VEGF-releasing suture material for enhancement of vascularization: development, in vitro and in vivo study.

As it has been demonstrated that bioactive substances can be delivered locally using coated surgical suture materials, the authors developed a vascular endothelial growth factor (VEGF)-releasing suture material that should promote vascularization and potentially wound healing. In this context, the study focused on the characterization of the developed suture material and the verification of its biological activity, as well as establishing a coating process that allows reproducible and stable coating of a commercially available polydioxanone suture material with poly(l-lactide) (PLLA) and 0.1µg and 1.0µg VEGF. The in vitro VEGF release kinetics was studied using a Sandwich ELISA. The biological activity of the released VEGF was investigated in vitro using human umbilical vein endothelial cells. The potential of the VEGF-releasing suture material was also studied in vivo 5days after implantation in the hind limb of Wistar rats, when the histological findings were analyzed. The essential results, enhanced cell viability in vitro as well as significantly increased vascularization in vivo, were achieved using PLLA/1.0µg VEGF-coated suture material. Furthermore, ELISA measurements revealed a high reproducibility of the VEGF release behavior. Based on the results achieved regarding the dose-effect relationship of VEGF, the stability during its processing and the release behavior, it can be predicted that a bioactive suture material would be successful in later in vivo studies. Therefore, this knowledge could be the basis for future studies, where bioactive substances with different modes of action are combined for targeted, overall enhancement of wound healing.

Chemical activation and changes in surface morphology of poly(ε-caprolactone) modulate VEGF responsiveness of human endothelial cells.

The high degree of clinical routine in percutaneous transluminal coronary angioplasty (PTCA) with and without stenting has not changed the fact that a large number of coronary heart disease patients are still affected by post-operative complications such as restenosis and thrombosis. Because re-endothelialization is the crucial aspect of wound healing after cardiovascular implant surgery, there is a need for modern biomaterials to aid endothelial cells in their adhesion and functional recovery post-stenting. This study systematically examines the potential of numerous chemical polymer modifications with regard to endothelialization. Poly(ε-caprolactone) (PCL) and its chemically activated forms are investigated in detail, as well as the impact of polymer surface morphology and precoating with matrix protein. Human umbilical vein endothelial cells (HUVECs) are used to characterize endothelial cell responses in terms of in vitro viability and adhesion. As a potential component in drug eluting implants, VEGF is applied as stimulus to boost endothelial cell proliferation on the polymer. In conclusion, plasma chemical activation of PCL combined with VEGF stimulation best enhances in vitro endothelialization. Examining the impact of morphological, chemical and biological modifications of PCL, this study makes an important new contribution towards the existing body of work on polymer endothelialization.

Permeability of the anterior lens capsule for large molecules and small drugs.

PURPOSE: For developing injectable lenses the retention properties of the capsular bag are important. Therefore the apparent permeability coefficients of sodium fluorescein and fluorescent dextrans of different sizes were determined for the human anterior lens capsule to calculate a molecular weight cutoff from these data. In addition, permeability coefficients of drugs helpful for the suppression of secondary cataract were determined. MATERIALS AND METHODS: Capsulorhexis specimens were fixed in a specially designed two compartment diffusion chamber to investigate the permeation of sodium fluorescein and fluorescent dextrans of different sizes (10, 40, 70 and 150 kDa) for 24 h (n ≥ 3) and of the antiproliferative drugs actinomycin D and methotrexate for 0.5, 24, 48 and 72 h (n ≥ 3). RESULTS: The molecular weight cutoff of the anterior lens capsule was found to be 166 ± 82 kDa. After 0.5 h, no passage of actinomycin D and methotrexate was detectable through the lens capsule. The apparent permeability coefficients for actinomycin D and methotrexate were calculated to 0.71 ± 0.02 µm/s and to 0.80 ± 0.13 µm/s, respectively. CONCLUSIONS: The capsular bag retains fluorescent dextrans with a molecular weight of >166 kDa. Hence, prepolymers are required to polymerize rapidly to be retained inside of the capsular bag. In addition, low-molecular substances intended as antiproliferative drugs for secondary cataract prevention should be applied within a time frame of five minutes in such a way that cells adjacent to the capsular bag will not be damaged.

