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In the early 1990's our team developed and patented one of the first percutaneous heart valve systems working with Dr. Ivan Casagrande and Dr. Domingos Moraes in Brazil. We learned in this experience that a patient is nearly always better off keeping their own heart valve it they can. The aim of

Follistatin (FST) and the related proteins FSTL1 and FSTL3 are crucial modulators of the transforming growth factor (TGF)-beta superfamily and function by neutralizing activins, a group of proteins implicated in many biologic processes, such as cell proliferation and differentiation, immune responses, various endocrine activities, wound repair, inflammation and fibrosis. Activins are increased in the serum of heart failure patients and in cardiomyocytes after experimental myocardial infarction, suggesting the involvement of activins in heart failure pathogenesis. FST is considered to be a key modulator in muscle development, differentiation and regeneration, and it has been implicated in the repair of mesodermal- and endodermal-derived tissues, promoting cell proliferation and hampering fibrogenesis. We have previously demonstrated that electrostimulation (ES) induces cardiomyocyte pre-commitment of both stem and non-stem cells in vitro. In this study, we evaluated whether applying ES to human mesenchymal stromal cells (hMSC) modulated FST production.hMSC were electrostimulated with 10 and 40 V for 12 h. FST production was assessed by immunostaining, Western blot and flow cytometry.FST was up-regulated in hMSC after ES and was associated with cardiomyogenic differentiation of hMSC by short-term ES.The possibility of stimulating the production of FST, a key regulator of mesodermal differentiation, in adult stem cells, while avoiding the drawbacks of conditioned media, dangerous drugs and gene delivery, has relevant potential therapeutic clinical applications. Additionally, this simple differentiation system could be useful for elucidating the molecular mechanisms driving the stem cell-differentiation process.

Electrostimulation represents a well-known trophic factor for different tissues. In vitro electrostimulation of non-stem and stem cells induces myogenic predifferentiation and may be a powerful tool to generate cells with the capacity to respond to local areas of injury. We evaluated the effects of in vivo electrostimulation on infarcted myocardium using a miniaturized multiparameter implantable stimulator in rats. Parameters of electrostimulation were organized to avoid a direct driving or pacing of native heart rhythm. Electrical stimuli were delivered for 14 days across the scar site. In situ electrostimulation used as a cell-free, cytokine-free stimulation system, improved myocardial function, and increased angiogenesis through endothelial progenitor cell migration and production of vascular endothelial growth factor (VEGF). In situ electrostimulation represents a novel means to stimulate repair of the heart and other organs, as well as to precondition tissues for treatment with cell-based therapies.

Stem cell therapy is a leading field of research worldwide given its promising potential for recovery or replacement of tissues and organs, especially for the treatment of cardiovascular pathologies. However, despite this enormous experimental effort and the reported positive results in different models, there is no conclusive demonstration of the mechanisms involved in tissue regeneration associated to adult stem cell treatment. This represents one of the major limitations for the clinical translation of stem cell therapy. A real regenerative medicine approach should consider the importance of the extracellular matrix (ECM) and the strong biological signals that it can provide. Connective tissue atmosphere in which cells are embedded exerts a number of actions affecting cells function and supporting their proliferation and differentiation. Polymeric electrospun matrices are among the most promising ECM-mimetic biomaterials, because of their physical structure closely resembling the fibrous proteins in native ECM. Moreover, electrospun materials can be easily functionalized with bioactive molecules providing localized biochemical stimuli to cells seeded therein. The idea of taking advantage of both stem cells plasticity and biomaterials that actively guide and provide the correct sequence of signals to allow ongoing lineage-specific differentiation is an attractive alternative and may represent a promising answer to the treatment limitations of cardiovascular severe diseases.

Tissue engineering of vascular grafts still presents several shortcomings. Aiming to vascular regeneration, we developed a biomimetic multilayered scaffold with a middle pivotal collagen lamina between two functionalized layers of poly-L-lactide by means of electrospinning technique, with oriented drug-delivery capacity for the differentiation of human mesenchymal stem cells seeded therein. Applying appropriate cytokines, the inner layer is able to act as a drug delivery system in order to generate a pro-angiogenic and anti-thrombotic environment and the outer one is used to induce the media and adventitia generation. Our findings are consistent with an adequate cell engrafting and a double type of differentiation in each side of the scaffold, in particular cells exhibited morphostructural changes resulting in the achievement of an endothelial-like phenotype in cells populating the inner side of the scaffold and SMA positivity with cell elongation resembling muscular phenotype in the cells of the outer layer. The proposed “smart” vascular bio-prosthesis will recapitulate the structure and microenvironment of native cardiovascular tissues. It could surmount many hurdles to clinical use and would be relevant for therapeutic applications in a variety of medical fields.

