Transient serum exposure regimes to support dual differentiation of human mesenchymal stem cells.

Human mesenchymal stem cells (MSCs), which can generate both osteoblasts and chondrocytes, represent an ideal resource for orthopaedic repair using tissue-engineering approaches. One major difficulty for the development of osteochondral constructs using undifferentiated MSCs is that serum is typically used in culture protocols to promote differentiation of the osteogenic component, whereas existing chondrogenic differentiation protocols rely on the use of serum-free conditions. In order to define conditions which could be compatible with both chondrogenic and osteogenic differentiation in a single bioreactor, we have analysed the efficiency of new biphasic differentiation regimes based on transient serum exposure followed by serum-free treatment. MSC differentiation was assessed either in serum-free medium or with a range of transient exposure to serum, and compared to continuous serum-containing treatment. Although osteogenic differentation was not supported in the complete absence of serum, marker expression and extensive mineralization analyses established that 5 days of transient exposure triggered a level of differentiation comparable to that observed when serum was present throughout. This initial phase of serum exposure was further shown to support the successful chondrogenic differentiation of MSCs, comparable to controls maintained in serum-free conditions throughout. This study indicates that a culture based on temporal serum exposure followed by serum-free treatment is compatible with both osteogenic and chondrogenic differentiation of MSCs. These results will allow the development of novel strategies for osteochondral tissue engineering approaches using MSCs for regenerative medicine.

The influence of coupling agents on mechanical property retention and long-term cytocompatibility of phosphate glass fibre reinforced PLA composites.

Completely resorbable composites are an attractive alternative for metallic bone-fracture fixation devices. However, failure of their interfacial integrity within aqueous environments, which can lead to a rapid loss of overall mechanical properties, has been reported in the literature. In this study coupling agents were investigated for phosphate glass fibre reinforced poly(lactic acid) composites. Three coupling agents with varying wettability were employed to improve initial mechanical properties and their retention in vitro via improvement of the interfacial bond between polymer matrix and fibres. Coupling agents were grafted onto the glass fibres by dip-coating in coupling agent solution at optimised concentrations. Three-aminopropyltriethoxy silane and sorbitol ended PLA oligomer treatments improved the initial flexural properties (27% strength with APS and 17% modulus via SPLA treatment) of the composites and 3-aminopropyltriethoxy silane and hexamethylene diisocyanate (HDI) treatments also decreased the loss of flexural strength and modulus during degradation. HDI treated samples retained 57.2% and 64.7% of their initial strength and modulus, respectively compared to control where only 34% of initial strength and 52% of initial modulus was retained after 28 days of degradation in PBS solution. Initial improvements in flexural properties were associated with improved shear bond strength at the interface due to covalent bonding between the glass fibres and polymer matrix provided by the coupling agents. Delay in mechanical property loss with degradation was suggested to be due to the hydrophobicity at the interface, which could have hindered the interfacial integrity loss and consequently loss of mechanical integrity of the composites. All coupling agent treated and control composites were tested for cytocompatibility using a primary human osteoblast cell line. A comparable response to the control, in terms of cell adhesion, proliferation and differentiation was observed supporting the use of these agents within implantable devices.

Cytocompatibility assessment of chemical surface treatments for phosphate glass to improve adhesion between glass and polyester.

Fully resorbable phosphate glass fiber reinforced polymer composites have shown real potential for replacing some of the existing metallic bone fracture fixation devices. However, some of these composites have not provided suitable mechanical strength profiles over the required healing period for bone. Typically, it has been seen that these composites can lose up to 50% or more of their strength within the first week of degradation. Functionalizing the glass surface to promote polymer adhesion or to introduce hydrophobicity at the glass surface could potentially introduce control over the mechanical properties of the composite and their retention. In this study eight chemical agents namely, Glycerol 2-phosphate disodium salt; 3-phosphonopropionic acid; 3-aminopropyltriethoxy silane; etidronic acid; hexamethylene diisocyanate; sorbitol/sodium ended PLA oligomers and amino phosphonic acid, were selected to functionalise the bulk phosphate glass surface. Selected chemical agents had one functional group (-OH or O C N) to react with the glass and another functionality (either -OH, NH2, or Na) to react with the polymer matrix and/or produce hydrophobicity at the fiber surface. Bulk phosphate glass surface-treated with the above agents were assessed for the cytotoxicity of degradation products cell-material interaction in short- and long-term direct cytocompatibility studies. Results obtained from these cytocompatibility studies (using human osteosarcoma (MG63) and primary human osteoblast cell lines) revealed no cytotoxicity from the degradation products and a response comparable to controls in terms of cell functions (attachment, viability, metabolic activity, proliferation, and differentiation) and morphology.

