[Show abstract][Hide abstract] ABSTRACT: Background: A bioartificial heart is a theoretical alternative to transplantation or mechanical left ventricular support. Native hearts decellularized with preserved architecture and vasculature may provide an acellular tissue platform for organ regeneration. We sought to develop a tissue-engineered whole-heart neoscaffold in human-sized porcine hearts.
Methods: We decellularized porcine hearts (n = 10) by coronary perfusion with ionic detergents in a modified Langendorff circuit. We confirmed decellularization by histology, transmission electron microscopy and fluorescence microscopy, quantified residual DNA by spectrophotometry, and evaluated biomechanical stability with ex-vivo left-ventricular pressure/ volume studies, all compared to controls. We then mounted the decellularized porcine hearts in a bioreactor and reseeded them with murine neonatal cardiac cells and human umbilical cord derived endothelial cells (HUVEC) under simulated physiological conditions.
Results: Decellularized hearts lacked intracellular components but retained specific collagen fibers, proteoglycan, elastin and mechanical integrity; quantitative DNA analysis demonstrated a significant reduction of DNA compared to controls (82.663.2 ng DNA/mg tissue vs. 473.2613.4 ng DNA/mg tissue, p,0.05). Recellularized porcine whole-heart neoscaffolds demonstrated re-endothelialization of coronary vasculature and measurable intrinsic myocardial electrical activity at 10 days, with perfused organ culture maintained for up to 3 weeks.
Conclusions: Human-sized decellularized porcine hearts provide a promising tissue-engineering platform that may lead to future clinical strategies in the treatment of heart failure.
[Show abstract][Hide abstract] ABSTRACT: Tissue-engineered tracheal transplants have been successfully performed clinically. However, before becoming a routine clinical procedure, further preclinical studies are necessary to determine the underlying mechanisms of in situ tissue regeneration. Here we describe a protocol using a tissue engineering strategy and orthotopic transplantation of either natural decellularized donor tracheae or artificial electrospun nanofiber scaffolds into a rat model. The protocol includes details regarding how to assess the scaffolds' biomechanical properties and cell viability before implantation. It is a reliable and reproducible model that can be used to investigate the crucial aspects and pathways of in situ tracheal tissue restoration and regeneration. The model can be established in <6 months, and it may also provide a means to investigate cell-surface interactions, cell differentiation and stem cell fate.
[Show abstract][Hide abstract] ABSTRACT: The development of tracheal scaffolds fabricated based on electrospinning technique by applying different ratios of polyethylene terephthalate (PET) and polyurethane (PU) is introduced here. Prior to clinical implantation, evaluations of biomechanical and morphological properties, as well as biocompatibility and cell adhesion verifications are required and extensively performed on each scaffold type. However, the need for bioreactors and large cell numbers may delay the verification process during the early assessment phase. Hence, we investigated the feasibility of performing biocompatibility verification using static instead of dynamic culture. We performed bioreactor seeding on 3-dimensional (3-D) tracheal scaffolds (PET/PU and PET) and correlated the quantitative and qualitative results with 2-dimensional (2-D) sheets seeded under static conditions. We found that an 8-fold reduction for 2-D static seeding density can essentially provide validation on the qualitative and quantitative evaluations for 3-D scaffolds. In vitro studies revealed that there was notably better cell attachment on PET sheets/scaffolds than with the polyblend. However, the in vivo outcomes of cell seeded PET/PU and PET scaffolds in an orthotopic transplantation model in rodents were similar. They showed that both the scaffold types satisfied biocompatibility requirements and integrated well with the adjacent tissue without any observation of necrosis within 30 days of implantation.
[Show abstract][Hide abstract] ABSTRACT: No definitive solution has been discovered for replacing long segments or the entire trachea in humans. Most of this challenge stems from the specific function and mechanics that are almost impossible to replicate except in the setting of an allotransplantation, which requires lifelong immunosuppressive medication. Recently, tissue engineering provided significant evidence concerning the next promising therapeutic alternative for tracheal replacement. Underlying mechanism and pathways of cell-surface interactions, cell migration, and differentiation are essential to understand the complexity of tracheal tissue regeneration. Tracheal replacement remains challenging but initial steps toward an ideal therapeutic concept have been made.
