Co-delivery of VEGF and amoxicillin using LP-coated co-axial electrospun fibres for the potential treatment of diabetic wounds.

Diabetic complications present throughout a wide range of body tissues, however one of the most widely recognised complications remains to be chronic diabetic wounds. Current treatment options largely rely on standard wound treatment routines which provide no promotion of wound healing mechanisms at different physiological stages of repair. Recently materials produced using novel additive manufacturing techniques have been receiving attention for applications in wound care and tissue repair. Additive manufacturing techniques have recently been used in the interest of targeted drug delivery and production of novel materials resembling characteristics of native tissues. The potential to exploit these highly tailorable manufacturing techniques for the design of novel wound care remedies is highly desirable. In the present study two additive manufacturing techniques are combined to produce a scaffold for the treatment of diabetic wounds. The combination of microfluidic manufacturing of an antimicrobial liposome (LP) formulation and a coaxial electrospinning method incorporating both antimicrobial and proangiogenic factors allowed dual delivery of therapeutics to target both infection and lack of vascularisation at wound sites. The coaxial fibres comprised of a polyvinyl alcohol (PVA) core containing vascular endothelial growth factor (VEGF) and a poly (l-lactide-co-ε-caprolactone) (PLCL) shell blended with amoxicillin (Amox). Additionally, a liposomal formulation was produced to incorporate Amox and adhered to the surface of fibres loaded with Amox and VEGF. The liposomal loading provided the potential to deliver a much higher, more clinically relevant dose of Amox without detrimentally changing the mechanical properties of the material. The growth factor release was sustained up to 7-days in vitro. The therapeutic effect of the antibiotic loading was analysed using a disk diffusion method with a significant increase in zone diameter following LP adhesion, proving the full scaffold system had improved efficacy against both Gram-positive and Gram-negative strains. Additionally, the dual-loaded scaffolds show enhanced potential for supporting vascular growth in vitro, as demonstrated via a viability assay and tubule formation studies. Results showed a significant increase in the average total number of tubes from 10 in control samples to 77 in samples fully-loaded with Amox and VEGF.

Liposomal encapsulation of amoxicillin via microfluidics with subsequent investigation of the significance of PEGylated therapeutics.

With an increasing concern of global antimicrobial resistance, the efforts to improve the formulation of a narrowing library of therapeutic antibiotics must be confronted. The liposomal encapsulation of antibiotics using a novel and sustainable microfluidic method has been employed in this study to address this pressing issue, via a targeted, lower-dose medical approach. The study focusses upon microfluidic parameter optimisation, formulation stability, cytotoxicity, and future applications. Particle sizes of circa. 130 nm, with viable short-term (28-day) physical stability were obtained, using two different non-cytotoxic liposomal formulations, both of which displayed suitable antibacterial efficacy. The microfluidic method allowed for high encapsulation efficiencies (≈77 %) and the subsequent in vitro release profile suggested high limits of antibiotic dissociation from the nanovessels, achieving 90% release within 72 h. In addition to the experimental data, the growing use of poly(ethylene) glycol (PEG) within lipid-based formulations is discussed in relation to anti-PEG antibodies, highlighting the key pharmacological differences between PEGylated and non-PEGylated formulations and their respective advantages and drawbacks. It's surmised that in the case of the formulations used in this study, the addition of PEG upon the liposomal membrane would still be a beneficial feature to possess owing to beneficial features such as stability, antibiotic efficacy and the capacity to further modify the liposomal membrane.

A comparison of gold nanoparticle surface co-functionalization approaches using Polyethylene Glycol (PEG) and the effect on stability, non-specific protein adsorption and internalization.

