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Purpose Cell-based therapies using lipoaspirate are gaining popularity in orthopaedics due to their hypothesised regenerative potential. Several ‘point-of-care’ lipoaspirate-processing devices/systems have become available to isolate cells for therapeutic use, with published evidence reporting their clinical relevance. However, few studies have analysed the composition of their ‘minimally-manipulated’ cellular products in parallel, information that is vital to understand the mechanisms by which these therapies may be efficacious. This scoping review aimed to identify devices/systems using mechanical-only processing of lipoaspirate, the constituents of their cell-based therapies and where available, clinical outcomes. Methods PRISMA extension for scoping reviews guidelines were followed. MEDLINE, Embase and PubMed databases were systematically searched to identify relevant articles until 21st April 2022. Information relating to cellular composition and clinical outcomes for devices/systems was extracted. Further information was also obtained by individually searching the devices/systems in the PubMed database, Google search engine and contacting manufacturers. Results 2895 studies were screened and a total of 15 articles (11 = Level 5 evidence) fulfilled the inclusion criteria. 13 unique devices/systems were identified from included studies. All the studies reported cell concentration (cell number regardless of phenotype per millilitre of lipoaspirate) for their devices/systems (range 0.005–21 × 106). Ten reported cell viability (the measure of live cells- range 60–98%), 11 performed immuno-phenotypic analysis of the cell-subtypes and four investigated clinical outcomes of their cellular products. Only two studies reported all four of these parameters. Conclusion When focussing on cell concentration, cell viability and MSC immuno-phenotypic analysis alone, the most effective manual devices/systems were ones using filtration and cutting/mincing. However, it was unclear whether high performance in these categories would translate to improved clinical outcomes. Due to the lack of standardisation and heterogeneity of the data, it was also not possible to draw any reliable conclusions and determine the role of these devices/systems in clinical practice at present. Level of Evidence Level V Therapeutic.

Aims Total hip arthroplasty (THA) and total knee arthroplasty (TKA) are common orthopaedic procedures requiring postoperative radiographs to confirm implant positioning and identify complications. Artificial intelligence (AI)-based image analysis has the potential to automate this postoperative surveillance. The aim of this study was to prepare a scoping review to investigate how AI is being used in the analysis of radiographs following THA and TKA, and how accurate these tools are. Methods The Embase, MEDLINE, and PubMed libraries were systematically searched to identify relevant articles. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for scoping reviews and Arksey and O’Malley framework were followed. Study quality was assessed using a modified Methodological Index for Non-Randomized Studies tool. AI performance was reported using either the area under the curve (AUC) or accuracy. Results Of the 455 studies identified, only 12 were suitable for inclusion. Nine reported implant identification and three described predicting risk of implant failure. Of the 12, three studies compared AI performance with orthopaedic surgeons. AI-based implant identification achieved AUC 0.992 to 1, and most algorithms reported an accuracy > 90%, using 550 to 320,000 training radiographs. AI prediction of dislocation risk post-THA, determined after five-year follow-up, was satisfactory (AUC 76.67; 8,500 training radiographs). Diagnosis of hip implant loosening was good (accuracy 88.3%; 420 training radiographs) and measurement of postoperative acetabular angles was comparable to humans (mean absolute difference 1.35° to 1.39°). However, 11 of the 12 studies had several methodological limitations introducing a high risk of bias. None of the studies were externally validated. Conclusion These studies show that AI is promising. While it already has the ability to analyze images with significant precision, there is currently insufficient high-level evidence to support its widespread clinical use. Further research to design robust studies that follow standard reporting guidelines should be encouraged to develop AI models that could be easily translated into real-world conditions. Cite this article: Bone Joint J 2022;104-B(8):929–937.

