Thermogelling Inclusion Complex System for Fine-Tuned Osteochondral Differentiation of Mesenchymal Stem Cells.

How to control osteochondral differentiation of mesenchymal stem cells at a proper stage is a key issue for articular cartilage regeneration. To solve this problem, injectable scaffolds with different chemical functional groups were designed by introducing one equivalent of α-cyclodextrin (α-CD) carboxylate and α-CD phosphate along poly(ethylene glycol)-poly(l-alanine) (PEG-L-PA) block copolymers. Dynamic light scattering, transmission electron microscopy images, and two-dimensional NMR spectra indicated that the PEG-L-PA block copolymers formed inclusion complexes with α-CD derivatives. Aqueous solutions of PEG-L-PA block copolymers (P), α-CD carboxylate/PEG-L-PA block copolymers (PCC), and α-CD phosphate/PEG-L-PA block copolymers (PCP) underwent sol-to-gel transition as the temperature increased. The storage moduli of P, PCC, and PCP gels ranged from 1000 to 1300 Pa at 37 °C. Tonsil-derived mesenchymal stem cells (TMSCs) were incorporated in situ in the gel during thermogelation of P, PCC, and PCP, which became the three-dimensional cell culture systems with different functional groups. After 21 days of incubation of TMSCs in the P, PCC, and PCP systems, the chondrogenic differentiation biomarker of type II collagen significantly increased in the P system, whereas the osteogenic biomarkers of osteocalcin and runt-related transcription factor 2 significantly increased in the PCP system. Both chondrogenic and osteogenic biomarkers were highly expressed in the PCC system. This study proved that thermogelling inclusion complex systems consisting of PEG-L-PA block copolymers and α-CD derivatives could be an excellent injectable matrix for fine-controlling osteochondral differentiation of mesenchymal stem cells.

Iron Ion-Releasing Polypeptide Thermogel for Neuronal Differentiation of Mesenchymal Stem Cells.

A poly(ethylene glycol)-based thermogel can capture an iron ion (Fe3+) through a crown ether-like coordination bond between the oxygen atom and metal ions, thus, providing a sustained Fe3+-releasing system. Poly(ethylene glycol)-l-poly(alanine) thermogel was used in this study. The polypeptide forms a rather robust gel, and the degradation products are a neutral amino acid, which provides cyto-compatible neutral pH environments during the cell culture. During the heat-induced sol-to-gel transition at 37 °C, tonsil-derived mesenchymal stem cells (TMSCs) and iron ions were incorporated, leading to the formation of a three-dimensional matrix toward neuronal differentiation of the incorporated TMSCs. The initial concentration of the iron ions was varied between 0, 15, 30, and 60 mM. About 10% of the loaded iron ions was released over 21 days, which continuously supplied iron ions to the cells. The incorporation of iron ions not only increased the gel modulus at 37 °C from 107 to 680 Pa, but also promoted cell aggregation with a significant secretion of the cell adhesion signal of FAK. Expression of biomarkers related to the neuronal differentiation of TMSCs, including NFM, MAP2, GFAP, NURR1, NSE, and TUBB3, increased 4-35-fold at the mRNA level in the Fe3+-containing system compared to that of the system without Fe3+. Immunofluorescence studies also confirmed pronounced cell aggregation and a significant increase in neuronal biomarkers at the protein level. This study suggests that an iron ion-releasing thermogelling system can be a promising injectable scaffold toward neuronal differentiation of stem cells.

Phosphorylcholine-Based Zwitterionic Biocompatible Thermogel.

Zwitterionic polymers have been investigated as surface-coating materials due to their low protein adsorption properties, which reduce immunogenicity, biofouling, and bacterial adsorption of coated materials. Most zwitterionic polymers, reported so far, are based on (meth)acrylate polymers which can induce toxicity by residual monomers or amines produced by degradation. Here, we report a new zwitterionic polymer consisting of phosphorylcholine (PC) and biocompatible poly(propylene glycol) (PPG) as a new thermogelling material. The PC-PPG-PC polymer aqueous solution undergoes unique multiple sol-gel transitions as the temperature increases. A heat-induced unimer-to-micelle transition, changes in ionic interactions, and dehydration of PPG are involved in the sol-gel transitions. Based on the broad gel window and low protein adsorption properties, the PC-PPG-PC thermogel is proved for sustained delivery of protein drugs and stem cells over 1 week.

Tubular nanomaterials for bone tissue engineering.

