Osteoarthritis (OA) of the equine distal interphalangeal joint (DIPJ) is a common cause of lameness. MicroRNAs (miRNAs) from biofluids such as plasma and synovial fluid make promising biomarker and therapeutic candidates.

The objectives of this study are (1) Identify differentially expressed (DE) miRNAs in mild and severe equine DIPJ OA synovial fluid samples and (2) Determine the effects of DE miRNAs on equine chondrocytes in monolayer culture.

Synovial fluid samples from five horses with mild and twelve horses with severe DIPJ OA were submitted for RNA-sequencing; OA diagnosis was made from MRI T2 mapping, macroscopic and histological evaluation. Transfection of equine chondrocytes (n=3) was performed using the Lipofectamine® RNAiMAX system with a negative control and a miR-92a mimic and inhibitor. qPCR was used to quantify target mRNA genes.

RNA-seq showed two miRNAs (miR-16 and miR-92a) were significantly DE (p<0.05). Ingenuity Pathway Analysis (IPA) identified important downstream targets of miR-92a involved in the pathogenesis of osteoarthritis and so this miRNA was used to transfect equine chondrocytes from three donor horses diagnosed with OA. Transfection was successfully demonstrated by a 1000-20000 fold increase in miR-92a expression in the equine chondrocytes. There was a significant (p<0.05) increase in COMP, COL3A1 and Sox9 in the miR-92a mimic treatment and there was no difference in ADAMTS-5 expression between the miR-92 mimic and inhibitor treatment.

RNA-seq demonstrated miR-92a was downregulated in severe OA synovial fluid samples which has not previously been reported in horses, however miR-92a is known to play a role in the pathogenesis of OA in other species. Over expression of miR-92a in equine chondrocytes led to significantly increased COMP and Sox9 expression, consistent with a chondrogenic phenotype which has been identified in human and murine chondrocytes.

Abstract

Abstract

Aim

To quantify the micro-motion at the fracture gap in a tibial fracture model stabilised with an external fixator.

Background:

Little is known about the forces carried by the Taylor Spatial Frame (TSF) hexapod fixator. Our aim was to measure the TSF resultant force and how this changed during the consolidation phase.

Purpose and background

Effective reassurance is an essential element of treatment for conditions that do not require further investigations, referrals and on-going monitoring. However, research defining what reassurance should consist of and how to deliver it is scarce. The aim of this review was to identify consultation-related processes that improved patients' outcomes, in order to build an evidence-based model of effective reassurance in primary care.

Early diagnosis of delayed- and non-union tibial fractures is difficult, but treatment options are available if timely data are available. Direct correlation between implant forces and healing status is difficult during stance phase loading due to soft tissue forces. This ongoing study seeks to find a minimal set of strain gauge sites needed to determine healing at any of several fracture sites, using isometric loading suitable for routine clinical usage. A series of instrumented tibial nails are being used to help determine whether an alternative technology can replace or augment existing routine methods for assessment of fracture healing.

In a prior study, a single strain gauge positioned close to the fracture site had produced mixed results. In the current study, a TRIGEN META NAIL, 10mm OD x 380mm long, was instrumented with 8 gauged sites spiraled down the nail at 34mm axial and 120deg angular separation (Gen1), and loaded in a Sawbone model in offset axial compression, 3 point bending and torque.

In order to gain early clinical results, and in a design informed by the Gen1 data, a set of instrumented nails have been made for an ovine wireless telemetry study (Gen3a), shortly to commence, in which the tibial nail has been over-gauged enabling multiple d.o.f. measurements to be made during gait, torque, axial compression and 3 point bending; the latter protocols offering more controlled patient postures. This study is to be followed by a similar human study (Gen3) involving five subjects (12 gauges per nail). Meanwhile, a parallel biomechanical study involving six nails with 20 gauges each is also planned.

