Histological study of stem-like cells in human colon adenocarcinoma at different stages of the disease.

The presence of stem-like cells in tumors reflects the invasive character of the disease; however, their identification is controversial. We investigated the distribution of CD133, CD44 and CD24 using histological sections and tissue microarrays (TMAs) of human colon adenocarcinoma obtained from patients with and without lymph node metastases and/or liver metastases. Immunohistochemical staining was combined with nuclear staining and evaluated quantitatively using image analysis software. Sections of normal colon mucosa, the primary tumor, lymph node, and liver also were analyzed qualitatively and compared to the quantitative method, which was more accurate. In most tissues, the expression of CD44 and CD24 was relatively low compared to CD133, with some variations. CD133 also was expressed in the normal colon mucosa and to a lesser degree in normal hepatic parenchyma. Liver metastases exhibited significantly greater CD133 staining compared to normal colon mucosa, primary tumor and lymph node metastases. Moreover, lymph node metastases obtained from patients with liver metastases expressed significantly greater CD133 staining than those obtained from patients without liver metastasis. Our data suggest that CD133 expression in lymph node metastases may be of value for prognosis of the disease.

Promotion of angiogenesis in tissue engineering: developing multicellular matrices with multiple capacities.

One of the aims of tissue engineering is to be able to develop multi-tissue organs in the future. This requires the optimization of conditions for the differentiation of multiple cell types and maintenance of the differentiated phenotype within complex engineered tissues. The goal of this study was to develop prototype tissue engineered matrices to support the simultaneous growth of different cell types with a particular focus on the angiogenic process. We examined two different matrix compositions for the promotion of blood vessel and tube formation. A fibrin-based matrix with the addition of a combination of growth factors supported vascular growth and the invasion of inflammatory cells. Using this fibrin matrix, in combination with a collagen-based hydrogel, a simple in vitro model of the cornea with adjacent sclera was developed that was complete with innervation and vascular structures. In addition, we showed that collagen-based matrices were effective in delivering mononuclear endothelial progenitor cells to ischemic tissue in vivo, and allowing these cells to incorporate into vascular structures. It is anticipated that with further development, these matrices have potential for use as delivery matrices for cell transplantation and for in vitro study purposes of multiple cell types.

Three-dimensional type I collagen cell culture systems for the study of bone pathophysiology.

Three-dimensional (3-D) type I collagen cell culture systems composed of reconstituted collagen fibres are able to support short- and long-term growth of various cell types, including cancer cell lines, endothelial cells, endometrial cells, hepatocytes, osteoblasts and fibroblasts and to sustain or even enhance cell differentiation, in vitro. In addition, 3-D culture systems have been successfully used in the investigation of complex biological processes, such as angiogenesis, wound healing, tumour invasion and metastasis. The latter suggested that 3-D culture systems have the potential to simulate cell-cell interactions, which take place in tissues under physiological and pathophysiological conditions. This review focuses on the investigational use of 3-D collagen cell culture systems in bone physiology and the pathophysiology of skeletal metastasis.

A collagen-based scaffold for a tissue engineered human cornea: physical and physiological properties.

Stabilized collagen-glycosaminoglycan scaffolds for tissue engineered human corneas were characterized. Hydrated matrices were constructed by blending type I collagen with chondroitin sulphates (CS), with glutaraldehyde crosslinking. A corneal keratocyte cell line was added to the scaffolds with or without corneal epithelial and endothelial cells. Constructs were grown with or without ascorbic acid. Wound-healing was evaluated in chemical-treated constructs. Native, noncrosslinked gels were soft with limited longevity. Crosslinking strengthened the matrix yet permitted cell growth. CS addition increased transparency. Keratocytes grown within the matrix had higher frequencies of K+ channel expression than keratocytes grown on plastic. Ascorbic acid increased uncrosslinked matrix degradation in the presence of keratocytes, while it enhanced keratocyte growth and endogenous collagen synthesis in crosslinked matrices. Wounded constructs showed recovery from exposure to chemical irritants. In conclusion, this study demonstrates that our engineered, stabilized matrix is well-suited to function as an in vitro corneal stroma.

