Objective:To compare in vivo versus in vitro fabrication of bone cement spacers in the treatment of bone defects by Masquelet technique. Methods:The data of 128 patients were analyzed retrospectively who had been treated for bone defects by Masquelet technique at Department of Orthopedics, Wuxi No. 9 People’s Hospital from January to August 2019. They were 74 males and 54 females, aged from 13 to 77 years. Their bone defects were traumatic in 54 cases and infectious in 74 cases. In 76 of them ( in vivo group), after a bone cement spacer was implanted into a bone defect during its dough phase, it was fabricated in vivo to form a cylindrical structure which was as large as or slightly larger than the defect size. In the other 52 cases ( in vitro group), before a bone cement spacer was implanted into a bone defect, it was fabricated in vivo during its dough phase into a cylindrical or block or bead chain or spherical form which was naturally solidificated at room temperature. The 2 groups were compared in terms of spacer filling time, bone healing time, delayed healing rate, infection control rate, spacer removal time, incidence of induced membrane or broken end bone lesion, as well as upper limb function evaluated by the Disability of the Arm, Shoulder and Hand Questionnaire (DASH) and the Paley lower limb grading at the last follow-up. Results:The 2 groups were comparable because there was no significant difference between them in gender, age, ratio of infected to non-infected cases, combined injuries, comorbidities or number of operations ( P>0.05). All the patients were followed up for 12 to 50 months (mean, 18.6 months). There were no significant differences between the 2 groups in spacer filling time, bone healing time, delayed healing rate, infection control rate or functional recovery for upper or lower limbs or for large or small bone defects (all P>0.05). In the in vivo group, for upper and lower limbs and for large and small bone defects respectively, the spacer removal time [(3.6±1.0) min, (4.1±1.1) min, (4.0±1.1) min and (3.9±1.0) min] and the incidence of induced membrane or broken end bone lesion [48.1%(13/27), 73.5%(36/49), 82.6%(39/46) and 66.7%(20/30)] were significantly longer or higher than those in the in vitro group [all (0.4±0.2) min; 3.2%(1/31), 9.5%(2/21), 0 (0/21) and 0 (0/31)] (all P<0.05). Conclusions:In the treatment of bone defects by Masquelet technique, in vivo and in vitro fabrication of bone cement spacers may lead to similar therapeutic effects. In vivo fabrication may be more suitable for lower limb, large or unstable bone defects but the spacer is not easy to remove and the induced membrane or bone ends are likely to get injured while in vitro fabrication may be more suitable for partial, small or upper limb defects because it may produce a variously shaped spacer.