Polypyrrole/Alginate Hybrid Hydrogels: Electrically Conductive and Soft Biomaterials for Human Mesenchymal Stem Cell Culture and Potential Neural Tissue Engineering Applications

Electrically conductive biomaterials that can efficiently deliver electrical signals to cells or improve electrical communication among cells have received considerable attention for potential tissue engineering applications. Conductive hydrogels are desirable particularly for neural applications, as they can provide electrical signals and soft microenvironments that can mimic native nerve tissues. In this study, conductive and soft polypyrrole/alginate (PPy/Alg) hydrogels are developed by chemically polymerizing PPy within ionically cross‐linked alginate hydrogel networks. The synthesized hydrogels exhibit a Young's modulus of 20–200 kPa. Electrical conductance of the PPy/Alg hydrogels could be enhanced by more than one order of magnitude compared to that of pristine alginate hydrogels. In vitro studies with human bone marrow‐derived mesenchymal stem cells (hMSCs) reveal that cell adhesion and growth are promoted on the PPy/Alg hydrogels. Additionally, the PPy/Alg hydrogels support and greatly enhance the expression of neural differentiation markers (i.e., Tuj1 and MAP2) of hMSCs compared to tissue culture plate controls. Subcutaneous implantation of the hydrogels for eight weeks induces mild inflammatory reactions. These soft and conductive hydrogels will serve as a useful platform to study the effects of electrical and mechanical signals on stem cells and/or neural cells and to develop multifunctional neural tissue engineering scaffolds.

     

Survival, migration and differentiation of retinal progenitor cells transplanted on micro-machined poly(methyl methacrylate) scaffolds to the subretinal space

Stem and progenitor cells can be combined with polymer substrates to generate tissue equivalents in culture. The replacement of retinal tissue lost to disease or trauma using retinal progenitor cells (RPCs) delivered on polymer scaffolds and transplanted into the sub-retinal space of the damaged retina is a promising therapeutic strategy. Micromachining-based, ultra-thin PMMA poly(methyl methacrylate) scaffolds may provide a suitable cytoarchitectural environment for tissue engineering and transplantation to the diseased eye. Here, adhesion of RPCs to polymer, as well as migration and differentiation in the host retina were compared for PMMA scaffolds (6 microm thickness) with either smooth or porous (11 microm diameter) surface topography. RPCs were cultured under identical conditions on smooth or porous laminin-coated polymer scaffolds and transplanted into the subretinal space of C57BL/6 mice. RPCs could be cultured on both scaffolds with similar results, although transplantation with non-porous scaffolds showed limited RPC retention. Porous scaffolds demonstrated enhanced RPC adherence during transplantation and allowed for greater process outgrowth and cell migration into the host retinal layers. Integrated cells expressed the mature neuronal marker neurofilament-200 (nf-200), the glial marker glial fibrillary acidic protein (GFAP) and the retinal-specific marker recoverin. No host foreign body response was seen. In conclusion, ultra-thin film PMMA scaffolds micromachined to contain through pores retain adherent RPCs to a considerably greater extent than unmachined versions during the transplantation process and can serve as a biocompatible substrate for cell delivery in vivo.     

A pneumatic micro cell chip for the differentiation of human mesenchymal stem cells under mechanical stimulation

A new micro cell chip which can induce stem cells to differentiate into specific body cell types has been designed and fabricated for tissue engineering. This paper presents the test results of a micro cell stimulator which can provide a new miniaturized tool in cell stimulation, culture and analysis for stem cell research. The micro cell stimulator is designed to apply compressive pressure to the hMSCs (human mesenchymal stem cells) for inducing osteogenesis. The micro cell stimulator is based on the pneumatic actuator with a flexible diaphragm which consists of an air chamber and cell chambers. The hMSCs under cyclic compressive stimulation for one week were observed and assessed by monitoring CD90 (Thy-1), actin, alkaline phosphatase (ALP) and alizarin red expression. The results suggest that cyclic mechanical stimulation is attributed to the different phenomenon of cultured hMSCs in cell proliferation and differentiation. These results are important for the feasibility of the micro cell stimulator to provide the reduction of the necessary quantity of cells, process cost and the increase of the throughput.     

