Cardiac tissue engineering for replacement therapy

Cell therapy is a new concept to repair diseased organs. For patients with myocardial infarction, heart failure, and congenital heart diseases cell based therapies might represent a potential cure. The field can be subdivided into two principally different approaches: (1) Implantation of isolated cells and (2) implantation of in vitro engineered tissue constructs. This review will focus on the latter approach. Cardiac tissue engineering comprises the fields of material sciences and cell biology. In general, scaffold materials such as gelatin, collagen, alginate, or synthetic polymers and cardiac cells are utilized to reconstitute tissue-like constructs in vitro. Ideally, these constructs display properties of native myocardium such as coherent contractions, low diastolic tension, and syncytial propagation of action potentials. To be applicable for surgical repair of diseased myocardium engineered tissue constructs should have the propensity to integrate and remain contractile in vivo. Size and mechanical properties of engineered constructs are critical for surgical repair of large tissue defects. Successful application of tissue engineering in men will depend on the utilization of an autologous or non-immunogeneic cell source and scaffold material to avoid life long immunosuppression. This review will give an overview of recent approaches in cardiac tissue engineering and its first applications in vivo. We will discuss materials and cell sources for cardiac tissue engineering. Further, principle obstacles will be addressed. Cardiac tissue engineering for replacement therapy has an intriguing perspective, but is in its early days. Its true value remains to be thoroughly evaluated.     

Evidence for nuclear modifier gene in mitochondrial cardiomyopathy

Mitochondrial DNA (mtDNA) inheritance and maintenance and function of the respiratory chain are the result of a synergistic action of the nuclear and the mitochondrial genomes. Mutations in either or both genomes can result in a wide range of multisystemic disorders. We have studied a homoplasmic mtDNA mutation in the tRNA^Ile gene that segregates exclusively with cardiomyopathy in two unrelated families. Cytochrome c oxidase (COX) deficiency was selectively observed only in the heart tissue and in patient's cardiomyocyte cultures and not in any other cell type, indicating that the defect is tissue specific. To understand the pathogenic mechanism of cardiomyopathy associated with a homoplasmic, tissue specific mtDNA mutation, we constructed transnuclear cardiomyocyte cell lines with normal or patient's nucleus and containing wild type or mutant mtDNA. Of the four cell lines analyzed, COX activity was low only in patient's cardiomyocytes illustrating that both the patient's nucleus and mitochondria are essential for expression of the phenotype. In cells with either wild type nucleus or wild type mtDNA, COX activity was normal. From these results it is evident that a tissue specific nuclear modifier gene may interact synergistically with the mtDNA mutation to cause COX deficiency.     

Cardiac tissue engineering,ex-vivo: design principles in biomaterials and bioreactors

Cardiac tissue engineering has emerged as a promising approach to replace or support an infarcted cardiac tissue and thus may hold a great potential to treat and save the lives of patients with heart diseases. By its broad definition, tissue engineering involves the construction of tissue equivalents from donor cells seeded within 3-D biomaterials, then culturing and implanting the cell-seeded scaffolds to induce and direct the growth of new, healthy tissue. In this review, we present an up-to-date summary of the research in cardiac tissue engineering, with an emphasis on the design principles and selection criteria that have been used in two key technologies employed in tissue engineering, (1) biomaterials technology, for the creation of 3-D porous scaffolds which are used to support and guide the tissue formation from dissociated cells, and (2) bioreactor cultivation of the 3-D cell constructs during ex-vivo tissue engineering, which aims to duplicate the normal stresses and flows experienced by the tissues.     

An expert consensus document on the management of cardiovascular manifestations of Fabry disease

Fabry disease (FD) is an X‐linked lysosomal storage disorder caused by pathogenic variants in the α‐galactosidase A (GLA) gene that leads to reduced or undetectable α‐galactosidase A enzyme activity and progressive accumulation of globotriaosylceramide and its deacylated form globotriaosylsphingosine in cells throughout the body. FD can be multisystemic with neurological, renal, cutaneous and cardiac involvement or be limited to the heart. Cardiac involvement is characterized by progressive cardiac hypertrophy, fibrosis, arrhythmias, heart failure and sudden cardiac death. The cardiac management of FD requires specific measures including enzyme replacement therapy or small pharmacological chaperones in patients carrying amenable pathogenic GLA gene variants and more general management of cardiac symptoms and complications. In this paper, we summarize current knowledge of FD‐related heart disease and expert consensus recommendations for its management.     

