Stromal Injury Research Articles

Corneal keratocytes, fibroblasts, and myofibroblasts exhibit distinct transcriptional profiles in vitro.

After stromal injury to the cornea, the release of growth factors and pro-inflammatory cytokines promotes the activation of quiescent keratocytes into a migratory fibroblast and/or fibrotic myofibroblast phenotype. Persistence of the myofibroblast phenotype can lead to corneal fibrosis and scarring, which are leading causes of blindness worldwide. This study aims to establish comprehensive transcriptional profiles for cultured corneal keratocytes, fibroblasts, and myofibroblasts to gain insights into the mechanisms through which these phenotypic changes occur. Primary rabbit corneal keratocytes were cultured in either defined serum-free media (SF), fetal bovine serum (FBS) containing media, or in the presence of TGF-β1 to induce keratocyte, fibroblast, or myofibroblast phenotypes, respectively. Bulk RNA sequencing followed by bioinformatic analyses was performed to identify significant differentially expressed genes (DEGs) and enriched biological pathways for each phenotype. Genes commonly associated with keratocytes, fibroblasts, or myofibroblasts showed high relative expression in SF, FBS, or TGF-β1 culture conditions, respectively. Differential expression and functional analyses revealed novel DEGs for each cell type, as well as enriched pathways indicative of differences in proliferation, apoptosis, extracellular matrix (ECM) synthesis, cell-ECM interactions, cytokine signaling, and cell mechanics. Overall, these data demonstrate distinct transcriptional differences among cultured corneal keratocytes, fibroblasts, and myofibroblasts. We have identified genes and signaling pathways that may play important roles in keratocyte differentiation, including many related to mechanotransduction and ECM biology. Our findings have revealed novel molecular markers for each cell type, as well as possible targets for modulating cell behavior and promoting physiological corneal wound healing.

Mild phototherapy strategies for efficient treatment of bacterial keratitis while avoiding side effects

Bacterial keratitis (BK) is one of the most threatening ocular diseases, leading to stromal injury, corneal perforation, or even blindness. Avoiding side effect is necessary for efficient BK treatment. Herein, we develop GDY-Ag@AgCl heterojunction catalysts to achieve BK treatment via mild phototherapy strategy. Mild photothermal therapy (PTT) can greatly enhance the generation of ROS produced by photocatalytic therapy (PCT), which can decrease the dosage of photosensitizer, as well as solve the issue of uncontrolled local overheating and the accumulation of PTT agents, minimizing side effects and avoiding secondary injuries. The results demonstrate that lower dosage of PTT/PCT agents, and milder and controlled thermal effect of GDY-Ag@AgCl exhibits excellent inactivation performance against ampicillin-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA). Moreover, this phototherapy strategy not only achieves more effective treatment of BK induced by MRSA with no side effects but also shows a superior ability to alleviate inflammation. This strategy offers a safe and promising theragnostic approach for treating ocular drug-resistant bacterial infections.

Patterned collagen films loaded with miR-133b@MBG-NH2 for potential applications in corneal stromal injury repair

Corneal stromal injury is a common surgical disease. With the development of tissue engineering materials, many artificial corneal scaffolds have been developed to replace allograft corneal transplantation and solve the problem of corneal donor shortage. However, few researchers have paid attention to corneal stromal wound healing. Herein, a nanocomposite of amino modified mesoporous bioactive glass (MBG-NH2) and microRNA-133b (miR-133b) was introduced into the patterned collagen films to achieve corneal stromal injury repair. MBG-NH2 nanoparticles as a nano delivery carrier could efficiently load miR-133b and achieve the slow release of miR-133b. The physicochemical properties of collagen films were characterized and found the microgrooved collagen films loaded with miR-133b@MBG-NH2 nanoparticles possessed similar swelling properties, optical clarity, and biodegradability to the natural cornea. In vitro cell experiments were also conducted and proved that the patterned collagen films with miR-133b@MBG-NH2 possessed good biocompatibility, and miR-133b@MBG-NH2 nanoparticles could be significantly uptake by rabbit corneal stromal cells (RCSCs) and have a significant impact on the orientation, proliferation, migration, and gene expression of RCSCs. More importantly, the patterned collagen films with miR-133b@MBG-NH2 could effectively promote the migration of RCSCs and accelerate wound healing process, and down-regulate the expression levels of α-SMA, COL-I, and CTGF genes associated with myofibroblast differentiation of corneal stromal cells, which has a potential application prospect in the repair of corneal stromal injury.

