Background

Hyperlaxity is associated with a high incidence of sporting injuries. Collagen V regulates the diameter of fibrils of the abundant collagen type I. Decorin and biglycan are members of the small leucine rich proteoglycans(SLRP's)family and play important roles in the regulation of collagen fibrillogenesis. The aim of this study was to identify if there was a link in hyperlaxity, tissue strength, collagen V and SLRP's expression.

Approximately 5 – 10% of all bone fractures are associated with impaired healing. It is thought that regenerative medicine has the potential to improve on existing treatments for non-union fractures, and the European market for such treatments is projected to reach £2.2 billion in 2010. The use of scaffolds for the delivery of both growth factors and human Marrow Stromal Cells (hMSCs) is thought to be a promising approach. It may be desirable to promote proliferation and chemotaxis of hMSCs at the defect site shortly after implantation, and differentiation in the longer term. This is likely to require a dual delivery system, capable of releasing multiple drugs with different release profiles. Our aim has been to develop a polymer scaffold capable of releasing bioactive molecules that are able to direct the differentiation of primary hMSCs down the osteoblastic lineage. We have examined two mutually compatible drug delivery systems: collagen coating for short term release, and polymer encapsulation for longer term release.

Polymer scaffolds were manufactured and coated with Type I Collagen containing BMP-7. hMSCs from three different patient sources were exposed to the scaffolds for 14 days. The cells were then histochemically stained for Alkaline Phosphatase (ALP) and photographed. The areas of ALP staining were then normalised against the total cell count.

Normalised ALP expression was increased compared to the controls for three different patients (‘110 ± 39% SE, n=6, p=0.005’, ‘540 ± 270% SE, n=6, p=0.001’, and ‘32 ± 17% SE, n=6’). Scaffolds were also manufactured either with 1,25 Vitamin D3 (another active compound) in a coating of Collagen, or encapsulated using proprietary methodologies. It was found that both treatments significantly increased normalised Alkaline Phosphatase expression within the 14d experimental period demonstrating release of the active 1,25 Vitamin D3 (’88 ± 37% SE, n=6, p=0.012’ and ‘100 ± 32% SE, n=6, p=0.012’ respectively).

Our findings suggest that, subject to future testing and development, such bioactive scaffolds could form the basis for a dual drug delivery system, suitable for applications in bone regenerative medicine.

Fracture repair is a wound healing process that in young healthy patients usually proceeds to uncomplicated union. However, the healing cascade is delayed with increasing age, medication and certain diseases such as rheumatoid arthritis.

Recently the important role of the immune system in fracture repair has become apparent within the emerging subject of Osteoimmunology. Patients with rheumatoid arthritis have an altered immune system and therefore we have investigated the hypothesis that patients with rheumatoid arthritis have a higher incidence of non-union after a fracture compared to patients without rheumatoid arthritis.

Method: The Edinburgh Royal Infirmary computer database was searched over a 10 year period (May 1996- May 2006) to identify all patients with non-union out of the total number of patients presenting with fractures. These patients groups were then subdivided into patients that had and did not have rheumatoid arthritis. Patients were excluded if they were lost to follow up, or if the fracture either occurred before the May 1996 or management continued passed May 2006. In this study non-union was defined as failure to heal within expected timescale and lack of progression at serial x-rays (all non-union were diagnosed at least 3 months from fracture).

Results: From May 1996 through to May 2006, 8,456 patients with fracture were defined. 71 of these patients with fractures had rheumatoid arthritis. Of these patients 63 had union of their fractures whilst 8 patients developed non-union of their fracture (11.3%). In a total of 8385 non rheumatoid arthritis patients 164 developed non-union of their fracture compared to 8221 patients who had union of their fractures (2%). Comparison between these two patient groups suggests rheumatoid arthritis patients are more likely to develop non-union of traumatic fractures (Chi squared test, p value < 0.001).

