A human retinal microvascular endothelial-pericyte co-culture model to study diabetic retinopathy in vitro.

This human primary co-culture model using human retinal microvascular endothelial cells (hREC) and human retinal pericyte cells (hRP) aims to improve current understanding of the cellular changes occurring in the retinal microvasculature during diabetic retinopathy (DR). Currently, patients often present in clinic with late-stage DR, only when vision becomes impaired. Therefore, new strategies for earlier detection in clinic, combined with novel pharmaceutical and cellular interventions are essential in order to slow or halt the progression of DR from background to sight-threatening stage. This co-culture model can be used as a simple, replicable in vitro tool to discover and assess novel drug therapies and improve fundamental understanding of alterations to cell behaviour in the human retinal microvasculature during DR. hRP and hREC were cultured for up to 21 days in normoxic (20%) or hypoxic (2%) oxygen levels and physiological (5.5 mM) or very high (33 mM) glucose, to maintain a healthy, or induce a diabetic-like phenotype in vitro. Mono- or co-cultured hREC and hRP were seeded 1:1 in healthy (20% oxygen and 5.5 mM glucose) or diabetic-like (2% oxygen and 33 mM glucose) conditions, on either side of untreated polyethylene terephthalate (PET) transwell inserts, and cultured for 21 days. Mono- and co-cultures were analysed for changes in metabolic activity, angiogenic response and junctional protein expression, using immunofluorescence antibody labelling, flow cytometry and multiplex ELISA technology. hRP and hREC were successfully co-cultured, and the glucose and oxygen concentrations selected for the in vitro healthy and diabetic-like conditions were sufficient for cell viability and EC monolayer integrity, with evidence of an angiogenic response in diabetic-like conditions within the 21 day timeframe. Angiopoietin-2 (Ang-2), vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) secretion were all increased, whilst hepatocyte growth factor (hHGF), tissue inhibitor for metalloproteinase-2 (TIMP-2) and interleukin-8 (IL-8) secretion were all reduced in the in vitro diabetic-like conditions. The secretion profile of co-cultures was different to mono-cultures, highlighting the importance of using co-culture models to collect data more reflective of the close relationship between hRP-hREC in vivo. Previous groups have developed useful co-culture models utilising non-human, immortalised or large vessel-sourced cells to explore changes to the vasculature during hypoxia and/or high glucose insult. In this study the use of human primary, retina-specific microvascular cells, mono- and co-cultured, collected over a longer culture period, has enabled detection of changes that may have been missed in previous models.

Poly-ε-lysine based hydrogels as synthetic substrates for the expansion of corneal endothelial cells for transplantation.

Dysfunction of the corneal endothelium (CE) resulting from progressive cell loss leads to corneal oedema and significant visual impairment. Current treatments rely upon donor allogeneic tissue to replace the damaged CE. A donor cornea shortage necessitates the development of biomaterials, enabling in vitro expansion of corneal endothelial cells (CECs). This study investigated the use of a synthetic peptide hydrogel using poly-ε-lysine (pεK), cross-linked with octanedioic-acid as a potential substrate for CECs expansion and CE grafts. PεK hydrogel properties were optimised to produce a substrate which was thin, transparent, porous and robust. A human corneal endothelial cell line (HCEC-12) attached and grew on pεK hydrogels as confluent monolayers after 7 days, whereas primary porcine CECs (pCECs) detached from the pεK hydrogel. Pre-adsorption of collagen I, collagen IV and fibronectin to the pεK hydrogel increased pCEC adhesion at 24 h and confluent monolayers formed at 7 days. Minimal cell adhesion was observed with pre-adsorbed laminin, chondroitin sulphate or commercial FNC coating mix (fibronectin, collagen and albumin). Functionalisation of the pεK hydrogel with synthetic cell binding peptide H-Gly-Gly-Arg-Gly-Asp-Gly-Gly-OH (RGD) or α2ß1 integrin recognition sequence H-Asp-Gly-Glu-Ala-OH (DGEA) resulted in enhanced pCEC adhesion with the RGD peptide only. pCECs grown in culture at 5 weeks on RGD pεK hydrogels showed zonula occludins 1 staining for tight junctions and expression of sodium-potassium adenosine triphosphase, suggesting a functional CE. These results demonstrate the pεK hydrogel can be tailored through covalent binding of RGD to provide a surface for CEC attachment and growth. Thus, providing a synthetic substrate with a therapeutic application for the expansion of allogenic CECs and replacement of damaged CE.

