Stefano Brizzola

Airway Fistula Closure after Stem-Cell Infusion

Investigators observed the healing of a broncholpeural fistula soon after the injection of mesenchymal stem cells into the area surrounding the fistula.

Stem Cell Transplantation Effectively Occludes Bronchopleural Fistula in an Animal Model

BACKGROUND Bronchopleural fistula after lung resection still represents a challenging life-threatening complication for thoracic surgeons. Considering its extremely high mortality rate, an effective treatment is urgently required. Our project investigated the hypothesis of experimental bronchopleural fistula closure by bronchoscopic injection of autologous bone marrow-derived mesenchymal stem cells into the cavity of the fistula, evaluating its feasibility and safety in a large animal model. METHODS An experimental bronchopleural fistula was created in 9 goats after right upper tracheal lobectomy. The animals were randomly assigned to two groups: one received autologous bone marrow-derived mesenchymal stem cell bronchoscopic transplantation; the other received standard bronchoscopic fibrin glue injection. RESULTS All animals receiving bronchoscopic stem cell transplantation presented fistula closure by extraluminal fibroblast proliferation and collagenous matrix development; none (0%) died during the study period. All animals receiving standard treatment still presented bronchopleural fistula; 2 of them (40%) died. Findings were confirmed by pathology examination, computed tomography, and magnetic resonance imaging. CONCLUSIONS Bronchoscopic transplantation of bone marrow-derived mesenchymal stem cells effectively closes experimental bronchopleural fistula by extraluminal fibroblast proliferation and collagenous matrix development. Stem cells may play a crucial role in the treatment of postresectional bronchopleural fistula after standard lung resection. Although these results provide a basis for the development of clinical therapeutic strategies, the exact mechanism by which they are obtained is not yet completely clear; further studies are required to understand exactly how stem cells work in this field.

Expression of C-Kit Proto-Oncogene in Canine Mastocytoma: A Kinetic Study Using Real-Time Polymerase Chain Reaction

KIT receptor, the c-kit gene product, is thought to play a major role in canine mastocytoma, one of the most common neoplastic diseases in dogs. In the present study, the expression of c-kit proto-oncogene in blood and in tumor biopsies from 41 dogs with histologically confirmed mastocytoma at different grades of cellular differentiation and 5 negative control dogs was investigated using real-time (quantitative) reverse transcription–polymerase chain reaction (RRT-PCR). The animals were followed up for over 1 year after surgery in order to characterize the kinetics of c-kit expression in blood. Transcript mRNAs extracted from blood at different time points after surgery and from tumor tissue surgically removed from each dog were used in a quantitative RRT-PCR assay targeting the extracellular coding region of the c-kit gene. Tissues constitutively expressing c-kit (brain and spleen) were used as positive controls. Levels of expression of c-kit were higher in tumor biopsies than in blood; the blood level decreased in the patients between 1 and 3 months after surgery. No KIT expression was detected in blood from the 5 dogs not affected by mastocytoma (negative controls). The RRT-PCR appears to be a suitable method for sensitive and quantitative detection of c-kit gene expression in canine blood and neoplastic tissues. Although c-kit expression levels measured by RRT-PCR do not correlate with prognosis, they confirm that surgery remains the main treatment to reduce circulating mastocytes and that circulating mast cells can be detected even in benign highly differentiated forms of mastocytoma such as grade I.

A dynamic distention protocol for whole-organ bladder decellularization: histological and biomechanical characterization of the acellular matrix

