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Biomedical subjects

John A Hunt

Publications and source records attributed to John A Hunt.

12 recordsLinked to original sources
PubMed2007

Effects of sterilisation method on surface topography and in-vitro cell behaviour of electrostatically spun scaffolds.

Electrostatic spinning is a potentially significant technique for scaffold production within the field of tissue engineering; however, the effect of sterilisation upon these structures is not known. This research investigated the extent of any topographical alteration to electrostatically spun scaffolds post-production through sterilisation, and examined any subsequent effect on contacting cells. Scaffolds made from Tecoflex SG-80A polyurethane were sterilised using ethylene oxide and UV-ozone. Scaffold topography was characterized in terms of inter-fibre separation (ifs), fibre diameter (f.dia) and surface roughness. Cell culture was performed over 7 days with both mouse L929 and human embryonic lung fibroblasts, the results of which were assessed using SEM, image analysis and confocal microscopy. Sterilisation by UV-ozone and ethylene oxide decreased ifs and increased f.dia; surface roughness was decreased by UV-ozone but increased by ethylene oxide. Possible mechanisms to explain these observations are discussed, namely photo-oxidative degradation in the case of UV-ozone and process-induced changes in surface roughness. UV-ozone sterilised scaffolds showed greater cell coverage than those treated with ethylene oxide, but lower coverage than all the controls. Changes in cell attachment and morphology were thought to be due to the changes in topography brought about by the sterilisation process. We conclude that surface modification by sterilisation could prove to be a useful tool at the final stage of scaffold production to enhance cell contact, phenotype or function.

Animals↗
PubMed2006

The guidance of human mesenchymal stem cell differentiation in vitro by controlled modifications to the cell substrate.

Material-driven control of bone-marrow-derived mesenchymal stem cell (MSC) behaviour and differentiation is a very exciting possibility. The aim of this study was to use silane-modified surfaces to control MSC adhesion and differentiation in vitro and evaluate the use of such techniques to control MSC behaviour both in basal and stimulated conditions. A range of characterised clean glass silane-modified surfaces, methyl (-CH(3)), amino (-NH(2)), silane (-SH), hydroxyl (-OH) and carboxyl (-COOH), were produced and cultured in contact with human MSC, in conjunction with a clean glass (TAAB) control, for time periods up to 28 days in basal, chondrogenic and osteogenic stimulated media. The samples were analysed for levels of viable cell adhesion, morphology and the production of various differentiation and transcription markers using both fluorescent immunohistochemistry (collagen I, II, osteocalcin, CBFA1) and real-time polymerase chain reaction (PCR) (collagen I, II, osteocalcin, osteopontin, osteonectin, CBFA1 and Sox 9). Analysis of the results demonstrated that the range of materials could be broken down into three distinct categories. Firstly, the -TAAB control and -CH(3) surfaces maintained the MSC phenotype; secondly, the -NH(2) and -SH-modified surfaces promoted and maintained osteogenesis both in the presence and absence of biological stimuli. These surfaces did not support long-term chondrogenesis under any test conditions. Finally, the -OH and -COOH-modified surfaces promoted and maintained chondrogenesis under both basal and chondrogenic stimulated conditions, but did not support osteogenesis. These results demonstrate that intricate material properties such as surface chemistry and energy can influence MSC behaviour in vitro. These results have implications not only in promoting the efficiency of tissue-engineered constructs, but also to the wider field of MSC isolation, maintenance and expansion.

Biocompatible Materials↗
PubMed2006

The use of poly(l-lactide) and RGD modified microspheres as cell carriers in a flow intermittency bioreactor for tissue engineering cartilage.

