RESUMO
Schwann cell (SC) transplantation represents a promising therapeutic approach for traumatic spinal cord injury but is frustrated by barrier formation, preventing cell migration, and axonal regeneration at the interface between grafted SCs and reactive resident astrocytes (ACs). Although regenerating axons successfully extend into SC grafts, only a few cross the SC-AC interface to re-enter lesioned neuropil. To date, research has focused on identifying and modifying the molecular mechanisms underlying such scarring cell-cell interactions, while the influence of substrate topography remains largely unexplored. Using a recently modified cell confrontation assay to model SC-AC barrier formation in vitro, highly oriented poly(ε-caprolactone) nanofibers were observed to reduce AC reactivity, induce extensive oriented intermingling between SCs and ACs, and ultimately enable substantial neurite outgrowth from the SC compartment into the AC territory. It is anticipated that these findings will have important implications for the future design of biomaterial-based scaffolds for nervous tissue repair.
Assuntos
Astrócitos , Neuritos , Humanos , Axônios , Regeneração Nervosa , Cicatriz/patologia , Células de Schwann/patologia , Células de Schwann/fisiologia , Células de Schwann/transplanteRESUMO
In the original publication, sixth author's surname was incorrectly published as "Llyod" instead of "Lloyd". The correct name should read as "Amy Lloyd".
RESUMO
Secondary damage following spinal cord injury leads to non-reversible lesions and hampering of the reparative process. The local production of pro-inflammatory cytokines such as TNF-α can exacerbate these events. Oligodendrocyte death also occurs, followed by progressive demyelination leading to significant tissue degeneration. Dental stem cells from human apical papilla (SCAP) can be easily obtained at the removal of an adult immature tooth. This offers a minimally invasive approach to re-use this tissue as a source of stem cells, as compared to biopsying neural tissue from a patient with a spinal cord injury. We assessed the potential of SCAP to exert neuroprotective effects by investigating two possible modes of action: modulation of neuro-inflammation and oligodendrocyte progenitor cell (OPC) differentiation. SCAP were co-cultured with LPS-activated microglia, LPS-activated rat spinal cord organotypic sections (SCOS), and LPS-activated co-cultures of SCOS and spinal cord adult OPC. We showed for the first time that SCAP can induce a reduction of TNF-α expression and secretion in inflamed spinal cord tissues and can stimulate OPC differentiation via activin-A secretion. This work underlines the potential therapeutic benefits of SCAP for spinal cord injury repair.
Assuntos
Ativinas/metabolismo , Diferenciação Celular/fisiologia , Papila Dentária/metabolismo , Inflamação/prevenção & controle , Células Precursoras de Oligodendrócitos/metabolismo , Células-Tronco/metabolismo , Adulto , Animais , Linhagem Celular , Doenças Desmielinizantes/metabolismo , Doenças Desmielinizantes/prevenção & controle , Papila Dentária/citologia , Humanos , Inflamação/metabolismo , Camundongos , Neurônios/metabolismo , Oligodendroglia/metabolismo , Ratos , Ratos Wistar , Medula Espinal/metabolismo , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/terapia , Células-Tronco/citologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The generation of complex three-dimensional bioengineered scaffolds that are capable of mimicking the molecular and topographical cues of the extracellular matrix found in native tissues is a field of expanding research. The systematic development of such scaffolds requires the characterisation of cell behaviour in response to the individual components of the scaffold. In the present investigation, we studied cell-substrate interactions between purified populations of Schwann cells and three-dimensional fibrin hydrogel scaffolds, in the presence or absence of multiple layers of highly orientated electrospun polycaprolactone nanofibres. Embedded Schwann cells remained viable within the fibrin hydrogel for up to 7 days (the longest time studied); however, cell behaviour in the hydrogel was somewhat different to that observed on the two-dimensional fibrin substrate: Schwann cells failed to proliferate in the fibrin hydrogel, whereas cell numbers increased steadily on the two-dimensional fibrin substrate. Schwann cells within the fibrin hydrogel developed complex process branching patterns, but, when presented with orientated nanofibres, showed a strong tendency to redistribute themselves onto the nanofibres, where they extended long processes that followed the longitudinal orientation of the nanofibres. The process length along nanofibre-containing fibrin hydrogel reached near-maximal levels (for the present experimental conditions) as early as 1 day after culturing. The ability of this three-dimensional, extracellular matrix-mimicking scaffold to support Schwann cell survival and provide topographical cues for rapid process extension suggest that it may be an appropriate device design for the bridging of experimental lesions of the peripheral nervous system.
