Horseradish peroxidase‐catalysed in situ‐forming hydrogels for tissue‐engineering applications

In situ‐forming hydrogels are an attractive class of implantable biomaterials that are used for biomedical applications. These injectable hydrogels are versatile and provide a convenient platform for delivering cells and drugs via minimally invasive surgery. Although several crosslinking methods for preparing in situ forming hydrogels have been developed over the past two decades, most hydrogels are not sufficiently versatile for use in a wide variety of tissue‐engineering applications. In recent years, enzyme‐catalysed crosslinking approaches have been emerged as a new approach for developing in situ‐forming hydrogels. In particular, the horseradish peroxidase (HRP)‐catalysed crosslinking approach has received increasing interest, due to its highly improved and tunable capacity to obtain hydrogels with desirable properties. The HRP‐catalysed crosslinking reaction immediately occurs upon mixing phenol‐rich polymers with HRP and hydrogen peroxide (H₂O₂) in aqueous media. Based on this unique gel‐forming feature, recent studies have shown that various properties of formed hydrogels, such as gelation time, stiffness and degradation rate, can be easily manipulated by varying the concentrations of HRP and H₂O₂. In this review, we outline the versatile properties of HRP‐catalysed in situ‐forming hydrogels, with a brief introduction to the crosslinking mechanisms involved. In addition, the recent biomedical applications of HRP‐catalysed in situ‐forming hydrogels for tissue regeneration are described. Copyright © 2014 John Wiley & Sons, Ltd.     

Design and performance of a poly(vinyl alcohol)/silk fibroin enzymatically crosslinked semi-interpenetrating hydrogel for a potential hydrophobic drug delivery

In this study, in order to obtain hydrogels with good properties for sustained release of hydrophobic drugs or for tissue engineering, poly(vinyl alcohol) (PVA)/silk fibroin (SF) semi-interpenetrating (semi-IPN) hydrogels with varied ratios of PVA/SF were enzymatically cross-linked using horseradish peroxidase. A vial inversion test determined approximate gelation times of PVA/SF hydrogels ranging from 5 to 10 min. The hydrogels with varied ratios showed differences in pore size and morphology. Mass loss rate of hydrogels increased from 15% to 58% with increasing PVA concentration. Stable hydrogels with PVA/SF at 0.5 : 1 w/w showed the best swelling ratio values in distilled water (7.36). FTIR analysis revealed that silk fibroin in these hydrogels exhibited the coexistence of amorphous and silk I crystalline structures and the SF and PVA molecules interacted with each other well. The mechanical properties of the composite hydrogels were controlled by the SF content. From the cell viability results, it was found that the hydrogels exerted very low cytotoxicity. Paeonol was chosen as the hydrophobic drug model for release studies from the hydrogels. Paeonol can be uniformly loaded into the composite hydrogels using the emulsifying property of PVA and paeonol release from the hydrogels was dependent on the PVA/SF ratio. This study applied a novel type of enzymatically crosslinked semi-IPN hydrogel that may have potential applications in drug delivery.

Enzymatically cross-linked PVA/SF semi-IPN hydrogels with tunable pore structure have potential applications in sustained release of hydrophobic drug.     

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.     

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.     

Cell encapsulation within PVA‐based hydrogels via freeze‐thawing: a one‐step scaffold formation and cell storage technique

Cryogelation is a physical hydrogel formation method for certain polymers, notably polyvinyl alcohol (PVA). The hypothesis of this study is that a PVA‐based solution with the necessary intracellular cryoprotectant and nutrient supply can be used, first for storage of vascular smooth muscle cells, and subsequently to form a suitable tissue‐engineering scaffold during the thawing process. Bovine arterial smooth muscle cells were encapsulated within PVA–gelatin hydrogels over a wide range of serum, DMSO and cell culture medium concentrations. Several parameters expected to affect gelation and cell viability (PVA viscosity, DMSO concentration, serum presence) were assessed with experimental designs and the optimal conditions for cell survival were determined. Cell viability can be improved by increasing concentration of DMSO and serum without compromising the gelation process. An additional crosslinking step using a coagulation bath was beneficial for hydrogel stability but caused peripheral accumulation of cells. In conclusion, a freeze–thaw process can be utilized to prepare and store cell‐laden hydrogels with adjustable mechanical properties. Copyright © 2009 John Wiley & Sons, Ltd.     

