Dual-source dual-power electrospinning and characteristics of multifunctional scaffolds for bone tissue engineering

Electrospun tissue engineering scaffolds are attractive due to their distinctive advantages over other types of scaffolds. As both osteoinductivity and osteoconductivity play crucial roles in bone tissue engineering, scaffolds possessing both properties are desirable. In this investigation, novel bicomponent scaffolds were constructed via dual-source dual-power electrospinning (DSDPES). One scaffold component was emulsion electrospun poly(d,l-lactic acid) (PDLLA) nanofibers containing recombinant human bone morphogenetic protein (rhBMP-2), and the other scaffold component was electrospun calcium phosphate (Ca–P) particle/poly(lactic-co-glycolic acid) (PLGA) nanocomposite fibers. The mass ratio of rhBMP-2/PDLLA fibers to Ca–P/PLGA fibers in bicomponent scaffolds could be controlled in the DSDPES process by adjusting the number of syringes used to supply solutions for electrospinning. Through process optimization, both types of fibers could be evenly distributed in bicomponent scaffolds. The structure and properties of each type of fibers in the scaffolds were studied. The morphological and structural properties and wettability of scaffolds were assessed. The effects of emulsion composition for rhBMP-2/PDLLA fibers and mass ratio of fibrous components in bicomponent scaffolds on in vitro release of rhBMP-2 from scaffolds were investigated. In vitro degradation of scaffolds was also studied by monitoring their morphological changes, weight losses and decreases in average molecular weight of fiber matrix polymers.     

Nerve growth factor-immobilized electrically conducting fibrous scaffolds for potential use in neural engineering applications

Engineered scaffolds simultaneously exhibiting multiple cues are highly desirable for neural tissue regeneration. To this end, we developed a neural tissue engineering scaffold that displays submicrometer-scale features, electrical conductivity, and neurotrophic activity. Specifically, electrospun poly(lactic acid-co-glycolic acid) (PLGA) nanofibers were layered with a nanometer thick coating of electrically conducting polypyrrole (PPy) presenting carboxylic groups. Then, nerve growth factor (NGF) was chemically immobilized onto the surface of the fibers. These NGF-immobilized PPy-coated PLGA (NGF-PPyPLGA) fibers supported PC12 neurite formation ( 28.0±3.0% of the cells) and neurite outgrowth (14.2 μm median length), which were comparable to that observed with NGF (50 ng/mL) in culture medium ( 29.0±1.3%, 14.4 μm). Electrical stimulation of PC12 cells on NGF-immobilized PPyPLGA fiber scaffolds was found to further improve neurite development and neurite length by 18% and 17%, respectively, compared to unstimulated cells on the NGF-immobilized fibers. Hence, submicrometer-scale fibrous scaffolds that incorporate neurotrophic and electroconducting activities may serve as promising neural tissue engineering scaffolds such as nerve guidance conduits.     

Evaluation of electrospun fibrous scaffolds of poly(dl-lactide) and poly(ethylene glycol) for skin tissue engineering

This study is derived from the innate concerns of electrospun poly(DL-lactide) (PDLLA) fibers as tissue engineering scaffolds: hydrophobic surface, surface erosion and dimensional shrinkage, which are not favorable to trigger the initial adhesion and further growth and population of cells. Blending electrospinning of PDLLA and poly(ethylene glycol) (PEG) with different PEG contents was evaluated for optimal tissue engineering scaffolds. The surface hydrophilicity was improved, and the degradation patterns of PDLLA/PEG mats changed from surface erosion to bulk degradation with the increase in PEG contents. The dimensional shrinkage was alleviated through the formation of crystal regions of PEG in the fiber matrix. The PDLLA/PEG fibrous mats were slightly weakened with the increase in the PEG contents, but a significant decrease in the tensile strength could be found for those with PEG contents of over 40%. Human dermal fibroblasts (HDFs) interacted and integrated well with the surrounding fibers containing 20 and 30% PEG, which provided significantly better environment for biological activities of HDFs than electrospun PDLLA mats. It indicated that electrospun mats containing 30% PEG exhibited the most balanced properties, including moderately hydrophilic surface, minimal dimensional changes, adaptable bulk biodegradation pattern and enhancement of cell penetration and growth within fibrous mats.     