Ocular fibroblast types differ in their mRNA profiles--implications for fibrosis prevention after aqueous shunt implantation.

PURPOSE: For an aqueous shunt draining from the anterior chamber into the choroidal space, fibroblasts from the choroidea and/or the sclera are most likely responsible for a fibrotic response around the outflow region of such a shunt. The prevention of fibrosis should extend the operating life of the shunt. A detailed characterization of fibroblasts derived from choroidea and sclera should provide information about whether a fibrosis reaction can be inhibited by cell type-specific agents. METHODS: We generated mRNA profiles of fibroblasts from the choroidea, sclera, and Tenon's space by gene array hybridization to provide a basis on which to search for potential pharmacological targets for fibrosis prevention. Hybridization data were analyzed by the Rosetta Resolver system and Limma to obtain mRNA profiles of the three fibroblast types. RESULTS: The three fibroblast types investigated shared fibroblast-specific gene expression patterns concerning extracellular matrix proteins as collagens and fibronectin, but also showed distinct mRNA patterns. CONCLUSIONS: Individual mRNA species overexpressed in one of the fibroblast types might serve as markers for the identification of the fibroblast type in histological analyses. Future in-depth analyses of the gene expression patterns might help identify pharmacological targets for fibrosis prevention.

New concepts for glaucoma implants--controlled aqueous humor drainage, encapsulation prevention and local drug delivery.

Glaucoma is a common cause of blindness in industrialized countries and is the most frequent cause of irreversible blindness worldwide. Since raised intraocular pressure (IOP) has been implicated as the major risk factor, the main goal of all glaucoma treatment is to reduce IOP sufficiently to prevent continuous irreversible retinal ganglion cell damage and progression of visual field loss. Pharmacological reduction of IOP is first-line therapy, followed by laser treatment of the trabecular meshwork and filtering glaucoma surgery, and cyclophotocoagulation of the ciliary body or allogenic implants. The most important glaucoma implants are presented (MOLTENO, AHMED, BAERVELDT, KRUPIN) together with more recent developments (Ex-Press, Eyepass, iStent, Gold micro shunt). Drainage into the suprachoroidal space is a promising option, but is also limited by scarring of the new created outflow route due to proliferation and adhesion of fibroblasts. A deeper understanding of fibroblasts in the related eye compartments is required. Characterization of scleral, choroidal, and, as a reference, Tenon fibroblast subtypes, is possible based on gene expression patterns. Alongside mitomycin C and 5-fluorouracil, newer drugs to prevent fibrosis have been proposed, offering effects that are more specific and more physiological. Effectors involved in wound healing phases and signaling pathways are potential targets for pharmaceutical intervention. Downregulation of growth factors like TGF-ß and their downstream effectors may suppress proliferation and differentiation of fibroblasts, extracellular matrix deposition, wound contraction, and neovascularization. Furthermore, current approaches to local drug delivery in glaucoma implant technology are briefly summarized.

Evaluation of protective effects of trehalose on desiccation of epithelial cells in three dimensional reconstructed human corneal epithelium.

