Building parameters linked with indoor transmission of SARS-CoV-2.

The rapid spread of Coronavirus Disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has emphasized the importance of understanding and adapting to the indoor remediation of transmissible diseases to decrease the risk for future pandemic threats. While there were many precautions in place to hinder the spread of COVID-19, there has also been a substantial increase of new research on SARS-CoV-2 that can be utilized to further mitigate the transmission risk of this novel virus. This review paper aims to identify the building parameters of indoor spaces that could have considerable influence on the transmission of SARS-CoV-2. The following building parameters have been identified and analyzed, emphasizing their link with the indoor transmission of SARS-CoV-2: temperature and relative humidity, temperature differences between rooms, ventilation rate and access to natural ventilation, occupant density, surface type and finish, airflow direction and speed, air stability, indoor air pollution, central air conditioning systems, capacity of air handling system and HVAC filter efficiency, edge sealing of air filters, room layout and interior design, and compartmentalization of interior space. This paper also explains the interactions of SARS-CoV-2 with indoor environments and its persistence. Furthermore, the modifications of the key building parameters have been discussed for controlling the transmission of SARS-CoV-2 in indoor spaces. Understanding the information provided in this paper is crucial to develop effective health and safety measures that will aid in infection prevention.

Development of a Spray-Dried Formulation of Peptide-DNA Nanoparticles into a Dry Powder for Pulmonary Delivery Using Factorial Design.

BACKGROUND: Gene therapy via pulmonary delivery holds the potential to treat various lung pathologies. To date, spray drying has been the most promising method to produce inhalable powders. The present study determined the parameters required to spray dry nanoparticles (NPs) that contain the delivery peptide, termed RALA (N-WEARLARALARALARHLARALARALRACEA-C), complexed with plasmid DNA into a dry powder form designed for inhalation. METHODS: The spray drying process was optimised using full factorial design with 19 randomly ordered experiments based on the combination of four parameters and three centre points per block. Specifically, mannitol concentration, inlet temperature, spray rate, and spray frequency were varied to observe their effects on process yield, moisture content, a median of particle size distribution, Z-average, zeta potential, encapsulation efficiency of DNA NPs, and DNA recovery. The impact of mannitol concentration was also examined on the spray-dried NPs and evaluated via biological functionality in vitro. RESULTS: The results demonstrated that mannitol concentration was the strongest variable impacting all responses apart from encapsulation efficiency. All measured responses demonstrated a strong dependency on the experimental variables. Furthermore, spray drying with the optimal variables in combination with a low mannitol concentration (1% and 3%, w/v) produced functional RALA/pDNA NPs. CONCLUSION: The optimal parameters have been determined to spray dry RALA/pDNA NPs into an dry powder with excellent biological functionality, which have the potential to be used for gene therapy applications via pulmonary delivery.

Exploiting the anticancer effects of a nitrogen bisphosphonate nanomedicine for glioblastoma multiforme.

Glioblastoma multiforme (GBM) is an incurable aggressive brain cancer in which current treatment strategies have demonstrated limited survival benefit. In recent years, nitrogen-containing bisphosphonates (N-BPs) have demonstrated direct anticancer effects in a number of tumour types including GBM. In this study, a nano-formulation with the RALA peptide was used to complex the N-BP, alendronate (ALN) into nanoparticles (NPs) < 200 nm for optimal endocytic uptake. Fluorescently labelled AlexaFluor®647 Risedronate was used as a fluorescent analogue to visualise the intracellular delivery of N-BPs in both LN229 and T98G GBM cells. RALA NPs were effectively taken up by GBM where a dose-dependent response was evidenced with potentiation factors of 14.96 and 13.4 relative to ALN alone after 72 h in LN229 and T98G cells, respectively. Furthermore, RALA/ALN NPs at the IC50, significantly decreased colony formation, induced apoptosis and slowed spheroid growth in vitro. In addition, H-Ras membrane localisation was significantly reduced in the RALA/ALN groups compared to ALN or controls, indicative of prenylation inhibition. The RALA/ALN NPs were lyophilised to enhance stability without compromising the physiochemical properties necessary for functionality, highlighting the suitability of the NPs for scale-up and in vivo application. Collectively, these data show the significant potential of RALA/ALN NPs as novel therapeutics in the treatment of GBM.

