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Significance: The spatial organization of collagen fibers has been used as a biomarker for assessing injury and disease progression. However, quantifying this organization for complex structures is challenging. Aim: To quantify and classify complex collagen fiber organizations. Approach: Using quantitative second-harmonic generation (SHG) microscopy, we show that collagen-fiber orientation can be viewed as pseudovector fields. Subsequently, we analyze them using fluid mechanic metrics, such as energy U, enstrophy E, and tortuosity tau. Results: We show that metrics used in fluid mechanics for analyzing fluid flow can be adapted to analyze complex collagen fiber organization. As examples, we consider SHG images of collagenous tissue for straight, wavy, and circular fiber structures. Conclusions: The results of this study show the utility of the chosen metrics to distinguish diverse and complex collagen organizations. We find that the distribution of values for E and U increases with collagen fiber disorganization, where they divide between low and high values corresponding to uniformly aligned fibers and disorganized collagen fibers, respectively. We also confirm that the values of tau cluster around 1 when the fibers are straight, and the range increases up to 1.5 when wavier fibers are present. (C) The Authors. Published by SPIE under a Creative Commons Attribution 4.0 International License. Published version

Fine particulate matter, a major air pollutant across the world, causes a series of pulmonary diseases. Vitamin D is a typical vitamin with emerging roles in inflammation and fibrosis. Different situations and diseases need different doses and modes of vitamin D administration, which challenges the existing vitamin D supplementary rules. Thus, studies of vitamin D applications and their mechanisms in various diseases are important for its future therapeutic applications. In this study, the therapeutic application of vitamin D3 in chronic particle-exposure-associated lung fibrosis and tissue remodeling was investigated. In vivo studies showed that vitamin D3 significantly attenuated fibrosis effects by decreasing α-smooth muscle actin-regulated extracellular matrix deposition and restoring expressions of E-cadherin and N-cadherin. With the importance of activated macrophage in the regulation of local epithelium and fibroblast in the process of tissue fibrosis, two separate in vitro systems of co-culture of macrophages with lung epithelium or fibroblast were built. The results confirmed that vitamin D3 promoted the proliferation of lung epithelium and depressed the fibrosis effects of fibroblasts as well. In addition, our results indicated that the therapeutic effects of vitamin D3 were through Nrf2 signals. Our work provides convincing experimental evidence for vitamin D therapeutic application to promote tissue repair and improve particle-associated lung fibrosis.

Significance: Collagen is the most abundant protein in vertebrates and is found in tissues that regularly experience tension, compression, and shear forces. However, the underlying mechanism of collagen fibril formation and remodeling is poorly understood. Aim: We explore how a collagen monomer is visualized using fluorescence microscopy and how its spatial orientation is determined. Defining the orientation of collagen monomers is not a trivial problem, as the monomer has a weak contrast and is relatively small. It is possible to attach fluorescence tags for contrast, but the size is still a problem for detecting orientation using fluorescence microscopy. Approach: We present two methods for detecting a monomer and classifying its orientation. A modified Gabor filter set and an automatic classifier trained by convolutional neural network based on a synthetic dataset were used. Results: By evaluating the performance of these two approaches with synthetic and experimental data, our results show that it is possible to determine the location and orientation with an error of ∼37 deg of a single monomer with fluorescence microscopy. Conclusions: These findings can contribute to our understanding of collagen monomers interaction with collagen fibrils surface during fibril formation and remodeling.

Cellular movement is essential in the formation and maintenance of healthy tissues as well as in disease progression such as tumor metastasis. In this work, we describe a paper-based Transwell assay capable of quantifying cellular invasion through an extracellular matrix. The paper-based Transwell assays generate similar datasets, with equivalent reproducibility, to commercially available Transwell assays. With different culture configurations, we quantify invasion: upon addition of an exogenous factor or in the presence of medium obtained from other cell types, in an indirect or direct co-culture format whose medium composition is dynamically changing, and in a single-zone or parallel (96-zone) format.

