The arcOGEN study identified the 9q33.1 locus as associated with hip osteoarthritis (OA) in females. TRIM32 lies within this locus and may have biological relevance to OA; it encodes a protein with E3 ubiquitin ligase activity.

Sanger sequencing of TRIM32 in the youngest 500 female patients with hip OA from the arcOGEN study identified genetic polymorphisms in the proximal promoter, and 3'untranslated region of TRIM32 that are disproportionately represented in female patients with hip OA compared to the control population.

Reduced expression of TRIM32 was identified in femoral head articular chondrocytes from patients with hip OA compared to control patients. Trim32 knockout resulted in increased aggrecanolysis in murine femoral head explants. Murine chondrocytes deficient in Trim32 exhibited increased expression of mature chondrocyte markers following anabolic cytokine stimulation, and increased expression of hypertrophic chondrocyte markers following catabolic cytokine stimulation.

Trim32 knockout mice demonstrated increased cartilage degradation and tibial subchondral bone changes after surgically-induced knee joint instability. Increased cartilage degradation and medial knee subchondral bone changes were also identified in aged Trim32 knockout mice.

These results further implicate TRIM32 in the genetic predisposition to OA, and indicate a role for TRIM32 in the joint degeneration evident in OA. These results support the further study of TRIM32 in the pathophysiology of OA and development of novel therapeutic strategies to manage OA.

A critical bone defect may be more frequently the consequence of a trauma, especially when a fracture occurs with wide exposure, but also of an infection, of a neoplasm or congenital deformities. This defect needs to be treated in order to restore the limb function. The treatments most commonly performed are represented by implantation of autologous or homologous bone, vascularized fibular grafting with autologous or use of external fixators; all these treatments are characterized by several limitations.

Nowadays bone tissue engineering is looking forward new solutions: magnetic scaffolds have recently attracted significant attention. These scaffolds can improve bone formation by acting as a “fixed station” able to accumulate/release targeted growth factors and other soluble mediators in the defect area under the influence of an external magnetic field. Further, magnetic scaffolds are envisaged to improve implant fixation when compared to not-magnetic implants.

We performed a series of experimental studies to evaluate bone regeneration in rabbit femoral condyle defect by implanting hydroxyapatite (HA), polycaprolactone (PCL) and collagen/HA hybrid scaffolds in combination with permanent magnets.

Our results showed that ostetoconductive properties of the scaffolds are well preserved despite the presence of a magnetic component. Interestingly, we noticed that, using bio-resorbable collagen/HA magnetic scaffolds, under the effect of the static magnetic field generated by the permanent magnet, the reorganization of the magnetized collagen fibers produces a highly-peculiar bone pattern, with highly-interconnected trabeculae orthogonally oriented with respect to the magnetic field lines. Only partial healing of the defect was seen within the not magnetic control groups.

Magnetic scaffolds developed open new perspectives on the possibility to exploiting magnetic forces to improve implant fixation, stimulate bone formation and control the bone morphology of regenerated bone by synergically combining static magnetic fields and magnetized biomaterials. Moreover magnetic forces can be exploited to guide targeted drug delivery of growth factors functionalized with nanoparticles.

TRIM32 is a candidate gene at the 9q33.1 genetic susceptibility locus for hip osteoarthritis (OA). Increased cartilage degradation typical of OA has previously been demonstrated in Trim32 knockout mice.

Our aim is to investigate the role of TRIM32 in human and murine articular tissue.

TRIM32 expression in human articular cartilage was examined by immunostaining. TRIM32 expression was compared in femoral head chondrocytes from patients with and without primary hip OA (n=6/group) and examined by Western blotting. Aggrecanolysis by femoral head explants from Trim32 knockout (T32KO) and wild-type (WT) mice was compared following stimulation with IL1α or retinoic acid (RA) and was assessed by DMMB assay (n=4/group). Expression of chondrocyte phenotype markers was measured by qPCR and compared between articular chondrocytes from WT and T32KO mice following catabolic (IL1α/TNFα) or anabolic (Oncostatin-M (OSM)/IGF1) stimulation.

