RESUMO
Intraocular drug delivery is a promising approach for treatment of ocular diseases. Chemotherapeutic drugs used in retinoblastoma (RB) treatment often lead to side effects and drug resistances. Therefore, new adjuvant therapies are needed to treat chemoresistant RBs. Biocompatible gold nanoparticles (GNPs) have unique antiangiogenic properties and can inhibit cancer progression. The combination of gold and low-molecular-weight hyaluronan (HA) enhances the stability of GNPs and promotes the distribution across ocular barriers. Attached to HA-GNPs, the atrial natriuretic peptide (ANP), which diminishes neovascularization in the eye, is a promising new therapeutic agent for RB treatment. In the study presented, we established ANP-coupled HA-GNPs and investigated their effect on the tumor formation potential of chemoresistant RB cells in an in ovo chicken chorioallantoic membrane model and an orthotopic in vivo RB rat eye model. Treatment of etoposide-resistant RB cells with ANP-HA-GNPs in ovo resulted in significantly reduced tumor growth and angiogenesis compared with controls. The antitumorigenic effect could be verified in the rat eye model, including a noninvasive application form via eye drops. Our data suggest that ANP-HA-GNPs represent a new minimally invasive, adjuvant treatment option for RB.
Assuntos
Nanopartículas Metálicas , Neoplasias da Retina , Retinoblastoma , Animais , Ratos , Fator Natriurético Atrial/farmacologia , Ouro/farmacologia , Ouro/química , Ácido Hialurônico/farmacologia , Nanopartículas Metálicas/química , Retinoblastoma/tratamento farmacológico , Retinoblastoma/patologiaRESUMO
Repurposing of the anthelminthic drug niclosamide was proposed as an effective treatment for inflammatory airway diseases such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease. Niclosamide may also be effective for the treatment of viral respiratory infections, such as SARS-CoV-2, respiratory syncytial virus, and influenza. While systemic application of niclosamide may lead to unwanted side effects, local administration via aerosol may circumvent these problems, particularly when the drug is encapsulated into small polyethylene glycol (PEG) hydrospheres. In the present study, we examined whether PEG-encapsulated niclosamide inhibits the production of mucus and affects the pro-inflammatory mediator CLCA1 in mouse airways in vivo, while effects on mucociliary clearance were assessed in excised mouse tracheas. The potential of encapsulated niclosamide to inhibit TMEM16A whole-cell Cl- currents and intracellular Ca2+ signalling was assessed in airway epithelial cells in vitro. We achieved encapsulation of niclosamide in PEG-microspheres and PEG-nanospheres (Niclo-spheres). When applied to asthmatic mice via intratracheal instillation, Niclo-spheres strongly attenuated overproduction of mucus, inhibited secretion of the major proinflammatory mediator CLCA1, and improved mucociliary clearance in tracheas ex vivo. These effects were comparable for niclosamide encapsulated in PEG-nanospheres and PEG-microspheres. Niclo-spheres inhibited the Ca2+ activated Cl- channel TMEM16A and attenuated mucus production in CFBE and Calu-3 human airway epithelial cells. Both inhibitory effects were explained by a pronounced inhibition of intracellular Ca2+ signals. The data indicate that poorly dissolvable compounds such as niclosamide can be encapsulated in PEG-microspheres/nanospheres and deposited locally on the airway epithelium as encapsulated drugs, which may be advantageous over systemic application.