To create clinically useful gold nanoparticle (AuNP) based cancer therapeutics it is necessary to co-functionalize the AuNP surface with a range of moieties; e.g. Polyethylene Glycol (PEG), peptides and drugs. AuNPs can be functionalized by creating either a mixed monolayer by attaching all the moieties directly to the surface using thiol chemistry, or by binding groups to the surface by means of a bifunctional polyethylene glycol (PEG) linker. The linker methodology has the potential to enhance bioavailability and the amount of functional agent that can be attached. While there is a large body of published work using both surface arrangements independently, the impact of attachment methodology on stability, non-specific protein adsorption and cellular uptake is not well understood, with no published studies directly comparing the two most frequently employed approaches. This paper compares the two methodologies by synthesizing and characterizing PEG and Receptor Mediated Endocytosis (RME) peptide co-functionalized AuNPs prepared using both the mixed monolayer and linker approaches. Successful attachment of both PEG and RME peptide using the two methods was confirmed using Dynamic Light Scattering, Fourier Transform Infrared Spectroscopy and gel electrophoresis. It was observed that while the 'as synthesized' citrate capped AuNPs agglomerated under physiological salt conditions, all the mixed monolayer and PEG linker capped samples remained stable at 1M NaCl, and were stable in PBS over extended periods. While it was noted that both functionalization methods inhibited non-specific protein attachment, the mixed monolayer samples did show some changes in gel electrophoresis migration profile after incubation with fetal calf serum. PEG renders the AuNP stable in-vivo however, studies with MDA-MB-231 and MCF 10A cell lines indicated that functionalization with PEG, blocks cellular uptake. It was observed that co-functionalization with RME peptide using both the mixed monolayer and PEG linker methods greatly enhanced cellular internalization compared to PEG capped AuNPs.

Surface Modified Biodegradable Electrospun Membranes as a Carrier for Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells.

Human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells are currently undergoing clinical trials to treat retinal degenerative diseases. Transplantation of hESC-RPE cells in conjuction with a supportive biomaterial carrier holds great potential as a future treatment for retinal degeneration. However, there has been no such biodegradable material that could support the growth and maturation of hESC-RPE cells so far. The primary aim of this work was to create a thin porous poly (L-lactide-co-caprolactone) (PLCL) membrane that could promote attachment, proliferation, and maturation of the hESC-RPE cells in serum-free culture conditions. The PLCL membranes were modified by atmospheric pressure plasma processing and coated with collagen IV to enhance cell growth and maturation. Permeability of the membranes was analyzed with an Ussing chamber system. Analysis with scanning electron microscopy, contact angle measurement, atomic force microscopy, and X-ray photoelectron spectroscopy demonstrated that plasma surface treatment augments the surface properties of the membrane, which enhances the binding and conformation of the protein. Cell proliferation assays, reverse transcription-polymerase chain reaction, indirect immunofluoresence staining, trans-epithelial electrical resistance measurements, and in vitro phagocytosis assay clearly demonstrated that the plasma treated PLCL membranes supported the adherence, proliferation, maturation and functionality of hESC-RPE cells in serum-free culture conditions. Here, we report for the first time, how PLCL membranes can be modified with atmospheric pressure plasma processing to enable the formation of a functional hESC-RPE monolayer on a porous biodegradable substrate, which have a potential as a tissue-engineered construct for regenerative retinal repair applications.

Calcium phosphate thin films enhance the response of human mesenchymal stem cells to nanostructured titanium surfaces.

