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Review
Crosslinking biopolymers for biomedical applications Narendra Reddy1, Roopa Reddy1, and Qiuran Jiang2,3 1
Center for Emerging Technologies, Jain University, Jakkasandra Post, Ramanagara District, Bengaluru 562112, India Key Laboratory of Textile Science and Technology, Ministry of Education, College of Textiles, Donghua University, Shanghai, P.R. China 3 Department of Technical Textiles, College of Textiles, Donghua University, Shanghai, P.R. China 2
Biomaterials made from proteins, polysaccharides, and synthetic biopolymers are preferred but lack the mechanical properties and stability in aqueous environments necessary for medical applications. Crosslinking improves the properties of the biomaterials, but most crosslinkers either cause undesirable changes to the functionality of the biopolymers or result in cytotoxicity. Glutaraldehyde, the most widely used crosslinking agent, is difficult to handle and contradictory views have been presented on the cytotoxicity of glutaraldehydecrosslinked materials. Recently, poly(carboxylic acids) that can crosslink in both dry and wet conditions have been shown to provide the desired improvements in tensile properties, increase in stability under aqueous conditions, and also promote cell attachment and proliferation. Green chemicals and newer crosslinking approaches are necessary to obtain biopolymeric materials with properties desired for medical applications. Biomaterials, crosslinkers, and the need for crosslinking Biomaterials have been used for a plethora of in vivo applications [1]. In this review we focus on biomaterials derived from biopolymers such as cellulose, starch, collagen, silk, chitosan, and poly(lactic acid) because of their advantageous features that include cytocompatibility and ability to degrade in the body without releasing harmful substances. Films, fibers, hydrogels, 2D and 3D structures, micro- and nanoparticles made from biopolymers are being used extensively for both in vitro and in vivo applications (Figure 1) [2–5]. Although several types of biopolymers have been used to fabricate biomaterials, proteins such as albumin, collagen, and silk are preferable for medical applications because of their better biocompatibility [2– 5]. In addition, proteins contain abundant functional groups that facilitate the loading and release of drugs, genes, and nutraceuticals [3–5]. Despite the known advantages and wide applicability of biomaterials, there are several limitations that restrict their use for biomedical applications [3]. Primarily, biopolymeric materials lack adequate mechanical properties Corresponding author: Reddy, N. ([email protected]). Keywords: biopolymers; biomaterials; crosslinking; physiological conditions; stability; carboxylic acids. 0167-7799/ ß 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2015.03.008
and in many instances the stability in aqueous and physiological environments required for medical applications [6]. For instance, films and electrospun structures made from proteins disintegrate at high humidities or in aqueous solutions [7,8]. Crosslinking has been the most common approach to overcome the limitations of biomaterials [9,10]. Crosslinkers interconnect molecules, increase molecular weight, and generally provide higher mechanical properties and improved stability. However, crosslinking also leads to decreased degradability, lower availability of functional groups in the crosslinked polymer, and changes the rheology of the polymers, leading to subsequent processing difficulties and potential increase in cytotoxicity [6]. Various types of crosslinkers and crosslinking techniques are used depending on the type of biopolymer to be crosslinked and the extent of improvement in properties desired (Figure 2, Table 1) [9,11,12]. Among the numerous chemical crosslinkers (Table 1) used, glutaraldehyde (Box 1) is predominantly used because it can react with functional groups in both proteins and carbohydrates, and can provide materials with substantial improvement in tensile properties [13,14]. Although glutaraldehyde provides good improvement in mechanical properties, contradictory evidence has been provided on the cytotoxicity of glutaraldehyde-crosslinked materials [7,8,13–15]. Nevertheless, cytotoxicity of glutaraldehyde is dependent on the concentration used, and up to 8% glutaraldehyde was shown to be non-cytotoxic [13]. Apart from glutaraldehyde, several other chemicals including carbodiimide, epichlorohydrin, and sodium metaphosphate have also been used for crosslinking biopolymers but with limited improvement in properties owing to their low crosslinking efficiency [8,16]. Recently, attempts have been made to use carboxylic acids (Box 2) such as citric acid to crosslink and improve the mechanical properties and stability of biomaterials without compromising the cytocompatibility [7,8]. Crosslinking biomaterials with citric acid provides pendant functionality and allows formation of ester bonds leading to better hemocompatibility and increased availability of binding sites for bioconjugation [17]. This review presents an overview of the chemicals and techniques used to crosslink biopolymeric materials intended for medical applications. Particular emphasis has been placed on protein-based biomaterials because they have better biocompatibility than synthetic polymer-based Trends in Biotechnology xx (2015) 1–8
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biomaterials but are less stable in aqueous environments and therefore inevitably need to be crosslinked.
Film
Hydrogel
2D electrospun fibers
3D electrospun fibers
Sponge
Nanoparcles
Ultrafine fibers TRENDS in Biotechnology
Figure 1. Schematic of the most common forms of scaffolds used for tissue engineering/controlled drug delivery. The relative ease with which cells can penetrate into the scaffolds is shown; the nanoparticles are depicted as being inside the cells.
Crosslinking biopolymers to form films and membranes Films are probably the easiest biomaterial structure to be fabricated and, therefore, most natural and synthetic polymers have been made into films for tissue engineering, controlled release, and other medical applications [18,19]. Films made from a majority of biopolymers including collagen, one of the most widely used proteins for medical applications, have relatively poor mechanical properties and are unstable and dissolve in water or aqueous solutions rapidly [20]. Therefore, primary requirement of crosslinking is the ability to improve mechanical properties and consequently resistance to degradation [21]. Crosslinking collagen films with EDC/NHS [N-ethyl-N0 -(3-(dimethylamino)propyl)carbodiimide/N-hydroxysuccinimide] or acid chlorides results in an increase in tensile strength up to 57% and an increase in modulus of nearly 17-fold, and the extent of increase could be controlled by varying the crosslinking conditions [21– 23]. Considerable decrease in swelling of the films was also observed after crosslinking. Although satisfactory improvement in mechanical properties and aqueous stability was
Chemical crosslinking Polymer I
(A)
Funconal group
Crosslinker Covalently bonded Polymer II Funconal group
(B) Crosslinker
Covalently bonded
Physical crosslinking (C) Non-covalently bonded
Enzymac crosslinking (D)
Enzyme
Covalently bonded
TRENDS in Biotechnology
Figure 2. Schematic of the three methods of crosslinking. (A) Chemical crosslinking with the crosslinker incorporated into the bond. (B) Chemical crosslinking with the crosslinker not incorporated into the bond. (C) Physical crosslinking. (D) Enzymatic crosslinking.
