In vivo confocal Raman spectroscopy: The window into the skin.

Development of in vivo confocal Raman spectroscopy (ICRS) methodology over the last 20 years has enabled previously unavailable capability to acquire molecular concentration gradients across the stratum corneum (SC), at the micron level and in a clinical setting. Professor Tony Rawlings has been a driving force in SC research for over 30 years, working with a wide range of teams across the world. Because a detailed knowledge of skin biochemistry was key to interpreting ICRS-acquired molecular concentration gradients, the authors formed a close working relationship with Professor Rawlings during the development of ICRS. This article, therefore, presents a summary of this process and how challenges raised by application of ICRS were tackled, towards the goal of validating the technique for clinical skin measurement.

Le développement de la méthodologie de spectroscopie confocale Raman in vivo (In vivo Confocal Raman Spectroscopy, ICRS) au cours des 20 dernières années a permis d'acquérir des gradients de concentration moléculaire dans l'ensemble du stratum corneum (SC), au niveau du micron et dans un contexte clinique, ce qui était impossible auparavant. Le professeur Tony Rawlings joue un rôle moteur dans la recherche sur le SC depuis plus de 30 ans et travaille avec de nombreuses équipes à travers le monde. Étant donné qu'une connaissance détaillée de la biochimie cutanée était essentielle à l'interprétation des gradients de concentration moléculaire acquis par l'ICRS, les auteurs ont établi une relation de travail étroite avec le professeur Rawlings pendant le développement de l'ICRS. Cet article présente donc un résumé de ce processus et de la manière dont les défis soulevés par l'application de l'ICRS ont été abordés dans le but de valider la technique de mesure clinique de la peau.

Preliminary In Vitro and In Vivo Insights of In Silico Candidate Repurposed Drugs for Alzheimer's Disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disease and is the most common type of dementia. Although a considerably large amount of money has been invested in drug development for AD, no disease modifying treatment has been detected so far. In our previous work, we developed a computational method to highlight stage-specific candidate repurposed drugs against AD. In this study, we tested the effect of the top 13 candidate repurposed drugs that we proposed in our previous work in a severity stage-specific manner using an in vitro BACE1 assay and the effect of a top-ranked drug from the list of our previous work, tetrabenazine (TBZ), in the 5XFAD as an AD mouse model. From our in vitro screening, we detected 2 compounds (clomiphene citrate and Pik-90) that showed statistically significant inhibition against the activity of the BACE1 enzyme. The administration of TBZ at the selected dose and therapeutic regimen in 5XFAD in male and female mice showed no significant effect in behavioral tests using the Y-maze and the ELISA immunoassay of Aß40. To our knowledge, this is the first time the drug tetrabenazine has been tested in the 5XFAD mouse model of AD in a sex-stratified manner. Our results highlight 2 drugs (clomiphene citrate and Pik-90) from our previous computational work for further investigation.

Effect of Hydrophilic Polymers on the Release Rate and Pharmacokinetics of Acyclovir Tablets Obtained by Wet Granulation: In Vitro and In Vivo Assays.

This study aims to evaluate the feasibility of producing acyclovir-containing modified release matrix tablets by a wet granulation method based on the type and concentration of two pharmaceutical-grade hydrophilic matrix polymers (i.e., hydroxypropyl methylcellulose (HPMC), carbomers, and their combinations) commonly used in biomedical applications. The mechanical properties of the tablets and in vitro and in vivo performance were studied. The physicochemical properties of the raw materials and corresponding physical mixtures were characterized by differential scanning calorimetry, showing that the hydrophilic polymers did not influence the physicochemical properties of the drug. The wet granulation process improved the flow and compression properties of the obtained granules. This method enabled the preparation of the matrix tablets of acyclovir with appropriate mechanical properties concerning hardness and friability. The drug release kinetics was governed by the type and concentration of the hydrophilic polymers composing the matrices. The study has proven that HPMC-composed tablets were superior in modified drug release properties compared to carbomer- and HPMC/carbomer-based tablets. Mathematical analysis of the release profiles, determined in a medium adjusted to pH 1.2 followed by pH 7.4, revealed that the drug released from the hydrophilic tablets followed non-Fickian first-order kinetics. An optimal HPMC-based formulation submitted to accelerated stability studies (40 °C, 75% RH) was stable for three months. A complete cross-over bioavailability study of the selected acyclovir-loaded sustained release tablets and marketed immediate-release tablets were compared in six healthy male volunteers. The extent of drug absorption from the sustained release tablets was significantly greater than that from immediate-release pills, which may improve the drug's antiviral properties attributed to the lower elimination rate and enhanced acyclovir half-life.

