3D Printing of Anisotropic Bone-Mimetic Structure with Controlled Fluid Flow Stimuli for Osteocytes: Flow Orientation Determines the Elongation of Dendrites

Aira Matsugaki, Tadaaki Matsuzaka, Ami Murakami, Pan Wang, Takayoshi Nakano

Article ID: 293
Vol 6, Issue 4, 2020, Article identifier:293

VIEWS - 921 (Abstract) 156 (PDF)


Although three-dimensional (3D) bioprinting techniques enable the construction of various living tissues and organs, the generation of bone-like oriented microstructures with anisotropic texture remains a challenge. Inside the mineralized bone matrix, osteocytes play mechanosensing roles in an ordered manner with a well-developed lacunar-canaliculi system. Therefore, control of cellular arrangement and dendritic processes is indispensable for construction of artificially controlled 3D bone-mimetic architecture. Herein, we propose an innovative methodology to induce controlled arrangement of osteocyte dendritic processes using the laminated layer method of oriented collagen sheets, combined with a custom-made fluid flow stimuli system. Osteocyte dendritic processes showed elongation depending on the competitive directional relationship between flow and substrate. To the best of our knowledge, this study is the first to report the successful construction of the anisotropic bone-mimetic microstructure and further demonstrate that the dendritic process formation in osteocytes can be controlled with selective fluid flow stimuli, specifically by regulating focal adhesion. Our results demonstrate how osteocytes adapt to mechanical stimuli by optimizing the anisotropic maturation of dendritic cell processes.


Bioprinting, Collagen substrate, Mineralization, Osteocyte, 3D arrangement of bone matrix

Full Text:



Ishimoto T, Nakano T, Umakoshi Y, et al., 2013, Degree of Biological Apatite c -axis Orientation Rather than Bone Mineral Density Controls Mechanical Function in Bone Regenerated Using Recombinant Bone Morphogenetic Protein-2. J Bone Miner Res, 28:1170–9. DOI: 10.1002/jbmr.1825.

Schaff F, Bech M, Zaslansky P, et al., 2015, Six-dimensional Real and Reciprocal Space Small-angle X-ray Scattering Tomography. Nature, 527:353–6. DOI: 10.1038/nature16060.

Nakano T, Kaibara K, Tabata Y, et al., 2002, Unique Alignment and Texture of Biological Apatite Crystallites in Typical Calcified Tissues Analyzed by Microbeam x-ray Diffractometer System. Bone, 31:479–87. DOI: 10.1016/s8756-3282(02)00850-5.

Nakano T, Kaibara K, Ishimoto T, et al., 2012, Biological Apatite (BAp) Crystallographic Orientation and Texture as a New Index for Assessing the Microstructure and Function of Bone Regenerated by Tissue Engineering. Bone, 51:741–7. DOI: 10.1016/j.bone.2012.07.003.

Hennessy KM, Pollot BE, Clem WC, et al., 2009, The Effect of Collagen I Mimetic Peptides on Mesenchymal Stem Cell Adhesion and Differentiation, and on Bone Formation at Hydroxyapatite Surfaces. Biomaterials, 30:1898–909. DOI: 10.1016/j.biomaterials.2008.12.053.

Lee S, Obata A, Brauer DS, et al., 2015, Dissolution Behavior and Cell Compatibility of Alkali-free MgO-CaO-SrO-TiO2-P2O5 Glasses for Biomedical Applications. Biomed Glasses, 1:151–8. DOI: 10.1515/bglass-2015-0015.

Prewitz MC, Seib FP, von Bonin M, et al., 2013, Tightly Anchored Tissue-mimetic Matrices as Instructive Stem Cell Microenvironments. Nat Methods, 10:788–794. DOI: 10.1038/nmeth.2523.

Bonewald LF, 2011, The Amazing Osteocyte. J Bone Miner Res, 26:229–38. DOI: 10.1002/jbmr.320.

Odagaki N, Ishihara Y, Wang Z, et al., 2018, Role of OsteocytePDL Crosstalk in Tooth Movement via SOST/Sclerostin. J Dent Res, 97:1374–82. DOI: 10.1177/0022034518771331.

Ganesh T, Laughrey LE, Niroobakhsh M, et al., 2020, Multiscale Finite Element Modeling of Mechanical Strains and Fluid Flow in Osteocyte Lacunocanalicular System. Bone, 2020:115328. DOI: 10.1016/j.bone.2020.115328.

Ng WL, Chua CK, Shen YF, 2016, Print Me An Organ! Why We Are Not There Yet. Prog Polym Sci, 2016:101145.

Ozbolat IT, Hospodiuk M, 2016, Current Advances and Future Perspectives in Extrusion-based Bioprinting. Biomaterials, 76:321–43. DOI: 10.1016/j.biomaterials.2015.10.076.

Gudapati H, Dey M, Ozbolat I, 2016, A Comprehensive Review on Droplet-based Bioprinting: Past, Present and Future, Biomaterials, 102:20–42. DOI: 10.1016/j.biomaterials.2016.06.012.

Ng WL, Lee JM, Yeong WY, et al., 2017, Microvalve-based Bioprinting Process, Bio-inks and Applications. Biomater Sci, 5:632–47. DOI: 10.1039/c6bm00861e.

Ng WL, Lee JM, Zhou M, et al., 2020, Vat Polymerizationbased Bioprinting Process, Materials, Applications and Regulatory Challenges. Biofabrication, 12:22001. DOI: 10.1088/1758-5090/ab6034.

Wang K, Le L, Chun BM, et al., 2019, A Novel Osteogenic Cell Line that Differentiates into GFP-Tagged Osteocytes and Forms Mineral with a Bone-Like Lacunocanalicular Structure. J Bone Miner Res, 34:979–95. DOI: 10.1002/jbmr.3720.

