Background & Objectives: Osteoporosis is one of the most prevalent bone diseases worldwide with fractures its major clinical consequence. Studies on the effect of osteoporosis on fracture repair are contradictory and although it might be expected for fracture repair to be delayed in osteoporotic individuals, a definitive answer still eludes us. Subsequently, the aim of this study was to attempt to clarify any such effect.

Methods: Osteoporosis was induced in 53 female Sprague-Dawley rats by ovariectomy (OVX) at 3 months. A femoral fracture was produced in these animals 12 weeks later {OVX+Fracture group (OVX+F)}. A control group received the fracture only group (F) at 6 months. The fracture consisted of an open osteotomy held with a unilateral external fixator. Outcome measures include histology, motion detector analysis, pQCT, biomechanical strength testing (BST) and digital radiography. Digital radiographs were taken at time of OVX, fracture (confirming satisfactory reduction) and sacrifice from which relative bone density (BMD) measurements were calculated.

Results: OVX+F animals were significantly heavier than F animals at fracture and sacrifice (p< 0.001 for both) and moved significantly less in days 1-4 (p=0.032) and 5-9 (p=0.020) post-fracture. Relative BMD measured in distal femur at fracture and sacrifice was significantly greater in F group (p< 0.001 for both). Furthermore, there was a significant decrease in relative BMD from fracture to sacrifice in OVX+F group (p< 0.001). pQCT showed a significantly greater total BMD {contralateral (p=0.021) and fractured femora (p< 0.001)} and trabecular BMD (p< 0.001 both limbs) in the distal femur of the F group. Histologically, no statistical differences were found, however, the F group generally displayed the most advanced repair. In the contralateral limb, the F group had significantly greater load to failure at 6 (p=0.026) and 8 (p=0.042) weeks and was significantly stiffer at 8 weeks (p=0.050). In the fractured leg, stiffness was significantly greater in the F group at 8 weeks (p=0.001).

Conclusion: OVX was linked to increased body weight, decreased motion, decreased BMD (with particular loss in trabecular BMD), and reduced mechanical properties. OVX did not have a significant effect on fracture healing and although there was no reduction in BMD at the fracture site, histology and reduced stiffness suggest it was delayed.

Background & Objectives: The objective of this study was to develop a rat model of fracture repair. Fixation of experimental fractures is generally internal {Kirschner wire/intramedullary (IM) nail} or external (single/double plane devices). Internal fixation using the IM-fixated model of a standard closed fracture is well described in rats. However, nail insertion can disrupt fracture site morphology and limit x-ray analysis. We planned to create an externally fixated femoral model, to optimise our outcome measures and facilitate the further investigation of bone healing within the department.

Methods: A simple four pin unilateral external fixator was designed and constructed from four stainless steel pins, secured to a stainless steel plate with nuts. Forty-one female Sprague-Dawley rats, (12–18wks), were used. Following anaesthesia the right femur was exposed and a mid-femoral osteotomy made prior to fixator application. Post-operative x-rays were taken to confirm reduction. Animals were assigned to groups for biomechanical strength testing (BST) or histology. Fifteen animals (fractured and contralateral limbs) were sacrificed at 4, 6 or 8 weeks for BST (four-point bending). Maximum load to failure was recorded and stiffness calculated from the load displacement curve obtained. Both parameters were standardised as a percentage of the contralateral limb. Twenty-five fractured limbs were used for histological analysis at day 4, and 1, 2, 4, 6 or 8 weeks.

Results: Satisfactory reduction was confirmed in all animals post operatively and no complications were noted. Histological assessment at day 4 demonstrated a predominantly lymphocytic inflammatory response within the fracture haematoma. This was replaced with endosteal and periosteal new bone between weeks 1 and 2. Bridging of the fracture gap was seen at week 6. Stiffness and load to failure increased with increasing time. There was a statistically significant improvement in the percentage stiffness (p=0.035) and load to failure (p=0.012) between 4 and 8 weeks.

Conclusion: A simple reproducible externally fixated rat model has been established and characterised by radiography, histology and four point bending. This model has since proven to be of value in the study of the role of lipid lowering and anti-inflammatory drugs as well as cell therapy on fracture repair.

