Item Open AccessIntegration of disease association and eQTL data using a Bayesian colocalisation approach highlights six candidate causal genes in immune-mediated diseases.(Oxford University Press (OUP), 2015-06-15) Guo, Hui; Fortune, Mary D; Burren, Oliver S; Schofield, Ellen; Todd, John A; Wallace, Chris; Fortune, Mary [0000-0002-6006-4343]; Burren, Oliver [0000-0002-3388-5760]; Schofield, Ellen [0000-0003-0648-1418]; Wallace, Chris [0000-0001-9755-1703]The genes and cells that mediate genetic associations identified through genome-wide association studies (GWAS) are only partially understood. Several studies that have investigated the genetic regulation of gene expression have shown that disease-associated variants are over-represented amongst expression quantitative trait loci (eQTL) variants. Evidence for colocalisation of eQTL and disease causal variants can suggest causal genes and cells for these genetic associations. Here, we used colocalisation analysis to investigate whether 595 genetic associations to ten immune-mediated diseases are consistent with a causal variant that regulates, in cis, gene expression in resting B cells, and in resting and stimulated monocytes. Previously published candidate causal genes were over-represented amongst genes exhibiting colocalisation (odds ratio > 1.5), and we identified evidence for colocalisation (posterior odds > 5) between cis eQTLs in at least one cell type and at least one disease for six genes: ADAM15, RGS1, CARD9, LTBR, CTSH and SYNGR1. We identified cell-specific effects, such as for CTSH, the expression of which in monocytes, but not in B cells, may mediate type 1 diabetes and narcolepsy associations in the chromosome 15q25.1 region. Our results demonstrate the utility of integrating genetic studies of disease and gene expression for highlighting causal genes and cell types. Item Open AccessIce-templated structures for biomedical tissue repair: From physics to final scaffolds(AIP Publishing, 2014) Pawelec, KM; Husmann, A; Best, SM; Cameron, RE; Husmann, Anke [0000-0001-5326-3785]; Best, Serena [0000-0001-7866-8607]; Cameron, Ruth [0000-0003-1573-4923]Ice-templating techniques, including freeze-drying and freeze casting, are extremely versatile and can be used with a variety of materials systems. The process relies on the freezing of a water based solution. During freezing, ice nucleates within the solution and concentrates the solute in the regions between the growing crystals. Once the ice is removed via sublimation, the solute remains in a porous structure, which is a negative of the ice. As the final structure of the ice relies on the freezing of the solution, the variables which influence ice nucleation and growth alter the structure of ice-templated scaffolds. Nucleation, the initial step of freezing, can be altered by the type and concentration of solutes within the solution, as well as the set cooling rate before freezing. After nucleation, crystal growth and annealing processes, such as Ostwald ripening, determine the features of the final scaffold. Both crystal growth and annealing are sensitive to many factors including the set freezing temperature and solutes. The porous structures created using ice-templating allow scaffolds to be used for many diverse applications, from microfluidics to biomedical tissue engineering. Within the field of tissue engineering, scaffold structure can influence cellular behavior, and is thus critical for determining the biological stimulus supplied by the scaffold. The research focusing on controlling the ice-templated structure serves as a model for how other ice-templating systems might be tailored, to expand the applications of ice-templated structures to their full potential. Item Open AccessProduction of zinc substituted hydroxyapatite using various precipitation routes.(IOP Publishing, 2013-04) Shepherd, David; Best, Serena M; Best, Serena [0000-0001-7866-8607]Substituted hydroxyapatites have been investigated for use as bone grafts and have been investigated for many years. Zinc is of interest due to its potential to reduce bone resorption and antibacterial properties. However, it has proven problematic to substitute biologically significant levels of zinc into the crystal structure through wet chemical routes, whilst retaining the high temperature phase stability required for processing. The aim of this study is to investigate two different precipitation routes used to synthesize zinc substituted hydroxyapatite and to explore the effects of ammonia used in the reactions on the levels of zinc substituted into the crystal