Aims

The aim of this study was to investigate the safety and efficacy of 3D-printed modular prostheses in patients who underwent joint-sparing limb salvage surgery (JSLSS) for malignant femoral diaphyseal bone tumours.

Aims

Monocyte-lymphocyte ratio (MLR) or neutrophil-lymphocyte ratio (NLR) are useful for diagnosing periprosthetic joint infection (PJI), but their diagnostic values are unclear for screening fixation-related infection (FRI) in patients for whom conversion total hip arthroplasty (THA) is planned after failed internal fixation for femoral neck fracture.

Aims

The morphology of medial malleolar fracture is highly variable and difficult to characterize without 3D reconstruction. There is also no universally accepeted classification system. Thus, we aimed to characterize fracture patterns of the medial malleolus and propose a classification scheme based on 3D CT reconstruction.

Aims

This study aimed to investigate the effect of solute carrier family 20 member 2 (SLC20A2) gene mutation (identified from a hereditary multiple exostoses family) on chondrocyte proliferation and differentiation.

Aims

The efficacy and safety of intrawound vancomycin for preventing surgical site infection in primary hip and knee arthroplasty is uncertain.

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.

Prosthetic reconstruction of high-riding hips is technically demanding. Insufficient bony coverage and osteopenic bone stock frequently necessitate transacetabular screw fixation to augment primary stability of the metal shell. We sought to determine the validity of the previously reported quadrant system, and if needed, to define a specialized safe zone for augmentation of screw fixation to avoid vascular injuries in acetabular cup reconstruction for high-riding hips.

Volumetric data from computed tomography enhancement scanning and CT angiography of eighteen hips (twelve patients) were obtained and input into a three-dimensional image-processing software. Bony and vascular structures were reconstructed three-dimensionally; we virtually reconstructed a cup in the original acetabulum and dynamically simulated transacetabular screw fixation. We mapped the hemispheric cup into several areas and, for each, measured the distance between the virtual screw and the blood vessel.

We found that the rotating centers of the cups shifted more anterior-inferiorly in high-riding hips than those in ordinary cases, and thus the safe zone shifted as well. Screw fixation guided by the quadrant system frequently injured the obturator blood vessels in high-riding hips. We then defined a specialized safe zone for transacetabular screw fixation for high-riding hips.

We conclude that the quadrant system can be misleading and of less value in guiding screw insertion to augment metal shells for high-riding hips. A new safe zone specific to high-riding hips should be used to guide transacetabular screw fixation in these cases.

We reviewed the outcome of a retrospective case series of eight patients with atlantoaxial instability who had been treated by percutaneous anterior transarticular screw fixation and grafting under image-intensifier guidance between December 2005 and June 2008.

The mean follow-up was 19 months (8 to 27). All eight patients had a solid C1–2 fusion. There were no breakages or displacement of screws. All the patients with pre-operative neck pain had immediate relief from their symptoms or considerable improvement. There were no major complications. Our preliminary clinical results suggest that percutaneous anterior transarticulation screw fixation is technically feasible, safe, useful and minimally invasive when using the appropriate instruments allied to intra-operative image intensification, and by selecting the correct puncture point, angle and depth of insertion.

Introduction: Recently, co-transplantion of mesenchy-mal stem cells (MSCs) with hematopoietic stem cells (HSCs) has been shown to alleviate complications such as GVHD and speeding recovery of HSCs. This in vivo finding suggests that coculture of MSCs and HSCs may enhance their growth potentials in vitro. As the large-scale expansion of HSCs has been achieved by NASA’s suspension culture system, we further examined the effects of this suspension culture system (rotary bio-reactor) on MSCs’ proliferation and differentiation potentials in vitro.

Methods: Mononuclear cell fractions (MNCs) of human bone marrow aspirates (n=6, ages 46–81) were collected by density gradient centrifugation. The cells were inoculated into bioreactor (RCCS, Synthecon Inc., Texas, USA) at the concentration 1x10⁶ cells/ml, in Myelocult^TM medium supplemented with 50ng/ml SCF, 20ng/ ml rhIL-3 and rhIL-6 (10ng/ml SCF, 2ng/ml IL-3 and IL-6 after the first feeding) and 10-6 M hydrocortisone for 8 days. The medium was fully exchanged after 3 days and 20% daily thereafter. Total cell numbers in the bioreactor were counted daily using hemacytometer. Cells from day 1, 4, and 8 cultures were subjected to tri-color flow cytometry examination using CD34, CD44, and Stro-1 antibodies. By the end of 8 day culture, the output cells were resuspended in DMEM medium with 10% FBS and cultured in T75 flasks at 1x10⁵ cells/cm2 for further 3 weeks. Upon harvest, half of the attached MSCs were prepared for western blotting assay using various antibodies. The other half was further cultured for 13–28 days in osteogenic, chondrogenic, and adipogenic induction medium respectively. Cell differentiation results were examined by histology staining, immunohistochemistry (ICC) and transmission electron microscope (TEM) examinations.

