Judith M. Curran

Introducing dip pen nanolithography as a tool for controlling stem cell behaviour: unlocking the potential of the next generation of smart materials in regenerative medicine.

Reproducible control of stem cell populations, regardless of their original source, is required for the true potential of these cells to be realised as medical therapies, cell biology research tools and in vitro assays. To date there is a lack of consistency in successful output when these cells are used in clinical trials and even simple in vitro experiments, due to cell and material variability. The successful combination of single chemistries in nanoarray format to control stem cell, or any cellular behaviour has not been previously reported. Here we report how homogenously nanopatterned chemically modified surfaces can be used to initiate a directed cellular response, particularly mesenchymal stem cell (MSC) differentiation, in a highly reproducible manner without the need for exogenous biological factors and heavily supplemented cell media. Successful acquisition of these data should lead to the optimisation of cell selective properties of materials, further enhancing the role of nanopatterned substrates in cell biology and regenerative medicine. The successful design and comparison of homogenously molecularly nanopatterned surfaces and their direct effect on human MSC adhesion and differentiation are reported in this paper. Planar gold surfaces were patterned by dip pen nanolithography (DPN) to produce arrays of nanodots with optimised fixed diameter of 70 nanometres separated by defined spacings, ranging from 140 to 1000 nm with terminal functionalities of simple chemistries including carboxyl, amino, methyl and hydroxyl. These nanopatterned surfaces exhibited unprecedented control of initial cell interactions and subsequent control of cell phenotype and offer significant potential for the future.

The use of dynamic surface chemistries to control msc isolation and function

Material modifications can be used to induce cell responses, in particular-CH(3) and -NH(2) have shown potential in enhancing the ability of a material to support mesenchymal stem cell (MSC) adhesion and differentiation. Currently this process is variable, due to the lack of definition of controlled contextual presentation of the chemical group of interest across the surface. This paper defines the potential of -CH(3) modified surfaces, with optimised dynamic surface chemistry, to manipulate initial MSC adhesive events, integrin binding, and subsequent cell function. An array of -CH(3) silane modified glass substrates was produced using different -CH(3) chain lengths and mechanisms of bonding to the base substrate. We show that changing the chain length affects the ability of the surfaces to support viable adult MSC adhesion, directly related to induced FGF release, and expression of STRO-1, CD29, 73, 90 and 105. Chlorodimethyloctylsilane (ODMCS) modified surfaces resulted in significant increases of associated adult MSC markers compared to all other -CH(3) modified and control substrates. In contrast Dichlorodimethylsilane (DMDCS) modified surfaces did not support adult MSC adhesion due to high levels of early FGF release, which had an inhibitory effect on adult MSC culture, but enhanced the efficiency and cell selective properties of the substrate in isolation of multi-potent progenitor/MSC from adult human whole blood. Incorporation of optimised -CH(3) groups is a cost effective route for producing substrates that significantly enhance MSC isolation and expansion, highlighting the potential of the optimised substrates to replace RGD and fibronectin modifications in selected applications.

The osteogenic response of mesenchymal stem cells to an injectable PLGA bone regeneration system.

The enrichment of substrates/surfaces with selected functional groups, methyl (-CH3), allyl amine (-NH2), allyl alcohol (-OH) and acrylic acid (-COOH), can be used to trigger mesenchymal stem (MSC) cell differentiation into specified lineages, minimising the need for exogenous biological supplementation. We present the successful translation of this research phenomenon to an injectable two phase injectable PLGA system, utilising plasma techniques, for the repair of bone defects. Modified microspheres were characterised using water contact angel (WCA), X-ray Photon Spectroscopy (XPS) and scanning electron microscopy (SEM). When cultured in contact with MSCs in vitro, the ability of the modified particles, within the 2 phase system, to induce differentiation was characterised using quantitative assays for cell viability and histological analysis for key markers of differentiation throughout the entirety of the three dimensional scaffold. Biological analysis proved that selected modified microspheres have the ability to induce MSC osteogenic (-NH2 modified scaffolds) and chondrogenic (-OH modified scaffolds) differentiation throughout the entirety of the formed scaffold. Therefore optimised plasma modification of microspheres is an effective tool for the production of injectable systems for the repair of bone and cartilage defects.

PLGA doping of PCL affects the plastic potential of human mesenchymal stem cells, both in the presence and absence of biological stimuli

A range of poly epsilon-caprolactone (PCL) films mixed/doped with poly(lactide-co-glycolide) (PLGA) (65:35) in 0, 10, 20, and 30 wt % were produced, sterilized using ethylene oxide, and analyzed using FTIR. Characterized human mesenchymal stem cells (hMSCs) were cultured in contact with the materials in basal, chondrogenic, and osteogenic medium for time periods up to 28 days, to determine if the materials could induce differentiation of MSC both in the presence and absence of biological stimuli. Viable cell adhesion was analyzed under all conditions. Collagen I, collagen II, sox-9, osteocalcin, osteopontin, osteonectin, and CBFA1 were evaluated at both the mRNA (real-time PCR) and protein production levels (fluorescent immunohistochemistry) and used to identify cell differentiation. Pure PCL and PCL mixed with PLGA demonstrated a chondrogenic potential. Only PCL 8 (80 wt % PCL, 20 wt % PLGA) facilitated osteogenic differentiation of MSCs under osteogenic conditions. This was attributed to the increased hydrophilic nature of the surface allowing sufficient homogeneous cell attachment and the formation of filamentous F-actin in the cells, allowing osteogenic differentiation. Of all materials tested, PCL 7 (70 wt % PCL, 30 wt % PLGA) demonstrated the greatest chondrogenic differentiation potential under basal and stimulated conditions at both the mRNA and protein production level.