lattice. It was found that considerable amounts of ammonia are required to maintain a pH sufficiently high for the production of stoichiometric hydroxyapatite using a reaction between calcium nitrate, zinc nitrate and ammonium phosphate. X-ray fluorescence analysis showed that a significant proportion of the zinc added did not substitute into the hydroxyapatite lattice. Fourier transform infrared spectroscopy revealed the existence of a zinc-ammonia complex that, it is proposed, inhibits zinc substitution for calcium. It was found that by reacting orthophosphoric acid with calcium nitrate and zinc nitrate, the volume of ammonia required in the reaction was reduced and higher levels of zinc substitution were achieved, with up to 0.58 wt% incorporated into the hydroxyapatite lattice. The resulting products were found to be stoichiometric hydroxyapatite and did not appear to contain any extraneous calcium phosphate phases after heat treatment up to 1100 °C. X-ray diffraction and Rietveld analysis revealed that the effect of substituting zinc into the HA lattice was to decrease the a-lattice parameter whilst increasing the c-lattice. Transmission electron microscopy also showed that the incorporation of zinc reduced both the length and width of the precipitated crystals. Item Open AccessHigh-speed camera characterization of voluntary eye blinking kinematics.(The Royal Society, 2013-08-06) Kwon, Kyung-Ah; Shipley, Rebecca J; Edirisinghe, Mohan; Ezra, Daniel G; Rose, Geoff; Best, Serena M; Cameron, Ruth E; Best, Serena [0000-0001-7866-8607]; Cameron, Ruth [0000-0003-1573-4923]Blinking is vital to maintain the integrity of the ocular surface and its characteristics such as blink duration and speed can vary significantly, depending on the health of the eyes. The blink is so rapid that special techniques are required to characterize it. In this study, a high-speed camera was used to record and characterize voluntary blinking. The blinking motion of 25 healthy volunteers was recorded at 600 frames per second. Master curves for the palpebral aperture and blinking speed were constructed using palpebral aperture versus time data taken from the high-speed camera recordings, which show that one blink can be divided into four phases; closing, closed, early opening and late opening. Analysis of data from the high-speed camera images was used to calculate the palpebral aperture, peak blinking speed, average blinking speed and duration of voluntary blinking and compare it with data generated by other methods previously used to evaluate voluntary blinking. The advantages of the high-speed camera method over the others are discussed, thereby supporting the high potential usefulness of the method in clinical research. Item Open AccessPreparation, characterization, and in vitro evaluation of nanostructured chitosan/apatite and chitosan/Si-doped apatite composites(Springer Science and Business Media LLC, 2013) Solís, Y; Davidenko, N; Carrodeguas, RG; Cruz, J; Hernández, A; Tomás, M; Cameron, RE; Peniche, C; Cameron, Ruth [0000-0003-1573-4923]Chitosan/apatite composites are attracting great attention as biomaterials for bone repair and regeneration procedures. The reason is their unique set of properties: bioactivity and osteoconductivity provided by apatite and resorbability supplied by chitosan among others. Thus, in this work chitosan/apatite and chitosan/Si-doped apatite composites were prepared and characterized. Particle size, surface area, in vitro physiological stability, enzymatic biodegradation and bioactivity were evaluated. Unimodal particle size distribution was obtained for composites with high chitosan/apatite ratios while bimodal distribution was present in composites with low chitosan/apatite ratio. Physiological stability decreased with Si-doping and with the chitosan content. Acetylation degree and molecular weight of chitosan did not affect in vitro stability. Rate of enzymatic degradation increased with the chitosan content in composites. Si-doped apatite composites also showed increased degradation with respect to non-doped ones. The bioactivity of the composites was evidenced by the deposition on their surface of a calcium phosphate layer with apatite morphology after immersion in simulated body fluid. Both, biodegradation and bioactivity were dependent on the molecular weight of the polymeric chitosan matrix. These results suggest that the chitosan/apatite composites obtained are promising materials for bone regeneration applications Item Open AccessCollagen fibre implant for tendon and ligament biological augmentation. In vivo study in an ovine model.