Tenomodulin knockout mice exhibit worse late healing outcomes with augmented trauma-induced heterotopic ossification of Achilles tendon.

Heterotopic ossification (HO) represents a common problem after tendon injury with no effective treatment yet being developed. Tenomodulin (Tnmd), the best-known mature marker for tendon lineage cells, has important effects in tendon tissue aging and function. We have reported that loss of Tnmd leads to inferior early tendon repair characterized by fibrovascular scaring and therefore hypothesized that its lack will persistently cause deficient repair during later stages. Tnmd knockout (Tnmd-/-) and wild-type (WT) animals were subjected to complete Achilles tendon surgical transection followed by end-to-end suture. Lineage tracing revealed a reduction in tendon-lineage cells marked by ScleraxisGFP, but an increase in alpha smooth muscle actin myofibroblasts in Tnmd-/- tendon scars. At the proliferative stage, more pro-inflammatory M1 macrophages and larger collagen II cartilaginous template were detected in this group. At the remodeling stage, histological scoring revealed lower repair quality in the injured Tnmd-/- tendons, which was coupled with higher HO quantified by micro-CT. Tendon biomechanical properties were compromised in both groups upon injury, however we identified an abnormal stiffening of non-injured Tnmd-/- tendons, which possessed higher static and dynamic E-moduli. Pathologically thicker and abnormally shaped collagen fibrils were observed by TEM in Tnmd-/- tendons and this, together with augmented HO, resulted in diminished running capacity of Tnmd-/- mice. These novel findings demonstrate that Tnmd plays a protecting role against trauma-induced endochondral HO and can inspire the generation of novel therapeutics to accelerate repair.

Intratumoral heterogeneity of second-harmonic generation scattering from tumor collagen and its effects on metastatic risk prediction.

BACKGROUND: Metastases are the leading cause of breast cancer-related deaths. The tumor microenvironment impacts cancer progression and metastatic ability. Fibrillar collagen, a major extracellular matrix component, can be studied using the light scattering phenomenon known as second-harmonic generation (SHG). The ratio of forward- to backward-scattered SHG photons (F/B) is sensitive to collagen fiber internal structure and has been shown to be an independent prognostic indicator of metastasis-free survival time (MFS). Here we assess the effects of heterogeneity in the tumor matrix on the possible use of F/B as a prognostic tool. METHODS: SHG imaging was performed on sectioned primary tumor excisions from 95 untreated, estrogen receptor-positive, lymph node negative invasive ductal carcinoma patients. We identified two distinct regions whose collagen displayed different average F/B values, indicative of spatial heterogeneity: the cellular tumor bulk and surrounding tumor-stroma interface. To evaluate the impact of heterogeneity on F/B's prognostic ability, we performed SHG imaging in the tumor bulk and tumor-stroma interface, calculated a 21-gene recurrence score (surrogate for OncotypeDX®, or S-ODX) for each patient and evaluated their combined prognostic ability. RESULTS: We found that F/B measured in tumor-stroma interface, but not tumor bulk, is prognostic of MFS using three methods to select pixels for analysis: an intensity threshold selected by a blinded observer, a histogram-based thresholding method, and an adaptive thresholding method. Using both regression trees and Random Survival Forests for MFS outcome, we obtained data-driven prediction rules that show F/B from tumor-stroma interface, but not tumor bulk, and S-ODX both contribute to predicting MFS in this patient cohort. We also separated patients into low-intermediate (S-ODX < 26) and high risk (S-ODX ≥26) groups. In the low-intermediate risk group, comprised of patients not typically recommended for adjuvant chemotherapy, we find that F/B from the tumor-stroma interface is prognostic of MFS and can identify a patient cohort with poor outcomes. CONCLUSIONS: These data demonstrate that intratumoral heterogeneity in F/B values can play an important role in its possible use as a prognostic marker, and that F/B from tumor-stroma interface of primary tumor excisions may provide useful information to stratify patients by metastatic risk.

Non-disruptive collagen characterization in clinical histopathology using cross-modality image synthesis.