[Show abstract][Hide abstract] ABSTRACT: A tissue-engineered oesophageal scaffold could be very useful for the treatment of pediatric and adult patients with benign or malignant diseases such as carcinomas, trauma or congenital malformations. Here we decellularize rat oesophagi inside a perfusion bioreactor to create biocompatible biological rat scaffolds that mimic native architecture, resist mechanical stress and induce angiogenesis. Seeded allogeneic mesenchymal stromal cells spontaneously differentiate (proven by gene-, protein and functional evaluations) into epithelial- and muscle-like cells. The reseeded scaffolds are used to orthotopically replace the entire cervical oesophagus in immunocompetent rats. All animals survive the 14-day study period, with patent and functional grafts, and gain significantly more weight than sham-operated animals. Explanted grafts show regeneration of all the major cell and tissue components of the oesophagus including functional epithelium, muscle fibres, nerves and vasculature. We consider the presented tissue-engineered oesophageal scaffolds a significant step towards the clinical application of bioengineered oesophagi.
[Show abstract][Hide abstract] ABSTRACT: Stem cells contribute to regeneration of tissues and organs. Cells with stem cell-like properties have been identified in tumors from a variety of origins, but to our knowledge there are yet no reports on tumor-related stem cells in the human upper respiratory tract. In the present study, we show that a tracheal mucoepidermoid tumor biopsy obtained from a 6 year-old patient contained a subpopulation of cells with morphology, clonogenicity and surface markers that overlapped with bone marrow mesenchymal stromal cells (BM-MSCs). These cells, designated as MEi (mesenchymal stem cell-like mucoepidermoid tumor) cells, could be differentiated towards mesenchymal lineages both with and without induction, and formed spheroids in vitro. The MEi cells shared several multipotent characteristics with BM-MSCs. However, they displayed differences to BM-MSCs in growth kinectics and gene expression profiles relating to cancer pathways and tube development. Despite this, the MEi cells did not possess in vivo tumor-initiating capacity, as proven by the absence of growth in situ after localized injection in immunocompromised mice. Our results provide an initial characterization of benign tracheal cancer-derived niche cells. We believe that this report could be of importance to further understand tracheal cancer initiation and progression as well as therapeutic development.
PLoS ONE 01/2014; 9(9):e107712. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aortic valve degeneration and dysfunction is one of the leading causes for morbidity and mortality. The conventional heart-valve prostheses have significant limitations with either life-long anticoagulation therapeutic associated bleeding complications (mechanical valves) or limited durability (biological valves). Tissue engineered valve replacement recently showed encouraging results, but the unpredictable outcome of tissue degeneration is likely associated to the extensive tissue processing methods. We believe that optimized decellularization procedures may provide aortic valve/root grafts improved durability. We present an improved/innovative decellularization approach using a detergent-enzymatic perfusion method, which is both quicker and has less exposure of matrix degenerating detergents, compared to previous protocols. The obtained graft was characterized for its architecture, extracellular matrix proteins, mechanical and immunological properties. We further analyzed the engineered aortic root for biocompatibility by cell adhesion and viability in vitro and heterotopic implantation in vivo. The developed decellularization protocol was substantially reduced in processing time whilst maintaining tissue integrity. Furthermore, the decellularized aortic root remained bioactive without eliciting any adverse immunological reaction. Cell adhesion and viability demonstrated the scaffold's biocompatibility. Our optimized decellularization protocol may be useful to develop the next generation of clinical valve prosthesis with a focus on improved mechanical properties and durability.
[Show abstract][Hide abstract] ABSTRACT: Pluripotent cells such as human embryonic stem cells and human induced pluripotent stem cells are useful in the field of regenerative medicine because they can proliferate indefinitely and differentiate into all cell types. However, a limiting factor for maintaining and propagating stem cells is the need for inactivated fibroblasts as a growth matrix, since these may potentially cause cross-contamination. In this study, we aimed to maintain stem cells on the extracellular matrix (ECM) of either nonirradiated or γ-irradiated fibroblasts. It has been demonstrated that the ECM contains factors and proteins vital for the adhesion, proliferation, and differentiation of pluripotent cells. In order to preserve the ECM, the cell layers of the fibroblasts were decellularized by treatment with 0.05% sodium dodecyl sulfate (SDS), which resulted in an absence of DNA as compared with conventional feeder culture. However, SDS treatment did not cause a detectable change in the ECM architecture and integrity. Furthermore, immunohistochemistry demonstrated that expressions of major ECM proteins, such as fibronectin, collagen, and laminin, remained unaltered. The human pluripotent cells cultured on this decellularized matrix maintained gene expression of the pluripotency markers NANOG and OCT4 and had the potency to differentiate to three germ layers. The in vitro culture system shown here has an excellent potential since the main allogeneic components (i.e., DNA of the feeder cells) are removed. It is also a technically easy, fast, safe, and cheap method for maintaining a refined feeder-free stem cell culture for further cell differentiation studies.
