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DECELLULARIZED AND ENGINEERED TENDONS WITH SHEEP AMNIOTIC EPITHELIAL STEM CELLS ALLO-TRANSPLANTED IN EXPERIMENTALLY INDUCED SHEEP TENDON RESECTION
No full textAchilles tendinopathy affects athletes, recreational exercisers and even inactive people. The tendon has a poor ability to regenerate, and even after healing the breaking point results in the scar tissue formation that impairs its mechanical properties. To overcome to the poor results of the current therapies, stem cell based treatments appears to be an innovative solution. In this context, amniotic epithelial stem cells (AECs) are receiving growing interest from scientist because of their pluripotency, easy accessibility, low immunogenicity and immunomodulatory properties making them ideal candidates in allo and xenotransplantation setting for stem cell therapy approaches. Preclinical model studies on experimentally tendon defects confirmed ovine AECs (oAECs) direct and indirect role in tendon regeneration also modulating the inflammatory response during tendon healing. In order to improve the partial or total tendon resection with the use of oAECs, this research aims to investigate the allo-transplantation of biological decellularized tendons or biocompatible electrospun scaffolds, both engineered with oAECs. Before starting with the above aims, to confirm oAECs in vitro capabilities to modulate the inflammatory response, this research focused on their in vivo immunomodulatory ability when transplanted into an allogeneic recipient. In particular, we investigated the allotransplanted oAECs immunomodulatory role during the early regeneration phase of sheep tendon experimentally induced defects. Specifically, it was evaluated the oAECs role exerted on macrophages (Mφ), in particular on M1Mφ pro-inflammatory and M2Mφ anti-inflammatory subpopulations involved in tissue regeneration. The in vivo experiments results indicate the oAECs modulate the M1Mφ pro-inflammatory switching toward the M2Mφ regenerative phenotype in the tendon lesion areas. Then, in this project, it has been developed a decellularization technique to obtain decellularized tendons that keep the extracellular matrix (ECM) bioactive components for their use as biological scaffolds for oAECs colonization. We conducted comparative studies of decellularization techniques either using detergents or decellularization of fetal tendon explants cultured in incubator, allowing cells to abandon the tissue and colonize the petri dish. The results on biological scaffolds indicated that the detergent decellularization method was effective for the complete cell components elimination but need to be improved in order to maintain the ECM bioactive components, while the decellularization culture method adopted has demonstrated to achieve a good conservation of the ECM components, but tendons were not completely decellularized. Thus, only the technique with detergents allowed obtaining decellularized tendons that soon will be cultured with oAECs obtaining biological engineered scaffolds. We also evaluated oAECs in vitro biocompatibility on biodegradable polymers, in particular on three electrospun biomaterials used for medical devices, Polylactic acid (PLA), Polycaprolactone (PCL), polylactic acid-co-glycolic (PLGA). This phase of the research was possible thanks to the collaboration of Dr. Tammaro (ENEA Research Center- Brindisi Italy).These scaffolds were treated with 3 different sterilization methods: 70% ethanol, ethylene oxide, ethanol 70% rehydrated for 24 hours, in order to verify which one was the best technique for the analysed scaffolds in terms of ultrastructural morphology. Ovine AECs biocompatibility on PLGA sterilized in ethanol 70% rehydrated revealed to be the best in terms of scaffolds ultrastructural stability, cell colonization and spatial organization around the microfibers. Tissue engineering represent a promising approach in regenerative medicine based on the synergy between biocompatible scaffolds miming ECM and oAECs, although the realization of the ideal scaffold should consider several factors, such as chemical and ultrastructural composition prior their use in allo- and xeno-transplantation setting. Next research will be focused on using aligned fibers PLGA scaffolds (in collaboration with Dr. Tammaro and INNOVENT enterprise), that mimic tendon structure, taking advantage of oAECs capacity to differentiate in tenocyte like cell and subsequently using PLGA engineered scaffolds on experimental of partial or total tendon resection preclinical studies to verify their augmented regenerative potential
