Mussel-Inspired Bioadhesives in Healthcare: Design Parameters, Current Trends, and Future Perspectives

Abstract

Mussels are well-known for their extraordinary capacity to adhere onto different surfaces in various hydrophillic conditions. Their unique adhesion ability under water or in wet conditions has generated considerable interest towards developing mussel inspired polymeric systems that can mimic the chemical mechanisms used by mussels for their adhesive properties. Catechols like 3,4 dihydroxy phenylalanine (DOPA) and their biochemical interactions have been largely implicated in mussels’ strong adhesion to various substrates and have been the centerpoint of research and development efforts towards creating superior tissue adhesives for surgical and tissue engineering applications. In this article, we review bioadhesion and adhesives from an enginnering standpoint, specifically the requirements of a good tissue glue, the relevance that DOPA and other catechols have in tissue adhesion, current trends in mussel-inspired bioadhesives, strategies to develop mussel-inspired tissue glues, and perspectives for future development of these materials.

Graphical Abstract

Mussel-inspired bioadhesives gain increasing interests in healthcare applications. In this review, adhesive mechanism, mussel-inspired bioadhesive synthesis and applications in healthcare are summarized and discussed.

1. Introduction

Tissue adhesives may facilitate the surgical reconnection of injured tissues or organs, in order to restore structure, function, and hemostasis. The conventional physical methods to connect tissues during surgeries involve the use of sutures, staples, and wires, which mechanically hold the tissues together to stop bleeding and fluid from leaking, while promoting faster healing and conferring resistance to tensile loads applied on the tissue. In spite of their widespread use, these methods have many limitations such as damaging the surrounding tissues, requiring secondary surgeries for removal, and interfering with tissue healing processes^1-5. In addition, the complete adherence of tissues using the aforementioned methods requires a high level of experience by the operating surgeon and is not easily applicable in every clinical scenario, specially at places and locations that are hard to reach internally. Furthermore, these mechanical methods are ineffective in adhering blood vessels and soft tissues like the liver, spleen and kidney.

Although mechanical methods present various limitations, they are still the most commonly used technique to close the estimated 114 million surgical wounds occurring worldwide yearly. In 2018, the global wound closure market was estimated at about $14 billion⁶. The growing healthcare costs associated with wound closure and the shortcomings of conventional methods make clear the necessity to develop better alternatives to adhere tissues together. In order to reduce the use of conventional methods and costs for patients and healthcare systems, applications of adhesive biomaterials to quickly and efficiently glue tissues together represent an interesting alternative.

These biomaterials are termed tissue adhesives, bioadhesives, or bioglues, and are generally defined as any substance that can polymerize in situ and help adhere two surfaces, one of which at least is a tissue². Tissue adhesives offer a simpler, quicker and suture-less way of reconnecting tissues and restoring their functionality. Their use promises a simplification of the complex wound closure traditional procedure, with the expectation of time saving and reduced costs and risks in surgical procedures.

Depending on the application, a bioadhesive can function as a glue to keep tissues closely attached until healing, as a hemostat to prevent bleeding and blood loss from tissues, or as a sealant to avoid fluid leakage. Bioglues may have the ability to facilitate wound healing while providing a needle free method of wound closure that protects the surgeon from the risk of needle stick injury and provides a better cosmetic outcome compared to sutures. In addition, bioadhesives also have the potential to perform as drug delivery carriers, such as the case of a slow and localized release of antibiotics and growth factors to prevent infections and to encourage cell growth and wound healing^{3, 4}. Around 1947, the application of tissue adhesives as drug delivery agents was demonstrated by delivering penicillin using a mucoadhesive formulated with tragacanth and a dental adhesive powder. This product was known as ‘Orabase’, and it targeted the oral mucosa to treat mouth ulcers ³.

The first use of modern synthetic tissue adhesives can be traced back to the cyanoacrylate glues developed in 1949 to close small skin cuts and lacerations, but its main drawbacks were the chronic inflammatory reactions and poor mechanical properties, which rendered the use of this product ineffective in the treatment of wounds.

Further attempts at improving these shortcomings led to the development of n-2-butylcyanoactylates. These adhesives, despite being purer and stronger, could not be clinically applied due to their low tensile strength and brittleness. Following this, many different types of tissue adhesives have been developed over the past 30 years, using many different types of materials.

To function as a surgical bioadhesive, an ideal material needs to demonstrate several different properties intended to satisfy the multiple demands of a clinical setting. Some of the most important are adhesion, curing in a wet environment, flowability, hydrophilicity, hydrogen bonding capability, appropriate molecular weight, low surface tension, and crosslinking formation capability. These clinical requirements are summarized in Table 1.

Table 1.

Summary of Desirable Properties Needed in an Effective Tissue Adhesive ^4,10.

Desired intrinsic property	Comments
Adhesion	Surgical adhesive must be able to hold two tissues together without external support until the wound heals, after which the adhesive should degrade into nontoxic biocompatible components.
Curing	The adhesive must have the ability to cure itself in the tissue environment, especially in a moist environment.
Flowability	The adhesive must have an ability to flow. Flowability of an adhesive relates to the movement of polymer chains and controls the amount of interpenetration between polymers and tissue surfaces.
Hydrophilicity	Hydrophilic polymers are preferred as they can form strong bonds with highly hydrophilic tissues.
Hydrogen bonding	The presence of strong hydrogen bonding groups like OH and COOH in the monomer can promote hydrogen bonding between polymer chains affecting the flowability and cohesive strength of the adhesive. A minimum cohesive strength is required for a good adhesive.
Molecular Weight	High molecular weight polymers (>100KDa) make for better adhesives as they provide a higher degree of entanglement.
Surface tension (Wettability)	For any adhesive to adhere to a substrate, its surface energy must be equal to or less than the substrate’s. Lower surface tensions promote the polymer to spread across the substrate for better adherence.
Crosslinking	Crosslink formation in an adhesive promotes an increased mechanical strength required by the adhesive to transfer shear stress across the adhered surfaces.

