Thixotropic Hydrogels Composed of Self-Assembled Nanofibers of Double-Hydrophobic Elastin-Like Block Polypeptides

Physically crosslinked hydrogels with thixotropic properties attract considerable attention in the biomedical research field because their self-healing nature is useful in cell encapsulation, as injectable gels, and as bioinks for three-dimensional (3D) bioprinting. Here, we report the formation of thixotropic hydrogels containing nanofibers of double-hydrophobic elastin-like polypeptides (ELPs). The hydrogels are obtained with the double-hydrophobic ELPs at 0.5 wt%, the concentration of which is an order of magnitude lower than those for previously reported ELP hydrogels. Although the kinetics of hydrogel formation is slower for the double-hydrophobic ELP with a cell-binding sequence, the storage moduli G′ of mature hydrogels are similar regardless of the presence of a cell-binding sequence. Reversible gel–sol transitions are demonstrated in step-strain rheological measurements. The degree of recovery of the storage modulus G′ after the removal of high shear stress is improved by chemical crosslinking of nanofibers when intermolecular crosslinking is successful. This work would provide deeper insight into the structure–property relationships of the self-assembling polypeptides and a better design strategy for hydrogels with desired viscoelastic properties.     

From Supramolecular Hydrogels to Multifunctional Carriers for Biologically Active Substances

Supramolecular hydrogels are 3D, elastic, water-swelled materials that are held together by reversible, non-covalent interactions, such as hydrogen bonds, hydrophobic, ionic, host–guest interactions, and metal–ligand coordination. These interactions determine the hydrogels’ unique properties: mechanical strength; stretchability; injectability; ability to self-heal; shear-thinning; and sensitivity to stimuli, e.g., pH, temperature, the presence of ions, and other chemical substances. For this reason, supramolecular hydrogels have attracted considerable attention as carriers for active substance delivery systems. In this paper, we focused on the various types of non-covalent interactions. The hydrogen bonds, hydrophobic, ionic, coordination, and host–guest interactions between hydrogel components have been described. We also provided an overview of the recent studies on supramolecular hydrogel applications, such as cancer therapy, anti-inflammatory gels, antimicrobial activity, controlled gene drug delivery, and tissue engineering.     

Enzymatic hydrogelation of small molecules

Enzymes, a class of highly efficient and specific catalysts in Nature, dictate a myriad of reactions that constitute various cascades in biological systems. Self-assembly, a process prevalent in Nature, also plays important roles in biology, from maintaining the integrity of cells to performing cellular functions and inducing abnormalities that cause disease. To explore enzyme-regulated molecular self-assembly in an aqueous medium will help to understand and control those important biological processes. On the other hand, certain small organic molecules self-assemble in water to form molecular nanofibers and result in a hydrogel, which is referred to as a "supramolecular hydrogel" (and the small molecules are referred to as "supramolecular hydrogelators"). Supramolecular hydrogelators share common features, such as amphiphilicity and supramolecular interactions (pi-pi interactions, hydrogen bonding, and charge interactions among the molecules, among others) that result in nanostructures and form the three-dimensional networks as the matrices of hydrogels. In this Account, we discuss the use of enzymes to trigger and control the self-assembly of small molecules for hydrogelation, which takes place in vitro or in vivo, extra- or intracellularly. Using phosphatase, thermolysin, beta-lactamase, and phosphatase/kinase as examples, we illustrate the design and application of enzyme-catalyzed or -regulated formation of supramolecular hydrogels that offer a new strategy for detecting the activity of enzymes, screening for enzyme inhibitors, typing bacteria, drug delivery systems, and controlling the fate of cells. Since the expression and distribution of enzymes differ by the types and states of cells, tissues, and organs, using an enzymatic reaction to convert precursors into hydrogelators that self-assemble into nanofibers as the matrices of the hydrogel, one can control the delivery, function, and response of a hydrogel according to a specific biological condition or environment, thus providing an accessible route to create sophisticated materials for biomedicine. Particularly, intracellular enzymatic hydrogelation of small molecules offers a unique means for scientists to integrate molecular self-assembly with inherent enzymatic reactions inside cells for developing new biomaterials and therapeutics at the supramolecular level and improving the basic understanding of dynamic molecular self-assembly in water.     

