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.

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.

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.

Electrohydrodynamic jetting behaviour of polyhedral oligomeric silsesquioxane nanocomposite.

In this paper, we investigate in detail the electrohydrodynamic spraying of a nonbiodegradable nanocomposite polyhedral oligomeric silsesquioxane polymer developed in our laboratories and currently being explored for coating metallic stent materials. Different concentrations of the polymer have been dissolved to prepare, characterise, and electrohydrodynamically deposit the polymer on stainless steel. From the experiments, the solution containing 15 wt% polymer was selected for further investigation. The variation of film/coating thickness as a function of spraying time was studied and the structural features of the film were assessed using microscopy. Films were also tensile tested. This study has identified a process and conditions which can be used in our stent coating research.

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.

Chemical group-dependent plasma polymerisation preferentially directs adipose stem cell differentiation towards osteogenic or chondrogenic lineages.

Human adipose derived stem cells (ADSCs) are being explored for the repair of craniofacial defects due to their multi-differentiation potential and ease of isolation and expansion. Crucial to using ADSCs for craniofacial repair is the availability of materials with appropriate biomechanical properties that can support their differentiation into bone and cartilage. We tested the hypothesis that different modifications of chemical groups on the surface of a nanocomposite polymer could increase human ADSC adhesion and selectively enhance their osteogenic and chondrogenic differentiation. We show that the COOH modification significantly promoted initial cell adhesion and proliferation over 14days compared to NH2 surfaces. Expression of focal adhesion kinase and vinculin was enhanced after plasma surface polymerisation at 24h. The COOH modification significantly enhanced chondrogenic differentiation as indicated by up-regulation of aggrecan and collagen II transcripts. In contrast, NH2 group functionalised scaffolds promoted osteogenic differentiation with significantly enhanced expression of collagen I, alkaline phosphatase and osteocalcin both at the gene and protein level. Finally, chorioallantoic membrane grafting demonstrated that both NH2 and COOH functionalised scaffolds seeded with ADSCs were biocompatible and supported vessel ingrowth apparently to a greater degree than unmodified scaffolds. In summary, our study shows the ability to direct ADSC chondrogenic and osteogenic differentiation by deposition of different chemical groups through plasma surface polymerisation. Hence this approach could be used to selectively enhance bone or cartilage formation before implantation in vivo to repair skeletal defects. STATEMENT OF SIGNIFICANCE: Human adipose derived stem cells (hADSCs) are an exciting stem cell source for regenerative medicine due to their plentiful supply and ease of isolation. However, the optimal environmental cues to direct stem cells towards certain lineages change have to has not been identified. We have shown that by modifying the surface of the scaffold with specific chemical groups using plasma surface polymerisation techniques we can control ADSCs differentiation. This study shows that ADSCs can be differentiated towards osteogenic and chondrogenic lineages on amine (NH2) and carboxyl (COOH) modified scaffolds respectively. Plasma polymerisation can be easily applied to other biomaterial surfaces to direct stem cell differentiation for the regeneration of bone and cartilage.

The mechanical behavior of vascular grafts: a review.

The development of intimal hyperplasia (IH) near the anastomosis of a vascular graft to artery is directly related to changes in the wall shear rate distribution. Mismatch in compliance and diameter at the end-to-end anastomosis of a compliant artery and rigid graft cause shear rate disturbances that may induce intimal hyperplasia and ultimately graft failure. The principal strategy being developed to prevent IH is based on the design and fabrication of compliant synthetic or innovative tissue-engineered grafts with viscoelastic properties that mirror those of the human artery. The goal of this review is to discuss how mechanical properties including compliance mismatch, diameter mismatch, Young's modulus and impedance phase angle affect graft failure due to intimal hyperplasia.

Cellular engineering of vascular bypass grafts: role of chemical coatings for enhancing endothelial cell attachment.

