Collagen Fibril Diameter Research Articles

Homeostatic maintenance via degradation and repair of elastic fibers under tension.

Cellular maintenance of the extracellular matrix requires an effective regulation that balances enzymatic degradation with the repair of collagen fibrils and fibers. Here, we investigate the long-term maintenance of elastic fibers under tension combined with diffusion of general degradative and regenerative particles associated with digestion and repair processes. Computational results show that homeostatic fiber stiffness can be achieved by assuming that cells periodically probe fiber stiffness to adjust the production and release of degradative and regenerative particles. However, this mechanism is unable to maintain a homogeneous fiber. To account for axial homogeneity, we introduce a robust control mechanism that is locally governed by how the binding affinity of particles is modulated by mechanical forces applied to the ends of the fiber. This model predicts diameter variations along the fiber that are in agreement with the axial distribution of collagen fibril diameters obtained from scanning electron microscopic images of normal rat thoracic aorta. The model predictions match the experiments only when the applied force on the fiber is in the range where the variance of local stiffness along the fiber takes a minimum value. Our model thus predicts that the biophysical properties of the fibers play an important role in the long-term regulatory maintenance of these fibers.

Collagen Fibril Ultrastructure in Mice Lacking Discoidin Domain Receptor 1.

The quantity and quality of collagen fibrils in the extracellular matrix (ECM) have a pivotal role in dictating biological processes. Several collagen-binding proteins (CBPs) are known to modulate collagen deposition and fibril diameter. However, limited studies exist on alterations in the fibril ultrastructure by CBPs. In this study, we elucidate how the collagen receptor, discoidin domain receptor 1 (DDR1) regulates the collagen content and ultrastructure in the adventitia of DDR1 knock-out (KO) mice. DDR1 KO mice exhibit increased collagen deposition as observed using Masson's trichrome. Collagen ultrastructure was evaluated in situ using transmission electron microscopy, scanning electron microscopy, and atomic force microscopy. Although the mean fibril diameter was not significantly different, DDR1 KO mice had a higher percentage of fibrils with larger diameter compared with their wild-type littermates. No significant differences were observed in the length of D-periods. In addition, collagen fibrils from DDR1 KO mice exhibited a small, but statistically significant, increase in the depth of the fibril D-periods. Consistent with these observations, a reduction in the depth of D-periods was observed in collagen fibrils reconstituted with recombinant DDR1-Fc. Our results elucidate how DDR1 modulates collagen fibril ultrastructure in vivo, which may have important consequences in the functional role(s) of the underlying ECM.

Effect of aging and exercise on the tendon.

Here, we review the literature on how tendons respond and adapt to ageing and exercise. With respect to aging, there are considerable changes early in life, but this seems to be maturation rather than aging per se. In vitro data indicate that aging is associated with a decreased potential for cell proliferation and a reduction in the number of stem/progenitor-like cells. Further, there is persuasive evidence that turnover in the core of the tendon after maturity is very slow or absent. Tendon fibril diameter, collagen content, and whole tendon size appear to be largely unchanged with aging, while glycation-derived cross-links increase substantially. Mechanically, aging appears to be associated with a reduction in modulus and strength. With respect to exercise, tendon cells respond by producing growth factors, and there is some support for a loading-induced increase in tendon collagen synthesis in humans, which likely reflects synthesis at the very periphery of the tendon rather than the core. Average collagen fibril diameter is largely unaffected by exercise, while there can be some hypertrophy of the whole tendon. In addition, it seems that resistance training can yield increased stiffness and modulus of the tendon and may reduce the amount of glycation. Exercise thereby tends to counteract the effects of aging.

Supramolecular hydrogel networks formed by molecular recognition of collagen and a peptide grafted to hyaluronic acid

