3D bioprinting of co-cultured osteogenic spheroids for bone tissue fabrication

TL;DR: A viable approach for 3D bioprinting of complex-shaped geometries using spheroids as building blocks, which can be used for various applications including but not limited to, tissue engineering, organ-on-a-chip and microfluidic devices, drug screening and, disease modeling.

Abstract: Conventional top-down approaches in tissue engineering involving cell seeding on scaffolds have been widely used in bone engineering applications. However, scaffold-based bone tissue constructs have had limited clinical translation due to constrains in supporting scaffolds, minimal flexibility in tuning scaffold degradation, and low achievable cell seeding density as compared with native bone tissue. Here, we demonstrate a pragmatic and scalable bottom-up method, inspired from embryonic developmental biology, to build three-dimensional (3D) scaffold-free constructs using spheroids as building blocks. Human umbilical vein endothelial cells (HUVECs) were introduced to human mesenchymal stem cells (hMSCs) (hMSC/HUVEC) and spheroids were fabricated by an aggregate culture system. Bone tissue was generated by induction of osteogenic differentiation in hMSC/HUVEC spheroids for 10 days, with enhanced osteogenic differentiation and cell viability in the core of the spheroids compared to hMSC-only spheroids. Aspiration-assisted bioprinting (AAB) is a new bioprinting technique which allows precise positioning of spheroids (11% with respect to the spheroid diameter) by employing aspiration to lift individual spheroids and bioprint them onto a hydrogel. AAB facilitated bioprinting of scaffold-free bone tissue constructs using the pre-differentiated hMSC/HUVEC spheroids. These constructs demonstrated negligible changes in their shape for two days after bioprinting owing to the reduced proliferative potential of differentiated stem cells. Bioprinted bone tissues showed interconnectivity with actin-filament formation and high expression of osteogenic and endothelial-specific gene factors. This study thus presents a viable approach for 3D bioprinting of complex-shaped geometries using spheroids as building blocks, which can be used for various applications including but not limited to, tissue engineering, organ-on-a-chip and microfluidic devices, drug screening and, disease modeling.

Summary

1. Introduction

• Compaction of spheroids leading to significant changes in geometry of tissue constructs compared to the desired has been a common issue post bioprinting, and hence, the authors utilize 3D bioprinting of pre-differentiated hMSC/HUVEC spheroid to minimize shape changes.
• Postbioprinting, the authors showed that they were able to control the shape of the bioprinted construct, using pre-differentiated hMSC/HUVEC spheroids.
• Thus, the authors attempted to address major limitations of spheroid bioprinting by bioprinting spheroids using AAB, fabricating complex-shaped bone tissue constructs, engineering the spheroids in a way to induce osteogenesis across the entire spheroid domain, and controlling osteogenic induction timelines before and after bioprinting in order to reduce spheroid compaction and increase retention of geometry of the tissue constructs.

2.1. Cell culture

• HMSCs (, Walkersville, MD) and HUVECs were used to fabricate of 3D cellular spheroids.
• HMSCs were cultured in all-in-one ready-to-use hMSC growth medium (Cell Applications, INC., San Diego, CA).
• Cells passages from three through seven were used for both hMSCs and HUVECs.

2.2. Spheroid fabrication

• HMSCs and HUVECs were harvested with trypsin and collected by centrifugation at 1600 rpm for 5 min for the fabrication of the spheroids.
• HMSC-only spheroids were fabricated similarly and used as control to understand the functionality of HUVECs in the spheroids.
• The cell medium was changed every three days.

2.5 Quantitative real-time polymerase chain reaction (real-time PCR)

• Real-time polymerase chain reaction (RT-qPCR) was performed after five days in growth media, and an additional 10 days in osteogenic media to evaluate the gene expression profiles of hMSC-only and hMSC/HUVEC spheroids.
• The total RNA of hMSC-only and hMSC/HUVEC spheroids was isolated using a RNeasy Plus Mini Kit (Qiagen, Germantown, MD) according to the manufacturer's instructions and quantified using a Nanodrop ND-1000 Spectrophotomer (Thermo Scientific, Wilmington, DE).
• RT-qPCR was analyzed using SsoFast™ EvaGreen ® Supermix (Bio-Rad, Hercules, CA) and all values were normalized by a house-keeping gene GAPDH.
• Threshold cycle values were calculated using a comparative cycle threshold method.
• The fold-change of hMSC-only was set at 1-fold, and the ratio of the normalized fold-change was calculated based on the standard condition.

2.6 3D bioprinting of spheroids

• AAB system was used to bioprint hMSC-only and hMSC/HUVEC spheroids as previously described [22] .
• Sodium alginate was dispensed on the glass substrate using microvalves, then the aerosol form of CaCl 2 was utilized to crosslink sodium alginate partially.
• Spheroids were collected into 1.5 ml conical tubes and transferred to the bioprinting platform.
• Afterwards, the top portion of the conical tubes were cut by a scissor.
• A customized glass pipette (~80 μm in diameter) was dipped into a conical tube and air pressure of 25 mmHg was applied to lift spheroids.

2.7 Characterization of osteogenic differentiation by immunocytochemistry and alizarin red S staining

• The calcium deposition was visualized by staining cross-sectioned slides with a 2 % alizarin red S staining solution for 10 min at room temperature.
• Stained samples were washed three times with DI water and imaged using optical microscopy.

