Argon plasma surface modification promotes the therapeutic angiogenesis and tissue formation of tissue-engineered scaffolds in vivo by adipose-derived stem cells.

BACKGROUND: Synthetic implants are being used to restore injured or damaged tissues following cancer resection and congenital diseases. However, the survival of large tissue implant replacements depends on their ability to support angiogenesis that if limited, causes extrusion and infection of the implant. This study assessed the beneficial effect of platelet-rich plasma (PRP) and adipose-derived stem cells (ADSCs) on synthetic biomaterials in combination with argon plasma surface modification to enhance vascularisation of tissue-engineered constructs. METHODS: Non-biodegradable polyurethane scaffolds were manufactured and modified with plasma surface modification using argon gas (PM). Donor rats were then used to extract ADSCs and PRP to modify the scaffolds further. Scaffolds with and without PM were modified with and without ADSCs and PRP and subcutaneously implanted in the dorsum of rats for 3 months. After 12 weeks, the scaffolds were excised and the degree of tissue integration using H&E staining and Masson's trichrome staining, angiogenesis by CD31 and immune response by CD45 and CD68 immunohistochemistry staining was examined. RESULTS: H&E and Masson's trichrome staining showed PM+PRP+ADSC and PM+ADSC scaffolds had the greatest tissue integration, but there was no significant difference between the two scaffolds (p < 0.05). The greatest vessel formation after 3 months was shown with PM+PRP+ADSC and PM+ADSC scaffolds using CD31 staining compared to all other scaffolds (p < 0.05). The CD45 and CD68 staining was similar between all scaffolds after 3 months showing the ADSCs or PRP had no effect on the immune response of the scaffolds. CONCLUSIONS: Argon plasma surface modification enhanced the effect of adipose-derived stem cells effect on angiogenesis and tissue integration of polyurethane scaffolds. The combination of ADSCs and argon plasma modification may improve the survival of large tissue implants for regenerative applications.

Biomechanical Characterisation of the Human Auricular Cartilages; Implications for Tissue Engineering.

Currently, autologous cartilage provides the gold standard for auricular reconstruction. However, synthetic biomaterials offer a number of advantages for ear reconstruction including decreased donor site morbidity and earlier surgery. Critical to implant success is the material's mechanical properties as this affects biocompatibility and extrusion. The aim of this study was to determine the biomechanical properties of human auricular cartilage. Auricular cartilage from fifteen cadavers was indented with displacement of 1 mm/s and load of 300 g to obtain a Young's modulus in compression. Histological analysis of the auricle was conducted according to glycoprotein, collagen, and elastin content. The compression modulus was calculated for each part of the auricle with the tragus at 1.67 ± 0.61 MPa, antitragus 1.79 ± 0.56 MPa, concha 2.08 ± 0.70 MPa, antihelix 1.71 ± 0.63 MPa, and helix 1.41 ± 0.67 MPa. The concha showed to have a significantly greater Young's Elastic Modulus than the helix in compression (p < 0.05). The histological analysis demonstrated that the auricle has a homogenous structure in terms of chondrocyte morphology, extracellular matrix and elastin content. This study provides new information on the compressive mechanical properties and histological analysis of the human auricular cartilage, allowing surgeons to have a better understanding of suitable replacements. This study has provided a reference, by which cartilage replacements should be developed for auricular reconstruction.

Development of resorbable nanocomposite tracheal and bronchial scaffolds for paediatric applications.

