Automatic Breathing Pattern Analysis from Reading-Speech Signals.

As the speech production mechanism is related to the breathing process, speech signals and breathing patterns impact each other. Breathing patterns are the physiological signals which help in understanding the psychological, physiological and cognitive states of an individual. Capturing such patterns relies on the availability of equipment such as respiratory belts, which are costly and uncomfortable to wear for long duration. In this paper, we attempt to extract the breathing patterns from speech signals, which are easily available and can be recorded using a smartphone's microphone. In the presented work, simultaneous speech and breath signals are captured from 100 Indians of the age group 20 to 25 years while they read a phonetically balanced passage in English language. We have identified five distinct breathing templates; following two broad speech-breath categories, exhibited by the speakers while they read the same passage. For one of the two categories, the time domain features with regression network can extract the breathing patterns from speech with a Pearson correlation coefficient of 0.70. By computational modelling, we distinguish these two breathing categories from speech with a classification accuracy of 79%.

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.

Proof-of-concept study of electrospun PLGA membrane in the treatment of limbal stem cell deficiency.

OBJECTIVE: The aim of this study was to assess the safety of poly-lactic co-glycolic acid (PLGA) electrospun membranes as carriers for limbal tissue explants for treatment of limbal stem cell deficiency (LSCD). METHODS AND ANALYSIS: Approval was obtained for a first in-man study from the Drug Controller General of India. PLGA membranes were applied to the affected eye of five patients after removal of the vascular pannus. Simple limbal epithelial transplantation was performed and limbal explants were secured on the membrane using fibrin glue followed by a bandage contact lens. Patients were followed up for 1 year with ocular exams including slit lamp exam, corneal thickness measurements, intraocular pressure measurements and recording of corneal vascularisation and visual acuity. Systemic examinations included pain grading, clinical laboratory assessment, blood chemistry and urine analysis at baseline, 3 and 6 months after surgery. RESULTS: PLGA membranes completely degraded by 8 weeks post-transplantation without any infection or inflammation. In all five patients, the epithelium regenerated by 3 months. In two in five patients, there was a sustained two-line improvement in vision. In one in five patients, the vision improvement was limited due to an underlying stromal scarring. There was recurrence of pannus and LSCD in two in five patients 6 months after surgery which was not attributable to the membrane. The ocular surface remained clear with no epithelial defects in three in five subjects at 12 months. CONCLUSION: PLGA electrospun membranes show promise as carrier for limbal epithelial cells in the treatment of LSCD.

Antimicrobial Activity of Poly-epsilon-lysine Peptide Hydrogels Against Pseudomonas aeruginosa.

Purpose: To determine the antimicrobial activity of poly-epsilon-lysine (pɛK) functionalization of hydrogels against Pseudomonas aeruginosa. Methods: Antimicrobial activities of pɛK and pɛK+ hydrogels were tested against both keratitis and a laboratory strain of Paeruginosa at a range of inocula sizes, over 4 and 24 hours. The number of viable CFU on pɛK and pɛK+ hydrogels or commercial contact lenses (CL) was investigated. Ex vivo porcine corneas were inoculated with Paeruginosa PAO1 (103 CFU) and incubated with pɛK+ hydrogels or commercial hydrogel CL for 24 hours and the effects of infection determined. Results: PɛK+ hydrogels showed log reductions in viable CFU compared with pɛK hydrogels for all Paeruginosa strains, depending on inocula sizes and incubation time. After 24 hours pɛK+ hydrogels showed >5 and >7.5 log reduction in CFU compared with commercial hydrogel CL at 103 and 106 CFU, respectively. In an ex vivo porcine corneal infection model, pɛK+ hydrogels led to a significant decrease in viable PAO1 CFU and histologic analysis indicated a decreased infiltration of PAO1 into the stroma. Conclusions: PɛK+ hydrogels demonstrated enhanced antimicrobial activity versus nonfunctionalized pɛK hydrogels against clinically relevant Paeruginosa strains. PɛK+ hydrogels have the potential to be used as a bandage CL with innate antimicrobial characteristics to minimize the risk of microbial keratitis.

A randomized, double-blind placebo-controlled study of intranasal standardized cinnamon bark extract for seasonal allergic rhinitis.

