A novel method for the extraction and culture of progenitor stem cells from human peripheral blood for use in regenerative medicine.

Human peripheral blood (HPB) contains both circulating endothelial cells (CECs) and endothelial progenitor stem cells (EPCs), which may be suitable for use in regenerative medicine. There has been considerable interest in using these cells, but there is no "gold standard" technique for isolating these cells. The aim of this study was to characterize and compare a number of different extraction and culture techniques to develop a system to isolate and culture cells. EPC and CEC were isolated from HPB using either Histopaque-1077 or Lymphoprep. The two isolation methods were compared for the number of cells isolated, cell metabolism, and RNA expression. Both isolations produced viable cells and were comparable. The tissue culture method employed does have a significant effect on the cell population with regard to medium choice, fetal bovine serum concentration, and surface modification of the culture surface. In conclusion, it can be seen that although this study and previous work can suggest a basis for culture, further work to develop an optimized and agreed "gold standard" culture regime for EPC from HPB is required to maximize the potential of this source of cells for regenerative medicine and to translate its clinical use in the future.

The long-term stability in gene expression of human endothelial cells permits the production of large numbers of cells suitable for use in regenerative medicine.

Tissue engineering has been conducted in the study of cardiovascular grafts for many years. Many obstacles have been overcome in this rapidly changing field, but one difficulty has remained until now: the large number of endothelial cells (ECs) needed for seeding the inner layer of bypass graft. Recent advances in endothelial progenitor cell (EPC) isolation and culture techniques have increased the interest in genetic studies. Despite these advances in EPC studies, the "gold standard" for the seeding of tissue engineering constructs or hybrid grafts remains mature human umbilical vein endothelial cells (HUVECs). This study investigates the ability of HUVECs to be expanded in culture to provide sufficient cells for graft seeding. The levels of gene expression of key genes are then examined to ensure that these cells retain the EC phenotype. This study demonstrates that HUVECs may be cultured for up to 12 passages without alteration in phenotype. Subsequent passage numbers are sufficiently similar to those preceding them to allow cells of different passages to be mixed without gene expression anomalies.

Inhibition of the p38 MAPK pathway sensitises human colon cancer cells to 5-fluorouracil treatment.

Colorectal cancer is the third most common cause of cancer-related deaths in the Western world. 5-Fluorouracil (5-FU) based chemotherapeutic regimes have been the mainstay of systemic treatment for disseminated colorectal cancer for many years. However, it only produces a 25% response rate due to the drug-resistance. The mitogen-activated protein kinase (MAPK) pathway is involved in the anti-apoptotic process; its activation provides cancer cells with a survival advantage to escape the apoptotic challenge. This study assessed whether the p38 MAPK pathway is involved in 5-FU resistance in colorectal cancer cells. 5-FU only or 5-FU combined with a p38 MAPK pathway inhibitor (SB203580) was used to treat 5-FU-resistant colorectal cancer cells. The effect of the treatment on cell viability, death and caspase activities was assessed. Western blotting was used to investigate the responses of apoptosis-related proteins following the treatment. Results showed that p38 MAPK inhibitor significantly increased colorectal cancer cell sensitivity to 5-FU. SB203580 in combination with 5-FU significantly reduced cell viability (P<0.01), and increased cell death and cellular caspase activity (P<0.01). Western blotting data revealed that SB203580 sensitises cancer cells to 5-FU due to an increase in Bax expression. These findings suggest that p38 MAPK is involved in cancer cell survival, and that the inhibition of p38 MAPK can enhance 5-FU to kill colorectal cancer cells.

Haemodynamic regulation of gene expression in vascular tissue engineering.

