Collagen Distribution Research Articles

Braided biomimetic PCL grafts for anterior cruciate ligament repair and regeneration

Anterior cruciate ligament (ACL) is a knee joint stabilizer with a limited regeneration capacity mainly because of low cellular content. State-of-the-art procedures are unable to restore the functions of the tissue as demonstrated by limited success rates. Regenerative engineering can offer a solution for restoring the functions of torn/ruptured ligaments provided that biomimetic grafts are available as grafts/scaffolds. However, a model construct to test behavior of cells to better understand the healing mechanism of ACL is still missing. This study, firstly, aimed at creating an injured rabbit ACL model. Then, the injured and healthy ACL tissues were characterized in terms of alignment and diameter distributions of collagen fibrils. Next, polycaprolactone (PCL) grafts were prepared from braided electrospun meshes and were characterized in terms of alignment and diameter distributions of fibers. Finally, biomechanical properties of ACL tissue and mechanical properties of PCL grafts were determined and compared. Findings demonstrated that distributions of the fiber diameters of PCL electrospun grafts were similar to diameter distribution of collagens of healthy and injured rabbit ACL. The novelty of this study relies on the determination of the diameter distribution of collagens of healthy and injured rabbit ACL tissues, and fabrication of PCL grafts with diameter distributions similar to that seen in healthy and injured ACLs. This study is significant because it addresses a worldwide clinical problem associated with millions of patients. The fibrous biomimetic graft designed in this study is different from the traditional grafts that exhibit unimodal distribution, and it is expected to have a significant contribution to ACL regeneration efforts.

New insights on collagen structural organization and spatial distribution around dental implants: a comparison between machined and laser-treated surfaces

BackgroundOne of the main factors for the osseointegration of dental implants is the development of an adequate soft tissue barrier, mainly composed by collagen, which protects the implant from bacterial development. The structural features of the peri-implant collagen are influenced by the implant components and, in particular, by the type of the surface. In the clinical practice, healing abutments are characterized by smooth surfaces, named machined. Recently, a new laser technique, Synthegra, has been developed to obtain a topography-controlled surface with micrometric regular pores that seems reducing the risk of peri-implantitis. Based on this background, this study aims investigating the structural organization and spatial distribution of collagen surrounding healing abutments characterized by laser-treated and machined surfaces.MethodsGingiva portions surrounding custom-made healing abutments (HA), characterized by alternated laser-treated and machined surfaces, were collected and analyzed by combining Fourier Transform InfraRed Imaging (FTIRI) spectroscopy, a non-invasive and high-resolution bidimensional analytical technique, with histological and multivariate analyses.ResultsMasson’s trichrome staining, specific for collagen, highlighted a massive presence of collagen in all the analyzed samples, evidencing a surface-related spatial distribution. The nature of collagen, investigated by the FTIRI spectroscopy, appeared more abundant close to the laser-treated surface, with a perpendicular disposition of the bundles respect to the HA; conversely, a parallel distribution was observed around the machined surface. A different secondary structure was also found, with a higher amount of triple helices and a lower quantity of random coils in collagen close to the laser treated surfaces.ConclusionsFTIRI spectroscopy demonstrates that the use of a laser treated transmucosal surface can improve the morphological organization of the peri-implant collagen, which presents a distribution more similar to that of natural teeth.Trial registration: This trial is registered with ClinicalTrials.gov Identifier: (Registration Number: NCT05754970). Registered 06/03/2023, retrospectively registered, https://clinicaltrials.gov/show/NCT05754970.

Is bladder outlet obstruction rat model to induce overactive bladder (OAB) has similarity to human OAB? Research on the events in smooth muscle, collagen, interstitial cell and telocyte distribution

