Quantitative Image Analysis of Fibrillar Collagens Reveals Novel Diagnostic and Prognostic Biomarkers and Histotype-Dependent Aberrant Mechanobiology in Lung Cancer.

Fibrillar collagens are the most abundant extracellular matrix components in non-small cell lung cancer (NSCLC). However, the potential of collagen fiber descriptors as a source of clinically relevant biomarkers in NSCLC is largely unknown. Similarly, our understanding of the aberrant collagen organization and associated tumor-promoting effects is very scarce. To address these limitations, we identified a digital pathology approach that can be easily implemented in pathology units based on CT-FIRE software (version 2; https://loci.wisc.edu/software/ctfire) analysis of Picrosirius red (PSR) stains of fibrillar collagens imaged with polarized light (PL). CT-FIRE settings were pre-optimized to assess a panel of collagen fiber descriptors in PSR-PL images of tissue microarrays from surgical NSCLC patients (106 adenocarcinomas [ADC] and 89 squamous cell carcinomas [SCC]). Using this approach, we identified straightness as the single high-accuracy diagnostic collagen fiber descriptor (average area under the curve = 0.92) and fiber density as the single descriptor consistently associated with a poor prognosis in both ADC and SCC independently of the gold standard based on the TNM staging (hazard ratio, 2.69; 95% CI, 1.55-4.66; P < .001). Moreover, we found that collagen fibers were markedly straighter, longer, and more aligned in tumor samples compared to paired samples from uninvolved pulmonary tissue, particularly in ADC, which is indicative of increased tumor stiffening. Consistently, we observed an increase in a panel of stiffness-associated processes in the high collagen fiber density patient group selectively in ADC, including venous/lymphatic invasion, fibroblast activation (α-smooth muscle actin), and immune evasion (programmed death-ligand 1). Similarly, a transcriptional correlation analysis supported the potential involvement of the major YAP/TAZ pathway in ADC. Our results provide a proof-of-principle to use CT-FIRE analysis of PSR-PL images to assess new collagen fiber-based diagnostic and prognostic biomarkers in pathology units, which may improve the clinical management of patients with surgical NSCLC. Our findings also unveil an aberrant stiff microenvironment in lung ADC that may foster immune evasion and dissemination, encouraging future work to identify therapeutic opportunities.

Biomechanical Properties of the Periaortic Abdominal Tissue: It is Not as Fragile as It Seems.

BACKGROUND: The perivascular adipose tissue has been studied as a critical element that could influence physiological and disease processes of the vessel covered by it. In terms of anatomy, during the abdominal aorta's dissection, it is possible to identify the periaortic adipose tissue and the periaortic parietal peritoneum lying over it, sealing the retroperitoneal space. They seem to be fragile layers, with apparently no biomechanical role in the abdomen. However, it is well known that most cases of ruptured abdominal aortic aneurysms (AAAs) that reach the emergency department still alive present retroperitoneal bleeding contained by the previously mentioned two-layer combination, eventually allowing time for surgical treatment. In previous studies about aortic wall stress, tension, and AAA rupture prediction, only information concerning the vessel wall itself is highlighted. Therefore, the present work aims to study the biomechanical and histological properties of the periaortic tissue, comparing them to the same variables measured in aortic wall samples described in the medical literature. MATERIALS AND METHODS: Samples of periaortic tissue were harvested from 27 individuals during necropsy. Smoking status and the presence of AAAs were observed. Biomechanical uniaxial destructive tests were performed up to samples' rupture. Values of failure stress, tension, and strain were obtained. Samples were also harvested for histological analysis. RESULTS: Periaortic tissue presented less amount of collagen in smokers than in nonsmokers (P = 0.017). The periaortic tissue seems to be more elastic than aortic walls described in the literature (strain: 0.75 ± 0.37). Analyzing periaortic tissue failure stress (56.8 ± 101.26 N/cm2) and tension (7.65 ± 4.99 N/cm), it has at least 52% and 55%, respectively, of the stress and tension described in the medical literature for AAA walls. CONCLUSIONS: The periaortic tissue presents less collagen fibers in smokers than in nonsmokers. The periaortic tissue seemed very delicate during an autopsy, but the study of its biomechanical properties showed that it presents more than half of the resistance of an AAA wall. This information suggests this tissue might have a mechanical protective role against massive bleeding when it comes to an aortic rupture. Therefore this tissue's biomechanical information should be included in computational models on enlargement and rupture prediction of AAAs.

