Preclinical study of experimental burns treated with photobiomodulation and Human Amniotic Membrane, both isolated and associated / Estudo pré-clínico de queimaduras experimentais tratadas com fotobiomodulação e membrana amniótica humana, isoladas e associadas / Estudio preclínico de quemaduras experimentales tratadas con fotobiomodulación y membrana amniótica humana, solas y combinadas

Abstract Objective: to evaluate the effect of photobiomodulation with low-level 660 nm laser alone or associated with Human Amniotic Membrane in the repair of partial-thickness burns in rats. Method: an experimental study conducted with 48 male Wistar rats, randomized into four groups: Control, Human Amniotic Membrane, Low-Level Laser Therapy, and Low-Level Laser Therapy associated with Human Amniotic Membrane. The histopathological characteristics of the skin samples were analyzed 7 and 14 days after the burn. The data obtained were submitted to the Kolmogorov-Smirnov and Mann-Whitney tests. Results: the histological analysis of the burn injuries showed a decrease in inflammation (p<0.0001) and an increase in proliferation of fibroblasts (p<0.0001) mainly at 7 days in all treatments related to the control group. At 14 days, the greater effectiveness in accelerating the healing process was significant (p<0.0001) in the Low-Level Laser Therapy group associated with the Human Amniotic Membrane. Conclusion: the association of photobiomodulation therapies with the Human Amniotic Membrane allowed verifying a reduction in the healing process time of the experimental lesions, stimulating its proposal as a treatment protocol in partial-thickness burns.

Resumo Objetivo: avaliar o efeito da fotobiomodulação com laser de baixa intensidade 660 nm isoladamente ou associada à membrana amniótica humana no reparo de queimaduras de espessura parcial em ratos. Método: estudo experimental com 48 ratos Wistar machos, randomizados em quatro grupos: Controle, Membrana Amniótica Humana, Terapia a Laser de Baixa Intensidade e Terapia a Laser de Baixa Intensidade associado à Membrana Amniótica Humana. As características histopatológicas das amostras de pele foram analisadas aos 7 e 14 dias após a queimadura. Os dados obtidos foram submetidos aos testes de Kolmogorov-Smirnov e Mann Whitney. Resultados: a análise histológica das lesões por queimadura mostrou a diminuição da inflamação (p <0,0001) e aumento da proliferação de fibroblastos (p <0,0001), principalmente nos 7 dias em todos os tratamentos relacionados ao grupo controle. Aos 14 dias, a maior efetividade na aceleração do processo cicatricial foi significativa (p<0,0001) no grupo Terapia a Laser de Baixa Intensidade associado à Membrana Amniótica Humana. Conclusão: a associação das terapias de fotobiomodulação à membrana amniótica humana permitiu comprovar redução no tempo do processo cicatricial das lesões experimentais, estimulando sua proposição como protocolo de tratamento em queimaduras de espessura parcial.

Resumen Objetivo: evaluar el efecto de la fotobiomodulación con láser de baja intensidad 660 nm de sola o combinada con la membrana amniótica humana en la reparación de quemaduras de espesor parcial en ratas. Método: estudio experimental con 48 ratas Wistar macho, aleatorizadas en cuatro grupos: Control, Membrana Amniótica Humana, Terapia con Láser de Baja Intensidad y Terapia con Láser de Baja Intensidad combinada con la Membrana Amniótica Humana. Las características histopatológicas de las muestras de piel fueron analizadas a los 7 y 14 días después de la quemadura. Los datos obtenidos fueron sometidos a las pruebas de Kolmogorov-Smirnov y Mann-Whitney. Resultados: el análisis histológico de las lesiones por quemadura mostró una disminución de la inflamación (p <0,0001) y un aumento de la proliferación de fibroblastos (p <0,0001) principalmente a los 7 días en todos los tratamientos en comparación con el grupo control; a los 14 días, en el grupo de Terapia con Láser de Baja Intensidad combinada con la Membrana Amniótica Humana la mayor efectividad en la aceleración del proceso de cicatrización fue significativa (p<0,0001). Conclusión: la asociación de terapias de fotobiomodulación con la membrana amniótica humana permitió comprobar que hubo una reducción en el tiempo del proceso de cicatrización de lesiones experimentales, lo cual favorece que se proponga como protocolo de tratamiento en quemaduras de espesor parcial.