Background The minimal clinically important difference, minimal important change, minimal detectable change and patient-acceptable symptom state are poorly defined for the Oxford Shoulder Score following shoulder arthroplasty. The study's aim was to calculate their values. Methods One hundred patients underwent shoulder arthroplasty and completed pre and 1-year postoperative Oxford Shoulder Score. Patient satisfaction was assessed at 1-year using a visual analogue scale from 0 to 100: ‘very satisfied’ (>80), ‘satisfied’ (>60–80), and ‘unsatisfied’ (≤60). The difference between patients recording ‘unsatisfied’ ( n = 11) and ‘satisfied’ ( n = 16) was used to define the minimal clinically important difference. MICcohort was calculated as the change in Oxford Shoulder Score for those satisfied (>60). Receiver-operating characteristic curve analysis was used to determine the MICindividual and patient-acceptable symptom state. Distribution-based methodology was used for the minimal detectable change. Results The minimal clinically important difference was 6.9 (95% confidence interval 0.7–13.1, p = 0.039). The MICcohort was 11.6 (95% confidence interval 6.8–16.4) and MICindividual 13. The minimal detectable change was 6.6 and the patient-acceptable symptom state was defined as ≥29. Discussion The minimal clinically important difference and minimal important change can assess whether there is a clinical difference between two groups and whether a cohort/patient has had a meaningful change in their Oxford Shoulder Score, respectively. These were greater than measurement error (minimal detectable change), suggesting a real change. The patient-acceptable symptom state can be used as a marker of achieving satisfaction.

Distributed systems pose unique challenges for software developers. Understanding the system’s communication topology and reasoning about concurrent activities of system hosts can be difficult. The standard approach, analyzing system logs, can be a tedious and complex process that involves reconstructing a system log from multiple hosts’ logs, reconciling timestamps among hosts with non-synchronized clocks, and understanding what took place during the execution encoded by the log. This article presents a novel approach for tackling three tasks frequently performed during analysis of distributed system executions: (1) understanding the relative ordering of events, (2) searching for specific patterns of interaction between hosts, and (3) identifying structural similarities and differences between pairs of executions. Our approach consists of XVector , which instruments distributed systems to capture partial ordering information that encodes the happens-before relation between events, and ShiViz , which processes the resulting logs and presents distributed system executions as interactive time-space diagrams. Two user studies with a total of 109 students and a case study with 2 developers showed that our method was effective, helping participants answer statistically significantly more system-comprehension questions correctly, with a very large effect size.

Background Chronic quadriceps tendon rupture is technically challenging for surgeons due to scarring and tendon retraction. The presence of concurrent ipsilateral knee osteoarthritis compounds the issue even further. Although a combined presentation is rare, treatment options to manage each coexisting pathology simultaneously are scarcely reported. We describe the case of a 67-year-old Caucasian male who had such a presentation, and was subsequently treated with a one-stage extensor mechanism autograft reconstruction and total knee replacement with computer navigation. Case The patient was a 67-year-old male Caucasian, who had previously sustained an acute rupture of his right quadriceps tendon that was adequately repaired 6 months prior. Despite an initial satisfactory result, he reported deterioration in his mobility in the few months thereafter, with worsening knee pain. His comorbidities consisted of hypertension, asthma, and a body mass index of 40.4 kg/m2. Otherwise, there were no risk factors for tendon rerupture. Clinical examination later revealed a large palpable gap in the right suprapatellar region and weakness of active knee extension. No traumatic cause for this new presentation was identified. Suspicion of a chronic quadriceps tendon rupture was confirmed on radiological imaging, but the investigations also noted the presence of severe tricompartmental osteoarthritis of the ipsilateral, native knee joint. The combined procedure took place in one surgical sitting. The total knee replacement with patella resurfacing was performed first and assisted by computer navigation. The quadriceps tendon reconstruction was then conducted sequentially using the patient’s hamstring tendons (semitendinosus and gracilis). The tensile strength was reinforced with use of a Ligament Augmentation and Reconstruction System (LARS) ligament. Initial outcomes were excellent, and these results were maintained at 6 months postoperatively, with the patient reporting no pain and having full range of movement. Conclusion Our techniques used have not previously been reported, but are successful options in treating coexisting chronic quadriceps tendon rupture and ipsilateral knee osteoarthritis. The advantage of using computer navigation with an extramedullary femoral jig can lead to improved accuracy of bone cuts, which is important in the presence of anatomical disruption. Chronic failures of the extensor mechanism require different approaches depending on the inherent and underlying pathology. We feel that the multidisciplinary team approach to the management and use of two surgeons with differing expertise added to the successful outcome of this complex case.

