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BACKGROUND: Traumatic peripheral nerve injury is common and incurs significant cost to individuals and society. Healing following direct nerve repair or repair with autograft is slow and can be incomplete. Several bioengineered nerve wraps or devices have become available as an alternative to direct repair or autologous nerve graft. Nerve wraps attempt to reduce axonal escape across a direct repair site and nerve devices negate the need for a donor site defect, required by an autologous nerve graft. Comparative evidence to guide clinicians in their potential use is lacking. We collated existing evidence to guide the clinical application of currently available nerve wraps and conduits. OBJECTIVES: To assess and compare the effects and complication rates of licensed bioengineered nerve conduits or wraps for surgical repair of traumatic peripheral nerve injuries of the upper limb. To compare effects and complications against the current gold surgical standard (direct repair or nerve autograft). SEARCH METHODS: We used standard, extensive Cochrane search methods. The latest search was 26 January 2022. We searched online and, where not accessible, contacted societies' secretariats to review abstracts from the British Surgical Society of the Hand, International Federation of Surgical Societies of the Hand, Federation of European Surgical Societies of the Hand, and the American Society for Peripheral Nerve from October 2007 to October 2018. SELECTION CRITERIA: We included parallel group randomised controlled trials (RCTs) and quasi-RCTs of nerve repair in the upper limb using a bioengineered wrap or conduit, with at least 12 months of follow-up. DATA COLLECTION AND ANALYSIS: We used standard Cochrane procedures. Our primary outcomes were 1. muscle strength and 2. sensory recovery at 24 months or more. Our secondary outcomes were 3. British Medical Research Council (BMRC) grading, 4. integrated functional outcome (Rosén Model Instrument (RMI)), 5. touch threshold, 6. two-point discrimination, 7. cold intolerance, 8. impact on daily living measured using the Disability of Arm Shoulder and Hand Patient-Reported Outcome Measure (DASH-PROM), 9. sensory nerve action potential, 10. cost of the device, and 11. adverse events (any and specific serious adverse events (further surgery)). We used GRADE to assess the certainty of the evidence. MAIN RESULTS: Five studies involving 213 participants and 257 nerve injuries reconstructed with wraps or conduits (129 participants) or standard repair (128 participants) met the inclusion criteria. Of those in the standard repair group, 119 nerve injuries were managed with direct epineurial repair, and nine autologous nerve grafts were performed. One study excluded the outcome data for the repair using an autologous nerve graft from their analysis, as it was the only autologous nerve graft in the study, so data were available for 127 standard repairs. There was variation in the functional outcome measures reported and the time postoperatively at which they were recorded. Mean sensory recovery, assessed with BMRC sensory grading (range S0 to S4, higher score considered better) was 0.03 points higher in the device group (range 0.43 lower to 0.49 higher; 1 RCT, 28 participants; very low-certainty evidence) than in the standard repair group (mean 2.75 points), which suggested little or no difference between the groups, but the evidence is very uncertain. There may be little or no difference at 24 months in mean touch thresholds between standard repair (0.81) and repair using devices, which was 0.01 higher but this evidence is also very uncertain (95% confidence interval (CI) 0.06 lower to 0.08 higher; 1 trial, 32 participants; very low-certainty evidence). Data were not available to assess BMRC motor grading at 24 months or more. Repair using bioengineered devices may not improve integrated functional outcome scores at 24 months more than standard techniques, as assessed by the Rosén Model Instrument (RMI; range 0 to 3, higher scores better); the CIs allow for both no important difference and a better outcome with standard repair (mean RMI 1.875), compared to the device group (0.17 lower, 95% CI 0.38 lower to 0.05 higher; P = 0.13; 2 trials, 60 participants; low-certainty evidence). Data from one study suggested that the five-year postoperative outcome of RMI may be slightly improved after repair using a device (mean difference (MD) 0.23, 95% CI 0.07 to 0.38; 1 trial, 28 participants; low-certainty evidence). No studies measured impact on daily living using DASH-PROM. The proportion of people with adverse events may be greater with nerve wraps or conduits than with standard techniques, but the evidence is very uncertain (risk ratio (RR) 7.15, 95% CI 1.74 to 29.42; 5 RCTs, 213 participants; very low-certainty evidence). This corresponds to 10 adverse events per 1000 people in the standard repair group