Mesenchymal Stem Cell-Laden In Situ-Forming Hydrogel for Preventing Corneal Stromal Opacity.

The aims of this study were to construct a mesenchymal stem cell (MSC)-laden in situ-forming hydrogel and study its effects on preventing corneal stromal opacity. The native gellan gum was modified by high temperature and pressure, and the rabbit bone marrow MSCs were encapsulated before adding Ca 2+ to initiate cross-linking. The effects of the hydrogel on 3D culture and gene expression of the rabbit bone marrow MSCs were observed in vitro. Then, the MSC-hydrogel was used to repair corneal stromal injury in New Zealand white rabbits within 28 days postoperation. The short-chain gellan gum solution has a very low viscosity (<0.1 Pa·s) that is ideal for encapsulating cells. Moreover, mRNA expressions of 3D-cultured MSCs coding for corneal stromal components (decorin, lumican, and keratocan) were upregulated (by 127.8, 165.5, and 25.4 times, respectively) ( P < 0.05) on day 21 in vitro and were verified by Western blotting results. For the in vivo study, the corneal densitometry of the experimental group was (20.73 ± 1.85) grayscale units which was lower than the other groups ( P < 0.05). The MSC-hydrogel downregulated mRNA expression coding for fibrosis markers (α-smooth muscle actin, vimentin, collagen type 5-α1, and collagen type 1-α1) in the rabbit corneal stroma. Furthermore, some of the 5-ethynyl-2'-deoxyuridine (EdU)-labeled MSCs integrated into the upper corneal stroma and expressed keratocyte-specific antigens on day 28 postoperation. The short-chain gellan gum allows MSCs to slowly release to the corneal stromal defect and prevent corneal stromal opacity. Some of the implanted MSCs can integrate into the corneal stroma and differentiate into keratocytes.

Good manufacturing practice production of human corneal limbus-derived stromal stem cells and in vitro quality screening for therapeutic inhibition of corneal scarring

BackgroundMesenchymal stem cells in the adult corneal stroma (named corneal stromal stem cells, CSSCs) inhibit corneal inflammation and scarring and restore corneal clarity in pre-clinical corneal injury models. This cell therapy could alleviate the heavy reliance on donor materials for corneal transplantation to treat corneal opacities. Herein, we established Good Manufacturing Practice (GMP) protocols for CSSC isolation, propagation, and cryostorage, and developed in vitro quality control (QC) metric for in vivo anti-scarring potency of CSSCs in treating corneal opacities.MethodsA total of 24 donor corneal rims with informed consent were used—18 were processed for the GMP optimization of CSSC culture and QC assay development, while CSSCs from the remaining 6 were raised under GMP-optimized conditions and used for QC validation. The cell viability, growth, substrate adhesion, stem cell phenotypes, and differentiation into stromal keratocytes were assayed by monitoring the electric impedance changes using xCELLigence real-time cell analyzer, quantitative PCR, and immunofluorescence. CSSC’s conditioned media were tested for the anti-inflammatory activity using an osteoclastogenesis assay with mouse macrophage RAW264.7 cells. In vivo scar inhibitory outcomes were verified using a mouse model of anterior stromal injury caused by mechanical ablation using an Algerbrush burring.ResultsBy comparatively assessing various GMP-compliant reagents with the corresponding non-GMP research-grade chemicals used in the laboratory-based protocols, we finalized GMP protocols covering donor limbal stromal tissue processing, enzymatic digestion, primary CSSC culture, and cryopreservation. In establishing the in vitro QC metric, two parameters—stemness stability of ABCG2 and nestin and anti-inflammatory ability (rate of inflammation)—were factored into a novel formula to calculate a Scarring Index (SI) for each CSSC batch. Correlating with the in vivo scar inhibitory outcomes, the CSSC batches with SI < 10 had a predicted 50% scar reduction potency, whereas cells with SI > 10 were ineffective to inhibit scarring.ConclusionsWe established a full GMP-compliant protocol for donor CSSC cultivation, which is essential toward clinical-grade cell manufacturing. A novel in vitro QC–in vivo potency correlation was developed to predict the anti-scarring efficacy of donor CSSCs in treating corneal opacities. This method is applicable to other cell-based therapies and pharmacological treatments.