Surgical treatment of vascular disease has become common. The use of synthetic materials is limited to grafts larger than 5-6mm, because of the frequency of occlusion observed with small-diameter prosthetics. An alternative would be a hybrid or tissue-engineered graft with the surface coated with a monolayer of the patient's own cells. Currently, to be effective, high-density seeding regimens have to be undertaken. This is because endothelial cells (ECs) are washed off the graft lumen once exposed to physiological blood flow. EC attachment has been shown to be significantly improved by pre-coating with substances known to attach ECs selectively. The review examines the various types of coating and bonding technology used to date to enhance endothelial cell attachment onto the surface of prosthetic vascular bypass grafts.

Synthesis and evaluation of amphiphilic RGD derivatives: uses for solvent casting in polymers and tissue engineering applications.

Derivatives containing arginine-glycine-aspartic acid (RGD) inhibit fibrinogen binding to activated platelets and promote endothelial and smooth muscle cell attachment. An amphiphilic derivative of RGD that can be dissolved in an organic solvent has potential in the development of non-thrombogenic biomaterials. Such a derivative, LA-GRGD, was synthesised by coupling glycine-arginine-glycine-aspartic acid (GRGD) with lauric acid (LA). Its solubility and antithrombotic, cytotoxic and cell-binding effects were then evaluated in comparison with heparin (which is used clinically) and a fibronectin-engineered protein polymer (FEPP). Thromboelastography (TEG) was used to measure blood clotting time using fresh whole blood from healthy volunteers. Tissue factor (TF) activity was measured using plasma with a standard prothrombin time assay (PT). Cytotoxicity was assessed on human umbilical cord endothelial cells (HUVECs) using an Alamar blue assay. Solubility of the conjugate was assessed in a co-solvent. These techniques were used to study LA-GRGD, using heparin and FEPP as controls. The amphiphilic property of LA-GRGD was dependent on the feed mole ratio of GRGD to LA. LA-GRGD was soluble in acetone:water and water. LA-GRGD inhibited TF by >90% and prolonged TEG-r by 8.2+/-3.3 min (200 microg ml(-1)). Heparin inhibited TF by >90%, but prolonged TEG-r by 97.4+/-1.6 min (1 U ml(-1)); FEPP inhibited TF by >90% (100 microg ml(-1)) and prolonged TEG-r by 73.7+/-8.4 min (10 microg ml(-1)). Heparin had no cytotoxic effect on EC metabolism and viability at the concentrations studied (0.1-100 U ml(-1)). No significant cytotoxic effect was produced by LA-GRGD or FEPP at concentrations ranging from 0.1 microg ml(-1) to 50 microg ml(-1), but, at higher concentrations (100 microg ml(-1) and 200 microg ml(-1)), a detrimental effect was observed. Cell binding studies showed that LA-GRGD bound 29% of ECs compared with FEPP (60%) and heparin (22%). This new approach for synthesising amphiphilic RGD and its analogues has potential as a drug delivery system for the manufacture of new polymer formulations for use in bypass grafts and other tissue-engineered devices.

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.

Dynamic protein adsorption at the polyurethane copolymer/water interface.

Polyurethanes (PU) and their polymeric derivatives are widely used in the manufacturing of medical devices. It is important to understand how protein adsorbs onto PU materials as this molecular process directly implicates surface biocompatibility. In this work, we compared protein adsorption at the PU film surfaces with that from the hydrophilic silicon oxide. Two PU polymers were used, a commercial polyurethane (PUA) and a novel poly(carbonate-urea)urethane matrix containing silsesquioxanes (PU4). AFM imaging revealed micro-domain segregation on both PU surfaces, but the incorporation of pendent silsesquioxanes made the PU4 surface much rougher, with the outer surface comprised of soft upper PU segments and lower PU-silsesquioxane hard segments. It appeared that fibrinogen was preferable to adsorb onto the upper soft PU segments. The spectroscopic ellipsometry (SE) measurements at the PU film/solution interface showed that human serum albumin (HSA) adsorption was little affected by surface chemistry whilst fibrinogen adsorption was much greater on the two PU surfaces indicating a strong surface effect. Further studies revealed that HSA adsorption was reversible on hydrophilic SiO(2) against changes in pH from 5 to 7, but irreversible on the two PU surfaces. In contrast, fibrinogen adsorption against the same pH cycling was found to be irreversible on all three surfaces. The different extent of irreversibility was clearly indicative of different interfacial interactions. Sequential protein adsorption revealed that the PU4 surface shared similar physiochemical properties to the SiO(2) surface, demonstrating the success in incorporating the siloxane pendant nanocages. The knowledge of protein surface structure and behaviour may lead to the development of effective means to control surface biocompatibility.