Assuntos
Niclosamida/administração & dosagem , Pneumonia/tratamento farmacológico , Sistema Respiratório/efeitos dos fármacos , Animais , Asma/tratamento farmacológico , Asma/metabolismo , Asma/patologia , COVID-19/complicações , Células Cultivadas , Modelos Animais de Doenças , Portadores de Fármacos/química , Composição de Medicamentos , Humanos , Hidrogéis/química , Instilação de Medicamentos , Camundongos , Microesferas , Muco/efeitos dos fármacos , Muco/metabolismo , Nanosferas/administração & dosagem , Nanosferas/química , Niclosamida/química , Niclosamida/farmacocinética , Pneumonia/patologia , Polietilenoglicóis/química , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/metabolismo , Sistema Respiratório/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Traqueia , Tratamento Farmacológico da COVID-19RESUMO
Dental pulp tissue engineering is possible after insertion of pulpal stem cells combined with a scaffold into empty root canals. Commonly used biomaterials are collagen or poly(lactic) acid, which are either difficult to modify or to insert into such a narrow space. New hydrogel scaffolds with bioactive, specifically tailored functions could optimize the conditions for this approach. Different synthetic and natural hydrogels were tested for their suitability to engineer dental pulp. Two functionalized modifications of polyethylene glycol were developed in this study and compared to a self-assembling peptide, as well as to collagen and fibrin. Cell viability of dental pulp stem cells in test materials was assessed over two weeks. Cells in selected test materials laden with dentin-derived growth factors were inserted into human tooth roots and implanted subcutaneously into immunocompromised mice. In vitro cell culture exhibited distinct differences between scaffold types, where viability was significantly higher in natural compared to synthetic materials. In vivo experiments showed considerable differences regarding scaffold degradation, soft tissue formation, vascularization, and odontoblast-like cell differentiation. Fibrin appeared most suitable to enable generation of a pulp-like tissue and differentiation of cells into odontoblasts at the cell-dentin interface. In conclusion, natural materials, especially fibrin, proved to be superior compared to synthetic scaffolds regarding cell viability and dental pulp-like tissue formation.
Assuntos
Materiais Biocompatíveis/química , Polpa Dentária/citologia , Engenharia Tecidual/métodos , Animais , Materiais Biocompatíveis/farmacologia , Diferenciação Celular/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Colágeno/química , Dentina/química , Feminino , Fibrina/química , Humanos , Hidrogéis/química , Hidrogéis/farmacologia , Camundongos , Odontoblastos/citologia , Polietilenoglicóis/química , Células-Tronco/citologia , Alicerces Teciduais/químicaRESUMO
Podocytes are an essential component of the glomerular filtration barrier and cover the outer aspect of glomerular capillaries. They form a complex actin-based cytoskeleton in vivo and show prominent motility in vitro, but whether podocytes are stationary or mobile in vivo is debated. To address this question, the pronephros of translucent zebrafish larvae (casper) expressing enhanced green fluorescent protein (eGFP) specifically in podocytes (wt1a:eGFP larvae) was observed by intravital two-photon microscopy over extended periods of time. Podocyte cell bodies and the interdigitating branching pattern of major processes could be resolved with a resolution of approximately 1 µm in the xy-plane. Time-lapse imaging of zebrafish larvae at 5-7 days after fertilization demonstrated that podocytes neither migrated nor changed the branching pattern of their major processes over a time period of up to 23 hours. In summary, we show by extended intravital two-photon microscopy that podocytes are stationary cells in the intact glomerulus of a translucent zebrafish with fluorescently-labeled podocytes.
Assuntos
Microscopia/métodos , Podócitos/citologia , Peixe-Zebra/anatomia & histologia , Animais , Larva/citologiaRESUMO
Polycations like poly(ethylene imine) (PEI) or poly(L-lysine) (pLL) form nanometer-sized complexes with nucleic acids (polyplexes) which can be used for gene delivery. It is known that the properties of these -carriers can be greatly improved by introducing disulfide bridges on the polymers, thus making them reduction sensitive. However, little is known about how such modified carriers behave intracellularly. Here, we describe a method that uses the reduction-sensitive fluorescent dye BODIPY FL L-cystine to label PEI and pLL. Our probe is activated under reductive conditions leading to strongly increased fluorescence intensity. Subsequently, we show how the intracellular route of polyplexes made from these labeled polymers can be monitored by flow cytometry.
Assuntos
Citometria de Fluxo/métodos , Vetores Genéticos , Espectrometria de Fluorescência/métodos , Animais , Células CHO , CricetinaeRESUMO
The delivery of nucleic acids to mammalian cells requires a potent particulate carrier system. The physicochemical properties of the used particles, such as size and surface charge, strongly influence the cellular uptake and thereby the extent of the subsequent biological effect. However the knowledge of this process is still fragmentary because heterogeneous particle collectives are applied. Therefore we present a strategy to synthesize carriers with a highly specific appearance on the basis of gold nanoparticles (AuNPs) and the Layer-by-Layer (LbL) technique. The LbL method is based on the alternate deposition of oppositely charged (bio-)polymers, in our case poly(ethylenimine) and nucleic acids. The size and surface charge of those particles can be easily modified and accordingly systematic studies on cellular uptake are accessible.