Sealing of polyester prostheses with autologous fibrin glue and bone marrow.

The purpose of this study was to develop a sealing technique for polyester prosthetic grafts able to promote healing and reduce intimal hyperplasia. The porcine experimental model was aortoiliac bypass with a 6-mm diameter knitted polyester prosthetic graft implanted for 14 and 90 days. Animals were divided into three groups according to sealing technique as follows: pre-clotting with blood (group I, n = 12), sealing with autologous fibrin glue (group II, n = 14), and sealing with autologous fibrin glue and bone marrow cells (group III, n = 16). Feasibility and quality of sealing were evaluated by scanning electron microscopy prior to implantation and by assessment of blood loss. After removal, prostheses were cut into three segments comprising the proximal anastomosis, midsection, and distal anastomosis. Pieces were fixed, embedded in paraffin, and serially sectioned for histologic study. Histological study focused on the degree of stenosis and hyperplasia of the neointima of each prosthesis. The results of this short-term study indicate that sealing of polyester vascular prosthetic grafts with autologous fibrin glue and bone marrow cells is effective in reducing intimal hyperplasia. However further study will be needed to assess long-term healing.

Functional human corneal equivalents constructed from cell lines.

Human corneal equivalents comprising the three main layers of the cornea (epithelium, stroma, and endothelium) were constructed. Each cellular layer was fabricated from immortalized human corneal cells that were screened for use on the basis of morphological, biochemical, and electrophysiological similarity to their natural counterparts. The resulting corneal equivalents mimicked human corneas in key physical and physiological functions, including morphology, biochemical marker expression, transparency, ion and fluid transport, and gene expression. Morphological and functional equivalents to human corneas that can be produced in vitro have immediate applications in toxicity and drug efficacy testing, and form the basis for future development of implantable tissues.

Fat- and bone marrow-impregnated small diameter PTFE grafts.

OBJECTIVES: to evaluate an alternative and simple technique which consists in impregnation of a synthetic prosthesis with either autogenic omental fat or bone marrow. These tissues have been selected based on previous works and because they contain multiple cellular and extracellular compounds with biological healing properties (i.e. angiogenesis, endothelialisation, etc.). DESIGN: PTFE grafts of Group 1 were impregnated with fatty tissue, those of Group 2 with bone marrow and those of Group 3 served as controls. MATERIALS: nine mongrel dogs divided among these three groups. PTFE grafts are 3 mm in diameter. METHODS: in each animal, both iliac arteries were submitted to an end-to-side ilio-iliac bypass. At 3 months, pathology assessment was performed. RESULTS: group 1: all grafts were thrombosed and intimal hyperplasia was found occluding the anastomotic sites. Group 2: 4/6 grafts were patent and their mid-portion presented a thin neointima which did not totally cover the anastomotic sites. Group 3: 2/5 grafts were patent and their mid-portion as well as the anastomotic sites were covered with neointima which was hyperplastic in some areas. CONCLUSIONS: addition of bone marrow cells may contribute to improve the quality of the healing process.

Development of a flow simulator to study haemodynamic behaviour of natural and artificial blood vessels under physiologic flow conditions.

A new computer-controlled flow simulator has been designed to study the haemodynamic behaviour of natural and artificial blood vessels under physiologic flow conditions. The simulator can generate well characterized and fully developed laminar flow properties. It includes a unique perfusion case that imposes an axial tension on the vessel segment, and a commercial programmable pump to reproduce pulsatile flow rates. Response to high frequency commands was greatly attenuated and displayed a frequency dependent phase angle. Thus, for complex pulsating flow rates containing different frequency components, the system response was significantly distinct from the command. To reproduce physiologic waveforms, the transfer function of the whole system was determined for different amplitudes and frequencies of flow rate excitations. Each input command was compared to the measured flow rate, and the values of the gain and phase angle were evaluated. If the desired flow rate was composed of a sum of n sine wave components, each has a frequency fj and an amplitude Aj, a corrected command signal was then reconstructed by amplifying the attenuated components and advancing those lagged in time. The corrected signal was finally applied as the new command to the pump. The results showed an excellent agreement with physiologic waveforms. Examples of different pulsatile flow experiments to investigate the effects of frequency, pressure, and wall elasticity are presented.