and 68 per 1000 (95% CI 17 to 280) in the device group. The use of nerve repair devices may be associated with a greater need for revision surgery but this evidence is also very uncertain (12/129 device repairs required revision surgery (removal) versus 0/127 standard repairs; RR 7.61, 95% CI 1.48 to 39.02; 5 RCTs, 256 nerve repairs; very low-certainty evidence). AUTHORS' CONCLUSIONS: Based on the available evidence, this review does not support use of currently available nerve repair devices over standard repair. There is significant heterogeneity in participants, injury pattern, repair timing, and outcome measures and their timing across studies of nerve repair using bioengineered devices, which make comparisons unreliable. Studies were generally small and at high or unclear risk of bias. These factors render the overall certainty of evidence for any outcome low or very low. The data reviewed here provide some evidence that more people may experience adverse events with use of currently available bioengineered devices than with standard repair techniques, and the need for revision surgery may also be greater. The evidence for sensory recovery is very uncertain and there are no data for muscle strength at 24 months (our primary outcome measures). We need further trials, adhering to a minimum standard of outcome reporting (with at least 12 months' follow-up, including integrated sensorimotor evaluation and patient-reported outcomes) to provide high-certainty evidence and facilitate more detailed analysis of effectiveness of emerging, increasingly sophisticated, bioengineered repair devices.
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Nervos Periféricos , Extremidade Superior , Humanos , Extremidade Superior/cirurgia , Nervos Periféricos/cirurgiaRESUMO
Tubular scaffolds which incorporate a variety of micro- and nanotopographies have a wide application potential in tissue engineering especially for the repair of spinal cord injury (SCI). We aim to produce metabolically active differentiated tissues within such tubes, as it is crucially important to evaluate the biological performance of the three-dimensional (3D) scaffold and optimize the bioprocesses for tissue culture. Because of the complex 3D configuration and the presence of various topographies, it is rarely possible to observe and analyze cells within such scaffolds in situ. Thus, we aim to develop scaled down mini-chambers as simplified in vitro simulation systems, to bridge the gap between two-dimensional (2D) cell cultures on structured substrates and three-dimensional (3D) tissue culture. The mini-chambers were manipulated to systematically simulate and evaluate the influences of gravity, topography, fluid flow, and scaffold dimension on three exemplary cell models that play a role in CNS repair (i.e., cortical astrocytes, fibroblasts, and myelinating cultures) within a tubular scaffold created by rolling up a microstructured membrane. Since we use CNS myelinating cultures, we can confirm that the scaffold does not affect neural cell differentiation. It was found that heterogeneous cell distribution within the tubular constructs was caused by a combination of gravity, fluid flow, topography, and scaffold configuration, while cell survival was influenced by scaffold length, porosity, and thickness. This research demonstrates that the mini-chambers represent a viable, novel, scale down approach for the evaluation of complex 3D scaffolds as well as providing a microbioprocessing strategy for tissue engineering and the potential repair of SCI.
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Técnicas de Cultura de Células/métodos , Sistema Nervoso Central/fisiologia , Traumatismos da Medula Espinal/fisiopatologia , Traumatismos da Medula Espinal/terapia , Engenharia Tecidual/métodos , Cicatrização/fisiologia , Astrócitos/fisiologia , Diferenciação Celular/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Fibroblastos/fisiologia , Humanos , Porosidade , Próteses e Implantes , Alicerces TeciduaisRESUMO
The function and fate of cells is influenced by many different factors, one of which is surface topography of the support culture substrate. Systematic studies of nanotopography and cell response have typically been limited to single cell types and a small set of topographical variations. Here, we show a radical expansion of experimental throughput using automated detection, measurement, and classification of co-cultured cells on a nanopillar array where feature height changes continuously from planar to 250 nm over 9 mm. Individual cells are identified and characterized by more than 200 descriptors, which are used to construct a set of rules for label-free segmentation into individual cell types. Using this approach we can achieve label-free segmentation with 84% confidence across large image data sets and suggest optimized surface parameters for nanostructuring of implant devices such as vascular stents.
