Hornhaut-Transplantation in Hochrisikosituationen

In diesem Kapitel werden Vorgehensweisen zur Durchführung von Keratoplastiken in Hochrisikosituationen sowie alternative (z.B. lamellierende) Ansätze vorgestellt. Die historischen und molekularen Hintergründe (Histokompatibilitätsantigene) sowie damit verbundene Risikofaktoren (Neovaskularisation der Hornhaut, Pathologie der Augenoberfläche, Hornhaut-Retransplantationen, Größe des Hornhaut-Transplantats, intraokuläre Chirurgie, Entzündung des vorderen Segments, Herpes simplex, Alter des Patienten) werden erläutert. Das postoperative Management, das die Behandlung von Herpes simplex, die systemische Behandlung von Hochrisikopatienten sowie die Auswahl des Spendergewebes durch Human-Leukozyten-Antigen-Matching einschließt, wird beschrieben. Lamellierende Keratoplastikverfahren werden als Alternativen zur konventionellen perforierenden Keratoplastik besprochen. Die Vor- und Nachteile sowohl der vorderen als auch der hinteren lamellierenden Keratoplastik werden skizziert

Examining the Transmission of Visible Light through Electrospun Nanofibrous PCL Scaffolds for Corneal Tissue Engineering

The transparency of nanofibrous scaffolds is of highest interest for potential applications like corneal wound dressings in corneal tissue engineering. In this study, we provide a detailed analysis of light transmission through electrospun polycaprolactone (PCL) scaffolds. PCL scaffolds were produced via electrospinning, with fiber diameters in the range from (35 ± 13) nm to (167 ± 35) nm. Light transmission measurements were conducted using UV–vis spectroscopy in the range of visible light and analyzed with respect to the influence of scaffold thickness, fiber diameter, and surrounding medium. Contour plots were compiled for a straightforward access to light transmission values for arbitrary scaffold thicknesses. Depending on the fiber diameter, transmission values between 15% and 75% were observed for scaffold thicknesses of 10 µm. With a decreasing fiber diameter, light transmission could be improved, as well as with matching refractive indices of fiber material and medium. For corneal tissue engineering, scaffolds should be designed as thin as possible and fabricated from polymers with a matching refractive index to that of the human cornea. Concerning fiber diameter, smaller fiber diameters should be favored for maximizing graft transparency. Finally, a novel, semi-empirical formulation of light transmission through nanofibrous scaffolds is presented

Electrospun PCL Scaffolds as Drug Carrier for Corneal Wound Dressing Using Layer-by-Layer Coating of Hyaluronic Acid and Heparin

Due to its ability to reduce scarring and inflammation, human amniotic membrane is a widely used graft for wound dressings after corneal surgery. To overcome donor dependency and biological variances in the donor tissue, artificial nanofibrous grafts acting as drug carrier systems are promising substitutes. Electrospun nanofibrous scaffolds seem to be an appropriate approach as they offer the properties of permeable scaffolds with a high specific surface, the latter one depending on the fiber diameter. Electrospun scaffolds with fiber diameter of 35 nm, 113 nm, 167 nm and 549 nm were manufactured and coated by the layer-by-layer (LbL) technology with either hyaluronic acid or heparin for enhanced regeneration of corneal tissue after surgery. Studies on drug loading capacity and release kinetics defined a lower limit for nanofibrous scaffolds for effective drug loading. Additionally, scaffold characteristics and resulting mechanical properties from the application-oriented characterization of suture pullout from suture retention tests were examined. Finally, scaffolds consisting of nanofibers with a mean fiber diameter of 113 nm were identified as the best-performing scaffolds, concerning drug loading efficiency and resistance against suture pullout

Self-Complementary Adeno-Associated Virus Vectors Improve Transduction Efficiency of Corneal Endothelial Cells.

Transplantation of a donor cornea to restore vision is the most frequently performed transplantation in the world. Corneal endothelial cells (CEC) are crucial for the outcome of a graft as they maintain corneal transparency and avoid graft failure due to corneal opaqueness. Given the characteristic of being a monolayer and in direct contact with culture medium during cultivation in eye banks, CEC are specifically suitable for gene therapeutic approaches prior to transplantation. Recombinant adeno-associated virus 2 (rAAV2) vectors represent a promising tool for gene therapy of CEC. However, high vector titers are needed to achieve sufficient gene expression. One of the rate-limiting steps for transgene expression is the conversion of single-stranded (ss-) DNA vector genome into double-stranded (ds-) DNA. This step can be bypassed by using self-complementary (sc-) AAV2 vectors. Aim of this study was to compare for the first time transduction efficiencies of ss- and scAAV2 vectors in CEC. For this purpose AAV2 vectors containing enhanced green fluorescent protein (GFP) as transgene were used. Both in CEC and in donor corneas, transduction with scAAV2 resulted in significantly higher transgene expression compared to ssAAV2. The difference in transduction efficiency decreased with increasing vector titer. In most cases, only half the vector titer of scAAV2 was required for equal or higher gene expression rates than those of ssAAV2. In human donor corneas, GFP expression was 64.7±11.3% (scAAV) and 38.0±8.6% (ssAAV) (p<0.001), respectively. Furthermore, transduced cells maintained their viability and showed regular morphology. Working together with regulatory authorities, a translation of AAV2 vector-mediated gene therapy to achieve a temporary protection of corneal allografts during cultivation and transplantation could therefore become more realistic

