Gamma-irradiated human amniotic membrane decellularised with sodium dodecyl sulfate is a more efficient substrate for the ex vivo expansion of limbal stem cells.

The gold standard substrate for the ex vivo expansion of human limbal stem cells (LSCs) remains the human amniotic membrane (HAM) but this is not a defined substrate and is subject to biological variability and the potential to transmit disease. To better define HAM and mitigate the risk of disease transmission, we sought to determine if decellularisation and/or γ-irradiation have an adverse effect on culture growth and LSC phenotype. Ex vivo limbal explant cultures were set up on fresh HAM, HAM decellularised with 0.5M NaOH, and 0.5% (w/v) sodium dodecyl sulfate (SDS) with or without γ-irradiation. Explant growth rate was measured and LSC phenotype was characterised by histology, immunostaining and qRT-PCR (ABCG2, ΔNp63, Ki67, CK12, and CK13). Ƴ-irradiation marginally stiffened HAM, as measured by Brillouin spectromicroscopy. HAM stiffness and γ-irradiation did not significantly affect the LSC phenotype, however LSCs expanded significantly faster on Ƴ-irradiated SDS decellularised HAM (p<0.05) which was also corroborated by the highest expression of Ki67 and putative LSC marker, ABCG2. Colony forming efficiency assays showed a greater yield and proportion of holoclones in cells cultured on Ƴ-irradiated SDS decellularised HAM. Together our data indicate that SDS decellularised HAM may be a more efficacious substrate for the expansion of LSCs and the use of a γ-irradiated HAM allows the user to start the manufacturing process with a sterile substrate, potentially making it safer. STATEMENT OF SIGNIFICANCE: Despite its disadvantages, including its biological variability and its ability to transfer disease, human amniotic membrane (HAM) remains the gold standard substrate for limbal stem cell (LSC) culture. To address these disadvantages, we used a decellularised HAM sterilised by gamma-irradiation for LSC culture. We cultured LSCs on fresh HAM, HAM decellularised with NaOH, HAM decellularised with sodium dodecyl sulfate (SDS) and HAM decellularised with SDS and sterilised with gamma-irradiation. We demonstrated that although HAM decellularised with SDS and sterilised with gamma-irradiation is significantly stiffer this does not affect LSC culture growth rate or the phenotype of cultured LSCs. We therefore recommend the use of SDS decellularised gamma-irradiated HAM in future LSC clinical trials.

Influence of elastase on alanine-rich peptide hydrogels.

The self-assembly of the alanine-rich amphiphilic peptides Lys(Ala)6Lys (KA6K) and Lys(Ala)6Glu (KA6E) with homotelechelic or heterotelechelic charged termini respectively has been investigated in aqueous solution. These peptides contain hexa-alanine sequences designed to serve as substrates for the enzyme elastase. Electrostatic repulsion of the lysine termini in KA6K prevents self-assembly, whereas in contrast KA6E is observed, through electron microscopy, to form tape-like fibrils, which based on X-ray scattering contain layers of thickness equal to the molecular length. The alanine residues enable efficient packing of the side-chains in a beta-sheet structure, as revealed by circular dichroism, FTIR and X-ray diffraction experiments. In buffer, KA6E is able to form hydrogels at sufficiently high concentration. These were used as substrates for elastase, and enzyme-induced de-gelation was observed due to the disruption of the beta-sheet fibrillar network. We propose that hydrogels of the simple designed amphiphilic peptide KA6E may serve as model substrates for elastase and this could ultimately lead to applications in biomedicine and regenerative medicine.

Alanine-rich amphiphilic peptide containing the RGD cell adhesion motif: a coating material for human fibroblast attachment and culture.