(Springer Science and Business Media LLC, 2013-08) Enea, Davide; Gwynne, Jessica; Kew, Simon; Arumugam, Meera; Shepherd, Jennifer; Brooks, Roger; Ghose, Siddhartha; Best, Serena; Cameron, Ruth; Rushton, Neil; Best, Serena [0000-0001-7866-8607]; Cameron, Ruth [0000-0003-1573-4923]PURPOSE: Although most in vitro studies indicate that collagen is a suitable biomaterial for tendon and ligament tissue engineering, in vivo studies of implanted collagen for regeneration of these tissues are still lacking. The objectives of this study were the following: (1) to investigate the regeneration of the central third of the ovine patellar tendon using implants made of an open array of collagen fibres (reconstituted, extruded bovine collagen); and (2) to compare two collagen crosslinking chemistries: carbodiimide and carbodiimide associated with ethyleneglycoldiglycidylether. METHODS: Forty-eight Welsh Mountain sheep were operated on their right hind leg. The central third of patellar tendon was removed and substituted with carbodiimide (n = 16) and carbodiimide-ethyleneglycoldiglycidylether-crosslinked implants (n = 16). In the control group the defect was left empty (n = 16). The central third of contralateral unoperated tendons was used as positive controls. Half of the sheep in each group were killed at 3- and 6-month time points. After proper dissection, tendon sub-units (medial, central and lateral) were tested to failure (n = 6 for each group), whilst 2 non-dissected samples were used for histology. RESULTS: Both the implants had significantly lower stress to failure and modulus with respect to native tendon at both 3- and at 6-month time points. The implants did not statistically differ in stress to failure, whilst carbodiimide-crosslinked implants had significantly higher modulus than carbodiimide-ethyleneglycoldiglycidylether-crosslinked implants both at 3 and at 6 months. Histology showed carbodiimide-crosslinked implants to have a better integration with the native tendon than carbodiimide-ethyleneglycoldiglycidylether-crosslinked implants. Carbodiimide-crosslinked implants appeared partially resorbed and showed increased tissue ingrowth with respect to carbodiimide-ethyleneglycoldiglycidylether-crosslinked implants. CONCLUSIONS: To deliver collagen implants as an open array of fibres allows optimal tendon-implant integration and good ingrowth of regenerated tissue. In the present study the resorption rate of both the examined implants was too low due to the high level of crosslinking. This led to only minor substitution of the implant with regenerated tissue, which in turn produced a low-strength implanted region. Further studies are needed to find the right balance between strength and resorption rate of collagen fibres. Item Open AccessA study of surface morphology and phase separation of polymer/cellulose liquid crystal composite membranes(Elsevier BV, 2012) Tu, M; Han, W; Zeng, R; Best, SM; Cameron, RE; Best, Serena [0000-0001-7866-8607]; Cameron, Ruth [0000-0003-1573-4923]This paper explores the effects of the incorporation of liquid crystalline phases into polyurethane (PU) matrix with the aim of creating composites with biomimetic surfaces. The surface morphology and phase separation structure of polyurethane/butyl hydroxypropyl cellulose ester (PU/BPC) composite membranes with different BPC contents and underwent different post-treatments were investigated by using polarized optical microscopy (POM), scanning electron microscopy (SEM) and small angle X-ray scattering (SAXS). Well-dispersed liquid crystal (LC) domains occurred on the PU/BPC composite membranes surfaces. As the increment of BPC content, the LC domains tended to form enlarged quasispherical aggregates with poorly molecular orientation, and low degree of regular phase separation occurred between the LC domains and PU substrate. Membranes with different LC contents underwent heat treating and cooling at three different conditions exhibited distinct surface morphologies, meanwhile, a sharp peak emerged in the SAXS pattern, which indicated that the ordered arrangement of BPC molecular chains existed in the LC domains and the phase separation structure between substrate and LC domain had changed. Sharper and more intense SAXS peaks were found in the membranes that annealing in oven, indicating more regular arrangement of LC domains and more obvious phase separation presented than those cooling to 20 °C or −20 °C respectively. Results suggested that the surface morphology of polymer/LC membranes could be controlled through adjusting LC contents or post treatment conditions. Item Open AccessEffect of fiber crosslinking on collagen-fiber reinforced collagen-chondroitin-6-sulfate materials for regenerating load-bearing soft tissues.