RESUMO
A 2D plane strain extended finite element method (XFEM) model was developed to simulate three-point bending fracture toughness tests for human bone conducted in hydrated and dehydrated conditions. Bone microstructures and crack paths observed by micro-CT imaging were simulated using an XFEM damage model. Critical damage strains for the osteons, matrix, and cement lines were deduced for both hydrated and dehydrated conditions and it was found that dehydration decreases the critical damage strains by about 50%. Subsequent parametric studies using the various microstructural models were performed to understand the impact of individual critical damage strain variations on the fracture behavior. The study revealed the significant impact of the cement line critical damage strains on the crack paths and fracture toughness during the early stages of crack growth. Furthermore, a significant sensitivity of crack growth resistance and crack paths on critical strain values of the cement lines was found to exist for the hydrated environments where a small change in critical strain values of the cement lines can alter the crack path to give a significant reduction in fracture resistance. In contrast, in the dehydrated state where toughness is low, the sensitivity to changes in critical strain values of the cement lines is low. Overall, our XFEM model was able to provide new insights into how dehydration affects the micromechanisms of fracture in bone and this approach could be further extended to study the effects of aging, disease, and medical therapies on bone fracture.
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Desidratação , Fraturas Ósseas , Humanos , Modelos Biológicos , Osso Cortical/diagnóstico por imagem , Osso e Ossos , Fraturas Ósseas/diagnóstico por imagemRESUMO
Chronic heavy alcohol consumption may influence the skeleton by suppressing intracortical bone remodeling which may impact the quality of bone and its mechanical properties. However, this aspect has not been thoroughly assessed in either humans or animal models whose cortical bone microstructure resembles the microstructure of human cortical bone. The current study is the first to investigate the effects of chronic heavy alcohol consumption on various mechanical properties of bone in a non-human primate model with intracortical remodeling. Male rhesus macaques (5.3 years old at the initiation of treatment) were induced to drink alcohol and then given the choice to voluntarily self-administer water or ethanol (4 % w/v) for approximately 14 months, followed by three abstinence phases (lasting 34, 41, and 39-46 days) with approximately 3 months of ethanol access in between. During the initial 14 months of open-access, monkeys in the alcohol group consumed an average of 2.9 ± 0.8 g/kg/d ethanol (mean ± SD) resulting in a blood ethanol concentration of 89 ± 47 mg/dl in longitudinal samples taken at 7 h after the daily sessions began. To understand the impact of alcohol consumption on material properties, various mechanical tests were conducted on the distal tibia diaphysis of 2-5 monkeys per test group, including dynamic mechanical analysis (DMA) testing, nano-indentation, microhardness testing, compression testing, and fracture resistance curve (R-curve) testing. Additionally, compositional analyses were performed using Fourier-transform infrared (FTIR) spectroscopy. Significant differences in microhardness, compressive stress-strain response, and composition were not observed with alcohol consumption, and only minor differences were detected in hardness and elastic modulus of the matrix and osteons from nanoindentation. Furthermore, the R-curves of both groups overlapped, with similar crack initiation toughness, despite a significant decrease in crack growth toughness (p = 0.032) with alcohol consumption. However, storage modulus (p = 0.029) and loss factor (p = 0.015) from DMA testing were significantly increased in the alcohol group compared to the control group, while loss modulus remained unchanged. These results indicate that heavy alcohol consumption may have only a minor influence on the material properties and the composition of cortical bone in young adult male rhesus macaques.
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Osso e Ossos , Osso Cortical , Animais , Masculino , Macaca mulatta , Consumo de Bebidas Alcoólicas , EtanolRESUMO
Sterilization of structural bone allografts is a critical process prior to their clinical use in large cortical bone defects. Gamma irradiation protocols are known to affect tissue integrity in a dose dependent manner. Alternative sterilization treatments, such as supercritical carbon dioxide (SCCO2 ), are gaining popularity due to advantages such as minimal exposure to denaturants, the lack of toxic residues, superior tissue penetration, and minor impacts on mechanical properties including strength and stiffness. The impact of SCCO2 on the fracture toughness of bone tissue, however, remains unknown. Here, we evaluate crack initiation and growth toughness after 2, 6, and 24 h SCCO2 -treatment using Novakill™ and ethanol as additives on ~11 samples per group obtained from a pair of femur diaphyses of a canine. All mechanical testing was performed at ambient air after 24 h soaking in Hanks' balanced salt solution (HBSS). Results show no statistically significant difference in the failure characteristics of the Novakill™-treated groups whereas crack growth toughness after 6 and 24 h of treatment with ethanol significantly increases by 37% (p = .010) and 34% (p = .038), respectively, compared to an untreated control group. In contrast, standard 25 kGy gamma irradiation causes significantly reduced crack growth resistance by 40% (p = .007) compared to untreated bone. FTIR vibrational spectroscopy, conducted after testing, reveals a consistent trend of statistically significant differences (p < .001) with fracture toughness. These trends align with variations in the ratios of enzymatic mature to immature crosslinks in the collagen structure, suggesting a potential association with fracture toughness. Additional Raman spectroscopy after testing shows a similar trend with statistically significant differences (p < .005), which further supports that collagen structural changes occur in the SCF-treated groups with ethanol after 6 and 24 h. Our work reveals the benefits of SCCO2 sterilization compared to gamma irradiation.
