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3D printing of osteocytic Dll4 integrated with PCL for cell fate determination towards osteoblasts in vitro

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Abstract

Since 3D printed hard materials could match the shape of bone, cell survival and fate determination towards osteoblasts in such materials have become a popular research target. In this study, a scaffold of hard material for 3D fabrication was designed to regulate developmental signal (Notch) transduction guiding osteoblast differentiation. We established a polycaprolactone (PCL) and cell-integrated 3D printing system (PCI3D) to reciprocally print the beams of PCL and cell-laden hydrogel for a module. This PCI3D module holds good cell viability of over 87%, whereas cells show about sixfold proliferation in a 7-day culture. The osteocytic MLO-Y4 was engineered to overexpress Notch ligand Dll4, making up 25% after mixing with 75% stromal cells in the PCI3D module. Osteocytic Dll4, unlike other delta-like family members such as Dll1 or Dll3, promotes osteoblast differentiation and the mineralization of primary mouse and a cell line of bone marrow stromal cells when cultured in a PCI3D module for up to 28 days. Mechanistically, osteocytic Dll4 could not promote osteogenic differentiation of the primary bone marrow stromal cells (BMSCs) after conditional deletion of the Notch transcription factor RBPjκ by Cre recombinase. These data indicate that osteocytic Dll4 activates RBPjκ-dependent canonical Notch signaling in BMSCs for their oriented differentiation towards osteoblasts. Additionally, osteocytic Dll4 holds a great potential for angiogenesis in human umbilical vein endothelial cells within modules. Our study reveals that osteocytic Dll4 could be the osteogenic niche determining cell fate towards osteoblasts. This will open a new avenue to overcome the current limitation of poor cell viability and low bioactivity of traditional orthopedic implants.

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Acknowledgements

The authors thank Professor Dr. Linda Bonewald for her kindly providing the MLO-Y4 cell line and appreciate Dr. Jun Li for his statistical analysis of data. This work is supported by the National Natural Science Foundation of China (Nos. U1601220, 82072450, and 81672118), Chongqing Science and Technology Commission-Basic Science and Frontier Technology Key Project (No. cstc2015jcyjBX0119), and Chongqing Medical University Intelligent Medicine Research Project (No.ZHYX202115).

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  1. Pengtao Wang, Xiaofang Wang, Bo Wang and Xian Li have contributed equally to this work.

Authors and Affiliations

  1. Laboratory of Skeletal Development and Regeneration, Institute of Life Sciences, Chongqing Medical University, Chongqing, 400016, China

    Pengtao Wang, Xiaofang Wang, Bo Wang, Zhengsong Xie, Jie Chen & Xiaolin Tu

  2. Medical Data Science Academy, Chongqing Medical University, Chongqing, 400016, China

    Xian Li

  3. Department of Immunology and Genomic Medicine, Graduate School of Medicine, Kyoto University, Yoshida Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan

    Tasuku Honjo

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  1. Pengtao Wang
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Contributions

XLT, PTW, and ZSX contributed to conceptualization; XLT, XFW, and JC provided methodology; PTW, XFW, BW, and ZSX performed investigation; PTW, BW, and XL carried out data analysis; PTW and XL performed writing—original draft; all authors performed writing—review and editing; XLT contributed to funding acquisition; XLT provided resources; XLT and XL performed supervision.

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Correspondence to Xiaolin Tu.

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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Ethical approval

All animal procedures in our study were approved by the Institutional Animal Care and Use Committee of Chongqing Medical University.

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Wang, P., Wang, X., Wang, B. et al. 3D printing of osteocytic Dll4 integrated with PCL for cell fate determination towards osteoblasts in vitro. Bio-des. Manuf. 5, 497–511 (2022). https://doi.org/10.1007/s42242-022-00196-1

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