The use of biodegradable microcarriers as initial supports for tissue engineering has been demonstrated to be advantageous for maintaining a differentiated cell phenotype; the high surface area also allows rapid cell expansion. Poly l-lactide (PLLA) is a significant member of a group of polymers regarded as bioresorbable and has been widely used for manufacturing 3D scaffolds for tissue engineering. In this study, the hypothesis that PLLA microspheres could be surface modified using RGD peptide sequences to improve the cell adhesion and function of those cells in contact with PLLA was tested. Using this type of approach it may be possible to generate larger structures that contain a high cell number relative to the amount of polymer, whilst remaining free from mass transport limitations. PLLA microspheres were prepared using an oil-in-water solvent-evaporation technique and then an RGD-motif was incorporated onto the microspheres surface by conjugation to improve cell attachment and function. Both PLLA and GRGDSPK modified PLLA microspheres were used as cell microcarriers for chondrocytes cultured in a flow intermittency bioreactor. At the same time, the degradation of the microspheres has been studied after 7, 14, 21, 28, 35, 49 and 56 days. The molecular weight of the PLLA microspheres was determined by Gel Permeation Chromatography. The morphology was assessed by scanning electron microscopy, and the thermal properties determined by Differential Scanning Calorimetry. It was demonstrated that the RGD modified and pure PLLA microspheres degraded gradually at a steady rate over the experimental period, which would provide a controlled degradation profile, both could serve as cell microcarriers because of their thermal and mechanical stabilities. The microspheres with RGD surface modification enhanced cell adhesion and increased the cell numbers in the microspheres aggregates.

Bioreactors↗
PubMed2006

The differentiation of bone marrow mesenchymal stem cells into chondrocyte-like cells on poly-L-lactic acid (PLLA) scaffolds.

While intervertebral disc (IVD) degeneration is associated with the majority of cases of low back pain, current treatments are symptomatic rather than curative. Tissue engineering offers a treatment that both cures the problem of disc degeneration and restores normal disc function. One of the major problems for any tissue engineering strategy, however, is ensuring that both the cells and matrices used are suitable for the target tissue. In this study, we have developed and studied a potential system for tissue engineering of the nucleus pulposus (NP) of the severely degenerate IVD. While cells from degenerate discs are not suitable for tissue engineering, bone-marrow-derived mesenchymal stem cells, which are capable of differentiating into chondrocyte-like cells such as those found within the NP of the disc, offer a potential source of cells. We have used transfection with adenoviral SOX-9, a transcription factor involved in differentiation of MSCs along the chondrogenic lineage, combined with culture in a specialised medium, to differentiate monolayer MSCs to NP-like (chondrocyte-like) cells, as shown by real-time quantitative polymerase chain reaction for NP-marker genes. We have also replicated these findings on porous, biodegradable three-dimensional (3D) poly-l-lactic acid scaffolds and shown expression and deposition of NP matrix markers such as type II collagen and aggrecan. We are therefore proposing pre-differentiation of human MSCs and seeding on porous, biodegradable 3D synthetic polymer scaffolds as a realistic tissue engineering strategy for regeneration of the degenerate human IVD.

Adenoviridae↗
PubMed2005

Intervertebral disc cell-mediated mesenchymal stem cell differentiation.

Low back pain is one of the largest health problems in the Western world today, and intervertebral disc degeneration has been identified as a main cause. Currently, treatments are symptomatic, but cell-based tissue engineering methods are realistic alternatives for tissue regeneration. However, the major problem for these strategies is the generation of a suitable population of cells. Adult bone marrow-derived mesenchymal stem cells (MSCs) are undifferentiated, multipotent cells that have the ability to differentiate into a number of cell types, including the chondrocyte-like cells found within the nucleus pulposus (NP) of the intervertebral disc; however, no method exists to differentiate these cells in an accessible monolayer environment. We have conducted coculture experiments to determine whether cells from the human NP can initiate the differentiation of human MSCs with or without cell-cell contact. Fluorescent labeling of the stem cell population and high-speed cell sorting after coculture with cell-cell contact allowed examination of individual cell populations. Real-time quantitative polymerase chain reaction showed significant increases in NP marker genes in stem cells when cells were cocultured with contact for 7 days, and this change was regulated by cell ratio. No significant change in NP marker gene expression in either NP cells or stem cells was observed when cells were cultured without contact, regardless of cell ratio. Thus, we have shown that human NP and MSC coculture with contact is a viable method for generating a large population of differentiated cells that could be used in cell-based tissue engineering therapies for regeneration of the degenerate intervertebral disc.