Assuntos
Fibrina/química , Hidrogéis/química , Nanofibras/química , Cultura Primária de Células/métodos , Células de Schwann/fisiologia , Alicerces Teciduais/química , Animais , Movimento Celular , Proliferação de Células , Células Cultivadas , Feminino , Hidrogéis/síntese química , Hidrogéis/farmacologia , Ratos , Ratos Sprague-Dawley , Células de Schwann/citologia , Células de Schwann/efeitos dos fármacosRESUMO
Recreating human tissues and organs in the petri dish to establish models as tools in biomedical sciences has gained momentum. These models can provide insight into mechanisms of human physiology, disease onset, and progression, and improve drug target validation, as well as the development of new medical therapeutics. Transformative materials play an important role in this evolution, as they can be programmed to direct cell behavior and fate by controlling the activity of bioactive molecules and material properties. Using nature as an inspiration, scientists are creating materials that incorporate specific biological processes observed during human organogenesis and tissue regeneration. This article presents the reader with state-of-the-art developments in the field of in vitro tissue engineering and the challenges related to the design, production, and translation of these transformative materials. Advances regarding (stem) cell sources, expansion, and differentiation, and how novel responsive materials, automated and large-scale fabrication processes, culture conditions, in situ monitoring systems, and computer simulations are required to create functional human tissue models that are relevant and efficient for drug discovery, are described. This paper illustrates how these different technologies need to converge to generate in vitro life-like human tissue models that provide a platform to answer health-based scientific questions.
Assuntos
Células-Tronco , Engenharia Tecidual , Humanos , Descoberta de Drogas , Sistemas de Liberação de Medicamentos , Materiais Biocompatíveis/farmacologiaRESUMO
In nerve regeneration, scaffolds play an important role in providing an artificial extracellular matrix with architectural, mechanical, and biochemical cues to bridge the site of injury. Directed nerve growth is a crucial aspect of nerve repair, often introduced by engineered scaffolds imparting linear tracks. The influence of physical cues, determined by well-defined architectures, has been mainly studied for implantable scaffolds and is usually limited to continuous guiding features. In this report, the potential of short anisometric microelements in inducing aligned neurite extension, their dimensions, and the role of vertical and horizontal distances between them, is investigated. This provides crucial information to create efficient injectable 3D materials with discontinuous, in situ magnetically oriented microstructures, like the Anisogel. By designing and fabricating periodic, anisometric, discreet guidance cues in a high-throughput 2D in vitro platform using two-photon lithography techniques, the authors are able to decipher the minimal guidance cues required for directed nerve growth along the major axis of the microelements. These features determine whether axons grow unidirectionally or cross paths via the open spaces between the elements, which is vital for the design of injectable Anisogels for enhanced nerve repair.