The extracellular matrix (ECM) is a nano-structured, highly complex hydrogel, in which the macromolecules are organized primarily by non-covalent interactions. Here, in a biomimetic approach, the decorin-derived collagen-binding peptide LSELRLHNN was grafted to hyaluronic acid (HA) in order to enable the formation of a supramolecular hydrogel network together with collagen. The storage modulus of a mixture of collagen and HA was increased by more than one order of magnitude (G′=157Pa) in the presence of the HA-grafted peptide compared to a mixture of collagen and HA (G′=6Pa). The collagen fibril diameter was decreased, as quantified using electron microscopy, in the presence of the HA-grafted peptide. Here, the peptide mimicked the function of decorin by spatially organizing collagen. The advantage of this approach is that the non-covalent crosslinks between collagen molecules and the HA chains created by the peptide form a reversible and dynamic hydrogel, which could be employed for a diverse range of applications in regenerative medicine. Statement of SignificanceBiopolymers of the extracellular matrix (ECM) like collagen or hyaluronan are attractive starting materials for biomaterials. While in biomaterial science covalent crosslinking is often employed, in the native ECM, stabilization and macromolecular organization is primarily based on non-covalent interactions, which allows dynamic changes of the materials. Here, we show that collagen-binding peptides, derived from the small proteoglycan decorin, grafted to hyaluronic acid enable supramolecular stabilization of collagen hydrogels. These hydrogels have storage moduli more than one order of magnitude higher than mixtures of collagen and hyaluronic acid. Furthermore, the peptide supported the structural organization of collagen. Such hydrogels could be employed for a diverse range of applications in regenerative medicine. Furthermore, the rational design helps in the understanding ECM structuring.

Ultrastructural evaluation of gingival connective tissue in hereditary gingival fibromatosis

To describe the ultrastructural features of hereditary gingival fibromatosis (HGF) in affected family members and compare microscopic findings with normal gingival (NG) tissue. Gingival tissue samples from nine patients with HGF from five unrelated families were evaluated by transmission electron microscopy. Nine NG tissue samples were used for comparison. Areas containing collagen fibrils forming loops and folds were observed in both groups, whereas oxytalan fibers were frequently identified in the HGF group. The diameter of collagen fibrils and the interfibrillar space among them were more uniform in the NG group than in the HGF group. Fibroblasts were the most common cells found in both the HGF and NG groups and exhibited enlarged, rough endoplasmic reticulum, mitochondria with well-preserved crests, conspicuous nucleoli, and euchromatic chromatin. Other cells, such as mast cells, plasma cells, and macrophages, were also observed. HGF tissues had ultrastructural characteristics that were very similar to those of NG tissues. Oxytalan fibers were observed more frequently in the HGF samples than in the NG samples. Other studies of HGF in patients from different families should be performed to better understand the pathogenesis of this hereditary condition.

Fibroblast-Derived MMP-14 Regulates Collagen Homeostasis in Adult Skin

Proteolytic activities in the extracellular matrix by the matrix metalloproteinase (MMP)-14 have been implicated in the remodeling of collagenous proteins during development. To analyze the function of fibroblast-derived MMP-14 in adult skin homeostasis, we generated mice with inducible deletion of MMP-14 in the dermal fibroblast (MMP-14Sf–/–). These mice are smaller and display a fibrosis-like phenotype in the skin. The skin of these mice showed increased stiffness and tensile strength but no altered collagen cross-links. In vivo, we measured a significantly increased amount of collagen type I accumulated in the skin of MMP-14Sf–/– mice without an increase in collagen fibril diameters. However, bleomycin-induced fibrosis in skin proceeded in a comparable manner in MMP-14Sf+/+ and MMP-14Sf–/– mice, but resolution over time was impaired in MMP-14Sf–/– mice. Increased accumulation of collagen type I was detected in MMP-14Sf–/– fibroblasts in culture without significant enhancement of collagen de novo synthesis. This points to a degradative but not synthetic phenotype. In support of this, MMP-14Sf–/– fibroblasts lost their ability to process fibrillar collagen type I and to activate proMMP-2. Taken together, these data indicate that MMP-14 expression in fibroblasts plays a crucial role in collagen remodeling in adult skin and largely contributes to dermal homeostasis underlying its pathogenic role in fibrotic skin disease.

Fibromodulin Interacts with Collagen Cross-linking Sites and Activates Lysyl Oxidase

The hallmark of fibrotic disorders is a highly cross-linked and dense collagen matrix, a property driven by the oxidative action of lysyl oxidase. Other fibrosis-associated proteins also contribute to the final collagen matrix properties, one of which is fibromodulin. Its interactions with collagen affect collagen cross-linking, packing, and fibril diameter. We investigated the possibility that a specific relationship exists between fibromodulin and lysyl oxidase, potentially imparting a specific collagen matrix phenotype. We mapped the fibromodulin-collagen interaction sites using the collagen II and III Toolkit peptide libraries. Fibromodulin interacted with the peptides containing the known collagen cross-linking sites and the MMP-1 cleavage site in collagens I and II. Interestingly, the interaction sites are closely aligned within the quarter-staggered collagen fibril, suggesting a multivalent interaction between fibromodulin and several collagen helices. Furthermore, we detected an interaction between fibromodulin and lysyl oxidase (a major collagen cross-linking enzyme) and mapped the interaction site to 12 N-terminal amino acids on fibromodulin. This interaction also increases the activity of lysyl oxidase. Together, the data suggest a fibromodulin-modulated collagen cross-linking mechanism where fibromodulin binds to a specific part of the collagen domain and also forms a complex with lysyl oxidase, targeting the enzyme toward specific cross-linking sites.

