Sviluppo di scaffolds biologici da omento e muscolo scheletrico mediante processi di decellularizzazione

ABSTRACT BACKGROUND The availability of organs and tissues, obtained in laboratory from biological material, is one of the main demands in the event of loss of substance, due to congenital or post-traumatic defects. For this purpose, bioengineered tissues have been developed through decellularization and recellularization processes. AIM OF THE STUDY The objectives of this study were: • Development of a new type of biological scaffold derived from omentum that preserves the structural integrity of the extracellular matrix and the vascular network of the native tissue even after a phase of decellularization. • Development of a further biological scaffold from skeletal muscle, derived from the decellularization of the rectus muscle of the rabbit, for the reconstruction of a defect in the abdominal wall. MATERIAL AND METHODS As regards the first part of the experiment, samples of omentum, from rat and human, were decellularized through physical reactions (freezing/thawing and mechanical agitation), chemical (EDTA and isopropanol) and enzymatic (trypsin, lipases and endonucleases) methods involving the removal of cells and their lipid content. Samples were analyzed at various stages by histological staining (hematoxylin and eosin, azan Mallory, van Gieson, PAS, Sudan, Oil Red), immunohistochemical reactions (anti- CD31, -CD34, α-smooth muscle actin) and stained with DAPI. Furthermore, evaluations about the quantitative concentration of genomic DNA at the end of decellularization were carried out with a spectrophotometer. Attempts have been made to recellularize scaffold by cells of the stromal vascular fraction (SVF), isolated from samples of human lipoaspirate. It has, also, developed an additional scaffolds by decellularization of samples of the rectus muscle of the rabbit. Samples were subjected to physical (freezing/thawing), chemical (EDTA and Triton X -100) and enzymatic (DNase) methods and results were analyzed by histological staining (hematoxylin and eosin, azan Mallory, van Gieson). Thereafter, the scaffold was implanted in a rabbit receiver in correspondence with a surgical defect in the abdominal wall with loss of substance. After three weeks, the implant was studied by ultrasound examination in vivo and, after sacrifice of the animal, it was analyzed with the histological stainings mentioned above. RESULTS Histological stainings confirmed the effectiveness of the decellularization process with the total loss of its lipid component of the omentum. This led to the creation of an acellular scaffold in which the three-dimensional organization of the collagen, elastic, and reticular fibers was preserved. Also, the preservation of the vascular network was highlighted. Preliminary studies have shown, moreover, the possibility of recellularization of the scaffold through the introduction of SVF cells. Regarding the second part of the project, it was shown that the muscle-derived scaffold has maintained the integrity of the extracellular matrix and the structure of the vascular channels, in the removal of cellular elements. Following implantation in the receiving rabbit, a solution of continuity with the adjacent tissues has been highlighted, by ultrasound in vivo, with the absence of herniation . After the sacrifice of the animal, histological analysis of the scaffold showed the presence of reparative tissue and new vascular channels in the seat of the abdominal defect. Future perspectives will be to recellularize the scaffold with progenitor cells to promote muscle regeneration. CONCLUSIONS We obtained a new type of acellular biological structure resulting from the omentum, through the application of the process of decellularization, which allows to minimize possible alterations of the extracellular matrix during the removal of the cells. The scaffold was preliminarily recellularized with cells of the stromal vascular fraction, but further experiments will focus on the further evolution of in vitro recellularization and in vivo implantation. The decellularization of the muscle tissue and its implantation in vivo has allowed to identify a biological structure capable of offering a valid alternative to the materials currently used for the defects repair in the abdominal wall. Also in this direction we will evaluate in the future the possibility of recellularization of the scaffold with progenitor cells

