Collagen Microarchitecture Research Articles

Improving HCC Prognostic Models after Liver Resection by AI-Extracted Tissue Fiber Framework Analytics.

Despite advances in diagnostic and treatment technologies, predicting outcomes of patients with hepatocellular carcinoma (HCC) remains a challenge. Prognostic models are further obscured by the variable impact of the tumor properties and the remaining liver parenchyma, often affected by cirrhosis or non-alcoholic fatty liver disease that tend to precede HCC. This study investigated the prognostic value of reticulin and collagen microarchitecture in liver resection samples. We analyzed 105 scanned tissue sections that were stained using a Gordon and Sweet's silver impregnation protocol combined with Picric Acid-Sirius Red. A convolutional neural network was utilized to segment the red-staining collagen and black linear reticulin strands, generating a detailed map of the fiber structure within the HCC and adjacent liver tissue. Subsequent hexagonal grid subsampling coupled with automated epithelial edge detection and computational fiber morphometry provided the foundation for region-specific tissue analysis. Two penalized Cox regression models using LASSO achieved a concordance index (C-index) greater than 0.7. These models incorporated variables such as patient age, tumor multifocality, and fiber-derived features from the epithelial edge in both the tumor and liver compartments. The prognostic value at the tumor edge was derived from the reticulin structure, while collagen characteristics were significant at the epithelial edge of peritumoral liver. The prognostic performance of these models was superior to models solely reliant on conventional clinicopathologic parameters, highlighting the utility of AI-extracted microarchitectural features for the management of HCC.

A Micro-anatomical Model of the Infarcted Left Ventricle Border Zone to Study the Influence of Collagen Undulation.

Myocardial infarction (MI) results in cardiac myocyte death and often initiates the formation of a fibrotic scar in the myocardium surrounded by a border zone. Myocyte loss and collagen-rich scar tissue heavily influence the biomechanical behavior of the myocardium which could lead to various cardiac diseases such as systolic heart failure and arrhythmias. Knowledge of how myocyte and collagen micro-architecture changes affect the passive mechanical behavior of the border zone remains limited. Computational modeling provides us with an invaluable tool to identify and study the mechanisms driving the biomechanical remodeling of the myocardium post-MI. We utilized a rodent model of MI and an image-based approach to characterize the three-dimensional (3-D) myocyte and collagen micro-architecture at various timepoints post-MI. Left ventricular free wall (LVFW) samples were obtained from infarcted hearts at 1-week and 4-week post-MI (n = 1 each). Samples were labeled using immunoassays to identify the extracellular matrix (ECM) and myocytes. 3-D reconstructions of the infarct border zone were developed from confocal imaging and meshed to develop high-fidelity micro-anatomically accurate finite element models. We performed a parametric study using these models to investigate the influence of collagen undulation on the passive micromechanical behavior of the myocardium under a diastolic load. Our results suggest that although parametric increases in collagen undulation elevate the strain amount experienced by the ECM in both early- and late-stage MI, the sensitivity of myocytes to such increases is reduced from early to late-stage MI. Our 3-D micro-anatomical modeling holds promise in identifying mechanisms of border zone maladaptation post-MI.

Comparison of the Effects of Tissue Processing on the Physicochemical Properties of Bone Allografts.

Purpose: To address the hypothesis that the tissue processing methods of solvent dehydration and freeze-drying would differentially affect the physicochemical characteristics of four commercially available bone allografts and the adhesion and differentiation of human bone marrow-derived mesenchymal stromal cells (hBMSCs) on such substrates in vitro. Materials and Methods: The surface morphology, surface area, and elemental composition of four commercially available cancellous bone allografts were examined using SEM, Brunauer-Emmett-Teller (BET) gas adsorption, and inductively coupled plasma (ICP) analyses. SEM was also employed to compare the allograft surfaces to that of human bone exposed by in vitro osteoclastic resorption. The allografts were seeded with hBMSCs, and the number of adhered cells was assessed at 3 and 7 days. Alkaline phosphatase (ALP) activity was quantified as a measure of osteogenic differentiation after 21 days. Results: Marked differences were seen between the physicochemical characteristics of the solvent-dehydrated and freeze-dried allografts, as well as between their resulting bone microarchitectures and that of osteoclast-resorbed human bone. Increased hBMSC adhesion and differentiation were observed on the solvent-dehydrated allografts compared to freeze-dried allografts, which suggests a higher putative osteogenic potential. The latter was attributed to better preservation of the bone collagen microarchitecture integrity, which may provide not only a more complex substrate architecture, but also a more favorable microenvironment to allow nutrients and oxygen to flow to the adhered cells. Conclusion: Commercially available cancellous bone allografts significantly differ in their physicochemical characteristics, stemming from differences in tissue processing and sterilization methods undertaken by tissue banks. These differences impact the response of MSCs in vitro and may alter the biologic performance of the grafts in vivo. Therefore, it is important to consider these characteristics when choosing a bone substitute for clinical application, as the physicochemical properties of the grafts play a crucial role in their interactions with the biologic environment and subsequent incorporation into the native bone.

