Aligned Nanofibrous Scaffolds Research Articles

Biophysical-driven piezoelectric and aligned nanofibrous scaffold promotes bone regeneration by re-establishing physiological electrical microenvironment

Biophysical-driven piezoelectric and aligned nanofibrous scaffold promotes bone regeneration by re-establishing physiological electrical microenvironment

Nanographene-Au fine-tuning to intensify plasmonic-resonance of polymeric hybrid bionanosystem for synergistic phototherapy and nerve photobiomodulation

Here, we report the multi-photo-bioactivity of the plasmonic-nano graphitic coordinated polycaprolactone-based aligned nanofibrous scaffolds-based bionanosystem for photothermal breast and colon cancer therapies and peripheral nerve photobiomodulation. The size-optimized colloidal reduced graphene oxide (nRGO, 180 nm) nanosheets, for enhanced photothermal impact, were surface-functionalized with gold nanospheres (AuNPs) to prepare the nRGO@AuNP monodispersed nano-composite and then doped 2.0 mg of nRGO@AuNP in biocompatible and biodegradable polymer polycaprolactone (PCL) to fabricate the nRGO@AuNP-PCL (2.0 mg) plasmonic aligned nanofibrous scaffolds. More than 90% of cancer cells, breast cancer (MCF-7) as well as colon cancer (CT-26), ablated after 5 min of low NIR (808 nm) laser power (0.72 W/cm2) illumination with nRGO@AuNP-PCL (2.0 mg) aligned nanofibrous scaffolds. Besides, the nRGO@AuNP-PCL (2.0 mg) provided an extraordinary microenvironment for adhesion, nerve growth, proliferation, and differentiation of PC12 and S42 cells which mimics the natural extracellular matrix. The 2.5-fold increase in neurite length was observed with NIR illumination after 3 days whereas 1.7-fold was found without NIR illumination after 7 days in comparison to PCL (pure). The current findings will be useful to provide a new crucial approach for preparing biocompatible multifunctional composite plasmonic nanofibers as a highly efficient distinct platform for photothermal therapies and promising bioimplants to overcome the loss of sensation after cancer surgery through nerve photobiomodulation.

Electrospinning of Polycaprolactone Nanofibrous Scaffolds Containing Folic Acid for Nerve Tissue Engineering

Objective: The aim of the study was to employ electrospinning technology to fabricate aligned nanofibrous scaffolds of polycaprolactone (PCL) containing folic acid (FA) for nerve tissue engineering. Material and Methods: Scanning electron microscopy (SEM) was used to assess the diameter distribution and degree of alignment of the nanofibers. Fourier transform infrared spectroscopy (FTIR) and powder X-ray diffraction (PXRD) were used to analyze the chemical and crystalline structures of the scaffold. Additionally, the content and release behavior of FA in the PCL fibrous scaffolds were examined. Finally, the biocompatibility of the scaffolds was evaluated using rat Schwann cells, assessing cell proliferation, alignment, and morphology. Results: The study revealed that the nanofiber diameters ranged from 210.07 to 227.36 nm, and the scaffolds maintained an amorphous form with no effects on their chemical structure following the electrospinning process. The investigation demonstrated that PCL fibers could accommodate FA loading within a range of 99.25-102.49% w/w and that the release profile of FA followed Higuchi model. Moreover, the FA-containing PCL nanofibrous scaffolds significantly enhanced rat Schwann cell proliferation during the initial two days of culture when compared to a normal PCL nanofiber scaffold. The hydrophilic properties of folic acid are thought to have facilitated directional growth along the electrospun nanofibers, contributing to the observed results. Conclusion: Finally, PCL-containing FA nanofibrous scaffolds may be applicable to nerve tissue engineering.

Integrated Manufacturing of Suspended and Aligned Nanofibrous Scaffold for Structural Maturation and Synchronous Contraction of HiPSC-Derived Cardiomyocytes.

