JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE J Tissue Eng Regen Med (2014) Published online in Wiley Online Library ( DOI: 10.1002/term.1917


Horseradish peroxidase-catalysed in situ-forming hydrogels for tissue-engineering applications Jin Woo Bae, Jong Hoon Choi, Yunki Lee and Ki Dong Park* Department of Molecular Science and Technology, Ajou University, Woncheon, Yeongtong, Suwon, Republic of Korea

Abstract 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. Received 11 September 2013; Revised 26 January 2014; Accepted 22 April 2014

Keywords in situ-forming hydrogel; horseradish peroxidase; hydrogen peroxide; enzymatic crosslinking; tissue regeneration; phenol-rich polymer

1. Introduction Tissue regeneration is an auto-healing process that occurs in the body. Tissue damage triggers the healing process that leads to several biological interactions. A complex healing process is systemically implemented for successful tissue renewal and growth. However, the process does not always work if tissue is damaged beyond repair. Tissue engineering can be used to improve the natural healing capability of the body and to replace unrecoverable tissue. Tissue engineering commonly involves the combinatorial use of the tissue-engineering triad: cells, signalling molecules and scaffolds. Previously published reviews highlight the importance of the triad constituents and the recent advances in the field of tissue engineering *Correspondence to: Ki Dong Park, Department of Molecular Science and Technology, Ajou University, 5 Woncheon, Yeongtong, Suwon 443-749, Republic of Korea. E-mail: [email protected] Copyright © 2014 John Wiley & Sons, Ltd.

(Cao et al., 2009; Brochhausen et al., 2009; Chan and Mooney, 2008; Hunt and Grover, 2010). There has been an increasing interest in the development of scaffold materials that can manipulate cell behaviours to promote tissue regeneration. From a biomimetic point of view, hydrogels are regarded as a scaffold platform with immense potential, owing to their mechanical and structural properties, which are similar to those of living tissues and extracellular matrix (ECM) (Annabi et al., 2010). Several recent studies have provided a good rationale for using hydrogels to regenerate tissue (Kim et al., 2011; Ye et al., 2011). Hydrogels that feature three-dimensional (3D) hydrophilic networks can be prepared by crosslinking reactions between polymer chains. The crosslinking reactions confer mechanical stability and structural integrity to the hydrogel matrices that is suitable for tissue regeneration. If the reactions occur rapidly, the gel precursor solution can be injected at the desired site to fill up a tissue

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defect for in situ hydrogel formation. This unique feature makes hydrogels more useful for local delivery of cells and/or drugs via a minimally invasive surgical procedure. Accordingly, the hydrogels featuring rapid gelation, typically referred to as in situ-forming hydrogels, hold great promise as a versatile injectable system in the field of tissue engineering. To prepare in situ-forming hydrogels, various methods have been used and they can be classified into physical and chemical crosslinking (Ko et al., 2013). Among them, an enzyme-catalysed chemical crosslinking method has received increasing interest as an alternative to the currently used crosslinking systems, due to its high energy-efficiency and selectivity (Kobayashi et al., 2001). In addition, it proceeds under mild conditions, without generating toxic side-products. There have been many attempts to produce in situ-forming hydrogels using various kinds of enzymes (e.g. transglutaminase, tyrosinase, phosphopantetheinyl transferase, lysyl oxidase, plasma amine oxidase and horseradish peroxidase), as recently reviewed by Teixeira et al. (2012). In most enzymatic crosslinking systems, however, slow gelation rates and poor mechanical properties of the formed hydrogels have limited their applications. In contrast, a horseradish peroxidase (HRP)-catalysed crosslinking system offers better control for producing the desired hydrogel properties, and thus is increasingly receiving attention for tissue regeneration. In this review, we introduce the various types of in situ-forming hydrogels produced using an HRP-catalysed crosslinking system and highlight their potential biomedical applications.

