Zinc-based subcuticular absorbable staples: An in vivo and in vitro study.

A zinc-nutrient element alloy (Zn-1.0Cu-0.5Ca) was developed into subcuticular absorbable staples (SAS) as a robust alternative to the commercially available poly(l-lactide-co-glycolide) (PLGA) SAS for the first time. The fixation properties of the Zn SAS were measured via pull-out tests and in-situ lap-shear pull-out test comparatively against the PLGA SAS. The Zn SAS exhibited fixation force of 18.9±0.2 N, which was over three times higher than that of PLGA SAS (5.5±0.1 N). The Zn SAS was used to close incision wounds in a SD rat model for biodegradability and biocompatibility characterisation at 1, 4 and 12 weeks. The Zn SAS showed uniform degradation behaviour after in vivo implantation at the average rate of 198±54, 112±28, and 70±24 µm/y after 1, 4, and 12 weeks, which reduced the fixation force to 16.8±1.1 N, 15.4±0.9 N, 12.7±0.7 N, respectively. These findings showed the potential of the Zn SAS for the closure of heavy loading and slowing healing tissues. The Zn SAS enabled successful closure and healing of the incision wound, similar to the PLGA staples. However, the slow long-term degradation rate of the Zn SAS may lead to unnecessary implant retention. In addition, the alloy SAS resulted in higher local foreign body responses due to their stiffness. Reducing the implant cross-section profile and applying low stiffness and a corrosion-accelerating coating are suggested as possible approaches to reduce post-service implant retention and improve the biocompatibility of the Zn SAS. STATEMENT OF SIGNIFICANCE: This work reports the fabrication of the first metallic subcuticular absorbable staples (SAS) made from ZnCuCa alloy for skin wound closure applications. The Zn-based SAS were characterised in vitro and in vivo (SD rat model) for biodegradability, fixation properties, biocompatibility and inflammatory responses, which were compared against the commercially available PLGA-based SAS. The Zn-based SAS provided a secure attachment of the full-thickness wounds on SD rats and allowed successful healing during the 12-week service period. In addition, the in vitro results showed that the Zn-based SAS provided more than three times higher fixation strength than the commercial PLGA, indicating the potential of the Zn-based SAS for load-bearing wound closure application.

Comparison of two formulations of alfaxalone for immersion anaesthesia in laboratory zebrafish (Daniorerio).

OBJECTIVE: To compare two commercial formulations of alfaxalone for immersion anaesthesia in laboratory zebrafish. STUDY DESIGN: Prospective, blinded, randomized study. ANIMALS: A total of 20 adult Danio rerio (Tuebingen strain). METHODS: Zebrafish were divided into two groups of 10 (five female, five male) and placed in individual immersion baths containing 10 mg L-1 of unpreserved alfaxalone (group 1) or preserved alfaxalone (group 2). Anaesthetists blinded to treatment used a composite score scale (CSS) (range 0-12) to assess fish every 30 seconds until induction of anaesthesia. Anaesthetic induction occurred when equilibrium and response to stimulus were lost. Fish were then placed in a clean water bath and scored every 60 seconds. Recovery from anaesthesia was defined as a CSS of ≤ 1. Time variables recorded were anaesthetic induction time (AIT), anaesthetic recovery time (ART) and total procedure time (TPT). Fish were observed for evidence of roupgross external pathology during the procedure. Following anaesthesia, four fish from each group were randomly chosen and euthanized for gill histopathology analysis immediately after recovery criteria were met. Data are presented as mean ± standard deviation. An independent t test was used to compare the difference in average anaesthetic time variables between groups (α = 0.05). RESULTS: There were no statistical differences between groups in reported variables. TPT, AIT and ART were 10.2 ± 1.2, 1.9 ± 0.9 and 8.3 ± 1.2 minutes for group 1 and 10.8 ± 2.9, 2.4 ± 1.2 and 8.4 ± 2.7 minutes for group 2. No gross external pathology was evident, and no fish died during the experimental period. Histopathology showed normal gill pathology and no difference between the groups. CONCLUSIONS AND CLINICAL RELEVANCE: Immersion anaesthesia using 10 mg L-1 of either formulation of alfaxalone resulted in anaesthesia of similar quality and duration.

In vivo performance of a rare earth free Mg-Zn-Ca alloy manufactured using twin roll casting for potential applications in the cranial and maxillofacial fixation devices.

