Soft tissue injuries (such as ligament, tendon, and meniscus tears) are the result of extracellular matrix damage from excessive tissue stretching. Deformation thresholds for soft tissues, however, remain largely unknown due to a lack of methods that can measure and compare the spatially heterogeneous damage and deformation that occurs in these materials. Here, we propose a full-field method for defining tissue injury criteria: multimodal strain limits for biological tissues analogous to yield criteria that exist for crystalline materials. Specifically, we developed a method for defining strain thresholds for mechanically-driven fibrillar collagen denaturation in soft tissues, using regional multimodal deformation and damage data. We established this new method using the murine medial collateral ligament (MCL) as our model tissue. Our findings revealed that multiple modes of deformation contribute to collagen denaturation in the murine MCL, contrary to the common assumption that collagen damage is driven only by strain in the direction of fibers. Remarkably, hydrostatic strain (computed here with an assumption of plane strain) was the best predictor of mechanically-driven collagen denaturation in ligament tissue, suggesting crosslink-mediated stress transfer plays a role in molecular damage accumulation. This work demonstrates that collagen denaturation can be driven by multiple modes of deformation and provides a method for defining deformation thresholds, or injury criteria, from spatially heterogeneous data. STATEMENT OF SIGNIFICANCE: Understanding the mechanics of soft tissue injuries is crucial for the development of new technology for injury detection, prevention, and treatment.  Yet, tissue-level deformation thresholds for injury are unknown, due to a lack of methods that combine full-field measurements of multimodal deformation and damage in mechanically loaded soft tissues. Here, we propose a method for defining tissue injury criteria: multimodal strain thresholds for biological tissues. Our findings reveal that multiple modes of deformation contribute to collagen denaturation, contrary to the common assumption that collagen damage is driven by strain in the fiber direction alone. The method will inform the development of new mechanics-based diagnostic imaging, improve computational modeling of injury, and be employed to study the role of tissue composition in injury susceptibility.

Solid tumors consist of tumor cells associated with stromal and immune cells, secreted factors and extracellular matrix (ECM), which together constitute the tumor microenvironment. Among stromal cells, activated fibroblasts, known as cancer-associated fibroblasts (CAFs) are of particular interest. CAFs secrete a plethora of ECM components including collagen and modulate the architecture of the ECM, thereby influencing cancer cell migration. The characterization of the collagen fibre network and its space and time-dependent microstructural modifications is key to investigating the interactions between cells and the ECM. Developing image analysis tools for that purpose is still a challenge because the structural complexity of the collagen network calls for specific statistical descriptors. Moreover, the low signal-to-noise ratio of imaging techniques available for time-resolved studies rules out standard methods based on image segmentation.
In this work, we develop a novel approach based on the stochastic modelling of the gel structure and on grey-tone image analysis. The method is then used to study the remodelling of a collagen matrix by migrating breast cancer-derived CAFs in a three-dimensional spheroid model of cellular invasion imaged by time-lapse confocal microscopy.
The structure of the collagen at the scale of a few microns consists in regions with high fibre density separated by depleted regions, which can be thought of as aggregates and pores. The approach developped captures this two-scale structure with a clipped Gaussian field model to describe the aggregates-and-pores large-scale structure, and a homogeneous Boolean model to describe the small-scale fibre network within the aggregates. The model parameters are identified by fitting the grey-tone histograms and correlation functions of the images. The method applies to unprocessed grey-tone images, and it can therefore be used with low magnification, noisy time-lapse reflectance images. When applied to the CAF spheroid time-resolved images, the method reveals different matrix densification mechanisms for the matrix in direct contact or far from the cells.
We developed a novel and multidisciplinary image analysis approach to investigate the remodelling of fibrillar collagen in a 3D spheroid model of cellular invasion. The specificity of the method is that it applies to the unprocessed grey-tone images, and it can therefore be used with noisy time-lapse reflectance images of non-fluorescent collagen. When applied to the CAF spheroid time-resolved images, the method reveals different matrix densification mechanisms for the matrix in direct contact or far from the cells.