Stromal Integrin α11β1 Affects RM11 Prostate and 4T1 Breast Xenograft Tumors Differently.

PurposeIt has been implied that the collagen binding integrin α11β1 plays a role in carcinogenesis. As still relatively little is known about how the stromal integrin α11β1 affects different aspects of tumor development, we wanted to examine the direct effects on primary tumor growth, fibrosis, tumor interstitial fluid pressure (PIF) and metastasis in murine 4T1 mammary and RM11 prostate tumors, using an in vivo SCID integrin α11-deficient mouse model.MethodsTumor growth was measured using a caliper, PIF by the wick-in-needle technique, activated fibroblasts by α-SMA immunofluorescence staining and fibrosis by transmission electron microscopy and picrosirius-red staining. Metastases were evaluated using hematoxylin and eosin stained sections.ResultsRM11 tumor growth was significantly reduced in the SCID integrin α11-deficient (α11-KO) compared to in SCID integrin α11 wild type (WT) mice, whereas there was no similar effect in the 4T1 tumor model. The 4T1 model demonstrated an alteration in collagen fibril diameter in the integrin α11-KO mice compared to WT, which was not found in the RM11 model. There were no significant differences in the amount of activated fibroblasts, total collagen content, collagen organization or PIF in the tumors in integrin α11-deficient mice compared to WT mice. There was also no difference in lung metastases between the two groups.ConclusionDeficiency of stromal integrin α11β1 showed different effects on tumor growth and collagen fibril diameter depending on tumor type, but no effect on tumor PIF or development of lung metastasis.

Changes in dermal matrix in the absence of Rac1 in keratinocytes.

Keratinocytes, in response to irritants, secrete pro-inflammatory mediators which recruit and activate immune and mesenchymal cells, including fibroblasts, to repair the skin. Fibroblasts respond by synthesising collagen and promoting the crosslinking extracellular matrix (ECM). We recently showed that the deletion of Rac1 in keratinocytes causes heightened inflammation due to aberrant crosstalk with immune cells. Indeed, the skin of these mice shows a higher inflammatory response to the induction of irritant contact dermatitis (ICD), and also even to treatment with a vehicle alone, compared with controls. As inflammation is intimately linked with fibrotic disease in the skin, this raised the question as to whether this deletion may also affect the deposition and arrangement of the dermal ECM. This study assessed the effects of Rac1 deletion in keratinocytes and of the heightened inflammatory status by induction of ICD on the tissue localisation and arrangements of dermal collagen. Qualitative analysis did not reveal evidence for the formation of pathologies in the dermis. However, quantitative analysis did reveal some perturbations in the dermal matrix, namely that only the combination of the lack of Rac1 and ICD affects the architectural organisation of the dermal collagen, and that a higher inflammatory state in the tissue (i.e. when Rac1 is deleted in the keratinocytes or ICD is induced in the skin, or a combination of both) influences the diameter of the collagen fibrils. It is proposed that this increase in the diameter of collagen fibrils due to inflammation may serve as pre-fibrotic marker enabling earlier determination of fibrosis and earlier treatment. This study has revealed previously unknown effects on the ECM due to the deletion of Rac1 in keratinocytes.

Osteogenesis imperfecta: Ultrastructural and histological findings on examination of skin revealing novel insights into genotype-phenotype correlation

ABSTRACTOsteogenesis imperfecta (OI) is a heterogeneous group of inherited disorders of bone formation, resulting in low bone mass and an increased propensity to fracture. Over 90% of patients with OI have a mutation in COL1A1/COL1A2, which shows an autosomal dominant pattern of inheritance. In-depth phenotyping and in particular, studies involving manifestations in the skin connective tissue have not previously been undertaken in OI. The aims of the study were to perform histological and ultrastructural examination of skin biopsies in a cohort of patients with OI; to identify common and distinguishing features in order to inform genotype-phenotype correlation; and to identify common and distinguishing features between the different subtypes of OI. As part of the RUDY (Rare Diseases in Bone, Joints and/or Blood Vessels) study, in collaboration with the NIHR Rare Diseases Translational Research Collaboration, we undertook a national study of skin biopsies in patients with OI. We studied the manifestations in the skin connective tissue and undertook in-depth clinical and molecular phenotyping of 16 patients with OI. We recruited 16 patients: analyses have shown that in type 1 collagen mutation positive patients (COL1A1/ COL1A2) (n-4/16) consistent findings included: variable collagen fibril diameter (CFD) and presence of collagen flowers. Histological examination in these patients showed an increase in elastic fibers that are frequently fragmented and clumped. These observations provide evidence that collagen flowers and CFD variability are consistent features in OI due to type 1 collagen defects and reinforce the need for accurate phenotyping in conjunction with genomic analyses.

