Justin J. Cooper-White

Directing osteogenic and myogenic differentiation of MSCs: interplay of stiffness and adhesive ligand presentation

The mechanical properties of the extracellular matrix (ECM) can exert significant influence in determining cell fate. Human mesenchymal stem cells (MSCs) grown on substrates with varying stiffness have been shown to express various cell lineage markers, without the use of toxic DNA demethylation agents or complex cocktails of expensive growth factors. Here we investigated the myogenic and osteogenic potential of various polyacrylamide gel substrates that were coated with covalently bound tissue-specific ECM proteins (collagen I, collagen IV, laminin, or fibronectin). The gel-protein substrates were shown to support the growth and proliferation of MSCs in a stiffness-dependent manner. Higher stiffness substrates encouraged up to a 10-fold increase in cell number over lower stiffness gels. There appears to be definitive interplay between substrate stiffness and ECM protein with regard to the expression of both osteogenic and myogenic transcription factors by MSCs. Of the 16 gel-protein combinations investigated, osteogenic differentiation was found to occur significantly only on collagen I-coated gels with the highest modulus gel tested (80 kPa). Myogenic differentiation occurred on all gel-protein combinations that had stiffnesses >9 kPa but to varying extents as ascertained by MyoD1 expression. Peak MyoD1 expression was seen on gels with a modulus of 25 kPa coated in fibronectin, with similar levels of expression observed on 80-kPa collagen I-coated gels. The modulation of myogenic and osteogenic transcription factors by various ECM proteins demonstrates that substrate stiffness alone does not direct stem cell lineage specification. This has important implications in the development of tailored biomaterial systems that more closely mimic the microenvironment found in native tissues.

Drop formation and breakup of low viscosity elastic fluids: Effects of molecular weight and concentration

The dynamics of drop formation and pinch-off have been investigated for a series of low viscosity elastic fluids possessing similar shear viscosities, but differing substantially in elastic properties. On initial approach to the pinch region, the viscoelastic fluids all exhibit the same global necking behavior that is observed for a Newtonian fluid of equivalent shear viscosity. For these low viscosity dilute polymer solutions, inertial and capillary forces form the dominant balance in this potential flow regime, with the viscous force being negligible. The approach to the pinch point, which corresponds to the point of rupture for a Newtonian fluid, is extremely rapid in such solutions, with the sudden increase in curvature producing very large extension rates at this location. In this region the polymer molecules are significantly extended, causing a localized increase in the elastic stresses, which grow to balance the capillary pressure. This prevents the necked fluid from breaking off, as would occur i...

New murine model of spontaneous autologous tissue engineering, combining an arteriovenous pedicle with matrix materials

The authors previously described a model of tissue engineering in rats that involves the insertion of a vascular pedicle and matrix material into a semirigid closed chamber, which is buried subcutaneously. The purpose of this study was to develop a comparable model in mice, which could enable genetic mutants to be used to more extensively study the mechanisms of the angiogenesis, matrix production, and cellular migration and differentiation that occur in these models. A model that involves placing a split silicone tube around blood vessels in the mouse groin was developed and was demonstrated to successfully induce the formation of new vascularized tissue. Two vessel configurations, namely, a flow-through pedicle (n = 18 for three time points) and a ligated vascular pedicle (n = 18), were compared. The suitability of chambers constructed from either polycarbonate or silicone and the effects of incorporating either Matrigel equivalent (n = 18) or poly(dl-lactic-co-glycolic acid) (n = 18) on angiogenesis and tissue production were also tested. Empty chambers, chambers with vessels only, and chambers with matrix only served as control chambers. The results demonstrated that a flow-through type of vascular pedicle, rather than a ligated pedicle, was more reliable in terms of patency, angiogenesis, and tissue production, as were silicone chambers, compared with polycarbonate chambers. Marked angiogenesis occurred with both types of extracellular matrix scaffolds, and there was evidence that native cells could migrate into and survive within the added matrix, generating a vascularized three-dimensional construct. When Matrigel was used as the matrix, the chambers filled with adipose tissue, creating a highly vascularized fat flap. In some cases, new breast-like acini and duct tissue appeared within the fat. When poly(dl-lactic-co-glycolic acid) was used, the chambers filled with granulation and fibrous tissue but no fat or breast tissue was observed. No significant amount of tissue was generated in the control chambers. Operative times were short (25 minutes), and two chambers could be inserted into each mouse. In summary, the authors have developed an in vivo murine model for studying angiogenesis and tissue-engineering applications that is technically simple and quick to establish, has a high patency rate, and is well tolerated by the animals.

The influence of substrate creep on mesenchymal stem cell behaviour and phenotype.