Assuntos
Sinais (Psicologia) , Neuritos , Axônios , Regeneração Nervosa , Neurogênese , Alicerces TeciduaisRESUMO
BACKGROUND: Molecular composition and topography of the extracellular matrix (ECM) influence regenerative cell migration following peripheral nerve injury (PNI). Advanced tissue engineering strategies for the repair of neurotmesis-type PNI include the development of nanofiber-containing implantable scaffolds that mimic features of the ECM to orchestrate regenerative growth. Reliable and quantifiable in vitro assays are required to assess the ability of such substrates to influence migration of the cell types of interest. However, most popular migration assays monitor cell migration into a cell exclusion zone (CEZ) but have dubious abilities to preserve the molecular and topographical cues of the substrate. NEW METHOD: Elastic band spacers (EBS), a simple, economical and standardized technique for the generation of well-defined CEZ based on the use of commercially available elastic bands, are introduced. RESULTS: EBS could sufficiently preserve ECM-derived molecular and poly(ε-caprolactone) (PCL) nanofiber-derived topographical cues. The application of EBS in the absence and presence of nanofiber-derived topographical cues was validated using perineurial cells and Schwann cells, both known to play key roles in peripheral nerve regeneration. COMPARISON WITH EXISTING METHODS: In contrast to EBS, commercial silicone inserts and the popular scratch assay caused substantial ECM substrate disruption, thereby preventing these techniques from being included in further investigations employing deposition of PCL nanofibers and cell migration analysis. CONCLUSIONS: EBS represent a useful addition to the existing repertoire of migration assays offering significant benefits in terms of substrate preservation. The simplicity and economy of the approach make it immediately accessible to research groups at minimal extra expense.
Assuntos
Nanofibras , Movimento Celular , Sinais (Psicologia) , Matriz Extracelular , Humanos , Nervos Periféricos , Alicerces TeciduaisRESUMO
Severe spinal cord injury (SCI) results in permanent functional deficits, which despite pre-clinical advances, remain untreatable. Combinational approaches, including the implantation of bioengineered scaffolds are likely to promote significant tissue repair. However, this critically depends on the extent to which host tissue can integrate with the implant. In the present paper, blood vessel formation and maturation were studied within and around implanted micro-structured type-I collagen scaffolds at 10 weeks post implantation in adult rat mid-cervical spinal cord lateral funiculotomy injuries. Morphometric analysis revealed that blood vessel density within the scaffold was similar to that of the lateral white matter tracts that the implant replaced. However, immunohistochemistry for zonula occludens-1 (ZO-1) and endothelial barrier antigen revealed that scaffold microvessels remained largely immature, suggesting poor blood-spinal cord barrier (BSB) reformation. Furthermore, a band of intense ZO-1-immunoreactive fibroblast-like cells isolated the implant. Spinal cord vessels outside the ZO-1-band demonstrated BSB-formation, while vessels within the scaffold generally did not. The formation of a double-layered fibrotic and astroglial scar around the collagen scaffold might explain the relatively poor implant-host integration and suggests a mechanism for failed microvessel maturation. Targeted strategies that improve implant-host integration for such biomaterials will be vital for future tissue engineering and regenerative medicine approaches for traumatic SCI.
Assuntos
Vasos Sanguíneos/patologia , Colágeno/química , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/terapia , Engenharia Tecidual/métodos , Alicerces Teciduais , Animais , Antígenos de Superfície/metabolismo , Materiais Biocompatíveis , Modelos Animais de Doenças , Feminino , Fibroblastos/metabolismo , Fibrose , Microcirculação , Ratos , Ratos Sprague-Dawley , Medicina Regenerativa , Medula Espinal/patologia , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
Tissue-engineered constructs have great potential in many intervention strategies. In order for these constructs to function optimally, they should ideally mimic the cellular alignment and orientation found in the tissues to be treated. Here we present a simple and reproducible method for the production of cell-laden pure fibrin micro-fibers with longitudinal topography. The micro-fibers were produced using a molding technique and longitudinal topography was induced by a single initial stretch. Using this method, fibers up to 1 m in length and with diameters of 0.2-3 mm could be produced. The micro-fibers were generated with embedded endothelial cells, smooth muscle cell/fibroblasts or Schwann cells. Polarized light and scanning electron microscopy imaging showed that the initial stretch was sufficient to induce longitudinal topography in the fibrin gel. Cells in the unstretched control micro-fibers elongated randomly in both the floating and encapsulated environments, whereas the cells in the stretched micro-fibers responded to the introduced topography by adopting a similar orientation. Proof of concept bottom-up tissue engineering (TE) constructs are shown, all displaying various anisotropic organization of cells within. This simple, economical, versatile and scalable approach for the production of highly orientated and cell-laden micro-fibers is easily transferrable to any TE laboratory.