BackgroundCellular and cytoskeletal events of overactive bladder (OAB) have not been sufficiently explored in human bladder due to different limitations. Bladder outlet obstruction (BOO) had been induced in different animal models with different methods to induce (OAB). Similarity of the animal models of BOO to the human OAB is postulated but has not been confirmed. The interstitial cells of Cajal (ICCs), and telocytes (TCs) are an important players in smooth muscles conductivity, they had not been well investigated in the previous BOO models. Objectives are to investigate the morphological pattern of cellular, cytoskeleton and telocytes distribution in BOO rat model and to match the events in two time periods and compare it to the findings in real-world human OAB.MethodsFemale Sprague-Dawley rats (Rattus norvegicus) were randomly divided into: sham (n = 10), BOO 6 W (n = 10), BOO 8 W (n = 10). Operative procedure to Induce BOO was done under anesthesia with intraperitoneal Ketamine administration. The Effect of induction of BOO was evaluated after 6 and 8 weeks. The rats were anesthetized, and the urinary bladder was removed, while the rat was unconscious under anaesthesia it was transferred to the inhalation anaesthesia cage for euthanasia, rats were sacrificed under light anesthesia using isoflurane. Care of animals, surgical procedure, and euthanasia adhered to Guide for the Care and Use of Laboratory Animals, and AVMA Guidelines for the Euthanasia of Animals. The retrieved bladder was processed for examination with histopathology, immunohistochemistry (IHC), and transmission electron microscopy (EM).ResultsHistological examination of the bladder shows thinner urothelium, condensation of collagen between muscle bundles. IHC with c-kit shows the excess distribution of ICCs between smooth muscle bundles. EM shows frequent distribution of TCs that were situated between collagen fibers. Finings in BOO 6 W group and BOO 8 W group were comparable.ConclusionThe animal model study demonstrated increased collagen/ smooth muscle ratio, high intensity of ICCs and presence of TCs. Findings show that a minimally invasive procedure to induce BOO in rats had resulted in an OAB that has morphological changes that were stable in 6 & 8 weeks. We demonstrated the distribution of TCs and ICCs in the rat animal model and defined them. The population of TCs in the BOO rat model is described for the first time, suggests that the TCs and ICCs may contribute to the pathophysiology of OAB. Similarity of animal model to human events OAB was demonstrated. These findings warrant further study to define the role of TCs in OAB.Clinical trial registryThe study does not require a clinical trial registration; it is an experimental animal study in basic science and does not include human subjects.

Synergistic Effects of SDS and H2O2 Combinations on Tracheal Scaffold Development: An In Vitro Study Using Goat Trachea.

Currently, a tissue-engineered trachea has been popularly used as a biological graft for tracheal replacement in severe respiratory diseases. In the development of tissue-engineered tracheal scaffolds, in vitro studies play a crucial role in allowing researchers to evaluate the efficacy and safety of scaffold designs and fabrication techniques before progressing to in vivo or clinical trials. This research involved the decellularization of goat trachea using SDS, H2O2, and their combinations. Various quantitative and qualitative assessments were performed, including histological analysis, immunohistochemistry, and biomechanical testing. Hematoxylin and eosin staining evaluated the cellular content, while safranin O-fast green and Masson's trichrome staining assessed glycosaminoglycan content and collagen distribution, respectively. The immunohistochemical analysis focused on detecting MHC-1 antigen presence. Tensile strength measurements were conducted to evaluate the biomechanical properties of the decellularized scaffolds. The results demonstrated that the combination of SDS and H2O2 for goat tracheal decellularization yielded scaffolds with minimal cellular remnants, low toxicity, preserved ECM, and high tensile strength and elasticity. This method holds promise for developing functional tracheal scaffolds to address severe respiratory diseases effectively.

Simvastatin Significantly Reduced Alcohol-Induced Cardiac Damage in Adolescent Mice

Alcohol abuse by adolescents is becoming a serious health concern as they often progress to becoming alcoholics later in life which may lead to heart problems. Chronic alcohol use alters the cardiac function and structure, such as haemodynamic changes, weakening and loss of cardiomyocytes, myocardial fibrosis, and inflammation. Simvastatin is a commonly used drug for the treatment and management of various cardiovascular problems but information on its protective effects against alcohol-induced cardiomyocyte hypertrophy, fibrosis, and inflammation is lacking in the literature. Four-week-old male (n = 5) and female (n = 5) C57BL/6 J mice were assigned to each experimental group: (I) NT—no administration of alcohol or Simvastatin; (II) ALC—2.5 g/Kg/day of 20% alcohol via intraperitoneal injection (i.p.); (III) SIM—5 mg/Kg/day of Simvastatin via oral gavage; (iv) ALC + SIM5—5 mg/Kg/day of Simvastatin via oral gavage followed by 2.5 g/Kg/day of 20% alcohol via i.p.; and (v) ALC + SIM15—15 mg/Kg/day Simvastatin via oral gavage followed by 2.5 g/Kg/day of 20% alcohol via i.p. After the 28-day treatment period, the heart was removed and processed for H&E, Masson’s trichrome, or TNF-α immunolabelling. The area and diameter of cardiomyocytes were measured on the H&E-stained sections. The distribution of collagen or TNF-α expression was quantified using the deconvolution tool of ImageJ software. The results confirmed alcohol-induced toxicity on the cardiomyocytes and Simvastatin reduced alcohol-induced cardiomyocyte hypertrophy, fibrosis, and inflammation in both sexes. This study demonstrated that Simvastatin, an FDA approved and easily accessible drug, may be beneficial in lowering the prevalence of alcohol-induced cardiovascular diseases (especially in adolescents) which will have a huge financial implication on health systems worldwide.