2015 Summer Biomechanics, Bioengineering, and Biotransport Conference Student Paper Competition.

As in past years, a Student Paper Competition was held at the 2015 Summer Biomechanics, Bioengineering, and Biotransport Conference (SB3C). There were three competition levels: Ph.D., M.S., and B.S. The competition was divided into multiple technical areas to provide for a fair evaluation of student work. There were also cash awards for the top three student papers in each area. We are pleased to recognize the following awardees from the 2015 SB3C.First Place: Kevin J. Warburton, Boise State University, “Wear Simulator for Canine Total Hip Replacements.”Second Place: Brett S. Klosterhoff, Purdue University, “Quantitative Visualization of Drug Response of Breast Cancer Cells Within a Three-Dimensional Extracellular Matrix.”Third Place: McKenna Drysdale, University of Utah, “Material Property Testing of Carboxymethylated Hyaluronic Acid Hydrogel Polymer.”First Place: Nicholas A. Bianco, University of Florida, “Can Measured Muscle Synergies Reconstruct Unmeasured Muscle Excitations?”Second Place: Justin A. Jones, University of Utah, “The Temporal Change in Protein Biomarkers of Vitreous Humor Following Blast Trauma of the Visual System.”Third Place: Nithya Vijayakumar, Stanford University, “A Mechanical Model for Cortical Folding During Brain Development.”First Place: Rosamaria Tricarico, University of Florida, “Elevated Wall Shear Stress Predicts Branch Graft Failure Following Chimney Endovascular Aortic Aneurysm Repair.”Second Place: Jazmine Aira, University of South Florida, “Image-Based 3D Morphometric Analysis of the Clavicle Intramedullary Canal in Male Population.”Third Place: Daniela Velez-Rendon, University of Illinois at Chicago, “Establishing in vivo Hemodynamic Baseline in a Normotensive Rat Model.”First Place: Erica E. Morrill, Boise State University, “Accurate Prediction of Collagen Fiber Distribution Using FFT: A Validation Study.”Second Place: Meghan Sylvia, California Polytechnic State University, San Luis Obispo, CA, “Development of a Human Knee Joint Finite Element Model to Investigate Cartilage Stress During Walking in Obese and Normal Weight Adults.”Third Place: Michael J. Raymond, Rensselaer Polytechnic Institute, “Individual Cell-Based Morphological Analysis to Determine Chirality of Epithelial Morphogenesis.”First Place: Priya Nair, Arizona State University, “Hemodynamic Characterization of Different Basilar Tip Aneurysm Templates Using Computational Fluid Dynamics.”Second Place: Samira Jamalian, Imperial College London, London, UK, “Suction Effect Produced by Active Contraction of Collecting Lymphatic Vessels Facilities Lymphatic Filling.”Third Place: David Schreier, University of Wisconsin–Madison, “Increased Red Blood Cell Stiffness Increases Pulmonary Vascular Resistance and Pulmonary Arterial Pressure.”First Place: M. Owais Khan, University of Toronto, Toronto, Canada, “CFD Simulation of Transition to Turbulence for Newtonian vs. Non-Newtonian Flow Through a Stenosis.”Second Place: Michael J. Weiler, Georgia Institute of Technology, “Reduced Lymphatic Function Correlates With Disease Progression in a Novel Single Vessel Ligation Model of Lymphedema.”Third Place: Anne M. Römgens, Eindhoven University of Technology, Eindhoven, The Netherlands, “Combined Experimental and Finite Element Analysis to Determine the Diffusion Coefficient Within and Between Human Skin Layers.”First Place: Feini Qu, University of Pennsylvania, “Interstitial Cell Migration in Dense Connective Tissues Is Modulated by Matrix Microstructure and Micromechanics.”Second Place: John T. Martin, University of Pennsylvania, “In Vitro Growth Trajectory and In Vivo Implantation of Cell-Seeded and Disc-Like Angle Ply Structures for Total Disc Replacement.”Third Place: Heather A. Cirka, Worcester Polytechnic Institute, “Cells Alter Traction Force and Orientation in Response to Long-Term Cyclic Stretch.”First Place: Jenell Smith, University of Pennsylvania, “Blocking Blood-Spinal Cord Barrier Breakdown Prevents the Development of Pain Following Nerve Root Compression Injury.”Second Place: Sijia Zhang, University of Pennsylvania, “Defining Collagen Fiber Mechanics in Neuron-Collagen Constructs Under Stretch Using Integrated Experimental & Modeling Approaches.”Third Place: Christopher F. Ferrigno, Rush University, “Redistribution of Knee Loads Using Auditory Feedback From Pressure Detecting Shoe Insoles.”First Place: Spencer E. Szczesny, University of Pennsylvania, “Evidence That Interfibrillar Load Transfer in Tendon Is Supported by a Network of Small Diameter Collagen Fibrils.”Second Place: Kara E. Garcia, Washington University in St. Louis, “Regional Contraction Shapes the Three-Dimensional Morphogenesis of the Embryonic Forebrain.”Third Place: Elias R. Pavlatos, The Ohio State University, “3D Strains in Posterior Sclera Using Ultrasound Speckle Tracking.”First Place: Devika M. Varma, The City College of New York, “Thermoresponsive, Redox-Crosslinked Cellulosic Hydrogels Undergo In Situ Gelation and Restore Nucleus Pulposus Biomechanical Properties Post Nucleotomy.”Second Place: Andrew P. Baumann, University of Notre Dame, “Influence of Intracortical Porosity on the Fracture Susceptibility of Human Cortical Bone.”Third Place: Robert J. Nims, Columbia University, “A Multigenerational Collagen Damage Model Explains Engineered Cartilage Growth and Remodeling Phenomena.”

