T. V. Anilkumar

The nature of cytotoxic drug-induced cell death in murine intestinal crypts.

The nature of cell death in murine small intestinal crypts caused by potentially lethal doses of four classes of cancer chemotherapeutic agents was studied. The drugs used were cytosine arabinoside, vincristine, adriamycin and nitrogen mustard. The compounds readily induced massive cell death in the proliferating compartment of the crypt. In each case, cell death was apparent within an hour, and the incidence of dead cells peaked during the following 4-8 h. By 24 h, little damage was discernible in the crypt systems. Remarkably, dead cells or dead cell fragments were phagocytosed rapidly (within about 1 h) by neighbouring healthy enterocytes. When examined by light microscopy, transmission electron microscopy and scanning electron microscopy, the dead cells showed the characteristic features of having succumbed to an apoptotic mode of cell death without any trace of cell and organelle oedema characteristic of necrosis. The study suggests that cell death by apoptosis operates even when the cells are exposed to severe pathological perturbation and that the phenomenon is not solely a process which operates in response to either physiological stimuli or to mild physical or chemical trauma.

Long term tissue response to titanium coated with diamond like carbon

Diamond like carbon (DLC) coatings were deposited on to Ti substrates by plasma enhanced chemical vapor deposition technique. Ti and DLC/Ti samples were implanted in skeletal muscle of rabbits. The samples were explanted after 1, 3, 6 and 12 months and the tissue-cell interaction was studied. Our data indicate both DLC/Ti and bare Ti to be compatible with skeletal muscle.

Advantages of hyaluronic acid as a component of fibrin sheet for care of acute wound

Skin injury is followed by accumulation of a fibrin based provisional matrix which normally drives the process of wound repair. Exogenous fibrin with extra cellular matrix (ECM) components can also favor the wound healing process. In a preliminary study we found that lyophilized fibrin sheet (FS) arrest bleeding from rabbit skin wound but it remained dry during the repair period and did not accelerate the healing process better than untreated control. In the current study, hyaluronic acid (HA) was incorporated into FS and the resultant HA-FS promoted water retention and improved wound healing process. Gross-morphology, histopathology and histomorphometry were employed to compare qualitative and quantitative difference of wound healing in treated group against controls. In experimental sites (HA-FS), re-epithelialization was completed by 14 days with neo-vascularization and deposition of wavy bundles of collagen in the treated sites. Rate of healing process was different in treated and untreated wounds and most striking difference was the appearance of appendages, sebaceous gland and hair follicle at some locations in HA-FS treated sites. Therefore, HA with fibrin can create an effective wound care matrix which promotes water retention and wound healing potential.

An analysis of apoptosis in lymphoid organs and lupus disease in murine systemic lupus erythematosus (SLE)

Apoptosis is a programmed cell death process that helps to regulate both T cell and B cell development. In this study, we have investigated the levels of apoptotic death in cells of the thymuses and spleens (white matter) of autoimmune MRL‐lpr/lpr mice with progressive lymphadenopathy and SLE disease activity; we also examined the renal pathology in these animals. Fas is a cell surface receptor, which when activated initiates the sequence of events that lead to apoptosis. In MRL‐lpr/lpr mice Fas is defective, so the competency for apoptosis may be reduced. In young animals of advancing age the thymuses enlarged until in 5‐month‐old females the average weight was three times that at 1 month, and spleen and kidney weights also increased in size disproportionately. At light microscope level apoptotic cells in tissue sections were counted using both routine eosin and haematoxylin staining (to identify them by their morphology) and in situ end‐labelling of cells with DNA strand breaks; their presence was further confirmed by electron microscopy. As the mice aged, the numbers of apoptotic cells in thymic cortex, thymic medulla and spleen white pulp areas reduced significantly (P < 0.01–0.001), whereas in BALB/c normal controls they increased significantly (P < 0.05). These changes were coincident with the development of severe lupus, whose activity was assessed by measuring serum anti‐ssDNA and anti‐dsDNA antibody titres and urinary protein (albumin) level which were elevated significantly by 5 months of age (P < 0.001 for both ssDNA and dsDNA and P < 0.01 for urine albumin) compared with their younger counterparts. Thus, lymphoid organ enlargement, decrease in apoptotic indices, elevated serum anti‐ssDNA and anti‐dsDNA antibody levels, and impaired renal function coincided with the onset and severity of lupus disease in lpr mice. It seems likely that there is a causal relationship between defective deletion of autoreactive lymphoid cells, imperfect Fas‐mediated apoptosis and development of murine SLE.

Transforming growth factor‐α immunoreactivity in a variety of epithelial tissues

Abstract. The immunohistochemical expression of transforming growth factor‐alpha (TGFα) has been examined in a range of normal adult epithelial tissues from both man and rat using an anti‐hTGFα 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 TGFα 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 TGFα 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 TGFα immunoreactivity.

Porcine cholecyst–derived scaffold promotes full-thickness wound healing in rabbit

Graft-assisted healing is an important strategy for treating full-thickness skin wounds. This study evaluated the properties of porcine cholecyst–derived scaffold and its use for treating full-thickness skin wound in rabbit. The physical properties of cholecyst-derived scaffold were congenial for skin-graft application. Compared to a commercially available skin-graft substitute made of porcine small intestinal submucosa, the cholecyst-derived scaffold was rich in natural biomolecules like elastin and glycosaminoglycans. When used as a xenograft, it promoted healing with excess cell proliferation at early phases and acceptable collagen deposition in the later remodelling phases.

