Incorporated-bFGF polycaprolactone/polyvinylidene fluoride nanocomposite scaffold promotes human induced pluripotent stem cells osteogenic differentiation

Bioactive scaffolds that can increase transplanted cell survival time at the defect site have a great promising potential to use clinically since tissue regeneration or secretions crucially depend on the transplanted cell survival. In this study embedded basic fibroblast growth factor (bFGF)-polycaprolactone-polyvinylidene fluoride (PCL-PVDF) hybrid was designed and fabricated by electrospinning as a bio-functional nanofibrous scaffold for bone tissue engineering. After morphological characterization of the PCL-PVDF (bFGF) scaffold, nanofibers biocompatibility was investigated by culturing of the human induced pluripotent stem cells (iPSCs). Then, the bone differentiation capacity of the iPSCs was evaluated when grown on the PCL-PVDF and PCL-PVDF (bFGF) scaffolds in comparison with culture plate as a control using evaluating of the common osteogenic markers. The viability assay displayed a significant increase in iPSCs survival rate when grown on the bFGF content scaffold. The highest alkaline phosphatase activity and mineralization were detected in the iPSCs while grown on the PCL-PVDF (bFGF) scaffolds. Obtained results from gene and protein expression were also demonstrated the higher osteoinductive property of the bFGF content scaffold compared with the scaffold without it. According to the results, the release of bFGF from PCL-PVDF nanofibers increased survival and proliferation rate of the iPSCs, which followed by an increase in its osteogenic differentiation potential. Taking together, PCL-PVDF (bFGF) nanofibrous scaffold demonstrated that can be noted as a promising candidate for treating the bone lesions by tissue engineering products.     

Improved infiltration of stem cells on electrospun nanofibers

Nanofibrous scaffolds have been recently used in the field of tissue engineering because of their nano-size structure which promotes cell attachment, function, proliferation and infiltration. In this study, nanofibrous polyethersulfone (PES) scaffolds was prepared via electrospinning. The scaffolds were surface modified by plasma treatment and collagen grafting. The surface changes then investigated by contact angle measurements and FTIR-ATR. The results proved grafting of the collagen on nanofibers surface and increased hydrophilicity after plasma treatment and collagen grafting. The cell interaction study was done using stem cells because of their ability to differentiate to different kinds of cell lines. The cells had normal morphology on nanofibers and showed very high infiltration through collagen grafted PES nanofibers. This infiltration capability is very useful and needed to make 3D scaffolds in tissue engineering.     

Nanofibers and their applications in tissue engineering

Developing scaffolds that mimic the architecture of tissue at the nanoscale is one of the major challenges in the field of tissue engineering. The development of nanofibers has greatly enhanced the scope for fabricating scaffolds that can potentially meet this challenge. Currently, there are three techniques available for the synthesis of nanofibers: electrospinning, self-assembly, and phase separation. Of these techniques, electrospinning is the most widely studied technique and has also demonstrated the most promising results in terms of tissue engineering applications. The availability of a wide range of natural and synthetic biomaterials has broadened the scope for development of nanofibrous scaffolds, especially using the electrospinning technique. The three dimensional synthetic biodegradable scaffolds designed using nanofibers serve as an excellent framework for cell adhesion, proliferation, and differentiation. Therefore, nanofibers, irrespective of their method of synthesis, have been used as scaffolds for musculoskeletal tissue engineering (including bone, cartilage, ligament, and skeletal muscle), skin tissue engineering, vascular tissue engineering, neural tissue engineering, and as carriers for the controlled delivery of drugs, proteins, and DNA. This review summarizes the currently available techniques for nanofiber synthesis and discusses the use of nanofibers in tissue engineering and drug delivery applications.     

Biomimetic and bioactive nanofibrous scaffolds from electrospun composite nanofibers

Electrospinning is an enabling technology that can architecturally (in terms of geometry, morphology or topography) and biochemically fabricate engineered cellular scaffolds that mimic the native extracellular matrix (ECM). This is especially important and forms one of the essential paradigms in the area of tissue engineering. While biomimesis of the physical dimensions of native ECM's major constituents (eg, collagen) is no longer a fabrication-related challenge in tissue engineering research, conveying bioactivity to electrospun nanofibrous structures will determine the efficiency of utilizing electrospun nanofibers for regenerating biologically functional tissues. This can certainly be achieved through developing composite nanofibers. This article gives a brief overview on the current development and application status of employing electrospun composite nanofibers for constructing biomimetic and bioactive tissue scaffolds. Considering that composites consist of at least two material components and phases, this review details three different configurations of nanofibrous composite structures by using hybridizing basic binary material systems as example. These are components blended composite nanofiber, core-shell structured composite nanofiber, and nanofibrous mingled structure.     

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.     