Forensic Clinical Anatomy of Spine in Child Abuse

Forensic Clinical Anatomy of Child Abuse includes studies of Functional and/or Biomechanical Anatomy which are performed on cadavers to verify compatibility of lesions with accidental dynamics. Moreover, some kinds of damages following Child Abuse are strictly anatomical in nature and require morphological/morphometric methods of investigation for adequate assessment. Problems of differential diagnosis between anatomical structures (normal or variant) and pathological findings also frequently arise [1]. In the present work, we focused on anatomical bases of spinal lesions in two autoptical cases of abusive head trauma, with particular reference to methodological issues. Both cases presented brain subdural haemorrhage and multiple bilateral retinal haemorrhages. In both cases, the spinal cord was sampled in continuity with the dura mater and it was subjected to complete sectioning. Spinal subdural haemorrhages were found along all the spinal levels. The histopathological characteristics of these haemorrhages also permitted to reveal different chronologies of the lesions, with consequent forensic implications. Hypoxic-ischaemic damages coexisted, mainly at the level of cervical and lumbar spinal cord, together with glio-mesodermic response. On the basis of in vivo imaging suggesting cervical sub-dislocations, portions of the vertebral column were also sampled and subjected to postmortem imaging before further hystopathological sampling. In one case, postmortem imaging permitted to confirm anterolisthesis of the second vertebral body over the third one. Histopathological analysis also showed the presence of haemorrhagic infiltrations of the epidural adipose tissue at the level of the atlanto-axial joints. A consistent methodology of analysis of the spinal structures should involve integration of postmortem imaging with detailed and exhaustive histopathological study

Brödel’s line: an anatomo-radiological study of the avascular kidney’s plane

The division in anterior and posterior branches of the renal artery implies the existence of an avascular plane, the so call Brödel’s line (1). This longitudinal zone is described along the convex renal border (2) or just posterior to the lateral aspect of the kidney (3). The aim of this study was to describe the extension of Brödel’s line with reference to the renal segments. 12 kidneys were injected with acrylic resins to obtain vascular corrosions casts that were analyzed also with computed tomography. We observed the presence of a relative avascular plane in all vascular casts, located on the posterior surface, ascribable to the Brodel’s line. In 33% of cases the line extended from the apical to the inferior segments, in the 33% of cases it extended from the superior to the inferior segments, in 33% of cases it is limited to the superior and middle segments. Since the Brödel’s line corresponds with the plane of the anterior surface of the posterior hilar calyces, the knowledge of its extension is relevant from the surgical point of view: this area permits a relatively safe access route to the pelvicalyceal system for nephrostomy insertion and incision within this plane results in significantly less blood loss than outside this plane

Distribution of cannabinoid receptor 1 (CB1) and 2 (CB2) in subcutaneous tissue and fasciae

The endocannabinoid system is constituted by the endocannabinoid receptors (CB1 and CB2), by the endocannabinoids and the machinery for their biosynthesis and metabolism. Cannabinoid receptors have been localized in the central and peripheral nervous system as well as on cells of the immune system but recently they are discovered at epidermis and dermis1. The endocannabinoid system has been involved in different physiological processes, in particular many works in animal models have discovered an antinociceptive activities in inflammatory state and chronic inflammatory disease. Those findings suggest the possibility that the endocannabinoid system interacts with different cells so it will be interesting to provide a description of endocannabinoid receptors distribution. Immunohistochemical and molecular investigation for CB1 and CB2 localization was carried out in human skin and subcutaneous tissue and direct analysis on fibroblasts isolated from deep fascia2 and subcutaneous tissue of human and rat samples. The majority of endocannabinoid receptors were found in the keratinocytes of skin and mast cells close to subcutaneous adipose tissue, whilst in the deep fascia the presence is scarse. The CB2 receptors were more frequently highlighted respect to CB1 receptors. The abundant distribution of cannabinoid receptors on skin, mast cells and subcutaneous tissue provides implications for an anti-inflammatory, and this suggests more studies to evaluate the therapeutic potential of endocannabinoids