Pleuroparenchymal fibroelastosis as a histological background of autoimmune diseases.

Patients with autoimmune disease-related interstitial lung disease (AID-ILD) occasionally develop radiologic pleuroparenchymal fibroelastosis (PPFE)-like lesions. However, the significance of AID as an etiology of PPFE has not been fully elucidated. The aim of this study is to verify the increase of elastic fibers in AID-ILD patients and evaluate the prevalence of histological PPFE in patients with AID-ILD. We selected cases of clinically diagnosed AID-ILD and idiopathic pulmonary fibrosis (IPF), in which an autopsy had been performed or in which the patient had undergone pneumonectomy for lung transplantation. We quantified the collagen fibers and elastic fibers in each lobe as the percentage of the non-aerated lung area (collagen fiber score and elastic fiber score, respectively) in histological specimens from a total of 73 patients (AID-ILD, n = 24; IPF, n = 49). There were no significant differences in the collagen fiber scores of the AID-ILD and IPF groups. Meanwhile, the elastic fiber scores of the AID-ILD group were significantly greater than those of the IPF group in the whole lung (17.3 ± 7.70 vs 11.6 ± 4.55), and the upper (16.6 ± 8.11 vs 11.2 ± 5.18), and lower (18.0 ± 9.68 vs 12.0 ± 5.55) lobes (all p < 0.01). Histological PPFE pattern was found in 12 of 24 AID-ILD patients (50%), and histological PPFE pattern as a dominant pattern of fibrosis was found in 2 of the 24 patients (8%). Thus, PPFE can be a manifestation of AID-ILD.

A targeted mass spectrometry method to screen collagen types I-V in the decellularized 3D extracellular matrix of the adult male rat thyroid.

Here we have developed and validated an original LC-MS/MS SRM procedure flexible enough to quantitatively screen collagen types I-V in copies of the same type of stromal matrix prepared with different protocols of cell removal to retain the native 3D architecture of the ECM. In a first step, identification of tryptic sequences exclusive to specific chains (either α1 or α2) of mammalian collagen standards types I-V was pursued using a combination of LC-LIT-Orbitrap XL and LC-MS/MS SRM analyses. In a second step, the adult male rat thyroid was decellularized using three different protocols specifically set for engineering of bioartificial 3D thyroid organoids. In a third step, DNA analysis of the decellularized 3D thyroid stroma was pursued to exclude contamination by cell nuclear debris. In a final step, collagen standards and 3D thyroid matrices were digested using the same mechanical / enzymatic protocol, and quantitative profiles of collagen types I-V ensued using comparisons of ionic intensities between tryptic peptides of collagen standards and matrices, as derived from targeted LC-MS/MS SRM analysis. Collectively, the procedure allowed for detection and quantitation of collagen types I-V at a femtomolar level in thyroid gland stromal matrices initially maintaining their original 3D architecture, tryptically digested through a method common to collagen standards and thyroid ECM, with satisfactory reproducibility of results, moderate procedural cost, and limited analytical time.

Development of Tissue-Engineered Ligaments: Elastin Promotes Regeneration of the Rabbit Medial Collateral Ligament.

When ligaments are injured, reconstructive surgery is sometimes required to restore function. Methods of reconstructive surgery include transplantation of an artificial ligament and autotransplantation of a tendon. However, these methods have limitations related to the strength of the bone-ligament insertion and biocompatibility of the transplanted tissue after surgery. Therefore, it is necessary to develop new reconstruction methods and pursue the development of artificial ligaments. Elastin is a major component of elastic fibers and ligaments. However, the role of elastin in ligament regeneration has not been described. Here, we developed a rabbit model of a medial collateral ligament (MCL) rupture and treated animal knees with exogenous elastin [100 µg/(0.5 mL·week)] for 6 or 12 weeks. Elastin treatment increased gene expression and protein content of collagen and elastin (gene expression, 6-fold and 42-fold, respectively; protein content, 1.6-fold and 1.9-fold, respectively), and also increased the elastic modulus of MCL increased with elastin treatment (2-fold) compared with the controls. Our data suggest that elastin is involved in the regeneration of damaged ligaments.

