A murine model for developmental dysplasia of the hip: ablation of CX3CR1 affects acetabular morphology and gait.

BACKGROUND: Developmental dysplasia of the hip (DDH) is a debilitating condition whose distinguishing signs include incomplete formation of the acetabulum leading to dislocation of the femur, accelerated wear of the articular cartilage and joint laxity resulting in osteoarthritis. It is a complex disorder having environmental and genetic causes. Existing techniques fail to detect milder forms of DDH in newborns leading to hip osteoarthritis in young adults. A sensitive, specific and cost effective test would allow identification of newborns that could be non-invasively corrected by the use of a Pavlik harness. Previously, we identified a 2.5 MB candidate region on human chromosome 3 by using linkage analysis of a 4 generation, 72 member family. Whole exome sequencing of the DNA of 4 severely affected members revealed a single nucleotide polymorphism variant, rs3732378 co-inherited by all 11 affected family members. This variant causes a threonine to methionine amino acid change in the coding sequence of the CX3CR1 chemokine receptor and is predicted to be harmful to the function of the protein To gain further insight into the function of this mutation we examined the effect of CX3CR1 ablation on the architecture of the mouse acetabulum and on the murine gait. METHODS: The hips of 5 and 8 weeks old wild type and CX3CR1 KO mice were analyzed using micro-CT to measure acetabular diameter and ten additional dimensional parameters. Eight week old mice were gait tested using an inclined treadmill with and without load and then underwent micro-CT analysis. RESULTS: (1) KO mice showed larger a 5-17% larger diameter left acetabula than WT mice at both ages. (2) At 8 weeks the normalized area of space (i.e. size discrepancy) between the femur head and acetabulum is significantly larger [38% (p = 0.001)-21% (p = 0.037)] in the KO mice. (3) At 8 weeks gait analysis of these same mice shows several metrics that are consistent with impairment in the KO but not the WT mice. These deficits are often seen in mice and humans who develop hip OA. CONCLUSION: The effect of CX3CR1 deletion on murine acetabular development provides suggestive evidence of a susceptibility inducing role of the CX3CR1 gene on DDH

Chemical modification of extracellular matrix by cold atmospheric plasma-generated reactive species affects chondrogenesis and bone formation.

The goal of this study was to investigate whether cold plasma generated by dielectric barrier discharge (DBD) modifies extracellular matrices (ECM) to influence chondrogenesis and endochondral ossification. Replacement of cartilage by bone during endochondral ossification is essential in fetal skeletal development, bone growth and fracture healing. Regulation of this process by the ECM occurs through matrix remodelling, involving a variety of cell attachment molecules and growth factors, which influence cell morphology and protein expression. The commercially available ECM, Matrigel, was treated with microsecond or nanosecond pulsed (μsp or nsp, respectively) DBD frequencies conditions at the equivalent frequencies (1 kHz) or power (~1 W). Recombinant human bone morphogenetic protein-2 was added and the mixture subcutaneously injected into mice to simulate ectopic endochondral ossification. Two weeks later, the masses were extracted and analysed by microcomputed tomography. A significant increase in bone formation was observed in Matrigel treated with μsp DBD compared with control, while a significant decrease in bone formation was observed for both nsp treatments. Histological and immunohistochemical analysis showed Matrigel treated with μsp plasma increased the number of invading cells, the amount of vascular endothelial growth factor and chondrogenesis while the opposite was true for Matrigel treated with nsp plasma. In support of the in vivo Matrigel study, 10 T1/2 cells cultured in vitro on μsp DBD-treated type I collagen showed increased expression of adhesion proteins and activation of survival pathways, which decreased with nsp plasma treatments. These results indicate DBD modification of ECM can influence cellular behaviours to accelerate or inhibit chondrogenesis and endochondral ossification. Copyright © 2016 John Wiley & Sons, Ltd

Mast cells and hypoxia drive tissue metaplasia and heterotopic ossification in idiopathic arthrofibrosis after total knee arthroplasty

