Critical size bone defects deriving from large bone loss are an unmet clinical challenge1. To account for disadvantages with clinical treatments, researchers focus on designing biological substitutes, which mimic endogenous healing through osteogenic differentiation promotion. Some studies have however suggested that this notion fails to consider the full complexity of native bone with respect to the interplay between osteoclast and osteoblasts, thus leading to the regeneration of less functional tissue2. The objective of this research is to assess the ability of our laboratory's previously developed 6-Bromoindirubin-3’-Oxime (BIO) incorporated guanosine diphosphate crosslinked chitosan scaffold in promoting multilineage differentiation of myoblastic C2C12 cells and monocytes into osteoblasts and osteoclasts1, 3, 4. BIO addition has been previously demonstrated to promote osteogenic differentiation in cell cultures5, but implementation of a co-culture model here is expected to encourage crosstalk thus further supporting differentiation, as well as the secretion of regulatory molecules and cytokines2.

Biocompatibility testing of both cell types is performed using AlamarBlue for metabolic activity, and nucleic acid staining for distribution. Osteoblastic differentiation is assessed through quantification of ALP and osteopontin secretion, as well as osteocalcin and mineralization staining. Differentiation into osteoclasts is verified using SEM and TEM, qPCR, and TRAP staining.

Cellular viability of C2C12 cells and monocytes was maintained when cultured separately in scaffolds with and without BIO for 21 days. Both scaffold variations showed a characteristic increase in ALP secretion from day 1 to 7, indicating early differentiation but BIO-incorporated sponges yielded higher values compared to controls. SEM and TEM imaging confirmed initial aggregation and fusion of monocytes on the scaffold's surface, but BIO addition appeared to result in smoother cell surfaces indicating a change in morphology. Late-stage differentiation assessment and co-culture work in the scaffold are ongoing, but initial results show promise in the material's ability to support multilineage differentiation.

Acknowledgements: The authors would like to acknowledge the financial support of the Collaborative Health Research Program (CHRP) through CIHR and NSERC, as well as Canada Research Chair – Tier 1 in Regenerative Medicine and Nanomedicine, and the FRQ-S.

Dynamization of fracture fixation is used clinically to improve the bone healing process. This study evaluated the effect of late dynamization on callus stiffness and size in a rat diaphyseal femoral osteotomy. The external unilateral fixator was dynamized by removal of the inner fixator bar, at three weeks (D3-group: n=8) or four weeks (D4-group: n=9) post-operation. Published data of a five week rigid (R-group: n=8) and flexible fixation group (F-group: n=8) were included for comparison. Preoperative and postoperative movements of the rats were measured using a motion detection system. After 5 weeks the rats were sacrificed and healing was evaluated by biomechanical and densitometric methods. By 34 days post-operation, rats from the four fixation groups had similar activity levels. There was no significant difference in flexural rigidity, callus volume or callus mineral density between the D3 and D4-groups. Both the D3-group and D4-group had significantly greater flexural rigidity (p< 0.01) and significantly lower callus total volume (p< 0.03) and callus bone volume (p< 0.03) compared to the F-group. There was no significant difference in flexural rigidity or callus mineral density between the dynamized groups compared to the R-group. However, the D3-group had less callus bone volume (p=0.06) compared to the R-group. The D4-group had significantly less callus bone volume (p=0.02) and less callus total volume (p=0.05) compared to the R-group. Late dynamization led to a stiffer callus with a smaller callus volume compared to continuously flexible fixation. The late dynamized groups had less callus volume than the continuously rigid group, but the stiffness and calcification and of the callus were similar. The late dynamized groups had undergone resorption processes, indicative of more advanced healing. Late dynamization enhanced fracture healing compared to the continuously rigid or flexible fixation.

Introduction: Dynamization is used to improve the healing process. The optimal time for dynamization however remains unknown. In this study we proved the hypothesis that an early dynamization will improve the fracture healing.

Material and Methods: Twenty-four rats underwent a diaphyseal femoral osteotomy, with a 1mm gap. The osteotomy was stabilized by either rigid (R-group; n=8) or flexible (F-group; n=8) external fixation. The dynamized group (D-group: n=8) had a rigid fixation for 1 week, and then a flexible fixation for the remaining 4 weeks. The flexible fixation design resulted in an axial stiffness of 10N/mm and the rigid fixation in 74N/mm. After 5 weeks, healing was evaluated by biomechanical, densitometric, and histological methods.

Results: The flexural rigidity was 47% higher in the R-group than in the F-group (p< 0.01). Also, the flexural rigidity was 45% higher in the R-group than in the D-group (p< 0.01) (Table 1). Mineralized callus tissue volume was 37% lower in the R-group than the D-group (p=0.002).

Conclusion: The hypothesis could not be supported, in that early dynamization did not improve healing compared to rigid or flexible fixation. The rigid fixation had a stiffer callus with smaller callus volume, and more calcified tissue in the whole callus. The rigid fixation had bridging in the gap more often, which explains the increased flexural rigidity measured. Dynamization utilized in previous studies allowed closure of the fracture gap and thereby enhanced the rate of healing, which was not the case in the present investigation.

The influence of controlled mechanical loading on osseointegration was investigated using an in vivo device implanted in the distal lateral femur of five male rabbits. Compressive loads (1 MPa, 1 Hz, 50 cycles/day, 4 weeks) were applied to a porous coated titanium cylindrical implant (5mm diameter, 2mm width, 75% porosity, 350ìm average pore diameter) and the underlying cancellous bone.. The contralateral limb served as an unloaded control. MicroCT scans at 28 μm resolution were taken of a 4 × 4mm cylindrical region of interest that included cancellous bone below the implant. A scanning electron microscope with a backscattered electron (BSE) detector was used to quantify the percent bone ingrowth and periprosthetic bone in undecalcified sections through the same region of interest. A mixed effects model was used to account for the correlation of the outcome measures within rabbits.. The percent bone ingrowth was significantly greater in the loaded limb (19 +/− 4%) compared to the unloaded control limb (16 +/− 4%, p=0.016) as measured by BSE imaging. The underlying cancellous periprosthetic tissue bone volume fraction was not different between the loaded (0.26 +/− 0.06) and unloaded control limb (0.27 +/− 0.07, p=0.81) by microCT. BSE imaging also showed no difference in the percent area of periprosthetic bone (27 +/− 10% loaded vs. 23 +/− 10% unloaded, p=0.25). Cyclic mechanical loading significantly enhanced bone ingrowth into a titanium porous coated surface compared to the unloaded controls.