Histochemical analysis of collagen reorganization at the invasive front of oral squamous cell carcinoma tumors.

AIM: The aim of the present study was to: (a) analyze the nature of collagen with respect to cohesive and discohesive invasive tumor front (ITF) of oral squamous cell carcinoma (OSCC); (b) analyze the clinicopathological correlation with the nature of collagen at the ITF of OSCC; and (c) correlate the nature of collagen with Broder's and Bryne's histological grading system of OSCC. METHODS: Tissue sections of 29 OSCC with ITF were stained with hematoxylin-eosin and picrosirius red staining for evaluation under a polarized microscope. RESULTS: Tumors with a cohesive front had a thick collagen fiber, are predominantly organized, red-yellow in color, well packed, and show strong birefringence (P<.005). A gradual change in the nature of the collagen fiber was observed in the discohesive tumor front, where the collagen fibers were thin, disorganized, yellow-orange to green-yellow in color, loosely packed, and with weak birefringence (P<.005). CONCLUSION: A cohesive tumor front with organized collagen fibers prevents tumor invasion and metastasis. As it is inhibits an increase in tumor size, it is associated with the initial stage of tumor (I & II), whereas in a discohesive tumor front, the fibers might enhance the movement of tumor cells, resulting in invasion and metastasis.

Fluorescence of Picrosirius Red Multiplexed With Immunohistochemistry for the Quantitative Assessment of Collagen in Tissue Sections.

The low cost and simplicity of picrosirius red (PSR) staining have driven its popularity for collagen detection in tissue sections. We extended the versatility of this method by using fluorescent imaging to detect the PSR signal and applying automated quantification tools. We also developed the first PSR protocol that is fully compatible with multiplex immunostaining, making it possible to test whether collagen structure differs across immunohistochemically labeled regions of the tissue landscape. We compared our imaging method with two gold standards in collagen imaging, linear polarized light microscopy and second harmonic generation imaging, and found that it is at least as sensitive and robust to changes in sample orientation. As proof of principle, we used a genetic approach to overexpress beta catenin in a patchy subset of mouse prostate epithelial cells distinguished only by immunolabeling. We showed that collagen fiber length is significantly greater near beta catenin overexpressing cells than near control cells. Our fluorescent PSR imaging method is sensitive, reproducible, and offers a new way to guide region of interest selection for quantifying collagen in tissue sections.

Fluoxetine effects on periodontogenesis: histomorphometrical and immunohistochemical analyses in rats.

OBJECTIVE: This study aimed to assess the effects of fluoxetine, a selective serotonin reuptake inhibitor, on the formation of the periodontal ligament during pregnancy and lactation in rat pups. MATERIAL AND METHODS: Twelve pregnant rats of Wistar lineage were divided into four study groups. In the control group, 0.9% sodium chloride solution was administered orally, throughout the entire period of the 21 days of pregnancy (CG group) and in the CGL group, it was administrated during pregnancy and lactation (from day 1 of pregnancy to the 21st day after birth). Fluoxetine was administered orally at the dose of 20 mg/kg in a group treated during pregnancy only (FG group), and during pregnancy and lactation (FGL group). Histometrical, histochemical and immunohistochemical analysis of the maxillary first molar periodontium region of the 24 rat pups was made under light microscopy, and periodontal ligament collagen was qualitatively evaluated under a polarizing light microscope. RESULTS: The quantity of fibroblasts (p=0.006), osteoblasts (p=0.027) and cementoblasts (p=0.001) was reduced in pups from the rats that received fluoxetine during pregnancy and lactation. No alterations were seen in the collagen fibers. CONCLUSION: These findings suggest that periodontal tissue may be sensitive to fluoxetine, and its interference in reducing periodontal cells depends on exposure time during lactation.

