Mild synthesis of poly(HEMA)-networks as well-defined nanoparticles in supercritical carbon dioxide.

Free-radical dispersion polymerisation of 2-hydroxyethyl methacrylate was carried out in supercritical carbon dioxide (scCO2) in the presence of stabilisers based on polyethylene oxide (PEO) and poly(heptadecafluorodecyl acrylate) (PFDA). Different architectures of copolymers (random, palm-tree and diblock) were tested for their surface tension, cloud point and as a stabilising agent. The diblock architecture was found to be the best candidate resulting in poly(HEMA) spherical particles with a size of 316 nm. Furthermore, the effect of the CO2-phobic block (PEO) in the diblock architecture was investigated by using three different chain lengths (1000, 2000, 5000 g mol-1). By optimizing the stabiliser composition and structure, mild reaction conditions have been identified allowing us to obtain well-defined spherical cross-linked poly(HEMA) particles with a mean diameter of unprecedented low size (216 nm) at a temperature as low as 35 °C.

Block copolymer synthesis by controlled/living radical polymerisation in heterogeneous systems.

Nanostructured soft materials open up new opportunities in material design and application, and block copolymer self-assembly is one particularly powerful phenomenon that can be exploited for their synthesis. The advent of controlled/living radical polymerisation (CLRP) has greatly simplified block copolymer synthesis, and versatility towards monomer types and polymer architectures across the different forms of CLRP has vastly expanded the range of functional materials accessible. CLRP-controlled synthesis of block copolymers has been applied in heterogeneous systems, motivated by the numerous process advantages and the position of emulsion polymerisation at the forefront of industrial latex synthesis. In addition to the inherent environmental advantages of heterogeneous routes, the incidence of block copolymer self-assembly within dispersed particles during polymerisation leads to novel nanostructured materials that offer enticing prospects for entirely new applications of block copolymers. Here, we review the range of block copolymers prepared by heterogeneous CLRP techniques, evaluate the methods applied to maximise purity of the products, and summarise the unique nanoscale morphologies resulting from in situ self-assembly, before discussing future opportunities within the field.

Porous inverse vulcanised polymers for mercury capture.

Supercritical carbon dioxide is used to generate macroporosity in an inverse vulcanised polymer, which shows excellent promise for enhanced mercury capture and filtration from water.

PEGylated Biodegradable Polyesters for PGSS Microparticles Formulation: Processability, Physical and Release Properties.

BACKGROUND: Particles from Gas Saturated Solution (PGSS) is an emergent method that employs supercritical carbon dioxide (scCO2) to produce microparticles. It is suitable for encapsulating biologically active compounds including therapeutic peptides and proteins. Poly(lactide acid) (PLA) and/or poly(lactic-coglycolic acid) (PLGA) are the most commonly used materials in PGSS, due to their good processability in scCO2. Previous studies demonstrated that the properties of the microparticles can be modulated by adding polyethylene glycol (PEG) or tri-block PEGylated copolymers. OBJECTIVE: In the present work, the effect of the addition of biodegradable PEGylated di-block copolymers on the physical properties and drug release performance of microparticles prepared by PGSS technique was evaluated. METHOD: mPEG5kDa-P(L)LA and mPEG5kDa-P(L)LGA with similar molecular weights were synthesized and their behaviour, when exposed to supercritical CO2, was investigated. Different microparticle formulations, composed of a high (81%) or low (9%) percentage of the synthesized copolymers were prepared and compared in terms of particle size distribution, morphology, yield and protein release. Drug release studies were performed using bovine serum albumin (BSA) as a model protein. RESULTS: PEGylated copolymers showed good processability in PGSS without significant changes to the physical properties of the microparticles. However, the addition of PEG exerted a modulating effect on the microparticle drug dissolution behaviour, increasing the rate of BSA release as a function of its content in the formulation. CONCLUSION: This study demonstrated the feasibility of producing microparticles by using PEGylated di-block copolymers through a PGSS technique at mild operating conditions (low operating pressure and temperature).

Green process for green materials: viable low-temperature lipase-catalysed synthesis of renewable telechelics in supercritical CO2.