Shore hardness is a more representative measurement of bulk tissue biomechanics than of skin biomechanics.

To support the effective use of Shore hardness (SH) in research and clinical practice this study investigates whether SH should be interpreted as a measurement of skin or of bulk tissue biomechanics. A 3D finite element model of the heel and a validated model of a Shore-00 durometer were used to simulate testing for different combinations of stiffness and thickness in the skin and subcutaneous tissue. The results of this numerical analysis showed that SH is significantly more sensitive to changes in skin thickness, relatively to subcutaneous tissue, but equally sensitive to changes in the stiffness of either tissue. Indicatively, 25% reduction in skin thickness (0.3 mm thickness change) or in subcutaneous tissue thickness (5.9 mm thickness change), reduced SH by 7% or increased SH by 2% respectively. At the same time, 25% reduction in skin stiffness (10.1 MPa change in initial shear modulus) or of subcutaneous tissue (4.1 MPa change in initial shear modulus) led to 11% or 8% reduction in SH respectively. In the literature, SH is commonly used to study skin biomechanics. However, this analysis indicates that SH quantifies the deformability of bulk tissue, not of skin. Measurements of skin thickness are also necessary for the correct interpretation of SH.

Recent progress and perspectives of continuous in vivo testing device.

Devices for continuous in-vivo testing (CIVT) can detect target substances in real time, thus providing a valuable window into a patient's condition, their response to therapeutics, metabolic activities, and neurotransmitter transmission in the brain. Therefore, CIVT devices have received increased attention because they are expected to greatly assist disease diagnosis and treatment and research on human pathogenesis. However, CIVT has been achieved for only a few markers, and it remains challenging to detect many key markers. Therefore, it is important to summarize the key technologies and methodologies of CIVT, and to examine the direction of future development of CIVT. We review recent progress in the development of CIVT devices, with consideration of the structure of these devices, principles governing continuous detection, and nanomaterials used for electrode modification. This detailed and comprehensive review of CIVT devices serves three purposes: (1) to summarize the advantages and disadvantages of existing devices, (2) to provide a reference for development of CIVT equipment to detect additional important markers, and (3) to discuss future prospects with emphasis on problems that must be overcome for further development of CIVT equipment. This review aims to promote progress in research on CIVT devices and contribute to future innovation in personalized medical treatments.

Effects of a grapeseed oil (Vitis vinifera L.) loaded dermocosmetic nanoemulgel on biophysical parameters of facial skin: A split-face, blinded, placebo-controlled study.

BACKGROUND: Worldwide, grapes (Vitis vinifera L.; family: Vitaceae) are one of the most important fruits. Grapeseed oil is rich in bioactive constituents that could be beneficial to the health and aesthetic features of human skin. OBJECTIVE: This study was conducted to evaluate the effects of a novel grapeseed oil-loaded dermocosmetic nanoemulgel on biophysical parameters of facial skin. METHODS: This was a split-face, blinded, placebo-controlled study. A novel grapeseed oil-loaded dermocosmetic nanoemulgel was developed, and its effects on the biophysical parameters of the facial skin were evaluated and compared to those of a placebo formulation on the cheeks of 15 healthy volunteers. Melanin, erythema, sebum production, fine and large facial pores, moisture, and elasticity levels were measured using Mexameter®, Corneometer®, Sebumeter®, Cutometer®, and VisioFace®. Measurements were made on weekly basis for 12 weeks. RESULTS: Compared to the placebo, the novel grapeseed oil-loaded dermocosmetic nanoemulgel received significantly higher sensory scores with regard to appearance, color, odor, consistency, adhesion, sensation, cohesiveness, and spreadability (p-value < 0.05). Additionally, the novel nanoemulgel continuously and significantly reduced skin melanin, erythema, sebum production, and fine and large pores (p-value < 0.05). On the contrary, the novel nanoemulgel continuously and significantly increased skin moisture contents and elasticity (p-value < 0.05). CONCLUSION: The novel grapeseed oil-loaded dermocosmetic nanoemulgel had attractive cosmetic attributes that could be useful for improving imperfections of the human skin. Future studies are still needed to test and evaluate the benefits of this novel grapeseed oil-loaded dermocosmetic nanoemulgel in disease conditions.

