Cardiac Stem Cell Characteristics in Physiological and Pathological Conditions.

For nearly a century, the adult heart was considered as a post-mitotic organ. The discovery of a resident cardiac stem cell (CSC) population in the heart has dramatically undermined this notion with the support of encouraging preclinical and clinical studies aiming to regenerate the damaged heart after a myocardial infarction (MI). There are two ways to obtain CSCs for transplantation: Allogeneic and autologous sources. Autologous cells may be obtained from the patients' own tissue. Obtaining cells from diseased patients may contain a risk for altered stem cell characteristics. In addition to MI, these patients may also suffer from pathological conditions such as hypertension, diabetes mellitus, heart failure, congenital heart disease or cancer, which are known to alter CSC characteristics. It is also known that physiological conditions such as aging and death affect CSC functions in the heart. Our knowledge about the CSC characteristics in various physiological and pathological conditions may shed light on our opinion about the regenerative capacity and biological activity of these cells in these situations. Defining these properties may guide the researchers and clinicians in choosing and obtaining the most qualified CSC populations for cardiac regenerative medicine therapies. The purpose of this review is to describe the alterations in CSC characteristics in various physiological and pathological conditions.

Decellularization of bovine small intestinal submucosa and its use for the healing of a critical-sized full-thickness skin defect, alone and in combination with stem cells, in a small rodent model.

In this study, we initially described an efficient decellularization protocol for bovine-derived small intestinal submucosa (bSIS), involving freeze-thaw cycles, acid/base treatment and alcohol and buffer systems. We compared the efficacy of our protocol to some previously established ones, based on DNA content and SEM and histochemical analyses. DNA content was reduced by ~89.4%, significantly higher than compared protocols. The sulphated GAG content of the remaining interconnected fibrous structure was 5.738 ± 0.207 µg/mg (55% retained). An in vitro study was performed to evaluate whether rat bone marrow mesenchymal stem cells (MSCs) could attach and survive on bSIS membranes. Our findings revealed that MSCs can preserve their viability and proliferate on bSIS for > 2 weeks in culture. We conducted in vivo applications for the treatment of an experimental rat model of critical sized (7 cm2 ) full-thickness skin defect. The wound models treated with either MSCs-seeded (1.5 × 106 cells/cm2 ) or non-seeded bSIS membranes were completely closed by week 7 without significant differences in closure time; on the other hand, the open wound control was closed at ~47% at this time point. Immunohistopathology results revealed that the group which received MSCs-seeded bSIS had less scarring at the end of the healing process and was in further stages of appendage formation in comparison with the non-seeded bSIS group. Copyright © 2015 John Wiley & Sons, Ltd.

Translational Applications of Tissue Engineering in Cardiovascular Medicine.

Cardiovascular diseases are the leading cause of global deaths. The current paradigm in medicine seeks novel approaches for the treatment of progressive or end-stage diseases. The organ transplantation option is limited in availability, and unfortunately, a significant number of patients are lost while waiting for donor organs. Animal studies have shown that upon myocardial infarction, it is possible to stop adverse remodeling in its tracks and reverse with tissue engineering methods. Regaining the myocardium function and avoiding further deterioration towards heart failure can benefit millions of people with a significantly lesser burden on healthcare systems worldwide. The advent of induced pluripotent stem cells brings the unique advantage of testing candidate drug molecules on organ-on-chip systems, which mimics human heart in vitro. Biomimetic three-dimensional constructs that contain disease-specific or normal cardiomyocytes derived from human induced pluripotent stem cells are a useful tool for screening drug molecules and studying dosage, mode of action and cardio-toxicity. Tissue engineering approach aims to develop the treatments for heart valve deficiency, ischemic heart disease and a wide range of vascular diseases. Translational research seeks to improve the patient's quality of life, progressing towards developing cures, rather than treatments. To this end, researchers are working on tissue engineered heart valves, blood vessels, cardiac patches, and injectable biomaterials, hence developing new ways for engineering bio-artificial organs or tissue parts that the body will adopt as its own. In this review, we summarize translational methods for cardiovascular tissue engineering and present useful tables on pre-clinical and clinical applications.

