We conducted a multicentre two arm double blind randomised controlled trial to assess efficacy of pulsed ultrasound for accelerating the rate of bone healing. Sixty-two skeletally mature adults undergoing limb lengthening, of between 2.5cm to 10cm by distraction osteogenesis, at the proximal tibia using an Ilizarov frame were randomised to either an active or a placebo (control) ultrasound device.

Primary outcome measure was time ready for removal of frame after adjusting for distraction length (days/cm) for both intension to treat (ITT) and per protocol (PP) patients. The time at which the frame was removed was determined by the maturation of the regenerate bone. Secondary outcomes were return to weight bearing and covariates affecting time to frame removal.

The baseline characteristics of the two groups were well balanced, and 90% of patients were managed and followed up as PP. There was no difference in the time to frame removal between the two groups for the ITT (5.0days/cm, p=0.23) or the PP (10.1days/cm, p=0.054). There was no difference in return to weight bearing between the two groups, after adjusting for distraction length, for the ITT or PP patients (p>0.5). Smoking was the only covariate identified to increase the frame removal time (hazard ratio 0.46, 95% confidence interval 0.22 to 0.96; p=0.04).

This trial demonstrated no difference in bone healing between those who underwent pulsed ultrasound and those who did not. Smoking was observed to have a significant inhibitory effect on bone healing.

Objective:

To assess efficacy of pulsed ultrasound for accelerating regenerate consolidation.

Introduction

With an ageing population comes an increased prevalence of osteoporosis and associated fracture. Whilst treatment of the condition following such a fracture is partially effective, primary prevention through screening and appropriate follow-up is the ideal. In order to assess a population's risk of fracture, paper questionnaires would traditionally have to be sent, however this is an wasteful and costly. A more efficient method may be to have patients assess their own FRAX score through a modified computer application.

To analyse the pain distribution in the acute and chronic phase following thoracolumbar fractures.

Prospective observational study

39 patients with fractures between T11 and L2, with no neurological deficit, were treated conservatively. Strict inclusion and exclusion criteria were applied. All had X-rays and MR imaging (whole spine) at post-injury and one-year follow-up.

The patients documented their pain distribution using pain drawing, along with 10 other domains of pain and functional outcomes for a period over 12 months. The pain distribution was analysed. The association of distal pain distribution to - other associated injury, resultant kyphosis, Pre-existing or increase in disc degeneration at the lower non-injured disc levels – were analysed and reviewed

The most common site of the pain distribution in both the acute (90%) and chronic phase (97%) was distal to the fracture (regions - iliac crest, lumbosacral junction and buttock). Factors mentioned above that could be related to distal pain distribution did not show any significant correlation (P>0.5) with different domains of pain outcome.

Some of the commonly believed reasons for distal pain distribution like resultant kyphosis and associated disc/facet pathologies were not supported by our study findings. The distal pain distribution corresponds to the scelerotomal referred pain mapping, which could be the probable explanation. Thoracolumbar pathologies could be the source of pain in patients complaining of low back symptoms. Distal pain distribution of spine pathologies should not be attributed as functional.

Stable thoracolumbar fracture is a common injury. The factors that determine its outcome are unclear. Aspects of injury severity were analysed for their ability to predict outcome by controlling other outcome-affecting factors (patient's pre-injury health status, legal aspects, associated injuries, etc.). No reliable disc injury severity grading system was available and therefore a new system was developed.

A prospective observational study of 44 conservatively treated patients with stable fractures between T11 and L5 was conducted. Bony injury severity was scored based on comminution, apposition and kyphosis parameters. Disc injury severity was scored by the new scale based on variables – Herniation, Indentation, Height decrease and Signal change – seen in MRI. Ten outcome domains (five domains of pain and function each) were assessed at 1 to 2 years from injury. The data was analysed by non-parametric correlation and stepwise-linear regression analysis to assess the predictive value of different variables (patient factors, injury factors and social factor) to outcome.