Assuntos
DNA/metabolismo , Portadores de Fármacos/química , Técnicas de Transferência de Genes , Ouro/química , Nanopartículas Metálicas/química , RNA/metabolismo , DNA/genética , Ácidos Graxos/química , Polietilenoimina/química , RNA/genética , Compostos de Sulfidrila/químicaRESUMO
The objective of the present study was to investigate ionic interactions between alginate and a monoclonal antibody (mAb1) and to utilize those interactions for the sustained release of mAb1. The existence of ionic interactions between alginate and mAb1 was strongly reflected by their rheological behavior. A 3-4 times increase in storage modulus (G') was observed by addition of 30 mg/mL mAb1 to a 20 mg/mL alginate solution. This increase was strongly dependent on pH and ionic strength. In vitro release studies revealed a marked pH-dependence of release rates and the reversibility of alginate-mAb1 complexation under physiological conditions. Two alginate-mAb1 sustained release formulations were developed by an internal gelation technique using CaCO(3) and CaHPO(4) as calcium sources for physical cross-linking. The CaCO(3) formulation provided a stable pH-environment, optimally suited for pH-sensitive proteins. CaHPO(4) led to a lower pH and stronger alginate-mAb1 interactions. The CaHPO(4) cross-linked alginate released mAb1 over a period of 10-15 days. The long release period and changes in viscoelastic properties of alginate, when being mixed with mAb1, indicate the incorporation of mAb1 molecules into a mixed network with alginate. The results of this study demonstrate that ionic interactions between polyanions and mAb1 are present and that they can be exploited for sustained release delivery of mAb1.
Assuntos
Anticorpos Monoclonais/metabolismo , Preparações de Ação Retardada/metabolismo , Polímeros/metabolismo , Proteínas Recombinantes/metabolismo , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/química , Preparações de Ação Retardada/administração & dosagem , Preparações de Ação Retardada/química , Humanos , Polieletrólitos , Polímeros/administração & dosagem , Polímeros/química , Ligação Proteica/fisiologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/química , Reologia/métodosRESUMO
Several nanocarrier systems are frequently used in modern pharmaceutical therapies. Within this study a potential toxicity risk of all nanoscaled drug delivery systems was found. An accumulation of several structurally different nanocarriers but not of soluble polymers was detected in rodent ovaries after intravenous (i.v.) administration. Studies in different mouse species and Wistar rats were conducted and a high local accumulation of nanoparticles, nanocapsules and nanoemulsions in specific locations of the ovaries was found in all animals. We characterised the enrichment by in vivo and ex vivo multispectral fluorescence imaging and confocal laser scanning microscopy. The findings of this study emphasise the role of early and comprehensive in vivo studies in pharmaceutical research. Nanocarrier accumulation in the ovaries may also comprise an important toxicity issue in humans but the results might as well open a new field of targeted ovarian therapies.
Assuntos
Portadores de Fármacos/toxicidade , Nanopartículas/toxicidade , Ovário/efeitos dos fármacos , Ovário/metabolismo , Animais , Portadores de Fármacos/farmacocinética , Feminino , Corantes Fluorescentes , Fluorometria , Injeções Intravenosas , Masculino , Camundongos , Camundongos Endogâmicos , Microscopia Confocal , Tamanho da Partícula , Ratos , Ratos Wistar , Risco , Especificidade da Espécie , Testículo/efeitos dos fármacos , Testículo/metabolismo , Distribuição TecidualRESUMO
The purpose of this feasibility study was to evaluate two novel techniques facilitating bone cement repair of ossicular discontinuity between the incus and stapes. An isolated damage of the long incus process can be repaired using bone cement. However, bridging of a large gap between incus remnant and stapes head with bone cement is difficult, since viscous cement is not stable and the wet cement bridge may collapse. Ten fresh-frozen cadaveric human temporal bones were used. The long process of the incus was subtotally resected. A novel instrument and polylactide acid (PLA) scaffolds were applied to support ossicular reconstruction with bone cement. Stability of cement bridging was tested by checking for a round window reflex or motion of the stapes by palpating the malleus handle. Both the instrument as well as the PLA scaffolds were relatively easy to insert into the middle ear. However, bone cement adhered to the instrument irrespective of cement viscosity and contact time of the instrument with the ossicles. The bone cement plug had to be detached and sculptured. By contrast, PLA scaffolds could be used in a standardized manner and generated stable cement reconstructions. Curved PLA scaffolds were superior to straight ones. Initial results in cadaveric human temporal bones suggest that implantable PLA scaffolds might be suitable to support bone cement repair, even in very large defects of the long incus process.
