We thank many contributors and users to CT-FIRE and CurveAlign over the years, including Dr. Rob Nowak, Dr. Carolyn Pehlke, Dr. Jeremy Bredfeldt, Guneet Mehta, Andrew Leicht, Dr. Adib Keikhosravi, Dr. Matt Conklin, Dr. Jayne Squirrell, Dr. Paolo Provenzano, Dr. Brenda Ogle, Dr. Patricia Keely, Dr. Joseph Szulczewski, Dr. Suzanne Ponik and additional technical contributions from Swati Anand and Curtis Rueden. This work was supported by funding from Semiconductor Research Corporation, Morgridge Institute for Research, and NIH grants R01CA199996, R01CA181385 and U54CA210190 to K.W.E.

The authors have nothing to disclose.

This protocol describes the use of CT-FIRE and CurveAlign for fibrillar collagen quantification and can be applied to any image with collagen fibers or other line-like or fiber-like elongated structures suitable for analysis by CT-FIRE or CurveAlign. For example, elastin or elastic fibers could be processed in a similar way on this platform. We have tested both tools on computationally generated synthetic fibers21. Depending on the application, users should choose the analysis mode that is most ap...

Fibrillar collagens are prominent, structural ECM components. Their organization changes impact tissue function and are likely associated with the progression of many diseases ranging from osteogenesis imperfecta1, cardiac dysfunction2, and wound healing3 to different types of cancer including breast4,5,6, ovarian7,8, kidney9, and pancreatic cancers10. Many established imaging modalities can be used to visualize fibrillar collagen such as second harmonic generation microscopy11, stains or dyes in conjunction with bright field or fluorescence microscopy or polarized light microscopy12, liquid crystal-based polarization microscopy (LC-PolScope)13, and electron microscopy14. As the importance of fibrillar collagen organization has become clearer, and the use of these methods has increased, the need for improved collagen fiber analysis approaches has also grown.
There have been many efforts to develop computational methods for automated measurement of fibrillar collagen. Freely available software tools are mainly focused on the calculation of fiber alignment or orientation by adopting either first derivative or structure tensor for pixels15,16, or Fourier transform-based spectrum analysis for image tiles17. All these tools are subject to limitations such as the requirement of manual steps, inaccuracy in detection of the fiber edge in noisy background, or lack of localized feature characterization.
Compared to other freely available open-source free software tools, the methods described in this protocol use CT&#8212;an optimal, multiscale, directional image representation method&#8212;to remove noise from fibrillar collagen images and enhance or track fiber edges. Information about location and orientation can be provided directly from a fiber rather than by using the indirect pixel-wise or window-wise information to infer the metrics of fiber organization. This CT-based framework18,19,20,21 can quantify fiber organization on a global, ROI, or fiber basis, mainly via two separate, but linked, packages named &#8220;CT-FIRE&#8221;18,21 and &#8220;CurveAlign&#8221;19,21. As far as the implementation of the software is concerned, in CT-FIRE, CT coefficients on multiple scales can be used to reconstruct an image that enhances edges and reduces noise. Then, an individual fiber extraction algorithm is applied to the CT-reconstructed image to track fibers for finding their representative center points, extending fiber branches from the center points, and linking fiber branches to form a fiber network. In CurveAlign, CT coefficients on a user-specified scale can be used to track local fiber orientation, where the orientation and locations of curvelets are extracted and grouped to estimate the fiber orientation at the corresponding locations. This resulting quantification framework has been developed for more than ten years and has evolved greatly in many aspects such as functionality, user interface, and modularity. For instance, this tool can visualize local fiber orientation and allows the user to measure up to thirty fiber features including individual fiber properties such as length, angle, width, and straightness, as well as bulk measurements such as density and alignment. Additionally, the user can measure fiber angle relative to manually or automatically segmented boundaries, which, for example, plays an important role in image-based biomarker development in breast cancer22 and pancreatic cancer studies10. This platform provides several feature modules including ones for ROI analysis, automatic boundary creation, and post-processing. The ROI module can be used to annotate different shapes of ROI and conduct corresponding&#160;ROI analysis. As an application example, the automatic boundary creation module can be used to register hematoxylin and eosin (H&#38;E) bright field images with second harmonic generation (SHG) images and generate the image mask of tumor boundaries from the registered H&#38;E images. The post-processing module can help facilitate the processing and integration of output data files from individual images for possible statistical analysis.
This quantification platform does not require prior experience of programming or image processing and can handle large datasets including hundreds or thousands of images, enabling efficient quantification of collagen organization for biological or biomedical applications. It has been widely used in different research fields by many researchers all over the world, including ourselves. There are four main publications on CT-FIRE and CurveAlign18,19,20,21, out of which the first three have been cited 272 times (as of 2020-05-04 according to Google Scholar). A review of the publications that cited this platform (CT-FIRE or CurveAlign) indicates that there are approximately 110 journal papers that directly used it for their analysis, in which approximately 35 publications were collaborative with our group, and the others (~ 75) were written by other groups. For instance, this platform was used for the following studies: breast cancer22,23,24, pancreatic cancer10,25, kidney cancer9,26, wound healing3,27,28,29,30, ovarian cancer8,31,7, uterosacral ligament32, hypophosphatemic dentin33, basal cell carcinoma34, hypoxic sarcoma35, cartilage tissue36, cardiac dysfunction37, neurons38, glioblastoma39, lymphatic contractions40, fibrous cacffolds41, gastric cancer42, microtubule43, and bladder fibrosis44. Figure 1 demonstrates the cancer imaging application of CurveAlign to find the tumor-associated collagen signatures of breast cancer19 from the SHG image. Figure 2 describes a typical schematic workflow of this platform. Although these tools have been reviewed technically18,19,21, and a regular protocol20 for alignment analysis with CurveAlign is also available, a visual protocol that demonstrates all the essential features could be useful. A visualized protocol, as presented here, will facilitate the learning process of using this platform as well as more efficiently address concerns and questions that users might have.

