combination-microstereolithography-electrospinning-to-produce

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 ...

L. V. Prasad Eye Institute

To investigate changes in the phosphorylation of myosin light chain (MLC) in response to histamine and its effect on the barrier integrity of corneal epithelial cells.

Histamine-induced myosin light chain phosphorylation breaks down the barrier integrity of cultured corneal epithelial cells.

Enhanced contractility of the actin cytoskeleton in trabecular meshwork (TM) cells is implicated in increased resistance to aqueous humor outflow. In this study, we have investigated effects of forskolin, which is known to elevate cAMP and also enhance aqueous humor outflow, on myosin light chain (MLC) phosphorylation, a biochemical marker of actin contractility.

Forskolin induces myosin light chain dephosphorylation in bovine trabecular meshwork cells.

Purpose. To determine the role of actin cytoskeleton in the disassembly and reformation of adherens junctions (AJs) and tight junctions (TJs) in bovine corneal endothelial monolayers. Methods. Disassembly and reformation of AJs and TJs were induced by extracellular Ca(2+) depletion and subsequent add-back of Ca(2+), respectively. Resultant changes in the transendothelial electrical resistance (TER), an indicator of integrity of TJs, were measured based on electrical cell-substrate impedance. Phosphorylated myosin light chain (ppMLC), a biochemical measure of actomyosin contraction, and activation of its upstream regulatory molecule RhoA-GTP were assessed by Western blot analysis. Results. Extracellular Ca(2+) depletion led to activation of RhoA, increase in ppMLC, decrease in TER, contraction of the perijunctional actomyosin ring (PAMR), and redistribution of zonula occludens-1 (ZO-1) and cadherins. These effects were reversed on Ca(2+) add-back. Pretreatment with Y-27632 and blebbistatin (as inhibitors of actomyosin contraction) reduced the rate of decline in TER, opposed the contraction of the PAMR, and blocked the redistribution of ZO-1 and cadherins. Both drugs reduced the recovery in TER and opposed the normal redistribution of ZO-1 and cadherins on Ca(2+) add-back. Cytochalasin D, which led to dissolution of the PAMR, also reduced the recovery of TER on Ca(2+) add-back. Conclusions. The (Ca(2+) depletion)-induced disassembly of AJs accelerates the breakdown of TJs through a concomitant increase in the actomyosin contraction of the PAMR. However, these data on reassembly show that a contractile tone of the PAMR is essential for assembly of the apical junctional complex.

Formation and disassembly of adherens and tight junctions in the corneal endothelium: regulation by actomyosin contraction.

Elevated cAMP in the trabecular meshwork (TM) cells increases the aqueous humor outflow facility. The authors investigated the mechanisms by which elevated cAMP opposes the RhoA-Rho kinase pathway, leading to the relaxation of the actomyosin system in bovine TM cells.

Effect of elevated intracellular cAMP levels on actomyosin contraction in bovine trabecular meshwork cells.

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.

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

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.

Charanya Ramachandran

L. V. Prasad Eye Institute

Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration