Biological performance of cell‐encapsulated methacrylated gellan gum‐based hydrogels for nucleus pulposus regeneration

Limitations of current treatments for intervertebral disc (IVD) degeneration have promoted interest in the development of tissue‐engineering approaches. Injectable hydrogels loaded with cells can be used as a substitute material for the inner IVD part, the nucleus pulposus (NP), and provide an opportunity for minimally invasive treatment of IVD degeneration. The NP is populated by chondrocyte‐like cells; therefore, chondrocytes and mesenchymal stem cells (MSCs), stimulated to differentiate along the chondrogenic lineage, could be used to promote NP regeneration. In this study, the in vitro and in vivo response of human bone marrow‐derived MSCs and nasal chondrocytes (NCs) to modified gellan gum‐based hydrogels was investigated. Both ionic‐ (iGG–MA) and photo‐crosslinked (phGG–MA) methacrylated gellan gum hydrogels show no cytotoxicity in extraction assays with MSCs and NCs. Furthermore, the materials do not induce pro‐inflammatory responses in endothelial cells. Moreover, MSCs and NCs can be encapsulated into the hydrogels and remain viable for at least 2 weeks, although apoptosis is observed in phGG–MA. Importantly, encapsulated MSCs and NCs show signs of in vivo chondrogenesis in a subcutaneous implantation of iGG–MA. Altogether, the data endorse the potential use of modified gellan gum‐based hydrogel as a suitable material in NP tissue engineering. Copyright © 2014 John Wiley & Sons, Ltd.     

Gellan gum-based hydrogels for intervertebral disc tissue-engineering applications

Intervertebral disc (IVD) degeneration is a challenging clinical problem that urgently demands viable nucleus pulposus (NP) implant materials. The best suited biomaterial for NP regeneration has yet to be identified, but it is believed that biodegradable hydrogel-based materials are promising candidates. In this work, we have developed ionic- and photo-crosslinked methacrylated gellan gum (GG-MA) hydrogels to be used in acellular and cellular tissue-engineering strategies for the regeneration of IVDs. The physicochemical properties of the developed hydrogels were investigated by Fourier-transform infrared spectroscopy, (1) H nuclear magnetic resonance and differential scanning calorimetry. The swelling ability and degradation rate of hydrogels were also analysed in phosphate-buffered saline solution at physiological pH for a period of 30 days. Additionally, the morphology and mechanical properties of the hydrogels were assessed under a scanning electron microscope and dynamic compression, respectively. An in vitro study was carried out to screen possible cytotoxicity of the gellan gum-based hydrogels by culturing rat lung fibroblasts (L929 cells) with hydrogel leachables up to 7 days. The results demonstrated that gellan gum was successfully methacrylated. We observed that the produced GG-MA hydrogels possess improved mechanical properties and lower water uptake ability and degradation rate as compared to gellan gum. This work also revealed that GG-MA hydrogels are non-cytotoxic in vitro, thus being promising biomaterials to be used in IVD tissue-engineering strategies.     

Enzymatic,urease‐mediated mineralization of gellan gum hydrogel with calcium carbonate,magnesium‐enriched calcium carbonate and magnesium carbonate for bone regeneration applications

Mineralization of hydrogel biomaterials is considered desirable to improve their suitability as materials for bone regeneration. Calcium carbonate (CaCO₃) has been successfully applied as a bone regeneration material, but hydrogel‐CaCO₃ composites have received less attention. Magnesium (Mg) has been used as a component of calcium phosphate biomaterials to stimulate bone‐forming cell adhesion and proliferation and bone regeneration in vivo, but its effect as a component of carbonate‐based biomaterials remains uninvestigated. In the present study, gellan gum (GG) hydrogels were mineralized enzymatically with CaCO₃, Mg‐enriched CaCO₃ and magnesium carbonate to generate composite biomaterials for bone regeneration. Hydrogels loaded with the enzyme urease were mineralized by incubation in mineralization media containing urea and different ratios of calcium and magnesium ions. Increasing the magnesium concentration decreased mineral crystallinity. At low magnesium concentrations calcite was formed, while at higher concentrations magnesian calcite was formed. Hydromagnesite (Mg₅(CO₃)₄(OH)₂.4H₂O) formed at high magnesium concentration in the absence of calcium. The amount of mineral formed and compressive strength decreased with increasing magnesium concentration in the mineralization medium. The calcium:magnesium elemental ratio in the mineral formed was higher than in the respective mineralization media. Mineralization of hydrogels with calcite or magnesian calcite promoted adhesion and growth of osteoblast‐like cells. Hydrogels mineralized with hydromagnesite displayed higher cytotoxicity. In conclusion, enzymatic mineralization of GG hydrogels with CaCO₃ in the form of calcite successfully reinforced hydrogels and promoted osteoblast‐like cell adhesion and growth, but magnesium enrichment had no definitive positive effect. Copyright © 2017 John Wiley & Sons, Ltd.     

