Structure Of Collagen Matrix Research Articles

Effects of mineralization on the hierarchical organization of collagen-a synchrotron X-ray scattering and polarized second harmonic generation study.

The process of mineralization fundamentally alters collagenous tissue biomechanics. While the structure and organization of mineral particles have been widely studied, the impact of mineralization on collagen matrix structure, particularly at the molecular scale, requires further investigation. In this study, synchrotron X-ray scattering (XRD) and polarization-resolved second harmonic generation microscopy (pSHG) were used to study normally mineralizing turkey leg tendon in tissue zones representing different stages of mineralization. XRD data demonstrated statistically significant differences in collagen D-period, intermolecular spacing, fibril and molecular dispersion and relative supramolecular twists between non-mineralizing, early mineralizing and late mineralizing zones. pSHG analysis of the same tendon zones showed the degree of collagen fibril organization was significantly greater in early and late mineralizing zones compared to non-mineralizing zones. The combination of XRD and pSHG data provide new insights into hierarchical collagen-mineral interactions, notably concerning possible cleavage of intra- or interfibrillar bonds, occlusion and reorganization of collagen by mineral with time. The complementary application of XRD and fast, label-free and non-destructive pSHG optical measurements presents a pathway for future investigations into the dynamics of molecular scale changes in collagen in the presence of increasing mineral deposition.

A Dentin Biomimetic Remineralization Material with an Ability to Stabilize Collagen.

The integrity of collagen matrix structure is a prerequisite for effectively inducing biomimetic remineralization. Repeated low pH stimulation activates matrix metalloproteinases (MMPs) in dental caries. Activated MMPs cause the breakdown of collagen fibrils. Collagen stabilization is a major obstacle to the clinical application of remineralization templates. Here, galardin-loaded poly(amido amine) (PAMAM)-NGV (PAMAM-NGV@galardin, PNG) is constructed to induce collagen stabilization and dentin biomimetic remineralization simultaneously, in order to combat early caries in dentin. PAMAM acts in the role of nucleation template for dentin remineralization, while galardin acts as the role of MMPs inhibitor. NGV peptides modified on the surface of dendrimer core can form small clusters with synergistic movement in short range, and those short-range clusters can form domain areas with different properties on the surface of PAMAM core and restrict the movement of collagen, favoring collagen crosslinking, which can be explained through the computational simulation analysis results. NGV peptides and galardin show a dual collagen-protective effect, laying the foundation for the dentin remineralization effect induced by PAMAM. PNG induces dentin remineralization in an environment with collagenase, meanwhile showsing anti-dentin caries efficacy in vivo. These findings indicate that PNG has great potential to combat early dentin caries for future clinical application.

Nanoscopic wear behavior of dentinogenesis imperfecta type II tooth dentin

ObjectivesThe aim of this study was to investigate the wear behavior of Dentinogenesis imperfecta type II (DGI-II) dentin and elucidate the correlation between its tribological properties and components. MethodsThe mid-coronal dentin of normal and DGI-II teeth were divided into two groups: perpendicular and parallel to the dentin tubules. The microstructure of dentin was detected using atomic force microscopy (AFM). The wear behavior of dentin was evaluated by nanoscratch tests and scanning electron microscopy (SEM). Meanwhile, changes in molecular groups and chemical composition were analyzed by Raman and Energy-Dispersive X-ray (EDX) tests, respectively. Nanohardness was also evaluated. ResultsAFM images of DGI-II dentin illustrated a decrease in the number of tubules and the tubule diameter. Nanoscratch test showed a higher friction coefficient and a greater depth-of-scratch in DGI-II dentin. The wear resistance of DGI-II dentin was reduced independent of tubule orientation. EDX results indicated that DGI-II dentin mineral content decreased and Raman spectra results showed DGI-II dentin had a decreased collagen matrix structure stability coupled with hypomineralization. Furthermore, a significant reduction in nanohardness and elastic modulus of DGI-II dentin was observed. Regression analysis revealed a close correlation between dentin components and inferior wear resistance. ConclusionsAll results indicated the wear behavior of DGI-II dentin was significantly deteriorated, presumably caused by the disorder in microstructures and the reduction of chemical composition.