Material characterisation and cytocompatibility assessment of quinternary phosphate glasses.

Six phosphate glass formulations (in the system P(2)O(5)-CaO-MgO-Na(2)O-Fe(2)O(3)) were produced with fixed magnesium and calcium content at 24 and 16 mol%, respectively. P(2)O(5) and Fe(2)O(3) were varied between 40-50 and 0-4 mol% respectively, with the balance being Na(2)O. EDX analyses confirmed the final composition of the glasses investigated to within a 1-2 % error margin. Thermal analyses showed a linear increase in T(g) with increasing Fe(2)O(3) and P(2)O(5) contents, with Fe(2)O(3) showing a greater effect than P(2)O(5). This was proposed to be due to the formation of Fe-O-P bonds and an increase in the cross-link density of the glass network enhancing the durability of the glass. The glasses that were investigated revealed a decrease in degradation rate with increasing Fe(2)O(3) and P(2)O(5) contents and again the effect of Fe(2)O(3) was greater. All the above characteristics correlated well with structural changes measured by IR and XPS analyses. Cytocompatibility studies showed good cellular (MG63) response to the glasses up to 168 h in terms of cell viability, proliferation and differentiation. Statistical analysis revealed that all the formulations with the exception of P50Fe4 gave a comparable response to the control (TCP), which suggested that after a threshold level of glass durability is achieved the degradation rate has no or minimal effect on biocompatibility. However, it was seen that the glass chemistry can also affect cellular response, since increasing the P(2)O(5) content promoted phenotypic expression that was not related to degradation rate but to the degradation products. This was supported using an elution assay.

Composites for bone repair: phosphate glass fibre reinforced PLA with varying fibre architecture.

Internal fixation for bone fractures with rigid metallic plates, screws and pins is a proven operative technique. However, refracture's have been observed after rigid internal fixation with metal plates and plate fixation has been known to cause localised osteopenia under and near the plate. In the present study, resorbable composites comprising a PLA matrix reinforced with iron doped phosphate glass fibres were investigated. Non-woven random mat laminates of approximately 30% and 45% fibre volume fraction (V(f)) were produced, along with unidirectional and 0°-90° samples of approximately 20% V(f). The non-woven composite laminates achieved maximum values of 10 GPa modulus and 120 MPa strength. The 0-90º samples showed unexpectedly low strengths close to matrix value (~50 MPa) although with a modulus of 7 GPa. The UD specimens exhibited values of 130 MPa and 11.5 GPa for strength and modulus respectively. All the modulus values observed were close to that expected from the rule of mixtures. Samples immersed in deionised water at 37°C revealed rapid mechanical property loss, more so for the UD and 0-90º samples. It was suggested that continuous fibres wicked the degradation media into the composite plates which sped up the deterioration of the fibre-matrix interface. The effect was less pronounced in the non-woven random mat laminates due to the discontinuous arrangement of fibres within the composite, making it less prone to wicking. Random mat composites revealed a higher mass loss than the UD and 0°-90° specimens, it was suggested this was due to the higher fibre volume fractions of these composites and SEM studies revealed voidage around the fibres by day 3. Studies of pH of the degradation media showed similar profiles for all the composites investigated. An initial decrease in pH was attributed to the release of phosphate ions into solution followed by a gradual return back to neutral.

Repair of calvarial defects in rats by prefabricated, degradable, long fibre composite implants.

We report results from an initial small animal study designed to provide information on the biocompatibility of a novel biodegradable composite designed for craniomaxillofacial reconstruction. Rat calvarium was chosen as a clinically analogous model, which allowed comparison between experimental groups (PCL alone, PCL/phosphate glass, or PCL/bioglass implants) and control groups (empty defects or bone grafted defects). All animals recovered well from surgery and no clinical complications were observed. Histological assessment indicated a lack of inflammatory response. The amount of new bone formation at the dural aspect of the implant was statistically significantly higher in the PCL/phosphate glass group than the other experimental groups. This study confirms, in a clinically analogous model, the promise of the novel PCL/phosphate glass composite material. Work is planned toward manufacturing scale up and clinical trials.