Assessment of the potential of progenitor stem cells extracted from human peripheral blood for seeding a novel vascular graft material.

OBJECTIVE: A novel nanocomposite has recently been developed based on polyhedral oligomeric silsesquioxane attached by direct reaction onto a urethane segment, as a potential vascular graft material; its trade name is UCL-Nano. The UCL-Nano has been demonstrated to have similar viscoelastic properties to the walls of a natural artery, to be resistant to degradation and to be able to sustain endothelial cell seeding. Human peripheral blood contains both circulating endothelial cells and endothelial progenitor cells, which may be suitable for conduit seeding. The aim of this study was to develop a system with the potential to deliver an endothelial cell-seeded bypass graft in a realistic time frame. MATERIALS AND METHODS: Endothelial progenitor cells and circulating endothelial cells were isolated from human peripheral blood and were characterized by fluorescent-activated cell sorting, reverse transcriptase-polymerase chain reaction and immunohistochemistry. Isolated cells were seeded on nanocomposite and were maintained in culture for 35 days. RESULTS: The UCL-Nano was successfully seeded with cells and a confluent cell layer was achieved after 14-day culture. Cells remained viable and confluent on the nanocomposite for 35 days. CONCLUSION: In conclusion, these results suggest that this process has potential both for a realistic and achievable two-stage seeding process for vascular bypass grafts and for the potential development of a device, with the aim of achieving in situ seeding once implanted.

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.

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.

Effect of prolonged pulsatile shear stress in vitro on endothelial cell seeded PTFE and compliant polyurethane vascular grafts.

OBJECTIVES: Compliance mismatch between graft and native artery, and failure of the graft to develop an endothelial lining are the two main factors in graft failure. The objective of this study was to assess a new compliant graft for effective cell attachment and cell retention at physiological levels of pulsatile shear stress over a 6-hour period of physiological pulsatile flow. DESIGN: Laboratory haemodynamic study. MATERIALS AND METHODS: Human umbilical vein endothelial cells labelled with 111In-oxine were seeded on compliant polyurethane (CPU) and polytetrafluoroethylene (PTFE) vascular grafts. These were then exposed to varying shear stresses of up to 13.8 +/- 0.6 dyn/cm2 using a pulsatile flow model. Dynamic scintigraphy images were acquired using a gamma camera linked to an on-line computer during 6 h of perfusion and data presented as mean +/- standard error of mean. RESULTS: Mean seeding efficiencies were significantly different at 4,316 +/- 505 and 825 +/- 504 CPM/cm2 on the CPU and PTFE grafts, respectively (p = 0.018). The flow experiment showed a higher percentage of cells retained on the CPU graft after exposure to shear stress caused by pulsatile flow compared to PTFE with respect to time. After 6 h pulsatile perfusion there was a significantly higher proportion of initial cells attached to CPU graft compared to PTFE graft (73 +/- 8% vs 42 +/- 8%, p = 0.018). The areas under the time activity curves over the 6-hour period were 280 +/- 26.4 for CPU and 176.0 +/- 30.0 for PTFE, confirming a significant greater total cell loss from PTFE compared with CPU grafts (51 +/- 7.0% vs 23 +/- 8.3%, p = 0.018, Wilcoxon matched-pairs signed-ranks test). CONCLUSIONS: This flow model provides an effective method of assessing cell retention on graft materials under physiological conditions over a 6-hour period; CPU combines both excellent compliance and endothelial cell attachment rates after 6 h exposure to shear stress.

Magnetic beads (Dynabead) toxicity to endothelial cells at high bead concentration: implication for tissue engineering of vascular prosthesis.