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Rastreamento de Células/métodos , Células Endoteliais/citologia , Fibroblastos/citologia , Nanoestruturas/química , Nanotecnologia/métodos , Rastreamento de Células/instrumentação , Técnicas de Cocultura , Fluorescência , Humanos , Nanotecnologia/instrumentaçãoRESUMO
By using different salts as a method to achieve gelation of two different amino-acid-functionalised perylene bisimides, we were able to tune reduction potentials while maintaining the mechanical and optical properties of the system all at pH 7.4.
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It is currently thought that treatment for spinal cord injury (SCI) will involve a combined pharmacological and biological approach; however, testing their efficacy in animal models of SCI is time-consuming and requires large animal cohorts. For this reason we have modified our myelinating cultures as an in vitro model of SCI and studied its potential as a prescreen for combined therapeutics. This culture comprises dissociated rat embryonic spinal cord cells plated onto a monolayer of astrocytes, which form myelinated axons interspaced with nodes of Ranvier. After cutting the culture, an initial cell-free area appears persistently devoid of neurites, accompanied over time by many features of SCI, including demyelination and reduced neurite density adjacent to the lesion, and infiltration of microglia and reactive astrocytes into the lesioned area. We tested a range of concentrations of the Rho inhibitor C3 transferase (C3) and ROCK inhibitor Y27632 that have been shown to promote SCI repair in vivo. C3 promoted neurite extension into the lesion and enhanced neurite density in surrounding areas but failed to induce remyelination. In contrast, while Y27632 did not induce significant neurite outgrowth, myelination adjacent to the lesion was dramatically enhanced. The effects of the inhibitors were concentration-dependent. Combined treatment with C3 and Y27632 had additive affects with an enhancement of neurite outgrowth and increased myelination adjacent to the lesion, demonstrating neither conflicting nor synergistic effects when coadministered. Overall, these results demonstrate that this culture serves as a useful tool to study combined strategies that promote CNS repair.
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Astrócitos/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Bainha de Mielina/fisiologia , Neuritos/efeitos dos fármacos , Medula Espinal/citologia , Amidas/farmacologia , Animais , Animais Recém-Nascidos , Antígeno CD11b/metabolismo , Células Cultivadas , Complemento C3/uso terapêutico , Doenças Desmielinizantes/tratamento farmacológico , Doenças Desmielinizantes/etiologia , Doenças Desmielinizantes/metabolismo , Embrião de Mamíferos , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo/metabolismo , Proteínas Ativadoras de GTPase/antagonistas & inibidores , Proteínas Ativadoras de GTPase/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Gliose/tratamento farmacológico , Gliose/etiologia , Gliose/patologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteína Proteolipídica de Mielina/metabolismo , Bainha de Mielina/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Antígenos O/metabolismo , Piridinas/farmacologia , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismoRESUMO
Cellular response to microgrooves is addressed using a new assay format, comprising orthogonal gradients of continuously varied groove pitch and depth. Dual layer etch masks are created using a combination of micropatterning and plasma polymer deposition. A silicon substrate with a constant groove width of 8 µm and with ridge width increasing from 8 µm in 0.5 µm steps across 10 mm is fabricated by photolithography. A plasma-polymerized hexane film which is 120 nm thick at one end of these grooves, and 10 nm at the other, is deposited under a diffusion mask. Reactive etching of the patterned sample transfers a gradient of groove pitch and groove depth into the silicon substrate. A silicon master with a gradient of groove depth spanning more than two orders of magnitude (less than 10 nm to over 1000 nm) is used to create an injection molding inlay for mass replication of the screening topography. Polycarbonate replicas are molded for use in cell culture studies, and the functionality of the topography as a high-throughput screening platform is investigated. The response of MDCK, h-TERT fibroblasts, and LE2 endothelial cells is examined, in terms of attachment and morphological response to the variation in topographical cues, with the aim of pinpointing the optimal combination of groove pitch and depth to elicit a tailored response from each cell type. When the range of topographical features screened on a single substrate is considered, this new assay represents a significant step forward in the parametric design and analysis of topographical cues at the biomaterial interface.