Directed neural stem cell differentiation on polyaniline-coated high strength hydrogels

Stem-cell-based neural regeneration has received significant attention, as it has potential to restore functionality to diseased or damaged neural tissues that have a limited ability to self-repair or regenerate. Culturing neural stem cells (NSCs) on hydrogel substrates has been shown to facilitate differentiation to neural progenitors, but this has only been achieved on very soft hydrogels, greatly increasing the difficulty of manufacture and limiting their wide applications. Here, we realized the differentiation of NSCs to neural and glial progenitors on high-strength hydrogels. Hydrogen-bonding-strengthened conductive hydrogels (PVV-PANI) were synthesized through one-pot copolymerization of 2-vinyl-4,6-diamino-1,3,5-triazine, 1-vinylimidazole and polyethylene glycol diacrylate, followed by post-coating with polyaniline (PANI). Diaminotriazine-diaminotriazine hydrogen bonding dramatically increases their mechanical strength, while copolymerization with VI pronouncedly promotes the adsorption of PANI particles, endowing the hydrogels with electrical conductivity. These hydrogels exhibit tensile strengths up to 1.16 MPa, a 559% breaking strain, a 9.9 MPa compressive strength and up to 16.7 mS/cm conductivity. Importantly, PVV-PANI hydrogels support the attachment, proliferation, and differentiation of NSCs, and allow the efficient induction of neural and glial differentiation via electrical stimulation. This work demonstrates high-strength conductive hydrogels can serve as an electroactive soft-wet platform for modulating the specific differentiation of NSCs, a significant step towards cell-based therapies for neurological diseases.     

Aminosilane Functionalized Aligned Fiber PCL Scaffolds for Peripheral Nerve Repair

Silane modification is a simple and cost-effective tool to modify existing biomaterials for tissue engineering applications. Aminosilane layer deposition has previously been shown to control NG108-15 neuronal cell and primary Schwann cell adhesion and differentiation by controlling deposition of ─NH₂ groups at the submicron scale across the entirety of a surface by varying silane chain length. This is the first study toreport depositing 11-aminoundecyltriethoxysilane (CL11) onto aligned Polycaprolactone (PCL) scaffolds for peripheral nerve regeneration. Fibers are manufactured via electrospinning and characterized using water contact angle measurements, atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS). Confirmed modified fibers are investigated using in vitro cell culture of NG108-15 neuronal cells and primary Schwann cells to determine cell viability, cell differentiation, and phenotype. CL11-modified fibers significantly support NG108-15 neuronal cell and Schwann cell viability. NG108-15 neuronal cell differentiation maintains Schwann cell phenotype compared to unmodified PCL fiber scaffolds. 3D ex vivo culture of Dorsal root ganglion explants (DRGs) confirms further Schwann cell migration and longer neurite outgrowth from DRG explants cultured on CL11 fiber scaffolds compared to unmodified scaffolds. Thus, a reproducible and cost-effective tool is reported to modify biomaterials with functional amine groups that can significantly improve nerve guidance devices and enhance nerve regeneration.     

Conductive stretchable shape memory elastomers combining with electrical stimulation for synergistic osteogenic differentiation

The natural extracellular matrix (ECM) possessed varying biomechanical properties which played important roles in the dynamic cellular microenvironment. However, for the conventional bone tissue engineering scaffolds, stretchability and shape memory property were normally absent. Thus, the behaviors of responsive changes required in dynamic physiological settings were unsatisfactory. Herein, a series of conductive polyurethane shape memory elastomers (PCL-IPDI-AT) were synthesized, which based on conductive amino capped aniline trimer (AT), isophorone diisocyanate (IPDI) and poly(ԑ-caprolactone) (PCL). The conductive elastomers possessed high elasticity and flexibility, especially, the breaking elongation of copolymer with 15% AT content was up to 570 ± 56%. The mechanical properties of elastomers could be adjusted by regulating the content of AT in copolymers. The conductive elastomers exhibited excellent shape fixity ratio and good shape recovery ability at 37 °C. The electrical conductivity of elastomers was measured via the standard van der Pauw four-probe method. They were all around 10⁻⁷ S/cm and similar to that in human physiological environments. On the one hand, excellent cytocompatibility was demonstrated by the viability and proliferation results of MC3T3-E1 pre-osteoblasts seeded on the elastomer. On the other hand, the elastomer could synergistically promote the osteogenic differentiation compared to PCL in terms of ALP activity, calcium deposition, and bone-related protein and gene expression levels as combined with electrical stimulation (ES). Specifically, the ALP activity for conductive elastomer under ES was notably improved by 1.4-fold compared to PCL at 7 days. Overall, the conductive elastomers displayed excellent stretchability, shape memory property, fatigue resistance and osteogenic bioactivity. They may be applied as bone substitutes for electrical-signal-sensitive bone tissue engineering.     