Supramolecular polymer gel with multi stimuli responsive,self-healing and erasable properties generated by host–guest interactions

A supramolecular polymer gel formed between triptycene-based bis(crown ether) and copolymer containing dibenzylammonium (DBA) moieties by host–guest interactions was described. We demonstrated the formation of the supramolecular polymer networks between the bis(crown ether) and the copolymer by the ¹H NMR spectroscopy and solution viscometry, and we also obtained a colorless and transparent supramolecular polymer gel at high concentration. Moreover, the supramolecular polymer gel showed multi-stimuli reversible responsiveness, such as thermo-, acid/base-, and chemo- induced gel–sol transitions. Furthermore, the result of rheological measurements showed the gel has an intrinsic self-healing property, and the thixotropic process could be repeated at least three times. Interestingly, when doped with spiropyran molecules, the supramolecular polymer gel could also be employed as erasable materials. Thus, these results could be highly anticipated to benefit for further construction of smart materials with high healing efficiency, and ultimately be used in practical application.     

Thermosensitive supramolecular hydrogels from atom transfer radical polymerization of polypseudorotaxanes self-assembled by triblock copolymer and α-cyclodextrins

We report novel thermosensitive supramolecular hydrogels with tunable crosslinking networks by using polypseudo-rotaxanes (PPRs) as macroinitiators in atom transfer radical polymerization and polyethylene glycol diacrylate (PEGDA) as bridge units. The PPRs were prepared by supramolecular self-assembly of α-cyclodextrin or 2-bromoisobutyryl bromide modified α-cyclodextrin (α-CD-BIBB) and triblock copolymer of F127 with bromopropionyl bromide terminals. The supramolecular hydrogels possess the crystal structure of inclusion complexes (ICs) and thermo-sensitive properties. The addition of mono-functional 2-hydroxyethyl methacrylate (HEMA) can enhance gelation. The swelling ratio (SR) was mainly influenced by the concentration of α-CD or modified α-CD, HEMA and PEGDA. The introduction of α-CDs was found to decrease the SR of hydrogels, while the introduction of α-CD-BIBBs could improve the SR. Meanwhile, gelation was tuned by regulating the concentration of HEMA. The SR of hydrogels was increased with the increase of the concentration of PEGDA, but decreased with the increase of that of HEMA. In the temperature range from 20 to 50 °C, the temperature sensitivity of hydrogels was mainly controlled by the state of gels. The introduction of PHEMA chains decreased the temperature responsivity of networks. The hydrogels prepared with α-CD-BIBBs threading onto the F127 chains showed the high thermo-sensitivity. The thermosensitive mechanism of hydrogels is due to the aggregation of PPO blocks in the networks.     

Thermo-sensitive hydrogels formed from the photocrosslinkable polypseudorotaxanes consisting of <Emphasis Type="Italic">β</Emphasis>-cyclodextrin and Pluronic F68/PCL macromer

To create thermo-sensitive supramolecular hydrogels with high mechanical strength, viscous gel precursors were first formed via block-selected inclusion complexation between β-cyclodextrin (β-CD) and Pluronic F68/poly(ε-caprolactone) block copolymer terminated with acryloyl groups in aqueous media, and subsequently in situ photocrosslinking was employed in the presence of a photoinitiator. The supramolecular assembly in photocrosslinked hydrogels was revealed by wide-angle X-ray diffraction (WXRD) and thermogravimetric analysis (TGA). The rheological studies demonstrated that in situ photocrosslinking could greatly improve the mechanical strength of the gellike precursors. The swelling measurements showed that as-obtained hydrogel displayed a thermo-responsive property. The temperature dependence of the hydrogels decreased with the increase of the β-CD amounts introduced. The resultant hydrogels have the potential to use as carriers for drug delivery and tissue engineering scaffolds.     

