Steven M. Howdle

Materials processing in supercritical carbon dioxide: surfactants, polymers and biomaterials

Supercritical carbon dioxide (scCO2) is a unique solvent with a wide range of interesting properties. This review focuses upon recent advances in the use of scCO2 in materials synthesis and materials processing. In particular, we consider the advances made in three major areas. First the design and application of new surfactants for use in scCO2, which enable the production of metal nanoparticles, porous polymers and polymers of high molecular weight with excellent morphology. Second the development of new polymer processing and polymer blend technologies in scCO2, which enable the synthesis of some very complex polymer composites and blends. Finally, the application of scCO2 in the preparation of novel biomedical materials, for example biodegradable polymer particles and scaffolds. The examples described here highlight that scCO2 allows facile synthesis and processing of materials, leading to new products with properties that would otherwise be very difficult to achieve.

The effect of the delivery of vascular endothelial growth factor and bone morphogenic protein-2 to osteoprogenitor cell populations on bone formation

Regenerating bone tissue involves complex, temporal and coordinated signal cascades of which bone morphogenic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF(165)) play a prominent role. The aim of this study was to determine if the delivery of human bone marrow stromal cells (HBMSC) seeded onto VEGF(165)/BMP-2 releasing composite scaffolds could enhance the bone regenerative capability in a critical sized femur defect. Alginate-VEGF(165)/P(DL)LA-BMP-2 scaffolds were fabricated using a supercritical CO(2) mixing technique and an alginate entrapment protocol. Increased release of VEGF(165) (750.4+/-596.8 rho g/ml) compared to BMP-2 (136.9+/-123.4 r hog/ml) was observed after 7-days in culture. Thereafter, up till 28 days, an increased rate of release of BMP-2 compared to VEGF(165) was observed. The alginate-VEGF(165)/P(DL)LA-BMP-2+HBMSC group showed a significant increase in the quantity of regenerated bone compared to the alginate-VEGF(165)/P(DL)LA-BMP-2 and alginate/P(DL)LA groups respectively in a critical sized femur defect study as indices measured by microCT. Histological examination confirmed significant new endochondral bone matrix in the HBMSC seeded alginate-VEGF(165)/P(DL)LA-BMP-2 defect group in comparison to the other groups. These studies demonstrate the ability to deliver a combination of HBMSC with angiogenic and osteogenic factors released from biodegradable scaffold composites enhances the repair and regeneration of critical sized bone defects.

Induction of human osteoprogenitor chemotaxis, proliferation, differentiation, and bone formation by osteoblast stimulating factor-1/pleiotrophin: osteoconductive biomimetic scaffolds for tissue engineering.

The process of bone growth, regeneration, and remodeling is mediated, in part, by the immediate cell‐matrix environment. Osteoblast stimulating factor‐1 (OSF‐1), more commonly known as pleiotrophin (PTN), is an extracellular matrix‐associated protein, present in matrices, which act as targets for the deposition of new bone. However, the actions of PTN on human bone progenitor cells remain unknown. We examined the effects of PTN on primary human bone marrow stromal cells chemotaxis, differentiation, and colony formation (colony forming unit‐fibroblastic) in vitro, and in particular, growth and differentiation on three‐dimensional biodegradable porous scaffolds adsorbed with PTN in vivo. Primary human bone marrow cells were cultured on tissue culture plastic or poly(DL‐lactic acid‐co‐glycolic acid) (PLGA; 75:25) porous scaffolds with or without addition of recombinant human PTN (1 pg‐50 ng/ml) in basal and osteogenic conditions. Negligible cellular growth was observed on PLGA scaffold alone, generated using a super‐critical fluid mixing method. PTN (50 μg/ml) was chemotactic to human osteoprogenitors and stimulated 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. The effects were time‐dependent. On three‐dimensional scaffolds adsorbed with PTN, alkaline phosphatase activity, type I collagen formation, and synthesis of cbfa‐1, osteocalcin, and PTN were observed by immunocytochemistry and PTN expression by in situ hybridization. PTN‐adsorbed constructs showed morphologic evidence of new bone matrix and cartilage formation after subcutaneous implantation as well as within diffusion chambers implanted into athymic mice. In summary, PTN has the ability to promote adhesion, migration, expansion, and differentiation of human osteoprogenitor cells, and these results indicate the potential to develop protocols for de novo bone formation for skeletal repair that exploit cell‐matrix interactions.

Supercritical fluid mixing: preparation of thermally sensitive polymer composites containing bioactive materials

We report the use of supercritical carbon dioxide (scCO2) to create a diverse range of polymeric composites incorporating thermal and solvent labile guest materials such as proteins; no additional co-solvents are required; the entire process can be carried out at near ambient conditions; polymer morphology is controllable; high loadings of guest species can be achieved and the protein function is retained.

Immunoselection and adenoviral genetic modulation of human osteoprogenitors: in vivo bone formation on PLA scaffold.