2. HRP-catalysed crosslinking mechanism HRP occurs in horseradish roots and is a haem-containing protein that catalyses the decomposition of hydrogen

peroxide (H2O2) at the expense of aromatic proton donors (Kobayashi et al., 2001). HRP-catalysed crosslinking reactions are outlined by the following equation, in which AH2 and AH indicate a reducing substrate and its radical product, respectively (Veitch, 2004). HRP

H2 O2 þ 2AH2 → 2H2 O þ 2AH Although several functional groups, such as aromatic phenols, amines and phenolic acids, can be used as the reducing substrate (Veitch, 2004), phenol has been widely studied because it is a common environmental pollutant in industrial waste. Earlier, HRP was used for phenol removal from wastewaters by precipitating insoluble polyphenols that are generated by HRP and H2O2 treatment (Tong et al., 1997; Nakamoto and Machida, 1992; Klibanov et al., 1980). Thereafter, HRP-catalysed crosslinking for in situ formation of hydrogels have been studied for biomedical applications. The HRP-mediated catalytic cycle of phenol, which is used as a reducing substrate, is illustrated in Figrue 1A. The catalytic cycle is initiated by an interaction between H2O2 and the resting ferric state of HRP [Fe(III)], which generates compound I. Compound I is a high oxidation-state intermediate with a cation radical. In the presence of phenol as a reducing substrate, compound I is converted to compound II after the first one-electron reduction, followed by an additional one-electron reduction step that returns HRP to its resting state. Consequently, the generated phenol radicals enable covalent bridge formation between aromatic rings (Figure 1B). The reaction equation and the catalytic cycle pathway indicate that one equivalent of H2O2 is consumed to create two equivalents of phenolic radicals, implying that varying the concentrations of both H2O2 and phenol can control the degree of crosslinking of polymer networks. All synthetic or natural polymers currently used for HRP-catalysed crosslinking utilize phenol as a reducing

Figure 1. The catalytic cycle of horseradish peroxidase (HRP) (A) and filling of tissue defects using HRP-catalysed in situ-forming hydrogel (B). For cell delivery, hydrogels can be fabricated using a dual-syringe kit equipped with a needle in which two different phenol-rich polymer solutions containing either HRP or H2O2 are loaded separately. The figure shows cells suspended in a compartment containing HRP and polymers. The mixing and injection of two separate solutions results in the in situ formation of hydrogels via HRP-catalysed crosslinking Copyright © 2014 John Wiley & Sons, Ltd.

J Tissue Eng Regen Med (2014) DOI: 10.1002/term

HRP-catalysed in situ-forming hydrogels

substrate, because the crosslinking reaction between phenol groups occurs much faster than other phenol derivatives, including aniline (Sakurada et al., 1990). The hydrogel phenol content can be increased by chemical conjugation of phenol derivatives, tyramine (TA) and hydroxyphenylpropionic acid (HPA), which enhance the hydrogel mechanical strength after HRP-catalysed crosslinking (Jin et al., 2007; Wang et al., 2010). However, the maximum threshold for the number of phenols available for crosslinking varies and depends on the number and type of functional groups available on polymer backbones. In addition, excessive conjugation of phenol derivatives may alter the water solubility of polymers before crosslinking (Lee et al., 2013).

3. Customizing hydrogel properties by using HRP-catalysed crosslinking In vivo interactions of cells delivered to tissue sites by using hydrogels may differ from their in vitro interactions. The delivered cells interact with the surrounding cells and/or tissues through multiple signalling pathways, which have not been completely characterized, and then start regenerating the damaged tissue. To instigate the tissue-repair process, implanted hydrogel matrices need to provide favourable environments for cell growth. In addition, increased control of cell environment would enable investigation of conditions suitable for tissue regeneration. Thus, in situ-forming hydrogels that can be efficiently customized might have broad tissue-engineering applications and can be improved for use as a delivery carrier. The HRP-catalysed reaction facilitates control of crosslinking rate and degree, both of which affect key hydrogel properties, such as gelation time, stiffness and degradation rate. Moreover, HRP-mediated crosslinking allows facile reactions with phenol-containing bioactive molecules.