PURPOSE: Trehalose has been shown to protect epithelial cells from desiccation damage in cell culture and the murine dry eye model. The present study evaluates the protective role of trehalose in reconstructed human corneal epithelium (3D-HCE) during desiccation. MATERIALS AND METHODS: The morphology of 3D-HCE was examined using in vivo an ex vivo confocal laser-scanning microscopy (CLSM). The 3D-HCE was desiccated with or without pre-treatment with trehalose. Evaluation of protective role of trehalose was conducted using different in vitro cell viability assays and CLSM. Tissue thickness for each condition was determined by optical coherence tomography (OCT). RESULTS: 3D-HCE tissue revealed similar features with human cornea at histological level. After desiccation the percentage of living cells was only 32% in 3D-HCE tissue without pre-incubation and 98% in trehalose-pre-incubated tissue, as shown by a cell viability assay. These findings were confirmed by using a Live-Dead assay. Also, the confocal immunofluorescence analysis revealed much better preservation of tight junctions in trehalose-pre-treated tissue. CONCLUSIONS: CLSM and an in vitro cell viability assays could be successfully used for the characterization of 3D-HCE tissue. We demonstrated the protective role of trehalose using reconstructed corneal epithelium (3D-HCE), which mimics HCE and has the potential to become a valuable model in ophthalmic research.

Different fibroblast subpopulations of the eye: a therapeutic target to prevent postoperative fibrosis in glaucoma therapy.

The aim of this study is the characterization of fibroblasts mainly responsible for fibrosis processes associated with trabeculectomy or microstent implantation for glaucoma therapy. Therefore we isolated human primary fibroblasts from choroidea, sclera, Tenon capsule, and orbital fat tissues. These fibroblast subpopulations were analysed in vitro for expression of the extracellular matrix components which are responsible for postoperative scarring in glaucoma therapy. For scarring the proteins of the collagen family are predominant and so we focused on the expression of collagen I, collagen III and collagen VI in every fibroblast subpopulation. Also, the extracellular matrix protein fibronectin which crosslinks collagen fibres or other extracellular matrix components and cell surfaces, was analyzed. Collagen I, III and VI were prominent in every fibroblast subpopulation. The highest amounts of collagen III were found in hCF and hOF, whereas the signal in hSF and hTF was negligible. Additionally, there is a link between scarring processes and proliferating potential of fibroblasts, in case of microstent implantation triggered through the infiltration of inflammatory cells. Thus we analyzed fibroblast subpopulations for the presence of TGF-ß1 which is one of the most important cytokines involved in proliferation processes. TGF-ß1 was prominent in all fibroblast subpopulations with lowest expression in hCF cultures. To prevent postoperative fibroblast proliferation we analyzed in vitro the proliferation-inhibitors paclitaxel and mitomycin C which are potential candidates in drug eluting drainage systems on ocular fibroblast subpopulations. These inhibitors arrest fibroblast proliferation and viability, being, however, not very specific and have a cytotoxic potential also on healthy tissues surrounding the microstent outflow area. Significant differences in protein synthesis of fibroblasts subpopulations which could be specific targets for inhibition may help to find out fibroblast specific inhibitors to prevent postoperative scarring and could prevent patients from secondary surgery after microstent implantation.

Surface functionalization of poly(ε-caprolactone) improves its biocompatibility as scaffold material for bioartificial vessel prostheses.

Within this study, chemically modified polymer surfaces were to be developed, which should enhance the subsequent immobilization of various bioactive substances. To improve the hemocompatibility and endothelialization of poly(ε-caprolactone) (PCL) intended as scaffold material for bioartificial vessel prostheses, terminal amino groups via ammonia (NH3) plasma, oxygen (O2) plasma/aminopropyltriethoxysilane (APTES), and 4,4'-methylenebis(phenyl isocyanate) (MDI)/water were provided. Then, immobilization of the anti-inflammatory and antithrombogenic model drug acetylsalicylic acid (ASA) and vascular endothelial growth factor (VEGF) were performed by employing N,N-disuccinimidyl carbonate (DSC) as crosslinker. Contact angle and fluorescence measurements, X-ray photoelectron spectroscopy and infrared spectroscopy confirmed the surface modification. Here the highest functionalization was observed for the O2 plasma/APTES modification. Furthermore, biocompatibility studies demonstrated that the surface reactions have no negative influence, neither on the viability of L929 mouse fibroblasts, nor on primary or secondary hemostasis. Release studies showed that the immobilization of ASA and VEGF on the modified PCL surface via DSC is greatly improved compared to the adsorption-only reference. The advantage of DSC is that it immobilizes both bioactive substances via non-hydrolyzable and/or hydrolyzable covalent bonding. The highest ASA loading and cumulative release was detected using NH3 plasma-activated PCL samples. For VEGF, the O2 plasma/APTES-modified PCL samples were most efficient with regard to loading and cumulative release. In conclusion, both modifications are promising methods to optimize PCL as scaffold material for bioartificial vessel prostheses.