In the Gen1 study, the strains diminished with distance from the fracture site and with out-of-plane sites during bending. During torque, however, the response was much more uniform for all strain sites. Significant increases in strains due to both loading regimes were seen in the fractured case vs. an intact bone.

Preliminary conclusions are that strains measured due to applied torque may offer a more sensitive and fracture site-independent means of assessing healing than induced bending. We now aim to confirm these observations in animal and human studies.

Tribology and wear of articular cartilage is associated with the mechanical properties, which are governed by the extracellular matrix (ECM). The ECM adapts to resist the loads and motions applied to the tissue. Most investigations take cartilage samples from quadrupeds, where the loading and motions are different to human. However, very few studies have investigated the differences between human and animal femoral head geometry and the mechanical properties of cartilage.

This study assessed the differences between human, porcine, ovine and bovine cartilage from the femoral head; in terms of anatomical geometry, thickness, equilibrium elastic modulus and permeability.

Diameter of porcine (3-6 months old), bovine (18-24 months old), ovine (4 years old) and human femoral heads were measured (n=6). Plugs taken out of the superior region of each femoral head and creep indentation was performed. The human femoral heads were obtained from surgery due to femoral neck fracture. Cartilage thickness was measured by monitoring the resistive force change as a needle traversed the cartilage and bone at a constant feed rate using a mechanical testing machine. The percentage deformation over time was determined by dividing deformation by thickness. A biphasic finite element model was used to obtain the intrinsic material properties of each plug. Data is presented as the mean ± 95% confidence limits. One-way ANOVA was used to test for significant differences (p < or = 0.05).

Significant differences in average femoral head diameter were observed between all animals, where bovine showed the largest femoral head. Human cartilage was found to be significantly thicker than cartilage from all quadrupedal hips. Human cartilage had a significantly larger equilibrium elastic modulus compared to porcine and bovine cartilage. Porcine articular cartilage was measured to be the most permeable which was significantly larger than all the other species. No significant difference in permeability was observed between human and the other two animals: bovine and ovine (Table 1).

The current study has shown that articular cartilage mechanical properties, thickness and geometry of the femoral heads differ significantly between different species. Therefore, it is necessary to consider these variations when choosing animal tissue to represent human.

Introduction: The non-invasive growing prosthesis continues to be used successfully for the treatment of limb salvage operations in tumour patients. We report our continued experience in 17 skeletally immature patients with osteosarcoma of the distal femur.

Methods: Patients had a mean age of 10.2 years (range 6 to 15) at the time of surgery. The endoprosthesis was lengthened at appropriate intervals in outpatient clinics without anaesthesia using the principle of electromagnetic induction.

Results: The mean follow-up was 28 months (range 2 to 55). The prostheses were lengthened by a mean of 47.4 mm (range 0.5 to 208) and maintained a mean knee flexion of 110 degrees (range 90 to 120 degrees).

Complications developed in seven patients: two implants failed requiring revision, one peri-prosthetic fracture occurred, one developed a flexion deformity of 25 degrees at the knee joint, which was subsequently overcome and three died of disseminated disease.

Discussion: The medium term results from patients treated with this device have continued to show a promising outcome. Four patients successfully completed desired lengthening, six patients are continuing with ongoing lengthening. The implant avoids multiple surgical procedures, general anaesthesia and assists in maintaining leg-length equality.

For the treatment of malignant bone tumours in immature patients, extending prostheses are used to maintain growth in the affected limb. This new prosthesis allows the implant to be lengthened by using electromagnetic coupling that is simple and easy to use.

Because of bone tumour, fourteen patients between the age of 8 and 15 years underwent bone replacement treatment and a further two patients, both male 18 and 61 years, received the same treatment to restore limb length discrepancy. These implants consisted of a telescoping shaft where the expansion is done by a power screw driven by a gearbox connected to a NdFeB magnet. This prosthesis is capable of being extended under an axial load of up to 1350N. This is in line with 76 distraction force measurements taken in 43 patients with growing prosthesis where extension was achieved by invasive procedure and where a force of up to 1513N for an extension of 6mm was recorded. Once implanted, the non-invasive prosthesis is extended by placing the limb through an external drive unit. As the drive unit is turned on, it produces a rotating magnetic field capturing the implant magnet causing it to rotate in synchronisation. At full speed, the implant grows at a rate of 0.23mm per minute.