Human mesenchymal stem cells (hMSCs) are capable of probing and responding to the mechanical properties of their substrate. Although most biological and synthetic matrices are viscoelastic materials, previous studies have primarily focused upon substrate compressive modulus (rigidity), neglecting the relative contributions that the storage (elastic) and loss (viscous) moduli make to the summed compressive modulus. In this study we aimed to isolate and identify the effects of the viscous component of a substrate on hMSC behaviour. Using a polyacrlyamide gel system with constant compressive modulus and varying loss modulus we determined that changes to substrate loss modulus substantially affected hMSC morphology, proliferation and differentiation potential. In addition, we showed that the effect of substrate loss modulus on hMSC behaviour is due to a reduction in both passive and actively generated isometric cytoskeletal tension caused by the inherent creep of substrates with a high loss modulus. These findings highlight substrate creep, or more explicitly substrate loss modulus, as an important mechanical property of a biomaterial system that can be tailored to encourage the growth and differentiation of specific cell types.

Enhanced chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells in low oxygen environment micropellet cultures

Chondrogenesis of mesenchymal stem cells (MSCs) is typically induced when they are condensed into a single aggregate and exposed to transforming growth factor-β (TGF-β). Hypoxia, like aggregation and TGF-β delivery, may be crucial for complete chondrogenesis. However, the pellet dimensions and associated self-induced oxygen gradients of current chondrogenic methods may limit the effectiveness of in vitro differentiation and subsequent therapeutic uses. Here we describe the use of embryoid body-forming technology to produce microscopic aggregates of human bone marrow MSCs (BM-MSCs) for chondrogenesis. The use of micropellets reduces the formation of gradients within the aggregates, resulting in a more homogeneous and controlled microenvironment. These micropellet cultures (~170 cells/micropellet) as well as conventional pellet cultures (~2 × 105 cells/pellet) were chondrogenically induced under 20% and 2% oxygen environments for 14 days. Compared to conventional pellets under both environments, micropellets differentiated under 2% O2 showed significantly increased sulfated glycosaminoglycan (sGAG) production and more homogeneous distribution of proteoglycans and collagen II. Aggrecan and collagen II gene expressions were increased in pellet cultures differentiated under 2% O2 relative to 20% O2 pellets but 2% O2 micropellets showed even greater increases in these genes, as well as increased SOX9. These results suggest a more advanced stage of chondrogenesis in the micropellets accompanied by more homogeneous differentiation. Thus, we present a new method for enhancing MSC chondrogenesis that reveals a unique relationship between oxygen tension and aggregate size. The inherent advantages of chondrogenic micropellets over a single macroscopic aggregate should allow for easy integration with a variety of cartilage engineering strategies.

Drop formation dynamics of constant low-viscosity, elastic fluids

The dynamics of drop formation under gravity has been investigated as a function of elasticity using a set of low-viscosity, ideal elastic fluids and an equivalent Newtonian glycerol-water solution. All solutions had the same shear viscosity, equilibrium surface tension, and density, but differed greatly in elasticity. The minimum drop radius in the early stages of drop formation (necking) was found to scale as expected from potential flow theory, independent of the elasticity of the solutions. Thus, during this stage of drop formation when viscous force is still weak, the dynamics are controlled by a balance between inertial and capillary forces, and there is no contribution of elastic stresses of the polymer. However, upon formation of the pinch regions, there is a large variation in the drop development to break-off observed between the various solutions. The elastic solutions formed secondary fluid threads either side of a secondary drop from the necked region of fluid between the upper and lower pinches, which were sustained for increasing amounts of time. The break-off lengths and times increase with increasing elasticity of the solutions. Evolution of the filament, length is, however, identical in shape and form for all of the polymer solutions tested, regardless of differing elasticity. This de-coupling between filament growth rate and break-up time (or equivalently, final filament length at break-up) is rationalised. A modified force balance to that of Jones and Rees [48] is capable of correctly predicting the filament growth of these low-viscosity, elastic fluids in the absence of any elastic contributions due to polymer extension within the elongating filament. The elongation of the necked region of fluid (which becomes the filament) is dominated by the inertia of the drop, and is independent of the elasticity of the solution. However, elasticity does strongly influence the resistance of the pinch regions to break-off, with rapid necking resulting in extremely high rates of surface contraction on approach to the pinch point, initiating extension of the polymer chains within the pinch regions. This de-coupling phenomenon is peculiar to low-viscosity, elastic fluids as extension does not occur prior to the formation of the pinch points (i.e. just prior to break-up), as opposed to the high viscosity counterparts in which extension of polymers in solution may occur even during necking. Once steady-state extension of the polymers is achieved within the pinch at high extension rates, the thread undergoes elasto-capillary break-up as the capillarity again overcomes the viscoelastic forces. The final length at detachment and time-to-break-off (relative to the equivalent Newtonian fluid) is shown to be linearly proportional to the longest relaxation time of the fluid

