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

Abstract

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.

Why Animal Experiments Are Still Indispensable in Bone Research: A Statement by the European Calcified Tissue Society

“…For example, several research groups include hypertrophy and mineralization regimes in their in vitro cultures, (47,48) as well as altering pH and oxygen tension. (49,50) The highly mineralized nature of bony tissue, in addition to anatomic structure, means that plastic or other planar surfaces are a poor environmental proxy. Importantly, these cultures are unable to maintain the phenotype of osteocytes and osteoclasts for more than several weeks.…”