Abstract. Objectives. Unicompartmental (UKA) and bicompartmental (BCA) knee arthroplasty are associated with improved functional outcomes compared to Total Knee Arthroplasty (TKA) in suitable patients, although the reason is poorly understood. The aim of this study was to measure how the different arthroplasties affect knee extensor function. Methods. Extensor function was measured for sixteen cadaveric knees and then re-tested following the different arthroplasties. Eight knees underwent medial UKA then BCA, then posterior-cruciate retaining TKA, and eight underwent the lateral equivalents then TKA. Extensor efficiency was calculated for ranges of knee flexion associated with common activities of daily living. Data were analyzed with repeated measures analysis of variance (α=0.05). Results. Compared to native, no differences were detected in either extensor moment or efficiency following UKA. Conversion to BCA resulted in a small drop in extensor moment between 70-90° flexion (p<0.05), but when examined in the context of daily activity ranges of flexion, extensor efficiency was largely unaffected. Following TKA, large decreases in extensor moment were measured at low knee flexion angles (p<0.05), resulting in 8–43% reduction in extensor efficiency for daily activity ranges. Conclusion. This cadaveric study found that TKA is associated with inferior extensor function compared to UKA and BCA. This may, in part, help explain the reported differences in function and satisfaction differences between partial and total knee arthroplasty. Declaration of Interest. (a) fully declare any financial or other potential conflict of interest

An innovative Kirschner (K-) wire point was developed and compared in fresh pig femora in terms of drilling efficiency and temperature elevation with the trochar and diamond points currently used in clinical practice. The tips of thermal couples were machined to the defined geometry and the temperature measured during drilling. Using the same drill speed (rev/min) and feed rate, the new K-wire point produced the lowest thrust force and torque as measured by a Kistler dynamometer. Drill point temperatures were highest with the trochar geometry (129 ± 6°C), followed by the diamond (98 ± 7°C). The lowest temperatures were recorded with the Medin K-wire (66 ± 2°C). On repeated drilling it could be used for up to 30 holes before reaching the less satisfactory drill performance of the diamond tip. The new K-wire provides a better alternative as it requires less effort for insertion, generates less heat and may be re-used

Background

The different biodegradable local antibiotic delivery systems are widely used in recent years. The aim of this study was to evaluate the bactericidal activity antibiotic loaded PerOssal pellet in vitro and its effectiveness in the treatment of Staphylococcus aureus induced chronic osteomyelitis.

Abstract. Objectives. Total hip arthroplasty (THA) procedures are physically demanding for surgeons. Repetitive mallet swings to impact a surgical handle (impactions), can lead to muscle fatigue, discomfort and injuries. The use of an automated surgical hammer may reduce fatigue and increase surgical efficiency. The aim of this study was to develop a method to quantify user's performance, by recording surface electromyography (sEMG), for automated and manual impactions. Methods. sEMG signals were recorded from eight muscle compartments (arm and back muscles) of an orthopaedic surgeon during repetitions of manual and automated impaction tasks, replicating femoral canal preparation (broaching) during a THA. Each task was repeated, randomly, four times manually and four times with the automated impaction device. The mechanical outcomes (broaching efficiency and broach advancement) were quantified by tracking the kinematics of the surgical instrumentation. Root mean square (RMS) values and median frequency (MDF) were calculated for each task to, respectively, investigate which muscles were mostly involved (higher RMS) in each task and to quantify the decrease in MDF, which is an indicator of muscle fatigue. Results. RMS for arm muscles was significantly higher (p-value=0.002) during manual impactions than during automated impactions and muscle fatigue was significantly reduced (p-value=0.011), for the same muscles, when the same tasks were performed with the automated surgical hammer. The time required to achieve the same mechanical outcome, in terms of broaching efficiency and broach advancement, was significantly reduced with the automated surgical hammer (p=0.019). Conclusions. Results from this study showed how with this methodology it was possible to discern muscle performance and fatigue, between impaction modalities. Moreover, the reduction in exposure time to automated impactions, could be a factor in muscle fatigue decrease. These results could therefore provide useful insights into the study of surgical ergonomic improvements, to reduce surgeons muscle fatigue and, potentially, injuries. Declaration of Interest. (a) fully declare any financial or other potential conflict of interest