The development of biomaterial surfaces possessing the topographical cues that can promote mesenchymal stem cell recruitment and, in particular, those capable of subsequently directing osteogenic differentiation is of increasing importance for the advancement of tissue engineering. While it is accepted that it is the interaction with specific nanoscale topography that induces mesenchymal stem cell differentiation, the potential for an attendant bioactive chemistry working in tandem with such nanoscale features to enhance this effect has not been considered to any great extent. This article presents a study of mesenchymal stem cell response to conformal bioactive calcium phosphate thin films sputter deposited onto a polycrystalline titanium nanostructured surface with proven capability to directly induce osteogenic differentiation in human bone marrow-derived mesenchymal stem cells. The sputter deposited surfaces supported high levels of human bone marrow-derived mesenchymal stem cell adherence and proliferation, as determined by DNA quantification. Furthermore, they were also found to be capable of directly promoting significant levels of osteogenic differentiation. Specifically, alkaline phosphatase activity, gene expression and immunocytochemical localisation of key osteogenic markers revealed that the nanostructured titanium surfaces and the bioactive calcium phosphate coatings could direct the differentiation towards an osteogenic lineage. Moreover, the addition of the calcium phosphate chemistry to the topographical profile of the titanium was found to induce increased human bone marrow-derived mesenchymal stem cell differentiation compared to that observed for either the titanium or calcium phosphate coating without an underlying nanostructure. Hence, the results presented here highlight that a clear benefit can be achieved from a surface engineering strategy that combines a defined surface topography with an attendant, conformal bioactive chemistry to enhance the direct osteogenic differentiation of human bone marrow-derived mesenchymal stem cells.

Mesenchymal stem cell response to conformal sputter deposited calcium phosphate thin films on nanostructured titanium surfaces.

Biomaterial surfaces that can directly induce the osteogenic differentiation of mesenchymal stem cells (MSCs) present an exciting strategy for bone tissue engineering and offers significant benefits for improving the repair or replacement of damaged or lost bone tissue. In this study, titanium nanostructures with distinctive topographical features were produced by radio frequency magnetron sputtering. The response of MSCs to the nanostructured titanium (Ti) surfaces before and after augmentation by a sputter deposited calcium phosphate (CaP) coating has been investigated. The sputtered CaP has the characteristics of a calcium enriched hydroxyapatite surface layer, as determined by X-ray photoelectron spectroscopy and X-ray diffraction studies. The sputter deposited Ti has a polycrystalline surface morphology, as confirmed by atomic force microscopy, and CaP layers deposited thereon (TiCaP) conform to this topography. The effects of these surfaces on MSC focal adhesion formation, actin cytoskeleton organization and Runx2 gene expression were examined. The Ti and TiCaP surfaces were found to promote changes in MSC morphology and adhesion known to be associated with subsequent downstream osteogenic differentiation; however, the equivalent events were not as pronounced on the CaP surface. A significant increase in Runx2 expression was observed for CaP compared to Ti, but no such difference was seen between either Ti and TiCaP, nor CaP and TiCaP. Importantly, the Ti surface engendered the expected contribution of nanoscale features to the MSC response; moreover, the CaP layer when used in combination with this topography has been found to cause no adverse effects in respect of MSC behavior.

Patterned cell culture substrates created by hot embossing of tissue culture treated polystyrene.

Patterning materials such that they elicit a different cell response in different regions would have significant implications in fields such as implantable biomaterials, in vitro cell culture and tissue engineering and regenerative medicine. Moreover, the ability to pattern polymers using inexpensive, currently available processes, without the need for adding proteins or other biochemical agents could lead to new opportunities in biomaterials research. The research reported here demonstrates that by combining the plasma surface treatments used to create commercial grade tissue culture treated polystyrene, with controlled hot embossing processes, that distinct regions can be created on a substrate that result in spatial control of endothelial cell adhesion and proliferation. As well as the topographical changes that result from hot embossing, significant changes in surface chemistry and wettability have been observed and characterised and the resultant effects on endothelial cell responses evaluated. By spatially controlling endothelial cell adhesion, proliferation and subsequent angiogenesis, the processes outlined here have the potential to be used to create a range of different substrates, with applications in the development of assays for high throughput screening, the patterning of implantable biomaterials or the development of smart scaffolds for tissue engineering.

Atmospheric pressure plasma induced grafting of poly(ethylene glycol) onto silicone elastomers for controlling biological response.