An appraisal of ultramicrotomy, FIBSEM and cryogenic FIBSEM techniques for the sectioning of biological cells on titanium substrates for TEM investigation.

Ultramicrotomy, focused ion beam scanning electron microscopy (FIBSEM) and cryogenic FIBSEM (cryo-FIBSEM) techniques, as developed for the controlled cross-sectioning of mesenchymal stem cells (MSCs) and human osteoblasts (HObs) on titanium (Ti) substrates for transmission electron microscopy (TEM) investigation, are compared. Conventional ultramicrotomy has been used to section cells on Ti-foil substrates embedded in resin, but significant problems with cell detachment using this technique restricted its general applicability. Conventional FIBSEM 'lift-out' procedures were found to be effective for the preparation of uniform sections of fixed and dehydrated cell/Ti specimens, but the control of cell staining remains an issue. Cryo-FIBSEM procedures used with an 'H-bar' sample geometry enabled the sectioning of fixed and hydrated cell/Ti specimens, but issues remain over ion beam-induced artefacts and control of frost on the sample foils.

Cytocompatibility and hemocompatibility of a novel chitosan-alginate gel system.

Two chitosan-alginate gel systems in the form of membranes were produced and evaluated. The first membrane was produced by a novel gel system formed after blending N-(methylsulfonic acid) chitosan with ammonium alginate (CAG1) and the second was an N-(methylsulfonic acid) chitosan-sodium alginate blend cross-linked with glutaraldehyde and calcium chloride (CAG2). The cytocompatibility and hemocompatibility of the gels were examined by assessing the cell viability of 3T3 Swiss mouse fibroblasts, whole blood hemolysis, and platelet activation. Cell viability was not significantly different by exposure to these gels compared to the controls. Both gel types had minimal effect on hemolysis of whole heparinized rabbit blood after 1-h exposure. Further platelet activation by the surfaces was also minimal. These results indicate that these novel gels merit further investigation for blood contact applications.

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.

Thin-film theories for two-phase reactive flow models of active cell motion.

The aim of this paper is to develop a broadly-applicable and self-consistent thin-film biphasic modelling framework for the full crawling cycle of a single animal cell. A hierarchy of thin-film two-phase 'reactive flow' models is derived; between them these cover a wide range of biologically relevant parameter regimes. The mathematical properties and biological implications of the resulting systems of high-order nonlinear degenerate parabolic-elliptic evolution equations are investigated. Linear-stability arguments suggest the formation of highly localized regions of high or low network density associated with small irregular oscillations or 'ruffling' of the plasma membrane. Local analyses at the contact line identify the classes of admissible contact-line conditions, through which we study for the first time the effect on the cell-scale motion of the 'mesoscopic' contact-line physics, which consists of the chemical and mechanical mechanisms for protrusive and retractive force generation near the outer cell periphery. One of the formulations is used to develop a minimal model for cell body translocation over a thin pseudopod, which predicts that myosin-driven contraction is not essential for rapid translocation. An analytic prediction for the translocation speed is given in terms of the network viscosity and slip coefficient (a parameter measuring the adhesion strength), of the membrane tension and of the thicknesses of the pseudopod and actin cortex; this is in good agreement with the translocation speed of osteoblasts on biomaterial substrates commonly used for orthopaedic implants. Limitations of the modelling approach and directions for future work are outlined.

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.

Preparation of poly(epsilon-caprolactone)/continuous bioglass fibre composite using monomer transfer moulding for bone implant.

Poly(epsilon-caprolactone) (PCL)/continuous bioglass fibre composite was prepared using the monomer transfer moulding technique coupled with a surface initiated polymerisation. The bioglass fibres were surface treated with an amine ended silane in order to initiate polymerisation of epsilon-caprolactone from the fibre surface. Surface initiated polymerisation significantly improved the Young's modulus and flexural strength and water resistance of the composite. Initial in vitro biocompatibility assessment suggests that amine ended silane treatment of bioglass fibres before their inclusion in the composite does not have a negative effect on the biological responses in terms of macrophage activation as measured by IL-1beta release and craniofacial osteoblast attachment.

The effect of production regime and crucible materials on the thermal properties of sodium phosphate glasses produced from salts.

