'I'm a failure again, I can't do it': Attitudes towards, and experiences of, exercise participation in adults with class III obesity.

OBJECTIVES: Living within a larger body brings unique challenges to exercise participation, which are poorly understood. This qualitative study explored the attitudes towards, and experiences of, exercise participation in adults with class III obesity. DESIGN: Individual semi-structured qualitative interviews. METHODS: We recruited 30 adults with class III obesity (body mass index: 45.8 ± 8.6 kg/m2) from a specialist multidisciplinary weight management service. Participants took part in semi-structured interviews while participating in a 6-month home-based aerobic and resistance exercise intervention. Open-ended questions were used flexibly to explore their views and experiences of exercise, encompassing barriers, motives and perceived benefits. Transcripts were analysed using reflexive thematic analysis. RESULTS: Three themes were developed: (1) a web of barriers; (2) tailored exercise facilitates positive experiences; and (3) a desire to live a normal life. People with class III obesity perceived that they were unable to do exercise; a view that was attributed to perceived judgement, low physical function, pain during everyday activities and failed weight loss attempts. These complex physical and psychosocial barriers to exercise were described as contributing to exercise avoidance. High value was placed on tailored exercise that accommodates the unique needs of moving in a larger body. A desire to carry out everyday tasks underpinned motivations for exercise. CONCLUSIONS: Our findings suggest that multi-component obesity interventions should move away from generic exercise prescriptions designed to maximize energy expenditure, and instead move towards addressing the unique physical and psychosocial needs of people who have class III obesity with tailored person-centred and weight-neutral exercise prescriptions.

Oxygenated Hydrogel Promotes Re-Epithelialization and Reduces Inflammation in a Perturbed Wound Model in Rat.

Chronic wounds can take months or even years to heal and require proper medical intervention. Normal wound healing processes require adequate oxygen supply. Accordingly, destroyed or inefficient vasculature leads to insufficient delivery to peripheral tissues and impair healing. Oxygen is critical for vital processes such as proliferation, collagen synthesis and antibacterial defense. Hyperbaric oxygen therapy (HBOT) is commonly used to accelerate healing however, this can be costly and requires specialized training and equipment. Efforts have turned to the development of topical oxygen delivery systems. Oxysolutions has developed oxygenated gels (P407, P407/P188, nanocellulose based gel (NCG)) with high levels of dissolved oxygen. This study aims to evaluate the efficacy of these newly developed oxygenated products by assessing their impact on healing rates in a rat perturbed wound model. Here, P407/P188 oxygenated gels demonstrated greater re-epithelialization distances compared to its controls at Day 3. In addition, all oxygenated gels had a higher proportion of wounds with complete wound closure. All three oxygenated gels also minimized further escalation in inflammation from Day 3 to Day 10. This highlights the potential of this newly-developed oxygenated gels as an alternative to existing oxygen therapies.

Targeting Cx43 to Reduce the Severity of Pressure Ulcer Progression.

In the skin, repeated incidents of ischemia followed by reperfusion can result in the breakdown of the skin and the formation of a pressure ulcer. Here we gently applied paired magnets to the backs of mice to cause ischemia for 1.5 h and then removed them to allow reperfusion. The sterile inflammatory response generated within 4 h causes a stage 1 pressure ulcer with an elevation of the gap junction protein Cx43 in the epidermis. If this process is repeated the insult will result in a more severe stage 2 pressure ulcer with a breakdown of the epidermis 2-3 days later. After a single pinch, the elevation of Cx43 in the epidermis is associated with the inflammatory response with an increased number of neutrophils, HMGB1 (marker of necrosis) and RIP3 (responsible for necroptosis). Delivering Cx43 specific antisense oligonucleotides sub-dermally after a single insult, was able to significantly reduce the elevation of epidermal Cx43 protein expression and reduce the number of neutrophils and prevent the elevation of HMGB1 and RIP3. In a double pinch model, the Cx43 antisense treatment was able to reduce the level of inflammation, necroptosis, and the extent of tissue damage and progression to an open wound. This approach may be useful in reducing the progression of stage 1 pressure ulcers to stage 2.