DNA vaccination for cervical cancer; a novel technology platform of RALA mediated gene delivery via polymeric microneedles.

HPV subtypes (16, 18) are associated with the development of cervical cancer, with oncoproteins E6 and E7 responsible for pathogenesis. The goal of this study was to evaluate our 'smart system' technology platform for DNA vaccination against cervical cancer. The vaccination platform brings together two main components; a peptide RALA which condenses DNA into cationic nanoparticles (NPs), and a polymeric polyvinylpyrrolidone (PVP) microneedle (MN) patch for cutaneous delivery of the loaded NPs. RALA condensed E6/E7 DNA into NPs not exceeding 100nm in diameter, and afforded the DNA protection from degradation in PVP. Sera from mice vaccinated with MN/RALA-E6/E7 were richer in E6/E7-specific IgGs, displayed a greater T-cell-mediated TC-1 cytotoxicity and contained more IFN-γ than sera from mice that received NPs intramuscularly. More importantly, MN/RALA-E6/E7 delayed TC-1 tumor initiation in a prophylactic model, and slowed tumor growth in a therapeutic model of vaccination, and was more potent than intramuscular vaccination.

Biocompatibility of calcium phosphate bone cement with optimised mechanical properties: an in vivo study.

This work establishes the in vivo performance of modified calcium phosphate bone cements for vertebroplasty of spinal fractures using a lapine model. A non-modified calcium phosphate bone cement and collagen-calcium phosphate bone cements composites with enhanced mechanical properties, utilising either bovine collagen or collagen from a marine sponge, were compared to a commercial poly(methyl methacrylate) cement. Conical cement samples (8 mm height × 4 mm base diameter) were press-fit into distal femoral condyle defects in New Zealand White rabbits and assessed after 5 and 10 weeks. Bone apposition and tartrate-resistant acid phosphatase activity around cements were assessed. All implants were well tolerated, but bone apposition was higher on calcium phosphate bone cements than on poly(methyl methacrylate) cement. Incorporation of collagen showed no evidence of inflammatory or immune reactions. Presence of positive tartrate-resistant acid phosphatase staining within cracks formed in calcium phosphate bone cements suggested active osteoclasts were present within the implants and were actively remodelling within the cements. Bone growth was also observed within these cracks. These findings confirm the biological advantages of calcium phosphate bone cements over poly(methyl methacrylate) and, coupled with previous work on enhancement of mechanical properties through collagen incorporation, suggest collagen-calcium phosphate bone cement composite may offer an alternative to calcium phosphate bone cements in applications where low setting times and higher mechanical stability are important.

Extent and mechanism of phase separation during the extrusion of calcium phosphate pastes.

The aim of this study was to increase understanding of the mechanism and dominant drivers influencing phase separation during ram extrusion of calcium phosphate (CaP) paste for orthopaedic applications. The liquid content of extrudate was determined, and the flow of liquid and powder phases within the syringe barrel during extrusion were observed, subject to various extrusion parameters. Increasing the initial liquid-to-powder mass ratio, LPR, (0.4-0.45), plunger rate (5-20 mm/min), and tapering the barrel exit (45°-90°) significantly reduced the extent of phase separation. Phase separation values ranged from (6.22 ± 0.69 to 18.94 ± 0.69 %). However altering needle geometry had no significant effect on phase separation. From powder tracing and liquid content determination, static zones of powder and a non-uniform liquid distribution was observed within the barrel. Measurements of extrudate and paste LPR within the barrel indicated that extrudate LPR remained constant during extrusion, while LPR of paste within the barrel decreased steadily. These observations indicate the mechanism of phase separation was located within the syringe barrel. Therefore phase separation can be attributed to either; (1) the liquid being forced downstream by an increase in pore pressure as a result of powder consolidation due to the pressure exerted by the plunger or (2) the liquid being drawn from paste within the barrel, due to suction, driven by dilation of the solids matrix at the barrel exit. Differentiating between these two mechanisms is difficult; however results obtained suggest that suction is the dominant phase separation mechanism occurring during extrusion of CaP paste.