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Dióxido de Carbono , Fraturas Ósseas , Animais , Cães , Dióxido de Carbono/farmacologia , Etanol/farmacologia , Osso e Ossos , Osso Cortical , Colágeno/farmacologiaRESUMO
Water is a crucial component of bone, affecting the interplay of collagen and minerals and contributing to bone's high strength and ductility. Dehydration has been shown to significantly effect osseous mechanical properties; however, studies comparing the effects of various dehydrating environments on fracture toughness of bone are scarce. Accordingly, the crack resistance curve (R-curve) behavior of human and sheep cortical bone was characterized in a bio-bath, in ambient pressure air, and in scanning electron microscopes (SEMs) under three different environmental conditions (water vapor pressure, air pressure, and high-vacuum). The aim of this work was to better understand the impact of test environment on both intrinsic and extrinsic toughening and hence crack initiation toughness, K0 and crack growth resistance, dK/dΔa. Results show significantly lower K0 values for samples that were tested inside SEMs combined with pronounced extrinsic toughening through microcracking and crack path deflections out of the mode I plane. Importantly, all three SEM test environments gave similar results, and thus it does not matter which type of SEM is used. Ex situ testing of hydrated samples revealed similar K0 for both environments but elevated crack growth resistance for testing in ambient air relative to the bio-bath. Our data reveals the experimental difficulties to directly observe microscale crack propagation in cortical bone that resembles the in vivo situation. Ex situ testing immersed in Hanks' Balanced Salt Solution (HBSS) with subsequent crack path analysis, while tedious, is thought to presents the most realistic picture of the in vivo structure-fracture property relations in biological tissue.
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Osso e Ossos , Fraturas Ósseas , Animais , Colágeno , Osso Cortical , Ovinos , Estresse Mecânico , Resistência à TraçãoRESUMO
Fabrication of three-dimensional (3D) constructs to model body tissues and organs can contribute to research into tissue development and models for studying disease, as well as supporting preclinical drug screening in vitro. Furthermore, 3D constructs can also be used for diagnosis and therapy of disease conditions via lab on a chip and microarrays for diagnosis and engineered products for tissue repair, replacement, and regeneration. While cell culture approaches for studying tissue development and disease in two dimensions are long-established, the translation of this knowledge into 3D environments remains a fertile field of research. In this Tutorial, we specifically focus on the application of biosynthetic hydrogels for neural cell encapsulation. The Tutorial briefly covers background on using biosynthetic hydrogels for cell encapsulation, as well as common fabrication techniques. The Methods section focuses on the hydrogel design and characterization, highlighting key elements and tips for more effective approaches. Coencapsulation of different cell types, and the challenges associated with different growth and maintenance requirements, is the main focus of this Tutorial. Much care is needed to blend different cell types, and this Tutorial provides tips and insights that have proven successful for 3D coculture in biosynthetic hydrogels.