We studied the self-assembly of peptide A6RGD (A: alanine, R: arginine, G: glycine, D: aspartic acid) in water, and the use of A6RGD substrates as coatings to promote the attachment of human cornea stromal fibroblasts (hCSFs). The self-assembled motif of A6RGD was shown to depend on the peptide concentration in water, where both vesicle and fibril formation were observed. Oligomers were detected for 0.7 wt% A6RGD, which evolved into short peptide fibres at 1.0 wt% A6RGD, while a co-existence of vesicles and long peptide fibres was revealed for 2-15 wt% A6RGD. A6RGD vesicle walls were shown to have a multilayer structure built out of highly interdigitated A6 units, while A6RGD fibres were based on ß-sheet assemblies. Changes in the self-assembly motif with concentration were reflected in the cell culture assay results. Films dried from 0.1-1.0 wt% A6RGD solutions allowed hCSFs to attach and significantly enhanced cell proliferation relative to the control. In contrast, films dried from 2.5 wt% A6RGD solutions were toxic to hCSFs.

Self-assembly of a peptide amphiphile containing L-carnosine and its mixtures with a multilamellar vesicle forming lipid.

The self-assembly of the peptide amphiphile (PA) hexadecyl-(ß-alanine-histidine) is examined in aqueous solution, along with its mixtures with multilamellar vesicles formed by DPPC (dipalmitoyl phosphatidylcholine). This PA, denoted C(16)-ßAH, contains a dipeptide headgroup corresponding to the bioactive molecule L-carnosine. It is found to self-assemble into nanotapes based on stacked layers of molecules. Bilayers are found to coexist with monolayers in which the PA molecules pack with alternating up-down arrangement so that the headgroups decorate both surfaces. The bilayers become dehydrated as PA concentration increases and the number of layers in the stack decreases to produce ultrathin nanotapes comprised of 2-3 bilayers. Addition of the PA to DPPC multilamellar vesicles leads to a transition to well-defined unilamellar vesicles. The unique ability to modulate the stacking of this PA as a function of concentration, combined with its ability to induce a multilamellar to unilamellar thinning of DPPC vesicles, may be useful in biomaterials applications where the presentation of the peptide function at the surface of self-assembled nanostructures is crucial.

The variation in transparency of amniotic membrane used in ocular surface regeneration.

BACKGROUND/AIMS: Scant consideration has been given to the variation in structure of the human amniotic membrane (AM) at source or to the significance such differences might have on its clinical transparency. Therefore, we applied our experience of quantifying corneal transparency to AM. METHODS: Following elective caesarean, AM from areas of the fetal sac distal and proximal (ie, adjacent) to the placenta was compared with freeze-dried AM. The transmission of light through the AM samples was quantified spectrophotometrically; also, tissue thickness was measured by light microscopy and refractive index by refractometry. RESULTS: Freeze-dried and freeze-thawed AM samples distal and proximal to the placenta differed significantly in thickness, percentage transmission of visible light and refractive index. The thinnest tissue (freeze-dried AM) had the highest transmission spectra. The thickest tissue (freeze-thawed AM proximal to the placenta) had the highest refractive index. Using the direct summation of fields method to predict transparency from an equivalent thickness of corneal tissue, AM was found to be up to 85% as transparent as human cornea. CONCLUSION: When preparing AM for ocular surface reconstruction within the visual field, consideration should be given to its original location from within the fetal sac and its method of preservation, as either can influence corneal transparency.

Transparency, swelling and scarring in the corneal stroma.

PURPOSE: This paper briefly reviews current explanations for corneal transparency and uses a well-developed model to try to explain the increased light scattering either accompanying corneal swelling or following phototherapeutic keratectomy (PTK). METHODS: The direct summation of fields (DSF) method was used to compute light transmission as a function of wavelength. The method requires input of a number of structural parameters. Some of these were obtained from electron micrographs and others were calculated from X-ray diffraction data. RESULTS: By swelling sections of stroma cut from different depths in the tissue, we have shown that fluid entering the cornea causes more swelling in the posterior lamellae than in the anterior lamellae. Furthermore, posterior lamellae can reach a higher final hydration than anterior lamellae. Collagen-free regions ('lakes') exist in corneas swollen in vitro and in Fuch's dystrophy corneas, many of which may be caused by the death of cells. The DSF method shows that local fibril disordering, increased refractive index mismatch, and increased corneal thickness together can account for a 20% increase in light scattering in a Fuch's dystrophy cornea at H=5.8 compared to the normal cornea. Additional scattering is probably caused by 'lakes'. The DSF method applied to PTK rabbit stroma with high levels of haze suggests that the newly deposited collagen is not the cause of the increased light scattering. CONCLUSIONS: Fluid is not uniformly distributed within the corneal stroma when the cornea swells. Increased hydration of posterior lamellae may be because of known differences in the glycosaminoglycans between the anterior and posterior stroma. Lamellar interweave in the anterior stroma probably limits the extent to which the constituent lamellae can swell. The DSF method can be used to account for increased light scattering in oedematous corneas but cannot account for haze following PTK.