STEM CELLS TRANSLATIONAL MEDICINE 10/2013; · 3.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In 2008, the first transplantation of a tissue-engineered trachea in a human being was done to replace an end-staged left main bronchus with malacia in a 30-year-old woman. We report 5 year follow-up results.
The patient was followed up approximately every 3 months with multidetector CT scan and bronchoscopic assessment. We obtained mucosal biopsy samples every 6 months for histological, immunohistochemical, and electron microscopy assessment. We also assessed quality of life, respiratory function, cough reflex test, and production and specificity of recipient antibodies against donor human leucocyte antigen.
By 12 months after transplantation, a progressive cicatricial stenosis had developed in the native trachea close to the tissue-engineered trachea anastomosis, which needed repeated endoluminal stenting. However, the tissue-engineered trachea itself remained open over its entire length, well vascularised, completely re-cellularised with respiratory epithelium, and had normal ciliary function and mucus clearance. Lung function and cough reflex were normal. No stem-cell-related teratoma formed and no anti-donor antibodies developed. Aside from intermittent bronchoscopic interventions, the patient had a normal social and working life.
These clinical results provide evidence that a tissue-engineering strategy including decellularisation of a human trachea, autologous epithelial and stem-cell culture and differentiation, and cell-scaffold seeding with a bioreactor is safe and promising.
European Commission, Knut and Alice Wallenberg Foundation, Swedish Research Council, ALF Medicine.
[Show abstract][Hide abstract] ABSTRACT: Development of novel prognostic, diagnostic, and treatment options will provide major benefits for millions of patients with acute or chronic respiratory dysfunction, cardiac-related disorders, esophageal problems, or other diseases in the thorax. Allogeneic organ transplant is currently available. However, it remains a trap because of its dependency on a very limited supply of donated organs, which may be needed for both initial and subsequent transplants. Furthermore, it requires lifelong treatment with immunosuppressants, which are associated with adverse effects. Despite early clinical applications of bioengineered organs and tissues, routine implementation is still far off. For this review, we searched the PubMed, MEDLINE, and Ovid databases for the following keywords for each tissue or organ: tissue engineering, biological and synthetic scaffold/graft, acellular and decelluar(ized), reseeding, bioreactor, tissue replacement, and transplantation. We identified the current state-of-the-art practices in tissue engineering with a focus on advances during the past 5 years. We discuss advantages and disadvantages of biological and synthetic solutions and introduce novel strategies and technologies for the field. The ethical challenges of innovation in this area are also reviewed.
Mayo Clinic Proceedings 10/2013; 88(10):1151-1166. · 5.79 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The clinical outcome of transplantations of bioartificial tissues and organs depends on the presence of living cells. There are still no standard operative protocols that are simple, fast and reliable for confirming the presence of viable cells on bioartificial scaffolds prior to transplantation. By using mathematical modeling, we have developed a colorimetric-based system (colorimetric scale bar) to predict the cell viability and density for sufficient surface coverage. First, we refined a method which can provide information about cell viability and numbers in an in vitro setting: i) immunohistological staining by Phalloidin/DAPI and ii) a modified colorimetric cell viability assay. These laboratory-based methods and the developed colorimetric-based system were then validated in rat transplantation studies of unseeded and seeded tracheal grafts. This was done to provide critical information on whether the graft would be suitable for transplantation or if additional cell seeding was necessary. The potential clinical impact of the colorimetric scale bar was confirmed using patient samples. In conclusion, we have developed a robust, fast and reproducible colorimetric tool that can verify and warrant viability and integrity of an engineered tissue/organ prior to transplantation. This should facilitate a successful transplantation outcome and ensure patient safety.