Study of proteoglycans in corneal stroma reconstructed by the self-assembly method

Event Abstract Back to Event Study of proteoglycans in corneal stroma reconstructed by the self-assembly method Gaëtan Le-Bel1, Jean-Michel Bourget1, Sébastien Larochelle2, Sylvain Guérin1, 3, Véronique Moulin2 and Lucie Germain1, 2, 3 1 CUO-Recherche, Centre de recherche du CHU de Québec, axe médecine régénératrice, Canada 2 Centre de recherche en organogenèse expérimentale de l’Université Laval/LOEX, Université Laval, Canada 3 Département d’ophtalmologie et d’ORL, Faculté de médecine, Université Laval, Canada Context and Objectives: Corneal transparency is due to a particular collagen fibril architecture defined by a constant diameter and a regular spacing of collagen fibrils within lamellae. Proteoglycans (PGs) are glycoproteins located between collagen fibrils involved in the determination of these two parameters. The objective of this study was to compare the expression profile of PGs of corneal and dermal tissue-engineered stromal models. Materials and Methods: Human corneal fibroblasts (HCFs) were isolated from donor cornea obtained at our local eye bank (n=2). Isolated human dermal fibroblasts (HDFs) were used as control (n=2). Cells were cultured in the presence of ascorbic acid to allow formation of extracellular matrix sheets. Gene expression of various PGs (lumican, keratocan, decorin, biglycan, mimecan, syndecan) was evaluated by PCR at different culture times. After 12 days, the mRNA was isolated for further transcriptome analysis by DNA microarray. The role of lumican was evaluated by knockdown using small interfering RNAs delivered by a retroviral vector to produce stable populations of HCFs (4 independent targets). Efficiency of the knockdown was evaluated by Western blot and immunofluorescence. Results: Expression of the majority of PGs analyzed was maximal at day 11 of culture with ascorbic acid. We found a similar gene expression profile for matrix proteins and PGs for all the populations, except for TNC, COL15A1 and COL5A3, which were higher in HDFs compared to HCFs. Decorin and lumican were highly expressed in cultured HCFs and HDFs while keratocan was not detected. HCF knockdowns for lumican were obtained successfully and the populations that were most inhibited were identified. Conclusion: These results demonstrated that HCFs present a dermal rather than a corneal stromal phenotype since expression of keratocan was not detected. Since the expression profiles of matrix proteins were similar between the HDFs and HCFs, differences in transparency of the reconstructed tissues are potentially due to differences in matrix organization rather than composition of these tissues. Next steps of the project will be to evaluate the diameter and spacing of collagen fibrils as well as transparency of the tissue-engineered corneal stroma with HCFs knockdown for lumican. These studies will allow to better define the role of lumican in the formation of a transparent corneal stroma. Keywords: Extracellular Matrix, Tissue Engineering, microstructure, complex tissue orgnization Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: Poster Topic: Biomaterials in constructing tissue substitutes Citation: Le-Bel G, Bourget J, Larochelle S, Guérin S, Moulin V and Germain L (2016). Study of proteoglycans in corneal stroma reconstructed by the self-assembly method. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.00343 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Gaëtan Le-Bel Jean-Michel Bourget Sébastien Larochelle Sylvain Guérin Véronique Moulin Lucie Germain Google Gaëtan Le-Bel Jean-Michel Bourget Sébastien Larochelle Sylvain Guérin Véronique Moulin Lucie Germain Google Scholar Gaëtan Le-Bel Jean-Michel Bourget Sébastien Larochelle Sylvain Guérin Véronique Moulin Lucie Germain PubMed Gaëtan Le-Bel Jean-Michel Bourget Sébastien Larochelle Sylvain Guérin Véronique Moulin Lucie Germain Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

