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

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

Research output: Contribution to journalArticlepeer-review

Abstract / Description of output

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.
Original languageEnglish
Pages (from-to)1-13
JournalFrontiers in Endocrinology
Publication statusPublished - 19 Dec 2023

Keywords / Materials (for Non-textual outputs)

  • bone
  • scaffold-free
  • bioassembly
  • spheroid
  • macrotissue
  • tissue engineering


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