RESUMEN
Melt extrusion-based additive manufacturing (ME-AM) is a promising technique to fabricate porous scaffolds for tissue engineering applications. However, most synthetic semicrystalline polymers do not possess the intrinsic biological activity required to control cell fate. Grafting of biomolecules on polymeric surfaces of AM scaffolds enhances the bioactivity of a construct; however, there are limited strategies available to control the surface density. Here, we report a strategy to tune the surface density of bioactive groups by blending a low molecular weight poly(ε-caprolactone)5k (PCL5k) containing orthogonally reactive azide groups with an unfunctionalized high molecular weight PCL75k at different ratios. Stable porous three-dimensional (3D) scaffolds were then fabricated using a high weight percentage (75 wt.%) of the low molecular weight PCL5k. As a proof-of-concept test, we prepared films of three different mass ratios of low and high molecular weight polymers with a thermopress and reacted with an alkynated fluorescent model compound on the surface, yielding a density of 201-561 pmol/cm2. Subsequently, a bone morphogenetic protein 2 (BMP-2)-derived peptide was grafted onto the films comprising different blend compositions, and the effect of peptide surface density on the osteogenic differentiation of human mesenchymal stromal cells (hMSCs) was assessed. After two weeks of culturing in a basic medium, cells expressed higher levels of BMP receptor II (BMPRII) on films with the conjugated peptide. In addition, we found that alkaline phosphatase activity was only significantly enhanced on films containing the highest peptide density (i.e., 561 pmol/cm2), indicating the importance of the surface density. Taken together, these results emphasize that the density of surface peptides on cell differentiation must be considered at the cell-material interface. Moreover, we have presented a viable strategy for ME-AM community that desires to tune the bulk and surface functionality via blending of (modified) polymers. Furthermore, the use of alkyne-azide "click" chemistry enables spatial control over bioconjugation of many tissue-specific moieties, making this approach a versatile strategy for tissue engineering applications. Supplementary Information: The online version contains supplementary material available at 10.1007/s42242-024-00286-2.
RESUMEN
Electrospinning PLLA solutions from two oppositely charged nozzles gives a triangle of fibers, also called E-triangle, that assemble into yarns at the convergence point. The formed yarn at the E-triangle was taken up by a unit comprising a take up roller and coupled twister plate, which twist rate can be varied. At all twist rates, uniform and smooth fibers without any beads were formed. The apex angle of the deposited fibers at the E-triangle was larger at higher twist rates. By increasing the twist rate from 80rpm to 320rpm the orientation angle of fibers in the yarn changes from 18.8° to 41.5°. Increasing the twist rate revealed a higher polymer crystallinity likely due to the polymer orientation by the applied tension to the fibers. The ultimate strength and modulus of electrospun yarns were higher when prepared at higher twist rates. However, at the highest twist rates, the strength and modulus of electrospun yarns leveled off and even decreased slightly. The results revealed that the mechanical properties not only depend on the polymer crystallinity but also on the alignment of the fibers in the yarn and the angle at which they were deposited. These biodegradable materials are promising materials to be used in a wide range of applications where environmentally friendly products are required.
Asunto(s)
Plásticos Biodegradables/análisis , Poliésteres/análisis , Plásticos Biodegradables/síntesis química , Poliésteres/síntesis química , PolímerosRESUMEN
In this study, twisted drug-loaded poly(L-lactide) (PLLA) and hybrid poly(L-lactide)/poly(vinyl alcohol) (PLLA/PVA) yarns were produced using an electrospinning technique based on two oppositely charged nozzles. Cefazolin, an antibiotic drug was incorporated in the yarn fibers by addition to the PLLA electrospinning solution. Morphological studies showed that independent of the twist rate, uniform and smooth fibers were formed. The diameter of the electrospun fibers in the yarns decreased at higher twist rates but produced yarns with larger diameters. At increasing twist rates the crystallinity of the fibers in the yarns increased. In the presence of cefazolin the fiber diameter, yarn diameter and crystallinity were always lower than in the non-drug loaded yarns. In addition the yarn mechanical properties revealed a slightly lower strength, modulus and elongation at break upon drug loading. The effect of the twist rate on the cefazolin in vitro release behavior from both PLLA and hybrid yarns revealed similar profiles for both types of drug-loaded yarns. However, the total amount of drug released from the hybrid PLLA/PVA yarns was significantly higher. The release kinetics over a period of 30 d were fitted to different mathematical models. Cefazolin release from electrospun PLLA yarns was governed by a diffusion mechanism and could best be fitted by Peppas and Higuchi models. The models that were found best to describe the drug release mechanism from the hybrid PLLA/PVA yarns were a first-order model and the Higuchi model.