PubMed: 31983145 Obesity is associated with a higher risk of developing breast cancer and with worse disease outcomes for women of all ages. The composition, density, and organization of the breast tissue stroma are also known to play an important role in the development and progression of the disease. However, the connections between obesity and stromal remodeling are not well understood. We sought to characterize detailed organization features of the collagen matrix within healthy and cancerous breast tissues acquired from mice exposed to either a normal or high fat (obesity inducing) diet. We performed second-harmonic generation and spectral two-photon excited fluorescence imaging, and we extracted the level of collagen-associated fluorescence (CAF) along with metrics of collagen content, three-dimensional, and two-dimensional organization. There were significant differences in the CAF intensity and overall collagen organization between normal and tumor tissues; however, obesity-enhanced changes in these metrics, especially when three-dimensional organization metrics were considered. Thus, our studies indicate that obesity impacts significantly collagen organization and structure and the related pathways of communication may be important future therapeutic targets..

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with poor prognosis with short lifespan following diagnosis as patients have limited effective treatment options. A fundamental limitation is a lack of knowledge of the underlying collagen alterations in the disease, as this could lead to better diagnostics, prognostics, and measures of treatment efficacy. While the fibroses is the primary presentation of the disease, the collagen architecture has not been well studied beyond standard histology. Here, we used several metrics based on second harmonic generation (SHG) microscopy and optical scattering measurements to characterize the subresolution collagen assembly in human IPF and normal lung tissues. Using SHG directional analysis, we found that while collagen synthesis is increased in IPF, the resulting average fibril architecture is more disordered than in normal tissue. Wavelength-dependent optical scattering measurements lead to the same conclusion, and both optical approaches are consistent with ultrastructural analysis. SHG circular dichroism revealed significant differences in the net chirality between the fibrotic and normal collagen, where the former has a more randomized helical structure. Collectively, the measurements reveal significant changes in the collagen macro/supramolecular structure in the abnormal fibrotic collagen, and we suggest these alterations can serve as biomarkers for IPF diagnosis and progression.

Paper-based scaffolds support the three-dimensional culture of mammalian cells in tissue-like environments. These Tissue Papers, a name that highlights the use of materials obtained from (plant) tissue to generate newly functioning (human) tissue structures, are a promising analytical tool to quantify cellular responses in physiologically relevant extracellular gradients and coculture architectures. Here, we highlight current examples of Tissue Papers, commonly used methods of analysis, and current measurement challenges.

Neoadjuvant chemotherapy (NACT) is routinely administered to subsets of breast cancer patients, including triple negative (TN) or human epidermal growth factor receptor 2-positive (HER2+) cancers. After NACT and subsequent surgical resection, 5% to 30% of patients have no residual invasive carcinoma, termed pathological complete response. Unfortunately, many patients experience little-to-no response after NACT and unnecessarily suffer its side effects. Methods are needed to predict an individual patient’s response to NACT. Core needle biopsies, taken before NACT, consist of tumor cells and the surrounding extracellular matrix. We performed second-harmonic generation (SHG) imaging of fibrillar collagen in core needle biopsy sections as a possible predictor of response to NACT. The ratio of forward-to-backward scattering (F/B) SHG was assessed in the “tumor bulk” and “tumor–host interface” in HER2+ and TN core needle biopsy sections. Patient response was classified post-treatment using the Residual Cancer Burden (RCB) score. In HER2+ biopsies, RCB class was associated with F/B derived from the tumor–stromal interface, but not tumor bulk. F/B was not associated with RCB class in TN biopsies. These findings suggest that F/B from needle biopsy sections may be a useful predictor of which patients will respond favorably to NACT, with the potential to help reduce overtreatment.