Assuntos
Cimentos Ósseos , Bigorna/cirurgia , Prótese Ossicular , Procedimentos de Cirurgia Plástica/métodos , Osso Temporal/cirurgia , Cadáver , Estudos de Viabilidade , Perda Auditiva Condutiva/patologia , Perda Auditiva Condutiva/cirurgia , Humanos , Bigorna/patologia , Desenho de PróteseRESUMO
Cancer therapies are often terminated due to serious side effects of the drugs. The cause is the nonspecific distribution of chemotherapeutic agents to both cancerous and normal cells. Therefore, drug carriers which deliver their toxic cargo specific to cancer cells are needed. Size is one key parameter for the nanoparticle accumulation in tumor tissues. In the present study the influence of the size of biodegradable nanoparticles was investigated in detail, combining in vivo and ex vivo analysis with comprehensive particle size characterizations. Polyethylene glycol-polyesters poly(lactide) block polymers were synthesized and used for the production of three defined, stable, and nontoxic near-infrared (NIR) dye-loaded nanoparticle batches. Size analysis based on asymmetrical field flow field fractionation coupled with multiangle laser light scattering and photon correlation spectroscopy (PCS) revealed narrow size distribution and permitted accurate size evaluations. Furthermore, this study demonstrates the constraints of particle size data only obtained by PCS. By the multispectral analysis of the Maestro in vivo imaging system the in vivo fate of the nanoparticles next to their accumulation in special red fluorescent DsRed2 expressing HT29 xenografts could be followed. This simultaneous imaging in addition to confocal microscopy studies revealed information about the accumulation characteristics of nanoparticles inside the tumor tissues. This knowledge was further combined with extended size-dependent fluorescence imaging studies at two different xenograft tumor types, the HT29 (colorectal carcinoma) and the A2780 (ovarian carcinoma) cell lines. The combination of two different size measurement methods allowed the characterization of the dependence of nanoparticle accumulation in the tumor on even rather small differences in the nanoparticle size. While two nanoparticle batches (111 and 141 nm in diameter) accumulated efficiently in the human xenograft tumor tissue, the slightly bigger nanoparticles (diameter 166 nm) were rapidly eliminated by the liver.
Assuntos
Transformação Celular Neoplásica , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Raios Infravermelhos , Lactatos/química , Nanopartículas/química , Tamanho da Partícula , Polietilenoglicóis/química , Animais , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Portadores de Fármacos/farmacocinética , Feminino , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Corantes Fluorescentes/farmacocinética , Fracionamento por Campo e Fluxo , Células HT29 , Humanos , Lasers , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Microscopia Confocal , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Espalhamento de Radiação , Análise EspectralRESUMO
PURPOSE: Detailed in vivo and ex vivo analysis of nanoparticle distribution, accumulation and elimination processes were combined with comprehensive particle size characterizations. METHODS: The in vivo fate of near infrared (NIR) nanoparticles in nude mice was carried out using the Maestro™ in vivo fluorescence imaging system. Asymmetrical field flow field fractionation (AF4) coupled with multi-angle laser light scattering (MALLS), photon correlation spectroscopy (PCS) and transmission electron microscopy (TEM) were employed for detailed in vitro characterization. RESULTS: PEG-PLA block polymers were synthesized and used for the production of defined, stable, nontoxic nanoparticles. Nanoparticle analysis revealed narrow size distribution; AF4/MALLS permitted further accurate size evaluation. Multispectral fluorescence imaging made it possible to follow the in vivo fate non-invasively even in deep tissues over several days. Detailed fluorescence ex vivo imaging studies were performed and allowed to establish a calculation method to compare nanoparticle batches with varying fluorescence intensities. CONCLUSION: We combined narrow-size distributed nanoparticle batches with detailed in vitro characterization and the understanding of their in vivo fate using fluorescence imaging, confirming the wide possibilities of the non-invasive technique and presenting the basis to evaluate future size-dependent passive tumor accumulation studies.