<table><tbody><tr><td>CT-FIRE</td><td>Univerity of Wisconsin-Madison</td><td>N/A</td><td>open source software available from https://eliceirilab.org/software/ctfire/</td></tr><tr><td>CurveAlign</td><td>University of Wisconsin-Madison</td><td>N/A</td><td>open source software available from https://eliceirilab.org/software/curvealign/</td></tr></tbody></table>

quantifying fibrillar collagen organization with curvelet transform-based tools

Fibrillar collagens are prominent extracellular matrix (ECM) components, and their topology changes have been shown to be associated with the progression of a wide range of diseases including breast, ovarian, kidney, and pancreatic cancers. Freely available fiber quantification software tools are mainly focused on the calculation of fiber alignment or orientation, and they are subject to limitations such as the requirement of manual steps, inaccuracy in detection of the fiber edge in noisy background, or lack of localized feature characterization. The collagen fiber quantitation tool described in this protocol is characterized by using an optimal multiscale image representation enabled by curvelet transform (CT). This algorithmic approach allows for the removal of noise from fibrillar collagen images and the enhancement of fiber edges to provide location and orientation information directly from a fiber, rather than using the indirect pixel-wise or window-wise information obtained from other tools. This CT-based framework contains two separate, but linked, packages named &#8220;CT-FIRE&#8221; and &#8220;CurveAlign&#8221; that can quantify fiber organization on a global, region of interest (ROI), or individual fiber basis. This quantification framework has been developed for more than ten years and has now evolved into a comprehensive and user-driven collagen quantification platform. Using this platform, one can measure up to about thirty fiber features including individual fiber properties such as length, angle, width, and straightness, as well as bulk measurements such as density and alignment. Additionally, the user can measure fiber angle relative to manually or automatically segmented boundaries. This platform also provides several additional modules including ones for ROI analysis, automatic boundary creation, and post-processing. Using this platform does not require prior experience of programming or image processing, and it can handle large datasets including hundreds or thousands of images, enabling efficient quantification of collagen fiber organization for biological or biomedical applications.