Bioactive, functional scaffolds are required to improve the regenerative potential of stem cells for tissue reconstruction and functional recovery of damaged tissues. Here, we report a paper-based bioactive scaffold platform for stem cell culture and transplantation for bone reconstruction. The paper scaffolds are surface-engineered by an initiated chemical vapor deposition process for serial coating of a water-repellent and cell-adhesive polymer film, which ensures the long-term stability in cell culture medium and induces efficient cell attachment. The prepared paper scaffolds are compatible with general stem cell culture and manipulation techniques. An optimal paper type is found to provide structural, physical, and mechanical cues to enhance the osteogenic differentiation of human adipose-derived stem cells (hADSCs). A bioactive paper scaffold significantly enhances in vivo bone regeneration of hADSCs in a critical-sized calvarial bone defect. Stacking the paper scaffolds with osteogenically differentiated hADSCs and human endothelial cells resulted in vascularized bone formation in vivo. Our study suggests that paper possesses great potential as a bioactive, functional, and cost-effective scaffold platform for stem cell-mediated bone tissue engineering. To the best of our knowledge, this is the first study reporting the feasibility of a paper material for stem cell application to repair tissue defects.

Considerable progress has been made toward a molecular understanding of how cells form lumen and tube structures in three-dimensional (3D) extracellular matrices (ECM). This progress has occurred through work performed with endothelial and epithelial cell models using both in vitro and in vivo approaches. Despite the apparent similarities between endothelial and epithelial cell lumen and tube formation mechanisms, there are clear distinctions that directly relate to their functional differences. This review will focus on endothelial cell (EC) lumen formation mechanisms which control blood vessel formation during development and postnatal life. Of great interest is that an EC lumen signaling complex has been identified which controls human EC lumen and tube formation in 3D matrices and which coordinates integrin-ECM contacts, cell surface proteolysis, cytoskeletal rearrangements, and cell polarity. This complex consists of the collagen-binding integrin α2β1, the collagen-degrading membrane-type 1 matrix metalloproteinase (MT1-MMP), junction adhesion molecule (Jam)C, JamB, polarity proteins Par3 and Par6b, and the Rho GTPase Cdc42-GTP. These interacting proteins are necessary to stimulate 3D matrix-specific signaling events (including activation of protein kinase cascades that regulate the actin and microtubule cytoskeletons) to control the formation of EC lumens and tube networks. Also, EC lumen formation is directly coupled to the generation of vascular guidance tunnels, enzymatically generated ECM conduits that facilitate EC tube remodeling and maturation. Mural cells such as pericytes are recruited along EC tubes within these tunnel spaces to control ECM remodeling events resulting in vascular basement membrane matrix assembly, a key step in tube maturation and stabilization.

The medial epithelial seam (MES) between the palatal shelves degrades during palatal fusion to achieve the confluence of palatal mesenchyme. Cellular mechanisms underlying the degradation of MES have been proposed, such as apoptosis, epithelial-mesenchymal transition (EMT) and migration of medial edge epithelia (MEE). Extracellular matrix components have been shown to play an important role in EMT in many model systems. Periostin (also known as osteoblast-specific factor-2) is a secreted mesenchymal extracellular matrix component that affects the ability of cells to migrate and/or facilitates EMT during both embryonic development and pathologic conditions. In this study, we evaluated the spatiotemporal expression patterns of periostin during mouse palatal fusion by in situ hybridization and immunofluorescence. Periostin mRNA and protein were present in the palatal mesenchyme, the protein being distributed in a fine fibrillar network and in the basement membrane, but absent from the epithelium. During MES degradation, the protein was strongly expressed in the basement membrane underlying the MES and in some select MEE. Confocal microscopic analysis using an EMT marker, twist1, and an epithelial marker, cytokeratin 14, provided evidence that select MEE were undergoing EMT in association with periostin. Moreover, the major extracellular matrix molecules in basement membrane, laminin and collagen type IV were degraded earlier than periostin. The result is that select MEE establish interactions with periostin in the mesenchymal extracellular matrix, and these new cell-matrix interactions may regulate MEE transdifferentiation during palatal fusion.

Biglycan (BGN) and WISP-1 are 2 extracellular matrix proteins that bind to each other and colocalize in mineralizing tissue. Here we show that WISP-1 abrogates the repression of proliferation in bone marrow stromal cells induced by BGN. We also demonstrate that WISP-1 and its variant WISP-1va can alleviate the repressed osteogenic differentiation caused by the absence of BGN. These preliminary data suggest that WISP-1 and BGN may functionally interact and control each other’s activity, thus regulating the differentiation and proliferation of osteogenic cells.