ABSTRACT: BACKGROUND: Idiopathic arthrofibrosis occurs in 3-4% of patients who undergo total knee arthroplasty (TKA). However, little is known about the cellular or molecular changes involved in the onset or progression of this condition. To classify the histomorphologic changes and evaluate potential contributing factors, periarticular tissues from the knees of patients with arthrofibrosis were analyzed for fibroblast and mast cell proliferation, heterotopic ossification, cellular apoptosis, hypoxia and oxidative stress. RESULTS: The arthrofibrotic tissue was composed of dense fibroblastic regions, with limited vascularity along the outer edges. Within the fibrotic regions, elevated numbers of chymase/fibroblast growth factor (FGF)-expressing mast cells were observed. In addition, this region contained fibrocartilage and associated heterotopic ossification, which quantitatively correlated with decreased range of motion (stiffness). Fibrotic, fibrocartilage and ossified regions contained few terminal dUTP nick end labeling (TUNEL)-positive or apoptotic cells, despite positive immunostaining for lactate dehydrogenase (LDH)5, a marker of hypoxia, and nitrotyrosine, a marker for protein nitrosylation. LDH5 and nitrotyrosine were found in the same tissue areas, indicating that hypoxic areas within the tissue were associated with increased production of reactive oxygen and nitrogen species. CONCLUSIONS: Taken together, we suggest that hypoxia-associated oxidative stress initiates mast cell proliferation and FGF secretion, spurring fibroblast proliferation and tissue fibrosis. Fibroblasts within this hypoxic environment undergo metaplastic transformation to fibrocartilage, followed by heterotopic ossification, resulting in increased joint stiffness. Thus, hypoxia and associated oxidative stress are potential therapeutic targets for fibrosis and metaplastic progression of idiopathic arthrofibrosis after TKA

Reactive oxygen and nitrogen species induce protein and DNA modifications driving arthrofibrosis following total knee arthroplasty

BACKGROUND: Arthrofibrosis, occurring in 3%-4% of patients following total knee arthroplasty (TKA), is a challenging condition for which there is no defined cause. The hypothesis for this study was that disregulated production of reactive oxygen species (ROS) and nitrogen species (RNS) mediates matrix protein and DNA modifications, which result in excessive fibroblastic proliferation. RESULTS: We found increased numbers of macrophages and lymphocytes, along with elevated amounts of myeloperoxidase (MPO) in arthrofibrotic tissues when compared to control tissues. MPO expression, an enzyme that generates ROS/RNS, is usually limited to neutrophils and some macrophages, but was found by immunohistochemistry to be expressed in both macrophages and fibroblasts in arthrofibrotic tissue. As direct measurement of ROS/RNS is not feasible, products including DNA hydroxylation (8-OHdG), and protein nitrosylation (nitrotyrosine) were measured by immunohistochemistry. Quantification of the staining showed that 8-OHdg was significantly increased in arthrofibrotic tissue. There was also a direct correlation between the intensity of inflammation and ROS/RNS to the amount of heterotopic ossification (HO). In order to investigate the aberrant expression of MPO, a real-time oxidative stress polymerase chain reaction array was performed on fibroblasts isolated from arthrofibrotic and control tissues. The results of this array confirmed the upregulation of MPO expression in arthrofibrotic fibroblasts and highlighted the downregulated expression of the antioxidants, superoxide dismutase1 and microsomal glutathione S-transferase 3, as well as the significant increase in thioredoxin reductase, a known promoter of cell proliferation, and polynucleotide kinase 3\u27-phosphatase, a key enzyme in the base excision repair pathway for oxidative DNA damage. CONCLUSION: Based on our current findings, we suggest that ROS/RNS initiate and sustain the arthrofibrotic response driving aggressive fibroblast proliferation and subsequent HO

Atmospheric Pressure Plasma Treatment of Skin: Penetration Into Hair Follicles

Sterilization of skin prior to surgery is challenged by the reservoir of bacteria that resides in hair follicles. Atmospheric pressure plasma jets (APPJs) have been proposed as a method to treat and deactivate these bacteria as atmospheric plasmas are able to penetrate into structures and crevices with dimensions similar to those found in hair follicles. In this paper, we discuss results from a computational investigation of an APPJ sustained in helium flowing into ambient air, and incident onto a layered dielectric similar to human skin in which there are idealized hair follicles. We found that, depending on the location of the follicle, the bulk ionization wave (IW) incident onto the skin, or the surface IW on the skin, are able to launch IWs into the follicle. The uniformity of treatment of the follicle depends on the location of the first entry of the plasma into the follicle on the top of the skin. Typically, only one side of the follicle is treated on for a given plasma pulse, with uniform treatment resulting from rastering the plasma jet across the follicle over many pulses. Plasma treatment of the follicle is sensitive to the angle of the follicle with respect to the skin, width of the follicle pocket, conductivity of the dermis and thickness of the underlying subcutaneous fat layer, the latter due to the change in capacitance of the tissue

