Abstract
Bioprinting is an emerging technology in which cell-laden biomaterials are precisely dispersed to engineer artificial tissues that mimic aspects of the anatomical and structural complexity of relatively soft tissues such as skin, vessels, and cartilage. However, reproducing the highly mineralized and cellular diversity of bone tissue is still not easily achievable and is yet to be demonstrated. Here, an extrusion-based 3D bioprinting strategy is utilized to fabricate 3D bone-like tissue constructs containing osteogenic cellular organization. A simple and low-cost bioink for 3D bioprinting of bone-like tissue is prepared based on two unmodified polymers (alginate and gelatin) and combined with human mesenchymal stem cells (hMSCs). To form 3D bone-like tissue and bone cell phenotype, the influence of different scaffold stiffness and cell density of 3D bioprinted cell-laden porous scaffolds on osteogenic differentiation and bone-like tissue formation was investigated over time. Our results showed that soft scaffolds (0.8%alg, 0.66 ± 0.08 kPa) had higher DNA content, enhanced ALP activity and stimulated osteogenic differentiation than stiff scaffolds (1.8%alg, 5.4 ± 1.2 kPa). At day 42, significantly more mineralized tissue was formed in soft scaffolds than in stiff scaffolds (43.5 ± 7.1 mm3 vs. 22.6 ± 6.0 mm3). Importantly, immunohistochemistry staining demonstrated more osteocalcin protein expression in high mineral compared to low mineral regions. Additionally, cells in soft scaffolds exhibited osteoblast- and early osteocyte-related gene expression and 3D cellular network within the mineralized matrix at day 42. Furthermore, the results showed that cell density in 15 M cells/ml can promote cell-cell connections at day 7 and mineral formation at day 14, while 5 M cells/ml had the significantly higher mineral formation rate than 15 M cells/ml from day 14 to day 21. In summary, this work reports the formation of 3D bioprinted bone-like tissue using a simple and low-cost cell-laden bioink, which was optimized for stiffness and cell density, showing great promise for bone tissue engineering applications. Statement of SignificanceIn this study, we presented for the first time a framework combining 3D bioprinting, bioreactor system and time-lapsed micro-CT monitoring to provide in vitro scaffold fabrication, maturation, and mineral visualization for bone tissue engineering. 3D bone-like tissue constructs have been formed via optimizing scaffold stiffness and cell density. The soft scaffolds had higher cell proliferation, enhanced alkaline phosphatase activity and stimulated osteogenic differentiation with 3D cellular network foramtion than stiff scaffolds. Significantly more mineralized bone-like tissue was formed in soft scaffolds than stiff scaffolds at day 42. Meanwhile, cell density in 15 M cells/ml can promote cell-cell connections and mineral formation in 14 days, while the higher mineral formation rate was found in 5 M cells/ml from day 14 to day 21.
Highlights
Bioprinting is a form of additive manufacturing that can spatially control the deposition of biomaterials, cells and/or biomolecules, the so-called bioink, in high resolution and predefined 3D patterns [1,2,3]
We presented for the first time a framework combining 3D bioprinting, bioreactor system and time-lapsed micro-computerized tomography (CT) monitoring to provide in vitro scaffold fabrication, maturation, and mineral visualization for bone tissue engineering. 3D bone-like tissue constructs have been formed via optimizing scaffold stiffness and cell density
To determine the optimal extracellular matrix (ECM) conditions for human mesenchymal stem cells (hMSCs) osteogenesis, we formulated a series of bioinks with different alginate concentration (0.8%alg and 1.8%alg) and different initial cell seeding density (1.67 M, 5 M and 15 M cells/ml) (Figs. 1 and S1)
Summary
Bioprinting is a form of additive manufacturing that can spatially control the deposition of biomaterials, cells and/or biomolecules, the so-called bioink, in high resolution and predefined 3D patterns [1,2,3]. Cells were embedded in the 3D bioprinted scaffolds and responded to an omnidirectional condition mimicking the 3D environment of cells in tissues and organs. 3D cell-laden scaffolds have been fabricated to mimic the 3D environment of bone cells with cell-cell and cell-matrix interactions representative of 3D bone tissue in its early stage of delopment, e.g. osteoid [10]. A number of 3D bioprinting approaches attempt to mimic this process, focusing on promoting the mineral formation and bone cell differentiation in hMSC-laden hydrogel scaffolds [16,17]. As the bioink acts as an extracellular matrix (ECM) providing mechanical and chemical stimuli to all cells in its environment, its correct selection is essential to induce hMSCs’ differentiation towards the osteogenic lineage and ECM mineralization
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