scispace - formally typeset
Search or ask a question
Posted Content•DOI•

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

Dong Nyoung Heo1, Dong Nyoung Heo2, Bugra Ayan2, Madhuri Dey2, Dishary Banerjee2, Hwabok Wee2, Gregory S. Lewis2, Ibrahim T. Ozbolat •
Kyung Hee University1, Pennsylvania State University2
17 Jun 2020-bioRxiv (Cold Spring Harbor Laboratory)-
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 (3 min read)

Jump to: [1. Introduction] – [2.1. Cell culture] – [2.2. Spheroid fabrication] – [2.5 Quantitative real-time polymerase chain reaction (real-time PCR)] – [2.6 3D bioprinting of spheroids] – [2.7 Characterization of osteogenic differentiation by immunocytochemistry and alizarin red S staining] – [2.8 Micro-computed tomography (μCT) measurements] – [2.9 Statistical analysis] – [3.1 Fabrication and characterization of hMSC-only and hMSC/HUVEC spheroids] – [3.2 Bioprinting of hMSC/HUVEC spheroids via AAB] – [3.3 Osteogenic differentiation of hMSC/HUVEC spheroids] – [3.4 3D bioprinting of osteogenic tissues] and [4. Conclusion]

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.

Did you find this useful? Give us your feedback

Figures (1)

Content maybe subject to copyright    Report

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
Citations
More filters
Organ Printing: Tissue Spheroids as Building Blocks

[...]

Vladimir Mironov, Jing Zhang, Carmine Gentile, K Brakke, Thomas C. Trusk, Karoly Jakab, Gabor Forgacs, Vladimirs Kasjanovs, Richard P. Visconti, Roger R. Markwald 
01 Jan 2009
TL;DR: Organ printing can be defined as layer-by-layer additive robotic biofabrication of three-dimensional functional living macrotissues and organ constructs using tissue spheroids as building blocks.
Abstract: Organ printing can be defined as layer-by-layer additive robotic biofabrication of three-dimensional functional living macrotissues and organ constructs using tissue spheroids as building blocks. The microtissues and tissue spheroids are living materials with certain measurable, evolving and potentially controllable composition, material and biological properties. Closely placed tissue spheroids undergo tissue fusion - a process that represents a fundamental biological and biophysical principle of developmental biology-inspired directed tissue self-assembly. It is possible to engineer small segments of an intraorgan branched vascular tree by using solid and lumenized vascular tissue spheroids. Organ printing could dramatically enhance and transform the field of tissue engineering by enabling large-scale industrial robotic biofabrication of living human organ constructs with "built-in" perfusable intraorgan branched vascular tree. Thus, organ printing is a new emerging enabling technology paradigm which represents a developmental biology-inspired alternative to classic biodegradable solid scaffold-based approaches in tissue engineering.

942 citations

Principles of the Kenzan Method for Robotic Cell Spheroid-Based Three-Dimensional Bioprinting

[...]

Nicanor I. Moldovan1, Narutoshi Hibino2, Koichi Nakayama3•
Indiana University – Purdue University Indianapolis1, Johns Hopkins University2, Saga University3
01 Jun 2017
TL;DR: Bioprinting is a technology with the prospect to change the way many diseases are treated, by replacing the damaged tissues with live de novo created biosimilar constructs as mentioned in this paper.
Abstract: Bioprinting is a technology with the prospect to change the way many diseases are treated, by replacing the damaged tissues with live de novo created biosimilar constructs. However, after more than...

90 citations

Posted Content•DOI•
Volumetric Tomographic 3D Bioprinting of Heterocellular Bone-like Tissues in Seconds

[...]

Jenny Gehlen1, Wanwan Qiu1, Ralph Mueller1, Xiao-Hua Qin1•
ETH Zurich1
15 Nov 2021-bioRxiv
TL;DR: In this paper, a volumetric tomographic photofabrication of centimeter-scale heterocellular bone models that enabled successful 3D osteocytic differentiation of human mesenchymal stem cells (hMSCs) within hydrogels after 42 days co-culture with human umbilical vein endothelial cell (HUVECs).
Abstract: 3D bioprinting has emerged as a powerful tool for custom fabrication of biomimetic hydrogel constructs that support the differentiation of stem cells into functional bone tissues. Existing stem cell-derived in vitro bone models, however, often lack terminally differentiated bone cells named osteocytes which are crucial for bone homeostasis. Here, we report ultrafast volumetric tomographic photofabrication of centimeter-scale heterocellular bone models that enabled successful 3D osteocytic differentiation of human mesenchymal stem cells (hMSCs) within hydrogels after 42 days co-culture with human umbilical vein endothelial cell (HUVECs). It is hypothesized that after 3D bioprinting the paracrine signaling between hMSCs and HUVECs will promote their differentiation into osteocytes while recreating the complex heterocellular bone microenvironment. To this, we formulated a series of bioinks with varying concentrations of gelatin methacryloyl (GelMA) and lithium Phenyl(2,4,6-trimethylbenzoyl)phosphinate (LAP). A bioink comprising 5% GelMA and 0.05% LAP was identified as an optimal material with high cell viability (>90%) and excellent structural fidelity. Increasing LAP concentration led to much lower degree of cell spreading, presumably due to phototoxicity effects. Biochemical assays evidenced significantly increased expression of both osteoblastic markers (collagen-I, ALP, osteocalcin) and osteocytic markers (Podoplanin, PDPN; dentin matrix acidic phosphoprotein 1, Dmp1) after 3D co-cultures for 42 days. Additionally, we demonstrate volumetric 3D bioprinting of perfusable, pre-vascularized bone models where HUVECs self-organized into an endothelium-lined channel within 2 days. Altogether, this work leverages the benefits of volumetric tomographic bioprinting and 3D co-culture, offering a promising platform for scaled biofabrication of 3D bone-like tissues with unprecedented long-term functionality.