Patients with rheumatoid arthritis who progressed to non-union were on the following medication, Gold (1), Indomethacin (1), Non steroidal anti-inflammatories (4), Combination analgesia (2), Antihypertensives (2), Omeprazole (1) and Thyroxine (1).

Discussion: The results from this study suggest that patients with rheumatoid arthritis have a greater incidence of non-union after a fracture compared to patients without rheumatoid arthritis. This maybe due to the abnormal immune system in rheumatoid arthritis patients. However rheumatoid arthritis patients are often on a number of medications and these drugs rather than the innate alteration of the immune system may be responsible for the altered healthy response. However whether as a result of the rheumatoid arthritis itself or the medication, our study demonstrates a higher non-union rate in the rheumatoid arthritis patients and this needs to be taken into account when treating rheumatoid arthritis patients with fractures.

Over 1 million fractures occur each year in the UK. Approximately 5-10% of these fractures have problems with healing. The treatments used for these patients often have a poor outcome and are associated with increased morbidity and disability. Application of synthetic peptides such as thrombin degradation peptide (TP508) has been shown to accelerate fracture repair in a closed rat femoral fracture model. Controlled release of TP508 using microspheres has been shown to enhance repair of articular cartilage defects and stimulate bone formation in segmental defects in rabbits. The aim of this study was to determine whether TP508 could bring about healing in an established fracture non-union model.

A validated rat model of fracture non-union was used. The model was created and left for 8 weeks in order to represent a clinically equivalent model of a non union of a fracture. Rats were randomised into two treatment groups receiving 10microg and 1microg doses of TP508 diluted in 50microL of microspheres and delivered directly to the non union site using percutaneous injection 8 weeks after surgery. The control group received no treatment. At 16 weeks post-surgery, osseous bridging was assessed both radiographically and histologically.

Radiographically there was no difference between the control and two treatment groups. However, histomor-phometric analysis demonstrated that bone formation increased by 43.9% in animals that received high dose of TP508 compared to the control animals. The analysis also indicated that administration of the low dose of TP508 increased the amount of bone formation compared to the control by 9.9 %.

Administration of TP508 has been shown to enhance healing of segmental defects in both critically and noncritically sized defects. However, in our model which is an established fracture non-union model, TP508 did not manage to achieve full osseous union. It has been suggested that the action of this peptide is concentration and environment dependent possibly indicating that TP508 might be less effective when administered in a chronic situation such as that associated with the established non-union fracture. However, even in this sub-optimal situation an increased amount of bone formation was observed.

Introduction: Atrophic nonunion is a well recognised complication of long bone fractures. Clinical trials show that BMP-2 accelerates healing and reduces nonunion in open tibial fractures. We are interested in a natural small molecule that has been previously demonstrated to stimulate angiogenesis in vivo. Our aim is to assess the two treatments in the prevention of nonunion. The small animal model we used is a non-critical size defect of the tibia deprived it of its blood supply by surgical stripping of the periosteum and curetting of the local endosteum thus closely reflecting the clinical situation. The outcomes were measured by radiographic assessment and histology.

Methods: Wistar rats were treated with either the angiogenic molecule (0.1% or 0.003%), BMP-2 or vehicle alone (PBS) soaked in a type I collagen sponge. All animals underwent a 2mm osteotomy, stripping of the periosteum and endosteum proximally and distally for the length of the diameter of the tibia. Fluorescent markers were injected at 2 weekly intervals. The rats were sacrificed at 8 weeks. Both tibiae were disarticulated; fixator and soft tissues were removed and AP and lateral X-rays were taken. Subjective assessment of the healing on X-ray was carried out in two ways; using a radiographic scoring system and by grey scale analysis. The samples were embedded, sectioned and stained for new bone formation.

Results: Bridging or potential to bridge was seen in a number of animals on x-ray. Bridging or potential to bridge was judged to be present in 72.22% of the BMP-2 group and 66.67% of the high dose group compared to 22.22% of the control group. Histological analysis is being performed to confirm these findings.