Fluoxetine effects on periodontogenesis: histomorphometrical and immunohistochemical analyses in rats

Abstract Reports have indicated that serotonin plays an important role in cell migration and differentiation during the organogenesis of several tissues, including the oral types. Administration of selective serotonin reuptake inhibitor (SSRI) drugs during pregnancy could affect the delivery of serotonin to embryonic tissues altering its development. Objective This study aimed to assess the effects of fluoxetine, a selective serotonin reuptake inhibitor, on the formation of the periodontal ligament during pregnancy and lactation in rat pups. Material and Methods Twelve pregnant rats of Wistar lineage were divided into four study groups. In the control group, 0.9% sodium chloride solution was administered orally, throughout the entire period of the 21 days of pregnancy (CG group) and in the CGL group, it was administrated during pregnancy and lactation (from day 1 of pregnancy to the 21st day after birth). Fluoxetine was administered orally at the dose of 20 mg/kg in a group treated during pregnancy only (FG group), and during pregnancy and lactation (FGL group). Histometrical, histochemical and immunohistochemical analysis of the maxillary first molar periodontium region of the 24 rat pups was made under light microscopy, and periodontal ligament collagen was qualitatively evaluated under a polarizing light microscope. Results The quantity of fibroblasts (p=0.006), osteoblasts (p=0.027) and cementoblasts (p=0.001) was reduced in pups from the rats that received fluoxetine during pregnancy and lactation. No alterations were seen in the collagen fibers. Conclusion These findings suggest that periodontal tissue may be sensitive to fluoxetine, and its interference in reducing periodontal cells depends on exposure time during lactation.

Label-free detection of fibrillar collagen deposition associated with vascular elements in glioblastoma multiforme by using multiphoton microscopy.

Glioblastoma multiforme (GBM-WHO grade IV) is the most common and the most aggressive form of brain tumors in adults with the median survival of 10-12 months. The diagnostic detection of extracellular matrix (ECM) component in the tumour microenvironment is of prognostic value. In this paper, the fibrillar collagen deposition associated with vascular elements in GBM were investigated in the fresh specimens and unstained histological slices by using multiphoton microscopy (MPM) based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG). Our study revealed the existence of fibrillar collagen deposition in the adventitia of remodelled large blood vessels and in glomeruloid vascular structures in GBM. The degree of fibrillar collagen deposition can be quantitatively evaluated by measuring the adventitial thickness of blood vessels or calculating the ratio of SHG pixel to the whole pixel of glomeruloid vascular structure in MPM images. These results indicated that MPM can not only be employed to perform a retrospective study in unstained histological slices but also has the potential to apply for in vivo brain imaging to understand correlations between malignancy of gliomas and fibrillar collagen deposition.

Morphological analysis of collagen and elastic fibers in oral squamous cell carcinoma using special stains and comparison with Broder's and Bryne's grading systems.

OBJECTIVES: Morphologic detection of connective tissue fiber changes in oral squamous cell carcinoma (OSCC) using special stains remains less documented. The aims of the present study were to study the collagen and elastic fibers in different stages of OSCC and to correlate these changes with two grading systems - Broder's and Bryne's. STUDY DESIGN: Forty-eight cases of OSCC were studied using hematoxylin and eosin, Verhoeff's - Van Gieson stain for elastic fibers and picrosirius red stain for collagen fibers. The changes were compared with all the grades of carcinoma. Normal mucosa was taken as control. RESULTS: Statistical analysis using Chi-square and ANOVA, showed significant association between the grades of carcinoma and extracellular matrix changes. Greenish-yellow collagen fibers were found to be significantly increased in the poorly differentiated/Grade 3 cases (P< 0.0001) where as well-differentiated/Grade 1 cases showed predominantly reddish-orange and yellowish-orange birefringence of collagen fibers. Chi-square analysis showed a significant amount of fragmented pattern of elastic fibers in poorly differentiated OSCC (χ2 = 104.45, P = 0.009)/Grade 3 OSCC (χ2 = 94.81, P = 0.016). CONCLUSION: The study of the connective tissue stromal changes can be used as an adjunct to histological grading and thereby helping the surgeon to determine the amount of marginal clearance.

Comparison of Picrosirius Red Staining With Second Harmonic Generation Imaging for the Quantification of Clinically Relevant Collagen Fiber Features in Histopathology Samples.

