Bone is a highly vascularized tissue, in which vascularization and mineralization are concurrent processes during skeletal development. Indeed, both components should be included in any reliable and adherent in vitro model platform for the study of bone physiology and pathogenesis of skeletal disorders. To this end, we developed an in vitro vascularized bone model, using a gelatin-nanohydroxyapatite (gel-nHA) 3D bioprinted scaffold. First, we seeded human mesenchymal stem cells (hMSCs) on the scaffold which underwent osteogenic differentiation for two weeks. Then, we included lentiviral-GFP transfected human umbilical vein endothelial cells (HUVECs) within the 3D bioprinted scaffold macropores to form a capillary-like network during two more weeks of culture. We tested three experimental conditions: Condition 1, bone constructs with HUVECs cultured in 1:1 osteogenic medium (OM):endothelial medium (EM); Condition 2, bone constructs without HUVECs cultured in 1:1 OM:EM; Condition 3: bone construct with HUVECs cultured in 1:1 growth medium:EM. All samples resulted in engineered bone matrix. In Conditions 1 and 3, HUVECs formed tubular structures within the bone constructs, with the assembly of a complex capillary-like network visible by fluorescence microscopy in the live tissue and histology. CD31 immunostaining confirmed significant vascular lumen formation. Quantitative real-time PCR was used to quantify osteogenic differentiation and endothelial response. Alkaline phosphatase and runt-related transcription factor 2 upregulation confirmed early osteogenic commitment of hMSCs. Even when OM was removed under Condition 3, we observed clear osteogenesis, which was notably accompanied by upregulation of osteopontin, vascular endothelial growth factor, and collagen type I. These findings indicate that we have successfully realized a bone model with robust vascularization in just four weeks of culture and we highlighted how the inclusion of endothelial cells more realistically supports osteogenesis. The approach reported here resulted in a biologically inspired in vitro model of bone vascularization, simulating de novo morphogenesis of capillary vessels occurring during tissue development.

Endothelial cells support osteogenesis in an in vitro vascularized bone model developed by 3D bioprinting

Chiesa, Irene;De Maria, Carmelo;Lapomarda, Anna;Fortunato, Gabriele Maria;Montemurro, Francesca;Vozzi, Giovanni
;
2020-01-01

Abstract

Bone is a highly vascularized tissue, in which vascularization and mineralization are concurrent processes during skeletal development. Indeed, both components should be included in any reliable and adherent in vitro model platform for the study of bone physiology and pathogenesis of skeletal disorders. To this end, we developed an in vitro vascularized bone model, using a gelatin-nanohydroxyapatite (gel-nHA) 3D bioprinted scaffold. First, we seeded human mesenchymal stem cells (hMSCs) on the scaffold which underwent osteogenic differentiation for two weeks. Then, we included lentiviral-GFP transfected human umbilical vein endothelial cells (HUVECs) within the 3D bioprinted scaffold macropores to form a capillary-like network during two more weeks of culture. We tested three experimental conditions: Condition 1, bone constructs with HUVECs cultured in 1:1 osteogenic medium (OM):endothelial medium (EM); Condition 2, bone constructs without HUVECs cultured in 1:1 OM:EM; Condition 3: bone construct with HUVECs cultured in 1:1 growth medium:EM. All samples resulted in engineered bone matrix. In Conditions 1 and 3, HUVECs formed tubular structures within the bone constructs, with the assembly of a complex capillary-like network visible by fluorescence microscopy in the live tissue and histology. CD31 immunostaining confirmed significant vascular lumen formation. Quantitative real-time PCR was used to quantify osteogenic differentiation and endothelial response. Alkaline phosphatase and runt-related transcription factor 2 upregulation confirmed early osteogenic commitment of hMSCs. Even when OM was removed under Condition 3, we observed clear osteogenesis, which was notably accompanied by upregulation of osteopontin, vascular endothelial growth factor, and collagen type I. These findings indicate that we have successfully realized a bone model with robust vascularization in just four weeks of culture and we highlighted how the inclusion of endothelial cells more realistically supports osteogenesis. The approach reported here resulted in a biologically inspired in vitro model of bone vascularization, simulating de novo morphogenesis of capillary vessels occurring during tissue development.
2020
Chiesa, Irene; De Maria, Carmelo; Lapomarda, Anna; Fortunato, Gabriele Maria; Montemurro, Francesca; Di Gesu, Roberto; Tuan, Rocky S; Vozzi, Giovanni;...espandi
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11568/1032352
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