These methods have been successfully applied in numerous studies. Some typical applications include: 1) Conklin et al.22 used CurveAlign to calculate tumor-associated collagen signatures, and found that collagen fibers were more frequently aligned perpendicularly to the duct perimeter in ductal carcinoma in situ (DCIS) lesions; 2) Drifka et al.10 used the CT-FIRE mode in CurveAlign to quantify the stromal collagen alignment for pancreatic ductal adenocarcinoma and normal/ch...

Watch this Scientific Journal Video about Quantifying Fibrillar Collagen Organization with Curvelet Transform-Based Tools at JoVE.com

Quantifying Fibrillar Collagen Organization with Curvelet Transform-Based Tools

Here, we present a protocol to use a curvelet transform-based, open-source MATLAB software tool for quantifying fibrillar collagen organization in the extracellular matrix of both normal and diseased tissues. This tool can be applied to images with collagen fibers or other types of line-like structures.

Fibrillar collagens are prominent extracellular matrix (ECM) components, and their topology changes have been shown to be associated with the progression ...

quantifying-fibrillar-collagen-organization-with-curvelet-transform

Research

JoVE Journal

Bioengineering

6.0K Views.  University of Wisconsin-Madison. Hier presenteren we een protocol om een curvelet transform-gebaseerde, open-source MATLAB-softwaretool te gebruiken voor het kwantificeren van fibrillaire collageenorganisatie in de extracellulaire matrix van zowel normale als zieke weefsels. Deze tool kan worden toegepast op afbeeldingen met collageenvezels of andere soorten lijnachtige structuren.

Video: Quantifying Fibrillar Collagen Organization with Curvelet Transform-Based Tools

Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global reorganization and realignment of the gel microstructure. This process proceeds in a complex manner that is dependent in part on the interplay between the location of the cells, the geometry of the gel, and the mechanical constraints on the gel. To better understand how these variables produce global fiber alignment patterns, we use time-lapse differential interference contrast (DIC) microscopy coupled with an environmentally controlled bioreactor to observe the compaction process between geometrically spaced explants (clusters of fibroblasts). The images are then analyzed with a custom image processing algorithm to obtain maps of the strain. The information obtained from this technique can be used to probe the mechanobiology of various cell-matrix interactions, which has important implications for understanding processes in wound healing, disease development, and tissue engineering applications.

Time-lapse microscopy and image processing techniques were used to observe and analyze fibroblast-mediated gel compaction and fibrin fiber realignment in an environmentally controlled bioreactor over a 48 hr&#160;period.

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global ...

Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim to use these principles to create tissues with predefined collagen architectures. A full understanding of the exact underlying processes of collagen remodeling to control the final tissue architecture, however, is lacking. In particular, little is known about the (re)orientation of collagen fibers in response to changes in tissue mechanical loading conditions. We developed an in&#160;vitro model system, consisting of biaxially-constrained myofibroblast-seeded fibrin constructs, to further elucidate collagen (re)orientation in response to i) reverting biaxial to uniaxial static loading conditions and ii) cyclic uniaxial loading of the biaxially-constrained constructs before and after a change in loading direction, with use of the Flexcell FX4000T loading device. Time-lapse confocal imaging is used to visualize collagen (re)orientation in a nondestructive manner.
Cell and collagen organization in the constructs can be visualized in real-time, and an internal reference system allows us to relocate cells and collagen structures for time-lapse analysis. Various aspects of the model system can be adjusted, like cell source or use of healthy and diseased cells. Additives can be used to further elucidate mechanisms underlying collagen remodeling, by for example adding MMPs or blocking integrins. Shape and size of the construct can be easily adapted to specific needs, resulting in a highly tunable model system to study cell and collagen (re)organization.

This model system starts from a myofibroblast-populated fibrin gel that can be used to study endogenous collagen (re)organization real-time in a nondestructive manner. The model system is very tunable, as it can be used with different cell sources, medium additives, and can be adapted easily to specific needs.

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim ...