Electrospun nanofiber constructs represent a promising alternative for mimicking the natural extracellular matrix in vitro and have significant potential for cardiac patch applications. While the effect of fiber orientation on the morphological structure of cardiomyocytes has been investigated, fibers only provide contact guidance without accounting for substrate stiffness due to their deposition on rigid substrates (e.g., glass or polystyrene). This paper introduces an in situ fabrication method for suspended and well aligned nanofibrous scaffolds via roller electrospinning, providing an anisotropic microenvironment with reduced stiffness for cardiac tissue engineering. A fiber surface modification strategy, utilizing oxygen plasma treatment combined with sodium dodecyl sulfate solution, was proposed to maintain the hydrophilicity of polycaprolactone (PCL) fibers, promoting cellular adhesion. Human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs), cultured on aligned fibers, exhibited an elongated morphology with extension along the fiber axis. In comparison to Petri dishes and suspended random fiber scaffolds, hiPSC-CMs on suspended aligned fiber scaffolds demonstrated enhanced sarcomere organization, spontaneous synchronous contraction, and gene expression indicative of maturation. This work demonstrates the suspended and aligned nano-fibrous scaffold provides a more realistic biomimetic environment for hiPSC-CMs, which promoted further research on the inducing effect of fiber scaffolds on hiPSC-CMs microstructure and gene-level expression.

Modulated plasmonic nanofibrous scaffold reinforced breast cancer photo-ablation and breast neurotization with resensation

Herein, we report the bioactivity of the hybrid plasmonic aligned nanofibrous scaffolds composed of reduced graphene oxide (rGO, 209 nm) nanosheets enfolded gold nanorods (AuNR) with polycaprolactone (PCL) for breast cancer photo-therapy and subsequent nerve regeneration. The strategic inclusion of 0.00068 wt% (OD = 4.0) of monodispersed AuNR-rGO in PCL (AuNR-rGO@PCL (4.0)) leads to significant higher physicochemical and optical properties such as higher mechanical strength (7-fold increase), electrical conductivity (∼6-times enhancement), larger surface area (6.4 times), and more NIR light absorbance. Hence, the AuNR-rGO@PCL (4.0) was found to be an excellent photo agent for breast tumor ablation with more than 90% cancer cell (MCF-7) death under 808 nm NIR irradiation at low laser power density (0.72 W/cm2). Also, the nanofibrous scaffold was observed to have a substantial effect on adhesion, proliferation, growth, and differentiation of PC12 and high affinity to S42 cell proliferation and migration which led to an exceptional nerve growth with a 2.5-fold (with NIR light after 3 days) and 2-fold (without NIR light after 7 days) increase in neurite length in comparison to PCL. The present study provides a new bioactive, biocompatible, non-toxic, and bifunctional aligned plasmonic scaffolds as an excellent single platform for breast cancer treatment and potential bioimplant to regain sensation loss after breast surgery and reconstruction.

Design of ECM Functionalized Polycaprolactone Aligned Nanofibers for Peripheral Nerve Tissue Engineering

PurposePeripheral nerve injury (PNI) and its regeneration continue to remain a significant medical burden worldwide. The current treatment strategies used to treat PNI are often associated with multiple complications and yet do not achieve complete motor and sensory functions. Recently, synthetic biodegradable nerve conduits have become one the most commonly used conduits to repair small gaps in nerve injury. But they have not shown better results than nerve grafts possibly because of the lack of biological microenvironment required for axonal growth. Schwann cells play a very crucial role in peripheral nerve regeneration where activated SCs produce multiple neurotrophic factors that help in remyelination and immune modulation during nerve repair. Studies have shown that nanofibrous scaffolds have better bioactivity and more closely mimic the native structure of the extracellular matrix. Therefore, the present study was focused on designing a nanofibrous scaffold that would cover the roles of both structural support for the cells that can provide a microenvironment with biological cues for nerve growth and regeneration.MethodsDecellularized Schwann cell ECM were spin-coated on polycaprolactone random and aligned nanofibrous scaffolds and their compatibility was evaluated using Schwann cells.ResultsSchwann cells displayed growth in the direction of the aligned PCL nanofibers and ACM treated exhibited appropriate bipolar morphology indicating that these modified fibers could provide directional cues making them highly suitable for neuronal cell growth.ConclusionOur results indicate that the fabricated aligned SC-ACM treated PCL scaffolds would be a potential biomaterial to treat peripheral nerve injuries and promote regeneration.Graphical

Fabrication of a Completely Biological and Anisotropic Human Mesenchymal Stem Cell-Based Vascular Graft.

