Electrical Stimulation of Human Mesenchymal Stem Cells on Conductive Substrates Promotes Neural Priming

Electrical stimulation (ES) within a conductive scaffold is potentially beneficial in encouraging the differentiation of stem cells toward a neuronal phenotype. To improve stem cell-based regenerative therapies, it is essential to use electroconductive scaffolds with appropriate stiffnesses to regulate the amount and location of ES delivery. Herein, biodegradable electroconductive substrates with different stiffnesses are fabricated from chitosan-grafted-polyaniline (CS-g-PANI) copolymers. Human mesenchymal stem cells (hMSCs) cultured on soft conductive scaffolds show a morphological change with significant filopodial elongation after electrically stimulated culture along with upregulation of neuronal markers and downregulation of glial markers. Compared to stiff conductive scaffolds and non-conductive CS scaffolds, soft conductive CS-g-PANI scaffolds promote increased expression of microtubule-associated protein 2 (MAP2) and neurofilament heavy chain (NF-H) after application of ES. At the same time, there is a decrease in the expression of the glial markers glial fibrillary acidic protein (GFAP) and vimentin after ES. Furthermore, the elevation of intracellular calcium [Ca²⁺] during spontaneous, cell-generated Ca²⁺ transients further suggests that electric field stimulation of hMSCs cultured on conductive substrates can promote a neural-like phenotype. The findings suggest that the combination of the soft conductive CS-g-PANI substrate and ES is a promising new tool for enhancing neuronal tissue engineering outcomes.     

3D Plotted PCL Scaffolds for Stem Cell Based Bone Tissue Engineering

The ability to control the architecture and strength of a bone tissue engineering scaffold is critical to achieve a harmony between the scaffold and the host tissue. Rapid prototyping (RP) technique is applied to tissue engineering to satisfy this need and to create a scaffold directly from the scanned and digitized image of the defect site. Design and construction of complex structures with different shapes and sizes, at micro and macro scale, with fully interconnected pore structure and appropriate mechanical properties are possible by using RP techniques. In this study, RP was used for the production of poly(ε-caprolactone) (PCL) scaffolds. Scaffolds with four different architectures were produced by using different configurations of the fibers (basic, basic-offset, crossed and crossed-offset) within the architecture of the scaffold. The structure of the prepared scaffolds were examined by scanning electron microscopy (SEM), porosity and its distribution were analyzed by micro-computed tomography (µ-CT), stiffness and modulus values were determined by dynamic mechanical analysis (DMA). It was observed that the scaffolds had very ordered structures with mean porosities about 60%, and having storage modulus values about 1 × 10⁷ Pa. These structures were then seeded with rat bone marrow origin mesenchymal stem cells (MSCs) in order to investigate the effect of scaffold structure on the cell behavior; the proliferation and differentiation of the cells on the scaffolds were studied. It was observed that cell proliferation was higher on offset scaffolds (262000 vs 235000 for basic, 287000 vs 222000 for crossed structure) and stainings for actin filaments of the cells reveal successful attachment and spreading at the surfaces of the fibers. Alkaline phosphatase (ALP) activity results were higher for the samples with lower cell proliferation, as expected. Highest MSC differentiation was observed for crossed scaffolds indicating the influence of scaffold structure on cellular activities.     

Conductive Biomaterials as Substrates for Neural Stem Cells Differentiation towards Neuronal Lineage Cells

The injuries and defects in the central nervous system are the causes of disability and death of an affected person. As of now, there are no clinically available methods to enhance neural structural regeneration and functional recovery of nerve injuries. Recently, some experimental studies claimed that the injuries in brain can be repaired by progenitor or neural stem cells located in the neurogenic sites of adult mammalian brain. Various attempts have been made to construct biomimetic physiological microenvironment for neural stem cells to control their ultimate fate. Conductive materials have been considered as one the best choices for nerve regeneration due to the capacity to mimic the microenvironment of stem cells and regulate the alignment, growth, and differentiation of neural stem cells. The review highlights the use of conductive biomaterials, e.g., polypyrrole, polyaniline, poly(3,4‐ethylenedioxythiophene), multi‐walled carbon nanotubes, single‐wall carbon nanotubes, graphene, and graphite oxide, for controlling the neural stem cells activities in terms of proliferation and neuronal differentiation. The effects of conductive biomaterials in axon elongation and synapse formation for optimal repair of central nervous system injuries are also discussed.     

