Engineering three-dimensional bone macro-tissues by guided fusion of cell spheroids

Vinothini Prabhakaran; Ferry P W Melchels; Lyndsay M Murray; Jennifer Z Paxton

doi:10.3389/fendo.2023.1308604

Engineering three-dimensional bone macro-tissues by guided fusion of cell spheroids

Front Endocrinol (Lausanne). 2023 Dec 19:14:1308604. doi: 10.3389/fendo.2023.1308604. eCollection 2023.

Authors

Vinothini Prabhakaran^{1

2}, Ferry P W Melchels^{3

4}, Lyndsay M Murray^{1

2

5}, Jennifer Z Paxton^{1

2}

Affiliations

¹ Anatomy@Edinburgh, Edinburgh Medical School, Biomedical Sciences, University of Edinburgh, Edinburgh, United Kingdom.
² Centre for Discovery Brain Sciences, College of Medicine and Veterinary Medicine, University of Edinburgh, Edinburgh, United Kingdom.
³ School of Engineering and Physical Sciences, Institute of Biological Chemistry, Biophysics and Bioengineering, Heriot-Watt University, Edinburgh, United Kingdom.
⁴ Future Industries Institute, University of South Australia, Adelaide, SA, Australia.
⁵ Euan McDonald Centre for Motor Neuron Disease Research, University of Edinburgh, Edinburgh, United Kingdom.

Abstract

Introduction: Bioassembly techniques for the application of scaffold-free tissue engineering approaches have evolved in recent years toward producing larger tissue equivalents that structurally and functionally mimic native tissues. This study aims to upscale a 3-dimensional bone in-vitro model through bioassembly of differentiated rat osteoblast (dROb) spheroids with the potential to develop and mature into a bone macrotissue.

Methods: dROb spheroids in control and mineralization media at different seeding densities (1 × 10⁴, 5 × 10⁴, and 1 × 10⁵ cells) were assessed for cell proliferation and viability by trypan blue staining, for necrotic core by hematoxylin and eosin staining, and for extracellular calcium by Alizarin red and Von Kossa staining. Then, a novel approach was developed to bioassemble dROb spheroids in pillar array supports using a customized bioassembly system. Pillar array supports were custom-designed and printed using Formlabs Clear Resin^® by Formlabs Form2 printer. These supports were used as temporary frameworks for spheroid bioassembly until fusion occurred. Supports were then removed to allow scaffold-free growth and maturation of fused spheroids. Morphological and molecular analyses were performed to understand their structural and functional aspects.

Results: Spheroids of all seeding densities proliferated till day 14, and mineralization began with the cessation of proliferation. Necrotic core size increased over time with increased spheroid size. After the bioassembly of spheroids, the morphological assessment revealed the fusion of spheroids over time into a single macrotissue of more than 2.5 mm in size with mineral formation. Molecular assessment at different time points revealed osteogenic maturation based on the presence of osteocalcin, downregulation of Runx2 (p < 0.001), and upregulated alkaline phosphatase (p < 0.01).

Discussion: With the novel bioassembly approach used here, 3D bone macrotissues were successfully fabricated which mimicked physiological osteogenesis both morphologically and molecularly. This biofabrication approach has potential applications in bone tissue engineering, contributing to research related to osteoporosis and other recurrent bone ailments.

Keywords: bioassembly; bone; macrotissue; scaffold-free; spheroid; tissue engineering.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Bone and Bones*
Cells, Cultured
Osteogenesis
Rats
Spheroids, Cellular*
Tissue Engineering / methods

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This research work was supported by Tenovus Scotland (grant no. E22-05BG). VP’s PhD was funded by Principal’s career development scholarship and Edinburgh global research scholarship, University of Edinburgh.