Stromal collagen alignment has been shown to have clinical significance in a variety of cancers and in other diseases accompanied by fibrosis. While much of the biological and clinical importance of collagen changes has been demonstrated using second harmonic generation (SHG) imaging in experimental settings, implementation into routine clinical pathology practice is currently prohibitive. To translate the assessment of collagen organization into routine pathology workflow, a surrogate visualization method needs to be examined. The objective of the present study was to quantitatively compare collagen metrics generated from SHG microscopy and commonly available picrosirius red stain with standard polarization microscopy (PSR-POL). Each technique was quantitatively compared with established image segmentation and fiber tracking algorithms using human pancreatic cancer as a model, which is characterized by a pronounced stroma with reorganized collagen fibers. Importantly, PSR-POL produced similar quantitative trends for most collagen metrics in benign and cancerous tissues as measured by SHG. We found it notable that PSR-POL detects higher fiber counts, alignment, length, straightness, and width compared with SHG imaging but still correlates well with SHG results. PSR-POL may provide sufficient and additional information in a conventional clinical pathology laboratory for certain types of collagen quantification.

Polarized Microscopy in Lesions With Altered Dermal Collagen.

Alterations in dermal collagen are noted in dermatofibroma, dermatofibrosarcoma protuberans, morphea, lichen sclerosus et atrophicus, hypertrophic scars, and keloids. The authors sought to determine whether variations in birefringence of collagen by polarized microscopy could be of help in diagnosing such conditions. Representative hematoxylin and eosin sections of 400 cases, including dermatofibroma, dermatofibrosarcoma protuberans, hypertrophic scars, keloid, morphea, and lichen sclerosus, were examined under polarized microscopy. Distinct patterns of birefringence of collagen for each disease were noted under polarized microscopy. This study highlights the use of polarized microscopy as adjunctive tool in differentiating different diseases with collagen alteration.

Polarimetric Interferometry for Assessment of Corneal Stromal Lamellae Orientation.

PURPOSE: To analyze corneal stromal lamellae orientation in normal subjects and patients with various corneal conditions using a polarimetric interferometer. METHODS: Thirty-two healthy control subjects (59 eyes) and 16 patients (22 eyes) with corneal conditions or postkeratoplasty were included in the study. All patients were imaged using the interferometer and slit lamp photography. The interferometer produces an orthogonal cross-like image of stromal lamellae by illuminating the cornea in phase-light polarization angle. Corneal haze was graded on a scale from 0 to 4. Interferometer cross-like images were graded on a scale from 0 to 5 by a masked observer. Keratometry, corneal central pachymetry, and optical densitometry were obtained with Scheimpflug corneal tomography. RESULTS: The cross-like image was observed in 31 of 32 healthy control subjects (58 of 59 eyes) and in 13 of 16 patients (19 of 22 eyes). The image was not detectable in 3 patients, 1 with total corneal neovascularization and scarring, 1 with central leukoma, and 1 with failed deep lamellar keratoplasty. Corneal haze was the main factor obscuring the cross-like image (P < 0.05). Clarity of the images was influenced by older age, steeper keratometry, higher pachymetry, and optical density (P < 0.05). There was no correlation between the orientation of the keratometric axes and the orientation of the 2 arms of the cross-like image (P > 0.05). CONCLUSIONS: Corneal stromal lamellae orientation can be efficiently assessed and displayed as a cross-like image by the polarimetric interferometer. Stromal lamellae orientation imaging may have potential implications in corneal diagnostics and surgery.

Histologic, histochemical, and biomechanical properties of fragments isolated from the anterior wall of abdominal aortic aneurysms.