Histological Assessment of the Bile Duct before Liver Transplantation: Does the Bile Duct Injury Score Predict Biliary Strictures?

Histological injury to the biliary tree during organ preservation leads to biliary strictures after liver transplantation. The Bile Duct Injury (BDI) score was developed to assess histological injury and identify the grafts most likely to develop biliary strictures. The BDI score evaluates the bile duct mural stroma, peribiliary vascular plexus (PVP) and deep peribiliary glands (DPGs), which were correlated with post-transplant biliary strictures. However, the BDI score has not been externally validated. The aim of this study was to verify whether the BDI score could predict biliary strictures at our transplant centre. Brain-dead donor liver grafts transplanted at a single institution from March 2015 to June 2016 were included in this analysis. Bile duct biopsies were collected immediately before transplantation and assessed for bile duct injury by two blinded pathologists. The primary outcome was the development of clinically significant biliary strictures within 24 months post-transplant. Fifty-seven grafts were included in the study which included 16 biliary strictures (28%). Using the BDI score, mural stromal, PVP and DPG injury did not correlate with biliary strictures including Non-Anastomotic Strictures. Severe inflammation (>50 leucocytes per HPF) was the only histological feature inversely correlated with the primary outcome (absent in the biliary stricture group vs. 41% in the no-stricture group, p = 0.001). The current study highlights limitations of the histological assessment of bile duct injury. Although all grafts had bile duct injury, only inflammation was associated with biliary strictures. The BDI score was unable to predict post-transplant biliary strictures in our patient population.

Taxane chemotherapy induces stromal injury that leads to breast cancer dormancy escape.

A major cause of cancer recurrence following chemotherapy is cancer dormancy escape. Taxane-based chemotherapy is standard of care in breast cancer treatment aimed at killing proliferating cancer cells. Here, we demonstrate that docetaxel injures stromal cells, which release protumor cytokines, IL-6 and granulocyte colony stimulating factor (G-CSF), that in turn invoke dormant cancer outgrowth both in vitro and in vivo. Single-cell transcriptomics shows a reprogramming of awakened cancer cells including several survival cues such as stemness, chemoresistance in a tumor stromal organoid (TSO) model, as well as an altered tumor microenvironment (TME) with augmented protumor immune signaling in a syngeneic mouse breast cancer model. IL-6 plays a role in cancer cell proliferation, whereas G-CSF mediates tumor immunosuppression. Pathways and differential expression analyses confirmed MEK as the key regulatory molecule in cancer cell outgrowth and survival. Antibody targeting of protumor cytokines (IL-6, G-CSF) or inhibition of cytokine signaling via MEK/ERK pathway using selumetinib prior to docetaxel treatment prevented cancer dormancy outgrowth suggesting a novel therapeutic strategy to prevent cancer recurrence.

Oxycodone alleviates mifepristone-stimulated human endometrial stromal cell injury by activating the Keap1/Nrf2/HO-1 signaling pathway.