[Show abstract][Hide abstract] ABSTRACT: Due to demographic changes end-stage organ failure is currently one of the major challenges. In the majority of the cases the only so far curable therapeutic option is an allogenic organ transplantation. However, conventional transplantation is associated with a shortage of donor organs and a lifetime of immunosuppressant, which is a non-ideal solution. Regenerative medicine, including tissue engineering (TE), cell therapy and pharmaceutical intervention represents a very promising alternative in this context. During the recent years several attempts has been made to engineer various tissue/ organs and initial evidence exists about the clinical potential of this method. Despite early clinical applications for the trachea, bladder, skin or heart valves, more solid organs can probably never be transferred to the patient regarding their demanding and complicated engineering process. For complex organs, such as heart, lung or the liver, cell therapy and pharmaceutical intervention can be an optimum alternative. RM aims to replace and regenerate damaged tissue to restore organ function. It is very common that all three aspects of RM, such as TE, cell therapy and pharmaceutical intervention are combined at some level and should not be categorical separated from each other. The following components are necessary to be considered: a) a scaffold/ matrix seeded with b) different cell type(s) usingc) the body or a device as a native or artificial bioreactor, respectively, d) bioactive molecules/Signaling and e) cell/gene therapy. We already learned that each target tissue requires specific components, and engineering processes need to be modified to distinctive needs. Early clinical application demonstrated the feasibility of using regenerative approaches to replace damaged tissue and organs with rather simple architecture. Preclinical findings suggest that even higher complex organs can be successfully partly engineered. However, due to the complexness of these solid organs cell therapy might be the more promising solution for the near clinical future. Notably, pharmaceutical strategies should be widely considered to increase endogenous regeneration and self-healing capacity of the recipient. The new field of RM rises novel ethical concerns and questions – these must be addressed and answered.
American Association for the Advancement of Science 2013 Annual Meeting; 02/2013
[Show abstract][Hide abstract] ABSTRACT: A mathematical model, in the form of an integro-partial differential equation, is presented to describe the dynamics of cells being deposited, attaching and growing in the form of a monolayer across an adherent surface. The model takes into account that the cells suspended in the media used for the seeding have a distribution of sizes, and that the attachment of cells restricts further deposition by fragmenting the parts of the domain unoccupied by cells. Once attached the cells are assumed to be able to grow and proliferate over the domain by a process of infilling of the interstitial gaps; it is shown that without cell proliferation there is a slow build up of the monolayer but if the surface is conducive to cell spreading and proliferation then complete coverage of the domain by the monolayer can be achieved more rapidly. Analytical solutions of the model equations are obtained for special cases, and numerical solutions are presented for parameter values derived from experiments of rat mesenchymal stromal cells seeded onto thin layers of collagen-coated polyethylene terephthalate electrospun fibers. The model represents a new approach to describing the deposition, attachment and growth of cells over adherent surfaces, and should prove useful for studying the dynamics of the seeding of biomaterials.
Journal of Mathematical Biology 02/2013; · 2.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: PURPOSE OF REVIEW: To summarize the so far applied clinical methods of tracheal replacement, comparing pros and cons of conventional and tissue-engineered approaches. RECENT FINDINGS: Several strategies have been most recently described to replace the trachea-like aortic homografts, allotransplantation, and tissue engineering. Allotransplantation requires lifelong immunosuppression and this may be ethically questioned being not a lifesaving procedure. Tissue-engineered tracheal transplantation has been clinically applied using biological or bioartificial tubular or bifurcated scaffolds reseeded with mesenchymal stromal cells, and bioactive molecules boosting regeneration and promoting neovascularization. SUMMARY: Tracheal tissue engineering may be a promising alternative to conventional allotransplantation in adults and children. Different methods have been developed and are currently under active clinical investigation, and await long-term results.