Discussion: Atrophic nonunion is a serious clinical complication, unfortunately BMP-2 is a highly costly treatment option and therefore alternative molecular therapies are much sought after. We describe here an angiogenic molecule has some potential in preventing formation of nonunion.

Thermonecrosis either results in bone loss which may weaken the purchase of surgically-inserted screws leading to loosening or the dead bone may remain in situ and become infected resulting in a ring sequestrum. The aim of this project was to measure the heat generated during drilling of bone. By using a novel realtime thermal camera the thermal events could be visualised topographically.

An experimental setup comprising a force table, an infrared camera, a power drill and a new surgical 2.5mm drill bit was constructed. This enabled measurements of the force applied and temperature changes in sheep cortical bone during a drilling operation. The temperature was observed throughout the drilling period and for further 15s after the drill bit was withdrawn. Images were grabbed using a LAND FTI Mv thermal camera which was driven by LIPS Mini software. Calibration was made in the range 20-200 degrees C, the upper value being provided by a high wattage resistor. Data was processed using routines written in MATLAB.

It was found that 12s were required to drill through a single cortex. Within one second of drilling, the maximum recorded temperature in the vicinity of the drill increased from the baseline of 20 to 170 degrees C. It remained above this temperature for 25s. Immediately after the drill bit was withdrawn, a region of approximately 15mm of diameter of cortical surface had a sustained temperature above 50 degrees C. After 15s of cooling, this diameter had only reduced to 10mm. By modelling the cooling curve, the maximum temperature at the drill tip was extrapolated to be between 500-600 degrees C.

Thermography has proven to be useful in the study of the thermal characteristics of bone during drilling. The process of drilling generates significant increase in temperature in the vicinity of the drill. This temperature elevation has been found to be sustained for a significant period of time.

Imaging of the musculoskeletal system is vital for delivering optimum treatment particularly in the assessment of fracture healing. X-ray and CT are adequate imaging methods for bone but, soft tissue needs other modalities such as MRI and Ultrasound. We propose the use of Freehand 3D Ultrasound to study the early stages of fracture healing by imaging the bone surfaces around the fracture site and monitoring changes in the surrounding soft tissue.

Freehand 3D ultrasound is acquired by attaching a position sensor to the probe of a conventional 2D diagnostic ultrasound machine. As the probe is moved, its position and orientation are recorded along with the 2D ultrasound images. This enables slices through the body to be viewed that would be inaccessible using a normal ultrasound system. Bone surfaces around a fracture site are scanned and the data reconstructed using the Stradx and Stradwin software developed by Cambridge University, to give a 3D visualization of the area.

To assess the feasibility of this proposed method the lower limbs of healthy volunteers were scanned using a 5–10MHz ultrasound probe. The scanning resolution of the system was evaluated using a phantom to ensure millimetre detail could be detected as would be required for imaging early fracture healing. It was found that detail down to 0.8mm could easily be resolved for measurement.

The 3D system could accurately profile the different soft tissue interfaces. The visible surfaces of the tibia were reconstructed to give 3D models. Additional layers of soft tissue interfaces could easily be added to these models to provide more detail.

This imaging modality can provided detailed 3D models of bone the bone surface and surrounding soft tissue. As ultrasound is non-ionizing, rescanning can be conducted more frequently than with CT or x-ray thus offering a more accurate assessment of a patient’s response to healing.

The response of the muscle is critical in determining the functional outcome of limb lengthening. We hypothesised that muscle response would vary with age and therefore studied the response of the muscles during tibial lengthening in ten young and ten mature rabbits. A bromodeoxyuridine technique was used to identify the dividing cells.

The young rabbits demonstrated a significantly greater proliferative response to the distraction stimulus than the mature ones. This was particularly pronounced at the myotendinous junction, but was also evident within the muscle belly.

Younger muscle adapted better to lengthening, suggesting that in patients in whom a large degree of muscle lengthening is required it may be beneficial to carry out this procedure when they are young, in order to achieve the optimal functional result.

