Deep-learning based classification of a tumor marker for prognosis on Hodgkin's disease.

PURPOSE: Hodgkin's disease is a common malignant disorder in adolescent patients. Although most patients are cured, approximately 10%-15% of patients experience a relapse or have resistant disease. Furthermore, there are no definitive molecular predictors for early identification of patients at high risk of treatment failure to first line therapy. The aim of this study was to evaluate the deep learning-based classifier model of medical image classification to predict clinical outcome that may help in appropriate therapeutic decisions. METHODS: Eighty-three FFPE biopsy specimens from patients with Hodgkin's disease were stratified according to the patient's qPET scores, stained with picrosirius red dye and digitalized by whole slide image scanning. The resulting whole slide images were cut into tiles and annotated by two classes based on the collagen fibers' degree of coloring with picrosirius red. The neural network (YOLOv4) was then trained with the annotated data. Training was performed with 30 cases. Prognostic power of the weakly stained picrosirius red fibers was evaluated with 53 cases. The same neural network was trained with MMP9 stained tissue slides from the same cases and the quantification results were compared with the variant from the picrosirius red cases. RESULTS: There was a weak monotonically increasing relationship by parametric ANOVA between the qPET groups and the percentages of weakly stained fibers (p = .0185). The qPET-positive cases showed an average of 18% of weakly stained fibers, and the qPET-negative cases 10%-14%. Detection performance showed an AUC of 0.79. CONCLUSIONS: Picrosirius red shows distinct associations as a prognostic metric candidate of disease progression in Hodgkin's disease cases using whole slide images but not sufficiently as a prognostic device.

A comparison of myocardial magnetic resonance extracellular volume mapping at 3 T against histology of tissue collagen in severe aortic valve stenosis and obstructive hypertrophic cardiomyopathy.

OBJECTIVE: Quantitative extracellular volume fraction (ECV) mapping with MRI is commonly used to investigate in vivo diffuse myocardial fibrosis. This study aimed to validate ECV measurements against ex vivo histology of myocardial tissue samples from patients with aortic valve stenosis or hypertrophic cardiomyopathy. MATERIALS AND METHODS: Sixteen patients underwent MRI examination at 3 T to acquire native T1 maps and post-contrast T1 maps after gadobutrol administration, from which hematocrit-corrected ECV maps were estimated. Intra-operatively obtained myocardial tissue samples from the same patients were stained with picrosirius red for quantitative histology of myocardial interstitial fibrosis. Correlations between in vivo ECV and ex vivo myocardial collagen content were evaluated with regression analyses. RESULTS: Septal ECV was 30.3% ± 4.6% and correlated strongly (n = 16, r = 0.70; p = 0.003) with myocardial collagen content. Myocardial native T1 values (1206 ± 36 ms) did not correlate with septal ECV (r = 0.41; p = 0.111) or with myocardial collagen content (r = 0.32; p = 0.227). DISCUSSION: We compared myocardial ECV mapping at 3 T against ex vivo histology of myocardial collagen content, adding evidence to the notion that ECV mapping is a surrogate marker for in vivo diffuse myocardial fibrosis.

Dynamic dentin: A quantitative microscopic assessment of age and spatial changes to matrix architecture, peritubular dentin, and collagens types I and III.

To investigate age and site-related changes to human dentin collagen, sound human teeth collected from donors aged 13-29 (young) and 50-74 (aged) years (n = 9/group) were cut to shallow and deep sites. Dentin collagen orientation and fibril bundling was investigated using the Picrosirius Red (PSR) stain observed under cross-polarized light microscopy (Pol), and collagen distribution was investigated using Confocal Laser Scanning Microscopy (CLSM). Collagen types III to I distribution in peritubular dentin (PTD) was revealed using Herovici stain and brightfield microscopy. Image analysis software and linear mixed modelling quantified outcomes. In situ dentin collagen was observed using Xenon Plasma Focussed Ion Beam Scanning Electron Microscopy (Xe PFIB-SEM). The PSR-Pol analysis revealed less coherently aligned and more bundled collagen fibrils in aged dentin (P = 0.005). Deep inner dentin collagen in both groups were less coherently aligned with reduced bundling. Regardless of age, CLSM showed collagen distribution remained stable; and more collagen type III was detectable in PTD located in inner dentin (Young: P = 0.006; Aged: P = 0.008). Observations following Xe PFIB-SEM cross-sectioning showed apatite-like deposits surrounding large intratubular collagen fibers, and evidence of smaller intertubular dentin collagen fibrils in situ. In conclusion, aging changes collagen network architecture, but not distribution or content.