Examining the Dynamics of Cellular Adhesion and Spreading of Epithelial Cells on Fibronectin During Oxidative Stress

The adhesion and spreading of cells onto the extracellular matrix (ECM) are essential cellular processes during organismal development and for the homeostasis of adult tissues. Interestingly, oxidative stress can alter these processes, thus contributing to the pathophysiology of diseases such as metastatic cancer. Therefore, understanding the mechanism(s) of how cells attach and spread on the ECM during perturbations in redox status can provide insight into normal and disease states. Described below is a step-wise protocol that utilizes an immunofluorescence-based assay to specifically quantify cell adhesion and spreading of immortalized fibroblast cells on fibronectin (FN) in vitro. Briefly, anchorage-dependent cells are held in suspension and exposed to the ATM kinase inhibitor Ku55933 to induce oxidative stress. Cells are then plated on FN-coated surface and allowed to attach for predetermined periods of time. Cells that remain attached are fixed and labeled with fluorescence-based antibody markers of adhesion (e.g., paxillin) and spreading (e.g., F-actin). Data acquisition and analysis are performed using commonly available laboratory equipment, including an epifluorescence microscope and freely available Fiji software. This procedure is highly versatile and can be modified for a variety of cell lines, ECM proteins, or inhibitors in order to examine a broad range of biological questions.

This method is useful for quantifying the early dynamics of cellular adhesion and spreading of anchorage-dependent cells onto the fibronectin. Furthermore, this assay can be used to investigate the effects of altered redox homeostasis on cell spreading and/or cell adhesion-related intracellular signaling pathways.

The adhesion and spreading of cells onto the extracellular matrix (ECM) are essential cellular processes during organismal development and for the ...

Biochemistry

Revealing the Cytoskeletal Organization of Invasive Cancer Cells in 3D

Cell migration has traditionally been studied in 2D substrates. However, it has become increasingly evident that there is a need to study cell migration in more appropriate 3D environments, which better resemble the dimensionality of the physiological processes in question. Migratory cells can substantially differ in their morphology and mode of migration depending on whether they are moving on 2D or 3D substrates. Due to technical difficulties and incompatibilities with most standard protocols, structural and functional analysis of cells embedded within 3D matrices still remains uncommon. This article describes methods for preparation and imaging of 3D cancer cell cultures, either as single cells or spheroids. As an appropriate ECM substrate for cancer cell migration, we use nonpepsinized rat tail collagen I polymerized at room-temperature and fluorescently labeled to facilitate visualization using standard confocal microscopes. This work also includes a protocol for 3D immunofluorescent labeling of endogenous cell cytoskeleton. Using these protocols we hope to contribute to a better description of the molecular composition, localization, and functions of cellular structures in 3D.

This article presents a method to fluorescently label collagen that can be further used for both fix and live imaging of 3D cell cultures. We also provide an optimized protocol to visualize endogenous cytoskeletal proteins of cells cultured in 3D environments.

Cell migration has traditionally been studied in 2D substrates. However, it has become increasingly evident that there is a need to study cell migration ...

Medicine

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Assessing Collagen and Elastin Pressure-dependent Microarchitectures in Live, Human Resistance Arteries by Label-free Fluorescence Microscopy

The pathogenic contribution of resistance artery remodeling is documented in essential hypertension, diabetes and the metabolic syndrome. Investigations and development of microstructurally motivated mathematical models for understanding the mechanical properties of human resistance arteries in health and disease have the potential to aid understanding how disease and medical treatments affect the human microcirculation. To develop these mathematical models, it is essential to decipher the relationship between the mechanical and microarchitectural properties of the microvascular wall. In this work, we describe an ex vivo method for passive mechanical testing and simultaneous label-free three-dimensional imaging of the microarchitecture of elastin and collagen in the arterial wall of isolated human resistance arteries. The imaging protocol can be applied to resistance arteries of any species of interest. Image analyses are described for quantifying i) pressure-induced changes in internal elastic lamina branching angles and adventitial collagen straightness using Fiji and ii) collagen and elastin volume densities determined using Ilastik software. Preferably all mechanical and imaging measurements are performed on live, perfused arteries, however, an alternative approach using standard video-microscopy pressure myography in combination with post-fixation imaging of re-pressurized vessels is discussed. This alternative method provides users with different options for analysis approaches. The inclusion of the mechanical and imaging data in mathematical models of the arterial wall mechanics is discussed, and future development and additions to the protocol are proposed.