Matsugaki A, Isobe Y, Saku T, et al., 2015, Quantitative Regulation of Bone-mimetic, Oriented Collagen/Apatite Matrix Structure Depends on the Degree of Osteoblast Alignment on Oriented Collagen Substrates: Anisotropic Construction of Cell-Produced Mineralized Matrix. J Biomed Mater Res A, 103:489–99. DOI: 10.1002/jbm.a.35189.

Nakanishi Y, Matsugaki A, Kawahara K, et al., 2019, Unique Arrangement of Bone Matrix Orthogonal to Osteoblast Alignment Controlled by Tspan11-Mediated Focal Adhesion Assembly. Biomaterials, 209:103–10. DOI: 10.1016/j.biomaterials.2019.04.016.

Kimura Y, Matsugaki A, Sekita A, et al., 2017, Alteration of Osteoblast Arrangement Via Direct Attack by Cancer Cells: New Insights into Bone Metastasis. Sci Rep, 7:44824. DOI: 10.1038/srep44824.

Wong G, Cohn DV, 1974, Separation of Parathyroid Hormone and Calcitonin-sensitive Cells from Non-responsive Bone Cells. Nature, 252:713–5. DOI: 10.1038/252713a0.

Stern AR, Stern MM, Van Dyke ME, et al., 2012, Isolation and Culture of Primary Osteocytes from the Long Bones of Skeletally Mature and Aged Mice. Biotechniques, 52:361–73. DOI: 10.2144/0000113876.

Matsugaki A, Yamazaki D, Nakano T, 2020, Selective Patterning of Netrin-1 as a Novel Guiding Cue for Anisotropic Dendrogenesis in Osteocytes. Mater Sci Eng C, 108:110391. DOI: 10.1016/j.msec.2019.110391.

Noyama Y, Nakano T, Ishimoto T, et al., 2013, Design and Optimization of the Oriented Groove on the Hip Implant Surface to Promote Bone Microstructure Integrity. Bone, 52:659–67. DOI: 10.1016/j.bone.2012.11.005.

Ozasa R, Ishimoto T, Miyabe S, et al., 2019, Osteoporosis Changes Collagen/Apatite Orientation and Young’s Modulus in Vertebral Cortical Bone of Rat. Calcif Tissue Int, 104:449–60. DOI: 10.1007/s00223-018-0508-z.

Matsugaki A, Harada T, Kimura Y, et al., 2018, Dynamic Collision Behavior between Osteoblasts and Tumor Cells Regulates the Disordered Arrangement of Collagen Fiber/Apatite Crystals in Metastasized Bone. Int J Mol Sci, 19:3474. DOI: 10.3390/ijms19113474.

Zeng J, Matsusaki M, 2019, Layer-by-Layer Assembly of Nanofilms to Control Cell Functions. Polym Chem, 10:2960–74. DOI: 10.1039/c9py00305c.

Lee S, Matsugaki A, Kasuga T, et al., 2019, Development of bifunctional oriented bioactive glass/poly(lactic acid) composite scaffolds to control osteoblast alignment and proliferation. J Biomed Mater Res A, 107:1031–41.

Ozasa R, Matsugaki A, Isobe Y, et al., 2018, Construction of Human Induced Pluripotent Stem Cell-derived Oriented Bone Matrix Microstructure by Using In Vitro Engineered Anisotropic Culture Model. J Biomed Mater Res A, 106:360–9. DOI: 10.1002/jbm.a.36238.

Matsugaki A, Fujiwara N, Nakano T, 2013, Continuous Cyclic Stretch Induces Osteoblast Alignment and Formation of Anisotropic Collagen Fiber Matrix. Acta Biomater, 9:7227–35. DOI: 10.1016/j.actbio.2013.03.015.

Wang L, Wang Y, Han Y, et al., 2005, In Situ Measurement of Solute Transport in the Bone Lacunar-canalicular System. Proc Natl Acad Sci U S A, 102:11911–6. DOI: 10.1073/pnas.0505193102.

Wang J, Ishimoto T, Nakano T, et al., 2017, Unloadinginduced Degradation of the Anisotropic Arrangement of Collagen/Apatite in Rat Femurs. Calcif Tissue Int, 100:87–94. DOI: 10.1007/s00223-016-0200-0.

Zhang K, Barragan-Adjemian C, Ye L, et al., 2006, E11/gp38 Selective Expression in Osteocytes: Regulation by Mechanical Strain and Role in Dendrite Elongation. Mol Cell Biol, 26:4539–52. DOI: 10.1128/mcb.02120-05.

Thi MM, Suadicani SO, Schaffler MB, et al., 2013, Spray Mechanosensory Responses of Osteocytes to Physiological Forces Occur Along Processes and not Cell Body and Require αVβ3 Integrin. Proc Natl Acad Sci U S A, 110:21012–7. DOI: 10.1073/pnas.1321210110.

Wang Y, Botvinick L, Zhao Y, et al., 2005, Visualizing the Mechanical Activation of SRC. Nature, 434:1040–5.

Schimmel L, Fukuhara D, Richards M, et al., 2020, c-SRC Controls Stability of Sprouting Blood Vessels in the Developing Retina Independently of Cell-cell Adhesion through Focal Adhesion Assembly. Development, 147:dev185405. DOI: 10.1242/dev.185405.

Skupien A, Konopka A, Trzaskoma P, et al., 2014, CD44 Regulates Dendrite Morphogenesis through SRC Tyrosine Kinase-dependent Positioning of the Golgi. J Cell Biol, 127:5038–51. DOI: 10.1242/jcs.154542.

DOI: http://dx.doi.org/10.18063/ijb.v6i4.293


  • There are currently no refbacks.

Copyright (c) 2020 Matsugaki, et al.

Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.