Anti‐angiogenic potential of VEGF blocker dendron loaded on to gellan gum hydrogels for tissue engineering applications

Damage of non‐vascularised tissues such as cartilage and cornea can result in healing processes accompanied by a non‐physiological angiogenesis. Peptidic aptamers have recently been reported to block the vascular endothelial growth factor (VEGF). However, the therapeutic applications of these aptamers are limited due to their short half‐life in vivo. In this work, an enhanced stability and bioavailability of a known VEGF blocker aptamer sequence (WHLPFKC) was pursued through its tethering of molecular scaffolds based on hyperbranched peptides, the poly(?‐lysine) dendrons, bearing three branching generations. The proposed design allowed simultaneous and orderly‐spaced exposure of 16 aptamers per dendrimer to the surrounding biological microenvironent, as well as a relatively hydrophobic core based on di‐phenylalanine aiming to promote an hydrophobic interaction with the hydrophobic moieties of ionically crosslinked methacrylated gellan gum (iGG‐MA) hydrogels. The VEGF blocker dendrons were entrapped in iGG‐MA hydrogels, and their capacity to prevent endothelial cell sprouting was assessed qualitatively and quantitatively using 3D in vitro models and the in vivo chick chorioallantoic membrane assay. The data demonstrate that at nanoscale concentrations, the dendronised structures were able to enhance control of the biological actvity of WHLPFKC at the material/tissue interface and hence the anti‐angiogenic capacity of iGG‐MA hydrogels not only preventing blood vessel invasion, but also inducing their regression at the tissue/iGG‐MA interface. The in ovo study confirmed that iGG‐MA functionalised with the dendron VEGF blockers do inhibit angiogenesis by controlling both size and ramifications of blood vessels in the proximity of the implanted gel surface. Copyright © 2016 John Wiley & Sons, Ltd.     

Generation of composites for bone tissue‐engineering applications consisting of gellan gum hydrogels mineralized with calcium and magnesium phosphate phases by enzymatic means

Mineralization of hydrogels, desirable for bone regeneration applications, may be achieved enzymatically by incorporation of alkaline phosphatase (ALP). ALP‐loaded gellan gum (GG) hydrogels were mineralized by incubation in mineralization media containing calcium and/or magnesium glycerophosphate (CaGP, MgGP). Mineralization media with CaGP:MgGP concentrations 0.1:0, 0.075:0.025, 0.05:0.05, 0.025:0.075 and 0:0.1 (all values mol/dm³, denoted A, B, C, D and E, respectively) were compared. Mineral formation was confirmed by IR and Raman, SEM, ICP‐OES, XRD, TEM, SAED, TGA and increases in the the mass fraction of the hydrogel not consisting of water. Ca was incorporated into mineral to a greater extent than Mg in samples mineralized in media A–D. Mg content and amorphicity of mineral formed increased in the order A < B < C < D. Mineral formed in media A and B was calcium‐deficient hydroxyapatite (CDHA). Mineral formed in medium C was a combination of CDHA and an amorphous phase. Mineral formed in medium D was an amorphous phase. Mineral formed in medium E was a combination of crystalline and amorphous MgP. Young's moduli and storage moduli decreased in dependence of mineralization medium in the order A > B > C > D, but were significantly higher for samples mineralized in medium E. The attachment and vitality of osteoblastic MC3T3‐E1 cells were higher on samples mineralized in media B–E (containing Mg) than in those mineralized in medium A (not containing Mg). All samples underwent degradation and supported the adhesion of RAW 264.7 monocytic cells, and samples mineralized in media A and B supported osteoclast‐like cell formation. Copyright © 2014 John Wiley & Sons, Ltd.     