The importance of fibrillar collagen topology and organization in disease progression and prognostication in different types of cancer has been characterized extensively in many research studies. These explorations have either used specialized imaging approaches, such as specific stains (e.g., picrosirius red), or advanced and costly imaging modalities (e.g., second harmonic generation imaging (SHG)) that are not currently in the clinical workflow. To facilitate the analysis of stromal biomarkers in clinical workflows, it would be ideal to have technical approaches that can characterize fibrillar collagen on standard H&E stained slides produced during routine diagnostic work. Here, we present a machine learning-based stromal collagen image synthesis algorithm that can be incorporated into existing H&E-based histopathology workflow. Specifically, this solution applies a convolutional neural network (CNN) directly onto clinically standard H&E bright field images to extract information about collagen fiber arrangement and alignment, without requiring additional specialized imaging stains, systems or equipment.

Collagen fibrillar structures in vocal fold scarring and repair using stem cell therapy: a detailed histological, immunohistochemical and atomic force microscopy study.

Regenerative medicine opens new opportunities in the repair of cicatricial lesions of the vocal folds. Here, we present a thorough morphological study, with the focus on the collagen structures in the mucosa of the vocal folds, dedicated to the effects of stem cells on the vocal folds repair after cicatricial lesions. We used a conventional experimental model of a mature scar of the rabbit vocal folds, which was surgically excised with a simultaneous implantation of autologous bone marrow-derived mesenchymal stem cells (MSC) into the defect. The restoration of the vocal folds was studied 3 months postimplantation of stem cells and 6 months after the first surgery. The collagen structure assessment included histology, immunohistochemistry and atomic force microscopy (AFM) studies. According to the data of optical microscopy and AFM, as well as to immunohistochemical analysis, MSC implantation into the vocal fold defect leads not only to the general reduction of scarring, normal ratio of collagens type I and type III, but also to a more complete restoration of architecture and ultrastructure of collagen fibres in the mucosa, as compared to the control. The collagen structures in the scar tissue in the vocal folds with implanted MSC are more similar to those in the normal mucosa of the vocal folds than to those of the untreated scars. AFM has proven to be an instrumental technique in the assessment of the ultrastructure restoration in such studies. LAY DESCRIPTION: Regenerative medicine opens new opportunities in the repair of the vocal fold scars. Because collagen is a main component in the vocal fold mucosa responsible for the scar formation and repair, we focus on the collagen structures in the mucosa of the vocal folds, using a thorough morphological study based on histology and atomic force microscopy (AFM). Atomic force microscopy is a scanning microscopic technique which allows revealing the internal structure of a tissue with a resolution up to nanometres. We used a conventional experimental model of a mature scar of the rabbit vocal folds, surgically excised and treated with a mesenchymal stem cells transplant. Our morphological study, primarily AFM, explicitly shows that the collagen structures in the scarred vocal folds almost completely restore after the stem cell treatment. Thus, the modern microscopic methods, and especially AFM are instrumental tools for monitoring the repair of the vocal folds scars.

Biomimetic intrafibrillar mineralized collagen promotes bone regeneration via activation of the Wnt signaling pathway.

PURPOSE: The purpose of this study was to assess the effects of biomimetic intrafibrillar mineralized collagen (IMC) bone scaffold materials on bone regeneration and the underlying biological mechanisms. MATERIALS AND METHODS: A critical-sized bone defect in the rat femur was created; then IMC, extrafibrillar mineralized collagen, and nano-hydroxyapatite bone scaffold materials were grafted into the defect. Ten weeks after implantation, micro-computed tomography and histology were applied to evaluate the bone regeneration. Furthermore, microarray technology was applied for transcriptional profile analysis at two postoperative time points (7 and 14 days). Subsequently, the critical genes involved in bone regeneration identified by transcriptional analysis were verified both in vivo through immunohistochemical analysis and in vitro by quantitative real-time transcription polymerase chain reaction evaluation. RESULTS: Significantly increased new bone formation was found in the IMC group based on micro-computed tomography and histological evaluation (P<0.05). Transcriptional analysis revealed that the early process of IMC-guided bone regeneration involves the overexpression of genes mainly associated with inflammation, immune response, skeletal development, angiogenesis, neurogenesis, and the Wnt signaling pathway. The roles of the Wnt signaling pathway-related factors Wnt5a, ß-catenin, and Axin2 were further confirmed both in vivo and in vitro. CONCLUSION: The IMC bone scaffold materials significantly enhanced bone regeneration via activation of the Wnt signaling pathway.