We present a novel near-ambient-temperature approach to telechelic renewable polyesters by exploiting the unique properties of supercritical CO(2) (scCO(2)). Bio-based commercially available monomers have been polymerized and functional telechelic materials with targeted molecular weight prepared by end-capping the chains with molecules containing reactive moieties in a one-pot reaction. The use of scCO(2) as a reaction medium facilitates the effective use of Candida antarctica Lipase B (CaLB) as a catalyst at a temperature as low as 35°C, hence avoiding side reactions, maintaining the end-capper functionality and preserving the enzyme activity. The functionalized polymer products have been characterized by (1)H nuclear magnetic resonance spectroscopy, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, gel permeation chromatography and differential scanning calorimetry in order to carefully assess their structural and thermal properties. We demonstrate that telechelic materials can be produced enzymatically at mild temperatures, in a solvent-free system and using renewably sourced monomers without pre-modification, by exploiting the unique properties of scCO(2). The macromolecules we prepare are ideal green precursors that can be further reacted to prepare useful bio-derived films and coatings.

A comparison of polymer and polymer-hydroxyapatite composite tissue engineered scaffolds for use in bone regeneration. An in vitro and in vivo study.

Previous in vitro work demonstrated porous PLA and PLGA both had the mechanical strength and sustained the excellent skeletal stem cell (SSC) growth required of an osteogenic bonegraft substitute, for use in impaction bone grafting. The purpose of this investigation was to assess the effects of the addition of hydroxyapatite (HA) to the scaffolds before clinical translation. PLA, PLA+10% HA, PLGA, and PLGA+10% HA were milled and impacted into discs before undergoing a standardized shear test. Cellular compatibility analysis followed 14 days incubation with human skeletal stems cells (SSC). The best two performing polymers were taken forward for in vivo analysis. SSC seeded polymer discs were implanted subcutaneously in mice. All polymers had superior mechanical shear strength compared with allograft (p < 0.01). Excellent SSC survival was demonstrated on all polymers, but the PLA polymers showed enhanced osteoblastic activity (ALP assay p < 0.01) and collagen-1 formation. In vivo analysis was performed on PLA and PLA+10% HA. MicroCT analysis revealed increased bone formation on the PLA HA (p < 0.01), and excellent neo-vessel formation in both samples. Histology confirmed evidence of de novo bone formation. PLA HA showed both enhanced osteoinductive and osteogenic capacity. This polymer composite has been selected for scaled-up experimentation before clinical translation.

Supercritical CO2 fluid-foaming of polymers to increase porosity: a method to improve the mechanical and biocompatibility characteristics for use as a potential alternative to allografts in impaction bone grafting?

Disease transmission, availability and cost of allografts have resulted in significant efforts to find an alternative for use in impaction bone grafting (IBG). Recent studies identified two polymers with both structural strength and biocompatibility characteristics as potential replacements. The aim of this study was to assess whether increasing the polymer porosity further enhanced the mechanical and cellular compatibility characteristics for use as an osteogenic biomaterial alternative to allografts in IBG. Solid and porous poly(DL-lactide) (P(DL)LA) and poly(DL-lactide-co-glycolide) (P(DL)LGA) scaffolds were produced via melt processing and supercritical CO(2) foaming, and the differences characterized using scanning electron microscopy (SEM). Mechanical testing included milling and impaction, with comparisons made using a shear testing rig as well as a novel agitation test for cohesion. Cellular compatibility tests for cell number, viability, and osteogenic differentiation using WST-1 assays, fluorostaining, and ALP assays were determined following 14 day culture with skeletal stem cells. SEM showed excellent porosity throughout both of the supercritical-foam-produced polymer scaffolds, with pores between 50 and 200 µm. Shear testing showed that the porous polymers exceeded the shear strength of allograft controls (P<0.001). Agitation testing showed greater cohesion between the particles of the porous polymers (P<0.05). Cellular studies showed increased cell number, viability, and osteogenic differentiation on the porous polymers compared to solid block polymers (P<0.05). The use of supercritical CO(2) to generate porous polymeric biodegradable scaffolds significantly improves the cellular compatibility and cohesion observed compared to non-porous counterparts, without substantial loss of mechanical shear strength. These improved characteristics are critical for clinical translation as a potential osteogenic composite for use in IBG.