Endometrial injury is a common disease in women caused by intrauterine inflammation, infections, and endocrine disorders. Human endometrial stromal cells (hEndoSCs) can maintain endometrial homeostasis and play an important role in repairing endometrial injury. Mifepristone, a steroidal anti-progesterone drug, is widely used in the field of reproductive medicine worldwide. Mifepristone-induced hEndoSC injury has been used to study endometrial injury in vitro. At present, the pathogenesis and potential regulatory mechanisms of oxycodone in endometrial injury remain unknown. We aimed to evaluate the functions of oxycodone in mifepristone-stimulated hEndoSC injury and analyze its potential molecular mechanism. hEndoSC viability, cytotoxicity, and apoptosis were analyzed using the methyl thiazolyl tetrazolium assay, the lactate dehydrogenase assay, and flow cytometry, respectively. Furthermore, the levels of cleaved-Caspase3, Keap1, Nrf2, HO-1, and NQO1 were assessed using reverse transcription quantitative polymerase chain reaction and western blot analysis, and the release of inflammatory cytokines was determined using the enzyme-linked immunosorbent assay. We observed that oxycodone had no adverse effects on hEndoSCs; rather, it protected hEndoSCs against mifepristone-induced endometrial damage, as confirmed by the enhanced cell viability, reduced number of apoptotic cells, decreased Caspase3 activity and inflammatory cytokine secretion, and increased Keap1/Nrf2/HO-1 pathway-related protein expression. In addition, we found that the protective effects of oxycodone on mifepristone-induced hEndoSC injury were inhibited by ML385 (a Keap1/Nrf2/HO-1 inhibitor). In summary, we confirmed that oxycodone alleviates mifepristone-induced hEndoSC injury by activating the Keap1/Nrf2/HO-1 signaling pathway.

Comparative analysis of the regenerative potential of blood derivatives on a cell model of corneal stromal injury

BACKGROUND: Agents based on blood derivatives contain substances that accelerate the healing of wound defects. Nowadays data have been accumulated that various blood derivatives have different effects on regeneration processes. Therefore, a comparative study of the effect of several types of such preparations on the corneal stromas cell culture is relevant.&#x0D; AIM: Impact assessment of 3 different blood derivatives on the processes of proliferation, migration and cell death of keratocytes in vitro.&#x0D; MATERIAL AND METHODS: This study was conducted on a primary cell culture of human corneal keratocytes. Blood samples were obtained from the cubital vein of healthy volunteers after signing informed consent to participate in the study. Monolayer wound healing test, formazan test to assess proliferation, staining for Annexin V to assess the processes of apoptosis and necrosis were perfomed. Moreover, effect evaluation of platelet-rich plasma, intact serum and processed serum were performed.&#x0D; RESULTS: It was shown that all 3 stimulants have a positive effect on the regeneration of the experimental stromal defect, but it is implemented in different ways. Processed whey showed the greatest stimulating effect on the process of cell migration. This serum reduced the level of cell death, but had almost no effect on proliferation processes. It is advisable to further study different types of stimulants to obtain processed serum. Intact serum increased the percentage of dividing cells more than others, but also proportionally increased the percentage of death in culture. Platelet-rich plasma showed the weakest effect on the migratory ability of keratocytes, but it still differed significantly from the control. This stimulator had little effect on the process of cell division, and had no effect on the overall level of cell death.&#x0D; CONCLUSION: Experimental data can be taken into account in choosing of the management in clinical practice and personalized usage of blood derivatives, specifically in prescribing some blood derivatives to accelerate regeneration, and other derivatives for reducing cell deaths processes.

What UV is what you get: skin injury by ultraviolet (UV) light results in renal cell loss and damage in the presence of a CD177high neutrophil population.

Abstract Immune-mediated communication between organs has been implicated in the pathogenesis of many inflammatory and autoimmune diseases. We previously showed that skin injury by UV light triggers neutrophil migration to the kidney, stimulating expression of renal injury genes. This skin-kidney axis is highly relevant to lupus patients, for whom skin exposure to UV sunlight rays can lead to kidney disease flares. Here, we applied 10X single-cell RNA-seq on saline-perfused digested kidneys and Ly6G-enriched neutrophils of B6 mice, prior to and 2 days after a single dose of UV, to define renal cell subsets damaged by skin exposure to UV and how neutrophils mediate the injury. Dimensional reduction analysis revealed 14 kidney cell types and showed that exposure to UV led to the loss of podocytes and distal tubular cells (DTCs) and an expansion in endothelial and stromal cells. Differential gene expression analysis showed that podocytes and DTCs in UV-exposed mice upregulated genes associated with renal injury, including type I interferon genes, and downregulated genes important for cellular integrity and function. Neutrophil scRNA-seq revealed a unique activated CD177 highneutrophil population in the kidneys of UV exposed mice, defined as the endpoint of the psuedotime trajectory by diffusion mapping. Ligand-receptor pair analyses showed skin injury by UV altered neutrophil interactions with renal structural cells, with expanded binding to stromal cells. While the CD177 highneutrophil cluster was dominant in the kidney, it was also found in the lung, but not in the skin and blood. These studies introduce a novel model by which CD177 highneutrophils may mediate podocyte, DTC, and stromal kidney injury secondary to sterile skin inflammation. Supported by grants from NIH (1R21AR079661 – 01, 5P20GM130454-02), The Department of Defense (W81XWH2110889), and Dartmouth Health Start Up Funds.