Enhanced Cell Penetration and Pluripotency Maintenance of hiPSCs in 3D Natural Chitosan Scaffolds

Human-induced pluripotent stem cells (hiPSCs) cultured in 3D matrices hold great promise in disease modeling, drug discovery, and tissue regeneration. Uniform cell distribution in a 3D structure is critical to the growth and function of hiPSCs, yet cell seeding in 3D matrices often remains superficial, leading to limited cell proliferation and compromised pluripotency. Here, an approach to improve cell penetration depth of hiPSCs in 3D scaffolds modified with hiPSCs conditioned medium (CM) is reported. It is shown that extracellular matrix components are successfully deposited onto the scaffold wall surface after CM treatment and promoted homogeneous cell adhesion during initial seeding. Compared to plain, unmodified scaffolds, the CM treated scaffold improves spatial cell distribution uniformity and upregulates pluripotency markers. Notably, the expression of 29 genes associated with 11 signaling pathways participated in the pluripotency maintenance of hiPSCs exhibits >2-fold change in hiPSCs grown in the CM treated scaffolds than 2D counterparts, demonstrating that CM treated scaffolds can support a more primitive and undifferentiated phenotype of hiPSCs. This study introduces a simple and effective method to enhance cell penetration and maintain cell pluripotency in 3D matrices.     

Mimicking Nanofibrous Hybrid Bone Substitute for Mesenchymal Stem Cells Differentiation into Osteogenesis

Mimicking hybrid extracellular matrix is one of the main challenges for bone tissue engineering (BTE). Biocompatible polycaprolactone/poly(α,β)‐DL ‐aspartic acid/collagen nanofibrous scaffolds were fabricated by electrospinning and nanohydroxyapatite (n‐HA) was deposited by calcium phosphate dipping method for BTE. Human mesenchymal stem cells (hMSCs) were cultured on these hybrid scaffolds to investigate the cell proliferation, osteogenic differentiation by alkaline phosphatase activity, mineralization, double immunofluorescent staining using CD90 and expression of osteocalcin. The present study indicated that the PCL/PAA/collagen/n‐HA scaffolds promoted greater osteogenic differentiation of hMSCs, proving to be a potential hybrid scaffolds for BTE.

     

Bone regeneration on macroporous aqueous-derived silk 3-D scaffolds

Spinner flask culture under osteogenic conditions was used to study osteogenic outcomes from human bone marrow-derived mesenchymal stem cells (hMSCs) seeded on aqueous-derived porous silk scaffolds. Of particular novelty was the use of larger sized scaffolds (15 mm diameter, 5 mm thick) and large pore sizes ( approximately 900-1 000 micron diameter). Cultures were maintained for 84 d in the spinner flasks and compared to static controls under otherwise similar conditions. The spinner flask cultures demonstrated enhanced cell proliferation compared to static cultures and the improved fluid flow promoted significantly improved osteogenic related outcomes based on elevated alkaline phosphatase (ALP) activity and the deposition of mineralized matrix. The expression of osteogenic differentiation associated markers based on real time PCR also demonstrated increased responses under the dynamic spinner flask culture conditions. Histological analysis showed organized bone-like structures in the constructs cultured in the spinner flasks after 56 d of culture. These structures stained intensely with von Kossa. The combination of improved transport due to spinner flask culture and the use of macroporous 3D aqueous-derived silk scaffolds with large pore sizes resulted in enhanced outcomes related to bone tissue engineering, even with the use of large sized scaffolds in the study. These results suggest the importance of the structure of the silk biomaterial substrate (water vs. solvent based preparation) and large pore sizes in improved bone-like outcomes during dynamic cultivation.     