A combined physical–chemical protocol for whole full‐thickness bladder decellularization is proposed, based on organ cyclic distention through repeated infusion/withdrawal of the decellularization agents through the urethra. The dynamic decellularization was intended to enhance cell removal efficiency, facilitating the delivery of detergents within the inner layers of the tissue and the removal of cell debris. The use of mild chemical detergents (hypotonic solution and non‐ionic detergent) was employed to limit adverse effects upon matrix 3D ultrastructure. Inspection of the presence of residual DNA and RNA was carried out on decellularized matrices to verify effective cell removal. Histological investigation was focused on assessing the retention of adequate structural and functional components that regulate the biomechanical behaviour of the acellular tissue. Biomechanical properties were evaluated through uniaxial tensile loading tests of tissue strips and through ex vivo filling cystometry to evaluate the whole‐organ mechanical response to a physiological‐like loading state. According to our results, a dynamic decellularization protocol of 17 h duration with a 5 ml/min detergent infusion flow rate revealed higher DNA removal efficiency than standard static decellularization, resulting in residual DNA content < 50 ng/mg dry tissue weight. Furthermore, the collagen network and elastic fibres distribution were preserved in the acellular ECM, which exhibited suitable biomechanical properties in the perspective of its future use as an implant for bladder augmentation. Copyright

Morphologic features of biocompatibility and neoangiogenesis onto a biodegradable tracheal prosthesis in an animal model

We evaluated a newly designed bioresorbable polymer (Degrapol) tracheal prosthesis in an in-vivo angiogenesis-inducing animal model focusing on the specific tissue reaction, the neo-angiogenesis and also the eventual cathepsin B role during the polymer degradation. Fifteen rabbits were divided into three groups (2, 6 and 8 weeks) and our tube-shaped porous prosthesis was implanted using the common carotid artery and the internal jugular vein as vascular pedicle. Optical and electron microscopy, immunohistochemistry and immunocytochemistry were performed at the end of each period, showing cells and fibrils, in direct contact with the Degrapol scaffold, strongly increased with time. Blood vessel neoformation was visible with CD31 expression localized at the endothelial cells forming the neovascular walls. Over time many of them differentiate in muscle fibers as validated by the expression of alpha-smooth muscle actin (SMA). Few inflammatory cells, expressing CD14, were visible while most cells adopting a pronounced spreading phenotype showed a strong positivity for cathepsin B. We concluded that this bioresorbable polymer provided a good substrate for fibrous tissue deposition with an excellent degree of neo-angiogenesis. Also, cathepsin B seems to contribute to the polymer degradation and particularly to neovascularization by stimulating capillary-like tubular structures and cell proliferation.

On the Use of the Platelet Activity State Assay for the In Vitro Quantification of Platelet Activation in Blood Recirculating Devices for Extracorporeal Circulation

We designed an experimental setup to characterize the thrombogenic potential associated with blood recirculating devices (BRDs) used in extracorporeal circulation (ECC). Our methodology relies on in vitro flow loop platelet recirculation experiments combined with the modified-prothrombinase platelet activity state (PAS) assay to quantify the bulk thrombin production rate of circulated platelets, which correlates to the platelet activation (PA) level. The method was applied to a commercial neonatal hollow fiber membrane oxygenator. In analogous hemodynamic environment, we compared the PA level resulting from multiple passes of platelets within devices provided with phosphorylcholine (PC)-coated and noncoated (NC) fibers to account for flow-related mechanical factors (i.e., fluid-induced shear stress) together with surface contact activation phenomena. We report for the first time that PAS assay is not significantly sensitive to the effect of material coating under clinically pertinent flow conditions (500 mL/min), while providing straightforward information on shear-mediated PA dynamics in ECC devices. Being that the latter is intimately dependent on local flow dynamics, according to our results, the rate of thrombin production as measured by the PAS assay is a valuable biochemical marker of the selective contribution of PA in BRDs induced by device design features. Thus, we recommend the use of PAS assay as a means of evaluating the effect of modification of specific device geometrical features and/or different design solutions for developing ECC devices providing flow conditions with reduced thrombogenic impact.

Epigenetic conversion of adult dog skin fibroblasts into insulin-secreting cells.