Clinical applications of decellularized extracellular matrices for tissue engineering and regenerative medicine.

Decellularization is the process of removing the cellular components from tissues or organs. It is a promising technology for obtaining a biomaterial with a highly preserved extracellular matrix (ECM), which may also act as a biological scaffold for tissue engineering and regenerative therapies. Decellularized products are gaining clinical importance and market space due to their ease of standardized production, constant availability for grafting and mechanical or biochemical superiority against competing clinical options, yielding clinical results ahead of the ones with autografts in some applications. Current drawbacks and limitations of traditional treatments and clinical applications can be overcome by using decellularized or acellular matrices. Several companies are leading the market with versatile acellular products designed for diverse use in the reconstruction of tissues and organs. This review describes ECM-based decellularized and acellular products that are currently in use for different branches of clinic.

Real-time monitoring of mesenchymal stem cell responses to biomaterial surfaces and to a model drug by using quartz crystal microbalance.

In this study, the mesenchymal stem cell (MSC) responses to biomaterial surfaces and to an anti-microtubule drug (vinblastine) were detected by using the quartz crystal microbalance (QCM). Gold electrodes with different coatings were subjected to MSCs under flow conditions; thus, crystal frequency decreased due to the adhesion of MSCs on the crystal. For evaluation of cell-drug interactions, vinblastine was introduced to MSCs attached onto the surfaces. The changes in frequency indicated the binding of drug to cell microtubules. The present study demonstrates the suitability of QCM as an invaluable tool for the real-time monitoring of cell-surface and cell-drug interactions.

Extracellular Matrix and Regenerative Therapies from the Cardiac Perspective.

Cardiovascular diseases are the leading cause of death and a major cause of financial burden. Regenerative therapies for heart diseases bring the promise of alternative treatment modalities for myocardial infarction, ischemic heart disease, and congestive heart failure. Although, clinical trials attest to the safety of stem cell injection therapies, researchers need to overcome the underlying mechanisms that are limiting the success of future regenerative options. This article aims to review the basic scientific concepts in the field of mechanobiology and the effects of extracellular functions on stem cell fate.

Differentiation of Human Embryonic Stem Cells on Periodontal Ligament Fibroblasts.

Human embryonic stem cells' (hESCs) unlimited proliferative potential and differentiation capability to all somatic cell types makes them one of the potential cell sources in cell-based tissue engineering strategies as well as various experimental applications in fields such as developmental biology, pharmacokinetics, toxicology, and genetics. Periodontal tissue engineering is an approach to reconstitute the ectomesenchymally derived alveolar bone, periodontal ligament apparatus, and cementum tissues lost as a result of periodontal diseases. Cell-based therapies may offer potential advantage in overcoming the inherent limitations associated with contemporary regenerative procedures, such as dependency on defect type and size and the pool and capacity of progenitor cells resident in the wound area. Further elucidation of developmental mechanisms associated with tooth formation may also contribute to valuable knowledge based upon which the future therapies can be designed. Protocols for the differentiation of pluripotent hESCs into periodontal ligament fibroblastic cells (PDLF) as common progenitors for ligament, cementum, and alveolar bone tissue represent an initial step in developing hESC-based experimental and tissue engineering strategies. The present protocol describes methods associated with the guided differentiation of hESCs by the use of coculture with adult PDLFs and the resulting change of morphotype and phenotype of the pluripotent embryonic stem cells toward fibroblastic and osteoblastic lineages.

In vitro evaluation of encapsulated primary rat hepatocytes pre- and post-cryopreservation at -80°C and in liquid nitrogen.

Encapsulation techniques have the potential to protect hepatocytes from cryoinjury. In this study, we comparatively evaluated the viability and metabolic function of primary rat hepatocytes encapsulated in calcium alginate microbeads, in chitosan tripolyphosphate beads, and in three-layered alginate-chitosan-alginate (ACA) microcapsules, before and after cryopreservation at -80°C and in liquid nitrogen (LN2) for 1 and 3 months. Findings demonstrated that LN2 was atop of -80°C in regard to preservation of viability (> 90%) and hepatic functions. LN2-cryopreserved hepatocytes encapsulated in ACA microcapsules retained metabolic function post-thawing, with > 90% of the albumin, total protein and urea syntheses activities, and > 80% of oxidative function.