Adolescent↗
PubMed2005

An in vivo study of the host response to starch-based polymers and composites subcutaneously implanted in rats.

Implant failure is one of the major concerns in the biomaterials field. Several factors have been related to the fail but in general these biomaterials do not exhibit comparable physical, chemical or biological properties to natural tissues and ultimately, these devices can lead to chronic inflammation and foreign-body reactions. Starch-based biodegradable materials and composites have shown promising properties for a wide range of biomedical applications as well as a reduced capacity to elicit a strong reaction from immune system cells in vitro. In this work, blends of corn starch with ethylene vinyl alcohol (SEVA-C), cellulose acetate (SCA) and polycaprolactone (SPCL), as well as hydroxyapatite (HA) reinforced starch-based composites, were investigated in vivo. The aim of the work was to assess the host response evoked for starch-based biomaterials, identifying the presence of key cell types. The tissues surrounding the implant were harvested together with the material and processed histologically for evaluation using immunohistochemistry. At implant retrieval there was no cellular exudate around the implants and no macroscopic signs of an inflammatory reaction in any of the animals. The histological analysis of the sectioned interface tissue after immunohistochemical staining using ED1, ED2, CD54, MHC class II and alpha/beta antibodies showed positively stained cells for all antibodies, except for alpha/beta for all the implantation periods, where it was different for the various polymers and for the period of implantation. SPCL and SCA composites were the materials that stimulated the greatest cellular tissue responses, but generally biodegradable starch-based materials did not induce a severe reaction for the studied implantation times, which contrasts with other types of degradable polymeric biomaterials.

Animals↗
PubMed2005

Inflammatory response to a novel series of siloxane-crosslinked polyurethane elastomers having controlled biodegradation.

A series of polyurethane polymers was synthesized with increasing proportions of silicone in the form of polydimethylsiloxane (PDMS) utilised as a cross-linking agent, based on an aromatic, non-biostable polyetherurethane (PEtU). Eight formulations ranging from 0-50% PDMS were constructed into porous and non-porous films. These were implanted subcutaneously in rats, both unstrained and 100% strained, for 3 and 6 months. Degradation was determined by FTIR-ATR. Porous films were implanted for 6 and 12 months intramuscularly in both rats and rabbits. These were explanted and examined for inflammatory cell markers by immunohistochemistry. Both low and high percentages of siloxane gave rise to increased degradation, with 20-40% PDMS resulting in the least degradation. Infrared spectral changes correlated well with both visual examination and observation by SEM. Changes to the concentration of siloxane gave rise to differences in the thickness of fibroblastic capsule and infiltration of inflammatory cells in both films & scaffolds. Cellular infiltration was greatest in the films with lower siloxane concentrations. Macrophage activation (MHC-I & MHC-II expression) was least in the higher siloxane variants. It is concluded that by varying the siloxane content in the PEtU matrix we can obtain an acceptable inflammatory response with a relatively short degradation time.

Animals↗
PubMed2004

Cytokine secretion from mononuclear cells cultured in vitro with starch-based polymers and poly-L-lactide.

The cytokine network is one of the major controlling systems of the inflammatory process, driving the magnitude and duration of the host response against invading microorganisms, foreign materials, or altered internal stimuli. Pro- and antiinflammatory cytokines were quantified after in vitro culture of a mixed population of monocytes/macrophages and lymphocytes with biodegradable polymers. Different blends of starch-based polymers and their composites filled with hydroxyapatite were studied and compared with poly-L-lactide. Interleukin (IL)-1beta, IL-6, and tumour necrosis factor-alpha were investigated as the markers of immunological reactivity because they are known to act at the early stages of injury/invasion. Interferon-gamma, recognized as a proinflammatory cytokine, although not present during early responses was also investigated. Contrarily, IL-4 derived from T lymphocytes, was investigated because it is an immunoregulator that counteracts some aspects of inflammation. T lymphocyte activation was also determined by quantifying IL-2. The results support the hypothesis that different biodegradable polymers can affect mononuclear cell activation and the production of several cytokines associated with the inflammatory process. No IL-2 or interferon-gamma was found in the culture supernatants after 3, 7, and 14 days in the presence of any of the materials. IL-6 was detected in the highest amounts, for all the conditions, followed by tumour necrosis factor-alpha. IL-1beta was produced in very low amounts, being undetectable with some of the starch-based materials. IL-4 was the only cytokine that did not demonstrate any significant difference within this group of materials. Starch-based polymers and composites induced lower production of proinflammatory cytokines in comparison to poly-L-lactide.