3.1. Adjustable gelation time Gelation time is defined as the time interval required for a solution to turn into either a solid or a semi-solid that cannot flow. The gelation time is a primary concern for the development of in situ-forming hydrogels and is closely associated with the crosslinking rate of the HRP-catalysed reaction. From a practical viewpoint, immediate gelation upon injection may lead to insufficient filling of the tissue defect. In contrast, a slow gelation rate may cause heterogeneous distribution of encapsulated entities as well as initial washing away of hydrogel constituents. Therefore, controlled gelation is necessary for efficient local delivery of cells and/or drugs. As discussed previously, HRP and H2O2 concentrations are essential parameters to modulate the crosslinking rate of phenol-rich polymers. The relationship between these parameters and gelation time has been well established. It has been reported that Copyright © 2014 John Wiley & Sons, Ltd.

the gelation time decreases with increasing HRP concentration, and vice versa (Kurisawa et al., 2005; Jin et al., 2007; Park et al., 2010). Although there are minor differences in the gelation time required for various material types, the gelation time can be controlled with an accuracy of few seconds to several minutes. Other parameters, such as molecular weight, phenol content, polymer concentration and water solubility, can also affect gelation time (Lee et al., 2002; Sakai et al., 2009; Park et al., 2010).

3.2. Hydrogel stiffness tuning There is increasing evidence that the mechanical stiffness or elasticity of hydrogels regulates a variety of cellular processes, such as adhesion, proliferation, spreading, migration and differentiation (Gilbert et al., 2010; Lutolf et al., 2009). Material surfaces with identical compositions can differ in stiffness, which can lead to unique cell type-dependent behaviours. Trappmann et al. (2012) demonstrated that hydrogels with varying stiffness can result in differing anchoring densities of ECM molecules, thereby altering the mechanical feedback of ECM. The hydrogel stiffness is attributed to crosslinking density, which determines nutrient permeability and transport of signalling molecules. The mechanical stiffness of hydrogels formed by the HRP-catalysed crosslinking is significantly influenced by the concentrations of HRP and H2O2, as well as the number of phenol groups available. Several reports demonstrate HRP and H2O2-dependent tendencies of hydrogel stiffness. We confirmed that increasing the concentration of HRP could lead to the increased storage modulus of tetronic–TA hydrogels (Park et al., 2010). The correlation of hydrogel stiffness to H2O2 concentration was also investigated using a range of H2O2 concentrations and a fixed HRP concentration. The data demonstrate that an increase in the H2O2 concentration resulted in increased storage modulus of the formed hydrogels. However, an excessive use of H2O2 resulted in H2O2-induced cell death, with a delayed gelation process. Similar findings were reported elsewhere (Jin et al., 2007; Lee et al., 2009; Park et al., 2011).

3.3. Controlled biodegradation For tissue-engineering applications, a suitable hydrogel degradation rate provides sufficient space for proliferation and migration of cells encapsulated within hydrogels. Biodegradable hydrogels also allow infiltration of surrounding tissues to remodel the artificial hydrogel matrices. In drug-delivery applications, controlled degradation of hydrogel matrices facilitates efficient local delivery of therapeutic drugs through temporally modulated drug release. To design biodegradable hydrogels, two strategies have been adopted for the HRP-catalysed in situ gelling systems: hydrolysis and enzymatic degradation. Most systems use natural polymers that are responsive to enzymatic hydrolysis, J Tissue Eng Regen Med (2014) DOI: 10.1002/term

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which include hyaluronic acid (Kurisawa et al., 2005), gelatin (Lee et al., 2013), chitosan (Tran et al., 2010) and dextran (Jin et al., 2007). These polymers can be degraded in vivo by several enzymes, such as hyaluronidase, lysozyme and collagenase. The second strategy for generating biodegradable gels is to use gels with hydrolysable molecular bonds, such as ester and urethane linkages (Jin et al., 2007; Park et al., 2010, 2012a). By chemically introducing the linkages between the conjugated phenol and the polymer backbone, the formed hydrogels can undergo hydrolytic degradation over time. Given that the materials themselves are degradable, the degradation kinetics of hydrogels formed by HRP-catalysed crosslinking can be controlled in situ by modulating the HRP and H2O2 concentrations, which is relevant to changes in crosslinking density. A low crosslinking density leads to a high hydrogel-degradation rate, whereas dense crosslinking results in a low degradation rate (Lee et al., 2013).