Optimisation of a two-liquid component pre-filled acrylic bone cement system: a design of experiments approach to optimise cement final properties.

The initial composition of acrylic bone cement along with the mixing and delivery technique used can influence its final properties and therefore its clinical success in vivo. The polymerisation of acrylic bone cement is complex with a number of processes happening simultaneously. Acrylic bone cement mixing and delivery systems have undergone several design changes in their advancement, although the cement constituents themselves have remained unchanged since they were first used. This study was conducted to determine the factors that had the greatest effect on the final properties of acrylic bone cement using a pre-filled bone cement mixing and delivery system. A design of experiments (DoE) approach was used to determine the impact of the factors associated with this mixing and delivery method on the final properties of the cement produced. The DoE illustrated that all factors present within this study had a significant impact on the final properties of the cement. An optimum cement composition was hypothesised and tested. This optimum recipe produced cement with final mechanical and thermal properties within the clinical guidelines and stated by ISO 5833 (International Standard Organisation (ISO), International standard 5833: implants for surgery-acrylic resin cements, 2002), however the low setting times observed would not be clinically viable and could result in complications during the surgical technique. As a result further development would be required to improve the setting time of the cement in order for it to be deemed suitable for use in total joint replacement surgery.

A numerical investigation of stress, strain, and bone density changes due to bone remodelling in the talus bone following total ankle arthroplasty.

Total ankle arthroplasty is the gold standard surgical treatment for severe ankle arthritis and fracture. However, revision surgeries due to the in vivo failure of the ankle implant are a serious concern. Extreme bone density loss due to bone remodelling is one of the main reasons for in situ implant loosening, with aseptic loosening of the talar component being one of the primary reasons for total ankle arthroplasty revisions. This study is aimed at determining the performance and potential causes of failure of the talar component. Herein, we investigated the stress, strain, and bone density changes that take place in the talus bone during the first 6 months of bone remodelling due to the total ankle arthroplasty procedure. Computed tomography scans were used to generate the 3D geometry used in the finite element (FE) model of the Intact and implanted ankle. The Scandinavian Total Ankle Replacement (STAR™) CAD files were generated, and virtual placement within bone models was done following surgical guidelines. The dorsiflexion physiological loading condition was investigated. The cortical region of the talus bone was found to demonstrate the highest values of stress (5.02 MPa). Next, the adaptive bone remodelling theory was used to predict bone density changes over the initial 6-month post-surgery. A significant change in bone density was observed in the talus bone due to bone remodelling. The observed quantitative changes in talus bone density over 6-month period underscore potential implications for implant stability and fracture susceptibility. These findings emphasise the importance of considering such biomechanical factors in ankle implant design and clinical management.

Multi-objective property optimisation of a phosphoserine-modified calcium phosphate cement for orthopaedic and dental applications using design of experiments methodology.