Effect of scaffold architecture and BMP-2/BMP-7 delivery on in vitro bone regeneration

The aim of this study was to develop 3-D tissue engineered constructs that mimic the in vivo conditions through a self-contained growth factor delivery system. A set of nanoparticles providing the release of BMP-2 initially followed by the release of BMP-7 were incorporated in poly(ε-caprolactone) scaffolds with different 3-D architectures produced by 3-D plotting and wet spinning. The release patterns were: each growth factor alone, simultaneous, and sequential. The orientation of the fibers did not have a significant effect on the kinetics of release of the model protein BSA; but affected proliferation of bone marrow mesenchymal stem cells. Cell proliferation on random scaffolds was significantly higher compared to the oriented ones. Delivery of BMP-2 alone suppressed MSC proliferation and increased the ALP activity to a higher level than that with BMP-7 delivery. Proliferation rate was suppressed the most by the sequential delivery of the two growth factors from the random scaffold on which the ALP activity was the highest. Results indicated the distinct effect of scaffold architecture and the mode of growth factor delivery on the proliferation and osteogenic differentiation of MSCs, enabling us to design multifunctional scaffolds capable of controlling bone healing.     

电纺丝聚乳酸纤维支架的制备及其细胞相容性研究

静电纺丝法制备的聚(D,L-乳酸)超细纤维具有孔隙率高、孔径/结构可调、生物相容性良好等特点,已成为组织工程支架材料研究的热点。探讨了纺丝溶液浓度、流量、电压、干燥方式等影响聚(D,L-乳酸)纤维支架形貌的因素,确定了最佳工艺参数为纺丝溶液浓度为0.20g/mL,流速为0.8mL/h,电压为12kV,经真空冷冻干燥,可获得分布比较均匀,直径大多为500～900nm的聚乳酸纤维。体外细胞形貌观察表明聚(D,L-乳酸)纤维支架具有良好的细胞相容性。     

Preparation and characterization of bioactive and biodegradable Wollastonite/poly(D,L-lactic acid) composite scaffolds

Composite scaffolds of poly(D,L-lactic acid) (PDLLA) with bioactive wollastonite were fabricated by the conventional solvent casting-particulate leaching method. The pore structures and morphology of the scaffolds were determined by scanning electron microscopy (SEM). The bioactivity of the composites was evaluated by soaking in a simulated body fluid (SBF), and the formation of the hydroxyapatite (HAp) layer was determined by SEM and energy-dispersive spectrometer. The results showed that the wollastonite/PDLLA composites were bioactive as it induced the formation of HAp on the surface of the composite scaffolds after soaking in SBF for seven days. In addition, pH and ion concentration changes of SBF solutions with composite scaffolds were examined. The results showed that the composites could release Ca and Si ions, which could neutralize the acidic degradation by-products of the PDLLA, and stabilize the pH of the SBF solutions between 6.7 and 7.2 within a three-week soaking period. Furthermore, the measurements of the water contact angles suggested that incorporation of wollastonite into PDLLA could improve the hydrophilicity of the composites and the enhancement was dependent on the wollastonite content. All these results suggest that incorporation of wollastonite into PDLLA might be a useful approach for the preparation of composite scaffolds for tissue repair and tissue-engineering applications.     

聚乳酸涂层改善网状羟基磷灰石陶瓷支架力学性能的研究

采用有机泡沫浸渍法制备出高孔隙率的三维网状羟基磷灰石(HA)生物陶瓷支架,并采用不同浓度的聚乳酸(PDLLA)溶液涂覆网状HA支架,以达到增强补韧网化陶瓷支架的目的.通过扫描电镜观察PDLLA涂覆网状HA陶瓷支架的显微结构和测试力学强度,研究了不同浓度PDLLA溶液涂覆对网状HA支架的涂层厚度、显微结构和力学性能的影响.实验结果表明:涂覆一定量的PDLLA涂层可以显著提高网状HA生物陶瓷支架的抗压强度,同时保持网状陶瓷支架的贯通性和孔隙率.     