Predicting chemical hazard across taxa through machine learning.

We applied machine learning methods to predict chemical hazards focusing on fish acute toxicity across taxa. We analyzed the relevance of taxonomy and experimental setup, showing that taking them into account can lead to considerable improvements in the classification performance. We quantified the gain obtained throught the introduction of taxonomic and experimental information, compared to classification based on chemical information alone. We used our approach with standard machine learning models (K-nearest neighbors, random forests and deep neural networks), as well as the recently proposed Read-Across Structure Activity Relationship (RASAR) models, which were very successful in predicting chemical hazards to mammals based on chemical similarity. We were able to obtain accuracies of over 93% on datasets where, due to noise in the data, the maximum achievable accuracy was expected to be below 96%. The best performances were obtained by random forests and RASAR models. We analyzed metrics to compare our results with animal test reproducibility, and despite most of our models "outperform animal test reproducibility" as measured through recently proposed metrics, we showed that the comparison between machine learning performance and animal test reproducibility should be addressed with particular care. While we focused on fish mortality, our approach, provided that the right data is available, is valid for any combination of chemicals, effects and taxa.

Synthesis, In Vitro Testing, and Biodistribution of Surfactant-Free Radioactive Nanoparticles for Cancer Treatment.

New forms of cancer treatment, which are effective, have simple manufacturing processes, and easily transportable, are of the utmost necessity. In this work, a methodology for the synthesis of radioactive Gold-198 nanoparticles without the use of surfactants was described. The nuclear activated Gold-198 foils were transformed into H198AuCl4 by dissolution using aqua regia, following a set of steps in a specially designed leak-tight setup. Gold-198 nanoparticles were synthesized using a citrate reduction stabilized with PEG. In addition, TEM results for the non-radioactive product presented an average size of 11.0 nm. The DLS and results for the radioactive 198AuNPs presented an average size of 8.7 nm. Moreover, the DLS results for the PEG-198AuNPs presented a 32.6 nm average size. Cell line tests showed no cytotoxic effect in any period and the concentrations were evaluated. Furthermore, in vivo testing showed a high biological uptake in the tumor and a cancer growth arrest.

Bony Stress and Its Association With Intervertebral Disc Degeneration in the Lumbar Spine: A Systematic Review of Clinical and Basic Science Studies.

STUDY DESIGN: Translational review encompassing basic science and clinical evidence. OBJECTIVES: Multiple components of the lumbar spine interact during its normal and pathological function. Bony stress in the lumbar spine is recognized as a factor in the development of pars interarticularis defect and stress fractures, but its relationship with intervertebral disc (IVD) degeneration is not well understood. Therefore, we conducted a systematic review to examine the relationship between bony stress and IVD degeneration. METHODS: Online databases Scopus, PubMed and MEDLINE via OVID were searched for relevant studies published between January 1980-February 2020, using PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-analyses) guidelines. Two authors independently analyzed the data, noting characteristics and biases in various studies. RESULTS: Thirty-two articles were included in the review: 8 clinical studies, 9 finite element modeling studies, 3 in-vivo biomechanical testing studies, and 12 in-vitro biomechanical testing studies. Of the 32 articles, 19 supported, 4 rejected and 9 made no conclusion on the hypothesis that there is a positive associative relationship between IVD degeneration and bony stress. However, sufficient evidence was not available to confirm or reject a causal relationship. CONCLUSIONS: Most studies suggest that the prevalence of IVD degeneration increases in the presence of bony stress; whether a causal relationship exists is unclear. The literature recommends early diagnosis and clinical suspicion of IVD degeneration and bony stress. Longitudinal studies are required to explore causal relationships between IVD degeneration and bony stress.

Galleria mellonella: The Versatile Host for Drug Discovery, In Vivo Toxicity Testing and Characterising Host-Pathogen Interactions.