3.4. Facile in situ conjugation An advantageous feature of the HRP-catalysed in situ gelling system is that the reaction can incorporate any compounds with phenol groups into the hydrogel. Thus, a compound of interest that has phenol groups can participate in the gelation reaction and covalently bind to other phenol-rich molecules. Park et al. (2012b) first reported a facile in situ conjugation of tyrosine-contained peptide, SVVYGLRGGY, to gelatin-poly(ethylene glycol)–tyramine (GPT) hydrogels. The peptide was simply mixed with an aqueous solution of GPT, followed by in situ crosslinking in the presence of HRP and H2O2. The results showed that the amount of conjugated peptides could be controlled by varying the peptide concentration and that the conjugated peptides were distributed throughout the hydrogel matrices. Furthermore, an increase in peptide

concentration significantly lowered the hydrogel stiffness, because GPT and peptides compete with each other for phenol–phenol coupling. The HRP-catalysed in situ gelling system can be also employed to fabricate a hydrogel hybrid composed of different phenol-rich polymers (Jin et al., 2011; Park et al., 2012a). By varying the mixing ratio of phenol-rich polymers, enzymatically crosslinked hydrogel hybrids can be created to have desired bioactivity or mechanical properties.

4. Potential biomedical applications Based on the above-described features, HRP-catalysed in situ-forming hydrogels are increasingly becoming an attractive class of implantable biomaterials. Significant advances in biomedical applications have been made using hydrogels; but major applications and findings of the HRP-catalysed in situ-forming hydrogels are focused on a few fields of research, as summarized in Table 1, probably due to a relatively short history. In the following section, we elaborate the most intensively studied applications using HRP-catalysed in situ-forming hydrogels.

4.1. Cartilage regeneration There have been numerous attempts to treat cartilage damaged by arthritis, trauma, sports injuries or inflammatory disorders. Among clinically-used cartilage treatments, autologous chondrocyte implantation (ACI) is the only restorative clinical treatment method and has been improved over the past few decades. Scaffold-assisted ACI is an advanced treatment method with potential for application in cartilage repair and is classified as a tissue-engineering approach. Scaffold-assisted ACI extensively uses hydrogels as scaffolds because of its mechanical properties, which

Table 1. Recent applications using HRP-catalysed in situ-forming hydrogels Hydrogels

Target applications

Dex-TA HA-g-Dex-TA Dex-TA/Hep-TA Dex-TA/platelet lysate GPT/PRP HA-Tyr CPT/rutin Gtn-HPA CPT GH, GHT Gtn-HPA

Cartilage regeneration Cartilage regeneration Cartilage regeneration Cartilage regeneration Cartilage regeneration Cartilage regeneration Wound healing Wound healing Wound healing Wound healing Brain tissue engineering


Skeletal muscle regeneration Adipose tissue engineering PAD treatment No specific applications Cancer treatment

References Jin et al., 2010 Jin et al., 2010 Moreira Teixeira et al., 2012a Moreira Teixeira et al., 2012b Lee et al., 2012a, 2012b Toh et al., 2012 Tran et al., 2011 Wang et al., 2012 Lih et al., 2012 Lee et al., 2013 Wang et al., 2010a, 2010b, Lim et al., 2012 Hwang et al., 2013 Ogushi et al., 2013 Kim et al., 2013a Kim et al., 2013b Xu et al., 2013

Dex-TA, dextran–tyramine; HA-g-Dex-TA, hyaluronic acid–grafted dextran–tyramine; Hep-TA, heparin–tyramine; GPT, gelatin–PEG–tyramine; CPT, chitosan–PEG–tyramine; Gtn-HPA and GT, gelatin–hydroxyphenylpropionic acid; GHT, gelatin–hydroxyphenylpropionic acid–tyramine; HA-Tyr, hyaluronic acid–tyramine; CMC-Ph, carboxymethylcellulose–tyramine; Tet-TA, tetronic tryamine; PAD, peripheral arterial disease; PRP, platelet-rich plasma. Copyright © 2014 John Wiley & Sons, Ltd.