Schwann Cells Promote Myogenic Differentiation of Myoblasts and Adipogenic Mesenchymal Stromal Cells on Poly-ɛ-Caprolactone-Collagen I-Nanofibers

For the purpose of skeletal muscle tissue engineering, different cell types have been investigated regarding their myogenic differentiation potential, including co-cultured myoblasts and adipogenic mesenchymal stromal cells (Mb/ADSC). As neural cells enhance synaptic junction formation, the aim of this study was to co-culture Schwann cells (SCs) with Mb/ADSC on biocompatible electrospun aligned poly-ε-polycaprolacton (PCL)-collagen I-nanofibers. It was hypothesized that SCs, as part of the peripheral nervous system, promote the myogenic differentiation of Mb/ADSC co-cultures. Mb/ADSC were compared to Mb/ADSC/SC regarding their capacity for myogenic differentiation via immunofluorescent staining and gene expression of myogenic markers. Mb/ADSC/SC showed more myotubes after 28 days of differentiation (p ≤ 0.05). After 28 days of differentiation on electrospun aligned PCL-collagen I-nanofibers, gene expression of myosin heavy chains (MYH2) and myogenin (MYOG) was upregulated in Mb/ADSC/SC compared to Mb/ADSC (p ≤ 0.01 and p ≤ 0.05, respectively). Immunofluorescent staining for MHC showed highly aligned multinucleated cells as possible myotube formation in Mb/ADSC/SC. In conclusion, SCs promote myogenic differentiation of Mb/ADSC. The co-culture of primary Mb/ADSC/SC on PCL-collagen I-nanofibers serves as a physiological model for skeletal muscle tissue engineering, applicable to future clinical applications

Effect of vector titer on transduction efficiency and cell viability (measured by flow cytometry two days after transduction).

<p>Means and standard deviations of three independent experiments are shown. (A) Transduced = GFP-positive cells. Transduction with scAAV2 resulted in significantly higher GFP expression rates compared to ssAAV2 (** = p<0.01, *** = p<0.001). (B) Dead = 7-AAD-positive cells. There were no significant differences between cells transduced with sc- and ssAAV2 or between transduced cells and negative controls (one-way ANOVA, p = 0.062). (C) Apoptotic = PO-PRO-1-positive cells. There were no significant differences between cells transduced with sc- and ssAAV2 or between transduced cells and negative controls (one-way ANOVA, p = 0.578).</p

Metabolic activity of transduced cells as a sign of cell viability (analyzed by MTT-assay two days after transduction).

<p>Means and standard deviations of three independent experiments are shown. There was no significant difference between transduced cells and negative controls (one-way ANOVA; p = 0.415).</p

Confocal microscopy images of endothelial cells of human donor corneas six days after transduction.

<p>(A) transduced with 100,000 GOI ssAAV2; (B) transduced with 100,000 GOI scAAV2. Six random microscopic fields per corneal piece were evaluated. For each vector, one exemplary microscopic field is shown (blue = DAPI, green = GFP, pink = Alexa 647, red = TUNEL).</p

Corneal Endothelial Cells Are Protected from Apoptosis by Gene Therapy

Corneal grafting is the most prevalent form of transplantation. Corneal endothelial cells (ECs), which form a monolayer of the cornea with minimal proliferative potential, are pivotal for maintenance of corneal clarity. Loss of EC viability and apoptosis leads to graft failure posttransplantation and reduces the quality of donor corneas in storage, such that up to 30% do not meet selection criteria and must be discarded. The current study investigates antiapoptotic effects of transduced mammalian Bcl-xL and baculoviral p35 on human ECs. Multiple apoptotic cell features are observed while inducing apoptosis either via the extrinsic (death receptor) or intrinsic (mitochondrial) apoptotic pathway. Human ECs were studied under three experimental conditions: (1) as an immortalized cell line, (2) as primary cells, and (3) in an intact cornea. Interestingly, in primary EC suspensions, Bcl-xL was protective against apoptosis mediated via both pathways. However, p35 was significantly more protective against apoptosis mediated via the intrinsic pathway compared with Bcl-xL. Our results provide critical insight into the role of apoptotic pathways in the maintenance of EC viability and the efficacy with which these protective proteins exert their effect. These observations could form the basis for future applications of antiapoptotic gene therapy to corneal preservation aiming to reduce both graft failure after transplantation as well as donor corneal damage during storage

GFP expression of HCEC-12 cells five days after transduction (flow cytometry).

<p>Means and standard deviations of three independent experiments are shown. GFP expression after transduction with scAAV was significantly higher compared to ssAAV2 (* = p<0.05, ** = p<0.01).</p