Assuntos
Nanopartículas/química , Polietilenoglicóis/química , Polietilenoglicóis/farmacocinética , Animais , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Portadores de Fármacos/síntese química , Portadores de Fármacos/química , Estabilidade de Medicamentos , Feminino , Fracionamento por Campo e Fluxo/métodos , Masculino , Camundongos , Camundongos Nus , Microscopia Eletrônica de Transmissão/métodos , Nanopartículas/administração & dosagem , Tamanho da Partícula , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Distribuição TecidualRESUMO
Interstrand thiazole orange (TO) dimers in RNA show a yellow colored emission that can be distinguished from the green TO monomer emission by confocal microscopy inside CHO cells.
Assuntos
Benzotiazóis/química , Corantes Fluorescentes/química , Microscopia Confocal/métodos , Quinolinas/química , RNA/administração & dosagem , RNA/análise , Animais , Células CHO , Cricetinae , Cricetulus , Lipídeos , RNA/químicaRESUMO
Adipose tissue engineering requires biomaterials that promote the differentiation of seeded adipocytes. Here, we report on the development and characterization of in situ forming, poly(ethylene glycol) (PEG) based hydrogels for soft tissue augmentation. Branched PEG-amines were modified with collagenase-sensitive peptides and cross-linked with branched PEG-succinimidyl propionates without the use of free-radical initiators (enzymatically degradable hydrogels). Alanine-modified PEG-amines were used for the preparation of non-degradable gels. Depending on the used polymer concentration, the strength of degradable gels after swelling ranged from 1708 to 7412 Pa; the strength of non-degradable hydrogels varied between 1496 and 7686 Pa. Enzyme mediated gel degradation occurred within 10, 16, and 19 days (5%, 10%, and 15% initial polymer content). To evaluate their suitability as scaffold materials for adipose tissue engineering, the hydrogels were functionalized with the laminin-derived adhesion peptide YIGSR, and seeded with 3T3-L1 preadipocytes. Compared to a standard two-dimensional cell culture model, the developed hydrogels significantly enhanced the intracellular triglyceride accumulation of encapsulated adipocytes. Functionalization with YIGSR further enhanced lipid synthesis within differentiating adipocytes. Long-term studies suggested that enzymatically degradable hydrogels furthermore promote the formation of coherent adipose tissue-like structures featuring many mature unilocular fat cells.
Assuntos
Tecido Adiposo/fisiologia , Colagenases/metabolismo , Hidrogéis/farmacologia , Polietilenoglicóis/farmacologia , Engenharia Tecidual/métodos , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Tecido Adiposo/efeitos dos fármacos , Animais , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Hidrogéis/química , Camundongos , Microscopia de Contraste de Fase , Peptídeos/farmacologia , Polietilenoglicóis/químicaRESUMO
Hydrogels are extensively studied as matrices for the controlled release of macromolecules. To evaluate the mobility of embedded molecules, these drug delivery systems are usually characterized by release studies. However, these experiments are time-consuming and their reliability is often poor. In this study, gels were prepared by step-growth polymerization of poly(ethylene glycol) (PEG) and loaded with fluoresceine isothiocyanate (FITC) labeled dextrans. Mechanical testing and swelling studies allowed prediction of the expected FITC-dextran diffusivity. The translational diffusion coefficients (D) of the incorporated FITC-dextrans were measured by fluorescence recovery after photobleaching (FRAP) and pulsed field gradient NMR spectroscopy. Because the determined values of D agreed well with those obtained from release studies, mechanical testing, FRAP, and pulsed field gradient NMR spectroscopy are proposed as alternatives to release experiments. The applied methods complemented each other and represented the relative differences between the tested samples correctly. Measuring D can therefore be used to rapidly evaluate the potential of newly developed drug delivery systems.