A magnesium alloy containing essential, non-toxic, biodegradable elements such as Ca and Zn has been fabricated using a novel twin-roll casting process (TRC). Microstructure, mechanical properties, in vivo corrosion and biocompatibility have been assessed and compared to the properties of the rare earth (RE) element containing WE43 alloy. TRC Mg-0.5 wt% Zn- 0.5 wt% Ca exhibited fine grains with an average grain size ranging from 70 to 150 µm. Mechanical properties of a TRC Mg-0.5Zn-0.5Ca alloy showed an ultimate tensile strength of 220 MPa and ductility of 9.3%. The TRC Mg-0.5Zn-0.5Ca alloy showed a degradation rate of 0.51 ± 0.07 mm/y similar to that of the WE43 alloy (0.47 ± 0.09 mm/y) in the rat model after 1 week of implantation. By week 4 the biodegradation rates of both alloys studied were lowered and stabilized with fewer gas pockets around the implant. The histological analysis shows that both WE43 and TRC Mg-0.5Zn-0.5Ca alloy triggered comparable tissue healing responses at respective times of implantation. The presence of more organized scarring tissue around the TRC Mg-0.5Zn-0.5Ca alloys suggests that the biodegradation of the RE-free alloy may be more conducive to the tissue proliferation and remodelling process.

Preclinical development of a molecular clamp-stabilised subunit vaccine for severe acute respiratory syndrome coronavirus 2.

OBJECTIVES: Efforts to develop and deploy effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue at pace. Here, we describe rational antigen design through to manufacturability and vaccine efficacy of a prefusion-stabilised spike (S) protein, Sclamp, in combination with the licensed adjuvant MF59 'MF59C.1' (Seqirus, Parkville, Australia). METHODS: A panel recombinant Sclamp proteins were produced in Chinese hamster ovary and screened in vitro to select a lead vaccine candidate. The structure of this antigen was determined by cryo-electron microscopy and assessed in mouse immunogenicity studies, hamster challenge studies and safety and toxicology studies in rat. RESULTS: In mice, the Sclamp vaccine elicits high levels of neutralising antibodies, as well as broadly reactive and polyfunctional S-specific CD4+ and cytotoxic CD8+ T cells in vivo. In the Syrian hamster challenge model (n = 70), vaccination results in reduced viral load within the lung, protection from pulmonary disease and decreased viral shedding in daily throat swabs which correlated strongly with the neutralising antibody level. CONCLUSION: The SARS-CoV-2 Sclamp vaccine candidate is compatible with large-scale commercial manufacture, stable at 2-8°C. When formulated with MF59 adjuvant, it elicits neutralising antibodies and T-cell responses and provides protection in animal challenge models.

In Vivo Evaluation of Bioabsorbable Fe-35Mn-1Ag: First Reports on In Vivo Hydrogen Gas Evolution in Fe-Based Implants.

This work investigates the influence of Ag (1 wt%) on the mechanical properties, in vitro and in vivo corrosion, and biocompatibility of Fe-35Mn. The microstructure of Fe-35Mn-1Ag possesses a uniform dispersion of discrete silver particles. Slight improvements in compressive properties are attributed to enhanced density and low porosity volume. Fe-35Mn-1Ag exhibits good in vitro and in vivo corrosion rate of Fe-35Mn due to an increase in microgalvanic corrosion. Gas pockets, which originate from an inflammatory response to the implants, are observed in the rats after 4 weeks implantation but are undetectable after 12 weeks. No chronic toxicity is observed with the Fe-35Mn-1Ag, suggesting acceptable in vivo biocompatibility. The high corrosion rate of the alloy triggers an increased level of nonadverse tissue inflammatory responses 4 weeks after implantation, which subsequently subsides at 12 weeks. The Fe-35Mn-1Ag displays properties that are suitable for orthopedic applications.

Absorbable Mg surgical tack: Proof of concept &in situ fixation strength.

A prototype magnesium (Mg) surgical tack is tested comparatively against commercially available tacks made of titanium (ProTacktm, Medtronic) and PLGA (AbsorbaTacktm, Medtronic). The pull-out force is measured in situ in a lap-shear pull-out test, using porcine abdominal muscle tissue as a model. The Mg tack had a pull-out force comparable to those of the commercially available tacks. The majority of the Mg tacks also had a more ductile failure mode (i.e. the tacks deformed prior to failure), compared to the commercial tacks which pulled directly from the tissue with no deformation. The Mg tacks deformed as they were removed from the tissue, causing less damage to the tissue in the process. This is the first reported use of a Mg alloy in this application, and the proof of concept indicates that this is an area that deserves further interest and study.

