In vivo tracking of unlabelled mesenchymal stromal cells by mannose-weighted chemical exchange saturation transfer MRI.

The tracking of the in vivo biodistribution of transplanted human mesenchymal stromal cells (hMSCs) relies on reporter genes or on the addition of exogenous imaging agents. However, reporter genes and exogenous labels may require bespoke manufacturing and regulatory processes if used in cell therapies, and the labels may alter the cells' properties and are diluted on cellular division. Here we show that high-mannose N-linked glycans, which are abundantly expressed on the surface of hMSCs, can serve as a biomarker for the label-free tracking of transplanted hMSCs by mannose-weighted chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI). For live mice with luciferase-transfected hMSCs transplanted into their brains, post-mortem fluorescence staining with a mannose-specific lectin showed that increases in the CEST MRI signal, which correlated well with the bioluminescence intensity of viable hMSCs for 14 days, corresponded to the presence of mannose. In vitro, osteogenically differentiated hMSCs led to lower CEST MRI signal intensities owing to the concomitantly reduced expression of mannose. The label-free imaging of hMSCs may facilitate the development and testing of cell therapies.

In Vivo Imaging of Implanted Hyaluronic Acid Hydrogel Biodegradation.

Although the use of stem cell therapy for central nervous system (CNS) repair has shown considerable promise, it is still limited by the immediate death of a large fraction of transplanted cells owing to cell handling procedures, injection stress and host immune attack leading to poor therapeutic outcomes. Scaffolding cells in hydrogels is known to protect cells from such immediate death by shielding them from mechanical damage and by averting an immune attack after transplantation. Implanted hydrogels must eventually degrade and facilitate a safe integration of the graft with the surrounding host tissue. Hence, serial monitoring of hydrogel degradation in vivo is pivotal to optimize hydrogel compositions and overall therapeutic efficacy of the graft. We present here methods and protocols to use chemical exchange saturation transfer magnetic resonance imaging (CEST MRI) as a non-invasive, label-free imaging paradigm to monitor the degradation of composite hydrogels made up of thiolated gelatin (Gel-SH), thiolated hyaluronic acid (HA-SH), and poly (ethylene glycol) diacrylate (PEGDA), of which the stiffness and CEST contrast can be fine-tuned by simply varying the composite concentrations and mixing ratios. By individually labeling Gel-S and HA-S with two distinct near-infrared (NIR) dyes, multispectral monitoring of the relative degradation of the components can be used for long-term validation of the CEST MRI findings.

In Vivo Imaging of Allografted Glial-Restricted Progenitor Cell Survival and Hydrogel Scaffold Biodegradation.

Transplanted glial-restricted progenitor (GRP) cells have potential to focally replace defunct astrocytes and produce remyelinating oligodendrocytes to avert neuronal death and dysfunction. However, most central nervous system cell therapeutic paradigms are hampered by high initial cell death and a host anti-graft immune response. We show here that composite hyaluronic acid-based hydrogels of tunable mechanical strengths can significantly improve transplanted GRP survival and differentiation. Allogeneic GRPs expressing green fluorescent protein and firefly luciferase were scaffolded in optimized hydrogel formulations and transplanted intracerebrally into immunocompetent BALB/c mice followed by serial in vivo bioluminescent imaging and chemical exchange saturation transfer magnetic resonance imaging (CEST MRI). We demonstrate that gelatin-sensitive CEST MRI can be exploited to monitor hydrogel scaffold degradation in vivo for ∼5 weeks post transplantation without necessitating exogenous labeling. Hydrogel scaffolding of GRPs resulted in a 4.5-fold increase in transplanted cell survival at day 32 post transplantation compared to naked cells. Histological analysis showed significant enhancement of cell proliferation as well as Olig2+ and GFAP+ cell differentiation for scaffolded cells compared to naked cells, with reduced host immunoreactivity. Hence, hydrogel scaffolding of transplanted GRPs in conjunction with serial in vivo imaging of cell survival and hydrogel degradation has potential for further advances in glial cell therapy.

In Vivo Imaging of Composite Hydrogel Scaffold Degradation Using CEST MRI and Two-Color NIR Imaging.

Hydrogel scaffolding of stem cells is a promising strategy to overcome initial cell loss and manipulate cell function post-transplantation. Matrix degradation is a requirement for downstream cell differentiation and functional tissue integration, which determines therapeutic outcome. Therefore, monitoring of hydrogel degradation is essential for scaffolded cell replacement therapies. We show here that chemical exchange saturation transfer magnetic resonance imaging (CEST MRI) can be used as a label-free imaging platform for monitoring the degradation of crosslinked hydrogels containing gelatin (Gel) and hyaluronic acid (HA), of which the stiffness can be fine-tuned by varying the ratio of the Gel:HA. By labeling Gel and HA with two different NIR dyes having distinct emission excitation frequencies, we show here that the HA signal remains stable for 42 days, while the Gel signal gradually decreases to <25% of its initial value at this time point. Both imaging modalities were in excellent agreement for both the time course and relative value of CEST MRI and NIR signals (R2=0.94). These findings support the further use of CEST MRI for monitoring biodegradation and optimizing of gelatin-containing hydrogels in a label-free manner.

