Abstract
Utility of plant-based materials in tissue engineering has exponentially increased over the years. Recent efforts in this area have been focused on substituting synthetic cross-linkers with natural ones derived from biological sources. These cross-linkers are essentially derived from the vegetative components of plants therefore suitably categorised as ‘green’ and renewable materials. Utilization of plant based cross-linkers in scaffolds and hydrogels offers several advantages compared to the synthetic ones. Natural compounds, like ferulic acid and genipin, when incorporated into scaffolds can promote cellular proliferation and growth, by regulation of growth factors. They participate in crucial activities, thus providing impetus for cell growth, function, differentiation and angiogenesis. Several natural compounds inherently possess anti-microbial, antioxidant and anti-inflammatory effects, which enhance the inherent characteristics of the scaffolds. Versatility of natural cross-linkers can be exploited for diverse applications. Integrating such potent molecules can enable the scaffold to display relevant characteristics for each function. This review article focuses on the recent developments with plant based cross-linkers that are employed for scaffold synthesis and their applications, which may be explored to synthesize scaffolds suitable for diverse biomedical applications.
Introduction
The term ‘tissue engineering’ was conceptualized by Dr. Y.C Fung, in the year 1987, during the meeting of National Science Foundation (NSF) at the University of San Diego. 1 Later, tissue engineering was defined as the ‘application of the principles and methods of engineering and life sciences to enable the fundamental understanding of the structure-function relationship in normal and pathological mammalian tissues and development of biological substitutes to restore, maintain and improve the tissue functions’. 2 Tissue engineering integrates concepts of cell transplantation and material design. This branch revolves around in vitro development of tissue constructs using suitable materials. The formed tissue constructs/scaffolds are further harnessed to promote pre-determined, endogenous regeneration in the human body. As limited number of organs are available for transplantation, this approach was primitively introduced to bridge the gap between the number of organs desired for urgent organ transplantations and those that were available.3,4 Tissue engineering withholds tremendous potential to revolutionize the bio-medical sector. It offers possibility of replacing the damaged tissues or organs due to accidental injuries or combat injury and organ damage due to aging and various diseases. Attempts are made time and again to alter or support functioning of both soft and hard tissue organs that are either damaged or diseased. In case of soft tissue engineering commercial scaffolds like Integra™ and Apligraf™ are available to cater needs of skin tissue engineering. Such grafts are in significant demand for wound/burn healing treatments. Further, 3 D bioprinting is also harnessed to produce required scaffolds made up of gelatin or polyurethane to construct or amend neural tissues restoring their cognitive functions. In case of fabricating harder tissues or organs like cartilage/bone tissues specific biomaterials like cellulose, hyaluronic acid, collagen and silk are used as materials of construction to obtain desired scaffold that would aid in constructing lost structural backbones of body. Exhaustive investigations in the area of tissue engineering have enabled emergence of novel therapies that present tremendous potential and can provide the ultimate remedy for chronic diseases.
Scaffolds, an integral part of tissue engineering, provide 3 D conformation and mechanical support to guide regeneration of different tissues. 5 The scaffolds recapitulate crucial roles and functions of the extracellular matrix (ECM). For instance, exogenous cells seeded on the scaffold, in combination with the endogenous cells, mediate regeneration and remodeling of the native tissue. In majority of the cases, the scaffolds are designed to degrade during the regeneration process as the cells commence assembling their own ECM. 6 Diverse fabrication techniques have facilitated the creation of porous 3 D scaffolds, using natural or synthetic materials. 7 The pre-requisite qualification for every hydrogel employed in such fabrication techniques is their spontaneity to form the scaffold. Hydrogels comprise of hydrophilic biomaterials, usually polymers that are cross-linked to enhance their mechanical strength and thus, improve their structural integrity. Cross-linking promotes formation of chemical links between the molecular chains of a polymer, generating a 3 D network. 8 Cross-linking is broadly categorized into two types, viz. physical and chemical cross-linking. The cross-linkers are further sub-divided into natural and synthetic. Natural, plant-based cross-linkers have exhibited huge potential in tissue engineering applications as they not only improve the mechanical strength and the physiochemical properties of scaffolds, but also enhance collagen deposition, cell attachment, cell differentiation and biocompatibility.9,10 Furthermore, they are non-toxic, easy to handle and are usually available at economic costs. 11 The aforementioned virtues displayed by the natural cross-linkers, as against their contemporary chemical counterparts, further demands their extensive investigation in scaffold manufacturing. This review article summarizes various natural, plant-based cross-linkers explored in tissue engineering, till date, primarily advocating their merits over synthetic materials. A wide range of natural cross-linkers, like polyphenols, organic acids, vitamins and plant extracts, have been elaborately discussed with respect to their specific applications.