2. Adhesion Theory and Mechanisms

Adhesion is related to the forces that make atoms or molecules stick together. The attachment of dissimilar atoms or molecules to each other is termed adhesion; whereas the bonding of similar atoms or molecules is termed cohesion. The former is caused by molecular interactions at the interface between materials, while the latter is dictated by intermolecular attractions between similar molecules or atoms within the material. Hence, bioadhesion is defined as a state in which a biological material is held together with another material, biological or not, by fact of forces acting at their interface³. This term was first used in the 1970’s to describe the capacity of different molecules of biological or synthetic origin to attach to biological tissues.

There are many examples of bioadhesion in nature such as the vertical motions of a gecko on upside down surfaces, and the extreme capacity of some molluscs to attach on wet or submerged substrates. Generally, this type of adhesion is the result of the natural secretion of bioadhesives made up of polysaccharides, lipids, and proteins, in addition to the manipulation of different superficial physical forces like Van der Walls, capillary, and suction induced by pressure differential⁷.

Bioadhesion is governed by interfacial phenomena, and the requirements and properties of the substrates being adhered differs from conventional adhesion³. To achieve bioadhesion, the substrate must be wet by the adhesive. Consequently, the bioglue must be able to flow, soak the substrate, and keep a certain amount of physiochemical intermolecular forces to allow adherence to the substrate³. Depending on the adhesive and substrate interactions, the nature of these intermolecular forces facilitating adhesion can vary, and this variance gives rise to the different mechanisms of bioadhesion that are reported in literature.

The mechanisms of bioadhesion may be classified into four main categories: intermolecular bonding, chain entanglement, mechanical interlocking, and electrostatic bonding. In most of the cases, the adhesive effect results from the action of more than one of these mechanisms. Figure 1 shows pictorial representations of the main mechanisms relevant to bioadhesion, which are also briefly discussed in the following paragraphs.

Figure 1.

The adhesion mechanisms may be classified into four main categories. Intermolecular bonding is considered the major mechanism in bioadhesion and is due to primary and secondary forces such as covalent bonds and van der Waals forces. Chain entanglement occurs at the interface, where macromolecules from adhesives adhere and diffuse and form an interpenetrated layer of about 1–100 nm. Electrostatic bonding is possibly the result of electron transfer at the interface, which might create a charged area resulting in the induction of electrostatic forces. Mechanical interlocking is due to adhesive infiltrating the adherent surface orifices and irregularities, creating a superficial binding ^4,8,11.

2.1. Mechanisms of Tissue Adhesion

2.1.1. Electrostatic bonding:

The proximity of tissue and adhesive surfaces can lead to the transfer of electrons between them due to differences in electron band structures, leading to the formation of an electric double-layered interface, whose adhesive strength is the result of the interaction among the attractive forces present in the dual layer, which accounts for the resistance to separation³. Although this mechanism is not relevant for non-metallic adhesion, it seems to play a role in bioadhesion, as in the case of electron transfer at the interface of mucoadhesion⁸.

2.1.2. Intermolecular bonding:

It is considered the main mechanism of bioadhesion⁸. According to the adsorption theory, the adhesion between adhesive and adherent is due to the surface forces acting between the chemical structures at the two surfaces when they are pressed together³. These surface forces are classified as primary and secondary. Examples of primary forces are the so called “Chemisorptions”, that encompass cationic, ionic, and metallic chemical bonds, which are high energy bonds that form at the interface and result in strong adhesion. Secondary forces form low energy bonds, but they have a significant effect resulting from the large number of bonding sites that may exist at any given interface. Some of these secondary forces are hydrogen bonding, van der Waals forces, London dispersion, and dipole-dipole interactions. Most of the time, the adhesive effect is due to primary forces, to secondary forces, or to a combination of both.

2.1.3. Chain entanglement

Semi-permanent bonds between the adhesive and tissue surface can form when polymer chains from the former permeate into the tissue surface and attain a depth enough penetration to hold the bonding. The interpenetration of the polymer chains is driven by concentration gradients where the polymer chains penetrate the tissue and glycoprotein chains from the tissue in turn penetrate the adhesive until an equilibrium depth is reached. The diffusive depth of the adhesive depends on the diffusion coefficient between tissue and adhesive, which in turn is related to the molecular weight of the polymeric chains and the crosslinking density of the adhesive^{9, 10}. A good example is the interpenetration and entanglement of glycoproteins and adhesive polymeric chains at the interface of mucous membranes that occurs in mucoadhesive drug delivery systems⁸.

2.1.4. Mechanical interlocking

The interaction between adhesive and tissue can result in the adhesive penetrating into the tissue micro irregularities and interlocking with the tissue to create binding¹¹. The tissue’s surface roughness plays an important role in mechanical interlocking as the adhesive locks onto microscopic roughness. An example of such kind of adhesion is the use of polymer based adhesives, which penetrate the tissue and later expand once inside the microscopic crevices; thus mechanically interlocking with the tissue¹². Another example is the use of polymeric microneedles based on poly(styrene) and poly(acrylic acid), which penetrate the tissue and then later swell in the presence of water inside, leading to local tissue deformation and consequent interlocking within the tissue surface¹³.