Nuclear entry of hyperbranched polylysine nanoparticles into cochlear cells.

BACKGROUND: Gene therapy is a potentially effective therapeutic modality for treating sensorineural hearing loss. Nonviral gene delivery vectors are expected to become extremely safe and convenient, and nanoparticles are the most promising types of vectors. However, infrequent nuclear localization in the cochlear cells limits their application for gene therapy. This study aimed to investigate the potential nuclear entry of hyperbranched polylysine nanoparticles (HPNPs) for gene delivery to cochlear targets. METHODS: Rat primary cochlear cells and cochlear explants generated from newborn rats were treated with different concentrations of HPNPs. For the in vivo study, HPNPs were administered to the rats' round window membranes. Subcellular distribution of HPNPs in different cell populations was observed with confocal microscope 24 hours after administration. RESULTS: Nuclear entry was observed in various cochlear cell types in vitro and in vivo. In the primary cochlear cell culture, concentration-dependent internalization was observed. In the cochlear organotypic culture, abundant HPNPs were found in the modiolus, including the spiral ganglion, organ of Corti, and lateral wall tissues. In the in vivo study, a gradient distribution of HPNPs through different layers of the round window membrane was observed. HPNPs were also distributed in the cells of the middle ear tissue. Additionally, efficient internalization of HPNPs was observed in the organ of Corti and spiral ganglion cells. In primary cochlear cells, HPNPs induced higher transfection efficiency than did Lipofectamine(™). CONCLUSION: These results suggest that HPNPs are potentially an ideal carrier for gene delivery into the cochlea.

Polymers and drugs suitable for the development of a drug delivery drainage system in glaucoma surgery.

Implantation of a glaucoma drainage system is an appropriate therapeutic intervention in some glaucoma patients. However, one drawback with this approach is the fibrotic tissue response to the implant material, leading to reduced flow of aqueous liquid or complete blockage of the drainage system. As a basis for developing an aqueous shunt we report here investigations with poly(3-hydroxybutyrate) (P(3HB)) and poly(4-hydroxybutyrate) (P(4HB)) as polymer matrices and with paclitaxel (PTX) and triamcinolone acetonide (TA) as drugs that might, in combination, delay or prevent the process of fibrosis by reducing fibroblast activity. P(3HB) and P(4HB) were fabricated into test prototypes with 500 µm outer and 200 µm inner diameter and ∼1 cm length. The antiproliferative agent PTX and the anti-inflammatory agent TA were incorporated into the polymer matrices and were released by diffusion. In vitro cell assays demonstrated that the polymers have the potential to reduce fibroblast viability, while TA showed differential inhibition of Tenon fibroblasts, but not cornea keratocytes. Implantation of polymer disks and prototype devices into rabbit eyes confirmed the good biocompatibility of the materials. The combined use of a poly(hydroxybutyrate) polymer with PTX or TA has the potential to reduce the fibrosis associated with conventional glaucoma drainage systems.

Suitability and calibration of a rebound tonometer to measure IOP in rabbit and pig eyes.

