RESUMEN
The increasing demand for functional materials and an efficient use of sustainable resources makes the search for new material systems an ever growing endeavor. With this respect, architected (meta-)materials attract considerable interest. Their fabrication at the micro- and nanoscale, however, remains a challenge, especially for composites with highly different phases and unmodified reinforcement fillers. This study demonstrates that it is possible to create a non-cytotoxic nanocomposite ink reinforced by a sustainable phase, cellulose nanocrystals (CNCs), to print and tune complex 3D architectures using two-photon polymerization, thus, advancing the state of knowledge toward the microscale. Micro-compression, high-res scanning electron microscopy, (polarised) Raman spectroscopy, and composite modeling are used to study the structure-property relationships. A 100% stiffness increase is observed already at 4.5 wt% CNC while reaching a high photo-polymerization degree of ≈80% for both neat polymers and CNC-composites. Polarized Raman and the Halpin-Tsai composite-model suggest a random CNC orientation within the polymer matrix. The microscale approach can be used to tune arbitrary small scale CNC-reinforced polymer-composites with comparable feature sizes. The new insights pave the way for future applications where the 3D printing of small structures is essential to improve performances of tissue-scaffolds, extend bio-electronics applications or tailor microscale energy-absorption devices.
Asunto(s)
Nanocompuestos , Nanopartículas , Polímeros/química , Celulosa/química , Nanopartículas/química , Nanocompuestos/química , Impresión TridimensionalRESUMEN
In the present work, gelatin type B is modified with highly reactive norbornene functionalities (Gel-NB) following a one-pot synthesis approach to enable subsequent thiol-ene photo-click crosslinking. The modification strategy displays close control over the amount of introduced functionalities. Additionally, Gel-NB exhibits considerably improved processing capabilities in terms of two-photon polymerization when benchmarked to earlier-reported crosslinkable gelatin derivatives (e.g., gelatin-methacrylamide (Gel-MOD) and gelatin-methacrylamide-aminoethylmethacrylate (Gel-MOD-AEMA)). The improvement is especially apparent in terms of minimally required laser power (20 mW vs ≥60 mW (Gel-MOD) vs ≥40 mW (Gel-MOD-AEMA) at 100 mm s-1 scan speed) and processable concentration range (≥5 w/v% vs ≥10 w/v% (Gel-MOD/Gel-MOD-AEMA)). Furthermore, the proposed functionalization scheme maintains the excellent biocompatibility and cell interactivity of gelatin. Additionally, the norbornene functionalities have potential for straightforward postprocessing "thiol-ene" surface grafting of active molecules. As a consequence, a very promising material toward tissue engineering applications and more specifically, biofabrication, is presented.
Asunto(s)
Materiales Biocompatibles/química , Hidrogeles/química , Norbornanos/química , Compuestos de Sulfhidrilo/química , Química Clic , Reactivos de Enlaces Cruzados/química , Gelatina/química , Luz , Polietilenglicoles/química , Polimerizacion , Ingeniería de TejidosRESUMEN
Photodegradable hydrogels have emerged as useful platforms for research on cell function, tissue engineering, and cell delivery as their physical and chemical properties can be dynamically controlled by the use of light. The photo-induced degradation of such hydrogel systems is commonly based on the integration of photolabile o-nitrobenzyl derivatives to the hydrogel backbone, because such linkers can be cleaved by means of one- and two-photon absorption. Herein we describe a cytocompatible click-based hydrogel containing o-nitrobenzyl ester linkages between a hyaluronic acid backbone, which is photodegradable in the presence of cells. It is demonstrated for the first time that by using a cyclic benzylidene ketone-based small molecule as photosensitizer the efficiency of the two-photon degradation process can be improved significantly. Biocompatibility of both the improved two-photon micropatterning process as well as the hydrogel itself is confirmed by cell culture studies.