Avaliação da remodelação tecidual prostática em ratos expostos ao bisfenol A e aos fitoestrógenos genisteína e indol-3-carbinol durante o período perinatal

ABSTRACT: Bisphenol A (BPA) is a compound known for its direct action on the prostate. Prostatic morphogenesis is a critical period when interference by any compound could permanently damage the organ. As such, the present study evaluated the morphological aspects resulting from gestational and lactational administration of BPA, indole-3-carbinol (I3C) and genistein (GEN) in prepubescent male rats. Pregnant Sprague Dawley females were allocated into 4 experimental groups and received the following: C: Control (no treatment); B: BPA (10 µg/Kg); BG: BPA+GEN (5 mg/Kg); BI: BPA+I3C (20 mg/Kg) from gestation day (GD) 17 to postnatal day (PND) 21. After euthanasia on PND22, the prostate was collected and processed. When administered alone, BPA reduced the stromal compartment when compared to group C (P = 0.039). This decline was reversed in the groups submitted to GEN (P = 0.019) or I3C (P = 0.017). The groups treated with BPA (P < 0.0001) and the phytoestrogens (P < 0.0001) exhibited decreased epithelial height in relation to the control group. These changes were observed in stereological and morphometric analyses, but not in fractal analysis (P = 0.569). The area occupied by collagen increased in groups treated with BPA (P < 0.0001) and phytoestrogens (P < 0.0001) in relation to controls, while collagen distribution values were higher in all the treated groups (P < 0.0001), according to fractal analysis. Thus, BPA induced prostrate stroma remodeling with no influence from the phytoestrogens, which may affect glandular development and cause histopathological changes in adulthood.

Ultrastructural Details of Epiretinal Membrane Foveoschisis

Introduction: The aim of this study was to describe differences in the vitreomacular interface (VMI) in idiopathic epiretinal membrane (ERM) foveoschisis compared to macular pseudohole (MPH) and lamellar macular hole (LMH). Methods: We analysed surgically excised epiretinal material and internal limiting membrane (ILM) specimens obtained from 16 eyes of 16 patients with ERM foveoschisis (6 eyes), MPH (5 eyes), and LMH (5 eyes) during standard pars plana vitrectomy (PPV) with membrane peeling. The three entities were classified according to the newly introduced optical coherence tomography (OCT) terminology. Transmission electron microscopy (TEM) was used to describe the ultrastructural features. Results: We found fibrocellular epiretinal tissues in all samples analysed. However, the cell and collagen composition of the VMI differed between groups. Eyes with ERM foveoschisis were characterized by a higher number of cells, multilayered membranes, and thick strands of vitreous collagen embedding the major cell types of myofibroblasts compared to MPH. Eyes with MPH also showed a predominance of myofibroblasts, but these were located directly on the ILM with no collagen between the cells and the ILM. Eyes with LMH showed a thick, multilayered epiretinal proliferation consisting mainly of non-tractional glial cells, corresponding to hypodense epiretinal proliferation on OCT. Eyes with ERM foveoschisis and MPH were more likely to have incomplete PVD compared to LMH in terms of posterior hyaloid status. Discussion/Conclusion: Tractional ERMs in eyes with ERM foveoschisis and MPH differ in their ultrastructure. The main difference is in the amount and topographical distribution of vitreous collagen. However, the epiretinal cell types are predominantly myofibroblasts in both entities. This highlights the importance of distinguishing ERM foveoschisis from both MPH and LMH in terms of pathogenesis and surgical peeling procedures.

Treprostinil inhibits functional activation of scleroderma (SSc) vascular smooth muscle cells (vSMCs) by inhibiting Yap and activating PPARG signaling.