OBJECTIVE: To analyze biomechanical, histologic, and histochemical properties of anterior fragments of abdominal aortic aneurysms (AAA) and to correlate them with the maximum transverse diameter (MTD) and symptoms associated to the aneurysms. METHODS: Fragments of the anterior aneurysm wall were obtained from 90 patients submitted to open repair of AAA of degenerative etiology from 2004 to 2009 in the Clinics Hospital of São Paulo University Medical School. Two specimens were produced from the fragments: one for histologic analysis for quantification of collagen fibers, elastic fibers, smooth muscle cells, and degree of inflammatory activity and the other for uniaxial tensile test to assess biomechanical failure properties of the material, such as strength, tension, and stress. Cases were classified according to symptoms and to the AAA MTD. RESULTS: Fragments from AAA with MTD < 5.5 cm showed higher values for biomechanical failure properties than those of AAA with MTD < 5.5 cm (strength, 5.32 ± 2.07 × 4.1 ± 2.41 N; tension, 13.83 ± 5.58 × 10.82 ± 6.48 N/cm; stress, 103.02 × 77.03 N/cm(2); P < .05). No differences were observed between the groups in relation to failure strain (0.41 ± 0.12 × 0.37 ± 0.14; P = .260) and thickness of the fragments (1.58 ± 0.41 × 1.53 ± 0.42 mm; P = .662). The average values of fiber compositions of all the fragments were as follows: collagen fibers, 44.34 ± 0.48% and 61.85 ± 10.14% (Masson trichrome staining and Picrosirius red staining, respectively); smooth muscle cells, 3.46 ± 2.23% (immunohistochemistry/alpha-actin); and elastic fibers, less than 1% (traces) (Verhoeff-van Gieson staining). No differences in fiber percentages (collagen, elastic, and smooth muscle) were observed in fragments from AAA with MTD <5.5 cm and <5.5 cm, but more intense inflammatory activity was seen in larger AAA (grade 3; 70% × 28.6%; P = .011). Compared with asymptomatic aneurysms, symptomatic aneurysms showed no differences in the biomechanical failure properties (strength, 5.32 ± 2.36 × 4.65 ± 2.05 N; P = .155; tension, 14.08 ± 6.11 × 12.81 ± 5.77 N/cm; P = .154; stress, 103.02 × 84.76 N/cm(2); P = .144), strain (0.38 ± 0.12 × 0.41 ± 0.13; P = .287), thickness of the fragments (1.56 ± 0.41 × 1.57 ± 0.41 mm; P = .848), and histologic composition (collagen fibers, 44.67 ± 11.17 × 44.02 ± 13.79%; P = .808; smooth muscle fibers, 2.52 × 2.35%; P = .751; elastic fibers, <1%) CONCLUSIONS: Fragments of the anterior wall from larger aneurysms were more resistant than those from smaller AAA, with no tissue properties that could explain this phenomenon in the histologic or histochemical analyses utilized. CLINICAL RELEVANCE: The fragments of the anterior midsection from larger aneurysms were more resistant than those from smaller abdominal aortic aneurysms, with no tissue properties that could explain this phenomenon in the histologic or histochemical analyses. Larger aneurysms, at least in this place may be stronger than smaller aneurysms. It could point toward regional differences (heterogeneity, localized pathologies) as an important player in aneurysm rupture. Uniaxial strain tests are an important tool for the comprehension of a complex behavior such as that from an aneurysmal aortic wall. However, these tests still have limitations in providing information that would allow the calculation of the risk of rupture for abdominal aortic aneurysms.

Multiphoton tomography visualizes collagen fibers in the tumor microenvironment that maintain cancer-cell anchorage and shape.

Second harmonic generation (SHG) multiphoton imaging can visualize fibrillar collagen in tissues. SHG has previously shown that fibrillar collagen is altered in various types of cancer. In the present study, in vivo high resolution SHG multi-photon tomography in living mice was used to study the relationship between cancer cells and intratumor collagen fibrils. Using green fluorescent protein (GFP) to visualize cancer cells and SHG to image collagen, we demonstrated that collagen fibrils provide a scaffold for cancer cells to align themselves and acquire optimal shape. These results suggest a new paradigm for a stromal element of tumors: their role in maintaining anchorage and shape of cancer cells that may enable them to proliferate.

Effectiveness of correction of bleomycin-induced lung injury by early and late administration of surfactant-BL.

Rats were exposed to inhalation of surfactant-BL starting from the first or eighth day after intratracheal administration of bleomycin. At the early stages, the preparation effectively attenuated damage to ultrastructural components of the lung tissue and reduced the severity and extent of subsequent pulmonary pathology.

In-vivo and in-vitro histological evaluation of two commercially available acellular dermal matrices.