Chitosan Hydrogel Supplemented with Metformin Promotes Neuron–like Cell Differentiation of Gingival Mesenchymal Stem Cells

Human gingival mesenchymal stem cells (GMSCs) are derived from migratory neural crest stem cells and have the potential to differentiate into neurons. Metformin can inhibit stem–cell aging and promotes the regeneration and development of neurons. In this study, we investigated the potential of metformin as an enhancer on neuronal differentiation of GMSCs in the growth environment of chitosan hydrogel. The crosslinked chitosan/β–glycerophosphate hydrogel can form a perforated microporous structure that is suitable for cell growth and channels to transport water and macromolecules. GMSCs have powerful osteogenic, adipogenic and chondrogenic abilities in the induction medium supplemented with metformin. After induction in an induction medium supplemented with metformin, Western blot and immunofluorescence results showed that GMSCs differentiated into neuron–like cells with a significantly enhanced expression of neuro–related markers, including Nestin (NES) and β–Tubulin (TUJ1). Proteomics was used to construct protein profiles in neural differentiation, and the results showed that chitosan hydrogels containing metformin promoted the upregulation of neural regeneration–related proteins, including ATP5F1, ATP5J, NADH dehydrogenase (ubiquinone) Fe–S protein 3 (NDUFS3), and Glutamate Dehydrogenase 1 (GLUD1). Our results help to promote the clinical application of stem–cell neural regeneration.     

Gelatin Methacryloyl–Riboflavin (GelMA–RF) Hydrogels for Bone Regeneration

Gelatin methacryloyl (GelMA) is a versatile biomaterial that has been used in various biomedical fields. UV light is commonly used to photocrosslink such materials; however, its use has raised several biosafety concerns. We investigated the mechanical and biological properties of a visible-wavelength (VW)-light-crosslinked gelatin-based hydrogel to evaluate its viability as a scaffold for bone regeneration in bone-destructive disease treatment. Irgacure2959 or riboflavin was added as a photoinitiator to create GelMA solutions. GelMA solutions were poured into a mold and exposed to either UV or VW light. KUSA-A1 cell-laden GelMA hydrogels were crosslinked and then cultured. Mechanical characterization revealed that the stiffness range of GelMA–RF hydrogel was suitable for osteoblast differentiation. KUSA-A1 cells encapsulated in GelMA hydrogels photopolymerized with VW light displayed significantly higher cell viability than cells encapsulated in hydrogels photopolymerized with UV light. We also show that the expression of osteogenesis-related genes at a late stage of osteoblast differentiation in osteoblasts encapsulated in GelMA–RF hydrogel was markedly increased under osteoblast differentiation-inducing conditions. The GelMA–RF hydrogel served as an excellent scaffold for the encapsulation of osteoblasts. GelMA–RF hydrogel-encapsulated osteoblasts have the potential not only to help regenerate bone mass but also to treat complex bone defects associated with bone-destructive diseases such as periodontitis.     

Injectable Thixotropic β–Cyclodextrin–Functionalized Hydrogels Based on Guanosine Quartet Assembly

Facile method for the preparation of β–cyclodextrin–functionalized hydrogels based on guanosine quartet assembly was described. A series of seven hydrogels were prepared by linking β–cyclodextrin molecules with guanosine moieties in different ratios through benzene–1,4–diboronic acid linker in the presence of potassium hydroxide. The potassium ions acted as a reticulation agent by forming guanosine quartets, leading to the formation of self–sustained transparent hydrogels. The ratios of the β–cyclodextrin and guanosine components have a significant effect on the internal structuration of the components and, correspondingly, on the mechanical properties of the final gels, offering a tunablity of the system by varying the components ratio. The insights into the hydrogels’ structuration were achieved by circular dichroism, scanning electron microscopy, atomic force microscopy, and X–ray diffraction. Rheological measurements revealed self–healing and thixotropic properties of all the investigated samples, which, in combination with available cyclodextrin cavities for active components loading, make them remarkable candidates for specific applications in biomedical and pharmaceutical fields. Moreover, all the prepared samples displayed selective antimicrobial properties against S. aureus in planktonic and biofilm phase, the activity also depending on the guanosine and cyclodextrin ratio within the hydrogel structure.     