Nanomaterial composition, morphology, and mechanical performance are critical parameters for tissue engineering. Within this rapidly expanding space, tubular nanomaterials (TNs), including carbon nanotubes (CNTs), titanium oxide nanotubes (TNTs), halloysite nanotubes (HNTs), silica nanotubes (SiNTs), and hydroxyapatite nanotubes (HANTs) have shown significant potential across a broad range of applications due to their high surface area, versatile surface chemistry, well-defined mechanical properties, excellent biocompatibility, and monodispersity. These include drug delivery vectors, imaging contrast agents, and scaffolds for bone tissue engineering. This review is centered on the recent developments in TN-based biomaterials for structural tissue engineering, with a strong focus on bone tissue regeneration. It includes a detailed literature review on TN-based orthopedic coatings for metallic implants and composite scaffolds to enhance in vivo bone regeneration.

Conducting polymer-based scaffolds for neuronal tissue engineering.

Neuronal tissue engineering has immense potential for treating neurological disorders and facilitating nerve regeneration. Conducting polymers (CPs) have emerged as a promising class of materials owing to their unique electrical conductivity and biocompatibility. CPs, such as poly(3,4-ethylenedioxythiophene) (PEDOT), poly(3-hexylthiophene) (P3HT), polypyrrole (PPy), and polyaniline (PANi), have been extensively explored for their ability to provide electrical cues to neural cells. These polymers are widely used in various forms, including porous scaffolds, hydrogels, and nanofibers, and offer an ideal platform for promoting cell adhesion, differentiation, and axonal outgrowth. CP-based scaffolds can also serve as drug delivery systems, enabling localized and controlled release of neurotrophic factors and therapeutic agents to enhance neural regeneration and repair. CP-based scaffolds have demonstrated improved neural regeneration, both in vitro and in vivo, for treating spinal cord and peripheral nerve injuries. In this review, we discuss synthesis and scaffold processing methods for CPs and their applications in neuronal tissue regeneration. We focused on a detailed literature review of the central and peripheral nervous systems.

Pralatrexate Sustainably Released from Polypeptide Thermogel Is Effective for Chondrogenic Differentiation of Mesenchymal Stem Cells.

Folic acid was reported to significantly improve chondrogenic differentiation of mesenchymal stem cells. In a similar mechanism of action, we investigated clinically approved antifolates by the U.S. Food and Drug Administration as chondrogenic-promoting compounds for tonsil-derived mesenchymal stem cells. A poly(ethylene glycol)-poly(l-alanine) thermogelling system was used as a three-dimensional cell culture matrix, where stem cells and antifolates could be incorporated simultaneously during a heat-induced in situ sol-to-gel transition. The antifolates could be supplied over several days by the sustained release of the drug from the thermogel. Initially, seven antifolates were prescreened based on cell viability and expression of a typical chondrogenic biomarker of type II collagen (COL II) at the mRNA level. Then, dapsone, pralatrexate, and trimethoprim were selected as candidate compounds in the second round screening, and detailed studies were carried out on the mRNA and protein expression of various chondrogenic biomarkers including COL II, SRY box transcription factor 9, and aggrecan. Three-dimensional cultures of stem cells in the thermogel in the absence of a chondrogenic promoter compound and in the presence of kartogenin (KGN) were performed as a negative control and positive control, respectively. The chondrogenic biomarkers were significantly increased in the selected antifolate-incorporating systems compared to the negative control system, without an increase in type I collagen (an osteogenic biomarker) expression. Pralatrexate was the best compound for inducing chondrogenic differentiation of the stem cells, even better than the positive control (KGN). Nuclear translocation of the core-binding factor ß subunit (CBFß) and enhanced nuclear runt-related transcription factor 1 (RUNX1) by antifolate treatment suggested that the chondrogenesis-enhancing mechanism is mediated by CBFß and RUNX1. An in silico modeling study confirmed the mechanism by proving the high binding affinity of pralatrexate to a target protein of filamin A compared with other antifolate candidates. To conclude, pralatrexate was rediscovered as a lead compound, and the polypeptide thermogel incorporating pralatrexate and mesenchymal stem cells can be a very effective system in promoting chondrogenic differentiation of stem cells and might be used in injectable tissue engineering for cartilage repair.

Poly(l-Ala-co-l-Lys) Exhibits Excellent Ice Recrystallization Inhibition Activity.