PURPOSE: Post-herniation abdominal wall repair can be performed with synthetic or biologic meshes. Synthetics have been associated with complications, so biologics are promising alternatives. The methods used to decellularize biological matrices may affect the extracellular components. This study evaluated the post-implantation biological response of two allogenic acellular dermal matrices (ADMs) in a hernia model. METHODS: Testing was conducted with two ADMs from different manufacturers: RTI Biologics (ADM-R) and LifeCell (ADM-L). Samples were evaluated for collagen IV, glycosaminoglycans (GAGs), and elastin before implantation. Samples were also used to repair bilateral full-thickness defects in rat abdominal walls. Pathologist evaluations included explant dimensions, inflammation, neovascularization, mature implant tissue, fibrosis, encapsulation, necrosis, mineralization, adhesions, granulomas, and hemorrhages at four and eight weeks post-implantation. RESULTS: GAG distribution in ADM-R samples was more consistent with native dermis than that in ADM-L samples. Collagen IV was visible in ADM-R, but not in ADM-L. The four-week ADM-R explants showed primarily lymphocytic infiltrates, and less inflammation at eight weeks. The four-week ADM-L explants showed primarily lymphocytic infiltrates, and sustained inflammation at eight weeks. Fibroplasia at four and eight weeks was higher in ADM-L than in ADM-R. Encapsulation, mature connective tissue, and vascular profile scores were comparable between groups. Picrosirius red image analysis showed no significant differences between groups. CONCLUSIONS: The post-processing matrix characterization and in-vivo response showed notable differences in these ADMs, despite similar allogenic origin. Future investigations into the different matrix composition with regard to fibrosis and inflammation are warranted.

Análise da composição do matriz extracelular das camadas urotelial e muscular da bexiga de mulheres em diferentes idades / Analysis of the extracellular matriz of urothelial and muscle layers of the bladder in women at different ages

Acomplacência da bexiga depende de músculos lisos, fibras colágenas, fibras elásticas e suas relações. O objetivo deste trabalho é determinar a composição da matriz extracelular em amostras de bexigas normais através de análise bioquímica de colágeno e glicosaminoglicanos em amostras obtidas de mulheres em diferentes grupos de idade, analisando separadamente as camadas urotelial e muscular. Avaliamos 17 amostras de bexiga divididas em três grupos: infância (N=5), menacme (N=6) e pós-menopausa (N=6). As bexigas foram analisadas para concentração de GAG total e colágeno e para análise qualitativa de GAG por eletroforese em gel de agarose. Na camada muscular, não houve diferença entre os grupos tanto para GAG quanto para colágeno. Na camada urotelial, a análise da concentração de colágeno não mostrou diferença entre os grupos, mas a concentração de GAG no grupo da pós-menopausa (0.21 +- 0.12 ug de ácido hexurônico/mg de tecido seco) apresentou diferença em relação aos grupos do menacme (1.78 +- 1.62 ug de ácido hexurônico/mg de tecido seco) e da infância (2.29 +- 1.32 ug de ácido hexurônico/mg de tecido seco). Nosso trabalho concluiu que a concentração de GAG está substancialmente diminuída na cadama urotelial da bexiga de mulheres na pós-menopausa.

Bladder compliance is dependent on smooth muscle, collagen fibers, elastic fiber and their ratios. The luminal surface of the urothelium is covered by an adhering glycosaminoglycan (GAG) layer. The aim of this study was to determine the composition of the extracellular matrix (ECM) in normal samples of women bladders through biochemistry analysis of collagen and GAG on samples obtained from individuals from different age groups, analyzing separately the urothelial and muscular layers. We studied samples taken from bladders of 17 patients divided in three different groups: childhood (N=5), menacme (N=6) and menopause (N=6). Bladders were analyzed for total GAG and collagen concentration per mg dry tissue and for the contents of GAG species, as determined by agarose electrophoresis and reported as the percent of total sulfated GAG. In muscular layer, collagen and GAG concentration showed no difference between groups. In urothelial layer, collagen concentration showed no difference between groups but GAG concentration in menopause (0.21 +- 0.12 ug hexuronic acid/mg dry tissue) was different from menacme (1.78 +- 1.62 ug hexuronic acid/mg dry tissue) and childhood (2.29 +- 1.32 ug hexuronic acid/mg dry tissue). There was no difference between sulfated GAG in three groups. In conclusion, GAG concentration in urothelial layer was substantially lower in menopause women.