Combined effects of multi-scale topographical cues on stable cell sheet formation and differentiation of mesenchymal stem cells.

To decipher specific cell responses to diverse and complex in vivo signals, it is essential to emulate specific surface chemicals, extra cellular matrix (ECM) components and topographical signals through reliable and easily reproducible in vitro systems. However, the effect of multiple cues such as micro-hole/pillar architectures under a common and easily tunable platform remains unexplored. Recently we have demonstrated the positive influence of surface chemical modification of polydimethylsiloxane (PDMS) surfaces on directing long-term adhesion, viability and potency of hMSCs. In this study, we include biophysical signals from diverse surface topographical elements along with biochemical influences to develop a holistic understanding of hMSC responses in complex tissue-like niches. We report the influence of chemically modified PDMS structures encompassing hole-, pillar- and groove-based multi-scale architectures on hMSC morphology, adhesion, proliferation and differentiation. The inclusion of hole and pillar features resulted in enhanced adhesion and proliferation of hMSCs. These effects were more pronounced with the inclusion of grooves, which resulted in the highest osteogenic differentiation among other substrates. Our study provides an additional basis for the chemical/physical regulation of hMSC behavior within controlled biomimetic architectures with an aim to foster efficient tissue regeneration strategies.

Fabrication and Characterization of Three-Dimensional (3D) Core-Shell Structure Nanofibers Designed for 3D Dynamic Cell Culture.

Three-dimensional elastic nanofibers (3D eNFs) can offer a suitable 3D dynamic microenvironment and sufficient flexibility to regulate cellular behavior and functional protein expression. In this study, we report a novel approach to prepare 3D nanofibers with excellent mechanical properties by solution-assisted electrospinning technology and in situ polymerization. The obtained 3D eNFs demonstrated excellent biocompatible properties to meet cell culture requirements under a dynamic environment in vitro. Moreover, these 3D eNFs also promoted human bone marrow mesenchymal stem cells (hMSCs) adhesion and collagen expression under biomechanical stimulation. The results demonstrated that this dynamic cell culture system could positively impact cellular collagen but has no significant effect on the proliferation of hMSCs grown in the 3D eNFs. This work may give rise to a new approach for constructing a 3D cell culture for tissue engineering.

A novel approach for fabricating highly tunable and fluffy bioinspired 3D poly(vinyl alcohol) (PVA) fiber scaffolds.

The excellent biocompatibility, biodegradability and chemo-thermal stability of poly(vinyl alcohol) (PVA) have been harnessed in diverse practical applications. These properties have motivated the fabrication of high performance PVA based nanofibers with adequate control over the micro and nano-architectures and surface chemical interactions. However, the high water solubility and hydrophilicity of the PVA polymer limits the application of the electrospun PVA nanofibers in aqueous environments owing to instantaneous dissolution. In this work, we report a novel yet facile concept for fabricating extremely light, fluffy, insoluble and stable three dimensional (3D) PVA fibrous scaffolds with/without coating for multifunctional purposes. While the solubility, morphology, fiber density and mechanical properties of nanofibers could be tuned by optimizing the cross-linking conditions, the surface chemical reactivity could be readily enhanced by coating with a polydopamine (pDA) bioinspired polymer without compromising the stability and innate properties of the native PVA fiber. The 3D pDA-PVA scaffolds exhibited super dye adsorption and constructive synergistic cell-material interactions by promoting healthy adhesion and viability of the human mesenchymal stem cells (hMSCs) within 3D micro-niches. We foresee the application of tunable PVA 3D as a highly adsorbent material and a scaffold material for tissue regeneration and drug delivery with close consideration of realistic in vivo parameters.

Fabrication, Characterization, and Biocompatibility of Polymer Cored Reduced Graphene Oxide Nanofibers.