An evaluation of factors affecting hazardous waste workers' use of respiratory protective equipment.

The purpose of this study was to describe the perceptions of a group of hazardous waste workers about their workplace hazards and to understand their beliefs and attitudes about the use of respiratory protective equipment (RPE). There were two phases of data collection: (1) interviews to identify factors that influence respirator use and (2) a written survey to evaluate the importance of these factors. This article describes the findings from the written survey completed by 255 eligible respondents (return rate = 46.5%, 255/548). Subjects used a weighting system to score 18 identified factors that influence the use of RPE. Scores were compared according to type of respirator, frequency of use, and associated health symptoms. The factors that had the most positive influence on respirator use were concern about work exposure, fit-testing, and training. The most negative influences were communication, personal comfort, effect on vision, structural environment, and fatigue. More frequent users (once per month or more) were significantly more likely to view fit-testing, health effects, and effects on vision negatively than were less frequent users. Persons who reported health symptoms associated with respirator use had more negative scores than persons without health symptoms. Workers categorized as laborers were more likely to be frequent respirator users and to wear supplied-air respirators, and were significantly more likely to view vulnerability to disease negatively, than were other worker groups. The findings from this study indicate that respiratory protection programs must extend beyond training and education; to be maximally effective, health professionals must be responsive to the specific concerns of the workers.

Hyaluronidase treatment, collagen fibril packing, and normal transparency in rabbit corneas.

PURPOSE: Hyaluronidase treatment is the initial step of corneaplasty, a treatment under development that induces stromal softening and involves the application of a custom designed forming lens to achieve modification of refractive error. The purpose of this investigation was to examine changes in the arrangement of stromal collagen fibrils after hyaluronidase treatment. METHODS: Rabbit corneas were evaluated by slit-lamp microscopy at 0, 2 and 7 days after treatment and haze was assessed by subjective observation. Molecular and interfibrillar Bragg spacing of corneal collagen were measured from synchrotron x-ray scattering patterns. Transmission electron microscopy and digital image analysis were used to calculate radial distribution functions from the positions of collagen fibrils. The calculated fibril sizes and positions were also used to predict the transmission of visible light through these corneas. RESULTS: Hyaluronidase-treated corneas were shown to have a decreased interfibrillar Bragg spacing of 15% to 21%. Fibril hydration did not change. Transparency of these corneas remained unaltered. CONCLUSIONS: Hyaluronidase reduced the hydration of the corneal stroma, which led to a more compacted collagen fibril arrangement. This compression was predicted to cause a small reduction in the transmission of visible light through the cornea but not to a point likely to cause visual impairment.

Structure and function of occupational health services within selected Department of Energy sites.

The mission of the United States Department of Energy sites has recently changed from nuclear weapons production to site remediation. Considering the mass of radiological and chemical contaminants at these sites, ensuring the health and safety of workers is a major challenge. This study used the findings from a written survey to describe occupational health services at 10 Department of Energy sites. The study aims were to describe and compare: (1) the primary hazards associated with the site activities; (2) the occupational safety and health structure, including service providers; and (3) the occupational health and safety functions, including surveillance, training, and service provision. Although explosions and radiological agents were identified as the hazards with the greatest associated risks, workers at these sites were most likely to be exposed to physical hazards, ergonomic hazards, and/or chemicals, including asbestos. Physicians accounted for 2.4% of service providers, nurses for 5.5%, industrial hygienists for 12.2%, safety personnel for 11.8%, and health physicists for 64.9%. It was concluded that there is an imbalance between the most important hazards and the types of health and safety personnel at these sites.