Tissue-engineered small-diameter vascular grafts are required to match mechanical properties as well as cellular and extracellular architecture of native blood vessels. Although various engineering technologies have been developed, the most reliable strategy highlights the needs for incorporating completely biological components and anisotropic cellular and biomolecular organization into the tissue-engineered vascular graft (TEVG). Based on the antithrombogenic, immunoregulatory, and regenerative properties of human mesenchymal stem cells (hMSCs), this chapter provides a step-by-step protocol for generating a completely biological and anisotropic TEVG that comprises of hMSCs and highly aligned extracellular matrix (ECM) nanofibers. The hMSCs were grown on an aligned nanofibrous ECM scaffold derived from an oriented human dermal fibroblast (hDF) sheet and then wrapped around a temporary mandrel to form a tubular assembly, followed by a maturation process in a rotating wall vessel (RWV) bioreactor. The resulting TEVG demonstrates anisotropic structural and mechanical properties similar to that of native blood vessels. A completely biological, anisotropic, and mechanically strong TEVG that incorporates immunoregulatory hMSCs is promising to meet the urgent needs of a surgical intervention for bypass grafting.

Preparation and characterization of polyurethane/chitosan/CNT nanofibrous scaffold for cardiac tissue engineering

Myocardial infarction of cardiomyocytes is a leading cause of heart failure (HF) worldwide. Since heart has very limited regeneration capacity, cardiac tissue engineering (TE) to produce a bioactive scaffold is considered. In this study, a series of polyurethane solutions (5–7%wt) in aqueous acetic acid were prepared using electrospinning. A variety of Polyurethane (PU)/Chitosan (Cs)/carbon nanotubes (CNT) composite nanofibrous scaffolds with random and aligned orientation were fabricated to structurally mimic the extracellular matrix (ECM). Electrospun nanofibers were then characterized using field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), water contact angle, degradation studies, tensile tests, electrical resistance measurement and cell viability assay. The biocompatibility of electrospun random and aligned nanofibrous scaffolds with H9C2 Cells was confirmed. The results revealed that fabricated PU/Cs/CNT composite nanofibrous scaffolds were electro-conductive and aligned nanofibers could be considered as promising scaffolds with nano-scale features for regeneration of infarcted myocardium.

Development of a Bioactive Scaffold based on NGF Containing PCL/Chitosan Nanofibers for Nerve Regeneration

Nanofibers are used in a wide range of applications, including scaffolds for tissue engineering. Electrospinning is a promising technique to fabricate nanofibrous scaffolds capable of supporting cell attachment and growth. Nanofibers with biomimetic alignment could also guide neural cell growth and orientation of cell processes necessary for peripheral nerve regeneration. This study fabricated aligned nanofibers of polycaprolactone/chitosan (PCL/CS) scaffolds and immobilized nerve growth factor (NGF) on them via dopamine coating mediated bonds to confer bioactivity to the scaffold and support attachment and growth of PC12 cells. The results showed that PCL/CS nanofibrous scaffolds revealed appropriate mechanical and surface properties. Cells remained viable on the scaffolds, and surface-modified aligned nanofibrous scaffolds interacted better with the cells, inducing neural morphology and orientation. Immobilization of NGF via polydopamine (PD) on nanofibers' surface proved to be a proper method to enhance PC12 cell attachment and proliferation. Thus, this construct could potentially be used as a scaffold for peripheral nerve regeneration.

Regeneration of the peripheral nerve via multifunctional electrospun scaffolds.

