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Biomedical subjects

S M Howdle

Publications and source records attributed to S M Howdle.

15 recordsLinked to original sources
PubMed2006

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.

Animals↗
PubMed2006

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.

Animals↗
PubMed2005

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.

Bone Substitutes↗
PubMed2004

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.

Animals↗
PubMed2004

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.

Animals↗
PubMed2003

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.

Journal Article↗
PubMed2003

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.

Aged↗
PubMed2001

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.

Alkaline Phosphatase↗
PubMed2001

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.

Aged↗
PubMed2001

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.

Bioartificial Organs↗
PubMed2000

Physical, chemical, and biological characterization of pulsed laser deposited and plasma sputtered hydroxyapatite thin films on titanium alloy.

The physical, chemical, and biological properties of pulsed laser deposited (PLD) and plasma sputtered (PS) hydroxyapatite (HA) coatings were compared. Human osteoblast-like cell responses to these coatings in vitro were assayed for proliferation and phenotypic expression. PS coatings formed smooth and continuous thin films that followed the contours of the substrate surface. PLD coatings consisted of numerous spheroidal micro- and macroparticles. The crystallinity of all coatings was quantified by comparison with the HA target used for both the PS and PLD processes. The XRD and FTIR results indicated that unannealed PLD coatings deposited at room temperature had X-ray spectra consistent with an amorphous structure and were found to dissolve after only a few hours in saline solution. Annealing at 400 degrees C increased the crystallinity (87-98%), which resulted in improved stability and cell activity. The PS coatings showed greater chemical stability than the unannealed PLD coatings and contained an approximate 15% crystalline phase, increasing to 65% postannealing. Cell proliferation and alkaline phosphatase production were significantly higher on unannealed PS specimens than the other coating treatments. There may be benefits in engineering the presence of a minor percentage of a microcrystalline phase in an amorphous or nanometer scale polycrystalline HA structure.

Alloys↗
PubMed2000

Dichloromaleic anhydride.

Although molecules of the title compound, 3,4-dichloro-2,5-dihydrofuran-2,5-dione (dichloromaleic anhydride, C(4)Cl(2)O(3)), (I), possess approximate non-crystallographic C(2v) symmetry, the two chlorine substituents deviate from the ring plane. Their deviations are in the same direction, but with values of 0.0356 (17) and 0.0167 (17) A, they differ significantly in magnitude. The closest intermolecular contact is of 2.888 (2) A between a carbonyl O atom and the C atom of a carbonyl group, with the O.C direction orthogonal to the C=O bond [O5.C2(i)=O2(i) 93.6 (2) degrees; symmetry code: (i) $3 \over 2$ - x, -(1/2) + y, z]. These contacts form infinite chains of molecules running parallel to the crystallographic b direction.

Journal Article↗
PubMed1997

Thermal and diffusion processes in laser-induced stress relaxation and reshaping of cartilage.

The thermodynamic characteristics of the 'bound-to-free' phase transformation of water are studied by means of differential microcalorimetry and FTIR spectroscopy. This phase transition controls the stress relaxation and reshaping of cartilage which we have observed previously under moderate laser heating. It is shown that the FTIR spectrum of bound water in cartilage differs from that of free water in cartilage, and that both show differences to the FTIR spectrum of pure water. The proportion of bound water in cartilage is found to be of the order 4%. We have examined water liberation and absorption kinetics and found them to be controlled by diffusion through the tissue and also by the bound-to-free water transformation. The theoretical modelling and experimental data allowed calculation of diffusion coefficients and the activation energy for water transfer. The latter was found to be close to the heat of phase transformation of water. We have established that the drying and wetting processes in cartilage are reversible (fully or in a part, depending on the temperature of preliminary drying) and can be described by identical solutions of the diffusion problem, with coefficients of water diffusion being the same. The mechanism of water diffusion in cartilaginous tissue is also discussed.

Animals↗
PubMed1997

Atomic force microscopic study of the surface morphology of apatite films deposited by pulsed laser ablation.

Atomic force microscopy (AFM) has been used to study the surface morphology of apatite films deposited on metallic and polyethylene substrates by laser ablation using KrF and transversely excited atmospheric CO2 lasers. The films are found to consist of a smooth apatite coating with macroparticles scattered on the surface. A wide variety of macroparticles, differing in size, shape and roughness, were found and analysed employing the high spatial resolution of AFM (< 1 nm). We have investigated the correlation between the apatite film morphology and the deposition conditions. Of particular importance are laser fluence, gas pressure, the nature of the target and the substrate temperature. We have explained these dependencies on the basis of a theoretical model which includes evaporation and a cluster-type laser ablation mechanism.

Apatites↗