Abstract 6096: The microarchitecture and fibrillar collagen expression of leiomyosarcoma are associated with malignancy

Abstract The expression, abundance, and microarchitecture of fibrillar collagens are associated with tumor development and aggressiveness in various carcinomas. However, the impact of fibrillar collagens on mesenchymal tumors is less understood. While uterine leiomyomas, also known as fibroids, are characterized by high fibrillar collagen deposition and depend on ECM signaling for cell proliferation, the impact of fibrillar collagens on malignant uterine leiomyosarcomas has not been explored. Thus, identifying malignancy and aggressiveness-associated features of the fibrillar collagen-leiomyosarcoma crosstalk may provide novel biomarkers and therapeutic targets for these aggressive tumors. We used publicly available RNAseq data and performed RNAseq and picrosirius red analysis of fiber microarchitecture in a cohort of normal myometrium (MM; n =68 ), leiomyoma (LM; n = 66), and leiomyosarcoma (LMS; n = 67) tissues. Furthermore, we cultured patient-derived primary cells (4 MM, 3 LM, and 4 LMS) on collagen I-functionalized polyacrylamide gels at stiffness ranging from 0.5 to 115 kPa, covering the physiological and pathological stiffness, to investigate distinct behaviors between cell types, including proliferation, migration, and activity of the ECM stiffness molecular rheostat YAP/TAZ. At the protein level, analysis of fibrillar collagen microarchitecture revealed that LMS tumors present reduced fibrillar collagen density and hyphal growth units and enhanced fiber endpoints compared to both MM and LM. At the gene expression level, however, LMS tumors did not show reduced fibrillar collagen expression, instead they exhibited enhanced matrix metalloproteinase expression, particularly of MMP14. Furthermore, COL11A1 was specifically upregulated in LMS tumors and its expression was associated with poor prognosis. Finally, in vitro response of MM, LM, and LMS cells to collagen I at defined stiffness showed that LMS cell migration, proliferation, and subcellular localization of YAP/TAZ are less sensitive to substrate stiffness than in MM and LM cells, although the response varied between distinct donors. In conclusion, we show that LMS tumors typically present low fibrillar collagen protein expression likely due to enhanced degradation. In addition, collagen I adhesion and stiffness have a lower impact on malignant LMS cells than on MM and LM, which may explain their ability to grow in low-collagen microenvironments. Furthermore, this study shows that COL11A1 is a potential biomarker with prognostic value in leiomyosarcoma. Citation Format: Jordi Gonzalez-Molina, Paula Hahn, Raul Maia Falcão, Georgia Kokaraki, Jorge Estefano de Souza, Tirzah Braz Petta Lajus, Kaisa Lehti, Joseph W. Carlson. The microarchitecture and fibrillar collagen expression of leiomyosarcoma are associated with malignancy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6096.

Poly-2-methyl-2-oxazoline–modified bioprosthetic heart valve leaflets have enhanced biocompatibility and resist structural degeneration