Changes in the thermal properties of sodium phosphate glasses during melt production have been investigated using Pt/Au and fused alumina crucibles. Glasses were produced from NaH(2)PO(4) as a starting material, providing an intrinsic Na(2)O:P(2)O(5) ratio of 1:1 and giving an O/P = 3, that is, a metaphosphate. In Pt/Au crucibles, glass transition temperatures rose to a plateau value of 295 degrees C at a rate determined by melt temperature. No contamination of the glass by platinum or gold was detected or indicated in the results. E(a) for the reaction was found to be 66.4 kJ mol(-1). In fused alumina crucibles, glass transition temperatures rose to over 450 degrees C, with these values showing some convergence at higher furnace temperatures. Extensive erosion of the alumina crucibles was observed. The amount of alumina incorporation within the glasses correlated well with the rise in glass transition temperature up to a maximum of 15.5 mol % Al(2)O(3) content. Al(2)O(3) incorporation above this value caused a reduction in the value of the T(g).

Synthesis and degradation of sodium iron phosphate glasses and their in vitro cell response.

The degradation profiles of six sodium iron phosphate glass formulations have been investigated using simple dissolution trials in deionized water. The glasses were produced from the appropriate phosphate salts by melting at 1200 degrees C in 5% Au/95% Pt crucibles. Dissolution rates varied from 0.2 gcm(-2)h(-1) for the 1% Fe glass to essentially zero over the 6-week test period for the 15% Fe and 20% Fe glasses. The overall degradation rate was found to vary according to the approximate relation: rate = 1.3e(-0.79x) gcm(-2)h(-1), where x is the percentage iron content of the glass. Glasses with 10% or greater iron content were observed to maintain a constant density over the course of the tests and thus appeared to degrade from the surface and not the bulk. In vitro cell response tests were conducted on the glasses using macrophages and primary craniofacial osteoblasts. These tests were performed on the glasses with 10% or greater iron content because glasses with lower iron content degraded too quickly. Confocal microscopy revealed a rounded macrophage morphology and IL-1beta production was low, suggesting little macrophage activation. However, a significant level of peroxide production was observed. Osteoblasts were observed to attach to the glass surfaces and spread, exhibiting a similar cytosketetal organization to the cells on the Thermanox controls, with a high level of F-actin organization. On balance, the 15% Fe glass performed slightly better than the 20% Fe glass in these assays.

Controlled degradation and macrophage responses of a fluoride-treated polycaprolactone.

We have developed a new bone replacement material based on polycaprolactone (PCL), which can act as a suitable matrix for monomer transfer molding of degradable composites. A boron trifluoride catalyst with glycerol additive was used to produce PCL with a degradation rate that can be altered by treatment with fluoride ions. The effect of cations on the degradation of the polymer and macrophage cell responses are discussed. We found that treatment with fluoride ions reduced the degradation rate. No significant difference between these three fluorides was observed although a general trend was seen where KF-treated PCL appeared to degrade slower than NaF-treated PCL which was slower than NH(4)F-treated PCL. Variation in solubilities of the salts was observed where the K(+) cation had the highest solubility and the Na(+) cation had the lowest solubility, which suggests that NaF was able to degrade the polymer more efficiently than the other fluorides. No significant macrophage activation was observed after culture on the polymer surfaces as determined by peroxide and IL-1 beta release, whereas some activation occurred after culture in degradation products.

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.

Craniofacial osteoblast responses to polycaprolactone produced using a novel boron polymerisation technique and potassium fluoride post-treatment.

There is no ideal material for craniofacial bone repair at present. The aim of this study was to test the biocompatibility of polycaprolactone (PCL) synthesised by a novel method allowing control of molecular weight and degradation rate, with regard to it being used as matrix for a biodegradable composite for craniofacial bone repair. Human primary craniofacial cells were used, isolated from paediatric skull after surgery. Cell responses were analysed using various assays and antibody staining. Cells attached and spread on the PCL in a similar manner to the Thermanox controls as shown by phalloidin staining of F-actin. Cells maintained the osteoblast phenotype as demonstrated by alkaline phosphatase assay and antibody staining throughout the time points studied, up to 28 days. Cells proliferated on the PCL as shown by a DNA assay. Collagen-1 staining showed extensive production of a collagen-1 containing extracellular matrix, which was also shown to be mineralised by alizarin red staining. Short-term (up to 48 h) attachment studies and long-term (up to 28 days) expression of markers of the osteoblast phenotype have been demonstrated on the PCL. This new method of synthesising PCL shows biocompatibility characteristics that give it potential to be used for craniofacial bone repair.