Electrospinning of PCL/PVP blends for tissue engineering scaffolds

Currently, one of the main drawbacks of using poly(ε-caprolactone) in the biomedical and pharmaceutical fields is represented by its low biodegradation rate. To overcome this limitation, electrospinning of PCL blended with a water-soluble poly(N-vinyl-2-pyrrolidone) was used to fabricate scaffolds with tunable fiber surface morphology and controllable degradation rates. Electrospun scaffolds revealed a highly immiscible blend state. The incorporated PVP phase was dispersed as inclusions within the electrospun fibers, and then easily extracted by immersing them in cell culture medium, exhibiting nanoporosity on the fiber surface. As a striking result, nanoporosity facilitated not only fiber biodegradation rates, but also improved cell attachment and spreading on the blend electrospun scaffolds. The present findings demonstrate that simultaneous electrospinning technique for PCL with water-soluble PVP provides important insights for successful tuning biodegradation rate for the PCL electrospun scaffolds but not limited to expand other high valuable biocompatible polymers for the future biomedical applications, ranging from tissue regeneration to controlled drug delivery.     

Neuroactive conducting scaffolds: nerve growth factor conjugation on active ester-functionalized polypyrrole

Electrically conductive and biologically active scaffolds are desirable for enhancing adhesion, proliferation and differentiation of a number of cell types such as neurons. Hence, the incorporation of neuroactive molecules into electroconductive polymers via a specific and stable method is essential for neuronal tissue engineering applications. Traditional conjugation approaches dramatically impair conductivities and/or stabilities of the scaffolds and ligands. In this study, we developed copolymers (PPy-NSE) of N-hydroxyl succinimidyl ester pyrrole and regular pyrrole, which can be immobilized with nerve growth factor (NGF) without significantly hindering electroconductivity. The presence of active ester groups was confirmed using reflectance infrared spectroscopy and X-ray photoelectron spectroscopy (XPS) from the copolymers prepared from different monomer compositions. We selected PPy-NSE₅₀ (polymerized from a 50 : 50 monomer ratio of pyrrole : pyrrole-NSE) for further modification with NGF because this copolymer retains good conductivity (approx. 8 S cm⁻¹) and presents active ester groups for NGF immobilization. We tethered NGF on the PPy-NSE₅₀ surface, and found that PC12 cells extended neurites similarly to cells cultured in NGF-containing medium. XPS and enzyme-linked immunosorbent assay confirmed that NGF immobilized via the active ester on the PPy-NSE₅₀ film was stable for up to 5 days in phosphate-buffered saline solution. Also, application of an external electrical potential to NGF-immobilized PPy films did not cause a significant release of NGF nor reduce their neurotrophic activity. This novel scaffold, providing electroconductive and neurotrophic activities, has potential for neural applications, such as tissue engineering scaffolds and biosensors.     

Controlled dual release of hydrophobic and hydrophilic drugs from electrospun poly(l-lactic acid) fiber mats loaded with chitosan microspheres

This work is to develop novel electrospun poly(l-lactic acid) (PLLA) fiber mats for controllable delivery of hydrophobic and hydrophilic drugs. For this aim, bovine serum albumin (BSA, used as a hydrophilic model drug) was firstly enveloped into chitosan microspheres by spray drying. Benzoin (used as a hydrophobic model drug) was directly dissolved in PLLA solution and then the chitosan microspheres were suspended into the PLLA solution, which was used to prepare PLLA fiber mats by electrospinning. Polyvinylpyrrolidone (PVP) was added into the PLLA solution to tune the drug release behaviors. The results showed that the chitosan microspheres were uniformly distributed in the fibers. BSA had a short-term release while benzoin had a long-term and sustained release in all the dual drug delivery systems, and the release of both hydrophobic and hydrophilic drugs could be adjusted by changing the ratio of PVP/PLLA.     