Human mesenchymal stem cell response to poly(ε-caprolactone/poly(methyl methacrylate) demixed thin films

Advances in material sciences have enabled the fabrication of biomaterials which are able to provide the requisite cues to stimulate cells to behave in a specific way. Nanoscale surface topographies are well known to be able to positively influence cell–substrate interactions. This study reports on a novel series of poly(ε-caprolactone) PCL and poly(methyl methacrylate) demixed nanotopographic films as non-biological cell-stimulating cues. The topographic features observed ranged from nanoislands to nanopits. PMMA was observed to segregate to the air interface, while PCL preferred the substrate interface. Preliminary response of human mesenchymal stem cells to these surfaces indicated that the substrate with nanoisland topography has the potential to differentiate to osteogenic, chondrogenic and adipogenic lineages.

Photoresponsive Hydrogels with Photoswitchable Mechanical Properties Allow Time-Resolved Analysis of Cellular Responses to Matrix Stiffening

As cell function and phenotype can be directed by the mechanical characteristics of the surrounding matrix, hydrogels have become important platforms for cell culture systems, with properties that can be tuned by external stimuli, such as divalent cations, enzymatic treatment, and pH. However, many of these stimuli can directly affect cell behavior, making it difficult to distinguish purely mechanical signaling events. This study reports on the development of a hydrogel that incorporates photoswitchable cross-linkers, which can reversibly alter their stiffness upon irradiation with the appropriate wavelength of light. Furthermore, this study reports the response of bone-marrow-derived mesenchymal stem cells (MSCs) on these hydrogels that were stiffened systematically by irradiation with blue light. The substrates were shown to be noncytotoxic, and crucially MSCs were not affected by blue-light exposure. Time-resolved analysis of cell morphology showed characteristic cell spreading and increased aspect ratios in response to greater substrate stiffness. This hydrogel provides a platform to study mechanosignaling in cells responding to dynamic changes in stiffness, offering a new way to study mechanotransduction signaling pathways and biological processes, with implicit changes to tissue mechanics, such as development, ageing, and fibrosis.

The optimisation and production of stable homogeneous amine enriched surfaces with characterised nanotopographical properties for enhanced osteoinduction of mesenchymal stem cells

Silane modification has been proposed as a powerful biomaterial surface modification tool. This is the first comprehensive investigation into the effect of silane chain length on the resultant properties of -NH2 silane monolayers and the associated osteoinductive properties of the surface. A range of -NH2 presenting silanes, chain length 3-11, were introduced to glass coverslips and characterized using water contact angles, atomic force microscopy, X-ray photoelectron spectroscopy, and Ninhydrin assays. The ability of the variation in chain length to form a homogenous layer across the entirety of the surfaces was also assessed. The osteoinductive potential of the resultant surfaces was evaluated by real-time polymerase chain reaction, immunocytochemistry, and von Kossa staining. Control of surface chemistry and topography was directly associated with changes in chain length. This resulted in the identification of a specific, chain length 11 (CL11) which significantly increased the osteoinductive properties of the modified materials. Only CL11 surfaces had a highly regular nano-topography/roughness which resulted in the formation of an appetite-like layer on the surface that induced a significantly enhanced osteoinductive response (increased expression of osteocalcin, CBFA1, sclerostin, and the production of a calcified matrix) across the entirety of the surface.

Fluorescence-Based Nano-Oxygen Particles for Spatiometric Monitoring of Cell Physiological Conditions.

Closed-loop artificial pancreas systems have recently been proposed as a solution for treating stage I diabetes by reproducing the function of the pancreas. However, there are many unresolved issues associated with their development, including monitoring and controlling oxygen, immune responses, and the optimization of glucose, all of which need to be monitored and controlled to produce an efficient and viable artificial organ that can become integrated in the patient and maintain homeostasis. This research focused on monitoring the oxygen concentration, specifically achieving this kinetically as the oxygen gradient in an artificial pancreas made of alginate spheres containing islet cells. Functional nanoparticles (NPs) for measuring the oxygen gradient in different hydrogel cellular environments using fluorescence-based (F) microscopy were developed and tested. By the ester bond, a linker Pluronic F127 was conjugated with a carboxylic acid-modified polystyrene NP (510 nm). A hydrophilic/hydrophobic interaction between the commercially available oxygen-sensitive fluorophore and F127 results in fluorescence-based nano-oxygen particles (FNOPs). The in-house synthesized FNOP was calibrated inside electrosprayed alginate-filled hydrogels and demonstrated a good broad dynamic range (2.73-22.23) mg/L as well as a resolution of -0.01 mg/L with an accuracy of ±4%. The calibrated FNOP was utilized for continuous measuring of the oxygen concentration gradient for cell lines RIN-m5F/HeLa for more than 5 days in alginate hydrogel spheres in vitro.