Scaffold: A vital component in tissue engineering
The ECM constitutes a highly versatile structural nexus of macromolecules, such as glucosaminoglycans (GAG's), proteoglycans and fibrous proteins, that undergoes continuous, remodeling and serves as an essential component for regulating all cellular homeostasis.12,13 It is predominantly responsible for the cellular attachment, growth, and multiplication. Apart from providing the mechanical support to the growing tissues, it is responsible for regeneration and healing processes. The ECM provides anchorage and regulates intricate arrangements of different cells, thereby determining cellular dynamics such as proliferation, adhesion, survival, migration etc. 14 In tissue culture, the ECM is responsible for formation of newer networks, which encourages tissue formation. 14 Mina Bissell and co-workers demonstrated the role of ECM in cellular growth, when incubated in two-dimensional (2 D) and three dimensional (3 D) environments. 15 Despite the extensive use of 2 D culture in cell biology, for studying cellular behavior and processes, recent studies have proven the need of 3 D cell culture for invoking in vivo tissue responses. Considering this complex mechanical and biochemical association between cells and the ECM, it is apparent that tissue regeneration is cumbersome in 2 D culture. Langer and Vacanti overcame this challenge while investigating a 3 D biodegradable synthetic scaffold as an artificial ECM. 16 Their findings were crucial and resulted in a paradigm shift in the field of tissue engineering, garnering serious attention towards this field. Since then, innumerable pre-clinical and clinical studies have been conducted using synthetic and bio-based materials for organ regeneration. Researchers have also constructed organs in situ by inoculating cells into scaffolds fabricated using synthetic biodegradable (α-hydroxy ester) polymers. These scaffolds were further transplanted into hosts, where they reinstated the functions of the lost tissues via inducing vascularization and cellular growth, and thus exhibited excellent results in animal models. This technique of cell transplantation was extremely efficient and the approach was termed as “Chimeric Neomorphogenesis”. 16 These explorations attested that the biodegradability and biocompatibility of scaffolds played a critical role in their successful application. Besides the material of construction, the 3 D conformation of scaffolds is of utmost importance to mimic the structure and functions of the host ECM. 17 Various techniques employed for fabrication of scaffolds have been meticulously reviewed by Eltom et al. 18
Top-down and bottom-up approach in tissue engineering
3D cell culture provides a better way to mimic natural tissue in vitro. This approach is advantageous over 2 D culture as it stimulates a complex environment, relevant to native tissues. For instance, a cancer treatment 3 D culture provides realistic approach which can be effective to validate new treatment methodologies. Further incorporating principles of microfluidic technique in such a 3 D cell culture allows for a better fluid flow having potential to develop barrier tissues which ideally mimic natural physiological condition for tissue of interest. A vital component of 3 D cell culture is the scaffold which provides a 3 D confinement and structural support to cells. Scaffolds potentiate extra cellular matrix (ECM) formation in which cells proliferate and differentiate. 19 Tissue engineering has two strategies for fabrication of 3 D construct as represented in Figure 1. In traditional top-down approach the scaffolds are fabricated first, followed by embedding cells within the scaffold. Top down approach for 3 D scaffold fabrication encompasses several techniques like electro-spinning, 20 phase separation, 21 freeze-drying 22 and self-assembly. Several thin and avascular tissues have been successfully engineered using the top-down approach, but fabrication of complex tissues via this technique still remains a challenge. On the other hand bottom-up approach leads to micro-scale tissue building generating blocks having specific micro-architecture and assembly which can form a larger tissue construct. 23 These specific building blocks can be fabricated via generation of bio-printed cellular sheets24,25 self-assembled cell aggregates, 26 cell encapsulation in hydrogels 27 or direct printing of cells. 28 Aforementioned methodologies can be employed to precisely control the shape and composition of the individual blocks, leading to formation of complex tissue constructs. 29
Top-down and bottom-up approach in tissue engineering.
Cross-linking of scaffolds and its significance in tissue engineering
Crosslinking is crucial and elementary step in tissue engineering as it plays a key role to impart and maintain desired architecture and structural integrity to the scaffold by forming firm network in the polymeric matrix. 30 Cross-linking of scaffolds is important for their application in tissue engineering as the process enhances physical parameters for the scaffold material like water contact angle, roughness, pore size of scaffold and viscoelasticity of hydrogels etc. Recent study performed by Indurkar et al. on guar gum-gelatin hydrogel crosslinked with glutaraldehyde highlighted the role of crosslinker in enhancing both storage modulus (G′) and loss modulus (G′) of the hydrogel with help of rheological analysis. 22 Enhancement in both the dynamic modulus and decrease in tan ∂ implicates elevation in strength and toughness of the hydrogel 31 as shown in Figure 2(a) and (b) respectively. Further, water contact angle analysis is very helpful to predict hydrophilicity of the scaffold which is primary requirement for cell attachment and media adsorption. A higher contact angle indicates hydrophobicity while a lower contact angle is characteristics of a hydrophilic material. Investigations performed by Chu and co-workers on collagen membranes crosslinked with Epigallocatechin-3-gallate (EGCG) have demonstrated increment in hydrophilicity with corresponding increase in EGCG concentration as represented in Figure 2(c). Additionally, uniformity in collagen fibers and fibre diameter was improved upon incorporation of EGCG in concentration dependent manner as shown in Figure 2(d1) to (d4). 32 Crosslinking makes scaffold compact, hence crosslinker concentration aids in getting desired pore size of the scaffold. Surface roughness is also important physical parameters of the scaffold on which cell attachments is dependent. Apte and co-workers evaluated effect of three crosslinkers (Calcium chloride, EDC/NHS and genipin) on alginate-chitosan films for their effect to modulate roughness of the formed scaffold. 33 Formed scaffolds when evaluated with atomic force microscopy revealed that surface roughness was increased by all crosslinkers as depicted in Figure 2(e). Crosslinkers are also reported to impact Tensile strength of formed scaffold which is important to maintain their structural integrity. Zhou et al. reported efficiency of Genipin (GP) to increase tensile strength of polyvinyl alcohol/silk/nano hydroxyapatite hydrogel from 0.48 MPa to 0.64 MPa as compared to others. 34
a) Rheological analysis of non-crosslinked (NCL) and crosslinked (CL) 2.5% w/v guar gum-gelatin hydrogel b) Tan ∂ analysis of non-crosslinked (NCL) and crosslinked (CL) 2.5% w/v guar gum-gelatin hydrogel [Adapted from Carbohydrate polymer technologies and applications 22 ] c) Contact angle of collagen membranes in which control represents uncross linked membranes while E-Col represents EGCG crosslinked collagen membranes d) Scanning electron microscopy of collagen fibbers (D1) Non-crosslinked, (D2-D4) crosslinked with increasing concentration of EGCG [Reproduced from Material Science and Engineering: C with permission of Elsevier 32 ] e) Surface roughness of alginate-chitosan films non-crosslinked and crosslinked with Calcium chloride, EDC/NHS and genipin respectively [Adapted from Macromolecular biosciences 33 ].
