Thick Collagen Fibers Research Articles

EXPRESSION OF TOTAL COLLAGEN IN NORMAL ORAL MUCOSA SUBMITTED TO THE TOPICAL APPLICATION OF CHAMOMILE

<h3>Objectives</h3> Characterize the pattern of collagen fiber expression and quantify the total collagen in the normal oral mucosa of rats after topical application of chamomile (<i>Matricaria recutita</i>). <h3>Study Design</h3> Thirty-five Wistar rats were allocated in experimental groups: control (G1), chamomile fluid extract (G2), chamomile infusion 2 times/day (G3), and chamomile infusion 3 times/day (G4). Euthanasia occurred on the 7th and 14th days after the application of the herbal medicine. The histologic sections of the buccal mucosa obtained were stained with hematoxylin and eosin and Sirius red for qualitative and quantitative analyses of collagen fibers at the lamina propria. <h3>Results</h3> The descriptive analysis showed a similar pattern for all groups, showing thick collagen fibers arranged in all directions. The percentage of total collagen on the 7th and 14th days (medians) was as follows: G2 (42.2 and 40.3), G3 (43.8 and 46.3), and G4 (49.6 and 49.9), respectively, with no significant differences between the groups and the periods studied. <h3>Conclusions</h3> There was no difference in the pattern of qualitative and quantitative expression of collagen fibers after topical application of chamomile in normal oral mucosa during the periods and groups studied, suggesting that the herbal medicine did not cause damage to the evaluated tissues.

P-458 Testosterone treatment induces changes in stromal collagen and elastin content of the ovaries of transgender men

Abstract Study question Does gender-affirming testosterone therapy alter the composition of the extra-cellular matrix (ECM) within the ovarian stroma and subsequently affect follicle activation in vivo Summary answer Ovarian stroma of trans men is more collagenous and less elastic, indicating fibrotic change. This may affect in vivo follicle growth activation What is known already Changes in the ovarian stroma have been demonstrated in the ovaries of transgender men taking testosterone, including thickening of the tunica albuginea, stromal cell hyperplasia and stromal cell luteinisation. Ovaries of trans men also have increased cortical stiffness. These changes are similar to those seen in female patients with PCOS and in physiological ovarian aging, which has been attributed to accumulation of collagen in the ECM. Increasing stiffness of the supportive follicular microenvironment has been shown to reduce follicle growth activation in vitro Study design, size, duration Whole ovaries were obtained from transgender men (mean age 27.6 ± 1.7 years, n = 8) with informed consent at oophorectomy. All patients had received 1000mg testosterone undecanoate intramuscularly at 12-16 week intervals for a minimum of 18 months pre-operatively (range 18 months-10 years). Cortical tissue was dissected into small fragments (≈1x1x0.5mm) and fixed for histological and immunohistochemical analysis. Testosterone-treated ovaries were compared to cortical biopsies from age-matched healthy women obtained at caesarean section (mean age 31.8±1.5, n = 8). Participants/materials, setting, methods Follicle number, classification of developmental stage, non-growing follicle density (NGFD) and stromal cell density were evaluated by histological analysis of ovarian cortical tissue. Sections were stained with Picrosirius red (PSR) to analyse total collagen content using brightfield microscopy. Polarised light was also used to analyse the collagen birefringence, which allows quantification of collagen fibre thickness into thick, medium or thin. Total elastin content was evaluated using immunofluorescence. Main results and the role of chance 4526 follicles were analysed. Transgender ovary showed a higher proportion of non-growing follicles found compared to control (93.9±1.2% vs 84.6±1.5% p &amp;lt; 0.05): the proportions of primary (4.7±0.9% vs 10.6±1.5%, p = 0.2) and secondary (1.4±0.4% vs 4.6±0.7%, p = 0.1) follicles tended to be lower. Stromal cell density was significantly higher in transgender ovarian cortex than control (2.5±0.1 x106cells/mm3 vs 1.7±0.1 x106cells/mm3), indicating stromal cell hyperplasia. Combined data from control and transgender groups showed a positive correlation between NGFD and stromal density (r = 0.64, p = 0.01). Transgender ovary had a higher total collagen content (77.2±1.2%) compared to control (31.3±3.3%, p &amp;lt; 0.005). Analysis of collagen birefringence showed that transgender ovaries had similar quantities of thick collagen fibres (0.014±0.005 vs 0.010±0.009, p = 0.1), more medium thickness collagen fibres (45.1±6.6%vs 14.4±4.9%, p &amp;lt; 0.05) and fewer thinner fibres (41.5±9.6% vs 27.7±2.8%, p = 0.08) than control. The total elastin content in transgender ovaries was lower than control (1.3±0.1% vs 3.6±0.6%, p &amp;lt; 0.005) and subsequently, the collagen/elastin ratio was significantly higher (63.1±7.9 vs 10±1.3, p &amp;lt; 0.005). Limitations, reasons for caution The impact of these findings on in vivo follicle growth are unclear. The effect of duration of testosterone treatment has not investigated. Wider implications of the findings More collagenous, less elastic ovarian stroma in trans men indicates fibrotic change; these findings are similar to women with PCOS and with reproductive ageing. These stromal changes may alter follicle growth activation and may contribute value to our understanding of the regulation of follicle function in a range of conditions. Trial registration number nil

