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.

The effect of sacral massage on meconium-stained amniotic fluid and the duration of fetal descent in labor: A randomized controlled trial.

OBJECTIVE: The aim of this study was to investigate the effect of sacral massage on the presence of meconium-stained amniotic fluid and the duration of fetal descent during labor in pregnant women. METHODS: A total of 220 nulliparous women with singleton low-risk pregnancies in the vertex position at term were recruited. Eligible women were randomly assigned to either massage group or a control group. The massages were performed with friction and tapotement protocol. The massage was applied for 10 min each time by a midwife who is also an expert in the field, at the onset of contractions during the intrapartum period, when cervical dilatation reached 10 cm. The Wong-Baker faces pain rating scale was evaluated. RESULTS: No significant difference was found between the length of the first stage of labor and the total length of delivery (p = 0.097 and 0.434), respectively. There was a significant difference between the two groups in terms of perineal injuries. Perineal injury was lower in the massaged group (p = 0.005). There was a low percentage of meconium-stained amniotic fluid in the massaged group. The difference between the groups was statistically significant. The duration of fetal descent was shorter in the massaged group (p < 0.001). A significant difference was found in Wong-Baker FACES pain rating scale scores. Lower scores were detected in the massaged group (p < 0.001). CONCLUSION: Sacral massage has beneficial effects on mothers and babies in obstetric practice. In addition, applying massage during labor plays a significant role in reducing the presence of meconium-stained amniotic fluid and the duration of fetal descent.

Amnion and chorion matrix maintain hMSC osteogenic response and enhance immunomodulatory and angiogenic potential in a mineralized collagen scaffold.

Craniomaxillofacial (CMF) bone injuries present a major surgical challenge and cannot heal naturally due to their large size and complex topography. We are developing a mineralized collagen scaffold that mimics extracellular matrix (ECM) features of bone. These scaffolds induce in vitro human mesenchymal stem cell (hMSC) osteogenic differentiation and in vivo bone formation without the need for exogenous osteogenic supplements. Here, we seek to enhance pro-regenerative potential via inclusion of placental-derived products in the scaffold architecture. The amnion and chorion membranes are distinct components of the placenta that each have displayed anti-inflammatory, immunomodulatory, and osteogenic properties. While potentially a powerful modification to our mineralized collagen scaffolds, the route of inclusion (matrix-immobilized or soluble) is not well understood. Here we compare the effect of introducing amnion and chorion membrane matrix versus soluble extracts derived from these membranes into the collagen scaffolds on scaffold biophysical features and resultant hMSC osteogenic activity. While inclusion of amnion and chorion matrix into the scaffold microarchitecture during fabrication does not influence their porosity, it does influence compression properties. Incorporating soluble extracts from the amnion membrane into the scaffold post-fabrication induces the highest levels of hMSC metabolic activity and equivalent mineral deposition and elution of the osteoclast inhibitor osteoprotegerin (OPG) compared to the conventional mineralized collagen scaffolds. Mineralized collagen-amnion composite scaffolds elicited enhanced early stage osteogenic gene expression (BGLAP, BMP2), increased immunomodulatory gene expression (CCL2, HGF, and MCSF) and increased angiogenic gene expression (ANGPT1, VEGFA) in hMSCs. Mineralized collagen-chorion composite scaffolds promoted immunomodulatory gene expression in hMSCs (CCL2, HGF, and IL6) while unaffecting osteogenic gene expression. Together, these findings suggest that mineralized collagen scaffolds modified using matrix derived from amnion and chorion membranes represent a promising environment conducive to craniomaxillofacial bone repair.

A minimally manipulated preservation and virus inactivation method for amnion/chorion.

