Ruth E. Cameron

Collagen-hyaluronic acid scaffolds for adipose tissue engineering.

Three-dimensional (3-D) in vitro models of the mammary gland require a scaffold matrix that supports the development of adipose stroma within a robust freely permeable matrix. 3-D porous collagen-hyaluronic acid (HA: 7.5% and 15%) scaffolds were produced by controlled freeze-drying technique and crosslinking with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride. All scaffolds displayed uniform, interconnected pore structure (total porosity approximately 85%). Physical and chemical analysis showed no signs of collagen denaturation during the formation process. The values of thermal characteristics indicated that crosslinking occurred and that its efficiency was enhanced by the presence of HA. Although the crosslinking reduced the swelling of the strut material in water, the collagen-HA matrix as a whole tended to swell more and show higher dissolution resistance than pure collagen samples. The compressive modulus and elastic collapse stress were higher for collagen-HA composites. All the scaffolds were shown to support the proliferation and differentiation 3T3-L1 preadipocytes while collagen-HA samples maintained a significantly increased proportion of cycling cells (Ki-67+). Furthermore, collagen-HA composites displayed significantly raised Adipsin gene expression with adipogenic culture supplementation for 8 days vs. control conditions. These results indicate that collagen-HA scaffolds may offer robust, freely permeable 3-D matrices that enhance mammary stromal tissue development in vitro.

Design of a multiphase osteochondral scaffold. I. Control of chemical composition.

This is the first in a series of articles that describe the design and development of a family of osteochondral scaffolds based on collagen-glycosaminoglycan (collagen-GAG) and calcium phosphate technologies, engineered for the regenerative repair of defects in articular cartilage. The osteochondral scaffolds consist of two layers: a mineralized type I collagen-GAG scaffold designed to regenerate the underlying subchondral bone and a nonmineralized type II collagen-GAG scaffold designed to regenerate cartilage. The subsequent articles in this series describe the fabrication and properties of a mineralized scaffold as well as a two-layer (one mineralized, the other not) osteochondral scaffold for regeneration of the underlying bone and cartilage, respectively. This article describes a technology through which the chemical composition-particularly the calcium phosphate mass fraction-of triple coprecipitated nanocomposites of collagen, glycosaminoglycan, and calcium phosphate can be accurately and reproducibly varied without the need for titrants or other additives. Here, we describe how the mineral:organic ratio can be altered over a range that includes that for articular cartilage (0 wt % mineral) and for bone (75 wt % mineral). This technology achieves the objective of mimicking the composition of two main tissue types found in articular joints, with particular emphasis on the osseous compartment of an osteochondral scaffold. Exclusion of titrants avoids the formation of potentially harmful contaminant phases during freeze-drying steps crucial for scaffold fabrication, ensuring that the potential for binding growth factors and drugs is maintained.

Magnetic resonance imaging and X-ray microtomography studies of a gel-forming tablet formulation

The capabilities of two methods for investigating tablet swelling are investigated, based on a study of a model gel-forming system. Results from magnetic resonance imaging (MRI) were compared with results from a novel application of X-ray microtomography (XmicroT) to track the movements of embedded glass microsphere tracers as the model tablets swelled. MRI provided information concerning the movement of hydration fronts into the tablets and the composition of the swollen gel layer, which formed at the tablet surface and progressively thickened with time. Conversely, XmicroT revealed significant axial expansion within the tablet core, at short times and ahead of the hydration fronts, where there was insufficient water to be observed by MRI (estimated to be around 15% by weight for the system used here). Thus, MRI and XmicroT may be regarded as complementary methods for studying the hydration and swelling behaviour of tablets.

Chitosan/apatite composite beads prepared by in situ generation of apatite or Si-apatite nanocrystals

