RESUMO
One of the main issues in orthopaedic implant design is the fabrication of scaffolds that closely mimic the biomechanical properties of the surrounding bone. This research reports on a multi-stage rapid prototyping technique that was successfully developed to produce porous titanium scaffolds with fully interconnected pore networks and reproducible porosity and pore size. The scaffolds' porous characteristics were governed by a sacrificial wax template, fabricated using a commercial 3D-printer. Powder metallurgy processes were employed to generate the titanium scaffolds by filling around the wax template with titanium slurry. In the attempt to optimise the powder metallurgy technique, variations in slurry concentration, compaction pressure and sintering temperature were investigated. By altering the wax design template, pore sizes ranging from 200 to 400 microm were achieved. Scaffolds with porosities of 66.8 +/- 3.6% revealed compression strengths of 104.4+/-22.5 MPa in the axial direction and 23.5 +/- 9.6 MPa in the transverse direction demonstrating their anisotropic nature. Scaffold topography was characterised using scanning electron microscopy and microcomputed tomography. Three-dimensional reconstruction enabled the main architectural parameters such as pore size, interconnecting porosity, level of anisotropy and level of structural disorder to be determined. The titanium scaffolds were compared to their intended designs, as governed by their sacrificial wax templates. Although discrepancies in architectural parameters existed between the intended and the actual scaffolds, overall the results indicate that the porous titanium scaffolds have the properties to be potentially employed in orthopaedic applications.
Assuntos
Titânio/química , Materiais Biocompatíveis , Linhagem Celular , Humanos , Metalurgia , Microscopia Eletrônica de Varredura , Pós , Engenharia TecidualRESUMO
In order to develop efficient design strategies for a tissue-engineered heart valve, in vivo and in vitro models of valvular structure and cellular function require extensive characterisation. Collagen and glycosaminoglycans (GAGs) provide unique functional characteristics to the heart valve structure. In the current study, type I collagen-GAG hydrogels were investigated as biomaterials for the creation of mitral valve tissue. Porcine mitral valve interstitial cells (VICs) and endothelial cells (VECs) were isolated and co-cultured for 4 weeks in hydrogel constructs composed of type I collagen. The metabolic activity and tissue organisation of mitral valve tissue constructs was evaluated in the presence and absence of chondroitin sulphate (CS) GAG, and comparisons were made with normal mitral valve tissue. Both collagen and collagen-CS mitral valve constructs contracted to form tissue-like structures in vitro. Biochemical assay demonstrated that over 75% of CS was retained within collagen-CS constructs. Morphological examination demonstrated enhanced VEC surface coverage in collagen-CS constructs compared to collagen constructs. Ultrastructural analysis revealed basement membrane synthesis and cell junction formation by construct VECs, with an increased matrix porosity observed in collagen-CS constructs. Immunohistochemical analyses demonstrated enhanced extracellular matrix production in collagen-CS constructs, including expression of elastin and laminin by VICs. Both native valve and collagen-CS construct VECs also expressed the vasoactive molecule, eNOS, which was absent from collagen construct VECs. The present study demonstrates that collagen gels can be used as matrices for the in vitro synthesis of tissue structures resembling mitral valve tissue. Addition of CS resulting in a more porous model was shown to positively influence the bioactivity of seeded valve cells and tissue remodelling. Collagen-GAG matrices may hold promise for a potential use in heart valve tissue engineering and improved understanding of heart valve biology.
Assuntos
Colágeno , Glicosaminoglicanos , Valva Mitral , Modelos Biológicos , Engenharia Tecidual/métodos , Animais , Materiais Biocompatíveis/química , Células Cultivadas , Técnicas de Cocultura , Colágeno/química , Células Endoteliais/ultraestrutura , Matriz Extracelular , Glicosaminoglicanos/química , Hidrogéis , Teste de Materiais , Valva Mitral/metabolismo , Valva Mitral/ultraestrutura , SuínosRESUMO
This study investigated the effect on the mechanical and physicochemical properties of type II collagen scaffolds after cross-linking with microbial transglutaminase (mTGase). It is intended to develop a collagen-based scaffold to be used for the treatment of degenerated intervertebral discs. By measuring the amount of epsilon-(gamma-glutamyl)lysine isodipeptide formed after cross-linking, it was determined that the optimal enzyme concentration was 0.005% (w/v). From the production of covalent bonds induced by mTGase cross-linking, the degradation resistance of type II collagen scaffolds can be enhanced. Rheological analysis revealed an almost sixfold increase in storage modulus (G') with 0.005% (w/v) mTGase cross-linked scaffolds (1.31 +/- 0.03 kPa) compared to controls (0.21 +/- 0.01 kPa). There was a significant reduction in the level of cell-mediated contraction of scaffolds with increased mTGase concentrations. Cell proliferation assays showed that mTGase crosslinked scaffolds exhibited similar cytocompatibility properties in comparison to non-cross-linked scaffolds. In summary, cross-linking type II collagen with mTGase imparted more desirable properties, making it more applicable for use as a scaffold in tissue engineering applications.
