Achieving net-zero in the dry eye disease care pathway.

Climate change is a threat to human health and wellbeing across the world. In recent years, there has been a surge in awareness of this crisis, leading to many countries and organisations setting "net-zero" targets. This entails minimising carbon emissions and neutralising remaining emissions by removing carbon from the atmosphere. At the 2022 United Nations Climate Change Conference (COP27), commitments to transition away from fossil fuels and augment climate targets were underwhelming. It is therefore imperative for public and private sector organisations to demonstrate successful implementation of net-zero and set a precedent for the global political consensus. As a top 10 world employer, the United Kingdom National Health Service (NHS) has pledged to reach net-zero by 2045. The NHS has already taken positive steps forward, but its scale and complexity as a health system means stakeholders in each of its services must highlight the specifications for further progress. Dry eye disease is a chronic illness with an estimated global prevalence of 29.5% and an environmentally damaging care pathway. Moreover, environmental damage is a known aggravator of dry eye disease. Worldwide management of this illness generates copious amounts of non-recyclable waste, utilises inefficient supply chains and involves recurrent follow-up appointments and prescriptions. By mapping the dry eye disease care pathway to environmental impact, in this review we will highlight seven key areas in which reduced emissions and pollution could be targeted. Examining these approaches for improved environmental sustainability is critical in driving the transformation needed to preserve our health and wellbeing.

Triggered metabolism of adenosine triphosphate as an explanation for the chemical heterogeneity of heterotopic ossification.

Heterotopic ossification (HO), the pathological formation of bone in soft tissues, is a debilitating condition, as well as one of the few instances of de novo bone formation in adults. Chemical mapping of HO tissue showed distinct islands of calcium phosphate within phosphate-deficient, calcium-rich regions, suggesting a transition to apatitic bone mineral from a non-phosphatic precursor. The transition of amorphous calcium carbonate (ACC), a generally suggested bone-mineral precursor, in physiological conditions was thus investigated. Here, we show that adenosine triphosphate (ATP), present in high amounts in forming bone, stabilised ACC for weeks in physiological conditions and that enzymatic degradation of ATP triggered rapid crystallisation into apatite, through an amorphous calcium phosphate phase. It is suggested that this localised enzymatic degradation could explain the chemical heterogeneity seen in HO and may also represent a pathway to physiological bone mineralisation.

Chemobrionic structures in tissue engineering: self-assembling calcium phosphate tubes as cellular scaffolds.

A diverse range of complex patterns and mineralised hierarchical microstructures can be derived from chemobrionic systems, with formation driven by complex reaction-diffusion mechanisms far from thermodynamic equilibrium. In these experiments, self-assembling calcium phosphate tubes are generated using hydrogels made with 1 M calcium solutions layered with solutions of dibasic sodium phosphate over a range of concentrations between 0.2-1 M. Self-assembling structures prepared using 0.8 M dibasic sodium phosphate solutions were selected to assess cell-material interactions. Candidate chemobrionic scaffolds were characterised by micro-X-Ray fluorescence (µ-XRF) spectroscopy, Raman spectroscopy, powder X-ray diffraction (XRD), helium pycnometry and scanning electron microscopy (SEM). As prepared tubes were formed from non-stoichiometric hydroxyapatite (HA, Ca10-x(PO4)6-x(HPO4)x(OH)2-x (0 ≤x≤ 1)), which was confirmed as calcium deficient hydroxyapatite (CDHA, Ca9(PO4)5HPO4OH). Thermal treatment of tubes in air at 650 °C for 4 h converted the structures to beta tricalcium phosphate (ß-TCP, ß-Ca3(PO4)2). The potential of these scaffolds to support the attachment of bone marrow derived mesenchymal stem cells (BMSCs) was investigated for the first time, and we demonstrate cell attachment and elongation on the fabricated tubular structures.

Organotypic Culture of Bone-Like Structures Using Composite Ceramic-Fibrin Scaffolds.

