Chondrogenic Pre-Differentiation Inhibits VEGF Angiogenic Effect in Pericranium-Derived Spheroids.

San-Marina, Serban; Prummer, Christopher; Voss, Stephen; Hunter, Danielle; Madden, Benjamin; Charlesworth, Mary Cristine; Ekbom, Dale; Janus, Jeffrey R

Craniofacial reconstruction of critical bone defects typically requires a bone graft. As graft availability may be restricted by disease or co-morbidities, tissue engineering approaches are actively sought. The pericranium could provide new bone graft material. During development and repair, bone transitions through a chondrogenic phase. However, with tissue engineering pluripotent cells can differentiation directly into bone cells. Does ability to recapitulate bone formation in vitro affect osteogenesis and vascularization of pericranium grafts? To answer this, we obtained tissue from nine patients with pre-planned craniotomy surgery and studied 3D osteogenesis and angiogenesis of pericranium-derived spheroids. First, we established growth and differentiation conditions on Matrigel. For each spheroid sample we investigated a) continuous osteogenic differentiation (COD), and b) osteogenic differentiation preceded by chondrogenesis (CDïƒ OD). The effect of VEGF was compared to VEGF supplemented with FGF, IL-1, IL-6, PDGF, and TNF-α, a growth factor mix (GFM) with possible synergistic effects. In this limited sample we observed no age or sex-related differences in cell expansion. Similarly, no statistically significant differences in osteogenic or angiogenic scores between COD or CDïƒ OD spheroids were noted with regular media. In COD however, VEGF statistically significantly increased angiogenesis compared to control media (p=0.007)). Also in COD, both VEGF and VEGF+GFM increased osteogenesis (p=0.047 and p=0.038, respectively). By contrast, in CDïƒ OD neither VEGF nor VEGF+GFM yielded statistically significant angiogenesis or osteogenesis scores compared to control media. To understand these results we characterized spheroid protein expression by nano-liquid chromatography coupled to tandem mass spectrometry (nLC-MS/MS). Nine angiogenic proteins were either uniquely expressed or upregulated in COD compared to CDïƒ OD: a) endothelial markers JUP, PTGIS, PTGS2, and TYMP, b) tissue remodeling factors CHI3L1 and MMP14, and c) metabolic pathways modulators ANGPTL4, ITGA5, and WNT5A. ANGPTL4, ITGA5, PTGIS, PTGS2, WNT5A define a conserved angiogenic network and were >2 fold increased in VEGF compared to VEGF+GFM. Finally, we examined bone formation on printable poly-(propylene-fumarate) (PPF) scaffolds for individualized grafting. Under COD+VEGF conditions, PPF scaffolds loaded with PDCs displayed hallmarks of spongiform-like bone formation. Thus, the human pericranium may be a potential repository for bone-generating cells with applications in craniofacial bone repair using tissue printing.