Structural changes in the collagen network of joint tissues in late stages of murine OA

Osteoarthritis (OA) is the most prevalent degenerative joint disease, resulting in joint pain, impaired movement, and structural changes. As the ability of joint tissue to resist stress is mainly imparted by fibrillar collagens in the extracellular matrix, changes in the composition and structure of collagen fibers contribute to the pathological remodeling observed in OA joints that includes cartilage degeneration, subchondral bone (SCB) sclerosis, and meniscal damage. Using the established OA model of destabilization of the medial meniscus (DMM) in C57BL/6J mice, we performed a comprehensive analysis of the content and structure of collagen fibers in the articular cartilage, subchondral bone, and menisci using complementary techniques, which included second harmonic generation microscopy and immunofluorescence staining. We found that regions exposed to increased mechanical stress in OA mice, typically closest to the site of injury, had increased collagen fiber thickness, dysregulated fiber formation, and tissue specific changes in collagen I and II (Col I and Col II) expression. In cartilage, OA was associated with decreased Col II expression in all regions, and increased Col I expression in the anterior and posterior regions. Col I fiber thickness was increased in all regions with disorganization in the center region. In the superficial SCB, all regions exhibited increased Col I expression and fiber thickness in OA mice; no changes were detected in the deeper regions of the subchondral bone except for increased Col I fiber thickness. In the menisci, OA led to increased Col I and Col II expression in the vascular and avascular regions of the anterior meniscus with increased Col I fiber thickness in these regions. Similar changes were observed only in the vascular region of the posterior meniscus. Our findings provide, for the first time, comprehensive insights into the microarchitectural changes of extracellular matrix in OA and serve as guidelines for studies investigating therapies that target collagenous changes as means to impede the progression of osteoarthritis.

Valproic acid modulates collagen architecture in the postoperative conjunctival scar.

Valproic acid (VPA), widely used for the treatment of neurological disorders, has anti-fibrotic activity by reducing collagen production in the postoperative conjunctiva. In this study, we investigated the capacity of VPA to modulate the postoperative collagen architecture. Histochemical examination revealed that VPA treatment was associated with the formation of thinner collagen fibers in the postoperative days 7 and 14 scars. At the micrometer scale, measurements by quantitative multiphoton microscopy indicated that VPA reduced mean collagen fiber thickness by 1.25-fold. At the nanometer scale, collagen fibril thickness and diameter measured by transmission electron microscopy were decreased by 1.08- and 1.20-fold, respectively. Moreover, delicate filamentous structures in random aggregates or closely associated with collagen fibrils were frequently observed in VPA-treated tissue. At the molecular level, VPA reduced Col1a1 but induced Matn2, Matn3, and Matn4 in the postoperative day 7 conjunctival tissue. Elevation of matrilin protein expression induced by VPA was sustained till at least postoperative day 14. In primary conjunctival fibroblasts, Matn2 expression was resistant to both VPA and TGF-β2, Matn3 was sensitive to both VPA and TGF-β2 individually and synergistically, while Matn4 was modulable by VPA but not TGF-β2. MATN2, MATN3, and MATN4 localized in close association with COL1A1 in the postoperative conjunctiva. These data indicate that VPA has the capacity to reduce collagen fiber thickness and potentially collagen assembly, in association with matrilin upregulation. These properties suggest potential VPA application for the prevention of fibrotic progression in the postoperative conjunctiva. KEY MESSAGES: VPA reduces collagen fiber and fibril thickness in the postoperative scar. VPA disrupts collagen fiber assembly in conjunctival wound healing. VPA induces MATN2, MATN3, and MATN4 in the postoperative scar.

