The use of nanovibration to discover specific and potent bioactive metabolites that stimulate osteogenic differentiation in mesenchymal stem cells

Bioactive metabolites have wide-ranging biological activities and are a potential source of future research and therapeutic tools. Here, we use nanovibrational stimulation to induce osteogenic differentiation of mesenchymal stem cells, in the absence of off-target, nonosteogenic differentiation. We show that this differentiation method, which does not rely on the addition of exogenous growth factors to culture media, provides an artifact-free approach to identifying bioactive metabolites that specifically and potently induce osteogenesis. We first identify a highly specific metabolite, cholesterol sulfate, an endogenous steroid. Next, a screen of other small molecules with a similar steroid scaffold identified fludrocortisone acetate with both specific and highly potent osteogenic-inducing activity. Further, we implicate cytoskeletal contractility as a measure of osteogenic potency and cell stiffness as a measure of specificity. These findings demonstrate that physical principles can be used to identify bioactive metabolites and then enable optimization of metabolite potency can be optimized by examining structure-function relationships

Nanotopography reveals metabolites that maintain the immunomodulatory phenotype of mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) are multipotent progenitor cells that are of considerable clinical potential in transplantation and anti-inflammatory therapies due to their capacity for tissue repair and immunomodulation. However, MSCs rapidly differentiate once in culture, making their large-scale expansion for use in immunomodulatory therapies challenging. Although the differentiation mechanisms of MSCs have been extensively investigated using materials, little is known about how materials can influence paracrine activities of MSCs. Here, we show that nanotopography can control the immunomodulatory capacity of MSCs through decreased intracellular tension and increasing oxidative glycolysis. We use nanotopography to identify bioactive metabolites that modulate intracellular tension, growth and immunomodulatory phenotype of MSCs in standard culture and during larger scale cell manufacture. Our findings demonstrate an effective route to support large-scale expansion of functional MSCs for therapeutic purposes

Rights, parents, children, and communities Some educational implications

SIGLEAvailable from British Library Document Supply Centre-DSC:DXN027710 / BLDSC - British Library Document Supply CentreGBUnited Kingdo

The use of microarrays and fluorescence in situ hybridization for the study of mechanotransduction from topography

The combination of transcriptomic analysis and fluorescence in situ hybridization (FISH) provides a robust methodology to study genomic changes in different biological conditions. Microarrays allow a global study of gene expression in response to the conditions of interest, with comparison between control(s) and one or more test condition(s). The messenger RNA amplification step permits detection of even low abundance transcripts, a critical advantage for applications such as biomaterials research, where the starting material may be limited. Different types of microarrays are commercially available that allow the investigation of specific features, such as exon arrays, microRNA arrays, and gene arrays. Microarrays are available for different model organisms, but we use Affymetrix ® HuGene ® ST (Sense Target) arrays, a type of gene array for analysis of human samples. FISH involves fluorescent detection of probe DNA hybridized to an in situ chromosomal target that can be either whole chromosomes or chromosomal segments. The overall hybridization is similar to labeling with radioactive probes but the incorporation of fluorescent detection of the probe sequences allows for high sensitivity in a simple and quick assay. FISH can be applied to a variety of specimen types depending on the study of interest. In this chapter, we describe the methodologies of these two techniques and provide technical tips that should help overcome challenges in carrying them out

Combinational approaches to improve outcomes following peripheral nerve repair

Introduction and aims: Peripheral nerve injury is common (1/1000) and can be functionally devastating. Despite advances in microsurgical repair axonal regrowth across the repair site, and functional outcome, remain unsatisfactory. The neurobiology of the nerve repair must be unraveled. Work at the University of Glasgow Centre for Cell Engineering and collaborating laboratories has previously demonstrated that healing nerves respond to both intrinsic and extrinsic factors. The directionality and rate of axonal regrowth following injury can be enhanced in vitro by growing cells on a nano/micropatterned surface. Exactly how these external factors exhibit their effect is unknown. This study aimed to demonstrate the downstream genetic effects of this extrinsic mechanical cue. Material and methods: Quantitative rt-PCR was used to demonstrate the downstream genetic effects of this extrinsic cue. Gene expression was measured at day 0, 1, 2, 5 and 10 following injury, of nerves grown on either the patterned surface or a smooth control. The Sprague-Dawley ex vivo model was used. Immunohistochemistry demonstrated axonal outgrowth. Results: Altering the topographical substrate on which nerves were grown resulted in a significant alteration in expression of the genes studied (MTOR, CRAT, MAP3K12), most pronounced at day 2 post injury (p < 0.05). Expression timelines and molecular pathways are discussed. Conclusion(s): This study further characterises a useful animal model and provides more detail on the complex interactions underlying nerve repair. It highlights some of the downstream effects of micropatterning, that could be harnessed and combined with other recent advances for the development of clincally useful nerve conduits

