Electrospun sodium alginate/poly(ethylene oxide) core-shell nanofibers scaffolds potential for tissue engineering applications

Core-shell structure nanofibers of sodium alginate/poly(ethylene oxide) were prepared via electrospinning their dispersions in water solution. The core-shell structure morphology of the obtained nanofibers was viewed under scanning electron microscope (SEM) and transmission electron microscope (TEM), and X-ray photoelectron spectroscopy (XPS) analysis was used to further quantify the chemical composition of the core-shell composite SA/PEO nanofibers surface in detail. Furthermore, one-step cross-linking method through being immersed in CaCl₂ solution was investigated to improve the anti-water property of the electrospun nanofibers mats in order to facilitate their practical applications as tissue engineering scaffolds, and the changes of the structural of nanofibers before and after cross-linking was characterized by Fourier transform infrared (FT-IR). Indirect cytotoxicity assessment indicated that SA/PEO nanofibers membrane was nontoxic to the fibroblasts cells, and cell culture suggested that SA/PEO nanofibers tended to promote fibroblasts cells attachment and proliferation. It was assumed that the nanofibers membrane of electrospun SA/PEO could be used for tissue engineering scaffolds.     

High yield of cells committed to the photoreceptor-like cells from conjunctiva mesenchymal stem cells on nanofibrous scaffolds

Transplantation of stem cells using biodegradable and biocompatible nanofibrous scaffolds is a promising therapeutic approach for treating inherited retinal degenerative diseases such as retinitis pigmentosa and age-related macular degeneration. In this study, conjunctiva mesenchymal stem cells (CJMSCs) were seeded onto poly-l-lactic acid (PLLA) nanofibrous scaffolds and were induced to differentiate toward photoreceptor cell lineages. Furthermore, the effects of orientation of scaffold on photoreceptor differentiation were examined. Scanning electron microscopy (SEM) imaging, quantitative real time RT-PCR (qPCR) and immunocytochemistry were used to analyze differentiated cells and their expression of photoreceptor-specific genes. Our observations demonstrated the differentiation of CJMSCs to photoreceptor cells on nanofibrous scaffolds and suggested their potential application in retinal regeneration. SEM imaging showed that CJMSCs were spindle shaped and well oriented on the aligned nanofiber scaffolds. The expression of rod photoreceptor-specific genes was significantly higher in CJMSCs differentiated on randomly-oriented nanofibers compared to those on aligned nanofibers. According to our results we may conclude that the nanofibrous PLLA scaffold reported herein could be used as a potential cell carrier for retinal tissue engineering and a combination of electrospun nanofiber scaffolds and MSC-derived conjunctiva stromal cells may have potential application in retinal regenerative therapy.     

Incorporation of SPION-casein core-shells into silk-fibroin nanofibers for cardiac tissue engineering

Mimicking the structure of extracellular matrix (ECM) of myocardium is necessary for fabrication of functional cardiac tissue. The superparamagnetic iron oxide nanoparticles (SPIONs, Fe₃O₄), as new generation of magnetic nanoparticles (NPs), are highly intended in biomedical studies. Here, SPION NPs (1 wt%) were synthesized and incorporated into silk-fibroin (SF) electrospun nanofibers to enhance mechanical properties and topography of the scaffolds. Then, the mouse embryonic cardiac cells (ECCs) were seeded on the scaffolds for in vitro studies. The SPION NPs were studied by scanning electron microscope (SEM), X-ray diffraction (XRD), and transmission electron microscope (TEM). SF nanofibers were characterized after incorporation of SPIONs by SEM, TEM, water contact angle measurement, and tensile test. Furthermore, cytocompatibility of scaffolds was confirmed by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. SEM images showed that ECCs attached to the scaffolds with elongated morphologies. Also, the real-time PCR and immunostaining studies approved upregulation of cardiac functional genes in ECCs seeded on the SF/SPION-casein scaffolds including GATA-4, cardiac troponin T, Nkx 2.5, and alpha-myosin heavy chain, compared with the ones in SF. In conclusion, incorporation of core-shells in SF supports cardiac differentiation, while has no negative impact on ECCs' proliferation and self-renewal capacity.     