Larvae of the greater wax moth, Galleria mellonella, are a convenient in vivo model for assessing the activity and toxicity of antimicrobial agents and for studying the immune response to pathogens and provide results similar to those from mammals. G. mellonella larvae are now widely used in academia and industry and their use can assist in the identification and evaluation of novel antimicrobial agents. Galleria larvae are inexpensive to purchase and house, easy to inoculate, generate results within 24-48 h and their use is not restricted by legal or ethical considerations. This review will highlight how Galleria larvae can be used to assess the efficacy of novel antimicrobial therapies (photodynamic therapy, phage therapy, metal-based drugs, triazole-amino acid hybrids) and for determining the in vivo toxicity of compounds (e.g., food preservatives, ionic liquids) and/or solvents (polysorbate 80). In addition, the disease development processes are associated with a variety of pathogens (e.g., Staphylococcus aureus, Listeria monocytogenes, Aspergillus fumigatus, Madurella mycotomatis) in mammals are also present in Galleria larvae thus providing a simple in vivo model for characterising disease progression. The use of Galleria larvae offers many advantages and can lead to an acceleration in the development of novel antimicrobials and may be a prerequisite to mammalian testing.

A Two-Photon Microimaging-Microdevice System for Four-Dimensional Imaging of Local Drug Delivery in Tissues.

Advances in the intratumor measurement of drug responses have included a pioneering biomedical microdevice for high throughput drug screening in vivo, which was further advanced by integrating a graded-index lens based two-dimensional fluorescence micro-endoscope to monitor tissue responses in situ across time. While the previous system provided a bulk measurement of both drug delivery and tissue response from a given region of the tumor, it was incapable of visualizing drug distribution and tissue responses in a three-dimensional (3D) way, thus missing the critical relationship between drug concentration and effect. Here we demonstrate a next-generation system that couples multiplexed intratumor drug release with continuous 3D spatial imaging of the tumor microenvironment via the integration of a miniaturized two-photon micro-endoscope. This enables optical sectioning within the live tissue microenvironment to effectively profile the entire tumor region adjacent to the microdevice across time. Using this novel microimaging-microdevice (MI-MD) system, we successfully demonstrated the four-dimensional imaging (3 spatial dimensions plus time) of local drug delivery in tissue phantom and tumors. Future studies include the use of the MI-MD system for monitoring of localized intra-tissue drug release and concurrent measurement of tissue responses in live organisms, with applications to study drug resistance due to nonuniform drug distribution in tumors, or immune cell responses to anti-cancer agents.

In Vivo Study of Osteochondral Defect Regeneration Using Innovative Composite Calcium Phosphate Biocement in a Sheep Model.

This study aimed to clarify the therapeutic effect and regenerative potential of the novel, amino acids-enriched acellular biocement (CAL) based on calcium phosphate on osteochondral defects in sheep. Eighteen sheep were divided into three groups, the treated group (osteochondral defects filled with a CAL biomaterial), the treated group with a biocement without amino acids (C cement), and the untreated group (spontaneous healing). Cartilages of all three groups were compared with natural cartilage (negative control). After six months, sheep were evaluated by gross appearance, histological staining, immunohistochemical staining, histological scores, X-ray, micro-CT, and MRI. Treatment of osteochondral defects by CAL resulted in efficient articular cartilage regeneration, with a predominant structural and histological characteristic of hyaline cartilage, contrary to fibrocartilage, fibrous tissue or disordered mixed tissue on untreated defect (p < 0.001, modified O'Driscoll score). MRI results of treated defects showed well-integrated and regenerated cartilage with similar signal intensity, regularity of the articular surface, and cartilage thickness with respect to adjacent native cartilage. We have demonstrated that the use of new biocement represents an effective solution for the successful treatment of osteochondral defects in a sheep animal model, can induce an endogenous regeneration of cartilage in situ, and provides several benefits for the design of future therapies supporting osteochondral defect healing.

Harm-Benefit Analyses Can Be Harmful.