Results: After 8-day culture in bioreactor, flow-cytometric analysis confirmed that two cell populations, CD34+CD44+ (HSCs) and Stro-1+CD44+ (MSCs), increased 8-fold and 29-fold respectively, when compared to the values of the MNCs prior to bioreactor treatment. Cell counting revealed that the total cell expansion over 8 days was 9-fold above the number of the input MNCs. Western blotting data confirmed that bioreactor-expanded MSCs population remained in their early-stage with the expression of primitive MSCs markers such as CD105 (endoglin, SH-2) and Vimentin, whereas no expression of differentiation markers including osteocalcin (osteogenesis), Type II collegen (chondrogenesis) and C/EBPα (adipogenesis). Upon differentiation induction, the bioreactor-expanded MSCs were capable of differentiating into osteocytes, chondrocytes, and adipocytes as evidenced by histology staining, ICC and TEM examinations.

Discussion: Our study has shown that the percentage of MSCs (Stro-1+CD44+) increased 29 folds in the bone marrow derived MNCs after they have been cultured with Myelocult¢â medium in bioreactor for 8 days. The suspension culture system did not affect the subsequent in vitro proliferation and differentiation potentials of MSCs. Current study indicates that rotary bioreactor may be used to rapidly expand the numbers of traditionally attachment-dependent MSCs from bone marrow-derived MNCs, which may be very useful in clinical tissue engineering applications.

Purpose: Osteolysis associated with periprosthetic loosening is generally associated with the presence of wear particle-associated macrophages which (i) release inflammatory cytokines (e.g. TNFα and IL-1α) and (ii) are capable of osteoclast differentiation and bone resorption. The recently identified molecule, RANKL (expressed on osteoblastic cells) has been shown to play a central role in the macrophage-osteoclast differentiation observed in aseptic loosening. However, as TNFα and IL-1α are abundant in periprosthetic tissues and have been shown to mediate wear particle (bone cement)-associated osteolysis in animal models, and as we have recently shown that TNFa can induce osteoclastogenesis in a manner independent of RANKL mechanism, the aim of the present study was to determine whether wear particles, in particular bone cement particles, can affect RANKL- and TNFα-induced osteoclast formation and bone resorption in vitro.

Methods: Murine monocytes were cultured on glass coverslips and dentine slices with or without PMMA particles in presence of:- (i) macrophage colony stimulating factor (M-CSF) alone, (ii) M-CSF + soluble RANKL (iii) M-CSF + TNFα or (iv) M-CSF + TNFα + IL-1a. All cultures were maintained for 7–10 days after which the extent of osteoclast differentiation was determined by the expression of specific osteoclast markers including tartrate-resistant acid phosphatase (TRAP) on coverslips and evidence of lacunar resorption on dentine slices.

Results: Extensive osteoclast formation and lacunar resorption was evident in monocyte cultures in the presence of soluble RANKL and M-CSF. Addition of PMMA in these cultures increased the extent of RANKL-induced lacunar resorption by about 2 fold. In the absence of soluble RANKL, but in the presence of TNFα (± IL-1α), murine monocytes were also capable of differentiating into active bone resorbing osteoclasts. Addition of PMMA particles to these cultures resulted in a marked increase in the TNFα-induced osteoclas-togenesis. It is worth noting that monocyte cultures containing M-CSF and PMMA particles only did not differentiate into bone resorbing osteoclasts.

Conclusion: These results indicate that PMMA particles can activate both RANKL- and cytokine-induced osteoclast formation and osteolysis. Although, we had previously shown the existence of these two distinct cellular mechanisms in periprosthetic loosening, this is the first report in which wear particles have directly been shown to stimulate these cellular mechanisms independently. Our findings could provide possible therapeutic approaches to control the wear particle-associated early failure of joint replacements.