BACKGROUND: Congenital tracheal defects and prolonged intubation following premature birth have resulted in an unmet clinical need for tracheal replacement. Advances in stem cell technology, tissue engineering and material sciences have inspired the development of a resorbable, nanocomposite tracheal and bronchial scaffold. METHODS: A bifurcated scaffold was designed and constructed using a novel, resorbable nanocomposite polymer, polyhedral oligomeric silsesquioxane poly(ϵ-caprolactone) urea urethane (POSS-PCL). Material characterization studies included tensile strength, suture retention and surface characteristics. Bone marrow-derived mesenchymal stem cells (bmMSCs) and human tracheobronchial epithelial cells (HBECs) were cultured on POSS-PCL for up to 14 days, and metabolic activity and cell morphology were assessed. Quantum dots conjugated to RGD (l-arginine, glycine and l-aspartic acid) tripeptides and anticollagen type I antibody were then employed to observe cell migration throughout the scaffold. RESULTS: POSS-PCL exhibited good mechanical properties, and the relationship between the solid elastomer and foam elastomer of POSS-PCL was comparable to that between the cartilaginous U-shaped rings and interconnective cartilage of the native human trachea. Good suture retention was also achieved. Cell attachment and a significant, steady increase in proliferation were observed for both cell types (bmMSCs, P = 0·001; HBECs, P = 0·003). Quantum dot imaging illustrated adequate cell penetration throughout the scaffold, which was confirmed by scanning electron microscopy. CONCLUSION: This mechanically viable scaffold successfully supports bmMSC and HBEC attachment and proliferation, demonstrating its potential as a tissue-engineered solution to tracheal replacement.

Nanotopography and plasma treatment: redesigning the surface for vascular graft endothelialisation.

INTRODUCTION: Vascular graft materials in clinical use, such as polytetrafluoroethylene (PTFE) and Dacron, do not endothelialise and have low patency rates. The importance of an endothelial cell layer on the luminal surface of a vascular graft is well-known with surface topography and chemistry playing an important role. The aim of this study was to investigate the potential of plasma treatment and topographical structures on the luminal graft surface to enhance the self-endothelialisation potential of a nanocomposite vascular graft. METHODS: POSS-PCU is a polycarbonate urea urethane (PCU) with a nanoparticle, polyhedral oligomeric silsesquioxane (POSS) incorporated within it. Planar, microgrooved, and nanopit patterned polymer films were fabricated using photolithography, electron beam lithography, reactive ion etching, and replication by solvent casting. Films were then exposed to oxygen plasma treatment at different powers for a fixed time (40 W, 60 W, 80 W/60 seconds). Effects of plasma treatment were assessed using scanning electron microscopy, atomic force microscopy and water contact angle analysis. Human umbilical vein endothelial cell (HUVEC) proliferation and morphology were characterised using immunostaining, live/dead staining, and Coomassie blue staining. RESULTS: Successful embossing of the micro- and nanostructures was confirmed. Oxygen plasma treatment of the different samples showed that increasing power significantly increased the hydrophilicity of the samples (p < .0001). Improved HUVEC adhesion was seen on plasma modified compared with untreated samples (p < .0001). Coomassie blue staining showed that after 5 days, cells started to form monolayers and live/dead staining showed the cells were viable. Immunostaining showed that HUVECs expressed nitric oxide synthase on all topographies with focal adhesions appearing more pronounced on nanopit surfaces, showing retention of morphology and function. CONCLUSION: These encouraging results indicate a future important role for plasma treatment and nanotopography in the development of endothelialised vascular grafts.

Luminal surface engineering, 'micro and nanopatterning': potential for self endothelialising vascular grafts?

OBJECTIVE: New technologies are being explored to meet the clinical need for an 'off-the-shelf' small diameter vascular graft with superior or at least equivalent properties to autologous vessel. The field of nanotechnology and fabrication promises major advances in biomaterial design and wall structure to deliver biomimetic grafts. This review brings together recent work on this topic. METHODS: A literature search was conducted of PubMed and ISI Web of Knowledge using relevant keywords. Articles published after January 2005 were given preference. Personal communications and PhD theses were also used as sources. RESULTS: An evolving focus on surface patterning of biomaterials has been found to carry great potential. Influencing cellular behaviour on prosthetic grafts using graft luminal surface modulation at the micro- and nano-levels is the basis of this recent concept in vascular graft development. CONCLUSION: This technology may deliver small diameter grafts with the potential for spontaneous in situ endothelialisation without the need for prior 'seeding', with the potential to open a new chapter in vascular graft development.