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Table 1. Assessment of the crosslinkers, crosslinking methods, and biomaterials that have been developed – cytotoxicity, mechanical properties, and aqueous stability for biomedical applicationsa Proteins Glutaraldehyde EDC/NHS Epichlorohydrin STMP Citric acid Dextran dialdehyde Genipin PA Glyoxal Chemical Physical (UV) Enzymatic Films Sponges, porous scaffolds Micro, nanoparticles Hydrogels Electrospun fibers Micro fibers In vitro cytotoxicity In vivo cytotoxicity Mechanical properties Stability
Gelatin U U U – U – U – U U U U U U U U U U U – Weak Poor
Carbohydrates BSA U U U – – – – – U U U – U U U U – – U – Weak Poor
Collagen U U U – U U U U U U U U U U U U U U U U Weak Poor
Zein U – – – U – – – – U – U U U U – U – U U Weak Poor
Soy protein U U – – – – U – U U U – U U U U U U U – Acceptable Weak
Keratin U U – – – – – – – U – U U U U – U – U U Acceptable Good
Cellulose U – U U U U – – – U – – U U – – – – U – Good Good
Starch U – – U – U – – U U U – U U U U – – U – Weak Poor
HA U U U – – U – – U U – U U U U U – – U – Weak Weak
Alginate U U – – – – U – U U – U U U – U – – U U Weak Weak
Chitosan U U U U – U U U U U U U U U U U U U U U Acceptable Weak
a
This table is based on widely reported literature specifically on developing biomaterials for medical applications and is by no means exhaustive.
obtained after crosslinking, the potential cytotoxicity of the crosslinked materials was not investigated. To avoid the undesirable changes and possible side effects of chemical crosslinkers, physical approaches of crosslinking collagen films have also been used. Collagen films were crosslinked with a combination of glucose and UV irradiation with the premise that UV-generated free radicals will form reactive, linear glucose molecules and enhance crosslinking [22]. Combining glucose and UV crosslinking provided a synergistic effect, and improved the mechanical properties and decreased enzymatic degradation [22]. Similarly, natural crosslinkers such as proanthocyanidin (PA) found in grape seeds increased the thermal resistance and resistance to enzymatic degradation of collagen films after crosslinking, without affecting their cytocompatibility [23]. Several weeks after subcutaneous implantation, the PA-crosslinked membranes showed considerably higher penetration of fibroblasts without any disintegration of tissue [23]. In addition to PA, several other natural crosslinkers (genipin, proanthocyadin, delmosine) are available, but only limited studies have been done on understanding the efficiency
Box 1. Glutaraldehyde Glutaraldehyde has been widely used to crosslink biopolymers for medical applications. However, contradictory results have been published on the cytotoxicity of glutaraldehyde-crosslinked biomaterials. Moreover, glutaraldehyde is difficult to handle during crosslinking owing to its pungent odor and low vapor pressure. Most results on glutaraldehyde crosslinking have been published based on in vitro studies, whereas in vivo evaluation of the crosslinked materials is necessary for meaningful understanding of the cytotoxicity and potential of glutaraldehyde-crosslinked materials in medical applications.
and cytotoxicity of these natural crosslinkers for crosslinking biomaterials. Crosslinking of biomaterials with porous and spongelike structures Sponges are 3D porous structures that have been used as scaffolds for culturing of osteoblasts for bone formation, tooth tissue engineering and several other applications [5,24]. Owing to their porous structure, sponges have poor mechanical properties and stability in aqueous environments and therefore need to be crosslinked [25]. Collagen sponges crosslinked with EDC/NHS with addition of lysine, glutamic acid, glycine or diphenylphosphorylazide (DPPA) had improved thermal stability and a lower rate of biodegradation [18,19,25,26]. Alternatively, combination of UV/glutaraldehyde crosslinking was able to provide good stability to collagen sponges, and also promoted cell
Box 2. Carboxylic acids Poly(carboxylic acids) can react with hydroxyl and amine groups, and therefore crosslink both polysaccharides and proteins. Proteins crosslinked with carboxylic acids have proved to be biocompatible and to provide the desired improvements in properties for both protein- and carbohydrate-based biomaterials. Conventionally, carboxylic acid (with at least three carboxylic groups) crosslinking was considered to occur only at high temperature (150–175 8C) and in the presence of catalysts. Recent studies have shown that carboxylic acids even with two carboxylic groups can crosslink biopolymer in wet and dry conditions and without the need for a potentially cytotoxic catalyst. In vitro studies have shown that fibers, films, electrospun, and phase-separated structures can all be crosslinked with citric acid. Further research using in vivo approaches need to be carried out to prove the biocompatibility and suitability of biomaterials crosslinked with carboxylic acids for medical applications. 3