OBJECTIVES: The aim of the study was to assess the safety and efficacy of a nasal spray containing a polyphenol-rich standardized extract of cinnamon bark (Cinnamomum zeylanicum) (IND02) for the treatment of seasonal allergic rhinitis (SAR). METHODS: This study was a randomized, double-blind, placebo-controlled study conducted in otherwise healthy men and women, aged between 18 and 75 years old, who were experiencing acute SAR symptoms. Participants were randomized in a 1:1 ratio to a nasal spray containing either IND02 (100 µg/100 µL) or matching placebo in each nostril, twice a day, for seven days. RESULTS: The outcome measures were the rhinoconjunctivitis quality of life questionnaire (RQLQ), the total daily symptom score comprising of day-time nasal, day-time eye, and night-time nasal symptom scores, the Work Productivity and Activities Impairment (WPAI:SHP), the Pittsburgh Sleep Quality Index (PSQI), the Perceived Stress Scale (PSS) and laboratory clinical parameters. RESULTS: The IND02 group showed a statistically and clinically significant reduction in total RQLQ and the sub-domains; activity limitation, sleep problems, nose symptoms, eye symptoms, non-nose/eye symptoms, practical problems and emotional function. There was a significant reduction in the total daily symptom score and sub-domains of total day-time nasal, total day-time eye and total night-time nasal symptoms scores, and total work impairment and regular activity impairment in the IND02 group compared with the placebo group after treatment. The laboratory clinical parameters remained within healthy normal reference range. CONCLUSION: The use of a nasal spray of a standardized extract of cinnamon bark (IND02) over seven days reduced symptom severity and improved quality of life, work productivity and regular daily activities in participants experiencing SAR.

Prenatal Developmental Toxicity Study of Glycosides-based Standardized Fenugreek Seed Extract in Rats.

CONTEXT: Glycoside-based standardized fenugreek seed extract (SFSE-G) demonstrated promising efficacy in animal models of immune-inflammatory conditions. AIM: The present study was aimed at embryo-fetal development toxicity evaluation of SFSE-G in Wistar rats as per guideline No. 414 of the Organization for Economic Co-operation and Development (OECD). MATERIAL AND METHODS: Mated female rats were randomized into four groups of 30 each and received oral doses of either SFSE-G at 250, 500, and 1000 mg/kg or vehicle (water) during the period of gestation (postconception) from gestational day 5 (GD5, an implantation day) until 1 day before cesarean sections (GD19). Maternal food consumption, body weights, and clinical signs were monitored throughout gestation. Cesarean sections were performed on GD20 and fetal observations (gravid uterine weight, implantation sites, early and late resorptions, live and dead fetuses) were recorded. Live fetuses were weighed and examined for external, visceral, and skeletal variations and malformations. RESULTS: None of the SFSE-G-treated groups showed maternal and embryo-fetal toxicity. Occasional and incidental skeletal and visceral malformations were observed and found to be spontaneous and unrelated to the treatment. CONCLUSION: Oral exposure of SFSE-G during the prenatal period did not show significant maternal and embryo-fetal toxicity up to a dose of 1000 mg/kg in rats. Therefore, the no-observed-adverse-effect level for SFSE-G for prenatal oral exposure was considered to be 1000 mg/kg. SUMMARY: Prenatal toxicity of glycoside-based standardized fenugreek seed extract (SFSE-G) was evaluated.SFSE-G was orally gavaged to rats on gestational days 5-19 with a limit dose of 1000 mg/kg.SFSE-G did not show maternal or developmental toxicity.SFSE-G showed NOAEL of 1000 mg/kg for prenatal exposure in female rats. Abbreviations used: CPCSEA: Committee for the Purpose of Control and Supervision of Experiments on Animals; GD: Gestational day; GRAS: Generally recognized as safe; HED: Human equivalent dose; NOAEL: No-observed adverse effect levels; OECD: Organization for Economic Co-operation and Development; SFSE-G: glycoside-based standardized fenugreek seed extract.

Preclinical Toxicological Evaluation of IDM01: The Botanical Composition of 4-Hydroxyisoleucine- and Trigonelline-based Standardized Fenugreek Seed Extract.