The corneal fibroblast: The Dr. Jekyll underappreciated overseer of the responses to stromal injury

PurposeTo review the functions of corneal fibroblasts in wound healing. MethodsLiterature review. ResultsCorneal fibroblasts arise in the corneal stroma after anterior, posterior or limbal injuries and are derived from keratocytes. Transforming growth factor (TGF) β1 and TGFβ2, along with platelet-derived growth factor (PDGF), are the major modulators of the keratocyte to corneal fibroblast transition, while fibroblast growth factor (FGF)-2, TGFβ3, and retinoic acid are thought to regulate the transition of corneal fibroblasts back to keratocytes. Adequate and sustained levels of TGFβ1 and/or TGFβ2, primarily from epithelium, tears, aqueous humor, and corneal endothelium, drive the development of corneal fibroblasts into myofibroblasts. Myofibroblasts have been shown in vitro to transition back to corneal fibroblasts, although apoptosis of myofibroblasts has been documented as a major contributor to the resolution of fibrosis in several in situ corneal injury models. Corneal fibroblasts, aside from their role as a major progenitor to myofibroblasts, also perform many critical functions in the injured cornea, including the production of critical basement membrane (BM) components during regeneration of the epithelial BM and Descemet's membrane, production of non-basement membrane-associated stromal collagen type IV to control and downregulate TGFβ effects on stromal cells, release of chemotactic chemokines that attract bone marrow-derived cells to the injured stroma, production of growth factors that modulate regeneration and maturation of the overlying epithelium, and production of collagens and other ECM components that contribute to stromal integrity after injury. ConclusionsCorneal fibroblasts are major contributors to and overseers of the corneal response to injuries.

Exosomes from bone mesenchymal stem cells alleviate mifepristone-induced human endometrial stromal cell injury by inhibiting TLR3 via delivering miR-941.

We aim to investigate the protective effect and underlying mechanisms of BMSCs-exo on human endometrial stromal cells (HESCs) induced by mifepristone in this study. BMSCs-exo were extracted and then identified by transmission electron microscopy and western-blot assay. RT-PCR assay was used to determine the level of miR-941. MiR-941 mimics or inhibitor were transfected into BMSCs and the exosomes were extracted. Then, Cell activity, apoptosis rate, cell migration and invasion, as well as the expression of angiogenic proteins were determined in HESCs stimulated by mifepristone and BMSCs-exo. Next, Dual-luciferase reporting assay was used to verify the targeted binding of miR-941 to TLR3, and the TLR3 expression in HESCs was detected by RT-PCR and western-blot. Finally, TLR3 was overexpressed to evaluate the effects of miR-941 from BMSCs-exo on cell apoptosis, cell invasion and angiogenesis in HESCs induced by mifepristone. miR-941 was highly expressed in BMSCs-exo. Exosome miR-941 in BMSCs-exo inhibited the cell apoptosis, and promoted cell activity, cell migration, invasion as well as angiogenesis were also improved in HESCs induced by mifepristone. TLR3 was a target of miR-941, which was up-regulated in mifepristonetreated HESCs. We further found that miR-941 derived from BMSCs-exo down-regulated the expression of TLR3 in HESCs treated by mifepristone. In addition, TLR3 overexpression blocked the inhibition of miR-941 on mifepristone-induced cell apoptosis, as well as cell migration and angiogenesis in HESCs. Thus, we concluded that BMSCs-exo has protective effect on mifepristone-induced cell damage by delivering miR-941 which targeted TLR3 and regulated cell activity, migration, and angiogenesis in HESCs.