17 citations

Book Chapter•DOI•
3D Printing and Bioprinting of Biomaterials and Bioceramic Scaffolds: Clinical Outcomes and Implications in Bone Tissue Engineering and Maxillofacial Reconstructive Surgery

[...]

Muhja Salah, Farhad B. Naini, Lobat Tayebi
01 Jan 2022-Springer series in biomaterials science and engineering

1 citations

Posted Content•DOI•
Repopulation of a 3D-simulated periapical lesion cavity with triggered osteoblastic-differentiated dental pulp stem cell spheroids.

[...]

Ribeiro Vl1, Janaina Dernowsek1, Roger Rodrigo Fernandes1, Dimitrius Leonardo Pitol1, João Paulo Mardegan Issa1, Chaves J1, Karina Fittipaldi Bombonato-Prado1, Sousa-Neto1, Geraldo Aleixo Silva Passos1 •
University of São Paulo1
04 Jul 2021-bioRxiv
TL;DR: In this article, a mesenchymal stem cell line isolated from the dental pulp of deciduous teeth (shed cells) was cultured in a 2D monolayer and then in 3D multicellular spheroids.
Abstract: We established a proof of concept model system for the biological repair of periapical lesions using stem cell spheroids. A mesenchymal stem cell line isolated from the dental pulp of deciduous teeth (shed cells) was cultured in a 2D monolayer and then in 3D multicellular spheroids. An image of a periapical lesion of an upper lateral incisor tooth was obtained by computed micro tomography, which was used as a model for photopolymer resin 3D printing to generate a negative frame of the lesion. The negative model served to prepare a positive model of the periapical lesion cavity in an agarose gel. Shed cells cultured in monolayers or as spheroids were seeded in the positive lesion mold before or after osteoblastic differentiation. The results showed that compared to cells cultured in monolayers, the spheroids featured uniform cellularity and had a greater viability within the lesion cavity, accompanied by a temporal reduction in the expression of mRNAs typically expressed by stem cells (Cd13, Cd29, Cd44, Cd73, and Cd90). Concomitantly, there was an increase in the expression of protein markers that characterize osteoblastic differentiation (RUNX2, ALP, and BGLAP). These results provide a new perspective for regenerative endodontics with the use of spheroids prepared with shed cells to repair periapical lesions.
References
More filters
Journal Article•DOI•
Prevascularization of 3D printed bone scaffolds by bioactive hydrogels and cell co-culture

[...]

Mitchell Kuss1, Shaohua Wu1, Ying Wang1, Jason B. Untrauer1, Wenlong Li2, Jung Yul Lim1, Bin Duan1 •
University of Nebraska Medical Center1, University of Nebraska–Lincoln2
01 Jul 2018-Journal of Biomedical Materials Research Part B
TL;DR: The potential of prevascularized 3D printed scaffolds with anatomical shape for the healing of larger bone defects is demonstrated, and the co-culture systems promoted in vitro vascularization, but had no significant effects on osteogenesis.
Abstract: Vascularization is a fundamental prerequisite for large bone construct development and remains one of the main challenges of bone tissue engineering. Our current study presents the combination of 3D printing technique with a hydrogel-based prevascularization strategy to generate prevascularized bone constructs. Human adipose derived mesenchymal stem cells (ADMSC) and human umbilical vein endothelial cells (HUVEC) were encapsulated within our bioactive hydrogels, and the effects of culture conditions on in vitro vascularization were determined. We further generated composite constructs by forming 3D printed polycaprolactone/hydroxyapatite scaffolds coated with cell-laden hydrogels and determined how the co-culture affected vascularization and osteogenesis. It was demonstrated that 3D co-cultured ADMSC-HUVEC generated capillary-like networks within the porous 3D printed scaffold. The co-culture systems promoted in vitro vascularization, but had no significant effects on osteogenesis. The prevascularized constructs were subcutaneously implanted into nude mice to evaluate the in vivo vascularization capacity and the functionality of engineered vessels. The hydrogel systems facilitated microvessel and lumen formation and promoted anastomosis of vascular networks of human origin with host murine vasculature. These findings demonstrate the potential of prevascularized 3D printed scaffolds with anatomical shape for the healing of larger bone defects. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1788-1798, 2018.