Asunto(s)
Compuestos de Bencilideno/química , Materiales Biocompatibles/química , Ácido Hialurónico/química , Hidrogeles/química , Fotólisis , Polietilenglicoles/química , Línea Celular , Química Clic , Humanos , Células Madre Mesenquimatosas/citología , Nitrobencenos/química , Fotones , Fármacos Fotosensibilizantes/química , Compuestos de Sulfhidrilo/química , Ingeniería de TejidosRESUMEN
The present work reports on the development of photo-cross-linkable gelatins sufficiently versatile to overcome current biopolymer two-photon polymerization (2PP) processing limitations. To this end, both the primary amines as well as the carboxylic acids of gelatin type B were functionalized with photo-cross-linkable moieties (up to 1 mmol/g) resulting in superior and tunable mechanical properties (G' from 5000 to 147000 Pa) enabling efficient 2PP processing. The materials were characterized in depth prior to and after photoinduced cross-linking using fully functionalized gelatin-methacrylamide (gel-MOD) as a benchmark to assess the effect of functionalization on the protein properties, cross-linking efficiency, and mechanical properties. In addition, preliminary experiments on hydrogel films indicated excellent in vitro biocompatibility (close to 100% viability) both in the presence of MC3T3 preosteoblasts and L929 fibroblasts. Moreover, 2PP processing of the novel derivative was superior in terms of applied laser power (≥40 vs ≥60 mW for gel-MOD at 100 mm/s) as well as post-production swelling (0-20% vs 75-100% for gel-MOD) compared to those of gel-MOD. The reported novel gelatin derivative (gel-MOD-AEMA) proves to be extremely suitable for direct laser writing as both superior mimicry of the applied computer-aided design (CAD) was obtained while maintaining the desired cellular interactivity of the biopolymer. It can be anticipated that the present work will also be applicable to alternative biopolymers mimicking the extracellular environment such as collagen, elastin, and glycosaminoglycans, thereby expanding current material-related processing limitations in the tissue engineering field.
Asunto(s)
Materiales Biocompatibles/síntesis química , Ácidos Carboxílicos/química , Gelatina/química , Hidrogeles/síntesis química , Fotones , Animales , Línea Celular , Reactivos de Enlaces Cruzados/química , Hidrogeles/química , Fenómenos Mecánicos , Ratones , PolimerizacionRESUMEN
Microneedle devices for transdermal delivery of nanoscale pharmacologic agents were fabricated out of organically-modified ceramic (Ormocer) materials using two photon polymerization. Out-of-plane hollow microneedle arrays with various aspect ratios were fabricated using this rapid prototyping process. Human epidermal keratinocyte (HEK) viability on Ormocer surfaces fabricated using two photon polymerization was similar to that on control surfaces. Nanoindentation studies were performed to determine hardness and Young's modulus values for Ormocer materials. Microneedies were shown to enable more rapid distribution of the PEG-amine quantum dot solution to the deep epidermis and dermis layers of porcine skin than topical administration. Our results suggest that two photon polymerization may be used to create microneedle arrays for transdermal delivery of nanoscale pharmacologic agents.
Asunto(s)
Sistemas de Liberación de Medicamentos/instrumentación , Microtecnología/métodos , Nanoestructuras/administración & dosificación , Agujas , Administración Cutánea , Animales , Materiales Biocompatibles/administración & dosificación , Materiales Biocompatibles/química , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Módulo de Elasticidad , Diseño de Equipo , Femenino , Dureza , Humanos , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Ensayo de Materiales , Microscopía Confocal , Microscopía Electrónica de Rastreo , Polimerizacion , Puntos Cuánticos , Piel/efectos de los fármacos , Piel/metabolismo , PorcinosRESUMEN
The introduction of two-photon polymerization (2PP) to the field of tissue engineering and regenerative medicine (TERM) has led to great expectations for the production of scaffolds with an unprecedented degree of complexity and tailorable architecture. Unfortunately, resolution and size are usually mutually exclusive when using 2PP, resulting in a lack of highly-detailed scaffolds with a relevant size for clinical application. Through the combination of using a highly reactive photopolymer and optimizing key printing parameters, we propose for the first time a biodegradable and biocompatible poly(trimethylene-carbonate) (PTMC)-based scaffold of large size (18 × 18 × 0.9 mm) with a volume of 292 mm3 produced using 2PP. This increase in size results in a significant volumetric increase by almost an order of magnitude compared to previously available large-scale structures (Stichel 2010 J. Laser Micro./Nanoeng. 5 209-12). The structure's detailed design resulted in a highly porous scaffold (96%) with excellent cytocompatibility, supporting the attachment, proliferation and differentiation of human adipose-derived mesenchymal stem cells towards their osteogenic and chondrogenic lineages. This work strongly attests that 2PP is becoming a highly suitable technique for producing large-sized scaffolds with a complex architecture. We show as a proof-of-concept that an arrayed design of repetitive units can be produced, but a further perspective will be to print scaffolds with anisotropic features that are more representative of human tissues.