Magic Bullets: The Coming Age of Meaningful Pharmacological Control of the Corneal Responses to Injury and Disease.

Corneal injuries from chemical burns, mechanical trauma, infections, immunological rejections, surgical complications, and some diseases are commonly associated with persistent epithelial defects (PED), neurotrophic epitheliopathy, scarring fibrosis, corneal neovascularization (CNV), and/or corneal endothelial damage that lead to vision loss. Several Food and Drug Administration (FDA) approved medications have recently become available, are currently in clinical trials, or are likely to enter clinical trials in the near future. For example, a 2-week course of topical human recombinant nerve growth factor is frequently an effective treatment for corneal neurotrophic epitheliopathy associated with PEDs. Topical losartan, an angiotensin converting enzyme II receptor antagonist that also inhibits TGF beta signaling, has been shown to effectively decrease myofibroblast generation and scarring fibrosis in alkali burn injury and Descemetorhexis rabbit models. Small molecule topical tyrosine kinase inhibitors, such as sunitinib and axitinib, FDA approved as chemotherapeutic agents to treat specific cancers, have also been found to be effective topical inhibitors of CNV in animal and human trials. Rho-kinase inhibitors, such as ripasudil and netarsudil, that are currently approved agents for the treatment of glaucoma in some countries, have been shown to stimulate corneal endothelial proliferation in animal studies and human trials, and may accelerate the regeneration of Descemet's membrane. These agents, as well as other drugs in development, will be used in targeted combinations to treat corneal pathophysiology associated with epithelial healing disorders, stromal scarring fibrosis, CNV, and corneal endothelial injury during the next decade.

Thinking Beyond the Marrow: Deceptive Presentation of a Rare Malignant Neoplasm

A middle-aged man presented with vague abdominal and chest pain. Laboratory results suggested anemia. Computed tomography of the chest and abdomen revealed mediastinal lymphadenopathy, sclerotic bone lesions, hypointense liver masses, and pancreatic body/tail mass. Left iliac bone biopsy revealed patchy bone marrow involvement by cords of atypical cells with eccentrically placed nuclei and plump eosinophilic cytoplasm (Figure A and B), associated with extensive “stromal injury” and residual trilineage hematopoiesis.

Combined Therapy Using Human Corneal Stromal Stem Cells and Quiescent Keratocytes to Prevent Corneal Scarring after Injury.

Corneal blindness due to scarring is conventionally treated by corneal transplantation, but the shortage of donor materials has been a major issue affecting the global success of treatment. Pre-clinical and clinical studies have shown that cell-based therapies using either corneal stromal stem cells (CSSC) or corneal stromal keratocytes (CSK) suppress corneal scarring at lower levels. Further treatments or strategies are required to improve the treatment efficacy. This study examined a combined cell-based treatment using CSSC and CSK in a mouse model of anterior stromal injury. We hypothesize that the immuno-regulatory nature of CSSC is effective to control tissue inflammation and delay the onset of fibrosis, and a subsequent intrastromal CSK treatment deposited collagens and stromal specific proteoglycans to recover a native stromal matrix. Using optimized cell doses, our results showed that the effect of CSSC treatment for suppressing corneal opacities was augmented by an additional intrastromal CSK injection, resulting in better corneal clarity. These in vivo effects were substantiated by a further downregulated expression of stromal fibrosis genes and the restoration of stromal fibrillar organization and regularity. Hence, a combined treatment of CSSC and CSK could achieve a higher clinical efficacy and restore corneal transparency, when compared to a single CSSC treatment.

Downregulation of collagen XI during late postnatal corneal development is followed by upregulation after injury.