Background:
 Progressive vascular wall thickness and fibrosis are the hallmarks of SSc vasculopathy. Overexpression of TGFB1 in SSc and activation of vSMCs are important steps in the pathogenesis of SSc vascular disease.
 Objectives:
 In this study, we examined the expression levels of COL1, PCNA, PFKP, IP, EP2, IP, and PTGIS in SSc skin, the effects of Treprostinil (prostacyclin analog) on cell proliferation, TGFB1-induced collagen expression in SSc-vSMCs, PPARG expression and Yap nuclear translocation.
 Methods: 
 SSc and control skin biopsies were fixed and 10uM serial sections were cut for histological examination. vSMCs were isolated from involved skin and matched healthy subjects. The expression and distribution of collagen, PCNA, PFKP, IP, EP2, PTGIS, PPARG, and Yap were measured by immunohistochemical staining or immunofluorescent staining. Cell proliferation was measured by MTT assay. The mRNA expression levels were detected by qPCR.
 Results:
 The protein expression levels of collagen, PCNA, PFKP, and EP2 were increased, while the expression levels of PTGIS and IP were decreased in vSMCs of SSc-skin, compared to the control. These results suggested that defective PGI2-IP signaling in SSc-vSMCs may contribute to vessel wall thickness and vascular fibrosis in SSc. Treprostinil inhibited vSMCs proliferation, COL1A1, and PFKP mRNA expression in SSc-vSMCs in a dose-dependent fashion. Treprostinil also inhibited TGFB1-induced COL1A1 mRNA expression in SSc-vSMCs via engagement of EP2. The PPARG expression was significantly increased in treprostinil-treated SSc-vSMCs. Treprostinil decreased the nuclear location of YAP which is induced by 10%FBS and TGFB1.
 Conclusion:
 Defective PGI2-IP signaling in SSc-vSMCs is associated with enhanced expression of collagen, PCNA, and PFKP in SSc vessel walls. The antiproliferative and antifibrotic activity of treprostinil is mediated through the inhibition of YAP nuclear translocation and enhanced PPARG expression. YAP and PPARG might be promising therapeutic targets for the treatment of SSc-related vasculopathy.

Strain inhibition of bacterial collagenase is consistent with a collagen fibril uncrimping mechanism in rat tail tendons

Mechanical strain inhibits bacterial collagenase from cleaving collagen. Additionally, the toe region of a soft tissue’s force–elongation curve arises from sequentially engaging collagen fibrils as the tissue lengthens. Together, these phenomena suggest that mechanical strain may gradually inhibit collagenase activity through a soft tissue’s toe region. Therefore, this investigation sought to test this hypothesis.92 rat tail tendon fascicles from 3 female sentinel animals underwent preliminary stiffness tests, and their force–elongation curves were fit to a collagen distribution model. This distribution-based model calculated the force magnitude corresponding to p% of collagen fibril engagement. Specimens were separated into one of five levels of p, and that level of force was maintained for two hours while being exposed to 0.054 U/mL of bacterial collagenase from C. histolyticum. The specimens were strained to failure following the creep test, and the relative reduction in stiffness was quantified to estimate the fraction of digested fibrils.Every 10% additional collagen engagement corresponded to a 6.3% (97% highest density interval: 4.3 – 8.4%) retention of stiffness, which indicated collagenase inhibition.The results of this investigation were consistent with a strain-inhibition hypothesis along with the established uncrimping mechanism in the toe region. These results support an interaction between mechanical strain and collagenolysis, which may be valuable for disease prevention or treatment.