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Biomimética , Neurônios/citologia , Alicerces Teciduais/química , Animais , Proliferação de Células , Forma Celular , Sobrevivência Celular , Células Imobilizadas/citologia , Técnicas de Cocultura , Fenômenos Eletrofisiológicos , Matriz Extracelular/metabolismo , Humanos , Hidrogéis/química , Crescimento Neuronal , Células PC12 , Álcool de Polivinil/química , Ratos , Células de Schwann/citologia , Esferoides Celulares/citologia , Tiramina/químicaRESUMO
The translation of growth factors (GFs) into clinical applications is limited by their low stability in physiological environments. Controlled GF delivery through biomaterial vehicles provides protection from proteases, targeted delivery, and longer term release profiles. However, current methods used to incorporate GFs into biomaterials still present limitations. While direct adsorption and encapsulation result in burst release, covalent incorporation provides a tailorable release profile but generally requires more complicated processes and chemical modification of the GFs. Bioaffinity methods provide long-term release profiles but fail in their specificity, resulting in GF-dependent applicability and release profiles. In the present study, we introduce tyraminated poly-vinyl-alcohol (PVA-Tyr) as a GF-delivery vehicle that can covalently incorporate native GFs through a photo-initiated cross-linking process via formation of bi-phenol bonds. Mass loss and release studies revealed that protein-loaded PVA-Tyr hydrogels had highly tailorable degradation times from 7 to 92 days, during which the covalently incorporated proteins were released in a linear fashion. The incorporation of bovine serum albumin (BSA), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), or brain-derived growth factor (BDNF) resulted in similar incorporation efficiencies and release profiles, demonstrating the low specificity and versatility of the system. Furthermore, functional studies demonstrated that VEGF, bFGF and BDNF released from the PVA-Tyr hydrogels retained the ability to increase the metabolic activity, migration, and 3D vessel formation of endothelial cells and mesenchymal stem cells. Taken together, this demonstrates that PVA-Tyr shows high potential as a highly tailorable GF delivery tool for a range of different regenerative medicine applications.
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Hidrogéis , Tiramina , Células Endoteliais , Luz , Fator A de Crescimento do Endotélio VascularRESUMO
Promoting nerve regeneration requires engineering cellular carriers to physically and biochemically support neuronal growth into a long lasting functional tissue. This study systematically evaluated the capacity of a biosynthetic poly(vinyl alcohol) (PVA) hydrogel to support growth and differentiation of co-encapsulated neurons and glia. A significant challenge is to understand the role of the dynamic degradable hydrogel mechanical properties on expression of relevant cellular morphologies and function. It was hypothesised that a carrier with mechanical properties akin to neural tissue will provide glia with conditions to thrive, and that glia in turn will support neuronal survival and development. PVA co-polymerised with biological macromolecules sericin and gelatin (PVA-SG) and with tailored nerve tissue-like mechanical properties were used to encapsulate Schwann cells (SCs) alone and subsequently a co-culture of SCs and neural-like PC12s. SCs were encapsulated within two PVA-SG gel variants with initial compressive moduli of 16â¯kPa and 2â¯kPa, spanning a range of reported mechanical properties for neural tissues. Both hydrogels were shown to support cell viability and expression of extracellular matrix proteins, however, SCs grown within the PVA-SG with a higher initial modulus were observed to present with greater physiologically relevant morphologies and increased expression of extracellular matrix proteins. The higher modulus PVA-SG was subsequently shown to support development of neuronal networks when SCs were co-encapsulated with PC12s. The lower modulus hydrogel was unable to support effective development of neural networks. This study demonstrates the critical link between hydrogel properties and glial cell phenotype on development of functional neural tissues. STATEMENT OF SIGNIFICANCE: Hydrogels as platforms for tissue regeneration must provide encapsulated cellular progenitors with physical and biochemical cues for initial survival and to support ongoing tissue formation as the artificial network degrades. While most research focuses on tailoring scaffold properties to suit neurons, this work aims to support glia SCs as the key cellular component that physically and biochemically supports the neuronal network. The challenge is to modify hydrogel properties to support growth and development of multiple cell types into a neuronal network. Given SCs ability to respond to substrate mechanical properties, the significance of this work lies in understanding the relationship between dynamic hydrogel mechanical properties and glia SCs development as the element that enables formation of mature, differentiated neural networks.