Over the last two decades, electrospun scaffolds have proved to be advantageous in the field of nerve tissue regeneration by connecting the cavity among the proximal and distal nerve stumps growth cones and leading to functional recovery after injury. Multifunctional nanofibrous structure of these scaffolds provides enormous potential by combining the advantages of nano-scale topography, and biological science. In these structures, selecting the appropriate materials, designing an optimized structure, modifying the surface to enhance biological functions and neurotrophic factors loading, and native cell-like stem cells should be considered as the essential factors. In this systematic review paper, the fabrication methods for the preparation of aligned nanofibrous scaffolds in yarn or conduit architecture are reviewed. Subsequently, the utilized polymeric materials, including natural, synthetic and blend are presented. Finally, their surface modification techniques, as well as, the recent advances and outcomes of the scaffolds, both in vitro and in vivo, are reviewed and discussed.

Pre-innervated tissue-engineered muscle promotes a pro-regenerative microenvironment following volumetric muscle loss

Volumetric muscle loss (VML) is the traumatic or surgical loss of skeletal muscle beyond the inherent regenerative capacity of the body, generally leading to severe functional deficit. Formation of appropriate somato-motor innervations remains one of the biggest challenges for both autologous grafts as well as tissue-engineered muscle constructs. We aim to address this challenge by developing pre-innervated tissue-engineered muscle comprised of long aligned networks of spinal motor neurons and skeletal myocytes on aligned nanofibrous scaffolds. Motor neurons led to enhanced differentiation and maturation of skeletal myocytes in vitro. These pre-innervated tissue-engineered muscle constructs when implanted in a rat VML model significantly increased satellite cell density, neuromuscular junction maintenance, graft revascularization, and muscle volume over three weeks as compared to myocyte-only constructs and nanofiber scaffolds alone. These pro-regenerative effects may enhance functional neuromuscular regeneration following VML, thereby improving the levels of functional recovery following these devastating injuries.

Efficient mesenchymal stem cell conversion to corneal keratocytes by the use of specific biomaterial and Cell Rev™ mSC diffKera media

Background & Aim Corneal blindness is a prevalent cause of vision loss, affecting approximately 4.9 million people worldwide (Flaxman et al, 2017). The demand for donor replacement corneas often exceeds the supply, requiring alternative treatment options. To achieve corneal function while minimizing the risks of donor rejection, we propose an autologous biomimetic cornea whereby patient-derived mesenchymal stem cells (MSCs) are converted to corneal-like cells on scaffolds that closely resemble the structure of the human cornea. We hypothesize that the effectiveness of cell differentiation depends on the biomaterials used and the cultured media composition. The more closely the environment resembles that of the human cornea, the higher the likelihood that a greater proportion of MSCs will differentiate into corneal keratocytes. Methods, Results & Conclusion Here, we investigated MSC differentiation into corneal keratocytes using various medium formulations on our electrospun scaffolds. Polycaprolactone-bovine dermis type I atelocollagen (PCL-atelocollagen) and polycaprolactone (PCL) solutions were electrospun to obtain the aligned nanofibrous scaffolds. MSCs were cultured on the scaffolds for 12 days in 3 different types of media: the expansion media described by Lynch & Ahearne (2007); keratocyte differentiation media used in the same study; and our Cell Rev™ mSC diffKera media. Immunofluorescence staining for keratocyte markers such as aldehyde dehydrogenase 1 and lumican was conducted on cells fixed on the scaffolds at days 6 and 12. The results confirmed that a greater proportion of MSCs differentiated into keratocytes in Cell Rev™ mSC diffKera media compared with the media used by Lynch & Ahearne (Figure 1). In addition, electrospun PCL-atelocollagen scaffolds are likely to support keratocyte differentiation better than electrospun PCL scaffolds (control) because corneal stromal collagen fibrils comprise mainly type I collagen. These findings demonstrate that the use of our electrospun PCL-atelocollagen scaffolds and Cell Rev™ mSC diffKera formulation effectively enhances MSC differentiation into keratocytes, and represents a novel step for the creation of artificial corneas. Corneal blindness is a prevalent cause of vision loss, affecting approximately 4.9 million people worldwide (Flaxman et al, 2017). The demand for donor replacement corneas often exceeds the supply, requiring alternative treatment options. To achieve corneal function while minimizing the risks of donor rejection, we propose an autologous biomimetic cornea whereby patient-derived mesenchymal stem cells (MSCs) are converted to corneal-like cells on scaffolds that closely resemble the structure of the human cornea. We hypothesize that the effectiveness of cell differentiation depends on the biomaterials used and the cultured media composition. The more closely the environment resembles that of the human cornea, the higher the likelihood that a greater proportion of MSCs will differentiate into corneal keratocytes. Here, we investigated MSC differentiation into corneal keratocytes using various medium formulations on our electrospun scaffolds. Polycaprolactone-bovine dermis type I atelocollagen (PCL-atelocollagen) and polycaprolactone (PCL) solutions were electrospun to obtain the aligned nanofibrous scaffolds. MSCs were cultured on the scaffolds for 12 days in 3 different types of media: the expansion media described by Lynch & Ahearne (2007); keratocyte differentiation media used in the same study; and our Cell Rev™ mSC diffKera media. Immunofluorescence staining for keratocyte markers such as aldehyde dehydrogenase 1 and lumican was conducted on cells fixed on the scaffolds at days 6 and 12. The results confirmed that a greater proportion of MSCs differentiated into keratocytes in Cell Rev™ mSC diffKera media compared with the media used by Lynch & Ahearne (Figure 1). In addition, electrospun PCL-atelocollagen scaffolds are likely to support keratocyte differentiation better than electrospun PCL scaffolds (control) because corneal stromal collagen fibrils comprise mainly type I collagen. These findings demonstrate that the use of our electrospun PCL-atelocollagen scaffolds and Cell Rev™ mSC diffKera formulation effectively enhances MSC differentiation into keratocytes, and represents a novel step for the creation of artificial corneas.