Label‐free Raman spectroscopy for the assessment of liver fibrosis

AbstractThe enormous incidence and dire consequences of liver fibrosis highlight the need for biologically plausible markers as readouts of pathology and drug efficacy. The classic reference standard for diagnosis of liver fibrosis is histopathological examinations. However, it is burdened by time‐consuming steps, subjectivity, and the static nature of information. Raman spectroscopy is a rapid, objective, and label‐free analytical tool that provides numerous molecular information. Thus, we utilized Raman spectroscopy to characterize the pathological features of liver fibrosis and to evaluate the efficacy of drugs (obeticholic acid [OCA] and gigantol [YS30]). Principal component analysis with subsequent linear discriminant analysis could basically discriminate between fibrotic mice and healthy controls and showed that animals with OCA or YS30 treatment resembled those with vehicle treatment. Moreover, we identified several potential biomarkers, including cytochrome C, for liver fibrosis development based on their Raman “fingerprints.” Raman imaging provided quantitative and spatial distribution of collagen and cytochrome C in situ. In addition, we demonstrated the role of OCA or YS30 in revitalizing mitochondrial function to alleviate liver fibrosis using Raman spectroscopy. Collectively, Raman spectroscopy succeeded in tracking liver fibrosis progression, assessing drug efficacy, and yielding significant molecular information useful for quantitatively linking pathological assessment and mechanism of drug action.

Melatonin acts through different mechanisms to control oxidative stress and primordial follicle activation and survival during in vitro culture of bovine ovarian tissue

This study aims to evaluate the effects of melatonin and its mechanisms of action on preantral follicle activation and survival, stromal cell density and collagen distribution in extracellular matrix (ECM). The involvement of melatonin receptors and mTORC1 pathway in these procedures were also investigated. To this end, ovarian fragments were cultured for six days in α‐MEM+ alone or supplemented with 1000 pM melatonin, 1000 pM melatonin with 1000 pM luzindole (inhibitor of melatonin receptors), or 1000 pM melatonin with 0.16 µg/ml rapamycin (mTORC1 inhibitor). At the end of culture period, tissues were processed for classical histology, and the follicles were classified as normal or degenerated, as well as in primordial or growing follicles. The ovarian stromal cell density and ECM collagen distribution were also evaluated. Samples of ovarian tissues were also destined to measure the levels of thiol and mRNA for CAT, SOD, GPX1 and PRDX1, as well as the activity of antioxidant enzymes CAT, SOD, and GPX1. The results demonstrated that ovarian tissues cultured with melatonin, melatonin with luzindole or melatonin with rapamycin had significantly higher percentage of morphologically normal follicles than those cultured in control medium (α‐MEM+). However, the presence of either luzindole or rapamycin, did not block the positive effects of melatonin on follicle survival (P > 0.05). Although the presence of melatonin in culture medium reduced the percentage of primordial follicles and increased the percentage of development follicles, these positive effects of melatonin were blocked by either luzindole or rapamycin (P < 0.05). Melatonin, melatonin with luzindole or melatonin with rapamycin did not influence the number of ovarian stromal cells. In contrast, melatonin significantly increased the percentages of collagen in ovarian tissues, but the positive effects of melatonin were blocked by either luzindole or rapamycin. Tissues cultured with melatonin and rapamycin had higher levels of mRNA for CAT and lower GPx activity when compared to those cultured in control medium. In conclusion, melatonin promotes primordial follicle activation, increases collagen fiber in ECM of in vitro cultured bovine ovarian tissue through its membrane-coupled receptors and mTORC1. Oppositely, melatonin increase follicles survival by acting through other pathways, since it can pass through cell membranes and directly regulate oxidative stress.

Does the Embryologic Limb Rotation Modify the Distribution of Dermal Collagen and Elastic Fibers in Different Planes of Human Extremities?

Does the Embryologic Limb Rotation Modify the Distribution of Dermal Collagen and Elastic Fibers in Different Planes of Human Extremities?

Beta-Sitosterol Alters Collagen Distribution in Prostate Fibroblasts

Herbal supplements containing several types of plant sterols, vitamins, and minerals, are marketed for prostate health. In the majority of these supplements, the most abundant plant sterol is saw palmetto extract or its’ principal component, beta-sitosterol. In terms of prostate health, previous work almost exclusively focused on the effects of beta-sitosterol on prostatic epithelium, with little attention paid to the effects on prostatic stroma. This omission is a concern, as the abnormal accumulation of collagen, or fibrosis, of the prostatic stroma has been identified as a factor contributing to lower urinary tract symptoms and dysfunction in aging men. To address whether beta-sitosterol may be promoting prostatic fibrosis, immortalized and primary prostate stromal fibroblasts were subjected to immunoblotting, immunofluorescence, qRT-PCR, ELISA, and image quantitation and analysis techniques to elucidate the effects of beta-sitosterol on cell viability and collagen expression and cellular localization. The results of these studies show that beta-sitosterol is nontoxic to prostatic fibroblasts and does not stimulate collagen production by these cells. However, beta-sitosterol alters collagen distribution and sequesters collagen within prostatic fibroblasts, likely in an age-dependent manner. This is a significant finding as prostate health supplements are used predominantly by middle aged and older men who may, then, be affected disproportionately by these effects.