Collagen Organization Within the Cartilage of Trpv4-/- Mice Studied with Two-Photon Microscopy and Polarized Second Harmonic Generation.

The polymodal channel TRPV4 has been shown to regulate development and maintenance of cartilage. Here we investigate whether TRPV4 activity regulates the early deposition and structure of collagen matrix in the femoral head cartilage by comparing the 3D morphology and the sub-micrometer organization of the collagen matrix between wild type and Trpv4 -/- mice pups four to five days old. Two-photon microscopy can be used to conduct label-free imaging of cartilage, as collagen generates a second harmonic signal (second harmonic generation [SHG]) under pulsed infrared excitation. In one set of measurements, we use circularly polarized laser light to reconstruct the 3D morphology of the femoral head cartilage and to measure the tissue thickness. Second, by rotating the direction of the linearly polarized light and using polarized SHG detection, we investigate the sub-micrometer orientation of collagen fibers in the cartilage. At this developmental stage, we cannot detect statistically significant differences between the two mice strains, although a tendency toward a more random orientation of collagen fibers and a higher thickness of the whole cartilage seems to characterize the Trpv4 -/- mice. We discuss possible reasons for these observations. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Silver improves collagen structure and stability at demineralized dentin: A dynamic-mechanical and Raman analysis

ObjectiveThis study aimed to evaluate the effect of silver loaded nanoparticles (NPs) application on dentin remineralization. MethodsPolymethylmetacrylate-based NPs and silver loaded NPs (Ag-NPs) were applied on demineralized dentin surfaces. Dentin was characterized morphologically by scanning electron microscopy, mechanically probed by a nanoindenter to test nanohardness and Young modulus, and chemically analyzed by Raman spectroscopy after 24 h and 7 d of storage. Untreated surfaces were used as control. Data were submitted to ANOVA and Student-Newman-Keuls multiple comparisons tests (P < 0.05). ResultsAfter Raman analysis, dentin treated with Ag-NPs obtained the lowest mineralization and intensity of stoichiometric hydroxyapatite when compared with specimens treated with undoped-NPs. The lowest relative mineral concentration, expressed as the ratio phosphate or carbonate/phenyl group, and crystallinity was attained by dentin treated with Ag-NPs, after 7 d. Ag-NPs application generated the highest values of collagen crosslinking (intensity at 1032 cm−1 band). The molecular conformation of the collagen’s polypeptide chains, amide-I and CH2 also attained the highest peaks in dentin treated with Ag-NPs. Staggered and demineralized collagen fibrils were observed covering the dentin surfaces treated with Ag-NPs, at both 24 h and 7 d. Samples treated with Ag-NPs attained the lowest values of nanohardness and Young’s modulus at 7 d of storage. ConclusionsPeritubular and intertubular dentin were remineralized when using undoped-NPs. After 7 d, collagen treated with NPs was remineralized but dentin treated with Ag-NPs attained an improved collagen matrix structure and stability but the lowest mineralization and crystallinity. Clinical significancePreservation of the demineralized organic matrix is fundamental in operative dentistry. Silver contributed to improve crosslinking, nature and secondary structure of demineralized dentin collagen, for further long-term intrafibrillar mineralization.

Abstract 174: Collagen fiber orientation influences tumor cell growth and Hedgehog signaling in the breast tumor microenvironment