Screening of Hydrogels for Human Pluripotent Stem Cell–Derived Neural Cells: Hyaluronan‐Polyvinyl Alcohol‐Collagen‐Based Interpenetrating Polymer Network Provides an Improved Hydrogel Scaffold

There is a clear need for novel in vitro models, especially for neuronal applications. Development of in vitro models is a multiparameter task consisting of cell‐, biomaterial‐, and environment‐related parameters. Here, three different human origin neuronal cell sources are studied and cultured in various hydrogel 3D scaffolds. For the efficient evaluation of complex results, an indexing method for data is developed and used in principal component analysis (PCA). It is found that no single hydrogel is superior to other hydrogels, and collagen I (Col1) and hyaluronan–poly(vinyl alcohol) (HA1‐PVA) gels are combined into an interpenetrating network (IPN) hydrogel. The IPN gel combines cell supportiveness of the collagen gel and stability of the HA1‐PVA gel. Moreover, cell adhesion is studied in particular and it is found that adhesion of neurons differs from that observed for fibroblasts. In conclusion, the HA1‐PVA‐col1 hydrogel is a suitable scaffold for neuronal cells and supports adhesion formation in 3D.     

Effect of Degree of Deacetylation of Chitosan/Chitin on Human Neural Stem Cell Culture

Stem cell therapy and research for neural diseases depends on reliable reproduction of neural stem cells. Chitosan-based materials have been proposed as a substrate for culturing human neural stem cells (hNSCs) in the pursuit of clinically compatible culture conditions that are chemically defined and compliant with good manufacturing practices. The physical and biochemical properties of chitosan and chitin are strongly regulated by the degree of deacetylation (DD). However, the effect of DD on hNSC behavior has not been systematically investigated. In this study, films with DD ranging from 93% to 14% are fabricated with chitosan and chitin. Under xeno-free conditions, hNSCs proliferate preferentially on films with a higher DD, exhibiting adherent morphology and retaining multipotency. Lowering the DD leads to formation of neural stem cell spheroids due to unsteady adhesion. The neural spheroids present NSC multipotency protein expression reduction and cytoplasmic translocation. This study provides an insight into the influence of the DD on hNSCs behavior and may serve as a guideline for hNSC research using chitosan-based biomaterials. It demonstrates the capability of controlling hNSC fate by simply tailoring the DD of chitosan.     

Injectable Nanohybrid Scaffold for Biopharmaceuticals Delivery and Soft Tissue Engineering

An injectable nanofibrous hydrogel scaffold integrated with growth factors (GFs) loaded polysaccharide nanoparticles was developed that specifically allows for targeted adipose‐derived stem cells (ASCs) encapsulation and soft tissue engineering. The nanofibrous hydrogel was produced via biological conjugation of biotin‐terminated star‐shaped poly(ethylene glycol) (PEG‐Biotin) and streptavidin‐functionalized hyaluronic acid (HA‐Streptavidin). The polysaccharide nanoparticles were noncovalently assembled via electrostatic interactions between low‐molecular‐weight heparin (LMWH) and N,N,N‐trimethylchitosan chloride (TMC). Vascular endothelial growth factor (VEGF) was entrapped in the LMWH/TMC nanoparticles by affinity interactions with LMWH.     

Polymeric Fibrous Matrices for Substrate‐Mediated Human Embryonic Stem Cell Lineage Differentiation

Chitosan‐based fibrous matrices are prepared to mimic the ECM architecture and elucidate substrate‐mediated hESC differentiation due to topographical scale and anisotropy without exogenic morphogens. Fibrous matrices support fewer pluripotent hESCs than films but enable topography‐mediated hESC differentiation. Matrices composed of 400 nm and 1.1 µm diameter fibers support increased expression of neural markers indicative of ectodermal commitment while matrices of 200 nm diameter fibers increase expression of osteogenic and hepatic markers indicative of endodermal and mesodermal commitment. The fibrous‐mediated hESC differentiation highlights the significant implication of tailored ECM‐like substrates for hESC‐based therapies.

     

Self‐Assembled Peptide‐Based Hydrogels as Scaffolds for Proliferation and Multi‐Differentiation of Mesenchymal Stem Cells