Construct and criterion validity of muscle ultrasonography for assessment of skeletal muscle in patients recovering from COVID-19.

Background: The purpose was to investigate the content, construct, and criterion validity of muscle ultrasound in a mixed cohort of participants recovering from mild and critical COVID-19. Methods: A secondary analysis of a prospective cross-sectional study was conducted on data obtained from a battery of muscle and physical function assessments including a muscle biopsy and muscle ultrasonography (US). Rectus femoris (RF) muscle thickness (mT), quadricep complex (QC) mT, RF muscle cross-sectional area (CSA) using 2D freeform trace and estimated from Feret's diameter, and RF echo intensity (EI) were assessed with US. Muscle fiber CSA, fiber type, protein content in muscle fibers, extracellular matrix content (ECM; wheat-germ agglutin), and percent area of collagen in ECM (picrosirius red) were examined from vastus lateralis muscle biopsies. Spearman rho correlations (r) were performed to assess validity of ultrasound parameters. Results: Thirty-three individuals participated including 11 patients surviving critical COVID-19, 15 individuals recovering from mild-COVID, and 7 controls. There were several significant correlations between RF mT, QC mT, RF CSA, and RF EI with age, comorbid burden, body-mass index, and measures of muscle strength, muscle power, and physical function (range r = 0.35-0.83). RF Feret's CSA correlated to CSA of type II muscle fibers (r = 0.41, p = 0.022) and the average size of all muscle fibers (r = 0.39, p = 0.031). RF EI was correlated with collagen in muscle ECM (r = 0.53, p = 0.003) and protein content in muscle tissue (r = -0.52, p = 0.012). Conclusion: Muscle size and quality measured using US has moderate content and construct validity, and to lesser extent, fair to moderate criterion validity in a mixed cohort of individuals recovering from COVID. Muscle ultrasound quality (EI) appears to be sensitive at detecting muscle dysfunction as it is associated with strength, power, physical function, and collagen distribution in a mixed group of individuals recovering from COVID-19.

Development of a hyaluronic acid—collagen bioink for shear-induced fibers and cells alignment

Human tissues are characterized by complex composition and cellular and extracellular matrix (ECM) organization at microscopic level. In most of human tissues, cells and ECM show an anisotropic arrangement, which confers them specific properties. In vitro, the ability to closely mimic this complexity is limited. However, in the last years, extrusion bioprinting showed a certain potential for aligning cells and biomolecules, due to the application of shear stress during the bio-fabrication process. In this work, we propose a strategy to combine collagen (col) with tyramine-modified hyaluronic acid (THA) to obtain a printable col-THA bioink for extrusion bioprinting, solely-based on natural-derived components. Collagen fibers formation within the hybrid hydrogel, as well as collagen distribution and spatial organization before and after printing, were studied. For the validation of the biological outcome, fibroblasts were selected as cellular model and embedded in the col-THA matrix. Cell metabolic activity and cell viability, as well as cell distribution and alignment, were studied in the bioink before and after bioprinting. Results demonstrated successful collagen fibers formation within the bioink, as well as collagen anisotropic alignment along the printing direction. Furthermore, results revealed suitable biological properties, with a slightly reduced metabolic activity at day 1, fully recovered within the first 3 d post-cell embedding. Finally, results showed fibroblasts elongation and alignment along the bioprinting direction. Altogether, results validated the potential to obtain collagen-based bioprinted constructs, with both cellular and ECM anisotropy, without detrimental effects of the fabrication process on the biological outcome. This bioink can be potentially used for a wide range of applications in tissue engineering and regenerative medicine in which anisotropy is required.