MO675: A New in Vivo Multi-Photon Microscopy Based Approach to Study the Peritoneal Membrane Changes Induced by Peritoneal Dialysis

Abstract BACKGROUND AND AIMS Peritoneal dialysis (PD) is a renal replacement therapy that allows the elimination of metabolic waste products and excess body fluid through the peritoneal membrane. The exposure to PD solution contributes to membrane aging and fibrosis resulting in ultrafiltration and clearance failure. The glucose, used as an osmotic factor, in PD dialysate triggers several processes involved in the pathogenesis of peritoneal fibrosis, angiogenesis and epithelial to mesenchymal transition. Studies on natural extracts, such as Oleuropein (Ole), a powerful antioxidant with remarkable antifibrotic and protective effects on the peritoneal membrane, are currently being validated. This study aims to develop a method based on multi-photon microscopy to study the physiology of the peritoneal membrane during dialysis exchange and to validate in vivo the effects of the Ole in animal models of fibrosis undergoing dialysis treatment. METHOD Multi-photon microscopy allows in vivo evaluation of the microcirculation that supply the peritoneum and also to study the framework of mesothelial cells and their underlying layer of collagen fibers that contributes to the sub-mesothelial space. We have implemented the surgical procedure in order to optimize the stability of a flap of parietal peritoneum to directly observe at the scope. With this approach the peritoneal membrane is evaluable at baseline condition and during exposure to dialysate solutions. RESULTS Our method allows building a three-dimensional render of the peritoneal membrane, with the evaluation of all the single layers without the use of specific markers. In this way we could assess specifically the phenomena induced by the fibrotic process: the thickening of the sub-mesothelial interstitium and the greater density of the vascular network. Furthermore, in vivo measurements of flow in the vessels of microcirculation (arterioles, capillaries, post-capillary venules) determine that exposure to hypotonic solutions increases significantly the flow in large-diameter vessels due to better permeability and hemodilution. Finally in order to evaluate the possibility to detect morphological and functional changes in pathogenic model we have used the well-established model of peritoneal fibrosis derived by 15 days’ long exposure of the peritoneal membrane with 3.86% glucose dialysate. Whth this approach we could detect the significant increase in the parameters of cellularity, vascularization, fibrosis and thickening of collagen fibers. In order to test the sensibility of our approach to the evaluation of the peritoneal membrane senescence parameters, we tested the effect of Ole in preventing the damage induced by high glucose–containing dialysate. Ole reduced both the thickness and the organization of the collagen fibers and the vascular network, including the number of branch points. CONCLUSION The developed method has potential for a dynamic and reliable in vivo approach. Studies are ongoing to validate the effects of drugs and dialysates with different osmotic and electrolytic compositions on the dialysis capacities of the peritoneal membrane and on the blood flow in peritoneal capillaries. This method offers great potential for testing new pharmacological approaches aimed at preserving the structural and functional integrity of the peritoneum and for the validation of substances, such as natural extracts with beneficial effects against the damage induced in the long term by conventional dialysis solutions.

The articular cartilage surface is impaired by a loss of thick collagen fibers and formation of type I collagen in early osteoarthritis

Osteoarthritis (OA) is a joint disease affecting millions of patients worldwide. During OA onset and progression, the articular cartilage is destroyed, but the underlying complex mechanisms remain unclear. Here, we uncover changes in the thickness of collagen fibers and their composition at the onset of OA. For articular cartilage explants from knee joints of OA patients, we find that type I collagen-rich fibrocartilage-like tissue was formed in macroscopically intact cartilage, distant from OA lesions. Importantly, the number of thick fibers (>100 nm) has decreased early in the disease, followed by complete absence of thick fibers in advanced OA. We have obtained these results by a combination of high-resolution atomic force microscopy imaging under near-native conditions, immunofluorescence, scanning electron microscopy and a fluorescence-based classification of the superficial chondrocyte spatial organization. Taken together, our data suggests that the loss of tissue functionality in early OA cartilage is caused by a reduction of thick type II collagen fibers, likely due to the formation of type I collagen-rich fibrocartilage, followed by the development of focal defects in later OA stages. We anticipate that such an integrative characterization will be very beneficial for an in-depth understanding of other native biological tissues and the development of sustainable biomaterials. Statement of significanceIn early osteoarthritis (OA) the cartilage appears macroscopically intact. However, this study demonstrates that the collagen network already changes in early OA by collagen fiber thinning and the formation of fibrocartilage-like tissue. Both nanoscopic deficiencies already occur in macroscopically intact regions of the human knee joint and are likely connected to processes that result in a weakened extracellular matrix. This study enhances the understanding of earliest progressive cartilage degeneration in the absence of external damage. The results suggest a determination of the mean collagen fiber thickness as a new target for the detection of early OA and a regulation of type I collagen synthesis as a new path for OA treatment.