Evaluation of dermal collagen stained with picrosirius red and examined under polarized light microscopy

Abstract: The special picrosirius red staining highlights the natural birefringence of collagen fibers when exposed to polarized light. The results from birefringence allow to evaluate the organization of the collagen fibers in the tissues. The authors intend to elucidate all steps to obtain and capture images of histological sections stained with picrosirius red and evaluated under polarized light microscopy, as well as possible artefacts that may occur.

Molecular and ultrastructural studies of a fibrillar collagen from octocoral (Cnidaria).

We report here the biochemical, molecular and ultrastructural features of a unique organization of fibrillar collagen extracted from the octocoral Sarcophyton ehrenbergi Collagen, the most abundant protein in the animal kingdom, is often defined as a structural component of extracellular matrices in metazoans. In the present study, collagen fibers were extracted from the mesenteries of S. ehrenbergi polyps. These fibers are organized as filaments and further compacted as coiled fibers. The fibers are uniquely long, reaching an unprecedented length of tens of centimeters. The diameter of these fibers is 9±0.37 µm. The amino acid content of these fibers was identified using chromatography and revealed close similarity in content to mammalian type I and II collagens. The ultrastructural organization of the fibers was characterized by means of high-resolution microscopy and X-ray diffraction. The fibers are composed of fibrils and fibril bundles in the range of 15 to 35 nm. These data indicate a fibrillar collagen possessing structural aspects of both types I and II collagen, a highly interesting and newly described form of fibrillar collagen organization.

Quantitative analysis of collagen fibrillar structure in canine uterus exposed to acetate of medroxyprogesterone using second harmonic generation microscopy / Análisis cuantitativo de la estructura de fibras colágenas en útero canino expuesto a acetato de medroxiprogesterona utilizando generación microscópica de un segundo harmónico

Collagen is the most abundant structural protein found in organs and is responsible for providing tissues with structure and function. In order to investigate in canine uteri the potential effect of medroxyprogesterone acetate (MPA) on the changes in collagen deposition were grouped as nulliparous (n=11), multiparous (n=11) and treated with MPA (n=11; nulliparous with two treatments; 5 mg/kg; i.m.). The amount of collagen was studied in the fold and basal regions of the endometrium and myometrium using second harmonic generation with a two-photon spectral confocal microscope, quantified using ImageJ software with a color segmentation plugin, was expressed as fraction area (%) and analyzed by ANOVA (p<0,05). No differences were observed between groups in the fold (p=0,3995) or base (p=0,7392) of the endometrium and myometrium (p=0,1781). In conclusion, our data demonstrate that two doses of MPA (5 mg/kg; i.m.) do not affect the total collagen deposition in canine uteri undergoing contraceptive treatment.

El colágeno es la proteína estructural más abundante presente en órganos y es responsable de proporcionar la sostén y función a los tejidos. Para investigar en caninos el efecto potencial del acetato de medroxiprogesterona (MPA) sobre cambios en el depósito de colágeno en útero, éstos fueron agrupados como nulíparos (n = 11), multíparos (n = 11) y tratados con MPA (n = 11, nulíparos con dos tratamientos 5 mg/kg, im). El colágeno fue evaluado en el pliegue y regiones basales del endometrio y en miometrio utilizando la Generación de un Segundo Harmónico con un microscopio confocal espectral y dos fotones y cuantificado utilizando el software ImageJ a partir de la segmentación de colores. Los resultados fueron expresados y analizados como fracción de área (%; ANOVA; p<0,05). No se observaron diferencias entre los grupos en el pliegue (p = 0,3995) y base (p=0,7392) del endometrio y tampoco en miometrio (p=0,1781). En conclusión, nuestra evidencia demuestra que dos dosis de MPA (5 mg/kg, i.m.) no afectan el depósito total de colágeno en úteros caninos expuestos a tratamiento anticonceptivo.