Assuntos
Fibrina/química , Fibroblastos/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Miócitos de Músculo Liso/metabolismo , Células de Schwann/metabolismo , Alicerces Teciduais/química , Humanos , Engenharia TecidualRESUMO
Severe traumatic spinal cord injury (SCI) results in a devastating and permanent loss of function, and is currently an incurable condition. It is generally accepted that future intervention strategies will require combinational approaches, including bioengineered scaffolds, to support axon growth across tissue scarring and cystic cavitation. Previously, we demonstrated that implantation of a microporous type-I collagen scaffold into an experimental model of SCI was capable of supporting functional recovery in the absence of extensive implant-host neural tissue integration. Here, we demonstrate the reactive host cellular responses that may be detrimental to neural tissue integration after implantation of collagen scaffolds into unilateral resection injuries of the adult rat spinal cord. Immunohistochemistry demonstrated scattered fibroblast-like cell infiltration throughout the scaffolds as well as the presence of variable layers of densely packed cells, the fine processes of which extended along the graft-host interface. Few reactive astroglial or regenerating axonal profiles could be seen traversing this layer. Such encapsulation-type behaviour around bioengineered scaffolds impedes the integration of host neural tissues and reduces the intended bridging role of the implant. Characterization of the cellular and molecular mechanisms underpinning this behaviour will be pivotal in the future design of collagen-based bridging scaffolds intended for regenerative medicine.
RESUMO
SIL1 acts as nucleotide exchange factor for the endoplasmic reticulum chaperone BiP. Mutations of SIL1 cause Marinesco-Sjögren syndrome (MSS), a neurodegenerative disorder. Moreover, a particular function of SIL1 for etiopathology of amyotrophic lateral sclerosis (ALS) was highlighted, thus declaring the functional SIL1-BiP complex as a modifier for neurodegenerative disorders. Thereby, depletion of SIL1 was associated with an earlier manifestation and in strengthened disease progression in ALS. Owing to the absence of appropriate in vitro models, the precise cellular pathophysiological mechanisms leading to neurodegeneration in MSS and triggering the same in further disorders like ALS are still elusive. We found that SIL1 depletion in human embryonic kidney 293 (HEK293) cells led to structural changes of the endoplasmic reticulum (ER) including the nuclear envelope and mitochondrial degeneration that closely mimic pathological alterations in MSS and ALS. Functional studies revealed disturbed protein transport, cytotoxicity with reduced proliferation and viability, accompanied by activation of cellular defense mechanisms including the unfolded protein response, ER-associated degradation pathway, proteolysis, and expression of apoptotic and survival factors. Our data moreover indicated that proteins involved in cytoskeletal organization, vesicular transport, mitochondrial function, and neurological processes contribute to SIL1 pathophysiology. Altered protein expression upon SIL1 depletion in vitro could be confirmed in Sil1-deficient motoneurones for paradigmatic proteins belonging to different functional classes. Our results demonstrate that SIL1-depleted HEK293 cells are an appropriate model to identify proteins modulated by SIL1 expression level and contributing to neurodegeneration in MSS and further disorders like ALS. Thereby, our combined results point out that proteins beyond such involved ER-related protein processing are affected by SIL1 depletion.