UNASSIGNED: Collagen protein plays a notable role maintaining firm skin. Topical creams containing collagen fibers are widely available, but their usefulness is questionable due to limited skin penetration. When applied in a cream, collagen does not penetrate the skin leaving the skin structure unaffected.
UNASSIGNED: We formulated micronized collagen in a cream base. Using human skin samples, we sought to investigate the ability of the micronized collagen cream to penetrate human skin.
UNASSIGNED: Particle sizes of micronized marine collagen were evaluated using electron microscopy. Optical profilometry was conducted to evaluate skin topography and roughness. The antioxidant activity of the collagen was evaluated using the electron paramagnetic resonance technique by measuring the changes in free radical production. Collagen penetration depth in human skin samples was monitored using a non-invasive optical technique known as iterative multiplane optical property extraction, which works based on the detection of laser light phase changes following the presence of collagen particles in deep skin layers.
UNASSIGNED: According to the electron microscopy, collagen particles were found to be of various sizes, the smallest being about 120nm in diameter. Skin topography measurements revealed that the treated collagen cream increased skin smoothness of the samples. Our results derived from the iterative multiplane optical property extraction indicated that micronized collagen in a cream base penetrates both the stratum corneum and the deep epidermal layers toward the dermis.
UNASSIGNED: Our investigation suggests that the collagen in the studied cream formulation was able to penetrate the stratum coreum and deep epidermal layers in human skin samples.

Vertebrates have distinct tissues which are not present in invertebrate chordates nor other metazoans. The rise of these tissues also coincided with at least one round of whole-genome duplication as well as a suite of lineage-specific segmental duplications. Understanding whether novel genes lead to the origin and diversification of novel cell types, therefore, is of great importance in vertebrate evolution. Here we were particularly interested in the evolution of the vertebrate musculoskeletal system, the muscles and connective tissues that support a diversity of body plans. A major component of the musculoskeletal extracellular matrix (ECM) is fibrillar collagens, a gene family which has been greatly expanded upon in vertebrates. We thus asked whether the repertoire of fibrillar collagens in vertebrates reflects differences in the musculoskeletal system. To test this, we explored the diversity of fibrillar collagens in lamprey, a jawless vertebrate which diverged from jawed vertebrates (gnathostomes) more than five hundred million years ago and has undergone its own gene duplications. Some of the principal components of vertebrate hyaline cartilage are the fibrillar collagens type II and XI, but their presence in cartilage development across all vertebrate taxa has been disputed. We particularly emphasized the characterization of genes in the lamprey hyaline cartilage, testing if its collagen repertoire was similar to that in gnathostomes. Overall, we discovered thirteen fibrillar collagens from all known gene subfamilies in lamprey and were able to identify several lineage-specific duplications. We found that, while the collagen loci have undergone rearrangement, the Clade A genes have remained linked with the hox clusters, a phenomenon also seen in gnathostomes. While the lamprey muscular tissue was largely similar to that seen in gnathostomes, we saw considerable differences in the larval lamprey skeletal tissue, with distinct collagen combinations pertaining to different cartilage types. Our gene expression analyses were unable to identify type II collagen in the sea lamprey hyaline cartilage nor any other fibrillar collagen during chondrogenesis at the stages observed, meaning that sea lamprey likely no longer require these genes during early cartilage development. Our findings suggest that fibrillar collagens were multifunctional across the musculoskeletal system in the last common ancestor of vertebrates and have been largely conserved, but these genes alone cannot explain the origin of novel cell types.

