Branched copolymer surfactants impart thermoreversible gelation to LAPONITE® gels.

This investigation seeks to integrate LAPONITE® clay gels with thermoresponsive branched copolymer surfactants (BCSs) to develop advanced functional materials with temperature-induced sol-gel behaviour. It is known that a diverse range of molecules adsorb strongly to clays which may be used to control liberation of the species in healthcare applications, and as such the development of polymer/clay hybrid materials which can add function to the native clay behaviour are of great interest. BCS were synthesised with a structure that encompasses poly(ethylene glycol)methacrylate (PEGMA), ethylene glycol dimethacrylate (EGDMA), and dodecanethiol (DDT), conferring versatile and tuneable thermoresponsive attributes. Systematic modulation of the monomer : DDT/initiator ratio was used to facilitate the synthesis of BCS architectures spanning a range of molecular weights. Through application of small-amplitude oscillatory shear (SAOS) rheology and small-angle neutron scattering (SANS) in conjunction with controlled temperature variations, the sol-gel transition dynamics of these nanocomposite materials were elucidated. Complementary insights into the mechanisms underpinning this transition and temperature-induced alterations in the constituents are gleaned through the utilization of SANS techniques employing contrast-matching methodologies to mitigate clay and polymer scattering interference. It is found that heating systems from room- to body- temperature induces self-assembly of BCS in the bulk aqueous phase with concurrent structuration of clay in gel-forming samples with lower number average molecular weight (Mn). SANS study unpicks this phenomenon to find that gelation occurs with concurrent aggregation of BCS in the bulk, inducing clay-clay interactions only in lower Mn BCS systems with large nanoaggregates.

Helicopter Emergency Medical Services Out-of-Hospital Cardiac Arrests During the Initial COVID-19 Lockdown Versus Nonpandemic: A Comparison.

OBJECTIVE: COVID-19 may have contributed to an excess of out-of-hospital cardiac arrests (OOHCAs). This observational study identified changes in OOHCA epidemiology pre- and post-COVID-19 lockdown in a single UK helicopter emergency medical service (HEMS). METHODS: A retrospective, single-center (Essex & Herts Air Ambulance), observational study was undertaken with anonymized OOHCA data (demographics, etiology, and outcomes) from March 23, 2020, to June 23, 2020, and comparative data from March 23, 2019, to June 23, 2019. Supplementary data (total OOHCAs and patient outcomes) were provided by the East of England Ambulance Service National Health Service Trust. Data were analyzed using the Mann-Whitney U test and chi-square test; P < .05 was statistically significant. RESULTS: Of the HEMS activations during national lockdown, 33.6% were for OOHCAs compared with 25.8% during the reference time frame. The frequency of young and female OOHCAs demonstrated a statistically significant increase. Statistically significant variations in medical etiology and initial cardiac rhythm were identified. CONCLUSION: During the initial UK-wide lockdown, the OOHCA characteristics attended by 1 HEMS team were altered. The changes seen may be due to the pathophysiology of COVID-19 or an alteration in dispatch due to the demand placed on the wider ambulance service; this may require further consideration for any future lockdowns or pandemics.

The Prehospital Emergency Anaesthetic in 2022.

Prehospital emergency anesthesia (PHEA) is a commonly performed prehospital procedure with inherent risks. The processes and drug regimens behind PHEA are continually updated by prehospital teams across the country as part of their governance structure. Essex & Herts Air Ambulance has recently updated this practice by reviewing the entire process of performing PHEA. Through experiential learning in a high-volume service, audit, and a contemporary literature review, a new standard operating procedure has been developed to combat common problems, such as hypotension, associated with the more traditional methods of performing PHEA. The aim of this article was to summarize the literature behind this new standard operating procedure, systematically breaking down the core components of performing a PHEA and the rationale behind them. The key components identified in the review are indications for PHEA, airway assessment, peri-intubation oxygenation, preparation for PHEA, drug dosing, special circumstances, and failed intubation. One significant change is the drug dosage regimen; 1 µg/kg fentanyl, 2 mg/kg ketamine, and 2 mg/kg rocuronium is recommended as the main drug dosing regimen for both medical and trauma patients. Other changes include preoxygenation with a nasal cannula in addition to the nonrebreather mask, optimizing patients in the preparation phase by considering inopressors or fluid bolus and ensuring a "sterile cockpit" to control the surrounding environment to ensure the first intubation attempt is the best attempt.

