Elastin-PLGA hybrid electrospun nanofiber scaffolds for salivary epithelial cell self-organization and polarization.
ABSTRACT: Development of electrospun nanofibers that mimic the structural, mechanical and biochemical properties of natural extracellular matrices (ECMs) is a promising approach for tissue regeneration. Electrospun fibers of synthetic polymers partially mimic the topography of the ECM, however, their high stiffness, poor hydrophilicity and lack of in vivo-like biochemical cues is not optimal for epithelial cell self-organization and function. In search of a biomimetic scaffold for salivary gland tissue regeneration, we investigated the potential of elastin, an ECM protein, to generate elastin hybrid nanofibers that have favorable physical and biochemical properties for regeneration of the salivary glands. Elastin was introduced to our previously developed poly-lactic-co-glycolic acid (PLGA) nanofiber scaffolds by two methods, blend electrospinning (EP-blend) and covalent conjugation (EP-covalent). Both methods for elastin incorporation into the nanofibers improved the wettability of the scaffolds while only blend electrospinning of elastin-PLGA nanofibers and not surface conjugation of elastin to PLGA fibers, conferred increased elasticity to the nanofibers measured by Young's modulus. After two days, only the blend electrospun nanofiber scaffolds facilitated epithelial cell self-organization into cell clusters, assessed with nuclear area and nearest neighbor distance measurements, leading to the apicobasal polarization of salivary gland epithelial cells after six days, which is vital for cell function. This study suggests that elastin electrospun nanofiber scaffolds have potential application in regenerative therapies for salivary glands and other epithelial organs. STATEMENT OF SIGNIFICANCE:Regenerating the salivary glands by mimicking the extracellular matrix (ECM) is a promising approach for long term treatment of salivary gland damage. Despite their topographic similarity to the ECM, electrospun fibers of synthetic polymers lack the biochemical complexity, elasticity and hydrophilicity of the ECM. Elastin is an ECM protein abundant in the salivary glands and responsible for tissue elasticity. Although it's widely used for tissue regeneration of other organs, little is known about its utility in regenerating the salivary tissue. This study describes the use of elastin to improve the elasticity, hydrophilicity and biochemical complexity of synthetic nanofibers and its potential in directing in vivo-like organization of epithelial salivary cells which helps the design of efficient salivary gland regeneration scaffolds.
Project description:Tissue engineering techniques using a combination of polymeric scaffolds and cells represent a promising approach for nerve regeneration. We fabricated electrospun scaffolds by blending of Poly (3-hydroxybutyrate) (PHB) and Poly (3-hydroxy butyrate-co-3- hydroxyvalerate) (PHBV) in different compositions in order to investigate their potential for the regeneration of the myelinic membrane. The thermal properties of the nanofibrous blends was analyzed by differential scanning calorimetry (DSC), which indicated that the melting and glass temperatures, and crystallization degree of the blends decreased as the PHBV weight ratio increased. Raman spectroscopy also revealed that the full width at half height of the band centered at 1725 cm(-1) can be used to estimate the crystalline degree of the electrospun meshes. Random and aligned nanofibrous scaffolds were also fabricated by electrospinning of PHB and PHBV with or without type I collagen. The influence of blend composition, fiber alignment and collagen incorporation on Schwann cell (SCs) organization and function was investigated. SCs attached and proliferated over all scaffolds formulations up to 14 days. SCs grown on aligned PHB/PHBV/collagen fibers exhibited a bipolar morphology that oriented along the fiber direction, while SCs grown on the randomly oriented fibers had a multipolar morphology. Incorporation of collagen within nanofibers increased SCs proliferation on day 14, GDNF gene expression on day 7 and NGF secretion on day 6. The results of this study demonstrate that aligned PHB/PHBV electrospun nanofibers could find potential use as scaffolds for nerve tissue engineering applications and that the presence of type I collagen in the nanofibers improves cell differentiation.
Project description:Nanofibrous scaffolds that are morphologically/structurally similar to natural ECM are highly interested for tissue engineering; however, the electrospinning technique has the difficulty in directly producing clinically relevant 3D nanofibrous scaffolds with desired structural properties. To address this challenge, we have developed an innovative technique of thermally induced nanofiber self-agglomeration (TISA) recently. The aim of this work was to prepare (via the TISA technique) and evaluate 3D electrospun PCL/PLA blend (mass ratio: 4/1) nanofibrous scaffolds having high porosity of ?95.8% as well as interconnected and hierarchically structured pores with sizes from sub-micrometers to ?300 ?m for bone tissue engineering. The hypothesis was that the incorporation of PLA (with higher mechanical stiffness/modulus and bioactivity) into PCL nanofibers would significantly improve human mesenchymal stem cells (hMSCs) osteogenic differentiation in vitro and bone formation in vivo. Compared to neat PCL-3D scaffolds, PCL/PLA-3D blend scaffolds had higher mechanical properties and in vitro bioactivity; as a result, they not only enhanced the cell viability of hMSCs but also promoted the osteogenic differentiation. Furthermore, our in vivo studies revealed that PCL/PLA-3D scaffolds considerably facilitated new bone formation in a critical-sized cranial bone defect mouse model. In summary, both in vitro and in vivo results indicated that novel 3D electrospun PCL/PLA blend nanofibrous scaffolds would be strongly favorable/desired for hMSCs osteogenic differentiation and cranial bone formation.