Asunto(s)
Antibacterianos/química , Cefazolina/química , Portadores de Fármacos/química , Antibacterianos/metabolismo , Rastreo Diferencial de Calorimetría , Cefazolina/metabolismo , Fuerza Compresiva , Difusión , Liberación de Fármacos , Módulo de Elasticidad , Cinética , Poliésteres/química , Alcohol Polivinílico/química , Espectrofotometría Ultravioleta , Factores de Tiempo , Temperatura de TransiciónRESUMEN
An electrospinning technique based on the use of two oppositely charged nozzles was applied to fabricate continuous twisted yarns of poly(L-lactide) (PLLA) nano/micro fibers. In this study, the effect of solvent on the electrospinning of PLLA fibrous yarns was investigated. For this purpose, yarns were electrospun using chloroform, dichloromethane or 2,2,2-trifluoroethanol as solvents at a PLLA concentration of 7 wt%. The analysis of the morphology, diameter, crystallinity and mechanical properties of electrospun yarns revealed that the vapor pressure of the solvent plays an important role. Whereas the fiber diameter decreased, the crystallinity of the fibers increased using a solvent with lower vapor pressure. In addition, mechanical properties (e.g., tensile strength and modulus) revealed that the yarns composed of fibers with smaller diameters showed higher tensile strength and modulus. In summary, fine-tuning solvent properties resulted in a modulation of fiber diameter, crystallinity, and thereby yarn mechanical properties, and are important factors to consider in the fabrication and application of electrospun yarns.
Asunto(s)
Nanofibras/química , Poliésteres/química , Ingeniería de Tejidos/instrumentación , Andamios del Tejido/química , Electroquímica/instrumentación , Electroquímica/métodos , Poliésteres/síntesis química , Porosidad , Solventes/química , Resistencia a la TracciónRESUMEN
Fully aliphatic segmented poly(ether ester amide) copolymers with uniform hard segments prepared by melt polycondensation of α,ω-hydroxyl end-functionalized polytetrahydrofuran and short glycine or ß-alanine bisester-bisoxalamide units hold promise for biomedical applications. For polymers with the hard block contents varying from 10% to 27%, differential scanning calorimetry and atomic force microscopy reveal a highly phase-separated morphology, with ribbon-like nanocrystals dispersed in the soft segment matrix. To relate the polymer properties to the structure of the hard segment, the monomers were prepared and studied by optical and X-ray diffraction measurements. It was shown that the glycine and ß-alanine carbonyl ester groups are tilted away from the oxalamide plane, which can affect the degradation rate via hydrolysis of the ester bond.