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Review attachment, but compromised differentiation and mineralization of osteoblastic cells compared to uncrosslinked sponges [27]. Because individual polymers may have several limitations in providing biomaterials with the desired properties, another approach to obtain biopolymeric materials with required properties is to use a blend of different polymers [24]. For instance, porous collagen/chitosan blend scaffolds were developed and treated with glutaraldehyde to improve their mechanical properties and stability [28]. An interconnected 3D structure of the scaffolds was maintained after crosslinking, but the mean pore size increased from 100 mM to >200 mM and a sheet-like structure was formed due to fusion of smaller pores into larger pores [28]. A similar phenomenon was observed when collagen/chitosan scaffolds were crosslinked with genipin, a natural crosslinking agent [29,30]. Reduced swelling and decrease in enzymatic degradation were seen without affecting the cell viability [31,32]. Similarly to the binary blends, a ternary composition of collagen, hyaluronic acid (HA), and poly(caprolactone) (PCL) was used to generate sponge-like dense membranes that were crosslinked with UV irradiation and EDC/NHS [33]. Developed hybrid crosslinking systems were able to preserve the native structure of collagen, and inclusion of PCL provided the ability to control the degradation and mechanical properties; this also made the sponges suitable for developing wound dressings and periodontal membranes [33]. Although crosslinked sponges have been shown to be biocompatible based on in vitro studies, it is necessary to implant the sponges and conduct in vivo biocompatibility studies. The application of sponges made from biopolymers may also be limited, unlike the porous structures made from ceramics, because of their inferior mechanical properties and stability. Crosslinking of biopolymeric hydrogels Hydrogels with the capacity to retain large amounts of water are used for tissue engineering and for the delivery of drugs, peptides, and proteins [34,35]. However, crosslinks must be present in a hydrogel to avoid dissolution and make the hydrogels usable for in vitro and in vivo applications [8]. Gelatin that readily forms gels was made into hydrogels and crosslinked with dextran dialdehyde; these were found to have a higher storage modulus than gels crosslinked with dextran aldehyde [36]. Another aldehyde (glutaraldehyde) was used in combination with a carboxylic acid (malic acid) and an dendrimer (EDC) to crosslink collagen gels; it was found that crosslinking decreased enzymatic degradation and promoted adhesion and growth of L 929 cells [37]. Instead of using a combination of crosslinkers, collagen hydrogels crosslinked with dendrimers such as EDC were able to provide similar resistance to collagenase degradation compared to glutaraldehyde [38]. In addition to the conventional approach of crosslinking with chemical crosslinking agents, collagen gels intended for controlled release of bovine serum albumin (BSA) have been crosslinked with the enzyme transglutaminase [11]. Crosslinking of the amine groups in collagen by transglutaminase was confirmed, and the denaturation 4
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temperature of the hydrogel increased from 38 to 668C – leading to reduced initial burst and sustained release of BSA after crosslinking [11]. Despite improvement in resistance to degradation after crosslinking, collagen gels do not have adequate properties for tissue engineering and many other applications [24]. To further improve the properties, collagen has been blended with other biopolymers to develop gels with improved stability and mechanical properties [28,39]. Blending collagen with gelatin and crosslinking the blend hydrogels with various concentrations of glutaraldehyde [41] increased the visoelastic properties of the gel and the breaking modulus without causing any toxicity to mouse fibroblast cells [39]. Chitosan/collagen hydrogels crosslinked with glyoxal and chitosan/BSA hydrogel crosslinked with genipin had enhanced mechanical properties and pH-dependent swelling, and were also cytocompatible [40,42]. Similarly to proteins, individual polysaccharides have also been made into hydrogels. Functional hydrogels made from dextran and crosslinked with epichlorohydrin and phosphorous oxychloride had low toxicity and good enzymatic degradability, but no cell culture or toxicity studies were carried out [43,44]. Using a unique approach of dual-crosslinking (butanedioldiglycidyl ether as the crosslinking agent) and photopatterning, hyaluronic acid was made into hydrogels with anisotropic swelling. Hydrogels were reported to swell to several times their weight but were able to maintain their morphology. Such highswelling hydrogels were suggested to be useful for ophthalmic, wound healing, and other medical applications [45]. Similarly, swelling (up to 720%) of hydrogels made from cellulose could be controlled by varying the conditions used for crosslinking with 1,2,3,4-butanetetracarboxylic acid dianhydride, a carboxylic acid with four carboxyl groups [46]. A double crosslinking approach was also used to first prepare aminated and oxidized hyaluronic acid that was further crosslinked with genipin. Crosslinked hydrogels were formed in vitro and also in vivo when the crosslinkercontaining polymer was injected into mice [47]. Higher compressive modulus, lower mass loss, and a compact microstructure were obtained after double crosslinking without decrease in biocompatibility [47]. These hydrogels were intended to be useful as biodegradable and injectable hydrogels for tissue engineering. Alginate hydrogels ionically crosslinked using Ca2+ ions had reduced swelling, and were able to maintain their mechanical properties and dimensional stability for up to 8 weeks in Ca2+ medium; the swelling was dependent on initial crosslinking density, alginate concentration, and chemical composition [35]. In most studies on hydrogels, the pH-dependent swelling of hydrogels and the resulting morphological studies have been reported, but the ability of the hydrogels to load and release drugs has only been reported to a limited extent. In addition, the ability of the hydrogels to support the attachment and growth of cells, and to resist degradation under physiological conditions, has not been thoroughly investigated. In addition, in vivo studies also need to be carried out to understand the potential of the crosslinked hydrogels for medical applications.