Lateral spacing of adhesion peptides influences human mesenchymal stem cell behaviour

Mesenchymal stem cells (MSCs) have attracted great interest in recent years for tissue engineering and regenerative medicine applications due to their ease of isolation and multipotent differentiation capacity. In the past, MSC research has focussed on the effects of soluble cues, such as growth factors and cytokines; however, there is now increasing interest in understanding how parameters such as substrate modulus, specific extracellular matrix (ECM) components and the ways in which these are presented to the cell can influence MSC properties. Here we use surfaces of self-assembled maleimide-functionalized polystyrene-block-poly(ethylene oxide) copolymers (PS-PEO-Ma) to investigate how the spatial arrangement of cell adhesion ligands affects MSC behaviour. By changing the ratio of PS-PEO-Ma in mixtures of block copolymer and polystyrene homopolymer, we can create surfaces with lateral spacing of the PEO-Ma domains ranging from 34 to 62 nm. Through subsequent binding of cysteine–GRGDS peptides to the maleimide-terminated end of the PEO chains in each of these domains, we are able to present tailored surfaces of controlled lateral spacing of RGD (arginine-glycine-aspartic acid) peptides to MSCs. We demonstrate that adhesion of MSCs to the RGD-functionalized block-copolymer surfaces is through specific attachment to the presented RGD motif and that this is mediated by α5, αV, β1 and β3 integrins. We show that as the lateral spacing of the peptides is increased, the ability of the MSCs to spread is diminished and that the morphology changes from well-spread cells with normal fibroblastic morphology and defined stress-fibres, to less-spread cells with numerous cell protrusions and few stress fibres. In addition, the ability of MSCs to form mature focal adhesions is reduced on substrates with increased lateral spacing. Finally, we investigate differentiation and use qRT-PCR determination of gene expression levels and a quantitative alkaline phosphatase assay to show that MSC osteogenesis is reduced on surfaces with increased lateral spacing while adipogenic differentiation is increased. We show here, for the first time, that the lateral spacing of adhesion peptides affects human MSC (hMSC) properties and might therefore be a useful parameter with which to modify hMSC behaviour in future tissue engineering strategies.

Palladium nanoparticles decorated carbon nanotubes: facile synthesis and their applications as highly efficient catalysts for the reduction of 4-nitrophenol

Two completely green and facile strategies have been developed to decorate Pd nanoparticles onto carbon nanotubes (CNT) sidewalls via non-covalent interactions assisted with newly synthesized hyperbranched polymers (PiHPs). The resultant CNT/PiHP/Pd heterogeneous catalysts exhibit ultrahigh catalytic reactivity towards the reduction of 4-nitrophenol.

A novel processing aid for polymer extrusion: Rheology and processing of polyethylene and hyperbranched polymer blends

The use of hyperbranched polymers (HBPs) as a processing aid for linear low density polyethylene (LLDPE) was investigated. Various generation (or pseudo-generation) HBPs were used which had either 16 carbon atom alkanes or a mixture of 20/22 carbon atom alkanes on the end groups. In addition, the degree of end group substitution was studied. Blends of up to 10% HBP content were mixed via extrusion at 170 °C to produce 1 mm diameter fibers. Processability, surface appearance and the rheological properties of the blends were evaluated. It was found the power requirement for extrusion was significantly decreased as a result of reduced blend viscosity, and also, the mass flow rate for a given extruder speed was greater than virgin LLDPE for all HBP blends. Melt fracture and sharkskin of the blends was successfully eliminated, and minimal preprocessing time was required for the effect to take place. Surface analysis using x-ray photoelectron spectroscopy and transmission electron microscope techniques were per...

Rheological properties of poly(lactides). Effect of molecular weight and temperature on the viscoelasticity of poly(l‐lactic acid)

The dynamic viscoelastic behavior of Poly(l-lactic acid) (PLLA), with molecular weights ranging from 2,000 to 360,000, have been studied over a broad range of reduced frequencies (approximately 1 × 10-3 s-1 to 1 × 103 s-1), using time-temperature superposition principle. Melts are shown to have a critical molecular weight, Mc, of approximately 16,000 g/mol, and an entanglement density of 0.16 mmol/cm3 (at 25°C). PLLA polymers are noted to require substantially larger molecular weights in order to display similar melt viscoelastic behavior, at a given temperature, as that for conventional non-biodegradable polymers such as polystyrene. The reason for this deviation is suspected to be due to steric hindrance, resulting from excessive coil expansion or other tertiary chain interactions. PLLA melts show a dependence of 0 on chain length to the 4.0 power (M[stack 4.0W ]), whilst J[stack 0e ] is independent of MW in the terminal region. Low molecular weight PLLA ( 40,000) shows Newtonian-like behavior at shear rates typical of those achieved during film extrusion.