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Hidrogéis/farmacologia , Rede Nervosa/fisiologia , Engenharia Tecidual/métodos , Animais , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Imobilizadas/citologia , Colágeno Tipo IV/metabolismo , Matriz Extracelular/química , Laminina/metabolismo , Rede Nervosa/efeitos dos fármacos , Células PC12 , Álcool de Polivinil/farmacologia , Ratos , Células de Schwann/citologia , Células de Schwann/efeitos dos fármacos , Alicerces Teciduais/químicaRESUMO
Variability in spider major ampullate (MA) silk properties at different scales has proven difficult to determine and remains an obstacle to the development of synthetic fibers mimicking MA silk performance. A multitude of techniques may be used to measure multiscale aspects of silk properties. Here we fed five species of Araneoid spider solutions that either contained protein or were protein deprived and performed silk tensile tests, small and wide-angle X-ray scattering (SAXS/WAXS), amino acid composition analyses, and silk gene expression analyses, to resolve persistent questions about how nutrient deprivation induces variations in MA silk mechanical properties across scales. Our analyses found that the properties of each spider's silk varied differently in response to variations in their protein intake. We found changes in the crystalline and non-crystalline nanostructures to play specific roles in inducing the property variations we found. Across treatment MaSp expression patterns differed in each of the five species. We found that in most species MaSp expression and amino acid composition variations did not conform with our predictions based on a traditional MaSp expression model. In general, changes to the silk's alanine and proline compositions influenced the alignment of the proteins within the silk's amorphous region, which influenced silk extensibility and toughness. Variations in structural alignment in the crystalline and non-crystalline regions influenced ultimate strength independent of genetic expression. Our study provides the deepest insights thus far into the mechanisms of how MA silk properties vary from gene expression to nanostructure formations to fiber mechanics. Such knowledge is imperative for promoting the production of synthetic silk fibers.
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Seda , Aranhas/metabolismo , Aminoácidos/análise , Animais , Expressão Gênica , Espalhamento de Radiação , Especificidade da Espécie , Aranhas/classificação , Resistência à TraçãoRESUMO
Like regular phenotypes, extended phenotypes have demonstrable fitness advantages and their properties may vary plastically across environments. However, the fitness advantages of plasticity are only known for a select few extended phenotypes. It is known that the form and functions of spider orb webs can be manipulated by laboratory experiments. For instance, the physical and chemical properties of the spiral and gluey silks vary in property as protein intake varies. Orb web spiders thus represent good models for extended phenotypic plasticity studies. We performed experiments manipulating the protein intake of two vertically aligned orb web building spiders to determine whether variations in the chemical and physical properties of their spiral and gluey silk affect prey retention in their webs. We found in both spider species that individuals deprived of protein had a greater gluey silk glycoprotein core volume, and this correlated strongly with spiral thread stickiness and increased prey retention by the webs. Moreover, we found strong positive correlations between glue droplet volume and glycoprotein core volume for spiders in the protein-deprived treatment, but weaker correlations for protein-fed spiders. We interpreted these findings as the spiders investing more in glycoprotein when nutrient deprived. We attribute the associated increase in prey retention capacity as a fitness consequence of plasticity in the spiral properties.
Assuntos
Adaptação Fisiológica , Aptidão Genética , Comportamento Predatório , Seda/química , Aranhas/fisiologia , Animais , Dieta , Feminino , Aranhas/químicaRESUMO
The adaptive benefits of extended phenotypic plasticity are imprecisely defined due to a paucity of experiments examining traits that are manipulable and measurable across environments. Spider webs are often used as models to explore the adaptive benefits of variations in extended phenotypes across environments. Nonetheless, our understanding of the adaptive nature of the plastic responses of spider webs is impeded when web architectures and silk physicochemical properties appear to co-vary. An opportunity to examine this co-variation is presented by modifying prey items while measuring web architectures and silk physiochemical properties. Here, we performed two experiments to assess the nature of the association between web architectures and gluey silk properties when the orb web spider Argiope keyserlingi was fed a diet that varied in either mass and energy or prey size and feeding frequency. We found web architectures and gluey silk physicochemical properties to co-vary across treatments in both experiments. Specifically, web capture area co-varied with gluey droplet morphometrics, thread stickiness and salt concentrations when prey mass and energy were manipulated, and spiral spacing co-varied with gluey silk salt concentrations when prey size and feeding frequency were manipulated. We explained our results as A. keyserlingi plastically shifting its foraging strategy as multiple prey parameters simultaneously varied. We confirmed and extended previous work by showing that spiders use a variety of prey cues to concurrently adjust web and silk traits across different feeding regimes.