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Bioensaio/métodos , Comunicação Celular , Membrana Celular/metabolismo , Animais , Forma Celular , Cães , Humanos , Células Madin Darby de Rim Canino , Gases em Plasma/química , Ratos , Propriedades de SuperfícieRESUMO
Saturation DiGE is a powerful but challenging technique for the characterisation of changes in protein expression between two or more scarce samples. In this paper, measures to prevent and troubleshoot artefacts in the saturation DiGE workflow are discussed, with illustration of some examples as they may be encountered in gel images or analysis.
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Proteínas/análise , Proteômica/métodos , Eletroforese em Gel Diferencial Bidimensional/métodos , Artefatos , Corantes Fluorescentes , Coloração e Rotulagem/métodosRESUMO
BACKGROUND: Despite the advancements in microsurgical techniques and noteworthy research in the last decade, peripheral nerve lesions have still weak functional outcomes in current clinical practice. However, cell transplantation of human adipose-derived stem cells (hADSC) in a bioengineered conduit has shown promising results in animal studies. Human platelet lysate (hPL) has been adopted to avoid fetal bovine serum (FBS) in consideration of the biosafety concerns inherent with the use of animal-derived products in tissue processing and cell culture steps for translational purposes. In this work, we investigate how the interplay between hPL-expanded hADSC (hADSChPL) and extracellular matrix (ECM) proteins influences key elements of nerve regeneration. METHODS: hADSC were seeded on different ECM coatings (laminin, LN; fibronectin, FN) in hPL (or FBS)-supplemented medium and co-cultured with primary dorsal root ganglion (DRG) to establish the intrinsic effects of cell-ECM contact on neural outgrowth. Co-cultures were performed "direct," where neural cells were seeded in contact with hADSC expanded on ECM-coated substrates (contact effect), or "indirect," where DRG was treated with their conditioned medium (secretome effect). Brain-derived nerve factor (BDNF) levels were quantified. Tissue culture plastic (TCPS) was used as the control substrate in all the experiments. RESULTS: hPL as supplement alone did not promote higher neurite elongation than FBS when combined with DRG on ECM substrates. However, in the presence of hADSC, hPL could dramatically enhance the stem cell effect with increased DRG neurite outgrowth when compared with FBS conditions, regardless of the ECM coating (in both indirect and direct co-cultures). The role of ECM substrates in influencing neurite outgrowth was less evident in the FBS conditions, while it was significantly amplified in the presence of hPL, showing better neural elongation in LN conditions when compared with FN and TCPS. Concerning hADSC growth factor secretion, ELISA showed significantly higher concentrations of BDNF when cells were expanded in hPL compared with FBS-added medium, without significant differences between cells cultured on the different ECM substrates. CONCLUSION: The data suggest how hADSC grown on LN and supplemented with hPL could be active and prone to support neuron-matrix interactions. hPL enhanced hADSC effects by increasing both proliferation and neurotrophic properties, including BDNF release.
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Separation and sorting of biological entities (viruses, bacteria, and cells) is a critical step in any microfluidic lab-on-a-chip device. Acoustofluidics platforms have demonstrated their ability to use physical characteristics of cells to perform label-free separation. Bandpass-type sorting methods of medium-sized entities from a mixture have been presented using acoustic techniques; however, they require multiple transducers, lack support for various target populations, can be sensitive to flow variations, or have not been verified for continuous flow sorting of biological cells. To our knowledge, this paper presents the first acoustic bandpass method that overcomes all these limitations and presents an inherently reconfigurable technique with a single transducer pair for stable continuous flow sorting of blood cells. The sorting method is first demonstrated for polystyrene particles of sizes 6, 10, and 14.5 µm in diameter with measured purity and efficiency coefficients above 75 ± 6% and 85 ± 9%, respectively. The sorting strategy was further validated in the separation of red blood cells from white blood cells and 1 µm polystyrene particles with 78 ± 8% efficiency and 74 ± 6% purity, respectively, at a flow rate of at least 1 µl/min, enabling to process finger prick blood samples within minutes.