A papain‐induced disc degeneration model for the assessment of thermo‐reversible hydrogel–cells therapeutic approach

Nucleus pulposus (NP) regeneration by the application of injectable cell‐embedded hydrogels is an appealing approach for tissue engineering. We investigated a thermo‐reversible hydrogel (TR‐HG), based on a modified polysaccharide with a thermo‐reversible polyamide [poly(N‐isopropylacrylamide), pNIPAM], which is made to behave as a liquid at room temperature and hardens at > 32 °C. In order to test the hydrogel, a papain‐induced bovine caudal disc degeneration model (PDDM), creating a cavity in the NP, was employed. Human mesenchymal stem cells (hMSCs) or autologous bovine NP cells (bNPCs) were seeded in TR‐HG; hMSCs were additionally preconditioned with rhGDF‐5 for 7 days. Then, TR‐HG was reversed to a fluid and the cell suspension injected into the PDDM and kept under static loading for 7 days. Experimental design was: (D1) fresh disc control + PBS injection; (D2) PDDM + PBS injection; (D3) PDDM + TR‐HG (material control); (D4) PDDM + TR‐HG + bNPCs; (D5) PDDM + TR‐HG + hMSCs. Magnetic resonance imaging performed before and after loading, on days 9 and 16, allowed imaging of the hydrogel‐filled PDDM and assessment of disc height and volume changes. In gel‐injected discs the NP region showed a major drop in volume and disc height during culture under static load. The RT–PCR results of injected hMSCs showed significant upregulation of ACAN, COL2A1, VCAN and SOX9 during culture in the disc cavity, whereas the gene expression profile of NP cells remained unchanged. The cell viability of injected cells (NPCs or hMSCs) was maintained at over 86% in 3D culture and dropped to ~72% after organ culture. Our results underline the need for load‐bearing hydrogels that are also cyto‐compatible. Copyright © 2013 John Wiley & Sons, Ltd.     

Development and characterization of novel agar and gelatin injectable hydrogel as filler for peripheral nerve guidance channels

Injectable hydrogels are becoming of increasing interest in the field of tissue engineering thanks to their versatile properties and to the possibility of being injected into tissues or devices during surgery. In peripheral nerve tissue engineering, injectable hydrogels having shear‐thinning properties are advantageous as filler of nerve guidance channels (NGCs) to improve the regeneration process. In the present work, gelatin‐based hydrogels were developed and specifically designed for the insertion into the lumen of hollow NGCs through a syringe during surgery. Injectable hydrogels were obtained using an agar–gelatin 20:80 weight ratio, (wt/wt) blend crosslinked by the addition of genipin (A/GL_GP). The physicochemical properties of the A/GL_GP hydrogels were analysed, including their injectability, rheological, swelling and dissolution behaviour, and their mechanical properties under compression. The hydrogel developed showed shear‐thinning properties and was applied as filler of NGCs. The A/GL_GP hydrogel was tested in vitro using different cell lines, among them Schwann cells which have been used because they have an important role in peripheral nerve regeneration. Viability assays demonstrated the lack of cytotoxicity. In vitro experiments showed that the hydrogel is able to promote cell adhesion and proliferation. Two‐ and three‐dimensional migration assays confirmed the capability of the cells to migrate both on the surface and within the internal framework of the hydrogel. These data show that A/GL_GP hydrogel has characteristics that make it a promising scaffold material for tissue engineering and nerve regeneration. Copyright © 2014 John Wiley & Sons, Ltd.     

