Mesenchymal stromal/stem cells (MSCs) are increasingly recognized as regulators of immune cells during disease or tissue repair. During these situations, the extracellular matrix (ECM) is very dynamic and therefore, our studies aim to understand how ECM influences the activity of MSCs. Human MSCs cultured on tissue culture plastic (TCP) and encapsulated within collagen type I, fibrin, or mixed Collagen-Fibrin were exposed to low dose TNFα and IFNɣ. Transcription profiles were examined using bulk RNA sequencing (RNAseq) after 24h of treatment. ELISA, Western blot, qPCR and immunofluorescence were employed to validate RNAseq results and to investigate the significance of transcriptional changes. Flow cytometry evaluated monocyte/macrophage phenotype.Abstract
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Controlled differentiation of Human mesenchymal stem cells (hMSCs) is required for timely induction of bone growth in implantable biomaterials. Differentiation of hMSCs towards a particular lineage depends upon their microenvironment, which is a complex mixture of various physical, chemical and biological parameters. The role of Bone morphogenic protein (BMP2) in early induction of bone formation is well established. Clinical experience and in vitro study has shown that presentation of this protein in small quantities by surface immobilisation significantly induces osteogenic differentiation compared to large quantities provided in solution. This project focuses on developing and understanding responsive micro/nano porous interfaces which deliver BMP2 in a dose dependent fashion to control osteogenic diffentiation of hMSCs. We hypothesise that use of porous membranes primed with LbL deposition of biomacromolecules such as COL and HA will help in induction of cell attachment and growth whilst controlled and localised delivery of BMP2 released from the layers of these porous constructs will induce sustained differentiation of hMSCs. By controlling pore size of membranes, rate of release of BMP2 can be controlled. We use fluorescently labelled Dextran (Flu-DEX) as model protein to study control release mechanism, which is of similar size to BMP2. Polycarbonate (PC) track etched membranes with various pore sizes were used for LbL assembly of COL/HA/Flu-DEX along with hydrolytically degradable polymer Poly-Beta amino ester (Poly2). Six bilayers were constructed into porous membranes with (COL-Flu-DEX)6 and (Poly2-Flu-DEX)6. Use of hydrolytically degradable polymer significantly enhances release of Flu-DEX compared to control (COL-Flu-DEX)6 assembly. Compared to flat (non porous) surface, release from porous samples maintained a relatively slow and steady release. We are currently investigating release of BMP2 using this approach and their influence on the differentiation of hMSCs in vitro
To control the growth and function of osteoblasts on Titanium alloy surfaces produced by electrochemical patterning. Samples of Ti6Al4V were prepared with three different finishes; no surface preparation following machining, polishing on a grinding wheel with sequential grit papers up to 4000 to achieve a mirror finish and treatment in a flat electrochemical cell with a 3M sulphuric acid in methanol using 9V supplied over 60 seconds to produce a surface with defined nano/microscale roughness. Glass coverslips were used as control surfaces. Surfaces were seeded with primary rat calvarial osteoblasts and incubated in Dulbecco's Modified Eagle Medium with 10% (v/v) sera for 24 hours before fixing and performing immunofluorescence staining with anti-vinculin antibody. Photomicrographs of the surfaces were analysed with Image J and analySIS FIVE programs. Results for cell number, cell area, focal adhesion area and polarity (lack of roundness) were analysed (using the Mann Whitney test) for ANOVA using SPSS.Aim
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