J Tissue Eng Regen Med (2014) DOI: 10.1002/term

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are similar to those of cartilage (Spiller et al., 2011). Jin et al. (2010) assessed whether dextran–tyramine (Dex-TA) hydrogels could be used as a 3D scaffold for cartilage tissue engineering. Dex-TA conjugates were prepared by varying in the molecular weight of Dex and the degree of substitution with TA. The results demonstrated that the gelation time and storage moduli of Dex-TA hydrogels could be controlled. The encapsulated bovine chondrocytes were viable for 2 weeks. In addition, the glycosaminoglycan and collagen type II content of Dex-TA hydrogels was higher than that of agarose. To further investigate Dex-TA hydrogel properties for tissue-engineering applications, Teixeira et al. (2012) studied the adhesion mechanism between cartilage and Dex-TA hydrogels and the cell recruitment pathways that were induced by heparin-TA (Hep-TA). Their study demonstrated that HRP-catalysed crosslinking enables covalent bonding of the hydroxyphenyl residues in Dex-TA and Hep-TA to tyrosine residues in cartilaginous matrix proteins. The mechanical analysis revealed that a high TA content resulted in strong binding with cartilage tissue. In addition, Hep-TA could enhance cell ingrowth by the high-binding affinity of heparin to growth factors and cytokines (Moreira Teixeira et al., 2012). Like other hydrogel systems, however, HRP-catalysed in situ-forming hydrogels lack intrinsic biological signals to promote tissue regeneration, although they are biocompatible. Recently, our group reported the effect of platelet-rich plasma (PRP)-carrying GPT hydrogels on articular cartilage repair (Lee et al., 2012a). A GPT solution containing rabbit chondrocytes was mixed with PRP, which is rich in growth factors and cytokines, followed by in situ gelation via HRP-catalysed crosslinking. In vivo results demonstrated that PRP–GPT hydrogels could improve cartilage remodelling by upregulating SOX-9, CB2, ChM-1 and aggrecan after a 7 day incubation period. We have also attempted to regenerate non-articular cartilage by the implantation of xyphoid chondrocytes encapsulated in PRP–GPT hydrogels (Lee et al., 2012b). A xyphoid defect rat model was used to show that the PRP–GPT hydrogels can induce proliferation and differentiation of chondrocytes in vitro and in vivo by inducing elevated levels of ChM-1 and CB1. Teixeira et al. (2012) performed studies with a similar approach and used Dex-TA hydrogels supplemented with platelet lysate for cartilage repair. The results showed that the addition of platelet lysate enhanced both proliferation and chondrogenic differentiation of MSCs encapsulated in the hydrogels.

4.2. Wound healing Hydrogels have been widely investigated as one of promising wound-healing materials because they possess many of the desirable features, particularly providing a moist environment around the wound (Boateng et al., 2008). A few attempts have been made recently to assess the wound-healing ability of hydrogels formed via HRP-catalysed crosslinking. Tran et al. (2011) have investigated the efficacy of rutin-releasing chitosan (RCPT) Copyright © 2014 John Wiley & Sons, Ltd.

hydrogels for dermal wound healing. In this study, rutin as an antioxidative drug was covalently bound to chitosan–PEG–tyramine (CPT) through a hydrolysable ester linkage. The RCPT solution formed a gel within 10 s, and the conjugated rutin was released in a controlled manner by varying the HRP and H2O2 concentrations. The released rutin enhanced fibroblast proliferation. Other in vivo wound-healing studies demonstrated that RCPT hydrogels exhibited improved wound healing effect with neo-epithelium formation and thicker granulation. Wang et al. (2012) tested gelatin–hydroxyphenyl propionic acid (Gtn-HPA) hydrogels as a wound-dressing material. They focused on hydrogel stiffness-dependent fibroblast proliferation in both 2D and 3D culture systems. The rheological properties of Gtn-HPA hydrogels were varied in the range 281–8172 Pa by increasing H2O2 concentration at a fixed HRP concentration. The 2D culture data showed that the proliferation rate of human fibroblasts HFF-1 increased with increasing the storage modulus, while it decreased with an increase in the hydrogel stiffness. In the 3D culture system, HFF-1 remained non-proliferative for 12 days and then started proliferating. The proliferation rate of HFF-1 cultured in the hydrogel with lower stiffness became much faster than the stiffer hydrogel. The results indicated that both culture dimensionality and hydrogel stiffness can accelerate the wound-healing process. Tissue adhesion of hydrogel-based wound dressing materials is an important physical property when applied to moist wound surfaces (Boateng et al., 2008). Chitosan–PEG-TA (CPT) was developed and then evaluated for its tissue-adhesive properties on porcine skin (Lih et al., 2012). CPT hydrogels had significantly higher adhesive strengths than that of fibrin glue, used as a control. The hydrogel prepared using 0.06 wt% H2O2 exhibited a maximum adhesive strength of 97 kPa on porcine skin, which was almost 20-fold higher than that (5 kPa) of fibrin glue. The highest elastic modulus was also obtained using the same hydrogel. The haemostatic and wound-closure material properties of CPT hydrogels were also assessed. In vivo results showed that hydrogel provides better haemostasis and wound-healing effects than those of sutures, fibrin glue and cyanoacrylate. Lee et al. (2013) reported a new injectable type of gelatinbased tissue adhesive. The study used two gelatin derivatives, GH and GHT, which varied in phenol contents and were prepared by conjugating HPA and TA. As expected, the crosslinking rate and degree of the hydrogel could be controlled by altering the H2O2 and HRP concentrations. An increase in the phenolic content of gelatin resulted in increased mechanical strength. Additionally, the hydrogel adhesive strength was two to three times higher than that of fibrin glue.

