Andrea J. O'Connor

Increasing the volume of vascularized tissue formation in engineered constructs: an experimental study in rats.

The authors have previously described a model of in vivo tissue generation based on an implanted, microsurgically created vessel loop in a plastic chamber (volume, 0.45 ml) containing a poly(DL-lactic-co-glycolic acid) (PLGA) scaffold. Tissue grew spontaneously in association with an intense angiogenic sprouting from the loop and almost filled the chamber, resulting in a mean amount of tissue in chambers of 0.23 g with no added matrix scaffold and 0.33 g of tissue in PLGA-filled chambers after 4 weeks of incubation. The aim of the present study was to investigate whether a greater volume of tissue could be generated when the same-size vessel loop was inserted into a larger (1.9 ml) chamber. In four groups of five rats, an arteriovenous shunt sandwiched between two disks of PLGA, used as a scaffold for structural support, was placed inside a large polycarbonate growth chamber. Tissue and PLGA weight and volume, as well as histological characteristics of the newly formed tissue, were assessed at 2, 4, 6, and 8 weeks. Tissue weight and volume showed a strong linear correlation. Tissue weight increased progressively from 0.13 +/- 0.04 g at 2 weeks to 0.57 +/- 0.06 g at 6 weeks (p < 0.0005). PLGA weight decreased progressively from 0.89 +/- 0.07 g at 2 weeks to 0.20 +/- 0.09 g at 8 weeks (p < 0.0005). Histological examination of the specimens confirmed increased tissue growth and maturation over time. It is concluded that larger quantities of tissue can be grown over a longer period of time by using larger-size growth chambers.

Multilayered microspheres for the controlled release of growth factors in tissue engineering.

Tissue regeneration may be stimulated by growth factors but to be effective, this delivery must be sustained and requires delivery vehicles that overcome the short half-life of these molecules in vivo. One promising approach is to couple growth factors to the biomaterial surface so that they are readily bioavailable. Here the layer-by-layer process was used to construct a multilayered polyelectrolyte delivery system on the surface of poly(lactic-co-glycolic) acid constructs. The system was first optimized on a planar surface before translation to a 3D microsphere system. The layers incorporated heparin to facilitate the loading of basic fibroblast growth factor and increase growth factor stability. Cross-linked capping layers also reduced any burst release. The model growth factor was released in a sustained manner and stimulated significantly higher cell proliferation in vitro on release compared with the addition of the growth factor heparin complex free in solution, demonstrating the promise of this approach.

Long-term stability of adipose tissue generated from a vascularized pedicled fat flap inside a chamber.

Background: Numerous studies demonstrate the generation and short-term survival of adipose tissue; however, long-term persistence remains elusive. This study evaluates long-term survival and transferability of de novo adipose constructs based on a ligated vascular pedicle and tissue engineering chamber combination. Methods: Defined adipose tissue flaps were implanted into rats in either intact or perforated domed chambers. In half of the groups, the chambers were removed after 10 weeks and the constructs transferred on their vascular pedicle to a new site, where they were observed for a further 10 weeks. In the remaining groups, the tissue construct was observed for 20 weeks inside the chamber. Tissue volume was assessed using magnetic resonance imaging and histologic measures, and constructs were assessed for stability and necrosis. Sections were assessed histologically and for proliferation using Ki-67. Results: At 20 weeks, volume analysis revealed an increase in adipose volume from 0.04 ± 0.001 ml at the time of insertion into the chambers to 0.27 ± 0.004 ml in the closed and 0.44 ± 0.014 ml in the perforated chambers. There was an additional increase of approximately 10 to 15 percent in tissue volume in flaps that remained in chambers for 20 weeks, whereas the volume of the transferred tissue not in chambers remained unaltered. Histomorphometric assessment of the tissues documented no signs of hypertrophy, fat necrosis, or atypical changes of the newly generated tissue. Conclusion: This study presents a promising new method of generating significant amounts of mature, vascularized, stable, and transferable adipose tissue for permanent autologous soft-tissue replacement.