The formation of a functional retinal pigment epithelium occurs on porous polytetrafluoroethylene substrates independently of the surface chemistry.

Subretinal transplantation of functioning retinal pigment epithelial (RPE) cells may have the potential to preserve or restore vision in patients affected by blinding diseases such as age-related macular degeneration (AMD). One of the critical steps in achieving this is the ability to grow a functioning retinal pigment epithelium, which may need a substrate on which to grow and to aid transplantation. Tailoring the physical and chemical properties of the substrate should help the engineered tissue to function in the long term. The purpose of the study was to determine whether a functioning monolayer of RPE cells could be produced on expanded polytetrafluoroethylene substrates modified by either an ammonia plasma treatment or an n-Heptylamine coating, and whether the difference in surface chemistries altered the extracellular matrix the cells produced. Primary human RPE cells were able to form a functional, cobblestone monolayer on both substrates, but the formation of an extracellular matrix to exhibit a network structure took months, whereas on non-porous substrates with the same surface chemistry, a similar appearance was observed after a few weeks. This study suggests that the surface chemistry of these materials may not be the most critical factor in the development of growth of a functional monolayer of RPE cells as long as the cells can attach and proliferate on the surface. This has important implications in the design of strategies to optimise the clinical outcomes of subretinal transplant procedures.

Computational fluid dynamic analysis of the effect of morphologic features on distraction forces in fenestrated stent grafts.

OBJECTIVE: Secure fixation of endovascular stent grafts is essential for successful endovascular aneurysm repair. Hemodynamic distraction forces are generated by blood pressure and blood flow and act against fixation force to encourage migration that may eventually lead to late stent graft failure. The aim of this in silico study was to determine which morphologic features were associated with greater distraction force. METHODS: Computer models of 54 in situ fenestrated stent grafts were constructed from postoperative computed tomography scans by use of image processing software. Computational fluid dynamic analysis was then performed by use of a commercial finite volume solver with boundary conditions representative of peak systole. Distraction force results were obtained for each component of the stent graft. Distraction force was correlated with lumen cross-sectional area (XSA) at the inlet and outlet of components and was compared between groups of components, depending on the magnitude of four predefined angles within the aortoiliac territory that we describe in detail. RESULTS: Median total resultant distraction force (RDF) acting on the fenestrated proximal bodies was 4.8 N (1.3-15.7 N); bifurcated distal bodies, 5.6N (1.0-8.0 N); and limb extensions, 1.7 N (0.6-8.4N). Inlet XSA exhibited strong, positive correlation with total RDF in proximal body and distal body components (Spearman correlation coefficient ρ, 0.883 and 0.802, respectively). Outlet XSA exhibited a similarly strong, positive correlation with total RDF in limb extension components (ρ, 0.822). Outlet angulation ≥ 45 degrees was associated with greater total RDF in the limb extension components only (P = .004). CONCLUSIONS: For a given blood pressure, XSA was the most important morphologic determinant of total RDF. Angulation within the aorta was not large enough to influence this, whereas iliac angulation affecting outlet angulation of limb extension components was associated with significantly greater total RDF.

Guided gingival fibroblast attachment to titanium surfaces: an in vitro study.

AIM: To assess the potential of gingival fibroblasts to attach in a predetermined linear orientation to a nano-topography of aligned fibres on titanium surfaces and determine the ability of such cells to deposit aligned collagen fibre matrix. MATERIALS AND METHODS: smooth glass and rough titanium substrates were coated with polytetrafluoroethylene (PTFE) nano-fibres. Ammonia plasma treatment was used to modify the surface chemistry. Human gingival fibroblasts were cultured on substrates and orientation and collagen deposition was assessed. RESULTS: Straight, unidirectional, parallel PTFE nano-fibres were deposited over the titanium features. By 7 days, the majority of cells were observed to orient to untreated fibres despite the presence of competing titanium surface features. On plasma-treated fibre-coated titanium substrates, cell orientation was mixed. On uncoated substrates, the majority of cells oriented to the titanium surface features. On fibre-coated glass substrates, cells oriented themselves with untreated and plasma-treated fibres and secreted collagen in the same direction after 1 week. On uncoated glass substrates, there was no preferred direction of collagen orientation. CONCLUSION: Polytetrafluoroethylene nano-fibres induced cell and collagen orientation. Surface chemistry appeared only to affect cell behaviour at early time points. An implant surface that controls cell orientation may also influence the orientation of collagen, providing improved gingival support.