Effectiveness Of Incision Wound Healing Of Pandanus Amaryllifolius Roxb In Wistar Rats

The incision is a condition of tissue discontinuity that is commonly encountered with the risk of prolonged healing time, imperfect wound healing, infection, etc. Assessment of the wound healing activity of the incision is the purpose of this research, which includes an examination of wound contraction, the thickness of collagen fibers, and the number of new blood vessels formation. In total, 25 rats will be divided into 5 test groups, each consisting of 5 rats; the control group (cream base treated), standard group (Povidone Iodine), followed by the treated group with 10%, 20%, and 30% pandan leaf extract ointment. The preliminary screening of phytochemical analysisshowed the presence of alkaloids, saponins, flavonoids, tannins, triterpenoids, and steroids and glycosides, with the total content of phenols and flavonoids from the ethanolic extract of Pandan Wangi is 20.95 GAE mg/gram extract, and 23.57 TAE mg/gram extract. Wound contraction examination result showed a significant difference between the treatment groups on day 16 and a sign the pairs of groups after being given the ointment with each concentration (p &lt;0.05), especially in the group with 30% fragrant leaves extract and the standard group. Examination of new blood vessels showed a significant difference (p &lt;0.05), especially with 30% of the leaves extract ointment. There was also a significant difference in collagen density in the group given pandanus ethanol ointment, especially at the 30% pandanus extract content against the group that was only given the ointment base. This research strongly suggests that the fragrant pandan leaf extract ointment is effective in healing incisional wounds and promises the potential for further studies on the pharmacological aspects.

βig-h3-structured collagen alters macrophage phenotype and function in pancreatic cancer

SummaryMacrophages play an important role in immune and matrix regulation during pancreatic adenocarcinoma (PDAC). Collagen deposition massively contributes to the physical and functional changes of the tissue during pathogenesis. We investigated the impact of thick collagen fibers on the phenotype and function of macrophages. We recently demonstrated that the extracellular protein βig-h3/TGFβi (Transforming growth factor-β-induced protein) plays an important role in modulating the stiffness of the pancreatic stroma. By using atomic force microscopy, we show that βig-h3 binds to type I collagen and establishes thicker fibers. Macrophages cultured on βig-h3-structured collagen layers display a different morphology and a pro-tumoral M2 phenotype and function compared to those cultured on non-structured collagen layers. In vivo injection of those instructed CD206+CD163+ macrophages was able to suppress T cell responses. These results reveal for the first time that the collagen structure impacts the phenotype and function of macrophages by potentiating their immunosuppressive features.

Effect of gelatin methacryloyl hydrogel on healing of the guinea pig vaginal wall with or without mesh augmentation.

The aims of this study were to evaluate the effectiveness of gelatin methacryloyl as an adjunct to anterior vaginal wall injury with or without vaginal mesh compared with traditional repair with suture. Virginal cycling Hartley strain guinea pigs (n= 60) were randomized to undergo surgical injury and repair using either polyglactin 910 suture or gelatin methacryloyl for epithelium re-approximation or anterior colporrhaphy with mesh augmentation using either polyglactin 910 suture or gelatin methacryloyl for mesh fixation and epithelium re-approximation. Noninjured controls (n= 5) were also evaluated. After 4 days, 4 weeks, or 3 months, tissues were analyzed by hematoxylin & eosin in addition to immunolabeling for macrophages, leukocytes, smooth muscle, and fibroblasts. Surgical injury repaired with suture was associated with increased inflammation and vessel density compared with gelatin methacryloyl. Vimentin and α-smooth muscle actin expression were increased with gelatin methacryloyl at 4 days (p= 0.0026, p= 0.0272). There were no differences in changes in smooth muscle or overall histomorphology after 3months between the two closure techniques. Mesh repair with suture was also associated with increased inflammation and vessel density relative to gelatin methacryloyl. Quantification of collagen content by picrosirius red staining revealed increased thick collagen fibers throughout the implanted mesh with gelatin methacryloyl compared with suture at 4 weeks (0.62 ± 0.01 μm2 vs 0.55 ± 0.01, p = 0.018). Even at the long-term time point of 3 months, mesh repair with suture resulted in a profibrotic encapsulation of the mesh fibers, which was minimal with gelatin methacryloyl. Smooth muscle density was suppressed after mesh implantation returning to baseline levels at 3 months regardless of fixation with suture or gelatin methacryloyl. These results suggest that gelatin methacryloyl might be a safe alternative to suture for epithelium re-approximation and anchoring of prolapse meshes to the vagina and may improve chronic inflammation in the vaginal wall associated with mesh complications.

