57 research outputs found
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 textCorrection: Antimicrobial resistance genotypes and phenotypes of Campylobacter jejuni isolated in Italy from humans, birds from wild and urban habitats, and poultry(PLoS ONE (2019) 14:10(e0223804)DOI: 10.1371/journal.pone.0223804)
No full textThe third author’s name is spelled incorrectly. The correct name is: Lisa Di Marcantonio. The correct citation is: Marotta F, Garofolo G, Di Marcantonio L, Di Serafino G, Neri D, Romantini R, et al. (2019) Antimicrobial resistance genotypes and phenotypes of Campylobacter jejuni isolated in Italy from humans, birds from wild and urban habitats, and poultry. PLoS ONE 14 (10): e0223804. https://doi.org/10.1371/journal.pone.0223804
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
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
Amniotic epithelial cell transplantation induced alternative M2 macrophage activation supporting tendon regeneration
No full textAmniotic epithelial cells (AECs) have a therapeutic potential in tissue repair because their multipotent
characteristics and ability to modulate the immune response (1,2). AECs, directly and secreting paracrine
factors, modulate tendon healing inducing a prompt regeneration in experimentally injured tendons (3). In
the present research, the role exerted of AECs on macrophages (Mφ) polarization from pro-inflammatory
M1Mφ to anti-inflammatory M2Mφ phenotype were studied and related to the early phases of tendon
healing.
Ovine PKH-26 labeled AECs were transplanted into experimentally induced calcaneal tendons defects (3).
Control (CTR) and AEC-tendon explants were explanted at 7, 14, and 28 days for morphological/molecular
analyses. Tendon healing was described using Hematoxylin and Eosin stain and immunohistochemical (IHC)
analyses to detect the expression of collagen type I an extracellular matrix protein (COL1). IHC and RT-PCR
analyses were carried out to identify Mφ (PanMφCD68) and the incidence of pro-inflammatory M1Mφ
(CD86, IL12) and anti-inflammatory M2Mφ (CD206, YM1, IL10) sub-populations.
AECs were always retrieved within the host tissue enhancing the early phase of tendon healing. In parallel,
tendons displayed a different evolution in inflammatory events. PanMφCD68 expression indicated that
inflammation progressively decreased in both tendons. However, while polarized Mφ and cytokines were
similarly expressed in AECs and CTR tendons until day 7, the M2Mφ became prevalent in the presence of
AECs at day 14 when the tissue started to recover its microarchitecture displaying COL1 parallel oriented
fibers. Analogously, the mRNA content of M2Mφ anti-inflammatory cytokines YM1, IL10, CD206 increased,
while M1Mφ pro-inflammatory markers IL12 and CD86 decreased. Mφ and cytokine expression were
considerably reduced in regenerated AEC-tendons after 28 days, while they were still expressed in CTR
tendons that maintained a disorganized tissue structure.
In summary, these findings suggest that AECs modulate macrophage recruitment enhancing the M2Mφ
regenerative phenotype and down regulating M1Mφ, pro-inflammatory phenotype contributing to tendon
healing. The present results suggests novel insights into potential mechanisms underlying tendon
regeneration, associated with extracellular matrix remodeling amelioration, induced by exogenous ovine
amnios epithelial layer stem cells delivery
Metagenomic Characterization and Volatile Compounds Determination in Rumen from Saanen Goat Kids Fed Olive Leaves
No full textThe accumulation and disposal of by-products deriving from the agro-food industry represents a problem both from an economic and environmental point of view. The use of these matrices in zootechnical nutrition could represent a feasible solution. The aim of the study was to examine the effect of a diet containing olive leaves (OL), a by-product of the olive industry, on the ruminal microbial community of Saanen goat kids and on volatile organic compounds (VOCs) produced during the digestion. Twenty goat kids were randomly divided into two groups of ten goat kids each. The control group (CTR) was fed with a standard diet, while the experimental group (OL+) received a custom-formulated diet containing 10 % OL on a dry matter (DM) basis. After 30 days of trial, genomic DNA was extracted from the rumen liquor and prepared for 16S rRNA-gene sequencing to characterize the rumen microbiota; furthermore, rumen VOCs were also characterized by solid-phase microextraction coupled with gas chromatography-mass spectrometry. The Shannon’s alpha index was not significantly different between the two groups, on the contrary, Bray-Curtis (p < 0.01) and Jaccard (p < 0.01) distances evidenced that feed affected microbial community. Eleven genera were influenced by OL supplementation, with a significant increase (p < 0.05) in Paludibacter, Fibrobacter, Sphaerochaeta Christensenella, Rikenella, Oligosphaera, Candidatus Endomicrobium, Anaerovorax, and Atopobium was observed, while the percentages of Bacteroides and Selenomonas were reduced (p < 0.05). Differences were also observed between the two groups at the family level (p < 0.004). Fibrobacteriaceae, Christensenellaceae, Coriobacteriaceae, Oligosphaeraceae, Candidatus Endomicrobium, and Planctomycetaceae were significantly higher (p < 0.05) in goat kids fed OL diet compared to CTR, while the levels of other identified families, Succinivibrionaceae and Bifidobacteriaceae, were opposite (p < 0.05). Finally, results showed that the main phyla in both groups were Bacteroidetes and Firmicutes; however, no significant differences in the relative abundance of any phyla were observed between the two groups. In addition to what has been reported, the analysis of VOCs at the rumen level showed the ability of the OL integration to induce an increase in hexanoic acid and a parallel decrease in decanal. Furthermore, only in OL+ samples there was the accumulation of α-terpineol to which a wide range of interesting biological properties is attributed. The presence of VOCs associated with health status suggests a favorable role of OL in preserving and improving animal welfare
Amniotic derived stem cells: from lab to regenerative medicine for the treatment of tendon disorders”
No full textMolecular Characterization and Antimicrobial Susceptibility of C. jejuni Isolates from Italian Wild Bird Populations
No full textPoultry is considered a major reservoir of human campylobacteriosis. It also been reported that not only poultry, but also wild birds, are capable of carrying C. jejuni, thus demonstrating to be a risk of spreading the bacteria in the environment. To gain insight into the population structure and investigate the antimicrobial resistance genotypes and phenotypes, we analyzed a collection of 135 C. jejuni from 15 species of wild birds in Italy. MLST revealed the presence of 41 sequence types (STs) and 13 clonal complexes (CCs). ST-179 complex and the generalist ST-45 complex were the most prevalent. Core genome MLST revealed that C. jejuni from ST-45 complex clustered according to the bird species, unlike the ST-179 complex which featured 3 different species in the same cluster. Overall we found a moderate prevalence of resistance to tetracycline (12.5%), ciprofloxacin and nalidixic acid (10%). The novel ST isolated from one pigeon showed resistance to all the antibiotics tested. The ST-179 complex (33.3%) was identified with significantly higher nalidixic acid resistance relative to other tested STs. Nine AMR genes (tet(O), cmeA, cmeB, cmeC, cmeR, aad, blaOXA-61, blaOXA-184 and erm(B)) and 23S rRNA and gyrA-associated point mutations were also described, indicating a concordance level between genotypic and phenotypic resistance of 23.3%, 23.4% and of 37.5% for streptomycin, tetracycline and quinolones/fluoroquinolones, respectively. We recommend that particular attention should be given to wild birds as key sentinel animals for the ecosystem contamination surveillance
Cellular 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
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