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A biodegradable substrate with a regular array of nanopillars fabricated by electron-beam lithography and hot embossing is used to address the mechanisms of nanotopographical control of cell behavior. Two different cell lines cultured on the nanopillars show striking differences in cell coverage. These changes are topography- and cell-dependent, and are not mediated by air bubbles trapped on the nanopattern. For the first time, a strong cell-selective effect of the same nanotopography has been clearly demonstrated on a large area; while fibroblast proliferation is inhibited, endothelial cell spreading is visibly enhanced. The reduced fibroblast proliferation indicates that a reduction of available surface area induced by nanotopography might be the main factor affecting cell growth on nanopatterns. The results presented herein pave the way towards the development of permanent vascular replacements, where non-adhesive, inert, surfaces will induce rapid in situ endothelialization to reduce thrombosis and occlusion.
Assuntos
Materiais Biocompatíveis , Técnicas de Cultura de Células/métodos , Nanotecnologia/métodos , Animais , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células , Células Cultivadas , Humanos , Camundongos , Microscopia de Força AtômicaRESUMO
The limited regenerative capacity of the CNS poses formidable challenges to the repair of spinal cord injury (SCI). Two key barriers to repair are (i) the physical gap left by the injury, and (ii) the inhibitory milieu surrounding the injury, the glial scar. Biomaterial implantation into the injury site can fill the cavity, provide a substrate for cell migration, and potentially attenuate the glial scar. We investigated the biological viability of a biocompatible and biodegradable poly-ε-lysine based biomaterial, Proliferate®, in low and high cross-linked forms and when coated with IKVAV peptide, for SCI implantation. We demonstrate altered astrocyte morphology and nestin expression on Proliferate® compared to conventional glass cell coverslips suggesting a less reactive phenotype. Moreover Proliferate® supported myelination in vitro, with myelination observed sooner on IKVAV-coated constructs compared with uncoated Proliferate®, and delayed overall compared with maintenance on glass coverslips. For in vivo implantation, parallel-aligned channels were fabricated into Proliferate® to provide cell guidance cues. Extensive vascularisation and cellular infiltration were observed in constructs implanted in vivo, along with an astrocyte border and microglial response. Axonal ingrowth was observed at the construct border and inside implants in intact channels. We conclude that Proliferate® is a promising biomaterial for implantation following SCI.
Assuntos
Materiais Biocompatíveis/química , Doenças do Sistema Nervoso Central/terapia , Polilisina/química , Próteses e Implantes , Traumatismos da Medula Espinal/terapia , Animais , Materiais Biocompatíveis/síntese química , Células Cultivadas , Polilisina/síntese química , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: The autologous nerve graft, despite its donor site morbidity and unpredictable functional recovery, continues to be the gold standard in peripheral nerve repair. Rodent research studies have shown promising results with cell transplantation of human adipose-derived stem cells (hADSC) in a bioengineered conduit, as an alternative strategy for nerve regeneration. To achieve meaningful clinical translation, cell therapy must comply with biosafety. Cell extraction and expansion methods that use animal-derived products, including enzymatic adipose tissue dissociation and the use of fetal bovine serum (FBS) as a culture medium supplement, have the potential for transmission of zoonotic infectious and immunogenicity. Human-platelet-lysate (hPL) serum has been used in recent years in human cell expansion, showing reliability in clinical applications. METHODS: We investigated whether hADSC can be routinely isolated and cultured in a completely xenogeneic-free way (using hPL culture medium supplement and avoiding collagenase digestion) without altering their physiology and stem properties. Outcomes in terms of stem marker expression (CD105, CD90, CD73) and the osteocyte/adipocyte differentiation capacity were compared with classical collagenase digestion and FBS-supplemented hADSC expansion. RESULTS: We found no significant differences between the two examined extraction and culture protocols in terms of cluster differentiation (CD) marker expression and stem cell plasticity, while hADSC in hPL showed a significantly higher proliferation rate when compared with the usual FBS-added medium. Considering the important key growth factors (particularly brain-derived growth factor (BDNF)) present in hPL, we investigated a possible neurogenic commitment of hADSC when cultured with hPL. Interestingly, hADSC cultured in hPL showed a statistically higher secretion of neurotrophic factors BDNF, glial cell-derived growth factor (GDNF), and nerve-derived growth factor (NFG) than FBS-cultured cells. When cocultured in the presence of primary neurons, hADSC which had been grown under hPL supplementation, showed significantly enhanced neurotrophic properties. CONCLUSIONS: The hPL-supplement medium could improve cell proliferation and neurotropism while maintaining stable cell properties, showing effectiveness in clinical translation and significant potential in peripheral nerve research.