Distribution of smooth muscle actin and collagen in superficial peritoneal endometriotic lesions varies from the surrounding microenvironment

Research questionDo different subtypes of superficial peritoneal endometriotic lesions exist, based on the presence and morphology of smooth muscle, collagen fibres and immune cell populations? DesignA retrospective cohort study of 24 patients, from across the menstrual cycle, with surgically and histologically confirmed endometriosis. Immunofluorescence was used to delineate the CD10 stromal area of lesions (n = 271 lesions from 67 endometriotic biopsies), and then smooth muscle actin (SMA) positive tissue and immune cell populations (CD45+ and CD68+) were quantified within and adjacent to these lesions. Second harmonic generation microscopy was used to evaluate the presence and morphology of type-1 collagen fibres within and surrounding lesions. ResultsOverall, immune cell numbers and the area of SMA and collagen within endometriotic lesions tended to be low, but a spectrum of presentations significantly varied, particularly in the adjacent tissue microenvironment, based on lesion locations, the morphology of endometriotic gland profiles, or both. Lesions in which collagen fibres formed well aligned capsules around the CD10+ stromal border were identified compared with lesions in which collagen fibre distribution was random. Considerable inter- and intra-patient variability in the morphology of SMA and collagen was observed within and surrounding lesions. ConclusionThese data demonstrate considerable diversity in the presence of immune cells and morphology of SMA and collagen within, but even more so, surrounding endometriotic lesions, even within individual patients. This heterogeneity, especially within individual patients, presents a challenge to incorporating these cell and tissue types into any new endometriosis classification systems or prognostic approaches.

Experimental research on mechanism of collagen remodeling and excellent properties of skin incisions under dual beam laser welding

For problems of water loss caused by rapid energy dissipation and insufficient depth of heat-affected zone (HAZ) during skin welding with single laser beam. A dual beam 1064 nm semiconductor laser was used to weld incisions on the back of porcine skin, and the dual beam laser was consisted of preliminary beam and the secondary beam. The main beam is vertically incident, and the secondary beam is deflected by 30° on a vertical basis. In this study, there are three types of energy ratios of dual beam laser, which are 1:1, 2:1 and 3:1. From 77.22 J/cm2 to 135.4 J/cm2 stepped increasing energy density, the fusion performances of skin incisions after welding were comprehensively analyzed and characterized. Using Matlab and high-precision dynamometer to evaluate thermal denaturation and biomechanical properties, combined with H&E staining, Masson and Sirius red staining to analyze the microstructure characteristics, collagen deposition behavior and distribution around the HAZ of incisions after welding. The results showed that when the energy density was 102.96 J/cm2, at the energy ratio of 1:1, the collagen network structure was dense, collagen deposition was 48 %, thermal damage value was 0.39, and the immediate tensile strength was 206.3 N/cm2, which was far above the standard of wound healing performances after surgical treatment, and the thermal denaturation was controlled at a low level, realizing the reconstruction of collagen after fracture, achieving the optimal dual-beam laser welding performance, in vivo test results found that above laser type and energy ratio can reduce healing cycle into 7 days, which provided important references for later study on laser tissue welding technology.

Versatile fiber-reinforced hydrogels to mimic the microstructure and mechanics of human vocal-fold upper layers

Human vocal folds are remarkable soft laryngeal structures that enable phonation due to their unique vibro-mechanical performances. These properties are tied to their specific fibrous architecture, especially in the upper layers, which comprise a gel-like composite called lamina propria. The lamina propria can withstand large and reversible deformations under various multiaxial loadings. Despite their importance, the relationships between the microstructure of vocal folds and their resulting macroscopic properties remain poorly understood. There is a need for versatile models that encompass their structural complexity while mimicking their mechanical features. In this study, we present a candidate model inspired by histological measurements of the upper layers of human vocal folds. Bi-photonic observations were used to quantify the distribution, orientation, width, and volume fraction of collagen and elastin fibers between histological layers. Using established biomaterials, polymer fiber-reinforced hydrogels were developed to replicate the fibrillar network and ground substance of native vocal fold tissue. To achieve this, jet-sprayed poly(ε-caprolactone) fibrillar mats were successfully impregnated with poly(L-lysine) dendrimers/polyethylene glycol hydrogels. The resulting composites exhibited versatile structural, physical and mechanical properties that could be customized through variations in the chemical formulation of their hydrogel matrix, the microstructural architecture of their fibrous networks (i.e., fiber diameter, orientation and volume fraction) and their assembly process. By mimicking the collagen network of the lamina propria with polymer fibers and the elastin/ground substance with the hydrogel composition, we successfully replicated the non-linear, anisotropic, and viscoelastic mechanical behavior of the vocal-fold upper layers, accounting for inter/intra-individual variations. The development of this mimetic model offers promising avenues for a better understanding of the complex mechanisms involved in voice production. Statement of significanceHuman vocal folds are outstanding vibrating soft living tissues allowing phonation. Simple physical models that take into account the histological structure of the vocal fold and recapitulate its mechanical features are scarce. As a result, the relations between tissue components, organisation and vibro-mechanical performances still remain an open question. We describe here the development and the characterization of fiber-reinforced hydrogels inspired from the vocal-fold microstructure. These systems are able to reproduce the mechanics of vocal-fold tissues upon realistic cyclic and large strains under various multi-axial loadings, thus providing a mimetic model to further understand the impact of the fibrous network microstructure in phonation.