Use of illness severity scores to predict mortality in interstitial lung disease patients hospitalised with acute respiratory deterioration.

INTRODUCTION: Hospitalisations relating to acute respiratory deteriorations (ARD) in Interstitial Lung Disease (ILD) have poor outcomes. Factors predicting adverse outcomes are not fully understood and data addressing the use of illness severity scores in prognostication are limited. OBJECTIVE: To investigate the use of CURB-65 and NEWS-2 severity scores in the prediction of mortality following ARD-ILD hospitalisation, using prospective methodology and to validate previously determined cut-offs, derived from a retrospective study cohort. METHODS: A dual-centre prospective observational cohort study of all adults (≥18y) hospitalised with ARD-ILD in Bristol, UK (n = 179). Gender-Age-Physiology (GAP), CURB-65 and NEWS-2 scores were calculated for each eligible admission. Receiver operating characteristics (ROC) curve analysis was used to quantify the strength of discrimination for NEWS-2 and CURB-65 scores. Univariable and multivariable logistic regression analyses were performed to explore the relationship between baseline severity scores and mortality. RESULTS: GAP showed some merit at predicting 30-day mortality (AUC = 0.64, P = 0.015); whereas CURB-65 showed modest predictive value for in-hospital (AUC = 0.72, P < 0.001) and 90-day mortality (AUC = 0.67, P < 0.001). NEWS-2 showed higher predictive value for in-hospital (AUC = 0.80, P < 0.001) and 90-day mortality (AUC = 0.75, P < 0.001), with an optimal derived cut-off ≥6.5 found to be sensitive and specific for predicting in-hospital (83% and 63%) and 90-day (73% and 72%) mortality. In exploratory analyses, GAP score addition improved the predictive ability of NEWS-2 against 30-day mortality and CURB-65 across all time-periods. CONCLUSION: NEWS-2 has good discriminatory value for predicting in-hospital mortality and moderate discriminatory value for predicting 90-day mortality. The optimal NEWS-2 cut-off value determined was the same as in a previous retrospective cohort, confirming the NEWS-2 score shows promise in predicting mortality following ARD-ILD hospitalisation.

The conjunctival extracellular matrix, related disorders and development of substrates for conjunctival restoration.

The conjunctiva can be damaged by numerous diseases with scarring, loss of tissue and dysfunction. Depending on extent of damage, restoration of function may require a conjunctival graft. A wide variety of biological and synthetic substrates have been tested in the search for optimal conditions for ex vivo culture of conjunctival epithelial cells as a route toward tissue grafts. Each substrate has specific advantages but also disadvantages related to their unique physical and biological characteristics, and identification and development of an improved substrate remains a priority. To achieve the goal of mimicking and restoring a biological material, requires information from the material. Specifically, extracellular matrix (ECM) derived from conjunctival tissue. Knowledge of the composition and structure of native ECM and identifying contributions of individual components to its function would enable using or mimicking those components to develop improved biological substrates. ECM is comprised of two components: basement membrane secreted predominantly by epithelial cells containing laminins and type IV collagens, which directly support epithelial and goblet cell adhesion differentiation and growth and, interstitial matrix secreted by fibroblasts in lamina propria, which provides mechanical and structural support. This review presents current knowledge on anatomy, composition of conjunctival ECM and related conjunctival disorders. Requirements of potential substrates for conjunctival tissue engineering and transplantation are discussed. Biological and synthetic substrates and their components are described in an accompanying review.

FluoroPi Device With SmartProbes: A Frugal Point-of-Care System for Fluorescent Detection of Bacteria From a Pre-Clinical Model of Microbial Keratitis.