Dehydropeptide Supramolecular Hydrogels and Nanostructures as Potential Peptidomimetic Biomedical Materials

Supramolecular peptide hydrogels are gaining increased attention, owing to their potential in a variety of biomedical applications. Their physical properties are similar to those of the extracellular matrix (ECM), which is key to their applications in the cell culture of specialized cells, tissue engineering, skin regeneration, and wound healing. The structure of these hydrogels usually consists of a di- or tripeptide capped on the N-terminus with a hydrophobic aromatic group, such as Fmoc or naphthalene. Although these peptide conjugates can offer advantages over other types of gelators such as cross-linked polymers, they usually possess the limitation of being particularly sensitive to proteolysis by endogenous proteases. One of the strategies reported that can overcome this barrier is to use a peptidomimetic strategy, in which natural amino acids are switched for non-proteinogenic analogues, such as D-amino acids, β-amino acids, or dehydroamino acids. Such peptides usually possess much greater resistance to enzymatic hydrolysis. Peptides containing dehydroamino acids, i.e., dehydropeptides, are particularly interesting, as the presence of the double bond also introduces a conformational restraint to the peptide backbone, resulting in (often predictable) changes to the secondary structure of the peptide. This review focuses on peptide hydrogels and related nanostructures, where α,β-didehydro-α-amino acids have been successfully incorporated into the structure of peptide hydrogelators, and the resulting properties are discussed in terms of their potential biomedical applications. Where appropriate, their properties are compared with those of the corresponding peptide hydrogelator composed of canonical amino acids. In a wider context, we consider the presence of dehydroamino acids in natural compounds and medicinally important compounds as well as their limitations, and we consider some of the synthetic strategies for obtaining dehydropeptides. Finally, we consider the future direction for this research area.     

Carbon nanotube‐hybridized supramolecular hydrogel based on PEO‐b‐PPO‐b‐PEO/α‐cyclodextrin as a potential biomaterial

In virtue of the potential biomedical application of carbon nanotube (CNT), the CNT was hybridized into a supramolecular hydrogel based on the selective inclusion of α‐cyclodextrin (α‐CD) onto poly(ethylene oxide) (PEO) segments of a triblock copolymer, i.e., PEO‐block‐poly(propylene oxide)‐block‐PEO. Different from the previous report, the content of α‐CD, in contrast to that of ethylene oxide unit, was decreased to decrease the network density in hydrogel and hence improve the diffusion of encapsulated substances. As a result, the modulus of the hydrogels climbed slightly after introducing CNT. Furthermore, as the essential properties for wound dressing, the antimicrobial activity, the skin‐adhesion, and water‐retention of such supramolecular hybrid hydrogels were also verified. On the other hand, the supramolecular hybrid hydrogels inherited the shear‐thinning property and are suitable as an injectable biomaterial. The cell viability assay confirmed the equivalent cytotoxicity of the supramolecular hybrid hydrogels to that of the native hydrogels without CNT. Consequently, such CNT‐hybridized supramolecular hydrogel shows a great potential in the biomedical application. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010     

Correlated Dynamics in Ionic Liquids by Means of NMR Relaxometry: Butyltriethylammonium bis(Trifluoromethanesulfonyl)imide as an Example