This study investigates the role that surface functionalisation of silicone elastomer (SE) by atmospheric pressure plasma induced graft immobilisation of poly(ethylene glycol) methyl ether methacrylate (PEGMA) plays in the attendant biological response. SE is used in modern ophthalmic medical devices and samples of the material were initially plasma treated using a dielectric barrier discharge reactor (DBD) to introduce reactive oxygen functionalities, prior to in situ grafting of two molecular weights of PEGMA (MW 1000 Da: PEGMA(1000), MW 2000 Da: PEGMA(2000)). The variously processed surfaces were characterised by water contact angle analysis, X-ray photoelectron spectroscopy, time-of-flight secondary ion mass spectrometry and atomic force microscopy. Lens epithelial cells were then cultured on the PEGMA grafted SE surfaces. It was found that cells on the pristine surface were not well spread and had shrunken morphology. On the DBD pre-treated surfaces, the cells were well spread. On the PEGMA(1000) surface, the cells displayed evidence of shrinkage and were on the verge of detaching. Remarkably, on the PEGMA(2000) surface, no cell adhesion was detection. Bacterial adhesion to the surfaces was studied using Staphylococcus aureus NTC8325. There was no difference in the number of bacteria adhering to any of the surfaces studied.

Assessment of an osteoblast-like cell line as a model for human primary osteoblasts using Raman spectroscopy.

Raman spectroscopy is employed to determine the suitability of the U20S osteoblast-like cell line for use as a model for human primary osteoblasts, with emphasis on the ability of these cell types to replicate their tissue of origin. It was found that both cell types demonstrated early stage mineral deposition that followed significantly different growth patterns. Analysis of the growth pattern and spectral data from primary cells revealed increasing bone quality ratios and a high crystallinity, consistent with previous reports. Conversely the investigation of the U20S osteoblast-like cell line provided evidence of dense multilayered mineralised regions that corresponded more closely to native bone in terms of its crystallinity and bone quality ratios. This finding contradicts previous reports on U20S osteoblast-like cells which have consistently described them as non-osteoinductive when cultured in various conditions on a number of substrates. This work demonstrates the successful application of Raman spectroscopy combined with biological and multivariate analysis for the investigation of osteoblast-like U20S cells and human primary osteoblasts, specifically with focus on the osteoinductive ability of the osteoblast-like cell line and the comparative differences in relation to the primary osteoblasts.

Osteoblast-like cell response to calcium phosphate coating chemistry and morphology on etched silicon surfaces.

Being able to control the behaviour of osteoblast-like cells on a surface may provide a genuine insight into the material surface characteristics and help in creating a successful coating/cell interface. The possibility of creating a micro-environment that can induce proliferation, differentiation and mineralisation of bone cells in vitro, by successfully combining both chemistry and topography of a micro-fabricated substrate is an area that requires a multi-disciplinary approach. Utilising sputter deposition, a process that lends itself to high processability, in conjunction with photolithography allowing for the creation of highly repeatable etched surfaces, we aim to provide a successful combination of chemistry and topography. Correlating the substrate conditions with resultant osteoblast biological function and activity can ultimately be used with a view to modulating the behavior of osteoblast-like cells in vitro.

Raman spectroscopic monitoring of the osteogenic differentiation of human mesenchymal stem cells.

The differentiation of stem cells into multi-lineages is essential to aid the development of tissue engineered materials that replicate the functionality of their tissue of origin. For this study, Raman spectroscopy was used to monitor the formation of a bone-like apatite mineral during the differentiation of human mesenchymal stem cells (hMSCs) towards an osteogenic lineage. Raman spectroscopy observed dramatic changes in the region dominated by the stretching of phosphate groups (950-970 cm(-1)) during the period of 7-28 days. Changes were also seen at 1030 cm(-1) and 1070 cm(-1), which are associated with the P-O symmetric stretch of PO(4)(3-) and the C-O vibration in the plane stretch of CO(3)(2-). Multivariate factor analysis revealed the presence of various mineral species throughout the 28 day culture period. Bone mineral formation was observed first at day 14 and was identified as a crystalline, non-substituted apatite. During the later stages of culture, different mineral species were observed, namely an amorphous apatite and a carbonate, substituted apatite, all of which are known to be Raman markers for a bone-like material. Band area ratios revealed that both the carbonate-to-phosphate and mineral-to-matrix ratios increased with age. When taken together, these findings suggest that the osteogenic differentiation of hMSCs at early stages resembles endochondral ossification. Due to the various mineral species observed, namely a disordered amorphous apatite, a B-type carbonate-substituted apatite and a crystalline non-substituted hydroxyapatite, it is suggested that the bone-like mineral observed here can be compared to native bone. This work demonstrates the successful application of Raman spectroscopy combined with biological and multivariate analyses for monitoring the various mineral species, degree of mineralisation and the crystallinity of hMSCs as they differentiate into osteoblasts.

