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Item Antimicrobial Effect of the Amniotic Membrane Isolated and Associated with Photodynamic Therapy(MDPI) Santos, Amanda Cerquearo Rodrigues dos; Teodoro, Guilherme Rodrigues; Ferreira-Strixino, Juliana; Sant’Anna, Luciana BarrosMicrobial control through alternative therapies, such as the amniotic membrane (AM) and antimicrobial photodynamic therapy (aPDT), has been gaining prominence with the advancement of bacterial resistance to conventional treatments. This study aimed to evaluate the antimicrobial effect of AM isolated and associated with aPDT using the PHTALOX® as a photosensitizer (PS) against Staphylococcus aureus and Pseudomonas aeruginosa biofilms. The groups studied were: C+; L; AM; AM+L; AM+PHTX; and AM+aPDT. The irradiation parameters were 660 nm, 50 J.cm−2, and 30 mW.cm−2. Two independent microbiological experiments were carried out in triplicate, and the results were analyzed by CFU/mL counting and a metabolic activity test, both statistically analyzed (p < 0.05). The integrity of the AM was verified after the treatments by a scanning electron microscope (SEM). The groups AM, AM+PHTX, and, mainly, AM+aPDT showed a statistical difference When compared to C+ regarding the decrease in CFU/mL and metabolic activity. SEM analysis showed significant morphological alterations in the AM+PHTX and AM+aPDT groups. The treatments with AM isolated or associated with PHTALOX® were adequate. The association had potentiated the biofilm effect, and the morphological differences presented by AM after treatment did not hinder its antimicrobial effect, encouraging its use in biofilm formation locals.Item Analysis of Movements in Spinal Cord Hemisection Treatment with Amniotic Membrane – Preclinical Study(Scientific Research Publishing Inc.) Neves, Marcele Florêncio das; Fonseca, Joyce Lilian Reis da; Carvalho, Paula Cristina Simões de; Pereira, Aline Priscila Campos; Polisel, Evandro Henrique Pintor; Proglhof, Stephanie de Medeiros Passos; Sant ́Anna, Luciana Barros; Lima, Mário Oliveira; Arisawa, Emília Ângela Lo SchiavoAims: This study aimed to evaluate the efficacy of Amniotic Membrane application in rats with Spinal Cord Injury induced by transverse hemisection using kinematic analysis and Sciatic Functional Index. Study design: True experimental research design. Place and Duration of Study: Instiuto de Pesquisa e Desenvolvimento (IP&D) of Universidade do Vale do Paraíba (UNIVAP), between September 2016 and December 2017. Methodology: Fifteen adult male rats were used, allocated into three equal groups: Control (the spinal cord injury and Amniotic Membrane application were simulated), Lesion (spinal cord injury not treated), Amniotic membrane (spinal cord injury treated by amniotic membrane). All animals underwent surgical procedures. A transverse hemisection was performed in groups Lesion and Amniotic Membrene. A fragment of the biomaterial was applied in group AM covering the hemisection area. Results: Sciatic Functional Index and motion analysis were performed by comparing images taken at pre- and postoperative time at 7, 14, 21, and 28 days. The kinematic analysis showed a significant difference between groups Control and Lesion at 7 days (p = 0.023) and 14 days (p = 0.015), and between groups Lesion and Amniotic Membrane at 14 days (p = 0.039), comparing the postoperative periods. The Sciatic Functional Index revealed significant differences between Groups Control and Lesion at 7 (p = -0.002), 14 (p = 0.003), and 21 days (p = 0.009), between Groups Control and Amniotic Membrane at 7 (p = 0.014), 14 (p = 0.007), and 28 days (p = 0; 013), and between Groups Lesion and Amniotic Membrane only at 14 days (p = 0.039). Conclusion: Application of amniotic membrane in spinal cord hemisection in rats induced gait recovery and improvement in SFI compared to the untreated group.Item Effects of Preservation Methods in the Composition of the Placental and Reflected Regions of the Human Amniotic Membrane(Karger) Moraes, Jéssica Tereza Guedes de Oliveira; Costa, Maíra Maftoum; Alves, Paula Cristina Santos; Sant’Anna, Luciana BarrosThe human amniotic membrane (AM) is emerging as an in- teresting biomaterial for regenerative medicine due to