In vivo biocompatibility of p(HPMAm‐lac)‐PEG hydrogels hybridized with hyaluronan

The present study reports on the biocompatibility in vivo after intramuscular and subcutaneous administration in Balb/c mice of vinyl sulphone bearing p(HPMAm‐lac1–2)‐PEG‐p(HPMAm‐lac1–2)/thiolated hyaluronic acid hydrogels, designed as novel injectable biomaterials for potential application in the fields of tissue engineering and regenerative medicine. Ultrasonography, used as a method to study hydrogel gelation and residence time in vivo, showed that, upon injection, the biomaterial efficiently formed a hydrogel by simultaneous thermal gelation and Michael Addition cross‐linking forming a viscoelastic spherical depot at the injection site. The residence time in vivo (20 days) was found to be shorter than that observed in vitro (32 days), indicating that the injected hydrogel was resorbed not only by chemical hydrolysis but also by cellular metabolism and/or enzymatic activity. Systemic biocompatibility was tested by analysing routine haematological parameters at different time‐points (7, 14 and 21 days after administration) and histology of the main organs, including the haematopoietic system. No statistically significant difference between parameters of the saline‐treated group and those of the hydrogel‐treated group was found. Importantly, a time‐dependent decrease of important pro‐inflammatory cytokines (TREM1 (Triggering Receptor Expressed on Myeloid cells‐1), tumour necrosis factor‐α and interleukin‐1β) in cultured bone marrow cells extracted from hydrogel treated mice was observed, possibly correlated to the anti‐inflammatory effect of hyaluronic acid released in time as hydrogel degraded. Copyright © 2016 John Wiley & Sons, Ltd.     

Assessing glucose and oxygen diffusion in hydrogels for the rational design of 3D stem cell scaffolds in regenerative medicine

Hydrogels are attractive biomaterials for replicating cellular microenvironments, but attention needs to be given to hydrogels diffusion properties. A large body of literature shows the promise of hydrogels as 3D culture models, cell expansion systems, cell delivery vehicles, and tissue constructs. Surprisingly, literature seems to have overlooked the important effects of nutrient diffusion on the viability of hydrogel‐encapsulated cells. In this paper, we present the methods and results of an investigation into glucose and oxygen diffusion into a silated‐hydroxypropylmethylcellulose (Si‐HPMC) hydrogel. Using both an implantable glucose sensor and implantable oxygen sensor, we continuously monitored core glucose concentration and oxygen concentration at the centre of hydrogels. We demonstrated that we could tune molecular transport in Si‐HPMC hydrogel by changing the polymer concentration. Specifically, the oxygen diffusion coefficient was found to significantly decrease from 3.4 × 10^?10 to 2.4 × 10^?10 m² s^?1 as the polymer concentration increased from 1% to 4% (w/v). Moreover, it was revealed during in vitro culture of cellularized hydrogels that oxygen depletion occurred before glucose depletion, suggesting oxygen diffusion is the major limiting factor for cell survival. Insight was also gained into the mechanism of action by which oxygen and glucose diffuse. Indeed, a direct correlation was found between the average polymer crosslinking node size and glucose parameters, and this correlation was not observed for oxygen. Overall, these experiments provide useful insights for the analysis of nutrient transport and gas exchange in hydrogels and for the development of future cellular microenvironments based on Si‐HPMC or similar polysaccharide hydrogels.     

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.     