Myosin phosphorylation on stress fibers predicts contact guidance behavior across diverse breast cancer cells.

During cancer progression the extracellular matrix is remodeled, forming aligned collagen fibers that proceed radially from the tumor, resulting in invasion. We have recently shown that different invasive breast cancer cells respond to epitaxially grown, aligned collagen fibrils differently. This article develops insight into why these cells differ in their contact guidance fidelity. Small changes in contractility or adhesion dramatically alter directional persistence on aligned collagen fibrils, while migration speed remains constant. The directionality of highly contractile and adhesive MDA-MB-231 cells can be diminished by inhibiting Rho kinase or ß1 integrin binding. Inversely, the directionality of less contractile and adhesive MTLn3 cells can be enhanced by activating contractility or integrins. Subtle, but quantifiable alterations in myosin II regulatory light chain phosphorylation on stress fibers explain the tuning of contact guidance fidelity, separate from migration per se indicating that the contractile and adhesive state of the cell in combination with collagen organization in the tumor microenvironment determine the efficiency of migration. Understanding how distinct cells respond to contact guidance cues will not only illuminate mechanisms for cancer invasion, but will also allow for the design of environments to separate specific subpopulations of cells from patient-derived tissues by leveraging differences in responses to directional migration cues.

Automated quantification of three-dimensional organization of fiber-like structures in biological tissues.

Fiber-like structures are prevalent in biological tissues, yet quantitative approaches to assess their three-dimensional (3D) organization are lacking. We develop 3D directional variance, as a quantitative biomarker of truly 3D fibrillar organization by extending the directional statistics formalism developed for describing circular data distributions (i.e. when 0° and 360° are equivalent) to axial ones (i.e. when 0° and 180° are equivalent). Significant advantages of this analysis include its time efficiency, sensitivity and ability to provide quantitative readouts of organization over different size scales of a given data set. We establish a broad range of applications for this method by characterizing collagen fibers, neuronal axons and fibroblasts in the context of cancer diagnostics, traumatic brain injury and cell-matrix interactions in developing engineered tissues. This method opens possibilities for unraveling in a sensitive, and quantitative manner the organization of essential fiber-like structures in tissues and ultimately its impact on tissue function.

Preparation of High-Density Fibrillar Collagen Matrices That Mimic Desmoplastic Tumor Stroma.

The stroma of invasive tumors becomes enriched in dense fibrillar collagen as a result of the desmoplastic reaction. This desmoplastic collagen exerts profound effects on tumor and normal cells. In view of these findings, it is important to develop novel in vitro cell systems that mimic this desmoplastic extracellular matrix in order to permit cell studies under in vivo-like conditions. This unit provides a protocol and troubleshooting guide for preparation of high-density fibrillar collagen (HDFC) matrices that closely model the desmoplastic collagenous matrix of malignant tumors. It then describes the use of this matrix for in vitro cell studies of invadopodia formation and function in extracellular matrix invasion. In addition, it provides a detailed protocol for immunolabeling of invadopodial proteins and detection of HDFC matrix degradation associated with invadopodia to permit visualization of invadopodia using fluorescence microscopy. © 2016 by John Wiley & Sons, Inc.

Transplantation of tendon-derived stem cells pre-treated with connective tissue growth factor and ascorbic acid in vitro promoted better tendon repair in a patellar tendon window injury rat model.