Closely knit polymeric networks, in the hydrogels formed with pre-determined levels of cross-linking, impart the hydrogels with an adequate resistance against disintegration in aqueous physiological fluids. 35 Moreover, it helps them to retain sufficient hydrophilicity and water-uptake capacity for interaction with cells, and thereby supports the structural integrity of the scaffolds in culture media/site of implantation. Crosslinking also imparts corrosion resistance to the scaffold/hydrogel. 36 A pictorial depiction of cross-linked scaffold and hydrogel is represented in Figure 3.
Illustration of cross-linking of polymers.
Depending upon nature of biomaterial and targeted profile of scaffolds to be fabricated several crosslinkers are explored for scaffold fabrication. More commonly during the crosslinking process active functional groups present on the biomaterial interacts with crosslinker forming a 3 D network. Till date variety of crosslinking techniques have been developed for scaffold fabrication which are categorized into three groups, as physical, chemical and enzymatic (represented graphically in Figure 4).
Mechanisms of chemical and physical cross-linking.
Physical cross-linking involves hydrogen bonding, ionic or electrostatic interactions, complex formation, hydrophobic interactions, and ultrasonic mediated sol-gel formation. For instance, Ionic integrations take place when alginate crosslinked with calcium chloride. The divalent ions react with gulcoronic units of alginate to form ionic bridges between different chains. Hydrogel of nano-crystalline cellulose and polyvinyl alcohol are crosslinked via hydrogen bonding when Tannic acid was used as crosslinker. 37 Irradiation of UV light is absorbed by double bonds, including aromatic ring present in tyrosine and phenyl aniline which leads in formation of free radical and intermolecular covenant bonds. 38 Use of ultrasound causes vibratory disturbances in the gel, thereby leading to generation of cavitation effects, inducing higher levels of shear, temperature and pressure locally. These local effects are responsible for self-assembly of hydrogels. 39 The prime advantage of using physical cross-linking is the avoidance of harsh chemicals which assists in circumventing any unwanted cytotoxicity arising due to any unreacted or residual chemicals. Further, physically cross-linked hydrogels have self-healing properties and are therefore employed for applications involving cell encapsulation and drug delivery. 40 Contrarily, chemical cross-linking is guided by principles of ‘click' chemistry, namely those involving oxime formation, Schiff base formation, Millard reaction, photo-polymerization etc. Compared to the physically cross-linked hydrogels, the chemically cross-linked hydrogels exhibits enhanced stability under physiological conditions, with outstanding mechanical properties and cost-effectiveness.41–43 The advantages and disadvantages of the three crosslinking approaches are indicated in Table 1.
Advantages and disadvantages of the physical, chemical and enzymatic crosslinking approaches.
Additionally, cross-linkers are also classified into two groups depending on their source as synthetic or natural. Some notable examples of synthetic cross-linkers used for cross-linking of scaffolds or hydrogels are illustrated in Table 2. Amongst chemical cross-linkers, glutaraldehyde (GA) has been the most extensively studied for fabrication of various hydrogels, scaffolds and composites. Although glutaraldehyde is majorly used to crosslink biomaterials in scaffold fabrication (eg. chitosan, gelatin etc), it has also been useful in crosslinking synthetic polymers like polyvinyl alcohol (PVA). GA predominantly reacts with the hydroxyl or amine groups present in the polymeric strands of a scaffold, via Schiff base reaction, generating inter or intra-molecular linkages. 60 Therefore, it can be safely considered as a prototype to evaluate the shortcomings of chemical cross-linkers. The absolute amount of GA employed in cross-linking is never consumed and the remaining unbound GA is highly toxic to the cells. Therefore, a procedure to remove the free GA, after cross-linking, is imperative. 61 Numerous findings related to poor biocompatibility, cell adhesion and apoptosis have been reported in case of matrices cross-linked with GA.62,63 Apart from its high toxicity, GA also initiates calcification in long-term implants. 64 Hence, owing to their toxicity profile and the untoward effects, time-consuming neutralization of the unreacted or free cross-linker is the major drawback offered by chemical cross-linkers like GA. EDGE is commercially available; epoxy based synthetic cross-linker that has been reported to hinder cell proliferation. It also possess poor resistance to enzymatic degradation, which raises concern over its timely elimination from physiological systems.65,66 Synthetic cross-linkers, therefore, confer diminished biocompatibility to the formulated scaffolds by imparting lack of conversion ability into biodegradable sub parts. Moreover, owing to their chemical origin, several organic solvents, initiators, stabilisers, emulsifiers, deployed for scaffold fabrication also pose a similar challenge. Furthermore, majority of the cross-linkers, such as glyoxal, glutaraldehyde, formaldehyde, isocyanides, and acryl amides etc, are precariously carcinogenic and noxious and thereby pose serious health hazards. Use of such reagents warrants employment of skilled and trained labour. Utmost precautions have to be exercised while working with such chemicals. However, despite their numerous shortcomings, the use of chemical crosslinkers dominate the tissue engineering field, mainly because they are easily accessible. Chemical crosslinkers, like glutaraldehyde, glyoxal etc, are suitable for industrial scale fabrication. Further, the synthetic routes of producing the chemical crosslinkers can be easily monitored, which aids in the production of materials having reproducible characteristics. When such crosslinkers are employed in scaffold manufacture, the resulting scaffolds exhibit reproducible performance. However, this provision, is not unavailable with natural crosslinkers, which may vary with their source of origin. Specific advantages and disadvantages of chemical and natural crosslinkers are summarized in Table 2.
Advantages and disadvantages of the chemical and natural crosslinkers.