2.8 Micro-computed tomography (μCT) measurements

• ΜCT scanner (VivaCT 40, Scanco Medical, Switzerland) was used with 10.5 μm voxel resolution, 55 kV energy, 145 μA intensity, 21.5mm diameter field-of-view, and 300 ms integration time to evaluate the mineralization of spheroids in bioprinted tissues.
• The samples were placed inside the μCT scanner and scanned.
• DICOM files were processed in Avizo software (FEI Company, Hillsboro, OR).
• A hydroxyapatite (HA) phantom (Micro-CT HA, (which was not certified by peer review) is the author/funder.
• Images were processed with a Gaussian smoothing filter (sigma 0.9) to reduce noise, and a threshold of 200 mgHA/ccm was used to visualize mineralized spheroids and quantify mineralized volume.

2.9 Statistical analysis

• All values are presented as mean (±) standard deviation.
• Multiple comparisons were analyzed using a one-way analysis of variance followed by Tukey's multiple comparison test.
• All statistical analysis was performed by Statistical Product and Service Solutions software (SPSS, IBM, Armonk, NY).

3.1 Fabrication and characterization of hMSC-only and hMSC/HUVEC spheroids

• 8 hMSC/HUVEC spheroids showed higher cell viability and mechanical properties (surface tension) compared to hMSC-only spheroids and demonstrated highest RNA content and pluripotency potential, also known as In summary, 92.
• Based on these results, the authors showed that introduction of only 8% HUVECs into the hMSCs is sufficient enough to enhance spheroid core cell viability with improved mechanical properties.
• Therefore, to accomplish their goal of building bone tissue and exploring the role of HUVECs in hMSCs spheroids on shape preservation of 3D constructs, the authors utilized 92:8 hMSC/HUVEC spheroids for the rest of the study, referred as hMSC/HUVEC spheroid henceforth.

3.2 Bioprinting of hMSC/HUVEC spheroids via AAB

• After bioprinting, the constructs were overlaid with alginate using the micro-valve dispenser, crosslinked with the aerosols of CaCl 2 , and then incubated for two days to facilitate fusion amongst spheroids.
• After removal of alginate and further incubation, the constructs were observed to undergo significant compaction, lost their designed configuration and turned into tissue balls , which is corroborated by previously published articles [14, 22, 30] .
• In order to direct stem cell differentiation towards bone tissue formation, constructs composed of stem cells should be inducted for about three weeks [14, 16, 31] .
• The bioprinted hMSC/HUVEC construct deformed into a tissue aggregate within four days of being guided into bone tissue differentiation.
• This led us to explore another strategy, by tuning the timelines of induction of osteogenic differentiation, for retaining the geometry of constructs while bioprinting using spheroids.

3.3 Osteogenic differentiation of hMSC/HUVEC spheroids

• Inducing the hMSC/HUVEC spheroids to differentiation media before bioprinting lowers the proliferative potential of hMSCs and allow the spheroids to be in their osteogenic differentiation pathway [41] .
• Hence, in this study, the authors utilized mid-term osteogenic hMSC/HUVEC spheroids for bioprinting scaffold-free bone-tissue constructs with an expectation of reduced deformation in the bioprinted geometry.

3.4 3D bioprinting of osteogenic tissues

• To demonstrate the ability of osteogenic hMSC/HUVEC spheroids to serve as building blocks in bioprinting, different topographies resembling triangle, hexagon, and diamond in a single layer were bioprinted using the AAB system.
• As shown in Figure 4A , hMSC/HUVEC spheroids were precisely arranged into a diamond shape and after incubation, fused into a single patch of tissue.
• Bioprinted single-layered osteogenic tissue construct exhibited high cell viability from the periphery to the core of the spheroid with a negligible number of dead cells, as indicated by LIVE/DEAD staining.
• The mineralized volume in the single-layered triangle, hexagon and diamond shaped configurations was measured to be 0.051, 0.056 and 0.081 mm 3 , respectively.
• The constructs retained the bioprinted shape and demonstrated uniform mineralization throughout the entire 3D configuration.

4. Conclusion

• In conclusion, the authors present an effective spheroid bioprinting strategy using the AAB system, which facilitates bioprinting of osteogenic hMSC/HUVEC spheroids to fabricate scaffoldfree 3D bone tissue constructs.
• HUVECs were introduced into hMSCs to investigate their influence on spheroid formation and compaction, osteogenic differentiation and reduction of shape deformation after differentiation.
• The authors findings demonstrate that osteogenically-differentiated hMSC/HUVEC spheroids, with as little as 8% of HUVECs, could be used as building blocks for bone tissue fabrication.
• These spheroids demonstrated reduced necrosis, increased cell viability in the core of the spheroid, enhanced differentiation into osteogenic lineage, and improved mechanical properties.
• Such a bioprinting approach, to facilitate fabrication of 3D geometries with negligible shape changes, using spheroids composed of differentiated stem cells and introduced with endothelial cells to enhance cell viability and differentiation, provides a new direction in bottom-up, scaffold-free bone tissue fabrication.

Figures

Figures 627 628

Full text

1
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

2
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

3
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.
The copyright holder for this preprintthis version posted June 17, 2020. ; https://doi.org/10.1101/2020.06.16.155143doi: bioRxiv preprint

4
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.
The copyright holder for this preprintthis version posted June 17, 2020. ; https://doi.org/10.1101/2020.06.16.155143doi: bioRxiv preprint

5
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

Frequently asked questions

Q1. What are the contributions in this paper?

In this paper, the authors investigated the role of endothelial cells in 3D bone regeneration using aspiration assisted bioprinting.