2.1.5. Surface wetting

Although not included into the four major mechanisms of adhesion, surface wetting is an important contributor to this phenomenon. The wetting theory is related to the capacity of the adhesive to disperse and create molecular binding with the tissue surface. The molecular contact between the adhesive and tissue results in the development of the previously discussed primary and secondary surface forces, which are the source of intermolecular bonding. Wetting is defined as the process of establishing a very close and continuous contact between the adhesive and the tissue. An adhesive can effectively wet an adherent if its surface tension is lower than the critical surface tension of the adherent. In order to have a high adhesive bond with the tissue, the adhesive must completely wet the tissue by maximizing contact with the tissue topography by flowing into the defects and crevices of the tissue. High quality wetting and adhesiveness are achieved when the angle of contact between the adhesive and adherent is minimal^{10, 14} as shown in Figure 2.

Figure 2.

Surface wetting is considered an important contributor to adhesiveness. If the adhesive spreads and makes extended molecular contact with the tissue surface, the contact angle will be close to zero, resulting in high quality wetting and an adhesive effect. ¹⁴

2.2. Role of DOPA in Mussel Inspired Tissue Adhesion

Nature employs adhesion in a host of ways and a variety of biological species such as mussels, barnacles, and some reptiles like geckos secrete substances with potential adhesiveness. These substances are usually proteins that help them stick to surfaces underwater¹⁵. As far as adhesiveness is concerned, mussels are the most fascinating species, which by the virtue of their adhesive secretions can stick onto rocks and ships underwater and can do so robustly under turbulent intertidal conditions. This adhesive ability of mussels has its biochemical basis in the secretion of a mixture of proteins called mussel adhesive proteins (MAPs).

Several researchers have studied the composition of these MAPs and have characterized the presence of at least six different proteins found in mussel byssus threads, termed mussel foot proteins Mfp-1 to Mfp-6^{16, 17}. These MAPs have an abundance of the particular catecholic amino acid 3,4 dihydroxy phenylalanine (DOPA), which has been implicated as a key factor behind mussel’s strong underwater adhesion^{18, 19}.It has been postulated that each particular type of Mfp has its own purpose and different contribution to the overall phenomenon of mussel adhesion. Proteins Mfp-3, Mfp-5, and Mfp-6 are mostly found at the adhesion interface and are known to contribute to the extreme wet adhesion²⁰. The exact mechanisms of mussels’ wet adhesion are not fully understood; however, it has been found that DOPA plays a vital role in the interfacial attachment and curing of the adhesive plaque proteins. DOPA molecules are capable of versatile chemistries enabling them to stick to many different surfaces, either organic or inorganic, by establishing reversible non covalent and irreversible covalent bonds as illustrated in Figure 3²⁰.

Figure 3.

The role of DOPA in mussel-inspired tissue adhesion. A) The oxidation state of DOPA controls adhesiveness. At a low pH, DOPA exists in its highly adhesive form conferred by the presence of two hydroxyl groups. At higher pH, approaching or exceeding pKa 9.3, these groups can autoxidize to quinone and even can tautomerize to α, β-dehydro-DOPA, both of which have reduced adhesive strengths. Reproduced with permission from reference 28, copyright 2018, John Wiley and Sons. B) Dopa and tissue interactions. DOPA molecules are capable of reversible non-covalent and irreversible covalent interactions, such as strong H-bonds via their dihydroxy functionality. Their benzene rings can interact with other aromatic rings via π-π interactions, or with positively charged ions through cation-π interactions. All these interactions can cause coacervation and subsequent increase in adhesion. Reproduced with permission from reference 20, copyright 2016, John Wiley and Sons.

DOPA can form strong hydrogen bonds via its dihydroxy functionality, promoting its absorption on tissue surfaces, especially mucosal tissues (Figure 3A). Catechol containing polymers have improved cohesive properties due to the benzene ring of catechol, which can interact with other aromatic rings via π-π interactions. In addition, the benzene ring can interact with positively charged ions through cation-π interactions, known to be among the more stable non-covalent interactions in water, which enables them to bind to positively charged surfaces and to substrates rich in cationic functional groups^{21, 22}. Additionally, catechol can form robust reversible bonds with metal ions such as Cu²⁺ and Zn²⁺, as well as interfacial bonds with metal oxide surfaces (Figure 3B). Furthermore, the highly reactive oxidized form of catechol can be involved in intermolecular covalent cross-linking that causes curing of DOPA containing adhesives. Catechol is usually oxidized into semi-quinone or quinone by one or two electron oxidation via enzymatic, chemical (periodate) or the presence of molecular oxygen. Quinone is capable of reacting with several nucleophilic functional groups found in tissues (─NH₂, ─SH, imidazole) to form interfacial covalent bonds, which can promote tissue adhesion (Figure 3A)²⁰. Although DOPA is the main adhesion contributor of Mfps, there are other mechanisms of interplay between DOPA and the Mfps, which contribute to the adhesion and these are reviewed in the next section.

2.2.1. Oxidation state of DOPA:

The oxidation state of catechols strictly controls their adhesive properties (Figure 3A)^23-25. Catechols exist in their highly adhesive reduced forms at an acidic pH. As pH changes and comes closer to pKa 9.3, the first dissociation constant of their -OH group, the catechol side chain autoxidizes to quinone, which exhibits a limited adhesive strength^{23, 26, 27}. Under alkaline sea water conditions, DOPA can be easily oxidized to DOPA-quinone, which can tautomerize to α, β-dehydro-DOPA. DOPA-quinone unlike DOPA and its tautomer, α, β-dehydro-DOPA, does not have the ability to form hydrogen bonds with surfaces, and thus, oxidation of DOPA to its quinone form must be limited for higher adhesion²⁸. Mussels are able to counteract DOPA’s oxidation through Mfp-3 and Mfp-6 proteins, which are present at the plaque surface interface and provide an antioxidant effect. Additionally, Mfp-3 also creates a hydrophobic environment, shielding the DOPA from oxidation and subsequently enhancing adhesion. Also, most mussel foot proteins with the exception of Mfp-1, have the non-polar amino acid glycine adjacent to DOPA²⁸. Many reports confirm that non-polar groups in close proximity to DOPA can inhibit its oxidation through hydrophobic shielding and electrostatic shielding effects^28-30.

