Cell proliferation rates in an artificial tissue-engineered environment.

Worldwide, and particularly in Europe, Japan and the USA, cardiovascular disease is a major killer. It can be treated using tissue or organ transplant surgery, but donor organs may be scarce. Tissue engineering is the integration of engineering principles and biology to produce satisfactory synthetic replacement body parts, using viable cells in a suitable matrix, for regenerative medicine. The aim of this study was to measure and compare cell proliferation kinetics after different time intervals of myofibroblasts in a synthetic matrix, thus to be able to deduce the period that a transplanted-cell population can be expected to survive in a tissue-engineered environment. Porcine aortic wall cells were grown in a porous sponge scaffold, that later could be fashioned into aortic or heart valve substitutes. Freshly acquired cells were seeded on identical sponges and were grown under normal culture conditions for a period of 4 weeks. Seeding concentration was a million cells per sponge. Cells progressively populated the sponges, both covering the surface and infiltrating the depth of the matrix, via sponge pores. Samples were taken at 1 week and at 4 weeks, and the rate of cell proliferation was determined by the metaphase arrest technique. Specimens were also taken for light and electron microscopy to determine whether these transplanted cells were capable of synthesizing their own extracellular matrix.

E1B-deleted adenovirus (dl1520) gene therapy for patients with primary and secondary liver tumors.

Clinical studies were performed with a recombinant mutant adenovirus with an E1B 55-kDa deletion, dl1520, to assess its toxicity and efficacy in patients with irresectable primary and secondary liver tumors. A phase I study showed that dl1520 was well tolerated when administered directly intratumorally, intraarterially, or intravenously up to a dose of 3 x 10(11) PFU. Ultrastructural examination of tissue showed the presence of adenovirus in cell cytoplasm around the nucleus and revealed two dissimilar end points of cell death after virus infection: a preapoptotic sequence and necrosis. A phase II study showed that the combination of dl1520 and 5-fluorouracil (5-FU), when infused into the hepatic artery, was well tolerated. Further improvement in the recombinant vector design will be needed in order to achieve better clinical response.

Pluripotential liver stem cells: facultative stem cells located in the biliary tree.

The ability of the liver to regenerate after parenchymal damage is usually accomplished by the ephemeral entry of normally proliferatively quiescent (G0) hepatocytes into the cell cycle. However, when hepatocyte regeneration is defective, arborizing ductules which are continuous with the biliary tree, proliferate and migrate into the surrounding parenchyma. In man these biliary cells have variously been referred to as ductular structures, neoductules and neocholangioles, and have been observed in many forms of chronic liver disease, including cancer. In experimental animals similar ductal cells are usually called oval cells, and their association with defective regeneration has led to the belief that these cells represent a progenitor cell population. Oval cells are thought to take over the burden of regenerative growth after substantial hepatocyte loss, suggesting that they are the progeny of facultative stem cells. The liver is not, however, generally considered as a stem cell-fed hierarchy, although this is disputed by others. Despite this, the subject of oval cells has aroused intense interest as these cells may represent a target population for hepatic carcinogens, and they may be useful vehicles for ex vivo gene therapy. This review proposes that the liver does harbour stem cells which are located throughout the biliary epithelium, and that oval cells represent the progeny of these stem cells and function as an amplification compartment for the generation of 'new' hepatocytes. This is a conditional process which only occurs when the regenerative capacity of hepatocytes is overwhelmed and thus, unlike the intestinal epithelium, the liver is not behaving as a classical continually renewing stem cell-fed lineage. We focus on the biliary network, not merely as a conduit for bile, but also as a cell compartment with the potential to proliferate under appropriate conditions and give rise to fully differentiated hepatocytes and other cell types.

Heart valve and arterial tissue engineering.

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.

Transforming growth factor-alpha immunoreactivity in a variety of epithelial tissues.