Distribution, Typical Structure and Self-Assembly Properties of Collagen from Fish Skin and Bone.

The source and type of collagen are crucial to its application, and both play a decisive role. Collagen was prepared from both tilapia skin and bone and skate skin and cartilage, named as CI-TI-s, CI-TI-b, CI-SK-s, and CII-SK-c, respectively. Types, distributions, structures, and self-assembly of collagen were studied. It showed that yellow collagen fibers from skin arranged longitudinally, while collagen fibers from skate cartilages displayed varying colors. CI-TI-s, CI-TI-b, CI-SK-s, and CII-SK-c showed the typical amide A (3316-3336 cm-1) and amide B (2929-2948 cm-1) in FTIR spectra. CI-TI-b and CII-SK-c showed 218-229 nm of UV absorption, 11.56-12.20 Å of d values in XRD, and 0.12-0.14 of Rpn values in CD. The thermal denaturation temperatures of CI-TI-s and CI-SK-s were 30.7 and 20.6 °C, respectively. The self-assembly of CI-TI-s and CII-SK-c were maximum at pH 7.2 and 7.4-7.6, respectively. The unique collagen peptides of tilapia and skate were GPSGPQGAVGATGPK, PAMPVPGPMGPMGPR, SPAMPVPGPMGPMGPR, GESGPSGPAGPAGPAGVR, SSGPPVPGPIGPMGPR, GLTGPIGVPGPPGAQGEK, GLAGPQGPR, and GLSGDPGVQGIK, respectively. The unique peptides of type I and type II collagen were GPTGEIGATGLAGAR, GVLGLTGMR, LGLTGMR, GEPGAAGPAGPSGPMGPR, SSGPPVPGPIGPMGPR, and GLSGDPGVQGIK, respectively.

Hypoxia-treated adipose mesenchymal stem cell-derived exosomes attenuate lumbar facet joint osteoarthritis

BackgroundLumbar facet joint osteoarthritis (LFJ OA) is a common disease, and there is still a lack of effective disease-modifying therapies. Our aim was to determine the therapeutic effect of hypoxia-treated adipose mesenchymal stem cell (ADSC)-derived exosomes (Hypo-ADSC-Exos) on the protective effect against LFJ OA.MethodsThe protective effect of Hypo-ADSC-Exos against LFJ OA was examined in lumbar spinal instability (LSI)-induced LFJ OA models. Spinal pain behavioural assessments and CGRP (Calcitonin Gene-Related Peptide positive) immunofluorescence were evaluated. Cartilage degradation and subchondral bone remodelling were assessed by histological methods, immunohistochemistry, synchrotron radiation-Fourier transform infrared spectroscopy (SR-FTIR), and 3D X-ray microscope scanning.ResultsHypoxia enhanced the protective effect of ADSC-Exos on LFJ OA. Specifically, tail vein injection of Hypo-ADSC-Exos protected articular cartilage from degradation, as demonstrated by lower FJ OA scores of articular cartilage and less proteoglycan loss in lumbar facet joint (LFJ) cartilage than in the ADSC-Exo group, and these parameters were significantly improved compared to those in the PBS group. In addition, the levels and distribution of collagen and proteoglycan in LFJ cartilage were increased in the Hypo-ADSC-Exo group compared to the ADSC-Exo or PBS group by SR-FTIR. Furthermore, Hypo-ADSC-Exos normalized uncoupled bone remodelling and aberrant H-type vessel formation in subchondral bone and effectively reduced symptomatic spinal pain caused by LFJ OA in mice compared with those in the ADSC-Exo or PBS group.ConclusionsOur results show that hypoxia is an effective method to improve the therapeutic effect of ADSC-Exos on ameliorating spinal pain and LFJ OA progression.