Combined effects of electrospun nanofibrous scaffold and electrical field on the neuronal outgrowth

Abstract Directed outgrowth and accelerated elongation are crucial for the neuronal regeneration and repair. Here we evaluated combined effects of nanofibrous scaffold and electrical field on the neuronal outgrowth. Aligned polycaprolactone (PCL) fibers provided a strong guidance cue for the neurite orientation, and the electrical field stimulation promoted the elongation and orientation of neurites. When two cues were applied simultaneously, neurite orientation was along the aligned fiber dominantly and neurite elongation was accelerated no matter the electrical field direction was parallel or vertical to the aligned fiber, indicating the synergistic effect of aligned nanofibrous scaffolds and electrical field. All the results suggested that combined application of physical contact guidance and electrical field have great prospects in the regeneration and repair of nerve system.

Synthesis and characterization of electrospun nanofibrous tissue engineering scaffolds generated from in situ polymerization of ionomeric polyurethane composites

Tissue scaffolds need to be engineered to be cell compatible, have timely biodegradable character, be functional with respect to providing niche cell support for tissue repair and regeneration, readily accommodate multiple cell types, and have mechanical properties that enable the simulation of the native tissue. In this study, electrospun degradable polar hydrophobic ionic polyurethane (D-PHI) scaffolds were generated in order to yield an extracellular matrix-like structure for tissue engineering applications. D-PHI oligomers were synthesized, blended with a degradable linear polycarbonate polyurethane (PCNU), and electrospun with simultaneous in situ UV cross-linking in order to generate aligned nanofibrous scaffolds in the form of elastomeric composite materials. The D-PHI/PCNU scaffold fibre morphology, cross-linking efficiency, surface nature, mechanical properties, in vivo degradation and integration, as well as in vitro cell compatibility were characterized. The results showed that D-PHI/PCNU scaffolds had a high cross-linking efficiency, stronger polar nature, and lower stiffness relative to PCNU scaffolds. In vivo, the D-PHI/PCNU scaffold degraded relatively slowly, thereby enabling new tissue time to form and yielding very good integration with the latter tissue. Based on a study with A10 vascular smooth muscle cells, the D-PHI/PCNU scaffold was able to support high cell viability, adhesion, and expression of typical smooth muscle cell markers after a 7-day culture period, which was comparable to PCNU scaffolds. These characterization results demonstrate that the unique properties of a D-PHI/PCNU scaffold, combined with the benefits of electrospinning, could allow for the generation of a tissue engineered scaffold that mimics important aspects of the native extracellular matrix and could be used for functional tissue regeneration. Statement of SignificanceTissue engineered scaffolds should recapitulate native extracellular matrix features. This study investigates the processing of a classical polycarbonate polyurethane (PCNU) with a cross-linked and degradable ionomeric polyurethane (D-PHI), polymerized via in situ rapid light curing to yield a 3-dimensional co-electrospun nanofibre matrix with chemical diversity and low modulus character. This research advances the use of D-PHI for tissue engineering applications by providing a facile means of changing physical and chemical properties in classical PCNUs without the need to adjust spinning viscosities of the base polymer. Further, the in vivo and cell culture findings set the stage for introducing unique elastic materials which inherently support wound healing, repair, and regeneration in tissues, for applications that require the recapitulation of native extracellular matrix physical features.