We describe simultaneous mechanical testing and 3D-imaging of the arterial wall of isolated, live human resistance arteries, and Fiji and Ilastik image analyses for the quantification of elastin and collagen spatial organization and volume densities. We discuss the use of these data in mathematical models of arterial wall mechanics.

The pathogenic contribution of resistance artery remodeling is documented in essential hypertension, diabetes and the metabolic syndrome. Investigations ...

Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of myosin heavy chain (MyHC) isoforms has emerged as a finer discriminator of fiber-type. Type I, type IIA, type IIX and type IIB fibers can now be identified with precision based on their MyHC profile; however, manual analysis of these data can be slow and down-right tedious. In this regard, rapid, accurate assessment of fiber-type composition and morphology is a very desirable tool. Here, we present a protocol for state-of-the-art immunohistochemical staining of MyHCs in frozen sections obtained from mouse hindlimb muscle in concert with a novel semi-automated algorithm that accelerates analysis of fiber-type and fiber morphology. As expected, the soleus muscle displayed staining for type I and type IIA fibers, but not for type IIX or type IIB fibers. On the other hand, the tibialis anterior muscle was composed predominantly of type IIX and type IIB fibers, a small fraction of type IIA fibers and little or no type I fibers. Several image transformations were used to generate probability maps for the purpose of measuring different aspects of fiber morphology (i.e., cross-sectional area (CSA), maximal and minimal Feret diameter). The values obtained for these parameters were then compared with manually-obtained values. No significant differences were observed between either mode of analysis with regards to CSA, maximal or minimal Feret diameter (all p &#62; 0.05), indicating the accuracy of our method. Thus, our immunostaining analysis protocol may be applied to the investigation of effects on muscle composition in many models of aging and myopathy.

Immunohistochemical staining of myosin heavy chain isoforms has emerged as the state-of-the-art discriminator of skeletal muscle fiber-type (i.e., type I, type IIA, type IIX, type IIB). Here, we present a staining protocol along with a novel semi-automated algorithm that facilitates rapid assessment of fiber-type and fiber morphology.

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of ...

A Method to Study the Correlation Between Local Collagen Structure and Mechanical Properties of Atherosclerotic Plaque Fibrous Tissue

The rupture of atherosclerotic plaques in coronary and carotid arteries is the primary cause of fatal cardiovascular events. However, the rupture mechanics of the heterogeneous, highly collagenous plaque tissue, and how this is related to the tissue's fibrous structure, are not known yet. Existing pipelines to study plaque mechanics are limited to obtaining only gross mechanical characteristics of the plaque tissue, based on the assumption of structural homogeneity of the tissue. However, fibrous plaque tissue is structurally heterogeneous, arguably mainly due to local variation in the collagen fiber architecture.
The mechano-imaging pipeline described here has been developed to study the heterogeneous structural and mechanical plaque properties. In this pipeline, the tissue's local collagen architecture is characterized using multiphoton microscopy (MPM) with second-harmonic generation (SHG), and the tissue's failure behavior is characterized under uniaxial tensile testing conditions using digital image correlation (DIC) analysis. This experimental pipeline enables correlation of the local predominant angle and dispersion of collagen fiber orientation, the rupture behavior, and the strain fingerprints of the fibrous plaque tissue. The obtained knowledge is key to better understand, predict, and prevent atherosclerotic plaque rupture events.

We have developed a mechano-imaging pipeline to study the heterogeneous structural and mechanical atherosclerotic plaque properties. This pipeline enables correlation of the local predominant angle and dispersion of collagen fiber orientation, the rupture behavior, and the strain fingerprints of the fibrous plaque tissue.

The rupture of atherosclerotic plaques in coronary and carotid arteries is the primary cause of fatal cardiovascular events. However, the rupture ...