Combined effects of chemical priming and mechanical stimulation on mesenchymal stem cell differentiation on nanofiber scaffolds

Functional tissue engineering of connective tissues such as the anterior cruciate ligament (ACL) remains a significant clinical challenge, largely due to the need for mechanically competent scaffold systems for grafting, as well as a reliable cell source for tissue formation. We have designed an aligned, polylactide-co-glycolide (PLGA) nanofiber-based scaffold with physiologically relevant mechanical properties for ligament regeneration. The objective of this study is to identify optimal tissue engineering strategies for fibroblastic induction of human mesenchymal stem cells (hMSC), testing the hypothesis that basic fibroblast growth factor (bFGF) priming coupled with tensile loading will enhance hMSC-mediated ligament regeneration. It was observed that compared to the unloaded, as well as growth factor-primed but unloaded controls, bFGF stimulation followed by physiologically relevant tensile loading enhanced hMSC proliferation, collagen production and subsequent differentiation into ligament fibroblast-like cells, upregulating the expression of types I and III collagen, as well as tenasin-C and tenomodulin. The results of this study suggest that bFGF priming increases cell proliferation, while mechanical stimulation of the hMSCs on the aligned nanofiber scaffold promotes fibroblastic induction of these cells. In addition to demonstrating the potential of nanofiber scaffolds for hMSC-mediated functional ligament tissue engineering, this study yields new insights into the interactive effects of chemical and mechanical stimuli on stem cell differentiation.     

Improved osteogenic differentiation of human induced pluripotent stem cells cultured on polyvinylidene fluoride/collagen/platelet-rich plasma composite nanofibers

Blood transfusion or blood products, such as plasma, have a long history in improving health, but today, platelet-rich plasma (PRP) is used in various medical areas such as surgery, orthopedics, and rheumatology in many ways. Considering the high efficiency of tissue engineering in repairing bone defects, in this study, we investigated the combined effect of nanofibrous scaffolds in combination with PRP on the osteogenic differentiation potential of human induced pluripotent stem cells (iPSCs). Electrospinning was used for fabricating nanofibrous scaffolds by polyvinylidene fluoride/collagen (PVDF/col) with and without PRP. After scaffold characterization, the osteoinductivity of the fabricated scaffolds was studied by culturing human iPSCs under osteogenic medium. The results showed that PRP has a considerable positive effect on the biocompatibility of the PVDF/col nanofibrous scaffold when examined by protein adsorption, cell attachment, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. In addition, the results obtained from alkaline phosphatase activity and calcium content assays demonstrated that nanofibers have higher osteoinductivity while grown on PRP-incorporated PVDF/col nanofibers. These results were also confirmed while the osteogenic differentiation of the iPSCs was more investigated by evaluating the most important bone-related genes expression level. According to the results, it can be concluded that PVDF/col/PRP has much more osteoinductivity while compared with the PVDF/col, and it can be introduced as a promising bone bio-implant for use in bone tissue engineering applications.     

Effect of chitin/silk fibroin nanofibrous bicomponent structures on interaction with human epidermal keratinocytes

To fabricate a biomimetic nanostructured bicomponent scaffolds, two types of chitin/silk fibroin (SF) nanofibrous scaffolds (blend scaffolds and hybrid scaffolds) were prepared by electrospinning or simultaneous electrospinning of chitin/SF solutions. The chitin/SF bicomponent scaffolds were after-treated with water vapor, and their nanofibrous structures were almost maintained. From the cytocompatibility and cell behavior on the chitin/SF blend or hybrid nanofibrous scaffolds, the hybrid matrix with 25% chitin and 75% SF as well as the chitin/SF blend nanofibers could be a potential candidate for tissue engineering scaffolds.     

Differentiation of bone marrow-derived mesenchymal stem cells into hepatocyte-like cells in an alginate scaffold

Objectives: Alginate scaffolds are the most frequently investigated biomaterials in tissue engineering. Tissue engineering techniques that generate liver tissue have become important for treatment of a number of liver diseases and recent studies indicate that bone marrow‐derived stem cells (BMSCs) can differentiate into hepatocyte‐like cells. The goal of the study described here, was to examine in vitro hepatic differentiation potential of BMSCs cultured in an alginate scaffold. Materials and methods: To investigate the potential of BMSCs to differentiate into hepatocyte‐like cells, we cultured BMSCs in alginate scaffolds in the presence of specific growth factors including hepatocyte growth factor, epidermal growth factor and fibroblast growth factor‐4. Results: We can demonstrate that alginate scaffolds are compatible for growth of BMSCs and when cultured in alginate scaffolds for several days they display several liver‐specific markers and functions. Specifically, they expressed genes encoding alpha‐foetoprotein, albumin (ALB), connexin 32 and CYP7A1. In addition, these BMSCs produced both ALB and urea, expressed cytokeratin‐18 (CK‐18) and were capable of glycogen storage. Percentage of CK‐18 positive cells, a marker of hepatocytes, was 56.7%. Conclusions: Our three‐dimensional alginate scaffolds were highly biocompatible with BMSCs. Furthermore, culturing induced their differentiation into hepatocyte‐like cells. Therefore, BMSCs cultured in alginate scaffolds may be applicable for hepatic tissue engineering.     