Project description:Smart biomaterials with an inherent capacity to elicit specific behaviors in lieu of biological prompts would be advantageous for regenerative medicine applications. In this work, we employ an electrospinning technique to model the in vivo nanofibrous extracellular matrix (ECM) of cartilage using a chondroinductive cellulose and silk polymer blend (75:25 ratio). This natural polymer composite is directly electrospun for the first time, into nanofibers without post-spun treatment, using a trifluoroacetic acid and acetic acid cosolvent system. Biocompatibility of the composite nanofibres with human mesenchymal stem cells (hMSCs) is demonstrated and its inherent capacity to direct chondrogenic stem cell differentiation, in the absence of stimulating growth factors, is confirmed. This chondrogenic stimulation could be countered biochemically using fibroblast growth factor-2, a growth factor used to enhance the proliferation of hMSCs. Furthermore, the potential mechanisms driving this chondroinduction at the cell-biomaterial interface is investigated. Composite substrates are fabricated as two-dimensional film surfaces and cultured with hMSCs in the presence of chemicals that interfere with their biochemical and mechanical signaling pathways. Preventing substrate surface elasticity transmission resulted in a significant downregulation of chondrogenic gene expression. Interference with the classical chondrogenic Smad2/3 phosphorylation pathway did not impact chondrogenesis. The results highlight the importance of substrate mechanical elasticity on hMSCs chondroinduction and its independence to known chondrogenic biochemical pathways. The newly fabricated scaffolds provide the foundation for designing a robust, self-inductive, and cost-effective biomimetic biomaterial for cartilage tissue engineering.
Project description:Electrospinning is a versatile technique that has gained popularity for various biomedical applications in recent years. Electrospinning is being used for fabricating nanofibers for various biomedical and dental applications such as tooth regeneration, wound healing and prevention of dental caries. Electrospun materials have the benefits of unique properties for instance, high surface area to volume ratio, enhanced cellular interactions, protein absorption to facilitate binding sites for cell receptors. Extensive research has been conducted to explore the potential of electrospun nanofibers for repair and regeneration of various dental and oral tissues including dental pulp, dentin, periodontal tissues, oral mucosa and skeletal tissues. However, there are a few limitations of electrospinning hindering the progress of these materials to practical or clinical applications. In terms of biomaterials aspects, the better understanding of controlled fabrication, properties and functioning of electrospun materials is required to overcome the limitations. More in vivo studies are definitely required to evaluate the biocompatibility of electrospun scaffolds. Furthermore, mechanical properties of such scaffolds should be enhanced so that they resist mechanical stresses during tissue regeneration applications. The objective of this article is to review the current progress of electrospun nanofibers for biomedical and dental applications. In addition, various aspects of electrospun materials in relation to potential dental applications have been discussed.
Project description:Optimization of nanofiber surface properties can lead to enhanced tissue regeneration outcomes in the context of bone tissue engineering. Herein, we developed a facile strategy to decorate elctrospun nanofibers using extracellular matrix (ECM) in order to improve their performance for bone tissue engineering. Electrospun PLLA nanofibers (PLLA NF) were seeded with MC3T3-E1 cells and allowed to grow for two weeks in order to harvest a layer of ECM on nanofiber surface. After decellularization, we found that ECM was successfully preserved on nanofiber surface while maintaining the nanostructure of electrospun fibers. ECM decorated on PLLA NF is biologically active, as evidenced by its ability to enhance mouse bone marrow stromal cells (mBMSCs) adhesion, support cell proliferation and promote early stage osteogenic differentiation of mBMSCs. Compared to PLLA NF without ECM, mBMSCs grown on ECM/PLLA NF exhibited a healthier morphology, faster proliferation profile, and more robust osteogenic differentiation. Therefore, our study suggests that ECM decoration on electrospun nanofibers could serve as an efficient approach to improving their performance for bone tissue engineering.
Project description:Nerve regeneration is a serious clinical challenge following peripheral nerve injury. Lycium barbarum polysaccharide (LBP) is the major component of wolfberry extract, which has been shown to be neuroprotective and promising in nerve recovery in many studies. Electrospun nanofibers, especially core-shell structured nanofibers being capable of serving as both drug delivery system and tissue engineering scaffolds, are well known to be suitable scaffolds for regeneration of peripheral nerve applications. In this study, LBP was incorporated into core-shell structured nanofibrous scaffolds via coaxial electrospinning. Alamar blue assays were performed to investigate the proliferation of both PC12 and Schwann cells cultured on the scaffolds. The neuronal differentiation of PC12 cells was evaluated by NF200 expression with immunostaining and morphology changes observed by SEM. The results indicated that the released LBP dramatically enhanced both proliferation and neuronal differentiation of PC12 cells induced by NGF. Additionally, the promotion of Schwann cells myelination and neurite outgrowth of DRG neurons were also observed on LBP loaded scaffolds by LSCM with immunostaining. In summary, LBP, as a drug with neuroprotection, encapsulated into electrospun nanofibers could be a potential candidate as tissue engineered scaffold for peripheral nerve regeneration.