Understanding Tendon Impingement &#8211; Murine Explant Model for Studying Mechanobiology

Murine Hind Limb Explant Model for Studying the Mechanobiology of Achilles Tendon Impingement

Tendon impingement upon bone generates a multiaxial mechanical strain environment with markedly elevated transverse compressive strain, which elicits a localized fibrocartilage phenotype characterized by accumulation of glycosaminoglycan (GAG)-rich matrix and remodeling of the collagen network. While fibrocartilage is a normal feature in impinged regions of healthy tendons, excess GAG deposition and disorganization of the collagen network are hallmark features of tendinopathy. Accordingly, impingement is clinically recognized as an important extrinsic factor in the initiation and progression of tendinopathy. Nevertheless, the mechanobiology underlying tendon impingement remains understudied. Prior efforts to elucidate the cellular response to tendon impingement have applied uniaxial compression to cells and excised tendon explants in vitro. However, isolated cells lack a three-dimensional extracellular environment crucial to mechanoresponse, and both in vitro and excised explant studies fail to recapitulate the multiaxial strain environment generated by tendon impingement in vivo, which depends on anatomical features of the impinged region. Moreover, in vivo models of tendon impingement lack control over the mechanical strain environment. To overcome these limitations, we present a novel murine hind limb explant model suitable for studying the mechanobiology of Achilles tendon impingement. This model maintains the Achilles tendon in situ to preserve local anatomy and reproduces the multiaxial strain environment generated by impingement of the Achilles tendon insertion upon the calcaneus during passively applied ankle dorsiflexion while retaining cells within their native environment. We describe a tissue culture protocol integral to this model and present data establishing sustained explant viability over 7 days. The representative results demonstrate enhanced histological GAG staining and decreased collagen fiber alignment secondary to impingement, suggesting elevated fibrocartilage formation. This model can easily be adapted to investigate different mechanical loading regimens and allows for the manipulation of molecular pathways of interest to identify mechanisms mediating phenotypic change in the Achilles tendon in response to impingement.

Author Spotlight: Unraveling the Mechanobiology of Tendon Impingement &#8211; A Multiaxial Murine Hind Limb Explant Model 

We present a custom experimental platform and tissue culture protocol that recreates fibrocartilaginous change driven by impingement of the Achilles tendon insertion in murine hind limb explants with sustained cell viability, providing a model suitable for exploring the mechanobiology of tendon impingement.

Tendon impingement upon bone generates a multiaxial mechanical strain environment with markedly elevated transverse compressive strain, which elicits a ...

Fabricating Engineered Nanofibrillar Collagen with Directed Patterning

Production of Nanofibrillar Patterned Collagen for Tissue Engineering

Regenerative biomaterials are designed to facilitate cell-material interactions to guide the repair of damaged tissues and organs. These materials are designed to emulate the biophysical properties of native tissue, providing cellular phenotypic and morphological guidance that contributes to the restoration of the regenerative tissue niche. Collagen, a prevalent extracellular matrix protein, is a common component of these regenerative biomaterials due to its biocompatibility and other favorable properties. The current study describes a novel and straightforward method for the fabrication of engineered nanofibrillar collagen with directed fibril patterning. Through the manipulation of shear stress, temperature, and pH, collagen fibrillogenesis and alignment are precisely controlled without requiring specialized equipment. This approach allows for the creation of nanofibrillar collagen biomaterials that mimic the native structure of tissues exhibiting either anisotropic or isotropic characteristics. The flexibility in collagen nanofibril patterning not only facilitates the study of nanoscale patterning on cell behavior but also offers diverse possibilities for patterned tissue engineering applications.

The following protocol presents a shear-based extrusion method for the fabrication of collagen hydrogels with nanoscale patterned fibrils, which can be applied to a broad range of tissue engineering applications.

Regenerative biomaterials are designed to facilitate cell-material interactions to guide the repair of damaged tissues and organs. These materials are ...

Quantifying Fibrillar Collagen Organization with Curvelet Transform-Based Tools

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