Fabio Acocella

Large animal models for cardiac stem cell therapies

Cardiovascular disease is the leading cause of death in developed countries and is one of the leading causes of disease burden in developing countries. Therapies have markedly increased survival in several categories of patients, nonetheless mortality still remains high. For this reason high hopes are associated with recent developments in stem cell biology and regenerative medicine that promise to replace damaged or lost cardiac muscle with healthy tissue, and thus to dramatically improve the quality of life and survival in patients with various cardiomyopathies. Much of our insight into the molecular and cellular basis of cardiovascular biology comes from small animal models, particularly mice. However, significant differences exist with regard to several cardiac characteristics when mice are compared with humans. For this reason, large animal models like dog, sheep and pig have a well established role in cardiac research. A distinct characteristic of cardiac stem cells is that they can either be endogenous or derive from outside the heart itself; they can originate as the natural course of their differentiation programme (e.g., embryonic stem cells) or can be the result of specific inductive conditions (e.g., mesenchymal stem cells). In this review we will summarize the current knowledge on the kind of heart-related stem cells currently available in large animal species and their relevance to human studies as pre-clinical models.

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.

Effects of abdominal distension on breathing pattern and respiratory mechanics in rabbits

The effects of acute abdominal distension (AD) on the electromechanical efficiency (Eff) of the inspiratory muscles were investigated in anesthetized rabbits by recording the electrical activity (A), pressure (P) exerted by the diaphragm (di) and parasternal intercostal muscles (ic), and lung volume changes when an abdominal balloon was inflated to various degrees. Eff,ic increased with increasing AD both in supine and upright postures. In upright rabbits Eff,di increased for intermediate but decreased at higher levels of AD, whilst it decreased at all levels of AD in supine rabbits. Tidal volume (VT) response followed that of Eff,di. Tonic Aic and Adi and inspiratory prolongation were elicited by AD. The effects of these neural mechanisms, acting to limit end-expiratory lung volume and VT changes, were however small since vagotomy prevented tonic Adi and inspiratory prolongation and reduced tonic Aic, but changed lung volume responses to AD only little. Hence, reduced respiratory system compliance and changes in inspiratory muscle electromechanical efficiency dominate lung volume responses to acute AD.

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

Finite element analysis of the mechanical behavior of preterm lamb tracheal bifurcation during total liquid ventilation

Knowledge of the mechanical behavior of immature airways is crucial to understand the effects exerted by ventilation treatments, namely by Total Liquid Ventilation (TLV). A computational approach was adopted to investigate preterm airways in the range of pressure applied during TLV. A 3D finite-element model of the tracheal bifurcation was developed. Structural analyses were performed using ABAQUS/Standard to evaluate airway deformation during TLV. The model consists of 7 rings, each composed of 3 tissues (cartilage, smooth muscle, connective tissue) modeled as hyperelastic materials. Biomechanical experimental tests were performed on lamb tracheae to obtain the stress-strain relationship for each tissue. Pressure load was applied on the internal surface of the model, reproducing the airway pressure tracing acquired during a TLV breath ending with a tracheal collapse phenomenon. Model reliability was verified by comparing the model outcomes to computer tomography scan images acquired during animal TLV trials. The simulations show progressive lumen narrowing during expiration, at increasing negative pressure until the occurrence of collapse; however not inducing complete airway occlusion. A reliable model was obtained to help setting ventilation parameters during TLV.

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.

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.

Tubular e-PTFE implants in glottal and neoglottal insufficiency: tolerance and stability in an animal model

The aim of our research is to develop a laryngeal tissue augmentation technique that will be useful in treating glottal and neoglottal insufficiency. In a previous study the feasibility of insertion of tubular expanded poly-tetrafluoroethylene (e-PTFE) tissue implants into the larynx of a porcine model was demonstrated. In this paper we report the clinical, endoscopic, computed tomography (CT) and magnetic resonance image (MRI) and histopathological follow-up findings. These results demonstrate host tolerance of the implant over the period of study (six months) with little tissue reactivity to tubular e-PTFE implants in the porcine model. The tubular shape of the implant enhances stabilization by the rapid and complete host tissue penetration of the implants interstices, making tubular e-PTFE a potentially useful soft tissue augmentation material for laryngeal incompetence.