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.     

Restraint of the Differentiation of Mesenchymal Stem Cells by a Nonfouling Zwitterionic Hydrogel

The success of human mesenchymal stem cell (hMSC) therapies is largely dependent on the ability to maintain the multipotency of cells and control their differentiation. External biochemical and biophysical cues can readily trigger hMSCs to spontaneously differentiate, thus resulting in a rapid decrease in the multipotent cell population and compromising their regenerative capacity. Herein, we demonstrate that nonfouling hydrogels composed of pure poly(carboxybetaine) (PCB) enable hMSCs to retain their stem‐cell phenotype and multipotency, independent of differentiation‐promoting media, cytoskeletal‐manipulation agents, and the stiffness of the hydrogel matrix. Moreover, encapsulated hMSCs can be specifically induced to differentiate down osteogenic or adipogenic pathways by controlling the content of fouling moieties in the PCB hydrogel. This study examines the critical role of nonspecific interactions in stem‐cell differentiation and highlights the importance of materials chemistry in maintaining stem‐cell multipotency and controlling differentiation.     

Regulation of Scaffold Cell Adhesion Using Artificial Membrane Binding Proteins

The rapid pace of development in biotechnology has placed great importance on controlling cell–material interactions. In practice, this involves attempting to decouple the contributions from adhesion molecules, cell membrane receptors, and scaffold surface chemistry and morphology, which is extremely challenging. Accordingly, a strategy is presented in which different chemical, biochemical, and morphological properties of 3D biomaterials are systematically varied to produce novel scaffolds with tuneable cell affinities. Specifically, cationized and surfactant‐conjugated proteins, recently shown to have non‐native membrane affinity, are covalently attached to 3D scaffolds of collagen or carboxymethyl‐dextran, yielding surface‐functionalized 3D architectures with predictable cell immobilization profiles. The artificial membrane‐binding proteins enhance cellular adhesion of human mesenchymal stem cells (hMSCs) via electrostatic and hydrophobic binding mechanisms. Furthermore, functionalizing the 3D scaffolds with cationized or surfactant‐conjugated myoglobin prevents a slowdown in proliferation of seeded hMSCs cultured for seven days under hypoxic conditions.

     

Bioactive Phenylboronic Acid-Functionalized Hyaluronic Acid Hydrogels Induce Chondro-Aggregates and Promote Chondrocyte Phenotype

Hydrogels are extensively investigated as biomimetic extracellular matrix (ECM) scaffolds in tissue engineering. The physiological properties of ECM affect cellular behaviors, which is an inspiration for cell-based therapies. Photocurable hyaluronic acid (HA) hydrogel (AHAMA-PBA) modified with 3-aminophenylboronic acid, sodium periodate, and methacrylic anhydride simultaneously is constructed in this study. Chondrocytes are then cultured on the surface of the hydrogels to evaluate the effect of the physicochemical properties of the hydrogels on modulating cellular behaviors. Cell viability assays demonstrate that the hydrogel is non-toxic to chondrocytes. The existence of phenylboronic acid (PBA) moieties enhances the interaction of chondrocytes and hydrogel, promoting cell adhesion and aggregation through filopodia. RT-PCR indicates that the gene expression levels of type II collagen, Aggrecan, and Sox9 are significantly up-regulated in chondrocytes cultured on hydrogels. Moreover, the mechanical properties of the hydrogels have a significant effect on the cell phenotype, with soft gels (≈2 kPa) promoting chondrocytes to exhibit a hyaline phenotype. Overall, PBA-functionalized HA hydrogel with low stiffness exhibits the best effect on promoting the chondrocyte phenotype, which is a promising biomaterial for cartilage regeneration.     