Bioprosthetic heart valves (BHV) fabricated from glutaraldehyde-fixed heterograft tissue, such as bovine pericardium (BP), are widely used for treating heart valve disease, a group of disorders that affects millions. Structural valve degeneration (SVD) of BHV due to both calcification and the accumulation of advanced glycation end products (AGE) with associated serum proteins limits durability. We hypothesized that BP modified with poly-2-methyl-2-oxazoline (POZ) to inhibit protein entry would demonstrate reduced accumulation of AGE and serum proteins, mitigating SVD. In vitro studies of POZ-modified BP demonstrated reduced accumulation of serum albumin and AGE. BP-POZ invitro maintained collagen microarchitecture per two-photon microscopy despite AGE incubation, and in cell culture studies was associated with no change in tumor necrosis factor-α after exposure to AGE and activated macrophages. Comparing POZ and polyethylene glycol (PEG)-modified BP invitro, BP-POZ was minimally affected by oxidative conditions, whereas BP-PEG was susceptible to oxidative deterioration. In juvenile rat subdermal implants, BP-POZ demonstrated reduced AGE formation and serum albumin infiltration, while calcification was not inhibited. However, BP-POZ rat subdermal implants with ethanol pretreatment demonstrated inhibition of both AGE accumulation and calcification. Ex vivo laminar flow studies with human blood demonstrated BP-POZ enhanced thromboresistance with reduced white blood cell accumulation. We conclude that SVD associated with AGE and serum protein accumulation can be mitigated through POZ functionalization that both enhances biocompatibility and facilitates ethanol pretreatment inhibition of BP calcification.

Mammary collagen architecture and its association with mammographic density and lesion severity among women undergoing image-guided breast biopsy

BackgroundElevated mammographic breast density is a strong breast cancer risk factor with poorly understood etiology. Increased deposition of collagen, one of the main fibrous proteins present in breast stroma, has been associated with increased mammographic density. Collagen fiber architecture has been linked to poor outcomes in breast cancer. However, relationships of quantitative collagen fiber features assessed in diagnostic biopsies with mammographic density and lesion severity are not well-established.MethodsClinically indicated breast biopsies from 65 in situ or invasive breast cancer cases and 73 frequency matched-controls with a benign biopsy result were used to measure collagen fiber features (length, straightness, width, alignment, orientation and density (fibers/µm2)) using second harmonic generation microscopy in up to three regions of interest (ROIs) per biopsy: normal, benign breast disease, and cancer. Local and global mammographic density volumes were quantified in the ipsilateral breast in pre-biopsy full-field digital mammograms. Associations of fibrillar collagen features with mammographic density and severity of biopsy diagnosis were evaluated using generalized estimating equation models with an independent correlation structure to account for multiple ROIs within each biopsy section.ResultsCollagen fiber density was positively associated with the proportion of stroma on the biopsy slide (p < 0.001) and with local percent mammographic density volume at both the biopsy target (p = 0.035) and within a 2 mm perilesional ring (p = 0.02), but not with global mammographic density measures. As severity of the breast biopsy diagnosis increased at the ROI level, collagen fibers tended to be less dense, shorter, straighter, thinner, and more aligned with one another (p < 0.05).ConclusionsCollagen fiber density was positively associated with local, but not global, mammographic density, suggesting that collagen microarchitecture may not translate into macroscopic mammographic features. However, collagen fiber features may be markers of cancer risk and/or progression among women referred for biopsy based on abnormal breast imaging.

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AbstractInstant polarized light microscopy (IPOL) allows for direct visualization of collagen with a single image encoding orientation and retardance in color and intensity, respectively, without post‐processing. The high spatiotemporal resolution of IPOL makes it well‐suited for quantitative imaging of collagen microarchitecture and behavior under dynamic loading. IPOL is therefore a powerful imaging tool that facilitates research on the biomechanics and pathophysiology of collagenous tissues. We demonstrate IPOL for sheep eyes, pig chordae tendineae and chicken tendon.Further details can be found in the article by Bin Yang, Po‐Yi Lee, Yi Hua, Bryn Brazile, Susannah Waxman, Fengting Ji, Ziyi Zhu, and Ian A. Sigal (e202000326). image

Phototunable interpenetrating polymer network hydrogels to stimulate the vasculogenesis of stem cell-derived endothelial progenitors