Purpose: Rapid and accurate diagnosis of microbial keratitis (MK) could greatly improve patient outcomes. Here, we present the development of a rapid, accessible multicolour fluorescence imaging device (FluoroPi) and evaluate its performance in combination with fluorescent optical reporters (SmartProbes) to distinguish bacterial Gram status. Furthermore, we show feasibility by imaging samples obtained by corneal scrape and minimally invasive corneal impression membrane (CIM) from ex vivo porcine corneal MK models. Methods: FluoroPi was built using a Raspberry Pi single-board computer and camera, light-emitting-diodes (LEDs), and filters for white-light and fluorescent imaging, with excitation and detection of bacterial optical SmartProbes: Gram-negative, NBD-PMX (exmax 488 nm); Gram positive, Merocy-Van (exmax 590 nm). We evaluated FluoroPi with bacteria (Pseudomonas aeruginosa and Staphylococcus aureus) isolated from ex vivo porcine corneal models of MK by scrape (needle) and CIM with the SmartProbes. Results: FluoroPi provides <1 µm resolution and was able to readily distinguish bacteria isolated from ex vivo models of MK from tissue debris when combined with SmartProbes, retrieved by both scrape and CIM. Single bacteria could be resolved within the field of view, with limits of detection demonstrated as 103 to 104 CFU/mL. Sample preparation prior to imaging was minimal (wash-free), and imaging and postprocessing with FluoroPi were straightforward, confirming ease of use. Conclusions: FluoroPi coupled with SmartProbes provides effective, low-cost bacterial imaging, delineating Gram-negative and Gram-positive bacteria directly sampled from a preclinical model of MK. Translational Relevance: This study provides a crucial stepping stone toward clinical translation of a rapid, minimally invasive diagnostic approach for MK.

Plasma polymer coatings to aid retinal pigment epithelial growth for transplantation in the treatment of age related macular degeneration.

Subretinal transplantation of functioning retinal pigment epithelial (RPE) cells grown on a synthetic substrate is a potential treatment for age-related macular degeneration (AMD), a common cause of irreversible vision loss in developed countries. Plasma polymers give the opportunity to tailor the surface chemistry of the artificial substrate whilst maintaining the bulk properties. In this study, plasma polymers with different functionalities were investigated in terms of their effect on RPE attachment and growth. Plasma polymers of acrylic acid (AC), allyl amine (AM) and allyl alcohol (AL) were fabricated and characterised using X-ray photoelectron spectroscopy (XPS) and water contact angle measurements. Octadiene (OD) hydrocarbon films and tissue culture polystyrene were used as controls. Wettability varied from hydrophobic OD to relatively hydrophilic AC. XPS demonstrated four very different surfaces with the expected functionalities. Attachment, proliferation and morphological examination of an RPE cell line and primary RPE cells were investigated. Both cell types grew on all surfaces, with the exception of OD, although the proliferation rate of primary cells was low. Good epithelial morphology was also demonstrated. Plasma polymerised films show potential as cell carrier surfaces for RPE cells in the treatment of AMD.

Antimicrobial Nitric Oxide-Releasing Electrospun Dressings for Wound Healing Applications.

Nonhealing and chronic wounds represent a major problem for the quality of life of patients and have cost implications for healthcare systems. The pathophysiological mechanisms that prevent wound healing are usually multifactorial and relate to patient overall health and nutrition, vascularity of the wound bed, and coexisting infection/colonization. Bacterial infections are one of the predominant issues that can stall a wound, causing it to become chronic. Successful wound healing often depends on weeks or months of antimicrobial therapy, but this is problematic given the rise in multidrug-resistant bacteria. As such, alternatives to antibiotics are desperately needed to aid the healing of chronic, and even acutely infected wounds. Nitric oxide (NO) kills bacteria through a variety of mechanisms, and thus, bacteria have shown no tendency to develop resistance to NO as a therapeutic agent and therefore can be a good alternative to antibiotic therapy. In this paper, we report on the development of NO-releasing electrospun membranes fabricated from polycaprolactone (PCL)/gelatin blends and optimized to reduce bacterial infection. The NO payload in the membranes was directly related to the number of amines (and hence the amount of gelatin) in the blend. Higher NO payloads corresponded with a higher degree of antimicrobial efficacy. No cytotoxicity was observed for electrospun membranes, and an in vitro wound closure assay demonstrated closure within 16 h. The results presented here clearly indicate that these NO-releasing electrospun membranes hold significant promise as wound dressings due to their antimicrobial activity and biocompatibility.

Amoebicidal Activity of Poly-Epsilon-Lysine Functionalized Hydrogels.