its biological and mechanical proprieties. The beneficial effects of the AM are probably related to its bioactive factors pro- duced by local cells and stored in the stromal matrix. How- ever, the search for a preservation method capable of pre- serving AM properties remains a challenge. The aim of this study was to evaluate important features of 2 anatomical re- gions of the human AM (reflected and placental amnion) af- ter different preservation methods. For this purpose, human placentas were harvested and processed for AM isolation and storage at 2 different conditions: room temperature for 18 h in DMEM (fresh AM) and −80°C in DMEM/glycerol solu- tion for 30 days (cryopreserved AM). After the storage peri- od, the structural integrity of the membrane was assessed by histological and Picrosirius polarization analysis, cellular vi- ability analysis was performed using the MTT assay, and the soluble proteins were quantified with the Qubit Protein Assay Kit. Both preservation protocols reduced the cell viabili- ty, mainly in the placental amnion region of the AM, but pre- served the morphology of epithelial and stromal layers, as well as the organization and distribution of collagen fibers. There was a reduction in soluble proteins only in fresh AM. Importantly, the cryopreserved AM group presented the same concentration as the control group. In conclusion, the cryopreservation using DMEM/glycerol was ideal for pre- serving the structural integrity and soluble protein content, indicating the feasibility of this method in preserving AM for its use in regenerative medicine.Item Evaluation of the antimicrobial action of plasma activated water on amniotic membrane(Springer) Almeida, Felipe Santos de; Doria, Anelise Cristina Osório Cesar; Sant’Anna, Luciana Barrosntroduction The potential use of the Amniotic Membrane (AM) in different applications has been widely studied and the need to guarantee the sterility of this material is essential for its use. Due to the increase in bacterial resistance, it is important to research new sterilization techniques. Material and methods The aim of this study was to analyze the antimicrobial action of plasma-activated reverse osmosis (RO) water on contaminated amniotic membrane using ATCC® strain of Escherichia coli (25922), Klebsiella pneumoniae (13883), Staphylococcus aureus (6538). The AM was contaminated, after which the PAW was placed in contact with the AM for 90 min. Plasma, gliding arc of argon and compressed air were used to activate the water. Results The results obtained were a cell viability of 11% with the refrigerated PAW and 15% with the ambient PAW for K. pneumoniae, 9% with the refrigerated PAW and 13% with the ambient PAW for S. aureus and 10% with the refrigerated PAW and 14% with the ambient PAW for E. coli, observing a better antimicrobial action of the refrigerated PAW. A greater reduction in CFU was observed when using refrigerated PAW + AM. Conclusion It is concluded that PAWs have a significant antimicrobial action, but not enough to perform AM sterilization.Item Amniotic membrane modulates MMP9 and MMP12 gene and protein expression in experimental model of the hepatic fibrosis(Academia Brasileira de Ciências) Alves, Ana Paula da Silva; Teixeira, Roberta Jenniffer Maciel; Silva, Raissa Monteiro da; Canevari, Renata de Azevedo; Sant’Anna, Luciana BarrosHepatic fibrosis is characterized by excessive deposition of collagen in the hepatic parenchyma, which disturbs the normal architecture and function. We have shown that human amniotic membrane (AM) can be used as a patch on the whole liver surface, resulting in an extremely significant reduction in collagen deposition. The aim of this study was to investigate the effects of AM on the matrix metalloproteinase 9 (MMP9) and matrix metalloproteinase 12 (MMP12) genes and proteins expression by real time quantitative PCR and immunohistochemistry, respectively, as well as image analysis on biliary fibrosis induced in rats by the bile duct ligation (BDL).Two weeks after the BDL, an AM fragment was applied onto the liver, and four weeks later, the liver samples were collected. MMP9 and MMP12 genes were significantly over expressed in group treated with AM. The immunoexpression of MMP9 and