RESUMO
Injectable biomimetic hydrogels have great potential for use in regenerative medicine as cellular delivery vectors. However, they can suffer from issues relating to hypoxia, including poor cell survival, differentiation, and functional integration owing to the lack of an established vascular network. Here we engineer a hybrid myoglobin:peptide hydrogel that can concomitantly deliver stem cells and oxygen to the brain to support engraftment until vascularisation can occur naturally. We show that this hybrid hydrogel can modulate cell fate specification within progenitor cell grafts, resulting in a significant increase in neuronal differentiation. We find that the addition of myoglobin to the hydrogel results in more extensive innervation within the host tissue from the grafted cells, which is essential for neuronal replacement strategies to ensure functional synaptic connectivity. This approach could result in greater functional integration of stem cell-derived grafts for the treatment of neural injuries and diseases affecting the central and peripheral nervous systems.
Assuntos
Hidrogéis , Células-Tronco Neurais , Hidrogéis/metabolismo , Oxigênio/metabolismo , Mioglobina/metabolismo , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Diferenciação CelularRESUMO
With the limited capacity for self-repair in the adult CNS, efforts to stimulate quiescent stem cell populations within discrete brain regions, as well as harness the potential of stem cell transplants, offer significant hope for neural repair. These new cells are capable of providing trophic cues to support residual host populations and/or replace those cells lost to the primary insult. However, issues with low-level adult neurogenesis, cell survival, directed differentiation and inadequate reinnervation of host tissue have impeded the full potential of these therapeutic approaches and their clinical advancement. Biomaterials offer novel approaches to stimulate endogenous neurogenesis, as well as for the delivery and support of neural progenitor transplants, providing a tissue-appropriate physical and trophic milieu for the newly integrating cells. In this review, we will discuss the various approaches by which bioengineered scaffolds may improve stem cell-based therapies for repair of the CNS.
Assuntos
Materiais Biocompatíveis/farmacologia , Sistema Nervoso Central/citologia , Sistema Nervoso Central/efeitos dos fármacos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Sistema Nervoso Central/patologia , HumanosRESUMO
Neurotrophic growth factors are effective in slowing progressive degeneration and/or promoting neural repair through the support of residual host and/or transplanted neurons. However, limitations including short half-life and enzyme susceptibility of growth factors highlight the need for alternative strategies to prolong localised delivery at a site of injury. Here, we establish the utility of minimalist N-fluorenylmethyloxycarbonyl (Fmoc) self-assembling peptides (SAPs) as growth factor delivery vehicle, targeted at supporting neural transplants in an animal model of Parkinson's disease. The neural tissue-specific SAP, Fmoc-DIKVAV, demonstrated sustained release of glial cell line derived neurotrophic factor, up to 172 hr after gel loading. This represents a significant advance in drug delivery, because its lifetime in phosphate buffered saline was less than 1 hr. In vivo transplantation of neural progenitor cells, together with our growth factor-loaded material, into the injured brain improved graft survival compared with cell transplants alone. We show for the first time the use of minimalist Fmoc-SAP in an in vivo disease model for sustaining the delivery of neurotrophic growth factors, facilitating their spatial and temporal delivery in vivo, whilst also providing an enhanced niche environment for transplanted cells.
Assuntos
Lesões Encefálicas/terapia , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Células-Tronco Neurais/transplante , Peptídeos/farmacologia , Alicerces Teciduais/química , Sequência de Aminoácidos , Animais , Lesões Encefálicas/patologia , Modelos Animais de Doenças , Feminino , Sobrevivência de Enxerto/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Neostriado/efeitos dos fármacos , Neostriado/patologia , Células-Tronco Neurais/efeitos dos fármacos , Doença de Parkinson/patologia , Doença de Parkinson/terapia , Peptídeos/químicaRESUMO
Astrocytes execute essential functions in the healthy CNS, whilst also being implicated as a limitation to functional regeneration and repair after injury. They respond to injury to minimize damage to healthy tissue whilst also attempting to seal the broken blood-brain-barrier, however, they impede recovery if they are persistent and form a permanent scar in the injured brain. As such, it is of great importance to understand the mechanism underlying the astrocytic response to injury, and this understanding is currently limited by the in vitro environments available to scientists. Biomaterials such as nanofibres and hydrogels offer great potential for the development of superior, 3D cell culture environments in which to study astrocyte behavior and phenotype. The implementation of such in vitro environments with a particularly interdisciplinary approach can improve the field's understanding of astrocytes, their role in central nervous system inflammation, and elucidate potential strategies to achieve functional regeneration.
