The potential importance of bone morphogenic proteins (BMPs) to improve fracture healing is of great interest to orthopaedic surgeons. Although the complex mechanisms leading from the presence of local BMP (either endogenous or exogenous) to form bone is increasingly understood, however most appropriate time to administer exogenous BMP has yet to be elucidated. The purpose of this study was to investigate when BMP may be administered to a fracture arena in order to best improve fracture healing. Forty mice were randomised into 4 groups; (group I) control, treated at day 0 with placebo; (groups II, III and IV) treated with BMP at days 0, 4 and 8, respectively. All animals underwent a previously validated surgical procedure involving the creation of an open femoral fracture which is stabilised using a 4 pin external fixator. Thirty microlitres of bovine serum albumin (BSA) alone was used in group I, and the other groups (II, III and IV) were treated with a combination of the BSA and 2.5 microgrames of rhBMP-2. The BSA and rhBMP were injected through a lateral approach immediately after operation, or at 4, or 8 days postoperatively. At days 0, 8, 16 and 22, sequential radiographs were taken using a digital x-ray machine and at day 22 all animals were sacrificed. Both femora were harvested and assessed biomechanically in 3-point bending prior to fixation for histological evaluation. All data were analysed using Mann-Whitney U tests (SPSS, Version 9, Chicago, Illinois) and differences were considered significant at p < 0.05. X-ray analysis indicated that healing of fractures treated with BMP at day 0(group II) or day 4(group III) was significantly greater than that at both days 16 and 22 (p < 0.05) than those animals in placebo (group I) and BMP day 8(group V) treatment groups. Although the administration of BMP at day 4 seemed to cause more bone formation than treatment at day 0, no significant difference were observed. There were no differences between group IV and group I. Biomechanically, group III exhibited ultimate load values closest to the contralateral unoperated femora followed by group II, then IV and finally the control group I. Significant differences (p < 0.05) were observed between the control animals (group I) and both groups II and III. Qualitative histology suggested that at 22 days after surgery, only groups II and III had healed with woven bone. Group I and group IV had considerable amounts of fibrous tissue and cartilage at the fracture gap. This study suggests that a single percutaneous injection of BMP has a positive effect on fracture healing in this model, when prescribed between the time of injury (day 0) and 4 days. Data suggests that the most effective timing of delivery of BMP may not be at the time of surgery but actually in the early healing phase. The day 4 time point in the mouse model is likely to equate to that of 7–10 days in larger animals or humans. This suggests that current human treatment practices may require further investigation in order to elucidate the most appropriate time of delivery for these important proteins. This work may negate the current requirements for carrier products and large doses of these expensive drugs.

Introduction: The potential of Bone Morphogenic Protein (BMP) to improve fracture healing is of great interest to orthopaedic surgeons. Although the complex mechanisms leading from the presence of local BMP to the fracture scenario has yet to be elucidated. The purpose of this study was to investigate whether introducing rhBMP-2 to the fracture arena, some days after fixation could be more beneficial to fracture repair.

Methods: 40 animals were randomised into 4 groups; namely control treatment at day 0 or rhBMP-2 treatment at days 0, 4 or 8 post-surgery. All animals underwent a previously validated surgical procedure involving creation of an open femoral fracture fixed using a 4-pin external fixator. 30 μl of bovine serum albumin (BSA) alone (control) or mixed with 2.5 μg of rhBMP-2 (treatments) was injected via a lateral approach directly into the fracture gap, either following closure of the wounds (day 0) or at 4 or 8 days postoperatively. Animals were assessed as to the outcome of surgery by digital sequential x-ray at days 0, 8, 16 and 22 using a Faxitron MX-20 camera; and y either biomechanical testing under a 3-point bending technique (Lloyd Instruments Ltd, UK) or histological examination following sacrifice at day 22. Data were analysed using Mann-Whitney U and Wilcoxon Tests for statistical differences (SPSS, Version 9). Differences were considered significant when p< 0.05.