Anatomic-radiologic study of the anterolateral ligament of the knee

Recent anatomic investigations of the lateral structures of the knee have identified a new ligament, called the anterolateral ligament (ALL). To date, the anterolateral ligament has not been microscopically analysed. A retrospective MRI study was carried out in 50 patients by two observers. MRI has been performed for various indications excluding trauma. 10 specimens of ALL were sampled from bodies of the Donation to Science program of the University of Padova. A thin linear structure originating at the lateral epicondyle, running obliquely downwards and forwards and inserting at the lateral aspect of the proximal tibia was observed in 18 cases (90%), with a mean length of 3.8 cm and mean thickness of 1.9 mm. The ALL was hyposignal on both T1- and T2-weighted sequences. From the microscopic point of view the ALL corresponds to a fibrous connective tissue, organised in 2-3 layers of collagen tissue (mean thickness 983+423 micron), with scarce elastic fibres, separated by a thin layer of fibroadipose tissue from the adjacent structures. The ALL appears as almost constantly depicted by MRI and shows a fibrous structure. Its layered organisation could account for its mechanical importance, as a presumed stabilizer of the medial rotation of the kne

Hyperoxia-induced changes in morphometric parameters of postnatal neurogenic sites in rat

In literature many works address the effects of hypoxia exposure on postnatal neurogenesis but few data are available about hyperoxia effects, although high oxygen concentrations are frequently used for ventilation of premature newborns. Thus, the aim of the present study was to compare with controls the morphometrical parameters of the main neurogenic sites (subventricular zone and dentate gyrus) in newborn Sprague-Dawley rats exposed to 60% or 95% oxygen for the first 14 postnatal days. Six rats were studied for each of the three groups. The unbiased quantitative method of the optical disector was applied to analyze neuronal densities, nuclear volumes, and total neuron numbers of the subventricular zone and hippocampal dentate gyrus. Apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling, TUNEL) and proliferation (Ki67) were also studied. The subventricular zone of newborn rats exposed to 95% hyperoxia showed statistically significant higher volume (mean value ± coefficient of variation: 0.40 ± 0.20 mm3) than subventricular zone of rats raised in normoxia (0.20 ± 0.11 mm3) or 60% hyperoxia (0.26 ± 0.18 mm3). Total neuron number was also significantly higher in 95% hyperoxia while neuronal densities did not reach statistically significant differences. TUNEL showed increased apoptotic indexes in hyperoxic rats. The percentage of proliferating KI67 positive cells was also higher in hyperoxia. The dentate gyrus granular layer of the normoxic rats showed higher volume (0.65 ± 0.11 mm3) than both the hyperoxic groups (60% hyperoxia: 0.39 ± 0.14 mm3; 95% hyperoxia: 0.36 ± 0.16 mm3). Total neuron numbers of hyperoxic dentate gyrus were also significantly reduced; neuronal densities were not modified. Hyperoxia-exposed rats also showed higher apoptotic and proliferating indexes in the dentate gyrus. Hyperoxia exposure in the first postnatal period may affect the main neurogenic areas (subventricular zone and dentate gyrus) increasing apoptosis but also inducing a certain reparative response consisting of increased proliferation. In particular, the increased volume of the subventricular zone may be ascribed to compensatory neurogenic response to the hyperoxic damage. Conversely, the decreased volume of the dentate gyrus granular layer could derive by a non sufficient neurogenic response to counterbalance the hyperoxic neuronal injury

Development and Implementation of the AIDA International Registry for Patients With Still's Disease