Plant based cross-linkers
Green crosslinkers are the class of crosslinker which are biocompatible and do not possess any environmental hazards. Innumerable such crosslinkers have been studied till date for formulating scaffolds for tissue regeneration. Plant derived materials constitute the bulk of the green cross-linkers. Other significant contributors to this domain include compounds like borax, 67 tripolyphosphate 68 and calcium chloride. 69 Notable examples of plant-based cross-linkers that have been extensively used for scaffold fabrication include epigallocatechin gallate, genipin, organic acids, polyphenols, plant extracts and vitamins etc. Plant-based cross-linkers provide greener and environmentally sustainable alternatives for producing suitable bio-composites. 11 In contrast to synthetic crosslinkers plant-based crosslinkers are user-friendly and non-toxic and do not possess any environmental hazard. 70 Further, these natural moieties overcome issues pertaining to the biocompatibility and toxicity of synthetic cross-linkers. Apart from being biocompatible, these natural cross-linkers promote cellular proliferation and adhesion by enhancing physiochemical properties of scaffold and are more economic.71–73 Plant based crosslinkers possess inherent medicinal properties and thereby furnish an addition benefit over synthetic crosslinkers. Forthcoming sections of this manuscript will be focused on case specific discussions related to plant-based cross-linkers that exhibit a huge potential in tissue engineering.
Cinnamaldehyde
Cinnamaldehyde (CA) is found as a thick viscous liquid in the bark of cinnamon tree and is extensively used for fragrance in aroma and essence industry. 74 Traditionally it has been used as a spice and herbal medicine. It has proven excellent anti-inflammatory, antioxidant, anticancer activity and also plays a key role in tissue repair. 75 Know et al. Investigated crosslinking ability of cinnamaldehyde for forming collagen scaffolds. The complex reaction of crosslinking occurs when aldehyde group of crosslinker reacts with the amine group of lysine residue present in collagen. Results highlighted that addition of crosslinker enhanced both the surface roughness and compressive strength of formed scaffold. Further enhancement in the cellular response by increasing cell attachment, proliferation and differentiation of human dental pulp cells was seen post incorporation of CA. 76
Epigallocatechin gallate
Epigallocatechin gallate (EGCG) is the major active constituent, primarily extracted from green tea. EGCG is composed of epigallocatechin and gallic acid moieties, linked together via ester linkages. 77 It inherently possesses anti-bacterial, anti-tumour, anti-inflammatory, anti-collagenase, anti-fibrotic, and anti-oxidant properties, that are exploited for specific applications. 78 EGCG initiates hydrogen bonding between the polymeric chains of the biomaterials (eg collagen, gelatin) used to fabricate scaffolds. This mechanism is primarily responsible for driving its cross-linking efficiency, leading to scaffold formation. 79 Kwon et al. reported synthesis of collagen scaffolds, cross-linked with EGCG, for understanding its potential in promoting proliferation and differentiation of human dental pulp cells (hDPC’s). Incorporation of EGCG as the cross-linker enhanced the mechanical properties and resulted in profound cell proliferation and differentiation. EGCG further imparted anti-bacterial properties to the formulated scaffold. 72 Similar to collagen, EGCG was used to fabricate cross-linked gelatin sponge. Matrix metalloproteinase-2 and matrix metalloproteinase-9 are well documented gelatinases that thwart the regeneration capability of gelatin-based scaffolds. Huang et al. proved the modulatory effect of EGCG towards matrix metalloproteinases, through analysing the comparative efficacy of gelatin sponges fabricated with and without EGCG. Results indicated improved bone formation ability of gelatin sponges as they maintained the necessary structural cohesiveness. EGCG arrested the negative effect of matrix metalloproteinase, leading to enhanced bone formation, which highlighted the importance of EGCG. 80
Genipin
Genipin (GP) is an iridoid compound with numerous reactive hydroxy and carboxyl functional groups. It is predominantly extracted from fruits of Genipa Americana and exhibits meagre toxicity issues when compared with GA. Several cellular studies endorse only a modest toxicity profile of GP, owing to which it has amassed significant recognition in tissue engineering applications. Cytotoxicity assays conducted with fibroblast cells, using MTT assay, revealed that GP was ten thousand times less toxic than GA. Also, colony forming assay with fibroblast cells indicated high cellular proliferation rate in scaffolds cross-linked with GE, in contrast to the scaffolds assembled with GA. 81 Despite the inferior cross-linking efficiency as compared to GA, GP was largely favoured due to its biocompatibility and negligible cytotoxicity. Gelatin scaffolds cross-linked with GP demonstrated improved differentiation of chondrocytes, as compared to those cross-linked using GA, when analysed through in vitro assays. 82 Further, GP was reported to spontaneously react with chitosan, proteins and amines etc. to form hydrogels, which bolstered it as a substitute for GA. 83 GP is chemically compatible with a wide range of biopolymers and therefore can be employed to cross-link multiple polymeric chains having distinct chemical features. Collagen and chitosan scaffolds, manufactured using GP, displayed enhanced stability during the in vitro studies conducted in PBS. GP distinctly controlled the pore size of the scaffolds. Further, the concentration of chitosan employed controlled the efficiency of Response: SEM stands for scanning electron microscopy. Initially SEM analysis indicated honey-comb structured pores; cell culture was performed on chondrocytes. SEM analysis of scaffold was also performed after 12 days of cell culturing which shows even spreading of cells, on surface and walls of the scaffold. Along with MTT analysis, SEM characterisation confirmed ability of GP cross-linked scaffolds to support adhesion and proliferation of chondrocytes, in vitro. 