The immunohistochemical expression of transforming growth factor-alpha (TGF alpha) has been examined in a range of normal adult epithelial tissues from both man and rat using an anti-hTGF alpha monoclonal antibody (GF10). No differences in distribution were apparent between man and rat. In the continually renewing epithelium of the gastrointestinal tract, no staining was seen within the proliferative compartments, but strong immunoexpression was noted in various differentiated populations. In the testis, the spermatogonia were unstained, but the more luminally orientated germ cells were strongly positive. In the gastrointestinal tract, at least, any mitogenic action of TGF alpha must be mediated through a relatively long paracrine loop. In contrast, the differentiated parenchyma of kidney, salivary gland and liver remained unstained apart from collecting ducts in the kidney, striated ducts in salivary glands and bile ducts in the liver. The association of TGF alpha with tubule formation was reinforced by the very strong staining of newly forming bile ducts in a model of liver oval cell proliferation. Thus, in all the epithelia studied there was a distinct spatial pattern of TGF alpha immunoreactivity.

Subcellular distribution of peptides associated with gastric mucosal healing and neoplasia.

The trefoil peptides pS2 and human spasmolytic peptide are putative growth factors, particularly associated with mucus-producing cells of the gastrointestinal tract including those of the stomach. The receptor for transforming growth factor alpha (TGF alpha) takes its name from one of its alternative ligands, epidermal growth factor, and is called the epidermal growth factor receptor. Although there is immunoreactive epidermal growth factor in the stomach, it is TGF alpha and the epidermal growth factor receptor that are abundant. Immunolabelling at electron microscope level allows for subcellular localisation of antigens; pS2 and human spasmolytic peptide co-localise to cytomembranes, including the Golgi apparatus, and thecae of surface/pit mucous cells. TGF alpha is abundant on the membranes of tubulovesicles of parietal cells and is also present in chief cells: in mucous producing cells it can be detected but not in association with mucous. The distribution of the epidermal growth factor receptor mimics that of TGF alpha but with preferential clustering on the basolateral membranes of gastric cells. The trefoil peptides are associated with healing and probably act, together with mucus, to protect the gastric mucosa and maintain a viable environment. TGF alpha, transduced via the epidermal growth factor receptor, inhibits gastric acid secretion, thus aids the trefoils in the maintenance of a gastric microenvironment conducive to healing after damage. TGF alpha, however, is also a potent mitogen; while this property plays a vital part in repairing mucosal defects, if this peptide or indeed its receptor are overexpressed, the result can be neoplasia.

Perioperative histologic and ultrastructural changes in the pericardium and adhesions.

The presence of pericardial adhesions prolongs the operation time and increases the risk of serious damage to the heart and other major vascular structures during resternotomy. The reported incidence of such damage is 2% to 6%. Pericardial mesothelial cells exhibit fibrinolytic activity, and therefore have an actual or potential role in the breakdown of the fibrinous adhesions that serve as the initial scaffolding for the firm collagenous adhesions seen at reoperation. Ten patients undergoing primary cardiac procedures were studied to assess the morphologic changes that take place within the pericardium and to relate these to accompanying changes in the pericardial plasminogen activating activity. Samples were taken at 0, 75, and 135 minutes after pericardiotomy. Compared with samples obtained at the time of pericardiotomy, those taken at 75 and 135 minutes demonstrated a significant progression in the mesothelial cell damage (p < 0.01), together with increasing evidence of pericardial inflammation (p < 0.01). The findings from electron microscope studies confirmed and supplemented these findings. Furthermore, compared with its initial levels (median, 2.06 IU/cm2; range, 1.28 to 6.48 IU/cm2), the plasminogen activating activity of pericardial biopsy specimens was significantly reduced at 75 minutes (median, 0.64 IU/cm2; range, 0.12 to 2.44 IU/cm2; P < 0.05), with some recovery at 135 minutes (median, 1.45 IU/cm2; range, 0.12 to 4.39 IU/cm2; p = 0.059). This study has revealed that, during cardiac procedures, the pericardium undergoes inflammatory changes with concomitant damage to its mesothelium, together with a reduction in the pericardial mesothelial fibrinolytic potential.(ABSTRACT TRUNCATED AT 250 WORDS)

Severe subcellular damage in the rat exocrine pancreas after pancreatobiliary diversion.

