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.

Introduction

Blood loss after TKA varied, but not uncommon with up to 1500 ml or a decrease in hemoglobin of 3–4 g/dL. In addition to improving prosthetic alignment, computer-assisted TKAs also contribute to reduced operative blood loss and systemic emboli. These observations imply that navigation TKAs may cause less microvascular endothelial damage than conventional TKAs. Cell adhesion molecules (CAMs) have been employed as markers for endothelial or vascular damage. We hypothesized serum levels of CAMs in patients receiving navigation TKAs may be different from those receiving conventional TKAs.

D-dimer is one of the useful laboratory tests to evaluate the incidence of venous thromboembolism (VTE) after the total knee arthroplasty (TKA). The most recent guideline for the prophylaxis of VTE points out the surgical procedure itself is a major risk factor for developing VTE.

Only a few literatures discuss the relationship of surgical procedures and the risk of venous thromboembolism. We therefore prospectively compare the difference of the perioperative plasma D-dimer levels between the patients undergoing navigation and convention TKA.

Two hundred consecutive total knee arthroplasties were performed between September 2011 and March 2013. The patients were randomised according to their registration to the orthopaedic clinic. Ninety-six patients (100 knees) underwent a navigation-assisted TKA and ninety-four patients (100 knees) had a conventional TKA. No intramedullary violation was done in the navigation-assisted TKA, while the intramedullary femoral guiding was adapted in the conventional group.

Pre-operative and post-operation day 1 plasma D-dimer levels were recorded and evaluated using Mann-Whitney U test. There was no difference in the demographic data and pre-operative D-dimer between the two groups (p=0.443). Significantly lower D-dimer levels on the post-operative day 1 were noted in the navigation group, when compared with the conventional group. (6.0 ± 4.4 mg/L vs 11.3 ± 9.6 mg/L, p = 0.000).

We demonstrated that lower D-dimer level is developed after the navigation-assisted TKA than the conventional one. Less incidence of VTE is expected and the finding may help to explain the fact that less systemic emboli in the navigation assisted TKA.

Background:

Full thickness cartilage defect of the knee frequently resulted in fibrous tissue formation, and larger lesions often lead to degenerative arthritis of the knee. Many techniques are designed to repair the cartilage defect including chondrocyte transplantation, microfracture and osteochondral graft. Each method has achieved some success but no universal results. Autologus osteochondral graft has gained in clinical popularity because of its technical feasibility and cost effectiveness.

Background:

Massive rotator cuff tear can cause functional disability due to instability and degenerative changes of the shoulder joint. In patients with massive irreparable rotator cuff tear, tendon transfer is often used as the salvage procedure. Latissimus dorsi and pectoris major transfer are technically demanding procedures and may incur complications. The biceps tendon transfer may provide a biologically superior tissue patch that improves the biomechanics of the shoulder joint in patients with irreparable rotator cuff tear. This study evaluated the functional outcomes of biceps tendon transfer for irreparable rotator cuff tear in 6 patients with two years and longer follow-up.