Assuntos
Retículo Endoplasmático/metabolismo , Precursores de Proteínas/metabolismo , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Degradação Associada com o Retículo Endoplasmático , Células HEK293 , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Membrana Nuclear/metabolismo , Membrana Nuclear/ultraestrutura , Proteoma/metabolismo , Frações Subcelulares/metabolismo , Resposta a Proteínas não DobradasRESUMO
The implantation of bioengineered scaffolds into lesion-induced gaps of the spinal cord is a promising strategy for promoting functional tissue repair because it can be combined with other intervention strategies. Our previous investigations showed that functional improvement following the implantation of a longitudinally microstructured collagen scaffold into unilateral mid-cervical spinal cord resection injuries of adult Lewis rats was associated with only poor axon regeneration within the scaffold. In an attempt to improve graft-host integration as well as functional recovery, scaffolds were seeded with highly enriched populations of syngeneic, olfactory bulb-derived ensheathing cells (OECs) prior to implantation into the same lesion model. Regenerating neurofilament-positive axons closely followed the trajectory of the donor OECs, as well as that of the migrating host cells within the scaffold. However, there was only a trend for increased numbers of regenerating axons above that supported by non-seeded scaffolds or in the untreated lesions. Nonetheless, significant functional recovery in skilled forelimb motor function was observed following the implantation of both seeded and non-seeded scaffolds which could not be correlated to the extent of axon regeneration within the scaffold. Mechanisms other than simple bridging of axon regeneration across the lesion must be responsible for the improved motor function.
RESUMO
Titanium-based alloys can be actively brazed onto bio-inert ceramics and potentially be used as biocompatible coatings. To further improve their bioactivity in vivo, introduction of calcium phosphate (CaP)-based granulates onto their surface layer is possible. For this, mechanically stable CaP-based granulates need to be able to withstand the demand of the brazing process. In this study, spherical granulates, made of a calcium phosphate composite composed primarily of ß-tricalcium phosphate and hydroxyapatite, a bioactive glass, and a mixture of the previous two, were manufactured by spray drying. The influence of organic additives (Dolapix CE64, trisodium citrate) and solids content (30-80 wt%) in the slurry on the physical characteristics of granulates was investigated. X-ray diffraction, Brunauer, Emmett, Teller specific surface area standard method, scanning electron microscopy, granulate size analysis, and single granule strength were performed. Our results showed that trisodium citrate permitted the production of granulates with regular morphology, high density, and increased failure stress values. The strong granules also withstood the brazing process. These results show that CaP bioactive agents can be generated and be integrated during the demanding metallurgical processes, allowing for one-step bioactivation of metal brazes.
Assuntos
Fosfatos de Cálcio/química , Cerâmica/química , Citratos/química , Materiais Revestidos Biocompatíveis/química , Titânio/química , Ligas , Vidro/química , Difração de Raios XRESUMO
Numerous in-vitro techniques exist for investigating the influence of 3D substrate topography on sensory axon growth. However, simple and cost-effective methods for studying post-natal motor axon interactions with such substrates are lacking. Here, spinal cord organotypic slice cultures (OSC) from post-natal day 7-9 rat pups were presented with spinal nerve roots, or blocks of fibrin hydrogel or 3D microporous collagen scaffolds to investigate motor axon-substrate interactions. By 7-14 days, axons from motor neuronal pools extended into the explanted nerve roots, growing along Schwann cell processes and demonstrating a full range of axon-Schwann cell interactions, from simple ensheathment to concentric wrapping by Schwann cell processes and the formation of compact myelin within a basal lamina sheath. Extensive motor axon regeneration and all stages of axon-Schwann interactions were also supported within the longitudinally orientated microporous framework of the 3D collagen scaffold. In stark contrast, the simple fibrin hydrogel only supported axon growth and cell migration over its surface. The relative ease of demonstrating such motor axon regeneration through the microporous 3D framework by immunofluorescence, two-photon microscopy and transmission electron microscopy strongly supports the adoption of this technique for assaying the influence of substrate topography and functionalization in regenerative bioengineering.