Background & Objectives: Statins have been shown to stimulate bone formation in vivo and in vitro in rodent models1 generating interest in the possibility that they may be useful therapeutic agents for osteoporosis. The major clinical consequence of osteoporosis are fractures that occur and although there is no firm evidence, there is a perceived associated delay in fracture repair. We examined the influence of atorvastatin on fracture repair in an ovariectomised rat fracture model.

Methods: 126 Sprague-Dawley rats had an ovariectomy (OVX) at three months and a femoral fracture (F) at six months. The fracture consisted of an open osteotomy held with an external fixator. All animals were randomly assigned into groups 1. OVX+F and early atorvastatin; 2. OVX+F and late atorvastatin; 3. OVX+F. Atorvas-tatin (5mg/kg) was given daily by oral gavage for three months in-group 1 between OVX and fracture and from time of fracture to sacrifice in-group 2. Outcome measures were histology, peripheral quantitative computed tomography (pQCT), biomechanical strength testing (BST) and digital radiography. Digital radiographs were taken at time of OVX, fracture (confirming satisfactory reduction) and sacrifice from which relative bone density (BMD) measurements were calculated.

Results: Non-statin treated animals moved significantly more in 4 days post-fracture (p=0.015), had signifi-cantly more relative (p=0.037) and total BMD (distal femur) than statin treated (p=0.040, early and p=0.036, late treatment). Total BMD at the fracture site was also significantly greater in the OVX+F than the late statin group (p=0.047) while in the adjacent site of the con-tralateral limb, the early statin group had significantly more (p=0.018) than the late statin group. However no differences were found between the early statin and OVX+F groups. Histologically, the rate of repair increased significantly in early statin (p=0.013) and OVX+F (p=0.011) groups. BST data showed no signifi-cant difference in stiffness at six or eight weeks.

Conclusion: Fractures healed in all three groups. Statins did not prevent OVX induced bone loss. Initial evidence suggests that early statin treatment may have a positive effect on early fracture, as shown by x-ray analysis and histology, however this effect was lost by week 8. Overall the evidence suggests that atorvastatin may have impaired fracture repair, particularly with late administration (relative BMD and pQCT results).

Aims An estimated 5–10% of fractures fail to heal adequately. Novel therapies in the treatment of problem fractures include the use of culture expanded cells. An animal model of delayed fracture union is required to parallel the clinical scenario so that variations in cell therapy techniques can be rapidly assessed.

Material and Methods A simple unilateral external fixator was designed for use in the rat. The fixator was applied following open osteotomy of the femur and a reproducible externally fixated femoral fracture model was established (n=41). Fracture union was assessed by digital radiography, histology and biomechanical strength testing (four point bending) at weeks 4, 6 and 8. Histological examination was also undertaken at day 4 and weeks 1 and 2. A delayed union in the fracture model was created by periosteal and endosteal stripping (n=14). Radiography and biomechanical strength testing were performed at week 8. The use of cell therapy was tested in the delayed union model. Osteogenic cells were culture expanded for 6 weeks before re-implantation. Reimplantation was facilitated by the use of a drill hole through the fracture site . Animals were randomized to one of three groups – i) drill hole & cells in a carrier ii) drill hole & carrier only iii) no drill hole, cells or carrier.

Results In the fracture model radiological and histological evidence of fracture union was apparent at week 6. Biomechanical testing showed a significant difference in load to failure and stiffness of the fracture between weeks 4 and 8 (p=0.009 and 0.008 respectively). There was also a significant difference in biomechanical properties between the fracture model and the delayed union model at week 8. Drilling with the injection of a carrier significantly improved the biomechanical properties (p=0.03) of a delayed union at week 14. Surprisingly this effect was negated by the introduction of cells.

Conclusion A fracture and delayed union model in the rat has been established for the testing of cell therapy. The application of cell therapy to a delayed union has been less advantageous in improving union than expected. This prompts the need for further work required in optimising cell culture techniques and cell delivery.