Assuntos
Dextranos/administração & dosagem , Sistemas de Liberação de Medicamentos , Fluoresceína-5-Isotiocianato/análogos & derivados , Hidrogéis/química , Dextranos/química , Difusão , Fluoresceína-5-Isotiocianato/administração & dosagem , Fluoresceína-5-Isotiocianato/química , Recuperação de Fluorescência Após Fotodegradação , Cinética , Espectroscopia de Ressonância MagnéticaRESUMO
BACKGROUND: This study is an evaluation of wound healing in an animal model for surgery of frontal sinusitis and treatment effect of topically released dexamethasone using a drug-releasing stent with special emphasis of osteoneogenesis. METHODS: A prospective, controlled, randomized, double-blinded animal study was performed. Nineteen New Zealand white rabbits were subjected to surgery via an external approach, a 4-mm circular wound was created on the medial side of the maxillary sinus and the underlying bone was denuded of periosteum. The wound was covered in a randomized fashion with either a silicone foil or a new dexamethasone-releasing stent system. Twelve to 30 days later, the animals were killed and a histological examination was performed. RESULTS: In comparison with the baseline bony thickness (40 micrometer) obtained in one animal, osteoneogenesis occurred on both paranasal sides but was significantly less if a dexamethasone-releasing stent was applied (117 [95% CI, 116-128]; 52 [95% CI, 43-64]; p < 0.001). Maximal bony thickness was observed in both treatment groups between days 20 and 25 with a tendency toward a higher percentage decrease in the dexamethasone-treated sides (p < 0.08). Using a visual analog scale (0-5) a significantly smoother bony surface was observed for dexamethasone (2 [95% CI, 1.1-1.9]; 2 [95% CI, 1.8-2.2]; p < 0.01). CONCLUSION: Using a new drug-releasing stent system, dexamethasone efficiently decreases postoperative osteoneogenesis in a standardized animal wound model for endoscopic sinus surgery. Therefore, the use of this system may be of value to decrease restenosis rates using corticosteroids in selected patients after frontal sinus surgery, especially the endoscopic modified Lothrop procedure.
Assuntos
Dexametasona/farmacologia , Drenagem/instrumentação , Stents Farmacológicos , Sinusite Frontal/cirurgia , Glucocorticoides/farmacologia , Osteogênese/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Seguimentos , Sinusite Frontal/patologia , Masculino , Periósteo/efeitos dos fármacos , Periósteo/patologia , Estudos Prospectivos , Coelhos , Resultado do TratamentoRESUMO
Nucleic acids are not only expected to assume a pivotal position as "drugs" in the treatment of genetic and acquired diseases, but could also act as molecular cues to control the microenvironment during tissue regeneration. Despite this promise, the efficient delivery of nucleic acids to their side of action is still the major hurdle. One among many prerequisites for a successful carrier system for nucleic acids is high stability in the extracellular environment, accompanied by an efficient release of the cargo in the intracellular compartment. A promising strategy to create such an interactive delivery system is to exploit the redox gradient between the extra- and intracellular compartments. In this review, emphasis is placed on the biological rationale for the synthesis of redox sensitive, disulfide-based carrier systems, as well as the extra- and intracellular processing of macromolecules containing disulfide bonds. Moreover, the basic synthetic approaches for introducing disulfide bonds into carrier molecules, together with examples that demonstrate the benefit of disulfides at the individual stages of nucleic acid delivery, will be presented.
RESUMO
Hyalocytes, the cells of the vitreous body, are assumed to be involved in physiological as well as patho-physiological processes within the eye. However, current knowledge about the cells is still limited. As different morphological types of hyalocytes are described in the literature, it seems reasonable to try to isolate individual populations prior to characterization of single cell types. To achieve this, the present study investigated the utility of fluorescence activated cell sorting (FACS) for hyalocyte separation. Subsequent to digestion of vitreous bodies using collagenase, the resulting cell suspension was analyzed and separated using FACS without any additional staining. Two-parameter dot plots of forward scatter (indicating size) against sideward scatter (indicating granularity) showed two distinct cell populations; staining with propidium iodide confirmed that both populations represent living cells. After sorting, cells of both populations were seeded on tissue culture plastic (tissue culture treated polystyrene). Only one population attached and proliferated, whereas the other population was non-adherent. Even when seeding the native cell mix, only one population of cells was observed after two passages, as indicated by FACS. Furthermore, ascorbic acid increased proliferation of these cells similarly to the proliferation of the separated cell population. These data point out that only one of the two populations adheres and proliferates on tissue culture plastic. To conclude, the established isolation technique allows for separation of clearly defined hyalocyte populations. Moreover, clear hints were obtained that only one of the two populations adheres and proliferates under the commonly applied culture conditions.