Of the sixteen patients, seven have been extended with one to its full capacity of 63mm. During extension, the patients have no sensations of vibration, heat, stretching or any other kind although the faint vibrations could be heard by placing a stethoscope on bony protrusions such as greater trochanter. At each sitting, the patients were extended by approximately 4mm during normal outpatient clinics and were able to walk as before immediately after the treatment. Patients with knee joint were functionally assessed before and after the treatment and showed approximately 10° to 15° of reduction in knee flexion/extension.

This new extending mechanism in these prostheses has provided the patients a treatment, which reduces trauma infection and discomfort. The mechanism of extension is reliable and effective.

We report our early experience with the use of a non-invasive distal femoral expandable endoprosthesis in seven skeletally immature patients with osteosarcoma of the distal femur. The patients had a mean age of 12.1 years (9 to 15) at the time of surgery. The prosthesis was lengthened at appropriate intervals in outpatient clinics, without anaesthesia, using the principle of electromagnetic induction. The patients were functionally evaluated using the Musculoskeletal Tumour Society scoring system. The mean follow-up was 20.2 months (14 to 30). The prostheses were lengthened by a mean of 25 mm (4.25 to 55) and maintained a mean knee flexion of 110° (100° to 120°). The mean Musculoskeletal Tumour Society score was 68% (11 to 29). Complications developed in two patients; one developed a flexion deformity of 25° at the knee joint, which was subsequently overcome and one died of disseminated disease. The early results from patients treated with this device have been encouraging. The implant avoids multiple surgical procedures, general anaesthesia and assists in maintaining leg-length equality.

Our study sets out to show whether vascular endothelial growth factor (VEGF) expression in stage 2B osteosarcomas around the knee influences disease-free and overall survival.

Fifty-two such patients treated in out unit were identified and followed-up for for a minimum of 92 months. All were treated according to the current MRC protocol and had resection of their tumour. Tissue from their resected tumours was stained for VEGF using immunohistochemical methods and the percentage of tumour cells staining for VEGF was assessed. The relationship between VEGF expression and survival was assessed using the log-rank test and Kaplan-Meier survival curves.

At follow-up 32 (62%) patients were dead, all from metastatic disease. Twenty-six (50%) tumours showed expression of VEGF. Statistical analysis showed that patients with tumours with VEGF expression in more than 25% of the cells had significantly shorter overall survival (p=0.019) and disease free intervals (p=0.009). Expression of VEGF also correlated with expression of the proteolytic enzyme MMP9 (p=0.02).

VEGF is peptide which acts as a stimulator of new blood vessel growth in normal tissues, as well as in some solid tumours and their metastases. A tumour which is able to induce a blood supply has an increased ability to grow, seed metastases and threaten life. Our study is the first to look at VEGF expression in the tumour cells surviving after chemotherapy. It is this population of cells which is important as it is these cells which may go on to develop into metastatic or locally recurrent tumours. The over-expression of VEGF by osteosarcoma cells is thought to be associated with a worse prognosis due to a number of mechanisms. This study shows that VEGF expression is an important prognostic factor in osteosarcomas and suggests that the mechanisms by which VEGF and MMP9 expression produce a poor prognosis may be linked. Suppression of tumour angiogenesis by inhibition of the action of VEGF has shown promise in animal models as a potential new treatment for osteosarcoma, and warrants further study.

Our study sets out to show whether vascular endothelial growth factor (VEGF) expression in stage 2B osteosarcomas around the knee influences disease-free and overall survival.