Improvement in growth performance and digestive function from amniotic injections of N-acetylglutamate in broiler chickens.

BACKGROUND: N-acetylglutamate (NAG) is the initial and essectial substrate in the process of de novo arginine synthesis, plays an important role in intestinal development. The aim of this study was to determine the effects of in ovo feeding of NAG, 1.5 mg/egg at 17.5 days of incubation (DOI) via amnion, on hatching performance, early intestinal histomorphometry, jejunal barrier, digestive function, and growth performance of broiler chickens between 1 and 14 days of age. RESULTS: Amniotic injection of NAG had no significant effect on hatching characteristics compared with the non-injected control group (NC group). Birds in the NAG solution-injected group (NAG group) exhibited lower average daily feed intake and better feed efficiency during a period of 1-14 days. In comparison with the NC group, the NAG group had decreased crypt depth (CD) in the ileum and increased villus height (VH) / CD in the jejunum at 7 days, and decreased CD in duodenum and significantly increased VH in the jejunum at 14 days. However, the effects of in ovo supplementation with NAG on the density of goblet cells, and gene expression of mucin 2 and alkaline phosphatase were not significant. Chicks in the NAG group had a significantly higher mRNA expression level of trypsin and maltase in jejunum at 7 days than the NC group but not at 14 days. CONCLUSION: Amniotic injections of NAG (1.5 mg/egg) at 17.5 DOI could improve early growth performance of broilers during 1-14 days after hatching by accelerating the development of the intestine and enhancing jejunal digestive function. © 2023 Society of Chemical Industry.

Investigating the regenerative effects of folic acid on human amniotic epithelial stem cells and amniotic pore culture technique (APCT) model in vitro using an integrated pharmacological-bioinformatic approach.

INTRODUCTION: Disruption of fetal membranes before the onset of labor is referred to as premature rupture of membranes (PROM). Lack of maternal folic acid (FA) supplementation reportedly leads to PROM. However, there is a lack of information on the location of FA receptors in the amniotic tissue. Additionally, the regulatory role and potential molecular targets of FA in PROM in vitro have rarely been investigated. METHODS: The three FA receptors (folate receptor α isoform [FRα], transporter of reduced folate [RFC], and proton-coupled folate transporter [PCFT]) in human amniotic epithelial stem cells (hAESCs) and amniotic tissue were localized using immunohistochemistry and immunocytochemistry staining. Effect and mechanism analyses of FA were performed in hAESCs and amniotic pore culture technique (APCT) models. An integrated pharmacological-bioinformatics approach was utilized to explore the potential targets of FA for the treatment of PROM. RESULTS: The three FA receptors were widely expressed in human amniotic tissue, especially in the hAESC cytoplasm. FA stimulated the amnion regeneration in the in vitro APCT model. This mimics the PROM status, in which cystathionine-ß-synthase, an FA metabolite enzyme, may play an important role. The top ten hub targets (STAT1, mTOR, PIK3R1, PTPN11, PDGFRB, ABL1, CXCR4, NFKB1, HDAC1, and HDAC2) of FA for preventing PROM were identified using an integrated pharmacological-bioinformatic approach. DISCUSSION: FRα, RFC, and PCFT are widely expressed in human amniotic tissue and hAESCs. FA aids the healing of ruptured membrane.

Preclinical study of experimental burns treated with photobiomodulation and Human Amniotic Membrane, both isolated and associated.

OBJECTIVE: to evaluate the effect of photobiomodulation with low-level 660 nm laser alone or associated with Human Amniotic Membrane in the repair of partial-thickness burns in rats. METHOD: an experimental study conducted with 48 male Wistar rats, randomized into four groups: Control, Human Amniotic Membrane, Low-Level Laser Therapy, and Low-Level Laser Therapy associated with Human Amniotic Membrane. The histopathological characteristics of the skin samples were analyzed 7 and 14 days after the burn. The data obtained were submitted to the Kolmogorov-Smirnov and Mann-Whitney tests. RESULTS: the histological analysis of the burn injuries showed a decrease in inflammation (p<0.0001) and an increase in proliferation of fibroblasts (p<0.0001) mainly at 7 days in all treatments related to the control group. At 14 days, the greater effectiveness in accelerating the healing process was significant (p<0.0001) in the Low-Level Laser Therapy group associated with the Human Amniotic Membrane. CONCLUSION: the association of photobiomodulation therapies with the Human Amniotic Membrane allowed verifying a reduction in the healing process time of the experimental lesions, stimulating its proposal as a treatment protocol in partial-thickness burns.