The objective of this work was to develop nanocrystalline apatite (Ap) dispersed in a chitosan (CHI) matrix as a material for applications in bone tissue engineering. CHI/Ap composites of different weight ratios (20/80, 50/50 and 80/20) and with CHI of different molecular weights were prepared by a biomimetic stepwise route. Firstly, CaHPO(4).2H(2)O (DCPD) crystals were precipitated from Ca(CH(3)COO)(2) and NaHPO(4) in the bulk CHI solution, followed by the formation of CHI/DCPD beads by coacervation. The beads were treated with Na(3)PO(4)/Na(5)P(3)O(10) solution (pH 12-13) to crosslink the CHI and to hydrolyse the DCPD to nanocrystalline Ap. This new experimental procedure ensured that complete conversion of DCPD into sodium-substituted apatite was achieved without appreciable increases in its crystallinity and particle size. In addition, composites with silicon-doped Ap were prepared by substituting Na(3)PO(4) by Na(2)SiO(3) in the crosslinking/hydrolysis step. Characterization of the resultant composites by scanning electron microscopy, X-ray powder diffraction (XRD), thermal analysis and Fourier transform infrared spectroscopy confirmed the formation, within the CHI matrix, of nanoparticles of sodium- and carbonate-substituted hydroxyapatite [Ca(10-x)Na(x)(PO(4))(6-x)(CO(3))(x)(OH)(2)] with diameters less than 20nm. Relatively good correspondence was shown between the experimentally determined inorganic content and that expected theoretically. Structural data obtained from its XRD patterns revealed a decrease in both crystal domain size and cell parameters of Ap formed in situ with increasing CHI content. It was found that the molecular weight of CHI and silicate doping both affected the nucleation and growth of apatite nanocrystallites. These effects are discussed in detail.

Synchrotron X-ray microtomographic study of tablet swelling

Tablet swelling behaviour was investigated by following the movements of embedded glass microsphere tracers, using X-ray microtomography (XmicroT) with intense illumination from a synchrotron. Specimens were prepared using combinations of hydroxypropyl-methyl-cellulose (HPMC) and microcrystalline cellulose (MCC) or pre-gelatinised starch (PGS), three materials commonly used as excipients for compacted tablets. The results revealed significant differences in swelling behaviour due to excipient type and compaction conditions. In particular, a sudden change was observed from gel-forming behaviour of formulations containing PGS or high HPMC content, to more rapid expansion and disintegration for formulations above 70% MCC. Although some radial expansion was observable with the higher PGS formulations and during later stages of swelling, axial expansion (i.e. the reverse of the compaction process) appeared to dominate in most cases. This was most pronounced for the 10/90 HPMC/MCC specimens, which rapidly increased in thickness, while the diameter remained almost unchanged. The expansion appeared to be initiated by hydration and may be due to the relaxation of residual compaction stress. This occurred within expansion zones, which initially appeared as thin bands close to the compacted (upper and lower) faces, but gradually advanced towards the centre and spread around the sides of the tablets. These zones exhibited lower X-ray absorbance, probably because they contained significant amounts of bubbles, which were formed by air released from the swelling excipients. Although, in most cases, these bubbles were too small to be resolved (<60 microm), larger bubbles (diameter up to 1mm) were clearly evident in the rapidly swelling 10/90 HPMC/MCC specimens. It is suggested that the presence of these bubbles may affect subsequent water ingress, by increasing the tortuosity and occluding part of the gel, which may affect the apparent diffusion kinetics (i.e. Fickian or Case II). These observations also suggested that axial expansion, initiated by water ingress, may be an important mechanism during tablet swelling.

Degradation properties of co-continuous calcium-phosphate-polyester composites.

Co-continuous composites consisting of a porous calcium phosphate matrix (hydroxyapatite, HA, or β-tricalcium phosphate, TCP) filled with poly(D,L-lactide) (PDLLA) were produced with two different methods: in situ polymerization of D,L-lactide monomer inside the matrix, or infiltration of the matrix with molten polymer. The influence of the calcium phosphate matrix as well as the manufacturing method on the degradation were investigated with accelerated in vitro studies at 42 °C in pH 7.4 phosphate-buffered saline (PBS), with some controls at 37 °C. The results show that samples produced with the infiltration method had higher initial molecular weights leading to a later onset of mass loss. Heterogenous polymer degradation was still present in the composites, as indicated by molecular weight distributions and glass transition temperature measurements. The calcium phosphate matrix delayed degradation, with evidence from X-ray microtomography suggesting that the polymer degrades more slowly in proximity to the matrix.