BACKGROUND AIMS: Treatment of tendon-derived stem cells (TDSCs) with connective tissue growth factor (CTGF) and ascorbic acid promoted their tenogenic differentiation. We investigated the effects of TDSCs pre-treated with CTGF and ascorbic acid on tendon repair in a patellar tendon window injury rat model. METHODS: Green fluorescent protein-TDSCs (GFP-TDSCs) were pre-treated with or without CTGF and ascorbic acid for 2 weeks before transplantation. The patellar tendons of rats were injured and divided into three groups: fibrin glue-only group (control group), untreated and treated TDSC group. The rats were followed up until week 16. RESULTS: The treated TDSCs accelerated and enhanced the quality of tendon repair compared with untreated TDSCs up to week 8, which was better than that in the controls up to week 16 as shown by histology, ultrasound imaging and biomechanical test. The fibrils in the treated TDSC group showed better alignment and larger size compared with those in the control group at week 8 (P = 0.004). There was lower risk of ectopic mineralization after transplantation of treated or untreated TDSCs (all P ≤ 0.050). The transplanted cells proliferated and could be detected in the window wound up to weeks 2 to 4 and week 8 for the untreated and treated TDSC groups, respectively. CONCLUSIONS: The transplantation of TDSCs promoted tendon repair up to week 16, with CTGF and ascorbic acid pre-treatment showing the best results up to week 8. Pre-treatment of TDSCs with CTGF and ascorbic acid may be used to further enhance the rate and quality of tendon repair after injury.

Histopathological Analysis of Internal Limiting Membrane Surgically Peeled From Eyes with Epiretinal Membrane.

PURPOSE: To investigate histopathological changes of internal limiting membrane (ILM) in patients with epiretinal membrane (ERM) Materials and Methods: Forty-two eyes of 42 patients who were diagnosed as ERM and enrolled for vitreoretinal surgery were included in this study. Brilliant Blue G (BBG) was used to stain the ILM in all patients. ILM was peeled in all subjects and analyzed by light microscopy (methylene blue-Azur II × 40). ILM samples were then fixed in 2.5% glutaraldehyde solution and examined in JEOL-JEM 1400 and 2100F electron microscope and photographed by CCD camera (Gatan Inc., Pleasanton, CA). RESULTS: Remained ERM fragments were observed on 80% of ILM's. Vacuolization of ILM was observed in a patient with diabetic ERM. There were cells and cellular fragments observed mostly at retinal side of ILM which was likely to be a fragment of Muller cells of retina. CONCLUSIONS: Most of the ILM's had residual ERM tissue and contained cells and cellular fragments at retinal side of ILM's. ILM peeling might have a role in decreasing ERM recurrence by removal of residual ERM tissues.

Structural analysis of adventitial collagen to feature aging and aneurysm formation in human aorta.

OBJECTIVE: Adventitial collagen structure provides the aorta with tensile strength. Like other collagen-rich tissues, it can be affected by internal factors including aging and location. We determined whether the structural characteristics of human aortic adventitial collagen change with aging, location, and aneurysm formation. METHODS: Nonatherosclerotic nonaneurysmal (NANA) human abdominal aortas were collected from 15 individuals who had died of noncardiovascular diseases (<40 years old, NANA young, n = 5; >60 years old, NANA old, n = 5). The architecture of adventitial collagen in the aortas was assessed by scanning electron microscopy, and fiber orientation was assessed by polarized microscopy with two-dimensional fast Fourier transform. We then analyzed retardation as an anisotropic property of adventitial collagen by polarized light microscopy. The orientation and retardation of NANA aortas were compared with those of abdominal aortic specimens from patients who were surgically treated for abdominal aortic aneurysm (AAA) (>60 years old, n = 11). RESULTS: Adventitial collagen of the abdominal aortas on scanning electron microscopy images appeared as wavy, ropy fibers in aortas from young individuals (NANA young, n = 5) and were essentially flattened in those from older patents (NANA old, n = 5) and from those with AAA. Collagen fibers were thicker but sparser in the adventitia of aortas with AAA. Orientation maintained in the collagen fibers of NANA aortas (n = 15) on two-dimensional fast Fourier transform analysis was unrelated to either location or age and did not differ between NANA aortas and those with AAA. However, collagen fibrils in NANA aortas (n = 15) were significantly less retarded only at the level of the inferior mesenteric artery compared with other aortic locations. In addition, retardation was significantly reduced in abdominal aortas with AAA at the level of the inferior mesenteric artery. CONCLUSIONS: The basic structure of adventitial collagen fiber was maintained in abdominal aortas regardless of location or age. Because the molecular structure at the subfibril level changed at abdominal aorta and enhanced in aortas with AAA, alterations in the molecular structure of adventitial collagen might be associated with aneurysmal formation.