Matrisome changes in Parkinson's disease.

Extracellular matrix (ECM) proteins, collectively known as the matrisome, include collagens, glycoproteins, and proteoglycans. Alterations in the matrisome have been implicated in the neurodegenerative pathologies including Parkinson's disease (PD). In this work, we utilized our previously published PD and control proteomics data from human prefrontal cortex and focused our analysis on the matrisome. Among matrisome proteins, we observed a significant enrichment in the expression of type I collagen in PD vs. control samples. We then performed histological analysis on the same samples used for proteomics study, and examined collagen expression using picrosirius red staining. Interestingly, we observed similar trends in collagen abundance in PD vs. control as in our matrisome analysis; thus, this and other histological analyses will be useful as a complementary technique in the future to study the matrisome in PD with a larger cohort, and it may aid in choosing regions of interest for proteomic analysis. Additionally, collagen hydroxyprolination was less variable in PD compared to controls. Glycoproteomic changes in matrisome molecules were also observed in PD relative to aged individuals, especially related to type VI collagen and versican. We further examined the list of differentially expressed matrisome molecules using network topology-based analysis and found that angiogenesis indicated by alterations in decorin and several members of the collagen family was affected in PD. These findings collectively identified matrisome changes associated with PD; further studies with a larger cohort are required to validate the current results.

A Histopathology-based Assessment of Biological Behavior in Oral Hyalinizing Odontogenic Tumors and Bone Lesions by Differential Stains.

AIM: Odontogenic tumors (OTs) and bone lesions of the oral cavity present diverse histological features and varying clinical behavior that makes predicting their biologic behavior difficult. The research undertaken in the current study aims to predict the biological behavior of oral hyalinizing odontogenic and bone lesions (OHO-BL) for the first time by employing four differential stains with clinicopathologic correlation. MATERIALS AND METHODS: The study was performed on retrospectively diagnosed formalin-fixed paraffin-embedded cases of OTs (n = 53) and bone lesions (n = 10). The severity of hyalinization (SOH) was assessed from stained tissue sections. Polarizing microscopy was used to analyze hyalinization in tissues stained with differential special stains, namely periodic acid-Schiff (PAS), Safranin-O, Alcian Blue, and Picrosirius red. SOH was also analyzed for possible correlation with recurrence and clinicopathologic correlation in OHO-BL. RESULTS: Intense staining was observed with PAS, Alcian Blue, and Safranin-O in OTs with increased SOH with a statistical significance. Polarizing greenish yellow color correlated significantly with the recurrence potential of the OT group. Recurrence in individual lesions of the OT group showed a statistically significant association with SOH. Such individual correlation was not observed in bone diseases. CONCLUSION: PAS, Alcian Blue, Safranin-O, and Picrosirius red are reliable stains to assess hyalinization in OHO-BL. Picrosirius red-polarizing microscopy is a dependable tool for identifying recurrent odontogenic lesions. CLINICAL SIGNIFICANCE: SOH can be considered a histological predictor of aggressive biologic behavior in oral hyalinizing odontogenic lesions that can enable the surgeon to arrive at an appropriate management protocol.

A Histopathology-based Assessment of Biological Behavior in Oral Hyalinizing Extraosseous Lesions by Differential Stains.

