Immunocytochemical characterization of ex vivo cultured conjunctival explants; marker validation for the identification of squamous epithelial cells and goblet cells

Tissue-engineered products are at the cutting edge of innovation considering their potential to functionally and structurally repair various tissue defects when the body’s own regenerative capacity is exhausted. At the ocular surface, the wound healing response to extensive conjunctival damage results in tissue repair with structural alterations or permanent scar formation rather than regeneration of the physiological conjunctiva. Conjunctival tissue engineering therefore represents a promising therapeutic option to reconstruct the ocular surface in severe cicatrizing pathologies. During the rapid race to be a pioneer, it seems that one of the fundamental steps of tissue engineering has been neglected; a proper cellular characterization of the tissue-engineered equivalents, both morphologically and functionally. Currently, no consensus has been reached on an identification strategy and/or markers for the characterization of cultured squamous epithelial and goblet cells. This study therefore evaluated the accuracy of promising markers to identify differentiated conjunctival-derived cells in human primary explant cultures through immunocytochemistry, including keratins (i.e., K7, K13, and K19) and mucins (i.e., MUC1, MUC5AC, and PAS-positivity). Comparison of the in vivo and in vitro cellular profiles revealed that the widely used goblet cell marker K7 does not function adequately in an in vitro setting. The other investigated markers offer a powerful tool to distinguish cultured squamous epithelial cells (i.e., MUC1 and K13), goblet cells (i.e., MUC5AC and PAS-staining), and conjunctival-derived cells in general (i.e., K19). In conclusion, this study emphasizes the power alongside potential pitfalls of conjunctival markers to assess the clinical safety and efficacy of conjunctival tissue-engineered products

Designer Descemet membranes containing PDLLA and functionalized gelatins as corneal endothelial scaffold

Corneal blindness is the fourth leading cause of visual impairment. Of specific interest is blindness due to a dysfunctional corneal endothelium which can only be treated by transplanting healthy tissue from a deceased donor. Unfortunately, corneal supply does not meet the demand with only one donor for every 70 patients. Therefore, there is a huge interest in tissue engineering of grafts consisting of an ultra-thin scaffold seeded with cultured endothelial cells. The present research describes the fabrication of such artificial Descemet membranes based on the combination of a biodegradable amorphous polyester (poly (d,l-lactic acid)) and crosslinkable gelatins. Four different crosslinkable gelatin derivatives are compared in terms of processing, membrane quality, and function, as well as biological performance in the presence of corneal endothelial cells. The membranes are fabricated through multi-step spincoating, including a sacrificial layer to allow for straightforward membrane detachment after production. As a consequence, ultrathin (90%), semi-permeable membranes could be obtained with high biological potential. The membranes supported the characteristic morphology and correct phenotype of corneal endothelial cells while exhibiting similar proliferation rates as the positive control. As a consequence, the proposed membranes prove to be a promising synthetic alternative to donor tissue

Fish scale-derived scaffolds for culturing human corneal endothelial cells

Purpose. To investigate the biocompatibility of fish scale-derived scaffolds (FSS) with primary human corneal endothelial cells (HCEnCs). Methods. HCEnCs were isolated from 30 donor corneas in a donor-matched study and plated in precoated Lab-Tek slides (n=15) and FSS (n=15). Cell morphology, proliferation/migration, and glucose uptake were studied (n=30). Hoechst, ethidium homodimer, and calcein AM (HEC) staining was performed to determine viability and toxicity (n=6). The cell surface area was calculated based on calcein AM staining. HCEnCs were stained for ZO-1 (n=6) to detect tight junctions and to measure cell morphology; Ki-67 (n=6) to measure proliferating cells; and vinculin to quantify focal adhesions (n=6). The formation of de novo extracellular matrix was analyzed using histology (n=6). Results. HCEnCs attach and grow faster on Lab-Tek slides compared to the undulating topography of the FSS. At day 11, HCEnCs on Lab-Tek slide grew 100% confluent, while FSS was only 65% confluent (p=0.0883), with no significant difference in glucose uptake between the two (p=0.5181) (2.2 μg/mL in Lab-Tek versus 2.05 μg/mL in FSS). HEC staining showed no toxicity. The surface area of the cells in Lab-Tek was 409.1 μm2 compared to 452.2 μm2 on FSS, which was not significant (p=0.5325). ZO-1 showed the presence of tight junctions in both conditions; however, hexagonality was higher (74% in Lab-Tek versus 45% in FSS; p=0.0006) with significantly less polymorphic cells on Lab-Tek slides (8% in Lab-Tek versus 16% in FSS; p=0.0041). Proliferative cells were detected in both conditions (4.6% in Lab-Tek versus 4.2% in FSS; p=0.5922). Vinculin expression was marginally higher in HCEnCs cultured on Lab-Tek (234 versus 199 focal adhesions; p=0.0507). Histological analysis did not show the formation of a basement membrane. Conclusions. HCEnCs cultured on precoated FSS form a monolayer, displaying correct morphology, cytocompatibility, and absence of toxicity. FSS needs further modification in terms of structure and surface chemistry before considering it as a potential carrier for cultured HCEnCs

Magnetron discharge volt-ampere characteristic investigation at thin film coating process

© Published under licence by IOP Publishing Ltd. Magnetron discharge at reactive and working gases mixture atmosphere current-voltage characteristic (I-U) for different sputtering parameters is investigated. It is shown, that form of volt-ampere characteristic doesn't depend on gas supply scheme at vacuum chamber pressure 4- 6.10-2 Pa. Reactive gas (oxygen) flow increasing leads to making I-U transition part wider and amplification of difference between top and bottom parts of hysteresis loop I-U. Discharge voltage is less at oxygen and argon gases mixture atmosphere than at argon atmosphere