Three-dimensional matrix fiber alignment modulates cell migration and MT1-MMP utility by spatially and temporally directing protrusions.

Multiple attributes of the three-dimensional (3D) extracellular matrix (ECM) have been independently implicated as regulators of cell motility, including pore size, crosslink density, structural organization, and stiffness. However, these parameters cannot be independently varied within a complex 3D ECM protein network. We present an integrated, quantitative study of these parameters across a broad range of complex matrix configurations using self-assembling 3D collagen and show how each parameter relates to the others and to cell motility. Increasing collagen density resulted in a decrease and then an increase in both pore size and fiber alignment, which both correlated significantly with cell motility but not bulk matrix stiffness within the range tested. However, using the crosslinking enzyme Transglutaminase II to alter microstructure independently of density revealed that motility is most significantly predicted by fiber alignment. Cellular protrusion rate, protrusion orientation, speed of migration, and invasion distance showed coupled biphasic responses to increasing collagen density not predicted by 2D models or by stiffness, but instead by fiber alignment. The requirement of matrix metalloproteinase (MMP) activity was also observed to depend on microstructure, and a threshold of MMP utility was identified. Our results suggest that fiber topography guides protrusions and thereby MMP activity and motility.

In vivo quantification of the structural changes of collagens in a melanoma microenvironment with second and third harmonic generation microscopy.

Using in vivo second harmonic generation (SHG) and third harmonic generation (THG) microscopies, we tracked the course of collagen remodeling over time in the same melanoma microenvironment within an individual mouse. The corresponding structural and morphological changes were quantitatively analyzed without labeling using an orientation index (OI), the gray level co-occurrence matrix (GLCM) method, and the intensity ratio of THG to SHG (RTHG/SHG). In the early stage of melanoma development, we found that collagen fibers adjacent to a melanoma have increased OI values and SHG intensities. In the late stages, these collagen networks have more directionality and less homogeneity. The corresponding GLCM traces showed oscillation features and the sum of squared fluctuation VarGLCM increased with the tumor sizes. In addition, the THG intensities of the extracellular matrices increased, indicating an enhanced optical inhomogeneity. Multiplying OI, VarGLCM, and RTHG/SHG together, the combinational collagen remodeling (CR) index at 4 weeks post melanoma implantation showed a 400-times higher value than normal ones. These results validate that our quantitative indices of SHG and THG microscopies are sensitive enough to diagnose the collagen remodeling in vivo. We believe these indices have the potential to help the diagnosis of skin cancers in clinical practice.

Collagen fibre and fibril ultrastructural arrangement of the superficial medial collateral ligament in the human knee.

PURPOSE: The aim of the study was to investigate the collagen fibre ultrastructural arrangement and collagen fibril diameters in the superficial medial collateral ligament (sMCL) in the human knee. Considering sMCL's distinctive functions at different angles of knee flexion, it was hypothesized a significant difference between the collagen fibril diameters of each portion of the sMCL. METHODS: Fourteen sMCL from seven fresh males (by chance because of the availability) cadavers (median age 40 years, range 34-59 years) were harvested within 12 h of death. sMCLs were separated into two orders of regions for analysis. The first order (divisions) was anterior, central and posterior. Thereafter, each division was split into three regions (femoral, intermediate and tibial), generating nine portions. One sMCL from each cadaver was used for transmission electron microscopy (TEM) and morphometric analyses, whereas the contralateral sMCL was processed for light microscopy (LM) or scanning electron microscopy (SEM). RESULTS: LM and SEM analyses showed a complex tridimensional architecture, with the presence of wavy collagen fibres or crimps. TEM analysis showed significant differences in median collagen fibril diameter among portions inside the anterior, central and posterior division of the sMCL (p < 0.0001 within each division). Significant differences were also present among the median [interquartile range] collagen fibril diameters of anterior (39.4 [47.8-32.9]), central (38.5 [44.4-34.0]) and posterior (41.7 [52.2-35.4]) division (p = 0.0001); femoral (38.2 [45.0-32.7]), intermediate (40.3 [47.3-36.1]) and tibial (40.7 [55.0-32.2]) region (p = 0.0001). CONCLUSIONS: Human sMCL showed a complex architecture that allows restraining different knee motions at different angles of knee flexion. The posterior division of sMCL accounted for the largest median collagen fibril diameter. The femoral region of sMCL accounted for the smallest median collagen fibril diameter. The presence of crimps in the medial collateral ligament, previously identified in the rat, was confirmed in humans (taking into consideration differences between these two species).