Surface and mechanical analysis of explanted Poly Implant Prosthèse silicone breast implants.

BACKGROUND: The recent events surrounding Poly Implant Prosthèse (PIP) breast implants have renewed the debate about the safety profile of silicone implants. The intentional use of industrial-grade instead of certified medical-grade silicone is thought to be responsible for reportedly higher frequencies of implant rupture in vivo. The differences in mechanical and viscoelastic properties between PIP and medical-grade silicone implant shells were investigated. Surface characterization of shells and gels was carried out to determine structural changes occurring after implantation. METHODS: Breast implants were obtained from women at the Royal Free Hospital (London, UK). PIP implants were compared with medical-grade control silicone implants. Tensile strength, tear resistance and elongation at break were assessed using a tensile tester. Surfaces were analysed using attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. Spearman correlation analyses and Kruskal-Wallis one-way statistical tests were performed for mechanical data. RESULTS: There were 18 PIP and four medical-grade silicone implants. PIP silicone shells had significantly weaker mechanical strength than control shells (P < 0·009). There were negative correlations between mechanical properties of PIP shells and implantation times, indicative of deterioration of PIP shells over time in vivo (r(s) = -0·75, P = 0·009 for tensile strength; r(s) = -0·76, P = 0·001 for maximal strain). Comparison of ATR-FTIR spectra of PIP and control silicones demonstrated changes in material characteristics during the period of implantation suggestive of time-dependent bond breakage and degradation of the material. CONCLUSION: This study demonstrated an increased weakness of PIP shells with time and therefore supports the argument for prophylactic removal of PIP breast implants.

Systematic review: the applications of nanotechnology in gastroenterology.

BACKGROUND: Over the past 30 years, nanotechnology has evolved dramatically. It has captured the interest of variety of fields from computing and electronics to biology and medicine. Recent discoveries have made invaluable changes to future prospects in nanomedicine; and introduced the concept of theranostics. This term offers a patient specific 'two in one' modality that comprises of diagnostic and therapeutic tools. Not only nanotechnology has shown great impact on improvements in drug delivery and imaging techniques, but also there have been several ground-breaking discoveries in regenerative medicine. AIM: Gastroenterology invites multidisciplinary approach owing to high complexity of gastrointestinal (GI) system; it includes physicians, surgeons, radiologists, pharmacologists and many more. In this article, we concentrate on current developments in nano-gastroenterology. METHODS: Literature search was performed using Web of Science and Pubmed search engines with terms--nanotechnology, nanomedicine and gastroenterology. Article search was concentrated on developments since 2005. RESULTS: We have described original and innovative approaches in gastrointestinal drug delivery, inflammatory disease and cancer-target treatments. Here, we have reviewed advances in GI imaging using nanoparticles as fluorescent contrast, and their potential for site-specific targeting. This review has also depicted various approaches and novel discoveries in GI regenerative medicine using nanomaterials for scaffold designs and induced pluripotent stem cells as cell source. CONCLUSIONS: Developments in nanotechnology have opened new range of possibilities to help our patients. This includes novel drug delivery vehicles, diagnostic tools for early and targeted disease detection and nanocomposite materials for tissue constructs to overcome cosmetic or physical disabilities.

Adipose-derived stem cells for clinical applications: a review.

The use of stem cells derived from adipose tissue as an autologous and self-replenishing source for a variety of differentiated cell phenotypes, provides a great deal of promise for reconstructive surgery. In this article, we review available literature encompassing methods of extraction of pluripotent adipose stem cells (ASCs) from lipoaspirate locations, their storage, options for culture, growth and differentiation, cryopreservation and its effect on stem cell survival and proliferation, and new technologies involving biomaterials and scaffolds. We will conclude by assessing potential avenues for developing this incredibly promising field.

Current trends in the application of nanoparticles in drug delivery.

