Advancing the 3Rs in bone tissue engineering: emerging in vitro, in silico, and refined in vivo strategies

Tissue engineering is an interdisciplinary field integrating materials science, biology, chemistry, and immunology to develop regenerative solutions for complex tissues. Validation of tissue-engineered therapies, however, remains challenging—particularly for bone, whose hierarchical organization governs vascularization, mechanical loading, and continuous remodeling. Historically, these complexities have necessitated extensive reliance on in vivo animal models. Here we examine contemporary advances in modeling and validating bone tissue engineering strategies through the framework of the 3Rs principles: Replacement, Reduction, and Refinement. This review summarizes recent progress in advanced in vitro platforms, including organoids, bioprinting, and organ-on-a-chip systems, as well as in silico modeling approaches designed to decrease reliance on animal experimentation. We also highlight evolving best practices in experimental design, longitudinal imaging, welfare optimization, and reporting standards that improve the ethical and scientific conduct of necessary animal studies. Drawing primarily from literature published since 2020, this review provides an updated assessment of how the 3Rs are being operationalized within bone tissue engineering. Although significant technical and biological limitations remain, particularly in replicating vascularized, mechanically competent, and immunologically integrated bone tissue, the field demonstrates measurable progress toward Reducing animal use and improving translational relevance. Collectively, current developments reflect a realistic yet optimistic trajectory in which non-animal methodologies increasingly Replace in vivo work while also highlighting achievable short-term strategies to Refine necessary animal research.