Early Human Pathophysiological Responses to Exertional Hypobaric Decompression Stress.

INTRODUCTION: Consistent blood biomarkers of hypobaric (altitude) decompression stress remain elusive. Recent laboratory investigation of decompression sickness risk at 25,000 ft (7620 m) enabled evaluation of early pathophysiological responses to exertional decompression stress.METHODS: In this study, 15 healthy men, aged 20-50 yr, undertook 2 consecutive (same-day) ascents to 25,000 ft (7620 m) for 60 and 90 min, breathing 100% oxygen, each following 1 h of prior denitrogenation. Venous blood was sampled at baseline (T0), immediately after the second ascent (T8), and next morning (T24). Analyses encompassed whole blood hematology, endothelial microparticles, and soluble markers of cytokine response, endothelial function, inflammation, coagulopathy, oxidative stress, and brain insult, plus cortisol and creatine kinase.RESULTS: Acute hematological effects on neutrophils (mean 72% increase), eosinophils (40% decrease), monocytes (37% increase), and platelets (7% increase) normalized by T24. Consistent elevation (mean five-fold) of the cytokine interleukin-6 (IL-6) at T8 was proinflammatory and associated with venous gas emboli (microbubble) load. Levels of C-reactive protein and complement peptide C5a were persistently elevated at T24, the former by 100% over baseline. Additionally, glial fibrillary acidic protein, a sensitive marker of traumatic brain injury, increased by a mean 10% at T24.CONCLUSIONS: This complex composite environmental stress, comprising the triad of hyperoxia, decompression, and moderate exertion at altitude, provoked pathophysiological changes consistent with an IL-6 cytokine-mediated inflammatory response. Multiple persistent biomarker disturbances at T24 imply incomplete recovery the day after exposure. The elevation of glial fibrillary acidic protein similarly implies incomplete resolution following recent neurological insult.Connolly DM, Madden LA, Edwards VC, D'Oyly TJ, Harridge SDR, Smith TG, Lee VM. Early human pathophysiological responses to exertional hypobaric decompression stress. Aerosp Med Hum Perform. 2023; 94(10):738-749.

Immobilised-enzyme microreactors for the identification and synthesis of conjugated drug metabolites.

The study of naturally circulating drug metabolites has been a focus of interest, since these metabolites may have different therapeutic and toxicological effects compared to the parent drug. The synthesis of metabolites outside of the human body is vital in order to conduct studies into the pharmacological activities of drugs and bioactive compounds. Current synthesis methods require significant purification and separation efforts or do not provide sufficient quantities for use in pharmacology experiments. Thus, there is a need for simple methods yielding high conversions whilst bypassing the requirement for a separation. Here we have developed and optimised flow chemistry methods in glass microfluidic reactors utilising surface-immobilised enzymes for sulfonation (SULT1a1) and glucuronidation (UGT1a1). Conversion occurs in flow, the precursor and co-factor are pumped through the device, react with the immobilised enzymes and the product is then simply collected at the outlet with no separation from a complex biological matrix required. Conversion only occurred when both the correct co-factor and enzyme were present within the microfluidic system. Yields of 0.97 ± 0.26 µg were obtained from the conversion of resorufin into resorufin sulfate over 2 h with the SULT1a1 enzyme and 0.47 µg of resorufin glucuronide over 4 h for UGT1a1. This was demonstrated to be significantly more than static test tube reactions at 0.22 µg (SULT1a1) and 0.19 µg (UGT1a1) over 4 h. With scaling out and parallelising, useable quantities of hundreds of micrograms for use in pharmacology studies can be synthesised simply.

Digital Health Solutions for Weight Loss and Obesity: A Narrative Review.

Personal exercise programmes have long been used and prescribed for weight loss and the improvement of quality of life in obese patients. While individualised programmes are usually the preferred option, they can be more costly and challenging to deliver in person. A move to digital programmes with a wider reach has commenced, and demand has increased due to the SARS-CoV-2 pandemic. In this review, we evaluate the current status of digital exercise programme delivery and its evolution over the past decade, with a focus on personalisation. We used specific keywords to search for articles that met our predetermined inclusion and exclusion criteria in order to provide valuable evidence and insights for future research. We identified 55 studies in total in four key areas of focus, from the more recent development of apps and personal digital assistants to web-based programmes and text or phone call interventions. In summary, we observed that apps may be useful for a low-intensity approach and can improve adherence to programmes through self-monitoring, but they are not always developed in an evidence-based manner. Engagement and adherence are important determinants of weight loss and subsequent weight maintenance. Generally, professional support is required to achieve weight loss goals.