Phosphoserine is a ubiquitous molecule found in numerous proteins and, when combined with alpha-tricalcium phosphate (α-TCP) powder, demonstrates the ability to generate an adhesive biomaterial capable of stabilising and repairing bone fractures. Design of Experiments (DoE) approach was able to optimise the composition of phosphoserine-modified calcium phosphate cement (PM-CPC) demonstrating that the liquid:powder ratio (LPR) and quantity of phosphoserine (wt%) significantly influenced the handling, mechanical, and adhesion properties. Subsequently, the DoE optimisation process identified the optimal PM-CPC formulation, exhibiting a compressive strength of 29.2 ± 4.9 MPa and bond/shear strength of 3.6 ± 0.9 MPa after a 24 h setting reaction. Moreover, the optimal PM-CPC composition necessitated a mixing time of 20 s and displayed an initial setting time between 3 and 4 min, thus enabling homogenous mixing and precise delivery within a surgical environment. Notably, the PM-CPC demonstrated a bone-to-bone bond strength of 1.05 ± 0.3 MPa under wet conditions, coupled with a slow degradation rate during the first five days. These findings highlight the ability of PM-CPC to effectively support and stabilise bone fragments during the initial stages of natural bone healing. The developed PM-CPC formulations fulfil the clinical requirements for working and setting times, static mechanical, degradation properties, and injectability, enabling surgeons to stabilise complex bone fractures. This innovative bioinspired adhesive represents a significant advancement in the treatment of challenging bone injuries, offering precise delivery within a surgical environment and the potential to enhance patient outcomes. STATEMENT OF SIGNIFICANCE: This manuscript presents a noteworthy contribution to the field of bone fracture healing and fixation by introducing a novel phosphoserine-modified calcium phosphate cement (PM-CPC) adhesive by incorporating phosphoserine and alpha-TCP. This study demonstrates the fabrication and extensive characterisation of this adhesive biomaterial that holds great promise for stabilising and repairing complex bone fractures. Design of Experiment (DoE) software was used to investigate the correlations between process, property, and structure of the adhesive, resulting in a cost-effective formulation with desirable physical and handling properties. The PM-CPC adhesive exhibited excellent adhesion and cohesion properties in wet-field conditions. This research offers significant potential for clinical translation and contributes to the ongoing advancements in bone tissue engineering.

Immunotherapy for HPV negative head and neck squamous cell carcinoma.

Head and neck cancer (HNSCC) is the 8th most common cancer in the UK, with incidence increasing due to lifestyle factors such as tobacco and alcohol abuse. HNSCC is an immune-suppressive disease characterised by impaired cytokine secretion and dysregulation of immune infiltrate. As such, immunotherapy is a potential treatment option, with therapeutic cancer vaccination demonstrating the greatest potential. The success of cancer vaccination is dependent on informed antigen selection: an ideal antigen must be either tumour-specific or tumour-associated, as well as highly immunogenic. Stratification of the patient population for antigen expression and validated biomarkers are also vital. This review focuses on the latest developments in immunotherapy, specifically the development of therapeutic vaccines, and highlights successes, potential drawbacks and areas for future development. Immunotherapy approaches considered for HNSCC include monoclonal antibodies (mAb), Oncolytic viral (OV) therapies, Immune Checkpoint Inhibitors (ICIs) and cancer vaccines.

Finite element analysis of vertebroplasty in the osteoporotic T11-L1 vertebral body: Effects of bone cement formulation.

Vertebral compression fractures are one of the most severe clinical consequences of osteoporosis and the most common fragility fracture afflicting 570 and 1070 out of 100,000 men and women worldwide, respectively. Vertebroplasty (VP), a minimally invasive surgical procedure that involves the percutaneous injection of bone cement, is one of the most efficacious methods to stabilise osteoporotic vertebral compression fractures. However, postoperative fracture has been observed in up to 30% of patients following VP. Therefore, this study aims to investigate the effect of different injectable bone cement formulations on the stress distribution within the vertebrae and intervertebral discs due to VP and consequently recommend the optimal cement formulation. To achieve this, a 3D finite element (FE) model of the T11-L1 vertebral body was developed from computed tomography scan data of the spine. Osteoporotic bone was modeled by reducing the Young's modulus by 20% in the cortical bone and 74% in cancellous bone. The FE model was subjected to different physiological movements, such as extension, flexion, bending, and compression. The osteoporotic model caused a reduction in the average von Mises stress compared with the normal model in the T12 cancellous bone and an increment in the average von Mises stress value at the T12 cortical bone. The effects of VP using different formulations of a novel injectable bone cement were modeled by replacing a region of T12 cancellous bone with the materials. Due to the injection of the bone cement at the T12 vertebra, the average von Mises stresses on cancellous bone increased and slightly decreased on the cortical bone under all loading conditions. The novel class of bone cements investigated herein demonstrated an effective restoration of stress distribution to physiological levels within treated vertebrae, which could offer a potential superior alternative for VP surgery as their anti-osteoclastogenic properties could further enhance the appeal of their fracture treatment and may contribute to improved patient recovery and long-term well-being.