Intervertebral disc degeneration can lead to physical disability and significant pain, while the present therapeutics still fail to biochemically and biomechanically restore the tissue. Stem cell-based therapy in treating intervertebral disc (IVD) degeneration is promising while transplanting cells alone might not be adequate for effective regeneration. Recently, gene modification and 3D-printing strategies represent promising strategies to enhanced therapeutic efficacy of MSC therapy. In this regard, we hypothesized that the combination of thermosensitive chitosan hydrogel and adipose derived stem cells (ADSCs) engineered with modRNA encoding Interleukin − 4 (IL-4) can inhibit inflammation and promote the regeneration of the degenerative IVD. Rat ADSCs were acquired from adipose tissue and transfected with modRNAs. First, the kinetics and efficacy of modRNA-mediated gene transfer in mouse ADSCs were analyzed in vitro. Next, we applied an indirect co-culture system to analyze the pro-anabolic potential of IL-4 modRNA engineered ADSCs (named as IL-4-ADSCs) on nucleus pulposus cells. ModRNA transfected mouse ADSCs with high efficiency and the IL-4 modRNA-transfected ADSCs facilitated burst-like production of bio-functional IL-4 protein. In vitro, IL-4-ADSCs induced increased anabolic markers expression of nucleus pulposus cells in inflammation environment compared to untreated ADSCs. These findings collectively supported the therapeutic potential of the combination of thermosensitive chitosan hydrogel and IL-4-ADSCs for intervertebral disc degeneration management. Histological and in vivo validation are now being conducted

Artificial Intelligence (AI) is becoming more powerful but is barely used to counter the growth in health care burden. AI applications to increase efficiency in orthopedics are rare. We questioned if (1) we could train machine learning (ML) algorithms, based on answers from digitalized history taking questionnaires, to predict treatment of hip osteoartritis (either conservative or surgical); (2) such an algorithm could streamline clinical consultation. Multiple ML models were trained on 600 annotated (80% training, 20% test) digital history taking questionnaires, acquired before consultation. Best performing models, based on balanced accuracy and optimized automated hyperparameter tuning, were build into our daily clinical orthopedic practice. Fifty patients with hip complaints (>45 years) were prospectively predicted and planned (partly blinded, partly unblinded) for consultation with the physician assistant (conservative) or orthopedic surgeon (operative). Tailored patient information based on the prediction was automatically sent to a smartphone app. Level of evidence: IV. Random Forest and BernoulliNB were the most accurate ML models (0.75 balanced accuracy). Treatment prediction was correct in 45 out of 50 consultations (90%), p<0.0001 (sign and binomial test). Specialized consultations where conservatively predicted patients were seen by the physician assistant and surgical patients by the orthopedic surgeon were highly appreciated and effective. Treatment strategy of hip osteoartritis based on answers from digital history taking questionnaires was accurately predicted before patients entered the hospital. This can make outpatient consultation scheduling more efficient and tailor pre-consultation patient education

Total knee and hip arthroplasty (TKA and THA) are the most commonly performed surgical procedures, the costs of which constitute a significant healthcare burden. Improving access to care for THA/TKA requires better efficiency. It is hypothesized that this may be possible through a two-stage approach that utilizes prediction of surgical time to enable optimization of operating room (OR) schedules. Data from 499,432 elective unilateral arthroplasty procedures, including 302,490 TKAs, and 196,942 THAs, performed from 2014-2019 was extracted from the American College of Surgeons (ACS) National Surgical and Quality Improvement (NSQIP) database. A deep multilayer perceptron model was trained to predict duration of surgery (DOS) based on pre-operative clinical and biochemical patient factors. A two-stage approach, utilizing predicted DOS from a held out “test” dataset, was utilized to inform the daily OR schedule. The objective function of the optimization was the total OR utilization, with a penalty for overtime. The scheduling problem and constraints were simulated based on a high-volume elective arthroplasty centre in Canada. This approach was compared to current patient scheduling based on mean procedure DOS. Approaches were compared by performing 1000 simulated OR schedules. The predict then optimize approach achieved an 18% increase in OR utilization over the mean regressor. The two-stage approach reduced overtime by 25-minutes per OR day, however it created a 7-minute increase in underutilization. Better objective value was seen in 85.1% of the simulations. With deep learning prediction and mathematical optimization of patient scheduling it is possible to improve overall OR utilization compared to typical scheduling practices. Maximizing utilization of existing healthcare resources can, in limited resource environments, improve patient's access to arthritis care by increasing patient throughput, reducing surgical wait times and in the immediate future, help clear the backlog associated with the COVID-19 pandemic