Results: X-ray analysis indicated that healing of fractures treated with rhBMP-2 at day 0 or day 4 was significantly greater than the two other groups at days 16 and 22. BMP given at day 4 tended to a greater effect than when given at day 0, though the range was too great to show a statistical difference. There were no differences between the BMP-8 and the BSA control groups. Mechanical testing showed that only animals that had received rhBMP-2 at day 4 had attained similar peak loads to failure to those of their contralateral unoperated leg. Bones from animals receiving rhBMP-2 at day 0 had attained the next greatest strength, which was followed by rhBMP-2 administration at day 8 animals, whereas the animals receiving BSA attaining the least strength. There was a statistical difference (p< 0.05) between both rhBMP-2 day 4 and day 0 groups compared to the BSA control group. Qualitative histology suggested that the rhBMP-2 day 0 and day 4 groups had almost fully healed with new bone whereas the BSA and rhBMP-2 day 8 groups still had considerable mounts of fibrous tissue and cartilage at the fracture gap 22 days following surgery.

Conclusions: The study demonstrates that a single percutaneous injection of rhBMP-2 has a positive effect on fracture healing, when prescribed at the time of injury or during the early period of fracture repair. Data suggests that the most effective timing of delivery of BMP may not be at the time of surgery but in the early healing phase. The day 4 time point in the mouse model is likely to equate to that of 7–10 days in larger animals or humans. Further investigation as to the most appropriate time for intervention using these proteins is warranted and may negate the current requirement for carried products and large doses of these expensive drugs.

The thrombin-related peptide, TP508, is a synthetic 23 amino acid peptide, which represents the receptor binding domain of thrombin. TP508 mimics thrombin by interacting with receptors on cells involved in tissue repair. TP508 has been shown to enhance revascularization of injured tissue, and promote soft tissue wound healing, cartilage repair, and fracture repair. The aim of this study is to (1) test the effect of TP508 on bone regeneration during distraction osteogenesis; (2) study the chemotactic effect of TP508 on human osteoblasts.

Unilateral tibial osteoectomies were performed and stabilized with MX100 Orthofix lengthener in 5 male adult NZW rabbits. After 7 days, distraction was initiated at rates of 1.4 mm / day for 6 days. TP508 (100 μg/ml, n=2; 10 μg/ml, n=1) or saline (300 μl, n=2) was injected into the osteotomy / lengthening gap at days 1, 7 and 14 post surgery. Animals were sacrificed at 2 weeks after leg lengthening. Bone formation in the regenerate was assessed by radiography, quantitative computed tomography (pQCT) and histology. For chemotaxis studies, MG63 cells were cultured on glass cover slips for three days, and then inverted onto a Dunn chamber slide and sealed with dental wax. Gradients of TP508 (1, 10, 100 μg/ml) were added to the outer well and plain medium to the inner well. A sequence of images of the cells between the wells was taken via a CCD camera for 9 hours at interval of 10 minutes. Movements of individual cells were tracked and statistically analysed by a specially written Macro program. The Rayleigh test for unimodal clustering was used to determine the directional chemotactic movements.

The radiographic evaluation indicated a significant increase in new bone in the distraction regenerate in the TP508 treated groups at 1 and 2 weeks. pQCT images at 2 weeks demonstrated more advanced bone formation in the TP508 treated animals compared to the control. The mean total bone mineral density (BMD) of the regenerate, obtained from 3 slices was significantly greater (p = 0.019, t-test) in the TP508 treated group (BMD = 479.20 +/− 35.57 mg/ccm) than that in the saline control group (BMD = 355 +/− 2.83 mg/ccm). The histological evaluation supported the radiographic and the pQCT results. For chemotaxis study, no directional movements of the cells were found in the controls, whereas the MG63 cells were strongly chemotactic to TP508 at 1, 10 and 100 μg/ml concentrations.

This preliminary study shows that administration of TP508 enhances bone formation during distraction osteogenesis in the rabbit. The findings also show that TP508 has a chemotactic effect on osteoblasts, consistent with the effect of TP508 on fracture repair. A large animal study is in the process to confirm these findings and explore the underlying mechanisms.