Low-High-Low Rotationally Pulse-Actuated Serial Dissolvable Film Valves Applied to Solid Phase Extraction and LAMP Isothermal Amplification for Plant Pathogen Detection on a Lab-on-a-Disc.

The ability of the centrifugal Lab-on-a-Disc (LoaD) platform to closely mimic the "on bench" liquid handling steps (laboratory unit operations (LUOs)) such as metering, mixing, and aliquoting supports on-disc automation of bioassay without the need for extensive biological optimization. Thus, well-established bioassays, normally conducted manually using pipettes or using liquid handling robots, can be relatively easily automated in self-contained microfluidic chips suitable for use in point-of-care or point-of-use settings. The LoaD's ease of automation is largely dependent on valves that can control liquid movement on the rotating disc. The optimum valving strategy for a true low-cost and portable device is rotationally actuated valves, which are actuated by changes in the disc spin-speed. However, due to tolerances in disc manufacturing and variations in reagent properties, most of these valving technologies have inherent variation in their actuation spin-speed. Most valves are actuated through stepped increases in disc spin-speed until the motor reaches its maximum speed (rarely more than 6000 rpm). These manufacturing tolerances combined with this "analogue" mechanism of valve actuation limits the number of LUOs that can be placed on-disc. In this work, we present a novel valving mechanism called low-high-low serial dissolvable film (DF) valves. In these valves, a DF membrane is placed in a dead-end pneumatic chamber. Below an actuation spin-speed, the trapped air prevents liquid wetting and dissolving the membrane. Above this spin-speed, the liquid will enter and wet the DF and open the valve. However, as DFs take ∼40 s to dissolve, the membrane can be wetted, and the disc spin-speed reduced before the film opens. Thus, by placing valves in a series, we can govern on which "digital pulse" in spin-speeding a reagent is released; a reservoir with one serial valve will open on the first pulse, a reservoir with two serial valves on the second, and so on. This "digital" flow control mechanism allows the automation of complex assays with high reliability. In this work, we first describe the operation of the valves, outline the theoretical basis for their operation, and support this analysis with an experiment. Next, we demonstrate how these valves can be used to automate the solid-phase extraction of DNA on on-disc LAMP amplification for applications in plant pathogen detection. The disc was successfully used to extract and detect, from a sample lysed off-disc, DNA indicating the presence of thermally inactivated Clavibacter michiganensis ssp. michiganensis (Cmm), a bacterial pathogen on tomato leaf samples.

Synthesis and characterisation of a nucleotide based pro-drug formulated with a peptide into a nano-chemotherapy for colorectal cancer.

Recent studies in colorectal cancer patients (CRC) have shown that increased resistance to thymidylate synthase (TS) inhibitors such as 5-fluorouracil (5-FU), reduce the efficacy of standard of care (SoC) treatment regimens. The nucleotide pool cleanser dUTPase is highly expressed in CRC and is an attractive target for potentiating anticancer activity of chemotherapy. The purpose of the current work was to investigate the activity of P1, P4-di(2',5'-dideoxy-5'-selenouridinyl)-tetraphosphate (P4-SedU2), a selenium-modified symmetrically capped dinucleoside with prodrug capabilities that is specifically activated by dUTPase. Using mechanochemistry, P4-SedU2 and the corresponding selenothymidine analogue P4-SeT2 were prepared with a yield of 19% and 30% respectively. The phosphate functionality facilitated complexation with the amphipathic cell-penetrating peptide RALA to produce nanoparticles (NPs). These NPs were designed to deliver P4-SedU2 intracellularly and thereby maximise in vivo activity. The NPs demonstrated effective anti-cancer activity and selectivity in the HCT116 CRC cell line, a cell line that overexpresses dUTPase; compared to HT29 CRC cells and NCTC-929 fibroblast cells which have reduced levels of dUTPase expression. In vivo studies in BALB/c SCID mice revealed no significant toxicity with respect to weight or organ histology. Pharmacokinetic analysis of blood serum showed that RALA facilitates effective delivery and rapid internalisation into surrounding tissues with NPs eliciting lower plasma Cmax than the equivalent injection of free P4-SedU2, translating the in vitro findings. Tumour growth delay studies have demonstrated significant inhibition of growth dynamics with the tumour doubling time extended by >2weeks. These studies demonstrate the functionality and action of a new pro-drug nucleotide for CRC.

