EPITHELIUM REPAIR

A new, easily prepared and highly reproducible collagen preparation could serve as an alternative to amniotic membrane as a substrate for the production of epithelial tissue for use in the treatment of limbal stem cell deficiency, said Hannah Levis PhD, UCL Institute of Ophthalmology, London UK.

[caption id='attachment_1309' align='aligncenter' width='620' caption='Figure 1. A schematic diagram showing the plastic compression process with fluid being expelled from the hydrogel in an unconfined manner (blue arrows)'][/caption]

 

[caption id='attachment_1310' align='aligncenter' width='620' caption='Figure 2. Light micrographs of haematoxylin and eosin stained histological cross sections of (A) human corneal epithelium and (B) human limbal epithelial cells on collagen constructs. (CC collagen construct, CS corneal stroma, CE, corneal epithelium CF corneal fibroblast, LF Limbal fibroblast. Scale bar 50mm)'][/caption]

The new substrate material consists of highly concentrated collagen imbedded with human limbal fibroblast cells. Laboratory studies conducted to date indicate that it provides a scaffold on which limbal epithelial stem cells can generate layered epithelial tissue that is similar in morphology and biochemical profile to that of the natural healthy corneal epithelium, Dr Levis said.

Dr Levis noted that limbal stem cell deficiency – whether arising from burns, chemical injury or disease, such as contact lens keratopathy or Stevens-Johnson syndrome – undermines the ability of the epithelium to maintain the integrity of the cornea. The current conventional treatment of the condition consists of the cell culture of autologous or allogeneic stem cells on an amniotic membrane substrate, and placing that on the eye, she noted.

She pointed out although amniotic membrane has many advantages as a substrate for limbal stem cell culture, including its anti-inflammatory and anti-angiogenic properties, it also has a lot of disadvantages including its semi-opacity, supply problems and biological variability between donors.

“In our hands, 40 per cent of those amniotic grafts with epithelial stem cells failed before they even got to the patients, so it’s not really an ideal carrier for these epithelial carrier cells in this context. Some of the alternatives that have been investigated include cross-linked collagen, electrospun collagen, collagen vitrigels, silk fibroin scaffolds and fibrin gels. They all have advantages but also many disadvantages, so we are trying to find a suitable alternative substrate.’

New substrate based on ideal criteria

Dr Levis said that the new substrate is designed to have all properties that an ideal material would need in order to generate functional epithelial tissue for transplantation to patients’ diseased or traumatised corneas. Those properties include reproducibility, ease of preparation under aseptic conditions, transparency and the ability to allow the formation and maintenance of a multilayered epithelium with a basement membrane and a mixed population of differentiated and undifferentiated cells. The cells also need to form cell-to-cell junctions so that the resulting tissue will act as a protective membrane.

Dr Levis and her associates prepare the collagen-based substrate through a process called plastic compression, wherein they compress the rat-tail derived collagen hydrogel between two nylon meshes, with a glass plate pressed down with a weight on top of it. The process reduces the thickness of the hyperhydrated collagen from around 6.0mm to about 100 microns thick. The result is a membrane that is strong and easily manipulated, unlike collagen hydrogels that have not been processed in this way.

“It’s a simple process, but the advantage of this plastic compressed collagen gel is that it is a highly reproducible substrate, because it’s just collagen and cells and the cells are seeded directly into the hydrogel before compression unlike other scaffolds, which usually require a lengthy wait for the cells to populate the scaffold. The process can be repeated every time; there is no variation as seen with amniotic membrane. This construct also has many tuneable properties, you can change the thickness, the collagen density, form it into any shape you like and you can also modulate the surface topography,†she noted.

Prior to performing the compression, Dr Levis and her team populate the collagen gel with human limbal fibroblasts. The fibroblasts appear to play an integral role in the development of the differentiated epithelial layers. The cells survive the compression process and continue to divide afterwards. Once the substrate is prepared, a single cell suspension of cultured limbal cells is placed on its surface, which stays submerged for three weeks after which it is brought to the surface and exposed to air.

“The resulting constructs are relatively transparent and they have enough mechanical strength to withstand being sutured to the surface of an eye and maintained even under quite harsh mechanical treatment,†Dr Levis noted.

Natural appearing epithelial layers

Experiments conducted thus far have demonstrated that the epithelial stem cells grown on the compressed, fibroblast-enriched collagen construct develop the same types of cells and cell layer arrangement as is the case in the natural corneal epithelium. That is, transmission electron microscopy and scanning electron microscopy confirmed that there were more cuboidal-like cells on the basal layer and more squamous-like cells on the surface. “The cells are highly interdigitated, and desmosome junctions form between the cells, which is an indication of a mature epithelium that will act as a barrier. The scanning electron microscopy images show that there are smooth polygonal cells on the surface with many microvilli, indicative of a healthy epithelium.â€

She noted that the cells’ immunohistochemistry mirrored their morphology. For example, cytokeratin 3, a differentiated corneal epithelial marker, was highly expressed in the squamous-like cells on the surface layer but was not expressed in the cells in basal layer. Similarly, p63-alpha, a putative stem cell marker was highly expressed in the cells of the basal layer but not in the surface squamous-like cells.

“We conclude that this plastic compressed collagen is a suitable substrate for limbal epithelial stem cell culture for transplantation and this could be a possible replacement for amniotic membrane and could also form the basis for a corneal epithelial model which would be very useful for studying the interactions between cell types.â€

This study was undertaken in collaboration with TAP Biosystems, funded by the Technology Strategy Board.