The correlation coefficients between injury severity and outcome were consistently higher with disc injury severity than bony. Disc injury severity showed highest predictive value for both pain (29%) and functional (16%) outcomes, whereas the bony injury severity parameters (kyphosis, etc.) and the posterior ligament injury severity provided no prediction of outcome. According to AO classification, the fractures were A1, A2, A3 and B1; in this spectrum of injuries, the AO classification had no prediction of outcome. The disc injury score also had a good predictive value for final disc degeneration.

Disc injury severity should be gauged in advising prognosis and treatment. The new disc injury severity grading system showed good construct validity.

Ten percent of fractures end in delayed or non-union. NSAIDs have been linked to an inhibitory action on fracture repair for three decades yet the mechanism of action remains to be elucidated. Cancer research has identified that NSAIDs impede cell proliferation by inhibiting angiogenesis. It is proposed that a similar mechanism occurs in the induction of NSAID induced non-union. We have investigated this hypothesis in a randomised placebo control trial of the NSAID rofecoxib using a murine femoral fracture.

All animals had an open femoral fracture treated using an external fixator. Outcomes measures included x-ray, histology and biomechanical testing, with laser Doppler used to assess blood flow across the fracture gap.

Radiology showed similar healing patterns in both groups; however, at the later stages (day 32) the NSAID group had significantly poorer healing. Histological analysis showed that controls healed quicker (days 24 and 32), with more callus (day 8) and less fibrous tissue (Day 32). Biomechanical testing showed controls were stronger at day 32. Both groups exhibited a similar pattern of blood flow; however NSAIDs exhibited a lower median flow from day 4 onwards (significant at days 4, 16 and 24).

Positive correlations were demonstrated between both histological and radiographic assessments of healing, with increasing blood flow. NSAID animals exhibited lower flows and poorer healing by all outcomes. Regression analysis demonstrates, however, that the negative effect of NSAIDs on fracture repair is independent of its inhibitory action on blood flow.

COX-2 inhibitors are marketed as having cleaner side effect profiles and are widely used in trauma patients. Following development of a novel method of analysing functional vascularity across a fracture gap, we have demonstrated that the COX-2 inhibitor rofecoxib has a significant negative effect on blood flow at the fracture gap alongside inhibiting fracture repair.

Introduction: Conflicting opinions exist as to whether bone healing is affected by the administration of bisphosphonates for osteoporosis. In an animal model, we assessed the effect of bisphosphonates on osteoporotic fracture healing and whether the timing of administration made a difference.

Methods: 36 female Wistar rats underwent a mid-diaphyseal femoral osteotomy six weeks after ovariectomy. They were then divided into 3 groups:

no treatment (control);

Results: Of the 36 rats, 8 were unable to complete the study. Group 3 differed from control in three respects: higher bone mineral content (BMC) and density (BMD); larger callus; lower torsional stiffness. Group 2 did not differ significantly from control. There was a significant positive correlation between stiffness and change in BMC in group 1 (r=0.85, p< 0.001) but not so for group 2 (r=0.2, p> 0.05) and group 3 (r=0.04, p> 0.05). A similar trend existed for all radiographic parameters in the three groups.

Conclusion: The results suggest that, with early bisphosphonate treatment, although there is an increase in the size of the callus, that callus is biomechanically inferior. Furthermore, administration of bisphosphonates at either stage destroys the relationship between radiographic and mechanical parameters used to assess fracture healing.

Introduction: Digital X-rays have become increasingly prevalent in Hospitals throughout the UK and Ireland in the past 10 years. We have devised a semi quantitative analysis of digital radiographs that measures the extent of healing across the fracture gap.

Methods: 48 CD 1 mice underwent a femoral fracture and subsequent fixation with an external fixator. A standardised radiograph was taken. A radiographic analysis was carried out. For each radiograph taken a pixel density graph was generated at five individual points across the fracture gap, along the longitudinal axis of the femur.