Osteoarthritis (OA) affects the whole joint and leads to chronic pain. The sympathetic nervous system (SNS) seems to be involved in OA pathogenesis, as indicated by in vitro studies as well as by our latest work demonstrating that sympathectomy in mice results in increased subchondral bone volume in the OA knee joint. We assume that chronic stress may lead to opposite effects, such as an increased bone loss in OA due to an elevated sympathetic tone. Therefore, we analyzed experimental OA progression in mice exposed to chronic stress. OA was induced in male C57BL/6J mice by surgical destabilization of the medial meniscus (DMM) and Sham as well as non-operated mice served as controls. Half of these groups were exposed to chronic unpredictable mild stress (CUMS). After 12 weeks, chronic stress efficiency was assessed using behavioral tests. In addition to measuring body weight and length, changes in subchondral bone were analyzed by μCT. Dynamic Weight Bearing system was used to monitor OA-related pain. Histological scoring will be conducted to investigate the severity cartilage degeneration and synovial inflammation. CUMS resulted in increased anxiety and significant decrease in body weight gain in all CUMS groups compared to non-CUMS groups. CUMS also increased serum corticosterone in healthy mice, with even higher levels in CUMS mice after DMM surgery. CUMS had no significant effect on subchondral bone, but subarticular bone mineral density and trabecular thickness were increased. Moreover, CUMS resulted in significant potentiation of DMM-associated pain. Our results suggest that the autonomic imbalance with increased sympathetic nervous activity induced by chronic stress exacerbates the severity of OA pain perception. We expect significantly increased cartilage degeneration as well as more severe synovial inflammation in CUMS DMM mice compared to DMM mice

Orthopaedic impaction-instruments are used to drive implants into the bone of the patient. Pre-clinical experimental testing protocols and computer models of those are used to assess robustness and functional efficiency of such instruments. This generally involves impaction of the instrument mounted on a substrate that should represent the mechanics of the patient. In this study, the effects of the substrate on stressing of the impaction-instruments were investigated using dynamic finite element analysis. Model results were compared with experimental data from lab protocols, which have been derived to recreate the mechanics of cadaveric implantations, which represent clinical conditions. FEA models of selected experimental protocols were created in which a simplified instrument was impacted on substrates with varying material properties and boundary conditions. After impaction, the instrument settled into a modal vibration which then decayed over time. The resulting axial strain data from the computational model was compared to strain-gauge data collected from experimental measurements. Strain signal amplitude, frequency and decay were compared. The damping-ratio was derived from the decay of the strain signal. The computational model slightly over-predicted the initial experimental strain amplitudes in all cases, but the frequency of the cyclic strain signals matched. However, the model underestimated the experimentally measured rate of signal decay. Inclusion of implant seating and soft-tissue conditions had little effect on decay. Clinical failures of impaction-instruments may be related to multiple fatigue cycles for each impaction and should be modelled accurately to allow failure prediction. Any soft substrate results in an impedance mismatch at the instrument interface, which reflects the pressure wave and causes vibration with a frequency related to the speed-of-sound in the instrument, and its geometry. While this could be accurately modelled computationally, signal decay was underestimated. Further experimental quantification of energy losses will be important to understand vibration decay

