Stromal vascular fraction (SVF) cells, isolated from adipose tissue are an abundant easily accessible stromal cell source for bone tissue engineering. These characteristics make them a good alternative to bone marrow derived mesenchymal stem cells (BM-MSCs). Previous studies from our group provided a proof-of-concept that Adipose-derived Stromal Cells (ASCs), resulting from the expansion of SVF-cells, can generate bone tissue through endochondral ossification (ECO) by forming hypertrophic cartilage tissue (HCT) in vitro which in turn remodels into bone tissue when implanted in vivo. However, little is known about the underling mechanisms of ASCs chondrogenesis and their in vivo remodeling process.
In this work, we hypothesized that (i) freshly isolated SVF-cells are better suited for generating mature HCTs than expanded ASCs (P0-ASCs) and (ii) that more mature HCTs are more prone to remodel into bone tissue in vivo.
To that aim, we assessed the effect of SVF-cells monolayer expansion and its consequences on the proteomic signature and chondrogenic potential. In addition, we sought out to determine which HCT-properties can predictively determine the in vivo bone forming capacities of ASCs.
SVF-cells or P0-ASCs (0.5x106) were first seeded onto collagen sponges (V=40 mm3) and then maintained in chondrogenic cultures for 3, 4, 5 or 6 weeks. The cartilage maturation of these adipose-derived HCTs (A-HCTs) was evaluated qualitatively by histological staining and quantitively using Elisa and compared to the ones generated by BM-MSCs (BM-HCTs). Next, these HCTs were implanted ectopically in nude mice for 12 weeks to evaluate their bone forming capacities. In addition, the proteomic profiles of SVF-cells and P0-ASCs were compared by mass spectromer.
SVF-cells from all tested donors formed mature HCT within 3 weeks whereas P0-ASCs needed at least 4 to 5 weeks (depending on the donor). Longer in vitro differentiation increased the degree of maturation of HCTs which was characterized by a denser cartilagenous matrix and more mineralization. Interestingly, the HCTs degree of maturation obtained in vitro was indicative of their bone forming capacity in vivo, with an optimal bone remodeling obtained not for the highest degree of maturation but for an average degree of maturation. In fact, excessive in vitro maturation resulted in mineralized tissue rather than bone in vivo. In contrast, insufficient maturation resulted in major scaffold resorption in vivo. A-HCTs showed more mineralization, higher content of IL-10 and TNF-alpha relative to BM-HCTs. However, A-HCTs formed mature bone organ in vivo similarly to BM-HCT when suitably matured in vitro. When looking at their proteomic profile, P0-ASCs presented a different profile to the ones of the SVF-cells; most notably regarding energy related pathways such as glycolysis, TCA-cycle and lipid metabolism.
Our data showed that SVF-cells possess a superior chondrogenic potential compared to P0-ASCs. In addition, we were able to not only control the degree of maturation of the HCTs but also modulate their in vivo fates.
We will assess whether a more physiological culture environment would better preserve the specific metabolic status of SVF-cells and thereby better retain their differentiation potential when expanded in vitro.