In situ formation of antimicrobial silver nanoparticles and the impregnation of hydrophobic polycaprolactone matrix for antimicrobial medical device applications

Bacterial infection associated with medical devices remains a challenge to modern medicine as more patients are being implanted with medical devices that provide surfaces and environment for bacteria colonization. In particular, bacteria are commonly found to adhere more preferably to hydrophobic materials and many of which are used to make medical devices. Bacteria are also becoming increasingly resistant to common antibiotic treatments as a result of misuse and abuse of antibiotics. There is an urgent need to find alternatives to antibiotics in the prevention and treatment of device-associated infections world-wide. Silver nanoparticles have emerged as a promising non-drug antimicrobial agent which has shown effectiveness against a wide range of both Gram-negative and Gram-positive pathogen. However, for silver nanoparticles to be clinically useful, they must be properly incorporated into medical device materials whose wetting properties could be detrimental to not only the incorporation of the hydrophilic Ag nanoparticles but also the release of active Ag ions. This study aimed at impregnating the hydrophobic polycaprolactone (PCL) polymer, which is a FDA-approved polymeric medical device material, with hydrophilic silver nanoparticles. Furthermore, a novel approach was employed to uniformly, incorporate silver nanoparticles into the PCL matrix in situ and to improve the release of Ag ions from the matrix so as to enhance antimicrobial efficacy.

Electrophoretic mobilities of proteins and protein mixtures in porous membranes

Abstract The electrophoresis of bovine serum albumin (BSA) and bovine haemoglobin (BHb) through microfiltration membranes has been investigated using an apparatus consisting of two well-stirred chambers separated by a microfiltration membrane, with electrode compartments at each end. The electrophoretic mobilities of the proteins were determined under conditions of zero concentration driving force and with negligible adsorption on to the membranes occurring during an experiment. Using a track-etched membrane of 9% porosity and 0.2 μm pore size, the initial mobilities of both proteins were close to published free-solution values at the same pH, after correction for the ionic strength of the buffer; this was so because the effect of reduced free area in the membrane was compensated by a corresponding increase in the potential gradient. Similar values were also obtained for BHb with four other microfiltration membranes of 70–75% porosity and 0.10 to 0.22 μm pore size, made from various materials; however BHb mobilities 26% lower were obtained with a membrane of 0.025 μm pore size. Data were also obtained for mixtures of the two proteins; these indicated, as expected, that interactions occurred when they were oppositely charged, changing the apparent mobilities from the free solution values. The single protein data were interpreted using a phenomenological model, which incorporated the effects of electroosmotic flow resulting from membrane surface charge, hydrodynamic drag, pressure-driven back-flow and back-diffusion. The mixed protein data were modelled using the Maxwell—Stefan relations, with promising results.

Amphiphilic core cross-linked star polymers as water-soluble, biocompatible and biodegradable unimolecular carriers for hydrophobic drugs

Unimolecular polymeric architectures are ideal candidates for drug encapsulation. In this study we report the facile yet well-controlled formation of a series of biocompatible and biodegradable core cross-linked star (CCS) polymers via the easily scalable, metal free, one- or two- step ring-opening polymerization (ROP) of e-caprolactone, using poly(ethylene glycol) (PEG) as initiator and [4,4′-bioxepane]-7,7′-dione (BOD) as cross-linker. The resulting CCS polymers, which exhibit hydrophilic PEG blocks in their outer shell and hydrophobic poly(e-caprolactone) (PCL) and BOD segments in their inner core, are water-soluble and amphiphilic and exist in a unimolecular state, both in organic solvents and in water. These properties provide the opportunity to easily stabilise water-insoluble, hydrophobic drugs in aqueous environments without the need for conjugation of the drug to the carrier and/or complex encapsulation techniques. The impact of hydrophilic/hydrophobic block length and core size on polymer properties was investigated via gel permeation chromatography (GPC) and dynamic light scattering (DLS). In addition, the change in drug encapsulation properties with varying hydrophilic/hydrophobic balance was studied using pirarubicin – a potent anthracycline – as a model hydrophobic drug. Formation of a drug–CCS polymer conjugate purely based on hydrophobic–hydrophobic interaction of the drug and the hydrophobic component of the CCS was verified by 1H NMR and UV-Vis measurements, and the size change confirmed by DLS and transmission electron microscopy (TEM). The in vitro study of drug–CCS conjugate demonstrated significantly faster release of anthracycline from the CCS polymer under acidic conditions (pH = 5.5) compared with normal physiological pH level (7.4). Furthermore, cytotoxicity and cellular uptake tests performed using Hela cells, demonstrated extremely low toxicity of the macroinitiators and CCS polymers even at high concentrations, while anthracycline-loaded CCS polymers exhibited similar IC50 values to the free drug. Confocal laser scanning microscopy and flow cytometry confirmed high uptake efficiency and intracellular localisation of the CCS polymers upon uptake, respectively.