AIM: The assessment of hyalinization to determine aggressive behavior in oral pathological lesions is a scarcely researched field that requires further exploration. The current study aims to predict the biological behavior of oral hyalinizing extraosseous lesions (OHEOL) by employing four differential stains with clinicopathologic correlation. MATERIALS AND METHODS: The study was performed on retrospectively diagnosed formalin-fixed paraffin-embedded cases of salivary gland tumors (SGTs) (n = 13), benign soft tissue (BST) lesions (n = 24), and oral submucous fibrosis (OSMF) (n = 53). The hematoxylin and eosin-stained sections were analyzed for the severity of hyalinization (SOH). Differential stains periodic acid Schiff (PAS), Alcian blue, safranin O, and picrosirius red with polarizing microscopy were used to assess the components of hyalinized tissue. The SOH was correlated with differential staining characteristics and clinicopathologic features to analyze possible correlation with aggressive potential in BST, advancement of disease in OSMF, and recurrence in SGT. RESULTS: Intensity of picrosirius red stain significantly correlated with SOH of SGTs (p = 0.044). The intensity of PAS stain (p = 0.040), picrosirius red polarizing greenish-yellow color (p = 0.002), and pattern of distribution of picrosirius red (p = 0.023) significantly correlated with recurrence of SGTs. The intensity of differential stains increased with the SOH in BST lesions indicating their correlation with SOH. The intensity (p = 0.008) and pattern (p = 0.010) of Alcian blue staining and intensity of safranin O stain (p = 0.003) significantly correlated with SOH in OSMF. Picrosirius red polarizing color reddish and yellowish red (p = 0.002) significantly correlated with SOH distinguishing early and advanced OSMF. CONCLUSION: Picrosirius red and PAS stains are reliable indicators of SOH and recurrence potential in SGT. Alcian blue, safranin O, and picrosirius red polarizing colors enable detection of SOH and accurately distinguish early from advanced OSMF. CLINICAL SIGNIFICANCE: SOH can be considered as a histological predictor of aggressive biologic behavior in OHEOL. These findings will result in appropriate management protocols.

Remodeling of collagen constituting interlobular septa of subcutaneous adipose tissue following microwaves application.

In this study, the application of a recently introduced device based on electromagnetic energy transfer by microwaves for fat reduction, permitted to study specifically the modifications of thick fibrous collagen interlobular septa in the subcutaneous adipose tissue, related to the formation of large clusters of adipocytes. The use of Picrosirius red staining associated with circularly polarized microscopy gave evidence of appreciable modifications of the fibrous connective tissue forming septa. Compact fibrotic bundles of collagen I forming interlobular septa appeared reduced or dissolved, in part substituted by the increase of more diffuse and finely reticular collagen III. Remodeling of fibrous collagen, which formed bridles involved in the appearance at the surface of the skin of dimpling/orange peer pattern typical of cellulite, was observed.

Diagnosis of Collagen Type III Glomerulopathy Using Picrosirius Red and PASH/Masson's Trichrome Stain.

Canine collagen type III glomerulopathy (Col3GP) is a rare juvenile nephropathy in which irregular type III collagen fibrils and fibronectin accumulate in glomerular capillary walls and the mesangium. Necropsy findings were reviewed from 5 puppies diagnosed with Col3GP at 6 to 18 weeks of age. Histologically, with hematoxylin and eosin stain, the glomerular capillary walls and mesangium were diffusely and globally expanded by homogeneous pale eosinophilic material. Ultrastructurally, the subendothelial zone and mesangium were expanded by fibronectin and cross-banded collagen type III fibrils, diagnostic of Col3GP. Two additional stains were employed to identify the material within glomeruli as fibrillar collagen using light microscopy. In all 5 cases, the material was red with picrosirius red and birefringent under polarized light, and was blue with periodic acid-Schiff/hematoxylin/trichrome (PASH/TRI), thereby identifying it as fibrillar collagen. Based on these unique staining characteristics with picrosirius red and PASH/TRI, Col3GP may be reliably diagnosed with light microscopy alone.

