Distinctive roles of fibrillar collagen I and collagen III in mediating fibroblast-matrix interaction: A nanoscopic study.

One major goal in tissue engineering is to create functional materials, mimicking scaffolds in native tissues, to modulate cell function for tissue repair. Collagen is the most abundant structural protein in human body. Though collagen I (COLI) and collagen III (COLIII) are the predominant collagen types in connective tissues and they form stable hybrid fibrils at varied ratios, cell responses to the hybrid matrices are underinvestigated. In this work, we aim to explicate the distinctive roles of COLI and COLIII in fibroblast activation. Unidirectionally aligned COLI, COLIII and COLI-COLIII hybrid nanofibrils were generated via epitaxial growth of collagen on mica. AFM analyses revealed that, with the increase of COLI/COLIII ratio, the fibril width and stiffness increased and the binding affinity of cells to the matrix decreased. A hybrid matrix was found to activate fibroblasts the most effectively, characterized by extensive cell polarization with rigid stress fiber bundles and high α-SMA expression, and by the highest-level of collagen synthesis. It is ascribed to the fine balance between biochemical and biophysical cues achieved on the hybrid matrix. Thus, matrices of aligned COLI-COLIII hybrid fibrils and their derived multifunctional composites can be good candidates of implantation scaffolds for tissue regeneration.

Collagen type III glomerulopathy: case report and review of the literature
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To summarize the clinical and pathological features of collagen type III glomerulopathy, the present report describes a case of collagen type III glomerulopathy and reviewed more than 60 cases recorded by other groups in English literature over the last few years. A 24-year-old female patient was admitted because of lower limbs edema, without any other discomforts. The lab examination of the patient reported proteinuria (2.42 g/24 h urine), microscopic hematuria, and the serum creatinine was 71 µmol/L. She received renal biopsy. The immunofluorescence staining results showed that collagen type III expression was positive. The electron microscopy examination showed that the mesangial regions were widened, with visible fine fibers in it. The periodic stripes of collagen fibers could be seen on some fine fiber-like substance under a high-magnification microscope, the diameters of the fiber-like substances were 40 - 70 nm. Serum collagen type III N-peptide (PIIIP N-P) was 97.94 ng/mL. After the patient received benazepril 10 mg per day and symptomatic treatments (Chinese drug, Cordyceps Capsules 0.5 g per day), her proteinuria resolved (~ 0.5 g/24 hour urine).

Clinical and morphological features of collagen type III glomerulopathy: a report of nine cases from a single institution.

AIMS: We report nine Chinese patients with collagen type III glomerulopathy. METHODS AND RESULTS: Two males and seven females were studied, ranging in age from 21 to 67 years. Proteinuria and hypertension were the most common symptoms, with incidences of 88.9 and 77.8%, respectively. Two patients had abnormal renal function. Their histological appearances varied. Massive eosinophilic and weakly periodic acid-Schiff (PAS)-positive substances were deposited along the capillary loops and in the mesangial area in three cases, while others had thickened capillary walls with a chain-like structure or double-contour appearance of the PAS- and silver-stained sections. Immunofluorescence analysis showed the abundant deposition of collagen type III. Electron microscopy revealed massive scattered or bundle-shaped fibre-like materials in the subendothelial and mesangial areas. During follow-up, 44.4% of the patients suffered a doubling of serum creatinine. The level of serum creatinine at biopsy was an independent predictor of this doubled serum creatinine value. CONCLUSIONS: Collagen type III deposits in the subendothelial and mesangial areas. Some patients show global nodular lesions, while others show subtle changes only via PAS/silver staining. Proteinuria and hypertension are the most common symptoms, and the serum creatinine level at biopsy is an independent predictor of the doubling of serum creatinine during follow-up.

3-D ultrastructure and collagen composition of healthy and overloaded human tendon: evidence of tenocyte and matrix buckling.

Achilles tendinopathies display focal tissue thickening with pain and ultrasonography changes. Whilst complete rupture might be expected to induce changes in tissue organization and protein composition, little is known about the consequences of non-rupture-associated tendinopathies, especially with regards to changes in the content of collagen type I and III (the major collagens in tendon), and changes in tendon fibroblast (tenocyte) shape and organization of the extracellular matrix (ECM). To gain new insights, we took biopsies from the tendinopathic region and flanking healthy region of Achilles tendons of six individuals with clinically diagnosed tendinopathy who had no evidence of cholesterol, uric acid and amyloid accumulation. Biochemical analyses of collagen III/I ratio were performed on all six individuals, and electron microscope analysis using transmission electron microscopy and serial block face-scanning electron microscopy were made on two individuals. In the tendinopathic regions, compared with the flanking healthy tissue, we observed: (i) an increase in the ratio of collagen III : I proteins; (ii) buckling of the collagen fascicles in the ECM; (iii) buckling of tenocytes and their nuclei; and (iv) an increase in the ratio of small-diameter : large-diameter collagen fibrils. In summary, load-induced non-rupture tendinopathy in humans is associated with localized biochemical changes, a shift from large- to small-diameter fibrils, buckling of the tendon ECM, and buckling of the cells and their nuclei.