Synthetic grafts, namely expanded polytetrafluoroethlene (ePTFE) and poly(ethylene terephthalate) (Dacron), used for cardiovascular bypass surgery are thrombogenic. Lining the inner lumen ("seeding") of synthetic grafts with endothelial cells (ECs) increases patency rates similar to those of autologous grafts (e.g. saphenous vein). The major drawback with seeding grafts is the retention of cells present on the graft after implantation in vivo, where large portions of cell wash off. Preconditioning the seeded EC monolayer with shear stress has been shown to promote the reorganisation of the EC cytoskeleton and production of extracellular matrix, resulting in higher EC retention after exposure to blood flow. Vascular ECs have a number of essential and complex roles. ECs synthesise and secrete vasoconstrictors, vasodilators, growth factors, fibrinolytic factors, cytokines, adhesion molecules, matrix proteins and mitogens that modulate many physiological processes such as wound healing, hemostasis, vascular remodelling, inflammatory and immune responses. Vascular cells in vivo are exposed to hemodynamic forces created by the pulsatile flow of blood through the vessel. Due to their unique anatomical position, ECs are constantly exposed to shear stress forces and allow the vessel wall to adapt to changes by modulating EC structure and function. This review describes the mainly in vitro and in vivo studies used to define the molecular role hemodynamics have in vascular disease and its usage in developing tissue engineered vascular bypass grafts.

Pretreatment with insulin-like growth factor I protects skeletal muscle cells against oxidative damage via PI3K/Akt and ERK1/2 MAPK pathways.

Oxidative stress has an important role in the pathogenesis of many muscle diseases. The major contributors to oxidative stress in muscle tissue are reactive oxygen species such as oxygen ions, free radicals, and peroxides. Insulin-like growth factor I (IGF-I) has been shown to increase muscle mass and promote muscle cell proliferation, differentiation, and survival. We, therefore, hypothesized that IGF-I might also be cytoprotective for muscle cells during oxidative stress. Exogenous hydrogen peroxide (H(2)O(2)) was used to induce oxidative stress/damage in two types of skeletal muscle cells. Apoptotic pathways were assessed after the oxidative damage and the effects of IGF-I on oxidative stress in muscle cells were examined. Different IGF-I sub-pathways were analyzed with measurement of the expression of pro-and anti-apoptotic proteins. It was found that H(2)O(2) diminishes muscle cell viability and induces a caspase-independent apoptotic cell death. Pretreatment with IGF-I protects muscle cells from H(2)O(2)-induced cell death and enhances muscle cells survival. This effect appears to result from the promotion of the anti-apoptotic protein, Bcl2. Further investigation shows that protection is via an IGF-I sub-pathway: PI3K/Akt and ERK1/2 MAPK pathways. Protecting muscle cells from oxidative damage presents a potential application in the treatment of the muscle wasting, which appears in many muscle pathologies including Duchenne muscle dystrophy and sarcopenia.

In vitro small intestinal epithelial cell growth on a nanocomposite polycaprolactone scaffold.

Tissue engineering of the small intestine remains experimental despite worldwide attempts to develop a functional substitute for short bowel syndrome. Most published studies have reported predominant use of PLLA (poly-L-lactide acid)/PGA (polyglycolic acid) copolymer as the scaffold material, and studies have been limited by in vivo experiments. This lack of progress has inspired a fresh perspective and provoked further investigation and development in this field of tissue engineering. In the present paper, we exploit a relatively new nanocomposite of POSS (polyhedral oligomeric silsesquioxane) and PCL [poly(caprolactone-urea)urethane] as a material to develop porous scaffolds using a solvent casting/particulate leaching technique to fabricate porous scaffolds in different pore sizes and porosities. Scaffolds were characterized for pore morphology and porosity using scanning electron microscopy and micro-computed tomography. Rat intestinal epithelial cells were then seeded on to the polymer scaffolds for an in vitro study of cell compatibility and proliferation, which was assessed by Alamar Blue and lactate dehydrogenase assays performed for 21 days post-seeding. The results obtained demonstrate that POSS-PCL nanocomposite was produced as a macroporous scaffold with porosity over the range of 40-80% and pore size over the range of 150-250 microm. This scaffold was shown to support epithelial cell proliferation and growth. In conclusion, as a further step in investigating small intestinal tissue engineering, the nanocomposite employed in this study may prove to be a useful alternative to poly(lactic-co-glycolic acid) in the future.