Challenges faced in developing an ideal chronic wound model.

INTRODUCTION: Chronic wounds are a major drain on healthcare resources and can lead to substantial reductions in quality of life for those affected. Moreover, they often precede serious events such as limb amputations and premature death. In the long run, this burden is likely to escalate with an ageing population and lifestyle diseases such as obesity. Thus far, the identification of beneficial therapeutics against chronic wounds have been hindered by the lack of an ideal chronic wound animal model. Although animal models of delayed healing have been developed, none of these models fully recapitulate the complexity of the human chronic wound condition. Furthermore, most animals do not develop chronic wounds. Only the thoroughbred racehorse develops chronic ulcers. AREAS COVERED: In this review, the different characteristics of chronic wounds that highlight its complexity are described. In addition, currently available models reflecting different aspects of chronic wound pathology and their relevance to human chronic wounds are discussed. This article concludes by listing relevant features representative of an ideal chronic wound model. Additionally, alternative approaches for the development of chronic wound models are discussed. EXPERT OPINION: Delayed models of healing, including the streptozotocin diabetic model, skin flap model and magnet-induced IR models have emerged. While these models have been widely adopted for preclinical therapeutic testing, their relevance towards human chronic wounds remains debatable. In particular, current delayed healing models often fail to fully incorporate the key characteristics of chronic ulcers. Ultimately, more representative models are required to expedite the advancement of novel therapeutics to the clinic.

Connexin43 in Post-Surgical Peritoneal Adhesion Formation.

OBJECTIVE: Post-surgical peritoneal adhesions are a serious problem for the quality of life and fertility. Yet there are no effective ways of preventing their occurrence. The gap junction protein Cx43 is known to be involved in fibrosis in several different organs and disease conditions often associated with inflammation. Here we examined the Cx43 dynamic expression in an ischemic button model of surgical adhesions. METHODS: Using the mouse ischemic button model, Cx43 antisense was delivered in Pluronic gel to attenuate Cx43 expression. The severity of button formation and immunofluorescence analysis of Cx43 and TGF-ß1 were performed. The concentration of tissue plasminogen activator via ELISA was also performed. RESULTS: As early as 6 h after button formation, the Cx43 levels were elevated in and around the button and some weak adhesions were formed. By 24 h Cx43 levels had increased further and adhesions were more defined. At 7 days the adhesions were much more robust, opaque, and vascularized, requiring blunt or sharp dissection to break them. Cx43 antisense attenuated its upregulation and, reduced the number and severity of adhesions that formed. CONCLUSION: Targeting Cx43 after surgical procedures may be a potential therapeutic strategy for preventing adhesion formation or at least reducing their severity.

Bio-Mimicking Acellular Wet Electrospun Scaffolds Promote Accelerated Integration and Re-Epithelialization of Full-Thickness Dermal Wounds.

Scaffolds can promote the healing of burns and chronic skin wounds but to date have suffered from issues with achieving full skin integration. Here, we characterise the wound response by both tissue integration and re-epithelialization to a scaffold using wet electrospinning to fabricate 3D fibrous structures. Two scaffold materials were investigated: poly(ε-caprolactone) (PCL) and PCL + 20% rat tail type 1 collagen (PCL/Coll). We assessed re-epithelisation, inflammatory responses, angiogenesis and the formation of new extracellular matrix (ECM) within the scaffolds in rat acute wounds. The 3D PCL/Coll scaffolds impeded wound re-epithelisation, inducing a thickening of wound-edge epidermis as opposed to a thin tongue of migratory keratinocytes as seen when 3D PCL scaffolds were implanted in the wounds. A significant inflammatory response was observed with 3D PCL/Coll scaffolds but not with 3D PCL scaffolds. Enhanced fibroblast migration and angiogenesis into 3D PCL scaffolds was observed with a significant deposition of new ECM. We observed that this deposition of new ECM within the scaffold was key to enabling re-epithelialization over the scaffold. Such scaffolds provide a biocompatible environment for cell integration to lay down new ECM and encourage re-epithelisation over the implanted scaffold.