An important negative regulator of factor VIIIa (FVIIIa) cofactor activity is A2 subunit dissociation. FVIII molecules with stabilized activity have been generated by elimination of charged residues at the A1-A2 and A2-A3 interfaces. These molecules exhibited reduced decay rates as part of the enzymatic factor Xa generation complex and retained their activities under thermal and chemical denaturing conditions. We describe here the potency and efficacy of 1 such stability variant, D519V/E665V, derived from B domain–deleted FVIII (BDD-FVIII). The major effect of A2 stabilization was on cofactor activity. D519V/E665V potency was increased twofold by the 2-stage chromogenic assay relative to BDD-FVIII. D519V/E665V demonstrated enhanced thrombin generation responses (fivefold by peak thrombin) relative to BDD-FVIII. In vivo consequences of enhanced cofactor activity of D519V/E665V included >fourfold increased maximal platelet-fibrin deposition after laser injury and twofold increased protection from bleeding in acute and prolonged vascular injury model in hemophilia A mice. These results demonstrate that noncovalent stabilization of the FVIII A2 subunit can prolong its cofactor activity, leading to differential enhancement in clot formation over protection from blood loss in hemophilia. The FVIII molecule described here is the first molecule with clear efficacy enhancement resulting from noncovalent stabilization of the A2 domain.

Vaso-occlusive crisis (VOC) is the most common and debilitating complication of sickle cell disease (SCD); recurrent episodes cause organ damage and contribute to early mortality. Plasma placental growth factor (PlGF) levels are elevated in SCD and can further increase under hypoxic conditions in SCD mice. Treatment with a PlGF-neutralizing antibody (anti-PlGF Ab) in SCD mice reduced levels of monocyte chemoattractant protein-3, eotaxin, macrophage colony-stimulating factor, and plasminogen activator inhibitor-1 significantly, and of macrophage-derived chemokine and macrophage inflammatory protein-3β moderately; this may contribute to inhibition of leukocyte recruitment, activation, and thrombosis. In subsequent experiments, anti-PlGF Ab treatment significantly reduced plasma lactate dehydrogenase levels, indicating possible reduction in cellular destruction and/or hemolysis. Histopathology studies revealed decreased incidence and severity of congestion in the lungs and spleen with repeated anti-PlGF Ab treatment. Furthermore, anti-PlGF Ab significantly reduced vaso-occlusion events under hypoxic conditions in a modified dorsal skinfold chamber model in SCD mice. Therefore, elevated PlGF levels may contribute to recruitment and activation of leukocytes. This can subsequently lead to increased pathology of affected organs in addition to mediating acute hypoxia/reoxygenation-triggered vaso-occlusion under SCD conditions. Thus, targeting PlGF may offer a therapeutic approach to reduce acute VOC and possibly alleviate long-term vascular complications in patients with SCD.

Experimental allergic encephalomyelitis (EAE) is a model of central nervous system (CNS) inflammation that follows immunization with certain CNS antigens. The course and clinical manifestations of EAE are similar to those of multiple sclerosis (MS) in humans; therefore, EAE has become an accepted animal model to study MS. The purpose of this study was to demonstrate that systemic expression of murine interferon-beta (IFN-beta) (MuIFN-beta), following intramuscular (i.m.) delivery of plasmid DNA encoding MuIFN-beta to the hind limb of mice, is effective in reducing the clinical manifestations of disease in a model of EAE. The results of the study demonstrate that gene-based delivery of MuIFN-beta caused significantly decreased clinical scores compared with delivery of the null vector. A single injection of the MuIFN-beta plasmid was as effective in reducing the severity of the disease as an every other day injection of MuIFN-beta protein.