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Células-Tronco Mesenquimais , Adipócitos , Tecido Adiposo , Animais , Plaquetas , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Humanos , Reprodutibilidade dos Testes , Células-TroncoRESUMO
Mn-Anderson based polyoxometalate clusters with different terminal groups have been patterned successfully onto self-assembled monolayer (SAM) using microcontact printing. Studies of the interactions between the designed SAMs and human fibroblast (hTERT-BJ1) cells have been reported, and it was observed that cells attach and spread efficiently for monolayer presenting a terminal aromatic pyrene platform with a polyoxometalate Mn-Anderson cluster as linker, demonstrating the crucial role played by the polyoxometalate metal oxide cluster as an intermediary in cell adhesion to the surface.
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Compostos de Tungstênio/química , Adesão Celular , Linhagem Celular , Humanos , Microscopia Eletrônica de Varredura , Modelos Moleculares , Estrutura Molecular , Estereoisomerismo , Propriedades de SuperfícieRESUMO
Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is a promising tool for subcellular chemical analysis of biological cells. However, to obtain relevant information, the method used for sample preparation is critical. In this work, we have used TOF-SIMS, scanning electron microscopy (SEM), and interference reflection microscopy (IRM) to study the effects of different fixation and drying methods on the morphology and chemical structure of human fibroblast cells (hTERT) adhered to a silicon surface. Specifically, two fixation techniques (chemical fixation with glutaraldehyde and cryofixation by plunge freezing) and two drying techniques (freeze drying and alcohol substitution drying) were investigated. Cryofixation followed by freeze drying was determined to produce dried cells with preserved cell morphology, intact cell membranes, and retained sodium/potassium ion concentration gradients across the plasma membrane. By washing samples in an aqueous solution of ammonium formate (AF) before cryofixation, the accumulation of salts on the sample surface during drying could be suppressed. IRM measurements showed that the cell morphology was preserved during washing with ammonium formate, although some swelling occurred. Compared with cryofixation, cells fixed with glutaraldehyde showed finer structures on the cell surface in SEM and similar lipid distributions in TOF-SIMS, but the sodium/potassium ion gradients were not retained. Alcohol drying was determined to remove cell membrane phospholipids significantly, although the use of osmium tetroxide as a post-fixative was shown to decrease this effect.
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Criopreservação/métodos , Dessecação/métodos , Fibroblastos/citologia , Fixadores , Liofilização/métodos , Espectrometria de Massa de Íon Secundário/métodos , Adesão Celular , Sobrevivência Celular , Células Cultivadas , Etanol , Fibroblastos/metabolismo , Formiatos , Glutaral , Humanos , Microscopia Eletrônica de Varredura/métodosRESUMO
Two numerical methods based on the Finite Element Method are presented for calculating the secondary acoustic radiation force between interacting spherical particles. The first model only considers the acoustic waves scattering off a single particle, while the second model includes re-scattering effects between the two interacting spheres. The 2D axisymmetric simplified model combines the Gor'kov potential approach with acoustic simulations to find the interacting forces between two small compressible spheres in an inviscid fluid. The second model is based on 3D simulations of the acoustic field and uses the tensor integral method for direct calculation of the force. The results obtained by both models are compared with analytical equations, showing good agreement between them. The 2D and 3D models take, respectively, seconds and tens of seconds to achieve a convergence error of less than 1%. In comparison with previous models, the numerical methods presented herein can be easily implemented in commercial Finite Element software packages, where surface integrals are available, making it a suitable tool for investigating interparticle forces in acoustic manipulation devices.