Asunto(s)
Impresión Tridimensional , Ingeniería de Tejidos , Andamios del Tejido , Carbonatos , Ciclopropanos , Dioxanos , Humanos , Fotones , Polimerizacion , Polímeros , PorosidadRESUMEN
An investigation of the shrinking behaviour of a zirconium-based sol-gel composite micro-structured by two-photon polymerization is presented and a simple, straightforward methodology allowing the evaluation of shrinkage is suggested. It is shown that volume reduction is directly related to the average laser power (irradiation dose) used for the microfabrication and becomes a critical issue near the polymerization threshold. It is demonstrated that this shrinkage can be employed beneficially to improve the structural resolution. This is demonstrated by the presence of stopbands in the photonic crystal nanostructures fabricated with controlled volume reduction. Well above the polymerization threshold, the studied material exhibits remarkably low shrinkage. Therefore, no additional effort for the pre-compensation of distortion and for the improvement of structural stability is required.
Asunto(s)
Modelos Químicos , Fotoquímica/métodos , Polímeros/química , Polímeros/efectos de la radiación , Circonio/química , Circonio/efectos de la radiación , Simulación por Computador , Ensayo de MaterialesRESUMEN
Gelatin is frequently used in various biomedical applications. However, gelatin is generally extracted from an animal source, which can result in issues with reproducibility as well as pathogen transmittance. Therefore, we have investigated the potential of a recombinant peptide based on collagen I (RCPhC1) for tissue engineering applications and more specifically for adipose tissue regeneration. In the current paper, RCPhC1 was functionalized with photo-crosslinkable methacrylamide moieties to enable subsequent UV-induced crosslinking in the presence of a photo-initiator. The resulting biomaterial (RCPhC1-MA) was characterized by evaluating the crosslinking behaviour, the mechanical properties, the gel fraction, the swelling properties and the biocompatibility. The obtained results were compared with the data obtained for methacrylamide-modified gelatin (Gel-MA). The results indicated that the properties of RCPhC1-MA networks are comparable to those of animal-derived Gel-MA. RCPhC1-MA is thus an attractive synthetic alternative for animal-derived Gel-MA and is envisioned to be applicable for a wide range of tissue engineering purposes.
Asunto(s)
Materiales Biocompatibles/química , Colágeno/química , Ingeniería de Tejidos/métodos , HumanosRESUMEN
Two-photon polymerization technique was applied to generate three-dimensional (3D) scaffold-like structures using the photosensitive organic-inorganic hybrid polymer ORMOCER. The structures were studied with respect to potential applications as scaffold for tissue engineering. Cell counting and comet assay, respectively, demonstrated that doubling time and DNA strand breaks of CHO cells, GFSHR-17 granulosa cells, GM-7373 endothelial cells, and SH-SY5Y neuroblastoma cells were not affected by ORMOCER. ORMOCER related alteration of formation of tissue specific cell-to-cell adhesions like gap junctions was ruled out by double whole-cell patch-clamp technique. Additionally, growth of cells on the vertical surfaces of 3D structures composed of ORMOCER is shown.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Cerámica/farmacología , Regeneración Tisular Dirigida/métodos , Fotoquímica/métodos , Silanos/farmacología , Ingeniería de Tejidos/métodos , Andamios del Tejido , Animales , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Materiales Biocompatibles/efectos de la radiación , Células CHO , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Comunicación Celular/efectos de los fármacos , Comunicación Celular/genética , Línea Celular Tumoral , Cerámica/química , Cerámica/efectos de la radiación , Diseño Asistido por Computadora/tendencias , Cricetinae , Cricetulus , Daño del ADN/efectos de los fármacos , Daño del ADN/fisiología , Uniones Comunicantes/efectos de los fármacos , Uniones Comunicantes/fisiología , Regeneración Tisular Dirigida/instrumentación , Humanos , Rayos Láser , Ensayo de Materiales/métodos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Cerámicas Modificadas Orgánicamente , Polímeros/química , Polímeros/farmacología , Polímeros/efectos de la radiación , Silanos/química , Silanos/efectos de la radiación , Ingeniería de Tejidos/instrumentaciónRESUMEN
