Recapitulating bone development events in a customised bioreactor through interplay of oxygen tension, medium pH, and systematic differentiation approaches

J Tissue Eng Regen Med. 2019 Sep;13(9):1672-1684. doi: 10.1002/term.2921. Epub 2019 Jul 15.


Bone development and homeostasis are intricate processes that require co-existence and dynamic interactions among multiple cell types. However, controlled dynamic niches that derive and support stable propagation of these cells from single stem cell source is not sustainable in conventional culturing vessels. In bioreactor cultures that support dynamic niches, the limited source and stability of growth factors are often a major limiting factor for long-term in vitro cultures. Hence, alternative growth factor-free differentiation approaches are designed and their efficacy to achieve different osteochondral cell types is investigated. Briefly, a dynamic niche is achieved by varying medium pH, oxygen tension (pO2 ) distribution in bioreactor, initiating chondrogenic differentiation with chondroitin sulphate A (CSA), and implementing systematic differentiation regimes. In this study, we demonstrated that CSA is a potent chondrogenic inducer, specifically in combination with acidic medium and low pO2 . Further, endochondral ossification is recapitulated through a systematic chondrogenic-osteogenic (ch-os) differentiation regime, and multiple osteochondral cell types are derived. Chondrogenic hypertrophy was also enhanced specifically in high pO2 regions. Consequently, mineralised constructs with higher structural integrity, volume, and tailored dimensions are achieved. In contrast, a continuous osteogenic differentiation regime (os-os) has derived compact and dense constructs, whereas a continuous chondrogenic differentiation regime (ch-ch) has attenuated construct mineralisation and impaired development. In conclusion, a growth factor-free differentiation approach is achieved through interplay of pO2 , medium pH, and systematic differentiation regimes. The controlled dynamic niches have recapitulated endochondral ossification and can potentially be exploited to derive larger bone constructs with near physiological properties.

Keywords: bone development; murine embryonic stem cells; nodules; perfusion bioreactor; stem cell differentiation; tissue engineering.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bioreactors*
  • Bone Development / drug effects*
  • Cell Aggregation / drug effects
  • Cell Differentiation / drug effects*
  • Cell Shape / drug effects
  • Culture Media / pharmacology*
  • Elastic Modulus
  • Gene Expression Regulation / drug effects
  • Hydrogen-Ion Concentration
  • Mice
  • Mouse Embryonic Stem Cells / cytology
  • Mouse Embryonic Stem Cells / drug effects
  • Mouse Embryonic Stem Cells / metabolism
  • Oxygen / pharmacology*
  • Perfusion
  • Spheroids, Cellular / cytology
  • Spheroids, Cellular / drug effects
  • Tissue Scaffolds / chemistry


  • Culture Media
  • Oxygen