Asunto(s)
Materiales Biocompatibles/química , Carbono/química , Nanopartículas/química , Nanopartículas/ultraestructura , Compuestos Orgánicos/química , Polímeros/química , Esterificación , Dureza , Enlace de Hidrógeno , Hidrólisis , Ensayo de MaterialesRESUMEN
Cell-based cartilage repair strategies such as matrix-induced autologous chondrocyte implantation (MACI) could be improved by enhancing cell performance. We hypothesised that micro-aggregates of chondrocytes generated in high-throughput prior to implantation in a defect could stimulate cartilaginous matrix deposition and remodelling. To address this issue, we designed a micro-mould to enable controlled high-throughput formation of micro-aggregates. Morphology, stability, gene expression profiles and chondrogenic potential of micro-aggregates of human and bovine chondrocytes were evaluated and compared to single-cells cultured in micro-wells and in 3D after encapsulation in Dextran-Tyramine (Dex-TA) hydrogels in vitro and in vivo. We successfully formed micro-aggregates of human and bovine chondrocytes with highly controlled size, stability and viability within 24 hours. Micro-aggregates of 100 cells presented a superior balance in Collagen type I and Collagen type II gene expression over single cells and micro-aggregates of 50 and 200 cells. Matrix metalloproteinases 1, 9 and 13 mRNA levels were decreased in micro-aggregates compared to single-cells. Histological and biochemical analysis demonstrated enhanced matrix deposition in constructs seeded with micro-aggregates cultured in vitro and in vivo, compared to single-cell seeded constructs. Whole genome microarray analysis and single gene expression profiles using human chondrocytes confirmed increased expression of cartilage-related genes when chondrocytes were cultured in micro-aggregates. In conclusion, we succeeded in controlled high-throughput formation of micro-aggregates of chondrocytes. Compared to single cell-seeded constructs, seeding of constructs with micro-aggregates greatly improved neo-cartilage formation. Therefore, micro-aggregation prior to chondrocyte implantation in current MACI procedures, may effectively accelerate hyaline cartilage formation.
Asunto(s)
Cartílago/crecimiento & desarrollo , Agregación Celular , Condrocitos/citología , Regulación de la Expresión Génica , Análisis de la Célula Individual , Agrecanos/metabolismo , Animales , Cartílago/metabolismo , Bovinos , Trasplante de Células/métodos , Condrocitos/metabolismo , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Metaloproteinasas de la Matriz/genética , Metaloproteinasas de la Matriz/metabolismo , Ratones , Ratones Desnudos , Análisis por MicromatricesRESUMEN
The mechanical properties of individual collagen fibrils of approximately 200 nm in diameter were determined using a slightly adapted AFM system. Single collagen fibrils immersed in PBS buffer were attached between an AFM cantilever and a glass surface to perform tensile tests at different strain rates and stress relaxation measurements. The stress-strain behavior of collagen fibrils immersed in PBS buffer comprises a toe region up to a stress of 5 MPa, followed by the heel and linear region at higher stresses. Hysteresis and strain-rate dependent stress-strain behavior of collagen fibrils were observed, which suggest that single collagen fibrils have viscoelastic properties. The stress relaxation process of individual collagen fibrils could be best fitted using a two-term Prony series. Furthermore, the influence of different cross-linking agents on the mechanical properties of single collagen fibrils was investigated. Based on these results, we propose that sliding of microfibrils with respect to each other plays a role in the viscoelastic behavior of collagen fibrils in addition to the sliding of collagen molecules with respect to each other. Our finding provides a better insight into the relationship between the structure and mechanical properties of collagen and the micro-mechanical behavior of tissues.
Asunto(s)
Colágeno Tipo I/química , Fenómenos Mecánicos , Microscopía de Fuerza Atómica , Animales , Tampones (Química) , Bovinos , Reactivos de Enlaces Cruzados/farmacología , Elasticidad , Vidrio/química , Modelos Moleculares , Estrés Mecánico , Propiedades de Superficie , Resistencia a la Tracción , ViscosidadRESUMEN
Poly(ε-caprolactone) (PCL) fibers ranging from 250 to 700 nm in diameter were produced by electrospinning a polymer tetrahydrofuran/N,N-dimethylformamide solution. The mechanical properties of the fibrous scaffolds and individual fibers were measured by different methods. The Young's moduli of the scaffolds were determined using macro-tensile testing equipment, whereas single fibers were mechanically tested using a nanoscale three-point bending method, based on atomic force microscopy and force spectroscopy analyses. The modulus obtained by tensile-testing eight different fiber scaffolds was 3.8±0.8 MPa. Assuming that PCL fibers can be described by the bending model of isotropic materials, a Young's modulus of 3.7±0.7 GPa was determined for single fibers. The difference of three orders of magnitude observed in the moduli of fiber scaffolds vs. single fibers can be explained by the lacunar and random structure of the scaffolds.