The discovery of new biologically active compounds that can be exploited therapeutically to treat disease has stalled, with fewer new drugs entering the market every year. The spotlight has now turned onto nanoparticles (NPs) as a versatile and multifaceted platform for the delivery of drugs. NPs offer better pharmacokinetic properties, controlled and sustained release, and targeting of specific cells, tissues or organs. All these features can improve the efficacy of existing drugs. The use of NPs can dramatically impact the treatment of many diseases. Many potential therapeutics that exist for alleviating brain diseases such as epilepsy, Alzheimer's disease and tumours are not feasible due to a lack of means to deliver drugs across the blood brain barrier. NPs offer an alternative solution, since they can be modified to cross the blood brain barrier. Additionally, NPs can also play a part in alternative methods of non-parental administration of drugs e.g. pulmonary and transdermally. Through active targeting and the enhanced permeation and retention effect, NPs reduce the systemic toxicity of chemotherapeutic drugs by ensuring delivery only to the site of the tumour, thus enhancing cancer treatment. We critically review the literature to provide a summary of current synthesis methodologies and applications of NPs in drug delivery.

Topical haemostatic agents.

BACKGROUND: A variety of local haemostatic agents is now available to stop troublesome bleeding. These agents are indicated for use during surgical interventions where conventional methods of haemostasis are not applicable because of the site of surgery or the degree of bleeding. METHOD: A literature search using the PubMed and ISI Web of Knowledge databases identified relevant studies on topical haemostatic agents. Manufacturers' recommendations were also sought through commercial websites. RESULTS AND CONCLUSION: A significant body of evidence now exists to support the use of topical haemostatic agents in a wide variety of clinical situations. The advantages and disadvantages of many of these agents are highlighted.

Tissue engineering of a hybrid bypass graft for coronary and lower limb bypass surgery.

Tissue-engineered blood vessels have largely relied on inelastic scaffolds or biological solutions with uncertain long-term in vivo durability. In this report we present for the first time a hybrid tissue-engineered bypass graft consisting of an elastic scaffold of compliant poly(carbonate-urea)urethane (CPU), incorporated with human smooth muscle cells (SMCs) and endothelial cells (ECs) from the same human source. Human vascular SMCs and ECs were extracted from umbilical cord vessels. The effect of shear stress preconditioning on cell retention on the hybrid bypass graft was investigated under pulsatile arterial flow conditions. Retention of ECs seeded onto CPU precoated with SMCs was significantly improved by a period of shear stress preconditioning, especially when the stress incrementally increased. This is probably because the mechanical stimuli orient cells and increase the release of matrix proteins and attachment factors. The stage is now set for developing a hybrid graft for in vivo studies.

Increased apoptosis and decreased proliferation of colorectal cancer cells using insulin-like growth factor binding protein-4 gene delivered locally by gene transfer.

OBJECTIVE: Insulin-like growth factor (IGF)-I induces proliferation of transformed cells. Its binding proteins (IGFBP) are involved in local regulation of IGF. This study assessed the effects of overexpression of IGFBP-4 on the development of cancer in vivo. METHOD: Nude mice were subcutaneously inoculated with HT-29 colorectal cancer cells (3 x 10(6)). When the tumour became visible (1 week after inoculation), animals received either 150 microg of mammalian expression vector containing IGFBP-4 cDNA or vector alone (n = 6 each) by peritumoural injection. Tumour size was measured during the growth. After 3 weeks of IGFBP-4 induction, animals were killed and tumour tissue samples were collected for examining the level of IGFBP-4 expression. Tumour mitotic activities were determined by counting numbers of mitotic cells on the tissue section. Apoptosis was investigated by terminal deoxynucleotidyl transferase-mediated dUDP nick end labelling assay. RESULTS: Following IGFBP-4 treatment, tumour showed large necrotic areas, significantly increased numbers of apoptotic cells (36.67 +/- 7.36 vs 7.07 +/- 1.91, P < 0.01 vs control), decreased cells undergoing mitosis (2.31 +/- 0.32 vs 3.61 +/- 0.27, P < 0.01 vs control) and higher expression of IGFBP-4 (P < 0.05 vs control). CONCLUSION: IGFBP-4 gene transfer increased apoptosis and decreased mitosis, but tumour volume was not significantly altered possibly due to cellular debris filling the centre of tumours.