Bioactive glass-ceramic bone repair associated or not with autogenous bone: a study of organic bone matrix organization in a rabbit critical-sized calvarial model.

OBJECTIVE: The aim of the study was to analyze bone matrix (BMX) organization after bone grafting and repair using a new bioactive glass-ceramic (Biosilicate®) associated or not with particulate autogenous bone graft. MATERIAL AND METHODS: Thirty rabbits underwent surgical bilateral parietal defects and divided into groups according to the materials used: (C) control-blood clot, (BG) particulate autogenous bone, (BS) bioactive glass-ceramic, and BG + BS. After 7, 14, and 30 days post-surgery, a fragment of each specimen was fixed in - 80 °C liquid nitrogen for zymographic evaluation, while the remaining was fixed in 10% formalin for histological birefringence analysis. RESULTS: The results of this study demonstrated that matrix organization in experimental groups was significantly improved compared to C considering collagenous organization. Zymographic analysis revealed pro-MMP-2, pro-MMP-9, and active (a)-MMP-2 in all groups, showing gradual decrease of total gelatinolytic activity during the periods. At day 7, BG presented more prominent gelatinolytic activity for pro-MMP-2 and 9 and a-MMP-2, when compared to the other groups. In addition, at day 7, a 53% activation ratio (active form/[active form + latent form]) was evident in C group, 33% in BS group, and 31% in BG group. CONCLUSION: In general, BS allowed the production of a BMX similar to BG, with organized collagen deposition and MMP-2 and MMP-9 disponibility, permitting satisfactory bone remodeling at the late period. CLINICAL RELEVANCE: The evaluation of new bone substitute, with favorable biological properties, opens the possibility for its use as a viable and efficient alternative to autologous bone graft.

Osteogenesis requires FAK-dependent collagen synthesis by fibroblasts and osteoblasts.

Focal adhesion kinase (FAK) is critical in adhesion-dependent signaling, but its role in osteogenesis in vivo is ill defined. We deleted Fak in fibroblasts and osteoblasts in Floxed-Fak mice bred with those expressing Cre-recombinase driven by 3.6-kb α1(I)-collagen promoter. Compared with wild-type (WT), conditional FAK-knockout (CFKO) mice were shorter (2-fold; P < 0.0001) and had crooked, shorter tails (50%; P < 0.0001). Microcomputed tomography analysis showed reduced bone volume (4-fold in tails; P < 0.0001; 2-fold in mandibles; P < 0.0001), whereas bone surface area/bone volume increased (3-fold in tails; P < 0.0001; 2.5-fold in mandibles; P < 0.001). Collagen density and fiber alignment in periodontal ligament were reduced by 4-fold (P < 0.0001) and 30% (P < 0.05), respectively, in CFKO mice. In cultured CFKO osteoblasts, mineralization at d 7 and mineralizing colony-forming units at d 21 were 30% (P < 0.0001) and >3-fold less than WT, respectively. Disruptions of FAK function in osteoblasts by conditional knockout, siRNA-knockdown, or FAK inhibitor reduced mRNA and protein expression of Runx2 (>30%), Osterix (>25%), and collagen-1 (2-fold). Collagen synthesis was abrogated in WT osteoblasts with Runx2 knockdown and in Fak-null fibroblasts transfected with an FAK kinase domain mutant or a kinase-impaired mutant (Y397F). These data indicate that FAK regulates osteogenesis through transcription factors that regulate collagen synthesis.-Rajshankar, D., Wang, Y., McCulloch, C. A. Osteogenesis requires FAK-dependent collagen synthesis by fibroblasts and osteoblasts.

Epigallocatechin Gallate-Modified Gelatins with Different Compositions Alter the Quality of Regenerated Bones.