Poly(l-lactide-co-glycolide) biodegradable microfibers and electrospun nanofibers for nerve tissue engineering: an in vitro study

For tissue engineering applications, the distribution and growth of cells on a scaffold are key requirements. The potential of biodegradable poly(l-lactide-co-glycolide) (PLGA) polymer with different microstructures, as scaffolds for nerve tissue engineering was investigated. In this study, an attempt was made to develop porous nanofibrous scaffolds by the electrospinning method. In this process, polymer fibers with diameters in the nanometer range are formed by subjecting a polymer fluid jet to a high electric field. Attempt was also made to develop microbraided and aligned microfiber scaffolds. A polymer film scaffold was made by solvent casting method. C17.2 nerve stem cells were seeded and cultured on all the four different types of scaffolds under static conditions for 3 days. Scanning electron micrographs showed that the nerve stem cells adhered and differentiated on all the scaffolds and supported neurite outgrowth. Interesting observation was seen in the aligned microfiber scaffolds, where the C17.2 nerve stem cells attached and differentiated along the direction of the fibers. The size and shape of the cell-polymer constructs remained intact. The present study suggests that PLGA is a potential scaffold for nerve tissue engineering and predicts the orientation and growth of nerve stem cells on the scaffold.     

Electrospun nanofibrous materials for tissue engineering and drug delivery

Abstract

The electrospinning technique, which was invented about 100 years ago, has attracted more attention in recent years due to its possible biomedical applications. Electrospun fibers with high surface area to volume ratio and structures mimicking extracellular matrix (ECM) have shown great potential in tissue engineering and drug delivery. In order to develop electrospun fibers for these applications, different biocompatible materials have been used to fabricate fibers with different structures and morphologies, such as single fibers with different composition and structures (blending and core-shell composite fibers) and fiber assemblies (fiber bundles, membranes and scaffolds). This review summarizes the electrospinning techniques which control the composition and structures of the nanofibrous materials. It also outlines possible applications of these fibrous materials in skin, blood vessels, nervous system and bone tissue engineering, as well as in drug delivery.     

Fiber scaffolds of polysialic acid via electrospinning for peripheral nerve regeneration

Fiber scaffolds of bioactive polysialic acid have been prepared via electrospinning for peripheral nerve regeneration. The diameter, morphology and alignment of fibers in scaffolds were adjusted by variation of electrospinning parameters, which are decisive for the cell-scaffold interaction. Due to the high water solubility of polysialic acid (poly-α-2,8-N-acetylneuraminic acid) a photoactive derivative (poly-α-2,8-N-pentenoylneuraminic acid) was used to obtain stable fiber scaffolds in water by photochemical crosslinking. At the optimized fiber scaffolds good cell viability and directed cell proliferation along the fibers was achieved by cell tests with immortalized Schwann cells.     

The comparison of the Wnt signaling pathway inhibitor delivered electrospun nanoyarn fabricated with two methods for the application of urethroplasty

Urethral strictures were common disease caused by over-expression of extracellular matrix from fibroblast. In this study, we compare two nanoyarn scaffolds for improving fibroblasts infiltration without inhibition the over-expression of extracellular matrix. Collagen/poly(L-lactide-co-caprolactone) (Col/P(LLA-CL)) nanoyarn scaffolds were prepared by conjugated electrospinning and dynamic liquid electrospinning, respectively. In addition, co-axial electrospinning technique was combined with the nanoyarn fabrication process to produce nanoyarn scaffolds loading Wnt signaling pathway inhibitor. The mechanical properties of the scaffolds were examined and morphology was observed by SEM. Cell morphology, proliferation and infiltration on the scaffolds were investigated by SEM, MTT assay and H&E staining, respectively. The release profiles of different scaffolds were determined using HPLC. The results indicated that cells showed an organized morphology along the nanoyarns and considerable infiltration into the nanoyarn scaffolds prepared by dynamic liquid electrospinning (DLY). It was also observed that the DLY significantly facilitate cell proliferation. The D-DLY could facilitate the infiltration of the fibroblasts and could be a promising scaffold for the treatment of urethra stricture while it may inhibit the collagen production.     