Project description:Engineering salivary glands is of interest due to the damaging effects of radiation therapy and the autoimmune disease Sjögren's syndrome on salivary gland function. One of the current problems in tissue engineering is that in vitro studies often fail to predict in vivo regeneration due to failure of cells to interact with scaffolds and of the single cell types that are typically used for these studies. Although poly (lactic co glycolic acid) (PLGA) nanofiber scaffolds have been used for in vitro growth of epithelial cells, PLGA has low compliance and cells do not penetrate the scaffolds. Using a core-shell electrospinning technique, we incorporated poly (glycerol sebacate) (PGS) into PLGA scaffolds to increase the compliance and decrease hydrophobicity. PGS/PLGA scaffolds promoted epithelial cell penetration into the scaffold and apical localization of tight junction proteins, which is necessary for epithelial cell function. Additionally, co-culture of the salivary epithelial cells with NIH3T3 mesenchymal cells on PGS/PLGA scaffolds facilitated epithelial tissue reorganization and apical localization of tight junction proteins significantly more than in the absence of the mesenchyme. These data demonstrate the applicability of PGS/PLGA nanofibers for epithelial cell self-organization and facilitation of co-culture cell interactions that promote tissue self-organization in vitro.
Project description:Electrospun nanofibers can be readily assembled into various types of scaffolds for applications in neural tissue engineering. The objective of this study is to examine and understand the unique patterns of neurite outgrowth from primary dorsal root ganglia (DRG) cultured on scaffolds of electrospun nanofibers having different orders, structures, and surface properties. We found that the neurites extended radially outward from the DRG main body without specific directionality when cultured on a nonwoven mat of randomly oriented nanofibers. In contrast, the neurites preferentially extended along the long axis of fiber when cultured on a parallel array of aligned nanofibers. When seeded at the border between regions of aligned and random nanofibers, the same DRG simultaneously expressed aligned and random neurite fields in response to the underlying nanofibers. When cultured on a double-layered scaffold where the nanofibers in each layer were aligned along a different direction, the neurites were found to be dependent on the fiber density in both layers. This biaxial pattern clearly demonstrates that neurite outgrowth can be influenced by nanofibers in different layers of a scaffold, rather than the topmost layer only. Taken together, these results will provide valuable information pertaining to the design of nanofiber scaffolds for neuroregenerative applications, as well as the effects of topology on neurite outgrowth, growth cone guidance, and axonal regeneration.
Project description:Here, we report the development of a new polyacrylamide (PAAm)/starch nanofibers' blend system and highlight its potential as substrate for efficient enzyme immobilization. PAAm was synthesized and blended with starch. The final blend was then electrospun into nanofibers. The response surface methodology was used to analyze the parameters that control nanofiber's diameter. Electrospun mat was then modified either by cross-linking or phytase immobilization using silane coupling agent and glutaraldehyde chemistry. Physico-chemical properties of blends were investigated using spectroscopic and thermal studies. The evaluation of immobilized enzyme kinetics on both pure and the starch blended PAAm nanofibers was performed using Michaelis-Menten kinetic curves. Fourier transform infrared spectroscopy results along with differential scanning and X-ray diffraction confirmed that blending was successfully accomplished. TGA analysis also demonstrated that the presence of starch enhances the thermal degradability of PAAm nanofibers. Finally, it was shown that addition of starch to PAAm increases the efficacies of enzyme loading and, therefore, significantly enhances the activity as well as kinetics of the immobilized enzyme on electrospun blend mats.
Project description:Biodegradable nanofibers simulate the fibril structure of natural extracellular matrix, and provide a cell-friendly microenvironment for tissue regeneration. However, the effects of nanofiber organization and immobilized biochemical factors on cell infiltration into three-dimensional scaffolds are not well understood. For example, cell infiltration into an electrospun nanofibrous matrix is often limited due to relatively small pore size between the fibers. Here we showed that biophysical and biochemical modification of nanofibrous scaffolds facilitated endothelial cell infiltration in three-dimensional scaffolds in vitro and in vivo. Aligned nanofibers significantly enhanced cell infiltration into the nanofibrous matrices in vitro. In a full-thickness dermal wound model, the nanofiber scaffolds enhanced epidermal skin cell migration across the wound when compared to a control group without scaffold. Aligned nanofibers promoted the infiltration of endothelial cells into the scaffolds. Furthermore, heparin-coated nanofibers also increased cell infiltration significantly. These results shed light on the importance of biophysical and biochemical properties of nanofibers in the regulation of cell infiltration into three-dimensional scaffolds and tissue remodeling.