Regenerative Neurology and Regenerative Cardiology: Shared Hurdles and Achievements.

From the first success in cultivation of cells in vitro, it became clear that developing cell and/or tissue specific cultures would open a myriad of new opportunities for medical research. Expertise in various in vitro models has been developing over decades, so nowadays we benefit from highly specific in vitro systems imitating every organ of the human body. Moreover, obtaining sufficient number of standardized cells allows for cell transplantation approach with the goal of improving the regeneration of injured/disease affected tissue. However, different cell types bring different needs and place various types of hurdles on the path of regenerative neurology and regenerative cardiology. In this review, written by European experts gathered in Cost European action dedicated to neurology and cardiology-Bioneca, we present the experience acquired by working on two rather different organs: the brain and the heart. When taken into account that diseases of these two organs, mostly ischemic in their nature (stroke and heart infarction), bring by far the largest burden of the medical systems around Europe, it is not surprising that in vitro models of nervous and heart muscle tissue were in the focus of biomedical research in the last decades. In this review we describe and discuss hurdles which still impair further progress of regenerative neurology and cardiology and we detect those ones which are common to both fields and some, which are field-specific. With the goal to elucidate strategies which might be shared between regenerative neurology and cardiology we discuss methodological solutions which can help each of the fields to accelerate their development.

Elastomeric Cardiowrap Scaffolds Functionalized with Mesenchymal Stem Cells-Derived Exosomes Induce a Positive Modulation in the Inflammatory and Wound Healing Response of Mesenchymal Stem Cell and Macrophage.

A challenge in contractile restoration of myocardial scars is one of the principal aims in cardiovascular surgery. Recently, a new potent biological tool used within healing processes is represented by exosomes derived from mesenchymal stem cells (MSCs). These cells are the well-known extracellular nanovesicles released from cells to facilitate cell function and communication. In this work, a combination of elastomeric membranes and exosomes was obtained and tested as a bioimplant. Mesenchymal stem cells (MSCs) and macrophages were seeded into the scaffold (polycaprolactone) and filled with exosomes derived from MSCs. Cells were tested for proliferation with an MTT test, and for wound healing properties and macrophage polarization by gene expression. Moreover, morphological analyses of their ability to colonize the scaffolds surfaces have been further evaluated. Results confirm that exosomes were easily entrapped onto the surface of the elastomeric scaffolds, increasing the wound healing properties and collagen type I and vitronectin of the MSC, and improving the M2 phenotype of the macrophages, mainly thanks to the increase in miRNA124 and decrease in miRNA 125. We can conclude that the enrichment of elastomeric scaffolds functionalized with exosomes is as an effective strategy to improve myocardial regeneration.

Electrospun PCL-Based Vascular Grafts: In Vitro Tests.

BACKGROUND: Electrospun fibers have attracted a lot of attention from researchers due to their several characteristics, such as a very thin diameter, three-dimensional topography, large surface area, flexible surface, good mechanical characteristics, suitable for widespread applications. Indeed, electro-spinning offers many benefits, such as great surface-to-volume ratio, adjustable porosity, and the ability of imitating the tissue extra-cellular matrix. METHODS: we processed Poly ε-caprolactone (PCL) via electrospinning for the production of bilayered tubular scaffolds for vascular tissue engineering application. Endothelial cells and fibroblasts were seeded into the two side of the scaffolds: endothelial cells onto the inner side composed of PCL/Gelatin fibers able to mimic the inner surface of the vessels, and fibroblasts onto the outer side only exposing PCL fibers. Extracellular matrix production and organization has been performed by means of classical immunofluorescence against collagen type I fibers, Scanning Electron-Microscopy (SEM) has been performed in order to evaluated ultrastructural morphology, gene expression by means gene expression has been performed to evaluate the phenotype of endothelial cells and fibroblasts. RESULTS AND CONCLUSION: results confirmed that both cells population are able to conserve their phenotype colonizing the surface supporting the hypothesis that PCL scaffolds based on electrospun fibers should be a good candidate for vascular surgery.

Elastomeric cardiopatch scaffold for myocardial repair and ventricular support.