Stem Cell-Based Cardiac Tissue Engineering

Cardiovascular diseases are the leading cause of death worldwide, and cell-based therapies represent a potential cure for patients with cardiac diseases such as myocardial infarction, heart failure, and congenital heart diseases. Towards this goal, cardiac tissue engineering is now being investigated as an approach to support cell-based therapies and enhance their efficacy. This review focuses on the latest research in cardiac tissue engineering based on the use of embryonic, induced pluripotent, or adult stem cells. We describe different strategies such as direct injection of cells and/or biomaterials as well as direct replacement therapies with tissue mimics. In this regard, the latest research has shown promising results demonstrating the improvement of cardiac function with different strategies. It is clear from recent studies that the most important consideration to be addressed by new therapeutic strategies is long-term functional improvement. For this goal to be realized, novel and efficient methods of cell delivery are required that enable high cell retention, followed by electrical integration and mechanical coupling of the injected cells or the engineered tissue to the host myocardium.     

Tissue engineering of heart valves: new opportunities and challenges

Replacement of heart valves appears to be prevailing method of surgical correction of end stage valvular heart defects. Main drawback of contemporary artificial valves is lack of growth, potential for remodeling, and inclination to degeneration. To overcome these limitations the modern science in the last decade focuses on tissue engineering of valves as an alternative to their prostheses. Basic idea of the technique is the use of decellularized xenogenic allogenic matrix or biopolymers seeded with autologous cells under special conditions created in bioreactor. This literature review is devoted to a novel direction in experimental cardiosurgery - tissue engineering of heart valves which in a unique way combines biological, engineering, and technological achievements.     

Tissue engineering of biological cardiovascular system surrogates

Cardiovascular diseases are common in ageing communities globally. This fact is most striking in the industrialised world where the aged population makes up a large proportion of society. Elderly patients are frequently treated surgically with grafts to replace damaged tissues and vessels. The number of human-donated components is insufficient and synthetic surrogates are sought. These might be wholly mechanical, wholly biological, or tissue engineered complexes of cells and their products growing in a scaffold. At present, many such composites exist with potential for use as substitutes for specific blood vessels. The challenges of producing tissue engineered heart valves are now being widely explored. Neotissues must provide an effective, durable, non-thrombogenic and non-immunogenic substitute that will fulfil the purpose of the natural tissue. The aims and scope of this paper are to review current and novel concepts in the field of tissue engineering of biological cardiovascular system surrogates. Mechanical stresses and strains on cardiovascular cells in vitro have been recognised and can be measured by a culture force monitor. Physiological stresses can be generated by a tensioning culture force monitor and applied to engineered tissue, aligning the cells and mimicking arterial wall architecture. The hydrostatic forces a vessel experiences and mechanical parameters of blood vessels can be studied in the tubular culture system of a multi-cue bioreactor.     

Cardiomyocyte transplantation into the failing heart-new therapeutic approach for heart failure?

Heart failure, frequently the consequence of irreversible myocardial damage with subsequent formation of akinetic scar tissue, is a highly prevalent disease, and in its advanced stages associated with high mortality. The transplantation of exogenous cells with the inherent ability to contract has been put forward as one potential treatment strategy to increase contractility and cardiac performance. Besides skeletal myoblasts or stem cells from various sources, immature cardiomyocytes, such as fetal or neonatal cardiomyocytes, have been transplanted into normal, cryoinjured, infarcted myocardium, as well as into models of global heart failure. Survival of transplanted immature cardiomyocytes has been demonstrated up to 6–7 months, accompanied by vascularization of the grafted tissue. Transplants developed sarcomeric structures and other morphological features of differentiation. The principal possibility of cell-to-cell coupling between graft and host cells was demonstrated after cardiomyocyte transplantation into normal hearts and in some studies in damaged myocardium. But most long-term follow-up investigations in models of myocardial infarction reported that optimal integration of the engrafted cells appeared to be hindered by scar tissue, separating the transplant from the host. Nonetheless, in several studies, improved parameters of cardiac performance were demonstrated ex-vivo and in vivo. Potential mechanisms might involve beneficial effects on the remodeling process. In this review, we critically evaluate the potential value of cardiomyocyte transplantation as a new approach in the treatment of the syndrome of heart failure.     

