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Development of a Novel Perfusion Rotating Wall Vessel Bioreactor with Ultrasound Stimulation for Mass-Production of Mineralized Tissue Constructs

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Tissue Engineering and Regenerative Medicine Aims and scope

Abstract

Background:

As stem cells are considered a promising cell source for tissue engineering, many culture strategies have been extensively studied to generate in vitro stem cell-based tissue constructs. However, most approaches using conventional tissue culture plates are limited by the lack of biological relevance in stem cell microenvironments required for neotissue formation. In this study, a novel perfusion rotating wall vessel (RWV) bioreactor was developed for mass-production of stem cell-based 3D tissue constructs.

Methods:

An automated RWV bioreactor was fabricated, which is capable of controlling continuous medium perfusion, highly efficient gas exchange with surrounding air, as well as low-intensity pulsed ultrasound (LIPUS) stimulation. Embryonic stem cells encapsulated in alginate/gelatin hydrogel were cultured in the osteogenic medium by using our bioreactor system. Cellular viability, growth kinetics, and osteogenesis/mineralization were thoroughly evaluated, and culture media were profiled at real time. The in vivo efficacy was examined by a rabbit cranial defect model.

Results:

Our bioreactor successfully maintained the optimal culture environments for stem cell proliferation, osteogenic differentiation, and mineralized tissue formation during the culture period. The mineralized tissue constructs produced by our bioreactor demonstrated higher void filling efficacy in the large bone defects compared to the group implanted with hydrogel beads only. In addition, the LIPUS modules mounted on our bioreactor successfully reached higher mineralization of the tissue constructs compared to the groups without LIPUS stimulation.

Conclusion:

This study suggests an effective biomanufacturing strategy for mass-production of implantable mineralized tissue constructs from stem cells that could be applicable to future clinical practice.

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Acknowledgements

This research was supported by Incheon National University (International Cooperative) Research Grant in 2019.

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Authors and Affiliations

  1. Department of Mechatronics Engineering, College of Engineering, Incheon National University, 119 Academy-ro, Yeonsu-gu, Incheon, 22012, Republic of Korea

    Jae Min Cha

  2. 3D Stem Cell Bioengineering Laboratory, Research Institute for Engineering and Technology, Incheon National University, Incheon, 22012, Republic of Korea

    Jae Min Cha

  3. Department of Maxillofacial Biomedical Engineering and Institute of Oral Biology, School of Dentistry, Kyung Hee University, Seoul, 02447, Republic of Korea

    Yu-Shik Hwang

  4. Department of Chemistry, Research Institute of Basic Sciences, Incheon National University, Incheon, 22012, Republic of Korea

    Dong-Ku Kang

  5. Wonkwang Bone Regeneration Research Institute, Wonkwang University, Iksan, 570-749, Republic of Korea

    Jun Lee

  6. Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, 30332, USA

    Elana S. Cooper & Athanasios Mantalaris

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  1. Jae Min Cha
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  2. Yu-Shik Hwang
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Contributions

Jae Min Cha, PhD and Athanasios Mantalaris, PhD: Administrative support, Conception and design, Manuscript writing, review & editing. Yu-Shik Hwang, PhD, Dong-Ku Kang, PhD, and Elana S Cooper, MSc: Manuscript writing, Data analysis and interpretation. Jun Lee, DDS, PhD: Collection and assembly of data.

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Correspondence to Jae Min Cha or Athanasios Mantalaris.

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No competing financial interests exist.

Ethical statement

The animal studies were performed after receiving approval of the Institutional Animal Care and Use Committee (IACUC) in Wonkwang University (IACUC approval No. WKU21-37).

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Supplementary Information

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13770_2022_447_MOESM1_ESM.tif

Supplementary Figure S1. Surgery procedures and biopsies of cranial vault at 1, 2, and 8 weeks after operation. After the central region was incised (A), four holes (diameter: 8 mm, depth: 2-3 mm) were generated as the standardized cranial defects (B). The defects were filled with osteogenic constructs generated from the BSEL bioreactor and alginate beads (about 10 beads each), respectively (C). Finally, the defects were covered with fibrin glue in order to fix all the grafts within the holes (D). The defected cranial vaults were isolated at 1, 2, and 8 weeks after the implant insertion, and photographed to visually examine the inflammation around the defected sites (E). (TIF 1454 KB)

13770_2022_447_MOESM2_ESM.tif

Supplementary Figure S2. (A-C) Customized-design of the BSEL bioreactor for mounting the LIPUS modules consisting of four ultrasound probes that were evenly placed to the front part of the culture chamber to ensure uniform transmission of ultrasound. The ultrasound probes were inserted to the culture vessel to ensure the maximum ultrasound transmitting level and attached on a bearing connected to the culture vessel to rotate simultaneously. In order to avoid the effect of ultrasound reflection, the back of the culture chamber was fitted with the ultrasound absorber material, HAM-A. (D) The controller is an analog box whereby the LIPUS intensity, PRF, duty ratio, and time can be adjusted. (E and F) The effective area was obtained from the number of pixels and the effective radius was calculated from the circle area equation, representing the ultrasound uniformity until reaching 20 mm in depth and then expanded without significant change of ultrasound intensity. (TIF 651 KB)

13770_2022_447_MOESM3_ESM.tif

Supplementary Figure S3. Results of a pilot animal testing with mineralized beads produced by the BSEL bioreactor. About ten mineralized beads were implanted in each cranial defect (diameter 8 mm × depth 2-3 mm). As a control, alginate beads without cells were implanted. At eight weeks, all implanted tissues were biopsied for 3D micro-CT scanning, H&E staining, and immunohistochemistry for osteocalcin. (A and B) 3D-reconstructed micro-CT scanning data of the bone defects implanted with mineralized beads and alginate beads, respectively. (C and D) Histological (I: H&E staining) and immunohistochemical (II: osteocalcin) data of the bone defects implanted with mineralized beads and alginate beads, respectively. (NB: new bone, FI: fibrous interzone, DM: dura mater, arrows: traces of vascularization). Scale bars represent 500 µm (×50) in H&E staining and 250 µm (×100) in immunohistochemical data. (TIF 1338 KB)

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Cha, J.M., Hwang, YS., Kang, DK. et al. Development of a Novel Perfusion Rotating Wall Vessel Bioreactor with Ultrasound Stimulation for Mass-Production of Mineralized Tissue Constructs. Tissue Eng Regen Med 19, 739–754 (2022). https://doi.org/10.1007/s13770-022-00447-3

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