OBJECTIVES: Prevention of postischaemic ventricular dilatation progressing towards pathological remodelling is necessary to decrease ventricular wall deterioration. Myocardial tissue engineering may play a therapeutic role due to its capacity to replace the extracellular matrix, thereby creating niches for cell homing. In this experimental animal study, a biomimetic cardiopatch was created with elastomeric scaffolds and nanotechnologies. METHODS: In an experimental animal study in 18 sheep, a cardiopatch was created with adipose tissue-derived progenitor cells seeded into an engineered bioimplant consisting of 3-dimensional bioabsorbable polycaprolactone scaffolds filled with a peptide hydrogel (PuraMatrix™). This patch was then transplanted to cover infarcted myocardium. Non-absorbable poly(ethyl) acrylate polymer scaffolds were used as controls. RESULTS: Fifteen sheep were followed with ultrasound scans at 6 months, including echocardiography scans, tissue Doppler and spectral flow analysis and speckle-tracking imaging, which showed a reduction in longitudinal left ventricular deformation in the cardiopatch-treated group. Magnetic resonance imaging (late gadolinium enhancement) showed reduction of infarct size relative to left ventricular mass in the cardiopatch group versus the controls. Histopathological analysis at 6 months showed that the cardiopatch was fully anchored and integrated to the infarct area with minimal fibrosis interface, thereby promoting angiogenesis and migration of adipose tissue-derived progenitor cells to surrounding tissues. CONCLUSIONS: This study shows the feasibility and effectiveness of a cardiopatch grafted onto myocardial infarction scars in an experimental animal model. This treatment decreased fibrosis, limited infarct scar expansion and reduced postischaemic ventricular deformity. A capillary network developed between our scaffold and the heart. The elastomeric cardiopatch seems to have a positive impact on ventricular remodelling and performance in patients with heart failure.

Compliance mismatch and compressive wall stresses drive anomalous remodelling of pulmonary trunks reinforced with Dacron grafts.

Synthetic grafts are often satisfactory employed in cardiac and vascular surgery, including expanded poly(ethylene terephthalate) or expanded poly(tetrafluoroethylene). However, accumulating evidences suggest the emergence of worrisome issues concerning the long-term fate of prosthetic grafts as large vessel replacement. Disadvantages related to the use of synthetic grafts can be traced in their inability of mimicking the elasto-mechanical characteristics of the native vascular tissue, local suture overstress leading to several prosthesis-related complications and retrograde deleterious effects on valve competence, cardiac function and perfusion. Motivated by this, in the present work it is analyzed - by means of both elemental biomechanical paradigms and more accurate in silico Finite Element simulations - the physical interaction among aorta, autograft and widely adopted synthetic (Dacron) prostheses utilized in transposition of pulmonary artery, highlighting the crucial role played by somehow unexpected stress fields kindled in the vessel walls and around suture regions, which could be traced as prodromal to the triggering of anomalous remodelling processes and alterations of needed surgical outcomes. Theoretical results are finally compared with histological and surgical data related to a significant experimental animal campaign conducted by performing pulmonary artery transpositions in 30 two-month old growing lambs, followed up during growth for six months. The in vivo observations demonstrate the effectiveness of the proposed biomechanical hypothesis and open the way for possible engineering-guided strategies to support and optimize surgical procedures.

Biomechanics drive histological wall remodeling of neoaortic root: A mathematical model to study the expression levels of ki 67, metalloprotease, and apoptosis transition.

The pulmonary artery autograft (PA) is the ideal substitute for aortic valve disease in children and young adult. However, it is harnessed by the issue of long-term dilation and regurgitation, often requiring surgery. PA implanted in aortic position during the growth phase in children undergoes a process of mechanical remodeling. We previously developed a semiresorbable armored prosthesis able to mechanically sustain the neoaorta preventing dilation and to gradually integrate with the PA wall inducing a progressive arterial-like tissue positive remodeling. We also described the mechanisms of growth, remodeling and stress shielding of the reinforced PA through a mathematical model. We sought to demonstrate the biological counterpart and the potential molecular mechanisms underlying this histological and mechanical remodeling. A specific mathematical model was developed to describe mechanical behavior of the PA. Mallory trichrome red staining and immunohistochemistry for MMP-9 were performed to elucidate extracellular matrix remodeling phenomena. Apoptosis and cell proliferation were determined by TUNEL assay and immunohistochemistry for Ki67, respectively. An histological remodeling phenomenon sustained by increased level of MMP-9, augmented cell proliferation and reduced apoptosis in the reinforced PA was demonstrated. The mathematical model predicted the biomechanical behavior subtended by the histological changes of the PA in these settings. Changes in metalloproteinases (MMP-9), cell proliferation and apoptosis are the main actors in the remodeling process occurring after transposition of the PA into systemic regimens. Use of semiresorbable reinforcements might induce a positive remodeling of the PA in the context of Ross operation. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2785-2793, 2016.