TRIM32 expression was demonstrated in human articular cartilage; TRIM32 expression by chondrocytes was reduced in patients with hip OA (p=0.03). Greater aggrecanolysis occurred in cartilage explants from T32KO mice after treatment with no stimulation (p=0.03), IL1α (p=0.02), and RA (p=0.001). Unstimulated T32KO chondrocytes expressed reduced Col2a1 (p=8.53×10⁻⁵), and Sox9 (p=2.35×10⁻⁶). Upon IL1α treatment, T32KO chondrocytes expressed increased Col10a1 (p=0.0003). Upon anabolic stimulation, T32KO chondrocytes expressed increased Col2a1 (OSM: p=0.001; IGF: p=0.001), and reduced Sox9 (OSM: p=0.0002; IGF: p=0.0006).

These results indicate that altered TRIM32 expression in human articular tissue is associated with OA, and that Trim32 knockout results in increased cartilage degradation in murine femoral head explants. Predisposition to cartilage degeneration with reduced Trim32 expression may involve increased chondrocyte hypertrophy upon catabolic cytokine stimulation and dysregulation of Col2a1 and Sox9 expression upon anabolic stimulation.

The cartilage diseases such as osteoarthritis and chondral injuries are considered irreversible and the result of recent treatments remains not optimal. One of the reasons is due to the poor understanding of chondrocyte behaviours. To understand more about cartilage, we designed a series of novel experiments. First, a total joint of bovine metatarsophalanges was isolated as our novel model. We chose it because the configuration and the healing potential were similar to human, and many variables of large animal studies could be controlled in laboratory. The model not only provided a good ex vivo platform for cartilage researches but also connected in vitro cellular studies and in vivo animal studies. To mimic joint movement a special driving machine was designed. To characterise the novel model viabilities of chondrocytes and contents of sulphated glycosaminoglycan (GAGs) in extracellular matrixes were measured every seven days. The preliminary results revealed the viabilities of chondrocytes remained above 80% alive in the middle zone after four-weeks culture. The GAGs contents decreased after this culturing period. The experiments still carry on going to compare the static and dynamic models which joint movement could be a determinative factor to the viability of chondrocytes. Cellular treatment is the recent mainstream for cartilage diseases. If advanced knowledge in chondrocyte behaviours could be obtained from this model, development of optimal treatment will be possible in the future.

Background

Hyperlaxity is associated with a high incidence of shoulder dislocations. 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, capsule strength, collagen V and SLRP's expression.

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.

Fractures repair by two mechanisms; direct fracture healing and indirect fracture healing via callus formation. Research concerning the effects of bisphosphonate on fracture repair has solely assessed indirect fracture healing. Patients with osteoporosis on bisphosphonates continue to sustain fragility fractures. A proportion of osteoporotic fractures require plate fixation. Bisphosphonates impair osteoclast activity and therefore, may adversely affect direct fracture healing that predominates with plate fixation.

Five skeletally mature Sprague-Dawley rats received daily subcutaneous injections of 1mg/kg Ibandronate (IBAN). Similarly, five control rats received saline (CONTROL). Three weeks following commencement of injections a tibial osteotomy was rigidly fixed with compression plating similar to that seen in routine clinical practice. Fracture healing was monitored with radiographs. Six weeks post plate fixation, animals were sacrificed. Radiographs were performed of the extricated tibiae following plate removal. The visibility of the osteotomy site was scored as totally visible, partially visible or absent as previously described. Mechanical testing was conducted on the healing osteotomies via 4-point bending.

Fractures healed without visible external callus. In the IBAN group three animals had totally visible osteotomy lines and two had partially visible osteotomy lines. The CONTROL group had three animals with absent osteotomy lines and two with partially visible osteotomy lines. The mean (±SD) stress at failure for the healing tibial osteotomies at 6 weeks was 28.8 (±23.97)MPa in the IBAN group and 37.4(±29.20) MPa in the CONTROL group (p=0.62)

Our results indicate that Ibandronate adversely affected direct fracture repair as demonstrated by the radiographic density of the fracture line. The strength of the repair was reduced but this did not reach statistical significance. Our results suggest that a sample size of 220 animals is required to detect a 15% difference (alpha 0.05, beta 0.2) which suggests the effect of bisphosphonates on direct fracture repair may be small.