The Impact of Limbal Mesenchymal Stromal Cells on Healing of Acute Ocular Surface Wounds Is Improved by Pre-cultivation and Implantation in the Presence of Limbal Epithelial Cells.

While limbal epithelial cells are used for treating ocular surface wounds, the therapeutic potential of mesenchymal cells cultivated from the limbal stroma (LMSC) is less clear. We have therefore examined the effects of LMSC when applied to acute ocular surface wounds. LMSC derived from male rabbits (RLMSC) were applied to the ocular surface of female rabbits immediately following removal of the corneal and limbal epithelium. Human amniotic membrane (HAM) was used as the vehicle for implanting the RLMSC. The effects of RLMSC were examined when applied alone (n = 3) and in conjunction with a stratified culture of human limbal epithelial cells (HLE) grown on the opposing surface of the HAM (n = 3). Outcomes were monitored over 3 months in comparison with animals receiving no treatment (n = 3) or treatment with HLE alone on HAM (n = 3). Animals treated with RLMSC (n = 6) displayed faster re-epithelialization (∼90% versus 70% healing after 12 weeks), with best results being observed when RLMSC were pre-cultivated and implanted in the presence of HLE (p < 0.01; 90% healing by 7 weeks). While all animals displayed conjunctival cells on the corneal surface (by presence of goblet cells and/or keratin 13 expression) and corneal neovascularization, evidence of corneal epithelial regeneration was observed in animals that received RLMSC in the presence of HLE (by staining for keratin 3 and the absence of goblet cells). Conversely, corneal neovascularization was significantly greater when RLMSC were applied in the absence of HLE (<0.05; 90% of cornea compared with 20-30% in other cohorts). Nevertheless, neither human nuclear antigen nor rabbit Y chromosome were detected within the regenerated epithelium. Our results demonstrate that while cultured LMSC encourage corneal re-epithelialization, healing is improved by the pre-cultivation and implantation of these mesenchymal cells in the presence of limbal epithelial cells.

Alfaxalone Causes Reduction of Glycinergic IPSCs, but Not Glutamatergic EPSCs, and Activates a Depolarizing Current in Rat Hypoglossal Motor Neurons.

We investigated effects of the neuroactive steroid anesthetic alfaxalone on intrinsic excitability, and on inhibitory and excitatory synaptic transmission to hypoglossal motor neurons (HMNs). Whole cell recordings were made from HMNs in brainstem slices from 7 to 14-day-old Wistar rats. Spontaneous, miniature, and evoked inhibitory post-synaptic currents (IPSCs), and spontaneous and evoked excitatory PSCs (EPSCs) were recorded at -60 mV. Alfaxalone did not alter spontaneous glycinergic IPSC peak amplitude, rise-time or half-width up to 10 µM, but reduced IPSC frequency from 3 µM. Evoked IPSC amplitude was reduced from 30 nM. Evoked IPSC rise-time was prolonged and evoked IPSC decay time was increased only by 10 µM alfaxalone. Alfaxalone also decreased evoked IPSC paired pulse ratio (PPR). Spontaneous glutamatergic EPSC amplitude and frequency were not altered by alfaxalone, and evoked EPSC amplitude and PPR was also unchanged. Alfaxalone did not alter HMN repetitive firing or action potential amplitude. Baseline holding current at -60 mV with a CsCl-based pipette solution was increased in an inward direction; this effect was not seen when tetrodotoxin (TTX) was present. These results suggest that alfaxalone modulates glycine receptors (GlyRs), causing a delayed and prolonged channel opening, as well as causing presynaptic reduction of glycine release, and activates a membrane current, which remains to be identified. Alfaxalone selectively reduces glycinergic inhibitory transmission to rat HMNs via a combination of pre- and post-synaptic mechanisms. The net effect of these responses to alfaxalone is to increase HMN excitability and may therefore underlie neuro-motor excitation during neurosteroid anesthesia.

Evaluation of waste isoflurane gas exposure during rodent surgery in an Australian university.

