Dentin matrix protein (DMP-1), a phosphoprotein highly linked to dentin formation, has recently been reported to have an important role in skeletal development. Previously we reported that adult mice lacking the gene for DMP-1 exhibit the characteristics of chondrodysplasia, osteoarthritis, and showed severe defects in mineralization. DMP-1 knock-out (KO) mice display a profound defect in mineralization, and this is not due to a systemic defect in calcium/phosphate metabolism because serum levels of calcium and phosphate are similar to those in the wild-type mice. Although KO neonates and newborns appear normal, upon closer examination, these animals exhibit skeletal abnormalities, which include delayed secondary ossification and impaired bone remodelling. Heterozygous DMP-1 (H) mice however, show no apparent differences to the wild-type mice. In this study, biomechanical assessment tests of bones from DMP-1 KO mice were performed. Fifteen heterozygous, H, (DMP-1 +/−) and 15 KO, (DMP-1 −/−) male mice were produced and used in this study. At 1, 3 and 7.5 months of age, the mice were sacrificed and 4–5 ulnae from each animal group were harvested and stored in 70% ethanol solution. Volumetric density (BMD) measurements of the intact ulnae were performed using peripheral quantitative computed tomography (XCT960M; Stratec, Pforzheim, Germany) and Norland Stratec software version 5.10. One millimetre thick slices were scanned at a distance of 1 mm under the articular cartilage surface of the elbow as identified by the scout view of the CT scan. BMD of the corticalis and subcortical bone were recorded. Cross-sectional area measurements were also made at the mid-diaphysis of the ulnae. Biomechanical tests were performed in 3-point bending, with supports 3.5 mm apart at a rate of 3 mm/min (Lloyd Instruments Ltd, UK). The ultimate load, yield load and stiffness were determined from the load-displacement curves. All data were analysed using Mann-Whitney U tests (SPSS, Version 9, Chicago, Illinois). Differences were considered significant at p < 0.05. Density studies revealed that H mice had higher BMD than KO mice at all ages (p < 0.001). In the H and KO mice, the cortical BMD peaked at 3 and 7.5 months, respectively. At 1 month, the mean cross-sectional areas of the ulnae were larger in H mice compared to KO mice (0.50 mm2 Vs 0.33 mm2). However at 7.5 months of age, the reverse was observed (H = 0.75 mm2 and KO = 0.98 mm2). Biomechanically, stiffness increased with age at a higher rate in H mice than KO mice. Significant differences were observed at 3 months (p< 0.01) and 7.5 months (p< 0.05) between the two animal groups. There were no significant differences between stiffness values at 1 month. This study has demonstrated that DMP-1 deficiency leads to:

severely compromised bone mineralization;

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.