Objective: Meniscus regeneration is limited, moreover, loss of meniscal tissue leads to osteoarthritis. A new biomaterial, consisting of hyaluronic acid and polycaprolactone was applied as a meniscus substitute device in a study in sheep.

Methods: 24 sheep received a total medial meniscal replacement. Group SCF (n=12) was treated with a cell free scaffold, Group SCS (n=12) with a scaffold seeded with autologous chondrocytes harvested from the contralateral joints, which served as sham controls (n=12). Further 12 non-operated and 2 menisectomy controls were included in the study.

The animals were sacrificed after 4 months. The implants and joint surfaces were evaluated on a macroscopic (Implant Gross Assessment Score; Gross Assessment of Joints Score) and histological level.

Results: There was no significant difference of the Implant Gross Assessment score between the SCF and SCS groups. All implants showed excellent capsular ingrowth at the periphery. Dislocation, extrusion and tears occurred in part of the specimen due to biomechanical problems caused by soft tissue quality. The mean Gross Assessment of Joint Changes Score of the groups SCF and SCS was not statistically different. Cartilage damage was significantly more severe in joints with implants than in non-operated joints and sham controls, but less pronounced than in menisectomy controls. Histological evaluation showed residual scaffold with an associated foreign body response in all implants. Fibrous tissue was present in all implants, in contrast small foci of cartilaginous differentiation were more common in the cell-seeded constructs.

Discussion: At 4 months regenerative meniscal tissue was present but immature. The present study showed that strong biomechanical scaffold properties are a required to allow guided tissue regeneration and maturation under loading conditions. Cell seeding of the scaffold encourages cartilaginous differentiation. Modification of the scaffold and the cell-seeding technique will be investigated in further studies.

The stress response to trauma is the summation of the physiological response to the injury (the ‘first hit’) and by the response to any on-going physiological disturbance or subsequent trauma surgery (the ‘second hit’).

Our animal model was developed in order to allow the study of each of these components of the stress response to major trauma. High-energy, comminuted fracture of the long bones and severe soft-tissue injuries in this model resulted in a significant tropotropic (depressor) cardiovascular response, transcardiac embolism of medullary contents and activation of the coagulation system. Subsequent stabilisation of the fractures using intramedullary nails did not significantly exacerbate any of these responses.

If an arthroplasty patient presents with wound breakdown, sinus formation or a hot, red joint the diagnosis of infection is straightforward. However, most total joint replacement (TJR) infections are difficult to distinguish from aseptic loosening. It is imperative to know if a painful TJR is infected to plan appropriate management.

In this prospective study of 204 patients we analysed the diagnostic accuracy of various tests for infection: Inflammatory Markers (CRP/ESR); Aspiration Microbiology; and the Polymerase Chain Reaction (PCR) – a novel technique in this situation. We used international criteria as the gold standard for infection, applied at the time of revision surgery. Any of – a sinus; frank pus in the wound; positive intra-operative microbiology; positive histology – classified the patient as infected. The sensitivity (Sens), specificity (Spec), positive predictive value (PPV) and negative predictive value (NPV) of each test were calculated.

52 patients with an original diagnosis of inflammatory arthritis were excluded, as histology may be inaccurate. The results for the remaining 152 patients are: CRP > 20mg/l: Sens 77%; Spec 76%; PPV 49%; NPV 92%. ESR > 30 mm/hr: Sens 61%; Spec 86%; PPV 57%; NPV 87%. Aspiration Microbiology: Sens 80%; Spec 83%; PPV 71%; NPV 88%. PCR: Sens 71%; Spec 78%; PPV 43%; NPV 89%.

Few patients with negative CRP/ESR were found to be infected; if positive, there was a 50/50 chance that the joint was infected. Positive aspiration microbiology was associated with underlying infection 3 times out of every 4, and negative results were correct 9 times out of 10. PCR was no more accurate than existing tests.

