Jie Tong

P2RX7 Purinoceptor::a therapeutic target for ameliorating the symptoms of Duchenne muscular dystrophy

Background Duchenne muscular dystrophy (DMD) is the most common inherited muscle disease, leading to severe disability and death in young men. Death is caused by the progressive degeneration of striated muscles aggravated by sterile inflammation. The pleiotropic effects of the mutant gene also include cognitive and behavioral impairments and low bone density. Current interventions in DMD are palliative only as no treatment improves the long-term outcome. Therefore, approaches with a translational potential should be investigated, and key abnormalities downstream from the absence of the DMD product, dystrophin, appear to be strong therapeutic targets. We and others have demonstrated that DMD mutations alter ATP signaling and have identified P2RX7 purinoceptor up-regulation as being responsible for the death of muscles in the mdx mouse model of DMD and human DMD lymphoblasts. Moreover, the ATP–P2RX7 axis, being a crucial activator of innate immune responses, can contribute to DMD pathology by stimulating chronic inflammation. We investigated whether ablation of P2RX7 attenuates the DMD model mouse phenotype to assess receptor suitability as a therapeutic target. Methods and Findings Using a combination of molecular, histological, and biochemical methods and behavioral analyses in vivo we demonstrate, to our knowledge for the first time, that genetic ablation of P2RX7 in the DMD model mouse produces a widespread functional attenuation of both muscle and non-muscle symptoms. In dystrophic muscles at 4 wk there was an evident recovery in key functional and molecular parameters such as improved muscle structure (minimum Feret diameter, p < 0.001), increased muscle strength in vitro (p < 0.001) and in vivo (p = 0.012), and pro-fibrotic molecular signatures. Serum creatine kinase (CK) levels were lower (p = 0.025), and reduced cognitive impairment (p = 0.006) and bone structure alterations (p < 0.001) were also apparent. Reduction of inflammation and fibrosis persisted at 20 mo in leg (p = 0.038), diaphragm (p = 0.042), and heart muscles (p < 0.001). We show that the amelioration of symptoms was proportional to the extent of receptor depletion and that improvements were observed following administration of two P2RX7 antagonists (CK, p = 0.030 and p = 0.050) without any detectable side effects. However, approaches successful in animal models still need to be proved effective in clinical practice. Conclusions These results are, to our knowledge, the first to establish that a single treatment can improve muscle function both short and long term and also correct cognitive impairment and bone loss in DMD model mice. The wide-ranging improvements reflect the convergence of P2RX7 ablation on multiple disease mechanisms affecting skeletal and cardiac muscles, inflammatory cells, brain, and bone. Given the impact of P2RX7 blockade in the DMD mouse model, this receptor is an attractive target for translational research: existing drugs with established safety records could potentially be repurposed for treatment of this lethal disease.

Microdamage assessment of bone-cement interfaces under monotonic and cyclic compression.

Bone-cement interface has been investigated under selected loading conditions, utilising experimental techniques such as in situ mechanical testing and digital image correlation (DIC). However, the role of bone type in the overall load transfer and mechanical behaviour of the bone-cement construct is yet to be fully quantified. Moreover, microdamage accumulation at the interface and in the cement mantle has only been assessed on the exterior surfaces of the samples, where no volumetric information could be obtained. In this study, some typical bone-cement interfaces, representative of different fixation scenarios for both hip and knee replacements, were constructed using mainly trabecular bone, a mixture of trabecular and cortical bone and mainly cortical bone, and tested under static and cyclic compression. Axial displacement and strain fields were obtained by means of digital volume correlation (DVC) and microdamage due to static compression was assessed using DVC and finite element (FE) analysis, where yielded volumes and strains (εzz) were evaluated. A significantly higher load was transferred into the cement region when mainly cortical bone was used to interdigitate with the cement, compared with the other two cases. In the former, progressive damage accumulation under cyclic loading was observed within both the bone-cement interdigitated and the cement regions, as evidenced by the initiation of microcracks associated with high residual strains (εzz_res).