Amniotic membrane attenuates heterotopic ossification following high-dose bone morphogenetic protein-2 treatment of segmental bone defects.

Treatment of large bone defects with supraphysiological doses of bone morphogenetic protein-2 (BMP-2) has been associated with complications including heterotopic ossification (HO), inflammation, and pain, presumably due to poor spatiotemporal control of BMP-2. We have previously recapitulated extensive HO in our rat femoral segmental defect model by treatment with high-dose BMP-2 (30 µg). Using this model and BMP-2 dose, our objective was to evaluate the utility of a clinically available human amniotic membrane (AM) around the defect space for guided bone regeneration and reduction of HO. We hypothesized that AM surrounding collagen sponge would attenuate heterotopic ossification compared with collagen sponge alone. In vitro, AM retained more BMP-2 than a synthetic poly(ε-caprolactone) membrane through 21 days. In vivo, as hypothesized, the collagen + AM resulted in significantly less heterotopic ossification and correspondingly, lower total bone volume (BV), compared with collagen sponge alone. Although bone formation within the defect was delayed with AM around the defect, by 12 weeks, defect BVs were equivalent. Torsional stiffness was significantly reduced with AM but was equivalent to that of intact bone. Collagen + AM resulted in the formation of dense fibrous tissue and mineralized tissue, while the collagen group contained primarily mineralized tissue surrounded by marrow-like structures. Especially in conjunction with high doses of growth factor delivered via collagen sponge, these findings suggest AM may be effective as an overlay adjacent to bone healing sites to spatially direct bone regeneration and minimize heterotopic ossification.

REPAIR OF THE EXIT WOUND OF A PERFORATING GLOBE INJURY WITH THE AMNIOTIC MEMBRANE: A SURGICAL ADJUVANT FOR EARLY VITRECTOMY.

PURPOSE: To describe the use of the amniotic membrane for the repair of the exit wound of a perforating injury involving the retina and the choroid. METHODS: Case report. RESULTS: A 46-year-old man presented one day after a perforating ocular injury with an exit wound close to the inferior temporal retinal vascular arcade. The next day, the patient underwent a combined vitrectomy and phacoemulsification with intraocular lens implantation. During vitrectomy, a retinochoroidectomy was performed at the exit wound and the internal limiting membrane was peeled over the macula and up to the exit wound. Laser retinopexy was followed by plugging of a piece of the amniotic membrane to the exit wound. A second piece of the amniotic membrane was used to cover the bare retinochoroidectomy area. The surgery was concluded with a silicone exchange. Postoperatively, no sign of proliferative vitreoretinopathy was observed, and at 3 months, the silicone oil was removed. The follow-up was uneventful, and the eye achieved a final visual acuity of 20/30. CONCLUSION: The amniotic membrane may offer a simple and safe solution for the repair of the exit wounds of perforating injuries involving the retina and the choroid. The use of the amniotic membrane for this purpose may afford the opportunity for early vitrectomy in the management of perforating ocular injuries.

SDF-1α loaded bioengineered human amniotic membrane-derived scaffold transplantation in combination with hyperbaric oxygen improved diabetic wound healing.

Based on its multifactorial nature, successful treatment of diabetic wounds requires combinatorial approach. In this regard, we hypothesized that engraftment of a bioengineered micro-porous three-dimensional human amniotic membrane-scaffold (HAMS) loaded by SDF-1α (SHAMS) in combination with hyperbaric oxygen (HBO), throughout mobilization and recruitment of endothelial progenitor cells (EPCs), could accelerate wound healing in rats with type 1 diabetes mellitus. To test this hypothesis, 30 days after inducting diabetes, an ischemic wound was created in rat skin and treatments were performed for 21 days. In addition to wounded non-diabetic (ND) group, diabetic animals were randomly divided into non-treated (NT-D), HBO-treated (HBO-D), HBO-treated plus HAMS transplantation (HBO+HAMS-D) or HBO-treated in combination with SHAMS transplantation (HBO+SHAMS-D) groups. Our results on post-wounding days 7, 14 and 21 showed that the wound closure, volume of new dermis and epidermis, numerical density of basal cells of epidermis, fibroblasts and blood vessels, number of proliferating cells, deposition of collagen and biomechanical properties of healed wound were considerably higher in both HBO+HAMS-D and HBO+SHAMS-D groups in comparison to those of the NT-D and HBO-D groups, and were the highest in HBO+SHAMS-D ones. The transcripts for Vegf, bFgf, and Tgf-ß genes were significantly upregulated in all treatment regimens compared to NT-D group and were the highest for HBO+SHAMS-D group. This is while expression of Tnf-α and Il-1ß as well as cell density of neutrophil and macrophage decreased more significantly in HBO+SHAMS-D group as compared with NT-D or HBO-D groups. Overall, it was found that using both HAMS transplantation and HBO treatment has more impact on diabetic wound healing. Moreover, SDF-1α loading on HAMS could transiently improve the wound healing process, as compared with the HBO+HAMS-D group on day 7 only.