Assuntos
Proteínas de Bactérias/fisiologia , Materiais Biocompatíveis , Colágeno Tipo II/metabolismo , Reagentes de Ligações Cruzadas/metabolismo , Dipeptídeos/metabolismo , Engenharia Tecidual , Transglutaminases/fisiologia , Células 3T3 , Animais , Proteínas de Bactérias/toxicidade , Proliferação de Células , Galinhas , Colágeno Tipo II/química , Reagentes de Ligações Cruzadas/química , Dipeptídeos/química , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Camundongos , Streptomyces/enzimologia , Transglutaminases/toxicidadeRESUMO
The definitive goal of this research is to develop protein-based scaffolds for use in soft tissue regeneration, particularly in the field of dermal healing. The premise of this investigation was to characterize the mechanical properties of gelatin cross-linked with microbial transglutaminase (mTGase) and to investigate the cytocompatibility of mTGase cross-linked gelatin. Dynamic rheological analysis revealed a significant increase in the storage modulus and thermal stability of gelatin after cross-linking with mTGase. Static, unconfined compression tests showed an increase in Young's modulus of gelatin gels after mTGase cross-linking. A comparable increase in gel strength was observed with 0.03% mTGase and 0.25% glutaraldehyde cross-linked gelatin gels. In vitro studies using 3T3 fibroblasts indicated cytotoxicity at a concentration of 0.05% mTGase after 72 h. However, no significant inhibition of cell proliferation was seen with cells grown on lower concentrations of mTGase cross-linked gelatin substrates. The mechanical improvement and cytocompatibility of mTGase cross-linked gelatin suggests mTGase has potential for use in stabilizing gelatin gels for tissue-engineering applications.
Assuntos
Gelatina/química , Gelatina/metabolismo , Transglutaminases/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Gelatina/farmacologia , Glutaral/metabolismo , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Hidrogel de Polietilenoglicol-Dimetacrilato/metabolismo , Camundongos , Células NIH 3T3 , Suínos , Temperatura , Fatores de Tempo , Engenharia Tecidual/métodosRESUMO
Realistic ex vivo anthropometric vascular environments are required for endovascular device optimization and for preclinical evaluation of interventional procedures. The objective of this research is to build an anthropomorphic model of the human carotid artery. The combination of magnetic resonance angiography image processing and computer-aided design and manufacturing techniques allowed fabrication of multicomponent morphologically precise casts of the carotid artery. The lost core technique was used to produce a hollow vessel prototype incorporating polyvinyl alcohol cryogel (PVA-C) as a tissue-mimicking vessel wall material. PVA-C was mechanically characterized by uniaxial tensile testing after different numbers of freeze/thaw cycles. The novel model construction approach outlined in this study accounts for the morphologic complexities of the human vasculature, and proved successful for the production of realistic compliant ex vivo arterial model.
Assuntos
Artérias Carótidas/fisiologia , Modelos Anatômicos , Materiais Biomiméticos/química , Artérias Carótidas/anatomia & histologia , Feminino , Humanos , Hidrogéis , Processamento de Imagem Assistida por Computador , Angiografia por Ressonância Magnética , Teste de Materiais , Pessoa de Meia-Idade , Álcool de Polivinil/química , Resistência à TraçãoRESUMO
Over the last twenty years there have been several reports on the use of nonviral vectors to facilitate gene transfer in the mammalian brain. Whilst a large emphasis has been placed on vector transfection efficiency, the study of the adverse effects upon the brain, caused by the vectors themselves, remains completely overshadowed. To this end, a study was undertaken to study the tissue response to three commercially available transfection agents in the brain of adult Sprague Dawley rats. The response to these transfection agents was compared to adeno-associated viral vector (AAV), PBS and naked DNA. Furthermore, the use of a collagen hollow sphere (CHS) sustained delivery system was analysed for its ability to reduce striatal toxicity of the most predominantly studied polymer vector, polyethyleneimine (PEI). The size of the gross tissue loss at the injection site was analysed after immunohistochemical staining and was used as an indication of acute toxicity. Polymeric vectors showed similar levels of acute brain toxicity as seen with AAV, and CHS were able to significantly reduce the toxicity of the PEI vector. In addition; the host response to the vectors was measured in terms of reactive astrocytes and microglial cell recruitment. To understand whether this gross tissue loss was caused by the direct toxicity of the vectors themselves an in vitro study on primary astrocytes was conducted. All vectors reduced the viability of the cells which is brought about by direct necrosis and apoptosis. The CHS delivery system reduced cell necrosis in the early stages of post administration. In conclusion, whilst polymeric gene vectors cause acute necrosis, administration in the brain causes adverse effects no worse than that of an AAV vector. Furthermore, packaging the PEI vector with CHS reduces surface charge and direct toxicity without elevating the host response.