A composite semiresorbable armoured scaffold stabilizes pulmonary autograft after the Ross operation: Mr Ross's dream fulfilled.

OBJECTIVES: Use of resorbable external reinforcement of the pulmonary autograft during the Ross operation has been suggested, but the differential regional potential for dilation of the aorta, mainly regarding the neo-root and the neo-Valsalva sinuses, represents an unresolved issue. Auxetic materials could be useful in preventing dilation given their favorable mechanical properties. We designed a composite semiresorbable armoured bioprosthesis constituted by polydioxanone and expanded polytetrafluoroethylene and evaluated its effectiveness as a pulmonary autograft reinforcement device in an animal model of the Ross procedure. METHODS: An experimental model of the Ross procedure was performed in 20 three-month-old growing lambs. The pulmonary autograft was alternatively nonreinforced (control group n = 10) or reinforced with composite bioprosthesis (reinforced group n = 10). Animals were followed up during growth for 6 months by angiography and echocardiography. Specific stainings for extracellular matrix and immunohistochemistry for metalloproteinase-9 were performed. RESULTS: Reference aortic diameter increased from 14 ± 1 mm to 19 ± 2 mm over 6 months of growth. In the control group, pulmonary autograft distension (28 ± 2 mm) was immediately noted, followed by aneurysm development at 6 months (40 ± 2 mm, P < .001 vs reference). In the reinforced group, an initial dilation to 18 ± 1 mm was detected and the final diameter was 27 ± 2 mm (42% increase). Two deaths due to pulmonary autograft rupture occurred in the control group. On histology, the control group showed medial disruption with connective fibrous replacement, whereas in the reinforced group compensatory intimal hyperplasia was present in the absence of intimal tears. The bioprosthesis promoted a positive matrix rearrangement process favoring neoarterialization and elastic remodeling as demonstrated on specific staining for elastin collagen and metalloproteinase-9. CONCLUSIONS: The device adapted and functionally compensated for the characteristics of autograft growth, guaranteeing a reasonable size of the autograft at 6 months, but more important, because the device is biocompatible, it did not disrupt the biological process of growth or cause inflammatory damage to the wall.

An experimental model of the Ross operation: Development of resorbable reinforcements for pulmonary autografts.

OBJECTIVES: To circumvent the issue of pulmonary autograft (PA) dilation after the Ross procedure, surgical reinforcement strategies have been suggested in clinical or anecdotal series. However, no preclinical large-animal model of the Ross procedure is available, which is needed to enable full comprehension of the pathologic mechanisms and the effectiveness of reinforcement techniques during growth. Our aim was to develop a large-animal model of the Ross operation, to reproduce the clinical scenario in which this procedure might be applied, and allow for development and testing of various devices and techniques to improve PA performance. In addition, we aimed to test the effectiveness of a bioresorbable mesh for PA reinforcement. METHODS: An experimental model of transposition of the pulmonary trunk as an autograft in the aortic position has been developed and performed under cardiopulmonary bypass in 20 growing lambs, aged 3 months. The experimental design included: a control group that underwent PA transposition; a group in which the PA was reinforced with an external, synthetic, nonresorbable, polypropylene grid; and a group that received various combinations of resorbable meshes. Animals were followed up during growth for 6 months by angiography and echocardiography and eventually killed for pathologic analysis. RESULTS: All animals survived the procedure with no complications. The model was easy and reproducible. Resorbable meshes prevented PA dilation and preserved its progressive growth process, aiding histologic remodelling. CONCLUSIONS: We developed an easy and reproducible model of the Ross procedure, allowing for a reliable simulation of the clinical scenario. Resorbable PA reinforcement may represent an interesting option in this context.

Bio-hybrid tissue engineering for cellular cardiomyoplasty: future directions.

Cardiomyopathy induces geometric alteration of the ventricular cavity, which changes from a natural elliptical (conical) to a spherical shape. Ventricular chamber dilatation and spherical deformation are important causes of morbidity and mortality among patients with congestive heart failure. In addition, diastolic dysfunction is an important clinical problem in these cases because there is no medical or surgical specific treatment. Myocardial tissue engineering associating stem cells represent a new road and fresh hope for this heart failure population.