A stastical analysis of intra and inter-observer variability was tested using the linearly-weighted kappa statistic for each of the 240 pixel density graphs taken and for the summation total in the 48 radiographs.

Results: For the individual pixel density graphs we expected an agreement of 67.82%. An agreement of 95.42% was recorded showing a kappa statistic of 0.8576 and a standard error of 0.0531.

On analysis of the summation scores we expected an agreement of 75.54% and observed an actual agreement of 96.30%. This showed a kappa statistic of 0.8545 and a standard error of 0.0849.

Conclusion: The results are very similar in the two analyses and indicate excellent agreements. As a result we offer a radiographic, semi-quantative analysis of bone healing across a fracture gap that is highly reproducible. Thus it has the potential for application to future research in this field and possibly to clinical practice with the increased use of digital radiographs in hospital departments

Thrombin related peptide (TP 508) is a 23 amino-acid synthetic peptide that mimics a portion of the receptor-binding domain of the human thrombin molecule.

Thrombin triggers both proteolytic activated receptors and non proteolytic activated receptors to bring about a mixture of responses ranging from tissue breakdown and clot formation, to new vessel formation and tissue repair. TP 508 stimulates only the non proteolytic activated receptors, and this initiates repair and angiogenesis but not clot formation or tissue breakdown Previous studies have shown that TP508 can stimulate repair in the dermal and musculoskeletal tissues by promoting angiogenesis and enhancing the proliferation and migration of cells.

High energy fractures are associated with a delay in healing. We hypothesized that high energy fracture healing would be improved with the use of TP508, and that the dose and site of application would have importance.

Methods: 80 CD 1 Mice were randomised into four groups; all underwent a high energy quadriceps muscle crush and a femoral fracture on the left hind limb. In each case the fracture was reduced and held with an external fixator. At the time of operation Group I received a dose of 100ìg TP 508 into the fracture, Group II 100ìg into the surrounding damaged soft tissue, Group III a dose of 10ìg into the fracture, and group IV (the control group) received PBS carrier into the fracture.

24 animals were sacrificed on day 21 and the remaining 56 mice on day 35. Of the 35 day old animals 8 in each group had both femora harvested and the biomechanical properties were tested using the 3-point bending technique. Specimens from the 21 day old animals and remaining 35 day old animals were used for histological analysis.

All 80 animals had digital radiographs taken each week. Using image analysis software five pixel density graphs were generated across each fracture gap. A validated semi quantitative analysis was used to score each graph and the total accumulated for each radiograph. The width of the fracture calus was measured and expressed as a ratio of the femur diameter.

Results: Mechanical testing showed significantly greater stiffness in group I when compared to control (p < 0.05), and a dose dependent trend of increasing strength.

Radiographic analysis showed greater healing of fracture and callus formation in Group I compared to Groups II, III, and IV, at both three and five weeks post-fracture (P< 0.05).

Histological analysis showed an increase in bone formation in group I compared to the other groups.

Conclusion: This data from this model, suggests that TP508 enhances healing in high energy fractures. The results also suggest that the effects of TP508 are dose dependant, and are greater when delivered into the fracture site.

We investigated the effect of neck dimension upon cervical range of movement. Data relating to 100 subjects healthy subjects aged between 20 and 40yrs was recorded with respect to age, gender and ranges of movement in three planes. Additionally two commonly used methods of measuring neck motion, chin-sternal distance and uniplanar goniometer, were assessed against a validated measurement tool the CROM goniometer (Performance Attainment Associates, Roseville, MN).

Using multiple linear regression analysis it was determined that sagittal flexion (P= 0.0021) and lateral rotation (P< 0.0001) were most closely related to neck circumference alone whereas lateral flexion (P< 0.0001) was most closely related to a ratio of circumference and length. The uniplanar goniometer has some usefulness when assessing neck motion, comparing favourably to chin-sternal distance that has almost no role.

Neck dimension should be incorporated into cervical functional assessment. One should be wary about recorded values for neck motion from non-validated measurement tools.