Cholecystokinin (CCK) may contribute to the genesis of both pancreatic carcinoma and acute pancreatitis. By transposing a long segment of jejunum to lie between the pylorus and the duodenal papilla, pancreatobiliary diversion (PBD) causes a persistent increase in circulating CCK levels, as the normal feedback inhibition of jejunal CCK release by pancreatic juice is evaded. A number of morphological, physiological, and acinar cytokinetic changes ensue. This investigation has examined the ultrastructural changes in pancreatic acinar cells after PBD in the presence and absence of CR1409 (lorglumide), a CCK receptor antagonist. After 14 days there was degranulation and vacuolation of acinar cells with involvement of the enzyme acid phosphatase. The presence of morphologically distinct extracisternal acid phosphatase indisputably predisposed acinar cells to severe damage. Treatment with CR1409 largely prevented degranulation after PBD, but vacuolation of acinar cells still occurred, indicating a possible toxic effect of the receptor antagonist. This is the first report of CCK itself, rather than one of its analogues, causing in vivo pancreatic damage that is generally considered as a forerunner to acute pancreatitis. This is of fundamental importance to the understanding of the earliest stages of the disease.

Immunolabeling for electron microscopy.

The protocols in this chapter concern postembedding immunolabeling for transmission electron microscopy; other schedules, such as pre-embedding methods, frozen tissue processes, and procedures for scanning electron microscopy, can be found elsewhere (1). In principle, immunolabeling at the electron microscope (EM) level follows the same precepts as immunolabeling at the light microscope level; in tissues or cells, the location of an antigen of interest is identified by a specific antibody, and must be visualized appropriately for investigation. Electron microscopy permits us to distinguish subcellu-lar organelles, and therefore ultrastructural localization of antigen position. At the EM level, however, the "visualizing step" needs to be provided by an electron-dense entity, most often a heavy metal, which reflects incident electrons; this is in contrast to the final step of light microscope level techniques, in which the final reaction product is sought to be colored (and where there is an element of choice of which color to use). Tissue processing for EM is considerably more severe than that for light microscopy, and thus maintenance of antigenicity in tissue is more taxing. Before immunolabeling for electron microscopy can be fruitful, the first step is to ensure that the antigen of interest is present (or is still present) in the tissue; this is done by performing a thorough procedure at the light microscope level on wax-embedded sections. Once a positive result has been obtained, studies can progress to the ultrastructural level. If the presence of an antigen cannot be demonstrated in a wax-embedded block, it will not be demonstrable in a resin-embedded EM block of the same tissue. In such a case, pre-embedding and frozen tissue techniques can be of use at both light and electron microscope levels.

Electron microscopic assessment of adenovirus-mediated transfer.

This chapter describes the method of preparing and observing hepatocellular carcinoma (HCC) and surrounding normal liver cells infected with therapeutically p (53)-transfected adenovirus (Ad-p (53)), so that morphology of the cells and viruses, and crucially their relationships to each other, are revealed. In standard practice, ultrastructural analysis of viruses carried in body fluids (e.g., stool or mucus) is sufficient for diagnosis, using the technique of phosphotungstate - dark field staining-of the aqueous extract. That method, however, is not suitable when one needs to examine precise subcellular location of viruses in situ, with tissue and cells intact, for complete pathological assessment; here, we describe our method (1) for transmission electron microscopy of the ultrastructure of virus-infected tumors. Tissue fixation, osmication, embedding, section cutting, and observation of Ad-p (53) infection will be included.

Squamous cell carcinoma of the feline thymus with widespread apoptosis.

The present report documents a case of squamous cell carcinoma originating from the thymus of a 12-year-old short-haired male cat. The tumour had metastasised to the sternal lymphoid tissue and to the left lung. To the authors' knowledge, thymic carcinoma has not been reported in domestic animals. Furthermore, extensive apoptosis was observed in the tumour mass.

Apoptosis: regulation and relevance to toxicology.

1. Apoptosis is a remarkably stereotyped morphological event across all tissues in response to a vast array of damaging agents. 2. Our very existence depends upon a willing exchange of old life for new: apoptotic cell death is our guardian and saviour from genetic damage. 3. There is a close link between cell proliferation and apoptosis: When a cell picks up the machinery to proliferate it also acquires an abort pathway--'better dead than wrong'. 4. A wide variety of highly conserved genes have been implicated in triggering apoptosis. 5. The release of DNA loops from the nuclear scaffold is a more crucial intracellular event than DNA 'laddering' in apoptotic cells. 6. The manipulation of apoptotic rates in many of the common diseases in man will be a major therapeutic strategy in the future.

