Objectives: Technologies on the spatial-temporal control of soluble growth factors such as bone morphogenetic protein 2(BMP-2)are essential for stem cell niche studies.Multi-photon-based biofabrication is an emerging technology able to improve the spatial resolution of conventional fabrication techniques by precisely fabricating protein structures utilizing femto-second lasers.In the present study,we aim to spatially control the presentation of BMP-2 by fabricating pre-designed BMP-2 patterns on protein structures via two-photon excited immobilization technique.Methods: The protein matrixes with dimension of 101μm × 101μm × 5μm were firstly fabricated by photo-crosslinking Bovine Serum Albumin(BSA)(300 mg/ml)in the presence of photosensitizer,Rose Bengal(RB)(0.1%,w/v),which was activated by femto-second laser with output power of 188 mW at wavelength of 800 nm.Then the protein matrixes were rinsed with phosphate buffer saline(PBS)followed by reloading with a mixture of recombinant human BMP-2(rhBMP-2)(5 μg/ml)and RB(0.1%,w/v).The photo-crosslinking of BMP-2 patterns with dimension of 20 μm × 20 μm × 2 μm were exerted on the BSA matrixes with different laser powers and scan cycles.Afterwards,the efficacy of BMP-2 photo-crosslinking was verified by immunofluorescence staining.Results: BMP-2 can be immobilized by two-photon laser in the presence of Rose Bengal in a dose-dependent manner of laser power and scan cycle.The range of laser power required for fabrication of visible BMP-2 patterns is from 20.6 mW to 62.0 mW and the according scan cycle is 1 and 5,respectively.Conclusions: Our current study provides a novel platform for spatial control the presentation of growth factor such as BMP-2.Investigation on the bioactivity of cross-linked BMP-2 is underway.