Introduction: Evidence exists concerning the anti-oxidant properties of oestrogen in protecting neuronal cells from oxidative stress. The withdrawal of oestrogen after menopause is the major factor determining age related bone loss and apoptotic death of osteocytes. While oestrogen replacement demonstrates clear oestrogen receptor mediated benefits to bone cells little is known regarding oestrogens’ anti-oxidant effects in bone.

Methods: Here we have used MLO-Y4 osteocyte-like cell line to determine whether oestrogen saving effects on osteocytes involves its activities as an anti-oxidant.

MLO-Y4 cells were treated with physiological doses (10⁻⁸)M of either 17-beta E₂ or the oestrogen receptor inactive stereoisomer 17-alpha E₂ with or without the specific oestrogen receptor antagonist ICI 182,780 prior to the addition of 0.4milliM 30% (v/v) H₂O₂. Cellular apoptosis was determined using morphological and biochemical criteria.

Results: H₂O₂ induced an increase in apoptosis of MLO-Y4 (14.3 ± 3 SD vs control 1.4 ± 0.9). Pre-treatment of the cells with 17-beta E₂ significantly reduced H₂O₂ induced apoptosis (2.4 ± 0.96). Pre-treatment of cells with 17-alpha E₂ or ICI 182,780 also reduced oxidant induced apoptosis to 3.4 ± 1.5 SD and 7.0 ± 2.3 respectively.

The cellular production of reactive oxygen species was determined using the free radical indicator 2′7′- dichlorodihydrofluorescein diacetate. H₂O₂ induced increases in the number of ROS positive cells (34.6 ± 9.07 SD vs control 0.22 ± 0.39 SD). In contrast pre-treatment with both 17-beta E₂ and 17-alpha E₂ reduced the number of ROS positive cells associated with H₂O₂ treatment (Fig 1).

Conclusion: These data suggest that oestrogens ability to save osteocytes from oxidant induced death is independent of the oestrogen receptor and may be related to oestrogens known activity as an anti-oxidant. This raises the possibility that loss of osteocytes during oestrogen insufficiency may occur through a failure to suppress the activity of naturally occurring or disease associated production of oxidant molecules.

Introduction: Oxidative stress occurs when reactive oxygen species (ROS) are produced faster than they can be removed by cellular defence mechanisms contributing to ageing, many chronic diseases, such as atherosclerosis, RA, Parkinson and Alzheimer’s disease and skeletal pathologies. Here we address the impact of ROS on the viability of early osteogenic precursors in the bone marrow and study the influence of estrogen on this interaction. Cells have a number of mechanisms to protect themselves from ROS, which are constantly being formed in the cell through normal metabolic pathways, such as Vitamin E, C and estrogen. Estrogen has been shown to prevent intracellular accumulation of peroxide and to attenuate oxidant-induced death of neuronal and endothelial cells. In addition, it contributes significantly to bone turnover and relieves postmenopausal symptoms. This study has focused on the potential anti-oxidant properties of estrogen against oxidative on bone marrow stromal cells. stress induced by H₂O₂

Methods: Primary bone marrow stromal cells were pre-treated with several different doses between 10⁻⁶M – 10⁻⁸M of estrogen prior to H₂O₂ administration at 0.08–0.4 mM 30% (v/v) for 2–24h. The cellular production of ROS was determined by using the free radical indicator DCFH-DA. Apoptosis was determined by morphological criteria.

Results: H₂O₂ induced an increase in apoptosis of osteoprogenitor cells (p< 0.05). Determination of apoptosis and cell number by nuclear staining, indicated that pre-treatment of bone marrow stromal cells with 17-beta estradiol reduced the apoptotic response induced by H₂O₂ (p< 0.05) and restored cell number to control levels. In order to test the anti-oxidant activity of estrogen, the dye DCFH-DA was introduced in a cell free system in the presence or absence of 17-beta estradiol and H₂O₂. The same experiment was repeated in the presence of bone marrow stromal cells. H₂O₂ increased both intracellularly and extracellularly oxidant activity and estradiol has the capacity of modifying this activity both inside and outside the cell.