Allogeneic amnion tissues have been widely used in tissue repair and regeneration, especially a remarkable trend of clinical uses in chronic wound repair. The virus inactivation procedures are necessary and required to be verified for the clinical use and approval of biological products. Cobalt-60 (Co-60) or electron-beam (e-beam) is the common procedure for virus and bacterial reduction, but the excessive dose of irradiation was reported to be harmful to biological products. Herein, we present a riboflavin (RB)-ultraviolet light (UV) method for virus inactivation of amnion and chorion tissues. We used the standard in vitro limiting dilution assay to test the viral reduction capacity of the RB-UV method on amnion or chorion tissues loaded with four types of model viruses. We found RB-UV was a very effective procedure for inactivating viruses of amnion and chorion tissues, which could be used as a complementary method to Co-60 irradiation. In addition, we also screened the washing solutions and drying methods for the retention of growth factors.

Biologic Therapies for the Treatment of Knee Osteoarthritis: An Updated Systematic Review.

BACKGROUND: Use of "orthobiologics" continues to expand for patients who have knee osteoarthritis (OA). We sought to perform a systemic review of biologic therapies relative to comparative groups, including the following: (1) platelet-rich plasma (PRP); (2) bone marrow-derived mesenchymal stem cells (BMSCs); (3) adipose-derived mesenchymal stem cells (ADSCs); and (4) amniotic-derived mesenchymal stem cells (AMSCs). We assessed the following: (1) study methodologies; (2) cell preparations and formulations; (3) patient-reported outcome scores (PROMs); and (4) structural changes. METHODS: PubMed, Cochrane Library, and Embase databases were queried (2013-2021) to conduct a systematic review of biologic therapies for knee OA, according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Eighty-two studies were included: PRP (51); BMSC (15); ADSC (11); and AMSC (5). Study evaluations were made using the Modified Coleman Methodology Score. PROMs included the Western Ontario and McMaster Universities Arthritis Index and the Visual Analog Scale. Structural change evaluations included ultrasounds, radiographs, or magnetic resonance imaging. RESULTS: PRP comprised a majority of the studies (n = 51), most with "fair" to "good" Modified Coleman Methodology Score. Studies had variable cell preparations and formulations, with comparison study results leading to inconsistent PROMs, and structural changes. A limited number of studies were included for BMSC, ADSC, and AMSC, all with similar findings to PRP. CONCLUSION: Available literature evaluating "orthobiologics" for knee OA remain nonsuperior to comparison cohorts. Higher level studies with larger sample sizes and improved methodologies are warranted to suggest differences. Despite a growth of "orthobiologics" in clinics, this updated systematic review highlights the uncertain efficacy for use in knee OA.

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.

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.

Differential Effector Response of Amnion- and Adipose-Derived Mesenchymal Stem Cells to Inflammation; Implications for Intradiscal Therapy.

Intervertebral disc degeneration (IVDD) is a progressive condition marked by tissue destruction and inflammation. The therapeutic effector functions of mesenchymal stem cells (MSCs) makes them an attractive therapy for patients with IVDD. While several sources of MSCs exist, the optimal choice for use in the inflamed IVD remains a significant question. Adipose (AD)- and amnion (AM)-derived MSCs have several advantages compared with other sources, however, no study has directly compared the impact of IVDD inflammation on their effector functions. Human MSCs were cultured in media with or without supplementation of interleukin-1ß (IL-1ß) and tumor necrosis factor-α at concentrations reportedly produced by IVDD cells. MSC proliferation and production of pro- and anti-inflammatory cytokines were quantified following 24 and 48 h of culture. Additionally, the osteogenic and chondrogenic potential of AD- and AM-MSCs was characterized via histology and biochemical analysis following 28 days of culture. In inflammatory culture, AM-MSCs produced significantly more anti-inflammatory IL-10 (14.47 ± 2.39 pg/ml; p = 0.004) and larger chondrogenic pellets (5.67 ± 0.26 mm2 ; p = 0.04) with greater percent area staining positively for glycosaminoglycan (82.03 ± 3.26%; p < 0.001) compared with AD-MSCs (0.00 ± 0.00 pg/ml; 2.76 ± 0.18 mm2 ; 34.75 ± 2.49%; respectively). Conversely, AD-MSCs proliferated more resulting in higher cell numbers (221,000 ± 8,021 cells; p = 0.048) and produced higher concentrations of pro-inflammatory cytokines prostaglandin E2 (1,118.30 ± 115.56 pg/ml; p = 0.030) and IL-1ß (185.40 ± 7.63 pg/ml; p = 0.010) compared with AM-MSCs (109,667 ± 5,696 cells; 1,291.40 ± 78.47 pg/ml; 144.10 ± 4.57 pg/ml; respectively). AD-MSCs produced more mineralized extracellular matrix (3.34 ± 0.05 relative absorbance units [RAU]; p < 0.001) compared with AM-MSCs (1.08 ± 0.06 RAU). Under identical inflammatory conditions, a different effector response was observed with AM-MSCs producing more anti-inflammatories and demonstrating enhanced chondrogenesis compared with AD-MSCs, which produced more pro-inflammatory cytokines and demonstrated enhanced osteogenesis. These findings may begin to help inform researchers which MSC source may be optimal for IVD regeneration. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2445-2456, 2019.