Vascularization of engineered scaffolds remains a critical obstacle hindering the translation of tissue engineering from the bench to the clinic. We previously demonstrated the robust micro-vascularization of collagen hydrogels with induced pluripotent stem cell (iPSC)-derived endothelial progenitors; however, physically cross-linked collagen hydrogels compact rapidly and exhibit limited strength. We have synthesized an interpenetrating polymer network (IPN) hydrogel comprised of collagen and norbornene-modified hyaluronic acid (NorHA) to address these challenges. This dual-network hydrogel combines the natural cues presented by collagen's binding sites and extracellular matrix (ECM)-mimicking fibrous architecture with the in situ modularity and chemical cross-linking of NorHA. We modulated the IPN hydrogel's stiffness and degradability by varying the concentration and sequence, respectively, of the NorHA peptide cross-linker. Rheological characterization of the photo-mediated gelation process revealed that the IPN hydrogel's stiffness increased with cross-linker concentration and was decoupled from the bulk NorHA content. Conversely, the swelling of the IPN hydrogel decreased linearly with increasing cross-linker concentration. Collagen microarchitecture remained relatively unchanged across cross-linking conditions, although the addition of NorHA delayed collagen fibrillogenesis. Upon iPSC-derived endothelial progenitor encapsulation, robust, lumenized microvascular networks developed in IPN hydrogels over two weeks. Subsequent computational analysis showed that an initial rise in stiffness increased the number of branch points and vessels, but vascular growth was suppressed in high stiffness IPN hydrogels. These results suggest that an IPN hydrogel consisting of collagen and NorHA is highly tunable, compaction resistant, and capable of supporting vasculogenesis.

Instant polarized light microscopy for imaging collagen microarchitecture and dynamics.

Collagen fibers are a primary load-bearing component of connective tissues and are therefore central to tissue biomechanics and pathophysiology. Understanding collagen architecture and behavior under dynamic loading requires a quantitative imaging technique with simultaneously high spatial and temporal resolutions. Suitable techniques are thus rare and often inaccessible. In this study, we present instant polarized light microscopy (IPOL), in which a single snapshot image encodes information on fiber orientation and retardance, thus fulfilling the requirement. We utilized both simulation and experimental data from collagenous tissues of chicken tendon, sheep eye, and porcine heart to evaluate the effectiveness of IPOL as a quantitative imaging technique. We demonstrate that IPOL allows quantitative characterization of micron-scale collagen fiber architecture at full camera frame rates (156 frames/second herein).

Probing the Role of Bone Lamellar Patterns through Collagen Microarchitecture Mapping, Numerical Modeling, and 3D‐Printing

Probing the Role of Bone Lamellar Patterns through Collagen Microarchitecture Mapping, Numerical Modeling, and 3D‐Printing

Collagen microarchitecture mechanically controls myofibroblast differentiation

Altered microarchitecture of collagen type I is a hallmark of wound healing and cancer that is commonly attributed to myofibroblasts. However, it remains unknown which effect collagen microarchitecture has on myofibroblast differentiation. Here, we combined experimental and computational approaches to investigate the hypothesis that the microarchitecture of fibrillar collagen networks mechanically regulates myofibroblast differentiation of adipose stromal cells (ASCs) independent of bulk stiffness. Collagen gels with controlled fiber thickness and pore size were microfabricated by adjusting the gelation temperature while keeping their concentration constant. Rheological characterization and simulation data indicated that networks with thicker fibers and larger pores exhibited increased strain-stiffening relative to networks with thinner fibers and smaller pores. Accordingly, ASCs cultured in scaffolds with thicker fibers were more contractile, expressed myofibroblast markers, and deposited more extended fibronectin fibers. Consistent with elevated myofibroblast differentiation, ASCs in scaffolds with thicker fibers exhibited a more proangiogenic phenotype that promoted endothelial sprouting in a contractility-dependent manner. Our findings suggest that changes of collagen microarchitecture regulate myofibroblast differentiation and fibrosis independent of collagen quantity and bulk stiffness by locally modulating cellular mechanosignaling. These findings have implications for regenerative medicine and anticancer treatments.

Probing the Role of Bone Lamellar Patterns through Collagen Microarchitecture Mapping, Numerical Modeling, and 3D‐Printing

The fibrillar organization is crucial for the mechanics of osteonal bone and for its anisotropy. Different collagen fiber orientation (CFO) patterns are observed in bone and correlated to the main local loading, leading to the hypothesis that the fibers are preferentially aligned to bear specific loading types. A heterogeneous distribution of osteon morphotypes (OMs) is noticed in long bones, although the relationship between the OM spatial distribution and the local predominant loading is still unclear. This study aims to shed light on the reasons why Nature uses specific OMs in diverse stress‐dominated regions. A multimodal approach is adopted, including collagen fiber orientation mapping, numerical modeling of different OMs under tensile and compressive loading, and 3D‐printing of OM‐inspired samples, to unravel the relationship between different OMs and their mechanical behavior. Simulation results suggest a better performance of the vertical OM (VOM) under tensile loading and of the twisted OM (TOM) under compression, confirming earlier hypotheses. The outcomes of mechanical testing, conducted on 3D‐printed samples, highlight a possible buckling‐induced failure of fibers with preferential vertical orientation and provide evidence that OMs are stress‐tailored, opening new venues for the design of stress‐tuned bioinspired composites.