Histological study of surface modified three dimensional poly (<Emphasis Type="SmallCaps">d</Emphasis>,<Emphasis Type="SmallCaps"> l</Emphasis>-lactic acid) scaffolds with chitosan in vivo

Biocompatibility and tissue regenerating capacity are essential for biomaterials that used in tissue engineering. The aim of this study was to histologically assess the tissue reactions and bone conductivities of surface modified three dimensional (3-D) poly (d, l-lactic acid) (PDLLA) scaffolds, which were coated with chitosan via a physical entrapment method. The native PDLLA scaffold was prepared via thermally induced phrase separation technique and was characterized by scanning electron microscopy (SEM) and differential scanning calorimetry (DSC). Osteocalcin assay, a method to evaluate the bone formation potential, has shown that the osteocalcin production in chitosan-modified 3-D PDLLA scaffold group was significantly higher (p < 0.05) than that of in control. The tissue reactions and bone conductivities between surface modified PDLLA and native PDLLA scaffolds were evaluated using a rabbit radialis defect model in vivo and compared at different implantation intervals (2, 4, 8 and 12 weeks). The histological results have shown a higher bone formation potential and better biocompatibility of chitosan-modified 3-D PDLLA scaffolds as compared with the control group scaffolds.     

Antimicrobial effects of nanofiber poly(caprolactone) tissue scaffolds releasing rifampicin

This study quantified the antibiotic release kinetics and subsequent bactericidal efficacy of rifampicin (RIF) against Gram-positive and Gram-negative bacteria under in vitro static conditions. Antibiotic-loaded scaffolds were fabricated by electrospinning poly(caprolactone) (PCL) with 10% or 20% (w/w) RIF. Scaffold fiber diameter and RIF loading were characterized, and RIF release kinetics were measured. RIF-releasing and RIF-free scaffolds were inoculated with Pseudomonas aeruginosa and Staphylococcus epidermidis, and the suspended concentration live and dead bacteria were determined by fluorescent microscopy. Adherent bacteria and biofilm formation were examined using scanning electron microscopy. Mean fiber diameters were 557 ± 399 nm for RIF-free, 402 ± 225 nm for 10% RIF, and 665 ± 402 nm for 20% RIF scaffolds. RIF release kinetics exhibited a short-burst release during the first hour, followed by a 7 h, zero-order release during which both RIF scaffolds released ~50% of their initial RIF mass loading. P. aeruginosa and S. epidermidis suspended cell populations proliferated in accordance with logarithmic growth models when exposed to control scaffolds; however both RIF-containing scaffolds completely inhibited bacterial growth in suspension and, subsequently, prevented biofilm formation within the scaffolds through the first 6 h.     

A new nanofiber fabrication technique based on coaxial electrospinning

In tissue engineering, nanofibrous scaffolds can achieve better biological responses than microfibrous scaffolds and electrospinning is a common method for producing fibrous scaffolds. However, not all biopolymers can be made into nanofibers through conventional electrospinning. The current investigation developed an innovative nanofiber fabrication technique based on coaxial electrospinning and used poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) as an example for achieving nanofibers. For obtaining PHBV nanofibers, core-shell structured fibers were fabricated first via coaxial electrospinning, with PHBV being the core and chitosan being the shell. The chitosan shell was then removed by washing electrospun scaffolds with water, leading to the formation of nanofibrous PHBV scaffolds. The PHBV nanofiber diameter was affected by the inner polymer (i.e., PHBV) solution concentration during coaxial electrospinning, which can be explained in terms of the coaxial electrospinning process and polymer solution viscosity. Compared to the approach of using a conductivity-enhancing salt in polymer solution to produce polymer nanofibers, the new technique not only eliminates the biocompatibility concerns but also provides a more effective way of reducing fiber diameters to the nano-size range.     