We have developed an organotypic culture system that allows the production of bone tissue features on a centimeter scale. A composite, calcium phosphate-strained fibrin gel system is able to organize itself in the presence of osteoblastic cells, creating basic hierarchical units as seen in vivo, and can be modified to produce a range of other tissues that require such directional structuring. Constructs evolve over time into multi-compositional structures containing a high mineral content and terminally differentiated, osteocyte-like cells. These tissues can be cultured over extended durations (exceeding 1 year) and are responsive to a variety of chemical and biological agents. The platform can reduce the number of animals used in experimentation by acting as an intermediate stage in which more personalized research conditions can be generated. We provide a thorough description of the protocol used to successfully culture and modify this system, as well as guidance on compositional characterization. © 2019 by John Wiley & Sons, Inc.

Formulation of a covalently bonded hydroxyapatite and poly(ether ether ketone) composite.

Spinal fusion devices can be fabricated from composites based on combining hydroxyapatite and poly(ether ether ketone) phases. These implants serve as load-bearing scaffolds for the formation of new bone tissue between adjacent vertebrae. In this work, we report a novel approach to covalently bond hydroxyapatite and poly(ether ether ketone) to produce a novel composite formulation with enhanced interfacial adhesion between phases. Compared to non-linked composites (HA_PEEK), covalently linked composites (HA_L_PEEK), loaded with 1.25 vol% hydroxyapatite, possessed a greater mean flexural strength (170 ± 5.4 vs 171.7 ± 14.8 MPa (mean ± SD)) and modulus (4.8 ± 0.2 vs 5.0 ± 0.3 GPa (mean ± SD)). Although the mechanical properties were not found to be significantly different (p > 0.05), PEEK_L_HA contained substantially larger hydroxyapatite inclusions (100-1000 µm) compared to HA_PEEK (50-200 µm), due to the inherently agglomerative nature of the covalently bonded hydroxyapatite and poly(ether ether ketone) additive. Larger inclusions would expectedly weaken the HA_L_PEEK composite; however, there is no significant difference between the flexural modulus of poly(ether ether ketone) with respect to HA_L_PEEK (p = 0.13). In addition, the flexural modulus of HA_PEEK is significantly lower compared to poly(ether ether ketone) (p = 0.03). Ultimately, covalent linking reduces hydroxyapatite particulate de-bonding from the polymeric matrix and inhibits micro-crack development, culminating in enhanced transfer of stiffness between hydroxyapatite and poly(ether ether ketone) under loading.

Sustained release of decorin to the surface of the eye enables scarless corneal regeneration.

Disorganization of the transparent collagenous matrix in the cornea, as a consequence of a variety of infections and inflammatory conditions, leads to corneal opacity and sight-loss. Such corneal opacities are a leading cause of blindness, according to the WHO. Public health programs target prevention of corneal scarring, but the only curative treatment of established scarring is through transplantation. Although attempts to minimize corneal scarring through aggressive control of infection and inflammation are made, there has been little progress in the development of anti-scarring therapies. This is owing to eye drop formulations using low viscosity or weak gelling materials having short retention times on the ocular surface. In this study, we report an innovative eye drop formulation that has the ability to provide sustained delivery of decorin, an anti-scarring agent. The novelty of this eye drop lies in the method of structuring during manufacture, which creates a material that can transition between solid and liquid states, allowing retention in a dynamic environment being slowly removed through blinking. In a murine model of Pseudomonas keratitis, applying the eye drop resulted in reductions of corneal opacity within 16 days. More remarkably, the addition of hrDecorin resulted in restoration of corneal epithelial integrity with minimal stromal opacity endorsed by reduced α-smooth muscle actin (αSMA), fibronectin, and laminin levels. We believe that this drug delivery system is an ideal non-invasive anti-fibrotic treatment for patients with microbial keratitis, potentially without recourse to surgery, saving the sight of many in the developing world, where corneal transplantation may not be available.

Structuring of Hydrogels across Multiple Length Scales for Biomedical Applications.