Single shot tetanus vaccine manufactured by a supercritical fluid encapsulation technology.

Single shot vaccines of tetanus toxoid (TT) were manufactured using the NanoMix process - a low temperature solvent free encapsulation technology using supercritical fluids. The formulations were injected into mice, and compared to multiple injections of a commercially available alum adsorbed TT vaccine. After 5 months the antibody titres were found to be similar for both the alum adsorbed and microparticle formulations, demonstrating for the first time the potential of formulating antigens in PLA microparticles using the supercritical fluid (NanoMix) technique to produce single shot vaccines. The results are likely to be due to the maintenance of toxoid bioactivity and some degree of sustained release of the encapsulated antigens, resulting in repeated stimulation of antigen presenting cells eliminating the need for multiple immunisations. This demonstrates the potential of this supercritical fluid processing technique to reduce the need for booster doses in a vaccine regimen.

Sustained release hGH microsphere formulation produced by a novel supercritical fluid technology: in vivo studies.

Novel sustained release formulations of hGH prepared by supercritical fluid processing of PLGA/PLA (the CriticalMix process) were produced in the form of microparticles for subcutaneous injection. The basis of the process is that PLGA/PLA polymers liquefy when exposed to supercritical CO(2), thereby allowing the hGH to be mixed efficiently into the polymers at an ambient temperature and in the absence of solvents. The CO(2) was removed from the mixture by depressurisation through a nozzle, resulting in the production of microparticles containing the hGH, which were collected in a cyclone. The best microparticle formulations showed an initial in vitro burst of around 35% and a sustained release over 14 days. When tested in the rat model, which displays a faster clearance rate of hGH than other animal models, two formulations showed prolonged release over 2-3 days with sustained plasma levels at 1-5 ng/ml whereas the soluble hGH formulation was cleared within 24h. Two selected sustained release formulations were tested in cynomolgus monkeys and compared to a single injection of soluble hGH. The burst release from the sustained release formulations was similar in magnitude to a daily dose of hGH and serum hGH levels were maintained for a seven day period. It is probable from the data that the sustained release would have continued for up to 14 days if sampling had been continued. The IGF-1 results showed there was no significant difference between the levels obtained for once daily injection of soluble hGH and the two sustained release formulations.

Effect of age and gender on the number and distribution of sites in Paget's disease of bone.

Paget's disease of bone (PDB) is reportedly declining in prevalence and severity, with increasing numbers of monostotic cases. Some accounts suggest that these findings are more evident in women, and that monostotic disease is unexpectedly frequent at certain sites. We have studied whether birth date or gender is associated with the number of sites affected and with the distribution of sites in monostotic disease and, by reviewing 100 follow-up 99Tc(m) methylene diphosphonate (MDP) scans, whether additional sites appear after initial diagnosis. Scintigraphic scans from 171 male (age 40-91 years) and 179 female (44-88 years) consecutive referrals with PDB were reviewed. Patients were analysed by referral date (1982-1992 and 1993-2001), and by their median date of birth (before (PRE21) and after (POST21) 1921). Mean age of pre-1993 patients was 69 years and 75 years for referrals after 1993. Younger patients had more monostotic disease (POST21 vs PRE21 subjects (47% vs 28%)), with a more marked trend in females (52% vs 25%), and POST21 females had fewer polyostotic sites than males (p<0.05), whereas the number in PRE21 males and females was similar. Monostotic females, but not males, showed an excess of tibial involvement. The spine was less involved in monostotic disease. Follow-up scans in 100 patients revealed no new sites. The incidence of monostotic disease has doubled over the last 30 years, but diminishing site involvement appears to be more marked in females. The lesser involvement at the axial sites in monostotic disease may lead to overestimation of the decline in PDB based on abdominal radiographs.