Bone quality is a significant indicator of the result of bone treatments. However, information regarding the quality of regenerated bones is limited. The study investigates the effect of different compositions of vacuum heated epigallocatechin gallate-modified gelatins sponge (vhEGCG-GS) on the quality of regenerated bones in critical size defects (9 mm) of rat calvariae. Five different compositions of vhEGCG-GSs containing the same amount of EGCG and different amounts of gelatin were tested. Following four weeks after implantation, the harvested regenerated bones were evaluated by using micro-computed tomography analysis, histological evaluation (hematoxylin-eosin and Villaneueva Goldner staining), picrosirius red-staining with polarized microscopic observation for collagen maturation, and Fourier transform infrared spectroscopy microscopy and imaging analysis for mineral-matrix ratio. The results indicated that increasing content of gelatin in the vhEGCG-GSs promoted bone and osteoid formation but yielded porous bones. Furthermore, tissue mineral density decreased and the maximum mineral-matrix ratio increased. In contrast, vhEGCG-GSs containing smaller amount of gelatin formed mature collagen matrix in the regenerated bones. These results suggest that the alteration of composition of vhEGCG-GSs affected the bone forming capability and quality of regenerated bone and provides valuable insight for the fabrication of new bone substitute materials.

Comparative measurement of collagen bundle orientation by Fourier analysis and semiquantitative evaluation: reliability and agreement in Masson's trichrome, Picrosirius red and confocal microscopy techniques.

Measurement of collagen bundle orientation in histopathological samples is a widely used and useful technique in many research and clinical scenarios. Fourier analysis is the preferred method for performing this measurement, but the most appropriate staining and microscopy technique remains unclear. Some authors advocate the use of Haematoxylin-Eosin (H&E) and confocal microscopy, but there are no studies comparing this technique with other classical collagen stainings. In our study, 46 human skin samples were collected, processed for histological analysis and stained with Masson's trichrome, Picrosirius red and H&E. Five microphotographs of the reticular dermis were taken with a 200× magnification with light microscopy, polarized microscopy and confocal microscopy, respectively. Two independent observers measured collagen bundle orientation with semiautomated Fourier analysis with the Image-Pro Plus 7.0 software and three independent observers performed a semiquantitative evaluation of the same parameter. The average orientation for each case was calculated with the values of the five pictures. We analyzed the interrater reliability, the consistency between Fourier analysis and average semiquantitative evaluation and the consistency between measurements in Masson's trichrome, Picrosirius red and H&E-confocal. Statistical analysis for reliability and agreement was performed with the SPSS 22.0 software and consisted of intraclass correlation coefficient (ICC), Bland-Altman plots and limits of agreement and coefficient of variation. Interrater reliability was almost perfect (ICC > 0.8) with all three histological and microscopy techniques and always superior in Fourier analysis than in average semiquantitative evaluation. Measurements were consistent between Fourier analysis by one observer and average semiquantitative evaluation by three observers, with an almost perfect agreement with Masson's trichrome and Picrosirius red techniques (ICC > 0.8) and a strong agreement with H&E-confocal (0.7 < ICC < 0.8). Comparison of measurements between the three techniques for the same observer showed an almost perfect agreement (ICC > 0.8), better with Fourier analysis than with semiquantitative evaluation (single and average). These results in nonpathological skin samples were also confirmed in a preliminary analysis in eight scleroderma skin samples. Our results show that Masson's trichrome and Picrosirius red are consistent with H&E-confocal for measuring collagen bundle orientation in histological samples and could thus be used indistinctly for this purpose. Fourier analysis is superior to average semiquantitative evaluation and should keep being used as the preferred method.

Comparison of collagen content in skin wounds evaluated by biochemical assay and by computer-aided histomorphometric analysis.