Assuntos
Colágeno/farmacologia , Vetores Genéticos/toxicidade , Microesferas , Neurotoxinas/toxicidade , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Sistemas Computacionais , Vetores Genéticos/efeitos adversos , Masculino , Polietilenoimina/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Multiple sclerosis (MS) is characterized by the presence of inflammatory demyelinating foci throughout the brain and spinal cord, accompanied by axonal and neuronal damage. Although inflammatory processes are thought to underlie the pathological changes, the individual mediators of this damage are unclear. In order to study the role of pro-inflammatory cytokines in demyelination in the central nervous system, we have utilized a hyperbranched poly(2-dimethyl-aminoethylmethacrylate) based non-viral gene transfection system to establish an inflammatory demyelinating model of MS in an ex-vivo environment. The synthesized non-viral gene transfection system was optimized for efficient transfection with minimal cytotoxicity. Organotypic brain slices were then successfully transfected with the TNF or IFNγ genes. TNF and IFNγ expression and release in cerebellar slices via non-viral gene delivery approach resulted in inflammation mediated myelin loss, thus making it a promising ex-vivo approach for studying the underlying mechanisms of demyelination in myelin-related diseases such as MS.
Assuntos
Doenças Desmielinizantes/patologia , Inflamação/patologia , Metacrilatos/farmacologia , Modelos Biológicos , Esclerose Múltipla/patologia , Polímeros/farmacologia , Animais , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Humanos , Interferon gama/metabolismo , Metacrilatos/síntese química , Metacrilatos/toxicidade , Proteína Básica da Mielina/metabolismo , Bainha de Mielina/metabolismo , Proteínas de Neurofilamentos/metabolismo , Polímeros/síntese química , Polímeros/toxicidade , Ratos , Ratos Sprague-Dawley , Transfecção , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The current microsurgical gold standard for repairing long gap nerve injuries is the autograft. Autograft provides a protective environment for repair and a natural internal architecture, which is essential for regeneration. Current clinically approved hollow nerve guidance conduits allow provision of this protective environment; however they fail to provide an essential internal architecture to the regenerating nerve. In the present study both structured and unstructured intraluminal collagen fibres are investigated to assess their ability to enhance conduit mediated nerve repair. This study presents a direct comparison of both structured and unstructured fibres in vivo. The addition of intraluminal guidance structures was shown to significantly decrease axonal dispersion within the conduit and reduced axonal mismatch of distal nerve targets (p < 0.05). The intraluminal fibres were shown to be successfully incorporated into the host regenerative process, acting as a platform for Schwann cell migration and axonal regeneration. Ultimately the fibres were able to provide a platform for nerve regeneration in a long term regeneration study (16 weeks) and facilitated increased guidance of regenerating axons towards their distal nerve targets.
Assuntos
Axônios/fisiologia , Colágeno/química , Regeneração Tecidual Guiada/métodos , Regeneração Nervosa/fisiologia , Traumatismos dos Nervos Periféricos/cirurgia , Nervos Periféricos/fisiologia , Animais , Materiais Biocompatíveis , Carbodi-Imidas/química , Comunicação Celular/fisiologia , Movimento Celular/fisiologia , Microambiente Celular/fisiologia , Colágeno/metabolismo , Colágeno/ultraestrutura , Feminino , Regeneração Tecidual Guiada/instrumentação , Regeneração Tecidual Guiada/tendências , Microscopia Eletrônica de Varredura , Procedimentos Neurocirúrgicos/métodos , Nervos Periféricos/ultraestrutura , Ratos , Ratos Endogâmicos Lew , Recuperação de Função Fisiológica , Células de Schwann/fisiologia , Nervo Isquiático/fisiologia , Succinimidas/química , Propriedades de Superfície , Transplante AutólogoRESUMO
An injectable hydrogel, acting as a reservoir for cell delivery and mimicking the native environment, offers promise for nucleus pulposus (NP) repair and regeneration. Herein, the potential of a stabilised type II collagen hydrogel using poly(ethylene glycol) ether tetrasuccinimidyl glutarate (4S-StarPEG) cross-linker, enriched with hyaluronic acid (HA) was investigated. The optimally stabilised type II collagen hydrogel was determined by assessing free amine groups, resistance to enzymatic degradation, gel point. The potential toxicity of the cross-linker was initially assessed against adipose-derived stem cells (ADSCs). After addition of HA (molar ratio type II collagen:HA 9:0, 9:1, 9:4.5, 9:9) within the hydrogel, the behaviour of the encapsulated NP cells was evaluated using cell proliferation assay, gene expression analysis, cell distribution and cell morphology. A significant decrease (p < 0.05) in the free amine groups of collagen was observed, confirming successful cross-linking. Gelation was independent of the concentration of 4S-StarPEG (8 min at 37 °C). The 1 mm cross-linked hydrogel yielded the most stable after enzymatic degradation (p < 0.05). No toxicity of the 4S-StarPEG was noted for the ADSCs. NP cell viability was high regardless of the concentration of HA (>80%). A cell proliferation was not seen after 14 days in its presence. At a gene expression level, HA did not influence NP cells phenotype after seven days in culture. After seven days in culture, the type I collagen mRNA expression was maintained (p > 0.05). The optimally stabilised and functionalised type II collagen/HA hydrogel system developed in this study shows promise as an injectable reservoir system for intervertebral disc regeneration.