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Review Crosslinking of coarse (regular) fibers In addition to films, hydrogels and sponges, fibers with diameters ranging from a few to several hundred micrometers have also been developed for medical applications, mostly as sutures and tissue engineering scaffolds [48]. These microdiameter fibers have been made using traditional fiber-spinning approaches, such as wet spinning and melt spinning, depending on the type of biopolymer used. A simple approach of developing fibers is to dissolve the polymers and extrude the polymers as fibers using a extruder or a syringe and needle [49]. Similarly, thermoplastic biopolymers can be melted and extruded using a orifice or spinneret. Type I collagen obtained from rat tail tendons was dissolved and made into fibers using a buffer. Because collagen fibers readily disintegrate in aqueous media, fibers obtained were crosslinked using EDC or sulfo/NHS, and later mineralized using calcium chloride and potassium phosphate [48,50]. Crosslinking with EDC and mineralization of the fibers lead to a nearly 50-fold increase in wet strength. A comparative study on the physical (UV treatment after severe dehydration) and chemical crosslinking (EDC) treatments on self-assembled collagen threads showed that physical treatments increased mechanical properties but decreased cell migration, whereas carbodiimide-crosslinked threads had a lower increase in strength but improved cell adhesion [50]. In addition to collagen, plant proteins such as wheat gluten and soyproteins have also been made into fibers for potential use as tissue engineering and drug delivery scaffolds [51–53]. These fibers have good mechanical properties under dry conditions but have inherently poor stability under aqueous conditions, and have therefore been crosslinked using carboxylic acids and other crosslinkers [51–53]. Highly water-stable protein fibers were obtained after crosslinking the fibers with carboxylic acids. It was suggested that the carboxylic acid crosslinking of proteins follows a pseudo-first-order reaction, and that the crosslinking conditions can be controlled to obtain fibers with desired mechanical properties and degradation rates [51,52]. When fibrous conduits for delivery of nerve growth factors (NGFs) made from gelatin were crosslinked with genipin, the rate of delivery of NGF could be controlled by varying the amount of crosslinking agent used, without compromising the biocompatibility [54]. The crosslinked conduits were considered ideal for regeneration of nerves. Crosslinking of ultrafine fibers Compared to regular fibers, ultrafine fibers possess many unique properties, and have been widely studied and adopted for medical applications [55]. Electrospinning is one of the most common methods to produce ultrafine fibers. Electrospun fibers developed from biopolymers resemble the ultrafine fibrous network in extracellular matrices (ECM) [56,57]. It has been demonstrated that the interconnected structures in electrospun scaffolds can promote the attachment and proliferation of cells [58]. However, electrospun fibers from biopolymers, especially proteins, have poor water stability (instantly dissolve or disintegrate in aqueous environments), particularly due to their fine structure and high surface area [55]. Therefore, crosslinking electrospun fibers
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requires special crosslinking techniques and approaches. A novel method of in situ crosslinking of electrospun collagen fibers (diameters of 0.42 0.11 mm) was developed using EDC/NHS as the crosslinking agent without the need for post-crosslinking [55]. Crosslinked samples maintained their morphology even after being in water, and the mechanical properties of the electrospun fibers were similar to those of native tissue [55]. Owing to the difficulties of using aqueous solutions for crosslinking electrospun fibers, electrospun collagen scaffolds have been crosslinked by exposure to saturated glutaraldehyde vapor [56,59]. Crosslinked scaffolds had higher tensile strength, and could resist degradation by collagenase, but had decreased porosity; contradictory results on the cytotoxicity of glutaraldehyde-crosslinked fibers have been reported [56,57,59,60]. To overcome the limitation of cytotoxicity associated with glutaraldehyde, citric acid has been used a biocompatible crosslinker for electrospun collagen fibers [6,7]. However, a crosslinking extender was necessary to crosslink electrospun collagen fibers with citric acid because of the limited availability of free functional groups. Addition of glycerol (as a crosslinking extender with large number of hydroxyl groups) assisted the formation of crosslinks, and improved the strength and stability of the electrospun fibers [7]. Similarly to collagen-based electrospun fibers, electrospun matrices made from zein have relatively weak tensile properties and also rapidly dissolve when immersed in aqueous solutions. Crosslinking electrospun zein matrices with citric acid of various concentrations provided matrices that retained their morphology even after 15 days of incubation in PBS at 37 8C. In addition, zein samples crosslinked with citric acid had a higher rate of attachment and proliferation of fibroblasts than did electrospun PLA scaffolds [6]. Similarly to electrospun protein fibers, chitosan, starch and other polysaccharides have also been used to produce electrospun structures and were crosslinked to improve their strength and stability. Electrospun chitosan fibers (143–334 nm) crosslinked with glycerol phosphate (GP), tripolyphosphate (TPP), and tannic acid (TA) [61] were insoluble even in 1 M acetic acid after immersion for 72 h, but their cytocompatibility was not assessed. In contrast to the usual methods for crosslinking fibers after electrospinning, the possibility of in situ crosslinking of pullan/dextran mixtures with trisodium metaphosphite (STMP) has also been demonstrated [62]. Crosslinking decreased swelling and also promoted viability of human dermal fibroblasts; actin stress fiber formation was also observed, suggesting potential applications of the crosslinked fibers in tissue engineering [62]. However, in situ crosslinking is not feasible with most crosslinkers or polymers, and may also lead to undesirable changes in the properties of the materials and decrease electrospinnability. In addition to electrospinning, phase separation is another method of fabricating ultrafine fibrous structures from biopolymers [63]. Unlike the layer-by-layer structure of electrospun materials, phase-separated structures have randomly oriented fibers, and this is closer to the 3D structure of ECMs than are structures produced by electrospinning [6]. However, maintaining the 3D structure of phase-separated fibers, especially in aqueous conditions, is 5
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Review challenging. Gelatin was made into ultrafine phase-separated fibers using thermally induced phase separation, and the scaffolds were crosslinked with EDC/NHS [63]. In another approach, an ultra-low concentration phase-separation method was used to produce 3D ultrafine gelatin fibers that were crosslinked with citric acid [64]. Better compatibility and cell infiltration was observed in the crosslinked gelatin fibers produced by phase separation compared to electrospinning [64]. Crosslinking micro- and nanoparticles Micro- and nanoparticles developed from biopolymers are preferable over metallic and synthetic polymers and have been used for in vivo delivery of drugs and other pharmaceuticals [65]. Several researchers have demonstrated that nanoparticles made from biopolymers can load high amounts of drugs, accumulate in tumors and other targeted organs, and provide efficient delivery of payloads [66,67]. Poor stability, consequent agglomeration and increase of particle size, and relatively quick degradation compared to metallic and synthetic polymer-based nanoparticles are some of the major limitations of biopolymeric nanoparticles [68,69]. Considerable physical and chemical modifications including crosslinking have been made to improve the performance of the polymeric nanoparticles [70]. Chitosan nanoparticles (