Assuntos
Comportamento Predatório , Seda/química , Aranhas/fisiologia , Adesividade , Ração Animal/análise , Fenômenos Fisiológicos da Nutrição Animal , Animais , Dieta , Metabolismo Energético , Comportamento Alimentar , Feminino , Seda/metabolismo , Aranhas/químicaRESUMO
Heparin-based hydrogels are attractive for controlled growth factor delivery, due to the native ability of heparin to bind and stabilize growth factors. Basic fibroblast growth factor and vascular endothelial growth factor are heparin-binding growth factors that synergistically enhance angiogenesis. Mild, in situ encapsulation of both basic fibroblast growth factor and vascular endothelial growth factor and subsequent bioactive dual release has not been demonstrated from heparin-crosslinked hydrogels, and the combined long-term delivery of both growth factors from biomaterials is still a major challenge. Both basic fibroblast growth factor and vascular endothelial growth factor were encapsulated in poly(vinyl alcohol)-heparin hydrogels and demonstrated controlled release. A model cell line, BaF32, was used to show bioactivity of heparin and basic fibroblast growth factor released from the gels over multiple days. Released basic fibroblast growth factor promoted higher human umbilical vein endothelial cell outgrowth over 24 h and proliferation for 3 days than the poly(vinyl alcohol)-heparin hydrogels alone. The release of vascular endothelial growth factor from poly(vinyl alcohol)-heparin hydrogels promoted human umbilical vein endothelial cell outgrowth but not significant proliferation. Dual-growth factor release of basic fibroblast growth factor and vascular endothelial growth factor from poly(vinyl alcohol)-heparin hydrogels resulted in a synergistic effect with significantly higher human umbilical vein endothelial cell outgrowth compared to basic fibroblast growth factor or vascular endothelial growth factor alone. Poly(vinyl alcohol)-heparin hydrogels allowed bioactive growth factor encapsulation and provided controlled release of multiple growth factors which is beneficial toward tissue regeneration applications.
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BACKGROUND: Dityrosine crosslinking in proteins is a bioinspired method of forming hydrogels. This study compares oxidative enzyme initiators for their relative crosslinking efficiency and cytocompatibility using the same phenol group and the same material platform. Four common enzyme and enzyme-like oxidative initiators were probed for resulting material properties and cell viability post-encapsulation. RESULTS: All four initiators can be used to form phenol-crosslinked hydrogels, however gelation rates are dependent on enzyme type, concentration, and the oxidant. Horseradish peroxidase (HRP) or hematin with hydrogen peroxide led to a more rapid poly (vinyl alcohol)-tyramine (PVA-Tyr) polymerization (10-60 min) because a high oxidant concentration was dissolved within the macromer solution at the onset of crosslinking, whereas laccase and tyrosinase require oxygen diffusion to crosslink phenol residues and therefore took longer to gel (2.5+ hours). The use of hydrogen peroxide as an oxidant reduced cell viability immediately post-encapsulation. Laccase- and tyrosinase-mediated encapsulation of cells resulted in higher cell viability immediately post-encapsulation and significantly higher cell proliferation after one week of culture. CONCLUSIONS: Overall this study demonstrates that HRP/H2O2, hematin/H2O2, laccase, and tyrosinase can create injectable, in situ phenol-crosslinked hydrogels, however oxidant type and concentration are critical parameters to assess when phenol crosslinking hydrogels for cell-based applications.
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The exceptional strength and extensibility of spider dragline silk have been thought to be facilitated by two spidroins, major ampullate spidroin 1 (MaSp1) and major ampullate spidroin 2 (MaSp2), under the assumption that protein secondary structures are coupled with the expressed spidroins. We tested this assumption for the dragline silk of three co-existing Australian spiders, Argiope keyserlingi, Latrodectus hasselti and Nephila plumipes. We found that silk amino acid compositions did not differ among spiders collected in May. We extended these analyses temporally and found the amino acid compositions of A. keyserlingi silks to differ when collected in May compared to November, while those of L. hasselti did not. To ascertain whether their secondary structures were decoupled from spidroin expression, we performed solid-state nuclear magnetic resonance spectroscopy (NMR) analysis on the silks of all spiders collected in May. We found the distribution of alanine toward ß-sheet and 3,10helix/random coil conformations differed between species, as did their relative crystallinities, with A. keyserlingi having the greatest 3,10helix/random coil composition and N. plumipes the greatest crystallinity. The protein secondary structures correlated with the mechanical properties for each of the silks better than the amino acid compositions. Our findings suggested that a differential distribution of alanine during spinning could decouple secondary structures from spidroin expression ensuring that silks of desirable mechanical properties are consistently produced. Alternative explanations include the possibility that other spidroins were incorporated into some silks.