Diabetic Foot Wounds Treated With Human Amniotic Membrane and Low-level Laser Therapy: A Pilot Clinical Study.

BACKGROUND: Low-level laser therapy (LLLT) and human amniotic membrane (HAM) application have been shown to be viable options for use in wound healing. PURPOSE: This study sought to compare LLLT and HAM to a control treatment (hydrogel, saline, and gauze) in persons with diabetes mellitus (DM) and foot ulcers. METHODS: Using a prospective pilot clinical study design, patients receiving care at a health center that specializes in the treatment of diabetic foot wounds between November 2016 and August 2017 were recruited. Eligible patients had to be 30 to 59 years of age; diagnosed with type 2 DM (postprandial capillary glucose levels between 140 and 350 mg/dL); and have uninfected, granulating stage 2 or 3 foot ulcers measuring less than 7 cm by 3 cm. Immunosuppressed and malnourished patients or those with neoplasms or in critical condition were not eligible to participate. Patients received the control treatment (2 mg hydrogel, saline, and gauze), HAM (patches of thawed HAM, applied with overlapping edges), or LLLT (phototherapy session, 2 mg hydrogel, saline, and gauze) for 28 days. Variables, wound area measurements, Pressure Ulcer Scale for Healing (PUSH) scores, and Visual Analog Scale (VAS) scores were used to assess wound improvement progress and pain on days 7, 14, 21, and 28. Descriptive statistics were used to analyze the participant anthropometric and clinical profiles. The Kolmogorov-Smirnov test was used to analyze the sample distribution. The Kruskal-Wallis test with Dunn's post-test was used to evaluate differences in PUSH and VAS scores and wound size for intergroup analysis, and the Mann-Whitney U test was used for the same outcomes in intragroup analysis. The level of significance was 5% (P < .05). RESULTS: Twenty-seven (27) patients participated (mean age, 51.4 years; mean body mass index, 26.5 kg/m2), with 9 patients in each treatment group. No statistically significant differences were noted in clinical or anthropometric variables among the groups, but mean baseline wound areas were different (2.6 cm² for the control, 1.9 cm² for the LLLT, and 5.5 cm² for the HAM groups). Intragroup comparisons showed a significant reduction in PUSH score in the LLT group between days 0 and 21 (8.2 vs 4.9; P < .01) and days 21 to 28 (4.9 vs 3.2; P < .001). In all treatment groups the percent reduction was significantly different between days 7 and 28. No outcomes were significantly different between groups. CONCLUSION: Diabetic foot ulcer wound area as well as PUSH and VAS scores showed more improvement for patients with DM receiving LLLT or HAM than for the control group, but the differences were not significant. Larger studies are needed to compare these treatment modalities.

Determining the efficacy of the bovine amniotic membrane homogenate during the healing process in rabbits' ex vivo corneas.

OBJECTIVE: To evaluate the efficacy of bovine amniotic membrane homogenate (BAMH) on wounded ex vivo rabbit corneas. PROCEDURE: Eighteen corneas obtained from normal rabbit eyes were wounded equally using a 6 mm trephine and cultured into an air-liquid interface model. Corneas were treated with phosphate-buffered saline (PBS) (n = 6, control group), 0.2% ethylenediaminetetraacetic acid (EDTA; n = 6), or BAMH (n = 6). All treatments were applied topically 6 times/day. Each cornea was macrophotographed daily with and without fluorescein stain to assess epithelialization and haziness. After 7 days, corneal transparency was evaluated, and the tissues prepared for histologic analysis of viability, and total and epithelial thickness. RESULTS: The mean epithelialization time was 6.2 ± 0.82 days for the control group, 6.2 ± 0.75 days for the EDTA-treated group, and 5.1 ± 0.40 days for the BAMH-treated group, demonstrating a significant difference between the BAMH and the other groups. The corneas that received EDTA had better transparency compared with the other groups. Histologically, all corneas had adequate morphology and architecture after healing. Analysis of corneal and epithelial thickness revealed no significant difference among groups. CONCLUSION: Bovine amniotic membrane homogenate is an effective and promising treatment for stromal and epithelial ulcers.