IGF-I activates caspases 3/7, 8 and 9 but does not induce cell death in colorectal cancer cells.

BACKGROUND: Colorectal cancer is the third most common cancer in the western world. Chemotherapy is often ineffective to treat the advanced colorectal cancers due to the chemo-resistance. A major contributor to chemo-resistance is tumour-derived inhibition or avoidance of apoptosis. Insulin-like growth factor I (IGF-I) has been known to play a prominent role in colorectal cancer development and progression. The role of IGF-I in cancer cell apoptosis is not completely understood. METHODS: Using three colorectal cancer cell lines and one muscle cell line, associations between IGF-I and activities of caspase 3/7, 8 and 9 have been examined; the role of insulin-like growth factor I receptor (IGF-IR) in the caspase activation has been investigated. RESULTS: The results show that exogenous IGF-I significantly increases activity of caspases 3/7, 8 and 9 in all cell lines used; blocking IGF-I receptor reduce IGF-I-induced caspase activation. Further studies demonstrate that IGF-I induced caspase activation does not result in cell death. This is the first report to show that while IGF-I activates caspases 3/7, 8 and 9 it does not cause colorectal cancer cell death. CONCLUSION: The study suggests that caspase activation is not synonymous with apoptosis and that activation of caspases may not necessarily induce cell death.

Apoptosis and colorectal cancer: implications for therapy.

Colorectal cancer (CRC) is characterized by the partial suppression of apoptosis, which in turn gives tumours a selective advantage for survival and can cause current chemotherapy approaches to be ineffective. Recent progress in understanding the mechanisms of apoptosis in colorectal carcinogenesis has provided potential new targets for therapy. Here, we review recent studies of the regulation of apoptosis and its role in CRC initiation and progression, and we discuss the relationship between chemoresistance and the suppression of apoptosis. Recent progress in targeting apoptotic pathways and their regulators provide strategies for the exploration of novel therapies for CRC.

Degradation studies on biodegradable nanocomposite based on polycaprolactone/polycarbonate (80:20%) polyhedral oligomeric silsesquioxane.

The development of biocompatible polymers has greatly advanced the field of tissue engineering. Some tissues can be propagated on a nondegradable scaffold. Tissue such as cartilage, however, is a complex tissue in which the chondrocytes require their own synthesized extracellular matrix (ECM) to function. Suitable scaffolds for tissue engineering cartilage should provide mechanical strength and degrade at a similar rate to that of cell growth and ECM production. We have developed a biodegradable nanocomposite based on polycaprolactone and polycarbonate polyurethane (PCU) with an incorporated polyhedral oligomeric silsesquioxane (POSS) (POSS modified Poly(caprolactone/carbonate) urethane/urea). Previous work on POSS incorporated into PCU (POSS-PCU) has been shown to possess good mechanical strength, elasticity and resistance to degradation. This series of experiments involved exposing this polymer to a selection of accelerated degradative solutions for up to 8 weeks. The samples were analyzed by infra-red spectroscopy, scanning electron microscopy, X-ray microanalysis, contact angle analysis, and stress-strain mechanical analysis. Degradation of hard and soft segments of the nanocomposite was evident by infra-red spectroscopy in all conditioned samples. POSS nanocage degradation was evident in some oxidative/peroxidative systems accompanied by gross changes in surface topography and significant changes in mechanical properties. The hydrophobic polymer became more hydrophilic in all conditions. This biodegradable nanocomposite demonstrated steady degradation with protection of mechanical properties when exposed to hydrolytic enzymes and plasma protein fractions and exhibited more dramatic degradation by oxidation.This pattern may be potentially employed in tissue engineering scaffolds where controlled degradation and retained structural stability of the scaffold is required.

Inducing apoptosis of human colon cancer cells by an IGF-I D domain analogue peptide.

