INTRODUCTION: Light-assisted additive bio-manufacturing has emerged as a powerful tool for custom fabrication of structurally complex 3D tissue models from stem cells.[1-2] Existing in vitro bone models, however, often lack terminally differentiated bone cells named osteocytes which are crucial for bone homeostasis and functional adaptation in response to mechanical loads. Here, we report ultrafast tomographic photofabrication of centimeter-scale heterocellular bone constructs that enabled osteocytic differentiation of human mesenchymal stem cells (hMSCs) within hydrogels after 42 days of 3D endothelial co-culture.
METHODS: A series of bioresins with varying amounts of gelatin methacryloyl (GelMA) and photoinitiator lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP) were formulated and printed on a Readily3D Tomolite bioprinter. Eosin Y staining and confocal imaging was used to assess the printing precision. Photo-rheology was applied to assess printability and gel mechanics. Cytocompatibility was evaluated by live-dead and actin-nuclei staining after 3D osteogenic culture for 7 days. Cell activity in 3D mono-culture and co-culture with human umbilical vein endothelial cells (HUVECs) was compared by weekly ALP and qPCR assays for up to 42 days.
RESULTS & DISCUSSION: To create a permissive environment for embedded cells, we screened a series of bioresin formulations in terms of photo-reactivity, gel mechanics, cell-compatibility, and the ability to support osteogenic differentiation. A variety of CAD models (femur, branched vasculature) can be printed by tomographic photofabrication within 30 seconds. The laser dose is dependent on LAP and GelMA concentrations. We identified an optimal bioresin comprising 5% GelMA, 0.03% LAP and 2 M/mL hMSCs. After printing cells were highly viable (>90%), indicating good biocompatibility of the resin and the printing process. However, increasing the LAP concentration led to a decrease in cell spreading. After 7 days cells showed long dendritic processes. Compared to a bioresin with 10% GelMA, our resin led to an upregulated expression of osteogenic gene markers. After 42 days, a significantly increased gene expression of both osteoblastic markers (collagen-I, ALP, osteocalcin) and osteocytic markers (Podoplanin; Dmp1) was evidenced in the 3D co-culture. The gel compressive moduli increased from 6 kPa to 46 kPa, indicating the increased matrix secretion. Finally, we developed a pre-vascularized model by injecting HUVECs into a printed gel construct containing hMSCs. After 2 days HUVECs self-organized into an endothelium lumen, forming a pre-vascularized tissue construct.
CONCLUSIONS: Using an optimized bioresin, this study leverages the benefits of ultrafast tomographic bioprinting and 3D co-culture for scaled additive photofabrication of 3D living bone tissues with an in vivo-like heterocellular environments and long-term functionality.
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 Gehlen et al. bioRxiv 2022, doi: 10.1101/2021.11.14.468504.