A dynamic mechanical thermal analysis study of the viscoelastic properties and glass transition temperature behaviour of bioresorbable polymer matrix nanocomposites

The application of bioresorbable polymer nanocomposites in orthopaedics offer the potential to address several of the limitations associated with the use of metallic implants. Their enhanced biological performance has been demonstrated recently, but until now relatively little work has been reported on their mechanical properties. To this end, the viscoelastic properties and Tg of bioresorbable polylactide-co-glycolide/α-tricalcium phosphate nanocomposites were investigated by dynamic mechanical thermal analysis. At room temperature of approximately 20°C, the storage moduli of the nanocomposites were generally higher than the storage modulus of the unfilled polymer due to the stiffening effect of the nano-particles. However at physiological temperature of approximately 37°C, the storage moduli of the nanocomposites decreased from 6.2 to 15.4% v/v nano-particle loadings. Similarly the Tg of the nanocomposites also decreased from 6.2 to 15.4% v/v nano-particle loadings. These effects were thought to be due to weak interfacial bonding between the nano-particles and polymer matrix. The storage moduli at 37°C and Tg increased from the minimum value when the particle loading was raised to 25.7 and 34.2% v/v loadings. SEM and particle size distribution histograms showed that at these loadings, there was a broad particle size distribution consisting of nano-particles and micro-particles and that some particle agglomeration was present. The consequent reduction in the interfacial area and the number of weak interfaces presumably accounts for the rise in the storage modulus at 37°C and the Tg.

Investigation into the intragranular structures of microcrystalline cellulose and pre-gelatinised starch

The internal structures of commercial spheronised microcrystalline cellulose (s-MCC) and pre-gelatinised starch (PGS) granules were investigated, using a range of methods. Results from scanning electron microscopy (SEM), transmission electron microscopy (TEM) and atomic force microscopy (AFM) revealed hierarchical structures, with dimensions ranging from nanometres to a few micrometres, for both materials. Residual fragments of plant cell walls, consisting largely of crystalline fibrillar bundles, were indicated within s-MCC granules, while PGS granules appeared to consist of densely packed spherical features. The lack of any obvious regular periodicity associated with the intragranular sub-structures was entirely consistent with the power-law behaviour of the small-angle X-ray scattering (SAXS) patterns from these materials. The presence of intragranular porosity was inferred from TEM, AFM and N(2)-adsorption measurements, while the ability to deform these structures was clearly indicated by the irregular force-displacement curves recorded by AFM on the granule surfaces. Hence, the intragranular sub-structures observed for s-MCC and PGS appeared to be consistent with the possibility of entire granules undergoing affine deformation during compaction. Since this mechanism was postulated to explain changes in SAXS patterns from these materials following compaction, as reported elsewhere, the work reported here provides a considerably stronger basis for using 2D-SAXS to investigate powder compaction behaviour.

Novel use of X-ray micro computed tomography to image rat sciatic nerve and integration into scaffold

This paper describes how specimens of nervous tissue can be prepared for successful imaging in X-ray Micro Computed Tomography (microCT), and how this method can be used to study the integration of nervous tissue into a polymeric scaffold. The sample preparation involves staining the biological tissue with osmium tetroxide to increase its X-ray attenuation, and a technique for maintaining the specimen in a moist environment during the experiment to prevent drying and shrinkage. Using this method it was possible to observe individual nerve fascicles and their relationship to the 3-D tissue structure. A scaffold supporting a regenerated sciatic nerve was similarly stained to distinguish the nervous tissue from the scaffold, and to observe how the nerve grew through a 2.5 mm long, 100 microm x 100 microm cross-section channel polyimide array. Furthermore, blood vessels could be identified in these images, and it was possible to monitor how a large proximal blood vessel split through the channel scaffold and proceeded down individual channels. This paper explains how microCT is a useful tool both for studying the location and extent of growth into a polymeric scaffold, and for determining whether the regenerated tissue has blood supply.

Variations in compaction behaviour for tablets of different size and shape, revealed by small‐angle X‐ray scattering

Local variations in compaction behaviour were investigated, for specimens of different shapes and thickness, comparing predictions from finite element (FE) modelling and results from a recently developed method using small-angle X-ray scattering (SAXS). Good agreement was generally obtained between these methods, in terms of the variations of density, compaction strain and principal strain direction within the specimens examined. The combination of SAXS and FE methods appeared particularly suitable for studying pharmaceutical tablets, revealing effects (such as nano-strain of intragranular morphology and strain direction) that are not easily observed by other methods, and which may have significant effects on tablet integrity or swelling and drug delivery characteristics.