Assuntos
Axônios/patologia , Neurônios Motores/patologia , Regeneração Nervosa , Medula Espinal/fisiopatologia , Alicerces Teciduais/química , Animais , Axônios/ultraestrutura , Técnicas de Cocultura , Colágeno/metabolismo , Fibrina/farmacologia , Hidrogel de Polietilenoglicol-Dimetacrilato/farmacologia , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Neurônios Motores/efeitos dos fármacos , Degeneração Neural/patologia , Degeneração Neural/fisiopatologia , Regeneração Nervosa/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Ratos , Medula Espinal/efeitos dos fármacos , Medula Espinal/patologia , Medula Espinal/ultraestrutura , Raízes Nervosas Espinhais/efeitos dos fármacos , Raízes Nervosas Espinhais/metabolismo , Raízes Nervosas Espinhais/patologia , Raízes Nervosas Espinhais/ultraestruturaRESUMO
Overexpression of the epidermal growth factor receptor (EGFR) is observed in a large number of neoplasms. The monoclonal antibody cetuximab/Erbitux is frequently applied to treat EGFR-expressing tumors. However, the application of cetuximab alone or in combination with radio- and/or chemotherapy often yields only little benefit for patients. In the present study, we describe a mechanism that explains resistance of both tumor cell lines and cultured primary human glioma cells to cetuximab. Treatment of these cells with cetuximab promoted DNA synthesis in the absence of increased proliferation, suggesting that DNA repair pathways were activated. Indeed, we observed that cetuximab promoted the activation of the DNA damage response pathway and prevented the degradation of essential meiotic endonuclease 1 homolog 1 (Eme1), a heterodimeric endonuclease involved in DNA repair. The increased levels of Eme1 were necessary for enhanced DNA repair, and the knockdown of Eme1 was sufficient to prevent efficient DNA repair in response to ultraviolet-C light or megavoltage irradiation. These treatments reduced the survival of tumor cells, an effect that was reversed by cetuximab application. Again, this protection was dependent on Eme1. Taken together, these results suggest that cetuximab initiates pathways that result in the stabilization of Eme1, thereby resulting in enhanced DNA repair. Accordingly, cetuximab enhances DNA repair, reducing the effectiveness of DNA-damaging therapies. This aspect should be considered when using cetuximab as an antitumor agent and suggests that Eme1 is a negative predictive marker.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Reparo do DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Endodesoxirribonucleases/metabolismo , Antineoplásicos/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/radioterapia , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Cetuximab , DNA/biossíntese , Endodesoxirribonucleases/genética , Receptores ErbB/antagonistas & inibidores , Técnicas de Silenciamento de Genes , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Glioblastoma/radioterapia , Humanos , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição STAT3/metabolismo , Células Tumorais CultivadasRESUMO
Glutamate-induced excitotoxicity is a major contributor to motor neuron (MN) degeneration in disorders such as amyotrophic lateral sclerosis (ALS), stroke and spinal cord injury. Numerous in vitro and in vivo models have been developed to evaluate the efficacy and mode of action of neuroprotective agents. However, the dominance of glutamate receptor-subtype in the different regions of the spinal cord in these models has generally been overlooked. This study first compared the neuroprotective effect of administering glutamate receptor antagonists, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), into a serum-free excitotoxic organotypic in vitro system, on the survival of MNs located in the lumbar area of spinal cord. The poor neuroprotection provided by MK-801 (NMDA (N-methyl-D-aspartate) antagonist) in comparison to CNQX (AMPA/KA (a-amino-3-hydroxy-5-methyl-4-isoxazole propionate/kainate) antagonist), raised the hypothesis that the extent of engagement by glutamate receptor sub-types in the mechanism of excitotoxicity may differ within different populations of MNs. The consequent examination of MN susceptibility to glutamate-induced excitotoxicity in relation to the rostro-caudal level from which MN originated revealed a differential glutamate receptor sub-type dominance at different spinal cord regions (i.e. cervical, thoracic and lumbar). In the cervical and lumbar regions, the AMPA receptor was the main contributor to MN excitotoxicity, whereas in thoracic regions, the NMDA receptor was the main contributor. This study provides a new way of looking at mechanisms leading to glutamate-induced excitotoxicity in MN and may therefore be important for the development of treatment strategies in protection of spinal MNs in neurodegenerative disease and traumatic injury.