The transcription factor mohawk homeobox regulates homeostasis of the periodontal ligament.

The periodontal ligament (PDL), which connects the teeth to the alveolar bone, is essential for periodontal tissue homeostasis. Although the significance of the PDL is recognized, molecular mechanisms underlying PDL function are not well known. We report that mohawk homeobox (Mkx), a tendon-specific transcription factor, regulates PDL homeostasis by preventing its degeneration. Mkx is expressed in the mouse PDL at the age of 10 weeks and expression remained at similar levels at 12 months. In Mkx-/- mice, age-dependent expansion of the PDL at the maxillary first molar (M1) furcation area was observed. Transmission electron microscopy (TEM) revealed that Mkx-/- mice presented collagen fibril degeneration in PDL with age, while the collagen fibril diameter gradually increased in Mkx+/+ mice. PDL cells lost their shape in Mkx-/- mice, suggesting changes in PDL properties. Microarray and quantitative polymerase chain reaction (qPCR) analyses of Mkx-/- PDL revealed an increase in osteogenic gene expression and no change in PDL- and inflammatory-related gene expression. Additionally, COL1A1 and COL1A2 were upregulated in Mkx-overexpressing human PDL fibroblasts, whereas osteogenic genes were downregulated. Our results indicate that Mkx prevents PDL degeneration by regulating osteogenesis.

Developmental Stage-dependent Regulation of Prolyl 3-Hydroxylation in Tendon Type I Collagen

3-Hydroxyproline (3-Hyp), which is unique to collagen, is a fairly rare post-translational modification. Recent studies have suggested a function of prolyl 3-hydroxylation in fibril assembly and its relationships with certain disorders, including recessive osteogenesis imperfecta and high myopia. However, no direct evidence for the physiological and pathological roles of 3-Hyp has been presented. In this study, we first estimated the overall alterations in prolyl hydroxylation in collagens purified from skin, bone, and tail tendon of 0.5-18-month-old rats by LC-MS analysis with stable isotope-labeled collagen, which was recently developed as an internal standard for highly accurate collagen analyses. 3-Hyp was found to significantly increase in tendon collagen until 3 months after birth and then remain constant, whereas increased prolyl 3-hydroxylation was not observed in skin and bone collagen. Site-specific analysis further revealed that 3-Hyp was increased in tendon type I collagen in a specific sequence region, including a previously known modification site at Pro(707) and newly identified sites at Pro(716) and Pro(719), at the early ages. The site-specific alterations in prolyl 3-hydroxylation with aging were also observed in bovine Achilles tendon. We postulate that significant increases in 3-Hyp at the consecutive modification sites are correlated with tissue development in tendon. The present findings suggest that prolyl 3-hydroxylation incrementally regulates collagen fibril diameter in tendon.

Adenomatous Polyposis Coli Mutation Leads to Myopia Development in Mice.