2.2.2. Coacervate formation:

Coacervation is the phase separation of fluids driven by columbic forces and charge neutralization between chains of opposite charges. An example is the liquid phase separation of ionic polymers from aqueous solutions. This process influences underwater adhesion by increasing the polymer concentration in the coacervates, improving surface wettability via decreasing interfacial energy and reducing viscosity of the phases to enhance adhesive delivery. In mussels, coacervation is not charge based as the cationic mussel foot proteins have not been reported to complex with any oppositely charged molecules, instead hydrophobic, π-π and cation-π interactions cause coacervation and subsequent increase in adhesion (Figure 3B)^{20, 28, 31, 32}.

2.2.3. Surface drying

In underwater adhesion, interfacial molecular contact between the substrate and the adhesive is necessary. This contact can be hindered due to the presence of a layer of adsorbed water, which gives rise to strong repulsive hydration forces that compromise adhesion. Several researchers have demonstrated that Mfp-3s have hydrophobic tryptophan side chains, which help in removal of the surface hydration layer. In addition, cationic residues in close proximity to DOPA such as lysine have been shown to seize the hydration layer, promoting DOPA’s adhesion to the underlying surface^{28, 33}. As a result, coacervates containing DOPA exhibit the capacity to remove interfacial water molecules and make molecular hydrogen bonds with the substrates, which increase the adhesion strength^{34, 35}.

2.2.4. Catechol modification:

Catechol modifications can significantly change their adhesive properties, e.g. their reactivity can be enhanced by the addition of a side electron withdrawing nitro-group at the para position, which also increases binding strength³⁶. The nitro functionalization also leads to better resistance to higher temperatures and oxidation as compared to an unmodified catechol^37-39. Also, nitro-catechol exhibits higher rates of covalent crosslinking and binds to biological substrates over an ample pH range³⁶. In addition, nitro- and chloro-functionalized catechols can present particular characteristics that allow them to resist light-induced degradation and improve their antimicrobial properties⁴⁰.

3. Current Non-Mussel Inspired Surgical Adhesives

3.1. Natural or Biological Adhesives

Some naturally occurring materials have intrinsic properties that make them interact and stick to tissues. Proteins, for instance, are rich in amine groups that allow them to connect with tissue functional groups and adhere to them. Similarly, polysaccharides are also mediators of adhesion because of the presence of copious amine-, carboxyl-, and hydroxyl- groups.

One of the first clinical proteinic adhesives still in use is fibrin glue, which is based on the bloodborne protein fibrin and works by mimicking the blood clot formation mechanism of the coagulation cascade⁶. Formulations of fibrin glue are clinically applied as wound closure agents, adhesives for wound healing, hemostasis promoters in cardiopulmonary bypass and liver resection, sealers of colonic anastomoses, and scaffolds for drug delivery and tissue regeneration. However, among its limitations are the possibility of viral infection and the eliciting of immune responses ⁴¹.

Over the past decade, collagen-based adhesives have become popular in clinical use. Collagen absorbs blood and coagulation products, binds to the wound, delivers fibrinogen, and causes the aggregation of platelets and activation of coagulation factors^{2, 4}. Collagen glues are used for vascular surgery hemostasis, to prevent cerebrospinal fluid leaks, and to control surgical bleeding^{2, 4, 32, 33}. Nevertheless, collagen-based hemostats can swell and cause tissue compression⁴². A hydrolyzed collagen derivative is the base of gelatine-based adhesives, one of the first biomaterials to be considered for internal surgery². The amino groups in gelatin are modified cholesteryl residues to improve their adhesive properties and tissue penetration, and the glue is often crosslinked to improve its adhesiveness and to control its degradability^{1, 2}. As an example, research efforts in gelatin-based bioadhesives have resulted in the development of visible light cross-linkable glues for the repair of corneal injuries, which also demonstrate regenerative capabilities⁴³.

In addition to gelatin and collagen, other proteins such as albumin and tropoelastin have also been used as adhesive bases. Albumin was combined with glutaraldehyde to prepare adhesive gels to seal lung tissue and repair large blood vessels⁴⁴ (Table 2). A similar use is that of MeTro sealant, prepared with recombinant human tropoelastin modified as methacryloyl-substituted tropoelastin, which exhibits highly elastic properties fitting the demanding application⁴⁵.

Table 2.

Summary of FDA Approved Tissue Adhesives and Sealants in the US.