Investigating the histological and structural properties of tendon gel as an artificial biomaterial using the film model method in rabbits

PurposeThis study aimed to evaluate the properties of tendon gel by investigating the histological and structural differences among tendon gels under different preservation periods using a rabbit model.MethodsForty mature female rabbits were divided into four groups, each containing ten rabbits, on the basis of in-vivo preservation periods of tendon gels (3, 5, 10, and 15 days). We created the Achilles tendon rupture models using the film model method to obtain tendon gels. Tensile stress was applied to the tendon gel to promote maturation. Histological and structural evaluations of the tendon gel were performed before and after applying the tensile force, and the results obtained from the four groups were compared.ResultsAlthough the day-3 and day-5 tendon gels before applying tensile stress were histologically more immature than the day-10 and day-15 gels, type I collagen fibers equivalent to those of normal tendons were observed in all groups after the tensile process. Based on the surface and molecular structural evaluations, the day-3 tendon gels after the tensile process were molecularly cross-linked, and thick collagen fibers similar to those present in normal tendons were observed. Structural maturation observed in the day-3 tendon gels caused by traction was hardly observed in the day-5, -10, and -15 tendon gels.ConclusionsThe day-3 tendon gel had the highest regenerative potential to become a normal tendon by applying a traction force.

Assessment of Changes in Birefringence and Orientation of Collagen Fibres in Different Grades of Oral Submucous Fibrosis and Oral Squamous Cell Carcinoma using Picrosirius Red and Polarising Microscopy

Introduction: Oral submucous fibrosis (OSMF) is a potentially malignant disease with a prevalence of 7-13%. It is characterised by the fibroelastic changes due to excessive deposition of collagen which results in dense fibrous bands and epithelial changes. About 90-95% of oral cancers are Oral Squamous Cell Carcinoma (OSCC). Stroma produced by the invading neoplastic cells are rich in collagen fibres. These collagen fibers have been the main focus of study to understand the pathogenesis of these lesions. Hence, these fibres have been evaluated under polarised microscopy following staining with Picrosirius red stain. Aim: To evaluate changes in birefringence, thickness and orientation of collagen fibres in different histopathological stages of OSMF and OSCC. Materials and Methods: This observational study involved sections of clinically and histopathologically confirmed cases of OSCC and OSMF from Department of Oral Pathology and Microbiology, Rajarajeswari Dental College and Hospital, Bangalore. The study was conducted for a duration of one year from April 2017 to March 2018. The study included total 60 cases among which 20 for each-normal mucosa, OSCC and OSMF. Tissue sections were stained with Picrosirius red stain and collagen fibres were analysed for colour, thickness and orientation under polarised microscope. Chi-square test was used for statistical analysis. Significance was set at p-value &lt;0.05. Results: Comparison of birefringence of collagen fibres between OSMF, OSCC and normal mucosa was not statistically significant (p-value=0.37). Orientation of collagen fibres between OSMF, OSCC and normal mucosa was statistically significant with (p-value=0.02). Conclusion: This study showed a change in colour from yellow orange to orange red in advanced OSMF cases which indicated progression of disease and tightly packing of collagen fibres, suggestive of presence of thick fibres in the extracellular matrix. In OSCC, the colour change from yellow orange to orange red and haphazardly arranged collagen fibres was indicative of transformation of preneoplastic to carcinoma stage.

Interplay of collagen and mast cells in periapical granulomas and periapical cysts: a comparative polarizing microscopic and immunohistochemical study.

ObjectivesThis pilot study aimed to establish the interrelationship between collagen and mast cells in periapical granulomas and periapical cysts.Materials and MethodsAn observational cross-sectional study was conducted on the paraffin-embedded tissue sections of 68 specimens (34 periapical granulomas and 34 periapical cysts). The specimens were stained with picrosirius to observe collagen fiber birefringence and anti-tryptase antibody to evaluate the mast cell count immunohistochemically. The mean number and birefringence of collagen fibers, as well as the mean number of mast cells (total, granulated, and degranulated), and the mean inflammatory cell density were calculated. The data obtained were analyzed using the Kruskal Wallis test, Mann Whitney U test, and Spearman correlation test (p < 0.05).ResultsThe mean number of thick collagen fibers was higher in periapical cysts, while that of thin fibers was higher in granulomas (p = 0.00). Cysts emitted orange-yellow to red birefringence, whereas periapical granulomas had predominantly green fibers (p = 0.00). The mean inflammatory cell density was comparable in all groups (p = 0.129). The number of total, degranulated, and granulated mast cells exhibited significant results (p = 0.00) in both groups. Thick cyst fibers showed significant inverse correlations with inflammation and degranulated mast cells (p = 0.041, 0.04 respectively).ConclusionsMast cells and inflammatory cells influenced the nature of collagen fiber formation and its birefringence. This finding may assist in the prediction of the nature, pathogenesis, and biological behavior of periapical lesions.