Hydrogels in acellular and cellular strategies for intervertebral disc regeneration

Low back pain is an extremely common illness syndrome that causes patient suffering and disability and requires urgent solutions to improve the quality of life of these patients. Treatment options aimed to regenerate the intervertebral disc (IVD) are still under development. The cellular complexity of IVD, and consequently its fine regulatory system, makes it a challenge to the scientific community. Biomaterials‐based therapies are the most interesting solutions to date, whereby tissue engineering and regenerative medicine (TE&RM) strategies are included. By using such strategies, i.e., combining biomaterials, cells, and biomolecules, the ultimate goal of reaching a complete integration between native and neo‐tissue can be achieved. Hydrogels are promising materials for restoring IVD, mainly nucleus pulposus (NP). This study presents an overview of the use of hydrogels in acellular and cellular strategies for intervertebral disc regeneration. To better understand IVD and its functioning, this study will focus on several aspects: anatomy, pathophysiology, cellular and biomolecular performance, intrinsic healing processes, and current therapies. In addition, the application of hydrogels as NP substitutes will be addressed due to their similarities to NP mechanical properties and extracellular matrix. These hydrogels can be used in cellular strategies when combined with cells from different sources, or in acellular strategies by performing the functionalization of the hydrogels with biomolecules. In addition, a brief summary of therapies based on simple injection for primary biological repair will be examined. Finally, special emphasis will focus on reviewing original studies reporting on the use of autologous cells and biomolecules such as platelet‐rich plasma and their potential clinical applications. Copyright © 2011 John Wiley & Sons, Ltd.     

Chondrogenic potential of injectable κ‐carrageenan hydrogel with encapsulated adipose stem cells for cartilage tissue‐engineering applications

Due to the limited self‐repair capacity of cartilage, regenerative medicine therapies for the treatment of cartilage defects must use a significant amount of cells, preferably applied using a hydrogel system that can promise their delivery and functionality at the specific site. This paper discusses the potential use of κ‐carrageenan hydrogels for the delivery of stem cells obtained from adipose tissue in the treatment of cartilage tissue defects. The developed hydrogels were produced by an ionotropic gelation method and human adipose stem cells (hASCs) were encapsulated in 1.5% w/v κ‐carrageenan solution at a cell density of 5 × 10⁶ cells/ml. The results from the analysis of the cell‐encapsulating hydrogels, cultured for up to 21 days, indicated that κ‐carrageenan hydrogels support the viability, proliferation and chondrogenic differentiation of hASCs. Additionally, the mechanical analysis demonstrated an increase in stiffness and viscoelastic properties of κ‐carrageenan gels with their encapsulated cells with increasing time in culture with chondrogenic medium. These results allowed the conclusion that κ‐carrageenan exhibits properties that enable the in vitro functionality of encapsulated hASCs and thus may provide the basis for new successful approaches for the treatment of cartilage defects. Copyright © 2013 John Wiley & Sons, Ltd.     

Development of a morphogenetically active scaffold for three‐dimensional growth of bone cells: biosilica–alginate hydrogel for SaOS‐2 cell cultivation

Polymeric silica is formed from ortho‐silicate during a sol–gel formation process, while biosilica is the product of an enzymatically driven bio‐polycondensation reaction. Both polymers have recently been described as a template that induces an increased expression of the genes encoding bone morphogenetic protein 2 (BMP‐2) and osteoprotegerin in osteoblast‐related SaOS‐2 cells; simultaneously or subsequently the cells respond with enhanced hydroxyapatite formation. In order to assess whether the biocompatible polymeric silica/biosilica can serve as a morphogenetically active matrix suitable for three‐dimensional (3D) cell growth, or even for 3D cell bioprinting, SaOS‐2 cells were embedded into a Na‐alginate‐based hydrogel. Four different gelatinous hydrogel matrices were used for suspending SaOS‐2 cells: (a) the hydrogel alone; (b) the hydrogel with 400 μm ortho‐silicate; (c) the hydrogel supplemented with 400 μm ortho‐silicate and recombinant silicatein to allow biosilica synthesis to occur; and (d) the hydrogel with ortho‐silicate and BSA. The SaOS‐2 cells showed an increased growth if silica/biosilica components were present in the hydrogel. Likewise intensified was the formation of hydroxyapatite nodules in the silica‐containing hydrogels. After an incubation period of 2 weeks, cells present in silica‐containing hydrogels showed a significantly higher expression of the genes encoding the cytokine BMP‐2, the major fibrillar structural protein collagen 1 and likewise of carbonic anhydrase. It is concluded that silica, and to a larger extent biosilica, retains its morphogenetic/osteogenic potential after addition to Na‐alginate‐based hydrogels. This property might qualify silica hydrogels to be also used as a matrix for 3D cell printing. Copyright © 2013 John Wiley & Sons, Ltd.     