Discussion: These data demonstrate the ability of estrogen, used at physiological doses, to block oxidant-induced apoptosis of osteoprogenitor cells. Estrogen appears to reduce the generation of ROS in these cells. These data could have important implications on the maintenance of osteogenic stem cells during fractures, ageing and disease.

Since cementless stem fixation in hip arthroplasty is becoming more and more common, the overall incidence of intraoperative femoral fractures has risen considerably. Depending on primary or revision arthroplasty, literature reports fracture rates between a few percent up to one third of the cases.

In this study, methods commonly applied in the field of structural testing were customized for this specified interference fit situation. A cementless hip system (ABG II, Stryker) was used on animal bones and biomechanical bones.

Transient excitation in the form of regular hammer strokes and sinusoidal excitation using a shaker served as an input. The output of the system under test was measured on the greater trochanter using a piezoelectric accelerometer.

The signals were digitized with a high-speed data acquisition system and analyzed in real-time with spectrum analysis software.

Analysis included threshold detection in the time domain to determine the time delay between the input and output transducer. Spectrum analysis in the frequency domain included FFT analysis and frequency response function analysis to identify shifts of fundamental frequencies and harmonics to describe the vibrational changes with increasing stability.

A digital imaging system was set up to take pictures of the metal-bone site to measure inducible displacement with each hammer impact and correlate it with the vibrometry results. Furthermore a strain gauge circularly mounted around the proximal femur monitored accurately any hairline fracture.

This study shows that changes of the vibrational spectrum are directly related to implant fit. The range of interest is well in the sonic range, which apparently is the reason for many surgeons to listen and ‘feel’ carefully during advancing the broach or the final implant into the femur.

The study is trying to extract critical vibrational parameters correlated with stability and femoral integrity. Due to the different dimensions of the tested animal bones and lack of soft tissue damping, further experiments on cadavers need to be carried out.

Vibrational spectrum analysis could prove to be a useful tool to readily assess implant stability and femoral integrity. It seems to be most beneficial in revision surgery or minimally invasive hip replacement, where the risk of femoral fractures is increased or fissures could easily be missed.

Distal radial fractures account for 17% of all fractures treated, with peaks in the bimodal distribution corresponding to young and senior patients. External fixation is one of the best techniques to allow quick patient recovery and is necessary for complex fractures, such as that of the distal radius. However, the safe removal time for these frames remains unclear. A conservative approach commonly leaves the external fixator in place for six weeks, which may be unnecessarily prolonged and lead to increased complications. The aim of this work is to develop a technique to quantify, objectively, a safe removal time for these frames. Studies have been conducted on external fixation of tibial fractures, however there are differences that do not allow transfer of these studies to the external fixation of distal radial fractures. These differences include configuration of the fixation frame, bone and fracture geometries, and the application and transfer of the load to the bone. In this work, the dynamic transfer of the load between the fractured bone and the fixator is investigated. An instrumented grip and a measuring device have been developed to monitor the axial force and displacement when the patient applies a load. Using measurements collected by the instrument and data specifying the frame geometry, a finite element model is used to calculate the load carried by the fixator and by the bone, and the rigidity of the new callus is determined. Plotting the rigidity on semi-logarithmic scale the healing rate can be established. This technique has been successfully verified in a laboratory simplified structure representative of bone fracture. The rigidity of several intra-gap materials has been estimated experimentally using the technique, and the results compared to the real value of the material. These measurements do not interfere in any way with the patient treatment and they can be collected from the first day after the operation. The technique has been tested on 14 volunteer patients and the increase in callus rigidity can be detected by measurements during treatment using the technique described. A randomised prospective study has been initiated to validate this technique and investigate the healing process. A positive outcome would enable the rigidity of the new callus bone and the healing rate to be monitored during clinical assessment. Any healing delay or non-union could be promptly detected, improving the quality of the treatment.