Coaxial electrospinning of (fluorescein isothiocyanate-conjugated bovine serum albumin)-encapsulated poly(epsilon-caprolactone) nanofibers for sustained release

As an aim toward developing biologically mimetic and functional nanofiber-based tissue engineering scaffolds, we demonstrated the encapsulation of a model protein, fluorescein isothiocyanate-conjugated bovine serum albumin (fitcBSA), along with a water-soluble polymer, poly(ethylene glycol) (PEG), within the biodegradable poly(epsilon-caprolactone) (PCL) nanofibers using a coaxial electrospinning technique. By variation of the inner flow rates from 0.2 to 0.6 mL/h with a constant outer flow rate of 1.8 mL/h, fitcBSA loadings of 0.85-2.17 mg/g of nanofibrous membranes were prepared. Variation of flow rates also resulted in increases of fiber sizes from ca. 270 nm to 380 nm. The encapsulation of fitcBSA/PEG within PCL was subsequently characterized by laser confocal scanning microscopy, transmission electron microscopy (TEM), and X-ray photoelectron spectroscopy (XPS) analysis. In vitro release studies were conducted to evaluate sustained release potential of the core-sheath-structured composite nanofiber PCL-r-fitcBSA/PEG. As a negative control, composite nanofiber PCL/fitcBSA/PEG blend was prepared from a normal electrospinning method. It was found that core-sheath nanofibers PCL-r-fitcBSA/PEG pronouncedly alleviated the initial burst release for higher protein loading and gave better sustainability compared to that of PCL/fitcBSA/PEG nanofibers. The present study would provide a basis for further design and optimization of processing conditions to control the nanostructure of core-sheath composite nanofibers and ultimately achieve desired release kinetics of bioactive proteins (e.g., growth factors) for practical tissue engineering applications.     

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.     

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.     

Fabrication and evaluation of biomimetic-synthetic nanofibrous composites for soft tissue regeneration

Electrospun scaffolds hold promise for the regeneration of dense connective tissues, given their nanoscale topographies, provision of directional cues for infiltrating cells and versatile composition. Synthetic slow-degrading scaffolds provide long-term mechanical support and nanoscale instructional cues; however, these scaffolds suffer from a poor infiltration rate. Alternatively, nanofibrous constructs formed from natural biomimetic materials (such as collagen) rapidly infiltrate but provide little mechanical support. To take advantage of the positive features of these constructs, we have developed a composite scaffold consisting in both a biomimetic fiber fraction (i.e., Type I collagen nanofibers) together with a traditional synthetic (i.e., poly-[ε-caprolactone], PCL) fiber fraction. We hypothesize that inclusion of biomimetic elements will improve initial cell adhesion and eventual scaffold infiltration, whereas the synthetic elements will provide controlled and long-term mechanical support. We have developed a method of forming and crosslinking collagen nanofibers by using the natural crosslinking agent genipin (GP). Further, we have formed composites from collagen and PCL and evaluated the long-term performance of these scaffolds when seeded with mesenchymal stem cells. Our results demonstrate that GP crosslinking is cytocompatible and generates stable nanofibrous type I collagen constructs. Composites with varying fractions of the biomimetic and synthetic fiber families are formed and retain their collagen fiber fractions during in vitro culture. However, at the maximum collagen fiber fractions (20%), cell ingress is limited compared with pure PCL scaffolds. These results provide a new foundation for the development and optimization of biomimetic/synthetic nanofibrous composites for in vivo tissue engineering.     

Biomimetic nanofibrous scaffolds: preparation and characterization of PGA/chitin blend nanofibers

Electrospinning of poly(glycolic acid) (PGA)/chitin blend solutions in 1,1,1,3,3,3-hexafluoro-2-propanol was investigated to fabricate biodegradable and biomimetic nanostructured scaffolds for tissue engineering. The morphology of the electrospun PGA/chitin blend nanofibers was investigated with a field emission scanning electron microscope. The PGA/chitin blend fibers have average diameters of around 140 nm, and their diameters have a distribution in the range 50-350 nm. The miscibility of PGA/chitin blend fibers was examined by differential scanning calorimetry. The PGA and chitin were immiscible in the as-spun nanofibrous structure. An in vitro degradation study of PGA/chitin blend nanofibers was conducted in phosphate-buffered saline, pH 7.2. It was found that the hydrolytic cleavage of PGA in the blend nanofibers was accelerated by the coexistence of hydrophilic chitin. To assay the cytocompatability and cell behavior on the PGA/chitin blend nanofibrous scaffolds, cell attachment and spreading of normal human epidermal fibroblasts seeded on the scaffolds were studied. Our results indicate that the PGA/chitin blend nanofibrous matrix, particularly the one that contained 25% PGA and 75% chitin with bovine serum albumin coating, could be a good candidate for tissue engineering scaffolds, because it has an excellent cell attachment and spreading for normal human fibroblasts.     