88 citations

Journal Article•DOI•
Role of neural-cadherin in early osteoblastic differentiation of human bone marrow stromal cells cocultured with human umbilical vein endothelial cells.

[...]

Haiyan Li1, Richard Daculsi1, Maritie Grellier1, Maritie Grellier2, Reine Bareille1, Chantal Bourget1, Joëlle Amédée1 •
French Institute of Health and Medical Research1, University of Porto2
01 Aug 2010-American Journal of Physiology-cell Physiology
TL;DR: It can be concluded that cocultures of HBMSCs with HUVECs increased N-cadherin expression and improved cell-cell adhesion, which indicated that the cell- cell adhesion was improved between cocultured cells.
Abstract: In our previous studies, roles of gap junction and vascular endothelial growth factor in the cross-talking of human bone marrow stromal cells (HBMSCs) and human umbilical vein endothelial cells (HU...

53 citations

Journal Article•DOI•
Effects of vascular endothelial cells on osteogenic differentiation of noncontact co-cultured periodontal ligament stem cells under hypoxia.

[...]

Yang Wu1, H. Cao1, Yi Yang1, Yuqiao Zhou1, Y. Gu1, Xia Zhao1, Yarong Zhang1, Zhihe Zhao1, Lei Zhang1, J. Yin1 •
Sichuan University1
01 Feb 2013-Journal of Periodontal Research
TL;DR: Investigation of the effects of vascular ECs on osteogenic differentiation, mineralization and the paracrine function of noncontact co-cultured periodontal ligament stem cells (PDLSCs) under hypoxia reveals the involvement of MEK/ERK and p38 MAPK pathways in the process.
Abstract: Background and Objective: During periodontitis or orthodontic tooth movement, the periodontal vasculature is severely impaired by chronic inflammation or excessive mechanical force. This leads to a hypoxic microenvironment of the periodontal cells and enhances the expression of various cytokines and growth factors that may regulate angiogenesis and alveolar bone remodeling. However, the role of hypoxia in regulating the communication between endothelial cells (ECs) and osteoblast progenitors during the remodeling and repair of periodontal tissue is still poorly defined. The aim of this study was to investigate the effects of vascular ECs on osteogenic differentiation, mineralization and the paracrine function of noncontact co-cultured periodontal ligament stem cells (PDLSCs) under hypoxia, and further reveal the involvement of MEK/ERK and p38 MAPK pathways in the process. Material and Methods: First, PDLSCs were obtained and a noncontact co-culture system of PDLSCs and human umbilical vein endothelial cells was established. Second, the effects of different time-periods of hypoxia (2% O2) on the osteogenic potential, mineralization and paracrine function of co-cultured PDLSCs were investigated. Third, ERK1/2 and p38 MAPK activities of PDLSCs under hypoxia were measured by western blotting. Finally, we employed specific MAPK inhibitors (PD98059 and SB20350) to investigate the involvement of ERK1/2 and p38 MAPK in PDLSC osteogenesis under hypoxia. Results: We observed further increased osteogenic differentiation of co-cultured PDLSCs, manifested by markedly enhanced alkaline phosphatase (ALP) activity and prostaglandin E2 (PGE2) levels, vascular endothelial growth factor (VEGF) release, runt-related transcription factor 2 (Runx2) and Sp7 transcriptional and protein levels and mineralized nodule formation, compared with PDLSCs cultured alone. ERK1/2 was phosphorylated in a rapid but transient manner, whereas p38 MAPK was activated in a slow and sustained way under hypoxia. Furthermore, hypoxia-stimulated transcription and expression of osteogenic regulators (hypoxia-inducible factor-1α, ALP, Runx2, Sp7, PGE2 and VEGF) were also inhibited by PD98059 and SB203580 to different degrees. Conclusion: Further increased osteogenic differentiation and mineralization of co-cultured PDLSCs under hypoxia were regulated by MEK/ERK and p38 MAPK pathways. And the ECs-mediated paracrine of PGE2 and VEGF may facilitate the unidirectional PDLSC-EC communication and promote PDLSCs osteogenesis.

52 citations

Journal Article•DOI•
Expression of OCT-4 and SOX-2 in Bone Marrow-Derived Human Mesenchymal Stem Cells during Osteogenic Differentiation

[...]

