Injectable hydrogel formulations to host adipose stem cell spheroids for stemness maintenance and bone and cartilage regeneration

Adipose-derived mesenchymal stem cells (ASCs) represent a great promise for tissue regeneration as fat is a very abundant source of stem cells (1) and owing to their ability to self-renew and differentiate into specific tissue types (2). In general, they are usually cultured as two-dimensional (2D) adherent monolayers, not representative of the in vivo condition, often entailing lower cell viability and, more in general, a lower “cell quality” in terms of regenerative potential (3, 4). When ASCs are cultured in low adhesion flasks and with a suitable culture medium, they aggregate in the form of three-dimensional spheroids (SASCs). The incorporation of these spheroids into injectable, in-situ gelling, polymer solutions can preserve the 3D structure of the cells from extraction to administration, avoid uncontrolled cell spreading, poor interaction and integration with the surrounding tissue. Moreover, in the presence of the right signaling molecules, SASCs are ready to differentiate in osteogenic (5) and chondrogenic (6) tissue, therefore, incorporated in the right scaffold, they can be very useful in the repair of both cartilage and bone defects that are still a challenge for modern medicine (7). As scaffold, hydrogels can meticulously look like the native ECM, due to their interconnected pore architecture, high water content and amenability to incorporate biomolecules or to provide specific biomechanical cues (8, 9). Therefore, hydrogels can be a fundamental element for tissue regeneration, maintenance of stem cell viability and stemess potential, induction of differentiation, support for cell proliferation and spreading with minimally invasive procedures. Our work explores the suitability of hydrogels obtained by temperature-triggered self-assembly of partially degalactosylated xyloglucan aqueous dispersions (dXGaq) as SASCs niches for stemness considtions maintenance or for their differentiation in either osteogenic or chondrogenic lineages. The influence of the different culture media mixed with dXGaq and the presence of cell spheroids in the mixtures on the flow properties before gelation of all the above and the extrusion process with a syringe on the formed physical networks were investigated by shear viscosity and small amplitude oscillatory rheological analyses. (1) Cheng, N. C., Wang, S., & Young, T. H. (2012). The influence of spheroid formation of human adipose-derived stem cells on chitosan films on stemness and differentiation capabilities. Biomaterials, 33(6), 1748-1758. (2) Nii, M., Lai, J. H., Keeney, M., Han, L. H., Behn, A., Imanbayev, G., & Yang, F. (2013). The effects of interactive mechanical and biochemical niche signaling on osteogenic differentiation of adipose-derived stem cells using combinatorial hydrogels. Acta Biomaterialia, 9(3), 5475-5483. (3) Di Stefano, A. B., Montesano, L., Belmonte, B., Gulino, A., Gagliardo, C., Florena, A. M., ... & Toia, F. (2020). Human Spheroids from Adipose-Derived Stem Cells Induce Calvarial Bone Production in a Xenogeneic Rabbit Model. Annals of Plastic Surgery. (4)Di Stefano, A. B., Grisafi, F., Perez-Alea, M., Castiglia, M., Di Simone, M., Meraviglia, S., ... & Toia, F. (2020). Cell quality evaluation with gene expression analysis of spheroids (3D) and adherent (2D) adipose stem cells. Gene, 145269. (5) Li, W., Liu, Y., Zhang, P., Tang, Y., Zhou, M., Jiang, W., ... & Zhou, Y. (2018). Tissue-engineered bone immobilized with human adipose stem cells-derived exosomes promotes bone regeneration. ACS applied materials & interfaces, 10(6), 5240-5254. (6) Ansari, S., Diniz, I. M., Chen, C., Aghaloo, T., Wu, B. M., Shi, S., & Moshaverinia, A. (2017). Alginate/hyaluronic acid hydrogel delivery system characteristics regulate the differentiation of periodontal ligament stem cells toward chondrogenic lineage. Journal of Materials Science: Materials in Medicine, 28(10), 162. (7) Kwon, H., Brown, W. E., Lee, C. A., Wang, D., Paschos, N., Hu, J. C., & Athanasiou, K. A. (2019). Surgical and tissue engineering strategies for articular cartilage and meniscus repair. Nature Reviews Rheumatology, 15(9), 550-570. (8) Geckil, H., Xu, F., Zhang, X., Moon, S., & Demirci, U. (2010). Engineering hydrogels as extracellular matrix mimics. Nanomedicine, 5(3), 469-484. (9) Hong, K. H., Kim, Y. M., & Song, S. C. (2019). Fine‐Tunable and Injectable 3D Hydrogel for On‐Demand Stem Cell Niche. Advanced Science, 6(17), 1900597.