Carbodiimide (EDC)-based dentin primers preserve hybrid layer (HL) integrity. However, aging >1 y has not been investigated. The present study examined whether the cross-linking effect of EDC was reflected in dentin bond strength, endogenous enzymatic activity, and the chemical profile of the HL after 5-y aging in artificial saliva. Noncarious human third molars (N = 42) were cut to expose middle/deep coronal dentin and treated as follows: group 1, dentin etched with 35% H3PO4, pretreated with a 0.3M aqueous EDC primer for 1 min and restored with XP Bond (Dentsply Sirona); group 2, as in group 1 but without EDC pretreatment; group 3, Clearfil SE Bond (Kuraray-Noritake) primer applied to dentin surface, followed by EDC pretreatment as in group 1 and application of bond; group 4, as in group 3 without EDC pretreatment. After composite buildup, the specimens were cut into sticks or slabs, depending on the experiment. All tests were performed at baseline (T0) and after 5 y of aging (T5) in artificial saliva at 37 °C. Microtensile bond strength (µTBS) was tested at a crosshead speed of 1 mm/min until failure. Endogenous enzymatic activity was investigated with in situ zymography. The chemical profile of HL was determined via Raman spectroscopy. Three-way analysis of variance and post hoc Tukey test were used to analyze µTBS and in situ zymography data (α = 0.05). EDC pretreatment and aging significantly influenced µTBS and in situ zymography results (P < 0.05). Higher bond strength and lower gelatinolytic activity were identified in the EDC-treated groups at T5 (P < 0.05), especially in the etch-and-rinse groups. Raman spectra revealed less defined amide III peaks in control specimens at T5. The EDC cross-linking effect persisted in the HL for 5 y in terms of bond strength, collagen structure preservation, and dentinal enzyme silencing.

Recent research has highlighted the importance of key tumor microenvironment features, notably the collagen-rich extracellular matrix (ECM) in characterizing tumor invasion and progression. This led to great interest from both basic researchers and clinicians, including pathologists, to include collagen fiber evaluation as part of the investigation of cancer development and progression. Fibrillar collagen is the most abundant in the normal extracellular matrix, and was revealed to be upregulated in many cancers. Recent studies suggested an emerging theme across multiple cancer types in which specific collagen fiber organization patterns differ between benign and malignant tissue and also appear to be associated with disease stage, prognosis, treatment response, and other clinical features. There is great potential for developing image-based collagen fiber biomarkers for clinical applications, but its adoption in standard clinical practice is dependent on further translational and clinical evaluations. Here, we offer a comprehensive review of the current literature of fibrillar collagen structure and organization as a candidate cancer biomarker, and new perspectives on the challenges and next steps for researchers and clinicians seeking to exploit this information in biomedical research and clinical workflows.

The mammary gland structurally and functionally remodels during pregnancy, during lactation and after weaning. There are three types of fibrillar collagens, types I, III, and V, in mammary stromal tissue. While the importance of the fibrillar structure of collagens for mammary morphogenesis has been suggested, the expression patterns of each type of fibrillar collagen in conjunction with mammary remodeling remain unclear. In this study, we investigated their expression patterns during pregnancy, parturition, lactation and involution. Type I collagen showed a well-developed fibril structure during pregnancy, but the fibrillar structure of type I collagen then became sparse at parturition and during lactation, which was concurrent with the downregulation of its mRNA and protein levels. The well-developed fibrillar structure of type I collagen reappeared after weaning. On the other hand, type V collagen showed a well-developed fibrillar structure and upregulation in the lactation period but not in the periods of pregnancy and involution. Type III collagen transiently developed a dense fibrillar network at the time of parturition and exhibited drastic increases in mRNA expression. These results indicate that each type of fibrillar collagen is distinctly involved in structural and functional remodeling in mammary glands during pregnancy, parturition, lactation, and involution after weaning. Furthermore, in vitro studies of mammary epithelial cells showed regulatory effects of type I collagen on cell adhesion, cell proliferation, ductal branching, and β-casein secretion. Each type of fibrillar collagen may have different roles in defining the cellular microenvironment in conjunction with structural and functional mammary gland remodeling.