Review
Crosslinking biopolymers for biomedical applications Narendra Reddy1, Roopa Reddy1, and Qiuran Jiang2,3 1
Center for Emerging Technologies, Jain University, Jakkasandra Post, Ramanagara District, Bengaluru 562112, India Key Laboratory of Textile Science and Technology, Ministry of Education, College of Textiles, Donghua University, Shanghai, P.R. China 3 Department of Technical Textiles, College of Textiles, Donghua University, Shanghai, P.R. China 2
Biomaterials made from proteins, polysaccharides, and synthetic biopolymers are preferred but lack the mechanical properties and stability in aqueous environments necessary for medical applications. Crosslinking improves the properties of the biomaterials, but most crosslinkers either cause undesirable changes to the functionality of the biopolymers or result in cytotoxicity. Glutaraldehyde, the most widely used crosslinking agent, is difficult to handle and contradictory views have been presented on the cytotoxicity of glutaraldehydecrosslinked materials. Recently, poly(carboxylic acids) that can crosslink in both dry and wet conditions have been shown to provide the desired improvements in tensile properties, increase in stability under aqueous conditions, and also promote cell attachment and proliferation. Green chemicals and newer crosslinking approaches are necessary to obtain biopolymeric materials with properties desired for medical applications. Biomaterials, crosslinkers, and the need for crosslinking Biomaterials have been used for a plethora of in vivo applications [1]. In this review we focus on biomaterials derived from biopolymers such as cellulose, starch, collagen, silk, chitosan, and poly(lactic acid) because of their advantageous features that include cytocompatibility and ability to degrade in the body without releasing harmful substances. Films, fibers, hydrogels, 2D and 3D structures, micro- and nanoparticles made from biopolymers are being used extensively for both in vitro and in vivo applications (Figure 1) [2–5]. Although several types of biopolymers have been used to fabricate biomaterials, proteins such as albumin, collagen, and silk are preferable for medical applications because of their better biocompatibility [2– 5]. In addition, proteins contain abundant functional groups that facilitate the loading and release of drugs, genes, and nutraceuticals [3–5]. Despite the known advantages and wide applicability of biomaterials, there are several limitations that restrict their use for biomedical applications [3]. Primarily, biopolymeric materials lack adequate mechanical properties Corresponding author: Reddy, N. ([email protected]). Keywords: biopolymers; biomaterials; crosslinking; physiological conditions; stability; carboxylic acids. 0167-7799/ ß 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2015.03.008
and in many instances the stability in aqueous and physiological environments required for medical applications [6]. For instance, films and electrospun structures made from proteins disintegrate at high humidities or in aqueous solutions [7,8]. Crosslinking has been the most common approach to overcome the limitations of biomaterials [9,10]. Crosslinkers interconnect molecules, increase molecular weight, and generally provide higher mechanical properties and improved stability. However, crosslinking also leads to decreased degradability, lower availability of functional groups in the crosslinked polymer, and changes the rheology of the polymers, leading to subsequent processing difficulties and potential increase in cytotoxicity [6]. Various types of crosslinkers and crosslinking techniques are used depending on the type of biopolymer to be crosslinked and the extent of improvement in properties desired (Figure 2, Table 1) [9,11,12]. Among the numerous chemical crosslinkers (Table 1) used, glutaraldehyde (Box 1) is predominantly used because it can react with functional groups in both proteins and carbohydrates, and can provide materials with substantial improvement in tensile properties [13,14]. Although glutaraldehyde provides good improvement in mechanical properties, contradictory evidence has been provided on the cytotoxicity of glutaraldehyde-crosslinked materials [7,8,13–15]. Nevertheless, cytotoxicity of glutaraldehyde is dependent on the concentration used, and up to 8% glutaraldehyde was shown to be non-cytotoxic [13]. Apart from glutaraldehyde, several other chemicals including carbodiimide, epichlorohydrin, and sodium metaphosphate have also been used for crosslinking biopolymers but with limited improvement in properties owing to their low crosslinking efficiency [8,16]. Recently, attempts have been made to use carboxylic acids (Box 2) such as citric acid to crosslink and improve the mechanical properties and stability of biomaterials without compromising the cytocompatibility [7,8]. Crosslinking biomaterials with citric acid provides pendant functionality and allows formation of ester bonds leading to better hemocompatibility and increased availability of binding sites for bioconjugation [17]. This review presents an overview of the chemicals and techniques used to crosslink biopolymeric materials intended for medical applications. Particular emphasis has been placed on protein-based biomaterials because they have better biocompatibility than synthetic polymer-based Trends in Biotechnology xx (2015) 1–8
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biomaterials but are less stable in aqueous environments and therefore inevitably need to be crosslinked.
Film
Hydrogel
2D electrospun fibers
3D electrospun fibers
Sponge
Nanoparcles
Ultrafine fibers TRENDS in Biotechnology
Figure 1. Schematic of the most common forms of scaffolds used for tissue engineering/controlled drug delivery. The relative ease with which cells can penetrate into the scaffolds is shown; the nanoparticles are depicted as being inside the cells.
Crosslinking biopolymers to form films and membranes Films are probably the easiest biomaterial structure to be fabricated and, therefore, most natural and synthetic polymers have been made into films for tissue engineering, controlled release, and other medical applications [18,19]. Films made from a majority of biopolymers including collagen, one of the most widely used proteins for medical applications, have relatively poor mechanical properties and are unstable and dissolve in water or aqueous solutions rapidly [20]. Therefore, primary requirement of crosslinking is the ability to improve mechanical properties and consequently resistance to degradation [21]. Crosslinking collagen films with EDC/NHS [N-ethyl-N0 -(3-(dimethylamino)propyl)carbodiimide/N-hydroxysuccinimide] or acid chlorides results in an increase in tensile strength up to 57% and an increase in modulus of nearly 17-fold, and the extent of increase could be controlled by varying the crosslinking conditions [21– 23]. Considerable decrease in swelling of the films was also observed after crosslinking. Although satisfactory improvement in mechanical properties and aqueous stability was
Chemical crosslinking Polymer I
(A)
Funconal group
Crosslinker Covalently bonded Polymer II Funconal group
(B) Crosslinker
Covalently bonded
Physical crosslinking (C) Non-covalently bonded
Enzymac crosslinking (D)
Enzyme
Covalently bonded
TRENDS in Biotechnology
Figure 2. Schematic of the three methods of crosslinking. (A) Chemical crosslinking with the crosslinker incorporated into the bond. (B) Chemical crosslinking with the crosslinker not incorporated into the bond. (C) Physical crosslinking. (D) Enzymatic crosslinking.