Putting the fizz into chemistry: applications of supercritical carbon dioxide in tissue engineering, drug delivery and synthesis of novel block copolymers.

This paper describes the recent progress at Nottingham towards the exploitation of the unique properties of scCO(2) (supercritical carbon dioxide) for the preparation of polymeric scaffolds for tissue engineering applications and new devices for controlled drug delivery, as well as the synthesis of novel block copolymers by the combination of eROP (enzymatic ring opening polymerization) and controlled polymerization methods for the potential use as drug carriers.

Supercritical carbon dioxide generated vascular endothelial growth factor encapsulated poly(DL-lactic acid) scaffolds induce angiogenesis in vitro.

The ability to deliver, over time, biologically active vascular endothelial growth factor-165 (VEGF) through tailored designed scaffolds offers tremendous therapeutic opportunities to tissue-engineered therapies. Porous biodegradable poly(DL-lactic) acid (PLA) scaffolds encapsulating VEGF have been generated using supercritical CO2 (scCO2) and the kinetic release and angiogenic activity of these scaffolds examined in vitro and in an ex vivo chick chorioallantoic membrane (CAM) angiogenesis model. After processing through scCO2, VEGF maintained its angiogenic activity as assessed by increased tubule formation of human umbilical vein endothelial cells (HUVEC) cultured on Matrigel (VEGF = 1937 +/- 205 microm; scCO2-VEGF = 2085 +/- 234 microm; control = 1237 +/- 179 microm). VEGF release kinetics from scCO2-VEGF incorporated PLA monolith scaffolds showed a cumulative release of VEGF (2837 +/- 761 rhog/ml) over a 21 day period in culture. In addition, VEGF encapsulated PLA scaffolds increased the blood vessel network in the CAM compared to controls; control, 24.8 +/- 9.6; VEGF/PLA, 44.1 +/- 12.1 (vessels/field). These studies demonstrate that the controlled release of growth factors encapsulated into three-dimensional PLA scaffolds can actively stimulate the rapid development of therapeutic neovascularisation to regenerate or engineer tissues.

The effect of anisotropic architecture on cell and tissue infiltration into tissue engineering scaffolds.

A common phenomenon in tissue engineering is rapid tissue formation on the outer edge of the scaffold which restricts cell penetration and nutrient exchange to the scaffold centre, resulting in a necrotic core. To address this problem, we generated scaffolds with both random and anisotropic open porous architectures to enhance cell and subsequent tissue infiltration throughout the scaffold for applications in bone and cartilage engineering. Hydroxyapatite (HA) and poly(D,L-lactic acid) (P(DL)LA) scaffolds with random open porosity were manufactured, using modified slip-casting and by supercritical fluid processing respectively, and subsequently characterised. An array of porous aligned channels (400 microm) was incorporated into both scaffold types and cell (human osteoblast sarcoma, for HA scaffolds; ovine meniscal fibrochondrocytes, for P(DL)LA scaffolds) and tissue infiltration into these modified scaffolds was assessed in vitro (cell penetration) and in vivo (tissue infiltration; HA scaffolds only). Scaffolds were shown to have an extensive random, open porous structure with an average porosity of 85%. Enhanced cell and tissue penetration was observed both in vitro and in vivo demonstrating that scaffold design alone can influence cell and tissue infiltration into the centre of tissue engineering scaffolds.

The influence of dispersant concentration on the pore morphology of hydroxyapatite ceramics for bone tissue engineering.