Biomimetic nanofibrous scaffolds: preparation and characterization of chitin/silk fibroin blend nanofibers

Electrospinning of chitin/silk fibroin (SF) blend solutions in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) was investigated to fabricate a biomimetic nanostructured scaffolds for tissue engineering. The morphology of the electrospun chitin/SF blend nanofibers was investigated with a field emission scanning electron microscope (FE-SEM). The average diameters of chitin/SF blend fibers decreased from 920 to 340 nm, with the increase of chitin content in blend compositions. The miscibility of chitin/SF blend fibers was examined by solution viscosity measurement. The chitin and SF were immiscible in the as-spun nanofibrous structure. The dimensional stability of chitin/SF blend nanofibers, with or without water vapor after-treatment, was conducted by immersing in water. As-spun SF-rich blend nanofibrous matrices were lost their fibrous structure after the water immersion for 24 h, and then changed into membrane-like structure. On the contrary, nanofibrous structures of water vapor-treated SF-rich blends were almost maintained. To assay the cytocompatibility and cell behavior on the chitin/SF blend nanofibrous scaffolds, cell attachment and spreading of normal human epidermal keratinocyte and fibroblasts seeded on the scaffolds were studied. Our results indicate that chitin/SF blend nanofibrous matrix, particularly the one that contained 75% chitin and 25% SF, could be a potential candidate for tissue engineering scaffolds because it has both biomimetic three-dimensional structure and an excellent cell attachment and spreading for NHEK and NHEF.     

Neurogenesis of bone marrow-derived mesenchymal stem cells onto β-mercaptoethanol-loaded PLGA film

Bone marrow-derived mesenchymal stem cells (BMSCs) are of particular interest in the field of tissue engineering because of their potential to differentiate into osteoblasts, chondrocytes, and neuronal cells. In order to promote the differentiation of BMSCs into specific cell types, appropriate scaffold biomaterials and bioactive molecules that can support the differentiation of BMSCs into specific cell types are needed. We hypothesized that β-mercaptoethanol (BME), which has been reported to induce the differentiation of BMSCs into neural-like cells, promotes BMSCs to differentiate into neural-like cells when BME is added to polymeric scaffolds containing the BMSCs. We fabricated biocompatible film shaped scaffolds composed of poly(lacti-co-glycolic) acid (PLGA) and various concentrations of BME to confirm that BME-promoted differentiation of BMSCs is concentration-dependent. Cell proliferation increased as the BME concentration in the films increased at the early stage, and the proliferation rate remained similar on the PLGA films for 3 weeks following the BMSC seeding. The expression of neuronal markers in differentiated BMSCs was assessed by RT-PCR. At 2- and 3-week time-points, mRNA expression of neurofilament and neuron specific enolase was significantly increased in PLGA/BME films containing 400 μM BME compared to PLGA films. Thus, we have identified BMSC-seeded PLGA/BME films with 200 μM and 400 μM BME as potentially useful candidates for neural tissue engineering applications by promoting BMSC proliferation and differentiation towards neural-like cells.     

Biochemical and biophysical aspects of collagen nanostructure in the extracellular matrix

ECM is composed of different collagenous and non-collagenous proteins. Collagen nanofibers play a dominant role in maintaining the biological and structural integrity of various tissues and organs, including bone, skin, tendon, blood vessels, and cartilage. Artificial collagen nanofibers are increasingly significant in numerous tissue engineering applications and seem to be ideal scaffolds for cell growth and proliferation. The modern tissue engineering task is to develop three-dimensional scaffolds of appropriate biological and biomechanical properties, at the same time mimicking the natural extracellular matrix and promoting tissue regeneration. Furthermore, it should be biodegradable, bioresorbable and non-inflammatory, should provide sufficient nutrient supply and have appropriate viscoelasticity and strength. Attributed to collagen features mentioned above, collagen fibers represent an obvious appropriate material for tissue engineering scaffolds. The aim of this minireview is, besides encapsulation of the basic biochemical and biophysical properties of collagen, to summarize the most promising modern methods and technologies for production of collagen nanofibers and scaffolds for artificial tissue development.