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Table 1. Assessment of the crosslinkers, crosslinking methods, and biomaterials that have been developed – cytotoxicity, mechanical properties, and aqueous stability for biomedical applicationsa Proteins Glutaraldehyde EDC/NHS Epichlorohydrin STMP Citric acid Dextran dialdehyde Genipin PA Glyoxal Chemical Physical (UV) Enzymatic Films Sponges, porous scaffolds Micro, nanoparticles Hydrogels Electrospun fibers Micro fibers In vitro cytotoxicity In vivo cytotoxicity Mechanical properties Stability
Gelatin U U U – U – U – U U U U U U U U U U U – Weak Poor
Carbohydrates BSA U U U – – – – – U U U – U U U U – – U – Weak Poor
Collagen U U U – U U U U U U U U U U U U U U U U Weak Poor
Zein U – – – U – – – – U – U U U U – U – U U Weak Poor
Soy protein U U – – – – U – U U U – U U U U U U U – Acceptable Weak
Keratin U U – – – – – – – U – U U U U – U – U U Acceptable Good
Cellulose U – U U U U – – – U – – U U – – – – U – Good Good
Starch U – – U – U – – U U U – U U U U – – U – Weak Poor
HA U U U – – U – – U U – U U U U U – – U – Weak Weak
Alginate U U – – – – U – U U – U U U – U – – U U Weak Weak
Chitosan U U U U – U U U U U U U U U U U U U U U Acceptable Weak
a
This table is based on widely reported literature specifically on developing biomaterials for medical applications and is by no means exhaustive.
obtained after crosslinking, the potential cytotoxicity of the crosslinked materials was not investigated. To avoid the undesirable changes and possible side effects of chemical crosslinkers, physical approaches of crosslinking collagen films have also been used. Collagen films were crosslinked with a combination of glucose and UV irradiation with the premise that UV-generated free radicals will form reactive, linear glucose molecules and enhance crosslinking [22]. Combining glucose and UV crosslinking provided a synergistic effect, and improved the mechanical properties and decreased enzymatic degradation [22]. Similarly, natural crosslinkers such as proanthocyanidin (PA) found in grape seeds increased the thermal resistance and resistance to enzymatic degradation of collagen films after crosslinking, without affecting their cytocompatibility [23]. Several weeks after subcutaneous implantation, the PA-crosslinked membranes showed considerably higher penetration of fibroblasts without any disintegration of tissue [23]. In addition to PA, several other natural crosslinkers (genipin, proanthocyadin, delmosine) are available, but only limited studies have been done on understanding the efficiency
Box 1. Glutaraldehyde Glutaraldehyde has been widely used to crosslink biopolymers for medical applications. However, contradictory results have been published on the cytotoxicity of glutaraldehyde-crosslinked biomaterials. Moreover, glutaraldehyde is difficult to handle during crosslinking owing to its pungent odor and low vapor pressure. Most results on glutaraldehyde crosslinking have been published based on in vitro studies, whereas in vivo evaluation of the crosslinked materials is necessary for meaningful understanding of the cytotoxicity and potential of glutaraldehyde-crosslinked materials in medical applications.
and cytotoxicity of these natural crosslinkers for crosslinking biomaterials. Crosslinking of biomaterials with porous and spongelike structures Sponges are 3D porous structures that have been used as scaffolds for culturing of osteoblasts for bone formation, tooth tissue engineering and several other applications [5,24]. Owing to their porous structure, sponges have poor mechanical properties and stability in aqueous environments and therefore need to be crosslinked [25]. Collagen sponges crosslinked with EDC/NHS with addition of lysine, glutamic acid, glycine or diphenylphosphorylazide (DPPA) had improved thermal stability and a lower rate of biodegradation [18,19,25,26]. Alternatively, combination of UV/glutaraldehyde crosslinking was able to provide good stability to collagen sponges, and also promoted cell
Box 2. Carboxylic acids Poly(carboxylic acids) can react with hydroxyl and amine groups, and therefore crosslink both polysaccharides and proteins. Proteins crosslinked with carboxylic acids have proved to be biocompatible and to provide the desired improvements in properties for both protein- and carbohydrate-based biomaterials. Conventionally, carboxylic acid (with at least three carboxylic groups) crosslinking was considered to occur only at high temperature (150–175 8C) and in the presence of catalysts. Recent studies have shown that carboxylic acids even with two carboxylic groups can crosslink biopolymer in wet and dry conditions and without the need for a potentially cytotoxic catalyst. In vitro studies have shown that fibers, films, electrospun, and phase-separated structures can all be crosslinked with citric acid. Further research using in vivo approaches need to be carried out to prove the biocompatibility and suitability of biomaterials crosslinked with carboxylic acids for medical applications. 3