Second-harmonic generation imaging of cancer.

The last 30 years has seen great advances in optical microscopy with the introduction of sophisticated fluorescence-based imaging methods such as confocal and multiphoton laser scanning microscopy. There is increasing interest in using these methods to quantitatively examine sources of intrinsic biological contrast including autofluorescent endogenous proteins and light interactions such as second-harmonic generation (SHG) in collagen. In particular, SHG-based microscopy has become a widely used quantitative modality for imaging noncentrosymmetric proteins such as collagen in a diverse range of tissues. Due to the underlying physical origin of the SHG signal, it is highly sensitive to collagen fibril/fiber structure and, importantly, to collagen-associated changes that occur in diseases such as cancer, fibrosis, and connective tissue disorders. An overview of SHG physics background and technologies is presented with a focused review on applications of SHG primarily as applied to cancer.

Observation of collagen fibrils produced by osteosarcoma cells using atomic force microscopy.

The present study examined the three-dimensional process of collagen fibril formation in the human osteosarcoma cell line NOS-1 by conventional scanning electron microscopy (SEM) and atomic force microscopy (AFM). SEM images showed collagen fibril formation on the bottom of culture dishes after 1 week of culture. The collagen fibrils had diameters of 30-100 nm. The surfaces of individual fibrils had characteristic grooves and ridges with periodicities of 60-70 nm. AFM images showed that the newly formed collagen fibrils were 30-300 nm in diameter and possessed characteristic grooves and ridges with periodicities of 60-70 nm. The thicker collagen fibrils contained thinner (approximately 30 nm thick) subfibrils that ran in a helical direction along the long axis of the thicker fibrils. Furthermore, twisted structures of collagen fibrils, which possessed a characteristic rope-like structure, were also identified. The ultrastructure of the collagen fibrils was clearly imaged in liquid medium by AFM, and the process of collagen fibril assembly was successfully analyzed under conditions much closer to the physiological state than those afforded by transmission electron microscopy or SEM. AFM also provided a precise morphological measurement, particularly of the vertical distance, of collagen fibrils with nanometer-scale resolution in liquid conditions.

Why do aortas cleave or dilate? Clues from an electronic scanning microscopy study in human ascending aortas.

In ascending aorta aneurysms (AscAA) the whole vessel wall dilates, while in aortic dissections (AD) the wall cleaves into two sheets. Both may present fine elastic fragmentation and a decrease in collagen. We analyzed whether alterations in the three-dimensional structure of these fibers could be involved in the pathogenesis of AscAA/AD. Specimens obtained at surgery for these diseases (n = 4 for each) and on coronary artery bypass surgery (controls, n = 4) were submitted to treatments which either preserve collagen or the elastic structure. These samples were examined by scanning electron microscopy. In all groups most of the collagen fibers were packed, forming laminar structures very similar to the elastic lamellae. In AscAA/AD, the fibers showed signs of degradation and/or fragmentation. Elastic tissue was distributed in large sheets with fenestrations, with smaller branches between them. In 1 of the dissection cases and 2 of the aneurysm cases elastic sheet fragmentation, which under light microscopy seems to be located at random, had a pattern of clefts which were irregular but approximately transversal to the main axis of the wall. The recognition of this pattern and the degradation/fragmentation of collagen and elastic fibrils facilitates understanding of why the wall is weak and affected by aneurysms and dissections.