Purpose: To determine the amoebicidal activity of functionalized poly-epsilon-lysine hydrogels (pɛK+) against Acanthamoeba castellanii. Methods: A. castellanii trophozoites and cysts were grown in the presence of pɛK solution (0-2.17 mM), pɛK or pɛK+ hydrogels, or commercial hydrogel contact lens (CL) for 24 hours or 7 days in PBS or Peptone-Yeast-Glucose (PYG) media (nutrient-deplete or nutrient-replete cultures, respectively). Toxicity was determined using propidium iodide and imaged using fluorescence microscopy. Ex vivo porcine corneas were inoculated with A. castellanii trophozoites ± pɛK, pɛK+ hydrogels or commercial hydrogel CL for 7 days. Corneal infection was assessed by periodic acid-Schiff staining and histologic analysis. Regrowth of A. castellanii from hydrogel lenses and corneal discs at 7 days was assessed using microscopy and enumeration. Results: The toxicity of pɛK+ hydrogels resulted in the death of 98.52% or 83.31% of the trophozoites at 24 hours or 7 days, respectively. The toxicity of pɛK+ hydrogels resulted in the death of 70.59% or 82.32% of the cysts in PBS at 24 hours or 7 days, respectively. Cysts exposed to pɛK+ hydrogels in PYG medium resulted in 75.37% and 87.14% death at 24 hours and 7 days. Ex vivo corneas infected with trophozoites and incubated with pɛK+ hydrogels showed the absence of A. castellanii in the stroma, with no regrowth from corneas or pɛK+ hydrogel, compared with infected-only corneas and those incubated in presence of commercial hydrogel CL. Conclusions: pɛK+ hydrogels demonstrated pronounced amoebicidal and cysticidal activity against A. castellanii. pɛK+ hydrogels have the potential for use as CLs that could minimize the risk of CL-associated Acanthamoeba keratitis.

Scaffold-Free Strategy Using a PEG-Dextran Aqueous Two-Phase-System for Corneal Tissue Repair.

Forming thin tissue constructs with minimal extracellular matrix surrounding them is important for tissue engineering applications. Here, we explore and optimize a strategy that enables rapid fabrication of scaffold-free corneal tissue constructs using the liquid-liquid interface of an aqueous two-phase system (ATPS) that is based on biocompatible polymers, dextran and polyethylene glycol. Intact tissue-like constructs, made of corneal epithelial or endothelial cells, can be formed on the interface between the two liquid phases of ATPS within hours and subsequently collected simply by removing the liquid phases. The formed corneal cell constructs express essential physiological markers and have preserved viability and proliferative ability in vitro. The corneal epithelial cell constructs are also able to re-epithelialize the corneal epithelial wound in vitro. The results suggest the promise of our reported strategy in corneal repair.

The impact of axial length on retinal tamponade for gas, silicone oil, and heavy silicone oil, using an in vitro model.

BACKGROUND: To investigate whether ocular axial length influences the tamponade efficacy of three commonly used agents: gas, silicone oil, and heavy silicone oil. METHODS: A series of filling experiments was conducted using 19-mm and 25-mm surface-modified spherical model eye chambers to mimic the vitreous cavity. For each agent, tamponade efficacy was assessed across a range of percentage fills, and comparison was made between the two model eye chambers. The behavior of each tamponade agent was quantified by measuring (1) the maximum height of the tamponade bubble, and calculating (2) the arc of retinal contact subtended by the tamponade bubble. RESULTS: Polynomial regression analysis found no statistically significant difference between the regression models for the different-sized model eye chambers for bubble height or arc of retinal contact subtended. This applied to all of the tamponade agents under investigation. CONCLUSIONS: Across the range of cavity sizes under investigation, no significant difference in tamponade efficacy (as reflected by the measured dimensions of the tamponade bubble) was identified.

3D reactive inkjet printing of poly-ɛ-lysine/gellan gum hydrogels for potential corneal constructs.