The control of ice recrystallization is very important in cryo-technological fields such as the food industry, biopharmaceuticals, and cell storage. Ice recrystallization inhibition (IRI) compounds are therefore designed to limit the growth of ice crystals, decrease the crystal size, and control the crystal shape. To improve the IRI activity of cryo-systems, various synthetic polymers such as biomimetic polypeptides from polar fish, facially amphiphilic polymers, polyampholytes, poly(vinyl alcohol) derivatives, and block copolymers with hydrophilic-hydrophobic balance have been developed. Except for graphene oxide, poly(vinyl alcohol) has thus far exhibited the best performance among these polymers. Herein, poly(l-alanine-co-l-lysine) (PAK) was shown to exhibit a similar IRI activity to that of poly(vinyl alcohol). Moreover, in contrast to the needle-shaped ice crystals generated by the aqueous PVA solution, the PAK solution was shown to generate cubic-to-spherical shaped ice crystals. Furthermore, neither poly(l-alanine-co-l-aspartic acid) (PAD) nor poly(ethylene glycol) (PEG) with a similar molecular weight provided any significant IRI activity. Examination by FTIR and circular dichroism spectroscopies indicated that the PAK forms α-helices, whereas the PAD forms random coils in water. Further, a dynamic ice shaping study suggested that PAK strongly interacts with ice crystals, whereas PAD and PEG only weakly interact. These results suggest that PAK is an important compound with superior IRI activity and that this activity is dependent upon the functional groups and secondary structure of the polypeptides.

Size and Shape Control of Ice Crystals by Amphiphilic Block Copolymers and Their Implication in the Cryoprotection of Mesenchymal Stem Cells.

Precise control over the size and shape of ice crystals is a key factor to consider in designing antifreezing and cryoprotecting molecules for cryopreservation of cells. Here, we report that a poly(ethylene glycol)-poly(l-alanine) (PEG-PA) block copolymer exhibits excellent cryoprotecting properties for stem cells and antifreezing properties for water. As the molecular weight of PA increased from 500, 760, and 1750 Da (P1, P2, and P3) at the same PEG molecular weight of 5000 Da, the ß-sheet content decreased and α-helix content increased. Comparing P2 (PEG-PA; 5000-760) and P4 (PEG-PA: 1000-750), ß-sheets increased as the PEG block length decreased. The critical micelle concentration of the PEG-PA block copolymers was in a range of 0.5-3.0 mg/mL and was proportional to the hydrophobicity of the PEG-PA block copolymers. The P1, P2, and P3 self-assembled into spherical micelles, whereas P4 formed micelles with cylindrical morphology. The difference in the block copolymer structure affected ice recrystallization inhibition (IRI) activity and cryopreservation of cells. IRI activity was assayed via mean largest grain size (MLGS), and interactions between polymers and ice crystal surfaces were studied by dynamic ice-shaping studies. The MLGS decreased to 58 → 53 → 45 → 35 → 23% of that of PBS, as the polymer (PEG-PA 5000-500) concentration increased from 0.0 (PBS; control) → 1.0 → 5.0 → 10 → 30 → 50 mg/mL. The MLGS of PEG 5k solutions (negative control) decreased to 74 → 71 → 64 → 44 → 37% of that of PBS in the same concentration range. P3 and P4 with a longer hydrophobic PA block developed elongated ice crystals at above 30 mg/mL. The dynamic ice-shaping study exhibited that ice crystals became needle-shaped, as the hydrophobicity of the polymer increased as in P2-P4. The cell recovery in the P1 system after cryopreservation at -196 °C for 7 days was 87% of that of the dimethyl sulfoxide (DMSO) 10% system (positive control). The cell recovery was 48% for the P2 system and drastically decreased to less than 30% of that of the DMSO 10% system in the P3, P4, PEG 5k, PEG 1k, PVA 80H, and PVA 100H systems. Current studies suggest that IRI activity, round ice crystal shaping, and membrane stabilization activity of P1 cooperatively provide excellent cell recovery among the candidate systems. Recovered stem cells exhibited excellent proliferation and multilineage differentiation into osteocytes, chondrocytes, and adipocytes. To conclude, the PEG-PA (5000-500) block copolymer is suggested to be a promising antifreezing cryoprotectant for stem cells.

Culture of neural stem cells on conductive and microgrooved polymeric scaffolds fabricated via electrospun fiber-template lithography.

We developed polymeric scaffolds that can provide both topographical and electrical stimuli on mouse neural stem cells (mNSCs) for potential use in nerve tissue engineering. In contrast to conventional patterning techniques such as imprinting, soft/photolithography, and three-dimensional printing, microgroove patterns were generated by using aligned electrospun fibers as templates, via a process denoted as electrospun fiber-template lithography. The preparation of polyvinylpyrrolidone fibers, followed by the deposition of poly(lactic-co-glycolic acid) (PLGA) and the removal of the fiber template, produced freestanding PLGA scaffolds with microgrooves having widths of 1.72 ± 0.24 µm. The subsequent deposition of polypyrrole (PPy) via chemical oxidative polymerization added conductivity to the microgrooved PLGA scaffolds. The resultant scaffolds were cytocompatible with mNSCs. The microgroove patterns enhanced the alignment and elongation of mNSCs, and the PPy layer promoted the interaction of cells with the surface by forming more and longer filopodia compared with the nonconductive surface. Finally, the neuron differentiation of mNSCs was evaluated by monitoring the Tuj-1 neuronal gene expression. The presence of both microgrooves and the conductive PPy layer enhanced the neuronal differentiation of mNSCs even without electrical stimulation, and the neuronal differentiation was further enhanced by stimulation with a sufficient electrical pulse (1.0 V).