Bioprinting is a process based on additive manufacturing from materials containing living cells. These materials, often referred to as bioink, are based on cytocompatible hydrogel precursor formulations, which gel in a manner compatible with different bioprinting approaches. The bioink properties before, during and after gelation are essential for its printability, comprising such features as achievable structural resolution, shape fidelity and cell survival. However, it is the final properties of the matured bioprinted tissue construct that are crucial for the end application. During tissue formation these properties are influenced by the amount of cells present in the construct, their proliferation, migration and interaction with the material. A calibrated computational framework is able to predict the tissue development and maturation and to optimize the bioprinting input parameters such as the starting material, the initial cell loading and the construct geometry. In this contribution relevant bioink properties are reviewed and discussed on the example of most popular bioprinting approaches. The effect of cells on hydrogel processing and vice versa is highlighted. Furthermore, numerical approaches were reviewed and implemented for depicting the cellular mechanics within the hydrogel as well as for prediction of mechanical properties to achieve the desired hydrogel construct considering cell density, distribution and material-cell interaction.
Asunto(s)
Bioimpresión/instrumentación , Impresión Tridimensional , Animales , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Humanos , Hidrogeles/química , Hidrogeles/farmacología , Ingeniería de Tejidos , Andamios del Tejido/químicaRESUMEN
A reproducible method is needed to fabricate 3D scaffold constructs that results in periodic and uniform structures with precise control at sub-micrometer and micrometer length scales. In this study, fabrication of scaffolds by two-photon polymerization (2PP) of a biodegradable urethane and acrylate-based photoelastomer is demonstrated. This material supports 2PP processing with sub-micrometer spatial resolution. The high photoreactivity of the biophotoelastomer permits 2PP processing at a scanning speed of 1000 mm s(-1), facilitating rapid fabrication of relatively large structures (>5 mm(3)). These structures are custom printed for in vitro assay screening in 96-well plates and are sufficiently flexible to enable facile handling and transplantation. These results indicate that stable scaffolds with porosities of greater than 60% can be produced using 2PP. Human bone marrow stromal cells grown on 3D scaffolds exhibit increased growth and proliferation compared to smooth 2D scaffold controls. 3D scaffolds adsorb larger amounts of protein than smooth 2D scaffolds due to their larger surface area; the scaffolds also allow cells to attach in multiple planes and to completely infiltrate the porous scaffolds. The flexible photoelastomer material is biocompatible in vitro and is associated with facile handling, making it a viable candidate for further study of complex 3D-printed scaffolds.
Asunto(s)
Células Madre Mesenquimatosas/citología , Impresión Tridimensional , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Fenómenos Biomecánicos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Elasticidad , Elastómeros/química , Elastómeros/farmacología , Humanos , PorosidadRESUMEN
Cells respond to topographical, mechanical and biochemical characteristics of the surrounding environment. Capability to reconstruct these factors individually, and also acting in accord, would facilitate systematic investigations of a multitude of related biological and tissue engineering questions. The subject of the present review is a group of technologies allowing realization of customized cell-culture matrices. These methods utilize photochemistry induced by multiphoton absorption and are carried out using essentially identical equipment. Fabrication of 2D microstructured substrates, complex 3D scaffolds, containing actively induced topographies, and immobilization of biomolecules in a spatially defined manner was demonstrated with these techniques. The reviewed reports indicate that multiphoton processing is a promising technology platform for the development of standard biomimetic microenvironments for 3D cell culture.