Collagen XI plays a role in nucleating collagen fibrils and in controlling fibril diameter. The aim of this research was to elucidate the role that collagen XI plays in corneal fibrillogenesis during development and following injury. The temporal and spatial expression of collagen XI was evaluated in C57BL/6 wild-type mice. For wound-healing studies in adult mice, stromal injuries were created using techniques that avoid caustic chemicals. The temporal expression and spatial localization of collagen XI was studied following injury in a Col11a1 inducible knockout mouse model. We found that collagen XI expression occurs during early maturation and is upregulated after stromal injury in areas of regeneration and remodeling. Abnormal fibrillogenesis with new fibrils of heterogeneous size and shape occurs after injury in a decreased collagen XI matrix. In conclusion, collagen XI is expressed in the stroma during development and following injury in adults, and is a regulator of collagen fibrillogenesis in regenerating corneal tissue.

The Effect of Collagen-Chitosan-Sodium Hyaluronate Implantation Composite on Inflammation Reaction (Flare) In New Zealand Rabbit Corneal Stroma

Corneal transplantation is a solution in treating visual disturbances due to corneal opacities, but this procedure is said to be limited. Various efforts have been made in the development of alternative therapies, one of which is by using composite biomaterials. For this reason, this study aimed to analyze the effect of the collagen-chitosan-sodium hyaluronate composite on the inflammatory reaction (flare) in the corneal stromal injury of New Zealand rabbits. The method used was thirty adult male New Zealand rabbits which were divided into three groups. The first group (negative control) was not treated, the second group (positive control) was subjected to intrastromal injuries by making a stromal pocket with the help of a crescent knife, and the third group had a collagen-chitosan-sodium hyaluronate biomaterial implanted in the stromal pocket. The results of this study showed that there were significant differences in inflammatory reactions (flares) in the collagen-chitosan-sodium hyaluronate composite implantation group with the control and positive control groups (p = 0.001, &gt;0.01). Therefore, it can be concluded that the inflammatory reaction (flare) in the implanted group was higher than the control group, this could occur due to the short observation period so that the biomaterial was not completely absorbed.

The anti-scarring role of Lycium barbarum polysaccharide on cornea epithelial-stromal injury

PurposeCornea epithelial-stromal scarring is related to the differentiation of fibroblasts into opaque myofibroblasts. Our study aims to assess the effectiveness of Lycium barbarum polysaccharide (LBP) solution as a pre-treatment in minimizing corneal scarring. MethodsHuman corneal fibroblasts were cultured in a three-dimensional collagen type I-based hydrogel in an eye-on-a-chip model. Fibroblasts were pre-treated with 2 mg/mL LBP for 24 h, followed by another 24-h incubation with 10 ng/mL transforming growth factor-beta 1 (TGF-β1) to induce relevant physiological events after stromal injury. Intracellular pro-fibrotic proteins, extracellular matrix proteins, and pro-inflammatory cytokines that involved in fibrosis, were assessed using immunocytochemistry and enzyme-linked immunosorbent assays. ResultsCompared to the positive control TGF-β1 group, LBP pre-treated cells had a significantly lower expression of alpha-smooth muscle actin, marker of myofibroblasts, vimentin (p < 0.05), and also extracellular matrix proteins both collagen type II and type III (p < 0.05) that can be found in scar tissues. Moreover, LBP pre-treated cells had a significantly lower secretion of pro-inflammatory cytokines interleukin-6 and interleukin-8 (p < 0.05). The cell-laden hydrogel contraction and stiffness showed no significant difference between LBP pre-treatment and control groups. Fibroblasts pretreated with LBP as well had reduced angiogenic factors expression and suppression of undesired proliferation (p < 0.05). ConclusionOur results showed that LBP reduced both pro-fibrotic proteins and pro-inflammatory cytokines on corneal injury in vitro. We suggest that LBP, as a natural Traditional Chinese Medicine, may potentially be a novel topical pre-treatment option prior to corneal refractive surgeries with an improved prognosis.