Morbidity and mortality of peripheral arterial occlusive disease significantly increases with age, often exhibiting more severe disease pathology and decreased treatment effectiveness. Therapeutic angiogenesis with angiogenic growth factors may represent a valuable treatment option for the severely ill, older adult patient population. Aging is considered an independent cardiovascular risk factor, but pathomechanistically it is not well understood. Diminished endothelial nitric oxide (EDNO) production has been considered as a major contributor to the aging process. To investigate the effect of age on postischemic revascularization independent of changes in EDNO, we used endothelial nitric oxide synthase-deficient (ecNOS-KO) mice. We found an age-dependent acceleration in ischemic injury following unilateral femoral artery ligation in these animals compared to C57BL/J6 mice. Postischemic revascularization, quantified by measuring von Willebrand factor expression, was significantly impaired, suggesting that factors other than progressive EDNO deterioration are also involved in the age-dependent severe disease phenotype. Ischemia led to an increase in the expression of vascular endothelial growth factor receptor-2, KDR, in younger ecNOS-KO; however, this increase in KDR expression was absent in the older animals. Lack of increased KDR expression may provide a mechanistic explanation for the severe ischemic injury and perhaps can be used as a clinical marker to identify severe, vascular endothelial growth factor refractory patient population.

Plasmid based gene delivery to skeletal muscle is an attractive method for the expression of therapeutic proteins. However, the level of transgene expression decreases over time and may be an obstacle to achieving sustained therapeutic levels of proteins. To investigate the mechanism responsible for loss of expression over time, a standard plasmid backbone containing the luciferase gene driven by the CMV promoter was injected into the skeletal muscle of the hind limb of C57Bl/6 mice at either a high dose (125|[mu]|g) or a low dose (25|[mu]|g) followed by electroporation. Transgene expression was monitored over time using in situ CCCD imaging. At the end of the study the quantity of transgene DNA and luciferase mRNA in the muscle was measured by quantitative PCR. At early time points (day 3 and day 7) luciferase activity in the high dose group was 2.5- fold higher than in the low dose group. However, luciferase activity in mice that received the high dose fell 230-fold between day 7 and day 42, while in mice that received the low dose of the same plasmid it fell only 4-fold. Vector DNA levels in the injected muscles at the end of the study (day 84) were not significantly different between the two doses indicating that the 27-fold lower luciferase activity in the high dose group compared to the low dose group at day 84 was not due to differences in vector DNA levels but rather to a difference in gene expression. Indeed, the luciferase mRNA to vector DNA ratio was 9-fold higher in the low dose group than in the high dose group at this time point. In the same study, a plasmid containing the identical CMV-luciferase expression cassette flanked by matrix attachment sites (MARs) and placed into a plasmid backbone from which all CpG dinucleotides had been removed, was tested at the same doses. At the low dose the CpG-depleted/MARs plasmid exhibited a similar luciferase expression profile to that of the standard plasmid at the same dose. However, in the high dose group the CpG- depleted/MARs plasmid partially prevented the decrease in luciferase expression over time that was observed with the standard plasmid at the high dose. At 28, 42 and 84 days after plasmid injection the CpG- depleted/MAR plasmid expressed 10-, 13- and 9-fold higher luciferase activity respectively than the standard plasmid. Vector DNA levels in the muscle at day 84 were only 2-fold higher in the CpG-depleted/ MAR group than in the mice that received the standard plasmid. These results demonstrate that reducing the plasmid dose can result in a significant improvement in the duration of transgene expression, and that this effect was not due only to improved persistence of the plasmid DNA in the muscle. In addition, use of a CpG-free plasmid backbone plus MARs partially prevented the decline of transgene expression that occurred at the high plasmid dose, suggesting that the presence of CpG's and/or absence of MARs may contribute to loss of expression over time.