Amniotic membrane extract eye drops for ocular surface diseases: use and clinical outcome in real-world practice.

PURPOSE: To study the indications and clinical outcomes, in a real-word setting, of amniotic membrane extract eye drops (AMEED) use for ocular surface disease (OSD). METHODS: A retrospective study of patients treated with topical AMEED between January 2018 and January 2020 was conducted. Patients were classified in two groups according to specific OSD-dry eye disease (DED) and wound healing delay (WHD) groups. Demographics, comorbidities, treatment duration and clinical outcomes were analysed. RESULTS: A total of 50 eyes of 36 patients with or without previous treatments were included. Patients in the DED group presented more systemic comorbidities (83 vs 22%; p < 0.001) and spent more mean time under AMEED treatment (10 vs 7.2 months average) than the WHD group (p = 0.0104). In four patients, long-term treatment (more than 24 months) was reported. Global similar symptomatic improvement was reported for both groups (DED 88.9% vs WHD 100%; p = 0.486), with the WHD group especially consisting in general relief (78%) and the DED group reporting more pain improvement (44%) (p = 0.011). Regarding patients with autologous serum as a previous treatment, no statistical differences were found in subjective or objective improvement. An overall success was achieved in 94.4% of the cases and no adverse events were found. CONCLUSION: AMEED administration is a promising mean to treat OSD such as dry eye, persistent epithelial defect and corneal ulcers. Although AMEED may be effective in the treatment of severe DED and persistent epithelial defect or corneal ulcers, conclusions are limited owing to the absence of controlled clinical trials.

Vitagenic Effect of Specific Bioactive Fractions of Rhodiola with Trachurus sp. Extract Against Oxidative Stress-Induced Aging in Human Amnion Derived Epithelial Cell Line: In View of a Novel Senolytic.

BACKGROUND: Rhodiola rosea is a herb that has been used in traditional medicine for several years, and LF is a class of lipoproteins derived from the fish Trachurus sp. (LF-T), which exhibits known anti-inflammatory activity. OBJECTIVE: Investigating the anti-aging effect of Rhodiola specific bioactive fractions cluster in combination with LF-T (R-L compound) in H2O2 mediated oxidative stress-induced human amnion derived epithelial cell line - FL cells as normal human cell line. METHODS: FL cells were treated with H2O2 to induce cellular aging, followed by treatment of the RL compound to study its anti-aging characteristics. Based on the proliferation rate, 0.05% and 0.1% concentration of R-L compound was determined using MTT assay. Anti-aging and anti-oxidant assays, ABTS, DPPH, Hyaluronidase activity Nitric Oxide, Lipid Peroxidase, and Superoxide Dismutase were performed. qPCR for anti-aging genes and matrix metalloproteinase genes were analyzed. RESULTS: FL cells treated with R-L compound exhibited increased proliferation rate and free-radical reduction. Decreased Hyaluronidase enzyme activity and regulation of genes such as SIRT1, KLOTHO, SERPINA 6, MMP 9, and MMP 2 expression depicted the anti-aging role of the R-L compound. Chemometric profiling of the R-L compound revealed that aromatic compounds and unsaturated fatty acids along with their derivatives, were present predominantly, which might have attributed to the potent oxidative stress impeded aging activity. CONCLUSION: Specific Bioactive Fractions of Rhodiola in combination with LF-T obtained from Trachurus sp. involve in the regulation of aging genes and might be a novel approach to prevent the cells from oxidative stress damage and also it might avert the aging of cells.

Commercial amniotic membrane extract for treatment of corneal ulcers in adult horses.