Stress shielding in periprosthetic bone following a total knee replacement: Effects of implant material, design and alignment

Periprosthetic bone strain distributions in some of the typical cases of total knee replacement (TKR) were studied with regard to the selection of material, design and the alignments of tibial components to examine which conditions are more forgiving than the others to stress shielding post a TKR. Four tibial components with two implant designs (cruciate sacrificing and cruciate retaining) and material properties (metal-backed (MB) and all-polyethylene (AP)) were considered in a specimen-specific finite element tibia bone model loaded in a neutral position. The influence of tibial material and design on the periprosthetic bone strain response was investigated under the peak loads of walking and stair descending/ascending. Two of the models were also modified to examine the effect of selected implant malalignment conditions (7° posterior, 5° valgus and 5° varus) on stress shielding in the bone, where the medio-lateral load share ratios were adjusted accordingly. The predicted increases of bone density due to implantation for the selected cases studied were also presented. For the cases examined, the effect of stress shielding on the periprosthetic bone seems to be more significantly influenced by the implant material than by the implant geometry. Significant stress shielding is found in MB cases, as opposed to increase in bone density found in AP cases, particularly in the bones immediately beneath the baseplate. The effect of stress shielding is reduced somewhat for the MB components in the malaligned positions compared with the neutral case. In AP cases, the effect of stress shielding is mostly low except in the varus position, possibly due to off-loading of lateral condyle. Increases in bone density are found in both MB and AP cases for the malaligned conditions.

Stress shielding in bone of a bone-cement interface

Cementation is one of the main fixation methods used in joint replacement surgeries such as Total Knee Replacement (TKR). This work was prompted by a recent retrieval study, which shows losses up to 75% of the bone stock at the bone-cement interface ten years post TKR. It aims to examine the effects of cementation on the stress shielding of the interfacing bone, when the influence of an implant is removed. A micromechanics finite element study of a generic bone-cement interface is presented here, where bone elements in the partially and the fully interdigitated regions were evaluated under selected load cases. The results revealed significant stress shielding effect in the bone of all bone-cement interface regions, particularly in fully interdigitated region. This finding may be useful in the studies of implant fixation and other related orthopedic treatment strategies.

Spatial resolution and measurement uncertainty of strains in bone and bone–cement interface using digital volume correlation

The measurement uncertainty of strains has been assessed in a bone analogue (sawbone), bovine trabecular bone and bone-cement interface specimens under zero load using the Digital Volume Correlation (DVC) method. The effects of sub-volume size, sample constraint and preload on the measured strain uncertainty have been examined. There is generally a trade-off between the measurement uncertainty and the spatial resolution. Suitable sub-volume sizes have been be selected based on a compromise between the measurement uncertainty and the spatial resolution of the cases considered. A ratio of sub-volume size to a microstructure characteristic (Tb.Sp) was introduced to reflect a suitable spatial resolution, and the measurement uncertainty associated was assessed. Specifically, ratios between 1.6 and 4 appear to give rise to standard deviations in the measured strains between 166 and 620 με in all the cases considered, which would seem to suffice for strain analysis in pre as well as post yield loading regimes. A microscale finite element (μFE) model was built from the CT images of the sawbone, and the results from the μFE model and a continuum FE model were compared with those from the DVC. The strain results were found to differ significantly between the two methods at tissue level, consistent in trend with the results found in human bones, indicating mainly a limitation of the current DVC method in mapping strains at this level.

Hip joint degeneration due to cam impingement: a finite element analysis

The goal of this study was to investigate the impact of cam impingement, a biomechanical risk factor, on hip joint degeneration and ultimately coxarthrosis. 3D finite element solid models of a healthy and a pathologic hip were developed based on clinical reports. The biphasic characteristics of cartilaginous tissues were considered to identify localised solid matrix overloading during normal walking and sitting down (SD). Localised femoral intrusion at the anterior-superior pelvic horn was revealed in the pathologic hip during SD, where the radial and meridional solid stresses in the acetabular cartilage and circumferential solid stresses within the acetabular labrum increased by 3.7, 1.5 and 2.7 times, respectively. The increased solid-on-solid stresses, reduction in fluid-load support and associated higher friction during articulation may result in joint wear and other degenerative changes in the hip.