Synthesis and Characterization of Electrospun Nanofibrous Tissue Engineering Scaffolds Generated from in Situ Polymerization of Ionomeric Polyurethane Composites

Tissue scaffolds need to be engineered to be cell compatible, have timely biodegradable character, be functional with respect to providing niche cell support for tissue repair and regeneration, readily accommodate multiple cell types, and have mechanical properties that enable the simulation of the native tissue. In this study, electrospun degradable polar hydrophobic ionic polyurethane (D-PHI) scaffolds were generated in order to yield an extracellular matrix-like structure for tissue engineering applications. D-PHI oligomers were synthesized, blended with a degradable linear polycarbonate polyurethane (PCNU), and electrospun with simultaneous in situ UV cross-linking in order to generate aligned nanofibrous scaffolds in the form of elastomeric composite materials. The D-PHI/PCNU scaffold fibre morphology, cross-linking efficiency, surface nature, mechanical properties, in vivo degradation and integration, as well as in vitro cell compatibility were characterized. The results showed that D-PHI/PCNU scaffolds had a high cross-linking efficiency, stronger polar nature, and lower stiffness relative to PCNU scaffolds. In vivo, the D-PHI/PCNU scaffold degraded relatively slowly, thereby enabling new tissue time to form and yielding very good integration with the latter tissue. Based on a study with A10 vascular smooth muscle cells, the D-PHI/PCNU scaffold was able to support high cell viability, adhesion, and expression of typical smooth muscle cell markers after a 7-day culture period, which was comparable to PCNU scaffolds. These characterization results demonstrate that the unique properties of a D-PHI/PCNU scaffold, combined with the benefits of electrospinning, could allow for the generation of a tissue engineered scaffold that mimics important aspects of the native extracellular matrix and could be used for functional tissue regeneration.

The cellular response of nerve cells on poly-l-lysine coated PLGA-MWCNTs aligned nanofibers under electrical stimulation

Tissue engineering scaffold provide an effective alternative for peripheral nerve repair. Nanofibrous nerve conduits fabricated with various synthetic and natural materials have great potential to support nerve regeneration as a bridge between adjacent ends. The physical, chemical and electrical properties of the scaffolds affect the outcome of nerve regeneration and recovery of function. In this paper, a surface modified, electrically conductive, aligned nanofibrous scaffold composed of poly(lactic-co-glycolic acid) (PLGA) and multi-walled carbon nanotubes (MWCNTs), referred to as L-PC_A was fabricated for nerve regeneration. The morphology, surface chemistry and hydrophilicity of nanofibers were characterized by Scanning Electron Microscopy (SEM), Energy-dispersive X-ray (EDX) and water contact angle, respectively. The mechanical property of the nanofibrous scaffold was also evaluated using a universal materials tester. The effects of these scaffolds on PC12 cell adhesion, proliferation and neuronal differentiation were all evaluated. A hydrophilic surface was created by poly-l-lysine coating, which was able to provide a better environment for cell attachment. Furthermore aligned fibers were proved to be able to guide PC12 cells and DRG neurons growing along the fiber direction and be beneficial for neurite outgrowth. The cellular responses of PC12 cells and DRG neurons on L-PC_A scaffold under electrical stimulation were evaluated by neurofilament proteins expression. As a result, the PC12 cells and DRG neurons stimulated with electrical shock showed longer neurite length, indicating that electrical stimulation with a voltage of 40 mV based on the scaffold with MWCNTs could enhance the neurite extension. Moreover, the cellular response of Schwann cells including cell attachment, proliferation and MBP expression were also enhanced with the synergistic effect of aligned nanofibers and electrical stimulation. In summary, the L-PC_A nanofibrous scaffold supported the cellular response of nerve cells in terms of cell proliferation, differentiation, neurite outgrowth, and myelination in the presence of electrical stimulation, which could be a potential candidate for nerve regeneration application.