A Comparative Study on In Vitro Osteogenic Priming Potential of Electron Spun Scaffold PLLA/HA/Col,PLLA/HA,and PLLA/Col for Tissue Engineering Application

A comparative study on the in vitro osteogenic potential of electrospun poly-L-lactide/hydroxyapatite/collagen (PLLA/HA/Col, PLLA/HA, and PLLA/Col) scaffolds was conducted. The morphology, chemical composition, and surface roughness of the fibrous scaffolds were examined. Furthermore, cell attachment, distribution, morphology, mineralization, extracellular matrix protein localization, and gene expression of human mesenchymal stromal cells (hMSCs) differentiated on the fibrous scaffolds PLLA/Col/HA, PLLA/Col, and PLLA/HA were also analyzed. The electrospun scaffolds with a diameter of 200–950 nm demonstrated well-formed interconnected fibrous network structure, which supported the growth of hMSCs. When compared with PLLA/H%A and PLLA/Col scaffolds, PLLA/Col/HA scaffolds presented a higher density of viable cells and significant upregulation of genes associated with osteogenic lineage, which were achieved without the use of specific medium or growth factors. These results were supported by the elevated levels of calcium, osteocalcin, and mineralization (P<0.05) observed at different time points (0, 7, 14, and 21 days). Furthermore, electron microscopic observations and fibronectin localization revealed that PLLA/Col/HA scaffolds exhibited superior osteoinductivity, when compared with PLLA/Col or PLLA/HA scaffolds. These findings indicated that the fibrous structure and synergistic action of Col and nano-HA with high-molecular-weight PLLA played a vital role in inducing osteogenic differentiation of hMSCs. The data obtained in this study demonstrated that the developed fibrous PLLA/Col/HA biocomposite scaffold may be supportive for stem cell based therapies for bone repair, when compared with the other two scaffolds.     

Three-dimensional hierarchical composite scaffolds consisting of polycaprolactone, β-tricalcium phosphate, and collagen nanofibers: fabrication, physical properties, and in vitro cell activity for bone tissue regeneration

β-Tricalcium phosphate (β-TCP) and collagen have been widely used to regenerate various hard tissues, but although Bioceramics and collagen have various biological advantages with respect to cellular activity, their usage has been limited due to β-TCP's inherent brittleness and low mechanical properties, along with the low shape-ability of the three-dimensional collagen. To overcome these material deficiencies, we fabricated a new hierarchical scaffold that consisted of a melt-plotted polycaprolactone (PCL)/β-TCP composite and embedded collagen nanofibers. The fabrication process was combined with general melt-plotting methods and electrospinning. To evaluate the capability of this hierarchical scaffold to act as a biomaterial for bone tissue regeneration, physical and biological assessments were performed. Scanning electron microscope (SEM) micrographs of the fabricated scaffolds indicated that the β-TCP particles were uniformly embedded in PCL struts and that electrospun collagen nanofibers (diameter = 160 nm) were well layered between the composite struts. By accommodating the β-TCP and collagen nanofibers, the hierarchical composite scaffolds showed dramatic water-absorption ability (100% increase), increased hydrophilic properties (20%), and good mechanical properties similar to PCL/β-TCP composite. MTT assay and SEM images of cell-seeded scaffolds showed that the initial attachment of osteoblast-like cells (MG63) in the hierarchical scaffold was 2.2 times higher than that on the PCL/β-TCP composite scaffold. Additionally, the proliferation rate of the cells was about two times higher than that of the composite scaffold after 7 days of cell culture. Based on these results, we conclude that the collagen nanofibers and β-TCP particles in the scaffold provide good synergistic effects for cell activity.     

Electrospun composite scaffolds containing poly(octanediol-co-citrate) for cardiac tissue engineering

A biocompatible and elastomeric nanofibrous scaffold is electrospun from a blend of poly(1,8-octanediol-co-citrate) [POC] and poly(L-lactic acid) -co-poly-(3-caprolactone) [PLCL] for application as a bioengineered patch for cardiac tissue engineering. The characterization of the scaffolds was carried out by Fourier transform infra red spectroscopy, scanning electron microscopy (SEM), and tensile measurement. The mechanical properties of the scaffolds are studied with regard to the percentage of POC incorporated with PLCL and the results of the study showed that the mechanical property and degradation behavior of the composites can be tuned with respect to the concentration of POC blended with PLCL. The composite scaffolds with POC: PLCL weight ratio of 40:60 [POC/PLCL4060] was found to have a tensile strength of 1.04 ± 0.11 MPa and Young's Modulus of 0.51 ± 0.10 MPa, comparable to the native cardiac tissue. The proliferation of cardiac myoblast cells on the electrospun POC/PLCL scaffolds was found to increase from Days 2 to 8, with the increasing concentration of POC in the composite. The morphology and cytoskeletal observation of the cells also demonstrated the biocompatibility of the POC containing scaffolds. Electrospun POC/PLCL4060 nanofibers are promising elastomeric substrates that might provide the necessary mechanical cues to cardiac muscle cells for regeneration of the heart.     