BACKGROUND: The resistance of tumour cells to apoptosis is a major contributor to the limited effectiveness of chemotherapies. Insulin-like growth factor I (IGF-I) has potential to protect cancer cells from variety of apoptotic challenges. This study was carried out to investigate the effect of a novel IGF-I receptor antagonist on apoptosis in colon cancer cells. RESULTS: We have designed and synthesised a novel antagonist of IGF-I receptor. The effect of this antagonist on human colon cancer cell proliferation was examined by a non-radioactive assay; the apoptosis was revealed by determining the activities of cellular caspases3/7, 8 and 9. The apoptosis pathways were investigated by examining the levels of pro-apoptosis proteins with Western blotting. Following 40 hours treatment with the novel antagonist peptide, colon cancer cell Caspase 3/7 activities increased 2-7 times; Caspase 8 activities increased 2-5 times and Caspase 9 increased 1.2-1.6 times. The proliferation of cancer cell was inhibited by 14-15%. The data showed that the antagonist induced colon cancer cell apoptosis and inhibited cancer cell proliferation. The different changes of Caspase 3/7, 8 and 9 activities suggested that the extrinsic pathways may play a major role in the antagonist peptide-induced apoptosis. CONCLUSION: This is the first report on this novel antagonist to induce human colon cancer cell apoptosis and inhibit cancer cell proliferation. These results suggest that IGF-I receptor antagonists may have the potential to be developed as a novel therapy for colon cancers in the future.

Integrins: a method of early intervention in the treatment of colorectal liver metastases.

The integrin family of cell surface receptors were principally thought to be involved in cell adhesion. Intense study has shown that these glycoproteins also regulate a diverse range of physiological processes. Inappropriate activation of integrins has been implicated in many pathological processes. Recent studies have shown that these molecules play a key role in the early stages of liver metastasis in colorectal cancer. In vivo experiments have demonstrated that integrins are involved in tumour cell targeting, arrest, adhesion and migration within the hepatic microcirculation. Indeed functional blocking of specific integrins has been shown to significantly impair these early stages of metastasis development. This review examines the current knowledge of integrin participation in this area and highlights the future therapeutic implications. Future targeted therapy against specific integrins would allow not only functional blocking but would provide the potential to deliver specific anti-cancer therapy.

Endothelial cell retention on a viscoelastic nanocomposite vascular conduit is improved by exposure to shear stress preconditioning prior to physiological flow.

In this study, endothelial cell (EC)-seeded nanocomposite grafts were preconditioned with 1-2 dynes/cm(2) in vitro to establish whether low shear stress resulted in improved cell adherence prior to physiological shear stress (15 dynes/cm(2)). Alamar blue cell viability was assessed. Polymerase chain reaction was conducted for glyceraldehyde-3-phosphate dehydrogenase, transforming growth factor beta-1 (TGFbeta-1), vascular endothelial growth factor receptor-1 (VEGFR-1), platelet EC adhesion molecule-1, and vascular endothelial growth factor receptor-2 (VEGFR-2). The Alamar blue results demonstrated improved cellular retention following preconditioning (P < 0.001). VEGFR-2 and TGFbeta-1 expression was up-regulated, and VEGFR-1 down-regulated following preconditioning. This investigation confirms previous findings regarding the potential benefits of preconditioning, and demonstrates that these benefits can be applied to ECs seeded on the nanocomposite employed. It also demonstrates further the suitability and potential of nanocomposite for future use in tissue-engineered cardiovascular devices.

Stem cells and cancer: an overview.

Definite evidence of the importance of cancer stem cells in the progression of cancer has now come to light. Key markers of these cells have been identified in many solid tumours as well as leukaemias. Specific studies modelling the tumour induction of specific cells isolated by surface antigens such as CD44 have demonstrated that these cells are not only present in tumours but that they are the key units in their tumourgenecity. These findings provide useful insight for disease progression, treatment and metastasis. The wide variety of proposed markers, and their similarity to endothelial progenitor cells found in angiogenesis, complicates these studies. Definite proof falls only in the induction of tumours in vivo. Here we review the developments in cancer stem cells and the markers that have been found for these cells.

Insulin-like growth factor binding protein-4 gene therapy increases apoptosis by altering Bcl-2 and Bax proteins and decreases angiogenesis in colorectal cancer.