¹H and ¹⁹F spin-lattice relaxation experiments have been performed for butyltriethylammonium bis(trifluoromethanesulfonyl)imide in the temperature range from 258 to 298 K and the frequency range from 10 kHz to 10 MHz. The results have thoroughly been analysed in terms of a relaxation model taking into account relaxation pathways associated with ¹H–¹H, ¹⁹F–¹⁹F and ¹H–¹⁹F dipole–dipole interactions, rendering relative translational diffusion coefficients for the pairs of ions: cation–cation, anion–anion and cation–anion, as well as the rotational correlation time of the cation. The relevance of the ¹H–¹⁹F relaxation contribution to the ¹H and ¹⁹F relaxation has been demonstrated. A comparison of the diffusion coefficients has revealed correlation effects in the relative cation–anion translational movement. It has also turned out that the translational movement of the anions is faster than of cations, especially at high temperatures. Moreover, the relative cation–cation diffusion coefficients have been compared with self-diffusion coefficients obtained by means of NMR (Nuclear Magnetic Resonance) gradient diffusometry. The comparison indicates correlation effects in the relative cation–cation translational dynamics—the effects become more pronounced with decreasing temperature.     

Sustained drug release using supramolecular hydrogels composed of cyclodextrin inclusion complexes with PCL/PEG multiple block copolymers

Methadone hydrochloride is a narcotic drug used to relieve chronic pain and control withdrawal symptoms in people undergoing detoxification for opiate addiction, but poses some limits. To reduce the limits and increase the drug’s effectiveness, supramolecular hydrogels composed of α-CD (α-cyclodextrin) and PCL/PEG (polycaprolactone/polyethylene glycol) copolymers, which gained attention due to their advantages, were chosen in this study as controlled-release formulations. PCL/PEG/PCL and PCL/PEG copolymers were synthesized by a microwave-assisted method and then supramolecular hydrogels were prepared by mixing the solutions of copolymers/drug and α-CD to make a methadone hydrochloride sustained-release system. Effects of the drug, copolymer, and α-CD concentrations and copolymer structure on gelation time were also investigated. The systems had rheological properties that depended on copolymer construction and component concentrations. The microwave-assisted method provides an accelerated synthesis of the copolymers with yield of 57 % and low level of impurities. The supramolecular hydrogels demonstrated shear thinning and thixotropic behavior and regained their networks quickly after passing through a fine needle. Due to the porous construction of supramolecular hydrogels, they swell in the presence of fluids and absorb large quantities of water, thereby making the system comprehensively biocompatible. In this study, supramolecular hydrogels composed of α-CD with tri- and di-block copolymers were investigated and found to have rheological properties that depended on copolymer construction and component concentrations. Changing one parameter alone like copolymer or α-CD concentration or the length of blocks could not significantly affect on the drug release, but combination of these factors was efficient. Results of the present study indicated that supramolecular hydrogels composed of α-CD and PCL/PEG copolymers are appropriate drug delivery systems that can release methadone hydrochloride in a sustained manner.     

3D Bioprinting of Gelatin–Xanthan Gum Composite Hydrogels for Growth of Human Skin Cells

In recent years, bioprinting has attracted much attention as a potential tool for generating complex 3D biological constructs capable of mimicking the native tissue microenvironment and promoting physiologically relevant cell–cell and cell–matrix interactions. The aim of the present study was to develop a crosslinked 3D printable hydrogel based on biocompatible natural polymers, gelatin and xanthan gum at different percentages to be used both as a scaffold for cell growth and as a wound dressing. The CellInk Inkredible 3D printer was used for the 3D printing of hydrogels, and a glutaraldehyde solution was tested for the crosslinking process. We were able to obtain two kinds of printable hydrogels with different porosity, swelling and degradation time. Subsequently, the printed hydrogels were characterized from the point of view of biocompatibility. Our results showed that gelatin/xanthan-gum bioprinted hydrogels were biocompatible materials, as they allowed both human keratinocyte and fibroblast in vitro growth for 14 days. These two bioprintable hydrogels could be also used as a helpful dressing material.     