Special issue: select papers from the 29th Northern Ireland Biomedical Engineering Conference, Belfast, UK, April 2009.

Each year, NIBES hosts a spring conference that is jointly organised by Queen's University of Belfast and University of Ulster. The 29th NIBES Spring meeting took place on 8th April 2009 at Queen's University of Belfast. NIBES 2009 had an impressive scientific program with two international leading plenary speakers and 28 oral presentations.

Lens epithelial cell response to atmospheric pressure plasma modified poly(methylmethacrylate) surfaces.

Selective control of cellular response to polymeric biomaterials is an important consideration for many ocular implant applications. In particular, there is often a need to have one surface of an ophthalmic implant capable of promoting cell attachment while the other needs to be resistant to this effect. In this study, an atmospheric pressure dielectric barrier discharge (DBD) has been used to modify the surface region of poly(methyl methacrylate) (PMMA), a well established ocular biomaterial, with the aim of promoting a controlled response to human lens epithelial cells (LEC) cultured thereon. The DBD plasma discharge environment has also been employed to chemically graft a layer of poly(ethylene glycol) methyl ether methacrylate (PEGMA) onto the PMMA and the response to LEC likewise determined. Two different molecular weights of PEGMA, namely 1000 and 2000 MW were used in these experiments. The LEC response to DBD treated polystyrene (PS) samples has also been examined as a positive control and to help to further elucidate the nature of the modified surfaces. The LEC adhered and proliferated readily on the DBD treated PMMA and PS surfaces when compared to the pristine polymer samples which showed little or no cell response. The PMMA and PS surfaces that had been DBD grafted with the PEGMA(1000) layer were found to have some adhered cells. However, on closer inspection, these cells were clearly on the verge of detaching. In the case of the PEGMA(2000) grafted surfaces no cells were observed indicating that the higher molecular weight PEGMA has been able to attain a surface conformation that is capable of resisting cell attachment in vitro.

Protein adhesion and cell response on atmospheric pressure dielectric barrier discharge-modified polymer surfaces.

Gaseous plasma discharges are one of the most common means to modify the surface of a polymer without affecting its bulk properties. However, this normally requires the materials to be processed in vacuo to create the active species required to permanently modify the surface chemistry. The ability to invoke such changes under normal ambient conditions in a cost-effective manner has much to offer to enhance the response of medical implants in vivo. It is therefore important to accurately determine the nature and scale of the effects derived from this technology. This paper reports on the modification of poly(styrene) (PS) and poly(methyl methacrylate) (PMMA) using atmospheric pressure plasma processing via exposure to a dielectric barrier discharge (DBD). The changes in surface chemistry and topography after DBD treatment were characterised using water contact angle, X-ray photoelectron spectroscopy (XPS) and atomic force microscopy. A marked increase in the surface oxygen concentration was observed for both PMMA and PS. An increase in surface roughness was observed for PMMA, but not for PS. These changes were found to result in an increase in surface wettability for both polymers. Adsorption of albumin (Alb) onto these substrates was studied using XPS and quartz crystal microbalance with dissipation (QCM-D). The rate of adsorption of Alb onto pristine PMMA and PS was faster than that on the DBD-treated polymers. XPS indicated that a similar concentration of Alb occurred on both of the treated surfaces. Deconvolution of the C1s XPS spectra showed that Alb is adsorbed differently on pristine (hydrophobic) compared to DBD-treated (hydrophilic) surfaces, with more polar functional groups oriented towards the upper surface in the latter case. The QCM-D data corroborates this finding, in that a more viscoelastic layer of Alb was formed on the DBD-treated surfaces relative to that on the pristine surfaces. It was also found that Alb was more easily replaced by larger proteins from foetal bovine serum on the DBD-treated surfaces. The viability of human lens epithelial cells on both of the DBD-treated polymer surface was significantly (P<0.05) greater than on the respective pristine surfaces. In addition, cells that adhered to the treated polymers exhibited a polygonal morphology with well spread actin stress fibres compared with the contracted shape displayed on the pristine surfaces. The results presented here clearly indicate that DBD surface modification has the capability to influence key protein and cell responses.

