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
Fatigue fractures which originate at stress-concentrating voids located at the implant-cement interface are a potential cause of septic loosening of cemented femoral components. Heating of the component to 44°C is known to reduce the porosity of the cement-prosthesis interface. The temperature of the cement-bone interface was recorded intra-operatively as 32.3°C. A simulated femoral model was devised to study the effect of heating of the component on the implant-cement interface. Heating of the implant and vacuum mixing have a synergistic effect on the porosity of the implant-cement interface, and heating also reverses the gradients of microhardness in the mantle. Heating of the implant also reduces porosity at the interface depending on the temperature. A minimum difference in temperature between the implant and the bone of 3°C was required to produce this effect. The optimal difference was 7°C, representing a balance between maximal reduction of porosity and an increased risk of thermal injury. Using contemporary cementing techniques, heating the implant to 40°C is recommended to produce an optimum effect.
Radiological assessment of the cement mantle is used routinely to determine the outcome of total hip replacement. We performed a simulated replacement arthroplasty on cadaver femora and took standard postoperative radiographs. The femora were then sectioned into 7 mm slices starting at the calcar, and high-resolution faxitron radiographs were taken of these sections. Analysis of the faxitron images showed that defects in the cement mantle were observed up to 100 times more frequently than on the standard films. We therefore encourage the search for a better technique in assessing the cement mantle.