A novel electrospun-aligned nanoyarn/three-dimensional porous nanofibrous hybrid scaffold for annulus fibrosus tissue engineering.

IntroductionHerniation of the nucleus pulposus (NP) because of defects in the annulus fibrosus (AF) is a well-known cause of low back pain. Defects in the AF thus remain a surgical challenge, and efforts have been made to develop new techniques for closure and repair. In this study, we developed an electrospun aligned nanoyarn scaffold (AYS) and nanoyarn/three-dimensional porous nanofibrous hybrid scaffold (HS) for AF tissue engineering.MethodsThe AYS was fabricated via conjugated electrospinning, while the aligned nanofibrous scaffold (AFS) was prepared by traditional electrospinning as a baseline scaffold. The HS was constructed by freeze-drying and cross-linking methods. Scanning electron microscopy and mechanical measurement were used to characterize the properties of these scaffolds. Bone marrow derived mesenchymal stem cells (BMSCs) were seeded on scaffolds, and cell proliferation was determined by CCK-8 assay, while cell infiltration and differentiation were assessed by histological measurement and quantitative real-time polymerase chain reaction, respectively.ResultsMorphological measurements showed that AYS presented a relatively better three-dimensional structure with larger pore sizes, higher porosity, and better fibers’ alignment compared to AFS. Mechanical testing demonstrated that the tensile property of AFS and AYS was qualitatively similar to the native AF tissue, albeit to a lesser extent. When BMSCs were seeded and cultured on these scaffolds, the number of cells cultured on HS and AYS was found to be significantly higher than that on AFS and culture plate after 7 days of culture (P<0.05). In addition, cell infiltration was significantly higher in HS when compared with AFS and AYS (P<0.05). A part of BMSCs ingressed into the inner part of AYS upon long-term in vitro culture. No significant difference was observed between AFS and AYS in terms of the median infiltration depth (P>0.05). BMSCs seeded on AYS demonstrated an increased expression of COL1A1, while the expression levels of SOX-9, COL2A1, and Aggrecan were higher in HS compared to other scaffolds (P<0.05).ConclusionThese findings indicate that HS makes a proper scaffold for the AF tissue engineering as it replicates the axial compression and tensile property of AF, thereby providing a better platform for cell infiltration and cell–scaffold interaction.

In vitro co-culture of epithelial cells and smooth muscle cells on aligned nanofibrous scaffolds

Esophagus is a complex, hollow organ consisting of epithelial cells in the inner mucosal layer and smooth muscle cells in the outer muscle layer. In the present study, we have evaluated the in vitro co-culture of epithelial cells and smooth muscle cells on the aligned nanofibrous scaffold made of PHBV, PHBV-gelatin, PCL and PCL-gelatin developed through electrospinning using rotating drum collector. Epithelial cells were labeled with cell tracker green while the smooth muscle cells were labeled with cell tracker red. Labeled cells were seeded on the aligned nanofibers matrices and tracked using laser scanning confocal microscopy. The results demonstrate that both epithelial and smooth muscle cells attach, extend, and proliferate over these nanofibrous matrices. Confocal z-sectioning shows that epithelial and smooth muscle cells tend to separate into two distinct layers on a single nanofiber system mimicking the in vivo anatomy. Cell viability assay showed that both types of cells are viable and also interact with each other. The functional gene expression of respective cell types demonstrates that both epithelial and smooth muscle cells are phenotypically as well as functionally active when they were co-cultured. Thus the study highlighted that aligned nanofibrous scaffolds could be potential alternative graft for esophageal tissue regeneration.

