are required. Amnionic membrane from cattle and horses were investigated for this purpose. The structure of these materials was characterized with synchrotron techniques and the strength was measured. A possible relationship between the structure and strength was identified. These amnion materials from animal sources are strong, thin, and elastic materials, although weaker than some other collagen tissues. They may be suitable for use in surgery as an alternative to material from human donors.Thin, strong scaffold materials are needed for surgical applications. New materials are required, particularly those readily available, such as from non-human sources. Bovine amniotic membrane (antepartum) and equine amniotic membrane (postpartum) were characterized with tear and tensile tests. The structural arrangement of the collagen fibrils was determined by small-angle X-ray scattering, scanning electron microscopy, and ultrasonic imaging. Bovine amnion had a thickness-normalized tear strength of 12.6 (3.8) N/mm, while equine amnion was 14.8 (5.3) N/mm. SAXS analysis of the collagen fibril arrangement yielded an orientation index of 0.587 (0.06) and 0.681 (0.05) for bovine and equine, respectively. This may indicate a relationship between more highly aligned collagen fibrils and greater strength, as seen in other materials. Amnion from bovine or equine sources are strong, thin, elastic materials, although weaker than other collagen tissue materials commonly used, that may find application in surgery as an alternative to material from human donors.

Rapid fabrication of tendon-like collagen gel via simultaneous fibre alignment and intermolecular cross-linking under mechanical loading

Artificial tissue replacement is a promising strategy for better healing outcomes for tendon and ligament injuries, due to the very limited self-regeneration capacity of these tissues in mammals, including humans. Because clinically available synthetic and biological scaffolds for tendon repair have performed more poorly than autografts, both biological and mechanical compatibility need to be improved. Here we propose a rapid fabrication method for tendon-like structure from collagen hydrogel, simultaneously achieving collagen fibre alignment and intermolecular cross-linking. Collagen gel, 24 h after polymerization, was subjected to mechanical loading in the presence of the chemical cross-linker, genipin, for 24 or 48 h. Mechanical loading during gel incubation oriented collagen fibres in the loading direction and made chemical cross-linking highly effective in a loading magnitude-dependent manner. Gel incubated with 4 g loading in the presence of genipin for 48 h possessed tensile strength of 4 MPa and tangent modulus of 60 MPa, respectively, which could fulfill the minimum biomechanical requirement for artificial tendon. Although mechanical properties of gels fabricated using the present method can be improved by using a larger amount of collagen in the starting material and through optimisation of mechanical loading and cross-linking, the method is a simple and effective for producing highly aligned collagen fibrils with excellent mechanical properties.

Insulin-Functionalized Bioactive Fiber Matrices with Bone Marrow-Derived Stem Cells in Rat Achilles Tendon Regeneration.

Approximately half of annual musculoskeletal injuries in the US involve tendon tears. The naturally hypocellular and hypovascular tendon environment makes tendons injury-prone and heal slowly. Tendon tissue engineering strategies often use biomimetic scaffolds combined with bioactive factors and/or cells to enhance healing. FDA-approved growth factors to promote tendon healing are lacking, which highlights the need for safe and effective bioactive factors. Our previous work evaluated insulin as a bioactive factor and identified an optimal dose to promote in vitro mesenchymal stem cell survival, division, and tenogenesis. The present work evaluates the ability of insulin-functionalized electrospun nanofiber matrices with or without mesenchymal stem cells to enhance tendon repair in a rat Achilles injury model. Electrospun nanofiber matrices were functionalized with insulin, cultured with or without mesenchymal stem cells, and sutured to transected Achilles tendons in rats. We analyzed rat tendons 4 and 8 weeks after surgery for the tendon morphology, collagen production, and mechanical properties. Bioactive insulin-functionalized fiber matrices with mesenchymal stem cells resulted in significantly increased collagen I and III at 4 and 8 weeks postsurgery. Additionally, these matrices supported highly aligned collagen fibrils in the regenerated tendon tissue at 8 weeks. However, treatment- and control-regenerated tissues had similar tensile properties at 8 weeks, which were less than that of the native Achilles tendon. Our preliminary results establish the benefits of insulin-functionalized fiber matrices in promoting higher levels of collagen synthesis and alignment needed for functional recovery of tendon repair.