There is a clinical need for synthetic scaffolds that will promote bone regeneration. Important factors include obtaining an optimal porosity and size of interconnecting windows whilst maintaining scaffold mechanical strength, enabling complete penetration of cells and nutrients throughout the scaffold, preventing the formation of necrotic tissue in the centre of the scaffold. To address this we investigated varying slip deflocculation in order to control the resulting porosity, pore size and interconnecting window size whilst maintaining mechanical strength. Hydroxyapatite (HA) porous ceramics were prepared using a modified slip casting process. Rheological measurements of the HA slips were used to identify deflocculation conditions which resulted in changes in the cell and window sizes of the resulting ceramics. Sintered ceramics were characterised by X-ray diffraction (XRD) and scanning electron microscopy (SEM). Pore and window size distribution was determined by SEM. XRD analysis confirmed that the crystal structure remained HA after the sintering process. SEM showed that HA porous ceramics presented a highly interconnected porous network with average pore sizes ranging from 391+/-39 to 495+/-25 microm. The average window size varied from 73+/-5 to 135+/-7 microm. Pore diameters obtained were controllable in the range 200-500 microm. Window sizes were in the range 30-250 microm. The use of dispersant concentration allows pore and window size to be modified whilst maintaining control over porosity demonstrated by a porosity of 85% for seven different dispersant concentrations. The advantage of this approach allows the correlation between the rheological conditions of the slip and the resultant sintered ceramic properties. In particular, optimising the ceramic strength by controlling the agglomeration during the casting process.

Laser stereolithography and supercritical fluid processing for custom-designed implant fabrication.

This paper describes the laser photopolymerization of a liquid mixture of polyfunctional acrylic monomers, photoinitiator and hydroxyapatite (HA). Pure polymeric and composite materials of specific shape and structure were fabricated by laser stereolithography based on images derived from three-dimensional (3D) computer modeling. The polymeric objects then were treated with supercritical carbon dioxide to remove potentially toxic residues (monomers, low molecular weight oligomers, etc.) and to provide interconnective microporosity. Finally, samples were implanted into white rats (diastolic epiphysis of femoral bone) to study living tissue response and processes of osteointegration and osteoinduction. It was shown that incorporation of HA into the composite implant structure encouraged periosteal as well as endosteal osteogenesis and improved their osteointegrative characteristics in particular. Supercritical carbon dioxide treatment significantly enhanced the biocompatibility of the materials, increasing the area of direct contact of the implant surface with regenerated bone tissue.

Porous methacrylate scaffolds: supercritical fluid fabrication and in vitro chondrocyte responses.

This paper describes a method of foaming a polymer system comprising poly(ethyl methacrylate)/tetrahydrofurfuryl methacrylate (PEMA/THFMA), characterisation of the resulting porosity and use of the foam for chondrocyte culture. The potential for this polymer system to support cartilage repair has been investigated previously, both in vivo and in vitro. PEMA/THFMA foamed created using supercritical carbon dioxide were characterised using scanning electron microscopy, mercury intrusion porosimetry and helium pycnometry. Foams were found to be 82% porous with open porosities of 57%. The mean pore diameter was found to be 99+60 microm. Bovine chondrocytes seeded directly onto the surface of the foamed and unfoamed PEMA/THFMA demonstrated lower proliferation on the foamed material, greater retention of the rounded cell morphology and increased glycosaminoglycan synthesis. In conclusion, this study has shown that a porous PEMA/THFMA system can further enhance the ability of the material to support chondrocytes in vitro. However, further modifications in processing are necessary to determine optimum conditions for cartilage tissue formation.

Novel osteoinductive biomimetic scaffolds stimulate human osteoprogenitor activity--implications for skeletal repair.

The development of new bone formation strategies offers tremendous therapeutic implications in a variety of musculoskeletal diseases. One approach involves harnessing the regenerative capacity of osteoprogenitor bone cells in combination with biomimetic scaffolds generated from appropriate scaffold matrices and osteoinductive factors. The aims of our study were to test the efficacy of two innovative osteoinductive agents: the osteoblast stimulating factor-1 (osf-1), an extracellular matrix-associated protein, and osteoinductive extracts of Saos-2 cells on human osteoprogenitor cells. Saos-2 extracted osteoinductive factors significantly stimulated alkaline phosphatase specific activity in basal and osteogenic conditions. Osf-1 significantly stimulated chemotaxis, total colony formation, alkaline phosphatase-positive colony formation, and alkaline phosphatase specific activity at concentrations as low as 10 pg/ml compared with control cultures. Osteoinductive factors present in Saos-2 cell extracts and osf-1 promoted adhesion, migration, expansion, and differentiation of human osteoprogenitor cells on 3-D scaffolds. The successful generation of 3-D biomimetic structures incorporating osf-1 or osteoinductive factors from Saos-2 cells indicates their potential for de novo bone formation that exploits cell-matrix interactions.