Gene editing advance re‐ignites debate on the merits and risks of animal to human transplantation

In Australia, and internationally, the shortage of organ and tissue donors significantly limits the number of patients with critical organ or tissue failure who are able to receive a transplant each year. The rationale for xenotransplantation – the transplantation of living cells, tissues or organs from one species to another – is to meet this shortfall in human donor material. While early clinical trials showed promise, particularly in patients with type I diabetes whose insulin dependence could be temporarily reversed by the transplantation of porcine islet cells, these benefits have been balanced with scientific, clinical and ethical concerns revolving around the risks of immune rejection and the potential transmission of porcine endogenous retroviruses or other infectious agents from porcine grafts to human recipients. However, the advent of CRISPR/Cas9, a revolutionary gene editing technology, has reignited interest in the field with the possibility of genetically engineering porcine organs and tissues that are less immunogenic and have virtually no risk of transmission of porcine endogenous retroviruses. At the same time, CRISPR/Cas9 may also open up a myriad of possibilities for tissue engineering and stem cell research, which may complement xenotransplantation research by providing an additional source of donor cells, tissues and organs for transplantation into patients. The recent international symposium on gene editing, organised by the US National Academy of Sciences, highlights both the enormous therapeutic potential of CRISPR/Cas9 and the raft of ethical and regulatory challenges that may follow its utilisation in transplantation and in medicine more generally.     

Tissue Engineering von Herzklappen und Myokard

Cardiac function, including the heart muscle and valves, can be severely altered by congenital and acquired heart diseases. Several graft materials are currently used to replace diseased cardiac tissue and valvular segments. Implantable grafts are either non-vital or can trigger an immune response which leads to graft calcification and degeneration. None of the existing grafts have the ability to remodel and grow in tandem with the physiological growth of a child and therefore require re-operation. Novel approaches such as tissue engineering have emerged as possible alternatives for cardiac reconstruction. The main concept of tissue engineering includes the use of biological and artificial scaffolds that form the shape of the organ structures for subsequent tissue replacement, which will provide absolute biocompatibility, no thrombogenicity, no teratogenicity, long-term durability and growth. Heart valve tissue engineering represents an important field especially in pediatric patients with valve pathologies. In order to create an autologous valve equivalent myofibroblasts and/or endothelial cells are seeded on specially designed scaffolds. Here we describe the different types of cell sources and different types of matrices currently used in heart valve tissue engineering. Valve manufacture is carried out in specially designed bioreactors providing physiological conditions. The number of clinical studies using tissue engineered valves is still limited; however, several promising results have already demonstrated their durability and ability to grow. Myocardial tissue engineering aims to repair, replace and regenerate damaged cardiac tissue using tissue constructs created ex vivo. Conceivable indications for clinical application of tissue engineered myocardial-implant substitutes include ischemic cardiomyopathies, as well as right ventricular outflow tract reconstruction in patients with congenital heart diseases. Therapeutic application of functional (contractile) tissue engineered heart muscle appears feasible once key issues such as identification of the suitable human cell source, large scale expansion and suitable scaffolds are solved. In addition, the present article discusses the importance of vascularization as an important prerequisite for successful bio-artificial myocardial tissue. Further experimental and clinical research on cardiovascular tissue engineering is felt to be of great importance for others as well as for us in order to create an ideal heart valve/myocardial substitute and help our patients with advanced cardiac pathologies.     

RNAi-mediated inhibition of the glucosylceramide synthase (GCS) gene: A preliminary study towards a therapeutic strategy for Gaucher disease and other glycosphingolipid storage diseases

Small interference RNAs (siRNAs) have recently been used in various experimental settings to silence gene expression. In some of them, chemically synthesized or in vitro transcribed siRNAs have been transfected into cells. In others, siRNAs have been expressed endogenously from siRNA expression vectors. Enzyme replacement and substrate deprivation therapies are currently used to treat Gaucher disease. Although good results have been reported, there are several limitations and side effects that make necessary to search for new alternatives. We present a new approach based on the inhibition of the GCS gene using siRNAs as a potential therapeutic strategy for Gaucher disease. We have designed four siRNAs for the human GCS gene and transfected them into HeLa cells. A clear reduction of GCS RNA levels and enzyme activity was obtained using two of the four siRNAs. Furthermore, a reduction in glucosylceramide synthesis was also observed. Similar results were obtained when plasmids expressing shRNAs (targeting the same sequences) were transfected into the cells. The inhibition of the mouse homolog Ugcg gene was also achieved, using a siRNA that targeted both human and mouse sequences.     