Laparoscopic end-to-end aortobifemoral bypass with reimplantation of the inferior mesenteric artery. An experimental study.

BACKGROUND: Colic ischemia is a serious complication that can occur after abdominal aortic surgery. It has been described in two patients after laparoscopic aortic surgery. The goal of the current experiment was to determine the feasibility of inferior mesenteric artery (IMA) reimplantation during laparoscopic aortobifemoral bypass (LAFB). METHODS: Six piglets were submitted to the laparoscopic approach according to the "apron" technique previously described. The infrarenal aorta was clamped and an LAFB was performed using a dacron graft. The IMA was reimplanted in the body of the graft with a running 5-0 polypropylene suture. RESULTS: Mean operation and dissection times were 282.5 min (range, 270-310 min) and 123 min (range, 110-140 min), respectively, with a mean blood loss of 108 ml (range, 80-150 ml). Aortic clamping and anastomotic times were 123 min (range, 110-135 min) and 33 min (range, 24-45 min), respectively. The IMA reimplantation took 55 min (range, 45-70 min). At autopsy, all anastomoses were patent with no stenosis nor leak. CONCLUSION: Laparoscopic IMA reimplantation during laparoscopic aortobifemoral bypass is feasible.

Peroxovanadium compounds as inhibitors of angiogenesis.

Angiogenesis is a complex process that involves the activation of endothelial cells through the triggering of several intracellular signaling pathways including those involving tyrosine phosphorylation. In the present study, we analyzed the angiogenic properties of two phosphotyrosyl phosphatase (PTP) inhibitors that are composed of a peroxovanadium core containing different ancillary ligands. In cell monolayer and 3D culture systems examined in this study, the administration of potassium bisperoxo(1,10-phenanthroline)oxovanadate(V) [bpV(phen)] or potassium bisperoxo(pyridine-2-carboxylato)oxovanadate(V) [bpV(pic)], but not oxovanadiums, interfered markedly with endothelial cell growth, organization, and terminal differentiation. This effect was dependent upon both the compound's dose and the nature of the ancillary ligand. Rat aortic ring assay showed a significant inhibition by low dose of bpV(phen) on cell migration. In addition, a chick embryo angiogenesis assay demonstrated that bpV(phen) is a potent inhibitor of angiogenesis. Among PTP inhibitors, bpV(phen) had powerful angiostatic properties at a low concentration.

Endothelial cells exposed to erythrocytes under shear stress: an in vitro study.

After injury and vascular replacement, endothelial cell recovery is limited and could lead to thrombosis. Seeding small diameter vascular prosthesis with endothelial cells has been proposed to fulfil cell lining and improve surface hemocompatibility. However, detachment of seeded cells occurs following implantation. Previous in vitro studies have looked at the fluid shear stress as a major cause of cell detachment. To our knowledge, the role of erythrocyte collisions has not been investigated. The present in vitro study aims at investigating whether endothelial cell adhesion depends on (i) the presence of erythrocytes in flow and (ii) the latent culture period (1, 24 and 48 h) between seeding and exposure to flow. Endothelial cells were exposed to culture media containing different erythrocyte concentrations using a steady laminar flow of 1350 ml min(-1) in a parallel plate flow chamber. Endothelial cell morphology in dynamic conditions was quantified and compared to that in static conditions. The projected area of cells were mostly found smaller under dynamic than static conditions, particularly at a wall shear stress of 23 dyn cm(-2). Cells from the 1 h latent culture period were oriented parallel to the flow axis and were more elongated than under static conditions. Conversely, endothelial cell shape was slightly modified when either the latent period or the wall shear stress was increased. Disparate orientation was observed on confluent endothelial cells (24-48 h latent period) exposed to shear stress with or without erythrocytes. Increasing fluid viscous forces due to erythrocytes play a critical role on the behaviour of freshly seeded endothelial cells upon exposure to blood flow.