Control of Osteogenic Differentiation and Mineralization of Human Mesenchymal Stem Cells on Composite Nanofibers Containing Poly[lactic‐co‐(glycolic acid)] and Hydroxyapatite

We fabricated composite fibrous scaffolds from blends of poly(lactide‐co‐glycolide) (PLGA) and nano‐sized hydroxyapatite (HA) via electrospinning. SEM‐EDX and AFM analysis demonstrated that HA was homogeneously dispersed in the nanofibers, and the roughness increased along with the amount of incorporated HA. When hMSCs were cultured on these PLGA/HA composite nanofibers, we found that incorporation of HA on the nanofibers did not affect cell viability whereas increased ALP activity and expression of osteogenic genes as well as the calcium mineralization of hMSCs. Our results indicate that the composite nanofibers can be offered as a potential bone regenerative biomaterial for stem cell based therapies.

     

Mass Transfer Analysis of Growth and Substance Metabolism of NSCs Cultured in Collagen-Based Scaffold In Vitro

The aim of this study is to analyze the growth and substance metabolism of neural stem cells (NSCs) cultured in biological collagen-based scaffolds. Mass transfer and metabolism model of glucose, lactic acid, and dissolved oxygen (DO) were established and solved on MATLAB platform to obtain the concentration distributions of DO, glucose, and lactic acid in culture system, respectively. Calculation results showed that the DO influenced their normal growth and metabolism of NSCs mostly in the in vitro culture within collagen-based scaffolds. This study also confirmed that 2-mm thickness of collagen scaffold was capable of in vitro cultivation and growth of NSCs with an inoculating density of 1?×?10⁶ cells/mL.     

In Situ Synthesis of Robust Conductive Cellulose/Polypyrrole Composite Aerogels and Their Potential Application in Nerve Regeneration

Nanostructured conductive polymers can offer analogous environments for extracellular matrix and induce cellular responses by electric stimulation, however, such materials often lack mechanical strength and tend to collapse under small stresses. We prepared electrically conductive nanoporous materials by coating nanoporous cellulose gels (NCG) with polypyrrole (PPy) nanoparticles, which were synthesized in situ from pyrrole monomers supplied as vapor. The resulting NCG/PPy composite hydrogels were converted to aerogels by drying with supercritical CO₂, giving a density of 0.41–0.53 g cm^?3, nitrogen adsorption surface areas of 264–303 m² g^?1, and high mechanical strength. The NCG/PPy composite hydrogels exhibited an electrical conductivity of up to 0.08 S cm^?1. In vitro studies showed that the incorporation of PPy into an NCG enhances the adhesion and proliferation of PC12 cells. Electrical stimulation demonstrated that PC12 cells attached and extended longer neurites when cultured on NCG/PPy composite gels with DBSA dopant. These materials are promising candidates for applications in nerve regeneration, carbon capture, catalyst supports, and many others.     

A Long‐Living Bioengineered Neural Tissue Platform to Study Neurodegeneration

The prevalence of dementia and other neurodegenerative diseases continues to rise as age demographics in the population shift, inspiring the development of long‐term tissue culture systems with which to study chronic brain disease. Here, it is investigated whether a 3D bioengineered neural tissue model derived from human induced pluripotent stem cells (hiPSCs) can remain stable and functional for multiple years in culture. Silk‐based scaffolds are seeded with neurons and glial cells derived from hiPSCs supplied by human donors who are either healthy or have been diagnosed with Alzheimer's disease. Cell retention and markers of stress remain stable for over 2 years. Diseased samples display decreased spontaneous electrical activity and a subset displays sporadic‐like indicators of increased pathological β‐amyloid and tau markers characteristic of Alzheimer's disease with concomitant increases in oxidative stress. It can be concluded that the long‐term stability of the platform is suited to study chronic brain disease including neurodegeneration.     

Human Mesenchymal Stem‐Cell Behaviour On Direct Laser Micropatterned Electrospun Scaffolds with Hierarchical Structures

Direct laser machining and electrospinning are utilized to obtain a bi‐layered hybrid scaffold with hierarchical topographical features to mimic extracellular matrix‐like microenvironment of cells. Adult bone marrow derived human mesenchymal stem cells (hMSCs) are cultured in vitro in these hybrid scaffolds, and cell orientation, proliferation, viability, and differentiation are evaluated. The results show that this novel hybrid scaffold not only supports cell growth like traditional scaffolds, but also elicits positive responses from the cells, like lineage commitment and alignment, which are essential features of future scaffolds.