Whole-Section Tumor Micro-Architecture Analysis by a Two-Dimensional Phasor-Based Approach Applied to Polarization-Dependent Second Harmonic Imaging.

Second Harmonic Generation (SHG) microscopy has gained much interest in the histopathology field since it allows label-free imaging of tissues simultaneously providing information on their morphology and on the collagen microarchitecture, thereby highlighting the onset of pathologies and diseases. A wide request of image analysis tools is growing, with the aim to increase the reliability of the analysis of the huge amount of acquired data and to assist pathologists in a user-independent way during their diagnosis. In this light, we exploit here a set of phasor-parameters that, coupled to a 2-dimensional phasor-based approach (μMAPPS, Microscopic Multiparametric Analysis by Phasor projection of Polarization-dependent SHG signal) and a clustering algorithm, allow to automatically recover different collagen microarchitectures in the tissues extracellular matrix. The collagen fibrils microscopic parameters (orientation and anisotropy) are analyzed at a mesoscopic level by quantifying their local spatial heterogeneity in histopathology sections (few mm in size) from two cancer xenografts in mice, in order to maximally discriminate different collagen organizations, allowing in this case to identify the tumor area with respect to the surrounding skin tissue. We show that the “fibril entropy” parameter, which describes the tissue order on a selected spatial scale, is the most effective in enlightening the tumor edges, opening the possibility of their automatic segmentation. Our method, therefore, combined with tissue morphology information, has the potential to become a support to standard histopathology in diseases diagnosis.

Impact of tissue preservation on collagen fiber architecture

ABSTRACTMicroarchitectural features of collagen-rich extracellular matrices provide the mechanical foundation for tissue function and exhibit topographical cues that influence cellular behavior including proliferation, migration and protein expression. Preservation of tissue microarchitecture is required for accurate evaluation of tissue characteristics and pathology. It is unclear whether common tissue preservation methods possess equal ability to preserve microarchitecture. We investigated collagen microarchitecture in samples that had been flash frozen, fixed in formalin or preserved in RNAlater®, and which contained both collagen-rich and collagen-sparse regions. Fibrillar collagen organization was characterized using picrosirius red staining and second harmonic generation (SHG) microscopy. Maintenance of collagen fiber characteristics compared to the gold standard of flash freezing depended on both the method of preservation and the local collagen content of the tissue. Both formalin fixation and RNAlater® preserved collagen fiber characteristics similar to flash freezing in collagen-rich areas of the tissue, but not in collagen-sparse regions. Analysis using picrosirius red staining indicated preservation-dependent changes in overall tissue architecture and suprafibrillar organization. Together with considerations of cost, ease of use, storage conditions and ability to use the preserved tissue for RNA or protein analysis, our quantitative characterization of the effects of preservation method on collagen microarchitecture may help investigators select the most appropriate preservation approach for their needs.

Decellularization and characterization of a whole intervertebral disk xenograft scaffold.

Intervertebral disk (IVD) degeneration is a multifactor process that results in the physical destruction of the nucleus pulposus (NP) and annulus fibrosus (AF). This compromises IVD function and causes significant disability and economic burden. Strategies to replace the entire composite structure of the IVD are limited and most approaches do not recapitulate the heterogenous biochemical composition, microarchitecture or mechanical properties of the native tissue. Our central hypothesis was that donor IVDs which resemble the size and biochemistry of human lumbar IVDs could be successfully decellularized while retaining the tissue's structure and function with the long-term goal of creating a composite scaffold for tissue engineering the human IVD. Accordingly, we optimized a procedure to decellularize bovine tail IVDs using a combination of detergents, ultrasonication, freeze-thaw cycles, and nucleases. The resultant decellularized whole IVD xenografts retained distinct AF and NP regions which contained no visible intact cell nuclei and minimal residual bovine deoxyribose nucleic acid (DNA; 65.98 ± 4.07 and 47.12 ± 13.22 ng/mg, respectively). Moreover, the NP region of decellularized IVDs contained 313.40 ± 50.67 µg/mg glycosaminoglycan. The presence of collagen type II was confirmed via immunohistochemistry. Additionally, histological analysis of the AF region of decellularized IVDs demonstrated retention of the native angle-ply collagen microarchitecture. Unconfined compression testing demonstrated no significant differences in swelling pressure and toe-region modulus between fresh and decellularized IVDs. However, linear region moduli, peak stress and equilibrium moduli were all significantly reduced. Together, this research demonstrates a successful initial step in developing a biomimetic acellular whole IVD xenograft scaffold for use in IVD tissue engineering. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A:2412-2423, 2018.

