Bone is a connective tissue that undergoes constant remodeling. Any disturbances during this process may result in undesired pathological conditions. A single nucleotide substitution (596T-A) in exon eight which leads to a M199K mutation in human RANKL was found to cause osteoclast-poor autosomal recessive osteopetrosis (ARO). Patients with ARO cannot be cured by hematopoietic stem cell transplantation and, without proper treatments, will die in their early age. To date, how this mutation alters RANKL function has not been characterized. We thus hypothesized that hRANKL M199 residue is a structural determinant for normal RANKL-RANK interaction and osteoclast differentiation. By sharing our findings, we aim to achieve an improved clinical outcome in treating bone-related diseases such as osteoporosis, ARO and osteoarthritis.

Site-directed mutagenesis was employed to create three rat RANKL mutants, replacing the methionine 200 (human M199 equivalent residue) with either lysine (M200K), alanine (M200A) or glutamic acid (M200E). Recombinant proteins were subsequently purified through affinity chromatography and visualized by Coomassie blue staining and western blot. MTS was carried out before osteoclastogenesis assay in vitro to measure the cellular toxicity. Bone resorption pit assay, immuno-fluorescent staining, luciferase reporter assay, RT-PCR, western blot and calcium oscillation detection were also conducted to explore the biological effect of rRANKL mutants. Computational modeling, thermal Shift Assay, western blot and protein binding affinity experiments were later carried out for structural analyses.

rRANKL mutants M200K/A/E showed a drastically reduced ability to induce osteoclast formation and did not demonstrate features of competitive inhibition against wild-type rRANKL. These mutants are all incapable of supporting osteoclastic polarization and bone resorption or activating RANKL-induced osteoclast marker gene transcription. Consistently, they were unable to induce calcium flux, and also showed a diminished induction of IκBa degradation and activation of NF-kB and NFATc1 transcriptional activity. Furthermore, the transcriptional activation of the antioxidant response element (ARE) crucial in modulating oxidative stress and providing cytoprotection was also unresponsive to stimulation with rM200s. Structural analyses showed that rM200 is located in a hydrophobic pocket critical for protein folding. Thermal shift and western blot assays suggested that rM200 mutants formed unstructured proteins, with disturbed trimerisation and the loss of affinity to its intrinsic receptors RANK and OPG.

Taken together, we first demonstrates the underlying cause of M199-meidated ARO in a cellular and molecular level by establishing a phenotype in BMMs similar to observed in human samples. Further investigation hints the structural significance of a hydrophobic pocket within the TNF-like region. Combined with pharmaceutical studies on small-molecule drugs, this finding may represent a therapeutic target motif for future development of anti-resorptive treatments.

Osteoarthritis (OA) is traditionally believed to affect the osteochondral unit by wear-and-tear from the superficial zone to the deep zone of cartilage and extended to subchondral plate. Obesity is commonly considered as a risk of OA development and hence total knee replacement (TKR), but the mechanism remains unclear. We hypothesized that obesity accelerated OA development by deteriorating tidemarks and increasing bone remodelling. 616,495 cases of TKR for OA from Australia and British joint replacement registries were collected, and data indicated that patients with higher BMI had TKR at earlier age. Specifically, patients with BMI ≤25kg/m² showed 8 years younger than patients with BMI ≥40kg/m² (P<0.0001) when they received TKR. We next examined tibia plateaus of 88 knee OA patients by micro-CT and histomorphometry. Linear regression showed that less cartilage degradation was associated with increased BMI in the load-bear compartment (p<0.05), while 58.3% of patients with BMI≥40kg/m² demonstrated a clear anatomical separation close to tidemarks filled with fibrosis, erythrocytes and bone fragments (compared to BMI ≤25kg/m² group: 7.7%, p<0.01). In subchondral bone, elevated bone formation was associated with increased BMI, as higher thickness of osteoid (p<0.01), percent osteoid volume (p<0.01), percent osteoid surface (p<0.01) were found in obese patients. However, no alteration of bone resorption and microstructural parameters was found to be associated with BMI. We suspected that the abnormal loading in knee joint due to high BMI led to the direct deterioration of binding site of osteochondral unit, which might be the mechanism of the rapid progression in obesity-related OA.

