Dynamic surfaces for mesenchymal stem cell self-renewal and differentiation

[摘要] It is thought that stem cells hold promise for use in future therapeutics.One such application is tissue engineering (TE) which aims to repair or replace diseased or damaged organs in vitro.Successful applications of TE, where the tissue is replaced and is functional, could improve a patients’ quality of life.Mesenchymal stem cells (MSCs) are a form of adult stem cell that are a precursor for fat, cartilage and bone cells.Bone is the second most transplanted tissue after blood therefore, enabling TE strategies through provision of high quality bone cells to facilitate bone repair would be beneficial.As MSCs are a precursor to bone, their use is attractive. Additionally, their proliferative potential and immunoregulatory properties make MSCs an ideal candidate cell for TE.MSCs require behavioural cues in vitro that direct phenotype in a targeted way.One method to direct stem cell behaviour is to utilise materials engineering.Static materials (examples include topography, chemistry and stiffness) have been employed but research has now moved towards stimuli responsive technologies to provide dual functionalities for culture and that emulate the properties of the stem cell niche. It is the intention of the work described in this thesis to utilise an enzyme responsive technology to promote MSC self-renewal and stimulate MSC differentiation to bone.Using solid phase peptide synthesis (SPPS) a biomimetic enzyme responsive material was made with the sequence PEG-GPAG↓LRGD tethered to a glass coverslip.Due to enzyme action on the sequence, the PEG cap is removed to create on demand adhesion to the peptide RGD.Further, the surface is designed to be under the control of cell secreted enzymes, rather than in response to enzymes added in by the user. The cell secreted enzymes that were investigated for this thesis were the matrix metalloproteases (MMPs). Here we confirm that the primary MMP secreted by MSCs was the gelatinase MMP-2 and a peptide sequence was designed to be cleaved by this MMP.It is known that redundancy can occur in MMP families and the role of MMP-9 was also investigated.The results show that MMP-9 is as efficient for surface cleavage, although cell supernatant concentration was 100-fold lower.MMP-2 concentration increased at week 3 specifically in response to peptides and so formed the original hypothesis that cleavage occurred at that time point.However, due to the potency of MMP-9 this may not be the case.Due to the limitations of manual synthesis and availability of materials, there was not enough evidence of MSC self-renewal.Further there was some indication of osteogenesis, specifically in response to the sequence at 4-6 weeks, however this is too long in culture to be therapeutically relevant.It may be better in the future to employ an enzyme responsive surface that can guarantee 100% efficiency of cleavage to ensure a synchronised population of end terminal cells.

[发布机构] University:University of Glasgow;Department:Institute of Molecular Cell and Systems Biology

[关键词] Mesenchymal stem cells, tissue engineering, materials, matrix metalloproteases, stimuli responsive technologies.