Dentin matrix protein 1 (DMP1) has been shown to be critical for the formation of dentin and bone. However, the precise pathway by which DMP1 participates in dentinogenesis and osteogenesis remains to be clarified. DMP1 is present in the extracellular matrix of dentin and bone as processed NH2- and COOH-terminal fragments. The NH2-terminal fragment occurs as a proteoglycan, whereas the COOH-terminal fragment is highly phosphorylated. The differences in biochemical properties suggest that these fragments may have different tissue and cell distribution in association with distinct functions. In this study, we analyzed the distribution of the NH2- and COOH-terminal fragments of DMP1 in tooth, bone, osteocytes as well as MC3T3-E1 and HEK-293 cells. Immunohistochemical analyses were performed using antibodies specific to the NH2- or COOH-terminal region of DMP1. Clear differences in the distribution of these fragments were observed. In the teeth and bone, the NH2-terminal fragment was primarily located in the nonmineralized predentin and cartilage of the growth plate, while the COOH-terminal fragment accumulated in the mineralized zones. In osteocytes, the NH2-terminal fragment appeared more abundant along cell membrane and processes of osteocytes, while the COOH-terminal fragment was often found in the nuclei. This pattern of distribution in cellular compartments was further confirmed by analyses on MC3T3-E1 and HEK-293 cells transfected with a construct containing DMP1 cDNA. In these cell lines, the COOH-terminal fragment accumulated in cell nuclei, while the NH2-terminal fragment was in the cytosol. The different distribution of DMP1 fragments indicates that these DMP1 variants must perform distinct functions.

Background —In vitro the Epstein-Barr virus (EBV) encoded latent membrane protein 1 (LMP-1) has been shown to upregulate expression of matrix metalloproteinase 9 (MMP-9), a member of a family of zinc dependent endopeptidases that is believed to facilitate tumour invasion and metastasis by degradation of the extracellular matrix. Aim —To test whether the expression of MMP-9 in Hodgkin9s disease correlates with EBV status and survival and to investigate whether LMP-1 expression affects MMP-9 concentrations in the Hodgkin9s disease cell line, L428. Methods —MMP-9 expression was measured by means of immunohistochemistry in a series of Hodgkin9s disease tumours and this expression was correlated with EBV status and survival. The influence of LMP-1 on MMP-9 expression was also investigated in the Hodgkin9s disease cell line, L428. Results —MMP-9 expression was demonstrated in the malignant Hodgkin and Reed-Sternberg cells of all (n = 86) formalin fixed, paraffin wax embedded Hodgkin9s disease tumours examined. Although the intensity of MMP-9 immunostaining varied between cases, there was no correlation between MMP-9 expression and EBV status or survival. MMP-9 expression was also detected in a variety of non-malignant cells, including fibroblasts. MMP-9 was detected by zymography in the L428 and KMH2 Hodgkin9s disease cell lines, whereas low or undetectable amounts of MMP-9 were found in the L591 Hodgkin9s disease cell line. Induction of LMP-1 expression in the Hodgkin9s disease cell line L428 did not result in a detectable increase in the values of MMP-9 as measured by zymography. Conclusions —These results demonstrate that MMP-9 is consistently expressed by the Hodgkin and Reed-Sternberg cells of Hodgkin9s disease tumours and by the Hodgkin9s disease cell lines, L428 and KMH2. However, this expression does not appear to be related either to LMP-1 values or to survival.