Auxin biosynthesis and cellular efflux act together to regulate leaf vein patterning.

Our current understanding of vein development in leaves is based on canalization of the plant hormone auxin into self-reinforcing streams which determine the sites of vascular cell differentiation. By comparison, how auxin biosynthesis affects leaf vein patterning is less well understood. Here, after observing that inhibiting polar auxin transport rescues the sparse leaf vein phenotype in auxin biosynthesis mutants, we propose that the processes of auxin biosynthesis and cellular auxin efflux work in concert during vein development. By using computational modeling, we show that localized auxin maxima are able to interact with mechanical forces generated by the morphological constraints which are imposed during early primordium development. This interaction is able to explain four fundamental characteristics of midvein morphology in a growing leaf: (i) distal cell division; (ii) coordinated cell elongation; (iii) a midvein positioned in the center of the primordium; and (iv) a midvein which is distally branched. Domains of auxin biosynthetic enzyme expression are not positioned by auxin canalization, as they are observed before auxin efflux proteins polarize. This suggests that the site-specific accumulation of auxin, as regulated by the balanced action of cellular auxin efflux and local auxin biosynthesis, is crucial for leaf vein formation.

Nanoclay-Polyamine Composite Hydrogel for Topical Delivery of Nitric Oxide Gas via Innate Gelation Characteristics of Laponite.

Because nitric oxide (NO) gas is an endogenously produced signaling molecule related to numerous physiological functions, manystudies have been conducted to develop NO delivery systems for potential biomedical applications. However, NO is a reactive radical gas molecule that has a very short life-time and readily transforms into nitrogen oxide species via reaction with oxygen species. Therefore, it is necessary to develop an NO delivery carrier that allows local release of the NO gas at the site of application. In this study, Laponite (LP) nanoclay was used to fabricate an NO delivery carrier through the formation of Laponite-polyamine (LP-PAn) composites. The Laponite clay and pentaethylenehexamine (PEHA) formed a macromolecular structure by electrostatic interaction and the nitric oxide donor, N-diazeniumdiolate (NONOates), was synthesized into the LP-PAn composite. We investigated the conformation of the LP-PAn composite structure and the NO donor formation by ζ potential, X-ray diffraction, and UV-vis and Fourier transform infrared (FT-IR) spectroscopies and also by analyzing the NO release profile. Additionally, we confirmed the applicability in biomedical applications via a cell viability and in vitro endothelial cell tube formation assay.

Polyetherketoneketone (PEKK), a framework material for complete fixed and removable dental prostheses: A clinical report.

This clinical report demonstrates the use of polyetherketoneketone (PEKK) as a framework material with individually luted heat-pressed lithium disilicate glass-ceramic crowns for an implant-supported complete fixed dental prosthesis (ICFDP) and a conventional complete removable dental prosthesis (CRDP). This prosthesis design provides a non-computer-aided design and computer-aided manufacturing (CAD-CAM) option for the fabrication of ICFDPs and CRDPs with individualized ceramic crowns for optimal esthetics. The performance of PEKK as a framework material needs to be assessed in clinical trials.

Concise review: bridging the gap: bone regeneration using skeletal stem cell-based strategies - where are we now?

Skeletal stem cells confer to bone its innate capacity for regeneration and repair. Bone regeneration strategies seek to harness and enhance this regenerative capacity for the replacement of tissue damaged or lost through congenital defects, trauma, functional/esthetic problems, and a broad range of diseases associated with an increasingly aged population. This review describes the state of the field and current steps to translate and apply skeletal stem cell biology in the clinic and the problems therein. Challenges are described along with key strategies including the isolation and ex vivo expansion of multipotential populations, the targeting/delivery of regenerative populations to sites of repair, and their differentiation toward bone lineages. Finally, preclinical models of bone repair are discussed along with their implications for clinical translation and the opportunities to harness that knowledge for musculoskeletal regeneration.

Biofabrication of nanocomposite-based scaffolds containing human bone extracellular matrix for the differentiation of skeletal stem and progenitor cells.