Harm-benefit analyses (HBAs) are becoming de rigueur with some governmental regulatory agencies and popular with local institutional animal care and use committees (or their equivalents), the latter due, in part, to the adoption of HBAs as an international accreditation standard. Such analyses are employed as an attempt to balance potential or actual pain or distress imposed on laboratory animals against scientists' justifications for those impositions. The outcomes of those analyses are then supposed to be included in an official assessment of whether a given animal protocol should be approved as proposed. While commendable in theory as a means to avoid or minimize animal suffering, HBAs come with a flawed premise. Establishing an accurate prediction of benefit, especially for so-called "basic" research (vs "applied" research, such as in vivo testing for product development or batch release), is often impossible given the uncertain nature of experimental outcomes and the eventual value of those results. That impossibility, in turn, risks disapproving a legitimate research proposal that might have yielded important new knowledge if it had been allowed to proceed. Separately, the anticipated harm to which the animal would be subjected should similarly be scrutinized with an aim to refine that harm regardless of purported benefits if the protocol is approved. The intentions of this essay are to reflect on the potential harm and benefit of the HBA itself, highlight how HBAs may be helpful in advancing refinements, and propose alternative approaches to both parts of the equation in the assessment process.

Efficacy of Natural Formulations in Bovine Mastitis Pathology: Alternative Solution to Antibiotic Treatment.

INTRODUCTION: Bovine mastitis is an inflammatory disease of the udder that causes important economic losses in the animal breeding and dairy product industries. Nowadays, the conventional livestock antibiotic treatments are slowly being replaced by alternative treatments. In this context, the main aim of this study was to evaluate the efficacy of natural products in alternative treatment of bovine mastitis. MATERIAL AND METHODS: Two natural formulations with previously suggested in vitro antimicrobial effect were tested in vivo on mastitic cows. Animals with a positive diagnosis for mastitis (n = 20) were divided into three treatment groups: two groups (n = 8) were administered formulations of propolis, alcoholic extracts of Brewers Gold and Perle hops, plum lichen, common mallow, marigold, absinthe wormwood, black poplar buds, lemon balm, and essential oils of oregano, lavender, and rosemary designated R4 and R7 (differing only in the latter being more concentrated) and one group (n = 4) a conventional antibiotic mixture. In vivo efficacy of treatments was evaluated by somatic cell and standard plate counts, the treatment being considered efficacious when both parameters were under the maximum limit. RESULTS: R7 was effective in the most cases, being therapeutically bactericidal in six out of eight cows, while R4 gave good results in three out of eight cows, and conventional antibiotics cured one out of four. CONCLUSION: These results suggest the possible therapeutic potential of these natural products in bovine mastitis.

Guidelines to Study and Develop Soft Electrode Systems for Neural Stimulation.

Electrical stimulation of nervous structures is a widely used experimental and clinical method to probe neural circuits, perform diagnostics, or treat neurological disorders. The recent introduction of soft materials to design electrodes that conform to and mimic neural tissue led to neural interfaces with improved functionality and biointegration. The shift from stiff to soft electrode materials requires adaptation of the models and characterization methods to understand and predict electrode performance. This guideline aims at providing (1) an overview of the most common techniques to test soft electrodes in vitro and in vivo; (2) a step-by-step design of a complete study protocol, from the lab bench to in vivo experiments; (3) a case study illustrating the characterization of soft spinal electrodes in rodents; and (4) examples of how interpreting characterization data can inform experimental decisions. Comprehensive characterization is paramount to advancing soft neurotechnology that meets the requisites for long-term functionality in vivo.

UtilisingGalleria mellonella larvae for studying in vivo activity of conventional and novel antimicrobial agents.

The immune response of insects displays many structural and functional similarities to the innate immune response of mammals. As a result of these conserved features, insects may be used for evaluating microbial virulence or for testing the in vivo efficacy and toxicity of antimicrobial compounds and results show strong similarities to those from mammals. Galleria mellonella larvae are widely used in this capacity and have the advantage of being easy to use, inexpensive to purchase and house, and being free from the ethical and legal restrictions that relate to the use of mammals in these tests. Galleria mellonella larvae may be used to assess the in vivo toxicity and efficacy of novel antimicrobial compounds. A wide range of antibacterial and antifungal therapies have been evaluated in G. mellonella larvae and results have informed subsequent experiments in mammals. While insect larvae are a convenient and reproducible model to use, care must be taken in their use to ensure accuracy of results. The objective of this review is to provide a comprehensive account of the use of G. mellonella larvae for assessing the in vivo toxicity and efficacy of a wide range of antibacterial and antifungal agents.