84 On similar lines, polyvinyl alcohol/silk fibroin and nano-hydroxyapatite scaffolds were cross-linked with GP and assessed as artificial substitutes for cornea. Besides upgrading the physical properties due to cross-linking, GP also improved the thermal stability of the resulting hydrogels. Biocompatibility experiments manifested negligible cellular toxicity of the formed scaffolds, which supported cellular proliferation and adhesion. 34 Decellularized biological matrices of hepatic tissues were hypothesized by researchers for regeneration of impaired or diseased liver. GP, by virtue of its low cytotoxicity, was directly integrated into the decellularized tissue to generate matrices. These matrices were found to be compatible in the porcine model, after four weeks of implantation, whereas matrices formed with GA elicited severe inflammatory reaction. 85 Validated reports have established the functionality of GP in scaffolds meant for wound-healing applications. Hydrogels formulated with gelatin, diosgenin and nanocellulose were cross-linked with GP. Efficient cross-linking of these diverse polymeric strands was achieved with GP, while maintaining sufficient hydrophilicity. 86
Organic acids
Organic acids (OA) are naturally occurring, weakly acidic compounds, largely derived from citrus plants. These are basically dicarboxylic acids recognised as primary metabolic products in plants, that play a key role in the Kreb’s cycle, and are further involved in the production of fatty acids. These are found in a variety of plants. For instance, Adipic acid (AD) is naturally found in beet roots and sugarcane, 87 whereas Azelaic acid (AZ) in grains such as wheat and barley. 87 Originally, dicarboxylic acids such as citric acid (CA), maleic acid (MA) and tartaric acid (TAA) etc have been widely explored for their efficacy in tissue engineering. 88 OA being diprotic acids contain two reactive ends and are water-soluble. These natural agents bear sufficient resistance towards oxidising agents and inherently exert anti-bacterial and anti-inflammatory effects. Therefore organic acids have favourably established their position as natural cross-linkers. 89 Mitra et al. have screened a variety of dicarboxylic acids, like adipic acid, Azelaic acid, maleic acid and succinic acid (SA), as cross-linking agents for coupling of collagen and chitosan (structural representation provided in supplementary information). In silico evaluations have provided mechanistic insights into their cross-linking behaviour. Dicarboxylic acids engage with collagen and chitosan by formation of both, ionic and multiple intermolecular hydrogen bonds. Cross-linking with these organic acids increased the mechanical strength of the scaffold. Cellular studies performed by culturing fibroblasts on each of the respective scaffolds, fabricated with different organic acids, displayed biocompatibility, and thus reflected the overall safety of the cross-linking agent.90–94 Identical examination was executed with CA, TAA and MA for estimating their ability to cross-link gelatin. Organic acids successfully formed scaffolds, imparting negative charge to the gelatin matrix and aiding in anti-thrombogenic and endothelialisation characteristics. This novel, cross-linked gelatin matrix was resistant to collagenases and promoted adhesion and proliferation of endothelial cells. This acquired property enhanced scope of the assembled scaffold for potential applications requiring mitigation of cardiovascular issues. 95 Numerous such concrete evidences have confirmed the usefulness of organic acids as green and potent cross-linking agents and as alternative to synthetic materials. However, specific applications still require in-depth analyses.
Citric acid
Citrus fruits, such as lime and lemon, are the major producers of the naturally occurring citric acid (CA). Over the past few years, CA has emerged as a non-toxic cross-linker for preparation of hydrogels.96–98 Temperature controls the cross-linking mechanism of citric acid. At elevated temperatures, CA forms a cyclic anhydride that esterifies the hydroxyl group present on the adjacent polymer chains. 99 CA is well documented in literature as a natural cross-linker for cellulose based hydrogels. Further studies indicated that induction of CA enhanced the rheological properties, hydrophilicity and surface roughness of the hydrogels, in turn supporting improved cell differentiation. 71 CA is adaptable with wide range of scaffold fabrication techniques and also displays inertness towards most of the natural polymers. Zein fibers were electrospun and cross-linked with citric acid in a study by Q. Jiang et al. 100 Experimental results indicated improved morphological stability and maintenance of fibrous structure even after immersion in PBS for 15 days, at 37°C. Further cross-linked zein fibres allowed excellent attachment and proliferation of fibroblasts cells, as opposed to non-crosslinked zein fibres. Investigations involving electrospun poly (vinyl) alcohol fibres, cross-linked with CA, revealed improved water resistance, thermal stability and tensile strength of the nanofibers. CA-PVA combination also provided conducive habitat for attachment and proliferation of fibroblast cells. 101
Polyphenols and tannins
Polyphenols and tannins are secondary plant metabolites which play a vital role in their defence mechanism. Prominent examples of this class of naturally occurring astringent compounds include ferulic acid, ellagic acid, gallic acid etc. Nearly 8000 such phenolic compounds possessing anti-microbial, anti-oxidant and anti-inflammatory activity and have been recognised till date.102,103 Polyphenols are preferably extracted from fruits, vegetables and tree barks for various applications pertaining to food, pharmaceutical or leather industry.104–106 These polyphenolic compounds are also being tried as substitutes to synthetic cross-linkers during scaffold manufacture. The following sections discuss about the recent investigations that have proved the efficiency of polyphenols in overcoming challenges associated with tissue engineering.