Collagen-based skin-like scaffolds (CBSS) are promising alternatives to skin grafts to repair wounds and injuries. In this work, we propose that the common marine invertebrate sea urchin represents a promising and eco-friendly source of native collagen to develop innovative CBSS for skin injury treatment. Sea urchin food waste after gonad removal was here used to extract fibrillar glycosaminoglycan (GAG)-rich collagen to produce bilayer (2D + 3D) CBSS. Microstructure, mechanical stability, permeability to water and proteins, ability to exclude bacteria and act as scaffolding for fibroblasts were evaluated. Our data show that the thin and dense 2D collagen membrane strongly reduces water evaporation (less than 5% of water passes through the membrane after 7 days) and protein diffusion (less than 2% of BSA passes after 7 days), and acts as a barrier against bacterial infiltration (more than 99% of the different tested bacterial species is retained by the 2D collagen membrane up to 48 h), thus functionally mimicking the epidermal layer. The thick sponge-like 3D collagen scaffold, structurally and functionally resembling the dermal layer, is mechanically stable in wet conditions, biocompatible in vitro (seeded fibroblasts are viable and proliferate), and efficiently acts as a scaffold for fibroblast infiltration. Thus, thanks to their chemical and biological properties, CBSS derived from sea urchins might represent a promising, eco-friendly, and economically sustainable biomaterial for tissue regenerative medicine.

Quantification of fibrillar collagen organization has given new insight into the possible role of collagen topology in many diseases and has also identified candidate image-based bio-markers in breast cancer and pancreatic cancer. We have been developing collagen quantification tools based on the curvelet transform (CT) algorithm and have demonstrated this to be a powerful multiscale image representation method due to its unique features in collagen image denoising and fiber edge enhancement. In this paper, we present our CT-based collagen quantification software platform with a focus on new features and also giving a detailed description of curvelet-based fiber representation. These new features include C++-based code optimization for fast individual fiber tracking, Java-based synthetic fiber generator module for method validation, automatic tumor boundary generation for fiber relative quantification, parallel computing for large-scale batch mode processing, region-of-interest analysis for user-specified quantification, and pre- and post-processing modules for individual fiber visualization. We present a validation of the tracking of individual fibers and fiber orientations by using synthesized fibers generated by the synthetic fiber generator. In addition, we provide a comparison of the fiber orientation calculation on pancreatic tissue images between our tool and three other quantitative approaches. Lastly, we demonstrate the use of our software tool for the automatic tumor boundary creation and the relative alignment quantification of collagen fibers in human breast cancer pathology images, as well as the alignment quantification of in vivo mouse xenograft breast cancer images.

Integrin α11β1 is a collagen receptor that has been reported to be overexpressed in the stroma of non-small cell lung cancer (NSCLC) and of head and neck squamous cell carcinoma (HNSCC). In the current study, we further analyzed integrin α11 expression in 14 tumor types by screening a tumor tissue array while using mAb 203E3, a newly developed monoclonal antibody to human α11. Different degrees of expression of integrin α11 were observed in the stroma of breast, ovary, skin, lung, uterus, stomach, and pancreatic ductal adenocarcinoma (PDAC) tumors. Co-expression queries with the myofibroblastic cancer-associated fibroblast (myCAF) marker, alpha smooth muscle actin (αSMA), demonstrated a moderate level of α11+ in myCAFs associated with PDAC and HNSCC tumors, and a lack of α11 expression in additional stromal cells (i.e., cells positive for fibroblast-specific protein 1 (FSP1) and NG2). The new function-blocking α11 antibody, mAb 203E1, inhibited cell adhesion to collagen I, partially hindered fibroblast-mediated collagen remodeling and obstructed the three-dimensional (3D) migration rates of PDAC myCAFs. Our data demonstrate that integrin α11 is expressed in a subset of non-pericyte-derived CAFs in a range of cancers and suggest that α11β1 constitutes an important receptor for collagen remodeling and CAF migration in the tumor microenvironment (TME).