Introduction: The aim of this research project was to establish a simple, reliable and repeatable externally fixed femoral fracture model. The rat was selected, as it was a suitable animal for use in a model of fracture repair and ovariectomy induced osteoporosis, both of which were to be investigated in future experiments. There are femoral fracture models described in the literature based on the insertion of an intramedullary nail prior to inducing a fracture. We felt, based on our experience of the unilateral externally fixed mouse fracture model, that external fixation would allow us to carry out radiographical and histological analysis of fracture healing without any of the tissue trauma caused by the insertion and removal of the intramedullary device.

Materials and Methods: A unilateral external fixator was chosen due to its simplicity. Four threaded stainless steel pins pass through holes in an aluminium plate with nuts placed on the pin above and below the plate. The holes in the plate were 0.1mm bigger than the pins and unthreaded allowing the plate to slide freely over the pins. Tightening of the upper nut compressed the plate against the lower nut holding the pin securely. 41 female Sprague-Dawley rats, aged between 12 and 18 weeks, were used. They were anaesthetised using a standard mixture of hypnorm and midazolam and analgesia, fluids and antibiotic were administered subcutaneously prior to surgery. The femur was exposed through a lateral approach and a standardised osteotomy was made prior to the application of the fixator plate. Accurate reduction was confirmed visually at the time of surgery and also by way of a post-op x-ray. 25 animals were sacrificed at 4 days and 1, 2, 4, 6 and 8 weeks for histology. The fractured limbs were harvested, fixed, decalcified and paraffin embedded as per standard protocol and serial sections were cut. These were stained with H& E and alcian blue and analysed 15 animals were sacrificed at 4,6 or 8 weeks for biomechanical strength testing. Four-point bending was carried out on freshly harvested femurs stored in normal saline between harvest and testing. Both limbs were tested and the fractured limbs were standardised relative to the unfractured limb. Maximum load to failure was recorded and stiffness was calculated from the load-displacement curve.

Results: No post-operative complications of fixation failure or infection occured. On histological assessment at D4 a predominantly lymphocytic inflammatory response was seen within the fracture haematoma. This inflammatory response was replaced with endosteal and periosteal new bone between wks 1 and 2. Bridging of the fracture gap was seen at week 6. Both stiffness and load to failure increased with increasing time. There was a statistically significant improvement in the percentage stiffness and percentage load to failure between 4 and 8 weeks (p=0.03 and p=0.018 respectively). The difference in load to failure between 6 and 8 weeks was also significantly different (p=0.042).

Discussion: A simple, reliable and repeatable externally fixed rat femoral fracture model has been established.

Introduction: During the development of an externally fixated femoral fracture model in the rat a single dose of Carpofen (Rimadyl) was administered as part of the pre-operative analgesia regime. The negative effect of a NSAID on fracture repair has been well documented.

Materials and Methods: The external fixator was designed and constructed from threaded stainless steel pins and a semi-cylindrical aluminum plate. The pins were passed through the four drill holes made in the plate and were secured by nuts above and below the plate. Forty-five female Sprague-Dawley rats, aged between twelve and eighteen weeks, were used in the model. Twenty-one animals received a single subcutaneous dose of Carpofen (4mg/kg) pre-operatively. Carpofen was then excluded from the pre-operative analgesia regime and the experiment was repeated. All animals received a dose of Buprenorphine hydrochloride (Temgesic, 0.03mg/kg) and a fluid bolus (40–80ml/ kg) both pre and post operatively and antibiotic pre-operatively. Femoral fractures were created after the animals had been anaesthetised. The right femur was then exposed and a mid femoral osteotomy was made prior to the application of the fixator. Post-operative digital x-rays were taken to confirm reduction. A minimum of four animals were assigned to a group for either biomechanical strength testing or histology. Thirty-one animals in total were sacrificed at 4, 6 or 8 weeks for biomechanical strength testing. The fractured limbs were freshly dissected and stored in saline prior to testing. Both the fractured and contralateral limbs were tested mechanically by four point bending. The maximum load to failure was recorded and stiffness was calculated from the load displacement curve obtained. The bending strength of each fractured femur was expressed as a percentage of the strength of the intact contralateral femur. Fourteen fractured limbs were fixed in formaldehyde, decalcified and paraffin embedded for histological analysis. Serial sections were cut and stained with haematoxylin, eosin and Alcin blue at 4, 6 or 8 weeks.