Impact of P2RX7 ablation on the morphological, mechanical and tissue properties of bones in a murine model of duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is an inherited, lethal disorder characterised by progressive muscle degeneration and associated bone abnormalities. We have previously demonstrated that P2RX7 purinergic receptors contribute to the pathogenesis of DMD, and found that P2RX7 ablation alleviated the severity of the disease. In this work we have used a dystrophic mdx mouse crossed with the global P2RX7 receptor to generate a knockout mouse (mdx/P2X7-/-), and compared its morphometric, mechanical and tissue properties against those of mdx, as well as the wild type (WT) and the P2RX7 knockout (P2X7-/-). Micro-computed tomography (µCT), three-point bending testing, scanning electron microscopy (SEM) and nano-indentation were utilised in the study. The bones were analysed at approximately 4 weeks of age to examine the impact of P2RX7 ablation on the bone properties during the acute disease phase, before muscle wasting is fully developed. The results show that P2RX7 purinoceptor ablation has produced improvement or significant improvement in some of the morphological, the mechanical and the tissue properties of the dystrophic bones examined. Specifically, although the ablation produced smaller bones with significantly lower total cross-section area (Tt.Ar) and Second Moment of Area (SMA), significantly higher cortical bone area (Ct.Ar), cortical area fraction (Ct.Ar/Tt.Ar) and trabecular bone volume fraction (BV/TV) are found in the mdx/P2X7-/- mice than in any other types. Further, the mdx/P2X7-/- bones have relatively higher average flexural strength, work-to-fracture and significantly higher strain to failure compared with those of mdx, suggesting greater resistance to fracture. Indentation modulus, elasticity and creep are also significantly improved in the knockout cortical bones over those of mdx. These findings seem to suggest that specific pharmacological blockade of P2RX7 may improve dystrophic bones, with a potential for therapeutic application in the treatment of the disease.

Near tip strain evolution of a growing fatigue crack

Near tip full-field strains in a growing fatigue crack have been studied in situ using the Digital Image Correlation (DIC) technique in a compact tension specimen of stainless steel 316L under tension-tension cyclic loading. An error analysis of displacements and strains has been carried out, and the results show that the precision of displacements and strains in the wake of the crack is worse than that in front of the crack. A method for the determination of crack tip location is proposed for the DIC analysis. Strain ratchetting is observed ahead of the growing fatigue crack tip and found to be dependent on the distance to the crack tip; whilst normal strains appear to stabilise behind the crack tip.

Mechanical characterisation of a scaffold under monotonic and cyclic loading conditions

Loading from daily activities or from intensive exercise can lead to increased risk of fracture. Implants designed for load bearing purposes, such as repair of articular cartilage and underlying subchondral bone in knees must have the necessary mechanical competence under long term physiological loadings. In this study, the mechanical behaviour of a polymer-based osteochondral scaffold was examined under monotonic and cyclic loading conditions in a phosphate buffered saline solution at 37°C. Monotonic compression tests at selected strain rates were performed in both confined and unconfined conditions to investigate the influence of confinement. The effects of strain rate and sample composition on mechanical properties were also studied. Multi-step cyclic tests were carried out with increasing compressive loads. Changes in secant modulus and residual strain accumulation are monitored. The secant modulus and the number of cycles to failure of the scaffold are obtained and compared with those of human trabecular bone (Topolinski et al., 2011).

Near tip strain evolution under cyclic loading

The concept of ratchetting strain as a crack driving force in controlling crack growth has previously been explored at Portsmouth using numerical approaches for nickel-based superalloys. In this paper, we report the first experimental observations of the near-tip strain evolution as captured by the Digital Image Correlation (DIC) technique on a compact tension specimen of stainless steel 316L. The evolution of the near-tip strains with loading cycles was studied whilst the crack tip was maintained stationary. The strains were monitored over the selected distances from the crack tip for a given number of cycles under an incremental loading regime. The results show that strain ratchetting does occur with load cycling, and is particularly evident close to the crack tip and under higher loads. A finite element model has been developed to simulate the experiments and the simulation results are compared with the DIC measurements.