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This work reports a bioinspired interlocking interface produced by electrostatic flocking. Such an interface can sustain cell growth, and the perpendicularly aligned flocked fibers can mitigate cell death caused by compression. In addition, the secreted extracellular matrix from seeded cells significantly enhances the mechanical properties of the interface.Drawing inspiration for biomaterials from biological systems has led to many biomedical innovations. One notable bioinspired device, Velcro, consists of two substrates with interlocking ability. Generating reversibly interlocking biomaterials is an area of investigation, as such devices can allow for modular tissue engineering, reversibly interlocking biomaterial interfaces, or friction?based coupling devices. Here, a biaxially interlocking interface generated using electrostatic flocking is reported. Two electrostatically flocked substrates are mechanically and reversibly interlocked with the ability to resist shearing and compression forces. An initial high?throughput screen of polyamide flock fibers with varying diameters and fiber lengths is conducted to elucidate the roles of different fiber parameters on scaffold mechanical properties. After determining the most desirable parameters via weight scoring, polylactic acid (PLA) fibers are used to emulate the ideal scaffold for in vitro use. PLA flocked scaffolds are populated with osteoblasts and interlocked. Interlocked flocked scaffolds improved cell survivorship under mechanical compression and sustained cell viability and proliferation. Additionally, the compression and shearing resistance of cell?seeded interlocking interfaces increased with increasing extracellular matrix deposition. The introduction of extracellular matrix?reinforced interlocking interfaces may serve as binders for modular tissue engineering, act as scaffolds for engineering tissue interfaces, or enable friction?based couplers for biomedical applications.