Tissue Adhesive Type	Product name	Manufacturer	Application	Advantages	Limitations
Cyanoacrylates	Histoacryl/Histoacryl Blue (n-Butyl-2-cyanoacrylate)47	B. Braun	Topical skin incisions and skin lacerations closure.	Fast curing. Strong dry adhesion. Barrier to microbial penetrations. Haemostatic and bacteriostatic properties.	Rapid degradation in moist conditions. Formaldehyde mediated cytotoxicity.
	Indermil (n-Butyl-2-cyanoacrylate)51	Henkel
	Dermabond (2-Octyl-2-cyanoacrylate)51, 52	Ethicon
	Omnex (n-Octyl-2-cyanoacrylate/butyl lactoyl-2-cyano acrylate)47	Ethicon	Blocking blood loss, bodily fluids, air leaks.
Fibrin glue	Tisseel (Human pooled plasma fibrinogen and thrombin)53	Baxter Inc.	Adjunct to hemostasis, used when control of bleeding by conventional techniques is ineffective or impractical. Adjunct for colostomy closure.	Fast curing. Good biocompatibility and biodegradability.	Risk of infection and allergic reaction. Ancillary equipment required. Poor tissue adhesion. Expensive.
	Evicel (Human pooled plasma fibrinogen and thrombin)53	Ethicon
	Vitagel (Autologous plasma fibrinogen and thrombin)44	Orthovita Inc.
	Cryoseal system (Autologous plasma fibrinogen and thrombin)54	ThermoGenesis Corp.
Poly (ethylene glycol) (PEG) based sealants	Coseal (2 four-armed PEGsped with glutaryl-succinimidyl ester and thiol)48	Baxter Inc.	Sealing suture lines and vascular graft.	Rapid gel formation. Fast hemostasis. Biocompatibility. Adhesion to tissue.	Risk of swelling. Possible allergic reaction. Expensive.
	Duraseal (PEG ester powder and trilysine amine solution with FD&C blue No.1 dye)49	Covidien Inc.	Sealing of cerebrospinal fluid (CSF).
	SprayGel44	Covidien	Adhesion barrier in gynecological and colorectal procedures.
	TissuGlu®50	Cohera Medical, Inc	Prevention of seroma formation under skin flaps.
	OcuSeal44	Hyperbranch Medical Technology	Dressing for corneal lacerations and bandage for corneal transplants.
	Adherus44		Sealant for hernia, spinal and cardiovascular applications.
Albumin and Glutaraldehyde	BioGlue (Bovine serum albumin and 10% glutaraldehyde)44	Cryolife Inc.	As adjunct to standard methods of achieving hemostasis in open surgical repair of large vessels.	Fast polymerization (20-30 sec). Full strength in 2 min. Good tissue adhesion.	Risk of glutaraldehyde toxicity.
	ProGel®44	NeoMend	Sealing air leaks on lung tissue after surgery.

Besides proteins, polysaccharides are also natural mediators of adhesion because of the presence of abundant amine-, carboxyl-, and hydroxyl- groups that bond to tissue amine groups via covalent interactions such as N-hydroxysuccinimide activation and Schiff base formation, among others¹. Dextran and chitosan are among the most widely studied polysaccharides for adhesive applications. Dextran contains an abundance of hydroxyl groups, which are used to generate aldehyde groups to react with the amine groups on tissue surfaces. It is often combined with amine containing polyethylene glycols (PEG) or gelatin to form intermolecular crosslinks. Various studies have shown dextran-PEG adhesives to be biocompatible and to have an adhesive strength greater than fibrin glues and similar to cyanoacrylate glues. On the other hand, chitosan is a cationic material that exhibits unique antioxidant and bacteriostatic properties. It can also react with anionic biopolymers like nucleic acids, glucose amino glycans, heparin and proteoglycans, which can be utilized to make soft tissue adhesives. However, chitosan adhesives show poor cohesion which results in inadequate adhesion, but this can be counteracted by the chemical modification of its amino and hydroxyl functional groups to improve crosslinking capabilities. Modified chitosan adhesives have been applied for nerve anastomosis and to seal defects in corneal tissue^{1, 35}.

The combined use of polysaccharides and proteins has also been explored, as previously mentioned with the dextran-gelatin combination. One example is that of the 3-dimensional adhesive and antimicrobial hydrogels prepared with methacrylated hyaluronic acid, elastin-like polypeptides, and zinc oxide nanoparticles. These hydrogels are intended for in situ application and cross-linking, in order to promote repair and regeneration of tissues due to their capabilities to support cell growth and proliferation⁴⁶.

3.2. Synthetic/Semi-synthetic adhesives

Synthetic tissue adhesives are composed of synthetic chemicals that, when applied to a tissue, undergo in situ polymerization or crosslinking to form insoluble adhesive matrices. These synthetic polymeric materials can make it possible to control the three-dimensional structure and chemical composition of the adhesive in order to expose functional groups apt for biological interaction and improved bioadhesion. Synthetic polymers also provide a better control over the final molecular weight, which can be used to tailor the degradation and elimination rates of the adhesive from the body^{1, 5}.

Cyanoacrylate-based adhesives are made from alkyl α-cyanoacrylates¹. They polymerize very quickly and provide a very high bonding strength^{1, 5}. Cyanoacrylate adheres to the tissue via two different mechanisms: (i) molecular interactions that result in covalent bonds with tissue surface proteins and (ii) mechanical interlocking of monomers onto the tissue surface. The adhesive properties of cyanoacrylates are reported to be superior, but their fast polymerization is related to tissue-damaging heat production. Furthermore, the degradation of cyanoacrylate-based adhesives can lead to the formation of formaldehyde, which can cause toxicity^{5, 47}. Additionally, cyanoacrylates may elicit severe immune responses. Despite the attractive properties and substantial strong adhesion of cyanoacrylate-based adhesives, their use is restricted to topical applications, because of the toxic characteristics of the degradation sub-products.

PEG based bioadhesives have also been manufactured as sealers for suture lines, vascular grafts, hernias, and cerebrospinal fluid. Additionally, they are employed as adhesive barriers in gynecological, colorectal, and corneal procedures ^{44, 48-50} (Table 2). They exhibit some advantages such as rapid gel formation, quick hemostasis, biocompatibility, and tissue adhesion. However, their main disadvantages besides elevated costs are risk of swelling and allergenic characteristics. PEG combined with the polysaccharide dextran has shown biocompatibility and adhesive strength similar to cyanoacrylate glues^{1, 35}, as previously mentioned. Some of the FDA approved protein-based and synthetic tissue adhesives are listed in Table 2, with additional information on their manufacturer, applications, advantages, and limitations.