(Wiley, 2013-01) Shepherd, JH; Ghose, S; Kew, SJ; Moavenian, A; Best, SM; Cameron, RE; Best, Serena [0000-0001-7866-8607]; Cameron, Ruth [0000-0003-1573-4923]Porous collagen-glycosaminoglycan structures are bioactive and exhibit a pore architecture favorable for both cellular infiltration and attachment; however, their inferior mechanical properties limit use, particularly in load-bearing situations. Reinforcement with collagen fibers may be a feasible route for enhancing the mechanical characteristics of these materials, providing potential for composites used for the repair and regeneration of soft tissue such as tendon, ligaments, and cartilage. Therefore, this study investigates the reinforcement of collagen-chondroitin-6-sulfate (C6S) porous structures with bundles of extruded, reconstituted type I collagen fibers. Fiber bundles were produced through extrusion and then, where applicable, crosslinked using a solution of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide. Fibers were then submerged in the collagen-C6S matrix slurry before being lyophilized. A second 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide crosslinking process was then applied to the composite material before a secondary lyophilization cycle. Where bundles had been previously crosslinked, composites withstood a load of approximately 60 N before failure, the reinforcing fibers remained dense and a favorable matrix pore structure resulted, with good interaction between fiber and matrix. Fibers that had not been crosslinked before lyophilization showed significant internal porosity and a channel existed between them and the matrix. Mechanical properties were significantly reduced, but the additional porosity could prove favorable for cell migration and has potential for directing aligned tissue growth. Item Open AccessSynthetic collagen fascicles for the regeneration of tendon tissue.(Elsevier BV, 2012-10) Kew, SJ; Gwynne, JH; Enea, D; Brookes, R; Rushton, N; Best, SM; Cameron, RE; Best, Serena [0000-0001-7866-8607]; Cameron, Ruth [0000-0003-1573-4923]The structure of an ideal scaffold for tendon regeneration must be designed to provide a mechanical, structural and chemotactic microenvironment for native cellular activity to synthesize functional (i.e. load bearing) tissue. Collagen fibre scaffolds for this application have shown some promise to date, although the microstructural control required to mimic the native tendon environment has yet to be achieved allowing for minimal control of critical in vivo properties such as degradation rate and mass transport. In this report we describe the fabrication of a novel multi-fibre collagen fascicle structure, based on type-I collagen with failure stress of 25-49 MPa, approximating the strength and structure of native tendon tissue. We demonstrate a microscopic fabrication process based on the automated assembly of type-I collagen fibres with the ability to produce a controllable fascicle-like, structural motif allowing variable numbers of fibres per fascicle. We have confirmed that the resulting post-fabrication type-I collagen structure retains the essential phase behaviour, alignment and spectral characteristics of aligned native type-I collagen. We have also shown that both ovine tendon fibroblasts and human white blood cells in whole blood readily infiltrate the matrix on a macroscopic scale and that these cells adhere to the fibre surface after seven days in culture. The study has indicated that the synthetic collagen fascicle system may be a suitable biomaterial scaffold to provide a rationally designed implantable matrix material to mediate tendon repair and regeneration. Item Open AccessThe interplay between physical and chemical properties of protein films affects their bioactivity.