Getting to the heart of cardiac remodeling; how collagen subtypes may contribute to phenotype.

The objective of this study was to investigate the nature and biomechanical properties of collagen fibers within the human myocardium. Targeting cardiac interstitial abnormalities will likely become a major focus of future preventative strategies with regard to the management of cardiac dysfunction. Current knowledge regarding the component structures of myocardial collagen networks is limited, further delineation of which will require application of more innovative technologies. We applied a novel methodology involving combined confocal laser scanning and atomic force microscopy to investigate myocardial collagen within ex-vivo right atrial tissue from 10 patients undergoing elective coronary bypass surgery. Immuno-fluorescent co-staining revealed discrete collagen I and III fibers. During single fiber deformation, overall median values of stiffness recorded in collagen III were 37±16% lower than in collagen I [p<0.001]. On fiber retraction, collagen I exhibited greater degrees of elastic recoil [p<0.001; relative percentage increase in elastic recoil 7±3%] and less energy dissipation than collagen III [p<0.001; relative percentage increase in work recovered 7±2%]. In atrial biopsies taken from patients in permanent atrial fibrillation (n=5) versus sinus rhythm (n=5), stiffness of both collagen fiber subtypes was augmented (p<0.008). Myocardial fibrillar collagen fibers organize in a discrete manner and possess distinct biomechanical differences; specifically, collagen I fibers exhibit relatively higher stiffness, contrasting with higher susceptibility to plastic deformation and less energy efficiency on deformation with collagen III fibers. Augmented stiffness of both collagen fiber subtypes in tissue samples from patients with atrial fibrillation compared to those in sinus rhythm are consistent with recent published findings of increased collagen cross-linking in this setting.

[Jejunum intersticium in mongolian gerbils after the flight on spacecraft Foton-M3].

Methods of light optical microscopy were used to explore histoarchitectonics, topography and tinctorial properties of the extracellular phase of fibers of jejunum wall intersticium in Mongolian gerbils following 12-day orbital flight aboard Foton-M3, ground-based simulation of the spaceflight factors in the KONTUR-L facility, and in the group of biological control Postflight destructive changes were found in reticulin fibers (type-III collagen) of villi stroma, intercrypt intersticium and submucosa. Local acidophilia and fiber homogenization formed in type I collagen present in the intestinal subserous layer, muscular layers endomysium and submucose against the background of progressing edema and arterial, venous plethora and lymphostasis. Elastic component of the intersticium was disarranged in the structures of internal elastic membrane of submucous vessels, fragmented and partly reduced. Simulation of the orbital factors, except for microgravity, in the KONTUR-L facility called forth similar, although less often and diffuse, changes in intersticium fibers. The results of examination of intestinal intersticium fibers in the vivarium control gerbils discovered expressed species characters that should be taken into account by investigators, especially when comparing with data obtained from other animal species.

Connective tissue alteration in abdominal wall hernia.

BACKGROUND: The aetiology and pathogenesis of abdominal wall hernia formation is complex. Optimal treatment of hernias depends on a full understanding of the pathophysiological mechanisms involved in their formation. The aim of this study was to review the literature on specific collagen alterations in abdominal wall hernia formation. METHODS: A computer-assisted search of the medical databases PubMed and Embase was performed, together with a cross-reference search of eligible papers. RESULTS: Fifty-two papers were included. Collagen alteration depended on the type of hernia; there were more pronounced changes in patients with a direct inguinal hernia than in those with an indirect inguinal hernia, recurrent inguinal hernia or incisional hernia. A consistent finding was a significant increase in immature type III collagen relative to the stronger type I collagen in patients with a hernia. This resulted in thinner collagen fibres with a correspondingly diminished biomechanical strength. It has been suggested that these alterations are due to variation in the synthesis, maturation or degradation of collagen by matrix metalloproteinases, in combination or alone. CONCLUSION: Hernia formation and recurrence is associated with altered collagen metabolism manifested by a decreased type I:III collagen ratio.