Gelatin‐based hydrogel for vascular endothelial growth factor release in peripheral nerve tissue engineering

Hydrogels are promising materials in regenerative medicine applications, due to their hydrophilicity, biocompatibility and capacity to release drugs and growth factors in a controlled manner. In this study, biocompatible and biodegradable hydrogels based on blends of natural polymers were used in in vitro and ex vivo experiments as a tool for VEGF‐controlled release to accelerate the nerve regeneration process. Among different candidates, the angiogenic factor VEGF was selected, since angiogenesis has been long recognized as an important and necessary step during tissue repair. Recent studies have pointed out that VEGF has a beneficial effect on motor neuron survival and Schwann cell vitality and proliferation. Moreover, VEGF administration can sustain and enhance the growth of regenerating peripheral nerve fibres. The hydrogel preparation process was optimized to allow functional incorporation of VEGF, while preventing its degradation and denaturation. VEGF release was quantified through ELISA assay, whereas released VEGF bioactivity was validated in human umbilical vein endothelial cells (HUVECs) and in a Schwann cell line (RT4‐D6P2T) by assessing VEGFR‐2 and downstream effectors Akt and Erk1/2 phosphorylation. Moreover, dorsal root ganglia explants cultured on VEGF‐releasing hydrogels displayed increased neurite outgrowth, providing confirmation that released VEGF maintained its effect, as also confirmed in a tubulogenesis assay. In conclusion, a gelatin‐based hydrogel system for bioactive VEGF delivery was developed and characterized for its applicability in neural tissue engineering. Copyright © 2014 John Wiley & Sons, Ltd.     

Ascorbic acid promotes extracellular matrix deposition while preserving valve interstitial cell quiescence within 3D hydrogel scaffolds

Current options for aortic valve replacements are non‐viable and thus lack the ability to grow and remodel, which can be problematic for paediatric applications. Toward the development of living valve substitutes that can grow and remodel, porcine aortic valve interstitial cells (VICs) were isolated and encapsulated within proteolytically degradable and cell‐adhesive poly(ethylene glycol) (PEG) hydrogels, in an effort to study their phenotypes and functions. The results showed that encapsulated VICs maintained high viability and proliferated within the hydrogels. The VICs actively remodelled the hydrogels via secretion of matrix metalloproteinase‐2 (MMP‐2) and deposition of new extracellular matrix (ECM) components, including collagens I and III. The soft hydrogels with compressive moduli of ~4.3 kPa quickly reverted VICs from an activated myofibroblastic phenotype to a quiescent, unactivated phenotype, evidenced by the loss of α‐smooth muscle actin expression upon encapsulation. In an effort to promote VIC‐mediated ECM production, ascorbic acid (AA) was supplemented in the medium to investigate its effects on VIC function and phenotype. AA treatment enhanced VIC spreading and proliferation, and inhibited apoptosis. AA treatment also promoted VIC‐mediated ECM remodelling by increasing MMP‐2 activity and depositing collagens I and III. AA treatment did not significantly influence the expression of α‐smooth muscle actin (myofibroblast activation marker) and alkaline phosphatase (osteogenic differentiation marker). No calcification or nodule formation was observed within the cell‐laden hydrogels, with or without AA treatment. These results suggest the potential of this system and the beneficial effect of AA in heart valve tissue engineering. Copyright © 2015 John Wiley & Sons, Ltd.     

Functionalized PVA–silk blended nanofibrous mats promote diabetic wound healing via regulation of extracellular matrix and tissue remodelling

Chronic cutaneous ulcers, a complex pathophysiological diabetic condition, represent a critical clinical challenge in the current diabetes mellitus pandemic. Consequently, there is a compelling need for bioactive dressings that can trigger healing processes for complete wound repair. Silk fibroin (SF), a natural protein polymer from mulberry and non‐mulberry silkworms, has properties that support accelerated wound healing rate. SF from non‐mulberry variety possesses additional cell‐binding motifs (arginine, glycine, and aspartate), offering cell–material interactions. This study is aimed to investigate wound healing efficacy of dressings made up of various SF varieties blended with poly(vinyl alcohol) biopolymer in alloxan‐induced diabetic rabbit model. The nanofibrous mats have been developed using electrospinning and functionalized with growth factors and LL‐37 antimicrobial peptide for sustained delivery. Following post 14‐day treatment, non‐mulberry SF (NMSF)‐based dressings healed the wounds faster, in comparison with their mulberry Bombyx mori SF, poly(vinyl alcohol), and control counterparts (p < .01). NMSF‐based dressings also supported faster granulation tissue development, angiogenesis, and reepithelialization of wounds. Gene expression study of matrix metalloproteinases and collagen proteins affirmed higher extent of tissue remodelling during the repair process. Furthermore, there was organized extracellular matrix deposition (collagen type I, collagen type III, elastin, and reticulin) and higher wound breaking strength in NMSF compared with other groups after 4 weeks. These results validated the potential of NMSF‐based bioactive dressings to regulate extracellular matrix deposition leading to faster and complete repair of chronic diabetic cutaneous wounds.     