Objective: Aim of this paper is to present the design, construction, and modalities of dissemination of the AutoInflammatory Disease Alliance (AIDA) International Registry for patients with systemic juvenile idiopathic arthritis (sJIA) and adult-onset Still's disease (AOSD), which are the pediatric and adult forms of the same autoinflammatory disorder. Methods: This Registry is a clinical, physician-driven, population- and electronic-based instrument implemented for the retrospective and prospective collection of real-world data. The collection of data is based on the Research Electronic Data Capture (REDCap) tool and is intended to obtain evidence drawn from routine patients' management. The collection of standardized data is thought to bring knowledge about real-life clinical research and potentially communicate with other existing and future Registries dedicated to Still's disease. Moreover, it has been conceived to be flexible enough to easily change according to future scientific acquisitions. Results: Starting from June 30th to February 7th, 2022, 110 Centers from 23 Countries in 4 continents have been involved. Fifty-four of these have already obtained the approval from their local Ethics Committees. Currently, the platform counts 290 users (111 Principal Investigators, 175 Site Investigators, 2 Lead Investigators, and 2 data managers). The Registry collects baseline and follow-up data using 4449 fields organized into 14 instruments, including patient's demographics, history, clinical manifestations and symptoms, trigger/risk factors, therapies and healthcare access. Conclusions: This international Registry for patients with Still's disease will allow a robust clinical research through collection of standardized data, international consultation, dissemination of knowledge, and implementation of observational studies based on wide cohorts of patients followed-up for very long periods. Solid evidence drawn from "real-life " data represents the ultimate goal of this Registry, which has been implemented to significantly improve the overall management of patients with Still's disease. NCT 05200715 available at

Clinical and laboratory features associated with macrophage activation syndrome in Still's disease: data from the international AIDA Network Still's Disease Registry

: To characterize clinical and laboratory signs of patients with still's disease experiencing macrophage activation syndrome (MAS) and identify factors associated with MAS development. patients with still's disease classified according to internationally accepted criteria were enrolled in the autoInflammatory disease alliance (AIDA) still's disease registry. clinical and laboratory features observed during the inflammatory attack complicated by MAS were included in univariate and multivariate logistic regression analysis to identify factors associated to MAS development. A total of 414 patients with Still's disease were included; 39 (9.4%) of them developed MAS during clinical history. At univariate analyses, the following variables were significantly associated with MAS: classification of arthritis based on the number of joints involved (p = 0.003), liver involvement (p = 0.04), hepatomegaly (p = 0.02), hepatic failure (p = 0.01), axillary lymphadenopathy (p = 0.04), pneumonia (p = 0.03), acute respiratory distress syndrome (p < 0.001), platelet abnormalities (p < 0.001), high serum ferritin levels (p = 0.009), abnormal liver function tests (p = 0.009), hypoalbuminemia (p = 0.002), increased LDH (p = 0.001), and LDH serum levels (p < 0.001). at multivariate analysis, hepatomegaly (OR 8.7, 95% CI 1.9-52.6, p = 0.007) and monoarthritis (OR 15.8, 95% CI 2.9-97.1, p = 0.001), were directly associated with MAS, while the decade of life at Still's disease onset (OR 0.6, 95% CI 0.4-0.9, p = 0.045), a normal platelet count (OR 0.1, 95% CI 0.01-0.8, p = 0.034) or thrombocytosis (OR 0.01, 95% CI 0.0-0.2, p = 0.008) resulted to be protective. clinical and laboratory factors associated with MAS development have been identified in a large cohort of patients based on real-life data

Sviluppo di scaffolds biologici da omento e muscolo scheletrico mediante processi di decellularizzazione