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Review attachment, but compromised differentiation and mineralization of osteoblastic cells compared to uncrosslinked sponges [27]. Because individual polymers may have several limitations in providing biomaterials with the desired properties, another approach to obtain biopolymeric materials with required properties is to use a blend of different polymers [24]. For instance, porous collagen/chitosan blend scaffolds were developed and treated with glutaraldehyde to improve their mechanical properties and stability [28]. An interconnected 3D structure of the scaffolds was maintained after crosslinking, but the mean pore size increased from 100 mM to >200 mM and a sheet-like structure was formed due to fusion of smaller pores into larger pores [28]. A similar phenomenon was observed when collagen/chitosan scaffolds were crosslinked with genipin, a natural crosslinking agent [29,30]. Reduced swelling and decrease in enzymatic degradation were seen without affecting the cell viability [31,32]. Similarly to the binary blends, a ternary composition of collagen, hyaluronic acid (HA), and poly(caprolactone) (PCL) was used to generate sponge-like dense membranes that were crosslinked with UV irradiation and EDC/NHS [33]. Developed hybrid crosslinking systems were able to preserve the native structure of collagen, and inclusion of PCL provided the ability to control the degradation and mechanical properties; this also made the sponges suitable for developing wound dressings and periodontal membranes [33]. Although crosslinked sponges have been shown to be biocompatible based on in vitro studies, it is necessary to implant the sponges and conduct in vivo biocompatibility studies. The application of sponges made from biopolymers may also be limited, unlike the porous structures made from ceramics, because of their inferior mechanical properties and stability. Crosslinking of biopolymeric hydrogels Hydrogels with the capacity to retain large amounts of water are used for tissue engineering and for the delivery of drugs, peptides, and proteins [34,35]. However, crosslinks must be present in a hydrogel to avoid dissolution and make the hydrogels usable for in vitro and in vivo applications [8]. Gelatin that readily forms gels was made into hydrogels and crosslinked with dextran dialdehyde; these were found to have a higher storage modulus than gels crosslinked with dextran aldehyde [36]. Another aldehyde (glutaraldehyde) was used in combination with a carboxylic acid (malic acid) and an dendrimer (EDC) to crosslink collagen gels; it was found that crosslinking decreased enzymatic degradation and promoted adhesion and growth of L 929 cells [37]. Instead of using a combination of crosslinkers, collagen hydrogels crosslinked with dendrimers such as EDC were able to provide similar resistance to collagenase degradation compared to glutaraldehyde [38]. In addition to the conventional approach of crosslinking with chemical crosslinking agents, collagen gels intended for controlled release of bovine serum albumin (BSA) have been crosslinked with the enzyme transglutaminase [11]. Crosslinking of the amine groups in collagen by transglutaminase was confirmed, and the denaturation 4
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temperature of the hydrogel increased from 38 to 668C – leading to reduced initial burst and sustained release of BSA after crosslinking [11]. Despite improvement in resistance to degradation after crosslinking, collagen gels do not have adequate properties for tissue engineering and many other applications [24]. To further improve the properties, collagen has been blended with other biopolymers to develop gels with improved stability and mechanical properties [28,39]. Blending collagen with gelatin and crosslinking the blend hydrogels with various concentrations of glutaraldehyde [41] increased the visoelastic properties of the gel and the breaking modulus without causing any toxicity to mouse fibroblast cells [39]. Chitosan/collagen hydrogels crosslinked with glyoxal and chitosan/BSA hydrogel crosslinked with genipin had enhanced mechanical properties and pH-dependent swelling, and were also cytocompatible [40,42]. Similarly to proteins, individual polysaccharides have also been made into hydrogels. Functional hydrogels made from dextran and crosslinked with epichlorohydrin and phosphorous oxychloride had low toxicity and good enzymatic degradability, but no cell culture or toxicity studies were carried out [43,44]. Using a unique approach of dual-crosslinking (butanedioldiglycidyl ether as the crosslinking agent) and photopatterning, hyaluronic acid was made into hydrogels with anisotropic swelling. Hydrogels were reported to swell to several times their weight but were able to maintain their morphology. Such highswelling hydrogels were suggested to be useful for ophthalmic, wound healing, and other medical applications [45]. Similarly, swelling (up to 720%) of hydrogels made from cellulose could be controlled by varying the conditions used for crosslinking with 1,2,3,4-butanetetracarboxylic acid dianhydride, a carboxylic acid with four carboxyl groups [46]. A double crosslinking approach was also used to first prepare aminated and oxidized hyaluronic acid that was further crosslinked with genipin. Crosslinked hydrogels were formed in vitro and also in vivo when the crosslinkercontaining polymer was injected into mice [47]. Higher compressive modulus, lower mass loss, and a compact microstructure were obtained after double crosslinking without decrease in biocompatibility [47]. These hydrogels were intended to be useful as biodegradable and injectable hydrogels for tissue engineering. Alginate hydrogels ionically crosslinked using Ca2+ ions had reduced swelling, and were able to maintain their mechanical properties and dimensional stability for up to 8 weeks in Ca2+ medium; the swelling was dependent on initial crosslinking density, alginate concentration, and chemical composition [35]. In most studies on hydrogels, the pH-dependent swelling of hydrogels and the resulting morphological studies have been reported, but the ability of the hydrogels to load and release drugs has only been reported to a limited extent. In addition, the ability of the hydrogels to support the attachment and growth of cells, and to resist degradation under physiological conditions, has not been thoroughly investigated. In addition, in vivo studies also need to be carried out to understand the potential of the crosslinked hydrogels for medical applications.