OBJECTIVE: To evaluate acute oral toxicity (AOT), subchronic (90-day repeated dose) toxicity, mutagenicity, and genotoxicity potential of IDM01, the botanical composition of 4-hydroxyisoleucine- and trigonelline-based standardized fenugreek (Trigonella foenum-graecum L) seed extract in laboratory rats. MATERIALS AND METHODS: The AOT and subchronic (90-day repeated dose) toxicity were evaluated using Sprague-Dawley rats as per the Organisation for Economic Co-operation and Development (OECD) guidelines No. 423 and No. 408, respectively. During the subchronic study, the effects on body weight, food and water consumption, organ weights with hematology, clinical biochemistry, and histology were studied. The mutagenicity and genotoxicity of IDM01 were evaluated by reverse mutation assay (Ames test, OECD guideline No. 471) and chromosome aberration test (OECD guideline No. 473), respectively. RESULTS: The IDM01 did not show mortality or treatment-related adverse signs during acute (limit dose of 2000 mg/kg) and subchronic (90-day repeated dose of 250, 500, and 1000 mg/kg with 28 days of recovery period) administration. The IDM01 showed oral median lethal dose (LD50) >2000 mg/kg during AOT study. The no-observed adverse effect level (NOAEL) of IDM01 was 500 mg/kg. IDM01 did not show mutagenicity up to a concentration of 5000 µg/plate during Ames test and did not induce structural chromosomal aberrations up to 50 mg/culture. CONCLUSIONS: IDM01 was found safe during preclinical acute and subchronic (90-day repeated dose) toxicity in rats without mutagenicity or genotoxicity. SUMMARY: Acute oral toxicity, subchronic (90-day) oral toxicity, mutagenicity and genotoxicity of IDM01 (4-hydroxyisoleucine- and trigonelline-based standardized fenugreek seed extract) was evaluated.The median lethal dose, LD50, of IDM01 was more than 2000 mg/kg of body weight in rats.No observed adverse effect level (NOAEL) of IDM01 was 500 mg/kg of body weight in rats.IDM01 was found safe during acute and subchronic oral toxicity studies in rats without mutagenicity or genotoxicity potetial. Abbreviations Used: 2-AA: 2-aminoanthracene; 2-AF: 2-aminofluorene; 4 NQNO: 4-nitroquinolene-N-oxide; 4HI: 4-hydroxyisoleucine; ANOVA: Analysis of variance; AOT: Acute oral toxicity; DM: Diabetes mellitus; IDM01: The Botanical composition of 4-hydroxyisoleucine- and trigonelline-based standardized fenugreek seed extract; LD50: Median lethal dose; MMS: Methyl methanesulfonate; NAD: No abnormality detected; OECD: Organisation for Economic Co-operation and Development; SD: Standard deviation; UV: Ultraviolet; VC: Vehicle control. 2-AA: 2-aminoanthracene; 2-AF: 2-aminofluorene; 4 NQNO: 4-nitroquinolene-N-oxide; 4HI: 4-hydroxyisoleucine; ANOVA: Analysis of variance; AOT: Acute oral toxicity; DM: Diabetes mellitus; IDM01: The Botanical composition of 4-hydroxyisoleucine- and trigonelline-based standardized fenugreek seed extract; LD50: Median lethal dose; MMS: Methyl methanesulfonate; NAD: No abnormality detected; OECD: Organisation for Economic Co-operation and Development; SD: Standard deviation; UV: Ultraviolet; VC: Vehicle control.

Preclinical Safety Assessment of Furostanol Glycoside-Based Standardized Fenugreek Seed Extract in Laboratory Rats.