In vitro mesenchymal stem cell differentiation after mechanical stimulation.

OBJECTIVES: Mesenchymal stem cells (MSC) are multipotent cells capable of differentiating into adipocytic, chondrocytic and osteocytic lineages on suitable stimulation. We have hypothesized that mechanical loading may influence MSC differentiation and alter their phenotype accordingly. MATERIALS AND METHODS: Mouse bone marrow-derived MSC were established in vitro by differential adherence to plastic culture plates and grown in low glucose medium with 10% foetal calf serum and growth factors. Cells grew out and were subcultured up to 20 times. Differentiation protocols were followed for several cell lineages. Clones with trilineage potential were seeded in type I collagen gels and incubated in a tensioning force bioreactor and real-time cell-derived forces were recorded. Gels were fixed and sectioned for light and electron microscopy. RESULTS: Cell monolayers of parent and cloned mouse bone marrow-derived MSC differentiated into adipocytes, osteocytes and chondrocytes, but not into cardiomyocytes, myotubes or neuronal cells. When cast into type I collagen gels and placed in tensioning bioreactors, MSC differentiated into fibroblast-like cells typical of tissue stroma, and upregulated α-smooth muscle actin, but rarely upregulated desmin. Electron microscopy showed collagen and elastin fibre synthesis into the matrix. CONCLUSIONS: These experiments confirmed that MSC cell fate choice depends on minute, cell-derived forces. Applied force could assist in commercial manufacture of cultured bio-engineered prostheses for regenerative medicine as it mimics tissue stresses and constitutes a good model for development of tissue substitutes.

Intestinal metaplasia in liver of rats after partial hepatectomy and treatment with acetylaminofluorene.

OBJECTIVES: The liver is widely recognized for its ability to self-regenerate after damage. Hepatocyte replication is the primary source of liver restoration, although hepatic stem cells (of one kind or another) may be a secondary font, only brought into effect when primary regeneration is severely compromised. MATERIALS AND METHODS: In experiments using small rodents, such an injury can be inflicted by surgically removing a large portion of the liver followed by treatment with hepatotoxin 2-acetylaminofluorene. Regeneration by hepatocyte replication is blocked and thus, stem cell involvement is promoted. However, other responses may be stimulated and this study describes the presence of mucinous glandular structures in the healing liver after two-thirds of its volume was removed via hepatectomy followed by treatment with 2-acetylaminofluorene. RESULTS: Unique observation of intestinal metaplastic cells was seen under alcian blue/periodic acid-Schiff staining. CONCLUSION: The existence of this phenotype (along with oval cells and small hepatocyte-like cells) is evidence of multipotency of progenitors involved in the hepatic healing response.

Proportions of mitotic and apoptotic cells in a range of untreated experimental tumours.

The quantitative aspect of apoptosis in experimental tumours is often neglected. In this study, the apoptotic and mitotic indices for a range of tumours have been determined at light microscope level. It has been found that the apoptotic levels fall into a consistent range for all tumour types and agree well with those described by previous workers. It is suggested that these might be basic parameters of tumour expansion, as relevant to growth kinetics as mitotic levels.

Kinetic studies on a murine sarcoma and an analysis of apoptosis.

A stathmokinetic method has been used to determine the cell cycle parameters, particularly the potential tumour doubling time, of a murine fat pad sarcoma. Additional information has been obtained by determining the percentage of labelled mitoses (PLM). A technique which simultaneously demonstrates autoradiographically labelled S phase nuclei and histochemically localized acid phosphatase activity has also been used at light microscope level to compare these parameters: acid phosphatase activity was demonstrated in tumour cells and macrophages. Single cell deletion by apoptosis has been investigated as distinct from necrosis. Condensed, dying apoptotic cells, have been found in proliferative areas of tumour that are not under physiological stress. The analysis of apoptosis indicated a previously unsuspected variation in apoptotic activity with tumour weight. Cell death by apoptosis initially rose as the tumour grew, but after the tumour reached a threshold weight it declined dramatically, and finally remained stable. This may reflect an initial attempt at autoregulation of tumour size which ultimately fails. Apoptosis was estimated to account for an average of 7% of the total cell loss rate in this tumour.