ABSTRACT BACKGROUND The availability of organs and tissues, obtained in laboratory from biological material, is one of the main demands in the event of loss of substance, due to congenital or post-traumatic defects. For this purpose, bioengineered tissues have been developed through decellularization and recellularization processes. AIM OF THE STUDY The objectives of this study were: • Development of a new type of biological scaffold derived from omentum that preserves the structural integrity of the extracellular matrix and the vascular network of the native tissue even after a phase of decellularization. • Development of a further biological scaffold from skeletal muscle, derived from the decellularization of the rectus muscle of the rabbit, for the reconstruction of a defect in the abdominal wall. MATERIAL AND METHODS As regards the first part of the experiment, samples of omentum, from rat and human, were decellularized through physical reactions (freezing/thawing and mechanical agitation), chemical (EDTA and isopropanol) and enzymatic (trypsin, lipases and endonucleases) methods involving the removal of cells and their lipid content. Samples were analyzed at various stages by histological staining (hematoxylin and eosin, azan Mallory, van Gieson, PAS, Sudan, Oil Red), immunohistochemical reactions (anti- CD31, -CD34, α-smooth muscle actin) and stained with DAPI. Furthermore, evaluations about the quantitative concentration of genomic DNA at the end of decellularization were carried out with a spectrophotometer. Attempts have been made to recellularize scaffold by cells of the stromal vascular fraction (SVF), isolated from samples of human lipoaspirate. It has, also, developed an additional scaffolds by decellularization of samples of the rectus muscle of the rabbit. Samples were subjected to physical (freezing/thawing), chemical (EDTA and Triton X -100) and enzymatic (DNase) methods and results were analyzed by histological staining (hematoxylin and eosin, azan Mallory, van Gieson). Thereafter, the scaffold was implanted in a rabbit receiver in correspondence with a surgical defect in the abdominal wall with loss of substance. After three weeks, the implant was studied by ultrasound examination in vivo and, after sacrifice of the animal, it was analyzed with the histological stainings mentioned above. RESULTS Histological stainings confirmed the effectiveness of the decellularization process with the total loss of its lipid component of the omentum. This led to the creation of an acellular scaffold in which the three-dimensional organization of the collagen, elastic, and reticular fibers was preserved. Also, the preservation of the vascular network was highlighted. Preliminary studies have shown, moreover, the possibility of recellularization of the scaffold through the introduction of SVF cells. Regarding the second part of the project, it was shown that the muscle-derived scaffold has maintained the integrity of the extracellular matrix and the structure of the vascular channels, in the removal of cellular elements. Following implantation in the receiving rabbit, a solution of continuity with the adjacent tissues has been highlighted, by ultrasound in vivo, with the absence of herniation . After the sacrifice of the animal, histological analysis of the scaffold showed the presence of reparative tissue and new vascular channels in the seat of the abdominal defect. Future perspectives will be to recellularize the scaffold with progenitor cells to promote muscle regeneration. CONCLUSIONS We obtained a new type of acellular biological structure resulting from the omentum, through the application of the process of decellularization, which allows to minimize possible alterations of the extracellular matrix during the removal of the cells. The scaffold was preliminarily recellularized with cells of the stromal vascular fraction, but further experiments will focus on the further evolution of in vitro recellularization and in vivo implantation. The decellularization of the muscle tissue and its implantation in vivo has allowed to identify a biological structure capable of offering a valid alternative to the materials currently used for the defects repair in the abdominal wall. Also in this direction we will evaluate in the future the possibility of recellularization of the scaffold with progenitor cells.RIASSUNTO PRESUPPOSTI DELLO STUDIO La disponibilità di organi e tessuti ottenuti in laboratorio da materiale biologico è, ad oggi, una delle maggiori richieste nei casi di perdita di sostanza a causa di difetti congeniti o post-traumatici. A fronte di