The present work is aimed at studying acute oral toxicity (AOT), subchronic oral toxicity, mutagenicity, and genotoxicity of furostanol glycosides-based standardized fenugreek seed extract (Fenu-FG) using the Organization for Economic Co-operation and Development (OECD) guidelines. The AOT and subchronic (90-day repeated dose) toxicity studies were performed on Wistar rats as per OECD 423 and OECD 408 guidelines, respectively. The mutagenicity (reverse mutation assay, Ames test) and genotoxicity (mammalian chromosome aberration test) were assessed in vitro using OECD 471 and OECD 473 guidelines, respectively. At an acute oral limit dose of 2,000 mg/kg, Fenu-FG did not show any mortality or treatment-related adverse signs. Ninety days of subchronic oral administration of Fenu-FG (250, 500, or 1,000 mg/kg) in rats did not induce any treatment-related significant changes with respect to body weight, hematology, blood biochemistry, urinalysis, gross pathology, or histopathology. The no-observed-adverse-effect-level of Fenu-FG was 1,000 mg/kg/day. Furthermore, Fenu-FG did not demonstrate mutagenic potential up to a concentration of 5,000 µg/plate (Ames test) and did not induce structural chromosome aberrations up to 2,000 µg/ml (in human lymphocyte cells in vitro). In conclusion, Fenu-FG was found safe during preclinical safety assessments.

Preclinical safety evaluation of low molecular weight galactomannans based standardized fenugreek seeds extract.

The objective of the present study was to evaluate acute oral toxicity, subchronic toxicity, and mutagenic potential of low molecular weight galactomannans based standardized fenugreek seeds extract (LMWGAL-TF) in laboratory animals rats as per Organization for Economic Co-operation and Development (OECD) guidelines. For the acute toxicity (AOT) study, LMWGAL-TF was orally administered to Sprague-Dawley (SD) rats at a dose of 2000 mg/kg with vehicle control (VC) group (n = 5 per sex per group) as per OECD guideline no. 423. For the repeated dose toxicity study, the SD rats were orally administered with a daily oral dose of LMWGAL-TF 250, 500 and 1000 mg/kg/day with VC group (n = 15 per sex) for a period of 90 days followed by a recovery period of 28 days as per OECD guideline no. 408. The effects on body weight, food and water consumption, organ weights with hematology, clinical biochemistry, and histology were studied. The mutagenic potential of LMWGAL-TF was tested using reverse mutation assay (AMES test, OECD guideline No. 471). The LMWGAL-TF did not show mortality or treatment-related adverse signs during acute (dose 2000 mg/kg) and subchronic (90-days repeated dose 250, 500 and 1000 mg/kg) administration. The LMWGAL-TF showed oral lethal dose (LD50) more than 2000 mg/kg during AOT study. The dose of 1000 mg/kg was found as no observed adverse effect level (NOAEL) in rats during subchronic toxicity study. Furthermore, LMWGAL-TF did not show mutagenic potential in vitro. In conclusion, LMWGAL-TF was found safe during acute and subchronic (90 days repeated dose) toxicity studies in rats with no mutagenicity.

Rocking media over ex vivo corneas improves this model and allows the study of the effect of proinflammatory cytokines on wound healing.

PURPOSE: The aim of this work was to develop an in vitro cornea model to study the effect of proinflammatory cytokines on wound healing. METHODS: Initial studies investigated how to maintain the ex vivo models for up to 4 weeks without loss of epithelium. To study the effect of cytokines, corneas were cultured with the interleukins IL-17A, IL-22, or a combination of IL-17A and IL-22, or lipopolysaccharide (LPS). The effect of IL-17A on wound healing was then examined. RESULTS: With static culture conditions, organ cultures deteriorated within 2 weeks. With gentle rocking of media over the corneas and carbon dioxide perfusion, the ex vivo models survived for up to 4 weeks without loss of epithelium. The cytokine that caused the most damage to the cornea was IL-17A. Under static conditions, wound healing of the central corneal epithelium occurred within 9 days, but only a single-layered epithelium formed whether the cornea was exposed to IL-17A or not. With rocking of media gently over the corneas, a multilayered epithelium was achieved 9 days after wounding. In the presence of IL-17A, however, there was no wound healing evident. Characterization of the cells showed that wherever epithelium was present, both differentiated cells and highly proliferative cells were present. CONCLUSIONS: We propose that introducing rocking to extend the effective working life of this model and the introduction of IL-17A to this model to induce aspects of inflammation extend its usefulness to study the effects of agents that influence corneal regeneration under normal and inflamed conditions.

Preclinical Safety Assessment of Standardized Extract of Centella asiatica (L.) Urban Leaves.