MMP12 was observed in all groups. However, the quantitative image analysis demonstrated an increase of the area occupied only by MMP12 in the livers of AM-treated rats with respect to BDL rats. These findings suggest that the AM exerts its beneficial effects on biliary fibrosis by increasing the MMP12, which in turn reduces the excessive collagen deposition on liver tissue.Item Histological analysis of Spinal Cord Injury treated with Amniotic Membrane(Universidade de São Paulo) Correia, Débora Campos Chaves; Lima, Leonardo Borges de; Sant'Anna, Luciana Barros; Lima, Mario Oliveira; Arisawa, Emilia Angela Lo SchiavoSpinal cord injury (SCI) is one of the most harmful syndromes that affects humans due to neuronal destruction and interruption of the nerve impulse transmission between axons. The conduction of motor, sensory, and autonomic responses below the level of the injury is seriously compromised, generating high treatment costs for the health system and a reduction in quality of life, stimulating research into new treatment protocols. This study aimed to evaluate the effectiveness of a biomaterial, the amniotic membrane (AM), to treat experimentally induced SCI. 15 adult rats were divided into three groups (n = 5): S (Sham), L (SCI without treatment), and AM (SCI treated with AM). Spinal cord injury was induced in the region T9-T10 by direct trauma, free-falling a weight (10 g, 2 mm flat edge) held on a mini guillotine, 25 mm above the exposed spinal cord. A fragment of AM, obtained from the human placenta after maternal consent, was applied to the injured area only in the AM group. After 28 days, specimens from the area of spinal cord injury were excised and subjected to routine histological procedures. Data from the semi-quantitative score, obtained from a scheme that assigned different scores to regions of the spinal cord, and from the quantitative analysis were subjected to parametric statistical analysis. Results showed that Group S presented medullary tissue without changes (score 0). In contrast, Group L presented numerous areas of cavitation in the dorsal and lateral regions of the white and gray matter (9.61 ± 6.60 p<0.001) with an intense inflammatory infiltrate. The AM group exhibited small areas of cavitation in the dorsal and lateral regions of the white matter and part of the dorsal columns in the gray matter (0.94 ± 1,03, p<0.001), with few inflammatory cells. The results suggest the effectiveness of AM in the treatment of induced SCI, characterized by a reduction in the evolution of inflammatory and degenerative processes in the central nervous tissue compared to the untreated group.Item Photobiomodulation and amniotic membrane for treat tendon injury in rats(Academia Brasileira de Ciências) Nicodemo, Mariana de Castro; Arisawa, Emilia Angela Lo Schiavo; Sant’Anna, Luciana Barros; Martins, Rodrigo Álvaro Brandão LopesTendons, complex fibrous structures, are subjected to great tensions, which can give rise to the so-called tendinopathies. This study aimed to evaluate photobiomodulation and human Amniotic Membrane applied as single or combined therapies to treat induced Achilles tendon lesions. Seventy-five rats were divided into five groups (n=15): C- control Sham surgery; I- tendon injury; LA- tendon injury treated with photobiomodulation; AM- tendon injury treated with Amniotic Membrane; LAM- tendon injury + photobiomodulation and Amniotic Membrane, subdivided into three groups (n=5) with analysis at 3, 7, and 14 days. The tendon injuries were made with a 20 g weight released from a mini guillotine onto the ankle in dorsiflexion. AM and LAM groups received an Amniotic Membrane fragment while LA and LAM groups received transcutaneous photobiomodulation, using a 660 nm wavelength laser. The inflammatory cells showed statistical differences between groups C and I (p<0.05), I and AM (p<0.01), I and LA (p<0.05), and I and LAM (p<0.01). Both photobiomodulation and Amniotic Membrane were shown to enhance tendon repair, and the association of photobiomodulation plus Amniotic Membrane was the most effective treatment. We conclude that the association of photobiomodulation plus Amniotic Membrane was effective in accelerating and improving the tendon regeneration process.