Igor Matić1, Maja Antunović1, Sime Brkic1, Pavle Josipovic1, Katarina Caput Mihalić1, Ivan Karlak, Alan Ivković, Inga Marijanović1 •
University of Zagreb1
18 Jan 2016-Open Access Macedonian Journal of Medical Sciences
TL;DR: In this article, the levels of expression of pluripotency genes OCT-4 and SOX-2 before and after osteogenic differentiation of human mesenchymal stem cells (hMSCs).
Abstract: AIM: Determine the levels of expression of pluripotency genes OCT-4 and SOX-2 before and after osteogenic differentiation of human mesenchymal stem cells (hMSCs).METHODS: Human MSCs were derived from the bone marrow and differentiated into osteoblasts. The analyses were performed on days 0 and 14 of the cell culture. In vitro differentiation was evaluated due to bone markers – alkaline phosphatase (AP) activity and the messenger RNA (mRNA) expression of AP and bone sialoprotein (BSP). The OCT-4 and SOX-2 expression was evaluated at mRNA level by real-time qPCR and at protein level by immunocytochemistry.RESULTS: In vitro cultures on day 14 showed an increase in AP activity and upregulation of AP and BSP gene expression. OCT-4 and SOX-2 in undifferentiated hMSCs on day 0 is detectable and very low compared to tumor cell lines as a positive control. Immunocytochemistry detected OCT-4 in the cell nuclei prior (day 0) and post differentiation (day 14). On the same time points, cultures were negative for SOX-2 protein.CONCLUSION: Messenger RNA for pluripotency markers OCT-4 and SOX-2 isolated from hMSCs was less present, while OCT-4 protein was detected in cell nuclei prior and post differentiation into osteoblast lineage.

52 citations

Journal Article•DOI•
Directed Assembly and Development of Material-Free Tissues with Complex Architectures

[...]

E.J. Vrij1, Jeroen Rouwkema2, Vanessa L.S. LaPointe1, Clemens van Blitterswijk1, Roman Truckenmüller1, Nicolas C. Rivron1 •
Maastricht University1, University of Twente2
01 Jun 2016-Advanced Materials
TL;DR: Material-free tissues are assembled using solely cells to form centimeter-scale tissues with precise architectures that contract autonomously, controllably shift shape, self-scaffold by secreting extracellular matrix, and undergo morphogenesis.
Abstract: Material-free tissues are assembled using solely cells. Microstructured hydrogel templates and high content screening allow the formation of centimeter-scale tissues with precise architectures. Similar to developing tissues, these contract autonomously, controllably shift shape, self-scaffold by secreting extracellular matrix, and undergo morphogenesis.

51 citations

Related Papers (5)
High-throughput fabrication of vascularized spheroids for bioprinting.

[...]

12 Jun 2018-Biofabrication
Lise De Moor, Idriz Merovci, Sarah Baetens, Julien Verstraeten, Paulina Kowalska, Dmitri V. Krysko, Winnok H. De Vos, Heidi Declercq 
Hybrid Bioprinting of Chondrogenically Induced Human Mesenchymal Stem Cell Spheroids.

[...]

25 May 2020-Frontiers in Bioengineering and Biotechnology
Lise De Moor, Sélina Fernandez, Chris Vercruysse, Liesbeth Tytgat, Mahtab Asadian, Nathalie De Geyter, Sandra Van Vlierberghe, Peter Dubruel, Heidi Declercq, Heidi Declercq 
Adult Stem Cells Spheroids to Optimize Cell Colonization in Scaffolds for Cartilage and Bone Tissue Engineering

[...]

25 Apr 2018-International Journal of Molecular Sciences
Leandra Santos Baptista, Gabriela S Kronemberger, Isis Côrtes, Letícia Emiliano Charelli, Renata Akemi Morais Matsui, Thiago Nunes Palhares, Jérôme Sohier, Alexandre Malta Rossi, José Mauro Granjeiro 
Synergistic interplay between human MSCs and HUVECs in 3D spheroids laden in collagen/fibrin hydrogels for bone tissue engineering

[...]

01 Sep 2019-Acta Biomaterialia
Dong Nyoung Heo, Monika Hospodiuk, Ibrahim T. Ozbolat
Bioprinting predifferentiated adipose-derived mesenchymal stem cell spheroids with methacrylated gelatin ink for adipose tissue engineering

[...]

23 Mar 2020-Journal of Materials Science: Materials in Medicine
Julien Colle, Phillip Blondeel, Axelle De Bruyne, Silke Bochar, Liesbeth Tytgat, Chris Vercruysse, Sandra Van Vlierberghe, Peter Dubruel, Heidi Declercq 
Frequently Asked Questions (1)
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.