Biomolecules as Model Indicators of In Vitro and In Vivo Cold Plasma Safety

The potential applications for cold plasma in medicine are extensive, from microbial inactivation and induction of apoptosis in cancer cells to stimulating wound healing and enhancing the blood coagulation cascade. The safe bio-medical application of cold plasma and subsequent effect on complex biological pathways requires precision and a distinct understanding of how physiological redox chemistry is manipulated. Chemical modification of biomolecules such as carbohydrates, proteins, and lipids treated with cold plasma have been characterized, however, the context of how alterations of these molecules affect cell behavior or in vivo functionality has not been determined. Thus, this study examines the cytotoxic and mutagenic effects of plasmatreated molecules in vitro using CHO-K1 cells and in vivo in Galleria mellonella larvae. Specifically, albumin, glucose, cholesterol, and arachidonic acid were chosen as representative biomolecules, with established involvement in diverse bioprocesses including; cellular respiration, intracellular transport, cell signaling or membrane structure. Long- and short-term effects depended strongly on the molecule type and the treatment milieu indicating the impact of chemical and physical modifications on downstream biological pathways. Importantly, absence of short-term toxicity did not always correlate with absence of longer-term effects, indicating the need to comprehensively assess ongoing effects for diverse biological applications

Protein S blocks the extrinsic apoptotic cascade in tissue plasminogen activator/N-methyl D-aspartate-treated neurons via Tyro3-Akt-FKHRL1 signaling pathway

<p>Abstract</p> <p>Background</p> <p>Thrombolytic therapy with tissue plasminogen activator (tPA) benefits patients with acute ischemic stroke. However, tPA increases the risk for intracerebral bleeding and enhances post-ischemic neuronal injury if administered 3-4 hours after stroke. Therefore, combination therapies with tPA and neuroprotective agents have been considered to increase tPA's therapeutic window and reduce toxicity. The anticoagulant factor protein S (PS) protects neurons from hypoxic/ischemic injury. PS also inhibits N-methyl-D-aspartate (NMDA) excitotoxicity by phosphorylating Bad and Mdm2 which blocks the downstream steps in the intrinsic apoptotic cascade. To test whether PS can protect neurons from tPA toxicity we studied its effects on tPA/NMDA combined injury which in contrast to NMDA alone kills neurons by activating the extrinsic apoptotic pathway. Neither Bad nor Mdm2 which are PS's targets and control the intrinsic apoptotic pathway can influence the extrinsic cascade. Thus, based on published data one cannot predict whether PS can protect neurons from tPA/NMDA injury by blocking the extrinsic pathway. Neurons express all three TAM (Tyro3, Axl, Mer) receptors that can potentially interact with PS. Therefore, we studied whether PS can activate TAM receptors during a tPA/NMDA insult.</p> <p>Results</p> <p>We show that PS protects neurons from tPA/NMDA-induced apoptosis by suppressing Fas-ligand (FasL) production and FasL-dependent caspase-8 activation within the extrinsic apoptotic pathway. By transducing neurons with adenoviral vectors expressing the kinase-deficient Akt mutant <it>Akt^K179A</it>and a triple FKHRL1 Akt phosphorylation site mutant (FKHRL1-TM), we show that Akt activation and Akt-mediated phosphorylation of FKHRL1, a member of the Forkhead family of transcription factors, are critical for FasL down-regulation and caspase-8 inhibition. Using cultured neurons from Tyro3, Axl and Mer mutants, we show that Tyro3, but not Axl and Mer, mediates phosphorylation of FHKRL1 that is required for PS-mediated neuronal protection after tPA/NMDA-induced injury.</p> <p>Conclusions</p> <p>PS blocks the extrinsic apoptotic cascade through a novel mechanism mediated by Tyro3-dependent FKHRL1 phosphorylation which inhibits FasL-dependent caspase-8 activation and can control tPA-induced neurotoxicity associated with pathologic activation of NMDA receptors. The present findings should encourage future studies in animal stroke models to determine whether PS can increase the therapeutic window of tPA by reducing its post-ischemic neuronal toxicity.</p