Novel injectable gellan gum hydrogel composites incorporating Zn‐ and Sr‐enriched bioactive glass microparticles: High‐resolution X‐ray microcomputed tomography,antibacterial and in vitro testing

Mineralization of hydrogel biomaterials is desirable to improve their suitability as materials for bone regeneration. In this study, gellan gum (GG) hydrogels were formed by simple mixing of GG solution with bioactive glass microparticles of 45S5 composition, leading to hydrogel formation by ion release from the amorphous bioactive glass microparticles. This resulted in novel injectable, self‐gelling composites of GG hydrogels containing 20% bioactive glass. Gelation occurred within 20 min. Composites containing the standard 45S5 bioactive glass preparation were markedly less stiff. X‐ray microcomputed tomography proved to be a highly sensitive technique capable of detecting microparticles of diameter approximately 8 μm, that is, individual microparticles, and accurately visualizing the size distribution of bioactive glass microparticles and their aggregates, and their distribution in GG hydrogels. The widely used melt‐derived 45S5 preparation served as a standard and was compared with a calcium‐rich, sol–gel derived preparation (A2), as well as A2 enriched with zinc (A2Zn5) and strontium (A2Sr5). A2, A2Zn, and A2Sr bioactive glass particles were more homogeneously dispersed in GG hydrogels than 45S5. Composites containing all four bioactive glass preparations exhibited antibacterial activity against methicillin‐resistant Staphylococcus aureus. Composites containing A2Zn5 and A2Sr5 bioactive glasses supported the adhesion and growth of osteoblast‐like cells and were considerably more cytocompatible than 45S5. All composites underwent mineralization with calcium‐deficient hydroxyapatite upon incubation in simulated body fluid. The extent of mineralization appeared to be greatest for composites containing A2Zn5 and 45S5. The results underline the importance of the choice of bioactive glass when preparing injectable, self‐gelling composites.     

Preparation of a “Branch-Fruit” structure chitosan nanofiber physical hydrogels with high mechanical strength and pH-responsive controlled drug release properties

The poor mechanical properties of chitosan physical hydrogels seriously hinder their application in the biomedical field. Inspired by the structure of cell tissues, a novel chitosan nanofiber (CSNF)/Hyaluronic acid (HA)/β-glycerophosphate disodium (β-GP) drug-loaded hydrogel was prepared by micro-dissolution and physical crosslinking. The hydrogel has a “Branch-Fruit” structure and exhibits excellent mechanical properties, good biocompatibility and cell-adhesion properties. Human cancer cells (HeLa) can adhere to the hydrogel surface, which might facilitate tumor site-specific administration of drugs. This material also exhibits high pH sensitivity, with which drug release can be triggered under acidic conditions at pH 4.00. The mechanical strength and drug release behavior of this hydrogel can be easily adjusted by varying the CSNF content.

Representation of the gelation mechanism of CSNF/HA/β-GP precursor solution.     

Mineralization of gellan gum hydrogels with calcium and magnesium carbonates by alternate soaking in solutions of calcium/magnesium and carbonate ion solutions