Fluorenyl‐9‐methoxycarbonyl (Fmoc)‐diphenylalanine (Fmoc‐FF) and Fmoc‐arginine‐glycine‐­aspartate (Fmoc‐RGD) peptides self‐assemble to form a 3D network of supramolecular hydrogel (Fmoc‐FF/Fmoc‐RGD), which provides a nanofibrous network that uniquely presents bioactive ligands at the fiber surface for cell attachment. In the present study, mesenchymal stem cells (MSCs) in Fmoc‐FF/Fmoc‐RGD hydrogel increase in proliferation and survival compared to those in Fmoc‐FF/Fmoc‐RGE hydrogel. Moreover, MSCs encapsulated in Fmoc‐FF/Fmoc‐RGD hydrogel and induced in each defined induction medium undergo in vitro osteogenic, adipogenic, and chondrogenic differentiation. For in vivo differentiation, MSCs encapsulated in hydrogel are induced in each defined medium for one week, followed by injection into gelatin sponges and transplantation into immunodeficient mice for four weeks. MSCs in Fmoc‐FF/Fmoc‐RGD hydrogel increase in differentiation into osteogenic, adipogenic, and chondrogenic differentiation, compared to those in Fmoc‐FF/Fmoc‐RGE hydrogel. This study concludes that nanofibers formed by the self‐assembly of Fmoc‐FF and Fmoc‐RGD are suitable for the attachment, proliferation, and multi‐differentiation of MSCs, and can be applied in musculoskeletal tissue engineering.

     

Electroactive Composite of FeCl3‐Doped P3HT/PLGA with Adjustable Electrical Conductivity for Potential Application in Neural Tissue Engineering

Conducting polymers (CPs) is one of intelligent biomaterials with the specific properties of reversible redox states, which have a significant effects on the cell behaviors and nerve tissue regeneration. However, the effects of CPs with different electrical conductivity on the behaviors of nerve cells are rarely reported. Therefore, a kind of Poly(3‐hexylthiophene) (P3HT) with certain molecular weight is synthesized by Kumada catalyst transfer polymerization (KCTP) method and employed to prepare bioabsorbable and electroactive intelligent composites of Poly(3‐hexylthiophene)/Poly(glycolide‐lactide) (P3HT/PLGA). FeCl₃ doping electroactive membranes with different electrical conductivities are prepared to investigate the cell behaviors. On the substrate with higher electrical conductivity, the proliferation of rat adrenal pheochromocytoma cells (PC12 cells) is significantly promoted and neurite length is increased obviously. In particular, the most significant improvements are the neuron gene expression of Synapsin 1 and microtubule‐associated protein 2 (MAP2) by the composites with high conductivity. These results suggest that P3HT/PLGA with suitable electrical conductivity have a positive role in promoting neural growth and differentiation, which is promising for advancing potential application of nerve repair and regeneration.     

Cell(MC3T3‐E1)‐Printed Poly(ϵ‐caprolactone)/Alginate Hybrid Scaffolds for Tissue Regeneration

A new cell‐printed scaffold consisting of poly(ϵ‐caprolactone) (PCL) and cell‐embedded alginate struts is designed. The PCL and alginate struts are stacked in an interdigitated pattern in successive layers to acquire a three‐dimensional (3D) shape. The hybrid scaffold exhibits a two‐phase structure consisting of cell (MC3T3‐E1)‐laden alginate struts able to support biological activity and PCL struts able to provide controllable mechanical support of the cell‐laden alginate struts. The hybrid scaffolds exhibit an impressive increase in tensile modulus and maximum strength compared to pure alginate scaffolds. Laden cells are homogeneously distributed throughout the alginate struts and the entire scaffold, resulting in cell viability of approximately 84%.     

3D Microfibrous Scaffolds Selectively Promotes Proliferation and Glial Differentiation of Adult Neural Stem Cells: A Platform to Tune Cellular Behavior in Neural Tissue Engineering

Biomaterials are essential for the development of innovative biomedical and therapeutic applications. Biomaterials‐based scaffolds can influence directed cell differentiation to improve cell‐based strategies. Using a novel microfluidics approach, poly (ε‐caprolactone) (PCL), is used to fabricate microfibers with varying diameters (3–40 µm) and topographies (straight and wavy). Multipotent adult rat hippocampal stem/progenitor cells (AHPCs) are cultured on 3D aligned PCL microfibrous scaffolds to investigate their ability to differentiate into neurons, astrocytes, and oligodendrocytes. The results indicate that the PCL microfibers significantly enhance proliferation of the AHPCs compared to control, 2D planar substrates. While the AHPCs maintained their multipotent differentiation capacity when cultured on the PCL scaffolds, there is a significant and dramatic increase in immunolabeling for astrocyte and oligodendrocyte differentiation when compared with growth on planar surfaces. Our results show a 3.5‐fold increase in proliferation and 23.4‐fold increase in astrocyte differentiation for cells on microfibers. Transplantation of neural stem/progenitor cells within a PCL microfiber scaffold may provide important biological and topographic cues that facilitate the survival, selective differentiation, and integration of transplanted cells to improve therapeutic strategies.     