Calcific Aortic Valve Disease Is Associated with Layer-Specific Alterations in Collagen Architecture.

Disorganization of the valve extracellular matrix (ECM) is a hallmark of calcific aortic valve disease (CAVD). However, while microarchitectural features of the ECM can strongly influence the biological and mechanical behavior of tissues, little is known about the ECM microarchitecture in CAVD. In this work, we apply advanced imaging techniques to quantify spatially heterogeneous changes in collagen microarchitecture in CAVD. Human aortic valves were obtained from individuals between 50 and 75 years old with no evidence of valvular disease (healthy) and individuals who underwent valve replacement surgery due to severe stenosis (diseased). Second Harmonic Generation microscopy and subsequent image quantification revealed layer-specific changes in fiber characteristics in healthy and diseased valves. Specifically, the majority of collagen fiber changes in CAVD were found to occur in the spongiosa, where collagen fiber number increased by over 2-fold, and fiber width and density also significantly increased. Relatively few fibrillar changes occurred in the fibrosa in CAVD, where fibers became significantly shorter, but did not otherwise change in terms of number, width, density, or alignment. Immunohistochemical staining for lysyl oxidase showed localized increased expression in the diseased fibrosa. These findings reveal a more complex picture of valvular collagen enrichment and arrangement in CAVD than has previously been described using traditional analysis methods. Changes in fiber architecture may play a role in regulating the pathobiological events and mechanical properties of valves during CAVD. Additionally, characterization of the ECM microarchitecture can inform the design of fibrous scaffolds for heart valve tissue engineering.

Collagen I hydrogel microstructure and composition conjointly regulate vascular network formation.

Neovascularization is a hallmark of physiological and pathological tissue remodeling that is regulated in part by the extracellular matrix (ECM). Collagen I hydrogels or Matrigel are frequently used to study vascular network formation; however, in isolation these materials do not typically mimic the integrated effects of ECM structure and composition that may influence endothelial cells in vivo. Here, we have utilized microfabricated 3D culture models to control collagen I microstructure in the presence and absence of Matrigel and tested the effect of these variations on vascular network formation by human cerebral microvascular endothelial cells (hCMECs). Varied collagen microarchitecture was achieved by adjusting the gelation temperature and subsequently confirmed by structural analysis. Casting at colder temperature increased collagen fiber thickness and length, and inclusion of Matrigel further pronounced these differences. Interestingly, the presence of Matrigel affected vascular network formation by modulating hCMEC growth, whereas altered collagen fiber structure impacted the morphology and maturity of the developed vascular network. These differences were related to substrate-dependent changes in interleukin-8 (IL-8) secretion and were functionally relevant as vascular networks preformed in more fibrillar, Matrigel-containing hydrogels promoted angiogenic sprouting. Our studies indicate that collagen hydrogel microstructure and composition conjointly regulate vascular network formation with implications for translational and basic science approaches. Neovascularization is a hallmark of both tissue homeostasis and disease and is in part regulated by cell remodeling that occurs in the extracellular matrix (ECM). The use of bio-mimetic hydrogel cell culture systems has been used to study the effects of the ECM on cell behavior. Here, we employ a hydrogel system that enables control over both the structure and composition of the ECM and subsequently investigated the effects that these have on blood vessel dynamics. Finally, we linked these differences to changes in protein secretion and the implications that this may play in scientific translation.

Increased peri-ductal collagen micro-organization may contribute to raised mammographic density.