Vascularized Co-Culture Clusteroids of Primary Endothelial and Hep-G2 Cells Based on Aqueous Two-Phase Pickering Emulsions.

Three-dimensional cell culture has been extensively involved in biomedical applications due to its high availability and relatively mature biochemical properties. However, single 3D cell culture models based on hydrogel or various scaffolds do not meet the more in-depth requirements of in vitro models. The necrotic core formation inhibits the utilization of the 3D cell culture ex vivo as oxygen permeation is impaired in the absence of blood vessels. We report a simple method to facilitate the formation of angiogenic HUVEC (human umbilical vein endothelial cells) and Hep-G2 (hepatocyte carcinoma model) co-culture 3D clusteroids in a water-in-water (w/w) Pickering emulsions template which can overcome this limitation. This method enabled us to manipulate the cells proportion in order to achieve the optimal condition for stimulating the production of various angiogenic protein markers in the co-cultured clusteroids. The HUVEC cells respond to the presence of Hep-G2 cells and their byproducts by forming endothelial cell sprouts in Matrigel without the exogenous addition of vascular endothelial growth factor (VEGF) or other angiogenesis inducers. This culture method can be easily replicated to produce other types of cell co-culture spheroids. The w/w Pickering emulsion template can facilitate the fabrication of 3D co-culture models to a great extent and be further utilized in drug testing and tissue engineering applications.

Fabrication of Angiogenic Sprouting Coculture of Cell Clusteroids Using an Aqueous Two-Phase Pickering Emulsion System.

Tumor cell spheroids and 3D cell culture have generated a lot of interest in the past decade due to their relative ease of production and biomedical research applications. To date, the frontier in tumor 3D models has been pushed to the level of personalized cancer treatment and customized tissue engineering applications. However, without vascularization, the central parts of these artificial constructs cannot survive without an adequate oxygen and nutrient supply. The formation of a necrotic core into in vitro 3D cell models still serves as the major obstacle in their wider practical application. Here, we propose a rapid formation protocol based on using a water-in-water (w/w) Pickering emulsion template to generate phenotypically endothelial/hepatic (ECV304/Hep-G2) coculture cell clusteroids with angiogenic capability. The w/w Pickering emulsion template was based on a dextran/poly(ethylene oxide) aqueous two-phase system stabilized by whey protein particles. The initial cell proportion in the coculture clusteroids can easily be manipulated for optimal performance. The cocultured pattern of the endothelial/hepatic cells could significantly promote the production of angiogenesis-related proteins. Our study confirmed that cocultured clusteroids can stimulate cell sprouting without the addition of vascular endothelial growth factor (VEGF) or other angiogenesis inducers at a 1:2 ratio of Hep-G2/ECV304. Angiogenesis gene production in the coculture clusteroids was enhanced with VEGF, urea, and insulin-like growth factor-binding protein along with angiogenesis-related marker CD34 levels, also indicating angiogenesis progress. Our aqueous two-phase Pickering emulsion templates provided a convenient approach to vascularize a target cell type in 3D cell coculture without additional stimulating factors, which could potentially apply to either cell lines or biopsy tissues, expanding the clusteroids downstream applications.

Development of a refined ex vivo model of peritoneal adhesion formation, and a role for connexin 43 in their development.