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).

The first aim of the study was to investigate if bacteria were implicated in non-union of fractures of the tibia and femur, which had been treated with intramedullary nailing. The second aim was to evaluate the antimicrobial susceptibility of bacteria isolated from the retrieved intramedullary nails.

Forty intramedullary nails removed from tibial and femoral fractures were retrieved for the purpose of the study. Twenty of these nails were from fractures, which had successfully united and 20 were removed from fractures which had failed to unite prior to further operative intervention. There was no evidence of clinical infection in either of the two groups. The nails were subjected to ultrasound in the research laboratory to dislodge adherent bacteria formed as biofilm from the surface of the nail. Using both standard culture techniques and non-culture techniques (Immunofluorescence microscopy and PCR analysis) any dislodged bacteria were isolated and identified.

Isolated bacteria were tested for antimicrobial susceptibility to commonly used antibiotics in orthopaedic practice according to NCCLS guidelines.

Bacteria were detected in 15 out of 20 [75%] of the nails removed from fractures, which had developed a non-union, and in 5 out of 20 [25%] of fractures that had united, using both standard culture techniques and non-culture techniques. The bacterial isolates identified were mainly Staphylococcus epidermidis and the Gram-positive anaerobe Proprionibacterium acnes.

Vancomycin was the most effective antibiotic, with 2 out of 34 [6%] isolates being resistant. Erythromycin was the least effective, with 21 out of 34 [62%] isolates being resistant. Based on overall Minimum Bactericidal Concentrations at which 90% of all strains were killed, Vancomycin was the most active bactericidal agent tested followed in decreasing order by fucidic acid, ciprofloxacin, gentamicin, cefamandole and erythromycin.

Bacteria were detected more commonly in the fracture non-union group than in the union group [p< 0.01]. Of the antibiotic agents tested Vancomycin was the most effective and Erythromycin was the least effective.

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.

NSAIDs inhibit fracture repair, yet the mechanism behind this effect is unknown. It is recognised that NSAIDs impede tumour growth via an inhibition of angiogenesis, primarily via a COX-2 pathway. We propose that the inhibition of fracture repair is via a similar mechanism and have investigated this hypothesis using a murine fracture model. 225 animals were randomised into either treatment (rofecoxib) or control groups and underwent a standard open femoral fracture treated using an external fixator. Outcomes measures involved assessment of healing using radiographic, histolological and biomechanical means; and measurement of blood flow across the fracture gap using Laser Doppler Flowmetry. X-ray analysis showed a similar healing pattern in both groups, however at days 16 and 32 the NSAID group had significantly poorer healing. Histological analysis showed that controls healed quicker (significant at days 24 and 32); and had more bone but less cartilage at day 8. Biomechanical testing showed controls were statistically stronger and stiffer at day 32, while NSAID animals had a significantly greater rate of fixation failure, leading to loss of pin-bone osseointegration; this occurred primarily before day 16. There was no difference in blood flow between the groups on the day of surgery, and both groups exhibited a similar flow pattern; NSAID animals however, exhibited a lower median flow from day 4 onwards, which was significantly poorer at days 4, 16 and 24. Positive correlations were demonstrated between a higher blood flow and both the histological and radiographic results. While NSAIDs were seen to inhibit fracture repair in all outcome measures; and were also noted to decrease blood flow at the fracture, with strong negative correlations being noted between NSAID prescription and fracture repair; multiple regression analysis suggest that this negative effect of NSAIDs on healing is independent of its inhibitory action on blood flow. COX-2 inhibitors are marketed as having cleaner side effect profiles and prescribing is on the rise. Recently however some of the newer COX-2 specific inhibitors have been removed from the market as their seemingly clean side effect profile has come under scrutiny. We have demonstrated that the COX-2 specific inhibitor rofecoxib does has a significant negative effect on fracture repair; and as hypothesised that it also has a significant negative effect on blood flow at the fracture site. While these outcomes strongly correlate, the mechanism behind the effect remains to be elucidated, as we have also demonstrated that these modalities are independent of each other.