Abstract Collagen matrix architecture is an important player in tumor transition to therapeutic resistance. Several studies have shown that high level of fiber orientation and density of collagen type I matrix enhances tumor cell invasion, decrease drug response and influence tumor metabolism, but the mechanisms of action that support transition to therapeutic resistance are not fully understood. We developed a 3D culture platform to evaluate the effects of collagen matrix architecture in the sensitivity of tumor and stromal cells to tumor promoting ligands and pharmacological inhibitors. Estrogen (E2) and Hedgehog (Hh) signaling pathways were selected for evaluation due to their relevance in breast cancer prognosis and tumor progression. In this 3D culture platform, electrospun collagen fiber sheets of random or aligned orientation are bound to double-sided medical grade tape to generate collagen fiber stickers. Automated cutting plotter allows razor-printing for precisely cutting shapes from collagen fiber sticker sheets. Round shapes were applied like a sticker to the bottom of culture well plates prior to cell seeding. The percentage of matrix porosity fiber orientation and diameter was quantified by Scanning Electron Microscopy (SEM) and Image J analysis. For estrogen signaling studies, MCF-7 cells were seeded in collagen fibers of random or aligned orientation using hormone free-culture conditions. Cells were treated +/- E2 (10nM) ligand and +/- estrogen receptor signaling inhibitors (Fulvestrant and Tamoxifen). Tumor cell proliferation was quantified at 48hrs via Image Cytometry. For Hh signaling studies, expression levels of GLI1, PTCH1 and SMO Hh target genes were monitored in triple negative breast cancer (MDA-MB-231 and MDA-MB-468) and mesenchymal cells. Cells were seeded on collagen fibers and treated with sonic hedgehog ligand for 24hrs. Tumor and mesenchymal cells were harvest for viability and qRT-PCR analysis respectively. Evaluation of cell growth in MCF-7 cultures shows that a fibrillar structure of collagen matrix in combination with high degree of fiber orientation can significantly increase cell proliferation as compared to gelatin or tissue culture plastic substrates. Collagen fibers significantly supported hormone-independent tumor growth at similar levels to those observed in E2 treatment. Increased proliferation levels were maintained in ER inhibition groups particularly when cultured in aligned collagen fibers. In Hh signaling studies, overexpression of GLI1 and SMO genes were observed for collagen fibers matrixes as compared to gelatin and tissue culture plastic. Overall our results show for the first time that collagen fiber architecture can promote hormone-independent growth dependence in breast cancer cells and activation of hedgehog signaling in tumor and mesenchymal cells. Citation Format: Ana M. Reyes, Jorge Almodovar, Maribella Domenech. Collagen fiber orientation influences tumor cell growth and Hedgehog signaling in the breast tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 174.

Comparison of different tanning agents on the stabilization of collagen via differential scanning calorimetry

Leather is composed of a three-dimensional weave of collagen fiber bundles. Collagen is a fibrous protein well organized in the formation of skin as building blocks. Leather production involves serial operations where the tanning plays the major role in improving the durability of leather products by stabilizing the triple helical structure of collagen matrix. In the leather-making industry, different tanning agents are used to produce different kind of leather goods. These tanning agents have varied efficiency on the stabilization of collagen. In this study, thermal stability of the leathers tanned with most commonly used tanning agents was evaluated by conventional shrinking test (CST) and differential scanning calorimetry (DSC) methods. The results showed that the thermal stability of leathers varied by the type of tanning agent which were in accordance with theoretical approaches. A distinct correlation was also observed between CST and DSC results of the tanned leathers.

Dermatan sulfate epimerase 1 deficient mice as a model for human abdominal wall defects.

BackgroundDermatan sulfate (DS) is a highly sulfated polysaccharide with a variety of biological functions in extracellular matrix organization and processes such as tumorigenesis and wound healing. A distinct feature of DS is the presence of iduronic acid, produced by the two enzymes, DS-epimerase 1 and 2, which are encoded by Dse and Dsel, respectively.MethodsWe have previously shown that Dse knockout (KO) mice in a mixed C57BL/6–129/SvJ background have an altered collagen matrix structure in skin. In the current work we studied Dse KO mice in a pure NFR genetic background.ResultsDse KO embryos and newborns had kinked tails and histological staining revealed significantly thicker epidermal layers in Dse KO mice when compared with heterozygote (Het) or wild-type (WT) littermates. Immunochemical analysis of the epidermal layers in newborn pups showed increased expression of keratin 5 in the basal layer and keratin 1 in the spinous layer. In addition, we observed an abdominal wall defect with herniated intestines in 16% of the Dse KO embryos. Other, less frequent, developmental defects were exencephaly and spina bifida.ConclusionWe conclude that the combination of defective collagen structure in the dermis and imbalanced keratinocyte maturation could be responsible for the observed developmental defects in Dse KO mice. In addition, we propose that Dse KO mice could be used as a model in pathogenetic studies of human fetal abdominal wall defects. Birth Defects Research (Part A) 100:712–720, 2014. © 2014 Wiley Periodicals, Inc.