Electrospun poly(lactic acid)/chitosan core-shell structure nanofibers from homogeneous solution

The core-shell structure nanofibers of poly(lactic acid)/chitosan with different weight ratios were successfully electrospun from homogeneous solution. The preparation process was more simple and effective than double-needle electrospinning. The nanofibers were obtained with chitosan in shell while poly(lactic acid) in core attributing to phase separation, which were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD) and energy dispersive spectrometer (EDS). The electrospun nanofibrous membrane was evaluated in vitro by using mouse fibroblasts (L929) as reference cell lines. Cell culture results indicated that these materials were good in promoting cell growth and attachment, thus they could be used for tissue engineering and wound healing dressing.     

Embryonic stem cells differentiated into neuron-like cells using SB431542 small molecule on nanofibrous PLA/CS/Wax scaffold

Electrospun nanofibrous scaffolds show huge potential to improve the neurological outcome in central nervous system disorders. In this study, we cultured mouse embryonic stem cells (mESCs) on an electrospun nanofibrous polylactic acid/Chitosan/Wax (PLA/CS/Wax) scaffold and surveyed the attachment, behavior, and differentiation of mESCs into neural cells. Differentiation in neural-like cells (NLCs) was investigated with a medium containing SB431542 as a small molecule and conjugated linolenic acid after 20 days. We used Immunocytochemistry and quantitative real-time polymerase chain reaction (RT-PCR) techniques to assess neural marker expression in differentiated cells. SEM imaging demonstrated that mESCs could strongly attach, stretch, and differentiate on PLA/CS/Wax scaffolds. MESCs that were cultured on PLA/CS/Wax scaffolds showed enhanced numbers of neural structures and neural markers including Nestin, NF-H, Tuj-1, and Map2 in neural induction medium compared to the control sample. These results revealed that electrospun PLA/CS/Wax scaffolds associated with the induction medium can assemble proper conditions for stem cell differentiation into NLCs. We hope that the development of new technologies in neural tissue engineering may pave a new avenue for neural tissue regeneration.     

Electrospinning of carboxymethyl chitin/poly(vinyl alcohol) nanofibrous scaffolds for tissue engineering applications

A novel fibrous membrane of carboxymethyl chitin (CMC)/poly(vinyl alcohol) (PVA) blend was successfully prepared by electrospinning technique. The concentration of CMC (7%) with PVA (8%) was optimized, blended in different ratios (0–100%) and electrospun to get nanofibers. Fibers were made water insoluble by chemical followed by thermal cross-linking. In vitro mineralization studies identified the ability of formation of hydroxyapatite deposits on the nanofibrous surfaces. Cytotoxicity of the nanofibrous scaffold was evaluated using human mesenchymal stem cells (hMSCs) by the MTT assays. The cell viability was not altered when these nanofibrous scaffolds were pre-washed with phosphate buffer containing saline (PBS) before seeding the cells. The SEM images also revealed that cells were able to attach and spread in the nanofibrous scaffolds. Thus our results indicate that the nanofibrous CMC/PVA scaffold supports cell adhesion/attachment and proliferation and hence this scaffold will be a promising candidate for tissue engineering applications.     

Electrospun water-soluble carboxyethyl chitosan/poly(vinyl alcohol) nanofibrous membrane as potential wound dressing for skin regeneration

Biocompatible carboxyethyl chitosan/poly(vinyl alcohol) (CECS/PVA) nanofibers were successfully prepared by electrospinning of aqueous CECS/PVA solution. The composite nanofibrous membranes were subjected to detailed analysis by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), and X-ray diffraction (XRD). SEM images showed that the morphology and diameter of the nanofibers were mainly affected by the weight ratio of CECS/PVA. XRD and DSC demonstrated that there was strong intermolecular hydrogen bonding between the molecules of CECS and PVA. The crystalline microstructure of the electrospun fibers was not well developed. The potential use of the CECS/PVA electrospun fiber mats as scaffolding materials for skin regeneration was evaluated in vitro using mouse fibroblasts (L929) as reference cell lines. Indirect cytotoxicity assessment of the fiber mats indicated that the CECS/PVA electrospun mat was nontoxic to the L929 cell. Cell culture results showed that fibrous mats were good in promoting the cell attachment and proliferation. This novel electrospun matrix would be used as potential wound dressing for skin regeneration.     