Myopia incidence in China is rapidly becoming a very serious sight compromising problem in a large segment of the general population. Therefore, delineating the underlying mechanisms leading to myopia will markedly lessen the likelihood of other sight compromising complications. In this regard, there is some evidence that patients afflicted with familial adenomatous polyposis (FAP), havean adenomatous polyposis coli (APC) mutation and a higher incidence of myopia. To clarify this possible association, we determined whether the changes in pertinent biometric and biochemical parameters underlying postnatal refractive error development in APCMin mice are relevant for gaining insight into the pathogenesis of this disease in humans. The refraction and biometrics in APCMin mice and age-matched wild-type (WT) littermates between postnatal days P28 and P84 were examined with eccentric infrared photorefraction (EIR) and customized optical coherence tomography (OCT). Compared with WT littermates, the APCMin mutated mice developed myopia (average -4.64 D) on P84 which was associated with increased vitreous chamber depth (VCD). Furthermore, retinal and scleral changes appear in these mice along with: 1) axial length shortening; 2) increased retinal cell proliferation; 3) and decreased tyrosine hydroxylase (TH) expression, the rate-limiting enzyme of DA synthesis. Scleral collagen fibril diameters became heterogeneous and irregularly organized in the APCMin mice. Western blot analysis showed that scleral alpha-1 type I collagen (col1α1) expression also decreased whereas MMP2 and MMP9 mRNA expression was invariant. These results indicate that defective APC gene function promotes refractive error development. By characterizing in APCMin mice ocular developmental changes, this approach provides novel insight into underlying pathophysiological mechanisms contributing to human myopia development.

A new method of detecting changes in corneal health in response to toxic insults

The size and arrangement of stromal collagen fibrils (CFs) influence the optical properties of the cornea and hence its function. The spatial arrangement of the collagen is still questionable in relation to the diameter of collagen fibril. In the present study, we introduce a new parameter, edge-fibrillar distance (EFD) to measure how two collagen fibrils are spaced with respect to their closest edges and their spatial distribution through normalized standard deviation of EFD (NSDEFD) accessed through the application of two commercially available multipurpose solutions (MPS): ReNu and Hippia. The corneal buttons were soaked separately in ReNu and Hippia MPS for five hours, fixed overnight in 2.5% glutaraldehyde containing cuprolinic blue and processed for transmission electron microscopy. The electron micrographs were processed using ImageJ user-coded plugin. Statistical analysis was performed to compare the image processed equivalent diameter (ED), inter-fibrillar distance (IFD), and EFD of the CFs of treated versus normal corneas. The ReNu-soaked cornea resulted in partly degenerated epithelium with loose hemidesmosomes and Bowman's collagen. In contrast, the epithelium of the cornea soaked in Hippia was degenerated or lost but showed closely packed Bowman's collagen. Soaking the corneas in both MPS caused a statistically significant decrease in the anterior collagen fibril, ED and a significant change in IFD, and EFD than those of the untreated corneas (p<0.05, for all comparisons). The introduction of EFD measurement in the study directly provided a sense of gap between periphery of the collagen bundles, their spatial distribution; and in combination with ED, they showed how the corneal collagen bundles are spaced in relation to their diameters. The spatial distribution parameter NSDEFD indicated that ReNu treated cornea fibrils were uniformly distributed spatially, followed by normal and Hippia. The EFD measurement with relatively lower standard deviation and NSDEFD, a characteristic of uniform CFs distribution, can be an additional parameter used in evaluating collagen organization and accessing the effects of various treatments on corneal health and transparency.

A rat model of radiation injury in the mandibular area.

BackgroundRadiation technology focuses on delivering the radiation as precisely as possible to the tumor, nonetheless both acute and long-term damage to surrounding normal tissue may develop. Injuries to the surrounding normal tissue after radiotherapy of head and neck cancer are difficult to manage. An animal model is needed to elucidate good treatment modalities. The aim of this study was to establish a rat model where a certain radiation dose gives reproducible tissue reactions in the mandibular area corresponding to injuries obtained in humans.MethodThe left mandible of male Sprague Dawley rats was irradiated by external radiotherapy (single fraction 15 Gy, total dose 75 Gy) every second week five times. Endpoint was six weeks after last radiation treatment, and the test group was compared to non-irradiated controls. Morphological alterations of the soft tissues, bone and tooth formation, as well as alterations of salivation, vascularity and collagen content were assessed. An unpaired, non-parametric Mann–Whitney test was used to compare the statistical differences between the groups.ResultsAnalysis of the soft tissues and mandible within the radiation field revealed severe unilateral alopecia and dermatitis of the skin, extensive inflammation of the submandibular gland with loss of serous secretory cells, hyperkeratinization and dense connective fiber bundles of the gingival tissue, and disturbed tooth development with necrosis of the pulp. Production of saliva and the vascularity of the soft tissues were significantly reduced. Furthermore, the collagen fibril diameter was larger and the collagen network denser compared to non-irradiated control rats.ConclusionWe have established an animal model of radiation injury demonstrating physiological and histological changes corresponding to human radiation injuries, which can be used for future therapeutic evaluations.