One Step e-Beam Radiation Cross-Linking of Quaternary Hydrogels Dressings Based on Chitosan-Poly(Vinyl-Pyrrolidone)-Poly(Ethylene Glycol)-Poly(Acrylic Acid)

We report on the successful preparation of wet dressings hydrogels based on Chitosan-Poly(N-Vinyl-Pyrrolidone)-Poly(ethylene glycol)-Poly(acrylic acid) and Poly(ethylene oxide) by e-beam cross-linking in weakly acidic media, to be used for rapid healing and pain release of infected skin wounds. The structure and compositions of hydrogels investigated according to sol-gel and swelling studies, network parameters, as well as FTIR and XPS analyses showed the efficient interaction of the hydrogel components upon irradiation, maintaining the bonding environment while the cross-linking degree increasing with the irradiation dose and the formation of a structure with the mesh size in the range 11–67 nm. Hydrogels with gel fraction above 85% and the best swelling properties in different pH solutions were obtained for hydrogels produced with 15 kGy. The hydrogels are stable in the simulated physiological condition of an infected wound and show appropriate moisture retention capability and the water vapor transmission rate up to 272.67 g m⁻² day⁻¹, to ensure fast healing. The hydrogels proved to have a significant loading capacity of ibuprofen (IBU), being able to incorporate a therapeutic dose for the treatment of severe pains. Simultaneously, IBU was released up to 25% in the first 2h, having a release maximum after 8 h.     

Alginate-g-PNIPAM-Based Thermo/Shear-Responsive Injectable Hydrogels: Tailoring the Rheological Properties by Adjusting the LCST of the Grafting Chains

Graft copolymers of alginate backbone and N-isopropylacrylamide/N-tert-butylacrylamide random copolymer, P(NIPAM_x-co-NtBAM_y), side chains (stickers) with various NtBAM content were designed and explored in aqueous media. Self-assembling thermoresponsive hydrogels are formed upon heating, in all cases, through the hydrophobic association of the P(NIPAM_x-co-NtBAM_y) sticky pendant chains. The rheological properties of the formulations depend remarkably on the NtBAM hydrophobic content, which regulates the lower critical solution temperature (LCST) and, in turn, the stickers’ thermo-responsiveness. The gelation point, T_gel, was shifted to lower temperatures from 38 to 20 °C by enriching the PNIPAM chains with 20 mol % NtBAM, shifting accordingly to the gelation temperature window. The consequences of the T_gel shift to the hydrogels’ rheological properties are significant at room and body temperature. For instance, at 37 °C, the storage modulus increases about two orders of magnitude and the terminal relaxation time increase about 10 orders of magnitude by enriching the stickers with 20 mol % hydrophobic moieties. Two main thermo-induced behaviors were revealed, characterized by a sol–gel and a weak gel–stiff gel transition for the copolymer with stickers of low (0.6 mol %) and high (14, 20 mol %) NtBAM content, respectively. The first type of hydrogels is easily injectable, while for the second one, the injectability is provided by shear-thinning effects. The influence of the type of media (phosphate buffer (PB), phosphate-buffered saline (PBS), Dulbecco’s modified Eagle’s medium (DMEM)) on the hydrogel properties was also explored and discussed. The 4 wt % NaALG-g-P(NIPAM₈₀-co-NtBAM₂₀)/DMEM formulation showed excellent shear-induced injectability at room temperature and instantaneous thermo-induced gel stiffening at body temperature, rendering it a good candidate for cell transplantation potential applications.     

Supramolecular hydrogels of polyaniline-poly(styrene sulfonate) prepared in concentrated solutions

Polyaniline-poly(styrene sulfonate) (PAn-PSS) hydrogels have been synthesized via supramolecular self-assembly between positively-charged PAn chains and negatively-charged PSS chains. Phase diagram is plotted to systematically investigate the gelation conditions for the PAn-PSS system. A hierarchical porous microstructure consisting of oriented 1D nanofibers is observed in the hydrogels, and the phase structure, molecular structure and crystal structure are also characterized. Based on the investigation of a unique transformation of the hydrogels to colloidal particles in alkaline solutions, the electrostatic interaction is proposed to be the origin force for the gelation of the materials. Additionally, in comparison with conventional PAn-PSS colloids, as-prepared PAn-PSS hydrogels are demonstrated to possess improved capacitance performance, such as higher energy density, higher power density and better electrochemical stability. The present study gives valuable hints for achieving controlled fabrication of supramolecular materials with designed structures and outstanding properties.     