Expression of chondro-osteogenic BMPs in clinical samples of patellar tendinopathy

The pathogenesis of patellar tendinopathy remains unclear. Expression of BMP-2/-4/-7 was reported in an ossified failed tendon healing animal model of patellar tendinopathy. This study aimed to investigate the expression of these chondro-osteogenic BMPs in clinical samples of patellar tendinopathy. Patellar tendon samples were collected from 16 consecutive patients with patellar tendinopathy and 16 consecutive controls undergoing anterior cruciate ligament reconstruction with bone-patellar tendon-bone autograft in the authors' hospital after getting their consent. The expression of BMP-2/-4/-7 was examined in all samples using immunohistochemistry. Ossification observed in two tendinopathy samples was characterized by histology, alizarin red S staining, alcian blue staining, TRAP staining and immunohistochemical staining of Sox9, osteopontin (OPN) and osteocalcin (OCN). Regions of hypo- and hyper-cellularity and vascularity, with loss of crimp structure of collagen matrix, were observed in patellar tendinopathy samples. Round cells and in some cases, cells with typical chondrocyte phenotype were observed. For the ossified tendinopathy samples with positive alizarin red S staining, OPN-positive and Sox9-positive chondrocyte-like cells in alcian blue-stained extracellular matrix, OCN-positive osteoblast-like cells and TRAP-positive multi-nucleated cells were observed around the ossified deposits. No expression of BMP-2/-4/-7 was observed in healthy patellar tendons. However, the expression of BMP-2/-4/-7 was observed in all patellar tendinopathy samples with or without ossification. Clinical samples of patellar tendinopathy showed ectopic expression of BMP-2/-4/-7. This was not evident in control samples from healthy patellar tendons. Prognostic studies, Level III.

Fish skin as a model membrane: structure and characteristics

Abstract Objectives Synthetic and cell-based membranes are frequently used during drug formulation development for the assessment of drug availability. However, most of the currently used membranes do not mimic mucosal membranes well, especially the aqueous mucous layer of the membranes. In this study we evaluated catfish (Anarichas lupus L) skin as a model membrane. Method Permeation of hydrocortisone, lidocaine hydrochloride, benzocaine, diethyl-stilbestrol, naproxen, picric acid and sodium nitrate through skin from a freshly caught catfish was determined in Franz diffusion cells. Key findings Both lipophilic and hydrophilic molecules permeate through catfish skin via hydrated channels or aqueous pores. No correlation was observed between the octanol/water partition coefficient of the permeating molecules and their permeability coefficient through the skin. Permeation through catfish skin was found to be diffusion controlled. Conclusions The results suggest that permeation through the fish skin proceeds via a diffusion-controlled process, a process that is similar to drug permeation through the aqueous mucous layer of a mucosal membrane. In addition, the fish skin, with its collagen matrix structure, appears to possess similar properties to the eye sclera.

Fish skin as a model membrane: structure and characteristics

Synthetic and cell-based membranes are frequently used during drug formulation development for the assessment of drug availability. However, most of the currently used membranes do not mimic mucosal membranes well, especially the aqueous mucous layer of the membranes. In this study we evaluated catfish (Anarichas lupus L) skin as a model membrane. Permeation of hydrocortisone, lidocaine hydrochloride, benzocaine, diethylstilbestrol, naproxen, picric acid and sodium nitrate through skin from a freshly caught catfish was determined in Franz diffusion cells. Both lipophilic and hydrophilic molecules permeate through catfish skin via hydrated channels or aqueous pores. No correlation was observed between the octanol/water partition coefficient of the permeating molecules and their permeability coefficient through the skin. Permeation through catfish skin was found to be diffusion controlled. The results suggest that permeation through the fish skin proceeds via a diffusion-controlled process, a process that is similar to drug permeation through the aqueous mucous layer of a mucosal membrane. In addition, the fish skin, with its collagen matrix structure, appears to possess similar properties to the eye sclera.