Asunto(s)
Biología/métodos , Técnicas de Cultivo de Célula/métodos , Fotones , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Materiales Biocompatibles/farmacología , HumanosRESUMEN
Due to their ability to serve as fluorophores and drug delivery vehicles, quantum dots are a powerful tool for theranostics-based clinical applications. In this study, microneedle devices for transdermal drug delivery were fabricated by means of two-photon polymerization of an acrylate-based polymer. We examined proliferation of cells on this polymer using neonatal human epidermal keratinocytes and human dermal fibroblasts. The microneedle device was used to inject quantum dots into porcine skin; imaging of the quantum dots was performed using multiphoton microscopy.
Asunto(s)
Sistemas de Liberación de Medicamentos/instrumentación , Agujas , Polímeros/química , Puntos Cuánticos , Piel/química , Administración Cutánea , Animales , Femenino , Humanos , Recién Nacido , Queratinocitos/química , Microinyecciones/métodos , Microscopía Fluorescente , Piel/citología , PorcinosRESUMEN
IMPORTANCE OF THE FIELD: Microneedles are small-scale devices that are finding use for transdermal delivery of protein-based pharmacologic agents and nucleic acid-based pharmacologic agents; however, microneedles prepared using conventional microelectronics-based technologies have several shortcomings, which have limited translation of these devices into widespread clinical use. AREAS COVERED IN THIS REVIEW: Two-photon polymerization is a laser-based rapid prototyping technique that has been used recently for direct fabrication of hollow microneedles with a wide variety of geometries. In addition, an indirect rapid prototyping method that involves two-photon polymerization and polydimethyl siloxane micromolding has been used for fabrication of solid microneedles with exceptional mechanical properties. WHAT THE READER WILL GAIN: In this review, the use of two-photon polymerization for fabricating in-plane and out-of-plane hollow microneedle arrays is described. The use of two-photon polymerization-micromolding for fabrication of solid microneedles is also reviewed. In addition, fabrication of microneedles with antimicrobial properties is discussed; antimicrobial microneedles may reduce the risk of infection associated with the formation of channels through the stratum corneum. TAKE HOME MESSAGE: It is anticipated that the use of two-photon polymerization as well as two-photon polymerization-micromolding for fabrication of microneedles and other microstructured drug delivery devices will increase over the coming years.
Asunto(s)
Sistemas de Liberación de Medicamentos/instrumentación , Microtecnología/métodos , Agujas , Preparaciones Farmacéuticas/administración & dosificación , Administración Cutánea , Materiales Biocompatibles , HumanosRESUMEN
BACKGROUND: Microneedle-mediated drug delivery is a promising method for transdermal delivery of insulin, incretin mimetics, and other protein-based pharmacologic agents for treatment of diabetes mellitus. One factor that has limited clinical application of conventional microneedle technology is the poor fracture behavior of microneedles that are created using conventional materials and methods. In this study polymer microneedles for transdermal delivery were created using a two-photon polymerization (2PP) microfabrication and subsequent polydimethylsiloxane (PDMS) micromolding process. METHODS: Solid microneedle arrays, fabricated by means of 2PP, were used to create negative molds from PDMS. Using these molds microneedle arrays were subsequently prepared by molding eShell 200, a photo-reactive acrylate-based polymer that exhibits water and perspiration resistance. RESULTS: The eShell 200 microneedle array demonstrated suitable compressive strength for use in transdermal drug delivery applications. Human epidermal keratinocyte viability on the eShell 200 polymer surfaces was similar to that on polystyrene control surfaces. In vitro studies demonstrated that eShell 200 microneedle arrays fabricated using the 2PP microfabrication and PDMS micromolding process technique successfully penetrated human stratum corneum and epidermis. CONCLUSIONS: Our results suggest that a 2PP microfabrication and subsequent PDMS micromolding process may be used to create microneedle structures with appropriate structural, mechanical, and biological properties for transdermal drug delivery of insulin and other protein-based pharmacologic agents for treatment of diabetes mellitus.
