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3D bioprinting of co-cultured osteogenic spheroids for bone tissue fabrication 1
Dong Nyoung Heo,
a,b,c,1
, Bugra Ayan,
a,b,1
, Madhuri Dey
b,d
, Dishary Banerjee,
a,b
, Hwabok 2
Wee,
e
Gregory S. Lewis,
e
Ibrahim T. Ozbolat
*a,b,f,g,h
3
4
a
Department of Engineering Science and Mechanics Department, Penn State University, 5
University Park, PA 16802, USA 6
b
The Huck Institutes of the Life Sciences, Penn State University, University Park, PA 16802, 7
USA 8
c
Department of Dental Materials, School of Dentistry, Kyung Hee University, 26 9
Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Republic of Korea 10
d
Department of Chemistry, Penn State University, University Park, PA 16802, USA 11
e
Department of Orthopedics and Rehabilitation, Penn State College of Medicine, Hershey, 12
PA 17033, USA 13
f
Biomedical Engineering Department, Penn State University, University Park, PA 16802, 14
USA 15
g
Materials Research Institute, Penn State University, University Park, PA 16802, USA 16
h
Department of Neurosurgery, Penn State College of Medicine, Hershey, PA 17033, USA 17
1
These authors contributed equally to this work 18
* Correspondence to Ibrahim T. Ozbolat, Ph. D. 19
Engineering Science and Mechanics Department, Biomedical Engineering Department, The 20
Huck Institutes of the Life Sciences, Materials Research Institute 21
The Pennsylvania State University 22
W313 Millennium Science Complex, University Park, PA 16802, USA 23
Contact: 1-814-863-5819/ito1@psu.edu
(Ibrahim T. Ozbolat). 24
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted June 17, 2020. ; https://doi.org/10.1101/2020.06.16.155143doi: bioRxiv preprint
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Abstract 25
Conventional top-down approaches in tissue engineering involving cell seeding on scaffolds 26
have been widely used in bone engineering applications. However, scaffold-based bone tissue 27
constructs have had limited clinical translation due to constrains in supporting scaffolds, 28
minimal flexibility in tuning scaffold degradation, and low achievable cell seeding density as 29
compared with native bone tissue. Here, we demonstrate a pragmatic and scalable bottom-up 30
method, inspired from embryonic developmental biology, to build three-dimensional (3D) 31
scaffold-free constructs using spheroids as building blocks. Human umbilical vein endothelial 32
cells (HUVECs) were introduced to human mesenchymal stem cells (hMSCs) 33
(hMSC/HUVEC) and spheroids were fabricated by an aggregate culture system. Bone tissue 34
was generated by induction of osteogenic differentiation in hMSC/HUVEC spheroids for 10 35
days, with enhanced osteogenic differentiation and cell viability in the core of the spheroids 36
compared to hMSC-only spheroids. Aspiration-assisted bioprinting (AAB) is a new 37
bioprinting technique which allows precise positioning of spheroids (11% with respect to the 38
spheroid diameter) by employing aspiration to lift individual spheroids and bioprint them 39
onto a hydrogel. AAB facilitated bioprinting of scaffold-free bone tissue constructs using the 40
pre-differentiated hMSC/HUVEC spheroids. These constructs demonstrated negligible 41
changes in their shape for two days after bioprinting owing to the reduced proliferative 42
potential of differentiated stem cells. Bioprinted bone tissues showed interconnectivity with 43
actin-filament formation and high expression of osteogenic and endothelial-specific gene 44
factors. This study thus presents a viable approach for 3D bioprinting of complex-shaped 45
geometries using spheroids as building blocks, which can be used for various applications 46
including but not limited to, tissue engineering, organ-on-a-chip and microfluidic devices, 47
drug screening and, disease modeling. 48
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted June 17, 2020. ; https://doi.org/10.1101/2020.06.16.155143doi: bioRxiv preprint
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Keywords: biofabrication, 3D bioprinting, aspiration-assisted bioprinting, osteogenic 49
spheroids, bone tissue regeneration 50
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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1. Introduction 51
Bone is a highly vascularized dynamic tissue, observed in a variety of shapes and sizes in 52