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Standard methods for seeding monolayer cell cultures in a multiwell plate or dish do not uniformly distribute cells on the surface. With traditional methods, users find aggregation around the circumference, in the centre, or a combination of the two. This variation is introduced due to the macro scale flow of the cell seeding suspension, and movement of the dish before cells can settle and attach to the surface. Reproducibility between labs, users, and experiments is hampered by this variability in cell seeding. We present a simple method for uniform and user-independent the cell seeding using an easily produced uniform cell seeder (UCS) device. This allows precise control of cell density in a reproducible manner. By containing the cell seeding suspension in a defined volume above the culture surface with the UCS, fluctuations in cell density are minimised. Seeding accuracy, as defined by the actual cell density versus the target seeding density is improved dramatically across users with various levels of expertise. We go on to demonstrate the impact of local variation in cell density on the lineage commitment of human embryonic stem cells (hESCs) towards pancreatic endoderm (PE). Variations in the differentiation profile of cells across a culture well closely mirror variations in cell density introduced by seeding method-with the UCS correcting variations in differentiation efficiency. The UCS device provides a simple and reproducible method for uniform seeding across multiple culture systems.
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Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Contagem de Células , Diferenciação Celular , Células Cultivadas , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Endoderma/citologia , Endoderma/fisiologia , Humanos , Pâncreas/citologia , Pâncreas/embriologia , Pâncreas/fisiologia , Impressão Tridimensional , Melhoria de QualidadeRESUMO
After spinal cord injury neuronal connections are not easily re-established. Success has been hampered by the lack of orientation of neurites inside scar tissue and a lack of neurites crossing out of the site of injury. Oriented scaffolds in biodegradable polymers could be an excellent way to support both the orientation of neurites within the injury site as well as aiding their crossing out of the lesion. To establish the validity of using grooved micro-topography in polycaprolactone in combination with glia we have studied the long-term (3 weeks) orientation of neuronal cells on monolayers of astrocytes on the top of grooved topographies of various dimensions. We find that neurites are significantly aligned by groove/ridge type topographies which are "buried" under a monolayer of astrocytes for up to 3 weeks. This alignment is significantly lower than that of neurites growing directly on the topography, but these neurons do not survive on the poly-l-lysine coated polymer for more than a week. The alignment of neurites on the astrocyte layer to the underlying topography decreases over time, and with groove width. Topographies with 12.5 or 25 microm lateral dimension appear optimal for the long-term alignment and can support myelination. We have shown for the first time that micro-topography can act through an overlaid astrocyte layer and results in aligned neurites in long-term culture and that these can be myelinated by endogenous oligodendrocytes.
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Astrócitos/citologia , Neuritos , Animais , Células Cultivadas , Imageamento Tridimensional , Fibras Nervosas Mielinizadas , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
Stainless Steel (SS), titanium (cpTi), and Ti-6Al-7Nb (TAN) are frequently used metals in fracture fixation, which contact not only bone, but also soft tissue. In previous soft tissue cytocompatibility studies, TAN was demonstrated to inhibit cell growth in its "standard" micro-roughened state. To elucidate a possible mechanism for this inhibition, cell area, shape, adhesion, and cytoskeletal integrity was studied. Only minor changes in spreading were observed for cells on electropolished SS, cpTi, and TAN. Cells on "standard" cpTi were similarly spread in comparison with electropolished cpTi and TAN, although the topography influenced the cell periphery and also resulted in lower numbers and shorter length of focal adhesions. On "standard" microrough TAN, cell spreading was significantly lower than all other surfaces, and cell morphology differed by being more elongated. In addition, focal adhesion numbers and mean length were significantly lower on standard TAN than on all other surfaces, with 80% of the measured adhesions below a 2-microm threshold. Focal adhesion site location and maturation and microtubule integrity were compromised by the presence of protruding beta-phase microspikes found solely on the surface of standard TAN. This led us to propose that the impairment of focal adhesion numbers, maturation (length), and cell spreading to a possibly sufficient threshold observed on standard TAN blocks cell cycle progress and eventually cell growth on the surface. We believe, as demonstrated with standard cpTi and TAN, that a difference in surface morphology is influential for controlling cell behavior on implant surfaces.