Assuntos
Antagonistas de Aminoácidos Excitatórios/farmacologia , Ácido Glutâmico/toxicidade , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Fármacos Neuroprotetores/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Vértebras Cervicais , Imuno-Histoquímica , Região Lombossacral , Técnicas de Cultura de Órgãos , Ratos , Ratos Sprague-Dawley , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Vértebras TorácicasRESUMO
Recent in vitro studies with electrospun nanofibers have used a range of techniques. The in vitro system presented in this article describes electrospun fibers deposited onto chemically reactive substrates to provide fiber adherence and surface chemistry control of the substrate. Fibers of poly(epsilon-caprolactone) (PCL) or of a blend of PCL and collagen type I (C/PCL) were electrospun directly onto collectors coated with isocyanate-terminated star (polyethylene glycol) (sPEG). Alternatively, parallel electrospun fibers were collected on dual collectors in "dilute" quantities and transferred onto sPEG-coated substrates. The initial reactive nature of the substrates allows the collection of very few fibers, which adhere well during frequent washes. Furthermore, the sPEG layer transforms into protein-repellent substrates with the additional potential to include specific cellular mediators such as glycine-arginine-glycine-aspartate-serine (GRGDS) peptides to promote cell adhesion. Therefore, the fiber and substrate chemistry can be modified independently, which is particularly useful for in vitro studies of guided migrating cells. In the present work, dissociated cells of dorsal root ganglia seeded onto the substrates were investigated to assess the influence of different combinations of fiber material, fiber orientation, and surface functionalization. Cell adhesion was observed predominantly on the nanofibers, except when the sPEG layer on the substrate contained GRGDS. On the cell-repellent sPEG substrates, neurites were aligned in direct contact with parallel C/PCL fibers and less so with PCL fibers. In contrast, neurite alignment showed less guidance effect with C/PCL electrospun fibers on the GRGDS/sPEG-coated substrates. Therefore, the combination of oriented biologically active fibers on cell-repellent surfaces enhanced the guidance of such cells. These reactive substrate systems provide a multitude of in vitro combinations for providing cells with specific mediators and, in turn, defining the optimum environment of regenerating devices for in vivo studies.
Assuntos
Nanoestruturas/química , Nanotecnologia/métodos , Animais , Axônios/metabolismo , Proliferação de Células , Sobrevivência Celular , Embrião de Galinha , Microscopia de Fluorescência , Nanoestruturas/ultraestrutura , Nervos Periféricos/citologia , Poliésteres/químicaRESUMO
AIM: Electrospun nanofibers represent potent guidance substrates for nervous tissue repair. Development of nanofiber-based scaffolds for CNS repair requires, as a first step, an understanding of appropriate neural cell type-substrate interactions. MATERIALS & METHODS: Astrocyte-nanofiber interactions (e.g., adhesion, proliferation, process extension and migration) were studied by comparing human neural progenitor-derived astrocytes (hNP-ACs) and a human astrocytoma cell line (U373) with aligned polycaprolactone (PCL) nanofibers or blended (25% type I collagen/75% PCL) nanofibers. Neuron-nanofiber interactions were assessed using a differentiated human neuroblastoma cell line (SH-SY5Y). RESULTS & DISCUSSION: U373 cells and hNP-AC showed similar process alignment and length when associated with PCL or Type I collagen/PCL nanofibers. Cell adhesion and migration by hNP-AC were clearly improved by functionalization of nanofiber surfaces with type I collagen. Functionalized nanofibers had no such effect on U373 cells. Another clear difference between the U373 cells and hNP-AC interactions with the nanofiber substrate was proliferation; the cell line demonstrating strong proliferation, whereas the hNP-AC line showed no proliferation on either type of nanofiber. Long axonal growth (up to 600 microm in length) of SH-SY5Y neurons followed the orientation of both types of nanofibers even though adhesion of the processes to the fibers was poor. CONCLUSION: The use of cell lines is of only limited predictive value when studying cell-substrate interactions but both morphology and alignment of human astrocytes were affected profoundly by nanofibers. Nanofiber surface functionalization with collagen significantly improved hNP-AC adhesion and migration. Alternative forms of functionalization may be required for optimal axon-nanofiber interactions.