OBJECTIVE: To utilize the Icare tonometer TAO1 for intraocular pressure (IOP) determination in experimental animals. To calculate true IOP calibration functions for rabbit and porcine eyes. ANIMALS: Enucleated eyes of 3-year-old healthy experimental rabbits (New Zealand white), and healthy 1 year old experimental pigs (Deutsche Landrasse) were used for the determination of IOP. PROCEDURES: Manometric (Geuder GmbH, Heidelberg/Germany) and rebound tonometry (Icare tonometer TAO1, Icare, Helsinki/Finland) were used to record IOP in enucleated animal eyes (rabbit n=2, pig n=3). RESULTS: The Icare tonometer TAO1 measurements underestimated true IOP by 37-60% in rabbit eyes and 17-63% in porcine eyes. IOP values obtained by both rebound and manometric tonometry for rabbit and porcine eyes followed a linear regression curve. Linear functions were calculated to correct the Icare tonometer TAO1 measurements to true IOP for both rabbit (p=1.4244p(ic) + 4.2421) and porcine eyes (p=1.0799p(ic) + 5.8557). CONCLUSIONS: The Icare tonometer TAO1 can be utilized for IOP determination in rabbit and porcine eyes when measured values are corrected with the appropriate linear function.

Biodegradable sirolimus-loaded poly(lactide) nanoparticles as drug delivery system for the prevention of in-stent restenosis in coronary stent application.

The administration of drugs using biodegradable polymer nanoparticles as carriers has generated immense interest due to their excellent biocompatibility and the prolonged drug release. The scope of this work was to determine the applicability of sirolimus-loaded biodegradable poly(D,L-lactide) (PDLLA) nanoparticles as drug carriers to prevent restenotic processes after stent implantation. The average 250 nm sized 20%(w/w) sirolimus-loaded nanoparticles were extensively characterized with regard to in vitro degradation, biocompatibility and in vitro drug release. The particles show biphasic release kinetics consisting of a short burst release of 50%(w/w) sirolimus payload, followed by a longer, slower release phase, which are desirable for the application as a drug delivery carrier. All presented results exhibit the potential of sirolimus-loaded PDLLA nanoparticles as promising local and sustained drug delivery systems administered intraluminally to reduce in-stent restenosis after stent implantation.

Inner ear biocompatibility of lipid nanocapsules after round window membrane application.

Nanoparticle-mediated drug delivery represents the future in terms of treating inner ear diseases. Lipid core nanocapsules (LNCs), 50 nm in size, were shown to pass though the round window membrane (RWM) and reached the spiral ganglion cells and nerve fibers, among other cell types in the inner ear. The present study aimed to evaluate the toxicity of the LNCs in vitro and in vivo, utilizing intact round window membrane delivery in rats. The primary cochlear cells and mouse fibroblast cells treated with LNCs displayed dosage dependant toxicity. In vivo study showed that administration of LNCs did not cause hearing loss, nanoparticle application-related cell death, or morphological changes in the inner ear, at up to 28 days of observation. The cochlear neural elements, such as synaptophysin, ribbon synapses, and S-100, were not affected by the administration of LNCs. However, expression of neurofilament-200 decreased in SGCs and in cochlear nerve in osseous spiral lamina canal after LNC delivery, a phenomenon that requires further investigation. LNCs are potential vectors for the delivery of drugs to the inner ear.

Drug-induced secondary cataract prevention: experimental ex vivo and in vivo results with disulfiram, methotrexate and actinomycin D.