The role of established and emerging risk factors in peripheral vascular graft occlusion.

Several studies have evaluated the association between established as well as emerging vascular risk factors with peripheral graft occlusion. There is evidence for a link between the risk for graft occlusion and total serum cholesterol, low-density lipoprotein cholesterol and triglyceride levels. The overall effect of smoking shows a 2.35-fold increase in risk of graft failure. Studies involving diabetic patients undergoing peripheral bypass may have failed to detect higher occlusion rates, possibly due to increased morbidity and mortality as well as higher amputation rates even if the graft is patent. Both antiplatelet agents and anticoagulation seem to be effective in the prevention of graft occlusion. Unconvincing data have been published with regards to the effect of hypertension on graft patency. Emerging factors such as fibrinogen, lipoprotein (a), C-reactive protein and homocysteine levels should also be considered when assessing the risk of graft occlusion. More research is needed to prevent graft occlusion due to the obvious clinical relevance, quality of life and cost issues.

Remote ischaemic preconditioning of the hind limb reduces experimental liver warm ischaemia-reperfusion injury.

BACKGROUND: Direct ischaemic preconditioning of the liver reduces ischaemia-reperfusion injury (IRI). Remote ischaemic preconditioning (RIPC) of a limb has been shown to reduce IRI to the heart. This study determined the effect of brief remote ischaemia to the limb in reducing early liver warm IRI. METHODS: Twenty-eight male rabbits were allocated to four groups: sham operated, RIPC alone, IRI alone, and RIPC plus IRI. RIPC was induced in the leg with a tourniquet, before liver IRI, by three alternate cycles of 10 min ischaemia followed by 10 min reperfusion. Liver IRI was produced by total inflow occlusion for 25 min. Markers of liver injury and systemic and hepatic haemodynamics were measured for 2 h after reperfusion. RESULTS: At 2 h, IRI alone was associated with increased serum levels of aminotransferases, and reduced mean arterial blood pressure, hepatic blood flow and peripheral oxygen saturation. There was significant improvement in these variables in animals that had RIPC before liver IRI, and hepatic venous nitrate/nitrite levels were also significantly higher. CONCLUSION: In this experimental model RIPC appeared to reduce liver IRI.

The mechanical properties of infrainguinal vascular bypass grafts: their role in influencing patency.

When autologous vein is unavailable, prosthetic graft materials, particularly expanded polytetrafluoroethylene are used for peripheral arterial revascularisation. Poor long term patency of prosthetic materials is due to distal anastomotic intimal hyperplasia. Intimal hyperplasia is directly linked to shear stress abnormalities at the vessel wall. Compliance and calibre mismatch between native vessel and graft, as well as anastomotic line stress concentration contribute towards unnatural wall shear stress. High porosity reduces graft compliance by causing fibrovascular infiltration, whereas low porosity discourages the development of an endothelial lining and hence effective antithrombogenicity. Therefore, consideration of mechanical properties is necessary in graft development. Current research into synthetic vascular grafts concentrates on simulating the mechanical properties of native arteries and tissue engineering aims to construct a new biological arterial conduit.

Tissue engineering of blood vessels.

BACKGROUND: Tissue engineering techniques have been employed successfully in the management of wounds, burns and cartilage repair. Current prosthetic alternatives to autologous vascular bypass grafts remain poor in terms of patency and infection risk. Growing biological blood vessels has been proposed as an alternative. METHODS: This review is based on a literature search using Medline, PubMed, ISIS and CAS of original articles and reviews, and unpublished material and abstracts. RESULTS AND CONCLUSIONS: Complete incorporation into host tissues and the maintenance of a viable and self-renewing endothelial layer are the fundamental goals to be achieved when developing a tissue-engineered blood vessel. Sourcing of cells and modulating their interaction with extracellular matrix and supporting scaffold have been the focus of intense research. Although the use of tissue-engineered blood vessels in humans is so far limited, advances in our knowledge of stem cell precursors and the development of new biomaterials should enable this technology to reach routine clinical practice within a decade.