Autograft or metal implants are routinely used in skeletal repair. However, they fail to provide long-term clinical resolution, necessitating a functional biomimetic tissue engineering alternative. The use of native human bone tissue for synthesizing a biomimetic material ink for three-dimensional (3D) bioprinting of skeletal tissue is an attractive strategy for tissue regeneration. Thus, human bone extracellular matrix (bone-ECM) offers an exciting potential for the development of an appropriate microenvironment for human bone marrow stromal cells (HBMSCs) to proliferate and differentiate along the osteogenic lineage. In this study, we engineered a novel material ink (LAB) by blending human bone-ECM (B) with nanoclay (L, Laponite®) and alginate (A) polymers using extrusion-based deposition. The inclusion of the nanofiller and polymeric material increased the rheology, printability, and drug retention properties and, critically, the preservation of HBMSCs viability upon printing. The composite of human bone-ECM-based 3D constructs containing vascular endothelial growth factor (VEGF) enhanced vascularization after implantation in an ex vivo chick chorioallantoic membrane (CAM) model. The inclusion of bone morphogenetic protein-2 (BMP-2) with the HBMSCs further enhanced vascularization and mineralization after only seven days. This study demonstrates the synergistic combination of nanoclay with biomimetic materials (alginate and bone-ECM) to support the formation of osteogenic tissue both in vitro and ex vivo and offers a promising novel 3D bioprinting approach to personalized skeletal tissue repair. Supplementary Information: The online version contains supplementary material available at 10.1007/s42242-023-00265-z.

Bioactive coatings on 3D printed scaffolds for bone regeneration: Use of Laponite® to deliver BMP-2 in an ovine femoral condyle defect model.

Biomaterial-based approaches for bone regeneration seek to explore alternative strategies to repair non-healing fractures and critical-sized bone defects. Fracture non-union occurs due to a number of factors resulting in the formation of bone defects. Rigorous evaluation of the biomaterials in relevant models and assessment of their potential to translate towards clinical use is vital. Large animal experimentation can be used to model fracture non-union while scaling-up materials for clinical use. Growth factors modulate cell phenotype, behaviour and initiate signalling pathways leading to changes in matrix deposition and tissue formation. Bone morphogenetic protein-2 (BMP-2) is a potent osteogenic growth factor, with a rapid clearance time in vivo necessitating clinical use at a high dose, with potential deleterious side-effects. The current studies have examined the potential for Laponite® nanoclay coated poly(caprolactone) trimethacrylate (PCL-TMA900) scaffolds to bind BMP-2 for enhanced osteoinduction in a large animal critical-sized bone defect. An ovine femoral condyle defect model confirmed PCL-TMA900 scaffolds coated with Laponite®/BMP-2 produced significant bone formation compared to the uncoated PCL-TMA 900 scaffold in vivo, assessed by micro-computed tomography (µCT) and histology. This indicated the ability of Laponite® to deliver the bioactive BMP-2 on the PCL-TMA900 scaffold. Bone formed around the Laponite®/BMP-2 coated PCL-TMA900 scaffold, with no erroneous bone formation observed away from the scaffold material confirming localisation of BMP-2 delivery. The current studies demonstrate the ability of a nanoclay to localise and deliver bioactive BMP-2 within a tailored octet-truss scaffold for efficacious bone defect repair in a large animal model with significant implications for translation to the clinic.

Enhancing the osteogenic efficacy of human bone marrow aspirate: concentrating osteoprogenitors using wave-assisted filtration.

BACKGROUND: Recent approaches have sought to harness the potential of stem cells to regenerate bone that is lost as a consequence of trauma or disease. Bone marrow aspirate (BMA) provides an autologous source of osteoprogenitors for such applications. However, previous studies indicated that the concentration of osteoprogenitors present in BMA is less than required for robust bone regeneration. We provide further evidence for the importance of BMA enrichment for skeletal tissue engineering strategies using a novel acoustic wave-facilitated filtration strategy to concentrate BMA for osteoprogenitors, clinically applicable for intraoperative orthopedic use. METHODS: Femoral BMA from 15 patients of an elderly cohort was concentrated for the nucleated cell fraction against erythrocytes and excess plasma volume via size exclusion filtration facilitated by acoustic agitation. The effect of aspirate concentration was assessed by assays for colony formation, flow cytometry, multilineage differentiation and scaffold seeding efficiency. RESULTS: BMA was filtered to achieve a mean 4.2-fold reduction in volume with a corresponding enrichment of viable and functional osteoprogenitors, indicated by flow cytometry and assays for colony formation. Enhanced osteogenic and chondrogenic differentiation was observed using concentrated aspirate and enhanced cell-seeding efficiency onto allogeneic bone graft as an effect of osteoprogenitor concentration relative specifically to the concentration of erythrocytes in the aspirate. CONCLUSIONS: These studies provide evidence for the importance of BMA nucleated cell concentration for both cell differentiation and cell seeding efficiency and demonstrate the potential of this approach for intraoperative application to enhance bone healing.