Chemical inactivators as sterilization agents for bovine collagen materials.

The use of collagen as a biomedical implant raises safety issues with regard to viruses and prions. Specific chemical agents that inactivate prion infectivity could be applied to collagen implants. The physicochemical changes and the in vitro and in vivo biocompatibility of collagen treated by formic acid (FA), trifluoroacetic acid (TFA), tetrafluorethanol (TFE), and hexafluoroisopropanol (HFIP) were investigated. In addition, the effects of these treatments on nucleic acids incorporated in collagen were analyzed. The molecules of FA and, more important, of TFA remained within collagen. FA, TFA, and HFIP treatments modify the secondary structure of collagen, as shown by Fourier transform infrared spectroscopy, while TFE does not. Differential scanning calorimetry measurements showed a decrease in the denaturation temperature compared to untreated collagen. However, resistance to collagenase was modified only after HFIP treatment. In vitro, cell growth was not impaired; in vivo, implants induced a temporary inflammatory reaction that was prolonged with TFA and HFIP treatments. TFE and FA-treated collagen were thoroughly infiltrated by fibroblasts. On the other hand, FA and TFA resulted in extensive depurination of nucleic acids while HFIP and TFE did so to a lesser degree. Among the investigated chemical scrapie inactivators, FA treatment could offer a safe and biocompatible collagen-derived material for biomedical use.

Stromal fibroblasts are required for PC-3 human prostate cancer cells to produce capillary-like formation of endothelial cells in a three-dimensional co-culture system.

The outcome of patients with prostate cancer is largely dependent on the ability of the primary tumor for local invasion, angiogenesis and metastasis. To better understand the cell-cell interactions that participate in prostate cancer neovascularization, we have developed a novel three-dimensional co-culture system. Capillary-like structures were induced in fibrin gel in which collagen gels containing fibroblasts and/or PC-3 human prostate adenocarcinoma cells were sandwiched together. In the presence of collagen-embedded fibroblasts, angiogenesis apparently occurred, while endothelial cells did not survive when only PC-3 cells were embedded in collagen. In contrast, when PC-3 cells were combined with fibroblasts in collagen gel an enhanced formation of capillary-like structure formation was noted, particularly using FGF-2-supplemented medium. In addition, we observed morphological evidence of PC-3 cells and fibroblast invasion into fibrin using this system. Therefore, we conclude that fibroblasts apparently play an important role in angiogenesis and tumor invasion. Furthermore, this novel three-dimensional co-culture is apparently a promising model for studying de novo angiogenesis and tumor invasion in vitro.

Biological molecule-impregnated polyester: an in vivo angiogenesis study.

Specific extracellular matrix molecules and growth factors (GFs) with angiogenic properties could be combined with biomaterials to enhance angiogenesis and subsequently tissue ingrowth through the wall of the porous structure. In this study, composite fibrin matrices containing hyaluronic acid (HA), fibronectin (FN) and/or fibroblast growth factor-1 (FGF-1), FGF-2 and an endothelial cell growth supplement (ECGS) were adsorbed onto Dacron meshes which were then implanted subcutaneously in mice. The release from the implants and the tissue distribution of implanted GFs were determined in vivo using radiolabelled FGF-2. Angiogenesis was quantified by counting the number of capillaries present in each Dacron histological serial section. Radiolabelled GF was rapidly released from matrices and was absent from them by day 28. A very low percentage of the implanted radiolabelled GFs was found in the kidneys and livers of the animals. The number of microvessels formed within fibrin-impregnated samples was increased in the presence of HA and ECGS at 14 d and of FN and ECGS or FGF-2 at 28 d. FGF-1 had no direct effect on angiogenesis in our model. These results indicate that enhancement of vascularization within prosthesis mesh may be achieved by using fibrin as a support for angiogenic molecules such as HA, FN and FGFs.

Human endometrial cells cultured in a type I collagen gel.