Cells, Cultured↗
PubMed2003

Can intrasac pressure monitoring reliably predict failure of endovascular aneurysm repair?

PURPOSE: To determine if pressure measured at a single location within aneurysm sac thrombus accurately reflects the force applied to the aneurysm wall and the risk of rupture by examining (1) if pressure is distributed uniformly within aneurysm thrombus, (2) the pressure transmission through aneurysm thrombus, and (3) the microstructural basis for pressure transmission. METHODS: Pressure within aneurysm thrombus was measured by direct puncture through the aneurysm wall at 121 sites in 26 patients during open abdominal aortic aneurysm repair. Measurements were taken prior to cross clamping and compared with intrasac pressure measured at 30 sites in 6 patients without aneurysm thrombus (controls). Transmission of pressure through aneurysm thrombus was further examined ex vivo by subjecting fresh thrombus to a pressure gradient in a custom-made pressure cell. Pressure transmission was correlated with matrix density as determined by light microscopy and image analysis. RESULTS: Mean pressure within aneurysm thrombus was higher than mean systemic pressure in 11 patients, lower in 1, and identical in 9. In 5 patients, the pressure was greater than systemic in some areas of the thrombus but less in others. Sac pressure was identical to systemic pressure at all sites in the controls. In 12 thrombus specimens (6 patients) examined in the pressure cell, pressure transmission varied significantly between specimens, correlating directly with matrix density (R(2)=0.747, p=0.001). CONCLUSIONS: Pressure transmission through aneurysm thrombus is variable and depends upon the microstructure of the thrombus. Pressure measured at a single location may not, therefore, accurately reflect the pressure acting on the aneurysm wall.

Angioplasty↗
PubMed2002

A comparison of reactive oxygen species generation by rat peritoneal macrophages and mast cells using the highly sensitive real-time chemiluminescent probe pholasin: inhibition of antigen-induced mast cell degranulation by macrophage-derived hydrogen peroxide.

Mast cells and macrophages live in close proximity in vivo and reciprocally regulate one another's function in various ways. Although activated macrophages possess a powerful reactive oxygen species (ROS) generating system, there is conflicting evidence regarding whether mast cells can produce ROS. We used the highly sensitive real-time chemiluminescent probe Pholasin to examine ROS release by peritoneal macrophages and mast cells isolated from OVA-sensitized rats. Macrophages stimulated with PMA (0.8 microM) or ionomycin (1 microM), but not OVA (1 microg/ml), released high-level ROS, levels of which peaked after 3-7 min and declined to baseline levels within 1 h. Superoxide was identified as the major ROS species induced by PMA but not by ionomycin. In contrast, purified mast cells stimulated with PMA released low-level ROS, which was entirely due to the contaminating (2%) macrophages, and did not release any detectable ROS in response to ionomycin or OVA at concentrations that induced degranulation. Stimulation of mixed cell populations with PMA to induce macrophage ROS release led to 50% inhibition of serotonin release from mast cells stimulated 5 min later with OVA. The PMA-induced inhibitory factor was identified as hydrogen peroxide. In conclusion, activated rat peritoneal macrophages but not mast cells produce ROS, and macrophage-derived hydrogen peroxide inhibits mast cell degranulation. The latter could be an important mechanism whereby phagocytic cells regulate mast cell activation and promote resolution of IgE-mediated inflammation.

Animals↗