The role of growth factors in gastrointestinal cell proliferation.

The epithelial lining of the gastrointestinal (GI) tract is in a state of continuous cell renewal, and the proliferating and differentiating/differentiated cell populations are spatially clearly demarcated. Members of the epidermal growth factor (EGF) family of peptides, the trefoil peptides and enteroglucagon appear to be the most important enterotrophic molecules for both normal cell renewal and healing after cell damage. Transforming growth factor-a (TGF-a) appears to be the primary physiological ligand for the EGF receptor (EGFR), promoting normal cell renewal, and TGF-a/EGFR are part of an autocrine loop in many intestinal cancers. In response to damage, a differentiating cell lineage arises from adjacent epithelium secreting EGF, TGF-a and trefoil peptides; this may be viewed as part of a 'repair kit' in damaged endodermally-derived tissue.

Apoptosis: a gene-directed programme of cell death.

Apoptosis is a particular type of programmed cell death which commonly occurs in the developing embryo, in normal healthy adult tissues and in many pathological settings. In contrast to necrosis, apoptosis is not a passive phenomenon but is gene-directed, usually requiring ongoing protein synthesis. The dying cell is characterised by having a raised level of cytosolic Ca2+; this activates a non-lysosomal Ca(2+)- and Mg(2+)-dependent endonuclease which digests the chromatin into oligonucleosome length fragments. The dying cell may or may not fragment into a number of apoptotic bodies, but in all cases the cell contents are bounded by a membrane which prevents the spillage of harmful substances such as DNA. Apoptotic cells are eliminated through phagocytosis by neighbouring cells and macrophages, and cell surface changes on apoptotic cells aid their recognition and engulfment by the phagocytosing cells. Extrinsic signals can both stimulate and inhibit apoptosis, and even direct damage to the cell can activate the process. Apoptosis is widely involved in organ formation in the embryo, and its occurrence in response to noxious stimuli such as cytotoxic drugs, irradiation and hyperthermia may be viewed as an altruistic suicide. Apoptosis provides a safe disposal mechanism for neutrophils at inflamed sites, and within the immune system it is considered responsible for eliminating self-reactive T-cell clones and for the affinity maturation of antibody producing cells. A failure to undergo apoptosis has been invoked in the pathogenesis of low-grade follicular lymphoma, and the triggering of apoptosis with monoclonal antibodies specifically in tumour cells has been achieved in one or two cases.

Liver stem cells: when the going gets tough they get going.

The ability of the liver to regenerate is widely acknowledged, and this is usually accomplished by the entry of normally proliferatively quiescent hepatocytes into the cell cycle. However, when hepatocyte regeneration is impaired, small bile ducts proliferate and invade into the adjacent hepatocyte parenchyma. In humans and experimental animals these ductal cells are referred to as oval cells, and their association with defective regeneration has led to the belief that they are the progeny of facultative stem cells. Oval cells are of great biological interest since they may represent a target population for hepatic carcinogens, and they may also be useful vehicles for ex vivo gene therapy for the correction of inborn errors of metabolism. The ability of oval cells to differentiate into hepatocytes has been demonstrated unequivocally. However, this process only occurs when the regenerative capacity of hepatocytes is overwhelmed, and thus, unlike the intestinal epithelium, the liver is not behaving as a classical continually renewing stem cell-fed lineage.

The biology and pathology of programmed cell death (apoptosis).

Apoptosis is a process by which cells die in a controlled and programmed manner in response to specific stimuli, often following extrinsic and intrinsic signals which ultimately cause the "switching on" of cell death regulatory genes. Condensation of chromatin and cytoplasm, fragmentation of the cell and formation of membrane-bound bodies containing intact organelles (apoptotic bodies), and phagocytosis of these bodies by resident cells are the major structural changes associated with apoptosis. Biochemically, activation of a nonlysosomal endonuclease is a cardinal feature of this mode of cell death. Several genes have been implicated in the execution of apoptosis. A signal transduction mechanism is suspected to regulate the phenomenon. Although apoptosis is widely considered as an adaptive response to physiological or near physiological stimuli, several noxious agents can initiate the reaction and thus it is often a toxicological response.