Processing of ovine cardiac valve allografts: 1. Effects of preservation method on structure and mechanical properties.

It is essential to have some method of preservation of allograft valves during the time between procurement and implantation. Cryopreservation is the most commonly-used storage method today but it has the major disadvantage of high cost, and because its aim is to preserve living cells only relatively gentle antimicrobial treatments are used. This study addresses two interrelated questions: Is it necessary to maintain living donor cells in the tissue graft?Can more effective measures be used to reduce the risk of transmission of diseases, especially viral diseases, via human tissue grafts. In this paper, were port an investigation of four preservation methods that could be combined with more effective disinfection: cryopreservation with dimethyl sulphoxide, storage at ～4 °C in a high concentration of glycerol as used for the preservation of skin, snap-freezing by immersion in liquid nitrogen and vitrification. Snap freezing was mechanically damaging and vitrification proved to be impracticable but two methods, cryopreservation and storage in 85%glycerol, were judged worthy of further study. Cryopreservation was shown to maintain cellular viability and excellent microscopic structure with unchangedmechanical properties. The glycerol-preserved valves did not contain any living cells but the connective tissue matrix and mechanical properties were well preserved. The importance of living cells in allograft valves is uncertain. If living cells are unimportant then either method could be combined with more effective disinfection methods: in that case the simplicity and economy of the glycerol method would be advantageous. These questions are addressed in the two later papers in this series.     

Brief History of the Tissue Bank, Charles University Hospital, Hradec Králové, Czech Republic

A brief history of the Tissue Bank (TB) of the University Hospital Hradec Králové, Czech Republic, established by Dr. R. Klen in 1952 is presented. In Dr. Klen's original concept the TB was defined as a department specialised in the harvesting, processing, preservation, storage and distribution of various kinds of tissue for clinical and experimental practice. The first kinds of tissue collected in cadaveric donors were corneas, bone and skin. Xenogeneic cartilage and bone grafts were prepared at the same time. Later, preparation of soft connective tissues and chorion–amnion was introduced. During the first 15 years of activity a total of 11,443 grafts preserved by hypothermy at +4°C or freezing in absence of cryoprotectants (–20°C) were prepared. In the 60's freeze-drying of tissue grafts was introduced and the bank of cryopreserved cell lines was established. In the 80's cryopreservation of haematopoietic progenitor cells for clinical transplantations was started and the spectrum of tissue grafts was enlarged (xenogeneic pericardium and allogeneic specially treated dura mater for neurosurgical operations, pigskin for burn treatment, demineralised bone for parodontology and implantology). In the 90's human keratinocyte culture for treatment of burns and chronic skin defects was started. The human milk bank and organ bank co-operating with the Regional Transplantation Centre are component parts of the TB as well. The TB is an institutional member of the European Association of Tissue Banks and annually delivers approximately 1000 grafts that are used in University and county hospitals as well as in surgeons' private practices. Health insurance companies reimburse all grafts on a non-profit and tax-free basis.     

Transfusion and transplantation of cryopreserved cells and tissues

The modern era of cryomedicine began in 1949 in London and developed world-wide in the second half of the 20th century based on the first report of a novel method of cryopreservation of sperm and erythrocytes using glycerol that was reported in 1949 and 1950 by Polge and Smith. In 1951 at Hradec Kralove, Czech. Klen initiated a "tissue bank" using his unique freeze-drying system. In 1964, the initial meeting of the Society for Cryobiology was organized by its first president. B. J. Luyet in Washington, DC. Cryobiology including cryopreservation and cryosurgery, contributed immense advances for clinical medicine. Cryomedicine will realize the goals of the New Millennium medicine: regeneration, plasticity, and minimally invasive therapy. I explained the first one, regeneration in this paper in detail.Cryomedicine involved subzero-temperatures to freeze the biological objects either for preservation or for destruction. Cryopreservation involves the cooling of the target biological materials to below the temperature of solidification by consumption of energy, through continuously supplying inert cryogens to attain the necessary cryo-temperatures by Joule-Thompson's effect. Therefore biological materials for cryopreservation should be carefully selected and once frozen purposefully kept in the frozen state to be used later to regenerate human cells, tissues and organs, and also to relaize "plasticity". Recently, lyophilization of human cells and tissues came back to the main street of cryopreservation to provide low cost economical and ecological banking of cells and tissues as a hope of the New Millennium. The first attempt of that was made by Prof. Dr. Rudolf Klen and his colleagues.Finally, physicians and related scientists who are going to be interested in cryomedicine should not worry about "freezing and thawing" as being time consuming and labor intensive, otherwise they will not share in the crucial benefits of cryomedicine.     