Background: Surgical reconstruction of congenital heart defects is often limited by the nonresorbable material used to approximate normal anatomy. In contrast, biologic scaffold materials composed of resorbable non-cross-linked extracellular matrix (ECM) have been used for tissue reconstruction of multiple organs and are replaced by host tissue. Preparation of whole organ ECM by decellularization through vascular perfusion can maintain much of the native three-dimensional (3D) structure, strength, and tissue-specific composition. A 3D cardiac ECM (C-ECM) biologic scaffold material would logically have structural and functional advantages over materials such as Dacron™ for myocardial repair, but the in vivo remodeling characteristics of C-ECM have not been investigated to date. Methods and Results: A porcine C-ECM patch or Dacron patch was used to reconstruct a full-thickness right ventricular outflow tract (RVOT) defect in a rat model with end points of structural remodeling function at 16 weeks. The Dacron patch was encapsulated by dense fibrous tissue and showed little cellular infiltration. Echocardiographic analysis showed that the right ventricle of the hearts patched with Dacron were dilated at 16 weeks compared to presurgery baseline values. The C-ECM patch remodeled into dense, cellular connective tissue with scattered small islands of cardiomyocytes. The hearts patched with C-ECM showed no difference in the size or function of the ventricles as compared to baseline values at both 4 and 16 weeks. Conclusions: The C-ECM patch was associated with better functional and histomorphological outcomes compared to the Dacron patch in this rat model of RVOT reconstruction.

Background: The extracellular matrix (ECM) is a critical determinant of neovessel integrity. Materials and Methods: Thirty-six (polyglycolic acid + polycaprolactone and poly lactic acid) tissue-engineered vascular grafts seeded with syngeneic bone marrow mononuclear cells were implanted as inferior vena cava interposition grafts in C57BL/6 mice. Specimens were characterized using immunohistochemical staining and qPCR for representative ECM components in addition to matrix metalloproteinases (MMPs). Total collagen, elastin, and glycosaminoglycan (GAG) contents were determined. MMP activity was measured using zymography. Results: Collagen production on histology demonstrated an initial increase in type III at 1 week followed by type I production at 2 weeks and type IV at 4 weeks. Gene expression of both type I and type III peaked at 2 weeks, whereas type IV continued to increase over the 4-week period. Histology demonstrated fibrillin-1 deposition at 1 week followed by elastin production at 4 weeks. Elastin gene expression significantly increased at 4 weeks, whereas fibrillin-1 decreased at 4 weeks. GAG demonstrated abundant production at each time point on histology. Gene expression of decorin significantly increased at 4 weeks, whereas versican decreased over time. Biochemical analysis showed that total collagen production was greatest at 2 weeks, and there was a significant increase in elastin and GAG production at 4 weeks. Histological characterization of MMPs showed abundant production of MMP-2 at each time point, while MMP-9 decreased over the 4-week period. Gene expression of MMP-2 significantly increased at 4 weeks, whereas MMP-9 significantly decreased at 4 weeks. Conclusions: ECM production during neovessel formation is characterized by early ECM deposition followed by extensive remodeling.

In vitro 3D culture is an important model for tissues in vivo. Cells in different locations of 3D tissues are physiologically different, because they are exposed to different concentrations of oxygen, nutrients, and signaling molecules, and to other environmental factors (temperature, mechanical stress, etc). The majority of high-throughput assays based on 3D cultures, however, can only detect the average behavior of cells in the whole 3D construct. Isolation of cells from specific regions of 3D cultures is possible, but relies on low-throughput techniques such as tissue sectioning and micromanipulation. Based on a procedure reported previously (‘‘cells-in-gels-in-paper’’ or CiGiP), this paper describes a simple method for culture of arrays of thin planar sections of tissues, either alone or stacked to create more complex 3D tissue structures. This procedure starts with sheets of paper patterned with hydrophobic regions that form 96 hydrophilic zones. Serial spotting of cells suspended in extracellular matrix (ECM) gel onto the patterned paper creates an array of 200 micron-thick slabs of ECM gel (supported mechanically by cellulose fibers) containing cells. Stacking the sheets with zones aligned on top of one another assembles 96 3D multilayer constructs. De-stacking the layers of the 3D culture, by peeling apart the sheets of paper, ‘‘sections’’ all 96 cultures at once. It is, thus, simple to isolate 200-micron-thick cell-containing slabs from each 3D culture in the 96-zone array. Because the 3D cultures are assembled from multiple layers, the number of cells plated initially in each layer determines the spatial distribution of cells in the stacked 3D cultures. This capability made it possible to compare the growth of 3D tumor models of different spatial composition, and to examine the migration of cells in these structures.