Long-term craniofacial osteoblast culture on a sodium phosphate and a calcium/sodium phosphate glass.

The aim of this study was to determine the characteristics of human craniofacial osteoblasts cultured on sodium phosphate glass and calcium-sodium phosphate glass in a long-term culture of up to 28 days. The characteristics studied were attachment, proliferation, alkaline phosphatase activity, collagen-1 production, and mineralization. A comparison of the degradation rate, measured by mass loss of the glasses, which are intended for use as a component of a novel degradable composite for craniofacial bone repair, was also performed. It was our hypothesis that the glass would be degradable with a change in degradation rate observed by calcium addition and support osteoblast proliferation and expression of the above characteristics. The inclusion of calcium into the reaction mixture significantly decreased the degradation rate, and it is suggested that the slower degradation is the result of pseudo crosslinking (ionic crosslinks rather than covalent bonding) of the polyphosphate chains by the calcium ions. Therefore, twice as many P-O bonds will need to be hydrolyzed for dissolution of the metal phosphate to occur, therefore greatly reducing the rate of hydrolysis. Osteoblasts were able to attach, spread, and proliferate in a manner comparable with the positive control, as shown by analysis of variance. Formation of a collagen-rich mineralized matrix was also observed. The results presented here suggest that a biocompatible soluble glass has been produced, which has potential to be included in a novel biodegradable craniofacial implant.

Seeding cells into needled felt scaffolds for tissue engineering applications.

Tissue engineering methods are under development that will enable the repair or replacement of a variety of tissues, including articular cartilage and bone. To engineer functional tissue it is necessary that scaffolds initially be seeded with a large number of cells distributed evenly throughout the scaffold structure. It previously has been shown that, compared to static seeding conditions, seeding scaffolds under dynamic conditions facilitates high seeding densities and even distributions of cells (Li et al., Biotechnology Progress 2001;17:935-944). The efficiency of seeding HOSTE85 cells and bovine chondrocytes into needled felt scaffolds following agitation at different speeds was determined. Seeding efficiency was determined using the Hoechst 33258 assay, and cell viability was assessed using the Alamar Blue trade mark assay. The distribution of cells within the scaffolds was imaged using scanning electron microscopy. It was found that the optimum seeding conditions varied for HOSTE85 cells and bovine chondrocytes, with different agitation speeds leading to different seeding efficiencies, cell viabilities, and distributions of cells within scaffolds. The optimum agitation speeds for seeding a high number of viable cells into scaffolds so that they were arranged evenly were 300 rpm for HOSTE85 cells and 200 rpm for bovine chondrocytes.

Chemically patterned, metal-oxide-based surfaces produced by photolithographic techniques for studying protein- and cell-interactions. II: Protein adsorption and early cell interactions.

Protein adsorption and adhesion of primary human osteoblasts on chemically patterned, metal-oxide-based surfaces comprising combinations of titanium, aluminium, vanadium and niobium were investigated. Single metal samples with a homogeneous surface and bimetal samples with a surface pattern produced by photolithographic techniques were used. The physical and chemical properties of the samples have been extensively characterised and are presented in a companion paper. Here, we describe their properties in terms of cell responses during the initial 24h of cell culture. Regarding the cell number and activity there was no significant difference between any of the single metal surfaces. However the morphology of cells on vanadium surfaces became spindle-like. In contrast to the behaviour on single metal samples, cells exhibited a pronounced reaction on bimetallic surfaces that contained aluminium. Cells tended to stay away from aluminium, which was the least favoured metal in all two-metal combinations. An initial cell alignment relative to the pattern geometry was detectable after 2h and was fully developed after 18h of incubation. The organisation of f-actin and microtubules as well as the localisation of vinculin were all more pronounced on non-aluminium regions. We hypothesised that the differences in cell response could be associated with differences in the adsorption of serum proteins onto the various metal oxides. Protein adsorption experiments were performed using microscopy in conjunction with immunofluorescent stains. They indicated that both fibronectin and albumin adsorption were significantly greater on the non-aluminium regions, suggesting that differences in cellular response correlate with a modulation of the concentration of serum proteins on the surface.