Assuntos
Fibroínas/química , Proteínas de Insetos/química , Seda/química , Aminoácidos/química , Animais , Cromatografia Líquida de Alta Pressão , Espectroscopia de Ressonância MagnéticaRESUMO
A photopolymerizable-tyraminated poly(vinyl alcohol) (PVA-Tyr) system that has the ability to covalently bind proteins in their native state was evaluated as a platform for cell encapsulation. However, a key hurdle to this system is the radicals generated during the cross-linking that can cause oxidative stress to the cells. This research hypothesized that incorporation of anti-oxidative proteins (sericin and gelatin) into PVA-Tyr gels would mitigate any toxicity caused by the radicals. The results showed that although incorporation of 1 wt% sericin promoted survival of the fibroblasts, both sericin and gelatin acted synergistically to facilitate long-term 3D cell function. The encapsulated cells formed clusters with deposition of laminin and collagen, as well as remaining metabolically active after 21 d.
Assuntos
Fibroblastos/citologia , Hidrogéis/farmacologia , Álcool de Polivinil/farmacologia , Tiramina/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Imobilizadas/citologia , Células Imobilizadas/efeitos dos fármacos , Células Imobilizadas/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Radicais Livres/química , Gelatina/farmacologia , Imuno-Histoquímica , Camundongos , Sericinas/farmacologia , Sus scrofaRESUMO
The presentation of multiple biological cues, which simulate the natural in vivo cell environment within artificial implants, has recently been identified as crucial for achieving complex cellular functions. The incorporation of two or more biological cues within a largely synthetic network can provide a simplified model of multifunctional ECM presentation to encapsulated cells. Therefore, the aim of this study was to examine the effects of simultaneously and covalently incorporating two dissimilar biological molecules, heparin and gelatin, within a PVA hydrogel. PVA was functionalized with 7 and 20 methacrylate functional groups per chain (FG/c) to tailor the permselectivity of UV photopolymerized hydrogels. Both heparin and gelatin were covalently incorporated into PVA at an equal ratio resulting in a final PVA:heparin:gelatin composition of 19:0.5:0.5. The combination of both heparin and gelatin within a PVA network has proven to be stable over time without compromising the PVA base characteristics including its permselectivity to different proteins. Most importantly, this combination of ECM analogues supplemented PVA with the dual functionalities of promoting cellular adhesion and sequestering growth factors essential for cellular proliferation. Multi-functional PVA hydrogels with synthetically controlled network characteristics and permselectivity show potential in various biomedical applications including artificial cell implants.
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Materiais Biocompatíveis/química , Gelatina/química , Heparina/química , Hidrogéis/química , Álcool de Polivinil/química , Animais , Materiais Biocompatíveis/metabolismo , Materiais Biocompatíveis/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Gelatina/metabolismo , Gelatina/farmacologia , Heparina/metabolismo , Heparina/farmacologia , Hidrogéis/metabolismo , Hidrogéis/farmacologia , Camundongos , Permeabilidade , Álcool de Polivinil/metabolismo , Álcool de Polivinil/farmacologiaRESUMO
Gene silencing using small interfering RNA has been proposed as a therapy for cancer, viral infections and other diseases. This study aimed to investigate whether layer-by-layer polymer surface modification could deliver small interfering RNA to decrease fibrotic processes associated with medical device implantation. Anti-green fluorescent protein labelled small interfering RNA was applied to tissue culture plates and polyurethane using a layer-by-layer technique with small interfering RNA and poly-L-lysine. In vitro studies showed that the level of down-regulation of green fluorescent protein was directly related to the number of coatings applied. This layer-by-layer coating technique was then used to generate Rhodamine-Flii small interfering RNA-coated implants for in vivo studies of small interfering RNA delivery via subcutaneous implantation in mice. After two days, Rh-positive cells were observed on the implants' surface indicating cellular uptake of the Rhodamine-Flii small interfering RNA. Decreased Flii gene expression was observed in tissue surrounding the Rhodamine-Flii small interfering RNA coated implants for up to seven days post implantation, returning to baseline by day 21. Genes downstream from Flii, including TGF-ß1 and TGF-ß3, showed significantly altered expression confirming a functional effect of the Rhodamine-Flii small interfering RNA on gene expression. This research demonstrates proof-of-principle that small interfering RNA can be delivered via layer-by-layer coatings on biomaterials and thereby can alter the fibrotic process.