Mineralization of hydrogels is desirable prior to applications in bone regeneration. CaCO₃ is a widely used bone regeneration material, and Mg, when used as a component of calcium phosphate biomaterials, has promoted bone‐forming cell adhesion and proliferation and bone regeneration. In this study, gellan gum hydrogels were mineralized with carbonates containing different amounts of calcium (Ca) and magnesium (Mg) by alternate soaking in, firstly, a calcium and/or magnesium ion solution and, secondly, a carbonate ion solution. This alternate soaking cycle was repeated five times. Five different calcium and/or magnesium ion solutions, containing different molar ratios of Ca to Mg ranging from Mg free to Ca free were compared. Carbonate mineral formed in all sample groups subjected to the alternate soaking cycle. Ca : Mg elemental ratio in the mineral formed was higher than in the respective mineralizing solution. Mineral formed in the absence of Mg was predominantly CaCO₃ in the form of a mixture of calcite and vaterite. Increasing the Mg content in the mineral formed led to the formation of magnesian calcite and decreased the total amount of the mineral formed and its crystallinity. Hydrogel mineralization and increasing Mg content in mineral formed did not obviously improve proliferation of MC3T3‐E1 osteoblast‐like cells or differentiation after 7 days.     

Hydrogel surfaces to promote attachment and spreading of endothelial progenitor cells

Endothelialization of artificial vascular grafts is a challenging process in cardiovascular tissue engineering. Functionalized biomaterials could be promising candidates to promote endothelialization in repair of cardiovascular injuries. The purpose of this study was to synthesize hyaluronic acid (HA) and heparin‐based hydrogels that could promote adhesion and spreading of endothelial progenitor cells (EPCs). We report that the addition of heparin into HA‐based hydrogels provides an attractive surface for EPCs promoting spreading and the formation of an endothelial monolayer on the hydrogel surface. To increase EPC adhesion and spreading, we covalently immobilized CD34 antibody (Ab) on HA–heparin hydrogels, using standard EDC/NHS amine‐coupling strategies. We found that EPC adhesion and spreading on CD34 Ab‐immobilized HA–heparin hydrogels was significantly higher than their non‐modified analogues. Once adhered, EPCs spread and formed an endothelial layer on both non‐modified and CD34 Ab‐modified HA–heparin hydrogels after 3 days of culture. We did not observe significant adhesion and spreading when heparin was not included in the control hydrogels. In addition to EPCs, we also used human umbilical cord vein endothelial cells (HUVECs), which adhered and spread on HA–heparin hydrogels. Macrophages exhibited significantly less adhesion compared to EPCs on the same hydrogels. This composite material could possibly be used to develop surface coatings for artificial cardiovascular implants, due to its specificity for EPC and endothelial cells on an otherwise non‐thrombogenic surface. Copyright © 2012 John Wiley & Sons, Ltd.     

Visualizing cell‐laden fibrin‐based hydrogels using cryogenic scanning electron microscopy and confocal microscopy

The present investigation explores the microscopic aspects of cell‐laden hydrogels at high resolutions, using three‐dimensional cell cultures in semi‐synthetic constructs that are of very high water content (>98% water). The study aims to provide an imaging strategy for these constructs, while minimizing artefacts. Constructs of poly(ethylene glycol)‐fibrinogen and fibrin hydrogels containing embedded mesenchymal cells (human dermal fibroblasts) were first imaged by confocal microscopy. Next, high‐resolution scanning electron microscopy (HR‐SEM) was used to provide images of the cells within the hydrogels, at submicron resolutions. Because it was not possible to obtain artefact‐free images of the hydrogels using room‐temperature HR‐SEM, a cryogenic HR‐SEM imaging methodology was employed to visualize the sample while preserving the natural hydrated state of the hydrogel. The ultrastructural details of the constructs were observed at subcellular resolutions, revealing numerous cellular components, the biomaterial in its native configuration, and the uninterrupted cell membrane as it relates with the biomaterial in the hydrated state of the construct. Constructs containing microscopic albumin microbubbles were also imaged using these methodologies to reveal fine details of the interaction between the cells, the microbubbles, and the hydrogel. Taken together with the confocal microscopy, this imaging strategy provides a more complete picture of the hydrated state of the hydrogel network with cells inside. As such, this methodology addresses some of the challenges of obtaining this information in amorphous hydrogel systems containing a very high water content (>98%) with embedded cells. Such insight may lead to better hydrogel‐based strategies for tissue engineering and regeneration.     