Nanocomposite containing bioactive peptides promote endothelialisation by circulating progenitor cells: an in vitro evaluation.

OBJECTIVE: The formation of an endothelial cell layer on the luminal surface of cardiovascular devices, especially bypass grafts, is an important attribute in order to improve their patency. Endothelial progenitor cells (EPCs) have a potential role in the endothelialisation of bypass grafts. We hypothesised that a novel approach to improve endothelialisation of bypass grafts by EPCs would be the creation on the graft lumen of a microenvironment that supports EPC adhesion and differentiation. METHODS: A new generation of nanocomposite based on silsesquioxane in the form of polyhedral oligomeric silsesquioxane (POSS) nanocages which incorporate bioactive peptides (RGD) was made into sheets. Peripheral blood mononuclear cells (PBMCs) containing EPCs isolated from six consenting young, healthy, adult volunteers were then plated both on (1) sheets of the nanocomposite with the bioactive peptide, (2) sheets of the nanocomposite without the bioactive peptide, (3) culture dishes as control and then cultured in presence of vascular endothelial growth factor (VEGF). Confirmation of endothelial and EPCs markers was carried out using fluorescence-activated cell sorter (FACS) analysis, reverse transcription polymerase chain reaction (RT-PCR) and immunostaining. RESULTS: One to two percent of PBMCs expressed CD34 as determined by FACS analysis. Cells were demonstrated to express mRNA for the EPC markers CD34, platelet-endothelial cell adhesion molecule-1 (CD31), CD133 and vascular endothelial growth factor receptor-2(FlK-1/KDR). Endothelial cell-colony forming units were formed between day 5 and day 7 after plating. Colonies were confirmed to be endothelial like cells by immunostaining. There were significantly greater numbers of EPC colonies on the bioactive nanocomposites as compared to the nanocomposite alone and the uncoated dishes. CONCLUSION: We report a new nanocomposite based biomaterial that has been demonstrated, in vitro, to promote endothelialisation from PBMCs containing EPCs.

Ischaemic preconditioning improves microvascular perfusion and oxygenation following reperfusion injury of the intestine.

BACKGROUND: Ischaemia-reperfusion (IR) injury of the intestine occurs commonly during abdominal surgery. Ischaemic preconditioning (IPC) provides a way of protecting the organ from damage inflicted by IR. This study was designed to evaluate the beneficial effect of IPC, focusing on the intestinal microcirculation and oxygenation in intestinal IR injury. METHODS: Rats were allocated to three groups. Animals in the IR and IPC groups underwent 30 min of intestinal ischaemia followed by 2 h of reperfusion. In the IPC group this was preceded by 10 min of ischaemia and 10 min of reperfusion. Animals in the third group underwent laparotomy but no vascular occlusion. Intestinal microvascular perfusion, oxygenation and portal venous blood flow (PVF) were monitored continuously. At the end of the reperfusion period, blood samples were obtained for measurement of lactate dehydrogenase (LDH) and biopsies of ileum for histological evaluation. RESULTS: : IPC improved intestinal microvascular perfusion and tissue oxygenation significantly at the end of the reperfusion period (P < 0.001). PVF improved significantly in the IPC compared with the IR group (P = 0.005). The serum LDH concentration was significantly lower in the IPC than the IR group (mean(s.e.m.) 667.1(86.8) versus 1973.8(306.5) U/l; P < 0.001) Histological examination showed that ileal mucosa was significantly less injured in the IPC group. CONCLUSIONS: This study demonstrated that IPC improves intestinal microvascular perfusion and oxygenation.