Emulsion electrospun nanofibers as substrates for cardiomyogenic differentiation of mesenchymal stem cells

The potential of cardiomyogenic differentiation of human mesenchymal stem cells (hMSCs) on emulsion electrospun scaffold containing poly(l-lactic acid)-co-poly-(ε-caprolactone), gelatin and vascular endothelial growth factor (PLCL/GV) was investigated in this study. The characterizations of the scaffold were carried out using scanning electron microscope (SEM), transmission electron microscope, water contact angle and porometer. The proliferation of hMSCs showed that 73.4 % higher cell proliferation on PLCL/GV scaffolds than that on PLCL scaffold after 20 days of cell culture. Results of 5-chloromethylfluorescein diacetate staining and SEM morphology analysis indicated that hMSCs differentiated on PLCL/GV scaffolds showed irregular morphology of cardiomyocyte phenotype compared to the typical long and thin hMSC phenotype. Immunostaining results showed the expression of alpha actinin and myosin heavy chain. Our studies identified emulsion electrospinning as a method for fabrication of core–shell fibers suitable for the differentiation of stem cells to cardiac cells, with potential application in cardiac regeneration.     

Biodegradable nanomats produced by electrospinning: expanding multifunctionality and potential for tissue engineering

With increasing interest in nanotechnology, development of nanofibers (n-fibers) by using the technique of electrospinning is gaining new momentum. Among important potential applications of n-fiber-based structures, scaffolds for tissue-engineering represent an advancing front. Nanoscaffolds (n-scaffolds) are closer to natural extracellular matrix (ECM) and its nanoscale fibrous structure. Although the technique of electrospinning is relatively old, various improvements have been made in the last decades to explore the spinning of submicron fibers from biodegradable polymers and to develop also multifunctional drug-releasing and bioactive scaffolds. Various factors can affect the properties of resulting nanostructures that can be classified into three main categories, namely: (1) Substrate related, (2) Apparatus related, and (3) Environment related factors. Developed n-scaffolds were tested for their cytocompatibility using different cell models and were seeded with cells for to develop tissue engineering constructs. Most importantly, studies have looked at the potential of using n-scaffolds for the development of blood vessels. There is a large area ahead for further applications and development of the field. For instance, multifunctional scaffolds that can be used as controlled delivery system do have a potential and have yet to be investigated for engineering of various tissues. So far, in vivo data on n-scaffolds are scarce, but in future reports are expected to emerge. With the convergence of the fields of nanotechnology, drug release and tissue engineering, new solutions could be found for the current limitations of tissue engineering scaffolds, which may enhance their functionality upon in vivo implantation. In this paper electrospinning process, factors affecting it, used polymers, developed n-scaffolds and their characterization are reviewed with focus on application in tissue engineering.     

Tissue engineering scaffolds for nerve regeneration manufactured by ink-jet technology

Nerve tissue engineering seeks to develop viable nerve substitutes by combining transplanted cells, bio-absorbable polymer scaffolds and growth factors. Ink-jet can be an attractive technology for manufacturing these scaffolds due to incorporation of data-driven, non-contact approaches that enable precise volumes of material to be deposited with high speed and accuracy at target sites. Cylindrical nerve conduits made of 80/20 poly (d,l-lactide-co-ε-caprolactone) copolymer were fabricated using an ink-jet system that digitally controlled various manufacturing parameters that ultimately dictate the physical and chemical properties of the scaffold. Selection of the scaffold materials was done by screening various biodegradable polymers based on their ability to be jetted and to promote human embryonic kidney cells attachment, survival and nerve growth factor (NGF) production. These cells were genetically modified to secrete NGF thus acting as a growth factor reservoir once seeded onto the polymeric conduit.