Assuntos
Astrócitos/citologia , Encéfalo/citologia , Técnicas de Cultura de Células , Doenças do Sistema Nervoso Central/patologia , Medula Espinal/citologia , Alicerces Teciduais , Animais , Astrócitos/fisiologia , Diferenciação Celular , Humanos , Hidrogéis , Inflamação/patologia , NanofibrasRESUMO
A simple strontium phosphate (SrP) conversion coating process was developed to protect magnesium (Mg) from the initial degradation post-implantation. The coating morphology, deposition rate and resultant phases are all dependent on the processing temperature, which determines the protective ability for Mg in minimum essential medium (MEM). Coatings produced at 80 °C are primarily made up of strontium apatite (SrAp) with a granular surface, a high degree of crystallinity and the highest protective ability, which arises from retarding anodic dissolution of Mg in MEM. Following 14 days' immersion in MEM, the SrAp coating maintained its integrity with only a small fraction of the surface corroded. The post-degradation effect of uncoated Mg and Mg coated at 40 and 80 °C on the proliferation and differentiation of human mesenchymal stem cells was also studied, revealing that the SrP coatings are biocompatible and permit proliferation to a level similar to that of pure Mg. The present study suggests that the SrP conversion coating is a promising option for controlling the early rapid degradation rate, and hence hydrogen gas evolution, of Mg implants without adverse effects on surrounding cells and tissues.
Assuntos
Materiais Revestidos Biocompatíveis/farmacologia , Magnésio/farmacologia , Fosfatos/farmacologia , Estrôncio/farmacologia , Fosfatase Alcalina/metabolismo , Diferenciação Celular/efeitos dos fármacos , Meios de Cultura , Técnicas Eletroquímicas , Humanos , Hidrogênio/análise , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/enzimologia , Microscopia Eletrônica de Varredura , Osteogênese/efeitos dos fármacos , Espectroscopia Fotoeletrônica , Propriedades de Superfície , Temperatura , Difração de Raios XRESUMO
Tissue specific scaffolds formed from minimalist N-fluorenylmethyloxycarbonyl self-assembling peptides (Fmoc-SAPs) have emerged as promising biomaterials due to their ease of synthesis and capacity to self-assemble via simple, non-covalent interactions into complex nanofibrous hydrogels. However, concerns remain over their biocompatibility and cytotoxicity for in vivo applications. Here, we demonstrate that these Fmoc-SAPs are biocompatible in vivo and well suited as a delivery vehicle for cell transplantation. In order to determine the effect of tissue specific parameters, we designed three Fmoc-SAPs containing varying bioactive peptide sequences derived from extracellular matrix proteins, laminin and fibronectin. Fmoc-SAPs delivering cortical neural progenitor cells into the mouse brain display a limited foreign body response, effective functionalization and low cytotoxicity for at least 28 days. These results highlight the suitability of Fmoc-SAPs for improved neural tissue repair through the support of grafted cells and adjacent host parenchyma. Overall, we illustrate that Fmoc-SAPs are easily engineered materials for use as a tool in cell transplantation, where biocompatibility is key to promoting cell survival, enhancing the graft-host interface and attenuation of the inflammatory response for improved tissue repair outcomes.
RESUMO
Patients who experience injury to the central or peripheral nervous systems invariably suffer from a range of dysfunctions due to the limited ability for repair and reconstruction of damaged neural tissue. Whilst some treatment strategies can provide symptomatic improvement of motor and cognitive function, they fail to repair the injured circuits and rarely offer long-term disease modification. To this end, the biological molecules, used in combination with neural tissue engineering scaffolds, may provide feasible means to repair damaged neural pathways. This review will focus on three promising classes of neural tissue engineering scaffolds, namely hydrogels, electrospun nanofibres and self-assembling peptides. Additionally, the importance and methods for presenting biologically relevant molecules such as, neurotrophins, extracellular matrix proteins and protein-derived sequences that promote neuronal survival, proliferation and neurite outgrowth into the lesion will be discussed.
Assuntos
Tecido Nervoso/citologia , Polímeros/química , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Animais , Humanos , Hidrogéis/química , Hidrogéis/metabolismo , Nanofibras/química , Peptídeos/química , Peptídeos/metabolismo , Polímeros/metabolismoRESUMO
In this study, thermoresponsive xyloglucan hydrogel scaffolds were investigated as candidates for neural tissue engineering of the spinal cord. The hydrogels were optimized to provide similar mechanical properties to that of native spinal cord, although also being functionalized through the immobilization of poly-D-lysine to promote neurone adhesion and neurite outgrowth. Under 2D and 3D culture conditions, xyloglucan scaffolds supported the differentiation of primary cortical neurones. Furthermore, functionalization provided a means of controlling and optimizing the cell diameter, number, migration and the neurite density, and the direction of growth. The interaction of neural stem cells (NSCs) was also investigated on the xyloglucan scaffolds in vitro. The survival of the NSCs and the axonal extensions on the scaffolds were similar to that of the primary cortical neurones. These findings suggest that xyloglucan-based materials are suitable for providing a neurotrophic milieu.