CONTEXT: Centella asiatica (CA) leaves extract has been shown therapeutic potential. However, safety information is lacking. AIMS: To evaluate acute oral toxicity (AOT), sub-chronic toxicity, and mutagenic potential of standardized extract of CA (L.) Urban leaves (INDCA). MATERIALS AND METHODS: For the acute toxicity study, INDCA was orally administered to Sprague-Dawley rats at a dose range of 0-2000 mg/kg. For the repeated dose toxicity study, the rats of either sex were orally administered with INDCA at the doses of 250, 500, and 1000 mg/kg/day for a period of 90 days. The effects on body weight, food and water consumption, organ weight, hematology, clinical chemistry as well as histology were studied. The mutagenic potential of INDCA was tested using reverse mutation assay (Ames test). STATISTICAL ANALYSIS USED: Data of each parameter were analyzed by one-way ANOVA followed by Dunnett's test to compare the difference between treated groups. RESULTS: The administration of INDCA did not produce mortality or significant changes in the clinical signs included but not limited to changes in the skin and fur, eyes and mucous membranes, and also respiratory, circulatory, autonomic and central nervous systems, somatomotor activity, and behavior pattern. The appearance, progress, and disappearance of these signs were recorded. The lethal dose and no observable adverse effect level of INDCA were 2000 mg/kg and 1000 mg/kg, respectively. There were no significant differences in the organ weights, hematological parameters, clinical chemistry values, or gross and microscopic appearance of the organs from the treatment groups as compared to the control group. It was found to be nonmutagenic in reverse mutation assay. CONCLUSIONS: INDCA was found safe in AOT, sub-chronic toxicity, and mutagenicity studies when tested in rats.

Combination of microstereolithography and electrospinning to produce membranes equipped with niches for corneal regeneration.

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as Aniridia or Steven Johnson Syndrome as well as by external factors such as chemical burns or radiation. Current treatments are (i) the use of corneal grafts and (ii) the use of stem cell expanded in the laboratory and delivered on carriers (e.g., amniotic membrane); these treatments are relatively successful but unfortunately they can fail after 3-5 years. There is a need to design and manufacture new corneal biomaterial devices able to mimic in detail the physiological environment where stem cells reside in the cornea. Limbal stem cells are located in the limbus (circular area between cornea and sclera) in specific niches known as the Palisades of Vogt. In this work we have developed a new platform technology which combines two cutting-edge manufacturing techniques (microstereolithography and electrospinning) for the fabrication of corneal membranes that mimic to a certain extent the limbus. Our membranes contain artificial micropockets which aim to provide cells with protection as the Palisades of Vogt do in the eye.

Amine functional hydrogels as selective substrates for corneal epithelialization.

The aim of this study was to develop a synthetic hydrogel to act as a corneal substitute capable of selectively supporting the adhesion and proliferation of limbal epithelial cells (LECs) while inhibiting growth of limbal fibroblasts. Deficiency of LECs causes conjunctival epithelial cells to move over the cornea, producing a thick scar pannus. Unilateral defects can be treated using LEC cultured from the unaffected eye, transplanting them to the affected cornea after scar tissue is removed. The underlying wound bed is often damaged, however, hence the need to develop a corneal inlay to aid in corneal re-epithelialization. Transparent epoxy-functional polymethacrylate networks were synthesized using a combination of glycerol monomethacrylate, ethylene glycol dimethacrylate, lauryl methacrylate and glycidyl methacrylate that produced two different bulk hydrogel compositions with different equilibrium water contents (EWCs): Base 1 and Base 2, EWC=55% and 35%, respectively. Two sets of amine-functional hydrogels were produced following reaction of the epoxide groups with excesses of either ammonia, 1,2-diamino ethane, 1,3-diamino propane, 1,4-diamino butane or 1,6-diamino hexane. Neither series of hydrogels supported the proliferation of limbal fibroblasts irrespective of amine functionalization but they both supported the adhesion and proliferation of limbal epithelial cells, particularly when functionalized with 1,4-diamino butane. With Base 1 hydrogels (less so with Base 2) a vigorous epithelial outgrowth was seen from small limbal explants and a confluent epithelial layer was achieved in vitro within 6days. The data support the development of hydrogels specific for epithelial formation.