Insulin-Transferrin-Selenium as a Novel Serum-free Media Supplement for the Culture of Human Amnion Mesenchymal Stem Cells

This study aimed to evaluate the use of Insulin-Transferrin-Selenium (ITS) medium in place of fetal bovine serum (FBS) to culture human amnion mesenchymal stem cells (hAMSCs). Cell morphology, ultrastructure, proliferation, migration and MSC related markers were assessed accordingly. The hAMSCs were induced to osteocyte, chondrocyte, adipocyte and keratinocyte by culturing in appropriate induction medium. hAMSCs mRNA expression was detected for the matrix metalloproteinases 2 (MMP2), keratinocyte growth factor (KGF), vascular endothelial growth factor (VEGF), insulin-like growth factor-I (IGF-I), Platelet-derived Growth Factor (PDGF), and transforming growth factor beta 1 (TGF-ß) by real-time quantitative RT-PCR. Our results showed that hAMSCs cultured in ITS medium exhibited similar proliferation rates, demonstrated a statistically significant increased migration and expressed similar levels of MSC markers(CD73+, CD90+, CD105+, CD45-, CD34-) compared with those cultured in FBS. Osteoblasts, chondrocytes, adipocytes and keratinocytes were differentiated. Results of transmission electron microscope (TEM) revealed that hAMSCs cultured in ITS medium underwent active metabolism. The mRNA expression of MMP2, VEGF, KGF, TGF-ß, IGF-I and PDGF upregulated in ITS medium. In conclusion, ITS medium has the potential to be used for the expansion of hAMSCs before clinical application.

Comparative proteomic analysis of amnion membrane transplantation and cross-linking treatments in an experimental alkali injury model.

PURPOSE: In this study, by using a two-dimensional (2D) electrophoresis-based experimental approach, we aimed at understanding the nature of alkali injuries and the underlying mechanisms. A secondary aim was to compare the effects of cross-linking (CXL) and amnion membrane transplantation (AMT) on corneal protein compositions at the end of the early repair phase after injured with alkali. METHOD: The right corneas of 24 rabbits were injured with a 1 N solution of NaOH. Groups were formed based on the adjuvant therapies as (1) healthy group, (2) control group, (3) CXL group, (4) AMT group. In addition to the therapies, a conventional medical treatment was applied to all groups. Left eyes were used as within-subject healthy corneas (1). The corneas were excised at day 21, and a comparative proteomic analysis was performed using 2D gel electrophoresis coupled with MALDI-TOF/TOF. RESULT: 2D gel electrophoresis revealed the presence seven protein spots whose abundance changed among the groups. Those proteins were SH3 domain-binding protein, plant homeodomain finger protein 23, S100 calcium binding protein A-11(S100 A11), keratin type 2 cytoskeletal 1 and 2, transketolase and glyceraldehyde 3-phosphate dehydrogenase. Ingenuity pathway analysis predicted that the observed changes may be linked to a central metabolic pathway, transforming growth factor beta 1. Canonical pathway analysis focused our attention to two different pathways, namely nicotinamide adenine dinucleotide repair pathway and non-oxidative branch of pentose phosphate pathway. CONCLUSION: Our results shed some light onto the molecular mechanisms affected by alkali injury and adjuvant treatments. Further research is needed to propose medically significant target molecules that may be used for novel drug developments for alkali injury.