the body. The skeleton serves crucial roles such as supporting the framework for the body 53
and protecting vital organs [1,2]. Large bone defects cannot self-regenerate regenerate, and 54
can be due to trauma, fracture nonunion, infection, bone tumor resections, and 55
removal/revision of joint replacements and other implant [3–5]. There is thus a substantial 56
demand for engineered constructs to restore and regenerate the diseased/excised part of the 57
bone tissue [4,6,7]. To-date, significant progress has been made using three-dimensional (3D) 58
bioprinting of living cells and tissues to recapitulate the bone tissue [8]. Most of these 59
bioprinting approaches rely on scaffold-based techniques where biodegradable hydrogels are 60
seeded or combined with mature osteoblasts or osteogenically-committed stem-cells [6,9,10]. 61
These scaffolds can provide mechanical support, serve as a template for cell attachment and 62
facilitate a conducive environment for cellular activities [3,6]. However, degradation of 63
scaffolds, limited cell density compared to native-tissues and limited communication among 64
cells are some of the major drawbacks in scaffold-based approaches [11]. 65
Scaffold-free bone tissue engineering pose a promising alternative to 3D bioprinting 66
approaches [12]. Stem-cell derived aggregates, in the form of spheroids, are considered as 67
building blocks for scaffold-free bioprinting and mimic the complex morphology and 68
physiology of native tissue by inducing cross-talk among cells and cell-extracellular cell 69
matrix (ECM) interactions [13,14]. In addition, osteogenic differentiation is observed to 70
increase due to stronger integrin-ECM interaction caused by the presence of both stroma and 71
structure within spheroids [15]. However, non-uniform bone tissue regeneration owing to the 72
non-homogeneous oxygen diffusion across the entire spheroid domain, especially to the core 73
of spheroids [15–17] forms the major roadblock in successful usage of spheroids in 3D 74
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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bioprinting applications. 2D co-culture systems with mature osteoblasts or stem cell derived 75
osteogenic progenitor cells with cells from endothelial lineage have shown potential to 76
address this limitation by enhanced secretion of endothelial cell-mediated paracrine factors 77
secretion under hypoxia [18]. In this study, we intended to exploit this potential of endothelial 78
cells and investigate their role in 3D on osteogenic differentiation. We achieved this by the 79
co-culture of a minimal number of human umbilical vein endothelial cells (HUVEC) in 80
human mesenchymal stem cells (hMSC) spheroids, and by induction of osteogenesis with 81
bone regeneration across the entire domain of spheroids. 82
Spheroids have been utilized in successful biofabrication of functional bone tissue substitutes 83
[19–22]. Although several approaches have been presented in the literature for bioprinting of 84
spheroids, most of these suffer from poor spatial control in 3D, significant damages to 85
spheroids with loss of viability and structural integrity, poor repeatability of the process while 86
using spheroids that are non-uniform in size, inability to form complex 3D shapes, inability to 87
maintain designed shape due to cell-mediated compaction post-bioprinting, and lack of 88
scalability for translation from bench to bedside [19–21]. Aspiration-assisted bioprinting 89
(AAB), a newly developed technique by our group, bioprints spheroids in 3D space using the 90
power of aspiration forces. AAB leverages the fact that spheroids can be formed from diverse 91
cell types at high densities [22]. When using human stem cells, these spheroids can 92
recapitulate aspects of embryonic development to self-assemble [21] into various organs. 93
Using this technique, in this study, we have demonstrated that spheroids with viscoelastic 94
properties can be lifted by aspiration forces, and then positioned precisely (11% with respect 95
to the spheroid diameter) into a hydrogel substrate, circumventing the limitations of the other 96
spheroid bioprinting techniques available. 97
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted June 17, 2020. ; https://doi.org/10.1101/2020.06.16.155143doi: bioRxiv preprint