CONTEXT: The quantification of total collagen is of major importance in a wide range of research areas, including the study of cutaneous wound healing and new drugs trials. OBJECTIVE: The total collagen content in skin biopsies was compared by biochemical hydroxyproline assay and by two computer-aided histomorphometric analyses of histological sections. MATERIALS AND METHODS: Two methods were used to evaluate collagen formation: the hydroxyproline assay, as the gold standard and histomorphometric image analysis of the filled areas by corresponding stained collagen fibres, using picrosirius and Gomori's trichrome staining. The image analyses were determined by digital densitometry recognition using computer-aided ImageJ software. One-way ANOVA, simple linear regression and ANCOVA were applied for the statistical analysis and correlation. RESULTS: In a simple linear regression analysis carried out on the 14th day period after the induction of skin injury, three techniques, picrosirius red (F = 33.57, p = 0.00), Gomori's trichrome (F = 81.61, p = 0.00) and hydroxyproline content (F = 16.85, p = 0.00) were able to detect collagen production. After scale adjustment, there were no significant differences among either the slopes (F = 1.17, p = 0.32) or the intercepts (F = 0.69, p = 0.51) of the estimated regression lines. It seems that a highly significant correlation exists between the histomorphometrical analysis and hydroxyproline assay. DISCUSSION AND CONCLUSION: The morphometric analysis proved to be adequate and can be used as a simple, rapid, low-cost technology for evaluating total collagen in cutaneous wound specimens, compared with the gold standard hydroxyproline assay.

Form and function of the intervertebral disc in health and disease: a morphological and stain comparison study.

Multiple histologic measurements are commonly used to assess degenerative changes in intervertebral disc (IVD) structure; however, there is no consensus on which stains offer the clearest visualization of specific areas within the IVD. The objective of this study was to compare multiple tinctorial stains, evaluate their ability to highlight structural features within the IVD, and investigate how they influence the capacity to implement a degeneration scoring system. Lumbar IVDs from seven human autopsy specimens were stained using six commonly used stains (Hematoxylin/Eosin, Toluidine Blue, Safranin-O/Fast Green, Extended FAST, modified Gomori's Trichrome, and Picrosirius Red Alcian Blue). All IVDs were evaluated by three separate graders to independently determine which stains (i) were most effective at discerning different structural features within different regions of the IVDs and (ii) allowed for the most reproducible assessment of degeneration grade, as assessed via the Rutges histological scoring system (Rutges et al. A validated new histological classification for intervertebral disc degeneration. Osteoarthritis Cartilage, 21, 2039-47). Although Trichrome, XFAST and PR/AB stains were all effective at highlighting different regions of whole IVDs, we recommend the use of PR/AB because it had the highest degree of rater agreement on assigned degeneration grade, allowed greater resolution of degeneration grade, has an inferential relationship between color and composition, and allowed clear differentiation of the different regions and structural disruptions within the IVD. The use of a standard set of stains together with a histological grading scheme can aid in the characterization of structural changes in different regions of the IVD and may simplify comparisons across the field. This collection of human IVD histological images highlights how IVD degeneration is not a single disease but a composite of multiple processes such as aging, injury, repair, and disease, each of which are unique to the individual.

Collagen and collagenases in mare's endometrium with endometrosis.

Equine endometrosis is a degenerative and predominantly fibrotic condition resulting from progressive and irreversible multifactorial causes that influence the endometrium of mare. Tissue remodeling in the equine endometrium occurs as part of the pathogenesis of endometrosis, a process characterized by a shift in extracellular matrix (ECM) components. The relationship between matrix metalloproteinases and their specific inhibitors is crucial for the remodeling process. Collagen play a significant role in maintaining a healthy uterus and may promote fibrotic processes. The aim of this study was to quantify endometrial collagen deposition using picrosirius 25 red (PSR) staining, and to evaluate gene expression of collagen type 2 (COL-2) and 3 (COL-3), matrix metalloproteinases 1 (MMP-1) and 2 (MMP-2), their tissue inhibitor (TIMP-2), and tumor necrosis factor (TNF-α) in the endometrium of mares with different grades of fibrosis. The samples (n = 34) were classified into three categories based on the frequency and distribution of fibrosis-related changes in the endometrium: Category I (healthy endometrium, n = 12), Category II (moderate fibrosis, n = 12), and Category III (severe fibrosis, n = 10). Collagen quantification demonstrate a substantial proportional increase (P < 0.0001) in collagen deposition across Category I (11.72 ± 1.39 %), Category II (17.76 ± 1.29 %), and Category III (24.15 ± 1.87 %). In transcript evaluations, higher COL-2 expression was found in Category II than in mares classified as Category I or III. MMP-1 showed increased transcript expression in Category II compared to Category III endometrial samples. Higher expression of MMP-2 was detected in Category III than in Category I and II. TIMP-2 showed lower mRNA expression in Category III vs Category I and II. However, TNF-α gene expression was higher in Category II than in Categories I and III. This study demonstrates that endometrial evaluation using PSR can play an important role in routine analyses for the detection and objective quantification of collagen in endometrial tissues. Additionally, this study demonstrated through gene expression analysis that MMP-1 may be linked to physiological endometrial remodeling. In contrast, MMP-2 could be associated with fibrogenesis in the endometrium, which is regulated by the inhibitor TIMP-2. Furthermore, COL-2 and TNF-α could be considered as biological markers involved in the progression endometrosis in mares. As such, the results of this study may contribute to the development of future antifibrotic therapies that aim to delay or even reverse the pathological remodeling of the extracellular matrix in the uterus, in addition to optimizing the diagnosis and prognosis of endometrial fibrosis in mares.