The development of new materials for clinical use is limited by an onerous regulatory framework, which means that taking a completely new material into the clinic can make translation economically unfeasible. One way to get around this issue is to structure materials that are already approved by the regulator, such that they exhibit very distinct physical properties and can be used in a broader range of clinical applications. Here, the focus is on the structuring of soft materials at multiple length scales by modifying processing conditions. By applying shear to newly forming materials, it is possible to trigger molecular reorganization of polymer chains, such that they aggregate to form particles and ribbon-like structures. These structures then weakly interact at zero shear forming a solid-like material. The resulting self-healing network is of particular use for a range of different biomedical applications. How these materials are used to allow the delivery of therapeutic entities (cells and proteins) and as a support for additive layer manufacturing of larger-scale tissue constructs is discussed. This technology enables the development of a range of novel materials and structures for tissue augmentation and regeneration.

Interfacial Mineral Fusion and Tubule Entanglement as a Means to Harden a Bone Augmentation Material.

A new bone augmenting material is reported, which is formed from calcium-loaded hydrogel-based spheres. On immersion of these spheres in a physiological medium, they become surrounded with a sheath of precipitate, which ruptures due to a build-up in osmotic pressure. This results in the formation of mineral tubes that protrude from the sphere surface. When brought into close contact with one another, these spheres become fused through the entanglement and subsequent interstitial mineralization of the mineral tubules. The tubular calcium phosphate induces the expression of osteogenic genes (runt-related transcription factor 2 (RUNX2), transcription factor SP7 (SP7), collagen type 1 alpha 1 (COL1A1), and bone gamma-carboxyglutamic acid-containing protein (BGLAP)) and promotes the formation of mineral nodules in preosteoblast cultures comparable to an apatitic calcium phosphate phase. Furthermore, alkaline phosphatase (ALP) is significantly upregulated in the presence of tubular materials after 10 d in culture compared with control groups (p < 0.001) and sintered apatite (p < 0.05). This is the first report of a bioceramic material that is formed in its entirety in situ and is therefore likely to provide a better proxy for biological mineral than other existing synthetic alternatives to bone grafts.

Effects of freezing, fixation and dehydration on surface roughness properties of porcine left anterior descending coronary arteries.

BACKGROUND: To allow measurements of surface roughness to be made of coronary arteries using various imaging techniques, chemical processing, such as fixation and dehydration, is commonly used. Standard protocols suggest storing fresh biological tissue at -40°C. The aim of this study was to quantify the changes caused by freezing and chemical processing to the surface roughness measurements of coronary arteries, and to determine whether correction factors are needed for surface roughness measurements of coronary arteries following chemical processes typically used before imaging these arteries. METHODS: Porcine left anterior descending coronary arteries were dissected ex vivo. Surface roughness was then calculated following three-dimensional reconstruction of surface images obtained using an optical microscope. Surface roughness was measured before and after a freeze cycle to assess changes during freezing, after chemical fixation, and again after dehydration, to determine changes during these steps of chemical processing. RESULTS: No significant difference was caused due to the freeze cycle (p>0.05). There was no significant difference in the longitudinally measured surface roughness (RaL=0.99±0.39µm; p>0.05) of coronary arteries following fixation and dehydration either. However, the circumferentially measured surface roughness increased significantly following a combined method of processing (RaC=1.36±0.40, compared 1.98±0.27µm, respectively; p<0.05). A correction factor can compensate for the change RaCß=RaC1+0.46in RaC due to processing of tissue, Where RaCß, the corrected RaC, had a mean of 1.31±0.21µm. CONCLUSIONS: Independently, freezing, fixation and dehydration do not alter the surface roughness of coronary arteries. Combined, however, fixation and dehydration significantly increase the circumferential, but not longitudinal, surface roughness of coronary arteries.

Calcium pre-conditioning substitution enhances viability and glucose sensitivity of pancreatic beta-cells encapsulated using polyelectrolyte multilayer coating method.