Drug insight: an overview of current anticoagulation therapy after heart valve replacement

Vitamin K antagonists, such as warfarin, are the gold standard approach for the long-term anticoagulant therapy of patients with mechanical heart valves. Management decisions are, however, based predominantly on expert consensus and on data from nonrandomized, follow-up studies, which have inherent limitations in their methods. Low-intensity anticoagulation therapy provides protection against thromboembolic complications in patients with most types of modern prosthetic heart valve. The addition of low-dose aspirin is safe if international normalized ratio values below 3.5 are maintained. A combined regimen should be considered in high-risk patients and those with coexistent coronary artery or cerebrovascular disease, and in patients who have suffered a thromboembolic event despite a therapeutic international normalized ratio. Thromboprophylaxis with unfractionated or low-molecular-weight heparins is restricted to specific situations, such as when a patient is intolerant to vitamin K antagonists, when surgical procedures require discontinuation of oral anticoagulation, or when the patient is pregnant. A lack of uniformity across practice guidelines make it difficult to reach treatment decisions. Each patient's preference, expressed after counseling about the risks and benefits of each treatment strategy, and an individual assessment of the patient's risk factors, should guide treatment decisions. At present, new anticoagulant agents such as factor Xa inhibitors do not represent a treatment option for heart valve recipients.     

Mycobacterium paratuberculosis in Intestinal Tissue from Patients with Crohn's Disease Demonstrated by a Nested Primer Polymerase Chain Reaction

Lisby G, Andersen J, Engbsek K, Binder V. Mycobacterium paratuberculosis in intestinal tissue from patients with Crohn's disease demonstrated by a nested primer polymerase chain reaction. Scand J Gastroenterol 1994;29:923-929.

Background: The etiology of Crohn's disease remains unknown, but current research has concentrated on autoimmunity and/or mycobacterial infection. The polymerase chain reaction (PCR) enables the detection of genetic material even when very few microorganisms are present. Methods: A nested primer PCR for detection of a multi-copy insertional element (IS900) specific for Mycobacterium paratuberculosis was applied to DNA extracted from fresh and from paraffin-embedded intestinal tissue obtained from patients undergoing surgery. Results: In fresh intestinal tissue from 11 of 24 patients with Crohn's disease, from 2 of 10 patients with ulcerative colitis, and from 3 of 28 patients with other colonic disorders, specific M. paratuberculosis DNA was found. In paraffin-embedded Crohn's disease tissue the presence of specific M. paratuberculosis DNA was also increased. Conclusions: Whether the presence of M. paratuberculosis is connected to the inflammatory bowel disease or is a mere coincidence cannot be stated. We find this presence interesting and encouraging for further investigations.     

Liver tissue engineering and cell sources: issues and challenges

Liver diseases are of major concern as they now account for millions of deaths annually. As a result of the increased incidence of liver disease, many patients die on the transplant waiting list, before a donor organ becomes available. To meet the huge demand for donor liver, alternative approaches using liver tissue engineering principles are being actively pursued. Even though adult hepatocytes, the primary cells of the liver are most preferred for tissue engineering of liver, their limited availability, isolation from diseased organs, lack of in vitro propagation and deterioration of function acts as a major drawback to their use. Various approaches have been taken to prevent the functional deterioration of hepatocytes including the provision of an adequate extracellular matrix and co‐culture with non‐parenchymal cells of liver. Great progress has also been made to differentiate human stem cells to hepatocytes and to use them for liver tissue engineering applications. This review provides an overview of recent challenges, issues and cell sources with regard to liver tissue engineering.     

Cryopreserved amniotic fluid-derived cells: a lifelong autologous fetal stem cell source for heart valve tissue engineering