Factors Affecting the Evaluation of Collagen Deposition and Fibrosis In Vitro.

Immune responses to biomedical implants, wound healing, and diseased tissues often involve collagen deposition by fibroblasts and other stromal cells. Dysregulated collagen deposition can lead to complications, such as biomaterial fibrosis, cardiac fibrosis, desmoplasia, liver fibrosis, and pulmonary fibrosis, which can ultimately result in losses of organ function or failure of biomedical implants. Current in vitro methods to induce collagen deposition include growing the cells under macromolecular crowding conditions or on fibronectin-coated surfaces. However, the majority of these methods have been demonstrated with a single cell line, and the combined impacts of culture conditions and postculture processing on collagen deposition have not been explored in detail. In this work, the effects of macromolecular crowding versus fibronectin coating, fixation with methanol versus fixation with paraformaldehyde, and use of plastic substrates versus glass substrates were evaluated using the WI-38 human lung fibroblast cell line. Fibronectin coating was found to provide enhanced collagen deposition under macromolecular crowding conditions, while a higher plating density led to improved collagen I deposition compared with macromolecular crowding. Collagen deposition was found to be more apparent on plastic substrates than on glass substrates. The effects of primary cells versus cell lines, and mouse cells versus human cells, were evaluated using WI-38 cells, primary human lung fibroblasts, primary human dermal fibroblasts, primary mouse lung fibroblasts, primary mouse dermal fibroblasts, and the L929 mouse fibroblast cell line. Cell lines exhibited enhanced collagen I deposition compared with primary cells. Furthermore, collagen deposition was quantified with picrosirius red staining, and plate-based drug screening through picrosirius red staining of decellularized extracellular matrices was demonstrated. The results of this study provide detailed conditions under which collagen deposition can be induced in vitro in multiple cell types, with applications including material development, development of potential antifibrotic therapies, and mechanistic investigation of disease pathways. Impact Statement This study demonstrated the effects of cell type, biological conditions, fixative, culture substrate, and staining method on in vitro collagen deposition and visualization. Further the utility of plate-based picrosirius red staining of decellularized extracellular matrices for drug screening through collagen quantification was demonstrated. These results should provide clarity and a path forward for researchers who aim to conduct in vitro experiments on collagen deposition.

Collagenase treatment reduces the anisotropy of ultrasonic backscatter in rat myocardium by reducing collagen crosslinks.