Type I diabetics are dependent on daily insulin injections. A therapy capable of immunoisolating pancreatic beta-cells and providing normoglycaemia is an alternative since it would avoid the late complications associated with insulin use. Here, 3D-concave agarose micro-wells were used to culture robust pancreatic MIN-6 cell spheroids within 24 hours that were shown to exhibit cell-cell contact and uniform size (201 ± 2 µm). A polyelectrolyte multilayer (PEM) approach using alginate and poly-l-lysine was employed to coat cell spheroids. In comparison to conventional PEM, use of a novel Ca2+ pre-coating step enhanced beta-cells viability (89 ± 6%) and metabolic activity since it reduced the toxic effect of the cationic polymer. Pre-coating was achieved by treating MIN-6 spheroids with calcium chloride, which enabled the adhesion of anionic polymer to the cells surface. Pre-coated cells coated with four bilayers of polymers were successfully immunoisolated from FITC-mouse antibody and pro-inflammatory cytokines. Novel PEM coated cells were shown to secret significantly (P < 0.05) different amounts of insulin in response to changes in glucose concentration (2 vs. 20 mM). This work presents a 3D culture model and novel PEM coating procedure that enhances viability, maintains functionality and immunoisolates beta-cells, which is a promising step towards an alternative therapy to insulin.

Biologically Analogous Calcium Phosphate Tubes from a Chemical Garden.

Calcium phosphate (CaPO4) tubes with features comparable to mineralized biological microstructures, such as Haversian canals, were grown from a calcium gel/phosphate solution chemical garden system. A significant difference in gel mass in response to high and low solute phosphate equivalent environments existed within 30 min of solution layering upon gel (p = 0.0067), suggesting that the nature of advective movement between gel and solution is dependent on the solution concentration. The transport of calcium cations (Ca2+) and phosphate anions (PO43-) was quantified and changes in pH were monitored to explain the preferential formation of tubes within a PO43- concentration range of 0.5-1.25 M. Ingress from the anionic solution phase into the gel followed by the liberation of Ca2+ ions from the gel was found to be essential for acquiring self-assembled tubular CaPO4 structures. Tube analysis by scanning electron microscopy (SEM), X-ray diffraction (XRD), and micro X-ray florescence (µ-XRF) revealed hydroxyapatite (HA, Ca10(PO4)6(OH)2) and dicalcium phosphate dihydrate (DCPD, CaHPO4·2H2O) phases organized in a hierarchical manner. Notably, the tubule diameters ranged from 100 to 150 µm, an ideal size for the permeation of vasculature in biological hard tissue.

Antimicrobial peptide coatings for hydroxyapatite: electrostatic and covalent attachment of antimicrobial peptides to surfaces.

The interface between implanted devices and their host tissue is complex and is often optimized for maximal integration and cell adhesion. However, this also gives a surface suitable for bacterial colonization. We have developed a novel method of modifying the surface at the material-tissue interface with an antimicrobial peptide (AMP) coating to allow cell attachment while inhibiting bacterial colonization. The technology reported here is a dual AMP coating. The dual coating consists of AMPs covalently bonded to the hydroxyapatite surface, followed by deposition of electrostatically bound AMPs. The dual approach gives an efficacious coating which is stable for over 12 months and can prevent colonization of the surface by both Gram-positive and Gram-negative bacteria.

Bedside, Benchtop, and Bioengineering: Physicochemical Imaging Techniques in Biomineralization.

The need to quantify physicochemical properties of mineralization spans many fields. Clinicians, mineralization researchers, and bone tissue bioengineers need to be able to measure the distribution, quantity, and the mechanical and chemical properties of mineralization within a wide variety of substrates from injured muscle to electrospun polymer scaffolds and everything in between. The techniques available to measure these properties are highly diverse in terms of their complexity and utility. Therefore it is of the utmost importance that those who intend to use them have a clear understanding of the advantages and disadvantages of each technique and its appropriateness to their specific application. This review provides all of this information for each technique and uses heterotopic ossification and engineered bone substitutes as examples to illustrate how these techniques have been applied. In addition, we provide novel data using advanced techniques to analyze human samples of combat related heterotopic ossification.