The suitability of reconstructed human epidermis models for medical device irritation assessment: A comparison of In Vitro and In Vivo testing results.

The ISO 10993 standards on biocompatibility assessment of medical devices discourage the use of animal tests when reliable and validated in vitro methods are available. A round robin validation study of in vitro reconstructed human epidermis (RhE) assays was performed as potential replacements for rabbit skin irritation testing. The RhE assays were able to accurately identify strong irritants in dilute medical device extracts. However, there was some uncertainty about whether RhE tissues accurately predicted the results of the rabbit skin patch or intracutaneous irritation test. To address that question, this paper presents in vivo data from the round robin and subsequent follow-up studies. The follow-up studies included simultaneous in vitro RhE model and in vivo testing of round robin polymer samples and the results of dual in vitro/in vivo testing of currently marketed medical device components/materials. Our results show for the first time that for both pure chemicals and medical device extracts the intracutaneous rabbit test is more sensitive to detect irritant activity than the rabbit skin patch test. The studies showed that the RhE models produced results that were essentially equivalent to those from the intracutaneous rabbit skin irritation test. Therefore, it is concluded that RhE in vitro models are acceptable replacements for the in vivo rabbit intracutaneous irritation test for evaluating the irritant potential of medical devices.

CRISPR-Generated Animal Models of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive neuromuscular disorder most commonly caused by mutations disrupting the reading frame of the dystrophin (DMD) gene. DMD codes for dystrophin, which is critical for maintaining the integrity of muscle cell membranes. Without dystrophin, muscle cells receive heightened mechanical stress, becoming more susceptible to damage. An active body of research continues to explore therapeutic treatments for DMD as well as to further our understanding of the disease. These efforts rely on having reliable animal models that accurately recapitulate disease presentation in humans. While current animal models of DMD have served this purpose well to some extent, each has its own limitations. To help overcome this, clustered regularly interspaced short palindromic repeat (CRISPR)-based technology has been extremely useful in creating novel animal models for DMD. This review focuses on animal models developed for DMD that have been created using CRISPR, their advantages and disadvantages as well as their applications in the DMD field.

Additively manufactured iron-manganese for biodegradable porous load-bearing bone scaffold applications.

Selective laser melting (SLM) can produce complex hierarchical architectures paving the way for highly customisable biodegradable load-bearing bone scaffolds. For the first time, an in-depth analysis on the performance of SLM-manufactured iron-manganese bone scaffolds suitable for load-bearing applications is presented. Microstructural, mechanical, corrosion and biological characterisations were performed on SLM-manufactured iron-manganese scaffolds. The microstructure of the scaffold consisted primarily of γ-austenite, leading to high ductility. The mechanical properties of the scaffold were sufficient for load-bearing applications even after 28 days immersion in simulated body fluids. Corrosion tests showed that the corrosion rate was much higher than bulk pure iron, attributed to a combination of the manufacturing method, the addition of Mn to the alloy and the design of the scaffold. In vitro cell testing showed that the scaffold had good biocompatibility and viability towards mammalian cells. Furthermore, the presence of filopodia showed good osteoblast adhesion. In vivo analysis showed successful bone integration with the scaffold, with new bone formation observed after 4 weeks of implantation. Overall the SLM manufactured porous Fe-35Mn implants showed promise for biodegradable load-bearing bone scaffold applications. STATEMENT OF SIGNIFICANCE: Biodegradable iron scaffolds are emerging as a promising treatment for critical bone defects. Within this field, selective laser melting (SLM) has become a popular method of manufacturing bespoke scaffolds. There is limited knowledge on SLM-manufactured iron bone scaffolds, and no knowledge on their application for load-bearing situations. The current manuscript is the first study to characterise SLM manufactured iron-manganese bone scaffolds for load-bearing applications and also the first study to perform In vivo testing on SLM produced biodegradable iron scaffolds. In this study, for the first time, the mechanical, corrosion and biological properties of an iron-manganese scaffold manufactured using SLM were investigated. In summary the SLM-manufactured porous iron-manganese implants displayed great potential for biodegradable load-bearing bone scaffolds.