4. Current Mussel-Inspired Tissue Adhesives: Synthesis and Applications

Mussels’ unique ability to accomplish strong underwater adhesion due to the interplay between mussel foot proteins and DOPA have led to considerable efforts to mimic this dynamic by conjugating catechol groups onto polymers. The general approaches and synthesis strategies employed are summarized. Some applications of mussel-inspired adhesives in relation to healthcare and biosciences as well as relevant examples are discussed.

4.1. Strategies to synthesize catechol functionalized polymers

Different approaches may be used to modify synthetic and natural polymers to contain catechol groups. In all cases, the adequate protection/deprotection of catechols along the synthetic route is vital to preserve their adhesive properties in the end product²⁰. Among the various strategies used to create Dopa-functionalized polymers, the most relevant to health care and biosciences are direct functionalization, polymerization of catechol-modified monomers, synthesis of Dopa-containing peptides, and genetic engineering. These strategies are briefly discussed in the following subsections and illustrated in Figure 4. Additional examples with performance parameters are shown in Table 3

Figure 4.

Strategies used to synthesize mussel-inspired adhesive polymers relevant to healthcare¹⁹. Catechols can be directly conjugated onto polymeric backbones for direct functionalization. Different monomers can be modified to include catechol groups and then polymerized via heat- or photo-initiation. Peptides containing catechol residues with their amine and catechol side chains protected can be prepared via solid-phase synthesis. Recombinant DNA technology may be used to replicate peptide sequences using bacteria such as Escherichia coli.

Table 3.

Polymeric Mussel-Inspired Bioadhesives along with their Crosslinking Conditions and Maximum Reported Adhesion Strengths.

Adhesive	Adhesion Test	Test Substrate	Crosslinker	Cure time/ Condition	Reported Adhesion Strength	Ref.
Direct functionalization of polymers with catechols (4.1.1)
iCMBA	lap shear	porcine, acellular SIS	sodium (meta) periodate (PI)	humid chamber, 2 hrs	123.2 ± 13.2 kPa	56
PEG-DOPA	lap shear	porcine skin	periodate	37 °C, 24 hrs	35.1 ± 12.5 kPa	57
catechol-Ala-Ala-PEG (cAAPEG)	lap shear	decellularized porcine dermis	periodate	RT, 2 h	30.4 ± 3.39 kPa	84
PEG-DOPAmine-PCL	lap shear	bovine pericardium	NaIO4	(1) RT, 2 hrs, (2) PBS, 37 °C, 1 hr.	107 ± 24.7 kPa	62
PEU (poly (CA-Tyr-co-Leu))	lap shear	porcine skin	Bu4N(IO4)	RT, 24 hrs	9 kPa	85
deacetylated chitosan; oxidized and DOPA-functionalized dextran	end-to-end	bovine cortical bone	ferric citrate	wet, 37 °C, 3 hrs	0.31 MPa	72
poly (AA-co-AANHS-co-MDOPA)	lap shear	porcine skin	thiol-PEG	RT, 10 min	~11.8 kPa	86
AbAf iCs	lap shear	porcine SIS	sodium (meta) periodate	humid chamber, 2 hrs	168.15 ± 17.02 kPa	59
poly (DOPAmine-co-acrylate) (PDA)	lap shear	porcine skin	horseradish peroxide (HRP)	RT, 1 day	76 ± 13.4 kPa	87
PEU (poly (CA-Ser- co-Leu-co-PPG))	lap shear	porcine skin	Bu4N(IO4)	wet, RT, 4 hrs	10.6 ± 2.1 kPa	88
DCTA (gelatin macromer, Fe3+, genipin)	lap shear	porcine skin	FeCl3 + genipin	37 °C, 2 hrs	24.7 ± 3.3 kPa	73
Polymerization of catechol-modified monomers (4.1.2)
DMA-APMA Mimetic copolymer	bond strength tests	bovine femur cortical bone	NaIO4	37 °C, 24 hrs	about 100 kPa	75
poly[(3,4-dihydroxystyrene)-co-styrene	lap shear	metal and wood adherends	FeCl3 NaIO4	RT, 1 hr. 55°C, 22 hrs	> 1 MPa	79
Synthesis of Catechol-containing polypeptides (4.1.3)
polytripeptide (Gly-Tyr-Lys)	shear test	porcine skin	tyrosinase	37 °C, 30 min	11.56 kPa	80
poly ((Lys.HBr) x-(DOPA)y)	lap shear	porcine skin	ferric citrate	dry, 25 °C, 12 hrs	0.21 ± 0.10 MPa	81
Genetic Engineering (4.1.4)
rfp-1 (MAP)	lap shear	porcine skin	FeCl3	2 hrs, in buffer solution (pH 8.2)	~130 kPa	82
LAMBA	lap shear	porcine skin	irradiation with dental curing lamp for 60 secs	RT, 2 hrs, PBS	72.2 ± 3.7 kPa	83