Injectable thermogel for 3D culture of stem cells.

Thermogel is an aqueous polymer solution that undergoes sol-to-gel transition as the temperature increases. Cells, growth factors, and signaling molecules can be incorporated simultaneously during the sol-to-gel transition. The cytocompatible procedure makes the thermogel an excellent platform for 3D culture of stem cells. This review focuses on the crucial questions that need to be addressed to achieve effective differentiation of stem cells into target cells, comprising low modulus, cell adhesion, and controlled supply of the growth factors. Recent progress in the use of thermogel as a 3D culture system of stem cells is summarized, and our perspectives on designing a new thermogel for 3D culture and its eventual application to injectable tissue engineering of stem cells are presented.

Composite System of Graphene Oxide and Polypeptide Thermogel As an Injectable 3D Scaffold for Adipogenic Differentiation of Tonsil-Derived Mesenchymal Stem Cells.

As two-dimensional (2D) nanomaterials, graphene (G) and graphene oxide (GO) have evolved into new platforms for biomedical research as biosensors, imaging agents, and drug delivery carriers. In particular, the unique surface properties of GO can be an important tool in modulating cellular behavior and various biological sequences. Here, we report that a composite system of graphene oxide/polypeptide thermogel (GO/P), prepared by temperature-sensitive sol-to-gel transition of a GO-suspended poly(ethylene glycol)-poly(L-alanine) (PEG-PA) aqueous solution significantly enhances the expression of adipogenic biomarkers, including PPAR-γ, CEBP-α, LPL, AP2, ELOVL3, and HSL, compared to both a pure hydrogel system and a composite system of G/P, graphene-incorporated hydrogel. We prove that insulin, an adipogenic differentiation factor, preferentially adhered to GO, is supplied to the incorporated stem cells in a sustained manner over the three-dimensional (3D) cell culture period. On the other hand, insulin is partially denatured in the presence of G and interferes with the adipogenic differentiation of the stem cells. The study suggests that a 2D/3D composite system is a promising platform as a 3D cell culture matrix, where the surface properties of 2D materials in modulating the fates of the stem cells are effectively transcribed in a 3D culture system.

Microsphere-Incorporated Hybrid Thermogel for Neuronal Differentiation of Tonsil Derived Mesenchymal Stem Cells.

Neuronal differentiation of tonsil-derived mesenchymal stem cells (TMSCs) is investigated in a 3D hybrid system. The hybrid system is prepared by increasing the temperature of poly(ethylene glycol)-poly(l-alanine) aqueous solution to 37 °C through the heat-induced sol-to-gel transition, in which TMSCs and growth factor releasing microspheres are suspended. The in situ formed gel exhibits a modulus of 800 Pa at 37 °C, similar to that of brain tissue, and it is robust enough to hold the microspheres and cells during the 3D culture of TMSCs. The neuronal growth factors are released over 12-18 d, and the TMSCs in a spherical shape initially undergo multipolar elongation during the 3D culture. Significantly higher expressions of the neuronal biomarkers such as nuclear receptor related protein (Nurr-1), neuron specific enolase, microtubule associated protein-2, neurofilament-M, and glial fibrillary acidic protein are observed in both mRNA level and protein level in the hybrid systems than in the control experiments. This study proves the significance of a controlled drug delivery concept in tissue engineering or regenerative medicine, and a 3D hybrid system with controlled release of growth factors from microspheres in a thermogel can be a very promising tool.

Bioinert membranes prepared from amphiphilic poly(vinyl chloride)-g-poly(oxyethylene methacrylate) graft copolymers.

Poly(vinyl chloride) (PVC) membrane was hydrophilically modified by grafting with poly(oxyethylene methacrylate) (POEM) using atom transfer radical polymerization (ATRP). The successful grafting of PVC main chain by POEM was characterized by Fourier transform infrared spectroscopy (FT-IR). The molecular weight and hydrophilicity of membranes increased with the amount of POEM grafting, as characterized by gel permeation chromatography (GPC) and contact angle measurement, respectively. Transmission electron microscope (TEM) and small angle X-ray scattering (SAXS) analysis revealed the microphase-separated structure of PVC-g-POEM and the domain spacing increased from 59.3 to 86.1 nm with increasing grafting degree. Scanning electron microscopy (SEM) was used for the direct visualization of the mouse embryonic fibroblast (MEF) cell and bacteria adhesion on the membrane surface. Protein adsorption and eukaryotic and prokaryotic cell adhesion tests showed that the bioinert properties of membranes were significantly increased with POEM content.