Assuntos
Hidrogel de Polietilenoglicol-Dimetacrilato/química , Células-Tronco Multipotentes/fisiologia , Regeneração Nervosa/fisiologia , Neuritos/fisiologia , Neurônios/citologia , Traumatismos da Medula Espinal , Alicerces Teciduais , Compostos de Anilina/química , Animais , Compostos Azo/química , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Diferenciação Celular , Células Cultivadas , Glucanos/química , Glucanos/metabolismo , Hidrogel de Polietilenoglicol-Dimetacrilato/metabolismo , Teste de Materiais , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Células-Tronco Multipotentes/citologia , Polilisina/química , Polímeros/química , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/terapia , Temperatura , Engenharia Tecidual/métodos , Xilanos/química , Xilanos/metabolismoRESUMO
Electrospinning has been employed extensively in tissue engineering to generate nanofibrous scaffolds from either natural or synthetic biodegradable polymers to simulate the cellular microenvironment. Electrospinning rapidly produces fibers of the nanolength scale and the process offers many opportunities to tailor the physical, chemical, and biological properties of a material for specific applications and cellular environments. There is growing evidence that nanofibers amplify certain biological responses such as contact guidance and differentiation, however this has not been fully exploited in tissue engineering. This review addresses the cellular interactions with electrospun scaffolds, with particular focus on neural, bone, cartilage, and vascular tissue regeneration. Some aspects of scaffold design, including architectural properties, surface functionalization and materials selection are also addressed.
Assuntos
Materiais Biocompatíveis/química , Fenômenos Fisiológicos Celulares , Técnicas Eletroquímicas/métodos , Nanoestruturas/química , Engenharia Tecidual/métodos , Animais , Técnicas de Cultura de Células/métodos , Humanos , Nanotecnologia/métodos , Medicina Regenerativa/métodos , Alicerces TeciduaisRESUMO
Development of biomaterials with specific characteristics to influence cell behaviour has played an important role in exploiting strategies to promote nerve regeneration. The effect of three-dimensional (3D) non-woven electrospun poly(epsilon-caprolactone) (PCL) scaffolds on the behaviour of rat brain-derived neural stem cells (NSCs) is reported. The interaction of NSCs on the randomly orientated submicron (PCL) fibrous scaffolds, with an average fibre diameter of 750 +/- 100 nm, was investigated. The PCL scaffolds were modified with ethylenediamine (ED) to determine if amino functionalisation and changes in surface tension of the fibrous scaffolds affected the proliferation and differentiation characteristics of NSCs. Surface tension of the fibrous scaffold increased upon treatment with ED which was attributed to amine moieties present on the surface of the fibres. Although surface treatment did not change the differentiation of the NSCs, the modified scaffolds were more hydrophilic, resulting in a significant increase in the number of adhered cells, and increased spreading throughout the entirety of the scaffold. When the NSCs were seeded on the PCL scaffolds in the presence of 10% FBS, the stem cells differentiated primarily into oligodendrocytes, indicating that electrospun PCL has the capacity to direct the differentiation of NSCs towards a specific lineage. The data presented here is useful for the development of electrospun biomaterial scaffolds for neural tissue engineering, to regulate the proliferation and differentiation of NSCs.
Assuntos
Diferenciação Celular/fisiologia , Neurônios/citologia , Oligodendroglia/citologia , Poliésteres/química , Células-Tronco/citologia , Animais , Adesão Celular/fisiologia , Proliferação de Células , Células Cultivadas , Neurônios/fisiologia , Oligodendroglia/fisiologia , Ratos , Células-Tronco/fisiologia , Propriedades de SuperfícieRESUMO
The interaction of murine embryonic cortical neurons on randomly orientated electrospun scaffolds of poly(L-lactide) (P(L)LA) and poly(lactide-co-glycolide) (PLGA) is investigated in this study. The scaffolds were surface treated with different concentrations of KOH to partially hydrolyze the surface and therefore change the surface tension. Hydrophilicity did not significantly influence the number of primary and secondary branches; however, it had a considerable effect on neurite extension. For scaffolds with surface tensions of 40-47 dyn cm(-1) there was a significantly greater overall neurite length for both the primary and secondary branches compared with more hydrophilic scaffolds. Another major finding of this work was that the interfibre distance influenced how the neurites extended. When the interfibre distance was greater than approximately 15 microm the neurites followed the fibres and avoided regions of very high fibre density. At interfibre distances less than approximately 15 microm, the neurites traversed between the fibres. Therefore, this study provided little evidence that contact guidance was the dominating cue in directing neurite extension, instead inferring that chemical cues, possibly from adjacent neurons had induced directional change.
