The mechanical properties of amniotic membrane influence its effect as a biomaterial for ocular surface repair

The human amniotic membrane (AM) is a tissue of fetal origin and has proven to be clinically useful as a biomaterial in the management of various ocular surface disorders including corneal stem cell transplantation. However, its success rate displays a degree of clinical unpredictability. We suggest that the measured variability inAMstiffness offers an explanation for the poor clinical reproducibility when it is used as a substrate for stem cell expansion and transplantation. Corneal epithelial stem cells were expanded upon AM samples possessing different mechanical stiffness. To investigate further the importance of biological substrate stiffness on cell phenotype we replaced AM with type I collagen gels of known stiffness. Substrate stiffness was measured using shear rheometry and surface topography was characterized using scanning electron microscopy and atomic force microscopy. The differentiation status of epithelial cells was examined using RT-PCR, immunohistochemistry and Western blotting. The level of corneal stem cell differentiation was increased in cells expanded upon AM with a high dynamic elastic shear modulus and cell expansion on type I collagen gels confirmed that the level of corneal epithelial stem cell differentiation was related to the substrate’s mechanical properties. In this paper we provide evidence to show that the preparatory method of AM for clinical use can affect its mechanical properties and that these measured differences can influence the level of differentiation within expanded corneal epithelial stem cells.

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.

Ex Vivo Construction of an Artificial Ocular Surface by Combination of Corneal Limbal Epithelial Cells and a Compressed Collagen Scaffold Containing Keratocytes

We have investigated the use of a laminin coated compressed collagen gel containing corneal fibroblasts (keratocytes) as a novel scaffold to support the growth of corneal limbal epithelial stem cells. The growth of limbal epithelial cells was compared between compressed collagen gel and a clinically proven conventional substrate, denuded amniotic membrane. Following compression of the collagen gel, encapsulated keratocytes remained viable and scanning electron microscopy showed that fibres within the compressed gel were dense, homogeneous and similar in structure to those within denuded amniotic membrane. Limbal epithelial cells were successfully expanded upon the compressed collagen resulting in stratified layers of cells containing desmosome and hemidesmosome structures. The resulting corneal constructs of both the groups shared a high degree of transparency, cell morphology and cell stratification. Similar protein expression profiles for cytokeratin 3 and cytokeratin 14 and no significant difference in cytokeratin 12 mRNA expression levels by real time PCR were also observed. This study provides the first line of evidence that a laminin coated compressed collagen gel containing keratocytes can adequately support limbal epithelial cell expansion, stratification and differentiation to a degree that is comparable to the leading conventional scaffold, denuded amniotic membrane.

Plastic compression of a collagen gel forms a much improved scaffold for ocular surface tissue engineering over conventional collagen gels

We compare the use of plastically compressed collagen gels to conventional collagen gels as scaffolds onto which corneal limbal epithelial cells (LECs) are seeded to construct an artificial corneal epithelium. LECs were isolated from bovine corneas (limbus) and seeded onto either conventional uncompressed or novel compressed collagen gels and grown in culture. Scanning electron microscopy (SEM) results showed that fibers within the uncompressed gel were loose and irregularly ordered, whereas the fibers within the compressed gel were densely packed and more evenly arranged. Quantitative analysis of LECs expansion across the surface of the two gels showed similar growth rates (p > 0.05). Under SEM, the LECs, expanded on uncompressed gels, showed a rough and heterogeneous morphology, whereas on the compressed gel, the cells displayed a smooth and homogeneous morphology. Transmission electron microscopy (TEM) results showed the compressed scaffold to contain collagen fibers of regular diameter and similar orientation resembling collagen fibers within the normal cornea. TEM and light microscopy also showed that cell–cell and cell–matrix attachment, stratification, and cell density were superior in LECs expanded upon compressed collagen gels. This study demonstrated that the compressed collagen gel was an excellent biomaterial scaffold highly suited to the construction of an artificial corneal epithelium and a significant improvement upon conventional collagen gels.

Influence of substrate on corneal epithelial cell viability within ocular surface models

Corneal tissue engineering has improved dramatically over recent years. It is now possible to apply these technological advancements to the development of superior in vitro ocular surface models to reduce animal testing. We aim to show the effect different substrates can have on the viability of expanded corneal epithelial cells and that those which more accurately mimic the stromal surface provide the most protection against toxic assault. Compressed collagen gel as a substrate for the expansion of a human epithelial cell line was compared against two well-known substrates for modeling the ocular surface (polycarbonate membrane and conventional collagen gel). Cells were expanded over 10 days at which point cell stratification, cell number and expression of junctional proteins were assessed by electron microscopy, immunohistochemistry and RT-PCR. The effect of increasing concentrations of sodium lauryl sulphate on epithelial cell viability was quantified by MTT assay. Results showed improvement in terms of stratification, cell number and tight junction expression in human epithelial cells expanded upon either the polycarbonate membrane or compressed collagen gel when compared to a the use of a conventional collagen gel. However, cell viability was significantly higher in cells expanded upon the compressed collagen gel. We conclude that the more naturalistic composition and mechanical properties of compressed collagen gels produces a more robust corneal model.