Effect of graphene oxide on the pH-responsive drug release from supramolecular hydrogels

Polypseudorotaxane (PPR) hydrogels formed by inclusion complexes between poly(ethylene glycol) (PEG) and α-cyclodextrin (α-CD) are highlighted as promising biomaterial for drug delivery. Here, we report a novel injectable PPR hydrogel containing graphene oxide (GO) for pH-responsive controlled release of doxorubicin hydrochloride (DOX). Our results showed that the gelation rates of the PEG/α-CD supramolecular structures could be tailored depending on the reagent concentrations. The formation of PEG/α-CD inclusion complexes was confirmed by TEM and XRD, the latter further confirming that GO restricts their formation. The supramolecular hydrogels were easily loaded with DOX by simple addition into the PEG solution before the complex formation with the α-CD solution. Noteworthy, disruption of ionic interactions between DOX and GO in the nanocomposite at pH = 5.5 resulted in higher DOX release than under physiological conditions (pH = 7.4). This pH dependence was barely observed in pure PPR hydrogel. These findings introduce DOX-loaded supramolecular hydrogels nanocomposites as promising carriers for pH-responsive and therefore localized, drug delivery systems.     

Impact of Endotoxins in Gelatine Hydrogels on Chondrogenic Differentiation and Inflammatory Cytokine Secretion In Vitro

Gelatine methacryloyl (GelMA) hydrogels are widely used in studies aimed at cartilage regeneration. However, the endotoxin content of commercially available GelMAs and gelatines used in these studies is often overlooked, even though endotoxins may influence several cellular functions. Moreover, regulations for clinical use of biomaterials dictate a stringent endotoxin limit. We determined the endotoxin level of five different GelMAs and evaluated the effect on the chondrogenic differentiation of equine mesenchymal stromal cells (MSCs). Cartilage-like matrix production was evaluated by biochemical assays and immunohistochemistry. Furthermore, equine peripheral blood mononuclear cells (PBMCs) were cultured on the hydrogels for 24 h, followed by the assessment of tumour necrosis factor (TNF)-α and C–C motif chemokine ligand (CCL)2 as inflammatory markers. The GelMAs were found to have widely varying endotoxin content (two with >1000 EU/mL and three with <10 EU/mL), however, this was not a critical factor determining in vitro cartilage-like matrix production of embedded MSCs. PBMCs did produce significantly higher TNF-α and CCL2 in response to the GelMA with the highest endotoxin level compared to the other GelMAs. Although limited effects on chondrogenic differentiation were found in this study, caution with the use of commercial hydrogels is warranted in the translation from in vitro to in vivo studies because of regulatory constraints and potential inflammatory effects of the content of these hydrogels.     

Injectable Human Hair Keratin–Fibrinogen Hydrogels for Engineering 3D Microenvironments to Accelerate Oral Tissue Regeneration

Traumatic injury of the oral cavity is atypical and often accompanied by uncontrolled bleeding and inflammation. Injectable hydrogels have been considered to be promising candidates for the treatment of oral injuries because of their simple formulation, minimally invasive application technique, and site-specific delivery. Fibrinogen-based hydrogels have been widely explored as effective materials for wound healing in tissue engineering due to their uniqueness. Recently, an injectable foam has taken the spotlight. However, the fibrin component of this biomaterial is relatively stiff. To address these challenges, we created keratin-conjugated fibrinogen (KRT-FIB). This study aimed to develop a novel keratin biomaterial and assess cell–biomaterial interactions. Consequently, a novel injectable KRT-FIB hydrogel was optimized through rheological measurements, and its injection performance, swelling behavior, and surface morphology were investigated. We observed an excellent cell viability, proliferation, and migration/cell–cell interaction, indicating that the novel KRT-FIB-injectable hydrogel is a promising platform for oral tissue regeneration with a high clinical applicability.