OBJECTIVE: To elaborate in vitro conditions that enable epithelial and stromal cells of human endometrium to grow within a gel of collagen. STUDY DESIGN: Primary cultures of epithelial cells derived from human endometrial biopsies were dissociated and mixed with a collagen solution, and the gel was allowed to form at physiologic pH. Control cultures were grown in plastic dishes. DNA replication was assessed by 3H-thymidine incorporation, morphology by histology and cell characterization by monoclonal antibodies to cytokeratins. RESULTS: Cells grown on plastic dishes exhibited a typical monolayer arrangement, and replication was increased 1.6-fold by the addition of stromal cell-conditioned medium (50% vol/vol). After a two- to three-week period of culture within the collagen gel in the presence of either stromal cells or stromal cell-conditioned medium, epithelial cells formed circular arrangements of cuboidal to columnar cells with open lumina resembling glands. These glandlike structures were cytokeratin positive as assessed by immunohistochemistry, thereby confirming their epithelial nature. CONCLUSION: The development of differentiated epithelial structures in a three-dimensional gel provides a promising method of studying various biochemical and cellular interactions of eutopic and ectopic endometrium.

Vascugraft microporous polyesterurethane arterial prosthesis as a thoraco-abdominal bypass in dogs.

In their progression towards clinical acceptance, any new synthetic vascular grafts under development must undisputedly prove that the chemistry and structure used in the construction of the prostheses is safe and that their biocompatibility and performance as arterial substitutes are satisfactory without degradation or weakening of the device. This study was conducted to evaluate the safety of the microporous polyesterurethane Vascugraft by investigating its biocompatibility in terms of cellular proliferation, morphology and adhesion of human fibroblasts on virgin and blood-soaked Vascugraft prostheses, and its performance in vivo as a large calibre graft in a canine thoraco-abdominal bypass model for periods of implantation ranging from 4 h to 6 months. After 3 d incubation, better cell proliferation and adhesion were observed on blood-soaked Vascugraft than on a non-porous polyurethane graft, Mitrathane, and two other polytetrafluorethylene prostheses, Impra and Goretex. Furthermore, no leachable cytotoxic contaminants were released from the prostheses. In vivo, the Vascugraft has demonstrated a good performance with the development of an endothelialised internal capsule at both anastomoses 2 weeks after implantation, reaching the medial portion of the graft at 4 months. During this period, the prostacyclin I2/thromboxane A2 ratio increased and was higher than 1.0 at 2 months. In addition, the Vascugraft exhibited low surface thrombogenicity in terms of radiolabelled platelets and fibrin deposited. Chemically, as revealed by ESCA and FTIR analyses, a slight decrease in carbonate content was observed on the external surface of the Vascugraft during the early post-implantation periods. Breaks in the microfibrous structure were also observed at 4 and 6 months, occurring mainly in the anastomotic regions and believed to be stress-related. This study shows that the polymer used in the Vascugraft is biocompatible in terms of fibroblast proliferation and promotes fair healing characteristics. However, the chemical and structural surface modifications noted in this study are disturbing and question the total inocuity of the Vascugraft. Consequently, the decision by B. Braun Melsungen AG to end this project is both highly conscientious and professional.

Experimental carbon dioxide pulmonary embolization after vena cava laceration under pneumoperitoneum.

The potential for pulmonary embolization following major venous laceration occurring during laparoscopic surgery has never been evaluated. Five anesthetized dogs were hemodynamically monitored with an arterial line and Swan-Ganz catheter. Observation by transesophageal echocardiography (TEE) allowed comparison with pulmonary artery pressure (PAP) recording. Under pneumoperitoneum, a 1-cm venotomy was performed in the infrarenal vena cava and a total of 11 events were evaluated upon unclamping the venotomy. These results were compared with intravenous (i.v.) bolus injections of 15 cc of CO2 (15 events) and of 100 cc of CO2 (12 events). The animals were maintained euvolemic. In 2 out of the 11 (18%) events which followed unclamping the venotomies, a few CO2 bubbles were seen in the right heart cavities. However, the quantity of gas was much less important than that seen after i.v. injection of 15 cc and 100 cc of CO2. There was no significant elevation of the PAP from pre-event values after venotomy or after i.v. injection of 15 cc of CO2. However, there was a significant difference (P < 0.05) when these results were compared to the PAP values recorded after i.v. injection of 100 cc of CO2. No dog died after these episodes of embolization. Massive i.v. injection of CO2 (> 300 cc) led to appearance of gas bubbles in the left heart cavities and death. This experiment suggests that caution should be exerted when laparoscopic surgery is performed beside large veins. Nevertheless, the observation that no gas embolism occurred in 82% of the cases after unclamping venotomies was unexpected. In contrast, many more gas bubbles were detected in the right heart after i.v. injection of only 15 cc of CO2. TEE is a more sensitive indicator of pulmonary embolization than elevation of PAP.