Vertebrae length and ultra-structure measurements of collagen fibrils and mineral content in the vertebrae of lordotic gilthead seabreams (Sparus aurata)

Skeletal deformities of gilthead seabream (Sparus aurata) are a major factor affecting the production cost, the external morphology and survival and growth of the fish. Adult individuals of S. aurata were collected from a commercial fish farm in Greece and were divided into two groups: one with the presence of lordosis, a skeletal deformity, and one without any skeletal deformity. Fishes were X-rayed, and cervical, abdominal and caudal vertebrae lengths were measured. Vertebrae were taken from the site of the vertebral column where lordosis occurred. One part was decalcified and prepared for collagen examination with transmission electron microscopy, and the rest were incinerated, and the Ca and P contents were measured. The stoichiometries of the samples were obtained by EDS (Energy Dispersive Spectroscopy). The same procedure was followed for fish without skeletal deformities (vertebrae were taken from the middle region of the vertebral column). The decalcified vertebrae parts were examined with TEM, collagen micrographs were taken and the fibrils’ periods and diameters were measured. There were no significant differences for both Ca and P or the collagen fibrils’ periods between the two fish groups. The mean lengths of the cervical, abdominal and caudal vertebrae where lordosis occurred were similar to the lengths of the respective regions of the individuals without the skeletal deformity. The TEM examination showed a significantly smaller mean vertebrae collagen fibril diameter from the fishes with lordosis compared with those from the controls, revealing the significance of collagen to bone structure.

Collagen Fibrils and Proteoglycans of Macular Dystrophy Cornea: Ultrastructure and 3D Transmission Electron Tomography.

We report the ultrastructure and 3D transmission electron tomography of collagen fibrils (CFs), proteoglycans (PGs), and microfibrils within the CF of corneas of patients with macular corneal dystrophy (MCD). Three normal corneas and three MCD corneas from three Saudi patients (aged 25, 31, and 49 years, respectively) were used for this study. The corneas were processed for light and electron microscopy studies. 3D images were composed from a set of 120 ultrastructural images using the program "Composer" and visualized using the program "Visuliser Kai". 3D image analysis of MCD cornea showed a clear organization of PGs around the CF at very high magnification and degeneration of the microfibrils within the CF. Within the MCD cornea, the PG area in the anterior stroma was significantly larger than in the middle and posterior stroma. The PG area in the MCD cornea was significantly larger compared with the PG area in the normal cornea. The CF diameter and inter-fibrillar spacing of the MCD cornea were significantly smaller compared with those of the normal cornea. Ultrastructural 3D imaging showed that the production of unsulfated keratin sulfate (KS) may lead to the degeneration of micro-CFs within the CFs. The effect of the unsulfated KS was higher in the anterior stroma compared with the posterior stroma.

Ultrastructural and histological findings on examination of skin in osteogenesis imperfecta: a novel study.

Osteogenesis imperfecta (OI) is a heterogeneous group of inherited disorders of bone formation, resulting in low bone mass and an increased propensity for fractures. It is a variable condition with a range of clinical severities. The histological and ultrastructural findings in the skin of patients with OI have not been described in detail in the previously published literature. Although protein analysis of cultured fibroblasts has historically been used in the diagnostic work-up of OI patients, other aspects of skin examination are not routinely performed as part of the diagnostic pathway in patients with OI. The aims of this study were to perform histological and ultrastructural examination of skin biopsies in patients with OI. This was to identify common and distinguishing features in the numerous genetically distinct subtypes of OI and compare the findings with those in patients who did not present with fractures, and to enable the use of the results thus obtained to aid in the diagnostic work-up of patients with OI. As part of a larger research study set-up to identify clinical features and natural history in patients with atypical features of OI, skin biopsy and examination (histology and electron microscopy) were undertaken. Genetic analysis and ancillary investigations were also performed to identify similarities within this group and to differentiate this group from the 'normal' population. At the end of this study, we were able to demonstrate that the histological and electron microscopic findings on a skin biopsy may be an indicator of the likelihood of identifying a pathogenic mutation in type 1 collagen genes. This is because patients with specific findings on examination, such as elastic fibre area fraction (on histological analysis), collagen fibril diameter variability, deviation from the expected mean and collagen flowers (on electron microscopy), are more likely to be positive on genetic analyses. This has, in turn, provided more insight into the pathways to direct gene testing and has reinforced the need for accurate phenotyping before undertaking further genetic investigations. The morphometric assessment of elastic fibre area fraction and ultrastructural findings from this study have provided us with a better understanding of OI and insights into the possible mechanism of these changes in the skin. Correlation of skin findings with the clinical phenotype as well as genetic testing has enabled understanding of the molecular pathogenesis and translation of changes at the genomic level to clinical phenotype.