Introduction: Sufficient quantity of osteogenic cells is an essential aspect for a successful cell therapy in the treatment of difficult bone fractures and defects. At present, this was achieved by culturing bone marrow and bone-derived cells in a relatively long duration. A large number of the non-adherent mesenchymal stem cells were discarded during medium change. We hypothesize that collecting the non-adherent cells and re-plating them may result in more osteogenic cells in the same duration of cell culture. The aim of this study was to investigate the possibility of enhancing number of osteogenic cells by collecting non-adherent cells in the pull-off media and to examine their osteognic potentials.

Methods: Mononuclear cells were isolated by density gradient centrifugation method from bone marrow washouts in the bone samples obtained from 5 patients undergone total hip replacement. Mononuclear cells were plated at a density of 1 x 10⁶/cm² in a T-75 flask with αMEM medium and 15% FCS. The first medium change was made at day 7 and every 3 days thereafter. For the first three times of medium change, the removed media were centrifuged at 250 g for 10 minutes and plated in a separate T-75 (first time change) and T-25 flask (for the 2nd and 3rd times change). The non-adherent cells from the second and the third puff-off flasks were also collected and plated in separate T-25 flasks. Thus, 1xT-75 flask and 4xT-25 flasks of non-adherent cells resulted from the original T-75 flask. The cells in all flasks were harvested at 21 days from the day when the original flask was set up. The total number of cells in all pull off flasks were counted and compared with that of the original T-75 flask. Rate of cell proliferation with or without osteogenic growth medium were also examined by MTT method for passage 1 of both cells types. Osteogenic differentiation was defined with immunocytochemistry of bone markers: ALP, type I collagen, Osteocalcin and cbfa1. It is planed that cells of passage 2 will be mixed with HA powders and to be implanted into the SCID mice to examine the in-vivo osteogenic potential of these cells.

Results: Mesenchymal stem cells (MSCs) derived from the non-adherent population of human bone marrow culture have demonstrated having similar cell proliferation and differentiation potential in vitro, when compared to the MSCs derived from the adherent cell population. These cells expressed bone markers such as: ALP, type I collagen, osteocalcin and cbfa1. When the non-adherent cells were collected and cultured accumulatively, the total number of MSCs was increased to an average of 39.7% (36.6%–42.9%), compared to the number of cells obtained from the original T-75 flask.

Conclusions: Collecting the non-adherent cell population in the bone marrow culture appeared to result in more MSCs. This harvesting method may be used as a non-invasive way for enhancing MSC numbers in a given period of time. Further in vitro and in vivo studies of these MSCs of non-adherent origins may provide information for optimizing cell culture protocols for rapid expanding the osteogenic cells in vitro. This will facilitate the clinical applications of human osteogenic cell therapy.

Introduction: Angiogenesis is essential during bone formation. Many studies have looked at the developing vascular network during normal and abnormal bone growth, using histological, immunohistological and contrast-radiological techniques; however all require sacrifice of animals to obtain tissue samples for examination and consequently chronological investigation of angiogenesis is not possible. We have endeavoured to produce an animal model, whereby quantitative assessment of blood flow, and callus formation across a fracture gap, can be repeatedly assessed.

Methods: The model is an adaptation of a 4-pin externally fixated murine femoral fracture previously developed in this department. Three extra conduits have been drilled onto the fixator cross-bar, such that it now links with an x-ray jig and implantable optical cable. The x-ray jig permits repeated lateral x-rays whereas the optical cable which is implanted adjacent to the fracture gap and connected to a laser, measures blood flow using the principle of the Doppler shift of light. Ten mice underwent surgery. Doppler readings and x-rays were taken on the day of surgery and subsequently at days 1, 2, 4, 8, 12, 16, 24 and 32.

