Speaker
Description
Introduction
The bone marrow (BM) is a dynamic organ whose main function is to support haematopoietic cells, which assure life-long production of blood. The haematopoietic compartment is supported by the stromal fraction of the BM that comprises mesenchymal stromal cells (MSCs), a heterogeneous cell population that undergoes osteogenic, chondrogenic, adipogenic differentiation. Following a tissue engineering approach, we combine the knowledge on BM-MSCs with biomaterials to dissect such cells heterogeneity and model BM biology. Our technology consists in encapsulating BM-MSCs in synthetic, biocompatible polyethylene glycole (PEG) hydrogels and following cells developmental potential in vitro and in vivo. We are currently focusing on the in vivo characterization of two murine BM-MSCs populations, Skeletal Stem Cells (SSCs) and Bone Cartilage Stromal Progenitors (BCSPs), aiming at generating new knowledge for bone and bone marrow regenerative strategies.
Methods
SSCs and BCSPs were mechanically and enzymatically extracted from limbs and sternum of post-natal day3 (P3) GFP+-C57/BL6 mice and separated by FACS using the marker panels CD45-Ter119-Tie2-AlphaV+Thy-6C3-CD105-CD200+ and CD45-Ter119-Tie2-AlphaV+Thy-6C3-CD105+, respectively. Due to the high amount of cells needed for in vivo trials, both cell types were expanded under standard culture conditions for 7 days. Subsequently, the cells were encapsulated at different cell densities in PEG hydrogels with or without minimal amounts of BMP-2. Such scaffolds, whose size is around 0.5 cm in diameter, were subcutaneously implanted at the back of NMRI-Foxn1nu immunocompromised mice and harvested after 8 weeks. Upon explantation, samples were macroscopically inspected, imaged and fixed. Bone formation was quantified through micro-CT analysis, followed by decalcification and histological characterization.
Results
The subcutaneous implantation of the scaffolds allowed to visually monitor their localization within the 8 weeks of implantation. At explantation, all the scaffolds were found back with their initial size, confirming biomaterial stability. Distinct scenarios in terms of BM organoids (or ossicles) were observed based on the different combinations of cell densities and BMP-2. A first macroscopic evaluation showed SSCs being more performant as they induced the formation of stiffer ossicles with high degree of vascularization both with and without BMP-2, at different cell densities. BCSPs generated ossicles only when in combination with BMP-2. Still-ongoing Micro-CT and histological analysis will precisely determine whether there is a direct proportionality between cell density, BMP-2 concentration at implantation and bone formation or marrow morphological complexity. Also, the GFP-labelling of the implanted cells will aid tracing them back in the ossicles, giving further information on their role in the BM organoids onset, thus on their biological function.
Conclusions
The results suggest that our technology can generate 3D in vivo BM organoids and so elegantly model such organ. Moreover, it proved to be a good instrument for investigating SSCs and BCSPs biological function in vivo, since even from a first macroscopical evaluation different outcomes from SSCs or BCSPs-loaded scaffolds were observed. Lastly, the biocompatibility of our biomaterial was demonstrated as most of the ossicles were found to be vascularized, thus suggesting their integration with the host vasculature and so the remodeling of the material by implanted or host cells.
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