Magnetic beads (Dynabeads) have been used for the purification of endothelial cells. One application for this procedure may be for single-stage seeding of bypass grafts. The number of endothelial cells (EC) isolated is crucial and therefore to increase the number of cells extracted, a higher number of Dynabeads per cell may need to be used. The effect of large numbers of CD31 Dynabeads on cell proliferation/metabolism is unknown. We undertook this study using CD31-coated Dynabeads and EC from human umbilical vein. EC were coated at concentrations of 4, 10, or 50 beads per cell. The cells were cultured for 6 days with control being normal EC. Cellular proliferation was assessed by trypsinization of cells and metabolism assessed with an Alamar blue viability assay. In a further experiment a compliant polyurethane graft was single-stage seeded with both coated Dynabeads and normal EC. The results showed that using a higher number of beads per cell resulted in a reduction in cell proliferation and a reduction in cell metabolism. The total number of Dynabeads-coated cells in culture compared to controls (%) by day 6 were 30.7 +/- 2.56, 41.3 +/- 9.8 and 59.2 +/- 7.3 for 50, 10, and 4 beads per cell, respectively. The corresponding results for Alamar blue were 43.7 +/- 1.2, 61.8 +/- 1.4, and 72.1 +/- 4.3. The seeded grafts showed reduced metabolism with the Dynabeads-coated EC. In conclusion, high numbers of beads per cell have a late detrimental effect on cell proliferation and metabolism. Therefore for single-stage seeding lower numbers of Dynabeads will need to be used with resultant reduction in the number of available EC.

Improving endothelial cell retention for single stage seeding of prosthetic grafts: use of polymer sequences of arginine-glycine-aspartate.

OBJECTIVE: single stage seeding within the timeframe of a typical vascular operation has not been successful. One reason for this is poor cell adherence to the graft lumen once exposed to pulsatile blood flow. In this study we have carried out investigations with the use of two different fibronectin-based peptides, fibronectin-like engineered protein polymer (FEPP) which contains multiple copies of arginine-glycine-aspartate (RGD) and fibronectin adhesion promoting peptide (FAPP) to improve cell adherence. MATERIALS AND METHODS: FAPP and FEPP were coated onto native polyurethane and heparinised polyurethane grafts. The grafts were then seeded for either 1 or 2h with human umbilical vein endothelial cells (HUVEC). After the incubation period the cells were washed off and cell retention was calculated. Cell metabolism was measured using Alamar Blue, and confirmed with scanning electron microscopy (SEM). RESULTS: heparinised grafts coated with FEPP showed the best cell retention after both 1 and 2h seeding (80+/-4% vs 81+/-3%). This graft had no significant difference in cell retention after both times whilst all the other grafts had better cell retention after a 2h seeding. The Alamar blue and SEM results confirmed cell viability and function for all graft types. CONCLUSION: heparinised graft coated with FEPP allows significant cell retention after only 1h of seeding and shows promise for single stage seeding.

A new technique for measuring the cell growth and metabolism of endothelial cells seeded on vascular prostheses.

For the improvement of vascular graft patency, an endothelial cell (EC) lining is desirable. It is essential that the EC remains viable after being seeded onto the prosthetic graft. The aim of this study was to adapt an Alamar redox assay (ABRA) as a technique to monitor the viability of ECs seeded on prosthetic grafts. To test the graft types, we seeded human umbilical vein ECs on compliant polyurethane (CPU), expanded polytetrafluoroethylene, and Dacron at a density of 2 x 10(5) cell/cm(2). After 24 h of incubation, ABRA was added, and the absorbance was measured at 4, 8, and 24 h. To assess seeded cell concentrations on grafts, we seeded CPU at densities ranging from 1 x 10(5) to 8 x 10(5) cell/cm(2). The validity of the test was assessed with sodium azide and mitomycin C, known physiological perturbators. ABRA reduction demonstrated that ECs were viable and functional postseeding on the prosthetic grafts. A significant correlation was observed with ABRA reduction and cell concentrations (p < 0.001). The acid phosphatase assay demonstrated enzyme activity in the cells, but they were not maintained under normal physiological conditions. The ABRA bioreduced product was soluble, stable, and noncytotoxic over 24 h. The assay is independent of the geometry or physiochemistry of the graft type. The technique allows the continuous assessment of the metabolism and viability of seeded cells, is simple to perform, and does not destroy the cells or graft materials.