Fifty-two such patients treated in out unit were identified and followed-up for for a minimum of 92 months. All were treated according to the current MRC protocol and had resection of their tumour. Tissue from their resected tumours was stained for VEGF using immunohistochemical methods and the percentage of tumour cells staining for VEGF was assessed. The relationship between VEGF expression and survival was assessed using the log-rank test and Kaplan-Meier survival curves.

At follow-up 32 (62%) patients were dead, all from metastatic disease. Twenty-six (50%) tumours showed expression of VEGF. Statistical analysis showed that patients with tumours with VEGF expression in more than 25% of the cells had significantly shorter overall survival (p=0.019) and disease free intervals (p=0.009).

VEGF is peptide which acts as a stimulator of new blood vessel growth in normal tissues, as well as in some solid tumours and their metastases. A tumour which is able to induce a blood supply has an increased ability to grow, seed metastases and threaten life. Our study is the first to look at VEGF expression in the tumour cells surviving after chemotherapy. It is this population of cells which is important as it is these cells which may go on to develop into metastatic or locally recurrent tumours. The over-expression of VEGF by osteosarcoma cells is thought to be associated with a worse prognosis due to a number of mechanisms. This study shows that VEGF expression is an important prognostic factor in osteosarcomas. Suppression of tumour angiogenesis by inhibition of the action of VEGF has shown promise in animal models as a potential new treatment for osteosarcoma, and warrants further study.

Aims: The purpose of this study is to investigate whether the expression of MMP-9 (matrix metalloproteinase-9) is a potentially useful marker in osteosarcomas. Methods: 55 patients with stage IIB knee osteosarcomas were treated in our unit and had a median follow-up of 68 months. In addition to clinical data, MMP-1, MMP,-2, MMP-3, MMP-7, MMP-9 and MMP-13 were studied in the resection specimens, using immunohistochemical methods. The importance of all factors was studied using the log-rank test, and the overall survival of patients was calculated using Kaplan-Meier survival curves. Multiple variable analysis was carried out using Cox regression models with variables chosen forward and backward stepwise methods with deviance statistics. Signiþcance was set at p< 0.05. Results: On multiple variable analysis only the MMP-9 status of the tumour cells had a signiþcant effect on overall (p=0.032) and disease free survival (p=0.014). Conclusions: Our study shows that some post-chemotherapy osteosarcoma specimens express MMP-9 in the surviving tumour cells after chemotherapy. We believe that MMP-9 in the osteosarcoma cells which survive chemotherapy, contributes to recurrence because of the ability of these cells, to stimulate a new vascular network. The relationship between osteosarcomas and MMP-9 is worthy of further study.

Ischaemic preconditioning is a process by which exposure of a tissue to a short period of non-damaging ischaemic stress leads to resistance to the deleterious effects of a subsequent prolonged ischaemic stress. It has been extensively described in the heart, but few studies have examined the possibility that it can occur in skeletal muscle. We have used a rat model of ischaemia of one limb to examine this possibility. Exposure of the hind limb to a period of ischaemia of five minutes and reperfusion for five minutes significantly protected the tibialis anterior muscle against the structural damage induced by a subsequent period of limb ischaemia for four hours and reperfusion for one hour. This protection was evident on examination of the muscle by both light and electron microscopy. Longer or shorter times of prior ischaemia had no effect.

We studied 55 patients with stage-IIB osteosarcoma around the knee with respect to the expression of matrix metalloproteinase (MMP)-9 in the surviving tumour cells in surgical resection specimens. They were followed up for a minimum of 2.5 years.

Factors significantly associated with poor overall survival were a high serum level of alkaline phosphatase at diagnosis and tumour cells expressing MMP-9 in the resection specimens. The only factor strongly associated with disease-free survival was the immunohistochemical status of tumour cells for MMP-9 in the resection specimens. The percentage of necrosis after chemotherapy failed marginally to reach statistical significance. On Cox regression analysis only MMP-9 remained significant for overall and disease-free survival.