Simple Radical Polymerization of Poly(Alginate‐Graft‐N‐Isopropylacrylamide) Injectable Thermoresponsive Hydrogel with the Potential for Localized and Sustained Delivery of Stem Cells and Bioactive Molecules

In this study, thermoresponsive copolymers that are fully injectable, biocompatible, and biodegradable and are synthesized via graft copolymerization of poly(N‐isopropylacrylamide) onto alginate using a free‐radical reaction are presented. This new synthesis method does not involve multisteps or associated toxicity issues, and has the potential to reduce scale‐up difficulties. Chemical and physical analyses verify the resultant graft copolymer structure. The lower critical solution temperature, which is a characteristic of sol–gel transition, is observed at 32 °C. The degradation properties indicate suitable degradation kinetics for drug delivery and bone tissue engineering applications. The synthesized P(Alg‐g‐NIPAAm) hydrogel is noncytotoxic with both human osteosarcoma (MG63) cells and porcine bone marrow derived mesenchymal stem cells (pBMSCs). pBMSCs encapsulated in the P(Alg‐g‐NIPAAm) hydrogel remain viable, show uniform distribution within the injected hydrogel, and undergo osteogenic and chondrogenic differentiation under appropriate culture conditions. Furthermore, for the first time, this work will explore the influence of alginate viscosity on the viscoelastic properties of the resulting copolymer hydrogels, which influences the rate of medical device formation and subsequent drug release. Together the results of this study indicate that the newly synthesized P(Alg‐g‐NIPAAm) hydrogel has potential to serve as a versatile and improved injectable platform for drug delivery and bone tissue engineering applications.     

Physically Crosslinked Nanocomposites from Silicate‐Crosslinked PEO: Mechanical Properties and Osteogenic Differentiation of Human Mesenchymal Stem Cells

The mechanical and biological properties of silicate‐crosslinked PEO nanocomposites are studied. A strong correlation is observed between silicate concentration and mechanical properties. In vitro cell culture studies reveal that an increase in silicate concentration enhances the attachment and proliferation of human mesenchymal stem cells significantly. An upregulation in the expression of osteocalcin on nanocomposites compared to the tissue culture polystyrene control is observed. Together, these results suggest that silicate‐based nanocomposites are bioactive and have the potential to be used in a range of biotechnological and biomedical applications such as injectable matrices, biomedical coatings, drug delivery, and regenerative medicine.

     

Hyaluronan/Collagen Hydrogels with Sulfated Hyaluronan for Improved Repair of Vascularized Tissue Tune the Binding of Proteins and Promote Endothelial Cell Growth

Innovative biomaterial‐based concepts are required to improve wound healing of damaged vascularized tissues especially in elderly multimorbid patients. To develop functional hydrogels as 3D cellular microenvironments and as carrier or scavenging systems, e.g., for mediator proteins or proinflammatory factors, collagen fibrils are embedded into a network of photo‐crosslinked acrylated hyaluronan (HA), chondroitin sulfate (CS), or sulfated HA (sHA). After lyophilization, the gels show a porous structure and an improved stability against degradation via hyaluronidase. Gels with CS and sHA bind significantly more lysozyme than HA/collagen gels and retard its release. The proliferation and metabolic activity of endothelial cells are significantly increased on sHA gels compared to CS‐ or only HA‐containing hydrogels. These findings highlight the potential of HA/collagen hydrogels with sulfated glycosaminoglycans to tune the protein binding and release behavior and to directly modulate cellular response. This can be easily translated into biomimetic biomaterials with defined properties to stimulate wound healing.     

Poly‐l‐Lactic Acid Nanofiber–Polyamidoamine Hydrogel Composites: Preparation,Properties, and Preliminary Evaluation as Scaffolds for Human Pluripotent Stem Cell Culturing

Electrospun poly‐l ‐lactic acid (PLLA) nanofiber mats carrying surface amine groups, previously introduced by nitrogen atmospheric pressure nonequilibrium plasma, are embedded into aqueous solutions of oligomeric acrylamide‐end capped AGMA1, a biocompatible polyamidoamine with arg‐gly‐asp (RGD)‐reminiscent repeating units. The resultant mixture is finally cured giving PLLA‐AGMA1 hydrogel composites that absorb large amounts of water and, in the swollen state, are translucent, soft, and pliable, yet as strong as the parent PLLA mat. They do not split apart from each other when swollen in water and remain highly flexible and resistant, since the hydrogel portion is covalently grafted onto the PLLA nanofibers via the addition reaction of the surface amine groups to a part of the terminal acrylic double bonds of AGMA1 oligomers. Preliminary tested as scaffolds, the composites prove capable of maintaining short‐term undifferentiated cultures of human pluripotent stem cells in feeder‐free conditions.