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Bone non-union after fracture, considered a therapeutic challenge for orthopedics, always needs a reversion surgery, including autograft transplantation (AGT). However, adverse events related to autograft harvest cannot be ignored. Our group designed a novel system called the bone marrow stem cell Screen-Enrich-Combine Circulating System (SECCS) by seeding mesenchymal stem cells (MSCs) into β-tricalcium phosphate (β-TCP) during surgery to thereafter rapidly process bioactive bone implantation. In this retrospective case-control study, 30 non-union patients who accepted SECCS therapy and 20 non-union patients who accepted AGT were enrolled. By SECCS therapy, the MSC-enriched β-TCP particles were implanted into the non-union gap. During the enrichment procedure, a significant proportion of MSCs were screened and enriched from bone marrow into porous β-TCP particles, and the cells possessed the capacity for three-line differentiation and were CD90 + /CD105 + /CD34 - /CD45 - . Approximately 82.0±10.7% of MSCs were enriched from 60 mL bone marrow without damaging cell viability, and approximately 11,444.0±6,018 MSCs were transplanted per patient. No implant-related infections occurred in any case. After 9 months of follow-up, 27 patients (90%) in the SECCS group acquired clinical union, compared with 18 patients (90%) in the AGT group (clinical union time, P = 0.064), and postoperative radiographic union score at 9 months post-operation was similar between the two groups. In conclusion, the SECCS could concentrate a large proportion of MSCs from bone marrow to acquire enough effective cells for therapy without in vitro cell culture. Bone substitutes processed by SECCS demonstrated encouraging promotion of bone regeneration and showed a satisfactory clinical curative effect for diaphyseal bone non-union, which was non-inferior to AGT.