Diabetes is among the most frequently diagnosed endocrine disorder in dogs and its prevalence continues to increase. Medical management of this pathology is lifelong and challenging because of the numerous serious complications. A therapy based on the use of autologous viable insulin-producing cells to replace the lost β cell mass would be very advantageous. A protocol to enable the epigenetic conversion of canine dermal fibroblasts, obtained from a skin biopsy, into insulin-producing cells (EpiCC) is described in the present manuscript. Cells were briefly exposed to the DNA methyltransferase inhibitor 5-azacytidine (5-aza-CR) in order to increase their plasticity. This was followed by a three-step differentiation protocol that directed the cells towards the pancreatic lineage. After 36 days, 38 ± 6.1% of the treated fibroblasts were converted into EpiCC that expressed insulin mRNA and protein. Furthermore, EpiCC were able to release insulin into the medium in response to an increased glucose concentration. This is the first evidence that generating a renewable autologous, functional source of insulin-secreting cells is possible in the dog. This procedure represents a novel and promising potential therapy for diabetes in dogs.

Prefabricated tracheal prosthesis with partial biodegradable materials: a surgical and tissue engineering evaluation in vivo

Large and circumferential tracheal defects remain at this time an unsolved problem for reconstructive surgery. Many types of prosthetic and tissue grafts have been used but with limited comfortable results. Major complications are anastomotic dehiscence, graft ischemia and stenosis due to the poor vascularization of the prosthetic complex. We studied the in vivo behaviour of a prefabricated flap composed of a partially bioresorbable tracheal prosthesis and an artero–venous vascular carrier. The prosthesis was made of a tubular skeleton of knitted Dacron (20 μm porosity) embedded within a bioresorbable poly-lactic-co-glycolic acid polymer (PLA75GA25) covering both sides. Fifteen New Zealand White rabbits were divided in three groups, depending on the time of examination (30, 90 and 180 days post-implantation). The prosthesis was implanted in the visceral space of the neck using the common carotid trunk and the internal jugular vein as vascular pedicle. The histological, immunohistochemical, and ESEM analyses of collected samples, showed a time-dependent process of tissue neoformation and neovascularization on the prosthetic material with a significant increase from 30 to 90 days post-implantation. In contrast, there was no statistically significant difference in the fibrovascular connective deposition from 90 to 180 days. This finding indicated the three months time as the best period for the tissue deposition and consequent hypothetical orthotopic transplantation of the prosthesis. Further in vivo studies are intended to confirm the results.

Mesenchymal Stem Cell Therapy for Airway Restoration Following Surgery

Post-resectional bronchopleural fistula is a pathological connection between the airway (bronchus) and the pleural space that may develop after lung resection; it may be caused by incomplete bronchial closure, impediment of bronchial stump wound healing, or stump destruction by residual neoplastic tissue. The clinical effect of impaired bronchial stump healing after anatomic lung resection may culminate in a life-threatening septic and ventilatory catastrophe.

Sheep head frame validation for CT and MRI studies

Abstract Introductions Aim of EDEN 2020 project’s Milestone 5 is the development of a steerable catheter for CED system in glioblastoma therapy. The VET group is involved in realization and validation of the proper animal model. Materials and methods In this part of the study two fresh sheep’s head from the local slaughter were used. The heads were located into an ad hoc Frame system based on anatomical measures and CT images, producted by Renishaw plc partner in this project. The frame was adapted and every components were checked for the ex vivo validation tests. CT imaging was taken in Lodi at Universita degli studi di Milano, Facolta di Medicina Veterinaria, with CT scanner and MRI imaging was taken in La Cittadina, Cremona Results System validation was approved by the ex vivo trial. The frame system doesn’t compromise the imaging acquisition in MRI and CT systems. Every system components are functional to their aims. Discussion The Frame system is adapted to the sheep head. It is composed by elements able to lock the head during the imaging acquisition. Frame system is characterized by a support base helpings the animals to keep the head straight forward during imaging time, under general anesthesia. The design of these device support the airways anatomy, avoiding damaging or obstruction of airflows during anesthesia period. The role of elements like mouth bar and ovine head pins is to lock the head in a stable position during imaging acquisition; fixing is guaranteed by V shape head pins, that are arranged against the zygomatic arches. Lateral compression forces to the cranium, and the V shape pins avoid the vertical shifting of the head and any kind of rotations. (fig. 1)