Guide to collagen characterization for biomaterial studies

The structure and remodeling of collagen in vivo is critical to the pathology and healing of many human diseases, as well as to normal tissue development and regeneration. In addition, collagen matrices in the form of fibers, coatings, and films are used extensively in biomaterial and biomedical applications. The specific properties of these matrices, both in terms of physical and chemical characteristics, have a direct impact on cellular adhesion, spreading, and proliferation rates, and ultimately on the rate and extent of new extracellular matrix formation in vitro or in vivo. In recent studies, it has also been shown that collagen matrix structure has a major impact on cell and tissue outcomes related to cellular aging and differentiation potential. Collagen structure is complex because of both diversity of source materials, chemistry, and structural hierarchy. With such significant impact of collagen features on biological outcomes, it becomes essential to consider an appropriate set of analytical tools, or guide, so that collagens attained from commercial vendors are characterized in a comparative manner as an integral part of studies focused on biological parameters. The analysis should include as a starting point: (a) structural detail-mainly focused on molecular mass, purity, helical content, and bulk thermal properties, (b) chemical features-mainly focused on surface elemental analysis and hydrophobicity, and (c) morphological features at different length scales. The application of these analytical techniques to the characterization of collagen biomaterial matrices is critical in order to appropriately correlate biological responses from different studies with experimental outcomes in vitro or in vivo. As a case study, the analytical tools employed for collagen biomaterial studies are reviewed in the context of collagen remodeling by fibroblasts. The goal is to highlight the necessity of understanding collagen biophysical and chemical features as a prerequisite to (a) studies with cells and tissue formation, and (b) suggest modes to establish comparative outcomes for studies conducted in different laboratories.

Novel Collagen Based Material for Local Drug Delivery Systems

AbstractIn a search for new drug delivery matrix, the synthesis of a novel collagen material based on tiopronin protected gold clusters (MPC) as a crosslinking agent is proposed. The structure of collagen matrix modified with MPC is studied using transmission electron microscope. The thermal properties are examined with differential scanning calorimetry. To assess the biocompatibility of the matrix, the cytotoxicity assays are conducted.

Impact of collagen structure on matrix trafficking by human fibroblasts.

Biodegradation of collagen biomaterial matrices and the deposition of new collagen extracellular matrix (ECM) are critical to the integration of in vitro bioengineered materials and tissues in vivo. In previous studies, we observed significant impact of collagen matrix structure on primary lung fibroblast behavior in vitro. In the present work, to begin to understand the mechanistic basis for our previous observation, the response of human fibroblasts (IMR-90) to the structural state of collagen matrices was studied with respect to cell proliferation, cell morphology, beta-galactosidase level, and transcript content for collagen (Col-1), matrix metalloproteinases (MMP-1, MMP-2), tissue inhibitors of matrix metalloproteinase (TIMP-1 and TIMP-2). Collagen digestion was assessed quantitatively by uptake of collagen-coated fluorescent beads incorporated in the preformed collagen matrix. Transcript levels related to the deposition of new ECM proteins varied as a function of the structure of the collagen matrix presented to the cells. Col-1 expression was 2-fold higher and expression for MMP-1, MMP-2, TIMP-1, and TIMP-2 increased for cells when grown on 156 microg/cm2 denatured collagen compared with cells grown on tissue culture (TC) plastic. On 156 microg/cm2 nondenatured (native) collagen, Col-1 expression was decreased by half and MMP-2 was increased by 2.5-fold compared with cells grown on TC plastic. On 78 microg/cm2 denatured collagen, Col-1 expression was 80% whereas the MMPs and TIMPs were increased by 1.25- to 2-fold compared with cells grown on TC plastic. On 78 microg/cm2 nondenatured collagen expression of all 5 transcripts was reduced 60-90% of the levels determined for the cells grown on TC plastic. Cell viability, based on cell morphology and beta-galactosidase activity, was improved on the denatured collagen. A higher level of collagen matrix incorporation was observed for cells grown on denatured collagen than on nondenatured collagen or TC plastic. These data suggest that tissue engineering matrices incorporating denatured collagen may promote more active remodeling toward new ECM in comparison to cells grown on nondenatured collagen or cells grown on TC plastic.