The goal of the present study was to explore the tropism of Virus-Like Particles (VLP) originating from the human polyoma JC- virus (JCV) in vitro and in vivo. VLP have been known for several years to be a promising alternative to the existing viral and non-viral DNA delivery systems. VLP are self-assembled structures composed only of proteins necessary for capsid formation, DNA packaging and gene delivery. They are replication deficient, exhibit a similar morphology and presumably share the same cell tropism, cellular uptake and intracellular trafficking mechanisms compared to their viral origin. VLP from JCV are being evaluated as a delivery vector for the central nervous system (CNS) because JCV preferentially infects both oligodendrocytes and astrocytes.

Interferon-|[szlig]| (IFN-|[szlig]|) is an immunoregulatory cytokine that has been approved as a protein therapeutic for multiple sclerosis (MS). Pharmacokinetic studies have shown that IFN-|[szlig]| has an extremely short half-life in the circulation. Within a few hours following bolus delivery of the recombinant protein, hIFN-|[szlig]| levels in serum are undetectable. Gene-based delivery of IFN-|[szlig]| would offer advantages over bolus delivery of protein in providing sustained long-term expression of the protein resulting in therapeutic efficacy while minimizing side effects.

Skeletal muscle has several features that make it attractive as a site for therapeutic gene expression - it is accessible to most of the current gene delivery systems, abundant, and displays a relatively slow turn over rate. A variety of gene delivery systems have been tested for high levels of sustainable gene expression from the skeletal muscle. Unfortunately, these studies have generally focused on optimization of one of the systems and have not made direct comparisons to other currently available gene delivery systems. To better understand the potential of these different systems to achieve high, sustained levels of gene expression from the skeletal muscle, we compared the viral-based gene delivery systems derived from human adenovirus (Ad) and adeno-associated virus (AAV) to optimized (electroporation, synthetic polymers) plasmid-based delivery. Using the HCMV promoter driven luciferase gene expression cassette as a reporter system for these studies, gene expression levels in the murine skeletal muscle were quantitated by both direct measurement of luciferase gene expression from excised skeletal muscle and by in vivo CCD camera intensity images. Initial expression levels (day 2 post infection/transduction) were highest from plasmid-based delivery by electroporation, slightly (approx. 2 fold ) higher than Ad and AAV-1 and significantly (100-1000X) higher than plasmid + poloxamer or plasmid alone. By day 6, the rank order of expression had changed, with AAV-1-based expression generating levels superior to the other systems tested (AAV-1>plasmid+electroporation>Ad>plasmid+poloxamer>plasmid). Gene expression has been detected out to 8 months for all the vectors examined. AAV-1 mediated expression levels at the 8 month time point are approximately 2-logs over both plasmid + electroporation and Ad as measured by CCD camera imaging. Consequently, AAV-1 based gene delivery results in superior expression levels and duration in the skeletal muscle relative to many of the other current gene delivery systems available.

Interferon beta (IFN-) has been shown to be an effective drug for patients with multiple sclerosis (MS). However dose-dependent side effects from administration of IFN- protein have limited its therapeutic efficacy and clinical utility. IFN- gene therapy offers an alternative approach to overcome these obstacles, and it would be advantageous over bolus delivery of protein. A critical issue in developing such a new approach is in vivo detection of biologically active IFN- in pre-clinical animal models. Several biomarkers have been established in humans undergoing IFN- therapy, including MxA (myxovirus resistant protein) an antiviral protein induced by IFN-. The goal of the present study was to establish and validate a quantitative PCR-based assay to measure RNA expression of Mx1, the murine homologue of human MxA, in peripheral blood mononuclear cells (PBMC) of mice following IFN- protein administration or gene-based delivery. In parallel it was investigated whether IP10, which was recently reported to be up-regulated following IFN- treatment in MS patients, could also be used as biomarker in mice. IP-10 has the advantage that it can be assayed directly by ELISA in plasma samples.