Fine structure and enzymatic degradation of poly[(R)-3-hydroxybutyrate] and stereocomplexed poly(lactide) nanofibers

Fiber morphology and crystalline structure of poly[(R)-3-hydroxybutyrate] (P(3HB)) and stereocomplexed poly(lactide) (PLA) nanofibers were investigated by using scanning and transmission electron microscopies and X-ray and electron diffractions. In the P(3HB) nanofibers spun from less than 1 wt% 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) solution, planar zigzag conformation (beta-form) as well as 2(1) helix conformation (alpha-form) structure was formed. Based on the electron diffraction measurement of single P(3HB) nanofiber, it was revealed that the molecular chains of P(3HB) align parallel to the fiber direction. From the enzymatic degradation test of P(3HB) nanofiber, it was shown that beta-form molecular chains are degraded more preferentially than alpha-form chains. Stereocomplexed PLA nanofibers were electrospun from 1 wt% poly(l-lactide)/poly(d-lactide) (PLLA/PDLA) solution in HFIP, which contains equal amounts of PLLA and PDLA. While as-spun stereocomplexed PLA nanofiber was amorphous, PLA nanofiber annealed at 100 degrees C contained only racemic crystal. It was supposed that the crystallization behavior of stereocomplexed PLA in the nanofiber is affected by the electrospinning process, which forcibly exerts the strain onto the polymer chains.     

Zinc oxide-doped poly(urethane) spider web nanofibrous scaffold via one-step electrospinning: a novel matrix for tissue engineering

Zinc oxide (ZnO) nanostructures have been commonly studied for electronic purposes due to their unique piezoelectric and catalytic properties; however, recently, they have been also exploited for biomedical applications. The purpose of this study was to fabricate ZnO-doped poly(urethane) (PU) nanocomposite via one-step electrospinning technique. The utilized nanocomposite was prepared by using colloidal gel composed of ZnO and PU, and the obtained mats were vacuum dried at 60 °C overnight. The physicochemical characterization of as-spun composite nanofibers was carried out by X-ray diffraction pattern, field emission scanning electron microscopy, energy-dispersive X-ray spectroscopy, electron probe microanalysis, and transmission electron microscopy, whereas the thermal behavior was analyzed by thermogravimetric analysis. The viability, attachment, and proliferation of NIH 3T3 mouse fibroblast cells on the ZnO/PU composite nanofibers were analyzed by in vitro cell compatibility test. The morphological features of the cells attached on nanofibers were examined by Bio-SEM. We conclude that the electrospun nanofibrous scaffolds with unique spider nets had good biocompatibility. Cytotoxicity experiments indicated that the mouse fibroblasts could attach to the nanocomposite after being cultured. Thus, the current work demonstrates that the as-synthesized ZnO/PU hybrid nanofibers represent a promising biomaterial to be exploited for various tissue engineering applications.     

Preparation, characterization and biocompatibility of electrospinning heparin-modified silk fibroin nanofibers

In this study, the electrospun silk fibroin nanofibrous scaffolds were modified with heparin by grafting after plasma treatment and blending electrospinning. Morphology, microstructure, chemical composition and grafting efficiency of the heparin-modified silk fibroin nanofibrous scaffolds were characterized to evaluate the effect of modification by means of scanning electron microscopy (SEM), Fourier transform infrared spectra (FTIR) and X-ray photoelectron spectrometer (XPS). The results showed that the heparin was successfully introduced to the silk fibroin nanofibrous scaffolds by both the two kinds of modification, and there was a hydrogen bonding between the silk fibroin and heparin. Moreover, the hydrophilicity, O-containing groups and negative charge density of the heparin-modified scaffolds were enhanced. In vitro coagulation time tests showed that the activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) of the heparin-modified scaffolds were much higher than those of the pure silk fibroin scaffolds. L929 fibroblasts and EVCs spread and proliferated better on the heparin-modified scaffolds than on the pure silk fibroin scaffolds. Macrophages, neutrophils and lymphocytes were not observed in the heparin-modified scaffolds, which indicated that the modified scaffolds could induce minor inflammation in vivo. The results indicated that the electrospun heparin-modified silk fibroin nanofibrous scaffolds could be considered as ideal candidates for tissue engineering scaffolds.     

