Project Summary/Abstract Nonunion fracture is a permanently broken bone that does not spontaneously heal and can occur in any bone tissue, causing severe pain and physical disability to patient. However, until now, there is no effective treatment for nonunion fracture and its pathological mechanism remains unidentified. To treat nonunion fracture, bone graft materials are often implanted into the damaged region to stimulate bone regeneration and to support bone structure. Among various bone implant materials, hydroxyapatite (HAP: Ca10(PO4)6(OH)2) and ?-tricalcium phosphate (?-TCP: Ca3(PO4)2) are the most widely used biomaterials due to excellent biocompatibility and bioresorbability, respectively. However, ?-TCP does not exist in our bone while it has microscale particle. In this respect, the current bone implant material, composed of HAP and ?-TCP, does not reflect composition and structure of bone at nanoscale and thus has a gap with natural bone tissue, that it is eventually purposed to be replaced by new bone. Therefore, to make a breakthrough in bone implant material, we propose to recreate an innovative bone implant material which can immediately harmonize with surrounding bone tissue after implantation, by utilizing the two major bone minerals, whitlockite (WH: Ca18Mg2(HPO4)2(PO4)12) and HAP. WH is the second most abundant crystal in bone and also known to exist with high ratio in younger aged and early stage of bone tissue formation. However, despite its significant distributions in bone, WH has been largely ignored from the research fields due to difficulty in its synthesis and analyses. Very recently, a facile synthetic method of WH nanoparticles has been reported and its beneficial effects on cellular proliferation and activities has been demonstrated. In this research, we aim to recreate bone-mimetic implant platform by controlling and optimizing the composition and the structure of the two major bone minerals in 3D hydrogel platform. We hypothesized that the two-phase WH/HAP composite will provide synergetic effects of material-, mechanical-, and biological properties on cellular proliferation and differentiation. This hypothesis will be verified by the following specific aims: 1) Assess the differentiation of MSCs into osteogenic cells in nanocomposite gels containing various ratios of WH and HAP nanoparticles; 2) Control the spatial architecture of MSC-laden WH/HAP nanocomposite gels by 3D printing; and 3) Assess the bone formation ability of WH/HAP nanocomposite gels in vivo. We expect that the new findings from the proposed research will provide better understandings of bone tissue and effective treatment for nonunion fractures.