BACKGROUND: Amniotic membrane extract enhances the rate of epithelialisation after corneal ulceration in several species but has not been studied in the equine cornea. OBJECTIVES: To evaluate the effect of amniotic membrane extract on re-epithelialisation of equine corneal ulcers compared with ulcers treated with antibiotic, antifungal and mydriatic medical therapy alone, and to evaluate equine corneal healing after experimentally induced superficial ulceration. STUDY DESIGN: Masked, randomised, controlled experimental trial. METHODS: Superficial, 8 mm corneal ulcers were created bilaterally in each horse. One eye was treated with amniotic membrane extract and the opposite was control. Both eyes were treated with medical therapy. Treatment eyes received amniotic membrane extract, and control eyes received the amniotic membrane extract vehicle. Ulcers were stained with fluorescein and photographed in 12-hour increments until completely healed. Ulcer surface area was determined by analysing photographs with ImageJ. A mixed linear model was used to compare ulcer surface area and hours until healing between treatment groups. A regression model was also used to calculate corneal re-epithelialisation rate over time. RESULTS: Regardless of therapy, healing occurred in two phases: an initial rapid phase of 0.88 mm2 /hr (95% CI: 0.81-0.94 mm2 /hr) for approximately 48-54 hours followed by a second, slow phase of 0.07 mm2 /hr (95% CI: 0.04-0.09 mm2 /hr). Most eyes healed within 135.5 ± 48.5 hours. Treatment (amniotic membrane extract vs. control) was not significantly associated with size of ulcers over time (P = .984). Discomfort was minimal to absent in all horses. MAIN LIMITATIONS: Results achieved experimental studies may differ from outcomes in the clinical setting. CONCLUSIONS: There was no significant difference in healing rate with addition of amniotic membrane extract to medical therapy for equine superficial corneal ulcers. A biphasic corneal healing process was observed, with an initial rapid phase followed by a slow phase. Further study will be needed to determine whether amniotic membrane extract will be helpful for infected or malacic equine corneal ulcers.

Alterations in the Intestinal Morphology, Gut Microbiota, and Trace Mineral Status Following Intra-Amniotic Administration (Gallus gallus) of Teff (Eragrostis tef) Seed Extracts.

The consumption of teff (Eragrostis tef), a gluten-free cereal grain, has increased due to its dense nutrient composition including complex carbohydrates, unsaturated fatty acids, trace minerals (especially Fe), and phytochemicals. This study utilized the clinically-validated Gallus gallus intra amniotic feeding model to assess the effects of intra-amniotic administration of teff extracts versus controls using seven groups: (1) non-injected; (2) 18Ω H2O injected; (3) 5% inulin; (4) teff extract 1%; (5) teff extract 2.5%; (6) teff extract 5%; and (7) teff extract 7.5%. The treatment groups were compared to each other and to controls. Our data demonstrated a significant improvement in hepatic iron (Fe) and zinc (Zn) concentration and LA:DGLA ratio without concomitant serum concentration changes, up-regulation of various Fe and Zn brush border membrane proteins, and beneficial morphological changes to duodenal villi and goblet cells. No significant taxonomic alterations were observed using 16S rRNA sequencing of the cecal microbiota. Several important bacterial metabolic pathways were differentially enriched in the teff group, likely due to teff's high relative fiber concentration, demonstrating an important bacterial-host interaction that contributed to improvements in the physiological status of Fe and Zn. Therefore, teff appeared to represent a promising staple food crop and should be further evaluated.

Anti-inflammatory effects of dexmedetomidine on human amnion-derived WISH cells.

Background: To maintain the normal pregnancy, suppression of inflammatory signaling pathway is a crucial physiologic response. Dexmedetomidine has been used for labor analgesia or supplement of inadequate regional analgesia during delivery. And it has been reported that dexmedetomidine has an anti-inflammatory effect. In this study, we examined the influence of dexmedetomidine on the expression of cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2) and inflammatory cytokines in lipopolysaccharide (LPS)-stimulated human amnion-derived WISH cells. In addition, we evaluated the association of mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) pathway in anti-inflammatory effect of dexmedetomidine. Methods: Human amnion-derived WISH cells were pretreated with various concentrations of dexmedetomidine (0.001-1 µg/ml) for 1 h and after then treated with LPS (1 µg/ml) for 24 h. MTT assay was conducted to evaluate the cytotoxicity. Nitric oxide (NO) production was analyzed using Griess-reaction microassay. RT-PCR was performed for analysis of mRNA expressions of COX-2, PGE2, tumor necrosis factor (TNF)-α and interlukin (IL)-1ß. Protein expressions of COX-2, PGE2, p38 and NF-κB were analyzed by western blotting. Results: LPS and dexmedetomidine had no cytotoxic effect on WISH cells. There was no difference in NO production after dexmedetomidine pretreatment. The mRNA and protein expressions of COX-2 and PGE2 were decreased by dexmedetomidine pretreatment in LPS-treated WISH cells. Dexmedetomidine also attenuated the LPS-induced mRNA expression of TNF-α and IL-1ß. The activation of p38 and NF-κB was suppressed by dexmedetomidine pretreatment in LPS-treated WISH cells. Conclusion: We demonstrated that dexmedetomidine pretreatment suppressed the expressions of inflammatory mediators increased by LPS. In addition, this study suggests that anti-inflammatory effect of dexmedetomidine on WISH cells was mediated by the inhibitions of p38 and NF-κB activation.

