Figure 4.

Development of prevascularized bone-like tissue analogues in Matrigel and gelPEG-based hydrogels. ECFC-MSC cocultures were cultured for 2 weeks under osteogenic culture conditions in the different hydrogels before analysis of osteogenesis, vasculogenesis, and presence of pericyte-like cells. Expression of BCLAP and SPP1 as osteogenic markers, CDH5 and PECAM1 were measured as endothelial markers and CSPG4 and ACTA2 as pericyte markers. a,e,i) Von Kossa staining was positive for gelPEG and gelPEG+LN111 hydrogels highlighting mineralization. b,f,j) Sections of hydrogels highlighted the presence of the osteogenic marker osteonectin in all groups. c,g,k) 100 μm z-projections of vascular-like networks (green) in the centre of the hydrogels. d,h,l) Vascular-like structures, stabilized by αSMA-positive pericyte-like cells (red). m) Total vessel length was equal in Matrigel and gelPEG+LN111, and significantly longer than in gelPEG hydrogels. n,o) mRNA expression for osteogenic genes encoding for osteocalcin and osteopontin were comparably expressed in Matrigel, gelPEG, and gelPEG+LN111. p,q) Vasculogenesis-associated genes encoding for CD31 and VE-cadherin were expressed in all hydrogels, with highest VE-cadherin expression in Matrigel, followed by gelPEG and gelPEG+LN111. r,s) Pericyte-associated genes encoding for αSMA and NG2 were equally expressed in all hydrogels. Data are depicted as mean + SD; N = 3, n = 3.