Graphene nanofibers have shown a promising potential across a wide spectrum of areas, including biology, energy, and the environment. However, fabrication of graphene nanofibers remains a challenging issue due to the broad size distribution and extremely poor solubility of graphene. Herein, we report a facile yet efficient approach for fabricating a novel class of polymer core-reduced graphene oxide shell nanofiber mat (RGO-CSNFM) by direct heat-driven self-assembly of graphene oxide sheets onto the surface of electrospun polymeric nanofibers without any requirement of surface treatment. Thus-prepared RGO-CSNFM demonstrated excellent mechanical, electrical, and biocompatible properties. RGO-CSNFM also promoted a higher cell anchorage and proliferation of human bone marrow mesenchymal stem cells (hMSCs) compared to the free-standing RGO film without the nanoscale fibrous structure. Further, cell viability of hMSCs was comparable to that on the tissue culture plates (TCPs) with a distinctive healthy morphology, indicating that the nanoscale fibrous architecture plays a critically constructive role in supporting cellular activities. In addition, the RGO-CSNFM exhibited excellent electrical conductivity, making them an ideal candidate for conductive cell culture, biosensing, and tissue engineering applications. These findings could provide a new benchmark for preparing well-defined graphene-based nanomaterial configurations and interfaces for biomedical applications.

Biocompatible, Free-Standing Film Composed of Bacterial Cellulose Nanofibers-Graphene Composite.

In recent years, graphene films have been used in a series of wide applications in the biomedical area, because of several advantageous characteristics. Currently, these films are derived from graphene oxide (GO) via chemical or physical reduction methods, which results in a significant decrease in surface hydrophilicity, although the electrical property could be greatly improved, because of the reduction process. Hence, the comprehensive performance of the graphene films showed practical limitations in the biomedical field, because of incompatibility of highly hydrophobic surfaces to support cell adhesion and growth. In this work, we present a novel fabrication of bacterial cellulose nanofibers/reduced graphene oxide (BC-RGO) film, using a bacterial reduction method. Thus-prepared BC-RGO films maintained excellent hydrophilicity, while electrical properties were improved by bacterial reduction of GO films in culture. Human marrow mesenchymal stem cells (hMSCs) cultured on these surfaces showed improved cellular response with higher cell proliferation on the BC-RGO film, compared to free-standing reduced graphene oxide film without the nanoscale fibrous structure. Furthermore, the cellular adhesion and proliferation were even comparable to that on the tissue culture plate, indicating that the bacterial cellulose nanofibers play a critically contructive role in supporting cellular activities. The novel fabrication method greatly enhanced the biochemical activity of the cells on the surface, which could aid in realizing several potential applications of graphene film in biomedical area, such as tissue engineering, bacterial devices, etc.

Synergistic Effects of Conductive Three-Dimensional Nanofibrous Microenvironments and Electrical Stimulation on the Viability and Proliferation of Mesenchymal Stem Cells.

In recent years, three-dimensional (3D) scaffolds have proven to be highly advantageous in mammalian cell culture and tissue engineering compared to 2D substrates. Herein, we demonstrated the fabrication of novel 3D core-shell nanofibers (3D-CSNFs) using an improved electrospinning process combined with in situ surface polymerization. The obtained 3D nanofibrous scaffold displayed excellent mechanical and electrical properties. Moreover, the cotton-like 3D structure with large internal connected pores (20-100 µm) enabled cells to easily infiltrate into the interior of the 3D scaffold with a good spatial distribution to mimic the ECM-like cell microenvironments. Stable cell-fiber composite constructs were formed in the 3D-CSNFs with relatively higher adhesion and viability compared to 2D-CSNFs. Furthermore, the human mesenchymal stem cells (hMSCs) cultured on conductive polymer coated electrically active 3D nanofibers responded with a healthy morphology and anchorage on the fibers with relatively higher viability and proliferation under electrical stimulation (ES). This study demonstrates the successful fabrication of 3D-CSNFs and the constructive interaction of the 3D microenvironment and subsequent electrical stimulations on hMSCs, thereby holding promising potential in tissue engineering and regenerative therapies aided by electro-stimulation-based differentiation strategies.

Enhanced In Vitro Biocompatibility of Chemically Modified Poly(dimethylsiloxane) Surfaces for Stable Adhesion and Long-term Investigation of Brain Cerebral Cortex Cells.

Studies on the mammalian brain cerebral cortex have gained increasing importance due to the relevance of the region in controlling critical higher brain functions. Interactions between the cortical cells and surface extracellular matrix (ECM) proteins play a pivotal role in promoting stable cell adhesion, growth, and function. Poly(dimethylsiloxane) (PDMS) based platforms have been increasingly used for on-chip in vitro cellular system analysis. However, the inherent hydrophobicity of the PDMS surface has been unfavorable for any long-term cell system investigations due to transitory physical adsorption of ECM proteins on PDMS surfaces followed by eventual cell dislodgement due to poor anchorage and viability. To address this critical issue, we employed the (3-aminopropyl)triethoxysilane (APTES) based cross-linking strategy to stabilize ECM protein immobilization on PDMS. The efficiency of surface modification in supporting adhesion and long-term viability of neuronal and glial cells was analyzed. The chemically modified surfaces showed a relatively higher cell survival with an increased neurite length and neurite branching. These changes were understood in terms of an increase in surface hydrophilicity, protein stability, and cell-ECM protein interactions. The modification strategy could be successfully applied for stable cortical cell culture on the PDMS microchip for up to 3 weeks in vitro.

