Juliane Teichmann

Human corneal endothelial cell sheets for transplantation: Thermo-responsive cell culture carriers to meet cell-specific requirements

Corneal endothelial diseases lead to severe vision impairment, motivating the transplantation of donor corneae or corneal endothelial lamellae, which is, however, impeded by endothelial cell loss during processing. Therefore, one prioritized aim in corneal tissue engineering is the generation of transplantable human corneal endothelial cell (HCEC) layers. Thermo-responsive cell culture carriers are widely used for non-enzymatic harvest of cell sheets. The current study presents a novel thermo-responsive carrier based on simultaneous electron beam immobilization and cross-linking of poly(vinyl methyl ether) (PVME) on polymeric surfaces, which allows one to adjust layer thickness, stiffness, switching amplitude and functionalization with bioactive molecules to meet cell type specific requirements. The efficacy of this approach for HCEC, which require elaborate cell culture conditions and are strongly adherent to the substratum, is demonstrated. The developed method may pave the way to tissue engineering of corneal endothelium and significantly improve therapeutic options.

Surface endotoxin contamination and hemocompatibility evaluation of materials

To evaluate the blood compatibility of new materials, a clear distinction between properties of the materials and effects due to surface contamination by adsorbed endotoxins is essential. This study compares direct contact approaches and elution methods with water, organic solvents, nonionic, and zwitterionic detergents for determination of surface-adsorbed endotoxin by the limulus amoebocyte lysate (LAL) test and determines the blood compatibility of various surfaces with controlled endotoxin contamination in vitro. The LAL test in direct contact with an endotoxin-contaminated surface was concluded to be not practicable for most devices and its sensitivity showed a high dependence on surface characteristics. Among the elution methods, 0.2% Tween-20 showed most stable elution characteristics and appears therefore preferable. Biological reactions at in vitro blood exposure were found to be only minimally influenced by adsorbed endotoxin during the time window of 2 h, allowing for a straightforward discrimination between materials and endotoxin-dependent reactions.

The control of endothelial cell adhesion and migration by shear stress and matrix-substrate anchorage

Endothelial cells constitute the natural inner lining of blood vessels and possess anti-thrombogenic properties. This characteristic is frequently used by seeding endothelial cells on vascular prostheses. As the type of anchorage of adhesion ligands to materials surfaces is known to determine the mechanical balance of adherent cells, we investigated herein the behaviour of endothelial cells under physiological shear stress conditions. The adhesion ligand fibronectin was anchored to polymer surfaces of four physicochemical characteristics exhibiting covalent and non-covalent attachment as well as high and low hydrophobicity. The in situ analysis combined with cell tracking of shear stress-induced effects on cultured isolated cells and monolayers under venous (0.5 dyn/cm(2)) and arterial (12 dyn/cm(2)) shear stress over a time period of 24 h revealed distinct differences in their morphological and migratory features. Most pronounced, unidirectional and bimodal migration patterns of endothelial cells in or against flow direction were found in dependence on the type of substrate-matrix anchorage. Combined by an immunofluorescent analysis of the actin cytoskeleton, cell-cell junctions, cell-matrix adhesions, and matrix reorganization these results revealed a distinct balance of laminar shear stress, cell-cell contacts and substrate-matrix anchorage in affecting endothelial cell fate under flow conditions. This analysis underlines the importance of materials surface parameters as well as primary and secondary adhesion ligand anchorage in the context of artificial blood vessels for future therapeutic devices.

Tissue Engineering of the Corneal Endothelium: A Review of Carrier Materials

Functional impairment of the human corneal endothelium can lead to corneal blindness. In order to meet the high demand for transplants with an appropriate human corneal endothelial cell density as a prerequisite for corneal function, several tissue engineering techniques have been developed to generate transplantable endothelial cell sheets. These approaches range from the use of natural membranes, biological polymers and biosynthetic material compositions, to completely synthetic materials as matrices for corneal endothelial cell sheet generation. This review gives an overview about currently used materials for the generation of transplantable corneal endothelial cell sheets with a special focus on thermo-responsive polymer coatings.

Biodegradable fiducial markers for X-ray imaging – soft tissue integration and biocompatibility

This study aims at the development of materials for biodegradable fiducial markers for X-ray based medical imaging and their anchorage in soft tissue. Towards this goal a degradable polymer matrix of poly(l-lactide-co-ε-caprolactone) (P[LAcoCL]) was combined with barium sulfate (BaSO4) and hydroxyapatite (HAp) as radio-opaque fillers. Low pressure plasma treatment was applied to the composite materials to improve cell adhesion and subsequent tissue integration. In particular, the effects of oxygen and ammonia plasmas were evaluated and compared using X-ray photoelectron spectroscopy, atomic force microscopy and dynamic water contact angle measurements as well as in vitro studies using the murine fibroblast cell line L929. To exclude the cytotoxic effects of degradation products of P[LAcoCL] and released BaSO4 or HAp cytotoxicity assays with the degradation products of the composite materials were conducted. The results obtained by this broad range of analytical techniques suggest the application of composites of P[LAcoCL] with BaSO4 and HAp as promising material systems for innovative fiducial markers for soft tissue in X-ray based medical imaging.

Thermo-responsive cell culture carriers based on poly(vinyl methyl ether)—the effect of biomolecular ligands to balance cell adhesion and stimulated detachment

Abstract Two established material systems for thermally stimulated detachment of adherent cells were combined in a cross-linked polymer blend to merge favorable properties. Through this approach poly(N-isopropylacrylamide) (PNiPAAm) with its superior switching characteristic was paired with a poly(vinyl methyl ether)-based composition that allows adjusting physico-chemical and biomolecular properties in a wide range. Beyond pure PNiPAAm, the proposed thermo-responsive coating provides thickness, stiffness and swelling behavior, as well as an apposite density of reactive sites for biomolecular functionalization, as effective tuning parameters to meet specific requirements of a particular cell type regarding initial adhesion and ease of detachment. To illustrate the strength of this approach, the novel cell culture carrier was applied to generate transplantable sheets of human corneal endothelial cells (HCEC). Sheets were grown, detached, and transferred onto planar targets. Cell morphology, viability and functionality were analyzed by immunocytochemistry and determination of transepithelial electrical resistance (TEER) before and after sheet detachment and transfer. HCEC layers showed regular morphology with appropriate TEER. Cells were positive for function-associated marker proteins ZO-1, Na+/K+-ATPase, and paxillin, and extracellular matrix proteins fibronectin, laminin and collagen type IV before and after transfer. Sheet detachment and transfer did not impair cell viability. Subsequently, a potential application in ophthalmology was demonstrated by transplantation onto de-endothelialized porcine corneas in vitro. The novel thermo-responsive cell culture carrier facilitates the generation and transfer of functional HCEC sheets. This paves the way to generate tissue engineered human corneal endothelium as an alternative transplant source for endothelial keratoplasty.