Tracking cellular uptake, intracellular trafficking and fate of nanoclay particles in human bone marrow stromal cells.

Clay nanoparticles, in particular synthetic smectites, have generated interest in the field of tissue engineering and regenerative medicine due to their utility as cross-linkers for polymers in biomaterial design and as protein release modifiers for growth factor delivery. In addition, recent studies have suggested a direct influence on the osteogenic differentiation of responsive stem and progenitor cell populations. Relatively little is known however about the mechanisms underlying nanoclay bioactivity and in particular the cellular processes involved in nanoclay-stem cell interactions. In this study we employed confocal microscopy, inductively coupled plasma mass spectrometry and transmission electron microscopy to track the interactions between clay nanoparticles and human bone marrow stromal cells (hBMSCs). In particular we studied nanoparticle cellular uptake mechanisms and uptake kinetics, intracellular trafficking pathways and the fate of endocytosed nanoclay. We found that nanoclay particles present on the cell surface as µm-sized aggregates, enter hBMSCs through clathrin-mediated endocytosis, and their uptake kinetics follow a linear increase with time during the first week of nanoclay addition. The endocytosed particles were observed within the endosomal/lysosomal compartments and we found evidence for both intracellular degradation of nanoclay and exocytosis as well as an increase in autophagosomal activity. Inhibitor studies indicated that endocytosis was required for nanoclay upregulation of alkaline phosphatase activity but a similar dependency was not observed for autophagy. This study into the nature of nanoclay-stem cell interactions, in particular the intracellular processing of nanosilicate, may provide insights into the mechanisms underlying nanoclay bioactivity and inform the successful utilisation of clay nanoparticles in biomaterial design.

Self-Assembly of Structured Colloidal Gels for High-Resolution 3D Micropatterning of Proteins at Scale.

Self-assembly, the spontaneous ordering of components into patterns, is widespread in nature and fundamental to generating function across length scales. Morphogen gradients in biological development are paradigmatic as both products and effectors of self-assembly and various attempts have been made to reproduce such gradients in biomaterial design. To date, approaches have typically utilized top-down fabrication techniques that, while allowing high-resolution control, are limited by scale and require chemical cross-linking steps to stabilize morphogen patterns in time. Here, a bottom-up approach to protein patterning is developed based on a novel binary reaction-diffusion process where proteins function as diffusive reactants to assemble a nanoclay-protein composite hydrogel. Using this approach, it is possible to generate scalable and highly stable 3D patterns of target proteins down to sub-cellular resolution through only physical interactions between clay nanoparticles and the proteins and ions present in blood. Patterned nanoclay gels are able to guide cell behavior to precisely template bone tissue formation in vivo. These results demonstrate the feasibility of stabilizing 3D gradients of biological signals through self-assembly processes and open up new possibilities for morphogen-based therapeutic strategies and models of biological development and repair.

Synthetic Notch Activation Patterns in a Proliferating Tissue.

Cell-cell communication through direct contact is essential during fundamental biological processes such as tissue repair and morphogenesis. Synthetic forms of contact-mediated cell-cell communication can generate custom gene expression outputs, making them valuable for tissue engineering and regenerative medicine. To precisely control the location and timing of synthetic signal outputs in growing tissues, it is necessary to understand the mechanisms underlying its spatiotemporal patterns. Towards this goal, we combine theory and experiments to study patterns of synthetic Notch (synNotch) activation - a custom synthetic gene circuit that we implement within Drosophila wing imaginal discs. We show that output synthesis and degradation rates together with cell division are the key minimal parameters that predict the heterogeneous spatiotemporal patterns of synNotch activation. Notably, synNotch output forms a graded exponential spatial profile that extends several cell diameters from the signal source, establishing evidence for signal propagation without diffusion or long range cell-cell communication. Furthermore, we discover that the shape of the interface between ligand and receptor cells is important in determining the synNotch output. Overall, we elucidate key biophysical principles that underlie complex emergent spatiotemporal patterns of synNotch output in a growing tissue.