Biomolecules as Model Indicators of In Vitro and In Vivo Cold Plasma Safety

The potential applications for cold plasma in medicine are extensive, from microbial inactivation and induction of apoptosis in cancer cells to stimulating wound healing and enhancing the blood coagulation cascade. The safe bio-medical application of cold plasma and subsequent effect on complex biological pathways requires precision and a distinct understanding of how physiological redox chemistry is manipulated. Chemical modification of biomolecules such as carbohydrates, proteins, and lipids treated with cold plasma have been characterized, however, the context of how alterations of these molecules affect cell behavior or in vivo functionality has not been determined. Thus, this study examines the cytotoxic and mutagenic effects of plasmatreated molecules in vitro using CHO-K1 cells and in vivo in Galleria mellonella larvae. Specifically, albumin, glucose, cholesterol, and arachidonic acid were chosen as representative biomolecules, with established involvement in diverse bioprocesses including; cellular respiration, intracellular transport, cell signaling or membrane structure. Long- and short-term effects depended strongly on the molecule type and the treatment milieu indicating the impact of chemical and physical modifications on downstream biological pathways. Importantly, absence of short-term toxicity did not always correlate with absence of longer-term effects, indicating the need to comprehensively assess ongoing effects for diverse biological applications

Nonthermal atmospheric pressure plasma enhances mouse limb bud survival, growth, and elongation.

The enhanced differentiation of mesenchymal cells into chondrocytes or osteoblasts is of paramount importance in tissue engineering and regenerative therapies. A newly emerging body of evidence demonstrates that appendage regeneration is dependent on reactive oxygen species (ROS) production and signaling. Thus, we hypothesized that mesenchymal cell stimulation by nonthermal (NT)-plasma, which produces and induces ROS, would (1) promote skeletal cell differentiation and (2) limb autopod development. Stimulation with a single treatment of NT-plasma enhanced survival, growth, and elongation of mouse limb autopods in an in vitro organ culture system. Noticeable changes included enhanced development of digit length and definition of digit separation. These changes were coordinated with enhanced Wnt signaling in the distal apical epidermal ridge (AER) and presumptive joint regions. Autopod development continued to advance for approximately 144 h in culture, seemingly overcoming the negative culture environment usually observed in this in vitro system. Real-time quantitative polymerase chain reaction analysis confirmed the up-regulation of chondrogenic transcripts. Mechanistically, NT-plasma increased the number of ROS positive cells in the dorsal epithelium, mesenchyme, and the distal tip of each phalange behind the AER, determined using dihydrorhodamine. The importance of ROS production/signaling during development was further demonstrated by the stunting of digital outgrowth when anti-oxidants were applied. Results of this study show NT-plasma initiated and amplified ROS intracellular signaling to enhance development of the autopod. Parallels between development and regeneration suggest that the potential use of NT-plasma could extend to both tissue engineering and clinical applications to enhance fracture healing, trauma repair, and bone fusion

The inhibition of \u3cem\u3eStaphylococcus epidermidis\u3c/em\u3e biofilm formation by vancomycinmodified titanium alloy and implications for the treatment of periprosthetic infection

Peri-prosthetic infections are notoriously difficult to treat as the biomaterial implant is ideal for bacterial adhesion and biofilm formation, resulting in decreased antibiotic sensitivity. Previously, we reported that vancomycin covalently attached to a Ti alloy surface (Vanc-Ti) could prevent bacterial colonization. Herein we examine the effect of this Vanc-Ti surface on Staphylococci epidermidis, a Gram-positive organism prevalent in orthopaedic infections. By direct colony counting and fluorescent visualization of live bacteria, S. epidermidis colonization was significantly inhibited on Vanc-Ti implants. In contrast, the gram-negative organism Escherichia coli readily colonized the Vanc-Ti rod, suggesting retention of antibiotic specificity. By histochemical and SEM analysis, Vanc-Ti prevented S. epidermidis biofilm formation, even in the presence of serum. Furthermore, when challenged multiple times with S. epidermidis, Vanc-Ti rods resisted bacterial colonization. Finally, when S. epidermidis was continuously cultured in the presence of Vanc-Ti, the bacteria maintained a Vanc sensitivity equivalent to the parent strain. These findings indicate that antibiotic derivatization of implants can result in a surface that can resist bacterial colonization. This technology holds great promise for the prevention and treatment of periprosthetic infections