Morphometric characteristics of the structural components of the intervertebral disc in rats at different times of the experimental opioid exposure and after its withdrawal

Background. The world and domestic literature from time to time report about the problems of uncontrolled drug abuse. This problem is associated not only with the negative impact on the morphological structure, but also is a serious factor which, if it has been exposing for a long time, leads to disability and death. Despite a significant number of studies in this area, the problem of changes in the morphometric parameters of the intervertebral disc’s structural components under chronic exposure of opioid agents still remains unresolved. That is why the study of morphometric characteristics of the intervertebral disc’s structural components under experimental opioid exposure will be interesting for both - morphologists and practical traumatologists. Objective. To study the morphometric parameters of the structural components of the intervertebral disc in rats at different times of the experimental opioid exposure and after its withdrawal. Methods. The objects of the study were 61 mature outbred male rats, weight - 80-135g, age - 4.5-7.5 months. Animals were injected with nalbuphine intramuscularly once daily (at 10-11 am) for 42 days. Histological specimens were stained with alcyan blue (for the study of the collagen fibers thickness of the anterior and posterior longitudinal ligaments) and PAS-reaction (for the study of the gelatinous nucleus and its capsule thickness). For measurements of collagen fiber thickness, were taken images at x100 magnification, for measurements of the gelatinous nucleus and its capsule thickness - at x40 magnification. Measurements were performed with ImageJver software. 1.51 using the tool "straight" for linear measurements. Results. Thus, changes in the collagen fibers thickness of the anterior and posterior longitudinal ligaments during the experiment were uneven and had different tendencies. The thickness of the posterior longitudinal fibers changed mostly within the central trend of the control group with a significant decrease only at the 2nd, 3rd, and 4th weeks. However, more significant, as for pathogenetic changes, was a sharp decrease in the number of fibers thicker than 25 μm in animals of the experimental group after 2 weeks of the study, and the maximum thickness at 5th and 6th week was only 31.41 μm and 35.24 μm whereas the maximum thickness of the control group was 81.48 μm. In the withdrawal group, only a decrease in sites with nuclear edema can be considered positive, which is displayed by morphometrical decrease in the maximum value, but the central trend remains much lower than in the control group, which also indicates the predominance of decompensatory changes. Conclusion. During the whole experiment, we observed similar changes in the nucleus capsule thickness: non-systemic fluctuations of the central parameters ​​up to the 5th week and a sharp decompensation at the 6th week, which was manifested by a sharp decrease in thickness. Simultaneously, we observed numerous sites of edema and thickening by more than 100 μm on the 2nd week. Despite a general decrease in median and quartile at this time, such sites were also noted at the 6th week. Thickness of the nucleus capsule in the withdrawal group did not demonstrate significant dynamics and remained similar to those at the 6th week. Although, this can be interpreted as the stabilization at a certain level without further negative dynamics.

Morphological prerequisites for the formation of fascial duplication in the elimination of damage to the anterior rectal wall during prostatectomy

Background Intraoperative rectal injury in prostatectomy patients is an uncommon but severe complication. Particular attention is paid to improving the results of healing damage to the anterior rectal wall during prostatectomy.Objective To study the morphological features of the parietal pelvic fascia and the rectal wall to substantiate the possibility of the formation of fascial duplication in the elimination of damage to the anterior rectal wall during prostatectomy.Material and Methods The authors carried out an intravital morphological analysis of the parietal pelvic fascia covering the levator rectum muscle and the anterior rectal wall in 10 men.Results The parietal pelvic fascia contains more powerful bundles of collagen fibers, which in certain areas are partially woven into the fibers of striated muscle tissue. The adventitia of the rectum is characterized by a looser arrangement of the interacting components of the formed connective and smooth muscle tissue. In the studied formations of the small pelvis, the thickness of collagen fibers separately and in the composition of bundles, as well as the cells of the differon and each fiber separately did not differ, which indicated the identity of their tinctorial properties in the compared zones.Conclusion Morphological analysis showed that when juxtaposing and touching the edges of the healing area of the surgical wound without tension, a stable and continuous scar of the fascial duplication is formed, which ensures reliable fusion of the stitched anatomical structures.

A human multi-lineage hepatic organoid model for liver fibrosis

To investigate the pathogenesis of a congenital form of hepatic fibrosis, human hepatic organoids were engineered to express the most common causative mutation for Autosomal Recessive Polycystic Kidney Disease (ARPKD). Here we show that these hepatic organoids develop the key features of ARPKD liver pathology (abnormal bile ducts and fibrosis) in only 21 days. The ARPKD mutation increases collagen abundance and thick collagen fiber production in hepatic organoids, which mirrors ARPKD liver tissue pathology. Transcriptomic and other analyses indicate that the ARPKD mutation generates cholangiocytes with increased TGFβ pathway activation, which are actively involved stimulating myofibroblasts to form collagen fibers. There is also an expansion of collagen-producing myofibroblasts with markedly increased PDGFRB protein expression and an activated STAT3 signaling pathway. Moreover, the transcriptome of ARPKD organoid myofibroblasts resemble those present in commonly occurring forms of liver fibrosis. PDGFRB pathway involvement was confirmed by the anti-fibrotic effect observed when ARPKD organoids were treated with PDGFRB inhibitors. Besides providing insight into the pathogenesis of congenital (and possibly acquired) forms of liver fibrosis, ARPKD organoids could also be used to test the anti-fibrotic efficacy of potential anti-fibrotic therapies.