Immunomodulatory role of amniotic epithelial stem cells in vivo: new perspectives in tendon regenerative medicine
No full textChallenges in Tendon Tissue Engineering: Innovative Electrospun Scaffolds Mimicking Tendon Extracellular Matrix are Teno-Inductive for Amniotic Epithelial Stem Cells
No full textCellular and molecular maturation in fetal and adult ovine calcaneal tendons
No full textProcesses of development during fetal life profoundly transform tendons from a plastic tissue into a highly differentiated structure, characterised by a very low ability to regenerate after injury in adulthood. Sheep tendon is frequently used as a translational model to investigate cell-based regenerative approaches. However, in contrast to other species, analytical and comparative baseline studies on the normal developmental maturation of sheep tendons from fetal through to adult life are not currently available. Thus, a detailed morphological and biochemical study was designed to characterise tissue maturation during mid- (2 months of pregnancy: 14 cm of length) and late fetal (4 months: 40 cm of length) life, through to adulthood. The results confirm that ovine tendon morphology undergoes profound transformations during this period. Endotenon was more developed in fetal tendons than in adult tissues, and its cell phenotype changed through tendon maturation. Indeed, groups of large rounded cells laying on smaller and more compacted ones expressing osteocalcin, vascular endothelial growth factor (VEGF) and nerve growth factor (NGF) were identified exclusively in fetal mid-stage tissues, and not in late fetal or adult tendons. VEGF, NGF as well as blood vessels and nerve fibers showed decreased expression during tendon development. Moreover, the endotenon of mid- and late fetuses contained identifiable cells that expressed several pluripotent stem cell markers [Telomerase Reverse Transcriptase (TERT), SRY Determining Region Y Box-2 (SOX2), Nanog Homeobox (NANOG) and Octamer Binding Transcription Factor-4A (OCT-4A)]. These cells were not identifiable in adult specimens. Ovine tendon development was also accompanied by morphological modifications to cell nuclei, and a progressive decrease in cellularity, proliferation index and expression of connexins 43 and 32. Tendon maturation was similarly characterised by modulation of several other gene expression profiles, including Collagen type I, Collagen type III, Scleraxis B, Tenomodulin, Trombospondin 4 and Osteocalcin. These gene profiles underwent a dramatic reduction in adult tissues. Transforming growth factor-β~1 expression (involved in collagen synthesis) underwent a similar decrease. In conclusion, these morphological studies carried out on sheep tendons at different stages of development and aging offer normal structural and molecular baseline data to allow accurate evaluation of data from subsequent interventional studies investigating tendon healing and regeneration in ovine experimental models
Electrospun poly(lactide-co-glycolide) scaffold with high grade of fibers alignment mimics tendon extracellular matrix influencing amniotic epithelial stem cells phenotype and orientation
No full textPoly(lactide-co-glycolide) (PLGA) is a copolymer known for its biodegradability and biocompatibility, and when electrospun, it becomes a fibrous device that can be engineered with stem cells. In this study, electrospun PLGA were engineered with amniotic derived stem cells (AECs). These type of stem cells are already known for their easy retrieval, non-ethical concerns, non-tumorigenic and immunomodulatory properties, thus ideal in allo and xenotransplantation settings. Moreover, they are able to differentiate toward the tenocyte linage when co-cultivated in vitro with tendon explants or when transplanted in vivo in a tendon injury model. In fact, when transplanted, oAECs are able to direct tissue remodeling either indirectly, thanks to their ability to release paracrine factors, and directly by producing Collagen Type 1 (COL1), which is the major protein expressed in a tendon. Thus, PLGA electrospun scaffolds were fabricated with a high degree of aligned fibers, in order to mimic tendon extracellular matrix (ECM), and with random fibers (control). Then, these