Asunto(s)
Materiales Biocompatibles/química , Poliésteres/química , Módulo de Elasticidad , Técnicas Electroquímicas , Ensayo de Materiales , Microscopía de Fuerza Atómica , Microscopía Electrónica de Rastreo , Estrés Mecánico , Resistencia a la TracciónRESUMEN
Currently, in bone tissue engineering research, the development of appropriate biomaterials for the regeneration of bony tissues is a major concern. Bone tissue is composed of a structural protein, collagen type I, on which calcium phosphate crystals are enclosed. For tissue engineering, one of the most applied strategies consists on the development and application of three dimensional porous scaffolds with similar composition to the bone. In this way, they can provide a physical support for cell attachment, proliferation, nutrient transport and new bone tissue infiltration. Hydroxyapatite is a calcium phosphate with a similar composition of bone and widely applied in several medical/dentistry fields. Therefore, in this study, hydroxyapatite three dimensional porous scaffolds were produced using the polymer replication method. Next, the porous scaffolds were homogeneously coated with a film of collagen type I by applying vacuum force. Yet, due to collagen degradability properties, it was necessary to perform an adequate crosslinking method. As a result, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) was employed as an efficient and non-toxic crosslinking method in this research. The composites were characterized by means of SEM, DSC and TNBS. Furthermore, heparin was incorporated in order to accomplish sustained delivery of a growth factor of interest namely, bone morphogenetic proteins (BMP-2). BMP-2 binding and release of non-heparinized and heparinized scaffolds was evaluated at specific time points. The incorporation of heparin leads to a reduced initial burst phase when compared to the non heparinized materials. The results show a beneficial effect with the incorporation of heparin and its potential as a localized drug delivery system for the sustained release of growth factors.
Asunto(s)
Colágeno/química , Durapatita/química , Heparina/química , Ingeniería de Tejidos/métodos , Andamios del Tejido , Tendón Calcáneo/química , Tendón Calcáneo/metabolismo , Animales , Materiales Biocompatibles/síntesis química , Materiales Biocompatibles/química , Sustitutos de Huesos/síntesis química , Sustitutos de Huesos/química , Sustitutos de Huesos/metabolismo , Huesos/efectos de los fármacos , Huesos/metabolismo , Bovinos , Reactivos de Enlaces Cruzados/farmacología , Microscopía Electrónica de Rastreo , Polímeros/síntesis química , Polímeros/química , Porosidad , Andamios del Tejido/químicaRESUMEN
Polysaccharide hybrids consisting of hyaluronic acid (HA) grafted with a dextran-tyramine conjugate (Dex-TA) were synthesized and investigated as injectable biomimetic hydrogels for cartilage tissue engineering. The design of these hybrids (denoted as HA-g-Dex-TA) is based on the molecular structure of proteoglycans present in the extracellular matrix of native cartilage. Hydrogels of HA-g-Dex-TA were rapidly formed within 2 min via enzymatic crosslinking of the tyramine residues in the presence of horseradish peroxidase and hydrogen peroxide. The gelation time, equilibrium swelling and storage modulus could be adjusted by varying the degree of substitution of tyramine residues and polymer concentration. Bovine chondrocytes incorporated in the HA-g-Dex-TA hydrogels remained viable, as shown by the Live-dead assay. Moreover, enhanced chondrocyte proliferation and matrix production were observed in the HA-g-Dex-TA hydrogels compared to Dex-TA hydrogels. These results suggest that HA-g-Dex-TA hydrogels have a high potential as injectable scaffolds for cartilage tissue engineering.