(Wiley, 2012-09) Grover, Chloe N; Farndale, Richard W; Best, Serena M; Cameron, Ruth E; Farndale, Richard [0000-0001-6130-8808]; Best, Serena [0000-0001-7866-8607]; Cameron, Ruth [0000-0003-1573-4923]Although mechanical properties, roughness, and receptor molecule expression have all been shown to influence the cellular reactivity of collagen-based biomaterials, their relative contribution, in a given system remains unclear. Here, we study films containing combinations of collagen, gelatin, and soluble and insoluble elastin, crosslinking of which results in altered film stiffness and roughness. Collagen and gelatin have similar amino acid sequences but altered cell-binding sites. We studied cell response with both C2C12 myoblast cells (which possess RGD-recognizing integrins α(V)β(3) and α(5)β(1)) and C2C12-α2+ cells (which, in addition, express the collagen-binding integrin α(2)β(1)) to establish the effect of altering the available binding sites on cell adhesion and spreading on films. Systematically altering the composition, crosslinking and cell type, allows us to deconvolute the effects of physical parameters and available binding sites on the cell reactivity of films in this system. Collagen-based films were rougher and stiffer and supported lower cell surface coverage than gelatin-based films. Additionally, C2C12-α2+ cells showed preferential attachment to collagen-based films compared with C2C12 cells, but no significant difference was seen using gelatin-based films. The cell count and surface coverage were found to decrease significantly on all films after crosslinking (Coll XL coverage = 2-6%, Gel XL coverage = 20-32%), but cell area and aspect ratio on collagen films were affected to a greater extent than on gelatin films. The results show that, in this system, the composition, and more significantly, crosslinking, of films affects the cell reactivity to a greater extent than their stiffness or roughness. Item Open AccessInvestigating the morphological, mechanical and degradation properties of scaffolds comprising collagen, gelatin and elastin for use in soft tissue engineering.(Elsevier BV, 2012-06) Grover, Chloe N; Cameron, Ruth E; Best, Serena M; Cameron, Ruth [0000-0003-1573-4923]; Best, Serena [0000-0001-7866-8607]Collagen-based scaffolds can be used to mimic the extracellular matrix (ECM) of soft tissues and provide support during tissue regeneration. To better match the native ECM composition and mechanical properties as well as tailor the degradation resistance and available cell binding motifs, other proteins or different collagen types may be added. The present study has explored the use of components such as gelatin or elastin and investigated their effect on the bulk physical properties of the resulting scaffolds compared to those made from pure collagen type I. The effect of altering the composition and crosslinking was evaluated in terms of the scaffold structure, mechanical properties, swelling, degradation and cell attachment. Results demonstrate that scaffolds based on gelatin had reduced tensile stiffness and degradation time compared with collagen. The addition of elastin reduced the overall strength and stiffness of the scaffolds, with electron microscopy results suggesting that insoluble elastin interacts best with collagen and soluble elastin interacts best with gelatin. Carbodiimide crosslinking was essential for structural stability, strength and degradation resistance for scaffolds of all compositions. In addition, preliminary cell adhesion studies showed these highly porous structures (pore size 130-160 μm) to be able to support HT1080 cell infiltration and growth. Therefore, this study suggests that the use of gelatin in place of collagen, with additions of elastin, can tailor the physical properties of scaffolds and could be a design strategy for reducing the overall material costs. Item Open AccessCrosslinking and composition influence the surface properties, mechanical stiffness and cell reactivity of collagen-based films.(Elsevier BV, 2012-08) Grover, Chloe N; Gwynne, Jessica H; Pugh, Nicholas; Hamaia, Samir; Farndale, Richard W; Best, Serena M; Cameron, Ruth E; Farndale, Richard [0000-0001-6130-8808]; Best, Serena [0000-0001-7866-8607]; Cameron, Ruth [0000-0003-1573-4923]This study focuses on determining the effect of varying the composition and crosslinking of collagen-based films on their physical properties and interaction with myoblasts. Films composed of collagen or gelatin and crosslinked with a carbodiimide were assessed for their surface roughness and stiffness. These samples are significant because they allow variation of physical properties as well as offering different recognition motifs for cell binding. Cell reactivity was determined by the ability of myoblastic C2C12 and C2C12-α2+ cell lines (with different integrin expression) to adhere to and spread on the films. Significantly, crosslinking reduced the cell reactivity of all films, irrespective of their initial composition, stiffness or roughness. Crosslinking resulted in a dramatic increase in the stiffness of the collagen film and also tended to reduce the roughness of the films (R(q) = 0.417 ± 0.035 μm, E = 31 ± 4.4 