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Review Crosslinking of coarse (regular) fibers In addition to films, hydrogels and sponges, fibers with diameters ranging from a few to several hundred micrometers have also been developed for medical applications, mostly as sutures and tissue engineering scaffolds [48]. These microdiameter fibers have been made using traditional fiber-spinning approaches, such as wet spinning and melt spinning, depending on the type of biopolymer used. A simple approach of developing fibers is to dissolve the polymers and extrude the polymers as fibers using a extruder or a syringe and needle [49]. Similarly, thermoplastic biopolymers can be melted and extruded using a orifice or spinneret. Type I collagen obtained from rat tail tendons was dissolved and made into fibers using a buffer. Because collagen fibers readily disintegrate in aqueous media, fibers obtained were crosslinked using EDC or sulfo/NHS, and later mineralized using calcium chloride and potassium phosphate [48,50]. Crosslinking with EDC and mineralization of the fibers lead to a nearly 50-fold increase in wet strength. A comparative study on the physical (UV treatment after severe dehydration) and chemical crosslinking (EDC) treatments on self-assembled collagen threads showed that physical treatments increased mechanical properties but decreased cell migration, whereas carbodiimide-crosslinked threads had a lower increase in strength but improved cell adhesion [50]. In addition to collagen, plant proteins such as wheat gluten and soyproteins have also been made into fibers for potential use as tissue engineering and drug delivery scaffolds [51–53]. These fibers have good mechanical properties under dry conditions but have inherently poor stability under aqueous conditions, and have therefore been crosslinked using carboxylic acids and other crosslinkers [51–53]. Highly water-stable protein fibers were obtained after crosslinking the fibers with carboxylic acids. It was suggested that the carboxylic acid crosslinking of proteins follows a pseudo-first-order reaction, and that the crosslinking conditions can be controlled to obtain fibers with desired mechanical properties and degradation rates [51,52]. When fibrous conduits for delivery of nerve growth factors (NGFs) made from gelatin were crosslinked with genipin, the rate of delivery of NGF could be controlled by varying the amount of crosslinking agent used, without compromising the biocompatibility [54]. The crosslinked conduits were considered ideal for regeneration of nerves. Crosslinking of ultrafine fibers Compared to regular fibers, ultrafine fibers possess many unique properties, and have been widely studied and adopted for medical applications [55]. Electrospinning is one of the most common methods to produce ultrafine fibers. Electrospun fibers developed from biopolymers resemble the ultrafine fibrous network in extracellular matrices (ECM) [56,57]. It has been demonstrated that the interconnected structures in electrospun scaffolds can promote the attachment and proliferation of cells [58]. However, electrospun fibers from biopolymers, especially proteins, have poor water stability (instantly dissolve or disintegrate in aqueous environments), particularly due to their fine structure and high surface area [55]. Therefore, crosslinking electrospun fibers
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requires special crosslinking techniques and approaches. A novel method of in situ crosslinking of electrospun collagen fibers (diameters of 0.42 0.11 mm) was developed using EDC/NHS as the crosslinking agent without the need for post-crosslinking [55]. Crosslinked samples maintained their morphology even after being in water, and the mechanical properties of the electrospun fibers were similar to those of native tissue [55]. Owing to the difficulties of using aqueous solutions for crosslinking electrospun fibers, electrospun collagen scaffolds have been crosslinked by exposure to saturated glutaraldehyde vapor [56,59]. Crosslinked scaffolds had higher tensile strength, and could resist degradation by collagenase, but had decreased porosity; contradictory results on the cytotoxicity of glutaraldehyde-crosslinked fibers have been reported [56,57,59,60]. To overcome the limitation of cytotoxicity associated with glutaraldehyde, citric acid has been used a biocompatible crosslinker for electrospun collagen fibers [6,7]. However, a crosslinking extender was necessary to crosslink electrospun collagen fibers with citric acid because of the limited availability of free functional groups. Addition of glycerol (as a crosslinking extender with large number of hydroxyl groups) assisted the formation of crosslinks, and improved the strength and stability of the electrospun fibers [7]. Similarly to collagen-based electrospun fibers, electrospun matrices made from zein have relatively weak tensile properties and also rapidly dissolve when immersed in aqueous solutions. Crosslinking electrospun zein matrices with citric acid of various concentrations provided matrices that retained their morphology even after 15 days of incubation in PBS at 37 8C. In addition, zein samples crosslinked with citric acid had a higher rate of attachment and proliferation of fibroblasts than did electrospun PLA scaffolds [6]. Similarly to electrospun protein fibers, chitosan, starch and other polysaccharides have also been used to produce electrospun structures and were crosslinked to improve their strength and stability. Electrospun chitosan fibers (143–334 nm) crosslinked with glycerol phosphate (GP), tripolyphosphate (TPP), and tannic acid (TA) [61] were insoluble even in 1 M acetic acid after immersion for 72 h, but their cytocompatibility was not assessed. In contrast to the usual methods for crosslinking fibers after electrospinning, the possibility of in situ crosslinking of pullan/dextran mixtures with trisodium metaphosphite (STMP) has also been demonstrated [62]. Crosslinking decreased swelling and also promoted viability of human dermal fibroblasts; actin stress fiber formation was also observed, suggesting potential applications of the crosslinked fibers in tissue engineering [62]. However, in situ crosslinking is not feasible with most crosslinkers or polymers, and may also lead to undesirable changes in the properties of the materials and decrease electrospinnability. In addition to electrospinning, phase separation is another method of fabricating ultrafine fibrous structures from biopolymers [63]. Unlike the layer-by-layer structure of electrospun materials, phase-separated structures have randomly oriented fibers, and this is closer to the 3D structure of ECMs than are structures produced by electrospinning [6]. However, maintaining the 3D structure of phase-separated fibers, especially in aqueous conditions, is 5
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Review challenging. Gelatin was made into ultrafine phase-separated fibers using thermally induced phase separation, and the scaffolds were crosslinked with EDC/NHS [63]. In another approach, an ultra-low concentration phase-separation method was used to produce 3D ultrafine gelatin fibers that were crosslinked with citric acid [64]. Better compatibility and cell infiltration was observed in the crosslinked gelatin fibers produced by phase separation compared to electrospinning [64]. Crosslinking micro- and nanoparticles Micro- and nanoparticles developed from biopolymers are preferable over metallic and synthetic polymers and have been used for in vivo delivery of drugs and other pharmaceuticals [65]. Several researchers have demonstrated that nanoparticles made from biopolymers can load high amounts of drugs, accumulate in tumors and other targeted organs, and provide efficient delivery of payloads [66,67]. Poor stability, consequent agglomeration and increase of particle size, and relatively quick degradation compared to metallic and synthetic polymer-based nanoparticles are some of the major limitations of biopolymeric nanoparticles [68,69]. Considerable physical and chemical modifications including crosslinking have been made to improve the performance of the polymeric nanoparticles [70]. Chitosan nanoparticles (