All patients with painful TJR’s should have inflammatory markers checked – if negative the clinician can be relatively reassured that the implant is not infected. If positive or suspicion remains, further investigation should be undertaken. Joint aspiration for microbiology is currently the best available second line investigation.

Introduction If an arthroplasty patient presents with wound breakdown, sinus formation or a hot, red, painful joint replacement the diagnosis of infection is relatively straightforward. However, most total joint replacement (TJR) infections present in an indolent fashion and are impossible to distinguish from aseptic loosening. It is imperative to know if pain in a TJR is due to infection to plan appropriate further management.

Methods In this prospective study of 204 patients we analysed the diagnostic accuracy of various tests for infection in the setting of TJR: Inflammatory Markers (CRP/ESR); Aspiration Microbiology; and the Polymerase Chain Reaction (PCR) – a novel technique in this situation. We used internationally agreed criteria as the gold standard for infection. The patient was deemed to be infected if any of the following were found at the time of revision surgery: a sinus; frank pus in the wound; positive microbiology or positive histology on intra-operative specimens. The sensitivity (Sens), specificity (Spec), positive predictive value (PPV) and negative predictive value (NPV) of each test were calculated.

Results 52 patients with an original diagnosis of inflammatory arthritis were excluded, as histology may be inaccurate. Their results have been presented elsewhere. The results for the remaining 152 patients are: CRP > 20mg/l: Sens 77%; Spec 76%; PPV 49%; NPV 92%. ESR > 30 mm/hr: Sens 61%; Spec 86%; PPV 57%; NPV 87%. Aspiration Microbiology: Sens 80%; Spec 83%; PPV 71%; NPV 88%. PCR: Sens 71%; Spec 78%; PPV 43%; NPV 89%.

Findings and Conclusions Only a few of the patients with negative inflammatory markers later turned out to be infected. If the inflammatory markers were positive, there was roughly a 50/50 chance that the joint was infected. Positive aspiration microbiology was associated with underlying infection approximately 3 times out of every 4, and negative results were correct 9 times out of 10. PCR was no more accurate than existing tests.

We recommend that all patients with painful TJRs have inflammatory markers checked as a screening test – if negative then the clinician can be relatively reassured that the implant is not infected. If positive, further investigation should be undertaken. Joint aspiration for microbiology is currently the best available second line investigation.

We investigated the use of PCR (the Polymerase Chain Reaction) to detect the presence of infection in a group of patients undergoing revision arthroplasty for loose TJR (total joint replacement), compared to internationally agreed criteria used as the ‘gold standard’ for infection.

We prospectively tested samples taken from 108 patients undergoing revision arthroplasty (76 hips, 32 knees). Antibiotics were omitted prior to obtaining samples. DNA was extracted by 2 methods – a previously published technique (reference) and a commercial extraction kit (Qiagen^®). PCR involved amplification of an 882 base pair segment of the universal bacterial 16S RNA gene. During revision arthroplasty multiple specimens were taken from around the joint for microbiological and histological examination and the presence or absence of pus was noted. The patient was deemed to be infected if one of the following criteria was found: presence of a sinus pre-operatively; 2 or more intra-operative cultures positive for the same organism; an acute inflammatory response on histology; pus in the joint at revision.

Using the published DNA extraction technique PCR had a sensitivity of 50%, specificity of 93%, positive predictive value of 67% and negative predictive value of 88%. Using commercial extraction the sensitivity improved to 60%, specificity to 98%, positive predictive value to 90% and negative predictive value to 90%.

The previous report stated that PCR had a high sensitivity but a low specificity for detecting low grade infection. However, when using the published technique we found the opposite results – a moderate sensitivity and a high specificity. Introduction of a new DNA extraction technique improved the sensitivity. The refined PCR technique had a high accuracy, but further work is needed to improve sensitivity before we would recommend this method for routine clinical use.