Intervertebral disc (IVD) degeneration occurs with aging, leading to low back pain (LBP), which is one of the leading conditions of disability worldwide. With the lack of effective treatment, decellularized extracellular matrix (dECM) – based biomaterials have been proposed for IVD regeneration. However, the impact of donor ages on tissue repair had never been explored before in the disc field. Therefore, we aimed to address this question. For that, a decellularization protocol for bovine nucleus pulposus (NP) of different aged donors (fetus, young and old) was optimized by testing several detergents (SDS and Triton). The process efficiency was evaluated in terms of DNA and cell removal, as well as ECM preservation. Afterwards, dECMs were repopulated with bovine NP cells and cultured ex vivo. At day 7, cell behavior, ECM de novo synthesis and remodeling were evaluated [1]. Moreover, dECMs’ inflammatory response was assessed after in vivo CAM assay. Finally, inflammatory and angiogenic cytokines were analyzed in the conditioned media-derived from dECMs by using a cytokine array. As results, an optimal decellularization protocol (SDS 0.1%, 1h), efficient at removing cells and DNA from bovine NPs, while preserving ECM cues of native tissues, was developed. After repopulation, aggrecan increased in younger NPs, while collagen 2 decreased which may be indicative of matrix remodeling [1]. After in vivo CAM assay, fetal dECMs showed the highest inflammatory response. Finally, no statistically significant changes of cytokines were detected in the matrices, despite for a trend of higher IFN-α, IFN-γ and LIF in fetal dECMs, IL-1β in young dECMs and Decorin in old dECMs. Overall, this work uncovered the importance of tissue donor ages for tissue regenerative purpose, opening new avenues for the development of appropriate therapeutic strategies for IVD degeneration. Acknowledgments: FCT, EUROSPINE, ON Foundation

Abstract. Background. 1. 63,284 patients presented with neck of femur fractures in England in 2020 (NHFD report 2021)2. To maximise theatre efficiency during the first wave of COVID-19, NHSE guidance recommended the use of HA for most patients requiring arthroplasty.3. The literature reports an incidence of Hemiarthroplasty dislocations of 1–15%. Aims. 1. To study the number and possible causes of dislocations in patients with Primary hemiarthroplasty for fracture neck of femur2. To compare our data with national and international data in terms of dislocation and revision rates for Hemiarthroplasty. Methods. Retrospective study Duration- 1st April 2021–31st March 20223. Inclusion criteria- Patients with neck of femur fracture treated with Hemiarthroplasty. Exclusion criteria- Patients treated with other surgical options for neck of femur fractures. Results. 1. No. of neck of femur patients- 4442. No. of patients treated with Hemiarthroplasty- 2143. No. of dislocations- 44. 75% were female, 75% had AMTS>7, 50% were operated within 36 hours of trauma, 75% dislocated within a month of surgery, 75% of the dislocations were revised. 5. One dislocation was due to >72 hour delay to surgery, second dislocation was due to smaller offset and shortening, third was due to acetabular dysplasia, fourth was due to larger head used. Discussion-1. Our 1.86% dislocation rate matches that in the literature of 1–6%2. 75% dislocated within a month of surgery matches that in the literature that maximum dislocations occur within one month. 3. Closed reduction as definitive method of management of dislocation 25% matches that in the literature of 22–25%4. 75% dislocations revised similar to literature of 75–80% revision rate. Conclusion. Pre-operative templating can reduce surgical error involving limb length discrepancy and shortening to prevent dislocations. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Matrix-bound vesicles (MBVs) are embedded within osteoid and function as the site of initial mineral formation. However, they remain insufficiently characterised in terms of biogenesis, composition and function while their relationship with secreted culture medium EVs (sEVs) such as exosomes remains debated. We aimed to define the biogenesis and pro-mineralisation capacity of MBVs and sEVs to understand their potential in regenerative orthopaedics. sEVs and MBVs isolated from conditioned medium (differential ultracentrifugation) and ECM (collagenase digestion and differential ultracentrifugation) of mineralising MC3T3 pre-osteoblast and human bone marrow MSC cultures were characterised by nanoparticle tracking analysis, western blotting, nano-flow cytometry, super resolution microscopy (ONI) and TEM. Immunoprecipitated populations positive for alkaline phosphatase (ALP), a putative marker of mineralisation capacity, were also characterised. Collagen binding efficiency was evaluated using MemGlow staining. Results reported were comparative across both cell lines. Western blots indicated MBV fractions were positive for markers of endosomal biogenesis (CD9, CD81, ALIX, TSG101) and pro-mineralising proteins (ALP, Pit1, Annexin II, Annexin V), with Annexin V and CD9 present in immunoprecipitated ALP-positive fractions. MBVs were significantly larger than sEVs (p<0.05) and contained a higher amount of ALP (p<0.05) with a significant increase from day 7 to day 14 of cellular mineralisation (p<0.05). This mirrored the pattern of electron-dense vesicles seen via TEM. Super resolution single vesicle analysis revealed for the first-time co-expression of ALP with markers of endosomal biogenesis (CD9, CD63, CD81, ALIX) and Annexin II in both vesicle types, with higher co-expression percentage in MBVs than sEVs. MBVs also exhibited preferential collagen binding. Advanced imaging methods demonstrated that contrary to opinions in the field, MBVs appear to possess exosomal markers and may arise via endosomal biogenesis. However, it was evident that a higher proportion of MBVs possessed machinery to induce mineralisation and were enriched in mineral-dense material

