Asian Journal of Dental Sciences

The evolution from conventional monolayer cell culture to sophisticated biomimetic multilayer constructs represents one of the most consequential paradigms shifts in contemporary biomedical engineering and regenerative medicine. Monolayer systems, whilst foundational to cell biology research, are increasingly recognised as inadequate surrogates for native tissue architecture, owing to their inability to recapitulate the three-dimensional, compositionally graded, and mechanically heterogeneous environments that govern cellular behaviour in vivo. The multilayer injection concept emerges as a transformative strategy that addresses these limitations by enabling the sequential or simultaneous deposition of compositionally distinct, bioactive material layers via injection-based platforms, thereby recreating the stratified complexity inherent to biological tissues. This review examines the conceptual progression from monolayer paradigms to biomimetic multilayer injectable architectures, exploring the biological rationale, material platforms, crosslinking strategies, and gradient engineering principles that underpin this approach. Applications spanning osteochondral tissue engineering, vascular repair, neural regeneration, and dermal reconstruction are critically evaluated, alongside an assessment of prevailing challenges related to vascularisation, mechanical integration, scalability, and clinical translation. The multilayer injection concept is contextualised within the broader trajectory of biofabrication, and future research priorities are identified to guide the field towards clinically viable biomimetic constructs capable of recapitulating not merely the gross composition but the full spatial and functional complexity of native tissues.