A description of factors affecting hazardous waste workers' use of respiratory protective equipment.

This article describes the first phase of a study that was designed to gain an understanding of hazardous waste workers' attitudes and beliefs about the use of respiratory protective equipment. Exploratory, open-ended interviews were conducted among 28 respirator users at a US Department of Energy facility. Subjects were asked to describe their knowledge, attitudes, and beliefs about their risks to hazards at their worksites and to discuss their use of respiratory protective equipment. A detailed content analysis of the interviews resulted in the generation of a taxonomy of issues and concerns which fell into three general categories: 1) Knowledge, Beliefs, and Attitudes, 2) Physical and Psychological Effects, and 3) External Influences. Knowledge, Beliefs, and Attitudes included Training, Fit Testing, Medical Clearance, Work Exposures, Respirator Use, and Vulnerability to Disease. Physical and Psychological Effects included Somatic/Health Effects, Personal Comfort, Visual Effects, Fatigue, Communication, and Anxiety. External Influences included Structural Environment, Quality and Availability of Equipment, Other PPEs, Co-Worker Influence, Supervisor Influence, and Organizational Culture. The findings from this study have important implications to training and education programs. Effective respiratory protection programs depend on a knowledge of the factors that affect workers' use of equipment. This study suggests that efforts to assure equipment comfort and fit, to assist workers who see and hear less well as a result of their equipment, and to develop strategies to allay worker anxiety when wearing equipment should all be components of a program. An organizational culture that supports and abets the appropriate use of equipment is also a critical element in a successful program.

Injured workers' perceptions of case management services. A descriptive study.

This article describes the findings from a study of injured workers conducted as part of a multifaceted evaluation study of a case management program. The sample consisted of workers who filed a workers' compensation claim between January 1 and September 30, 1995. Data collection consisted of written surveys (n = 45), personal interviews (n = 27), and telephone interviews (n = 16). The findings from this study provided many insights into the injured workers' personal and work experiences, and, in particular, their perceptions of their experience with the nurse case management program. Workers satisfied with services described the nurse case manager (NCM) as having the ability to see the "big picture," to develop appropriate goals, and to anticipate client needs. Dissatisfied workers reported feeling unimportant in terms of service provision. They reported feeling that "the system" did not respond to their needs, and that the NCM was uninterested and disrespectful. This vivid portrayal of workers' experiences and perceptions of case management services provides valuable information about the world view of the injured worker.

Health and safety hazards associated with farming.

1. Farming is a dangerous occupation with many potential physical, chemical, and biological hazards. Hazards include noise, machinery, motor vehicles, electricity, temperature extremes, pressurized hydraulic fluids, grain storage facilities, hand and power tools, repetitive motion, vibration, chemicals, dusts, gases, and infectious agents. 2. Psychosocial factors related to agriculture include stress, economic considerations, poor access to health care, and injuries to minors who begin working at a young age. 3. Many who farm also work at other jobs. Farm related illnesses and injuries can be costly to non-farm employers in terms of lost work time, medical insurance, and life insurance. 4. Occupational health nurses can promote agricultural occupational health through companies that employ farmers and through community organizations, thereby contributing to a better understanding of farm related hazards and developing strategies for reducing these hazards.

The Occupational Health Nurses in Agricultural Communities program: identifying and preventing agriculturally related illnesses and injuries.

1. The Occupational Health Nurses in Agricultural Communities (OHNAC) program is a national surveillance program to identify and prevent agriculturally related illnesses and injuries. Locally based nurses work closely with health departments, hospitals, physicians, and other providers to identify and report sentinel health events. 2. Through OHNAC, sentinel health events are assessed and evaluated to identify occupational risk factors that may be applicable to the larger community of agricultural workers. This information is then disseminated nationally, locally, and to the individual workers. 3. The surveillance and investigation information is used to develop programs to prevent agricultural illnesses and injuries. The use of case based surveillance and subsequent data driven interventions has broad applicability for occupational health nurses in other industrial sectors.