* In Vitro Generated Intervertebral Discs: Toward Engineering Tissue Integration.

The intervertebral disc (IVD) is composed of nucleus pulposus (NP) surrounded by multilamellated annulus fibrosus (AF), and is located between the vertebral bodies. Current treatments for chronic neck or low back pain do not completely restore the functionality of degenerated IVDs. Thus, developing biological disc replacements is an approach of great interest. Given the complex structure of the IVD, tissue engineering of the individual IVD components and then combining them together may be the only way to achieve this. The engineered disc must then be able to integrate into the host spine to ensure mechanical stability. The goal of this study was to generate an integrated model of an IVD in vitro. Multilamellated AF tissues were generated in vitro using aligned nanofibrous polycarbonate urethane scaffolds and AF cells. After 3 weeks in culture, it was placed around NP tissue formed on and integrated with a porous bone substitute material (calcium polyphosphate). The two tissues were cocultured to fabricate the IVD model. The AF tissue composed of six lamellae containing type I collagen-rich extracellular matrix (ECM) and the NP tissue had type II collagen- and aggrecan-rich ECM. Immunofluorescence studies showed both type I and II collagen at the AF-NP interface. There was evidence of integration of the tissues. The peel test for AF lamellae showed an interlamellar shear stress of 0.03 N/mm. The AF and NP were integrated as the pushout test demonstrated that the AF-NP interface had significantly increased mechanical stability by 2 weeks of coculture. To evaluate if these tissues remained integrated, allogeneic IVD model constructs were implanted into defects freshly made in the NP-inner AF and bone of the bovine coccygeal spine. One month postimplantation, the interfaces between the AF lamellae remained intact and there was integration with the host AF tissue. No inflammatory reaction was noted at this time period. In summary, an engineered IVD implant with mechanically stable integration between AF lamellae and AF-NP can be generated in vitro. Further study is required to scale up the size of this construct and evaluate its ability to serve as a biological disc replacement.

Heparin/collagen encapsulating nerve growth factor multilayers coated aligned PLLA nanofibrous scaffolds for nerve tissue engineering.

Biomimicing topological structure of natural nerve tissue to direct axon growth and controlling sustained release of moderate neurotrophic factors are extremely propitious to the functional recovery of damaged nervous systems. In this study, the heparin/collagen encapsulating nerve growth factor (NGF) multilayers were coated onto the aligned poly-L-lactide (PLLA) nanofibrous scaffolds via a layer-by-layer (LbL) self-assembly technique to combine biomolecular signals, and physical guidance cues for peripheral nerve regeneration. Scanning electronic microscopy (SEM) revealed that the surface of aligned PLLA nanofibrous scaffolds coated with heparin/collagen multilayers became rougher and appeared some net-like filaments and protuberances in comparison with PLLA nanofibrous scaffolds. The heparin/collagen multilayers did not destroy the alignment of nanofibers. X-ray photoelectron spectroscopy and water contact angles displayed that heparin and collagen were successfully coated onto the aligned PLLA nanofibrous scaffolds and improved its hydrophilicity. Three-dimensional (3 D) confocal microscopy images further demonstrated that collagen, heparin, and NGF were not only coated onto the surface of aligned PLLA nanofibrous scaffolds but also permeated into the inner of scaffolds. Moreover, NGF presented a sustained release for 2 weeks from aligned nanofibrous scaffolds coated with 5.5 bilayers or above and remained good bioactivity. The heparin/collagen encapsulating NGF multilayers coated aligned nanofibrous scaffolds, in particular 5.5 bilayers or above, was more beneficial to Schwann cells (SCs) proliferation and PC12 cells differentiation as well as the SC cytoskeleton and neurite growth along the direction of nanofibrous alignment compared to the aligned PLLA nanofibrous scaffolds. This novel scaffolds combining sustained release of bioactive NGF and aligned nanofibrous topography presented an excellent potential in peripheral nerve regeneration. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1900-1910, 2017.