Composites of gellan gum hydrogel enzymatically mineralized with calcium–zinc phosphate for bone regeneration with antibacterial activity

Gellan gum hydrogels functionalized with alkaline phosphatase were enzymatically mineralized with phosphates in mineralization medium containing calcium (Ca) and zinc (Zn) to improve their suitability as biomaterials for bone regeneration. The aims of the study were to endow mineralized hydrogels with antibacterial activity by incorporation of Zn in the inorganic phase, and to investigate the effect of Zn incorporation on the amount and type of mineral formed, the compressive modulus of the mineralized hydrogels and on their ability to support adhesion and growth of MC3T3‐E1 osteoblast‐like cells. Mineralization medium contained glycerophosphate (0.05 m) and three different molar Ca:Zn ratios, 0.05:0, 0.04:0.01 and 0.025:0.025 (all mol/dm³), hereafter referred to as A, B and C, respectively. FTIR, SAED and TEM analysis revealed that incubation for 14 days caused the formation of predominantly amorphous mineral phases in sample groups A, B and C. The presence of Zn in sample groups B and C was associated with a drop in the amount of mineral formed and a smaller mineral deposit morphology, as observed by SEM. ICP–OES revealed that Zn was preferentially incorporated into mineral compared to Ca. Mechanical testing revealed a decrease in compressive modulus in sample group C. Sample groups B and C, but not A, showed antibacterial activity against biofilm‐forming, methicillin‐resistant Staphylococcus aureus. All sample groups supported cell growth. Zn incorporation increased the viable cell number. The highest values were seen on sample group C. In conclusion, the sample group containing the most Zn, i.e. group C, appears to be the most promising. Copyright © 2015 John Wiley & Sons, Ltd.     

RGD‐mimic polyamidoamine–montmorillonite composites with tunable stiffness as scaffolds for bone tissue‐engineering applications

This paper reports on the development of montmorillonite (MMT)‐reinforced hydrogels, based on a peptidomimetic polyamidoamine carrying guanidine pendants (AGMA1), as substrates for the osteo‐induction of osteoblast precursor cells. AGMA1 hydrogels of various degrees of crosslinking responded favourably to MMT reinforcement, giving rise to composite hydrogels with shear storage modulus G′, when fully swollen in water, up to 200 kPa, i.e. 20 times higher than the virgin hydrogels and of the same order or higher than other hydrogel‐based composites proposed for orthopaedic applications. This significant improvement was ascribed to the effective interpenetration between the polymer matrix and the inorganic filler. AGMA1–MMT hydrogels, when evaluated as scaffolds for the osteogenic differentiation of mouse calvaria‐derived pre‐osteoblastic MC3T3‐E1 cells, proved able to support cell adhesion and proliferation and clearly induced differentiation towards the osteoblastic phenotype, as indicated by different markers. In addition, AGMA1–MMT hydrogels proved completely degradable in aqueous media at pH 7.4 and did not provide any evidence of cytotoxicity. The experimental evidence suggests that AGMA1–MMT composites definitely warrant potential as scaffolds for osteoblast culture and bone grafts. Copyright © 2016 John Wiley & Sons, Ltd.     

Viability and neuronal differentiation of neural stem cells encapsulated in silk fibroin hydrogel functionalized with an IKVAV peptide

Three‐dimensional (3D) porous scaffolds combined with therapeutic stem cells play vital roles in tissue engineering. The adult brain has very limited regeneration ability after injuries such as trauma and stroke. In this study, injectable 3D silk fibroin‐based hydrogel scaffolds with encapsulated neural stem cells were developed, aiming at supporting brain regeneration. To improve the function of the hydrogel towards neural stem cells, silk fibroin was modified by an IKVAV peptide through covalent binding. Both unmodified and modified silk fibroin hydrogels were obtained, through sonication, with mechanical stiffness comparable to that of brain tissue. Human neural stem cells were encapsulated in both hydrogels and the effects of IKVAV peptide conjugation on cell viability and neural differentiation were assessed. The silk fibroin hydrogel modified by IKVAV peptide showed increased cell viability and an enhanced neuronal differentiation capability, which contributed to understanding the effects of IKVAV peptide on the behaviour of neural stem cells. For these reasons, IKVAV‐modified silk fibroin is a promising material for brain tissue engineering. Copyright © 2015 John Wiley & Sons, Ltd.