Insulin-like growth factors are known to inhibit apoptosis and promote tumour angiogenesis. Previously we have shown that insulin-like growth factor binding protein-4 (IGFBP-4) gene therapy increased apoptosis and decreased mitosis in colon cancer. In this experiment we used HT-29 colon cancer cells to induce subcutaneous cancers in nude mice and administered either the mammalian expression vector with IGFBP-4 insert or vector only around the tumour site. Three weeks after gene transfer, tumours were harvested and expressions of Bax, Bcl-2 and IGF-I receptor in tumours were determined by Western blotting and immunofluorescence. Micro-vessel counting was performed by immunostaining with CD34 and von Willebrand antibodies. Results showed that tumours treated with IGFBP-4 gene had higher expression of Bax, lower expression of Bcl-2 and IGF-I receptor. Bcl-2 was localised to tumour cell cytoplasm while Bax was expressed both in the interstitial area and cytoplasm. IGFBP-4 treatment also decreased micro-vessel count in tumour tissues. Micro-vessels were mainly located in the periphery and interstitial area. This experiment shows that IGFBP-4 gene therapy increases tumour apoptosis via altering the expressions of Bcl-2 and Bax and decreasing the angiogenesis in colorectal cancer.

Biomaterials and scaffold design: key to tissue-engineering cartilage.

Cartilage remains one of the most challenging tissues to reconstruct or replace, owing to its complex geometry in facial structures and mechanical strength at articular surfaces in joints. This non-vascular tissue has poor replicative capacity and damage results in its functionally inferior repair tissue, fibrocartilage. This has led to a drive for advancements in tissue engineering. The variety of polymers and fabrication techniques available continues to expand. Pore size, porosity, biocompatibility, shape specificity, integration with native tissue, degradation tailored to rate of neocartilage formation and cost efficiency are important factors which need consideration in the development of a scaffold. The present review considers the current polymers and fabrication methodologies used in scaffold engineering for cartilage and postulates whether we are closer to developing the ideal scaffold for clinical application.

Addressing thrombogenicity in vascular graft construction.

Thrombosis is a major cause of poor patency in synthetic vascular grafts for small diameter vessel (< 6 mm) bypass. Arteries have a host of structural mechanisms by which they prevent triggering of platelet activation and the clotting cascade. Many of these are present in vascular endothelial cells. These mechanisms act together with perpetual feedback at different levels, providing a constantly fine-tuned non-thrombogenic environment. The arterial wall anatomy also serves to promote thrombosis as a healing mechanism when it has been severely injured. Surface modification of synthetic graft surfaces to attenuate the coagulation cascade has reduced thrombosis levels and improved patency in vitro and in animal models. Success in this endeavor is critically dependent on the methods used to modify the surface. Platelets adhere to positively charged surfaces due to their own negative charge. They also preferentially attach to hydrophobic surfaces. Therefore synthetic graft development is concerned with hydrophilic materials with negative surface charge. However, fibrinogen has both hydrophilic and hydrophobic binding sites-amphiphilic materials reduce its adhesion and subsequent platelet activation. The self-endothelializing synthetic graft is an attractive proposition as a confluent endothelial layer incorporates many of the anti-thrombogenic properties of arteries. Surface modification to promote this has shown good results in animal models. The difficulties experienced in achieving spontaneous endothelialisation in humans have lead to the investigation of pre-implantation in vitro endothelial cell seeding. These approaches ultimately aim to result in novel synthetic grafts which are anti-thrombogenic and hence suitable for coronary and distal infrainguinal bypass.

Quantitating therapeutic disruption of tumor blood flow with intravital video microscopy.

Vascular-disrupting agents (VDA) kill tumor cells by selectively disrupting blood circulation in tumors. In vivo analysis of this intensely studied class of anticancer agents is invaluable for preclinical assessment of pharmacodynamic end points and effective therapeutic windows. In this review, we consider the role of intravital video microscopy in measuring tumor vascular response to VDAs, the potential of which lies in the opportunity to quantitate specific variables and to obtain real-time information on how VDAs affect tumor microcirculation.

Tissue engineering of small intestine--current status.

