RESUMO
Chronic wasting disease is a fatal, horizontally transmissible prion disease of cervid species that has been reported in free-ranging and farmed animals in North America, Scandinavia, and Korea. Like other prion diseases, CWD susceptibility is partly dependent on the sequence of the prion protein encoded by the host's PRNP gene; it is unknown if variations in PRNP have any meaningful effects on other aspects of health. Conventional diagnosis of CWD relies on ELISA or IHC testing of samples collected post-mortem, with recent efforts focused on antemortem testing approaches. We report on the conclusions of a study evaluating the role of antemortem testing of rectal biopsies collected from over 570 elk in a privately managed herd, and the results of both an amplification assay (RT-QuIC) and conventional IHC among animals with a several PRNP genotypes. Links between PRNP genotype and potential markers of evolutionary fitness, including pregnancy rates, body condition, and annual return rates were also examined. We found that the RT-QuIC assay identified significantly more CWD positive animals than conventional IHC across the course of the study, and was less affected by factors known to influence IHC sensitivity - including follicle count and PRNP genotype. We also found that several evolutionary markers of fitness were not adversely correlated with specific PRNP genotypes. While the financial burden of the disease in this herd was ultimately unsustainable for the herd owners, our scientific findings and the hurdles encountered will assist future CWD management strategies in both wild and farmed elk and deer.
Assuntos
Cervos/fisiologia , Doença de Emaciação Crônica/terapia , Envelhecimento/patologia , Animais , Colorado/epidemiologia , Feminino , Genótipo , Imuno-Histoquímica , Estudos Longitudinais , Tecido Linfoide/patologia , Gravidez , Prevalência , Proteínas Priônicas/metabolismo , Análise de Sobrevida , Doença de Emaciação Crônica/epidemiologia , Doença de Emaciação Crônica/genética , Doença de Emaciação Crônica/patologiaRESUMO
Corneal substitutes are being developed to address the shortage of human donor tissues as well as the current disadvantages in some clinical indications, which include immune rejection. In the past few years, there have been significant developments in bioengineered corneas that are designed to replace part or the full thickness of damaged or diseased corneas that range from keratoprostheses that solely address the replacement of the cornea's function, through tissue-engineered hydrogels that permit regeneration of host tissues. We describe examples of corneal substitutes that encourage regeneration of the host tissue. We also contend that it is unlikely that there will be a single "one-size-fits-all" corneal substitute for all indications. Instead, there will most likely be a small range of corneal substitutes ranging from prostheses to tissue-engineered matrix substitutes that are tailored to different clusters of clinical indications. The tissue-engineered matrices can either be produced as sterile acellular matrices, or complete with functional cells, ready for implantation.
Assuntos
Órgãos Artificiais , Córnea , Transplante de Córnea , Medicina Regenerativa/métodos , Engenharia Tecidual/métodos , Adulto , Idoso , Engenharia Biomédica , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Porcine and recombinant human atelocollagen I solutions were cross-linked with a water soluble carbodiimide at various stoichiometries and collagen concentrations (5-20 w/w %). The resulting hydrogels were clear and, when used as cell growth matrices, allowed cell and nerve visualization in vitro and in vivo. We have previously reported that, after six months of implantation in pigs' and rabbits' corneas, these robust hydrogels allowed regeneration of host cells and nerves to give optically clear corneas with no detected loss in thickness, indicating stable engraftment. Here, the biocompatible hydrogel formulations leading to this novel in vivo performance were characterized for amine consumption, gel hydration, thermal properties, optical clarity, refractive index, nutrient diffusion, biodegradation, tensile measurements, and average pore diameters. Gels with excellent in vitro (epithelial overgrowth, neurite penetration) and in vivo performance (clarity, touch sensitivity regeneration) had 4-11 nm pores, yet had glucose and albumin diffusive coefficients similar to mammalian corneas and allowed neurite extension through the gels.