Cultivation of limbal epithelial cells on electrospun poly (lactide-co-glycolide) scaffolds for delivery to the cornea.

In delivering tissues to the body, both natural and synthetic materials have been used. Currently, a natural membrane, the human amniotic membrane (AM), is used to deliver limbal epithelial cells (LEC) to the cornea. AM presents inherent problems with structural variation and requires extensive serological screening before use. Therefore alternatives are required to improve the predictability in clinical outcomes and economic costs associated with the use of this biological substrate. In this chapter, we describe the development of an alternative, structurally simple, synthetic biodegradable electrospun scaffold based on poly(lactide-co-glycolide) (PLGA: materials used in dissolvable sutures) to replace AM.

Development of a microfabricated artificial limbus with micropockets for cell delivery to the cornea.

The aim of this study was to develop a synthetic alternative to the human corneal limbus for use initially as an ex vivo model in which to study corneal stem cell function within a niche environment and ultimately to develop an implantable limbus for future clinical use. Microstereolithography was used for the fabrication of polyethylene glycol diacrylate (PEGDA) based rings on a macroscopic (1.2 cm) scale containing unique microfeatures (pockets) which were then modified with fibronectin to promote cell adhesion. These rings were designed to mimic the limbal area of the eye containing structures of the approximate size and shape of the stem cell microenvironments found in the palisades of Vogt. The attachment of rabbit limbal fibroblasts and rabbit limbal epithelial cells to the PEGDA rings was increased by pretreating the microfabricated structures with biotinylated fibronectin. Cell outgrowth from fibronectin coated microfabricated structures was 50% greater than from rings without structures or fibronectin coating. The cell loaded rings were then placed on an ex vivo wounded cornea model and the outgrowth of cells to form a multilayered epithelium was observed. We suggest this is a new approach to investigating limbal stem cells niches and the first steps towards a new approach for corneal regeneration.

Simplifying corneal surface regeneration using a biodegradable synthetic membrane and limbal tissue explants.

Currently, damage to the ocular surface can be repaired by transferring laboratory cultured limbal epithelial cells (LECs) to the cornea using donor human amniotic membrane as the cell carrier. We describe the development of a synthetic biodegradable membrane of Poly D,L-lactide-co-glycolide (PLGA) with a 50:50 ratio of lactide and glycolide for the delivery of both isolated LECs and of cells grown out from limbal tissue explants. Both isolated LECs and limbal explants produced confluent limbal cultures within 2 weeks of culture on the membranes without the need for fibroblast feeder layers. Outgrowth of cells from explants was promoted by the inclusion of fibrin. Membranes with cells on them broke down predictably within 4-6 weeks in vitro and the breakdown was faster for a lower molecular weight (MW) (44 kg/mol) rather than a higher MW (153 kg/mol) PLGA. Membranes could be reproducibly produced, sterilised with gamma irradiation and stored dry at -20 °C for at least 12 months, and the ability to support cell outgrowth from explants was retained. We demonstrate transfer of cells (both isolated LECs and of cells grown out from limbal explants) from the membranes to an ex vivo rabbit cornea model. Characterisations of the cells by immunohistochemistry showed both differentiated and stem cell populations. A synthetic membrane combined with limbal explants in theatre would avoid the need for tissue banked human amniotic membrane and also avoid the need for specialist laboratory facilities for LEC expansion making this more accessible to many more surgeons and patients.

Combined microfabrication and electrospinning to produce 3-D architectures for corneal repair.

Corneal stem cell niches are located within the limbus of the eye and are believed to play an important role in corneal regeneration. These niches are often lost in corneal disease or trauma. Our work explores the design of artificial limbal stem cell niches by the fabrication of biodegradable electrospun rings containing bespoke microfeatures. In creating artificial niches, we seek to provide a physically protective environment for limbal cells to act as a cell reservoir for tissue regeneration purposes. This study describes the first step in this challenge to produce structures which structurally approximate to the limbal niches. This was achieved using a combination of electrospinning and microfabrication. Initial microfabricated structures were developed using microstereolithography via a layer-by-layer photocuring approach based on the patterning of photocurable polymers, in this case polyethylene glycol diacrylate. This was then used as a template on which to electrospin a biodegradable membrane of poly(lactic-co-glycolic acid) 50:50, which incorporates the features of the underlying microfabricated structures. The study describes preliminary evaluation of these constructs using rabbit limbal epithelial and stromal cells.