Modification of gellan gum with nanocrystalline hydroxyapatite facilitates cell expansion and spontaneous osteogenesis.

Nanocomposites composed of hydrogels and calcium phosphates are of great interest in the development of bone graft replacements since they may have a structural and compositional resemblance to bone. Culture beads formed from such materials could be used in stirred tank culture and thereby enable cell expansion in a sufficiently efficient manner to allow for the generation of enough large number of cells for large-scale bone reconstruction. Although combinations of materials such as alginate, collagens, and various calcium phosphates have been investigated as culture beads, these materials are unsuitable for application since they have been shown to rapidly degrade in physiological conditions and enable relatively little tailoring of mechanical properties. In this study, gellan gum-nano sized hydroxyapatite (nHA) composites, which have been shown to be resistant to degradation and easily modified with respect to modulus, were formulated and characterized as regards their ability to enable cell attachment and proliferation. It was shown that the addition of 5 wt% of nHA to the culture beads enabled cell attachment and that an increase in nHA concentration to up to 25 wt% enhanced the rate of cell proliferation. Most importantly, it was demonstrated that the addition of nHA to the cell culture beads enabled the formation of nodules in culture of MC3T3-E1 cells and strikingly stimulated the osteogenic differentiation of bone marrow stromal cells in the absence of osteogenic media when compared with tissue culture plastic (TCP) with the same condition. Biotechnol. Bioeng. 2016;113: 1568-1576. © 2016 Wiley Periodicals, Inc.

The effects of cobalt-chromium-molybdenum wear debris in vitro on serum cytokine profiles and T cell repertoire.

Cobalt-chromium-molybdenum (CoCrMo) alloy-based metal-on-metal prostheses have been the implant of choice for total hip replacement in younger patients. However 6.2% of patients require revision of their CoCrMo total hip replacement (THR) implant within five years of surgery and their use was restricted in 2013. We aimed to determine if there were individual differences in the immune response to wear debris that might indicate a poor outcome with a CoCrMo prosthesis. Blood from 22 donors was incubated with CoCrMo particles (>99.9% less than 10 µm diameter) generated by a wear simulator for 24 h. T cell phenotype was assessed by immunostaining and secretion of 8 different pro- and anti-inflammatory cytokines was measured using multiplex technology. Clear differences were seen between individuals in the induction of Th17 and Th1 responses, with some donors showing pro-inflammatory responses (increased IL17 or IFNγ) and others showing anti-inflammatory responses (decreased IL17 or IFNγ). The only differences seen for gender and age related to increased IL-10 expression from T cells in females (p = 0.008) and a trend towards decreased IL-6 expression systemically for older donors (p = 0.058). We conclude that individuals show differential responses to CoCrMo wear debris and that these responses could give early indications of the suitability of the patient for a metal-on-metal prosthesis.

Nanoscale crystallinity modulates cell proliferation on plasma sprayed surfaces.

Calcium phosphate coatings have been applied to the surface of metallic prostheses to mediate hard and soft tissue attachment for more than 40years. Most coatings are formed of high purity hydroxyapatite, and coating methods are often designed to produce highly crystalline surfaces. It is likely however, that coatings of lower crystallinity can facilitate more rapid tissue attachment since the surface will exhibit a higher specific surface area and will be considerably more reactive than a comparable highly crystalline surface. Here we test this hypothesis by growing a population of MC3T3 osteoblast-like cells on the surface of two types of hip prosthesis with similar composition, but with differing crystallinity. The surfaces with lower crystallinity facilitated more rapid cell attachment and increased proliferation rate, despite having a less heterogeneous surface topography. This work highlights that the influence of the crystallinity of HA at the nano-scale is dominant over macro-scale topography for cell adhesion and growth. Furthermore, crystallinity could be easily adjusted by without compromising coating purity. These findings could facilitate designing novel coated calcium phosphate surfaces that more rapidly bond tissue following implantation.