Morphological and biochemical analysis of intact and opaque cornea in dogs.

The arrangement of collagen fibrils and glycosaminoglycans (GAGs) in substantia propria are important for maintaining transparency of the cornea. Interferences in collagen fibrils and GAG production could be adversative to corneal integrity. In this study, six dogs consisting of four Beagles with normal cornea (normal), one Beagles with opaque cornea (sample No. 1) and one Shih Tzu with neovascularization opaque cornea (sample No.2) were used. All samples were observed morphologically by light and electron microscopes to obtain diameter and distribution of collagen fibrils in substantia propria and were performed biochemically to investigate into GAGs and collagen types. The average diameter of collagen fibrils in the intact cornea of normal, sample No.1 and No.2 was 33.2, 35.0 and 25.0 nm, respectively. The percentage of matrix per unit area was 67% in normal, 87% in sample No.1 and 28.3% in sample No.2. The type III collagen ratio was 25.3% in normal, 21.3% in sample No.1 and 35.8% in sample No.2. The relative amount of heparan sulfate, chondroitin sulfate, dermatan sulfate and keratin sulfate was 1.5, 9.7, 51.1 and 37.7% in normal, 3.3, 26.0, 45.7 and 23.7% in sample No.1 and 1.2, 18.0, 16.6 and 54.1% in sample No.2. Hyaluronic acid was found only in sample No.1 with a relative amount of 1.3%. Since there was some relationship between collagen formation and GAGs composition, it might be speculated that disturbance in arrangement of collagen fibrils and GAG metabolism especially in substantia propria would bring up opacity of the cornea.

Lamina propria cellularity and collagen composition: an integrated assessment of structure in humans.

OBJECTIVES: In this study, we quantitatively examined cell density, collagen types I and III, and regional variations in collagen fiber thickness and orientation in the human midmembranous vocal fold lamina propria (LP). METHODS: Lamina propria samples were solubilized with proteinase K or with cyanogen bromide. Cell density was assessed in proteinase K digests by measuring DNA and normalizing it to tissue total protein. Collagen types I and III were quantified by enzyme-linked immunosorbent assay-based detection of collagen type-specific peptides generated by cyanogen bromide digestion. In addition, LP total collagen was determined by measuring sample hydroxyproline levels. Variations in collagen fiber thickness and orientation with LP region were evaluated by examining picrosirius red-stained LP sections with circularly polarized light. RESULTS: The mean (+/-SEM) cell density in the LP and associated epithelium was approximately 0.57 +/- 0.09 million cells per milligram of tissue total protein. Collagen type III composed an average of 34% to 40% of LP total collagen. Quantitative histology indicated that the superficial LP contained an average of 70% thin, 26% intermediate, and 4% thick collagen fibers. This is in contrast to the intermediate and deep LPs, each of which contained less than 25% thin and more than 50% thick collagen fibers. The angular deviations in collagen fiber orientation were relatively large and were similar in magnitude across all LP layers. CONCLUSIONS: The total cell density of the LP and associated epithelium was intermediate between that of hyaline cartilage and dermis. The ratio of collagen type III to total collagen in the LP was similar to that of highly elastic lung parenchyma and roughly twice that of the comparatively less-elastic dermis. The average thickness of collagen fibers increased markedly with increasing LP depth, and the relatively large angular deviations in fiber orientation appeared to correspond in part to the crimped nature of LP collagen fibers.

Simulation of the mechanical strength of a single collagen molecule.

We perform atomistic simulations on a single collagen molecule to determine its intrinsic molecular strength. A tensile pull simulation to determine the tensile strength and Young's modulus is performed, and a simulation that separates two of the three helices of collagen examines the internal strength of the molecule. The magnitude of the calculated tensile forces is consistent with the strong forces of bond stretching and angle bending that are involved in the tensile deformation. The triple helix unwinds with increasing tensile force. Pulling apart the triple helix has a smaller, oscillatory force. The oscillations are due to the sequential separation of the hydrogen-bonded helices. The force rises due to reorienting the residues in the direction of the separation force. The force drop occurs once the hydrogen bond between residues on different helices break and the residues separate.

Preparation of biomolecule gel matrices for electron microscopy.

We report a new sample preparation method that allows the direct transmission electron microscopy evaluation of the architectural characteristics of biomolecules entrapped in gel matrices. We demonstrate that this sample preparation technique can be used for the identification and ultrastructural characterization of liposomes, collagen I and collagen III embedded in gel matrices, and has the potential to be useful for transmission electron microscopy (TEM) characterization of other biomolecule-gel matrix systems.