Top of pageAbstract At present, the most methods to evaluate in vivo gene expression are limited to the use of tissue obtained from sacrificed animals. To develop a simple, non-invasive and quantitative method for the detection of gene expression in the mouse central nervous system (CNS) while the animal is living, we evaluated a method of repetitively tracking in vivo gene expression of firefly luciferase (Luc) using a Cooled Charged Couple Detector (CCCD) camera. To evaluate the sensitivity of the bioluminescence imaging system, the initial experiment was performed by stereotactic injection of different doses of recombinant E1-deleted type 5 adenovirus expressing the Luc, Ad5.Luc, into the striatum of anesthetized adult C57BL/6 mice. The real-time in vivo gene expression of luciferase was monitored by CCCD camera at day 2, 7, 14, 28, and day 60 post gene transfer. The results indicated that mice treated with middle dose of Ad5.Luc (4|[times]|107 VP), a bioluminescence signal was detected at the site of injection as early as 2 days post injection. In contrast, the mice treated with higher dose of adenovirus (4|[times]|108 VP), the expression level reached its peak at day 7 and declined after day14. There was no detectable signal with the lowest dose of adenovirus (4|[times]|106 VP) at any of the time point post injection. To evaluated two recombinant adeno-associated virus (AAV) serotypes (AAV2/2, and AAV 2/5) with the bioluminescence imaging system in the mouse CNS, a subsequent experiment was performed using the similar protocol, stereotactic-guide injected 5|[times]|109 VP of AAV.Luc in 4 ul of PBS into the striatum of C57BL/6 mice. The results showed comparable expression of Luc in the mice treated with both AAV 2/2 and AAV 2/5 on day 2 post gene transfer. Between day 7 to day 28, the luciferase imaging in the mice treated with AAV 2/5 was two to five fold higher than in the mice treated with AAV 2/2. Gene expression of both AAV 2/2 and AAV2/5 have been persistent over two months in the live mice. Our results demonstrate that real-time in vivo bioluminescence imaging is a promising method to measure gene expression in the CNS of mice.

Plasmid based delivery of foreign transgenes often results in transient expression in vivo. To investigate the cause of this transient expression we used quantitative PCR to measure the copy number of transgene RNA and vector DNA in the skeletal muscle of C57BL6 mice after electroporation of various plasmids. Gene transfer of a plasmid encoding the mouse interferon beta (mIFN-|[beta]|) gene driven by the CMV promoter (pCMV-mIFN) resulted in mIFN RNA levels between 1 and 5 |[times]| 106 copies per microgram (|[mu]|g) of total RNA that were stable for the first 10 days then dropped by only 10-fold by day 44. Plasmid vector DNA levels were also stable for the first 10 days then dropped 7.5-fold between day 10 and day 44. The ratio of mIFN RNA copies per vector DNA copy reached a peak of 1.8 at day 10 and declined slightly to 0.9 at day 44. These results show that following delivery of a non-foreign transgene, a slow decline in RNA levels occurred that was due to a gradual loss of plasmid DNA. Next, we compared plasmids encoding either the mIFN-|[beta]| or human interferon beta (hIFN-|[beta]|) genes driven by identical CMV promoters (pCMV-hIFN and pCMV-mIFN). At 50 days after electroporation of mouse skeletal muscle, the mean copy number of transgene RNA in the muscles that received the mouse or human IFN-|[beta]| plasmids was 1.1 |[times]| 105 and 7.4 |[times]| 103 copies per |[mu]|g of total RNA, respectively. In contrast, the mean plasmid DNA level was slightly higher in the muscles that received pCMV-hIFN. The mean IFN-|[beta]| RNA to vector DNA ratio was 1.3 in the muscles that received pCMV-mIFN, but only 0.04 (31-fold lower, p