Exosome-shuttled miR-7162-3p from human umbilical cord derived mesenchymal stem cells repair endometrial stromal cell injury by restricting APOL6

BackgroundRecent studies have shown that exosomes (Exos) derived from stem cells can be used as paracrine factors to regenerate cells and tissues via shuttling miRNAs. Exos derived from human umbilical cord derived mesenchymal stem cells (UCMSCs) have been found to alleviate mifepristone-induced endometrial stromal cell (ESC) injury in vitro. Information on the functions and mechanisms of Exos from UCMSC-induced endometrial repair is limited and requires more study. MethodsUCMSC-Exos were isolated and identified by Transmission Electron Microscopy, Nanoparticle Tracking Analysis software, and western blot assays. The damaged-ESC model and the UCMSC co-culture system were established, while GW4869, a noncompetitive neutral sphingomyelinase (N-SMase) inhibitor, was used to investigate the effects of UCMSC-Exos on mifepristone-induced ESC injury. Cell apoptosis of damaged ESCs treated with UCMSCs was detected using the TUNEL assay and flow cytometry analysis. Then, miRNA microarrays were performed to detect differentially expressed miRNA profiles in both UCMSCs and ESCs after co-culturing. A subset of upregulated miRNAs was validated by qRT-PCR, and miRNA mimics/inhibitor were used to investigate the functions of miR-7162-3p. The miRNA-mRNA interactions were predicted by Targetscan software, while the miRNA binding sites were predicted by miRcode software. Moreover, dual-luciferase reporter, western blot assays and qPCR were conducted to identify the regulatory mechanisms between miR-7162-3p and APOL6. ResultsUCMSCs attenuated mifepristone-induced endometrial stromal cell apoptosis by Exos, while three miRNAs (miR-6831-5p, miR-4669, and miR-7162-3p) were both upregulated in UCMSCs and ESCs after co-culture, and were candidate effectors of UCMSC-Exos-mediated endometrial repair. We showed that miR-7162-3p was shuttled by Exos from UCMSCs and regulated the expression of APOL6 by targeting its 3ʹ-UTR in ESCs. ConclusionsThese results showed UCMSC-Exos protected ESCs from mifepristone-induced apoptosis and played an active role in repairing the damaged ESCs by in vitro shuttling of miR-7162-3p. The miR-7162-3p-overexpressed UCMSC-Exos may therefore be used in cell-free therapy of endometrial injury.

Creation and grading of experimental corneal scars in mice models

PurposeTo develop a stromal wound healing model and a reliable scar classification score system that correlates photographic evaluation with changes in the structure and organization of the extracellular matrix. Materials and methodsWe tested three stromal injury techniques in adult C57BL/6 mice. Technique 1, a lineal partial thickness keratotomy in the horizontal axis. Technique 2, corneal epithelial and stromal debridement using a diamond burr in the horizontal axis, and technique 3, a combination of techniques 1 and 2. To assess intra-observer and inter-observer agreement between two examiners evaluating formed stromal scars, stereo microscopic photographs of anterior segment were scored by two masked examiners at around 1-month. Depending on the severity of opacification and the area of involvement, scars were classified on a scale from 0 to 3 based on a modified Fantes haze scale. Extracellular matrix composition as well as matrix organization, macrophage infiltration and neovascularization were evaluated with immunofluorescence and second harmonic generation (SHG) microscopy. ResultsTechnique 1 created mild scars, with a score of 0.5 ± 0.43, while techniques 2 (score 2.1 ± 0.45) and 3 (score 2 ± 0.66), created dense scars with a higher score. A significant difference in scar severity score was noted between the 3 techniques (one way ANOVA, p < 0.0001). Masked graders demonstrated excellent agreement (intraclass correlation = 0.927 [95% confidence interval: 0.87–0.96]). The severity of scars noted at stereo microscopy correlated with the severity of changes in extracellular matrix in the stroma as demonstrated by the expression of collagens I, IV and fibronectin and evaluation of matrix hierarchical organization. In contrast to mild scarring, moderate and severe scars had increased expression of CD31 and CD68, markers of vascular endothelial cells and macrophages, respectively. ConclusionMouse models of stromal scarring using simple surgical techniques are described. Corneal scars can be consistently classified by two observers. Grading of scar severity positively correlates with changes in extracellular matrix composition, disorganization and cell infiltration.