In current practice in the UK there are three main approaches to investigating suspected scaphoid fractures not seen on initial plain film x-rays. Early MRI of all cases. Review all cases in clinic at two weeks with repeat x-rays. Hybrid model. Virtual Fracture Clinic (VFC) triage to reduce those who are seen in clinic at two weeks by:. ∘. Organising early MRI for those with high-risk presentation. ∘. Discharging those with an alternative more likely diagnosis. Our unit uses the VFC model. We aimed to evaluate its efficiency, safety, clinical outcomes and economic viability. All patients attending the emergency department with either a confirmed or suspected scaphoid fracture between March and December 2020 were included (n=305). Of these 297 were referred to the VFC: 33 had a confirmed fracture on x-ray and 264 had a suspected fracture. Of the suspected fractures reviewed in VFC 14% had an MRI organised directly owing to a high-risk presentation, 79% were brought for fracture clinic review and 17% discharged with an alternative diagnosis such as osteoarthritis. Of those subsequently reviewed in fracture clinic at two weeks: 9% were treated as scaphoid fractures (based on clinical suspicion and repeat x-rays), 17% had MRI or CT imaging organised, 5% did not attend and 69% were discharged. Overall, 17% of cases initially triaged, had further imaging – 41 MRIs and 5 CTs. MRI detected: 5% scaphoid fracture, 17% other fracture, 24% bone contusion, complete ligament tear 10%, partial ligament tear 39% and normal study 10%. The results of MRI minimally affected management. 3 patients were taken out of plaster early, 1 patient was immobilized who was not previously and no patients underwent operative management. In the following 12-month period one patient re-presented with a hand or wrist issue. This approach avoided 218 MRIs, equating to £24000 and 109 hours of scanner time. VFC triage and selective use of MRI scanning is a safe, efficient and cost-effective method for the management suspected scaphoid fractures. This can be implemented in units without the resource to MRI all suspected scaphoid fractures from the emergency department

To create a comprehensive, user-friendly, database that facilitates selection of optimized animal models for fracture research. Preclinical testing using research animal models can expedite effective and safe interventions for clinical fracture patients but ethical considerations (e.g., adherence to 3R humane principles) and failure to meet critical review (e.g., clinical translation, reproducibility) currently complicate the model selection process. English language publications (1980-2021) were derived from PubMed® using the search-term ‘bone and fracture and animal’. Clinical cases, reviews, and cadaver studies were excluded. Qualifying papers reporting use of fracture models had the following data transcribed: Author, journal, abstract, summary data, animal data, bone, focus (e.g., allograft) and model (e.g., articular fracture). Publications were quantitatively scored (1 star [very poor] – 5 stars [excellent]) for reproducibility, clinical translation and animal welfare. 4602 papers were derived from 677 journals from 177 publishers. Number of annual publications progressively increased from 18 (1980), peaking in 2015 (250) before substantially declining in 2020 (121) and 2021 (51). Descriptors (low to high) included 15 species (frog [1]–rat [1586]), 24 bones (phalanx [1]–femur [1646]), 134 research foci (bioprinting [4]–fracture healing [3533]), and 37 fracture models (avulsion [4]–diaphyseal [2113]). Percent of total publications scoring 1 or more stars for reproducibility, clinical translation and animal welfare ranged from: 1.0–5.8% (1 star), 5.9–30.6% (2 star), 21.3–42.8% (3 star), 19.2–44.4% (4 stars), and 1.3–26.7% (5 stars). FRAMD provides a dedicated resource that enhances selection of animal models that pertain to researchers’ fracture focus while being clinically relevant, reproducible and humane. FRAMD will help improve scientific data, reduce unnecessary use of animals, heighten workplace efficiency, and reduce cost by avoiding ill-suited or outdated models. FRAMD may particularly benefit grant writers and organizations seeking ‘best-practice’ assurance (e.g., funding agencies, academic research societies, CROs)