Results: Fracture gap pixel density was seen to rise steadily and plateau at day 24, with significant statistical differences between the day of surgery and early time points, and then again between these early time-points (days 2, 4 and 8) and the late time-point day 24. Blood flow was noted to fall following the day of surgery and then slowly increase, with a rapid rise in flow at day 8 until day 16, when levels began to fall again to resting levels.

Conclusion: The data correlates with previous histo-morphological work performed in this department and also with early results from immunohistochemical studies. The above graph for blood flow conforms to that expected in a murine model of fracture healing, with a short initial drop in flow followed by a large rise as angiogenesis follows chondrocyte hypertrophy at the end of the first week, leading to callus formation. This in vivo model may be used to assess the effects on angiogenesis and callus formation of osteogenic compounds and investigate possible antiangiogenic mechanisms of action of medications such as NSAIDs that are known to be detrimental to fracture repair.

Introduction: The existence of peripheral blood (PB) derived mesenchymal stem cells (PB-MSCs) have been documented in different mammalian species including young and adult human. However, the number of PB-MSCs is low in normal adult human blood. We have demonstrated previously that there was an increase in the number of PB-MSCs following long bone fracture and in the patients suffering from fracture non-union. The present study was to compare the biological characteristics of the PB-MSCs from fracture non-union patients, with human bone marrow derived MSCs (BM-MSCs).

Methods: 200 mls PB was collected from 9 patients suffering from fracture non-union. The mononuclear cells (MNCs) were isolated by density gradients centrifugation and cultured in á-MEM containing 15% FBS. The PB-MNCs from normal donors (n=8) and BM-MSCs from patients underwent total hip replacement were used as controls. The colony forming efficiency (CFE) of the PB-MSCs was calculated, and the phenotypes of PB-MSCs and BM-MSCs were compared using immunocytochemistry and flow cytometry methods. Their multipotent differentiation potentials into osteoblasts, chondrocytes, adipocytes, neurogenic and angiogenic cells were examined under specific inductive culture media. The in vivo osteogenic potential of PB-MSCs was examined by implanting the HA-TCP blocks seeded with PB-MSCs into the SCID mice for 12 weeks.

Results: After 28 days in culture, fibroblastic colonies were formed in the PB-MNCs cultures in 5 of 9 fracture non-union patients, with CFE ranging from 2.08–2.86 per 10^8 MNCs. No fibroblastic colony was seen in PB-MNCs cultures of the 8 normal donors. Under flow cytometry examination, PB-MSCs and BM-MSCs were CD34 (low) and CD105+, but PB-MSCs were CD29-, CD44-, and ALP (low), whereas BM-MSCs were CD29+, CD44+, and ALP (high). Under specific differentiation inductions, the PB-MSCs differentiated into osteoblastic cells (ALP+, type I collagen+, osteocalcin+ and Alizarin red+; chondrocytes (type II collagen+ and Alcian Blue nodules formation); adipocytes (Oil red-O positive lipid accumulation). Neurogenic differentiation was confirmed by positive neuro-filament staining, and differentiation into endothelial cells was evident with tube formation in 2D culture, and positive staining for VW factor and CD31. After implantation in the SCID mice for 12 weeks, newly formed woven bones were found in the biomaterials seeded with PB-MSCs, and they were positive for human osteocalcin immunostaining.

Discussion: This study indicated that there were more PB-MSCs in the peripheral circulation of the fracture non-union patients than that in the normal subjects. This may be due to a continous systemic response for recruiting MSCs from remote bone marrow sites, with attempt to repair the fracture(s). The PB-MSCs were clearly multi-potential cells, which had shared some common phenotypic markers with BM-MSCs, as well as many distinguishable makers from the BM-MSCs. The recruitment of the PB-MSCs through circulation might be a general phenomenon of systemic responses in many pathological conditions, such as fracture or wound healing and other systemic diseases. Further understanding the roles of PB-MSCs in diseases and repair may lead to novel therapeutic strategies.