For the treatment of ununited fractures, we developed a system of delivering magnetic labelled mesenchymal stromal cells (MSCs) using an extracorporeal magnetic device. In this study, we transplanted ferucarbotran-labelled and luciferase-positive bone marrow-derived MSCs into a non-healing femoral fracture rat model in the presence of a magnetic field. The biological fate of the transplanted MSCs was observed using luciferase-based bioluminescence imaging and we found that the number of MSC derived photons increased from day one to day three and thereafter decreased over time. The magnetic cell delivery system induced the accumulation of photons at the fracture site, while also retaining higher photon intensity from day three to week four. Furthermore, radiological and histological findings suggested improved callus formation and endochondral ossification. We therefore believe that this delivery system may be a promising option for bone regeneration.

Summary Statement. This study demonstrated that Sclerostin monoclonal antibody (Scl-Ab) enhanced bone healing in the rat osteotomy model. Scl-Ab increased callus size, callus bone volume fraction, rate of callus bone formation and fracture callus strength. Introduction. Sclerostin is a protein secreted by osteocytes and is characterized as a key inhibitor of osteoblast-mediated bone formation. Previous studies demonstrated that treatment with a sclerostin monoclonal antibody (Scl-Ab) results in significantly increased bone formation, bone mass and strength in rat closed fracture model (1–2). However, the effects of Scl-Ab on healing of open fracture model have not yet been reported in rats. Previously in ORS and ASBMR Annual Meeting, we have reported that Scl-Ab promoted the open fracture healing at week 3 and week 6 post-fracture. Here we extended our investigation for up to week 9 with additional histological assessments and dynamic histomorphometric analysis to investigate the effects of systemic administration of Scl-Ab on a later phase of fracture repair. Patients & Methods. Animal research ethics approval was obtained from our institute (reference No. 09/042/MIS), and the institute's guidelines for the care and use of laboratory animals were followed. In total, 120 six-month-old male SD rats were randomly divided into Scl-Ab group and vehicle group after a transverse osteotomy performed at the mid-shaft of right femur with internal fixation. One day post-surgery, rats were treated with a rodent Scl-Ab (Scl-Ab IV, s.c. injection, 25 mg/kg, 2 times per week) or vehicle for 3, 6 or 9 weeks. The progress of fracture healing for each animal was monitored weekly by digital radiography. Images acquired 3, 6 and 9 weeks post-operation were analyzed by ImageJ to quantify the total area of the fracture calluses. After euthanasia, femora were collected and subjected to the following analyses: micro-CT for bone mineral density (BMD) and callus volume fraction (BV/TV), micro-CT-based angiography for angiogenesis, histological evaluation and dynamic histomorphometry, and four-point mechanical testing for ultimate load, energy to failure and stiffness (3–6). Two-way ANOVA with Bonferroni post-hoc test was used to analyze the data. Significance level was set at P<0.05. Results. Radiographically, Scl-Ab treatment groups had significantly larger fracture calluses compared with respective vehicle group starting from week 3 post-fracture by quantitative analysis. Micro-CT analysis showed that Scl-Ab treatment groups had significantly higher callus bone volume fraction (+16–23%, P<0.01) and BMD (+15–16%, P<0.01) compared with respective vehicle groups at all time points post-fracture. Histological analysis also revealed more bone and less cartilage tissue in calluses in Scl-Ab group starting at week 3, which is explained by faster in the rate of new bone formation in fluorescence microscopy. Micro-CT based angiography demonstrated that Scl-Ab significantly enhanced neovasculation at the fracture calluses at week 3. Four-point bending test showed significantly higher ultimate load in Scl-Ab group than vehicle group at week 6 (+98%, P<0.01) and week 9 (+45%, P<0.05) post-fracture. In addition, ultimate load at week 6 of Scl-Ab group was at the similar level as seen at week 9 of the vehicle group, indicating the increased healing by Scl-Ab in this model. Stiffness (week 6 and 9) and energy to failure (week 6) were also tended higher in Scl-Ab group. Discussion/Conclusion. This study demonstrated that Scl-Ab enhanced bone healing in the rat osteotomy model. Scl-Ab increased callus size, callus bone volume fraction, rate of callus bone formation and fracture callus strength. Neovasculation was enhanced in the Scl-Ab group at week 3, implying Scl-Ab may enhance coupling of osteogenesis and angiogenesis. Scl-Ab treatment also resulted in more bone and less cartilage tissue in fracture calluses. Our results indicated that the systemic administration of Scl-Ab enhanced open fracture healing in rat femoral osteotomy model

We investigated the effect of locally administered bisphosphonate on distraction osteogenesis in a rabbit model and evaluated its systemic effect. An osteotomy on the right tibia followed by distraction for four weeks was performed on 47 immature rabbits. They were divided into seven equal groups, with each group receiving a different treatment regime. Saline and three types of dosage of alendronate (low, 0.75 μg/kg; mid, 7.5 μg/kg and high 75 μg/kg) were given by systemic injection in four groups, and saline and two dosages (low and mild) were delivered by local injection to the distraction gap in the remaining three groups. The injections were performed five times weekly during the period of distraction.

After nine weeks the animals were killed and image analysis and mechanical testing were performed on the distracted right tibiae and the left tibiae which served as a control group. The local low-dose alendronate group showed a mean increase in bone mineral density of 124.3 mg/cm³ over the local saline group (analysis of variance, p < 0.05) without any adverse effect on the left control tibiae.

The findings indicate that the administration of local low-dose alendronate could be an effective pharmacological means of improving bone formation in distraction osteogenesis.