Carbon nanodot decorated acellular dermal matrix hydrogel augments chronic wound closure.

Impaired skin regeneration in chronic wounds like in diabetes corresponds to high oxidative stress, poor angiogenesis and insufficient collagen hyperplasia. Therefore, a multifaceted strategy for treatment is required to address critical issues associated with chronic wound healing. Fascinating application of nanomaterials in chronic wounds is still limited; hence, in the present work bioactive solubilized decellularized dermal matrix (sADM) was employed to form a hydrogel with chitosan (CTS) at physiological pH/temperature and modified with reactive oxygen species (ROS) scavenging carbon nanodots (ND). A detailed in vitro investigation found that the ND modified bioactive hydrogel (CsADMND) is suitable for human amniotic membrane derived stem cell (hAMSC) delivery. Also, CsADMND was observed to possess a good ROS scavenging property, hemocompatibility and pro-angiogenic potential as demonstrated by 2,2-diphenyl-1-picrylhydrazyl (DPPH), haemolysis and chick chorioallantoic membrane (CAM) assay, respectively. The hybrid hydrogel promoted migration of cells in vitro in scratch assay owing to its antioxidant potential and the presence of bioactive moieties. Further, its efficacy in healing full thickness (FT) chronic wounds was evaluated in a streptozotocin (STZ) induced diabetic model. The CsADMND hydrogel after association with hAMSCs led to stimulation of early angiogenesis, superior collagen deposition, rapid wound closure, complete reepithelialisation, and formation of distinct organized dermal epidermal junctions (DEJ) post 21 days of healing. These results suggest that the hAMSC laden CsADMND hydrogel may serve as a promising therapeutic strategy for the management of chronic wounds.

Effects of doxorubicin associated with amniotic membrane stem cells in the treatment of canine inflammatory breast carcinoma (IPC-366) cells.

BACKGROUND: Tumours in mammary glands represent the most common neoplasia in bitches, as in humans. This high incidence results in part from the stimulation of sex hormones on these glands. Among mammary tumours, inflammatory carcinoma is the most aggressive, presenting a poor prognosis to surgical treatment and chemotherapy. One of the most widely used chemotherapy drugs for breast cancer treatment is doxorubicin (DOXO). Alternative therapies have been introduced in order to assist in these treatments; studies on treatments using stem cells have emerged, since they have anti-inflammatory and immunomodulatory properties. The aim of this study was to evaluate the effects of DOXO and canine amniotic membrane stem cells (AMCs) on the triple-negative canine inflammatory mammary carcinoma cell line IPC-366. METHODS: Four experimental groups were analysed: a control group without treatment; Group I with DOXO, Group II with AMC and Group III with an association of DOXO and AMCs. We performed the MTT assay with DOXO in order to select the best concentration for the experiments. The growth curve was performed with all groups (I-III) in order to verify the potential of treatments to reduce the growth of IPC-366. For the cell cycle, all groups (I-III) were tested using propidium iodide. While in the flow cytometry, antibodies to progesterone receptor (PR), estrogen receptor (ER), PCNA, VEGF, IL-10 and TGF-ß1 were used. For steroidogenic pathway hormones, an ELISA assay was performed. RESULTS: The results showed that cells treated with 10 µg/mL DOXO showed a 71.64% reduction in cellular growth after 72 h of treatment. Reductions in the expression of VEGF and PCNA-3 were observed by flow cytometry in all treatments when compared to the control. The intracellular levels of ERs were also significantly increased in Group III (4.67% vs. 27.1%). Regarding to the levels of steroid hormones, significant increases in the levels of estradiol (E2) and estrone sulphate (S04E1) were observed in Groups I and III. On the other hand, Group II did not show differences in steroid hormone levels in relation to the control. We conclude that the association of DOXO with AMCs (Group III) promoted a reduction in cell growth and in the expression of proteins related to proliferation and angiogenesis in IPC-366 triple-negative cells. CONCLUSIONS: This treatment promoted ER positive expression, suggesting that the accumulated oestrogen conducted these cells to a synergistic state, rendering these tumour cells responsive to ERs and susceptible to new hormonal cancer therapies.