Corneal opacities are the 4th leading cause of blindness, and the only current treatment method is the replacement of damaged tissue with a donor cornea. The worldwide shortage of donor eye bank tissue has influenced research into biomaterial substrates for both partial and full thickness corneal implantation. Here, polymer hydrogels based on natural peptides, poly-ɛ-lysine and gellan gum, can be manufactured using reactive inkjet printing (RIJ). The inks used for printing were optimised based on their rheological properties. Printing alternating layers of ink forms a unique surface pattern, based on the immediate formation of ionic bonds between polymers of opposing charges. This surface pattern resembles a repeating honeycomb-like structure, visible by both optical and scanning electron microscopy. The structure of the printed hydrogels can be modified to include pores, a feature of interest for the tissue engineering of full thickness corneal constructs. Printed poly-ɛ-lysine/gellan gum hydrogels demonstrated a transparency of 80% and cyto-compatibility with both corneal epithelial and endothelial cells. Both corneal cell types demonstrated cell attachment across the surface of the printed hydrogel arrays, displaying their typical cell morphology. This gives confidence of the cyto-compatibility of these hydrogels in vitro. Reactive inkjet printing can produce 3D structures with a high resolution, producing printed tracks in the micron range. Additionally, RIJ demonstrates versatility, as constructs can be tailored to meet various dimension and thickness requirements. Furthermore, this work demonstrates for the first time that reactive inkjet printing can been used to produce hydrogel constructs based on these two inks, with the aim of producing constructs for corneal tissue engineering.

Characterization of Tunable Poly-ε-Lysine-Based Hydrogels for Corneal Tissue Engineering.

A family of poly-ε-lysine hydrogels can be synthesized by crosslinking with bis-carboxylic acids using carbodiimide chemistry. In addition to creating hydrogels using a simple cast method, a fragmented method is used to introduce increased porosity within the hydrogel structure. Both methods have created tunable characteristics ranging in their mechanical properties, transparency, and water content, which is of interest to corneal tissue engineering and can be tailored to specific cellular needs and applications. With a worldwide shortage of cornea donor tissue available for transplant and limitations including rejection and potential infection, a synthetic material that can be used as a graft, or a partial thickness corneal replacement, would be an advantageous treatment method. These hydrogels can be tuned to have similar mechanical and transparency properties to the human cornea. They also support the attachment and growth of corneal epithelial cells and the integration of corneal stromal cells.

Melt electro-written scaffolds with box-architecture support orthogonally oriented collagen.

Melt electro-writing (MEW) is a state-of-the-art technique that supports fabrication of 3D, precisely controlled and reproducible fiber structures. A standard MEW scaffold design is a box-structure, where a repeat layer of 90° boxes is produced from a single fiber. In 3D form (i.e. multiple layers), this structure has the potential to mimic orthogonal arrangements of collagen, as observed in the corneal stroma. In this study, we determined the response of human primary corneal stromal cells and their deposited fibrillar collagen (detected using a CNA35 probe) following six weeksin vitroculture on these box-structures made from poly(ϵ-caprolactone) (PCL). Comparison was also made to glass substrates (topography-free) and electrospun PCL fibers (aligned topography). Cell orientation and collagen deposition were non-uniform on glass substrates. Electrospun scaffolds supported an excellent parallel arrangement of cells and deposited collagen to the underlying architecture of aligned fibers, but there was no evidence of bidirectional collagen. In contrast, MEW scaffolds encouraged the formation of a dense, interconnected cellular network and deposited fibrillar collagen layers with a distinct orthogonal-arrangement. Collagen fibrils were particularly dominant through the middle layers of the MEW scaffolds' total thickness and closer examination revealed these fibrils to be concentrated within the pores' central regions. With the demand for donor corneas far exceeding the supply-leaving many with visual impairment-the application of MEW as a potential technique to recreate the corneal stroma with spontaneous, bidirectional collagen organization warrants further study.

Material Characterisation and Stratification of Conjunctival Epithelial Cells on Electrospun Poly(ε-Caprolactone) Fibres Loaded with Decellularised Tissue Matrices.

The conjunctiva, an under-researched yet incredibly important tissue, plays key roles in providing protection to the eye and maintaining homeostasis of its ocular surface. Multiple diseases can impair conjunctival function leading to severe consequences that require surgical intervention. Small conjunctival defects can be repaired relatively easily, but larger defects rely on tissue grafts which generally do not provide adequate healing. A tissue engineering approach involving a biomaterial substrate capable of supporting a stratified epithelium with embedded, mucin-secreting goblet cells offers a potential solution. As a first step, this study aimed to induce stratification of human conjunctival epithelial cells cultured on electrospun scaffolds composed from poly(ε-caprolactone) (PCL) and decellularised tissue matrix (small intestinal submucosa (SIS) or urinary bladder matrix (UBM)) and held at the air/liquid interface. Stratification, up to 5 cell layers, occurred more frequently on scaffolds containing PCL + UBM. Incorporation of these decellularised tissue matrices also impacted material properties, with significant changes occurring to their fibre diameter, tensile properties, and chemical composition throughout the scaffold structure compared to PCL alone. These matrix containing scaffolds warrant further long-term investigation as a potential advanced therapy medicinal product for conjunctiva repair and regeneration.