Evaluation of adenoviral vascular endothelial growth factor-activated chitosan/hydroxyapatite scaffold for engineering vascularized bone tissue using human osteoblasts: In vitro and in vivo studies.

Bone tissue is dependent on an efficient blood supply to ensure delivery of nutrients and oxygen. One method to acquire a vascular-engineered bone tissue could be the use of an angiogenic gene-activated scaffold. In the current study, porous chitosan/hydroxyapatite (C/HA) scaffolds were fabricated via freeze-drying with desired pore size, and then combined with the adenoviral vector encoding vascular endothelial growth factor and green fluorescence protein (Ad-VEGF). Human osteoblasts were cultured and seeded on characterized scaffolds. The attachment, proliferation, and differentiation of cells on gene-activated and unactivated C/HA scaffolds were evaluated in vitro and in vivo by histo- and immunohistochemistry. Findings confirmed that human osteoblasts cultured on gene-activated C/HA scaffold secreted vascular endothelial growth factor, besides maintaining its characteristic phenotype with specific extracellular matrix production. In vivo experiments indicated that scaffolds were tissue biocompatible, and that gene-activated scaffold provided a suitable environment for neovessel formation by recruiting host endothelial cells into the newly forming ectopic bone-like tissue. This study revealed that the Ad-VEGF-activated C/HA composite scaffold has potential for vascular bone regeneration applications.

In vitro cytotoxicity of hydrothermally synthesized ZnO nanoparticles on human periodontal ligament fibroblast and mouse dermal fibroblast cells.

The use of metal oxide nanoparticles (NPs) in industrial applications has been expanding, as a consequence, risk of human exposure increases. In this study, the potential toxic effects of zinc oxide (ZnO) NPs on human periodontal ligament fibroblast cells (hPDLFs) and on mouse dermal fibroblast cells (mDFs) were evaluated in vitro. We synthesized ZnO NPs (particle size; 7-8 nm) by the hydrothermal method. Characterization assays were performed with atomic force microscopy, Braun-Emmet-Teller analysis, and dynamic light scattering. The hPDLFs and mDFs were incubated with the NPs with concentrations of 0.1, 1, 10, 50 and 100 µg/mL for 6, 24 and 48h. Under the control and NP-exposed conditions, we have made different types of measurements for cell viability and morphology, membrane leakage and intracellular reactive oxygen species generation. Also, we monitored cell responses to ZnO NPs using an impedance measurement system in real-time. While the morphological changes were visualized using scanning electron microscopy, the subcellular localization of NPs was investigated by transmission electron microscopy. Results indicated that ZnO NPs have significant toxic effects on both of the primary fibroblastic cells at concentrations of ∼50-100 µg/mL. The cytotoxicity of ZnO NPs on fibroblasts depended on concentration and duration of exposure.

Isolation and characterization of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have drawn great interest in the field of regenerative medicine, for cell replacement, immunomodulatory, and gene therapies. It has been shown that these multipotent stromal cells can be isolated from tissues such as bone marrow, adipose tissue, trimester amniotic tissue, umbilical cord blood, and deciduous teeth and can be expanded in adherent culture. They have the capacity to differentiate into cells of the connective tissue lineages in vitro and contribute to tissue parenchyma in vivo. However, proper in vitro manipulation of MSCs is a key issue to reveal a potential therapeutic benefit following transplantation into the patients. This chapter summarizes some of the essential protocols and assays used at our laboratory for the isolation, culture, differentiation, and characterization of mesenchymal stem cells from the bone marrow and adipose tissue.

Stem cells in tooth tissue regeneration--challenges and limitations.