Microarray analysis of verbenalin-treated human amniotic epithelial cells reveals therapeutic potential for Alzheimer's Disease.

Alzheimer's disease (AD) has become a major world health problem as the population ages. There is still no available treatment that can stop or reverse the progression of AD. Human amnion epithelial cells (hAECs), an alternative source for stem cells, have shown neuroprotective and neurorestorative potentials when transplanted in vivo. Besides, studies have suggested that stem cell priming with plant-derived bioactive compounds can enhance stem cell proliferation and differentiation and improve the disease-treating capability of stem cells. Verbenalin is an iridoid glucoside found in medicinal herbs of Verbenaceae family. In the present study, we have conducted microarray gene expression profiling of verbenalin-treated hAECs to explore its therapeutic potential for AD. Gene set enrichment analysis revealed verbenalin treatment significantly enriched AD-associated gene sets. Genes associated with lysosomal dysfunction, pathologic angiogenesis, pathologic protein aggregation, circadian rhythm, age-related neurometabolism, and neurogenesis were differentially expressed in the verbenalin-treated hAECs compared to control cells. Additionally, the neuroprotective effect of verbenalin was confirmed against amyloid beta-induced neurotoxicity in human neuroblastoma SH-SY5Y cells. Our present study is the first to report the therapeutic potential of verbenalin for AD; however, further in-depth research in the in vitro and in vivo models are required to confirm our preliminary findings.

Comparative study between topically applied irradiated human amniotic membrane in combination with tea tree oil versus topical tioconazole in pityraisis versicolor treatment.

Pityriasis versicolor (PV) is a chronic skin disease caused by virulence activities of Malassezia, a genus of skin-associated yeasts. Traditionally, Tioconazole is used as a topical antifungal for curing PV. Previous investigations cited that human amniotic membrane (HAM), a placental tissue, has antimicrobial and anti-inflammatory activities and is useful as a dressing for healing skin lesions. Moreover, tea tree oil (TTO) has a potent antifungal efficacy. This clinical trial aims to achieve an alternative therapeutic treatment able to kill Malassezia and heal PV lesions using TTO-saturated HAM (TOSHAM), with little application times. This study subjected 120 patients with hypopigmented or hyperpigmented PV lesions; half patients were treated weekly with TOSHAM compared with the others who applying 1% Tioconazole cream daily as a traditional treatment. Microbiological evaluation of in vitro fungicidal activity of TOSHAM versus Tioconazole was carried out against Malassezia furfur culture. The clinical outcomes of this study proved the superior activity of TOSHAM to heal PV lesions than Tioconazole; this was in harmony with microbiological findings. This study approached a novel therapeutic treatment of PV with great outcomes by using TOSHAM.

The Relative Permittivity Changes of EGF by 50 Hz MF Exposure Neither Affect the Interaction of EGF With EGFR Nor Its Biological Effects.

The biophysical mechanism of magnetic fields (MFs) acting on living systems is not clear. Previous research showed that, similar to epidermal growth factor (EGF), MF exposure induced EGF receptor (EGFR) clustering and activated EGFR signaling. In this study, we investigated whether MF exposure induced the changes in physical characteristics of EGF and downstream effects of EGF and EGFR interaction. The phase-interrogation surface plasmon resonance (SPR) sensing analyses showed that 50 Hz MF exposure at 4.0 mT for 1 h induced reversible relative permittivity changes of EGF solution. However, compared with sham-exposed EGF solution, the MF-exposed EGF solution did not affect the binding of EGF to EGFR, nor the cell viability and EGFR clustering in human amniotic epithelial cells (FL cells). Our data suggest that cellular EGFR clustering response to MF exposure might not be a result of changes in relative permittivity of EGF in cell culture solution. Bioelectromagnetics. © 2020 Bioelectromagnetics Society.