The relevance of ice crystal formation for the cryopreservation of tissues and organs

This paper discusses the role of ice crystal formation in causing or contributing to the difficulties that have been encountered in attempts to develop effective methods for the cryopreservation of some tissues and all organs. It is shown that extracellular ice can be severely damaging but also that cells in situ in tissues can behave quite differently from similar cells in a suspension with respect to intracellular freezing. It is concluded that techniques that avoid the formation of ice altogether are most likely to yield effective methods for the cryopreservation of recalcitrant tissues and vascularised organs.     

Mechanisms of intracellular ice formation.

The phenomenon of intracellular freezing in cells was investigated by designing experiments with cultured mouse fibroblasts on a cryomicroscope to critically assess the current hypotheses describing the genesis of intracellular ice: (a) intracellular freezing is a result of critical undercooling; (b) the cytoplasm is nucleated through aqueous pores in the plasma membrane; and (c) intracellular freezing is a result of membrane damage caused by electrical transients at the ice interface. The experimental data did not support any of these theories, but was consistent with the hypothesis that the plasma membrane is damaged at a critical gradient in osmotic pressure across the membrane, and intracellular freezing occurs as a result of this damage. An implication of this hypothesis is that mathematical models can be used to design protocols to avoid damaging gradients in osmotic pressure, allowing new approaches to the preservation of cells, tissues, and organs by rapid cooling.     

Scaffolds in tissue engineering of blood vessels

Tissue engineering of small diameter (<5?mm) blood vessels is a promising approach for developing viable alternatives to autologous vascular grafts. It involves in vitro seeding of cells onto a scaffold on which the cells attach, proliferate, and differentiate while secreting the components of extracellular matrix that are required for creating the tissue. The scaffold should provide the initial requisite mechanical strength to withstand in vivo hemodynamic forces until vascular smooth muscle cells and fibroblasts reinforce the extracellular matrix of the vessel wall. Hence, the choice of scaffold is crucial for providing guidance cues to the cells to behave in the required manner to produce tissues and organs of the desired shape and size. Several types of scaffolds have been used for the reconstruction of blood vessels. They can be broadly classified as biological scaffolds, decellularized matrices, and polymeric biodegradable scaffolds. This review focuses on the different types of scaffolds that have been designed, developed, and tested for tissue engineering of blood vessels, including use of stem cells in vascular tissue engineering.     

The cryopreservation of composite tissues: Principles and recent advancement on cryopreservation of different type of tissues

Cryopreservation of human cells and tissue has generated great interest in the scientific community since 1949, when the cryoprotective activity of glycerol was discovered. Nowadays, it is possible to reach the optimal conditions for the cryopreservation of a homogeneous cell population or a one cell-layer tissue with the preservation of a high pourcentage of the initial cells. Success is attained when there is a high recovery rate of cell structures and tissue components after thawing. It is more delicate to obtain cryopreservation of composite tissues and much more a whole organ. The present work deals with fundamental principles of the cryobiology of biological structures, with special attention to the transfer of liquids between intra and extracellular compartments and the initiation of the formation and aggregation of ice during freezing. The consequences of various physical and chemical reactions on biological tissue are described for different cryoprotective agents. Finally, we report a review of results on cyropreservation of various tissues, on the one hand, and various organs, on the other. We also report immunomodulation of antigenic responses to cryopreserved cells and organs.     

The cryopreservation of composite tissues

Cryopreservation of human cells and tissue has generated great interest in the scientific community since 1949, when the cryoprotective activity of glycerol was discovered. Nowadays, it is possible to reach the optimal conditions for the cryopreservation of a homogeneous cell population or a one cell-layer tissue with the preservation of a high pourcentage of the initial cells. Success is attained when there is a high recovery rate of cell structures and tissue components after thawing. It is more delicate to obtain cryopreservation of composite tissues and much more a whole organ. The present work deals with fundamental principles of the cryobiology of biological structures, with special attention to the transfer of liquids between intra and extracellular compartments and the initiation of the formation and aggregation of ice during freezing. The consequences of various physical and chemical reactions on biological tissue are described for different cryoprotective agents. Finally, we report a review of results on cyropreservation of various tissues, on the one hand, and various organs, on the other. We also report immunomodulation of antigenic responses to cryopreserved cells and organs.     