The accelerated pace of research in the stem cell field in recent decades and the accumulated body of knowledge has spurred the interest in potential clinical applications of stem cells in all branches of medicine including regenerative dentistry. In humans, embryonic and adult stem cells are two major groups of cells that can serve as a donor source in tissue engineering strategies based on ex-vivo cellular expansion. It has been shown that adult stem cell populations are present in all examined living tissues of the organism, thus being a crucial source of tissue homeostasis and regeneration, and offering a target population for in situ stimulation of extensive tissue regeneration. Experimental findings indicate that in the complex structure of the tooth organ, both periodontal and endodontic tissues harbour adult stem cells with characteristics peculiar to early stages of cellular differentiation. Myriad of strategies incorporating both embryonic and adult stem cells for the regeneration of a particular tooth structure or the whole teeth were proposed; however their successful application to solve real problems encountered in the clinical practice of dentistry remains an elusive and challenging objective.

Neovascularization by bFGF releasing hyaluronic acid-gelatin microspheres: in vitro and in vivo studies.

Therapeutic angiogenesis with angiogenic growth factors has been described as a promising approach for tissue engineering, wound healing, and for treating ischemic tissues. Here, we assessed the merit of heparin-entrapped hyaluronic acid-gelatin (HA-G) microspheres for the sustained release of recombinant basic fibroblast growth factor (rbFGF) to promote localized neovascularization. HA-G microspheres were prepared by a water-in-oil emulsion method, and the in vitro release kinetics were first examined using three model proteins. Then, bFGF was incorporated into microspheres, and the bioactivity of the in vitro-released rbFGF was tested on human umbilical vein endothelial cell cultures. The ability to promote microvessel growth was assessed in vivo, at the subcutaneous groin fascia of Wistar rats after 3, 7, 14, and 21 days. Histological and morphometrical analysis indicated that heparin-entrapped HA-G microspheres have the capacity to release bioactive rbFGF, leading to localized neovascularization in the rat subcutaneous tissue.

Proteome analysis of rat bone marrow mesenchymal stem cell differentiation.

Bone marrow multipotent stromal cells (or mesenchymal stem cells; MSCs) have the capacity for renewal and the potential to differentiate in culture into several cell types including osteoblasts, chondrocytes, adipocytes, cardiomyocytes, and neurons. This study was designed to investigate the protein expression profiles of rat bone marrow MSCs during differentiation into adipogenic (by dexamethasone, isobutylmethylxanthine, insulin, and indomethacin), cardiomyogenic (by 5-azacytidine), chondrogenic (by ascorbic acid, insulin-transferrin-selenous acid, and transforming growth factor-ß1), and osteogenic (by dexamethasone, ß-glycerophosphate, and ascorbic acid) lineages by well-known differentiation inducers. Proteins extracted from differentiated MSCs were separated using two-dimensional gel electrophoresis (2-DE) and protein spots were detected using Sypro Ruby dye. Protein spots that were determined to be up- or down-regulated when the expression of corresponding spots (between weeks 1 and 2, 1 and 3, 1 and 4) showed an increase (≥2-fold) or decrease (≤0.5-fold) were successfully identified by MALDI-TOF-MS. In summary, 23 new proteins were identified either up- or down-regulated during differentiation experiments.

Human embryonic stem cell differentiation on periodontal ligament fibroblasts.

Human embryonic stem cells' (hESCs) unlimited proliferative potential and differentiation capability to all somatic cell types made them potential cell source in different cell-based tissue engineering strategies as well as various experimental applications in fields such as developmental biology, pharmacokinetics, toxicology, and genetics. Periodontal tissue engineering aims to improve the outcome of regenerative therapies which have variable success rates when contemporary techniques are used. Cell-based therapies may offer potential advantage in overcoming the inherent limitations associated with guided tissue-regeneration procedures, such as dependency on defect type and size and the pool and capacity of progenitor cells resident in the wound area. Elucidation of developmental mechanisms of different periodontal tissues may also contribute to valuable knowledge based upon which the future therapies can be designed. Prior to the realization of such a potential, protocols for the differentiation of pluripotent hESCs into periodontal ligament fibroblastic cells (PDLF) as common progenitors for ligament, cementum, and alveolar bone tissue need to be developed. The present protocol describes methods associated with the guided differentiation of hESCs by the use of coculture with adult PDLFs, and the resulting change of morphotype and phenotype of the pluripotent embryonic stem cells toward fibroblastic and osteoblastic lineages.

In vitro differentiation and attachment of human embryonic stem cells on periodontal tooth root surfaces.