Assuntos
Separação Celular/métodos , Corpo Vítreo/citologia , Animais , Adesão Celular , Proliferação de Células , Células Cultivadas , Citometria de Fluxo/métodos , Sus scrofaRESUMO
OBJECTIVE/HYPOTHESIS: Evaluation of the impact of continuously topically released dexamethasone using a drug-releasing stent on quality of regenerated mucosa after full thickness injury in the paranasal sinuses. STUDY DESIGN: Prospective, controlled, randomized, double-blinded animal study. METHODS: Nineteen New Zealand white rabbits were subjected to surgery: via an external approach, a 4 mm circular wound was created on the medial side of the maxillary sinus. The wound was covered in a randomized fashion with either a silicone foil or a new drug releasing stent system. Twelve to 30 days later, the animals were killed and histology and electron microscopy were performed. One animal was used for baseline comparisons at day 0. RESULTS: No animals were lost due to infection or dislocation of the stent, leaving 18 animals for evaluation of postoperative healing quality. According to macroscopic examination, extent of granulations was smaller in the treatment group (dexamethasone: median 0 [95% confidence interval: 0.1-0.6]) than the silicone group (2 [1.5-2.3]; P < or = .05). Epithelial wound healing was complete in all specimens, whereas the stroma was significantly thinner in the dexamethasone-group (44 [37-60]; 178 [148-214]). Improved healing quality was achieved significantly more often on the treatment, than on the control side. Scanning electron microscopy revealed no difference between both groups. CONCLUSIONS: Using a new drug-releasing stent system, dexamethasone efficiently decreases granulation formation and stroma thickness without impeding epithelial differentiation. Therefore, the use of this system may be of value to decrease restenosis rates in selected patients after frontal sinus surgery.
Assuntos
Materiais Revestidos Biocompatíveis , Dexametasona/administração & dosagem , Seio Maxilar/patologia , Mucosa Nasal/patologia , Implantação de Prótese/instrumentação , Stents , Cicatrização/efeitos dos fármacos , Administração Tópica , Animais , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Seguimentos , Glucocorticoides/administração & dosagem , Masculino , Seio Maxilar/efeitos dos fármacos , Seio Maxilar/cirurgia , Sinusite Maxilar/patologia , Sinusite Maxilar/cirurgia , Mucosa Nasal/efeitos dos fármacos , Estudos Prospectivos , Coelhos , Resultado do TratamentoRESUMO
PURPOSE: In cases of severe retinal diseases, the vitreous body has to be removed and replaced by a suitable biomaterial. Currently, however, no satisfying long-term vitreous substitute is in clinical use. A novel therapeutic concept represents the combination of hyalocytes with suitable biomaterials. The goal of the present study was to evaluate the potential of bFGF and TGF-beta1 as tools to control hyalocyte proliferation and the accumulation of extracellular matrix (ECM). METHODS: In vitro investigation on the influence of different concentrations of bFGF and TGF-beta1 on hyalocyte morphology, proliferation and ECM production. RESULTS: Both growth factors affected hyalocyte morphology; small, round cells could be observed after bFGF supplementation, whereas the cells appeared more completely spread when cultured with TGF-beta1. Hyalocyte proliferation was increased 3-fold by 10 ng/ml bFGF; 1 ng/ml TGF-beta1 in contrast reduced cell proliferation to about 40% of the control. Converse effects of the growth factors could also be observed on the ECM accumulation of hyalocytes; whereas bFGF halved ECM accumulation, TGF-beta1 enhanced the ECM production up to 3-fold. Precultivation of hyalocytes with bFGF for two passages had no influence on their subsequent accumulation of glycosaminoglycans (GAG). However, cells precultivated with bFGF exhibited a doubled accumulation of collagen compared to controls. CONCLUSIONS: The observed opposite effects of bFGF and TGF-beta1 on hyalocyte proliferation and ECM accumulation may allow for the control of hyaloycte properties. Therefore, these two growth factors seem to be valuable tools towards the development of a cell-based vitreous substitute.