Coating and release of an anti-inflammatory hormone from PLGA microspheres for tissue engineering †

Many biomaterials used in tissue engineering cause a foreign body response in vivo, which left untreated can severely reduce the effectiveness of tissue regeneration. In this study, an anti-inflammatory hormone α-melanocyte stimulating hormone (α-MSH) was physically adsorbed to the surface of biodegradable poly (lactic-co-glycolic) acid (PLGA) microspheres to reduce inflammatory responses to this material. The stability and adsorption isotherm of peptide binding were characterized. The peptide secondary structure was not perturbed by the adsorption and subsequent desorption process. The α-MSH payload was released over 72 h and reduced the expression of the inflammatory cytokine, Tumor necrosis factor-α (TNF-α) in lipopolysaccharide activated RAW 264.7 macrophage cells, indicating that the biological activity of α-MSH was preserved. α-MSH coated PLGA microspheres also appeared to reduce the influx of inflammatory cells in a subcutaneous implantation model in rats. This study demonstrates the potential of α-MSH coatings for anti-inflammatory delivery and this approach may be applied to other tissue engineering applications.

Porous PLGA microspheres tailored for dual delivery of biomolecules via layer-by-layer assembly

Tissue engineering is a complex and dynamic process that requires varied biomolecular cues to promote optimal tissue growth. Consequently, the development of delivery systems capable of sequestering more than one biomolecule with controllable release profiles is a key step in the advancement of this field. This study develops multilayered polyelectrolyte films incorporating alpha-melanocyte stimulating hormone (α-MSH), an anti-inflammatory molecule, and basic fibroblast growth factor (bFGF). The layers were successfully formed on macroporous poly lactic-co-glycolic acid microspheres produced using a combined inkjet and thermally induced phase separation technique. Release profiles could be varied by altering layer properties including the number of layers and concentrations of layering molecules. α-MSH and bFGF were released in a sustained manner and the bioactivity of α-MSH was shown to be preserved using an activated macrophage cell assay in vitro. The system performance was also tested in vivo subcutaneously in rats. The multilayered microspheres reduced the inflammatory response induced by a carrageenan stimulus 6 weeks after implantation compared to the non-layered microspheres without the anti-inflammatory and growth factors, demonstrating the potential of such multilayered constructs for the controlled delivery of bioactive molecules.

Adsorption of lysozyme and trypsin onto mesoporous silica materials

Abstract The adsorption of lysozyme and trypsin by the mesoporous materials MCM-41 and SB A- 15 with different pore volumes has been studied as a model protein adsorption system. The amounts adsorbed depend on the pore volumes and surface chemistry of the mesoporous materials as well as the binding mechanisms of particular proteins. The adsorbed amounts of lysozyme on MCM-41 were 90-110 mg/g over a wide range of lysozyme solution concentrations, whereas the adsorption isotherm of trypsin on MCM-41 fitted the Langmuir model and adsorbed amounts of up to 200 mg/g were recorded.

Physicochemical and cytotoxicity analysis of glycerol monoolein-based nanoparticles

Lyotropic liquid crystalline dispersions, such as cubosomes, have been proposed as potential drug delivery vehicles. A recently described ‘salt induced’ method of cubosome production may be suitable for the encapsulation of macromolecular bioactive therapeutics, such as proteins, within the cubic phase. Here, we develop and characterise glycerol-monoolein (GMO)-based cubosomes using this novel method of cubosome production. Using the anionic biological lipid 1,2-dipalmitoyl phosphatidylserine (DPPS) to prevent GMO from forming its natural cubic-phase, we validate that addition of phosphate buffered saline (PBS) can be used to reverse the effects of DPPS. However, this transition is dependent on the type of Pluronic® block copolymer stabiliser used to prevent re-flocculation of the cubosome dispersions. Using small angle X-ray scattering (SAXS) and cryogenic transmission electron microscopy, we show that the ‘salt induced’ phase transition, from small unilamellar vesicles to cubosomes, is inhibited when using Pluronic® F127. In contrast, using the larger, more hydrophilic stabiliser Pluronic® F108, cubosomes can be formed, although further analysis using SAXS suggests these GMO-based cubosomes are less thermally stable than those comprising GMO alone. In addition, we find no significant difference in the in vitro cytotoxicity of cubosome dispersions formed using either of these stabilisers, or between those containing DPPS and those without. The ability to control cubic phase transitions may present an opportunity for the incorporation of therapeutically relevant proteins in these nanoparticles.