Dysregulation of collagen deposition, degradation, and crosslinking in the heart occur in response to increased physiological stress. Collagen content has been associated with ultrasonic backscatter (brightness), and we have shown that the anisotropy of backscatter can be used to measure myofiber alignment, that is, variation in the brightness of a left ventricular short-axis ultrasound. This study investigated collagen's role in anisotropy of ultrasonic backscatter; female Sprague-Dawley rat hearts were treated with a collagenase-containing solution, for either 10 or 30 min, or control solution for 30 min. Serial ultrasound images were acquired at 2.5-min intervals throughout collagenase treatment. Ultrasonic backscatter was assessed from anterior and posterior walls, where collagen fibrils are predominately aligned perpendicular to the angle of insonification, and the lateral and septal walls, where collagen is predominately aligned parallel to the angle of insonification. Collagenase digestion reduced backscatter anisotropy within the myocardium. Collagen remains present in the myocardium throughout collagenase treatment, but crosslinking is altered within 10 min. These data suggest that crosslinking of collagen modulates the anisotropy of ultrasonic backscatter. An Anisotropy Index, derived from differences in backscatter from parallel and perpendicularly aligned fibers, may provide a noninvasive index to monitor the progression and state of myocardial fibrosis.

Reparative potential of mesenchymal stem cells and platelet-rich plasma on irradiated submandibular glands of male albino rats.

OBJECTIVE: To appraise and compare the reparative role of bone marrow-mesenchymal stem cells (BM-MSCs) and platelet-rich plasma (PRP) against irradiation damage on albino rats' submandibular gland. DESIGN: Seventy four male albino rats were used, one for BM-MSCs harvesting, 10 for PRP preparation, seven as control group (Group 1). The remaining 56 rats were subjected to single dose (6 Gy) gamma irradiation and were divided into equal four groups; (Group 2): received no treatment, (Group 3): each rat was injected with 1 × 105 BM-MSCs, (Group 4): each rat was injected with 0.5 ml/kg PRP, and (Group 5): each rat was injected with 1 × 105 BM-MSCs and 0.5 ml/kg PRP. Each group was further subdivided into two subgroups in which rats sacrificed after one and two weeks from irradiation. Any structural changes were examined histopathologically, immunohistochemically using proliferating cell nuclear antigen (PCNA) and CD31 primary antibodies and histochemically using picrosirius red (PSR) stain, then analyzed statistically. RESULTS: Histopathological examination of Group 2 showed atrophied acini, with nuclear changes and signs of degeneration in duct systems. Treated groups revealed signs of regeneration in form of uniform acini and regenerated duct systems especially in Group 5 and in a time depended manner. Immunohistochemical examination revealed increased immunoexpression of PCNA and CD31, while histochemical examination showed decreased PSR in all treated groups in relation to the irradiated group and this was proved statistically. CONCLUSIONS: BM-MSCs and PRP are effective as treatment for irradiation-induced submandibular gland damage. However, the combined therapy is recommended over each one separately.

Morphometric Analysis of Collagen in Different Grades of Oral Squamous Cell Carcinoma Using Picrosirius Red Stain and Spectrophotometry.

Introduction: Oral squamous cell carcinoma (OSCC) is one of the most formidable health problems for mankind. These carcinomas are characterized by invasion of epithelial tumor cells into the stroma, which get embedded in extracellular matrix and collagen producing reactive changes. Such changes in the stroma may alter the biological aggressiveness of the tumor. An attempt was made to evaluate the collagen changes in different grades of OSCC which can contribute to understanding the biologic behavior of oral cancer and predict clinical outcomes. Aims and Objectives: To assess the quantitative changes in collagen in different grades of OSCC using hematoxylin and eosin (H and E) and Picrosirius red (PSR) stain through spectrophotometry and to compare the efficacy of these stains for estimation of collagen. Materials and Methods: The study comprised a total sample size of 60, which were distributed under 4 different groups, each containing 15 samples. Group I to IV consisted of normal buccal mucosa, with well-, moderately-, and poorly-differentiated OSCC, respectively. The tissues of 10 µm thickness were stained with H and E and PSR for spectrophotometric analysis. Results: The quantity of collagen decreased with increasing grades of OSCC. Comparison between two stains showed that PSR can provide a more reliable and accurate result than H and E. Conclusion: Collagen estimation is one of the methods to assess the progression of tumor. The method used in the present study for collagen estimation in different grades of OSCC is reliable and accurate.