Controversies about the chromosomal stability of cultivated mesenchymal stem cells: their clinical use is it safe?

The usefulness of adult stem cells in research and therapeutic applications highly relies on their genomic integrity and stability. Many laboratories including ours have addressed this concern using methods such as karyotyping, Qbanding, fluorescent in situ hybridization, array CGH, flow cytometry and Pap test to evaluate number and structure of chromosomes and cellular phenotype. This review attempts to summarize the findings reported so far for the studies on chromosomal aberrations in adult stem cells and warrant to perform certain basic tests before transplantation to avoid any adverse reactions, which will thus aid in better therapeutic output after cellular transplantation in the treatment of various diseases.

Matrix therapy with RGTA OTR4120 improves healing time and quality in hairless rats with deep second-degree burns.

BACKGROUND: ReGeneraTing Agents (RGTAs) are biodegradable polymers engineered to mimic heparan-sulfate in the extracellular matrix of damaged tissue. RGTAs improve tissue healing in several animal models by stabilizing and protecting heparin-binding growth factors and matrix proteins. RGTA restores the normal matrix architecture and supports tissue regeneration. In this study, the authors evaluated the effects of RGTA on epidermal repair and dermal remodeling in a rat burn model. METHODS: Deep second-degree burns were induced in 156 hairless rats, of which half (n = 78) received topical and intramuscular RGTA immediately after the burn followed by intramuscular RGTA weekly for 1 month. The controls (n = 78) received saline according to the same protocol. Rats were killed starting on each day of the first week and on days 14, 28, 60, 120, 240, and 365. The burns were evaluated by photography, histology, and immunohistochemistry. RESULTS: Coagulation necrosis involved the entire epidermis and superficial adnexa. Compared with the controls, speed of epidermal repair, as assessed between days 3 and 7 based on cell-layer number and anticytokeratin-14 staining, was faster in the RGTA group; and the zone of stasis, as assessed based on secondary vascular lesions in the dermis, was smaller. On day 7, reepithelialization was complete in both groups. On days 14 and 28, the remodeled dermal zone was smaller in the RGTA group. CONCLUSION: RGTA accelerated epidermal repair and protected the dermis from secondary effects of heat as quantified by zone-of-stasis size and extent of dermal remodeling.

Alternatives to heart transplantation: integration of biology with surgery.

Chronic heart failure is one of the major health care issues in terms of increasing number of patients, rate of hospitalizations and costs. Heart transplantation is the best established therapy for patients with severe heart failure. However, the number of donors limits the activity to 5000 heart transplants performed annually worldwide. This limitation has generated alternative treatments. The increase of the interest in the reversibility of the heart failure and the application of new biological alternatives has generated therapeutic strategies designed to integrate biology and medical technologies in order to act to the biomechanical, the molecular and the neurohormonal mechanisms of heart failure. These treatments include cellular cardiomyoplasty, tissue engineering, surgical left ventricular restoration as well as passive and active mechanical ventricular assistance as destination therapy, bridge to recovery or bridge to transplantation. The integrated development of these approaches could offer hopeful treatments, although there is still much to be learned regarding the optimal use of these strategies.

Development of bioartificial myocardium using stem cells and nanobiotechnology templates.

Cell-based regenerative therapy is undergoing experimental and clinical trials in cardiology, in order to limit the consequences of decreased contractile function and compliance of damaged ventricles following myocardial infarction. Over 1000 patients have been treated worldwide with cell-based procedures for myocardial regeneration. Cellular cardiomyoplasty seems to reduce the size and fibrosis of infarct scars, limit adverse postischemic remodelling, and improve diastolic function. The development of a bioartificial myocardium is a new challenge; in this approach, tissue-engineered procedures are associated with cell therapy. Organ decellularization for bioscaffolds fabrication is a new investigated concept. Nanomaterials are emerging as the main candidates to ensure the achievement of a proper instructive cellular niche with good drug release/administration properties. Investigating the electrophysiological properties of bioartificial myocardium is the challenging objective of future research, associating a multielectrode network to provide electrical stimulation could improve the coupling of grafted cells and scaffolds with host cardiomyocytes. In summary, until now stem cell transplantation has not achieved clear hemodynamic benefits for myocardial diseases. Supported by relevant scientific background, the development of myocardial tissue engineering may constitute a new avenue and hope for the treatment of myocardial diseases.

Animal models of heart failure: what is new?