Exploiting cell-mediated contraction and adhesion to structure tissues in vitro.

Progress in tissue engineering is now impacting beyond the field of regenerative medicine. Engineered tissues are now used as tools to evaluate the toxicity of compounds or even to enable the modelling of disease. While many of the materials that are used to facilitate tissue growth are designed to enable cell attachment, many researchers consider that the contraction and modification of these matrices by attached cells is not desirable and take measures to prevent this from occurring. Where substantial alignment of the molecules within tissues, however, is a feature of structure the process of contraction can be exploited to guide new matrix deposition. In this paper, we will demonstrate how we have used the cell contraction process to generate tissues with high levels of organization. The tissues that have been grown in the laboratory have been characterized using a suite of analytical techniques to demonstrate significant levels of matrix organization and mechanical behaviour analogous to natural tissues. This paper provides an overview of research that has been undertaken to determine how tissues have been grown in vitro with structuring from the molecular, right through to the macroscopic level.

Quantification of volume and size distribution of internalised calcium phosphate particles and their influence on cell fate.

We report preliminary findings suggesting that the diameter of the internalised calcium phosphate particles is critical to cell fate with particles/aggregates of particles of larger than 1.5 µm not processed by lysosomes leading to cell death. This has significant implications for the design of medical materials even from those consisting of non-toxic calcium phosphate salts.

Thiol modification of silicon-substituted hydroxyapatite nanocrystals facilitates fluorescent labelling and visualisation of cellular internalisation.

Calcium phosphates are used widely as orthopaedic implants and in nanocrystalline form to enable the transfer of genetic material into cells. Despite widespread use, little is known about their fate after they have crossed the cell membrane. Here we present a method of surface modification of silicon-substituted hydroxyapatite (SiHA) through a silane group, which enables the engraftment of a fluorescent dye to facilitate real-time biological tracking. Surface modification of the nanocrystal surface was undertaken using (3-mercaptopropyl)trimethoxysilane (MPTS), which presented a thiol for the further attachment of a fluorophore. Successful modification of the surface was demonstrated using zeta potential measurements and fluorescence microscopy and the number of thiol groups at the surface was quantified using Ellman's reagent. In vitro experiments using the fluorescently modified particles enabled the discrimination of the calcium phosphate particulate from other biological debris following internalisation by a population of MC3T3 (pre-osteoblast) cells and the particles were shown to maintain fluorescence for 24 hours without quenching. The successful modification of the surface of SiHA with thiol groups offers the tantalising possibility of the intracellular growth factor delivery.

An analytical Micro CT methodology for quantifying inorganic dentine debris following internal tooth preparation.

OBJECTIVES: MicroCT allows the complex canal network of teeth to be mapped but does not readily distinguish between structural tissue (dentine) and the debris generated during cleaning. The aim was to introduce a validated approach for identifying debris following routine instrumentation and disinfection. METHODS: The mesial canals of 12 mandibular molars were instrumented, and irrigated with EDTA and NaOCl. MicroCT images before and after instrumentation and images were assessed qualitatively and quantitatively. RESULTS: Debris in the canal space was identified through morphological image analysis and superimposition of the images before and after instrumentation. This revealed that the removal of debris is prohibited by protrusions and micro-canals within the tooth creating areas which are inaccessible to the irrigant. Although the results arising from the analytical methodology did provide measurements of debris produced, biological differences in the canals resulted in variances. Both irrigants reduced debris compared to the control which decreased with EDTA and further with NaOCl. However, anatomical variation did not allow definitive conclusions on which irrigant was best to use although both reduced debris build up. CONCLUSIONS: This work presents a new approach for distinguishing between debris and structural inorganic tissue in root canals of teeth. The application may prove useful in other calcified tissue shape determination. CLINICAL SIGNIFICANCE: Remaining debris may contain bacteria and obstruct the flow of irrigating solutions into lateral canal anatomy. This new approach for detecting the amount of remaining debris in canal systems following instrumentation provides a clearer methodology of the identification of such debris.