Therapeutic effects of human amnion-derived mesenchymal stem cell transplantation and conditioned medium enema in rats with trinitrobenzene sulfonic acid-induced colitis.

Cell therapy with mesenchymal stem cells (MSCs) is expected to provide a new strategy for the treatment of inflammatory bowel disease (IBD). Large amounts of MSCs can be obtained from human amnion. Therefore, we investigated the effect of transplantation of human amnion-derived MSCs (hAMSCs) or enema of conditioned medium (CM) from hAMSCs into rats with 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. In the first experiment, 10-week-old male Sprague-Dawley rats were intravenously injected with hAMSCs (1 × 106 cells) 3 h after rectal administration of TNBS (45 mg/kg). In the second experiment, rats with TNBS-induced colitis received CM by enema into the colon for 3 days. Colitis was investigated by endoscopy, histology, immunohistochemistry, and by measuring mRNA expression of inflammatory mediators. Administration of hAMSCs or CM enema significantly improved the endoscopic score. In addition, these two interventions resulted in significantly decreased infiltration of neutrophils and monocytes/macrophages and decreased expression levels of TNF-α, CXCL1, and CCL2. In conclusion, transplantation of hAMSCs and CM enema provided significant improvement in rats with TNBS-induced colitis. CM from hAMSCs and hAMSCs may be new strategies for the treatment of IBD.

Human amnion epithelial cells modulate hyperoxia-induced neonatal lung injury in mice.

BACKGROUND AIMS: Human amnion epithelial cells (hAECs) prevent pulmonary inflammation and injury in fetal sheep exposed to intrauterine lipopolysaccharide. We hypothesized that hAECs would similarly mitigate hyperoxia-induced neonatal lung injury. METHODS: Newborn mouse pups were randomized to either normoxia (inspired O2 content (FiO2) = 0.21, n = 60) or hyperoxia (FiO2 = 0.85, n = 57). On postnatal days (PND) 5, 6 and 7, hAECs or sterile saline (control) was administered intraperitoneally. All animals were assessed at PND 14. RESULTS: Hyperoxia was associated with lung inflammation, alveolar simplification and reduced postnatal growth. Administration of hAECs to hyperoxia-exposed mice normalized body weight and significantly attenuated some aspects of hyperoxia-induced lung injury (mean linear intercept and septal crest density) and inflammation (interleukin-1α, interleukin-6, transforming growth factor-ß and platelet-derived growth factor-ß). However, hAECs did not significantly alter changes to alveolar airspace volume, septal tissue volume, tissue-to-airspace ratio, collagen content or leukocyte infiltration induced by hyperoxia. CONCLUSIONS: Intraperitoneal administration of hAECs to neonatal mice partially reduced hyperoxia-induced lung inflammation and structural lung damage. These observations suggest that hAECs may be a potential therapy for neonatal lung disease.

New Treatment for Band Keratopathy: Superficial Lamellar Keratectomy, EDTA Chelation and Amniotic Membrane Transplantation

We report two cases of band keratopathy who were treated with thick amniotic membrane that contained a basement membrane structure as a graft, after ethylenediaminetetraacetic acid chelation with trephination and blunt superficial lamellar keratectomy in the anterior stroma. In each case, basement membrane was destroyed and calcium plaque invaded into anterior stroma beneath Bowman's membrane. The calcified lesions were removed surgically, resulting in a smooth ocular surface, and the fine structures of band keratopathy were confirmed by pathologic findings. After that, amniotic membrane transplantation was performed to replace the excised epithelium and stroma. Wound healing was completed within 10 days. Stable ocular surface was restored without pain or inflammation. During the mean follow-up period of 13.5 months, no recurrence of band keratopathy was observed. This combined treatment is a safe and effective method for the removal of deep-situated calcium plaque and allowing the recovery of a stable ocular surface.