Detection of infection in total joint replacements (TJR) is notoriously difficult. Ideally the diagnosis should be known before revision arthroplasty is undertaken. The level of C-reactive protein (CRP) is one readily available test. Sanzen et al. reported sensitivity of 78% and specificity of 100% for CRP in distinguishing infection in 23 infected TJRs and 33 non-infected TJRs undergoing revision, using a cut off of 2mg/dl¹. However, they used only intra-operative cultures as the standard to compare the CRP against. We have analysed the reliability of CRP to diagnose infection pre-operatively in a group of patients undergoing revision arthroplasty, using the following criteria as the reference standard for infection: 2 or more intra-operative cultures positive for the same organism; presence of acute inflammatory response on histology; presence of pus in the joint at revision (1/3 positive indicates true infection), as described by Hanssen et al.²

The results of CRP and the operative investigations of 26 patients undergoing revision arthroplasty (15 hips and 11 knees) were studied prospectively. In our unit CRP is assayed in mg/dl serum by an automated machine. During revision arthroplasty, multiple specimens were taken from around the joint for microbiological and histological examination. Microbiological cultures were carried out on solid media and broth in aerobic and anaerobic conditions. Histological analysis assessed the level of neutrophils present in the tissue. The presence or absence of pus was noted. The results were analysed graphically and a cut off level of CRP was then chosen for analysis of reliability.

Thirteen patients were infected and 13 were not. Eleven of the 13 infected patients had a CRP greater than 2 mg/dl, and 10 of the 13 non-infected patients had a CRP less than 2 mg/dl. Using 2 mg/dl as a cut off, CRP had a sensitivity of 85% and a specificity of 77%. If 4mg/dl is taken as the threshold for infection, then CRP is 100% specific but only 61% sensitive.

CRP is a useful investigation in the diagnosis of infection in joint replacements. However we have shown that a cut off of 2mg/dl is not 100% specific for non-infected patients. Increasing the threshold improves the specificity, but reduces the sensitivity. Unfortunately there is no single investigation that is 100% accurate in this setting. CRP results must be interpreted in the light of the clinical picture and other investigations. These patients are part of an ongoing study to identify the most reliable criteria for diagnosing the presence of infection in total joint replacement.

Pre-revision detection of infection in failed total joint replacements (TJR) is essential to allow appropriate management planning. Unfortunately, low-grade infection is often difficult to detect. The use of molecular biology may offer increased sensitivity in this setting. We have analysed the use of the Polymerase Chain Reaction (PCR) to diagnose infection in pre-operative aspirates in a group of patients undergoing revision arthroplasty. We prospectively tested 50 aspirates in 50 patients with failed TJR (34 hips and 16 knees). Antibiotics were omitted for 2 weeks prior to aspiration. The aspirate was sent for microbiological culture in aerobic and anaerobic conditions. An aliquot was retained for PCR analysis which involved DNA extraction then amplification of an 882 base pair segment of the Universal 16S RNA gene. In 33 patients who subsequently underwent revision arthroplasty multiple specimens were taken from around the joint for microbiological and histological examination and the presence or absence of pus was noted. The patient was deemed to be infected if one of these criteria was found: 2 or more intra-operative cultures positive for the same organism; an acute inflammatory response on histology; pus in the joint at revision ¹.

PCR was positive in 29 cases. Aspiration microbiology was positive in 13 cases. Of the 33 cases revised, 15 patients were deemed to be infected using the previously established criteria, described above. Compared to preoperative aspiration microbiology PCR had a sensitivity of 92% and specificity of 54%. Compared to the published criteria for infection, PCR was 93% sensitive and 61% specific. If rheumatoid cases are excluded the specificity improves to 71%.

It was concluded that PCR has the ability to amplify very small amounts of target DNA. The apparently high false positive rate compared to aspiration microbiology may indicate that PCR is picking up DNA from contaminating or non-viable organisms (treated or phagocytosed), giving poor specificity. However, microbiology is known to have poor sensitivity on pre-operative aspiration samples, and some of the microbiology results may be false negative. Compared to the criteria for infection after revision our results for PCR are more encouraging, especially for non-rheumatoid patients. These patients are part of an ongoing study to identify the most reliable criteria for pre-operative diagnosis of infection in total joint replacement.