Asunto(s)
Diabetes Mellitus/tratamiento farmacológico , Sistemas de Liberación de Medicamentos/instrumentación , Insulina/administración & dosificación , Agujas , Administración Cutánea , Materiales Biocompatibles , Dimetilpolisiloxanos/química , Humanos , Microtecnología/métodos , Ácidos Polimetacrílicos/síntesis químicaRESUMEN
Two-photon polymerization has been employed to fabricate three-dimensional structures using the biodegradable triblock copolymer poly(epsilon-caprolactone-co-trimethylenecarbonate)-b-poly(ethylene glycol)-b-poly(epsilon-caprolactone-co-trimethylenecarbonate) with 4,4'-bis(diethylamino)benzophenone as the photoinitiator. The fabricated structures were of good quality and had four micron resolution. Initial cytotoxicity tests show that the material does not affect cell proliferation. These studies demonstrate the potential of two-photon polymerization as a technology for the fabrication of biodegradable scaffolds for tissue engineering.
Asunto(s)
Materiales Biocompatibles/química , Polímeros/química , Animales , Biodegradación Ambiental , Proliferación Celular , Diseño de Equipo , Luz , Ratones , Ratones Endogámicos BALB C , Modelos Químicos , Células 3T3 NIH , Fotoquímica/métodos , Fotones , Propiedades de Superficie , Ingeniería de Tejidos/métodosRESUMEN
In this study, a novel rapid prototyping technology was used to fabricate scaphoid and lunate bone prostheses, two carpal bones that are prone to avascular necrosis. Carpal prostheses were fabricated with an Envisiontec Perfactory SXGA stereolithography system using Envisiontec eShell 200 photocurable polymer. Fabrication was guided using 3-D models, which were generated using Mimics software (Materialise NV, Leuven, Belgium) from patient computer tomography data. The prostheses were fabricated in a layer-by-layer manner; approximately 50-microm thick layers were observed in the prostheses. Hardness and Young's modulus values of polymerized eShell 200 material were 93.8 +/- 7.25 MPa and 3050 +/- 90 MPa, respectively. The minimum compressive force required for fracture was 1360 N for the scaphoid prosthesis and 1248 N for the lunate prosthesis. Polymerized Envisiontec eShell material exhibited high human neonatal epidermal keratinocyte cell viability rate in an MTT assay. The results of this study indicate that small bone prostheses fabricated by stereolithography using eShell 200 polymer may have suitable geometry, mechanical properties, and cytocompatibility properties for in vivo use.
Asunto(s)
Hueso Semilunar/anatomía & histología , Diseño de Prótesis/métodos , Hueso Escafoides/anatomía & histología , Análisis de Varianza , Supervivencia Celular , Fuerza Compresiva , Diseño Asistido por Computadora , Módulo de Elasticidad , Humanos , Queratinocitos/metabolismo , Ensayo de Materiales , Polímeros/química , Prótesis e Implantes , Resinas Sintéticas/química , Tomografía Computarizada por Rayos XRESUMEN
We report on recent advances in the fabrication of three-dimensional (3D) scaffolds for tissue engineering and regenerative medicine constructs using a two-photon polymerization technique (2PP). 2PP is a novel CAD/CAM technology allowing the fabrication of any computer-designed 3D structure from a photosensitive polymeric material. The flexibility of this technology and the ability to precisely define 3D construct geometry allows issues associated with vascularization and patient-specific tissue fabrication to be directly addressed. The fabrication of reproducible scaffold structures by 2PP is important for systematic studies of cellular processes and better understanding of in vitro tissue formation. In this study, 2PP was applied for the generation of 3D scaffold-like structures, using the photosensitive organic-inorganic hybrid polymer ORMOCER (ORganically MOdified CERamics) and epoxy-based SU8 materials. By comparing the proliferation rates of cells grown on flat material surfaces and under control conditions, it was demonstrated that ORMOCER and SU8 are not cytotoxic. Additional tests show that the DNA strand breaking of GFSHR-17 granulosa cells was not affected by the presence of ORMOCER. Furthermore, gap junction conductance measurements revealed that ORMOCER did not alter the formation of cell-cell junctions, critical for functional tissue growth. The possibilities of seeding 3D structures with cells were analysed. These studies demonstrate the great potential of 2PP technique for the manufacturing of scaffolds with controlled topology and properties.