Engineering an Enzymatically Active Granular Matrix for On‐Chip Modeling of Bone‐Like Mineralization

ABSTRACT Controlled biomineralization is central to engineering physiologically relevant hard‐tissue models, yet achieving spatially organized, three‐dimensional (3D) mineral deposition in microfluidic on‐chip systems remains challenging. While cell‐based bone‐on‐chip platforms offer biological complexity, they couple mineral initiation to matrix remodeling and paracrine signaling, complicating isolation of the earliest biochemical drivers of mineralization. Inspired by bottom‐up synthetic biology, we engineered an enzymatically active granular matrix that recapitulates a key osteogenic function within a perfusable 3D microenvironment. Alkaline phosphatase (ALP), the key driver of native bone formation, was covalently conjugated to poly(ethylene glycol)‐based (PEG) microgels via thiol–ene photochemistry, retaining over 90% of its initial activity after 24 h. These monodisperse microgels were assembled into a jammed, perfusable matrix on‐chip, enabling independent control over enzyme loading and substrate delivery. The system supported in situ mineralization (24–48 h), yielding a calcium‐deficient, poorly crystalline apatite‐like phase characteristic of early‐stage bone mineral. We demonstrate 3D localization of enzymatic activity to the granular matrix, which, coupled with interstitial perfusion, enabled mineral formation whose spatial distribution depended on the perfusion regime. This mechanistically defined, acellular platform provides a tunable foundation for investigating ALP‐driven 3D mineralization and establishes a modular route toward hybrid biosynthetic models of (patho)physiological tissue mineralization.