Antigen removal for the production of biomechanically functional,xenogeneic tissue grafts

Xenogeneic tissues are derived from other animal species and provide a source of material for engineering mechanically functional tissue grafts, such as heart valves, tendons, ligaments, and cartilage. Xenogeneic tissues, however, contain molecules, known as antigens, which invoke an immune reaction following implantation into a patient. Therefore, it is necessary to remove the antigens from a xenogeneic tissue to prevent immune rejection of the graft. Antigen removal can be accomplished by treating a tissue with solutions and/or physical processes that disrupt cells and solubilize, degrade, or mask antigens. However, processes used for cell and antigen removal from tissues often have deleterious effects on the extracellular matrix (ECM) of the tissue, rendering the tissue unsuitable for implantation due to poor mechanical properties. Thus, the goal of an antigen removal process should be to reduce the antigen content of a xenogeneic tissue while preserving its mechanical functionality. To expand the clinical use of antigen-removed xenogeneic tissues as biomechanically functional grafts, it is essential that researchers examine tissue antigen content, ECM composition and architecture, and mechanical properties as new antigen removal processes are developed.     

Heart valve and arterial tissue engineering

Abstract.  In the industrialized world, cardiovascular disease alone is responsible for almost half of all deaths. Many of the conditions can be treated successfully with surgery, often using transplantation techniques; however, autologous vessels or human-donated organs are in short supply. Tissue engineering aims to create specific, matching grafts by growing cells on appropriate matrices, but there are many steps between the research laboratory and the operating theatre. Neo-tissues must be effective, durable, non-thrombogenic and non-immunogenic. Scaffolds should be bio-compatible, porous (to allow cell/cell communication) and amenable to surgery. In the early days of cardiovascular tissue engineering, autologous or allogenic cells were grown on inert matrices, but patency and thrombogenicity of grafts were disappointing. The current ethos is toward appropriate cell types grown in (most often) a polymeric matrix that degrades at a rate compatible with the cells' production of their own extracellular matrical proteins, thus gradually replacing the graft with a living counterpart. The geometry is crucial. Computer models have been made of valves, and these are used as three-dimensional patterns for mass-production of implant scaffolds. Vessel walls have integral connective tissue architecture, and application of physiological level mechanical forces conditions bio-engineered components to align in precise orientation. This article reviews the concepts involved and successes achieved to date.     

Fetal Tissue Banking for Transplantation: Characteristics of the Donor Population and Considerations for Donor and Tissue Screening

We initiated this study to evaluate the suitability for therapeutic use in transplantation of tissues obtained from human abortuses. We have developed protocols for the collection, handling and preservation of hepatic stem cells from electively aborted embryos and have developed methods for assessment of the cells so derived and processed. In this paper we present our findings regarding screening of potential donors, acquisition of fetal tissues, and assessment of the tissues for potentially infectious contaminants. We assess the suitability of the tissue donors according to current standards used for donors of commonly transplanted tissues (e.g., bone grafts, skin grafts and heart valves) and present data regarding the real availability of tissues from elective abortion procedures that would meet those standard tissue banking criteria.We specifically evaluated the donor's willingness to provide a blood sample for testing, conducted a detailed interview similar to those used for typical organ and tissue donors, and assessed the type and incidence of contamination in collected tissues. We find that although many women are willing to consent to use of the tissues for transplantation, attrition from the study for various reasons results in few fetal organs ultimately realistically available for transplantation. Typical reasons for attrition include: unwillingness to have a blood sample drawn or tested, positive serology results, social/medical high risk factors for acquisition of transmissible disease, no identifiable organs available, and unacceptable microbial contamination. Thus, although it might seem that due to the numbers of abortions performed annually, that there would be substantial numbers of suitable tissues available, only a small proportion are truly suitable for transplantation.     

Processing of ovine cardiac valve allografts: 3. Implantation following antimicrobial treatment and preservation.

It is known that a satisfactory clinical outcome can follow the implantation of cardiac valve allografts in spite of the loss of living cells in the tissue. If viable cells are not required for long term graft function, then effective disinfection of the tissue might become possible. In an earlier paper in this series we reported that peracetic acid (PAA) is an effective antimicrobial agent for the treatment of valve allografts; it was lethal to the cells but at a concentration of 0.21% had little effect on the mechanical properties or extracellular morphology of the valve leaflets. It was also found that PAA-treatment could be combined with storage in 85% glycerol at 4 °C, or cryopreservation with 10%Me₂SO, without substantial further impairment of microscopic structure or mechanical properties. In this paper we describe the implantation of processed ovine aortic valves in the descending thoracic aorta of sheep. The experimental groups included control untreated valves and valves that had been treated with antibiotics or PAA and either cryopreserved, or stored in 85%glycerol. The recipient sheep showed good clinical appearances until the experiment was terminated at six months. The explanted grafts were examined by standard morphological and mechanical testing methods. The PAA-treated valves were clearly recognisable as valves: the leaflets had fair to medium morphology in both the unpreserved and the cryopreserved groups. All leaflets had a superficial overgrowth of cells. Microsatellite analysis for allelic differences were performed on samples of donor and recipient tissues using three markers of tissue source. Only one valve, which had been treated with PAA, revealed allelic differences between donor and recipient. It is suggested that DNA-fragments may have remained after the destruction of donor cells and six months of implantation: the overgrowing cells were almost certainly of recipient origin. We conclude that our experiments, in which PAA-treatment was combined with preservation, are sufficiently encouraging to justify further studies to refine the technique, but in our opinion they are not sufficient to justify a clinical trial at this time. This revised version was published online in July 2006 with corrections to the Cover Date.     