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.     

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.     

Preparing 3D-printable silk fibroin hydrogels with robustness by a two-step crosslinking method

Regenerated silk fibroin (RSF) features excellent biocompatibility and high-strength mechanical properties. However, traditional RSF-based materials can hardly be applied in 3D printing, which has shown great potential in producing artificial implants. In this work, we report a 3D printable RSF hydrogel formed by a weak, chemically crosslinked network. After the 3D printing process, the mechanical properties of the above hydrogel can be remarkably improved by a ripening process. The maximum compressive modulus of this RSF hydrogel is 2.5 MPa, reaching the same order of magnitude as natural elastomers such as cartilage. The mechanical properties of this hydrogel are superior to most RSF-based 3D printed hydrogels. The investigation of gelation mechanism reveals that the chemically crosslinked network can constrain the growth of β-sheet structures of RSF to form a dense and uniform physical network. Such a physically crosslinked network endows the high strength and good resilience of RSF hydrogels. With both good biocompatibility and mechanical properties, this double-network hydrogel has potential in producing 3D printed scaffolds for tissue engineering.

Schematic showing the fabrication process of the 3D-printed robust double-network RSF hydrogels.     

Core‐shell silk hydrogels with spatially tuned conformations as drug‐delivery system

Hydrogels of spatially controlled physicochemical properties are appealing platforms for tissue engineering and drug delivery. In this study, core‐shell silk fibroin (SF) hydrogels of spatially controlled conformation were developed. The core‐shell structure in the hydrogels was formed by means of soaking the preformed (enzymatically crosslinked) random coil SF hydrogels in methanol. When increasing the methanol treatment time from 1 to 10 min, the thickness of the shell layer can be tuned from about 200 to about 850 μm as measured in wet status. After lyophilization of the rehydrated core‐shell hydrogels, the shell layer displayed compact morphology and the core layer presented porous structure, when observed by scanning electron microscopy. The conformation of the hydrogels was evaluated by Fourier transform infrared spectroscopy in wet status. The results revealed that the shell layer possessed dominant β‐sheet conformation and the core layer maintained mainly random coil conformation. Enzymatic degradation data showed that the shell layers presented superior stability to the core layer. The mechanical analysis displayed that the compressive modulus of the core‐shell hydrogels ranged from about 25 kPa to about 1.1 MPa by increasing the immersion time in methanol. When incorporated with albumin, the core‐shell SF hydrogels demonstrated slower and more controllable release profiles compared with the non‐treated hydrogel. These core‐shell SF hydrogels of highly tuned properties are useful systems as drug‐delivery system and may be applied as cartilage substitute. Copyright © 2016 John Wiley & Sons, Ltd.     

A straightforward method to produce decellularized dermis‐based matrices for tumour cell cultures

Decellularized matrices are steadily gaining popularity to study the biology of cells and tissues, as they represent a biomimetic environment in which cells can recapitulate certain behaviours that share similarities with those observed in vivo. Basically, biochemistry, microstructure and mechanics of the decellularized matrices are the most valuable properties that differentiate these culturing systems from conventional bidimensional models. Several procedures to decellularize tissues have been proposed so far, with the common aim to preserve the tissue chemical/physical properties of the original tissue. However, these processes are complex, time‐consuming and expensive. In this work, we propose a cost‐effective, easy‐to‐produce decellularized dermal matrix, derived from animal skin. The chemical/physical processes to obtain the matrices proved to not alter matrix structure and did not induce cytotoxicity issues. To test the validity of the decellularized matrices as a model to study the behaviour of tumour cells in vitro, we performed microstructural and mechanical investigations as well as cell proliferation assays. In particular, three different tumour cell lines were used, which proliferated and invaded the matrix with no additional treatments. Decellularized skin scaffold, presented in this work, could be a strong competitor for conventional 3D systems like synthetic porous scaffolds or hydrogels. Copyright © 2016 John Wiley & Sons, Ltd.     