Fundamental investigations of human biology, and the development of therapeutics, commonly rely on 2D cell-culture systems that do not accurately recapitulate the structure, function, or physiology of living tissues. Systems for 3D cultures exist but do not replicate the spatial distributions of oxygen, metabolites, and signaling molecules found in tissues. Microfabrication can create architecturally complex scaffolds for 3D cell cultures that circumvent some of these limitations; unfortunately, these approaches require instrumentation not commonly available in biology laboratories. Here we report that stacking and destacking layers of paper impregnated with suspensions of cells in extracellular matrix hydrogel makes it possible to control oxygen and nutrient gradients in 3D and to analyze molecular and genetic responses. Stacking assembles the “tissue”, whereas destacking disassembles it, and allows its analysis. Breast cancer cells cultured within stacks of layered paper recapitulate behaviors observed both in 3D tumor spheroids in vitro and in tumors in vivo: Proliferating cells in the stacks localize in an outer layer a few hundreds of microns thick, and growth-arrested, apoptotic, and necrotic cells concentrate in the hypoxic core where hypoxia-sensitive genes are overexpressed. Altering gas permeability at the ends of stacks controlled the gradient in the concentration of the O 2 and was sufficient by itself to determine the distribution of viable cells in 3D. Cell cultures in stacked, paper-supported gels offer a uniquely flexible approach to study cell responses to 3D molecular gradients and to mimic tissue- and organ-level functions.

Aims —To determine mechanisms regulating the production of connective tissue growth factor (CCN2; CTGF) and transforming growth factor β1 (TGF-β1) in the mouse uterus. Methods —In situ hybridisation and immunohistochemistry were used to localise CCN2 (CTGF) and TGF-β1 in uteri from sexually mature female mice that had either been (1) mated with sterile males to induce pseudopregnancy or (2) ovariectomised (OVX) and administered estradiol-17β (E 2 ) or progesterone (P 4 ), either alone or in combination. Uteri collected on days 0.5, 1.5, 2.5, 3.5, 4.5, or 5.5 of pseudopregnancy or at one, three, six, 12, or 24 hours after steroid administration were fixed, sectioned, and incubated with specific riboprobes or antibodies to permit detection and localisation of mRNA or protein for CTGF and TGF-β1. Results —On days 0.5–2.5 of pseudopregnancy, CCN2 (CTGF) and TGF-β1 were principally colocalised to uterine epithelial cells, with much smaller amounts in the stroma. On days 3.5–4.5, there was a reduction of CCN2 (CTGF) and TGF-β1 in the epithelium but an increase in stromal and endothelial cells, corresponding to a period of extracellular matrix remodelling and neovascularisation within the endometrium. In OVX mice, epithelial cells were weakly positive for both CCN2 (CTGF) and TGF-β1 in the absence of steroid hormones. Epithelial CTGF mRNA production were strongly but transiently stimulated in OVX mice cells by E 2 . These effects were antagonised by P 4 , which itself transiently stimulated epithelial CCN2 (CTGF) production, although less robustly than E 2 . CTGF and TGF-β1 protein amounts were high in epithelial cells throughout steroid treatment and were increased in the stroma, where they were relatively long lived. Stromal CCN2 (CTGF) and TGF-β1 were lower after co-administration of E 2 and P 4 than in response to each hormone individually. Although ccn2 (ctgf) is a TGF-β1 inducible gene in other systems, and both growth factors were often co-localised in uterine tissues in these studies, several treatment regimens resulted in high amounts of TGF-β1 protein in stromal cells without the concomitant production of ccn2 (ctgf) mRNA. Conclusions— Maternal factors are principal cues for CCN2 (CTGF) and TGF-β1 production in the uterus because (1) their expression during pseudopregnancy is comparable to that seen in pregnancy and (2) they are regulated by ovarian steroids. TGF-β dependent and independent mechanisms of ccn2 (ctgf) gene transcription exist in the uterus that are variably regulated by steroid hormones. Collectively, the data support a role for CCN2 (CTGF) in mediating the effects of steroid hormones and TGF-β on endometrial function.