Theoretical considerations regarding the cell membrane function during cold-induced preservation of tissues and organs: new possibilities for optimizing the process

An analysis of the effects of temperature variation on the function of the mammalian cell membrane strongly suggests that during the cold-induced preservation of living tissues and organs the passive flow of micromolecules and ions across the cell membrane is the main factor in determining the state of the cellular biological system. Practically, the optimal organ (tissue) storage could be obtained at low temperatures (exceeding the freezing point) by using preservation fluids with constitutive substances in concentrations equal to those existing in the normal cytoplasm. Consequently, the formulae for adequate preservation fluids must be calculated according to the peculiar cytoplasmic constitution of the cells of each organ and tissue.     

Determination of mechanical stress distribution in Drosophila wing discs using photoelasticity

Morphogenesis, the process by which all complex biological structures are formed, is driven by an intricate interplay between genes, growth, as well as intra- and intercellular forces. While the expression of different genes changes the mechanical properties and shapes of cells, growth exerts forces in response to which tissues, organs and more complex structures are shaped. This is exemplified by a number of recent findings for instance in meristem formation in Arabidopsis and tracheal tube formation in Drosophila. However, growth not only generates forces, mechanical forces can also have an effect on growth rates, as is seen in mammalian tissues or bone growth. In fact, mechanical forces can influence the expression levels of patterning genes, allowing control of morphogenesis via mechanical feedback. In order to study the connections between mechanical stress, growth control and morphogenesis, information about the distribution of stress in a tissue is invaluable. Here, we applied stress-birefringence to the wing imaginal disc of Drosophila melanogaster, a commonly used model system for organ growth and patterning, in order to assess the stress distribution present in this tissue. For this purpose, stress-related differences in retardance are measured using a custom-built optical set-up. Applying this method, we found that the stresses are inhomogeneously distributed in the wing disc, with maximum compression in the centre of the wing pouch. This compression increases with wing disc size, showing that mechanical forces vary with the age of the tissue. These results are discussed in light of recent models proposing mechanical regulation of wing disc growth.     

The mechanics of development: Models and methods for tissue morphogenesis

Embryonic development is a physical process during which groups of cells are sculpted into functional organs. The mechanical properties of tissues and the forces exerted on them serve as epigenetic regulators of morphogenesis. Understanding these mechanobiological effects in the embryo requires new experimental approaches. Here we focus on branching of the lung airways and bending of the heart tube to describe examples of mechanical and physical cues that guide cell fate decisions and organogenesis. We highlight recent technological advances to measure tissue elasticity and endogenous mechanical stresses in real time during organ development. We also discuss recent progress in manipulating forces in intact embryos. Birth Defects Research (Part C) 90:193–202, 2010. © 2010 Wiley‐Liss, Inc.     

Cryo-scanning electron microscopic study on freezing behaviors of tissue cells in dormant buds of larch (Larix kaempferi)

The freezing behavior of dormant buds in larch, especially at the cellular level, was examined by a Cryo-SEM. The dormant buds exhibited typical extraorgan freezing. Extracellular ice crystals accumulated only in basal areas of scales and beneath crown tissues, areas in which only these living cells had thick walls unlike other tissue cells. By slow cooling (5 °C/day) of dormant buds to −50 °C, all living cells in bud tissues exhibited distinct shrinkage without intracellular ice formation detectable by Cryo-SEM. However, the recrystallization experiment of these slowly cooled tissue cells, which was done by further freezing of slowly cooled buds with LN and then rewarming to −20 °C, confirmed that some of the cells in the leaf primordia, shoot primordia and apical meristem, areas in which cells had thin walls and in which no extracellular ice accumulated, lost freezable water with slow cooling to −30 °C, indicating ability of these cells to adapt by extracellular freezing, whereas other cells in these tissues retained freezable water with slow cooling even to −50 °C, indicating adaptation of these cells by deep supercooling. On the other hand, all cells in crown tissues and in basal areas of scales, areas in which cells had thick walls and in which large masses of ice accumulated, had the ability to adapt by extracellular freezing. It is thought that the presence of two types of cells exhibiting different freezing adaptation abilities within a bud tissue is quite unique and may reflect sophisticated freezing adaptation mechanisms in dormant buds.     