The aim of this study was to examine the potential of immunoselected genetically modified human osteoprogenitors to form bone in vivo on porous PLA scaffolds. Human osteoprogenitors from bone marrow were selected using the antibody STRO-1 utilising a magnetically activated cell separation system. The STRO-1(+) fraction isolated 7% of nucleated marrow cells and increased fibroblastic colony formation by 300% and alkaline phosphatase activity by 190% over unselected marrow cell cultures. To engineer bone tissue, STRO-1(+) culture-expanded cells were transduced with AxCAOBMP-2, an adenovirus carrying the human BMP-2 gene, injected into diffusion chambers containing porous PLA scaffolds, and implanted in vivo. After 11 weeks the presence of bone mineral was observed by X-ray analysis and confirmed for mineral by von Kossa, as well as bone matrix composition by Sirius red staining, birefringence, and type I collagen immunohistochemistry. Bone formation in vivo indicates the potential of using immunoselected progenitor cells and ex vivo gene transfer with biodegradable scaffolds, for the development of protocols for the treatment of a wide variety of musculo-skeletal disorders.

Supercritical carbon dioxide: putting the fizz into biomaterials

This paper describes recent progress made in the use of high pressure or supercritical fluids to process polymers into three-dimensional tissue engineering scaffolds. Three current examples are highlighted: foaming of acrylates for use in cartilage tissue engineering; plasticization and encapsulation of bioactive species into biodegradable polyesters for bone tissue engineering; and a novel laser sintering process used to fabricate three-dimensional biodegradable polyester structures from particles prepared via a supercritical route.

Clean preparation of nanoparticulate metals in porous supports: a supercritical route

Here we present the synthesis of nanometre sized silver particles which have been trapped within porous substrates; poly(styrene-divinylbenzene) beads and silica aerogels. This is the first time that supercritical carbon dioxide has been used to impregnate such porous materials with silver coordination complexes. In this paper we demonstrate that control over the resultant nanoparticles with respect to size, loading and distribution in the support material has been achieved by simple choice of the precursor complex. The solubility of the precursor complexes in the supercritical solvent is shown to be one of the key parameters in determining the size of the nanoparticles, their distribution and their homogeneity within the support matrix. Moreover, we demonstrate that the same methodology can be applied to two very different substrate materials. In the particular case of aerogels, conventional organic solvents could not be used to prepare nanoparticles because the surface tension of the solvent would lead to fracturing of the aerogel structure.Controlled decomposition of the coordination complexes in situ leads to metallic silver nanoparticles with a narrow size distribution, typically 10–100 nm that are homogeneously dispersed throughout the porous substrate. The whole process is carried out at near ambient temperature and no solvent residues are introduced into the porous media. The silver precursors are specifically designed to be both CO2 soluble and sufficiently labile to ensure facile decomposition to the metal. In-depth characterisation by X-ray diffraction and transmission electron microscopy has been applied to illustrate the homogeneous dispersion of particles throughout the composite material, determine the range and variation in particle size within the solid matrices and fully identify the resultant particles as metallic silver. This enables visualisation of dispersion and concentration, and control over particle size of the fabricated nanocomposite materials.

Successful dispersion polymerization in supercritical CO2 using polyvinylalkylate hydrocarbon surfactants synthesized and anchored via RAFT.

New CO2-philic hydrocarbon molecules were synthesized by reversible addition fragmentation chain-transfer polymerization. These poly(vinyl alkylates) show the highest solubility in supercritical CO2 of any hydrocarbon reported to date. By utilizing the anchoring ability of the thiocarbonylthio end group, the dispersion polymerization of N-vinyl pyrrolidone was successfully achieved in scCO2 leading to high yields of well-defined spherical polymer particles.

Osteoblast growth on titanium foils coated with hydroxyapatite by pulsed laser ablation.

Pulsed laser ablation is a new method for deposition of thin layers of hydroxyapatite (HA) on to biomaterial surfaces. In this paper, we report activity and morphology of osteoblasts grown on HA surfaces fabricated using different laser conditions. Two sets of films were deposited from dense HA targets, at three different laser fluences: 3, 6 and 9 Jcm(-2). One set of the surfaces was annealed at 575 degrees C to increase the crystallinity of the deposited films. Primary human osteoblasts were seeded onto the material surfaces and cytoskeletal actin organisation was examined using confocal laser scanning microscopy. The annealed surfaces supported greater cell attachment and more defined cytoskeletal actin organisation. Cell activity, measured using the alamar Blue assay, was also found to be significantly higher on the annealed samples. In addition, our results show distinct trends that correlate with the laser fluence used for deposition. The cell activity increases with increasing fluence. This pattern was repeated for alkaline phosphatase production by the cells. Differences in cell spreading were apparent which were correlated with the fluence used to deposit the HA. The optimum surface for initial attachment and spreading of osteoblasts was one of the HA films deposited using 9 J cm(-2) laser fluence and subsequently annealed at 575 degrees C.

A highly effective gene delivery vector – hyperbranched poly(2-(dimethylamino)ethyl methacrylate) from in situ deactivation enhanced ATRP

A hyperbranched 2-(dimethylamino)ethyl methacrylate (DMAEMA) based polymer has been synthesised by a one-pot in situ deactivation enhanced atom transfer radical polymerisation (DE-ATRP); it exhibits much higher transfection ability than linear poly(DMAEMA) and is comparable to the well known branched poly(ethylene imine) (PEI) and the SuperFect dendrimer but with lower cytotoxicity.