4.1.1. Direct functionalization of polymers with DOPA

The use of linear or branched PEG polymers grafted with DOPA has gained ground over the past few years. These polymers provide an inert and biocompatible macromolecular support to impart cohesive strength while DOPA molecules confer adhesive properties to the crosslinked hydrogels¹⁸. DOPA and dopamine can be directly conjugated onto polymeric backbones via the formation of amide, urethane, and ester linkages with the functional groups -NH₂, -COOH, and -OH^{55, 56}. In addition, mussel inspired PEG based polymers with tunable adhesive and mechanical (degradation, swelling rate, curing rate) properties can be synthesized by varying the PEG molecular weight, PEG architecture, number of DOPA residues, and the type of linkage¹⁸. For instance, researchers investigated the stimuli responsive gel formation of PEG-Dopa polymers. They employed a mixture of dipalmitoyl and dimyristoyl phosphatidylcholine to create lipid vesicles able to release their contents at 37 °C. These liposomes were then combined with a Dopa-functionalized PEG to form fast crosslinking hydrogels. Lap shear strength tests measured an adhesive strength of 35.1 ± 12.5 kPa, several times higher than that of fibrin glue⁵⁷. More recently, PEG-catechol hydrogels with reported adhesive strengths on blood-covered skin above 40 kPa were prepared by reinforcing the material with the addition of collagen fibers and hydroxyapatite nanoparticles⁵⁸. In another example, researchers aimed to develop injectable PEG and citrate-based mussel-inspired bio adhesives (iCMBAs) for internal organs. These materials exhibited a considerably stronger wet adhesion as compared to fibrin glue and demonstrated adequate elastomeric compliance, degradability, and cytocompatibility. In vivo studies showed iCMBAs could stop bleeding and close model surgical wounds without the need of suturing⁵⁶. Furthermore, iCMBAs were improved by the addition of click chemistry. A copper-catalyzed azide-alkyne cycloaddition reaction was used in combination with the chelating agent 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, to overcome the inhibitory effect between the catechol crosslinker and the copper reductant catalyst. The products exhibited enhanced cohesive and wet adhesive strength, about 13 times higher than that of fibrin glue. Additionally, they also showed antibacterial, antifungal, and haemostatic capabilities⁵⁹.

Different architectures of PEG have also been combined with polymers such as polycaprolactone (PCL) and polypropylene oxide (PPO) to form block copolymers, which then may be combined with catechol and conjugated onto polymer chains^{55, 60, 61}. For example, dopamine was combined with 4-arm PEG and PCL to prepare an adhesive polymer to coat a hernia repair biologic mesh. Further mechanical tests on bovine pericardium demonstrated a lap shear strength of 107 ± 24.7 kPa⁶². DOPA has also been conjugated with PPO and poly (ethylene oxide) PEO. For instance, catechol groups were conjugated to PEO-PPO-PEO block copolymers with a coupling efficiency about 80%. The product exhibited temperature-induced gelation, which was able to be tailored to specific conditions. Additionally, the increased mucoadhesive characteristics of this product were demonstrated by viscosity measurements⁵⁵. Additionally, PCL with glycidyl methacrylate (GMA) and oligo (ethylene glycol) methacrylate (OEGMA) on the backbone has been utilized to combine with catechol grafts. As an example, a DOPA-based mimetic adhesive with good adherence to tissues and metals was formulated by immobilizing catechol groups on the epoxy rings of GMA. The results showed a stable bio glue at physiological conditions for at least one week. The material was also proven to be enzymatically degradable and exhibited low toxicity. However, its reported adhesion strength is 9 kPa, below that of PEG-DOPA materials⁶³.

Apart from synthetic polymers, biopolymers such as dextran, chitosan, hyaluronic acid, gelatin, and alginate have been covalently attached with acid- or amine-functionalized catechols^64-69 and crosslinked to form hydrogels with strong tissue adhesive properties^{13, 66, 70, 71}. In one case, hyaluronic acid (HA) was modified by dopamine conjugation via carbodiimide chemistry. Using a layer-by-layer (LbL) technique, this material was combined with chitosan to form multilayer films with enhanced nanotopography⁶⁶. The films exhibited improved cell adhesion, proliferation, and viability. Similarly, and focusing on developing a bone bio-adhesive, researchers prepared a two-component glue using chitosan and dextran-DOPA. They combined both materials and allowed crosslinking by Schiff’s base formation. The characterization studies demonstrated a biocompatible product with adherence of 0.31 MPa, superior to fibrin glue, but less than that of cyanoacrylates⁷². Furthermore, a double-crosslink tissue adhesive (DCTA) was developed using a dopamine-conjugated gelatin macromer. The first gelation was attained using Fe⁺³ as the fast-acting crosslinker and genipin as the long-term one. The formation of a catechol─Fe⁺³ complex and later genipin-induced covalent crosslinking allowed a two-prong approach permitting an enhanced instant adhesive effect and posterior extended effectivity. The DCTA exhibited an adhesive strength of 24.7 ± 3.3 kPa, higher than that of fibrin-based commercially available bio-adhesives, as well as elasticity, appropriate degradability, and biocompatibility⁷³.

4.1.2. Polymerization of catechol-modified monomers

Dopamine methacrylamide (DMA) is a catechol containing monomer, which can undergo free radical polymerization when activated by heat or light to form acrylate-based polymers²⁰. DMA has been combined with other monomers such as oligomeric ethylene glycol, monoacryloxyethyl phosphate, and methoxyethyl acrylate to form Dopamine grafted polymers with varying catechol contents^74-76. In one study, phosphate was combined with DMA to mimic the natural glue proteins secreted by marine species. These materials formed coacervates at neutral pH and were used to adhere samples of wet cortical bone. They demonstrated the formation of bonding with strength about 40% of that of cyanoacrylates⁷⁵. In a different study, polyacrylamide (PAAm) was used to form nanocomposite hydrogels by crosslinking with DMA. These hydrogels showed a drastic adhesiveness increase that correlated with DMA contents. Furthermore, not only did they exhibited better fracture resistance, but also could resist repeated compression loads⁷⁷. An additional example is the three-dimensional polymer network formed by the copolymerization of DMA with PAAm using a bi-functional crosslinker like N, N′-methylenebisacrylamide. In this case, photopolymerizable DMA monomer was added to polyacrylamide (PAAm) hydrogels crosslinked with different amounts of Laponite (nano-silicate). These materials showed viscous dissipation properties as well as increased stiffness, rendering them able to release fracture energy while reducing any kind of lasting deterioration of the three-dimensional architecture⁷⁷.