Stabilized imaging of immune surveillance in the mouse lung

Real-time imaging of cellular and subcellular dynamics in vascularized organs requires image resolution and image registration to be simultaneously optimized without perturbing normal physiology. This problem is particularly pronounced in the lung, in which cells may transit at speeds >1 mm s(-1) and in which normal respiration results in large-scale tissue movements that prevent image registration. Here we report video-rate, two-photon imaging of a physiologically intact preparation of the mouse lung that is stabilizing and nondisruptive. Using our method, we obtained evidence for differential trapping of T cells and neutrophils in mouse pulmonary capillaries, and observed neutrophil mobilization and dynamic vascular leak in response to stretch and inflammatory models of lung injury in mice. The system permits physiological measurement of motility rates of >1 mm s(-1), observation of detailed cellular morphology and could be applied in the future to other organs and tissues while maintaining intact physiology.     

Biomimetic approach to tissue engineering

The overall goal of tissue engineering is to create functional tissue grafts that can regenerate or replace our defective or worn out tissues and organs. Examples of grafts that are now in pre-clinical studies or clinical use include engineered skin, cartilage, bone, blood vessels, skeletal muscle, bladder, trachea, and myocardium. Engineered tissues are also finding applications as platforms for pharmacological and physiological studies in vitro. To fully mobilize the cell's biological potential, a new generation of tissue engineering systems is now being developed to more closely recapitulate the native developmental milieu, and mimic the physiologic mechanisms of transport and signaling. We discuss the interactions between regenerative biology and engineering, in the context of (i) creation of functional tissue grafts for regenerative medicine (where biological input is critical), and (ii) studies of stem cells, development and disease (where engineered tissues can serve as advanced 3D models).     

Glycation of collagen: the basis of its central role in the late complications of ageing and diabetes

The most serious late complications of ageing and diabetes mellitus follow similar patterns in the dysfunction of retinal capillaries, renal tissue, and the cardiovascular system. The changes are accelerated in diabetic patients owing to hyperglycaemia and are the major cause of premature morbidity and mortality. These tissues and their optimal functioning are dependent on the integrity of their supporting framework of collagen. It is the modification of these properties by glycation that results in many of the damaging late complications. Initially glycation affects the interactions of collagen with cells and other matrix components, but the most damaging effects are caused by the formation of glucose-mediated intermolecular cross-links. These cross-links decrease the critical flexibility and permeability of the tissues and reduce turnover. In contrast to the renal and retinal tissue, the cardiovascular system also contains a significant proportion of the other fibrous connective tissue protein elastin, and its properties are similarly modified by glycation. The nature of these glycation cross-links is now being unravelled and this knowledge is crucial in any attempt to inhibit these deleterious glycation reactions.     

Vitrification of large tissues with dielectric warming: biological problems and some approaches to their solution

If large pieces of tissue and organs are to be successfully stored at low temperatures, some means must be found to minimize the disruption of extracellular structures by the ice that develops during conventional cryopreservation methods. The use of sufficiently high concentrations of cryoprotectant (CPA) to vitrify rather than freeze the tissue is a possible solution to this problem, and the retention of function of embryos and elastic arteries after vitrification suggests that some cells and tissues at least can withstand exposure to the high concentrations of CPA necessary for this process to occur. There are, however, additional problems in applying vitrifying techniques to bulky tissues and organs. These are related to the additional time required for tissue equilibration of CPA to occur and the consequences for toxic injury, the difficulty in achieving sufficiently rapid and uniform cooling rates to produce the required glassy state, and the even more rapid and uniform warming rates that are necessary to avoid devitrification. Non-uniformity of temperature will increase the risk of mechanical stresses and fractures developing in the glass during rapid warming. This paper reviews possible strategies and the progress that has been made in overcoming these problems. This will include the permeation of CPA mixtures into whole tissues and possibilities for reducing their toxicity by the inclusion of adjuncts such as ice inhibitors and sugars. The warming of tissues by dielectric heating is currently the only practical means by which sufficiently rapid rates can be achieved in bulky tissues given that the tolerable limits of CPA concentration will most likely be insufficient to prevent the development of ice nuclei during cooling. The biological effects of microwaves are reviewed and their effectiveness in producing the required uniformity in warming of tissue models of various shapes are discussed.