Many age-related diseases affect our skeletal system, but bone health-targeting drug development strategies still largely rely on 2D in vitro screenings. We aimed at developing a scaffold-free progenitor cell-based 3D biomineralization model for more physiological high-throughput screenings. MC3T3-E1 pre-osteoblast spheroids were cultured in V-shaped plates for 28 days in alpha-MEM (10% FCS, 1% L-Gln, 1X NEAA) with 1% pen/strep, changed every two days, and differentiation was induced by 10mM b-glycerophosphate and 50µg/ml ascorbic-acid. Osteogenic cell differentiation was assessed through profiling mRNA expression of selected osteogenic markers by efficiency corrected normalized 2^DDCq RT-qPCR. Biomineralization in spheroids was evaluated by histochemistry (Alizarin Red/von Kossa staining), Alkaline phosphatase (Alp) activity, Fourier transform infrared spectroscopy (FTIR) analyses, micro-CT analyses, and scanning electron microscopy on critical point-dried samples. GraphPad Prism 9 analyses comprised Shapiro-Wilk and Brown-Forsythe tests as well as 2-way ANOVA with Tukey post-hoc and non-parametric Kruskal-Wallis with Dunn post-hoc tests. During mineralization, as opposed to non-mineralizing conditions, characteristic mRNA expression profiles of selected early and late osteoblast differentiation markers (e.g., RunX, Alp, Col1a1, Bglap) were observed between day 0 and 28 of culture; Alp was strongly upregulated (p<0.001) from day 7 on, followed by its enzymatic activity (p<0.001). Bglap and Col1a1 expression peaked on (p<0.001) and from day 14 on (p<0.05), respectively. IHC revealed osteocalcin staining in the spheroid core regions at day 14, while type I collagen staining of the cores was most prominent from day 21 on. Alizarin Red and Von Kossa confirmed central and radially outwards expanding mineralization patterns between day 14 and day 28, which was accompanied by a steady increase in extracellular calcium deposition over time (p<0.001). Micro-CT analyses allowed quantitative appreciation of the overall increase in mineral density over time (day21, p<0.05; d28, p<0.001), while SEM-EDX and FTIR ultimately confirmed a bone-like hydroxyapatite mineral deposition in 3D. A novel and thoroughly characterized versatile bone-like 3D biomineralization in vitro model was established, which allows for studying effects of pharmacological interventions on bone mineralization ex vivo under physiomimetic conditions. Ongoing studies currently aim at elucidating in how far it specifically recapitulates intramembranous ossification