scaffolds were cultured with ovine AECs in order to verify their biocompatibility and if the high degree of fiber alignment could influence cell phenotype and orientation mimicking a tendon tissue structure. To this aim, oAECs were seeded on scaffolds and cultivated for 48h. The results obtained in this study demonstrate that oAECs are biocompatible with the analyzed scaffolds. In fact, Calcein AM and PKH26 vital dyes and Ki67, a cell proliferation marker, immunostaining show that nearly all cells were alive and able to proliferate on electrospun PLGA. Additionally, these fluorescent dyes proved that oAECs spatial distribution and orientation was influenced by scaffold fibers’ alignment. In fact, when oAECs were cultivated on these highly aligned electrospun PLGA fibers they changed their morphology acquiring a spindle tenocyte-like shape, and were able to align along the longitudinal axis of the fibers, whereas in random electrospun PLGA scaffolds oAECs maintained their cuboidal morphology. Moreover, several of these oAECs, were able to express in their cytoplasm COL1 after 48h of culture only on aligned fibers scaffolds and not on the random oriented fibers ones. These findings indicate that when oAECs are seeded on electrospun PLGA scaffolds with highly aligned fibers, their phenotype and orientation are influenced by this artificial tendon ECM structure, thus acquiring an early tenogenic-like phenotype. In conclusion, electrospun PLGA scaffolds engineered with oAECs appears to be a good synergy that can be used for future clinical application in the treatment of tendon disorders
Cellule staminali di derivazione amniotica: dal laboratorio alla medicina rigenerativa delle lesioni del tendine
No full textLe tendinopatie hanno una elevata incidenza e rappresentano un importante problema per la Sanità Pubblica. Infatti, lesioni a carico del tendine insorgono non solo in soggetti praticanti sport ma anche in persone anziane causando dolore e, di conseguenza, una forte riduzione della qualità della vita. Gli scarsi risultati clinici ottenuti sulla riparazione delle lesioni tendinee e la limitata capacità rigenerativa di questo tessuto hanno aumentato l'interesse dell'utilizzo della terapia cellulare sia negli animali che nell'uomo. A tal riguardo, le cellule staminali di derivazione amniotica sono oggetto di un crescente interesse scientifico a causa della loro proprietà di pluripotenza, facile accessibilità, bassa immunogenicità e proprietà immunomodulatorie. Tutti questi aspetti le rendono candidate ideali per poter essere utilizzate anche in approcci di terapia cellulare: cellule immature dotate di proprietà immunomodulatorie che consentono un loro utilizzo anche in situazioni di allo-trapianto e xeno-trapianto. Studi su modelli preclinici eseguiti su difetti tendinei sperimentali hanno confermato il loro ruolo tenorigenerativo in modo sia indiretto che diretto. Queste cellule, una volta trapiantate, sono in grado di modulare la produzione di biomolecole cruciali per la rigenerazione tendinea, come il TGF beta 1, che regola la sintesi del collagene di tipo I, ed il VEGF, che modula la vasolarizzazione tissutale, ed inibiscono il processo infiammatorio locale. Le cellule staminali di derivazione amniotica hanno anche il potenziale di differenziarsi in senso tenogenico nel tessuto ospite producendo collagene di tipo I. Per confermare il loro transdifferenziamento osservato in vivo, abbiamo sviluppato sistemi colturali che simulassero in vitro il microambiente tenodifferenziativo ottenendo strutture tridimensionali tendino-simili. Sulla base di questi promettenti risultati nasce ora la necessità di traslare gli studi preclinici alla pratica clinica. Proprio perché una delle patologie più frequenti è la resezione parziale/totale del tendine, sarà necessario ampliare la conoscenza dei meccanismi di ingegnerizzazione tessutale attraverso l’uso di matrici biologiche o scaffold di polimeri sintetici biocompatibili coltivati con le cellule staminali di derivazione amniotica allo scopo di creare strutture tridimensionali immunologicamente accettate dall’ospite senza l’uso di farmaci anti-infiammatori ed immunosoppressivi
Amniotic derived stem cells: from lab to regenerative medicine for the treatment of tendon disorders”
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Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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