Despite many advances across the surgical sciences, post-surgical peritoneal adhesions still pose a considerable risk in modern-day procedures and are highly undesirable. We have developed a novel mouse peritoneal strip ex vivo adhesion model which may serve to bridge the gap between single cell culture systems and in vivo animal drug testing for the assessment of potential anti-adhesion agents, and study of causality of the process. We investigated the optimal conditions for adhesion formation with mouse peritoneal tissue strips by modifying an existing ex vivo rat model of peritoneal adhesions. We assessed the impact of the following conditions on the formation of adhesions: contact pressure, abrasions, and the presence of clotted blood. Macroscopic adhesions were detected in all mouse peritoneal strips exposed to specific conditions, namely abrasions and clotted blood, where peritoneal surfaces were kept in contact with pressure using cotton gauze in a tissue cassette. Adhesions were confirmed microscopically. Interestingly, connexin 43, a gap junction protein, was found to be upregulated at sites of adhesions. Key features of this model were the use of padding the abraded tissue with gauze and the use of a standardised volume of clotted blood. Using this model, peritoneal strips cultured with clotted blood between abraded surfaces were found to reproducibly develop adhesion bands at 72 h. Our goal is to develop a model that can be used in genetically modified mice in order to dissect out the role of particular genes in adhesion formation and to test drugs to prevent adhesion formation.

A wireless and battery-free wound infection sensor based on DNA hydrogel.

The confluence of wireless technology and biosensors offers the possibility to detect and manage medical conditions outside of clinical settings. Wound infections represent a major clinical challenge in which timely detection is critical for effective interventions, but this is currently hindered by the lack of a monitoring technology that can interface with wounds, detect pathogenic bacteria, and wirelessly transmit data. Here, we report a flexible, wireless, and battery-free sensor that provides smartphone-based detection of wound infection using a bacteria-responsive DNA hydrogel. The engineered DNA hydrogels respond selectively to deoxyribonucleases associated with pathogenic bacteria through tunable dielectric changes, which can be wirelessly detected using near-field communication. In a mouse acute wound model, we demonstrate that the wireless sensor can detect physiologically relevant amounts of Staphylococcus aureus even before visible manifestation of infection. These results demonstrate strategies for continuous infection monitoring, which may facilitate improved management of surgical or chronic wounds.

Enhanced clearing of Candida biofilms on a 3D urothelial cell in vitro model using lysozyme-functionalized fluconazole-loaded shellac nanoparticles.

Candida urinary tract biofilms are increasingly witnessed in nosocomial infections due to reduced immunity of patients and the hospital ecosystem. The indwelling devices utilized to support patients with urethral diseases that connect the unsterilized external environment with the internal environment of the patient are another significant source of urinary tract biofilm infections. Recently, nanoparticle (NP)-associated therapeutics have gained traction in a number of areas, including fighting antibiotic-resistant bacterial biofilm infection. However, most studies on nanotherapeutic delivery have only been carried out in laboratory settings rather than in clinical trials due to the lack of precise in vitro and in vivo models for testing their efficiency. Here we develop a novel biofilm-infected 3D human urothelial cell culture model to test the efficiency of nanoparticle (NP)-based antifungal therapeutics. The NPs were designed based on shellac cores, loaded with fluconazole and coated with the cationic enzyme lysozyme. Our formulation of 0.2 wt% lysozyme-coated 0.02 wt% fluconazole-loaded 0.2 wt% shellac NPs, sterically stabilised by 0.25 wt% poloxamer 407, showed an enhanced efficiency in removing Candida albicans biofilms formed on 3D layer of urothelial cell clusteroids. The NP formulation exhibited low toxicity to urothelial cells. This study provides a reliable in vitro model for Candida urinary tract biofilm infections, which could potentially replace animal models in the testing of such antifungal nanotechnologies. The reproducibility and availability of a well-defined biofilm-infected 3D urothelial cell culture model give valuable insights into the formation and clearing of fungal biofilms and could accelerate the clinical use of antifungal nanotherapeutics.

Biofilm-Infected Human Clusteroid Three-Dimensional Coculture Platform to Replace Animal Models in Testing Antimicrobial Nanotechnologies.