Ferulic acid
Ferulic acid (FA) is a phenolic phytochemical abundantly found in the cell wall of plants. ferulic acid is also a promising anti-bacterial, anti-diabetic and an anti-inflammatory agent. 107 Therefore, owing to its bio-medical importance ferulic acid has been often investigated in various in vitro studies for its ability to promote proliferation of cultured cells. Modulation of cyclic D1 and vascular endothelial growth factor (VEGF) was identified as the primary mechanism by which ferulic acid induced proliferation of endothelial cells (ECV304). 108 The cross-linking reaction of ferulic acid is complex and occurs by different mechanisms. 109 Firstly, it can react with amino and thiol groups of proteins, when oxidised and present in quinone form. In other cases, the resonance stabilised free radical form of ferulic acid reacts with amino acid residues, such as tyrosine, lysine and cysteine etc. of the involved substrates. Ferulic acid can undergo self-dimerization to bridge protein molecules. Another well documented cross-linking mechanism exhibited by ferulic acid involves interactions with carboxylic or hydroxy moieties, under non-oxidative environment, by forming esters. Given the multi-faceted interactive prospects of ferulic acid, it was successful utilized to cross-link gelatin by Jing et al., which improved its intrinsic viscosity improving the manufacturability of the scaffold to be formed. 110 Polyurethane scaffolds fabricated using ferulic acid exhibited haemolytic activity and fibrinogen generation, along with lower platelet adhesion. Further, FA also aided in successful proliferation of endothelial cells on the scaffold, which directly implied its utility in regeneration of vascular tissues. 111
Gallic acid
Gallic acid, chemically known as 3, 4, 5-trihydroxybenzoic acid, is a phenolic compound mainly found in the leaves of bear-berries, pomegranate, root bark and many other plants. 112 Gallic acid is a molecule of therapeutic importance due to its intrinsic antioxidant, anti-inflammatory and anti-neoplastic activities. It therefore offers necessary benefits for alleviation of cardiovascular, gastrointestinal, metabolic and neuropsychological disorders. 113 Collagen has numerous and well-reported applications in the bio-medical field. It is prominently employed as a matrix forming agent due to its structural analogy with the connective tissues in human or animal models. Molecular docking experiments have presented conclusive evidence for hydrogen bonding interactions amongst Gallic acid and collagen. 114 Gallic acid, alongside its synthetic equivalents like Glutaraldehyde, is reported to assist 1-ethyl-3–(3-dimethylaminopropyl) carbodiimide (EDC)/N-hydroxy succinimide (NHS) in cross-linking of collagen. The fabricated matrix acquired elevated resistance to collagenase activity and improved its overall stability. 115 Further, chitosan scaffolds were efficiently fabricated using Gallic acid as the cross-linker by Thagavel et al. The resulting scaffolds exhibited enhanced thermal stability, anti-bacterial activity and enhanced attachment of fibroblast cells. 116
Hesperidin
Hesperidin (HE) is a bioflavonoid found in unripe citrus fruits. Basically, HE is a plant pigment that demonstrates anti-inflammatory and anti-oxidant properties. 117 Hesperidin displays a competitive edge over other natural cross-linkers, like riboflavin-5-phosphate and sodium ascorbate, for its ability to cross-link collagen. 118 Limited studies have been conducted with this promising molecule and further in vitro and in vivo investigations are required to confirm its suitability and biocompatibility in tissue engineering.
Nordihydroguaiaretic acid
Nordihydroguaiaretic acid (NGDA) is an anti-oxidant with lipoxygenase inhibitor activity and which is mainly extracted from the leaves of creosote bush. 119 Decellularized heart cells were used with collagen to construct a matrix, which was cross-linked with NGDA. 120 Scaffolds cross-linked with NDGA possessed higher mechanical strength than the scaffold cross-linked with glutaraldehyde. The resulting scaffolds were non-toxic to the cultures of endothelial cells and valvular interstitial cells, in vitro. Scaffolds cross-linked with NDGA retained morphology of endothelial cells and promoted their adhesion and proliferation. Thus, scaffolds cross-linked with NGDA were more beneficial than those cross-linked with glutaraldehyde. NGDA by virtue of its natural origin was relatively non-toxic as compared to glutaraldehyde and possessed 30 times lower values of lethal concentration 50 (LC50). Better cross-linking ability, coupled with low toxicity, and rendered NGDA as an excellent alternative to synthetic cross-linkers like glutaraldehyde. Another investigation employed NGDA for cross-linking collagen to evaluate its effect on adhesion and migration of fibroblast cells, which revealed a higher strength of the resulting collagen matrix. Results indicated that NGDA slightly reduced cellular adhesion strength and lower migration rates were recorded; however this limitation was largely compensated by superior mechanical properties that it imparted to the collagen scaffold. 121
Oleuropein
Oleuropein (OP) is an iridoid glycoside commonly found in olive leaf extract. This bio-material is known for its anti-bacterial, anti-angiogenic, anti-cancer, anti-inflammatory, as well as its anti-oxidant activities. 122 Oleuropein produces aglycones upon hydrolysis by β-glucosidase, which plays an important role in cross-linking of proteins. Literature reports have indicated the better ability of oleuropein to cross-link amino acids, as compared to glutaraldehyde. In tissue engineering applications, oleuropein was primarily used to fabricate scaffolds of zein in order to evaluate its effect on fibroblast cells. 123 Collagen scaffold was also successfully fabricated and cross-linked with oleuropein for bone tissue engineering. The resulting scaffolds did not exhibit any toxic or untoward effects towards osteoblasts, but promoted their adhesion and proliferation. 124
Proanthocyanidin
Proanthocyanidins (PA) are chemically oligomeric flavonoids and natural plant metabolites extracted from grape seeds. Proanthocyanidin are made up of esters of gallic acid with catechin or epicatechin. These complex compounds, analogous to other polyphenolic equivalents, possess multiple biological applications as a result of their anti-oxidant, anti-microbial, anti-diabetic and neuroprotective properties. 125 Hon et al. conducted cytotoxicity studies with collagen scaffolds, cross-linked with proanthocyanidins. The study revealed that the resulting scaffold was 120 times less toxic towards fibroblasts cells than the one cross-linked using glutaraldehyde. In vitro analysis demonstrated the protective nature of proanthocyanidins when used to cross-link collagen. Inclusion of proanthocyanidins imparted enhanced resistance to the collagen scaffold against bacterial collagenase 126 Collagen hydrogels formed using proanthocyanidins as the cross-linking reagent resulted in excellent adhesion and proliferation of human periodontal ligament cells, when evaluated in vitro. 10 Chen et al. formulated collagen-konjac glucomannan hydrogel using proanthocyanidins. Profound anti-oxidant and anti-calcification activity was induced by the scaffold containing proanthocyanidins, along with a controlled biodegradability. Proanthocyanidins also aided in accelerated wound healing, when used for cross-linking gamma-poly (glutamate) and gelatin scaffold. Proanthocyanidins based hydrogels were found to be non-allergic in guinea pig models, which emphasized their safety and biocompatibility. The resulting scaffolds resulted in accelerated wound-healing and reepithelialisation in murine models, within 21 days of evaluation, as compared to the untreated animals. 127
Quercetin