Dynamics of pathomorphological changes in the structural organization of the intervertebral disc at the end of the seventh and fourteenth day of experimental opioid exposure at the ultrastructural level

In general, the modern literature pays attention to the issues of spine pathology and intervertebral discs. A significant percentage of vertebral disorders - scoliosis, osteochondrosis, spinal disc herniation, etc., occur as a result of exposure to various factors and manifest in changes of the intervertebral discs. The aim of our work was to study at the ultrastructural level the features of pathomorphological manifestations in the structural components of the intervertebral disc at the end of the seventh and fourteenth days of experimental opioid exposure. Materials and methods of research. The objects of the study were 32 sexually mature, white, male rats, weighing 92 - 103 g, aged 4.5 months. Animals were injected with nalbupine intramuscularly once daily (at 10-11 a.m.) for 14 days. The initial dose of nalbuphine was 8 mg/kg during the first week, 15 mg/kg during the second week. It created the conditions of chronic opioid exposure. Before sampling, the animals were withdrawn from the experiment using dibutyl ether. Intervertebral discs of rats were used as a material for ultrastructural study. Ultrastructural specimens were prepared according to the accepted methods. The results of the study. As a result of the sampling after 7 days of opioid exposure we found inhomogeneous osmiophilicity and compaction of the nucleus pulposus matrix in which intensively accumulated osmiophilic grains of glycogen proteoglycans, increased the number of collagen fibers, some of them were heterogeneous. It was also noted the development of moderate degenerative changes in some notochondral cells, which was accompanied by increased vacuolization of the cytoplasm by inhomogeneous compaction of the nucleus and an increase of heterochromatin there. After 14 days necrotic changes in the cells of the nucleus pulposus, as well as the destruction of collagen fibers of the annulus fibrosus were found. In particular, an increase in the amount of heterochromatin in the nucleui of notochondral cells, which was accompanied by a decrease in the volume of the nucleui and inhomogeneous swelling of the cytoplasm. Active fibroblasts were often visualized in the annulus fibrosus. Intense osmiophilicity and thickening of collagen fibers of the annulus fibrosus were observed in some areas of the fibrous ring. Focal destruction of collagen fibers was also noted. In the areas of destruction the fibrils of collagen fibers disintegrated into an inhomogeneous fine-grained stratified mass and were located loosely. Conclusions. At the end of the first week we found that the cytoplasmic processes of chondrocytes decreased in volume, shortened, underwent fragmentation and destruction, some of them detached from the surface of the plasmolemma. At the end of the second week signs of opioid exposure progressed and manifested by an increase in the destruction of cytoplasmic processes in chondrocytes. Also focal destruction of collagen fibers was noted.

Autologous nanofat injection in treatment of scars: A clinico-histopathological study.

Scars are the unfortunate outcome of most injuries and some diseases. Its psychological impact on patients can deeply affect their quality of life. The aim of this study was to evaluate the efficacy of autologous nanofat injection in improving the aesthetic outcome of scars, combined with histopathological correlation of the response. Thirty patients with scars of different etiologies undergone one session of nanofat injection and evaluation was done 6months after the session. Efficacy of treatment was assessed clinically using Vancouver scar scale by two independent blinded dermatologists and histopathologically using image analysis system. The age of enrolled patients ranged from 18 to 40years old. There was a statistically significant improvement on the total Vancouver scar scale regarding the height and pliability of the scars. Pathological evaluation showed an increase in epidermal thickness, increased number and density of collagen and elastic fibers along with neovascularization. Evidenced by clinical and pathological improvement, autologous nanofat injection is an effective strategy for treating scars of different etiologies.