Properties of calcium phosphate coatings deposited and modified with lasers.

Physical, chemical and biological properties of calcium phosphate coatings fabricated by a pulse laser deposition method at room temperature (RT PLD) have been studied. In vitro evaluation of RT PLD coatings on bioresorbable polymers (Poly-epsilon-caprolactone and Poly-L-lactide) have been carried out. It was shown that both polymers support osteoblast growth, with increased cell activity, alkaline phosphatase activity and total protein content on those surfaces that have been coated. The advantages of RT PLD coatings in biomaterials surface optimization are discussed.

Growth factor release from tissue engineering scaffolds.

Synthetic scaffold materials are used in tissue engineering for a variety of applications, including physical supports for the creation of functional tissues, protective gels to aid in wound healing and to encapsulate cells for localized hormone-delivery therapies. In order to encourage successful tissue growth, these scaffold materials must incorporate vital growth factors that are released to control their development. A major challenge lies in the requirement for these growth factor delivery mechanisms to mimic the in-vivo release profiles of factors produced during natural tissue morphogenesis or repair. This review highlights some of the major strategies for creating scaffold constructs reported thus far, along with the approaches taken to incorporate growth factors within the materials and the benefits of combining tissue engineering and drug delivery expertise.

Human osteoprogenitor growth and differentiation on synthetic biodegradable structures after surface modification.

The ability to generate new bone for skeletal use is a major clinical need. Biomimetic scaffolds that interact and promote osteoblast differentiation and osteogenesis offer a promising approach to the generation of skeletal tissue to resolve this major health-care issue. In this study we examine the ability of surface-modified poly(lactic acid) (PLA) films and poly(lactic-co-/glycolic acid) (PLGA) (75:25) porous structures to promote human osteoprogenitor adhesion, spreading, growth, and differentiation. Cell spreading and adhesion were examined using Cell Tracker green fluorescence and confocal microscopy. Osteogenic differentiation was confirmed with alkaline phosphatase activity as well as immunocytochemistry for type I collagen, core binding factor-1 (Cbfa-1), and osteocalcin. Poor cell growth was observed on nonmodified PLA films and PLGA scaffolds. The polymers were then coupled with RGD peptides [using poly(L-lysine), or PLL] and physical adsorption as well as PLA films presenting adsorbed fibronectin (FN). Both modifications enhanced cell attachment and spreading. On PLA-FN and PLA-PLL-GRGDS films, the osteoblast response was dose dependent (20 pmol/L to 0.2 micromol/L FN and 30 nmol/L to 30 micromol/L PLL-GRGDS) and significant at concentrations as low as 2 nmol/L FN and 30 nmol/L PLL-GRGDS. With optimal concentrations of FN or RGD, adhesion and cell spreading were comparable to tissue culture plastic serum controls. In PLGA (75:25) biodegradable porous scaffolds, coated with FN, PLL-GRGDS, or fetal calf serum for 24 h in alpha MEM alone, prior to growth in dexamethasone and ascorbate-2-phosphate for 4-6 weeks, extensive osteoblast impregnation was observed by confocal and fluorescence microscopy. Cell viability in extended culture was maintained as analyzed by expression of Cell Tracker green and negligible ethidium homodimer-1 (a marker of cell necrosis) staining. Alkaline phosphatase activity, type I collagen, Cbfa-1, and osteocalcin expression were observed by immunocytochemistry. Mineralization of collagenous matrix took place after 4 weeks, which confirmed the expression of the mature osteogenic phenotype. These observations demonstrate successful adhesion and growth of human osteoprogenitors on protein- and peptide-coupled polymer films as well as migration, expansion, and differentiation on three-dimensional biodegradable PLGA scaffolds. The use of peptides/proteins and three-dimensional structures that provide positional and environmental information indicate the potential for biomimetic structures coupled with appropriate factors in the development of protocols for de novo bone formation.