MPa). Gelatin films were generally smoother and more compliant than comparable collagen films (R(q) = 7.9 ± 1.5 nm, E = 15 ± 3.1 MPa). The adhesion of α2-positive cells was enhanced relative to the parental C2C12 cells on collagen compared with gelatin films. These results indicate that the detrimental effect of crosslinking on cell response may be due to the altered physical properties of the films as well as a reduction in the number of available cell binding sites. Hence, although crosslinking can be used to enhance the mechanical stiffness and reduce the roughness of films, it reduces their capacity to support cell activity and could potentially limit the effectiveness of the collagen-based films and scaffolds. Item Accepted version Open AccessOsteoinduction by combining bone morphogenetic protein (BMP)-2 with a bioactive novel nanocomposite.(British Editorial Society of Bone & Joint Surgery, 2012-07) Sharma, A; Meyer, F; Hyvonen, M; Best, SM; Cameron, RE; Rushton, N; Hyvonen, Marko [0000-0001-8683-4070]; Best, Serena [0000-0001-7866-8607]; Cameron, Ruth [0000-0003-1573-4923]OBJECTIVES: There is increasing application of bone morphogenetic proteins (BMPs) owing to their role in promoting fracture healing and bone fusion. However, an optimal delivery system has yet to be identified. The aims of this study were to synthesise bioactive BMP-2, combine it with a novel α-tricalcium phosphate/poly(D,L-lactide-co-glycolide) (α-TCP/PLGA) nanocomposite and study its release from the composite. METHODS: BMP-2 was synthesised using an Escherichia coli expression system and purified. In vitro bioactivity was confirmed using C2C12 cells and an alkaline phosphatase assay. The modified solution-evaporation method was used to fabricate α-TCP/PLGA nanocomposite and this was characterised using X-ray diffraction and scanning electron microscopy. Functionalisation of α-TCP/PLGA nanocomposite by adsorption of BMP-2 was performed and release of BMP-2 was characterised using an enzyme-linked immunosorbent assay (ELISA). RESULTS: Alkaline phosphatase activity of C2C12 cells was increased by the presence of all BMP-2/nanocomposite discs compared with the presence of a blank disc (p = 0.0022), and increased with increasing incubation concentrations of BMP-2, showing successful adsorption and bioactivity of BMP-2. A burst release profile was observed for BMP-2 from the nanocomposite. CONCLUSIONS: Functionalisation of α-TCP/PLGA with BMP-2 produced osteoinduction and was dose-dependent. This material therefore has potential application as an osteoinductive agent in regenerative medicine. Item Open AccessCollagen-hyaluronic acid scaffolds for adipose tissue engineering.(Elsevier BV, 2010-10) Davidenko, N; Campbell, JJ; Thian, ES; Watson, CJ; Cameron, RE; Watson, Christine [0000-0002-8548-5902]; Cameron, Ruth [0000-0003-1573-4923]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. Item Open AccessBiomimetic collagen scaffolds with anisotropic pore architecture(Elsevier, 2011-01-10) Davidenko, N; Gibb, T; Schuster, C; Best, SM; Campbell, JJ; Watson, CJ; Cameron, RE; Best, Serena [0000-0001-7866-8607]; Watson, Christine [0000-0002-8548-5902]; Cameron, Ruth [0000-0003-1573-4923]Sponge-like matrices with a specific 3D structural design resembling the actual extracellular matrix (ECM) of a particular tissue show significant potential for the regeneration and repair of a broad range of damaged anisotropic tissues. The manipulation of the structure of collagen scaffolds using a freeze-drying technique was explored in this work as an intrinsically biocompatible way of tailoring the inner architecture of the scaffold. The research was focused on the influence of temperature gradients, imposed during the phase of crystallisation of collagen suspensions, upon the degree of anisotropy in the microstructures of the scaffolds produced. Moulding technology was employed to achieve differences in heat transfer rates during the freezing processes. For this purpose various moulds with different configurations were developed with a view to producing uni-axial and multidirectional temperature gradients across the sample during this process. SEM analysis of different cross-sections (longitudinal and horizontal) of scaffolds revealed that highly aligned matrices with axially directed pore architecture were obtained where single unidirectional temperature gradient was induced. Alteration of freezing conditions by the introduction of multi-temperature gradients allowed collagen scaffolds to be produced with complex pore orientations, and anisotropy in pore size and alignment.