Microbial biofilms are a major concern in wound care, implant devices, and organ infections. Biofilms allow higher tolerance to antimicrobial drugs, can impair wound healing, and potentially lead to sepsis. There has been a recent focus on developing novel nanocarrier-based delivery vehicles to enhance the biofilm penetration of traditional antibacterial drugs. However, a feasible in vitro human skin model to mimic the biofilm formation and its treatment for clearance have not yet been reported. This study describes the benefits of using an innovative bacterial biofilm-infected keratinocyte clusteroid model for the first time. It paves a new way for testing innovative nanomedicine delivery systems in a rapid and reproducible way on a realistic human cell-based platform, free of any animal testing. Herein, we have developed a novel composite 3D biofilm/human keratinocyte clusteroid coculture platform, which was used to measure biofilm clearance efficiency of nanoparticle (NP)-based therapeutics. We tested this model by treating the biofilm-infected 3D coculture layers by a ciprofloxacin-loaded Carbopol nanogel particles, surface-functionalized by the cationic protease Alcalase. We measured the antibacterial efficiency of the NP treatment on clearing Staphylococcus aureus and Pseudomonas aeruginosa biofilms on the 3D keratinocyte clusteroid/biofilm coculture model. Our experiments showed that these bacteria can infect the 3D layer of keratinocyte clusteroids and produce a stable biofilm. The biofilms were efficiently cleared by treatment with a formulation of 0.0032 wt % ciprofloxacin-loaded in 0.2 wt % Carbopol NPs surface-functionalized with 0.2 wt % Alcalase. Taken together, these promising results demonstrate that our coculture model can be exploited as a novel platform for testing the biofilm-eliminating efficiency of various NP formulations emulating skin and wound infections and could have wider applicability to replace animal models in similar experiments. This 3D cell culture-based platform could help in developing and testing of more effective antibacterial agents for clinical applications of antiplaque dental treatments, implants, infection control, and wound dressings.

A flexible multiplexed immunosensor for point-of-care in situ wound monitoring.

Chronic wounds arise from interruption of normal healing due to many potential pathophysiological factors. Monitoring these multivariate factors can provide personalized diagnostic information for wound management, but current sensing technologies use complex laboratory tests or track a limited number of wound parameters. We report a flexible biosensing platform for multiplexed profiling of the wound microenvironment, inflammation, and infection state at the point of care. This platform integrates a sensor array for measuring inflammatory mediators [tumor necrosis factor-α, interleukin-6 (IL-6), IL-8, and transforming growth factor-ß1], microbial burden (Staphylococcus aureus), and physicochemical parameters (temperature and pH) with a microfluidic wound exudate collector and flexible electronics for wireless, smartphone-based data readout. We demonstrate in situ multiplexed monitoring in a mouse wound model and also profile wound exudates from patients with venous leg ulcers. This technology may facilitate more timely and personalized wound management to improve chronic wound healing outcomes.

Characterisation of an ischemia reperfusion model for the formation of a stage I pressure ulcer in mouse skin.

Pairs of magnets were applied to the loose skin on the backs of mice in order to cause ischemia for periods of 1.5, 2, 2.5 and 3 h followed by reperfusion. We found 1.5 h of ischemia resulted in the most reliable outcome of blanched skin but no redness or skin breakdown. Histological analysis at 4 h of reperfusion showed, in the centre of the insult, condensed nuclei in the epidermis and sebaceous glands with a build up of neutrophils in the blood vessels, and a reduction in the number of fibroblasts. At 24 h, spongiosis was seen in the epidermis and pockets of neutrophils began to accumulate under it, as well as being scatted through the dermis. In the centre of the insult there was a loss of sebaceous gland nuclei and fibroblasts. Four days after the insult, spongiosis was reduced in the epidermis at the edge of the insult but enhanced in the centre and in hair follicles. Leukocytes were seen throughout the central dermis. At 8 days, spongiosis and epidermal thickness had reduced and fibroblasts were reappearing. However, blood vessels still had leukocytes lining the lumen. The gap junction protein connexin 43 was significantly elevated in the epidermis at 4 h and 24 h reperfusion. Ischemia of 1.5 h generates a sterile inflammatory reaction causing the loss of some cell types but leaving the epidermis intact reminiscent of a stage I pressure ulcer.

The effects of Staphylococcus aureus biofilm conditioned media on 3T3 fibroblasts.