Topoarchitected polymer networks expand the space of material properties

Many living tissues achieve functions through architected constituents with strong adhesion. An Achilles tendon, for example, transmits force, elastically and repeatedly, from a muscle to a bone through staggered alignment of stiff collagen fibrils in a soft proteoglycan matrix. The collagen fibrils align orderly and adhere to the proteoglycan strongly. However, synthesizing architected materials with strong adhesion has been challenging. Here we fabricate architected polymer networks by sequential polymerization and photolithography, and attain adherent interface by topological entanglement. We fabricate tendon-inspired hydrogels by embedding hard blocks in topological entanglement with a soft matrix. The staggered architecture and strong adhesion enable high elastic limit strain and high toughness simultaneously. This combination of attributes is commonly desired in applications, but rarely achieved in synthetic materials. We further demonstrate architected polymer networks of various geometric patterns and material combinations to show the potential for expanding the space of material properties.

DDR1 associates with TRPV4 in cell-matrix adhesions to enable calcium-regulated myosin activity and collagen compaction.

Tissue fibrosis manifests as excessive deposition of compacted, highly aligned collagen fibrils, which interfere with organ structure and function. Cells in collagen-rich lesions often exhibit marked overexpression of discoidin domain receptor 1 (DDR1), which is linked to increased collagen compaction through the association of DDR1 with the Ca2+ -dependent nonmuscle myosin IIA (NMIIA). We examined the functional relationship between DDR1 and the transient receptor potential vanilloid type 4 (TRPV4) channel, a Ca2+ -permeable ion channel that is implicated in collagen compaction. Fibroblasts expressing high levels of DDR1 were used to model cells in lesions with collagen compaction. In these cells, the expression of the β1 integrin was deleted to simplify studies of DDR1 function. Compared with DDR1 wild-type cells, high DDR1 expression was associated with increased Ca2+ influx through TRPV4, enrichment of TRPV4 in collagen adhesions, and enhanced contractile activity mediated by NMIIA. At cell adhesion sites to collagen, DDR1 associated with TRPV4, which enhanced DDR1-mediated collagen alignment and compaction. We conclude that DDR1 regulates Ca2+ influx through the TRPV4 channel to promote critical, DDR1-mediated processes that are important in lesions with collagen compaction and alignment.

Skeletal muscle progenitors are sensitive to collagen architectural features of fibril size and cross linking.

Muscle stem cells (MuSCs) are essential for the robust regenerative capacity of skeletal muscle. However, in fibrotic environments marked by abundant collagen and altered collagen organization, the regenerative capability of MuSCs is diminished. MuSCs are sensitive to their extracellular matrix environment but their response to collagen architecture is largely unknown. The present study aimed to systematically test the effect of underlying collagen structures on MuSC functions. Collagen hydrogels were engineered with varied architectures: collagen concentration, cross linking, fibril size, and fibril alignment, and the changes were validated with second harmonic generation imaging and rheology. Proliferation and differentiation responses of primary mouse MuSCs and immortal myoblasts (C2C12s) were assessed using EdU assays and immunolabeling skeletal muscle myosin expression, respectively. Changing collagen concentration and the corresponding hydrogel stiffness did not have a significant influence on MuSC proliferation or differentiation. However, MuSC differentiation on atelocollagen gels, which do not form mature pyridinoline cross links, was increased compared with the cross-linked control. In addition, MuSCs and C2C12 myoblasts showed greater differentiation on gels with smaller collagen fibrils. Proliferation rates of C2C12 myoblasts were also higher on gels with smaller collagen fibrils, whereas MuSCs did not show a significant difference. Surprisingly, collagen alignment did not have significant effects on muscle progenitor function. This study demonstrates that MuSCs are capable of sensing their underlying extracellular matrix (ECM) structures and enhancing differentiation on substrates with less collagen cross linking or smaller collagen fibrils. Thus, in fibrotic muscle, targeting cross linking and fibril size rather than collagen expression may more effectively support MuSC-based regeneration.