From hurdle to springboard: The macrophage as target in biomaterial-based bone regeneration strategies.

The past decade has seen a growing appreciation for the role of the innate immune response in mediating repair and biomaterial directed tissue regeneration. The long-held view of the host immune/inflammatory response as an obstacle limiting stem cell regenerative activity, has given way to a fresh appreciation of the pivotal role the macrophage plays in orchestrating the resolution of inflammation and launching the process of remodelling and repair. In the context of bone, work over the past decade has established an essential coordinating role for macrophages in supporting bone repair and sustaining biomaterial driven osteogenesis. In this review evidence for the role of the macrophage in bone regeneration and repair is surveyed before discussing recent biomaterial and drug-delivery based approaches that target macrophage modulation with the goal of accelerating and enhancing bone tissue regeneration.

Cell-cell interactions and fluctuations in the direction of motility promote directed migration of osteoblasts in direct current electrotaxis.

Under both physiological (development, regeneration) and pathological conditions (cancer metastasis), cells migrate while sensing environmental cues in the form of mechanical, chemical or electrical stimuli. In the case of bone tissue, osteoblast migration is essential in bone regeneration. Although it is known that osteoblasts respond to exogenous electric fields, the underlying mechanism of electrotactic collective movement of human osteoblasts is unclear. Here, we present a computational model that describes the osteoblast cell migration in a direct current electric field as the motion of a collection of active self-propelled particles and takes into account fluctuations in the direction of single-cell migration, finite-range cell-cell interactions, and the interaction of a cell with the external electric field. By comparing this model with in vitro experiments in which human primary osteoblasts are exposed to a direct current electric field of different field strengths, we show that cell-cell interactions and fluctuations in the migration direction promote anode-directed collective migration of osteoblasts.

Comparison of bone formation mediated by bone morphogenetic protein delivered by nanoclay gels with clinical techniques (autograft and InductOs®) in an ovine bone model.

Development of a growth factor delivery vehicle providing appropriate temporal-spatial release together with an appropriate preclinical large animal model to evaluate bone formation is critical in the development of delivery strategies for bone tissue regeneration. Smectite nanoclays such as LAPONITE™ possess unique thixotropic and protein retention properties offering promise for use in growth factor delivery in bone repair and regeneration. This study has examined bone formation mediated by a clinically approved growth factor delivery system (InductOs®) in combination with Laponite gel in an aged female ovine femoral condyle defect preclinical model (10 weeks). Two different designs, one containing a low volume of Laponite gel (LLG) in combination with the InductOs® absorbable collagen sponge (ACS), the other in which Laponite gel formed the implant (HLG), were compared against InductOs® alone and an autograft positive control. Thus, five groups: (i) empty defect, (ii) autograft, (iii) BMP2 + ACS, (iv) BMP2 + ACS + LLG and (v) BMP2 + HLG + ACS were examined in 9 mm × 12 mm defects performed bilaterally in the medial femoral condyles of 24 aged (>5 years) sheep. Bone formation within the defect was assessed using micro-computed tomography (micro-CT), digital volume correlation (DVC) for biomechanical characterisation as well as histology. The autograft and InductOs® mediated enhanced bone formation (p < 0001) compared to blank controls, while no significant differences were observed between the Laponite/Collagen/BMP delivery vehicles. However, the current study illustrated the excellent biocompatibility of Laponite and its ability to deliver localised active BMP-2, with the opportunity for improved efficacy with further optimisation. Interestingly, DVC-computed strain distributions indicated that the regenerated bone structure is mechanically adapted to bear external loads from the early remodelling stages of the bone reparation cascade. The current studies of selected nanoclay delivery platforms for BMP, assessed in a clinically relevant large animal model auger well for the development of bone fracture therapeutics for an ageing population.