Collagen types I, III, and V constitute the thick collagen fibrils of the mouse decidua.

A mammal's endometrium is deeply remodeled while receiving and implanting an embryo. In addition to cell proliferation and growth, endometrial remodeling also comprises synthesis and degradation of several molecular components of the extracellular matrix. All of these events are orchestrated by a precise sequence of ovarian hormones and influenced by several types of cytokines. As we have previously reported, an intriguing and rapid increase in collagen fibril diameter occurs in the decidualized areas of the endometrium, surrounding the implantation crypt, whereas collagen fibrils situated far from the embryo remain unchanged. Collagen fibrilogenesis is a complex molecular process coordinated by a number of factors, such as the types and amounts of glycosaminoglycans and proteoglycans associated with collagen molecules. Collagen genetic type, mechanical stress, aging, and other factors not yet identified also contribute to this development. A recent study suggests that thick fibrils from mouse decidua are formed, at least in part, by aggregation of thin fibrils existing in the stroma before the onset of decidualization. In the present ultrastructural study using single and double immunogold localization, we showed that both thin and thick collagen fibrils present in the mouse pregnant endometrium endometrium are heterotypic structures formed at least by type I, type III, and type V collagens. However, type V collagen predominates in the thick collagen fibrils, whereas it is almost absent of the thin collagen fibrils. The putative role of type V homotrimer in the rapid increase of the diameter of collagen fibrils of the mouse decidua is discussed.

Graded arrangement of collagen fibrils in the equine superficial digital flexor tendon.

By using ultramorphological and biochemical methods, we analyzed the regional differences between the three parts of the equine superficial digital flexor tendon (SDFT), namely, the myotendinous junction (MTJ), middle metacarpal (mM), and osteotendinous junction (OTJ). Cross-sectional images showed unique distributions of collagen fibrils of varying diameters in each region. Small collagen fibrils (diameter <100 nm) were distributed predominantly in the MTJ region, and the OTJ region was relatively rich in large collagen fibrils (diameter >200 nm). In the mM region, the collagen fibrils were intermediately distributed between the MTJ and OTJ. The results indicate a graded arrangement of collagen fibrils in the tendon. Type V collagen was detected preferentially in the MTJ region. Since type V collagen is believed to be one of the collagens regulating collagen fibril formation, its possible functionality in the MTJ region in terms of fibril formation and fibril arrangement in the tendon has been discussed here.

Immuno-scanning electron microscopy of collagen types I and III in human vocal fold lamina propria.

OBJECTIVES: This study was undertaken to identify the types of collagen fibrils in the extracellular matrix of the human vocal fold lamina propria. METHODS: Human vocal folds were obtained from 3 autopsy cases less than 65 years of age. The vocal fold specimens were labeled by primary antibodies of anti-type I and anti-type III collagens, and then by secondary antibody conjugated with 15 nm colloidal gold. The specimens were observed with a scanning electron microscope. Secondary electron imaging and backscatter electron imaging of high-resolution field emission scanning electron microscopy were used to detect gold particles indicating immunolabeling. RESULTS: Type III collagen-labeling gold particles were abundant on the fibrils constructing collagenous fibers, whereas type I collagen-labeling gold particles were sparsely present on fibrils in collagenous fibers. A few reticular fibers were labeled by both collagen type I and collagen type III. CONCLUSIONS: The results suggest that collagen type I coexists with collagen type III in fibrils of both collagenous fibers and reticular fibers.

In vitro fibrillogenesis of tropocollagen type III in collagen type I affects its relative fibrillar topology and mechanics.

Tropocollagen types I and III were simultaneously fibrilized in vitro, and the differences between the geometric and mechanical properties of the heterotypic fibrils with different mixing ratios of tropocollagen III to I were investigated. Transmission electron microscopy was used to confirm the simultaneous presence of both tropocollagen types within the heterotypic fibrils. The incorporation of collagen III in I caused the fibrils to be thinner with a shorter D-banding than pure collagen I. Hertzian contact model was used to obtain the elastic moduli from atomic force microscope indentation testing using a force volume analysis. The results indicated that an increase in the percentage of tropocollagen III reduced the mechanical stiffness of the obtained fibrils. The mechanical stiffness of the collagen fibrils was found to be greater at higher loading frequencies. This observation might explain the dominance of collagen III over I in soft distensible organs such as human vocal folds.

