Developing optimized methods for cGMP compliance in the isolation of human adipose-derived stromal/stem cells.

Objectives: This work aims to establish Platelet Lysates (PL) as optimal source¹ of growth factors and other molecules that are vital for promoting cell proliferation and differentiation pathways, eventually allowing the substitution FBS and/or osteogenic supplements in culture media in bone tissue engineering strategies. Furthermore we intent to design new approaches to incorporate PLs in a scaffold material, as a hydrogel encapsulating the cells or as a coating for 3D porous structures, thus developing a tissue engineered construct with enhanced/multiple functionalities.

Methods: Starch–polycaprolactone (SPCL) meshes were obtained by a fiber bonding method as previously described².  PL gels were obtained by activation of platelets coagulation cascade using thrombin  dissolved in a calcium chloride solution. 

Human adipose stem cells (hASCs) were obtained by enzymatic digestion of lipoaspirates samples. hASCs were either seeded directly into the SPCL scaffolds (control group) or into the scaffolds previously coated with PL gels or suspended in the PL and then seeded in the scaffold and gellified.  hASCs proliferation and differentiation was assessed after  different culturing time points of the constructs, by DNA  and ALP quantification and by RT-PCR and immunohystological analysis.

Results: The preliminary results obtained sustain the hypothesis that growth factors and other signaling molecules present in PL groups are actually active and vital to initiate proliferation and osteogenic differentiation of hASCs. DNA quantification and cell viability were similar and even higher in PL groups, as well as early markers of osteogenic differentiation, such as ALP activity. Latest time-points revealed less noteworthy differences especially due to the progressive degradation of the PL gel.

Conclusions: PL represents a substrate and a delivery system of important growth factors and other signaling molecules, and therefore making these molecules available for cells within a tissue engineering construct provides an important enhancement of autologous  bone tissue engineering strategies.