Development of an in vitro 3D bone-muscle co-culture model using emulsion templated microporous constructs

Musculoskeletal (MSK) conditions are a leading cause of disability affecting ~22% of the global population. Current treatments provide limited relief, and progress is hindered by the poor clinical relevance of animal models. In vitro 3D MSK models can potentially offer biologically relevant, ethical, and cost-effective alternatives by mimicking native cell interactions, thereby improving our understanding of disease mechanisms and accelerating therapeutic innovation.
This study reports the development of in vitro 3D bone and skeletal muscle tissue models using poly(glycerol sebacate) methacrylate (PGSM), a biocompatible, photocurable, and elastomeric polymer with tunable mechanical properties. Microporous polyHIPE (high internal phase emulsion) scaffolds, PGSM-50 and PGSM-80-HA (nanohydroxyapatite functionalised), were fabricated via emulsion templating. Both scaffolds revealed interconnected porous architectures, with PGSM-50 and PGSM-80-HA exhibiting soft and stiffer mechanical properties, respectively.
Mouse myoblasts (C2C12) cultured on PGSM-50 showed efficient adhesion, growth, migration, and myogenic differentiation into multicellular myotubes. Under uniaxial cyclic stretching, these cells exhibited enhanced maturation, even in high-serum conditions, compared to static controls, demonstrating the importance of mechanical cues in regulating cell fate. These findings highlight PGSM-50 polyHIPEs' potential to function as physiologically relevant 3D muscle models responsive to mechanical cues.
3D bone microtissues were developed by culturing mouse osteoblasts (MLO-A5) on PGSM-80-HA and compared with PGSM-80 (non-HA) scaffolds. PGSM-80-HA promoted higher cell metabolic activity, upregulated osteocytic markers (E-11, SOST), and showed mineral deposition comparable to PGSM-80, demonstrating its osteoinductive potential, required for modelling bone microtissues to study osteogenesis and related disorders.
Finally, a co-culture study employing the developed bone and muscle constructs was performed to examine the influence of inter-tissue biochemical crosstalk on tissue maturation. Transwell inserts restricted physical contact between the microtissues while enabling communication. Shared medium composition strongly influenced tissue growth, phenotype, and crosstalk-mediated tissue functionality. These findings help progress the development of physiologically relevant MSK co-culture systems and identify biomolecular factors with therapeutic potential for aging-associated MSK disorders.