Periodontal tissue engineering based on cell replacement therapies is a promising field for improved regeneration of tooth supporting structures lost as a result of destructive periodontal diseases. Human embryonic stem cells (hESCs) could become adequate cell source for tissue engineering because of their unlimited proliferative potential and ability to differentiate to all somatic cell types. The aim of this study was to analyze the differentiation capacity of hESCs toward periodontal compartment cells and their relationship with tooth root surfaces in vitro. Periodontal ligament fibroblastic cell (PDLF) cultures were established and characterized; hESCs (HUES-9 line) were expanded in undifferentiated state and characterized for pluripotency morphologically and immunohistochemically. Extracted tooth root slices (RS) of 300 microm thickness, prepared with both periodontal and endodontic instrumentation, were used. Three different experimental groups were established: (i) undifferentiated hESC colonies cultured on and around the RS; (ii) undifferentiated hESC colonies cultured on and around RS with PDLF coculture, and (iii) undifferentiated hESC colonies cultured on and around RS with PDLF coculture in osteoinductive medium for 3 weeks. The fibrogenic and osteogenic marker expression was assessed with immunohistochemistry; histological staining and scanning electron microscopy were utilized to determine the relationship between differentiating hESCs and mineralized tooth root structures. Results demonstrate that hESC differentiation is influenced by tooth structures, PDLFs, and osteogenic medium, resulting with increased propensity toward mesenchymal lineage commitment, and formation of soft-hard tissue relationship in close contact areas. The proposed experimental system may facilitate further understanding in development of periodontal structures and contribute to realization of hESCs as a cell source in periodontal tissue engineering applications.

Proteome analysis of rat bone marrow mesenchymal stem cell subcultures.

Bone marrow mesenchymal stem cells (BM-MSCs) have the capacity for renewal and the potential to differentiate in culture into several cell types including osteoblasts, chondrocytes, adipocytes, astrocytes, myocytes, oligodendrocytes, and neurons. Albeit previous reports demonstrated some of the effects of extensive subculturing on MSCs, the results still remain controversial. The aim of this study was to generate proteome maps of undifferentiated rat BM-MSCs, and identify differentially regulated proteins during serial subcultures within the first 10 passages. Proteins extracted from Wistar rat BM-MSCs were separated by two-dimensional gel electrophoresis and about 1000 protein spots were detected using the Sypro Ruby dye. Among them, 106 selected spots were digested with trypsin for mass spectrometry analysis, and 31 proteins were successfully identified by MALDI-TOF-MS. Here, 18 differentially expressed proteins are reported for the first time; these proteins are classified into 8 functional categories: metabolism, signal transduction, cell adhesion and growth, cytoskeleton, cell cycle, protein degradation, cell-cell interaction, and ion transfer. These proteins are reported to be involved in cell proliferation and differentiation through different signaling pathways. These studies suggest that differentially regulated passage-specific proteins may play a role in the decrease of proliferation potential under serial subculturing. The molecular mechanisms of rat BM-MSCs are discussed at the proteome level.

Periodontal ligament cellular structures engineered with electrospun poly(DL-lactide-co-glycolide) nanofibrous membrane scaffolds.

Periodontal tissue engineering is expected to overcome the limitations associated with the existing regenerative techniques for the treatment of periodontal defects involving alveolar bone, cementum, and periodontal ligament. Cell-based tissue engineering approaches involve the utilization of in vitro expanded cells with regenerative capacity and their delivery to the appropriate sites via biomaterial scaffolds. The aim of this study was to establish living periodontal ligament cell-containing structures on electrospun poly(DL-lactic-co-glycolic acid) (PLGA) nanofiber membrane scaffolds, assess their viability and characteristics, and engineer multilayered structures amenable to easy handling. Human periodontal ligament (hPDL) cells were expanded in explant culture and then characterized morphologically and immunohistochemically. PLGA nanofiber membranes were prepared by the electrospinning process; mechanical tensile properties were determined, surface topography, nanofiber size, and porosity status were investigated with SEM. Cells were seeded on the membranes at approximately 50,000 cell/cm(2) and cultured for 21 days either in expansion or in osteogenic induction medium. Cell adhesion and viability were demonstrated using SEM and MTT, respectively, and osteogenic differentiation was determined with IHC and immunohistomorphometric evaluation of osteopontin, osteocalcin, and bone sialoprotein marker expression. At days 3, 6, 9, and 12 additional cell/membrane layers were deposited on the existing ones and multilayered hybrid structures were established. Results indicate the feasibility of periodontal ligament cell-containing tissue-like structures engineering with PDL cells and electrospun nanofiber PLGA scaffolds supporting cell adhesion, viability and osteogenic differentiation properties of cells in hybrid structures amenable to macroscopic handling.