4.3. Stem cell-based tissue engineering Stem cells possessing the capacity to divide and differentiate into diverse specialized cell types are regarded as the most versatile and promising cell source in regenerative J Tissue Eng Regen Med (2014) DOI: 10.1002/term

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medicine (Lutolf et al., 2009). At an early stage of stem cell research, many researchers have put a great deal of effort into regulating stem cell fate with various biochemical signals. In spite of these efforts, the stem cell-regulatory mechanisms were not yet fully unveiled. A new paradigm for stem cell fate determination has lately emerged, which is based on the mechanical properties of the hydrogel microenvironment in which the stem cells reside. Despite the marked differences in use of material types and the crosslinking methods used, several studies have shown that hydrogel stiffness directs stem cell fate in the absence of other biochemical factors (Banerjee et al., 2009; Tse and Engler, 2011; Choi and Harley, 2012). As previously stated, an HRP-catalysed in situ crosslinking method facilitates easy and effective control over the hydrogel stiffness by varying HRP and H2O2 concentrations. Kurisawa’s group provided the first evidence for stiffness-dependent differentiation of human mesenchymal stem cells (hMSCs) on hydrogels that were prepared by HRP-catalysed crosslinking (Wang et al., 2010a). To create Gtn-HPA hydrogels with varying stiffnesses in the range 629–12780 Pa, a range of H2O2 concentrations were used with a fixed HRP concentration. The gelation time was barely affected by a change in H2O2 concentration. Additionally, hMSCs that were cultured on stiffer hydrogels had spread across large areas, had organized cytoskeletons, stable focal adhesion, high migration rate and a high proliferation rate. Notably, the hydrogel stiffness stimulated neurogenesis and myogenesis of hMSCs. Wang et al. (2010b) also investigated the differentiation behaviours of hMSCs cultured in 3D Gtn-HPA hydrogels with different stiffness properties. The study showed that hMSCs encapsulated in soft hydrogels (281 Pa) had an increased proliferation rate. After a 3 week culture period, hMSCs cultured in soft hydrogels showed higher neuronal protein marker expression than that of hMSCs cultured in stiffer hydrogels (841 Pa). Lim et al. (2012) adopted the hydrogel system designed by Wang et al. for 3D culture of adult neural stem cells (aNSCs) that can self-renew and differentiate into central nervous system cell types. The data showed that Gtn-HPA hydrogels had good cell adhesion properties, enabled increased cell resistance to oxidative stress and influenced cell proliferation and migration. Interestingly, the enhanced differentiation of aNSCs into the neuronal lineage was observed in stiffer hydrogels. Toh et al. (2012) used HA-Tyr hydrogels for chondrogenic modulation of caprine MSCs; MSCs cultured in hydrogels with low stiffness enhanced chondrogenesis, whereas high hydrogel stiffness yielded MSCs that differentiated into fibrous phenotypes with fibrocartilage formation. Spatiotemporal delivery of signalling molecules, such as growth factors and cytokines, plays key roles in regulating cellular functions and improving tissue repair, and it also affects stem cell fate decisions (Lee et al., 2011). Recently, we reported a combination therapy of human adipose-derived stem cells (hADSCs) and basic fibroblast growth factor (bFGF) encapsulated into GPT hydrogels for skeletal muscle regeneration (Hwang et al., 2013). Copyright © 2014 John Wiley & Sons, Ltd.