Biological responses to hydroxyapatite surfaces deposited via a co-incident microblasting technique.

Hydroxyapatite (HA) is routinely used as a coating on a range of press-fit (cementless) orthopaedic implants to enhance their osseointegration. The standard plasma spraying method used to deposit a HA surface layer on such implants often contains unwanted crystal phases that can lead to coating delamination in vivo. Consequently, there has been a continuous drive to develop alternate surface modification technologies that can eliminate the problems caused by a non-optimal coating process. In this study two methods for creating a HA layer on metal alloys that employ micro-blasting have been evaluated to determine if the inclusion of an abrasive agent can enhance the in vitro and in vivo performance of the modified surface. The first method employs direct micro-blasting using HA as the abrasive media, while the second employs a simultaneous blasting with an alumina abrasive and coincident blasting with HA as a dopant. Whereas, both methods were found to produce a surface which was enriched with HA, the respective microstructures created were significantly different. Detailed surface characterisation revealed that the use of the abrasive produced disruption of the metal surface without producing detectable incorporation of alumina particles. Roughening of the metal surface in this way breached the passivating oxide layer and created sites which subsequently provided for impregnation, mechanical interlocking and chemical bonding of HA. The co-incident use of an alumina abrasive and a HA dopant resulted in a stable surface that demonstrated enhanced in vitro osteoblast attachment and viability as compared to the response to the surface produced using HA alone or the metal substrate control. Implantation of the surface produced by co-incident blasting with alumina and HA in a rabbit model confirmed that this surface promoted the in vivo formation of early stage lamellar bone growth.

Patients with long bone fracture have altered Caveolin-1 expression in their peripheral blood mononuclear cells.

INTRODUCTION: Fracture triggers a cascade of systemic and local responses including inflammatory mediator signaling, chemotaxis, osteogenic cell recruitment, differentiation and proliferation at the fracture site. Early signaling between immune cells and repair cells in fracture repair is not well understood. Caveolin-1, a 21-24 kDa membrane protein plays key roles in transmembrane signaling. This study was to investigate the expression of caveolin-1 in human peripheral blood mononuclear cells (PBMNCs) following long bone fracture. METHODS: PBMNCs were obtained from healthy volunteers or fracture patients at three time points following fracture by density-gradient-centrifugation procedure. Caveolin-1 gene expression and protein characterization was examined by semi-quantitative RT-PCR, immunocytochemistry and Western blot analysis. RESULTS: Caveolin-1 mRNA and protein was expressed at low levels in the PBMNCs of non-fracture samples. In contrast, caveolin-1 expression was greatly increased in the PBMNCs of fracture patients 9-12 days and reduced at 16-21 days following long bone fracture. CONCLUSION: The identification of caveolin-1 in PBMNCs and osteoblasts makes this cellular domain a new focus for further investigation. We speculate that caveolin-1 expression in PBMNCs and osteoblasts play an important role in signal transduction during the early stages of fracture healing and may be an indicator for normal or abnormal fracture repair.