Biomaterial properties of cholecyst‐derived scaffold recovered by a non‐detergent/enzymatic method

Isolation procedures for the recovery of extracellular matrices (ECMs) from animal organs/tissues that are useful in regenerative medicine involve multiple sequential steps/stages including collection of the source organ at slaughter, their transportation to laboratory, decellularization, decontamination, stabilization, and sterilization. Most of these steps require extensive use of chemicals/reagents/enzymes which may also adversely affect the quality of the scaffold. With an effort to minimize the use of chemicals/reagents/enzymes, while extracting biomaterial-grade ECM from porcine cholecyst (gall bladder), we performed preisolation ex situ incubation of the organ in a stabilizing agent that also caused in situ crosslinking of tissue-components and delaminated the collagen-rich ECM from the tissue-layer beneath the mucosa. The physical, chemical, and biological properties of the isolated scaffolds were similar to that of a commercially available porcine small intestinal submucosa. The cholecyst-derived scaffold not only satisfied preclinical safety-test procedures such as cytotoxicity, local response, and endotoxin load but also showed the potential to promote healing of full-thickness skin wound in a rabbit model. The procedure was also suitable for isolating scaffolds from other hollow organs such as jejunum and urinary bladder. It was concluded that enzyme/detergent treatment may be an avoidable step while isolating biomaterial-grade scaffolds from hollow organs.

Biocompatibility and Immunophenotypic Characterization of a Porcine Cholecyst–derived Scaffold Implanted in Rats

Comparative histomorphological assessment of local response to implanted reference biomaterial, also called biocompatibility testing/evaluation, in an appropriate animal model is a widely practiced safety evaluation procedure performed on biomaterials before clinical use. Standardized protocols and procedures, originally designed for testing synthetic materials, available for the testing/evaluation do not account for the immunogenic potential of a candidate biomaterial. Therefore, it is appropriate to supplement the routine biocompatibility test reports with adjunct data that may provide insight into the immunogenic potential of candidate biomaterials, especially when testing biomaterials that are derived from mammalian sources. This article presents expanded safety evaluation data of a porcine cholecyst–derived scaffold (CDS) intended as a xenogeneic graft. The biocompatibility was tested in rat subcutaneous model in comparison with a reference material and the CDS was found biocompatible. However, when studied by immunohistochemistry and real-time reverse transcription polymerase chain reaction for the number and/or polarization of M1 macrophage, M2 macrophage, cytotoxic T-cell, helper T cell, TH1 cell, and TH2 cell, the CDS appeared to induce a differential local immunopathological tissue reaction despite the similarity in biocompatibility with the reference material. The adjunct data collected were useful for objectively assessing the safety of CDS as a xenograft.

The anti-proliferative effect of plasma from rats with acute fulminant hepatic failure

BACKGROUND During fulminant hepatic failure (FHF) metabolites normally cleared by the liver accumulate in the circulation and cause hepatic coma. It is believed that the plasma of FHF patients has an inhibitory effect on liver regeneration. Plasma exchange was used to study the effect of plasma collected from donor FHF rats on liver regeneration in two-thirds partially hepatectomized syngeneic animals. METHODS FHF and hepatic coma were induced in donors by administration of galactosamine at a dose of 1.85 g/kg. Plasma from donors in either grade-II or -IV coma was transfused by plasma exchange into partially hepatectomized animals 2h after resection. RESULTS The livers from donor animals showed evidence of oval cell activation 1-2 days after galactosamine, but differentiation of oval cells to hepatocytes did not occur before the development of coma. The plasma collected from animals in grade-IV coma totally abolished regeneration in the partially hepatectomized recipients. CONCLUSION These results support the hypothesis that metabolites present in the plasma during FHF inhibit liver regeneration.

A gold nanoparticle coated porcine cholecyst-derived bioscaffold for cardiac tissue engineering

Extracellular matrices of xenogeneic origin have been extensively used for biomedical applications, despite the possibility of heterogeneity in structure. Surface modification of biologically derived biomaterials using nanoparticles is an emerging strategy for improving topographical homogeneity when employing these scaffolds for sophisticated tissue engineering applications. Recently, as a tissue engineering scaffold, cholecyst derived extracellular matrix (C-ECM) has been shown to have several advantages over extracellular matrices derived from other organs such as jejunum and urinary bladder. This study explored the possibility of adding gold nanoparticles, which have a large surface area to volume ratio on C-ECM for achieving homogeneity in surface architecture, a requirement for cardiac tissue engineering. In the current study, gold nanoparticles (AuNPs) were synthesized and functionalised for conjugating with a porcine cholecystic extracellular matrix scaffold. The conjugation of nanoparticles to C-ECM was achieved by 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide/N-hydroxysuccinimide chemistry and further characterized by Fourier transform infrared spectroscopy, environmental scanning electron microscopy, energy dispersive X-ray spectroscopy and thermogravimetric analysis. The physical properties of the modified scaffold were similar to the original C-ECM. Biological properties were evaluated by using H9c2 cells, a cardiomyoblast cell line commonly used for cellular and molecular studies of cardiac cells. The modified scaffold was found to be a suitable substrate for the growth and proliferation of the cardiomyoblasts. Further, the non-cytotoxic nature of the modified scaffold was established by direct contact cytotoxicity testing and live/dead staining. Thus, the modified C-ECM appears to be a potential biomaterial for cardiac tissue engineering.