Hepatic differentiation from human mesenchymal stem cells on a novel nanofiber scaffold

The emerging fields of tissue engineering and biomaterials have begun to provide potential treatment options for liver failure. The goal of the present study is to investigate the ability of a poly L-lactic acid (PLLA) nanofiber scaffold to support and enhance hepatic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs). A scaffold composed of poly L-lactic acid and collagen was fabricated by the electrospinning technique. After characterizing isolated hMSCs, they were seeded onto PLLA nanofiber scaffolds and induced to differentiate into a hepatocyte lineage. The mRNA levels and protein expression of several important hepatic genes were determined using RT-PCR, immunocytochemistry and ELISA. Flow cytometry revealed that the isolated bone marrow-derived stem cells were positive for hMSC-specific markers CD73, CD44, CD105 and CD166 and negative for hematopoietic markers CD34 and CD45. The differentiation of these stem cells into adipocytes and osteoblasts demonstrated their multipotency. Scanning electron microscopy showed adherence of cells in the nanofiber scaffold during differentiation towards hepatocytes. Our results showed that expression levels of liver-specific markers such as albumin, α-fetoprotein, and cytokeratins 8 and 18 were higher in differentiated cells on the nanofibers than when cultured on plates. Importantly, liver functioning serum proteins, albumin and α-1 antitrypsin were secreted into the culture medium at higher levels by the differentiated cells on the nanofibers than on the plates, demonstrating that our nanofibrous scaffolds promoted and enhanced hepatic differentiation under our culture conditions. Our results show that the engineered PLLA nanofibrous scaffold is a conducive matrix for the differentiation of MSCs into functional hepatocyte-like cells. This represents the first step for the use of this nanofibrous scaffold for culture and differentiation of stem cells that may be employed for tissue engineering and cell-based therapy applications.     

Fabrication,Characterization and Cellular Compatibility of Poly(Hydroxy Alkanoate) Composite Nanofibrous Scaffolds for Nerve Tissue Engineering

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.     

Carbon nanotube reinforced Bombyx mori silk nanofibers by the electrospinning process

Nanocomposite fibers of Bombyx mori silk and single wall carbon nanotubes (SWNT) were produced by the electrospinning process. Regenerated silk fibroin dissolved in a dispersion of carbon nanotubes in formic acid was electrospun into nanofibers. The morphology, structure, and mechanical properties of the electrospun nanofibers were examined by field emission environmental scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectroscopy, Raman spectroscopy, and microtensile testing. TEM of the reinforced fibers shows that the single wall carbon nanotubes are embedded in the fibers. The mechanical properties of the SWNT reinforced fiber show an increase in Young's modulus up to 460% in comparison with the un-reinforced aligned fiber, but at the expense of the strength and strain to failure.     

Structure and morphology changes during in vitro degradation of electrospun poly(glycolide-co-lactide) nanofiber membrane

Electrospun poly(glycolide-co-lactide) (PLA10GA90, LA/GA ratio 10/90) biodegradable nanofiber membranes possessed very high surface area to volume ratios and were completely noncrystalline with a relatively lowered glass transition temperature. These characteristics led to very different structure, morphology, and property changes during in vitro degradation, which were examined systematically. A shrinkage study showed that the electrospun crystallizable but amorphous PLA10GA90 membranes exhibited a very small shrinkage percentage when compared with the electrospun membranes of noncrystallizable poly(lactide-co-glycolide) (PLA75GA25, LA/GA 75/25) and poly(d,l-lactide). Although the weight loss of electrospun PLA10GA90 membranes exhibited a similar degradation behavior as cast thin films, detailed studies showed that the structure and morphology changes in electrospun membranes followed different pathways during the hydrolytic degradation. After 1 day of degradation in buffer solution at 37 degrees C, electrospun PLA10GA90 membranes exhibited a sudden increase in crystallinity and glass transition temperature, due to the fast thermally induced crystallization process. The continuous increase in crystallinity and apparent crystal size, as well as the decrease in long period and lamellae thickness, indicated that the thermally induced crystallization was followed by a chain cleavage induced crystallization process. The mass loss rate was accelerated after 6 days of degradation. The increase in glass transition temperature during this period further confirmed that the degradation of PLA10GA90 nanofibers was initiated from the amorphous region within the lamellar superstructures. A mechanism of structure and morphology changes during in vitro degradation of electrospun PLA10GA90 nanofibers is proposed.     