Delivery of a peptide/microRNA blend via electrospun antimicrobial nanofibres for wound repair.

Downregulation of microRNA-31 (miR-31) and microRNA-132 (miR-132) has been associated with delayed wound healing. Therefore, it was hypothesised that intracellular delivery of miR-31 and miR-132, both as individual and blend formulations, could promote tissue repair. The use of a blend could minimise potential toxicity and achieve synergistic effects, thus maximising the therapeutic effect. miR-31 and miR-132 were condensed with a 30-mer positively charged amphipathic peptide, RALA, to form nanocomplexes with an average size <200 nm and zeta-potential ≥10 designed to facilitate cellular internalisation. This enabled a fold increase in miR-31 and miR-132 expression of ≥100,000 in a murine fibroblast cell line (NCTC-929) and a skin human keratinocyte cell line (HaCaT), with intracellular delivery >70% for individual and blend formulations. Moreover, incubation with the nanocomplexes increased the migration of HaCaT cells ≥25% at 4 and 8 h post-incubation, as well as downregulation of EMP-1 and RASA1 and HB-EGF and RASA1, target genes for miR-31 and miR-132, respectively. Electrospinning was then employed to produce an alginate/polyvinyl alcohol/ciprofloxacin nanofibrous wound patch to facilitate the controlled delivery of the nanocomplexes. Nanofibres were crosslinked with glutaraldehyde to improve stability in aqueous solvents, and they were proven to be biocompatible with antimicrobial activity without cellular attachment to avoid injury upon removal. RALA/miR nanoparticles were incorporated to the nanofibrous wound dressing and in vivo wound healing studies using C57BL/6J mice demonstrated a >60% acceleration in the wound closure rate at Day 7 post-wounding, a ≥1.5 increase in epidermal thickness, and a ≥2 increase in blood vessel count with respect to commercial and untreated controls. Taken together, this data proves that delivery of RALA/miR-31 and RALA/miR-132 from an alginate/polyvinyl alcohol/ciprofloxacin nanofibrous wound dressing constitutes an advanced therapy for wound healing, by accelerating wound closure and improving healed tissue quality. STATEMENT OF SIGNIFICANCE: In this study, we report for the first time the use of the RALA peptide to deliver two miRNA 31 & 132 simultaneously from an electrospun patch. Both miRs have been shown to be downregulated in wounds and this study endeavoured to deliver a blend of the miRs from a nanofibre patch. Electrospinning was used to produce an alginate/polyvinyl alcohol/ciprofloxacin wound patch to enable controlled delivery of the miRs without cellular attachment to the wound with the added benefit of anti-microbial activity. Application of the nanofibre patch loaded with the blended RALA/miR nanoparticles demonstrated a synergistic effect with acceleration of wound closure rate, a significant increase in epidermal thickness and blood vessel count with respect to commercial and untreated controls.

Optimization of an Injectable, Resorbable, Bioactive Cement Able to Release the Anti-Osteoclastogenic Biomolecule ICOS-Fc for the Treatment of Osteoporotic Vertebral Compression Fractures.