Quercetin (QT) is a flavonoid widely distributed in number of fruits such as grapes, tomatoes, berries and leafy vegetables and exhibits strong anti-oxidant properties. 128 Effect of cross-linking with quercetin on decellularized porcine heart valve ECM was studied by Zhai et al. 129 Scaffold cross-linked with quercetin expressed improved tensile strength, elasticity and enzyme resistance than the scaffolds cross-linked with glutaraldehyde. Cells studies further indicated that quercetin containing scaffolds supported adhesion and proliferation of vascular endothelial cells, as well as protected the ECM against mineral deposition Quercetin was also reported to cross-link nano-hydroxyapatite (nHA) and decellularized lung tissue. In silico experiments revealed the ability of quercetin to form multiple intermolecular hydrogen bonds with the collagen of decellularized tissue. nHA modified, quercetin - crosslinked lung tissues provided natural homing sites for bone marrow derived mesenchymal stem cells, facilitating their conversion into osteoblasts having a rapid proliferation rate. 130
Tannic acid
Tannic acid (TA) is a naturally occurring polyphenol obtained from chestnut and oak trees, which displays remarkable anti-viral, anti-inflammatory, anti-oxidant and anti-bacterial properties. 131 Owing to is polyphenolic structure, tannic acid contains ample hydroxyl or acid functionalities, which enables it to cross-link gelatin, collagen, albumin and chitosan, via hydrogen bonding interaction. 132 Tannic acid effectively cross-links a wide range of polymers and also does not intervene with the normal functioning of cells. The anti-cancer activity collagen scaffold, cross-linked with tannic acid, was assessed using breast cancer cell line (MCF-7). The resulting scaffolds effectively induced apoptosis in these cells. Normal homeostasis of lipid production by D1 cells was not altered in presence of tannic acid cross-linked collagen scaffolds, which underlined its potential to be used as a breast implant. 133 In vivo studies in murine model demonstrated the ability of tannic acid cross-linked collagen matrices for wound-healing application. 134 Tannic acid was also utilized to cross-link synthetic polymers, like polyethylene glycol. The resulting composite was found to demonstrate excellent activity against both, gram positive (eg. Staphylococcus aureus) as well as gram negative bacteria (eg. Escherichia coli), advocating its potential in biomedical arena. 135
Plant extracts
Traditionally, plant extracts (PE) have been indispensably employed as components of medicinal importance. The term PE refers to a multi-component mixture of bioactive compounds, such as phenolics, flavonoids, tannins etc. Several of these components individually possess the capacity to cross-link hydrogels, as reported in existing scientific literature. Therefore, collective potential of PE is undoubtedly high as a feasible and effective approach for cross-linking of scaffolds having application in the biomedical sector. Till date, only limited plant extracts have been used as cross-linking agents, for example grape seed extract and Myrica Rubra extract were used for cross-linking of collagen. Further, Galla chensis and tea extracts were used for cross-linking of gelatin.136–139 Inclusion of these extracts improved the denaturation temperature of matrix materials collagen and gelatin employed for scaffold preparation as proven by DSC analysis also augmenting their physical strength as compared to synthetic crosslinkers like glutaraldehyde and formaldehyde. Further, to improve exploration of plant extracts in tissue engineering applications, in depth investigations related to their safety and efficacy with respect to different cell lines needs to be evaluated.
Vitamins
Vitamins are essential micronutrients required by the body to maintain daily homeostasis. Broadly these are divided into two categories depending upon their aqueous solubility, as being water soluble or fat soluble. 140 The former group of vitamins are widely preferred in tissue engineering owing to their solubility in physiological aqueous fluids. These organic compounds are biocompatible and are essentially required by the body in small quantities for normal growth and well-being.
Riboflavin (RF) is the water soluble vitamin B2 essential for human health, which is obtained from grains, plants and dairy products. 141 Riboflavin is a photo-crosslinker and therefore utilizes normal UV light for activation. Being biocompatible and cost-effective, RF formed an excellent alternative for synthetic photo-crosslinker. Riboflavin was extensively used in corneal cross-linking for the treatment of keratoconus. 142 Riboflavin’s ability to cross-link collagen ultra-violet (UV)-A light was harnessed in tissue engineering applications. Scaffolds made up of collagen, chondroitin sulphate and fibronectins were fabricated by crosslinking with riboflavin in presence of (UV)-A light. Investigational results indicated that photo-crosslinked scaffolds exhibited no cytotoxicity towards gingival fibroblast cells and imparted resistance to collagenase activity, which in turn enhanced its bio-stability. 143 Riboflavin cross-linked collagen scaffold was also successfully employed to encapsulate fibrochondrocytes. The resulting scaffold was biocompatible and efficiently upregulated the gene expression levels pertaining to Type II Collagen. It could also repair the meniscus tissue in case of knee injury. 144 Riboflavin was equally effective in cross-linking and strengthening Type I collagen, which unarguably is largest component of human dentin. Cross-linking of this collagen type in human dentin is crucial with respect to prevention or correction of dental carries. R. Uemura et. al. identified that Riboflavin and UV treatments, under simulated oral microenvironments, appreciably corrected the mineral loss and improved lesions in the human teeth, as compared to the control group. Further treated surfaces, when exposed to enzymatic and acidic treatments, displayed enhanced resistance. Further, scaffolds cross-linked using RF protected the human dentin from acid and enzymatic degradation. 145
Biocompatibility tests
Biocompatibility evaluation for the fabricated scaffolds is essentially performed using both In vitro as well as In vivo assays in order to check their toxicity profile for the intended applications. In vitro biocompatibility for the formed scaffold is analysed by performing 3–(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) MTT assay. It is a calorimetric assay measuring the reduction of yellow coloured 3–(4, 5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide by mitochondrial succinate dehydrogenase. This reduction is only catalysed by live cells, making possible to calculate the percentage live cells. 146 Another method regularly used in to analyse biocompatibility is the live/dead assay. It consists of two fluorescent dyes Calcian-AM which stains cells green by recognising esterase activity and integrity of plasma membrane. The enzyme converts Calcein-AM to fluorescent Calcein. The dye retains in live cell producing green fluorescence. Ethelene homodimer enters damaged cell producing red fluorescence. 22 Some examples of biocompatibility tests for scaffold manufactured using plant based crosslinkers have been listed in Table 3. Biocompatibility and safety of crosslinkers cannot be certified using any single in vitro test from amongst MTT, alkaline phosphatase (ALP), live/dead assay or ELISA etc. These merely signify the series of potential tests to be performed. Moreover, as there is no standard protocol that may be followed to ascertain the biocompatibility of material. Thus, it is a common observation that the results obtained from in vitro testing do not correlate well with the behaviour of material inside the body, under physiological conditions, which warrants their in vivo evaluation. In vivo evaluation of the scaffolds is directly performed on suitable animal models to prove their safety and efficacy in physiological milieu. In vivo assays directly help analyse ability of scaffold to elicit the expected regenerative ability inside living body. For instance, in case of bone tissue regeneration immunosuppressant murine models has exhibited usefulness in representing bone related malignancies, whereas bone defects in larger bones like tibia or femoral defects are suitably evaluated using larger animals’ models like goat or pig. 150 For skin tissue engineering or cartilage tissue reconstruction small rodent and lapine models are preferred owing to feasibility in handling and utility in generating proof of concepts. Details of such animal models are extensively reviewed by Nyambat et al. and Chu et al. respectively.151,152
Biocompatibility of scaffold crosslinked with plant based crosslinkers.