Results: Satisfactory reduction of the fracture was confirmed post-operatively by faxtitron x-ray imaging in all animals. Preliminary data showed that there was a significant difference in stiffness at 8 weeks between the two groups (p= 0.008). Although not a significant difference, stiffness and load to failure were lower in the NSAID group at each of the three time points.

Conclusion: This data suggests that a single pre-operative dose of a NSAID is sufficient to delay fracture repair. The clinical relevance of this finding is that frequently in acute fracture patients a single dose of NSAID is given peri-operatively as it is felt that this will have no effect on fracture repair. This practice may need to be reviewed. On qualitative histology endosteal and periosteal bridging was evident in the group that did not receive NSAID at 1 and 2 weeks. Healing within the NSAID group at 4 weeks was poor.

NSAID’s cycle-oxygenase (COX) inhibitory characteristics are either non-specific, COX-1 preferential or recently COX-2 preferential. NSAID’s have been widely reported to delay fracture repair however the mechanism of this affect remains unclear.

Left femoral osteotomies were performed in 54 male 3 month old CFLP mice immobilised with uniplanar external fixators. 27 externally fixated mice received 4mg/kg meloxicam,b.d., from the day of surgery, by gavage. The control group received the carrier alone. 18 mice had external fixators applied to intact femurs and received no meloxicam as a sham control. Individual mouse movement, was quantified each day by autocounters using an infrared beam motion detection system. Plasma was obtained by right ventricular aspiration under anaesthesia on days 2,4,8 and 16-post surgery.

A validated bioassay and a slot blotting immunoassay were employed to determine the plasma concentration of 11-6 and relative TNF-α levels to normal mouse serum.

TNF-α levels peaked at day 4 and were suppressed by COX-2 inhibition. Both the control and treatment groups had higher levels of TNF-α than the non-fractured controls. The plasma concentration of 11-6 was elevated by COX-2 inhibition at all time points. The levels of TNF-α and 11-6 correlated in fracture control and treatment groups (Spearman’s 0.039 and 0.002 respectively). The 11-6 plasma concentration correlated to the animal motion in the treatment group alone (Spearman’s 0.017).

As it has been shown that TNF-α induces 11-6 production and that this inhibits TNF-α production a possible model for these interaction is shown below.

Hypothesis: Early initiation of COX-2 inhibition is more detrimental to fracture healing than later, irrespective of the analgesia-permitted biomechanical stimulation of the fracture by movement.

Model: A validated externally fixated murine femoral fracture model.

Intervention: Left femoral osteotomies, immobilised with sagittal uniplanar external fixators. Treated mice received 4mg/kg meloxicam from the day of surgery, day 4 or day 8 post op until sacrifice, by gavage. Control mice received carrier alone.

Outcome Measurements: Mouse movement was quantified each day until sacrifice. Fracture geometry was determined from post-sacrifice orthogonal x-rays. Animals were sacrificed on day 4,8,16 and 24. Blind computer aided histomorphometric analysis was performed, on six coronal sections per specimen, determining the medial, intramedullary and lateral areas of total callus, mesenchymal tissue, cartilage and new bone.

Results: No difference existed between the treated groups and control fracture fragments overlap or alignment nor in post-operative movement. Meloxicam treated groups showed decreased callus areas on day 4, 8, and 16, although it was noted that callus remodelling had commenced after day 16 in the control specimens only. New bone areas were reduced in all treated groups at all time points examined relative to the controls with the reduction being proportional to duration of COX-2 inhibition. Mesenchymal tissue differentiation was maximally affected in the earliest treatment group.

On day 24, day 0 treated specimens demonstrated significantly more mesenchymal tissue. No correlation was demonstrated between post-operative motion and callus area or new bone area. The care of cartilage present however, was significantly correlated to the amount of post-operative movement in all groups.

Conclusions: COX-2 inhibition inhibits new bone formation in proportion to the proximity of its commencement the fractures occurrence, irrespective of fracture stimulation by motion. Cartilage production in the healing fracture is not altered by COX-2 inhibition but is proportional to fracture stimulation by motion.