Introduction: There are no current estimates of the risk of transmission of HIV, HBV, HCV, or HTLV by musculoskeletal tissue transplantation. Such accurate data would be helpful to determine the effectiveness of current and proposed screening and processing procedures, and contribute to increased confidence in the use of musculoskeletal tissue products.

Methods: The prevalence rates of HIV, HBV, HCV, and HTLV were determined from 12.245 musculoskeletal tissue donors from three bone tissue banks across Australia from the period 1993 to 2004. The incidence rates among tissue donors were estimated by comparing the data with age-specific incidence rates of first-time blood donors. We estimated the probability of a tissue donor was within the window period when infection was undetected by serological screening procedures by the modified incidence-window period model. Further we calculated the projected probability of viremia with the addition of nucleic-acid amplification testing (NAT).

Results: The prevalence (per 100,000 persons) of confirmed positive tests among musculoskeletal tissue donors was 169.15 for HIV, 427.68 for HBV, 534.63 for HCV, and 121.66 for HTLV. This is greater than the prevalence among first-time blood donors during the same period (6.47 for HIV, 136.00 for HBV, 215.29 for HCV, and 3.46 for HTLV). The incidence rate among musculoskeletal donors were estimated to be 15.81, 0.68, 3.53, and 4.85 per 100,000 person-years, respectively. The estimated probability of viremia (per 100,000 persons) at the time of donation was 1.38 for HIV, 0.46 for HBV, 1.82 for HCV, and 0.85 for HTLV. These estimations would be even lower with the addition of NAT – 0.57, 0.23, and 0.20 respectively.

Conclusions: The prevalence and incidence of HIV, HBV, HCV, and HTLV among musculoskeletal tissue donors, although low are significantly higher than those of first-time blood donors. Current screening and processing measures are effective, though the probability of viremia can be reduced further by nucleic-acid amplification testing.

Introduction We believe minimally invasive surgery should be defined by the extent of soft tissue dissection rather than incision length. We describe a new technique that is truly soft-tissue sparing and report our early results.

The surgical approach The landmarks for the incision are identified and a 6–8cm oblique incision is made over the posterior aspect of the greater trochanter. Longer incisions are required in more difficult cases. Piriformis and the proximal insertion of gluteus maximus are preserved. After implant insertion, meticulous capsular repair is performed through drill holes into bone to reconstruct the posterior envelope. There are no restrictions to mobility. No specialised instruments are required.

Method The standard posterior approach (group 1) was compared with the PSMI approach (group 2) in a prospective cohort study of 200 consecutive patients over 60 years of age. In the standard approach the external rrotators were dettached. The capsule was repaired to bone, and the piriformis tendon reattached to the Gluteus Medius tendon. Routine restrictions to mobility were imposed. Patients were scored pre-operatively and followed up prospectively, by a blinded observer.

Results Demographics and functional scores were similar. Mean operation time was about 1 hour in both groups. Mean incision length was 21.5 cm (range 15 – 25) in group 1 and 8.4 cm (range 6 – 16) in group 2. Mean blood loss in group 1 was significantly higher than group 2 (P< 0.0001, 95%CI 191–547). Mean inpatient stay was 8.0 days in group 1, and 4.8 days in group 2 (P< 0.0001, 95%CI 3.4–6.0).

Minimum follow-up was 3 years in group 1 and 1.5 years in group 2. There were 3 dislocations in group 1, and none in group 2. There were 2 re-operations in both groups. The relative improvement in WOMAC scores was significantly greater in group 2 at 3 months and 1 year (P< 0.05).

Conclusion The PSMI approach to the hip is truly soft-tissue sparing. It is safe and relatively easy to perform. The stability and minimal morbidity allow early mobilisation. This is the first study to suggest the benefits of minimally invasive surgery may be prolonged. Cosmesis is a by-product rather than primary objective.