Palomares Cabeza, Virginia

English

Bone defects that remain unresolved represent a clinical challenge. Ideally, the use of the own patient’s bone to fill in the defect (autologous transplantation) would be the gold standard. However, the use of this approach is, in reality, not ideal, as it requires an extra surgical procedure and poses the limitation of the amount of bone that can be harvested.Tissue engineering approaches aim to overcome this problem, by employing marrow stromal cells (MSCs). These cells are able to differentiate into bone through an intermediate cartilage anlage when chondrogenically stimulated with TGF- β3. This laboratory-induced process mimics the endochondral ossification route that occurs naturally in the human body when bone formation takes place. The choice of cells to follow this approach remains crucial, as creating a bone ‘mould’ requires a high number of cells that are able to differentiate into the desired tissue upon the right stimuli. However, in aged patients this might be not an option, as these abilities decay with age. Prior evidence suggests that MSCs derived from young, healthy sources outperform adult cells for differentiation purposes. Therefore, finding the best source of cells that is able to be expanded and differentiated into a bone graft rapidly would be the starting point to make this approach a reality. In addition, guaranteeing the absence of a potential immune response that might arise when using an allogeneic source of cells would be key in order to guarantee the long-term stability of the bone allograft. These topics are discussed in chapter 1, which provides a general introduction of this thesis.In chapter 2, we focus on investigating the differentiation and proliferation properties of a source of paediatric MSCs (pMSCs) in comparison with adult MSCs (aMSCs). Here we find that, under the same conditions, paediatric MSCs are more suitable than adult MSCs for tissue engineering purposes in terms of faster proliferation, more consistent multilineage differentiation, including chondrogenesis, and less senescent. Similar to adult MSCs, these cells did not seem to be immunogenic in an initial co-culture assay with T cells. Hence, they were chosen as the ideal candidates for the rest of the studies.As previously mentioned, investigating the behaviour of MSCs in an allogeneic immune system is essential to avoid a potential immune rejection process that could destroy the bone allograft. In particular, the adaptive fraction of the immune response composed of T and B cells has been shown to play a fundamental role in allogeneic-derived rejection processes. In chapter 3, the adaptive immune response towards undifferentiated pMSCs is investigated. To mimic the most realistic scenario that would occur when implanting these bone allografts in a surgery, we incorporated the pro-inflammatory cytokine IFN‐γ. Our results show that IFN‐γ does not alter the immunomodulatory abilities of pMSCs towards T cells. However, IFN‐γ primed pMSCs but not unprimed pMSCs strongly inhibited B cell proliferation, as well as antibody-producing plasmablast formation and immunoglobulin G (IgG) antibody production. Therefore, in the case of pro-inflammatory signals, such as IFN-γ produced by activated T cells, pMSCs safeguard their immunomodulatory status towards T cells, and gain anti-proliferative abilities upon B cells. This could indicate that an allo-antibody immune response would therefore not develop in vivo, even under inflammatory conditions.It is to mimic a more in vivo realistic situation that in chapters 4 and 5 we move towards mouse models. In chapter 4, we used an immunodeficient animal model humanised with allogeneic immune cells to study the immune responses towards chondrogenically differentiated pMSCs (ch-pMSCs). These ch-pMSC-derived grafts did not show immunogeneic responses in vitro. Upon subcutaneous implantation, they were able to mineralise in the presence of an allogeneic immune system for 3 weeks without the onset of immune responses. Moreover, ch-pMSCs generated mature bone after 8 weeks of implantation. This bone persisted for up to 6 more weeks in the presence of an allogeneic immune system, suggesting that these bone allografts do not get cleared by the host. However, the intrinsic limitations of the humanised animal model, namely the lack of engraftment of certain immune subsets such as antigen-presenting cells (APCs) or B cells, made us look for other potential animal models. In chapter 5, we made use of an immunocompetent mouse model to study the potential of our ch-pMSCs to form bone in the presence of a fully functional xenogeneic immune system. In that scenario, we found that the percentages of CD4+ and CD8+ T cells, B cells and classical/non-classical monocyte subsets were not systemically altered in these animals after two weeks. However, ch-pMSC-implanted mice had significantly higher levels of serum total IgG compared to sham-operated mice at this timepoint. After 12 weeks of implantation, we found low levels of T cell and macrophage infiltration in the xenografts. Despite this, ch-pMSC constructs persisted and did not show signs of resorption. In addition, constructs became mineralised, and their bone volume increased from 4 weeks post implantation to 12 weeks.The findings of this thesis are discussed in chapter 6. We also suggest ways of improving the existent animal models towards mimicking more realistic biological situations for bone repair and osteoimmunology. All in all, our results indicate that pMSCs are excellent candidates for bone repair, proving that initiation and maintenance of bone formation occurs without a substantial immune response in both a xenogeneic and allogeneic scenario.<br/

EUR Research Repository

Down to the bone:Investigating the immune responses in bone tissue engineering approaches using paediatric mesenchymal stem cells

https://pure.eur.nl/files/54613549/thesis_palomares_cabeza_final_version_62750f91ef6fa.pdf

Down to the bone:Investigating the immune responses in bone tissue engineering approaches using paediatric mesenchymal stem cells

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