Asunto(s)
Biomimética , Cartílago/química , Dextranos/química , Ácido Hialurónico/química , Hidrogeles/química , Ingeniería de Tejidos , Animales , Materiales Biocompatibles/química , Conformación de Carbohidratos , Secuencia de Carbohidratos , Cartílago/patología , Bovinos , Proliferación Celular , Células Cultivadas , Condrocitos/química , Condrocitos/citología , Reactivos de Enlaces Cruzados/química , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Inyecciones , Ensayo de Materiales , Modelos Moleculares , Datos de Secuencia Molecular , Simpatomiméticos/química , Ingeniería de Tejidos/instrumentación , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Tiramina/químicaRESUMEN
Injectable hydrogels based on hyaluronic acid (HA) and poly(ethylene glycol) (PEG) were designed as biodegradable matrices for cartilage tissue engineering. Solutions of HA conjugates containing thiol functional groups (HA-SH) and PEG vinylsulfone (PEG-VS) macromers were cross-linked via Michael addition to form a three-dimensional network under physiological conditions. Gelation times varied from 14min to less than 1min, depending on the molecular weights of HA-SH and PEG-VS, degree of substitution (DS) of HA-SH and total polymer concentration. When the polymer concentration was increased from 2% to 6% (w/v) in the presence of 100Uml(-1) hyaluronidase the degradation time increased from 3 to 15days. Hydrogels with a homogeneous distribution of cells were obtained when chondrocytes were mixed with the precursor solutions. Culturing cell-hydrogel constructs prepared from HA185k-SH with a DS of 28 and cross-linked with PEG5k-4VS for 3weeks in vitro revealed that the cells were viable and that cell division took place. Gel-cell matrices degraded in approximately 3weeks, as shown by a significant decrease in dry gel mass. At day 21 glycosaminoglycans and collagen type II were found to have accumulated in hydrogels. These results indicate that these injectable hydrogels have a high potential for cartilage tissue engineering.
Asunto(s)
Materiales Biocompatibles/síntesis química , Condrocitos/citología , Condrocitos/fisiología , Ácido Hialurónico/síntesis química , Hidrogeles/síntesis química , Polietilenglicoles/síntesis química , Animales , Cartílago Articular/lesiones , Cartílago Articular/cirugía , Bovinos , Células Cultivadas , Hidrogeles/administración & dosificación , Inyecciones Intraarticulares , Ensayo de MaterialesRESUMEN
Water-soluble chitosan derivatives, chitosan-graft-glycolic acid (GA) and phloretic acid (PA) (CH-GA/PA), were designed to obtain biodegradable injectable chitosan hydrogels through enzymatic crosslinking with horseradish peroxidase (HRP) and H2O2. CH-GA/PA polymers were synthesized by first conjugating glycolic acid (GA) to native chitosan to render the polymer soluble at pH 7.4, and subsequent modification with phloretic acid (PA). The CH-GA43/PA10 with a degree of substitution (DS, defined as the number of substituted NH2 groups per 100 glucopyranose rings of chitosan) of GA of 43 and DS of PA of 10 showed a good solubility at pH values up to 10. Short gelation times (e.g. 10 s at a polymer concentration of 3 wt%), as recorded by the vial tilting method, were observed for the CH-GA43/PA10 hydrogels using HRP and H2O2. It was shown that these hydrogels can be readily degraded by lysozyme. In vitro culturing of chondrocytes in CH-GA43/PA10 hydrogels revealed that after 2 weeks the cells were viable and retained their round shape. These features indicate that CH-GA/PA hydrogels are promising as an artificial extracellular matrix for cartilage tissue engineering.
Asunto(s)
Cartílago/metabolismo , Quitosano/química , Quitosano/metabolismo , Hidrogeles/química , Hidrogeles/metabolismo , Animales , Bovinos , Supervivencia Celular , Células Cultivadas , Concentración de Iones de Hidrógeno , Inyecciones , Estructura Molecular , Muramidasa/metabolismo , Reología , Factores de Tiempo , Ingeniería de Tejidos , Agua/químicaRESUMEN
Meshes of collagen and/or elastin were successfully prepared by means of electrospinning from aqueous solutions. Flow rate, applied electric field, collecting distance and composition of the starting solutions determined the morphology of the obtained fibres. Addition of PEO (M(w)=8 x 10(6)) and NaCl was always necessary to spin continuous and homogeneous fibres. Spinning a mixture of collagen and elastin resulted in fibres in which the single components could not be distinguished by SEM. Increasing the elastin content determined an increase in fibres diameters from 220 to 600 nm. The voltage necessary for a continuous production of fibres was dependent on the composition of the starting solution, but always between 10 and 25 kV. Under these conditions, non-woven meshes could be formed and a partial orientation of the fibres constituting the mesh was obtained by using a rotating tubular mandrel as collector. Collagen/elastin (1:1) meshes were stabilized by crosslinking with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). This treatment afforded materials with a high thermal stability (T(d)=79 degrees C) without altering their original morphology. Upon crosslinking PEO and NaCl were fully leached out. Smooth muscle cells grew as a confluent layer on top of the crosslinked meshes after 14 d of culture.