Synthetic poly(amino acid) hydrogels with incorporated cell‐adhesion peptides for tissue engineering

Preparation of soft poly(amino acid) hydrogels containing biomimetic cell‐adhesive peptides was investigated. Covalently crosslinked gels were formed by radical co‐polymerization of methacryloylated macromonomer poly[N⁵‐(2‐hydroxyethyl)‐L ‐glutamine‐stat‐L ‐alanine‐stat‐methacryloyllysine] with 2‐hydroxyethyl methacrylate (HEMA) as minor co‐monomer. Hydrogels carrying biomimetic peptides were prepared by using methacryloylated peptides, such as methacryloyl–GGGRGDSG–OH and methacryloyl–GGGYIGSR–OH, as additional monomers in the polymerization mixture. Mechanical stability and swelling in water of the hydrogels obtained for different solid:water and polypeptide:HEMA ratios were evaluated. The microporosity of gels (5–20 µm), dependent on the polyHEMA phase separation in water, was followed by low‐vacuum SEM. The effect of biomimetic modification of hydrogels with RGDS and YIGSR peptides on the seeding efficiency of porcine mesenchymal stem cells (MSCs) was studied in vitro. While unmodified hydrogels showed very low cell adhesion, due to their highly hydrophilic nature, the incorporation of adhesive peptides significantly improved the adhesion and viability of seeded cells. Copyright © 2010 John Wiley & Sons, Ltd.     

TGF‐β1‐activated type 2 dendritic cells promote wound healing and induce fibroblasts to express tenascin c following corneal full‐thickness hydrogel transplantation

We showed previously that 1‐ethyl‐3‐(3‐dimethylamino‐propyl)‐carbodiimide hydrochloride (EDC) cross‐linked recombinant human collagen III hydrogels promoted stable regeneration of the human cornea (continued nerve and stromal cell repopulation) for over 4 years. However, as EDC cross linking kinetics were difficult to control, we additionally tested a sterically bulky carbodiimide. Here, we compared the effects of two carbodiimide cross linkers—bulky, aromatic N‐cyclohexyl‐N0‐(2‐morpholinoethyl)‐carbodiimide (CMC), and nonbulky EDC—in a mouse corneal graft model. Murine corneas undergoing full‐thickness implantation with these gels became opaque due to dense retro‐corneal membranes (RCM). Corneal epithelial cytokeratin 12 and alpha smooth muscle actin indicative of functional tissue regeneration and wound contraction were observed in RCM surrounding both hydrogel types. However, quantitatively different levels of infiltrating CD11c⁺ dendritic cells (DC) were found, suggesting a hydrogel‐specific innate immune response. More DC infiltrated the stroma surrounding EDC‐N‐hydroxysuccinimide (NHS) hydrogels concurrently with higher fibrosis‐associated tenascin c expression. The opposite was true for CMC‐NHS gels that had previously been shown to be more tolerising to DC. In vitro studies showed that DC cultured with transforming growth factor β1 (TGF‐β1) induced fibroblasts to secrete more tenascin c than those cultured with lipopolysaccharide and this effect was blocked by TGF‐β1 neutralisation. Furthermore, tenascin c staining was found in 40‐ to 50μm long membrane nanotubes formed in fibroblast/DC cocultures. We suggest that TGF‐β1 alternatively activated (tolerising) DC regulate fibroblast‐mediated tenascin c secretion, possibly via local production of TGF‐β1 in early wound contraction, and that this is indirectly modulated by different hydrogel chemistries.