Wavelength-Dependent Second Harmonic Generation Circular Dichroism for Differentiation of Col I and Col III Isoforms in Stromal Models of Ovarian Cancer Based on Intrinsic Chirality Differences.

Extensive remodeling of the extracellular matrix (ECM) occurs in many epithelial cancers. For example, in ovarian cancer, upregulation of collagen isoform type III has been linked to invasive forms of the disease, and this change may be a potential biomarker. To examine this possibility, we implemented wavelength-dependent second harmonic generation circular dichroism (SHG-CD) imaging microscopy to quantitatively determine changes in chirality in ECM models comprised of different Col I/Col III composition. In these models, Col III was varied between 0 and 40%, and we found increasing Col III results in reduced net chirality, consistent with structural biology studies of Col I and III in tissues where the isoforms comingle in the same fibrils. We further examined the wavelength dependence of the SHG-CD to both optimize the response and gain insight into the underlying mechanism. We found using shorter SHG excitation wavelengths resulted in increased SHG-CD sensitivity, where this is consistent with the electric-dipole-coupled oscillator model suggested previously for the nonlinear chirality response from thin films. Moreover, the sensitivity is further consistent with the wavelength dependency of SHG intensity fit to a two-state model of the two-photon absorption in collagen. We also provide experimental calibration protocols to implement the SHG-CD modality on a laser scanning microscope. We last suggest that the technique has broad applicability in probing a wide range of diseased states with changes in collagen molecular structure.

Monitoring mesenchymal stromal cell developmental stage to apply on-time mechanical stimulation for ligament tissue engineering.

To evaluate the appropriate time frame for applying mechanical stimuli to induce mesenchymal stromal cell (MSC) differentiation for ligament tissue engineering, developmental cell phenotypes were monitored during a period of in vitro culture. MSCs were seeded onto surface-modified silk fibroin fiber matrices and cultured in Petri dishes for 15 days. Cell metabolic activity, morphology, and gene expression of extracellular matrix (ECM) proteins (collagen type I and III and fibronectin), ECM receptors (integrins alpha-2, alpha-5, and beta-1), and heat-shock protein 70 (HSP-70) were monitored during the culture of MSC. MSCs showed fluctuations in cell metabolic activity, ECM, integrin, and HSP-70 transcription potentially correlating to innate developmental processes. Cellular response to mechanical stimulation was dependent on the stage of cell development. At day 9, when levels of cell metabolic activity, ECM, integrin, and HSP-70 transcription peaked, mechanical stimulation increased MSC metabolic activity, alignment, and collagen production. Mechanical stimulation applied at day 1 and 3 showed detrimental effects on MSCs seeded on silk matrices. The results presented in this study identify a unique correlation between innate MSC development processes on a surface-modified silk matrix and dynamic environmental signaling.

An automated approach for analyzing D-periods in collagen fibril images.

This paper considers an approach for analyzing fibrillar collagen structures based on fundamental concepts of pattern recognition. It focuses on the quantitative comparison between collagen structural data (electron-optical data) and chemical data. Theoretical models in the form of sequence-generated histograms are used as reference for extracting and analyzing the structural unit in images from collagen fibrils. In this respect, collagen provides a valuable model system for studying the chemical basis of ultrastructure, as well as detecting the alterations in collagen fibril structure produced by a disorder. Application examples are presented and the results are compared with biochemical studies.

The structural and optical properties of type III human collagen biosynthetic corneal substitutes.

The structural and optical properties of clinically biocompatible, cell-free hydrogels comprised of synthetically cross-linked and moulded recombinant human collagen type III (RHCIII) with and without the incorporation of 2-methacryloyloxyethyl phosphorylcholine (MPC) were assessed using transmission electron microscopy (TEM), X-ray scattering, spectroscopy and refractometry. These findings were examined alongside similarly obtained data from 21 human donor corneas. TEM demonstrated the presence of loosely bundled aggregates of fine collagen filaments within both RHCIII and RHCIII-MPC implants, which X-ray scattering showed to lack D-banding and be preferentially aligned in a uniaxial orientation throughout. This arrangement differs from the predominantly biaxial alignment of collagen fibrils that exists in the human cornea. By virtue of their high water content (90%), very fine collagen filaments (2-9 nm) and lack of cells, the collagen hydrogels were found to transmit almost all incident light in the visible spectrum. They also transmitted a large proportion of UV light compared to the cornea which acts as an effective UV filter. Patients implanted with these hydrogels should be cautious about UV exposure prior to regrowth of the epithelium and in-growth of corneal cells into the implants.