The effects of poly(dimethylsiloxane) surface silanization on the mesenchymal stem cell fate.

In recent years, poly(dimethylsiloxane) (PDMS)-based microfluidic devices have become very popular for on-chip cell investigation. Maintenance of mammalian cell adhesion on the substrate surface is crucial in determining the cell viability, proliferation and differentiation. However, the inherent hydrophobicity of PDMS is unfavourable for cell culture, causing cells to eventually dislodge from the surface. Although physically adsorbed matrix proteins can promote initial cell adhesion, this effect is usually short-lived. To address this critical issue, in this study, we employed (3-aminopropyl) triethoxy silane (APTES) and cross-linker glutaraldehyde (GA) chemistry to immobilize collagen type 1 (Col1) on PDMS. These modified surfaces are highly efficient to support the adhesion of mesenchymal stem cells (MSCs) with no deterioration of their potency. Significant changes of the native PDMS surface properties were observed with the proposed surface functionalization, and MSC adhesion was improved on PDMS surfaces modified with APTES + GA + Protein. Therefore, this covalent surface modification could generate a more biocompatible platform for stabilized cell adhesion. Furthermore, this modification method facilitated long-term cell attachment, which is favourable for successful induction of osteogenesis and cell sheet formation with an increased expression of osteogenic biomarkers and comparable extracellular matrix (ECM) constituent biomarkers, respectively. The surface silanization can be applied to PDMS-based microfluidic systems for long-term study of cellular development. Similar strategies could also be applied to several other substrate materials by appropriate combinations of self-assembled monolayers (SAMs) and ECM proteins.

Synergistic effects of a novel free-standing reduced graphene oxide film and surface coating fibronectin on morphology, adhesion and proliferation of mesenchymal stem cells.

Graphene films have broad use in engineering, energy and biomedical applications. The cost-effective, eco-friendly and easy to scale-up fabrication methods of graphene films are always highly desired. In this work, we develop a novel fabrication method of free-standing reduced graphene oxide (RGO) films by vacuum filtration of graphene oxide aqueous solution through a nanofiber membrane in combination with chemical reduction. Instead of the smooth surface, the generated RGO films have nanoscale patterns transferred from the nanofiber membrane and controlled in a large range by varying the parameters of the electrospinning process. The cellular culture results of the human marrow mesenchymal stem cells (hMSCs) show that the fibronectin modified RGO films could exhibit excellent biocompatibility, which could be attributed to the synergistic effects of the RGO films including both surface morphology and fibronectin modification. The novel fabrication method greatly enhances the fabrication capability and the potential of graphene films for application in cell culture, tissue engineering as well as in other engineering and biomedical applications.

Surface chemical modification of poly(dimethylsiloxane) for the enhanced adhesion and proliferation of mesenchymal stem cells.

The surface chemistry of materials has an interactive influence on cell behavior. The optimal adhesion of mammalian cells is critical in determining the cell viability and proliferation on substrate surfaces. Because of the inherent high hydrophobicity of a poly(dimethylsiloxane) (PDMS) surface, cell culture on these surfaces is unfavorable, causing cells to eventually dislodge from the surface. Although physically adsorbed matrix proteins can promote initial cell adhesion, this effect is usually short-lived. Here, (3-aminopropyl)triethoxy silane (APTES) and cross-linker glutaraldehyde (GA) chemistry was employed to immobilize either fibronectin (FN) or collagen type 1 (C1) on PDMS. The efficiency of these surfaces to support the adhesion and viability of mesenchymal stem cells (MSCs) was analyzed. The hydrophobicity of the native PDMS decreased significantly with the mentioned surface functionalization. The adhesion of MSCs was mostly favorable on chemically modified PDMS surfaces with APTES + GA + protein. Additionally, the spreading area of MSCs was significantly higher on APTES + GA + C1 surfaces than on other unmodified/modified PDMS surfaces with C1 adsorption. However, there were no significant differences in the MSC spreading area on the unmodified/modified PDMS surfaces with FN adsorption. Furthermore, there was a significant increase in cell proliferation on the PDMS surface with APTES + GA + protein functionalization as compared to the PDMS surface with protein adsorption only. Therefore, the covalent surface chemical modification of PDMS with APTES + GA + protein could offer a more biocompatible platform for the enhanced adhesion and proliferation of MSCs. Similar strategies can be applied for other substrates and cell lines by appropriate combinations of self-assembly monolayers (SAMs) and extracellular matrix proteins.