Staphylococcus aureus (SA) is the most common bacterial species in chronic wounds. However, there is a lack of understanding of how SA secretions affect the cell biology during the healing process. We studied the effects of biofilm-secretions from SA strain SA29213 on 3T3 fibroblasts. SA29213 is a chronic wound isolate and widely used as a reference strain. We used a series of concentrations of biofilm-conditioned media (BCM) and found 100% BCM is lethal within 10 h. Cells survived in ≤75% BCM but the rate of closure in scratch wound assays was reduced. Treatment with 75% and 50% BCM caused fibroblasts to change shape and develop dendrite like processes. Prolonged treatment with 75% and 50% BCM reduced cell proliferation and increased the 4n deoxyribonucleic acid cell population with cell cycle arrest. There was also an elevation in the senescence marker beta galactosidase and the number of multinucleated cells. Shorter treatments with 75% and 50% SA BCM caused an increase in cell-cell adhesion and a redistribution of ß-catenin from the cell membrane to the cytoplasm along with a change in the appearance and decrease in size of ZO-1, vinculin and paxillin structures. Fibroblasts in the edge of chronic wounds exposed to the secretions of SA may suffer similar effects such as induction of senescence, reduced proliferation and migration, which may contribute to the delayed healing of these chronic infected wounds.

Pulpal upregulation of connexin 43 during pulpitis.

OBJECTIVES: Connexins are building blocks of membranous channels that form gap junctions and hemichannels. These channels are essential portals for information exchange and coordination during inflammation. Pathologic levels of these conduits may result in excessive inflammation and collateral destruction. This study aimed to analyse temporospatial levels of connexin 43 (Cx43) during pulpitis in extracted human teeth and in a rodent model. A specific interest was directed at the pulpal stroma as it is conserved during vital pulp therapy. MATERIALS AND METHODS: Pulpal tissues were attained from human extracted teeth of various pulpal inflammatory stages and fixed for cryosections. Pulpal exposures were created in bilateral maxillary molars in Sprague-Dawley rats. Rats were sacrificed at days 1 to 5 post-exposure. Immunofluorescence histology was performed to detect Cx43, markers for inflammation, and cell death. Immunofluorescent levels in the pulpal stroma at 3 sites (wound/near/far) were matched to pulpal condition (human) or days post-exposure (rodent). RESULTS: Cx43 upregulation was observed with increased severity of pulpitis both in humans and rodent model. The upregulation appeared to be global and included distant regions. Elevated levels of neutrophils were present in advanced pulpitis. Apoptosis and necroptosis seem to be upregulated in human samples as Cx43 levels rose. CONCLUSIONS: We observed a disseminated upregulation of Cx43 throughout the pulpal stroma as inflammation became advanced. This observation may facilitate cell death signal transfer or represent overt levels of purinergic signalling that leads to pro-inflammatory conditions. CLINICAL RELEVANCE: Cx43 downregulation may represent a potential therapeutic approach to enable resolution of pulpal inflammation.

Advanced biomedical applications based on emerging 3D cell culturing platforms.

It is of great value to develop reliable in vitro models for cell biology and toxicology. However, ethical issues and the decreasing number of donors restrict the further use of traditional animal models in various fields, including the emerging fields of tissue engineering and regenerative medicine. The huge gap created by the restrictions in animal models has pushed the development of the increasingly recognized three-dimensional (3D) cell culture, which enables cells to closely simulate authentic cellular behaviour such as close cell-to-cell interactions and can achieve higher functionality. Furthermore, 3D cell culturing is superior to the traditional 2D cell culture, which has obvious limitations and cannot closely mimic the structure and architecture of tissues. In this study, we review several methods used to form 3D multicellular spheroids. The extracellular microenvironment of 3D spheroids plays a role in many aspects of biological sciences, including cell signalling, cell growth, cancer cell generation, and anti-cancer drugs. More recently, they have been explored as basic construction units for tissue and organ engineering. We review this field with a focus on the previous research in different areas using spheroid models, emphasizing aqueous two-phase system (ATPS)-based techniques. Multi-cellular spheroids have great potential in the study of biological systems and can closely mimic the in vivo environment. New technologies to form and analyse spheroids such as the aqueous two-phase system and magnetic levitation are rapidly overcoming the technical limitations of spheroids and expanding their applications in tissue engineering and regenerative medicine.