Material-driven fibronectin and vitronectin assembly enhances BMP-2 presentation and osteogenesis

Mesenchymal stem cell (MSC)-based tissue engineering strategies are of interest in the field of bone tissue regenerative medicine. MSCs are commonly investigated in combination with growth factors (GFs) and biomaterials to provide a regenerative environment for the cells. However, optimizing how biomaterials interact with MSCs and efficiently delivering GFs, remains a challenge. Here, via plasma polymerization, tissue culture plates are coated with a layer of poly (ethyl acrylate) (PEA), which is able to spontaneously permit fibronectin (FN) to form fibrillar nanonetworks. However, vitronectin (VN), another important extracellular matrix (ECM) protein forms multimeric globules on the polymer, thus not displaying function groups to cells. Interestingly, when FN and VN are co-absorbed onto PEA surfaces, VN can be entrapped within the FN fibrillar nanonetwork in the monomeric form providing a heterogeneous, open ECM network. The combination of FN and VN enhances MSC adhesion and leads to enhanced GF binding; here we demonstrate this with bone morphogenetic protein-2 (BMP2). Moreover, MSC differentiation into osteoblasts is enhanced, with elevated expression of osteopontin (OPN) and osteocalcin (OCN) quantified by immunostaining, and increased mineralization observed by von Kossa staining. Osteogenic intracellular signalling is also induced, with increased activity in the SMAD pathway. The study emphasizes the need of recapitulating the complexity of native ECM to achieve optimal cell-material interactions

Osteogenesis of Mesenchymal Stem Cells by Nanoscale Mechanotransduction

It is likely that mesenchymal stem cells will find use in many autologous regenerative therapies. However, our ability to control cell stem growth and differentiation is presently limited, and this is a major hurdle to the clinical use of these multipotent cells especially when considering the desire not to use soluble factors or complex media formulations in culture. Also, the large number of cells required to be clinically useful is currently a hurdle to using materials-based (stiffness, chemistry, nanotopography, <i>etc.</i>) culture substrates. Here we give a first demonstration of using nanoscale sinusoidal mechanotransductive protocols (10–14 nm displacements at 1 kHz frequency), “nanokicking”, to promote osteoblastogenesis in human mesenchymal stem cell cultures. On the basis of application of the reverse piezo effect, we use interferometry to develop the optimal stem cell stimulation conditions, allowing delivery of nanoscale cues across the entire surface of the Petri dishes used. A combination of immunofluorescence, PCR, and microarray has then been used to demonstrate osteoblastogenesis, and the arrays implicate RhoA as central to osteoblastic differentiation in agreement with materials-based strategies. We validate this with pharmacological inhibition of RhoA kinase. It is easy to envisage such stimulation protocols being up-scaled to form large-scale osteoblast bioreactors as standard cell culture plates and incubators are used in the protocol

Using nanotopography and metabolomics to identify biochemical effectors of multipotency

It is emerging that mesenchymal stem cell (MSC) metabolic activity may be a key regulator of multipotency. The metabolome represents a "snapshot" of the stem cell phenotype, and therefore metabolic profiling could, through a systems biology approach, offer and highlight critical biochemical pathways for investigation. To date, however, it has remained difficult to undertake unbiased experiments to study MSC multipotency in the absence of strategies to retain multipotency without recourse to soluble factors that can add artifact to experiments. Here we apply a nanotopographical systems approach linked to metabolomics to regulate plasticity and demonstrate rapid metabolite reorganization, allowing rational selection of key biochemical targets of self-renewal (ERK1/2, LDL, and Jnk). We then show that these signaling effectors regulate functional multipotency