Ex vivo expansion of limbal stem cells is affected by substrate properties

Limbal epithelial stem cells play a key role in the maintenance and regulation of the corneal surface. Damage or destruction of these cells results in vascularisation and corneal opacity. Subsequent limbal stem cell transplantation requires an ex vivo expansion step and preserving cells in an undifferentiated state remains vital. In this report we seek to control the phenotype of limbal epithelial stem cells by the novel application of compressed collagen substrates. We have characterised the mechanical and surface properties of conventional collagen gels using shear rheology and scanning electron microscopy. In doing so, we provide evidence to show that compressive load can improve the stiffness of collagen substrates. In addition Western blotting and immunohistochemistry display increased cytokeratin 3 (CK3) protein expression relating to limbal epithelial cell differentiation on stiff collagen substrates. Such gels with an elastic modulus of 2900 Pa supported a significantly higher number of cells than less stiff collagen gels (3 Pa). These findings have substantial influence in the development of ocular surface constructs or experimental models particularly in the fields of stem cell research, tissue engineering and regenerative medicine.

Towards the use of hydrogels in the treatment of limbal stem cell deficiency

Corneal blindness caused by limbal stem cell deficiency (LSCD) is a prevailing disorder worldwide. Clinical outcomes for LSCD therapy using amniotic membrane (AM) are unpredictable. Hydrogels can eliminate limitations of standard therapy for LSCD, because they present all the advantages of AM (i.e. biocompatibility, inertness and a biodegradable structure) but unlike AM, they are structurally uniform and can be easily manipulated to alter mechanical and physical properties. Hydrogels can be delivered with minimum trauma to the ocular surface and do not require extensive serological screening before clinical application. The hydrogel structure is also amenable to modifications which direct stem cell fate. In this focussed review we highlight hydrogels as biomaterial substrates which may replace and/or complement AM in the treatment of LSCD.

In vivo study of the biocompatibility of a novel compressed collagen hydrogel scaffold for artificial corneas

The experiments were designed to evaluate the biocompatibility of a plastically compressed collagen scaffold (PCCS). The ultrastructure of the PCCS was observed via scanning electron microscopy. Twenty New Zealand white rabbits were randomly divided into experimental and control groups that received corneal pocket transplantation with PCCS and an amniotic membrane, respectively. And the contralateral eye of the implanted rabbit served as the normal group. On the 1st, 7th, 14th, 21st, 30th, 60th, 90th, and 120th postoperative day, the eyes were observed via a slit lamp. On the 120th postoperative day, the rabbit eyes were enucleated to examine the tissue compatibility of the implanted stroma. The PCCS was white and translucent. The scanning electron microscopy results showed that fibers within the PCCS were densely packed and evenly arranged. No edema, inflammation, or neovascularization was observed on ocular surface under a slit lamp and few lymphocytes were observed in the stroma of rabbit cornea after histological study. In conclusion, the PCCS has extremely high biocompatibility and is a promising corneal scaffold for an artificial cornea. (c) 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Decomposition of surface electromyographic signal using Hidden Markov Model

The detection of physiological signals from the motor system (electromyographic signals) is being utilized in the practice clinic to guide the therapist in a more precise and accurate diagnosis of motor disorders. In this context, the process of decomposition of EMG (electromyographic) signals that includes the identification and classification of MUAP (Motor Unit Action Potential) of a EMG signal, is very important to help the therapist in the evaluation of motor disorders. The EMG decomposition is a complex task due to EMG features depend on the electrode type (needle or surface), its placement related to the muscle, the contraction level and the health of the Neuromuscular System. To date, the majority of researches on EMG decomposition utilize EMG signals acquired by needle electrodes, due to their advantages in processing this type of signal. However, relatively few researches have been conducted using surface EMG signals. Thus, this article aims to contribute to the clinical practice by presenting a technique that permit the decomposition of surface EMG signal via the use of Hidden Markov Models. This process is supported by the use of differential evolution and spectral clustering techniques. The developed system presented coherent results in: (1) identification of the number of Motor Units actives in the EMG signal; (2) presentation of the morphological patterns of MUAPs in the EMG signal; (3) identification of the firing sequence of the Motor Units. The model proposed in this work is an advance in the research area of decomposition of surface EMG signals.