Asunto(s)
Colágeno/química , Elastina/química , Ingeniería de Tejidos , Animales , Bovinos , Colágeno/ultraestructura , Elastina/ultraestructura , Electrones , Microscopía Electrónica de Rastreo , ViscosidadRESUMEN
Porous scaffolds composed of collagen or collagen and elastin were prepared by freeze drying at temperatures between -18 and -196 degrees C. All scaffolds had a porosity of 90-98% and a homogeneous distribution of pores. Freeze drying at -18 degrees C afforded collagen and collagen/elastin matrices with average pore sizes of 340 and 130 mum, respectively. After 20 successive cycles up to 10% of strain, collagen/elastin dense films had a total degree of strain recovery of 70% +/- 5%, which was higher than that of collagen films (42% +/- 6%). Crosslinking of collagen/elastin matrices either in water or ethanol/water (40% v/v) was carried out using a carbodiimide (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, EDC) in combination with a succinimide (N-hydroxysuccinimide, NHS) in the presence or absence of a diamine (J230) or by reaction with butanediol diglycidylether (BDGE), followed by EDC/NHS. Crosslinking with EDC/NHS or EDC/NHS/J230 resulted in matrices with increased stiffness as compared to noncrosslinked matrices, whereas sequential crosslinking with the diglycidylether and EDC/NHS yielded very brittle scaffolds. Ethanol/water was the preferred solvent in the crosslinking process because of its ability to preserve the open porous structure during crosslinking. Smooth muscle cells were seeded on the (crosslinked) scaffolds and could be expanded during 14 days of culturing.
Asunto(s)
Prótesis Vascular , Colágeno/uso terapéutico , Elastina/uso terapéutico , Ingeniería de Tejidos/métodos , Fenómenos Biomecánicos , Técnicas de Cultivo de Célula , Reactivos de Enlaces Cruzados , Liofilización , Humanos , Miocitos del Músculo Liso/citología , Porosidad , SolventesAsunto(s)
ADN/administración & dosificación , Técnicas de Transferencia de Gen , Terapia Genética , Polietilenglicoles/síntesis química , Polietileneimina/síntesis química , Animales , Células COS , Supervivencia Celular , Polietilenglicoles/administración & dosificación , Polietileneimina/administración & dosificación , TransfecciónRESUMEN
Calcification limits the long-term durability of xenograft glutaraldehyde (GA)-crosslinked heart valves. Previously, a study in rats showed that epoxy-crosslinked heart valves reduced lymphocyte reactions to the same extent as the GA-crosslinked control and induced a similar foreign-body response and calcification reaction. The present study was aimed at reducing the occurrence of calcification of epoxy-crosslinked tissue. Two modifications were carried out and their influence on cellular reactions and the extent of calcification after 8 weeks' implantation in weanling rats was evaluated. First, epoxy-crosslinked valves were post-treated with two detergents to remove cellular elements, phospholipids and small soluble proteins, known to act as nucleation sites for calcification. The second approach was to study the effect of the impaired balance between negatively and positively charged amino acids by an additional crosslinking step with a dicarboxylic acid. The detergent treatment resulted in a washed-out appearance of especially the cusp tissue. With the dicarboxylic acid, both the cusps and the walls had a limited washed-out appearance. The wall also demonstrated some detachment of the subendothelium. After implantation, both detergent and dicarboxylic acid post-treatment histologically resulted in reduced calcification at the edges of cusps and walls. However, total amounts of calcification, measured by atomic emission spectroscopy, were not significantly reduced. Data concerning the presence of lymphocytes varied slightly, but were in the same range as the GA-crosslinked control, i.e., clearly reduced compared with a noncrosslinked control. It is concluded that both the double detergent and the dicarboxylic acid post-treatment of epoxy-crosslinked heart valve tissue do not represent a sound alternative in the fabrication of heart valve bioprostheses.