ciò, si è cercato di realizzare dei tessuti bioingegnerizzati mediante l’applicazione di processi di decellularizzazione e successiva ricellularizzazione. SCOPO DELLO STUDIO Gli obiettivi del presente lavoro sono stati i seguenti: • Sviluppare un nuovo tipo di scaffold biologico derivato dall'omento che conservi l’integrità strutturale della matrice extracellulare e della rete vascolare del tessuto nativo anche dopo una fase di decellularizzazione. • Sviluppare un ulteriore scaffold biologico da muscolo scheletrico, derivato dalla decellularizzazione del muscolo retto del coniglio, per la ricostruzione di un difetto della parete addominale. MATERIALI E METODI Per quanto riguarda la prima parte sperimentale, campioni di omento, di ratto e di uomo, sono stati decellularizzati mediante l'impiego di una serie di reazioni fisiche (congelamento/scongelamento ed agitazione meccanica), chimiche (EDTA e isopropanolo) ed enzimatiche (tripsina, endonucleasi e lipasi) che comportano la rimozione delle cellule e della loro componente lipidica. I campioni sono stati analizzati nelle varie fasi mediante colorazioni istologiche (ematossilina ed eosina, azan Mallory, van Gieson, PAS, Sudan, Oil Red), reazioni immunoistochimiche (anti-CD31, -CD34, -α actina del muscolo liscio) e colorazione con DAPI. Inoltre, sono state effettuate valutazioni quantitative allo spettrofotometro della concentrazione di DNA genomico al termine della decellularizzazione. Sono stati effettuati tentativi di ricellularizzazione dello scaffold mediante cellule della frazione stromale vascolare (SVF), isolate da campioni di lipoaspirato umano. È stato altresì sviluppato un ulteriore scaffold mediante decellularizzazione di campioni del muscolo retto di coniglio. Questi sono stati sottoposti a reazioni fisiche (congelamento/scongelamento), chimiche (EDTA e Triton X-100) ed enzimatiche (DNasi) ed i risultati sono stati analizzati mediante colorazioni istologiche (ematossilina ed eosina, azan Mallory, van Gieson). In seguito, lo scaffold è stato impiantato a tutto spessore in un coniglio ricevente in corrispondenza di un difetto della parete addominale con perdita di sostanza prodotto chirurgicamente. Dopo tre settimane, l’impianto è stato studiato mediante esame ecografico in vivo e, dopo sacrificio dell’animale, è stato analizzato mediante le colorazioni istologiche di cui sopra. RISULTATI Le colorazioni istologiche hanno confermato l'efficacia del procedimento di decellularizzazione con la perdita totale della componente lipidica propria dell'omento. Ciò ha portato alla realizzazione di uno scaffold acellulare nel quale è stata mantenuta l’organizzazione tridimensionale delle fibre collagene, elastiche e reticolari. Inoltre è stata evidenziata la preservazione della rete di canali vascolari. Studi preliminari hanno dimostrato, inoltre, la possibilità di ricellularizzare lo scaffold mediante introduzione di cellule appartenente alla SVF. Riguardo la seconda parte del progetto, è stato dimostrato che lo scaffold di derivazione muscolare ha mantenuto l'integrità della matrice extracellulare e della struttura dei canali vascolari, di cui rimangono apprezzabili i diversi strati che li compongono. A seguito dell’impianto nel coniglio ricevente è stata evidenziata, mediante ecografia in vivo, l’integrazione dello scaffold con i tessuti adiacenti con buone proprietà meccaniche. Dopo il sacrificio dell’animale l’analisi istologica dello scaffold ha delineato la presenza di tessuto riparativo e di nuovi canali vascolari nella sede del difetto addominale. Obiettivo futuro sarà quello di ricellularizzare lo scaffold con cellule progenitrici muscolari per favorire la rigenerazione in senso muscolare dello scaffold stesso. CONCLUSIONI Abbiamo ottenuto un nuovo tipo di struttura acellulare biologica derivante dall'omento, mediante l'applicazione del processo di decellularizzazione, il quale permette di minimizzare il più possibile le alterazioni della matrice extracellulare durante la rimozione delle cellule. In via preliminare lo scaffold è stato ricellularizzato con cellule appartenenti alla frazione stromale vascolare ma ulteriori esperimenti si focalizzeranno sul potenziamento della ricellularizzazione in vitro e su impianto in vivo. La decellularizzazione del tessuto muscolare ed il suo impianto in vivo ha permesso di identificare una struttura biologica in grado di offrire una valida alternativa ai materiali attualmente impiegati per la riparazione dei difetti di parete addominale. Anche in questa direzione verrà valutata in futuro la possibilità di ricellularizzare lo scaffold con cellule progenitrici