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Materiais Biocompatíveis , Proteínas do Citoesqueleto/genética , Polímeros/química , RNA Interferente Pequeno/administração & dosagem , Animais , Proteínas de Transporte , Linhagem Celular , Camundongos , Camundongos Endogâmicos BALB C , Proteínas dos Microfilamentos , Células NIH 3T3 , Propriedades de Superfície , TransativadoresRESUMO
Hydrogels hold significant promise for supporting cell based therapies in the field of bioelectrodes. It has been proposed that tissue engineering principles can be used to improve the integration of neural interfacing electrodes. Degradable hydrogels based on poly (vinyl alcohol) functionalised with tyramine (PVA-Tyr) have been shown to support covalent incorporation of non-modified tyrosine rich proteins within synthetic hydrogels. PVA-Tyr crosslinked with such proteins, were explored as a scaffold for supporting development of neural tissue in a three dimensional (3D) environment. In this study a model neural cell line (PC12) and glial accessory cell line, Schwann cell (SC) were encapsulated in PVA-Tyr crosslinked with gelatin and sericin. Specifically, this study aimed to examine the growth and function of SC and PC12 co-cultures when translated from a two dimensional (2D) environment to a 3D environment. PC12 differentiation was successfully promoted in both 2D and 3D at 25 days post-culture. SC encapsulated as a single cell line and in co-culture were able to produce both laminin and collagen-IV which are required to support neuronal development. Neurite outgrowth in the 3D environment was confirmed by immunocytochemical staining. PVA-Tyr/sericin/gelatin hydrogel showed mechanical properties similar to nerve tissue elastic modulus. It is suggested that the mechanical properties of the PVA-Tyr hydrogels with native protein components are providing with a compliant substrate that can be used to support the survival and differentiation of neural networks.
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Técnicas de Cocultura/métodos , Hidrogéis/química , Animais , Diferenciação Celular , Linhagem Celular , Sobrevivência Celular , Técnicas de Cocultura/instrumentação , Colágeno Tipo IV/metabolismo , Módulo de Elasticidade , Gelatina/química , Laminina/metabolismo , Células PC12 , Álcool de Polivinil/química , Ratos , Engenharia TecidualRESUMO
5,6-Dihydroxy-1H-indazole (DHI) is able to self-polymerize through the same mussel-inspired chemistry responsible for generating poly(dopamine) (PDA), demonstrating the potential to expand this class of catecholamine-exclusive chemistry onto heterocyclic catechol derivatives for the preparation of functional materials. Although DHI exhibits slower polymerization kinetics compared to dopamine, the two chemical species are compatibly polymerizable under the same reaction conditions and allow the preparation of copolymer coatings in different molar ratios. Of these copolymers, the 1 : 3-copolymer (DHI-to-dopamine ratio) has demonstrated adequate structural stability as a polymer coating. While PDA performs as an intact framework, the incorporated DHI enhances the colloidal stability and provides additional coordinating functionality through the pyrazole moieties. The 1 : 3-copolymer was fabricated into polymer capsules which exhibit negligible cytotoxicity towards murine dermal fibroblasts (L929) and enhanced binding behaviour towards copper(ii). This represents a new channel for fabricating cargo carriers for biomedical applications that involve the use of transition metal-based species.
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Heparin-based hydrogels are attractive for cell encapsulation and drug delivery because of the ability of heparin to bind native proteins. However, heparin-based hydrogels have received little attention for their potential as stimuli-sensitive materials. Biosynthetic, poly(vinyl alcohol) (PVA)-heparin hydrogels were formed using dynamic, covalent cross-linking. Hydrogel stimuli-sensitivity was tailored by tuning the concentration of heparin to PVA. Relatively thermally and pH stable hydrogels were produced when formed from only the synthetic, nonionic PVA polymer cross-linked via hydrazone bonds. Cross-linking in the ionic biopolymer heparin, to form PVA-heparin gels, has a profound impact on thermal stability, with degradation ranging from over 6 months to only 4 days across 25-50 °C. PVA-heparin hydrogels degrade within 18 days at basic pH (10), while not fully degrading over 6 months at lower pH (4, 7.4). This finding is attributed to the anionic repulsion of carboxyls and sulfates in heparin. PVA-heparin macromers were cytocompatible and enabled mild cell encapsulation, in addition to providing pH-controlled growth factor release. Overall, it is demonstrated that the biopolymer heparin can be used to create pH and temperature-responsive hydrogel biomaterials for cell and drug delivery.