Cartilage and bone tissue engineering has been widely investigated but is still hampered by cell differentiation and transplant integration issues within the constructs. Scaffolds represent the pivotal structure of the engineered tissue and establish an environment for neo-extracellular matrix synthesis. They can be associated to signals to modulate cell activity. In this study, considering the well reported role of hydroxyapatite (HA) in cartilage repair, we focused on the putative chondrogenic differentiation of human mesenchymal stem cells (hMSCs) following culture on membranes of electrospun fibers of poly-l-lactic acid (PLLA) loaded with nanoparticles of HA. hMSCs were seeded on PLLA/HA and bare PLLA membranes and cultured in basal medium, using chondrogenic differentiation medium as a positive control. After 14 days of culture, SOX-9 positive cells could be detected in the PLLA/HA group. Cartilage specific proteoglycan immunostain confirmed the presence of neo-extracellular-matrix production. Co-expression of CD29, a typical surface marker of MSCs and SOX-9, suggested different degrees in the differentiation process. We developed a hydroxyapatite functionalized scaffold with the aim to recapitulate the native histoarchitecture and the molecular signaling of osteochondral tissue to facilitate cell differentiation toward chondrocyte. PLLA/HA nanocomposites induced differentiation of hMSCs in a chondrocyte-like phenotype with generation of a proteoglycan based matrix. This nanocomposite could be an amenable alternative scaffold for cartilage tissue engineering using hMSCs.

Stem-cell therapy has become a promising therapeutic tool for myocardial repair. Cardiac pre-committed cells, which complete their differentiation in the myocardium, may reduce fibrosis and restore muscle function. However, many questions concerning a precise, functional integration of injected cells remain unanswered. Fibroblasts regulate the cardiac extracellular matrix and are the most abundant cell population in an infarcted area. Electrostimulation is a well-known trophic factor and can induce phenotypic changes in myoblasts. The objective of this study was to evaluate the effectiveness of electrical stimulation to induce pre-commitment of fibroblasts into cardiomyocytes in vitro. Using short-time electrostimulation in a cytokine-free culture system, we induced pre-commitment of two fibroblast cell lines to a cardiomyocyte phenotype. This partial differentiation in vitro may facilitate further differentiation within the cardiac environment and result in better electro-mechanical integration of the therapeutically introduced cells.

Angiogenesis and vasculogenesis are fundamental processes in embryonic development enabling multiorgan characteristics. Through the capillary net, the nutrient diffusion process is successfully achieved in complex cellular organisms. In adults the establishment, maintenance, and renewal of an efficient vascular net are required for the maintenance of normal, viable tissues. This requirement is especially important in the cardiovascular system, where vascular supply efficiency and endothelium normal function are affected in many different pathological entities. Impaired angiogenesis and endothelial dysfunction are the bases of many cardiovascular diseases such as hypertension, coronary artery disease, myocardial infarction, and chronic heart failure.

Background: Current clinical experience with cellular cardiomyoplasty (using serum bovine-cultivated myoblasts) has demonstrated significant malignant ventricular arrhythmias and sudden deaths in patients. In some ongoing clinical trials the implantation of cardioverterdefibrillator is mandatory. We have hypothesized that contact of human cells with fetal bovine serum results after 3-week fixation of animal proteins on the cell surface, representing an antigenic substrate for immunological and inflammatory adverse events. Methods and Results: Autologous myoblasts were transplanted into infarcted LV in 20 patients (90% males, mean age 62±8 years). Cells were cultivated in a complete human medium during 3 weeks, using the patients' own serum obtained from a blood sample or from plasmapheresis. Injections were performed during CABG (2.1 grafts/pt). All patients had an uneventful recovery. At a mean follow-up of 14±5 months without mortality, no malignant cardiac arrhythmias are reported. LV ejection fraction improved from 28±3% to 52:k4.7% (p = 0.03), and regional wall motion score index (WMSI) from 3.1 to 1.4 (p = 0.04) in the cell-treated segments. Myocardial viability tests showed areas of regeneration. Patients moved from mean NYHA class 2.5 to class 1.2. Conclusions: A total autologous cell culture procedure was used in cellular cardiomyoplasty reducing the risk of arrhythmia. Human-autologous-serum cell expansion avoids the risk of prion, viral or zoonoses contamination. Since patients treated with noncultivated bone marrow cells are free of arrhythmia, the bovine-culture medium seems to be responsible for this complication. Cellular cardiomyoplasty may be efficient to avoid progression of ventricular remodeling and subsequent heart failure in ischemic heart disease.

Targeting wound repair, we developed an electrospun poly-L-lactide fibrous scaffold functionalized with G-CSF, a growth factor which is widely recognized as important in wound healing homeostasis. The scaffold was characterized in terms of morphology, mechanical properties and in vitro capacity to induce organization of co-cultures of murine fibroblasts and keratinocytes into a dermo-epidermal multilayered structure. Our findings are consistent with the promotion of a nonhostile environment, in which seeded cells could arrange themselves in an appropriate topographic distribution of elements at different levels of maturation up to a cornified epithelium on the top layer, resembling native skin.