BACKGROUND/AIMS: A clinical approach to prevent secondary cataract after lens implantation involves the intraocular application of pharmacological agents. The goals of our study were to develop an ex vivo model to test the drug effectiveness for lens epithelial cell ablation from the basal membrane and to verify the data in rabbit intraocular lens implantation experiments. METHODS: Human capsular rhexis specimens were incubated with drugs and the residual cells were differentiated by use of the Live-Dead assay and quantified by staining with Hoechst dye. After phakoemulsification of rabbit eyes and before intraocular lens implantation, capsular bags were filled with drug-loaded hyaluronic acid for 5 min. RESULTS: An ex vivo model was established which allows the testing of drugs on lens epithelial cell ablation. Drug treatment reduced the number of viable cells on the specimens drastically, ranging between 0.44 ± 0.53% (6.0 ± 7.3 cells/mm²) for disulfiram, 0.27 ± 0.50% (3.7 ± 6.9 cells/mm²) for methotrexate and 0.07 ± 0.19% (0.1 ± 0.27 cells/mm²) for actinomycin D. Rabbit eyes treated with a mixture of methotrexate/actinomycin D showed no posterior capsule opacification at 4 months and a low opacification 6 months postoperatively. Without drug treatment low opacification starts 6 weeks postoperatively. CONCLUSIONS: The drug screening in the described ex vivo model can help to reduce the number of preclinical studies for secondary cataract prevention. The successful ex vivo cell ablation by methotrexate/actinomycin D was confirmed by a delayed in vivo secondary cataract formation.

Expression of connexin26 in islets of Langerhans is associated with impaired glucose tolerance in patients with pancreatic adenocarcinoma.

OBJECTIVES: Impairment of glucose tolerance is one of the leading clinical presentations in patients with pancreatic carcinoma. The mechanism of disturbed glucose metabolism, however, is still under debate. Using microarray technology, key mechanisms of deregulated molecular functions of cancer cell-specific mRNAs and tumor-induced mRNAs in peritumorous tissue should be identified in pancreatic ductal adenocarcinoma (PDAC) by comparison to chronic pancreatitis and normal pancreas. METHODS: Forty-three mRNAs were abundant in tissue specimens of patients operated due to pancreatic carcinoma but absent or of low abundance in chronic pancreatitis and normal pancreas. One of these mRNAs encodes the gap junction protein connexin26, known as a tumor suppressor, which was 10.8- and 6.9-fold more abundant in pancreatic carcinoma than in normal pancreas and chronic pancreatitis, respectively. Quantitative RT-PCR was performed for connexin26, with mRNA being expressed 26.7- and 2.9-fold more than in normal pancreas (n = 6), in pancreatic carcinoma (n = 7), and chronic pancreatitis (n = 8), respectively. RESULTS: By immunohistochemistry, connexin26 was predominantly localized to the islets in the vicinity of the pancreatic carcinoma tissue. Control sections of tissue with chronic pancreatitis and normal pancreas show connexin26 expression in the islets as well. Interestingly, the level of mRNA abundance (fold over normal pancreas) in RT-PCR correlates (r = 0.62) with the 2h value of the pre-operative oral glucose tolerance test of these patients. CONCLUSION: Whether overexpressed connexin26 in pancreatic cancer is a cause of impaired glucose tolerance remains to be elucidated in further experimental studies.

Biomaterial implants induce the inflammation marker CRP at the site of implantation.

Following implantation of biomaterial patches into the gastrointestinal tract, we analyzed the host's response towards the foreign material. Asymmetric patches of polydioxanone covered Vicryl or poly-3-hydroxybutyrate were sutured onto the rat stomach. Tissue samples were generated at distinct time intervals after surgery, and RNA profiles were compared by Differential Display. RT-PCR analysis of gene candidates that seemed differentially expressed showed that vitamin D binding protein mRNA was induced in stomach tissue after implantation of the biomaterial patches. In parallel, the amount of C-reactive protein mRNA was found to be increased transiently as well. Implants induce a tissue response that is specific for a given material.

Biomaterial patches sutured onto the rat stomach induce a set of genes encoding pancreatic enzymes.

Asymmetric patches of polyhydroxybutyric acid with one smooth and one rough surface were produced by a dipping procedure. These patches were implanted into the rat gastrointestine and tissue samples were generated at distinct time intervals after surgery. The host's response towards the foreign material was analyzed by Differential Display and RNA profiles were compared to each other. One to two weeks after surgery a group of mRNAs encoding pancreatic enzymes was transiently present after biomaterial implantation.