Current opinion in otolaryngology & head and neck surgery 02/2013;
[Show abstract][Hide abstract] ABSTRACT: INTRODUCTION: Inflammatory and autoimmune diseases have been associated with the tumor necrosis factor superfamily member "A PRoliferation Inducing Ligand" (APRIL). However, up to now, APRIL has not been investigated in critical illness or sepsis. We therefore analyzed APRIL serum concentrations in a large cohort of well-characterized intensive care unit patients. METHODS: Serum concentrations of APRIL were measured in 246 critically ill patients, of which 157 fulfilled sepsis criteria in comparison with 81 healthy controls. Clinical data were recorded and correlated with APRIL serum levels. RESULTS: We detected strongly elevated serum levels of APRIL in critically ill patients compared with healthy controls. Levels of APRIL were further elevated in sepsis and significantly correlated with classical markers of inflammation, bacterial infection, or multiorgan failure. Consequently, high APRIL levels were associated with an unfavorable prognosis and predicted mortality with higher diagnostic accuracy than established prognostic scoring systems such as the Acute Physiology and Chronic Health Evaluation II score. CONCLUSION: Serum levels of APRIL were significantly elevated in intensive care unit patients, with the highest concentrations in septic patients, and associated with unfavorable outcome. Besides being used as a single marker, APRIL may be implemented into established scoring systems to further improve their sensitivity and specificity in predicting patient's prognosis.
Journal of critical care 01/2013; · 2.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Stem cells possess the unique properties of unlimited self-renewal capability and a broad differentiation spectrum to produce multiple different cell types. This provides many platforms to explore novel multidisciplinary approaches to create and/or restore functional three-dimensional tissues or organs for the treatment of a range of diseases. In this chapter, in the context of respiratory diseases, we review the unique properties of stem cells, and how they have been studied for their therapeutic potential in cell therapy and tissue engineering. In addition, we give a brief overview of the current clinical studies on the use of stem cells for both acute and chronic respiratory diseases.
Advances in biochemical engineering/biotechnology 08/2012; · 1.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Objectives:Jehovah's Witnesses present a challenge to cardiac surgeons, as quality of care is not only defined by mortality and morbidity, but also by the avoidance of blood transfusions. Over the last years, minimized perfusion circuits (MPC) have contributed substantially to the achievement of this goal in our clinic. Presented is a retrospective analysis of our experience. METHODS: Twenty-nine Jehovah's Witnesses, aged 69 ± 10 years, have undergone cardiac surgery with a MPC in our institution since 2005. The ROCsafe (Reservoir Optional Circuit) MPC was used in most of these patients (n=27) as it offers the unique possibility of a speedy integration of a reservoir in the event of a major air leak, thereby, negligating any safety concerns. RESULTS: There was no in-hospital or 30-day postoperative mortality. Mean ICU stay was 1.6 ± 2 days with a mean intubation time of 11.3 ± 9.1 hrs. Postoperative complications included one myocardial infarction with accompanying low cardiac output, one stroke, one transient delirium, one idiopathic thrombocytopenia and three re-operations (one sternal infection, one postoperative bleeding and one delayed tamponade). The mean postoperative hospital stay was 9.9 ± 2.3 days. Mean decrease in hemoglobin was 2.1 ± 1.3 g/dl during cardiopulmonary bypass and 3.4 ±1.4 g/dl at discharge. The lowest postoperative hemoglobin level was 9.3 ±1.8 (Range 6-12.9). CONCLUSIONS: These encouraging results emphasize the role MPCs can play in optimizing the quality of patient care. We hope that this report can serve as a stimulus for similar experiences.
[Show abstract][Hide abstract] ABSTRACT: In 2011, the first in-man successful transplantation of a tissue engineered trachea-bronchial graft, using a synthetic POSS-PCU nanocomposite construct seeded with autologous stem cells, was performed. To further improve this technology, we investigated the feasibility of using polymers with a three dimensional structure more closely mimicking the morphology and size scale of native extracellular matrix (ECM) fibers. We therefore investigated the in vitro biocompatibility of electrospun polyethylene terephthalate (PET) and polyurethane (PU) scaffolds, and determined the effects on cell attachment by conditioning the fibers with adhesion proteins. Rat mesenchymal stromal cells (MSCs) were seeded on either PET or PU fiber-layered culture plates coated with laminin, collagen I, fibronectin, poly-D-lysine or gelatin. Cell density, proliferation, viability, morphology and mRNA expression were evaluated. MSC cultures on PET and PU resulted in similar cell densities and amounts of proliferating cells, with retained MSC phenotype compared to data obtained from tissue culture plate cultures. Coating the scaffolds with adhesion proteins did not increase cell density or cell proliferation. Our data suggest that both PET and PU mats, matching the dimensions of ECM fibers, are biomimetic scaffolds and, because of their high surface area-to-volume provided by the electrospinning procedure, makes them per se suitable for cell attachment and proliferation without any additional coating.