Although 80% of fractures typically heal without any problems, there is a small proportion (<20%) that suffer complications such as delayed healing and potential progression to non-union. In patients with healing complications, the coordinated regulation between pro- and anti-inflammatory cytokines, such as interleukin-1β (IL-1β) and interleukin-1 receptor antagonist (IL-1Ra) respectively, is often dysregulated. The aim of this study is to develop a therapeutic strategy based on the local delivery of genes to reparative mesenchymal stromal cells (MSCs) migrating into the local fracture microenvironment, thereby promoting a more favourable healing environment to enhance fracture repair. Our approach involves the local delivery of nanoparticles complexing the non-viral vector polyethyleneimine (PEI) with therapeutic plasmid DNA (pDNA) encoding for IL-1Ra. pDNA encoding green fluorescent protein and Gaussia luciferase were used as reporter genes to determine the transfection efficiency of both rat and human MSCs using flow cytometry and to assess the transgene expression profile using a luciferase expression assay. The effect of transfection with PEI on the viability of MSCs was assessed using the metabolic assay Cell Titer Blue and dsDNA quantification. Levels of IL-1Ra produced by cells following transfection with nanoparticles encoding IL-1Ra was assessed using enzyme-linked immunosorbent assays (ELISA). HEK-Blue IL-1β reporter cells, which secrete alkaline phosphatase in response to IL-1β stimulation, were used to confirm that the IL-1Ra produced by transfected cells is functionally active, i.e. the successful antagonism of IL-1β bioactivity. We have determined that using PEI-based nanoparticles we can achieve a transfection efficiency of 14.8 + 1.8% in rat MSCs. Transgene expression was found to be transient, with a peak in expression at 7 days post-transfection and a gradual decrease over time, which was maintained for up to 4 weeks. Using an optimized concentration of PEI, the impact of the nanoparticles on MSC viability was limited, with no significant difference in cellular metabolic activity compared to non-transfected cells at 10 days post-transfection. We have additionally demonstrated the capacity to successfully transfect both rat and human MSCs with pDNA encoding for IL-1Ra, resulting in enhanced levels of IL-1Ra, which is functionally active. The use of non-viral gene therapy to locally deliver immunomodulatory genes, such as IL-1Ra, to MSCs presents a promising strategy to enhance bone healing. Specifically, the transgene expression levels achieved with such an approach can remain therapeutically effective and are transient in nature, presenting an advantage over other methods such as recombinant protein delivery and viral-based gene delivery methodologies

Summary Statement. Calcium phosphate (CaP) particles have attracted great interest as transfection reagents, yet little is known about their mechanism of internalisation. We report live cell time-course tracking of CaP particles during internalisation and the influence of Ca:P ratio on transfection efficiency. Introduction. Relatively recent work has seen calcium phosphate (CaP) salts used for the delivery of biological materials into cells in the form of peptides, polymers and DNA sequences. Calcium phosphate salts have a critical safety advantage over other vectors such as viruses in that they pose no risk of pathogenicity due to mutation and show no apparent cytotoxicity. Previous work within the group showed that Ca:P ratio influenced the transfection efficiency, but the fate of the particles on internalisation is yet unknown. The difficulty in tracking the particles can be related to the visual similarity to granulation within the cells. Using a surface modification method that enables the fluorescent labeling of silicon-substituted hydroxyapatite (SiHA) particles, we have tracked the internalisation of the particles to understand their mechanism of entry and how particle composition may influence transfection efficiency. Patients & Methods. SiHA particles were synthesised by the dropwise addition of an aqueous solution of diammonium hydrogen phosphate and silicon tetraacetate to an aqueous solution of calcium nitrate while under mixing and maintained at pH10. The particles were functionalised with thiol groups using (3-mercaptopropyl)trimethoxysilane and dye-labelled with fluorescein-5-maleimide. MC3T3 osteoblast precursor cells were incubated in cell culture media containing labelled particles at a concentration of 0.6μg/mL for 12 hours. Confocal images were obtained with a Zeiss LSM 710 ConfoCor 3 system based around a Zeiss AxioObserverZ1 microscope. Results. DNA binding efficiency between 79 to 94%, the lowest being the CaP sample of new CaP route at Ca/P ratio of 0.33 by SEDS processing, which was 79% and the highest was the HAp SEDS processed sample at 40°C, solvent flowrate of 1 ml/min and antisolvent flowrate of 60 g/min (particle size of 131 nm). From the fluorescence microscopy images, localised regions of particles measuring around 500–1000nm were detected. With a typical SiHA particle size of 50–70nm in length, these regions contain 10's of particles. Discussion/Conclusion. Thiol functionalisation enabled the internalised SiHA to be visually discriminated from the other cellular material with similar morphology and optical contrast as shown in the bright field image. HA particles (Ca:P of 1.67) showed a strong affinity for the cell membrane despite extensive washing with PBS and their higher calcium content may enhance the binding of the DNA to the particle surface, therefore improving transfection efficiency

