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Orthopaedic Proceedings
Vol. 102-B, Issue SUPP_7 | Pages 59 - 59
1 Jul 2020
Qiu H Cheng T Chim SM Zhu S Xu H Qin A Wang C Teguh D Zhang G Tickner J Yao F Vrielink A Smithers L Pavlos N Xu J
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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.


Orthopaedic Proceedings
Vol. 101-B, Issue SUPP_4 | Pages 142 - 142
1 Apr 2019
Murphy W Lane P Lin B Cheng T Terry D Murphy S
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INTRODUCTION

In the United States, the Centers for Medicare and Medicaid Services consider rates of unplanned hospital readmissions to be indicators of provider quality. Understanding the common reasons for readmission following total joint arthroplasty will allow for improved standards of care and better outcomes for patients. The current study seeks to evaluate the rates, reasons, and Medicare costs for readmission after total hip and total knee arthroplasty.

METHODS

This study used the Limited Data Set (LDS) from the Centers for Medicare and Medicaid Services (CMS) to identify all primary, elective Total Knee Arthroplasties (TKA) and Total Hip Arthroplasties (THA) performed from January 2013 through June 2016. The data were limited to Diagnosis-Related Group (DRG) 470, which is comprised of major joint replacements without major complications or comorbidities. Readmissions were classified by corresponding DRG. Readmission rates, causes, and associated Medicare Part A payments were aggregated over a ninety-day post-discharge period for 804,448 TKA and 409,844 THA.