Возможность применения криопротекторов для хранения роговичных тканеинженерных конструкций

Purpose. Conduct a comparative analysis of corneal tissue–engineered constructs that have undergone cryopreservation of three different cryoprotectants. Materials and methods. After refractive surgery Relex SMILE, corneal tissue was obtained which is called a lenticule. Corneal tissue engineering constructs (RTK) have created using decellularization methods that relate to the technologies of tissue and regenerative medicine. For decellularization of the lenticules, a protocol with a 1.5 M solution of Sodium Chloride with DNase 5 U / ml and RNase 5 U / ml was used. After decellularization, RTK was cryopreserved using: 1) 10% DMSO and 90% “corneal storage solution” (Borzenok–Moroz medium), 2) «Cryoderm» cell cryopreservation medium, 3) 100% glycerin. Transparency was assessed using spectrophotometry. The thickness of collagen fibers was assessed by scanning electron microscopy (SEM). Results. When assessing the transparency of the control and experimental groups, a statistically significant decrease in transparency was revealed in the groups using cryoprotectants – Glycerin and Cryoderm (p&lt;0.001), the groups using DMSO did not differ from the control (p=0.99). Also, statistically significant differences were found between the experimental groups «Cryoderm»–DMSO (p=0.02), «Cryoderm»–Glycerin (p&lt;0.001) and Glycerin–DMSO (p&lt;0.001). When evaluating the SEM data, it was found that the thickness of collagen fibers did not differ in the DMSO–Control and Cryoderm–Control groups (p&gt;0.05). The Glycerol–Control group was statistically different (p&lt;0.001). Conclusions. In our work, we have shown the use of DMSO for storing RTK causes thickening of collagen fibers after decellularization of similar to native lenticules. This protocol can be useful for long–term storage and creation of the RTK cryobank. Key words: storage, lenticule, cryoprotectants, cryopreservation, cornea.

Morphometric analysis of the intercellular substance of hypertrophic scars after anti-scar treatment

AIM: This study aimed to assess the formation of scar tissue after burns under the influence of an anti-scar gel. Understanding of the processes involving scars and the morphofunctional features of the tissue at different stages of development allows targeted selection of therapy and prevention of scars.&#x0D; MATERIALS AND METHODS: We conducted a comparative prospective analysis from 2005 to 2020. Of which two groups were identified. In group 1 (n = 47), burns were treated according to the standard scheme without the use of modern wound coverings. In group 2 (n = 41), early primary prevention of pathological scarring was performed, where the Contractubex gel was applied to the area of burn injury from the moment of epithelialization. Histological examination included the analysis of skin biopsies in the area of damage before and after conservative treatment.&#x0D; RESULTS: Histological examination showed quantitative changes in the cellular composition of the scar tissue in all groups. The average quantitative index of the fibroblast activity was significantly reduced in group 2 using Contractubex gel. Thickness of collagen fibers, according to the morphometric analysis, is most reduced in all layers of the dermis in group 2 (p 0.05). In group 1, collagen fibers are represented as nodular clusters; in some areas of the reticular layer of the dermis, fibers have a more fragmented appearance. In group 2, the use of Contractubex leads to a significant decrease in the level of tumor growth factor- in the papillary and reticular layers of the dermis.&#x0D; CONCLUSION: The use of Contractubex gel in the early prevention of pathological scarring significantly reduces the need for subsequent reconstructive surgical interventions

The effect of redox signaling on extracellular matrix changes in diabetic wounds leading to amputation

Introduction& Objectives: Redox signaling is a critical regulator in the process of wound healing. This signaling pathway can be effective in the development or healing of diabetic ulcers through the ECM.In this study, the structure of extracellular matrix investigated in relation to redox signaling in the tissue of patients with diabetic ulcers that lead to organ amputation. Materials and methodsThe case-control design on diabetic patients ulcers as case group and non-diabetic limb ischemia as control were used.Hematoxylin-eosin, trichrome, and elastin staining methods were used for pathological evaluations of ECM. MDA, total thiol, and SOD levels were measured using ELISA kits to assess the oxidative stress level. Also, NO level was measured by using ELISA kits in both groups. Expression levels of genes MMP2, MMP9, and HIF were detected using real-time PCR with SYBR-green assay. ResultsThe pathological results showed an increase in the thickness of collagen and elastin fibers. Lipids atrophy was visible in the tissue isolated from the diabetic wound group. The amount of MAD to evaluate the level of lipid oxidation in patients with diabetic Ulcer was significantly higher than the control group(p < 0.01). Thiol level was significantly lower in the diabetic ulcer group than in the control group(p < 0.0001). The expression of metalloproteinases 2 and 9 genes in the tissues isolated from diabetic ulcers was lower than the control group(p < 0.0001). While the expression of the HIF gene in this group was higher than the control group(p < 0.0001). ConclutionIn the diabetic wound, the HIF secretion due to hypoxic conditions is beneficial for matrix deposition and prevents protease activity, but if the hypoxia persists, it can lead to ECM deposition subsequently increases the tissue pressure, increases of the collagen I-to-collagen III ratio in collagen accumulation that due to more hypoxia , lipidsAtrophy and eventually amputation.