Heart failure is the major cause of mortality in Western countries. Medical treatment of heart failure is associated with 50% survival at 5 years. Experimental models are required to better understand the progression of the disease and elaborate new therapy. Heart transplantation, left ventricular assist devices, artificial hearts, and cardiac bioassist techniques require animal models for testing and optimizing before they are implemented on human patients. The perfect model of heart failure that reproduces every aspect of the natural disease does not exist. Acute and chronic heart failure models have been developed to reproduce different aspect of the pathology.

Induction of chronic cardiac insufficiency by arteriovenous fistula and doxorubicin administration.

OBJECTIVES: Large animal experimental models of chronic heart failure (HF) permit repeated invasive assessment of cardiovascular function, and evaluation of new medical or surgical therapies. The existing models however fail to achieve stable and long-term HF. The aim of this study was to create a simple and stable chronic model of HF in goat, using both arteriovenous fistula and weekly intravenous doxorubicin injections. METHODS: After a preliminary experiment on four goats receiving weekly 1 to 2 mg/kg of doxorubicin, six adult female goats, having had an arteriovenous fistula without signs or symptoms of heart failure, received weekly two different dosages of doxorubicin for 13 weeks: group A (n = 3) received 0.5 mg/kg and group B (n = 3) received 1 mg/kg. After a three-month period without medication, both groups received again 1 mg/kg for four weeks. Cardiac function was assessed by repeated electrocardiographic and echocardiographic examinations. RESULTS: Four goats died during the medication period (one in group A, three in group B). During the period without medication a stable ventricular hypocontractility with left ventricular dilation was observed. Left ventricular dysfunction was more pronounced in group B, and was associated with clinical symptoms of HF. CONCLUSIONS: Arteriovenous fistula alone did not produce HF. Its association with doxorubicin injections provides a simple and stable chronic model of HF. This association allows reduction of the required doxorubicin dose and toxicity in animals and in the environment. Depending of the dose of doxorubicin, it is possible to obtain a model of heart dilatation and ventricular hypokinesia with or without clinical symptoms of HF, with a different mortality.

Cardiomioplastia celular / Cellular cardiomyoplasty

El objetivo de la cardiomioplastia celular es el de limitar las consecuencias de la disminución en la función contráctil que produce el infarto de miocardio. La terapéutica celular para el tratamiento de la escara posisquémica es particularmente atractiva debido a la potencialidad en la regeneración miocárdica que una variedad de células miogénicas y angiogénicas presentan, como los mioblastos esqueléticos, células madre mesenquimáticas de la médula ósea, células progenitoras circulantes en sangre, células musculares lisas, células endoteliales vasculares y células madre embrionarias. La cardiomioplastia celular con empleo de mioblastos esqueléticos autólogos se realizó en nuestros centros en 18 pacientes. Hasta el momento, en todo el mundo se ha efectuado en más de cien casos. El criterio de inclusión consiste en pacientes con fracción de eyección del ventrículo izquierdo baja y con escaras posinfartos acinéticas y no viables. La terapia celular con células miogénicas puede reducir el tamaño y la fibrosis del área infartada, lo cual limita el remodelado posisquémico y restaura la contractilidad regional miocárdica en pacientes que han sufrido infartos extensos. Se han desarrollado técnicas de cultivo de mioblastos equeléticos expandidos ex vivo con suero autólogo obtenido por plasmaféresis. Estas técnicas son capaces de producir 300 millones de células en tres semanas, las cuales contienen más del 70 por ciento de mioblastos. La viabilidad celular en el momento del implante es mayor del 90 por ciento. El beneficio de usar suero humano autólogo en el cultivo es el de que evita el riesgo de contaminación con priones, virus o zoonosis. Tradicionalmente, las técnicas de cultivo emplearon el suero fetal bovino como medio de crecimiento. El contacto de células humanas con suero fetal bovino luego de tres semanas da por resultado la fijación de proteínas animales sobre la superficie celular, que conforman antígenos para que se produzcan efectos inmunológicos adversos. En estos casos se produce una reacción inflamatoria con la posterior fibrosis. De esta manera, en pacientes cuyas células fueron cultivadas con suero fetal bovino se ha observado arritmia ventricular severa y muerte súbita. En muchas oportunidades esta complicación requirió el implante de desfibriladores. En este artículo presentamos las técnicas para regeneración cardíaca y sus resultados clínicos preliminares