a3–(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide).
bAlkaline Phosphatase activity.
cAlizarin red S staining.
dEnzyme-linked immunosorbent assay.
eReverse transcription polymerase chain reaction.
fReactive oxygen species.
Efficiency of plant-based crosslinked compared to glutaraldehyde
Majority of synthetic crosslinkers possess toxicity which results in incidence of biocompatibility issues with the scaffolds. Natural plant-based crosslinkers have huge potential to overcome this drawback of synthetic crosslinkers. Generally, plant based crosslinkers are secondary metabolites of plants, exploited for their medicinal properties since ancient times. These properties inculcate additional benefit to crosslinkers utilized in tissue engineering applications. Lien et al. studied efficiency of genipin over glutaraldehyde by fabricating gelatin scaffolds crosslinked individually with glutaraldehyde and genipin. Biocompatibility assays were performed on chondrocytes from Wistar rat’s joint. Uniform cell distribution and tissue development was confirmed using E staining within 9 days in genipin crosslinked scaffold which cellular response was poor with glutaraldehyde as represented in Figure 5(a) and (b). Whereas, results from Masson staining revealed that collagen secretion was more in genipin crosslinked scaffolds as shown in Figure 5(c) and (d). Additionally, safranin-O staining indicate that glucosaminoglycans secretion was more enhanced in genipin crosslinked scaffold than glutaraldehyde crosslinked scaffold 82 shown in Figure 5(e) and (f). In another study performed by Pinheiro and co-workers compared effect of Glutaraldehyde (GA) with Genipin (GP), Epigallocatechin gallate (EGCG) and Proanthocyanidin (PA) with regards to physical and mechanical properties of crosslinked decellularized cartilage. Porcine articular cartilage was decellularized by Sodium Dodecyl Sulphate (SDS) treatment and further subjected to crosslinking treatment. Extent of crosslinking for natural crosslinkers ranged from 50% in case of EGCG to 78% for GP as against the glutaraldehyde control which exhibited 83% extent of crosslinking. Collagenase resistance was found to improve with incorporation of natural crosslinkers. Further the study also confirmed that natural crosslinkers utilized would impart similar mechanical characteristics to the formed scaffold as compared to GA. 11
H&E staining of gelatin scaffold after speeding for 9 days a) Glutaraldehyde crosslinked scaffold b) Genipin crosslinked scaffold. Masson staining of gelatin scaffold after speeding for 9 days c) GA crosslinked scaffold d) GE crosslinked scaffold. Safranin-O staining of gelatin scaffold after speeding for 9 days e) GA crosslinked scaffold f) GE crosslinked scaffold. [Reproduced from Material Science and Engineering: C with permission of Elsevier 82 ].
Conclusions
Bio-based cross-linkers derived from vegetative sources are inherently safe and are therefore regarded as environmentally sustainable and renewable alternative to their chemical counterparts. Many plant-based materials are well categorised as polyphenols, organic acids that distinctly possess anti-microbial activity, which is pivotal to ensure elevated cellular response. Bedsides improving mechanical strength and imparting structural integrity, these natural cross-linkers display several crucial advantages over the synthetic cross-linkers. Many synthetic cross-linkers are toxic and carcinogenic compared to the natural ones; the latter provide ecological and user-friendly alternatives. Because of these advantages, employment of plant-based cross-linkers is increasing in biomedical and tissue engineering applications. However, incomplete investigations have marred the potential applications of numerous plant-based materials. Using plant extracts is another greener and commercially viable approach for cross-linking. However, limited exploration has been conducted in this domain. Although in-depth exploration is warranted, recent years have demonstrated a positive inclination towards the use of natural biomaterials in fabrication of hydrogels and scaffolds for tissue engineering. Prospective applications of plant-based cross-linkers will be a fertile area of research.
Supplemental Material
sj-pdf-1-jba-10.1177_0885328220979273 - Supplemental material for Plant based cross-linkers for tissue engineering applications
Supplemental material, sj-pdf-1-jba-10.1177_0885328220979273 for Plant based cross-linkers for tissue engineering applications by Abhishek Indurkar, Ashish Pandit, Ratnesh Jain and Prajakta Dandekar in Journal of Biomaterials Applications
Supplemental Material
sj-pdf-2-jba-10.1177_0885328220979273 - Supplemental material for Plant based cross-linkers for tissue engineering applications
Supplemental material, sj-pdf-2-jba-10.1177_0885328220979273 for Plant based cross-linkers for tissue engineering applications by Abhishek Indurkar, Ashish Pandit, Ratnesh Jain and Prajakta Dandekar in Journal of Biomaterials Applications
Footnotes
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