Poly(ethylene glycol)-serum albumin hydrogel as matrix for enzyme immobilization: biomedical applications.

Poly(ethylene glycol)-albumin hydrogels were implanted in mice in subcutaneous position to study their biocompatibility. After one month of implantation, the fibrous capsule formed around the implant was thin and the inflammatory tissue was limited. Acid phosphatase (AP) was selected to evaluate the hydrogel as matrix for enzyme immobilization. AP-hydrogels were prepared using activated PEG (PEGa) of different molecular weights (M.W. 4,600 to 20,000) to evaluate the effect of the matrix composition on the activity of AP. The apparent Km of the immobilized AP was 16 to 20 times higher than the Km of the soluble enzyme. The apparent Km value decreases with the increase of the chain length of the PEGa used. This can be correlated to an increase in the hydrogel porosity. The operational stability of the AP was markedly improved after immobilization by 110 to 160 times according to the PEGa molecular weight involved. Also, asparaginase (ASNase) was immobilized in PEGa (M.W. 10,000)-albumin-hydrogel as a model for in vivo bioreactor. ASNase hydrogels were implanted in the peritoneal cavity of rats; 7 days later, 75% of the initial enzyme activity were retrieved.

Three-dimensional type I collagen gel system for the study of osteoblastic metastases produced by metastatic prostate cancer.

We developed a three-dimensional type I collagen gel cell culture system that allows coculturing of human MG-63 osteoblast-like cells and various human cancer cells. Inoculation of human PC-3 metastatic prostate cancer cells into this type I collagen gel containing human MG-63 osteoblast-like cells produced an osteoblastic-like reaction that presented as an increased number of MG-63 cells and increased density of type I collagen around MG-63 cells adjacent to inoculated PC-3 cells by microscope analysis. Under identical experimental conditions, inoculation of cell-free medium, human KLE endometrial adenocarcinoma cells, and Calu-1 lung cancer cells did not produce this blastic-like reaction. In situ hybridization documented the uniform expression of insulin-like growth factor I (IGF-I) and of urokinase-type plasminogen activator (uPA) mRNA in MG-63 and PC-3 cells separately cultured in this substrata. The uniform expression of uPA was also documented by immunocytochemistry using a monoclonal and a polyclonal antihuman uPA antibody. The relative expression of uPA was higher in PC-3 cells than in MG-63, KLE, and Calu-1 cancer cells. We conclude that this novel cell culture system may become a useful model to study the pathophysiology of the osteoblastic reaction in vitro.

Heparin-fibroblast growth factor-fibrin complex: in vitro and in vivo applications to collagen-based materials.

Biological molecules such as fibrin and growth factors could have interesting features to design bioactive biomaterials and particularly collagen-based materials used as connective tissue replacement. Different combinations of fibroblast growth factor (FGF) and heparin complexed to fibrin were analysed. In vitro, FGF bound to matrix was rapidly, but partially released, specifically with heparin. Heparin concentrations were progressively equilibrated between matrix and medium. DNA replication of fibroblasts grown either on or within fibrin matrices was increased in the presence of both FGF and high doses of heparin incorporated in fibrin. Subcutaneous implantations of collagen sponges impregnated with composite fibrin matrices showed qualitative and quantitative tissue ingrowth within the sponges. The uncross-linked collagen of fibrin-impregnated sponges swelled after implantation. The resulting fibroblast-infiltrated tissue resembled a normal dense connective tissue that was observed particularly in the presence of high doses of heparin and FGF incorporated in fibrin.