With a cumulative bFGF release over 2 weeks, significantly improved muscle contraction and reduced fibrosis were observed in the hADSCs and bFGF encapsulated GPT hydrogel group applied to the lacerated gastrocnemius muscle of mice. The immunohistochemistry and western blot results demonstrated skeletal muscle differentiation of the hADSCs. Moreover, we confirmed that the combination therapy led to functional recovery, revascularization and re-innervation with minimal fibrosis. A similar study was reported by Ogushi et al. (2013); they found that subcutaneous delivery of ASCs and bFGF using carboxymethylcellulose with phenols (CMC-Ph) resulted in enhanced adipogenesis and neovascularization at the injection site.

4.4. Other applications In addition to several applications described above, HRP-catalysed in situ-forming hydrogels have shown great potential for various biomedical applications. Peripheral arterial disease (PAD) is caused by the atherosclerotic accumulation of blood vessel plaque and can lead to acute or chronic ischaemia, due to lack of blood supply. Kim et al. (2013a) adopted a cell sheet-engineering approach for the reconstruction of ischaemia-damaged myocardium; they cultured C2C12 myoblasts on thermosensitive tetronic– tyramine hydrogel surfaces, incorporating cell adhesive GRGDGGGGGY peptides. After 1 day of culture, confluent sheet-like myoblast monolayers were obtained by decreasing the temperature to 4°C. It was found that the C2C12 myoblast monolayers had high retention ability, anti-apoptotic and angiogenic potentials in vivo, maintaining the stable ECM structure in vitro and in vivo. In addition, the cell layer transplanted into hindlimb ischaemia of nude mice could improve the therapeutic effect by inducing angiogenesis. HRP-catalysed crosslinking can be used to stabilize self-assembled nanoconstructs that may be dissociated by continuous dilution. Recently we have developed hybrid polymeric micelles composed of tetronic-TA and heparinTA that were sterically stabilized by HRP-catalysed shell crosslinking (Kim et al., 2013b). The shell crosslinking between TA groups of each conjugate resulted in changes in the micelle size and surface charge. All shell crosslinked micelles appeared to be stable over 4 weeks, retaining their original sizes and zeta potentials. Using this approach, we were able to design a new micellar platform for simultaneous release of bFGF and indomethacin. Hydrogels can be used as an injectable depot for the sustained release of drugs, especially therapeutic proteins, because their aqueous environment can prevent denaturation of proteins. Xu et al. (2013) investigated HA-Tyr hydrogels that incorporate interferon-α2a (IFN-α 2a) for liver cancer treatment. IFN-α2a release profiles from hydrogel matrices were strongly influenced by hydrogel stiffness, but showed the well-maintained activity of IFN-α2a in the hydrogel with lower stiffness. Subcutaneously injected IFN-α2a-incorporated hydrogels to the backs of tumour-bearing mice effectively inhibited tumour growth and angiogenesis of tumour tissues. J Tissue Eng Regen Med (2014) DOI: 10.1002/term

HRP-catalysed in situ-forming hydrogels

5. Concluding remarks The crucial role of cells, scaffolds and signalling molecules in effective tissue regeneration and repair has been previously established. With the tremendous efforts devoted to the combinatorial use of the triad, various tissue-engineering products have been marketed and are under clinical trial. Nevertheless, further research is necessary to develop advanced strategies aiming at complete and accelerated tissue regeneration. In this review, we briefly discussed the recent progress of HRP-catalysed in situ-forming hydrogels and various techniques for manipulating hydrogel properties that influence tissue repair. Hydrogel properties, such as gelation time, stiffness and degradation rate, can be easily manipulated by varying HRP and H2O2 concentrations. Moreover, the HRP-catalysed crosslinking method enables conjugation or hybridization of materials of interest for building customized cell-friendly microenvironments that induce favourable cellular behaviours. However, HRP-catalysed crosslinking may have a potential safety issue related to

HRP entrapment inside hydrogel matrices, which inevitably occurs during gelation (Lee et al., 2002). Therefore, careful consideration is required before HRP-catalysed hydrogel systems are used in clinics. If the residual HRP issue is resolved, HRP-catalysed in situ-forming hydrogels could serve as an efficient clinically translatable scaffold platform with broad tissue-engineering applications.

Conflict of interest The authors have declared that there is no conflict of interest.

Acknowledgements This study was supported by the Basic Science Research Programme (Grant No. NRF-2012R1A2A2A06046885) and the Priority Research Centres Programme (Grant No. 2012-0006687) through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT and Future Planning.

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J Tissue Eng Regen Med (2014) DOI: 10.1002/term

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 ar...
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