Abstract
Here, we aimed to investigate osteogenic differentiation of human adipose-derived stem cells (hASCs) in three-dimensional (3D) bioprinted tissue constructs in vitro and in vivo. A 3D Bio-plotter dispensing system was used for building 3D constructs. Cell viability was determined using live/dead cell staining. After 7 and 14 days of culture, real-time quantitative polymerase chain reaction (PCR) was performed to analyze the expression of osteogenesis-related genes (RUNX2, OSX, and OCN). Western blotting for RUNX2 and immunofluorescent staining for OCN and RUNX2 were also performed. At 8 weeks after surgery, osteoids secreted by osteogenically differentiated cells were assessed by hematoxylin-eosin (H&E) staining, Masson trichrome staining, and OCN immunohistochemical staining. Results from live/dead cell staining showed that most of the cells remained alive, with a cell viability of 89%, on day 1 after printing. In vitro osteogenic induction of the 3D construct showed that the expression levels of RUNX2, OSX, and OCN were significantly increased on days 7 and 14 after printing in cells cultured in osteogenic medium (OM) compared with that in normal proliferation medium (PM). Fluorescence microscopy and western blotting showed that the expression of osteogenesis-related proteins was significantly higher in cells cultured in OM than in cells cultured in PM. In vivo studies demonstrated obvious bone matrix formation in the 3D bioprinted constructs. These results indicated that 3D bioprinted constructs consisting of hASCs had the ability to promote mineralized matrix formation and that hASCs could be used in 3D bioprinted constructs for the repair of large bone tissue defects.
Highlights
Dental caries, periodontal disease, dental trauma, cancer, and other diseases can lead to maxillofacial bone defects, which are commonly encountered by dentists [1]
This study focused on osteogenic differentiation of 3D bioprinted constructs consisting of human ASCs (hASCs)
We optimized the key parameters for hASCs-based 3D bioprinting
Summary
Periodontal disease, dental trauma, cancer, and other diseases can lead to maxillofacial bone defects, which are commonly encountered by dentists [1]. Tissue engineering involves three necessary elements [2,3,4]: cells with high osteogenic potential; osteogenic growth factors, and a 3D scaffold that is porous for vascularization and gives the 3D construct sufficient mechanical properties for loading. To form an ideal construct, the seeded cells should be autologous and easy to obtain; the scaffold should be biodegradable and derived from homologous materials [5]. Stem cells derived from bone marrow (BMSCs) and adipose tissue (ASCs) possess the capabilities of self-renewal and differentiation into osteoblasts. The minimal invasive capacity, ease of access, and abundance of hASCs in adipose tissue provide clear advantages over BMSCs and make these stem cells an ideal source for tissue engineering therapies [6]. Human ASCs (hASCs) may have applications in tissue engineering as seed cells
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