As near-infrared (NIR) photothermal agents, copper sulfide nanoparticles (CuSNP) offer several advantages over plasmonic gold nanoparticles (GNP), the most widely used photothermal nanotransducers in biomedical applications. CuSNP exhibit strong optical absorption at NIR wavelengths (650–1100 nm) and convert it into heat due excitation of electronic transitions or plasmonic photoexcitation. In contrast with GNP, CuSNP are degradable, readily prepared, inexpensive to produce, efficiently cleared from the body and their photothermal efficiency is less sensitive to the dielectric constant of the surrounding medium. We explored the feasibility of CuSNP to function as degradable NIR nanotransducers within fibrin-based cellular scaffolds, paying great attention to the stability and photothermal efficiency of the composite. We tested in vitro and in vivo whether NIR-responsive fibrin hydrogels comprising CuSNP (CuSNP hydrogels) are reliable platforms for triggering transgene expression in cells harboring a gene circuit activatable by heat and dependent of rapamycin. NIR laser irradiation of the CuSNP hydrogels increased local temperature and, in the presence of rapamycin, triggers the gene switch based on the promoter of the highly heat-inducible HSP70B gene (HSPA7). After implantation of such a cell-containing CuSNP hydrogel, transgenic expression can be remotely triggered by NIR-irradiation. Interestingly, we found that CuSNP hydrogels induce remodeling activity in stem cells and stimulate an angiogenic response. In short, CuSNP hydrogels offer compelling features for tissue engineering applications, as fully degradable implants with enhanced integration capacity in host tissues that can provide for remote control in the deployment of therapeutic gene products

In the treatment of bone non-unions an alternative to bone autografts is the use of bone morphogenetic proteins (BMP-2, BMP-7) with powerful osteoinductive and osteogenic properties. In clinical settings, BMPs are applied using absorbable collagen sponges. Supraphysiological doses are needed and major side effects may occur as induce ectopic bone formation, chronic inflammation and excessive bone resorption. In order to increase the efficiency of the delivered for BMPs we designed cryostructured collagen scaffolds functionalized with hydroxyapatite, mimicking the structure of cortical bone (aligned porosity, anisotropic, ANI) or trabecular bone (random distributed porosity, isotropic, ISO). We hypothesize that anisotropic structure would enhance osteoconductive properties of the scaffolds increasing rhBMP-2 regenerative properties. In vitro, both scaffolds presented similar mechanical properties, rhBMP-2 retention and delivery capacity. For in vivo testing, a rat femoral critical size defect model was created. Four groups were assessed depending on the implant applied to the bone defect: ISO, unloaded isotropic sponge; ISO-BMP, isotropic sponge loaded with 5 μg of hrBMP-2; ANI, unloaded anisotropic sponge; and ANI-BMP, anisotropic sponge loaded with 5 μg of hrBMP-2. Regeneration was allowed for 10 weeks. X-ray, μCT, biomechanical testing and histology were used to evaluate repair. Independently of their structure, sponges loaded with rhBMP-2 demonstrate increased bone volume, and biomechanical properties than their controls (p<0.01 and p<0.05 respectively). Globally, ANI-BMP group demonstrated better bone regeneration outputs with increased defect bridging (p<0.05 when compared ANI-BMP vs ISO-BMP groups). In conclusion, anisotropic cryostructured collagen scaffolds improve the efficiency of rhBMP-2 in bone regeneration

Theatre efficiency is an increasingly important factor as the health service is faced with an ever greater number of patients, but tighter fiscal restrictions. We carried out an audit was to utilise data collected routinely on the Opera Surgical Management System (CHCA, Canada) to look at the efficiency of orthopaedic theatre usage, and to look for potential areas of improvement. The data related to all elective procedures carried out by a single Orthopaedic Consultant in a city hospital, over a one year period. We found that lists frequently started late, with the first patient of the day entering the anaesthetic room after 9 am on over 50% of occasions. The reasons for this were not regularly recorded. There was a wide variability in the length of time taken to anaesthetise and position the patients. Although there was a weak association with the underlying health of the patients (ASA score), the seniority of the anaesthetist was not available for correlation. The turnaround time between cases was variable, with a tendency for it to take longer over the lunchtime hours. Almost a third of lists finished before 4.30 pm, mainly due to patient cancellations, however nearly a third of lists finished after 5.30 pm. We found that data routinely collected on our theatre management system provides useful information that could potentially be used to fine-tune our peri-operative processes, however greater detail about specific timings and delays affecting the patient journey would be required before any recommendations could be made to improve theatre efficiency