Assuntos
Materiais Biocompatíveis/química , Colágeno/química , Óptica e Fotônica , Engenharia Tecidual , Aminas/química , Animais , Carbodi-Imidas/química , Córnea/química , Reagentes de Ligações Cruzadas/química , Géis/química , Humanos , Hidrogéis/química , Teste de Materiais , Proteínas Recombinantes/química , Suínos , Temperatura , Água/químicaRESUMO
The downgrowth of corneal epithelial cells at the interface of an artificial cornea and the host eye tissue poses a significant problem to be overcome in developing a successful implant. As a means of inhibiting the proliferation of corneal epithelial cells on the stromal surface of the implant, we examined the immobilization of transforming growth factor beta-2 (TGF-beta2) via a bifunctional poly ethylene glycol (PEG) spacer to poly dimethyl siloxane (PDMS) surfaces. Growth factor immobilization was confirmed by modification with (125)I-labeled TGF-beta 2. The modified surfaces were also characterized by advancing water contact angles, X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). Although the amount of growth factor covalently bound to the surface was difficult to quantify apparently due to strong interactions between the growth factor and the PEG layer and high levels of adsorption, differences in the modified surfaces, suggestive of the presence of a significant amount of TGF-beta 2, were found. In vitro interactions of the modified surfaces with human corneal epithelial and stromal cells were examined. Growth factor surface concentrations as well as culture in the absence and presence of serum and other adhesive proteins were examined. Corneal stromal and epithelial cells cultured on the TGF-beta 2-modified surfaces consistently gave results opposite to those expected. Likely, the most notable and surprising result was the almost complete lack of adhesion of the stromal cells, with coverage averaging between 3 and 5%. In comparison, corneal epithelial cell growth appeared to be promoted by the presence of the immobilized growth factor, with cell coverage averaging 50-60% at 7 days of culture. A TGF-beta 2 concentration effect was noted with both cell types in the absence of serum, with increases in the coverage at higher TGF-beta 2 concentrations. The observed cell growth appeared to be the result of interactions between the cells and active growth factor, because the addition of anti-TGF-beta 2 to the culture medium reduced cell coverage to levels similar to those noted on control surfaces. Therefore, although TGF-beta 2-modified surfaces may not be suitable as corneal epithelial cell inhibiting surfaces, interactions of surface immobilized growth factor and corneal cells are complex and should be further examined.
Assuntos
Córnea/citologia , Sistemas de Liberação de Medicamentos , Fator de Crescimento Transformador beta/administração & dosagem , Materiais Biocompatíveis , Proteínas Sanguíneas/análise , Proteínas Sanguíneas/metabolismo , Adesão Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Córnea/efeitos dos fármacos , Substância Própria/citologia , Transplante de Córnea , Dimetilpolisiloxanos , Epitélio Corneano/citologia , Humanos , Modelos Biológicos , Silicones , Propriedades de Superfície , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta2RESUMO
Surface properties have an enormous effect on the success or failure of a biomaterial device, thus signifying the considerable importance of and the need for adequate characterization of the biomaterial surface. Microscopy techniques used in the analysis of biomaterial surfaces include scanning electron microscopy, transmission electron microscopy, atomic force microscopy, and confocal microscopy. Spectroscopic techniques include X-ray photoelectron spectroscopy, Fourier Transform infrared attenuated total reflection and secondary ion mass spectrometry. The measurement of contact angles, although one of the earlier techniques developed remains a very useful tool in the evaluation of surface hydrophobicity/hydrophilicity. This paper provides a brief, easy to understand synopsis of these and other techniques including emerging techniques, which are proving useful in the analysis of the surface properties of polymeric biomaterials. Cautionary statements have been made, numerous authors referenced and examples used to show the specific type of information that can be acquired from the different techniques used in the characterization of polymeric biomaterials surfaces.
Assuntos
Materiais Biocompatíveis/química , Teste de Materiais/métodos , Polímeros/química , Microscopia de Força Atômica , Microscopia Confocal , Microscopia Eletrônica de Varredura , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massa de Íon Secundário , Espectroscopia de Infravermelho com Transformada de Fourier , Propriedades de SuperfícieRESUMO
Epithelialization of a corneal implant is a desirable property. In this study we compared surface modification of poly (2-hydroxyethyl methacrylate) (pHEMA) with the cell adhesion peptides RGDS and YIGSR. Various parameters in the tresyl chloride activation and modification reactions were considered in order to maximize surface coverage with the peptide including tresyl chloride reaction solvent. tresyl chloride reaction time, tresyl chloride concentration, peptide concentration, and peptide reaction pH. Surface chemistry and corneal epithelial cell adhesion to the modified surfaces were examined. X-ray photoelectron spectroscopy data suggested that while peptide modification had occurred, surface coverage with the peptide was incomplete. Acetone was found to result in a higher fraction of nitrogen and surface bound carboxyl groups compared to dioxane and ether. Furthermore, corneal epithelial cell adhesion to the surfaces for which acetone was used for the activation reaction was significantly greater. Statistical analysis of the various samples suggests that lower peptide concentrations and higher tresyl chloride reaction times result in better cell adhesion. Furthermore, modification with YIGSR resulted in higher surface concentrations and better cell adhesion than modification with RGDS. Little or no cell adhesion was noted on the unmodified pHEMA controls. Protein adsorption results suggest that the differences in cell adhesion cannot be attributed to differences in serum protein adsorption from the culture medium. We conclude that YIGSR modified surfaces have significant potential for further development in corneal applications.