Biological tissues and components, and synthetic substrates for conjunctival cell transplantation.

The conjunctiva is the largest component of the ocular surface. It can be damaged by various pathological processes leading to scarring, loss of tissue and dysfunction. Depending on the amount of damage, restoration of function may require a conjunctival graft. Numerous studies have investigated biological and synthetic substrates in the search for optimal conditions for the ex vivo culture of conjunctival epithelial cells that can be used as tissue grafts for transplantation. These substrates have advantages and disadvantages that are specific to the characteristics of each material; the development of an improved material remains a priority. This review is the second of a two-part review in The Ocular Surface. In the first review, the structure and function of the conjunctiva was evaluated with a focus on the extracellular matrix and the basement membrane, and biological and mechanical characteristics of the ideal substrate with recommendations for further studies. In this review the types of biological and synthetic substrates used for conjunctival transplantation are discussed including substrates based on the extracellular matrix. .

Plasma polymer surface modified expanded polytetrafluoroethylene promotes epithelial monolayer formation in vitro and can be transplanted into the dystrophic rat subretinal space.

The aim of this study was to evaluate whether the surface modification of expanded polytetrafluoroethylene (ePTFE) using an n-heptylamine (HA) plasma polymer would allow for functional epithelial monolayer formation suitable for subretinal transplant into a non-dystrophic rat model. Freshly isolated iris pigment epithelial (IPE) cells from two rat strains (Long Evans [LE] and Dark Agouti [DA]) were seeded onto HA, fibronectin-coated n-heptylamine modified (F-HA) and unmodified ePFTE and fibronectin-coated tissue culture (F-TCPS) substrates. Both F-HA ePTFE and F-TCPS substrates enabled functional monolayer formation with both strains of rat. Without fibronectin coating, only LE IPE formed a monolayer on HA-treated ePTFE. Functional assessment of both IPE strains on F-HA ePTFE demonstrated uptake of POS that increased significantly with time that was greater than control F-TCPS. Surgical optimization using Healon GV and mixtures of Healon GV: phosphate buffered saline (PBS) to induce retinal detachment demonstrated that only Healon GV:PBS allowed F-HA ePTFE substrates to be successfully transplanted into the subretinal space of Royal College of Surgeons rats, where they remained flat beneath the neural retina for up to 4 weeks. No apparent substrate-induced inflammatory response was observed by fundus microscopy or immunohistochemical analysis, indicating the potential of this substrate for future clinical applications.

Increasing the extensional viscosity of silicone oil reduces the tendency for emulsification.

PURPOSE: The purpose was to study the emulsification of silicone oil tamponade agents that cause clinical complications. This study aimed to increase the emulsification resistance of silicone oil 1,000 to be at least as resistant as silicone oil 5,000 while maintaining the shear viscosity <5,000 mPa/s to aid injection and removal. METHODS: High-molecular-weight (423 kDa) poly(dimethyl siloxane) was added to silicone oil 1,000 at 5% and 10% w/w concentration. The shear and extensional viscosity of 1,000 and 5,000, a 50:50 mixture of 1,000 and 5,000 and 5% and 10% w/w additive blends, respectively, of silicone oil were measured using capillary breakup extensional, rotational shear, and capillary extrusion rheometry. In vitro emulsification was assessed qualitatively after agitation using Pluronic F68 or a protein solution as the emulsion stabilizer. RESULTS: The addition of high-molecular-weight polymer increased the extensional viscosity of the blends at high strain rates to levels equal to or greater than silicone oil 5,000. In all cases, the shear viscosity of the blends was lower than that of silicone oil 5,000. The additive blends were qualitatively as emulsification-resistant as silicone oil 5,000. CONCLUSION: The addition of low concentrations of very-high-molecular-weight polymers of the same chemistry as the bulk oil has the potential to increase the emulsification resistance of the tamponade agents while maintaining ease of injection and removal.