Biomedical researchers use of inhalational anesthetics has increased in recent years. Use of isoflurane as an inhalational anesthetic may result in human exposure to waste anesthetic gas. Potential health effects from exposure include genotoxic and hepatotoxic effects with some evidence of teratogenic and reproductive effects. Research suggests that exposure to waste anesthetic gas within human hospital settings has improved substantially but exposures to biomedical researchers and veterinarians still requires improvement. A number of biomedical research facilities are located at The University of Queensland, Australia, where researchers and animal handlers are potentially exposed to waste isoflurane gas. There is limited published data on the exposures received by biomedical researchers performing routine procedures. This project aimed to assess isoflurane exposure received during routine rodent anesthetic protocols performed at the university. Atmospheric concentrations of isoflurane were assessed via two methods-personal active gas sampling using sorbent tubes and direct readings using infrared spectroscopy. Total procedure and isoflurane exposure times ranged from 135-268 min. Personal sorbent tube sampling detected isoflurane levels from below detectable limits (<0.01 ppm) to a Time Weighted Average for the task (TWA-Task) of 6.20 ppm (0.73 ± 9.13). Participants were not exposed to isoflurane outside of the sampling period during the remainder of the workday. TWA-8 hr adjusted levels ranged from below the limit of detection to 1.76 ppm isoflurane (0.69 ppm ± 0.61 ppm). The infrared spectroscopy readings taken in the breathing zone of participants ranged from 0.1-68 ppm. Results indicate that if adequately controlled through good room ventilation, effective active gas scavenging and well constructed anesthetic equipment, waste anesthetic exposures are minimal. However, where industry standards are not met exposures may occur, including some high peak exposures.

Evaluation of the AlgerBrush II rotating burr as a tool for inducing ocular surface failure in the New Zealand White rabbit.

The New Zealand White rabbit has been widely used as a model of limbal stem cell deficiency (LSCD). Current techniques for experimental induction of LSCD utilize caustic chemicals, or organic solvents applied in conjunction with a surgical limbectomy. While generally successful in depleting epithelial progenitors, the depth and severity of injury is difficult to control using chemical-based methods. Moreover, the anterior chamber can be easily perforated while surgically excising the corneal limbus. In the interest of creating a safer and more defined LSCD model, we have therefore evaluated a mechanical debridement technique based upon use of the AlgerBrush II rotating burr. An initial comparison of debridement techniques was conducted in situ using 24 eyes in freshly acquired New Zealand White rabbit cadavers. Techniques for comparison (4 eyes each) included: (1) non-wounded control, (2) surgical limbectomy followed by treatment with 100% (v/v) n-heptanol to remove the corneal epithelium (1-2 min), (3) treatment of both limbus and cornea with n-heptanol alone, (4) treatment of both limbus and cornea with 20% (v/v) ethanol (2-3 min), (5) a 2.5-mm rounded burr applied to both the limbus and cornea, and (6) a 1-mm pointed burr applied to the limbus, followed by the 2.5-mm rounded burr applied to the cornea. All corneas were excised and processed for histology immediately following debridement. A panel of four assessors subsequently scored the degree of epithelial debridement within the cornea and limbus using masked slides. The 2.5-mm burr most consistently removed the corneal and limbal epithelia. Islands of limbal epithelial cells were occasionally retained following surgical limbectomy/heptanol treatment, or use of the 1-mm burr. Limbal epithelial cells were consistently retained following treatment with either ethanol or n-heptanol alone, with ethanol being the least effective treatment overall. The 2.5-mm burr method was subsequently evaluated in the right eye of 3 live rabbits by weekly clinical assessments (photography and slit lamp examination) for up to 5 weeks, followed by histological analyses (hematoxylin & eosin stain, periodic acid-Schiff stain and immunohistochemistry for keratin 3 and 13). All 3 eyes that had been completely debrided using the 2.5-mm burr displayed symptoms of ocular surface failure as defined by retention of a prominent epithelial defect (∼40% of corneal surface at 5 weeks), corneal neovascularization (2-3 quadrants), reduced corneal transparency and conjunctivalization of the corneal surface (demonstrated by the presence of goblet cells and/or staining for keratin 13). In conclusion, our findings indicate that the AlgerBrush II rotating burr is an effective method for the establishment of ocular surface failure in New Zealand White rabbits. In particular, we recommend use of the 2.5-mm rotating burr for improved efficiency of epithelial debridement and safety compared to surgical limbectomy.