Short bowel syndrome (SBS) has always posed a great threat to patients and has been one of the biggest challenges for doctors due to its high morbidity and mortality. So far, parenteral nutrition (PN) and small bowel transplantation remain the only viable therapeutic options. However, sepsis and liver failure associated with PN and limited availability of the donor organs and high graft rejection rates associated with transplantation have limited their use to a nonpermanent solution. Clearly, there is a need for an alternative therapy whereby increasing the absorptive surface area would help neonates and adults suffering from permanent intestinal failure. Techniques such as sequential intestinal lengthening are being explored in animal models with little success. Attempts to engineer small intestine since the late 1980s have achieved varying degrees of success in animal models with evolving refinements in biotechnology. The most encouraging results so far have been the generation of intestinal neomucosa in the form of cysts when intestinal epithelial organoid units isolated from neonatal rats were seeded onto biodegradable polymers before implantation in syngeneic adult rats' omentum. Although still experimental, continued attempts worldwide using cultured stem cells and improved polymer technology offer promise to provide an off-the-shelf artificial intestine as a novel therapy for patients with SBS. This article reviews the current status of progress in the field of small intestinal tissue engineering and addresses various types of cell sources and scaffold material having potential to be used in this field.

The endothelialization of polyhedral oligomeric silsesquioxane nanocomposites: an in vitro study.

It has been recognized that seeding vascular bypass grafts with endothelial cells is the ideal method of improving their long-term patency rates. The aim of this study was to assess the in vitro cytocompatibility of a novel silica nanocomposite, polyhedral oligomeric silsesquioxane-poly(carbonate-urea)urethane (POSS-PCU) and hence elicit its feasibility at the vascular interface for potential use in cardiovascular devices such as vascular grafts. Using primary human umbilical vein endothelial cells (HUVEC), cell viability and adhesion were studied using AlamarBlue assays, whereas cell proliferation on the polymer was assessed using the PicoGreen dye assay. Cellular confluence and morphology on the nanocomposite were analyzed using light and electron microscopy, respectively. Our results showed that there was no significant difference between cell viability in standard culture media and POSS-PCU. Endothelial cells were capable of adhering to the polymer within 30 min of contact (Student's t-test, p < 0.05) with no difference between POSS-PCU and control cell culture plates. POSSPCU was also capable of sustaining good cell proliferation for up to 14 d even from low seeding densities (1.0 x 10(3) cells/cm(2)) and reaching saturation by 21 d. Microscopic analysis showed evidence of optimal endothelial cell adsorption morphology with the absence of impaired motility and morphogenesis. In conclusion, these results support the application of POSS-PCU as a suitable biomaterial scaffold in bio-hybrid vascular prostheses and biomedical devices.

The effect of shear stress on human endothelial cells seeded on cylindrical viscoelastic conduits: an investigation of gene expression.

The present study assesses the effect of physiological shear stress on gene expression from human ECs (endothelial cells) seeded on a small-diameter cylindrical bypass graft constructed from nanocomposite based on poly(carbonate-silsesquioxane-bridge-urea)urethane. ECs were seeded on to 5-mm-diameter conduits, placed in a physiological flow circuit and exposed to 1 or 4 h of shear stress at 1.4+/-0.3 Pa. Subsets of conduits were incubated at 37 degrees C and 5% CO2/95% O2 for a further 4 h to determine if gene expression returned to basal levels. PCR was conducted for glyceraldehyde-3-phosphate dehydrogenase, TGFbeta-1 (transforming growth factor beta-1), COL-1 (collagen-1) and PECAM-1 (platelet/EC adhesion molecule-1). Increases in gene expression were seen following flow in nanocomposite conduits. These were significant at 4 h for TGFbeta-1, COL-1 and PECAM-1. After a 4 h recovery period, there were no significant differences in gene intensity, suggesting that this change is transient. These data prove that mRNA can be obtained from ECs seeded on tubular conduits and exposed to shear stress and that gene-expression studies can be successfully carried out. We believe this is a substantial improvement on studies based on flat sheets.