Vertebral compression fractures are typical of osteoporosis and their treatment can require the injection of a cement through a minimally invasive procedure to restore vertebral body height. This study reports the development of an injectable calcium sulphate-based composite cement able to stimulate bone regeneration while inhibiting osteoclast bone resorption. To this aim, different types of strontium-containing mesoporous glass particles (Sr-MBG) were added to calcium sulphate powder to impart a pro-osteogenic effect, and the influence of their size and textural features on the cement properties was investigated. Anti-osteoclastogenic properties were conferred by incorporating into poly(lactic-co-glycolic)acid (PLGA) nanoparticles, a recombinant protein able to inhibit osteoclast activity (i.e., ICOS-Fc). Radiopaque zirconia nanoparticles (ZrO2) were also added to the formulation to visualize the cement injection under fluoroscopy. The measured cement setting times were suitable for the clinical practice, and static mechanical testing determined a compressive strength of ca. 8 MPa, comparable to that of human vertebral bodies. In vitro release experiments indicated a sustained release of ICOS-Fc and Sr2+ ions up to 28 days. Overall, the developed cement is promising for the treatment of vertebral compression fractures and has the potential to stimulate bone regeneration while releasing a biomolecule able to limit bone resorption.

Peptide delivery of a multivalent mRNA SARS-CoV-2 vaccine.

Lipid nanoparticles (LNP) have been instrumental in the success of mRNA vaccines and have opened up the field to a new wave of therapeutics. However, what is ahead beyond the LNP? The approach herein used a nanoparticle containing a blend of Spike, Membrane and Envelope antigens complexed for the first time with the RALA peptide (RALA-SME). The physicochemical characteristics and functionality of RALA-SME were assessed. With >99% encapsulation, RALA-SME was administered via intradermal injection in vivo, and all three antigen-specific IgG antibodies were highly significant. The IgG2a:IgG1 ratio were all >1.2, indicating a robust TH1 response, and this was further confirmed with the T-Cell response in mice. A complete safety panel of markers from mice were all within normal range, supported by safety data in hamsters. Vaccination of Syrian Golden hamsters with RALA-SME derivatives produced functional antibodies capable of neutralising SARS-CoV-2 from both Wuhan-Hu-1 and Omicron BA.1 lineages after two doses. Antibody levels increased over the study period and provided protection from disease-specific weight loss, with inhibition of viral migration down the respiratory tract. This peptide technology enables the flexibility to interchange and add antigens as required, which is essential for the next generation of adaptable mRNA vaccines.

The osteogenic and angiogenic potential of microRNA-26a delivered via a non-viral delivery peptide for bone repair.

Bone-related injuries and diseases are among the most common causes of morbidity worldwide. Current bone-regenerative strategies such as auto- and allografts are invasive by nature, with adverse effects such as pain, infection and donor site morbidity. MicroRNA (miRNA) gene therapy has emerged as a promising area of research, with miRNAs capable of regulating multiple gene pathways simultaneously through the repression of post-transcriptional mRNAs. miR-26a is a key regulator of osteogenesis and has been found to be upregulated following bone injury, where it induces osteodifferentiation of mesenchymal stem cells (MSCs) and facilitates bone formation. This study demonstrates, for the first time, that the amphipathic, cell-penetrating peptide RALA can efficiently deliver miR-26a to MSCs in vitro to regulate osteogenic signalling. Transfection with miR-26a significantly increased expression of osteogenic and angiogenic markers at both gene and protein level. Using a rat calvarial defect model with a critical size defect, RALA/miR-26a NPs were delivered via an injectable, thermo-responsive Cs-g-PNIPAAm hydrogel to assess the impact on both rate and quality of bone healing. Critical defects treated with the RALA/miR-26a nanoparticles (NPs) had significantly increased bone volume and bone mineral density at 8 weeks, with increased blood vessel formation and mechanical properties. This study highlights the utility of RALA to deliver miR-26a for the purpose of bone healing within an injectable biomaterial, warranting further investigation of dose-related efficacy of the therapeutic across a range of in vivo models.

Effect of Uniaxial Compression Frequency on Osteogenic Cell Responses in Dynamic 3D Cultures.