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Materiais Biocompatíveis , Fibroblastos/citologia , Aço Inoxidável , Titânio , Actinas/metabolismo , Biomarcadores/metabolismo , Adesão Celular/fisiologia , Linhagem Celular Transformada , Forma Celular/fisiologia , Tamanho Celular , Citoesqueleto/fisiologia , Fibroblastos/fisiologia , Fibroblastos/ultraestrutura , Humanos , Processamento de Imagem Assistida por Computador , Microscopia de Força Atômica , Propriedades de Superfície , Tubulina (Proteína)/metabolismo , Vinculina/metabolismoRESUMO
Despite microsurgical repair, recovery of function following peripheral nerve injury is slow and often incomplete. Outcomes could be improved by an increased understanding of the molecular biology of regeneration and by translation of experimental bioengineering strategies. Topographical cues have been shown to be powerful regulators of the rate and directionality of neurite regeneration, and in this study we investigated the downstream molecular effects of linear micropatterned structures in an organotypic explant model. Linear topographical cues enhanced neurite outgrowth and our results demonstrated that the mTOR pathway is important in regulating these responses. mTOR gene expression peaked between 48 and 72h, coincident with the onset of rapid neurite outgrowth and glial migration, and correlated with neurite length at 48h. mTOR protein was located to glia and in a punctate distribution along neurites. mTOR levels peaked at 72h and were significantly increased by patterned topography (p<0.05). Furthermore, the topographical cues could override pharmacological inhibition. Downstream phosphorylation assays and inhibition of mTORC1 using rapamycin highlighted mTORC2 as an important mediator, and more specific therapeutic target. Quantitative immunohistochemistry confirmed the presence of the mTORC2 component rictor at the regenerating front where it co-localised with F-actin and vinculin. Collectively, these results provide a deeper understanding of the mechanism of action of topography on neural regeneration, and support the incorporation of topographical patterning in combination with pharmacological mTORC2 potentiation within biomaterial constructs used to repair peripheral nerves. STATEMENT OF SIGNIFICANCE: Peripheral nerve injury is common and functionally devastating. Despite microsurgical repair, healing is slow and incomplete, with lasting functional deficit. There is a clear need to translate bioengineering approaches and increase our knowledge of the molecular processes controlling nerve regeneration to improve the rate and success of healing. Topographical cues are powerful determinants of neurite outgrowth and represent a highly translatable engineering strategy. Here we demonstrate, for the first time, that microtopography potentiates neurite outgrowth via the mTOR pathway, with the mTORC2 subtype being of particular importance. These results give further evidence for the incorporation of microtopographical cues into peripheral nerve regeneration conduits and indicate that mTORC2 may be a suitable therapeutic target to potentiate nerve regeneration.
Assuntos
Regulação da Expressão Gênica , Alvo Mecanístico do Complexo 2 de Rapamicina/biossíntese , Regeneração Nervosa , Traumatismos dos Nervos Periféricos/metabolismo , Nervos Periféricos/fisiologia , Serina-Treonina Quinases TOR/biossíntese , Animais , Modelos Animais de Doenças , Traumatismos dos Nervos Periféricos/patologia , Nervos Periféricos/patologia , Ratos , Ratos Sprague-DawleyRESUMO
It is counterintuitive that invertebrate shells can induce bone formation, yet nacre, or mother of pearl, from marine shells is both osteoinductive and osteointegrative. Nacre is composed of aragonite (calcium carbonate) and induces production of vertebrate bone (calcium phosphate). Exploited by the Mayans for dental implants, this remarkable phenomenon has been confirmed in vitro and in vivo, yet the characteristic of nacre that induces bone formation remains unknown. By isolating nacre topography from its inherent chemistry in the production of polycaprolactone (PCL) nacre replica, we show that, for mesenchymal stem cells, nacre topography is osteoinductive. Gene expression of specific bone marker proteins, osteopontin, osteocalcin, osteonectin, and osterix, is increased 10-, 2-, 1.7-, and 1.8-fold, respectively, when compared to planar PCL. Furthermore, we demonstrate that bone tissue that forms in response to the physical topographical features of nacre has a higher crystallinity than bone formed in response to chemical cues with a full width half-maximum for PO43- Raman shift of 7.6 ± 0.7 for mineral produced in response to nacre replica compared to a much broader 34.6 ± 10.1 in response to standard osteoinductive medium. These differences in mineral product are underpinned by differences in cellular metabolism. This observation can be exploited in the design of bone therapies; a matter that is most pressing in light of a rapidly aging human population.