BackgroundHigh mammographic density is a therapeutically modifiable risk factor for breast cancer. Although mammographic density is correlated with the relative abundance of collagen-rich fibroglandular tissue, the causative mechanisms, associated structural remodelling and mechanical consequences remain poorly defined. In this study we have developed a new collaborative bedside-to-bench workflow to determine the relationship between mammographic density, collagen abundance and alignment, tissue stiffness and the expression of extracellular matrix organising proteins.MethodsMammographic density was assessed in 22 post-menopausal women (aged 54–66 y). A radiologist and a pathologist identified and excised regions of elevated non-cancerous X-ray density prior to laboratory characterization. Collagen abundance was determined by both Masson’s trichrome and Picrosirius red staining (which enhances collagen birefringence when viewed under polarised light). The structural specificity of these collagen visualisation methods was determined by comparing the relative birefringence and ultrastructure (visualised by atomic force microscopy) of unaligned collagen I fibrils in reconstituted gels with the highly aligned collagen fibrils in rat tail tendon. Localised collagen fibril organisation and stiffness was also evaluated in tissue sections by atomic force microscopy/spectroscopy and the abundance of key extracellular proteins was assessed using mass spectrometry.ResultsMammographic density was positively correlated with the abundance of aligned periductal fibrils rather than with the abundance of amorphous collagen. Compared with matched tissue resected from the breasts of low mammographic density patients, the highly birefringent tissue in mammographically dense breasts was both significantly stiffer and characterised by large (>80 μm long) fibrillar collagen bundles. Subsequent proteomic analyses not only confirmed the absence of collagen fibrosis in high mammographic density tissue, but additionally identified the up-regulation of periostin and collagen XVI (regulators of collagen fibril structure and architecture) as potential mediators of localised mechanical stiffness.ConclusionsThese preliminary data suggest that remodelling, and hence stiffening, of the existing stromal collagen microarchitecture promotes high mammographic density within the breast. In turn, this aberrant mechanical environment may trigger neoplasia-associated mechanotransduction pathways within the epithelial cell population.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0664-2) contains supplementary material, which is available to authorized users.

Bone Cell Biology Assessed by Microscopic Approach. The effects of active vitamin D3 such as alfacalcidol and eldecalcitol on bone quality

Active vitamin D3 is used for the treatment for osteoporosis in Japan. Recently, data have accumulated that collagen cross-link formation in bone affect bone strength. In fact, impaired enzymatic cross-linking, over-hydroxylation of crosslinks, and an increase in non-enzymatic crosslinking advanced glycation end products (AGEs) such as pentosidine, in bone collagen have been proposed as a major cause of bone fragility in osteoporosis. We reported that alfacalcidol and eldecalcitol improves bone material properties such as collagen cross-link formation, microarchitecture, and microcrack resulting in the increase of bone strength (Saito M, Bone 2010;46:1170-1179, Calcif Tissue Int 2011;88:314-324, Bone, 2015;73:8-15). In this review, we described how active vitamin D3 improve bone collagen cross-link formation and mineral qualities.

Increased susceptibility of skin from HERDA (Hereditary Equine Regional Dermal Asthenia)-affected horses to bacterial collagenase degradation: a potential contributing factor to the clinical signs of HERDA.

Hereditary equine regional dermal asthenia (HERDA) is a genetic disorder of collagen resulting in fragile, hyper-extensible skin and ulcerative lesions. The predominance of skin lesions have been shown to occur on the dorsum of HERDA-affected horses. While this has been postulated to be due to increased exposure to sunlight of these areas, the precise pathological mechanism which causes this to occur is unclear. We hypothesized that an increase in collagenase activity, that has been associated with the exposure of dermal fibroblasts to sunlight, will significantly degrade the material properties of skin from HERDA-affected horses when compared to unaffected controls. Six unaffected and seven HERDA-affected horses, all euthanized for other reasons. Full-thickness skin samples from similar locations on each horse were collected and cut into uniform strips and their material properties (tensile modulus) determined by mechanical testing before (n = 12 samples/horse) or after (n = 12 samples/horse) incubation in bacterial collagenase at 37°C for 6 h. The change in modulus following treatment was then compared between HERDA-affected and unaffected horses using a Student's t-test. The modulus of skin from HERDA-affected horses decreased significantly more than that from unaffected horses following collagenase treatment (54 ± 7% versus 30 ± 16%, P = 0.004). The significant decrease in the modulus of skin from HERDA-affected horses following collagenase exposure suggests that their altered collagen microarchitecture is more susceptible to enzymatic degradation and may explain the localization of skin lesions in HERDA-affected horses to those areas of the body most exposed to sunlight. These findings appear to support the previously reported benefits of sunlight restriction in HERDA-affected horses.