The application of mechanical stimulation on bone tissue engineering constructs aims to mimic the native dynamic nature of bone. Although many attempts have been made to evaluate the effect of applied mechanical stimuli on osteogenic differentiation, the conditions that govern this process have not yet been fully explored. In this study, pre-osteoblastic cells were seeded on PLLA/PCL/PHBV (90/5/5 wt.%) polymeric blend scaffolds. The constructs were subjected every day to cyclic uniaxial compression for 40 min at a displacement of 400 µm, using three frequency values, 0.5, 1, and 1.5 Hz, for up to 21 days, and their osteogenic response was compared to that of static cultures. Finite element simulation was performed to validate the scaffold design and the loading direction, and to assure that cells inside the scaffolds would be subjected to significant levels of strain during stimulation. None of the applied loading conditions negatively affected the cell viability. The alkaline phosphatase activity data indicated significantly higher values at all dynamic conditions compared to the static ones at day 7, with the highest response being observed at 0.5 Hz. Collagen and calcium production were significantly increased compared to static controls. These results indicate that all of the examined frequencies substantially promoted the osteogenic capacity.

A computational analysis of a novel therapeutic approach combining an advanced medicinal therapeutic device and a fracture fixation assembly for the treatment of osteoporotic fractures: Effects of physiological loading, interface conditions, and fracture fixation materials.

The occurrence of periprosthetic femoral fractures (PFF) has increased in people with osteoporosis due to decreased bone density, poor bone quality, and stress shielding from prosthetic implants. PFF treatment in the elderly is a genuine concern for orthopaedic surgeons as no effective solution currently exists. Therefore, the goal of this study was to determine whether the design of a novel advanced medicinal therapeutic device (AMTD) manufactured from a polymeric blend in combination with a fracture fixation plate in the femur is capable of withstanding physiological loads without failure during the bone regenerative process. This was achieved by developing a finite element (FE) model of the AMTD together with a fracture fixation assembly, and a femur with an implanted femoral stem. The response of both normal and osteoporotic bone was investigated by implementing their respective material properties in the model. Physiological loading simulating the peak load during standing, walking, and stair climbing was investigated. The results showed that the fixation assembly was the prime load bearing component for this configuration of devices. Within the fixation assembly, the bone screws were found to have the highest stresses in the fixation assembly for all the loading conditions. Whereas the stresses within the AMTD were significantly below the maximum yield strength of the device's polymeric blend material. Furthermore, this study also investigated the performance of different fixation assembly materials and found Ti-6Al-4V to be the optimal material choice from those included in this study.

Development of miR-26a-activated scaffold to promote healing of critical-sized bone defects through angiogenic and osteogenic mechanisms.

Very large bone defects significantly diminish the vascular, blood, and nutrient supply to the injured site, reducing the bone's ability to self-regenerate and complicating treatment. Delivering nanomedicines from biomaterial scaffolds that induce host cells to produce bone-healing proteins is emerging as an appealing solution for treating these challenging defects. In this context, microRNA-26a mimics (miR-26a) are particularly interesting as they target the two most relevant processes in bone regeneration-angiogenesis and osteogenesis. However, the main limitation of microRNAs is their poor stability and issues with cytosolic delivery. Thus, utilising a collagen-nanohydroxyapatite (coll-nHA) scaffold in combination with cell-penetrating peptide (RALA) nanoparticles, we aimed to develop an effective system to deliver miR-26a nanoparticles to regenerate bone defects in vivo. The microRNA-26a complexed RALA nanoparticles, which showed the highest transfection efficiency, were incorporated into collagen-nanohydroxyapatite scaffolds and in vitro assessment demonstrated the miR-26a-activated scaffolds effectively transfected human mesenchymal stem cells (hMSCs) resulting in enhanced production of vascular endothelial growth factor, increased alkaline phosphatase activity, and greater mineralisation. After implantation in critical-sized rat calvarial defects, micro CT and histomorphological analysis revealed that the miR-26a-activated scaffolds improved bone repair in vivo, producing new bone of superior quality, which was highly mineralised and vascularised compared to a miR-free scaffold. This innovative combination of osteogenic collagen-nanohydroxyapatite scaffolds with multifunctional microRNA-26a complexed nanoparticles provides an effective carrier delivering nanoparticles locally with high efficacy and minimal off-target effects and demonstrates the potential of targeting osteogenic-angiogenic coupling using scaffold-based nanomedicine delivery as a new "off-the-shelf" product capable of healing complex bone injuries.