BACKGROUND AND AIM OF THE STUDY: Fetal stem cells represent a promising cell source for heart valve tissue engineering. In particular, amniotic fluid-derived cells (AFDC) have been shown to lead to autologous fetal-like heart valve tissues in vitro for pediatric application. In order to expand the versatility of these cells also for adult application, cryopreserved AFDC were investigated as a potential life-long available cell source for heart valve tissue engineering. METHODS: Human AFDC were isolated using CD133 magnetic beads, and then differentiated and analyzed. After expansion of CD133- as well as CD133+ cells up to passage 7, a part of the cells was cryopreserved. After four months, the cells were re-cultured and phenotyped by flow cytometry and immunohistochemistry, including expression of CD44, CD105, CD90, CD34, CD31, CD141, eNOS and vWF, and compared to their non-cryopreserved counterparts. The stem cell potential was investigated in differentiation assays. The viability of cryopreserved AFDC for heart valve tissue engineering was assessed by creating heart valve leaflets in vitro. RESULTS: After cryopreservation, amniotic fluid-derived CD133- and CD133+ cells retained their stem cell-like phenotype, expressing mainly CD44, CD90 and CD105. This staining pattern was comparable to that of their non-cryopreserved counterparts. Moreover, CD133- cells demonstrated differentiation potential into osteoblast-like and adipocyte-like cells. CD133+ cells showed characteristics of endothelial-like cells by eNOS, CD141 and beginning vWF expression. When used for the fabrication of heart valve leaflets, cryopreserved CD133- cells produced extracellular matrix elements comparable to their non-cryopreserved counterparts. Moreover, the resulting tissues showed a cellular layered tissue formation covered by functional endothelia. The mechanical properties were similar to those of tissues fabricated from non-cryopreserved cells. CONCLUSION: The study results suggest that the use of cell bank technology fetal amniotic fluid-derived stem cells might represent a life-long available autologous cell source for heart valve tissue engineering, and also for adult application.     

组织工程心脏瓣膜支架材料进展

当前应用于临床的瓣膜置换物(生物瓣和机械瓣)都不同程度的存在着一定缺陷。组织工程心脏瓣膜为理想生物瓣膜替代物的出现带来了希望。组织工程心脏瓣膜的构建包括:⑴支架的选材及制备;⑵种子细胞的选择、分离与培养;⑶种子细胞在支架上的种植与培养三部分。现重点综述近年来国内外在组织工程心脏瓣膜支架材料研究上的新进展新突破,展望了支架材料发展的方向和前景。     

Calcium Current in Single Human Cardiac Myocytes

Calcium Current in Human Heart. Introduction: Significant species-, issue-, and age-dependent differences have been described for the L-type calcium current (I_Ca). Therefore, extrapolation data obtained from the many animal models to human cardiac physiology is difficult. In this study, we have characterized the voltage-dependent properties of I_Ca from pediatric and adult, atrial and ventricular human heart tissue. Methods and Results: I_Ca, was measured in single human heart muscle cells using the “whole cell,” voltage clamp method. Single myocytes were isolated from myocardial specimens obtained intraoperatively from both pediatric and adult patients (ages 3 months to 75 years) undergoing cardiac surgery. Cells obtained for these experiments appeared to be healthy; the resting potential was between -80 and -85 mV. The action potential shape and duration and current-voltage relationship for 1_Ca were similar to that reported by others for human heart cells. The steady-state activation variable, d_x was found to be similar in both pediatric atrial and ventricular cells but shifted approximately 5 mV negative in the adult atrial and ventricular cells. I, of all cells displayed biex-ponential inactivation and steady-state inactivation was incomplete at positive potentials (steady-state inactivation curves turned up at positive potentials) consistent with inactivation arising from voltage-dependent and calcium-dependent processes as reported in heart cells from many species. The potential of maximal inactivation was more negative for adult cells (around -10 mV) than pediatric cells (around 0 mV). Estimates of the calcium “window” current, using a modified Hodgkin-Huxlcy model, could explain measured differences in action potential shape and duration. Conclusion: Human cardiac I, can be investigated using whole cell, voltage clamp methods and a modified Hodgkin-Huxley model. Quantitative characterization of many of the properties of I_Ca in human heart tissue suggests that important species differences do exist and that further investigations are required to characterize the dependence of inactivation on [Ca²⁺]_i in human heart cells. Since the array of characteristics of I_Ca in different species varies, the study of human myocardial cells per se continues to be important when examining human cardiac physiology.     

Tissue engineering in musculoskeletal problems related to haemophilia

Summary.  This article is a review of how advances in tissue engineering can be applied to the musculoskeletal pathology of patients with haemophilia. This article will also explain the theory that the deterioration of joints in patients with haemophilia is due to biological and mechanical causes.
Current concepts of tissue engineering would be to replace the degenerated and damaged tissue by live cells, using them as a biological implant. However, before these new technologies are applied, an appropriate control of their indication and results is required.