Biocompatible hydrogels based on hyaluronic acid cross-linked with a polyaspartamide derivative as delivery systems for epithelial limbal cells.

The aim of this work was to evaluate the potential use of hydrogels based on hyaluronic acid (HA) chemically cross-linked with α,ß-poly(N-2-hydroxyethyl) (2-aminoethylcarbamate)-D,L-aspartamide (PHEA-EDA) as substitutes for the amniotic membrane able to release limbal cells for corneal regeneration. Hydrogels, shaped as films, with three different molar ratios (X) between PHEA-EDA and HA (X = 0.5, 1.0 and 1.5) have been investigated. First, it has been evaluated their swelling ability, hydrolytic resistance in simulated physiological fluid and cell compatibility by using human dermal fibroblasts chosen as a model cell line. Then adhesion studies in comparison with collagen gel, have been performed by using immortalized cells, such as human corneal epithelial cells (HCEC) or primary cells, such as rabbit limbal epithelial cells (RLEC) and/or rabbit limbal fibroblasts (RLF). HA/PHEA-EDA hydrogels allow a moderate/poor adhesion of all investigated cells thus suggesting their potential ability to act as cell delivery systems. Finally, commercial contact lenses have been coated, in their inner surface, with each HA/PHEA-EDA film and it has been found that in these conditions, a greater cell adhesion occurs, particularly when RLEC are in co-culture with RLF. However, this adhesion is only transitory, in fact after three days, viable cells are released in the culture medium thus suggesting a potential application of HA/PHEA-EDA hydrogels, for delivering limbal cells in the treatment of corneal damage.

Using poly(lactide-co-glycolide) electrospun scaffolds to deliver cultured epithelial cells to the cornea.

AIMS: To assess the potential of electrospun poly(lactide-co-glycolide) membranes to provide a biodegradable cell carrier system for limbal epithelial cells. MATERIAL & METHODS: 50:50 poly(lactide-co-glycolide) scaffolds were spun, sterilized and seeded with primary rabbit limbal epithelial cells. Cells were cultured on the scaffolds for 2 weeks and then examined by confocal microscopy, cryosectioning and scanning-electron microscopy. The tensile strength of scaffolds before and after annealing and sterilization was also studied. RESULTS: The limbal cells had formed a continuous multilayer of cells on either side of the scaffold. Scaffolds with cells showed signs of the onset of degradation within 2 weeks in culture media at 37 degrees C. Scaffolds that were annealed resulted in a more brittle and stiff mat. CONCLUSIONS: We suggest this carrier membrane could be used as a replacement for the human amniotic membrane in the treatment of limbal stem cell deficiency, lowering the risk of disease transmission to the patient.

Development of a surface-modified contact lens for the transfer of cultured limbal epithelial cells to the cornea for ocular surface diseases.

Our aim was to develop an improved cell transfer system for delivering laboratory-cultured human limbal epithelial cells to the cornea, which would be low risk for the patient and convenient to use for the surgeon. We took a standard contact lens and developed a plasma polymer layer for coating this for attachment of cells to the lens and subsequent transfer of cells to the cornea. A range of plasma polymer surfaces were examined for initial cell attachment using three different combinations of human and rabbit epithelial and stromal cells, initially expanding cells both with and without bovine serum. The most promising surfaces, based on acrylic acid, were then coated onto contact lenses. Cell transfer from the lenses to the denuded surface of a 3D rabbit organ culture model was then used to make a second selection of substrates, which permitted reliable cell transfer. Primary rabbit and human corneal cells attached and proliferated well on acrylic acid-coated surfaces. Reliable transfer of primary epithelial cells from the coated contact lenses to a rabbit cornea was achieved by coating lenses with acrylic acid at 5 W/10 cm(3)/min and using cell densities of 1 x 10(5)/lens and above.