Assuntos
Córtex Cerebral/embriologia , Regeneração Tecidual Guiada/métodos , Ácido Láctico/química , Nanoestruturas/química , Neurônios/citologia , Neurônios/fisiologia , Ácido Poliglicólico/química , Polímeros/química , Engenharia Tecidual/métodos , Animais , Materiais Biocompatíveis/química , Técnicas de Cultura de Células/métodos , Crescimento Celular , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/fisiologia , Regeneração Tecidual Guiada/instrumentação , Camundongos , Camundongos Endogâmicos C57BL , Nanoestruturas/ultraestrutura , Tamanho da Partícula , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Propriedades de SuperfícieRESUMO
The aim of this study was to investigate the interaction of mouse embryonic cortical neurons on P(L)LA and PLGA substrates, which were partially hydrolysed using potassium hydroxide (KOH). The chemical and topographical properties of the surfaces were characterized, and it was discovered that there was a decrease in the hydrophilicity for the P(L)LA with increasing concentration of KOH. This was due to chemical modifications to the surfaces of the substrates. Alternatively for the PLGA substrate, only the 0.1 M KOH treated sample had a significantly different hydrophilicity highlighting that surface erosion resulted at higher concentrations. The morphology of the neurons grown on the two substrates were compared to poly(D)lysine (positive control). The neurons formed colonies on all of the substrates, but were dramatically reduced in size in the case of the 0.1 M KOH treated substrates. This finding was attributed to the increases in cell spreading and the size of the cells, as they were larger, more elongated and bipolar like those on the positive control. However, there was a significant decrease in the total number of live cells per unit area. Therefore, on these materials when there was increased cellular spreading there was significantly higher cell death. Furthermore, unlike the 0, 0.2, and 0.4 M KOH treated substrates, there was an absence of large bundles of axons that extended between colonies on the 0.1 M sample, instead exhibiting short axons that grew in free space.
Assuntos
Córtex Cerebral/embriologia , Neurônios/fisiologia , Animais , Materiais Biocompatíveis , Técnicas de Cultura de Células , Divisão Celular , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Concentração de Íons de Hidrogênio , Hidróxidos , Ácido Láctico , Camundongos , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Neurônios/citologia , Poliésteres , Ácido Poliglicólico , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Polímeros , Compostos de Potássio , Propriedades de SuperfícieRESUMO
Galactose modified xyloglucan is a thermally reversible hydrogel that is increasingly used in the biomedical field due to the ease of altering the gelation time and temperature by modifying the galactose removal ratio. However there is little information concerning the morphology and rheological properties of the hydrogel under physiological conditions. Differential scanning microcalorimetry (DSmicroC) showed the thermal gelation process to occur over a broad temperature range (5-50 degrees C). The rheological properties of the hydrogels were investigated as a function of concentration, temperature and ionic strength. The final elastic moduli of the hydrogels increased with increases in concentration. Isothermal rheology suggests that the gelation occurred in two distinct stages, which was influenced by the solution media. Scanning electron microscopy (SEM) was used to characterize the morphology of the xyloglucan which were thermally gelled at 37 degrees C. The resultant morphology was strongly dependent on the concentration of the hydrogel. Strong hydrogels were only obtained at 3 wt.% at 37 degrees C, and the morphology characterized by an open 3-dimensional network, comprised of thin membranes. It is proposed that the first stage of the isothermal gelation is the formation and growth of the thin membranes, followed by the formation of a three dimensional network.
Assuntos
Glucanos/química , Hidrogéis/química , Xilanos/química , Varredura Diferencial de Calorimetria , Glucanos/ultraestrutura , Estrutura Molecular , Reologia , Temperatura , Xilanos/ultraestruturaRESUMO
The aim of this study was to determine the ability of various poly(alpha-hydroxy esters) to support the in vitro propagation of murine embryonic stem (ES) cells in an undifferentiated state. To this end, ES cell colonization, growth and Oct-4 immunoreactivity following a 48 h culture period upon poly((D,L)-lactide), poly((L)-lactide), poly(glycolide) and poly((D,L)-lactide-co-glycolide) (PLGA) were assessed. By the analysis of live and dead cell number indices and Oct-4 immunoreactivity, ES cell colonization rate during a 48 h culture period was found to be significantly greater on PLGA compared to all the other unmodified poly(alpha-hydroxy esters) tested. Surface treatment of all polymers with 0.1m potassium hydroxide revealed a significant increase in ES cell live numbers when compared to all unmodified polymers, thus revealing a correlation between polymer content, hydrophilicity and colonization rate. These data suggest that surface treated poly(alpha-hydroxy esters) may be employed for ES cell scale up procedures and in tissue engineering applications requiring the colonization of scaffolds by ES cells in an undifferentiated state. According to such applications, once the designated scaffold has been colonized, ES cell directed differentiation into the desired and fully differentiated, functional adult tissue may then be effected.