Hydrogels and electrospun nanofibrous scaffolds of N-methylene phosphonic chitosan as bioinspired osteoconductive materials for bone grafting

In this work, N-methylene phosphonic chitosan (NMPC) based hydrogels and electrospun nanofibrous scaffolds are reported with objective to obtain osteoconductive and osteoinductive matrixes for bone grafting applications. NMPC, a phosphorylated derivative of chitosan, is known to mimic the function of non collagenous phosphoproteins in providing nucleation sites for biomineralization. NMPC hydrogels were prepared by crosslinking between NMPC and genipin. A detailed investigation of physicochemical properties of NMPC solutions is also carried out in order to obtain beads free nanofibers. Both NMPC gels and nanofibers were further evaluated for their biomineralization potential and biocompatibility with human osteoblast like cells. Results indicated that hydrogels and nanofibrous scaffolds NMPC are biocompatible and significantly osteoinductive compared to tissue culture plate controls. However, cells seeded on nanofibrous scaffolds exhibited greater proliferation measured by MTT assay, and higher expression of early markers for osteogenic differentiation proving the superior applicability of nanofibrous scaffolds for bone grafting applications.     

Silk–PVA Hybrid Nanofibrous Scaffolds for Enhanced Primary Human Meniscal Cell Proliferation

In this study, silk fibroin nanofibrous scaffolds were developed to investigate the attachment and proliferation of primary human meniscal cells. Silk fibroin (SF)–polyvinyl alcohol (PVA) blended electrospun nanofibrous scaffolds with different blend ratios (2:1, 3:1, and 4:1) were prepared. Morphology of the scaffolds was characterized using atomic force microscopy (AFM). The hybrid nanofibrous mats were crosslinked using 25 % (v/v) glutaraldehyde vapor. In degradation study, the crosslinked nanofiber showed slow degradation of 20 % on weight after 35 days of incubation in simulated body fluid (SBF). The scaffolds were characterized with suitable techniques for its functional groups, porosity, and swelling ratio. Among the nanofibers, 3:1 SF:PVA blend showed uniform morphology and fiber diameter. The blended scaffolds had fluid uptake and swelling ratio of 80 % and 458 ± 21 %, respectively. Primary meniscal cells isolated from surgical debris after meniscectomy were subcultured and seeded onto these hybrid nanofibrous scaffolds. Meniscal cell attachment studies confirmed that 3:1 SF:PVA nanofibrous scaffolds supported better cell attachment and growth. The DNA and collagen content increased significantly with 3:1 SF:PVA. These results clearly indicate that a blend of SF:PVA at 3:1 ratio is suitable for meniscus cell proliferation when compared to pure SF-PVA nanofibers.     

Electrospun poly(epsilon-caprolactone) microfiber and multilayer nanofiber/microfiber scaffolds: characterization of scaffolds and measurement of cellular infiltration

The physical and spatial architectural geometries of electrospun scaffolds are important to their application in tissue engineering strategies. In this work, poly(epsilon-caprolactone) microfiber scaffolds with average fiber diameters ranging from 2 to 10 microm were individually electrospun to determine the parameters required for reproducibly fabricating scaffolds. As fiber diameter increased, the average pore size of the scaffolds, as measured by mercury porosimetry, increased (values ranging from 20 to 45 microm), while a constant porosity was observed. To capitalize on both the larger pore sizes of the microfiber layers and the nanoscale dimensions of the nanofiber layers, layered scaffolds were fabricated by sequential electrospinning. These scaffolds consisted of alternating layers of poly(epsilon-caprolactone) microfibers and poly(epsilon-caprolactone) nanofibers. By electrospinning the nanofiber layers for different lengths of time, the thickness of the nanofiber layers could be modulated. Bilayered constructs consisting of microfiber scaffolds with varying thicknesses of nanofibers on top were generated and evaluated for their potential to affect rat marrow stromal cell attachment, spreading, and infiltration. Cell attachment after 24 h did not increase with increasing number of nanofibers, but the presence of nanofibers enhanced cell spreading as evidenced by stronger F-actin staining. Additionally, increasing the thickness of the nanofiber layer reduced the infiltration of cells into the scaffolds under both static and flow perfusion culture for the specific conditions tested. The scaffold design presented in this study allows for cellular infiltration into the scaffolds while at the same time providing nanofibers as a physical mimicry of extracellular matrix.