Engineering of rat articular cartilage on porous sponges: effects of tgf-beta 1 and microgravity bioreactor culture.

The objective of this study was to develop an engineered rat hyaline cartilage by culturing articular chondrocytes on three-dimensional (3D) macroporous poly(DL-lactic-co-glycolic acid) (PLGA) sponges under chondrogenic induction and microgravity bioreactor conditions. Experimental groups consisted of 3D static and dynamic cultures, while a single cell monolayer (2D) served as the control. The effect of seeding conditions (static vs. dynamic) on cellularization of the scaffolds was investigated. MTT assay was used to evaluate the number of viable cells in each group at different time points. Formation of a hyaline-like cartilage was evaluated for up to 4 weeks in vitro. While 2D culture resulted in cell sheets with very poor matrix production, 3D culture was in the favor of tissue formation. A higher yield of cell attachment and spatially uniform cell distribution was achieved when dynamic seeding technique was used. Dynamic culture promoted cell growth and infiltration throughout the sponge structure and showed the formation of cartilage tissue, while chondrogenesis appeared attenuated more towards the outer region of the constructs in the static culture group. Medium supplemented with TGF-beta 1 (5 ng/ml) had a positive impact on proteoglycan production as confirmed by histochemical analyses with Alcian blue and Safranin-O stainings. Formation of hyaline-like tissue was demonstrated by immunohistochemistry performed with antibodies against type II collagen and aggrecan. SEM confirmed higher level of cellularization and cartilage tissue formation in bioreactor cultures induced by TGF-beta 1. The data suggest that PLGA sponge inside rotating bioreactor with chondrogenic medium provides an environment that mediates isolated rat chondrocytes to redifferentiate and form hyaline-like rat cartilage, in vitro.

Human embryonic stem cell differentiation on tissue engineering scaffolds: effects of NGF and retinoic acid induction.

The indefinite proliferative capacity and ability to differentiate into all somatic cell types can make human embryonic stem cells (hESCs) useful in experimental and applied studies in embryonic development, tissue engineering, genetic engineering, pharmacokinetics, and the like. Cellular differentiation dynamics can be studied in monolayer cell cultures; however, it proceeds in three-dimensional (3D) organization in vivo. The aim of this study was to assess the effects of retinoic acid (RA) and nerve growth factor (NGF) on the differentiation patterns of hESCs in 3D culture environment and to compare it with the monolayer culture. Expanded hESCs (HUES-9) were differentiated in two experimental groups for 21 days: (i) two-dimensional (2D) monolayer cultures of hESC colonies, and (ii) 3D culture of hES single cells in poly(DL-lactic-co-glycolic acid) scaffolds. The media used were embryonic stem cell expansion medium (ES-EM), embryonic stem cell differentiation medium containing fetal calf serum (ES-DM), ES-EM containing either 10 ng/mL NGF or 10(-6) M RA, and their combination. Fixed specimens were analyzed with scanning electron microscopy, and expression of nestin, pan-cytokeratin, troponin, and alpha-fetoprotein at days 7, 14, and 21 was evaluated by immunohistomorphometry and reverse transcriptase--polymerase chain reaction. Results indicate different patterns of ectodermal, mesodermal, and endodermal marker expressions between groups, where NGF and RA preferentially favors the differentiation toward ectodermal and mesodermal lineages. While troponin and nestin expression is significantly elevated in 3D culture environment, pan-cytokeratin expression is favored by 2D culture instead. The effects of 3D scaffold culture imply the usefulness of testing in vitro differentiation properties of hESCs in various culture settings designed as models in prospective tissue engineering applications.