Gelatin Methacryloyl Hydrogels for Musculoskeletal Tissue Regeneration.

Musculoskeletal disorders are a significant burden on the global economy and public health. Hydrogels have significant potential for enhancing the repair of damaged and injured musculoskeletal tissues as cell or drug delivery systems. Hydrogels have unique physicochemical properties which make them promising platforms for controlling cell functions. Gelatin methacryloyl (GelMA) hydrogel in particular has been extensively investigated as a promising biomaterial due to its tuneable and beneficial properties and has been widely used in different biomedical applications. In this review, a detailed overview of GelMA synthesis, hydrogel design and applications in regenerative medicine is provided. After summarising recent progress in hydrogels more broadly, we highlight recent advances of GelMA hydrogels in the emerging fields of musculoskeletal drug delivery, involving therapeutic drugs (e.g., growth factors, antimicrobial molecules, immunomodulatory drugs and cells), delivery approaches (e.g., single-, dual-release system), and material design (e.g., addition of organic or inorganic materials, 3D printing). The review concludes with future perspectives and associated challenges for developing local drug delivery for musculoskeletal applications.

Determination of protoplast growth properties using quantitative single-cell tracking analysis.

BACKGROUND: Although quantitative single-cell analysis is frequently applied in animal systems, e.g. to identify novel drugs, similar applications on plant single cells are largely missing. We have exploited the applicability of high-throughput microscopic image analysis on plant single cells using tobacco leaf protoplasts, cell-wall free single cells isolated by lytic digestion. Protoplasts regenerate their cell wall within several days after isolation and have the potential to expand and proliferate, generating microcalli and finally whole plants after the application of suitable regeneration conditions. RESULTS: High-throughput automated microscopy coupled with the development of image processing pipelines allowed to quantify various developmental properties of thousands of protoplasts during the initial days following cultivation by immobilization in multi-well-plates. The focus on early protoplast responses allowed to study cell expansion prior to the initiation of proliferation and without the effects of shape-compromising cell walls. We compared growth parameters of wild-type tobacco cells with cells expressing the antiapoptotic protein Bcl2-associated athanogene 4 from Arabidopsis (AtBAG4). CONCLUSIONS: AtBAG4-expressing protoplasts showed a higher proportion of cells responding with positive area increases than the wild type and showed increased growth rates as well as increased proliferation rates upon continued cultivation. These features are associated with reported observations on a BAG4-mediated increased resilience to various stress responses and improved cellular survival rates following transformation approaches. Moreover, our single-cell expansion results suggest a BAG4-mediated, cell-independent increase of potassium channel abundance which was hitherto reported for guard cells only. The possibility to explain plant phenotypes with single-cell properties, extracted with the single-cell processing and analysis pipeline developed, allows to envision novel biotechnological screening strategies able to determine improved plant properties via single-cell analysis.

Multi-Scale Analysis of the Composition, Structure, and Function of Decellularized Extracellular Matrix for Human Skin and Wound Healing Models.

The extracellular matrix (ECM) is a complex mixture of structural proteins, proteoglycans, and signaling molecules that are essential for tissue integrity and homeostasis. While a number of recent studies have explored the use of decellularized ECM (dECM) as a biomaterial for tissue engineering, the complete composition, structure, and mechanics of these materials remain incompletely understood. In this study, we performed an in-depth characterization of skin-derived dECM biomaterials for human skin equivalent (HSE) models. The dECM materials were purified from porcine skin, and through mass spectrometry profiling, we quantified the presence of major ECM molecules, including types I, III, and VI collagen, fibrillin, and lumican. Rheological analysis demonstrated the sol-gel and shear-thinning properties of dECM materials, indicating their physical suitability as a tissue scaffold, while electron microscopy revealed a complex, hierarchical structure of nanofibers in dECM hydrogels. The dECM materials were compatible with advanced biofabrication techniques, including 3D printing within a gelatin microparticle support bath, printing with a sacrificial material, or blending with other ECM molecules to achieve more complex compositions and structures. As a proof of concept, we also demonstrate how dECM materials can be fabricated into a 3D skin wound healing model using 3D printing. Skin-derived dECM therefore represents a complex and versatile biomaterial with advantageous properties for the fabrication of next-generation HSEs.