Similarly, catechol containing vinyl groups like 3,4-dihyroxystyrene, 4-vinylcatechol acetonide, and 3-vinylcatechol acetonide can also be polymerized with monomeric styrene to produce polystyrene based catecholic polymers^{78, 79}. A research group developed a biomimetic polymeric adhesive based on styrene–catechol copolymers. Catechol groups were crosslinked to a polystyrene backbone to mimic L-3,4-dihydroxyphenylalanine (DOPA). The resulting poly[(3,4-dimethoxystyrene)-co-styrene] polymers demonstrated curing properties similar to those of natural adhesive proteins. They also exhibited increased adhesion after cross-linking⁷⁸. Likewise, another group prepared poly[(3,4-dihydroxystyrene)-co-styrene by distributing pendant catechol groups at different concentrations onto polystyrene backbones. Mechanical tests demonstrated a bioglue with balanced cohesive and adhesive characteristics. The materials’ adhesive performance was similar to that of cyanoacrylate-based commercial glues when tested on low and high energy surfaces⁷⁹.

4.1.3. Synthesis of Catechol-containing peptides

Another approach to mimic MAPs is the synthesis of Dopa-containing peptides. For instance, several mussel-inspired polypeptides were synthesized from (X-Tyr-Lys)n, (Y-Lys)n, (X-Tyr-Lys)n, (XGly, Ala, Pro, Ser, Leu, Ile, Phe), (Y-Lys)n, (YGly, Tyr), and (Gly-Tyr)n. These materials were crosslinked with tyrosinase and subject to shear adhesive strength tests. It was demonstrated that their adhesive strengths were similar to that of the synthetic polydecapeptides (Ala-Lys-Pro-Ser-Tyr-Pro-Pro-Thr-Tyr-Lys)n and (Gly-Pro-Lys-Thr-Tyr-Pro-Pro-Thr-Tyr-Lys)n, the former inspired by Blue mussel and the late by Californian mussel⁸⁰. In another case, monomers of DOPA and L-lysine were prepared using ring-opening polymerization. Then poly ((Lys.HBr) x-(DOPA)y) was prepared as a synthetic mimic of natural MAPs and cured with ferric citrate. The adhesive properties of the polypeptide were tested and exhibited strengths of 0.21 ± 0.10 MPa. Additional studies also demonstrated adequate cell compatibility⁸¹.

4.1.4. Recombinant genetic engineering

Genetically engineered organisms such as E. coli are used to replicate dopa-containing peptide sequences similar to those found in MAPs. The microorganisms secrete these sequences employing their naturally occurring tyrosine residues, which by the action of tyrosinase are then transformed into Dopa. For example, the gelation of a hydrogel system based on DOPA-containing recombinant MAPs was obtained by Fe⁺³-mediated noncovalent cross-linking. Rheological tests showed a hydrogel with deformable and self-healing properties, with adequate adhesive strength⁸². Furthermore, recombinant technology is unique in that it allows the combination of different polypeptide sequences to generate products with hybrid characteristics. Using this strategy, a bioadhesive was developed based on a recombinant hybrid MAP mimicking Mfp-1 and Mfp-5. This protein was cured with a light-activated dityrosine crosslinking process. The resulting adhesive exhibited wet tissue adhesion values reported as 72.2 ± 3.7 kPa, with good biocompatibility⁸³.

4.2. Applications of Mussel-Inspired Bioadhesives in Relation to Healthcare

Bioadhesives have found fascinating applications in many different fields. Given the broad scope of those applications, we will focus only in healthcare and biosciences (Figure 5). In the following subsections, we will discuss examples of surgical, tissue engineering, drug delivery, cellular engineering, and other related biomedical research applications. It is interesting to note that as of today, no mussel-inspired bioadhesive has been approved by FDA or is reported in clinical trials. According to a PubMed search, just a few years ago the number of articles related to mussel-inspired adhesives began an increasing trend, going from 20 papers published in 2010 up to 85 in 2019. Given the incipient nature of the research on this field, most probably it will take a few more years before these research efforts trickle down to real-life applications.

Figure 5.

Applications of mussel-inspired bioadhesives in relation to Healthcare. Tissue engineering applications seem to be mostly directed towards regeneration, in which case the bioadhesive functions also as a carrier of biomolecules and/or therapeutic agents. Surgical glues are mainly employed to ensure a faster wound closing with minimal scare formation, to stop and seal bleeding, and to adhere implants to bone as in the case of hip implants. Drug delivery applications include the dual function of bioadhesive and drug carrier, the most common example being mucoadhesive drug delivery. Cellular engineering attempts to use bioadhesives for different purposes, from controlling the cell division process to improving cell-surface interactions. Other uses of bioadhesives include antimicrobial materials, biosensing, and ionoprinting.

4.2.1. Surgical glues

In order to stop bleeding and to ensure faster suture-less wound closing during surgical procedures, mussel-inspired bioadhesives are the subject of continuous research. For instance, injectable citrate-based adhesives were prepared and exhibited the promotion of wound healing and total biodegradation without significant inflammation⁵⁶. Also, poly (γ-glutamic acid) was combined with dopamine to form hydrogels that exhibited 10-fold wet tissue adhesion strength as compared to commercially available fibrin glue⁸⁷. In another experiment, recombinant MAPs were photo-crosslinked using a dityrosine-based strategy inspired in the natural phenomena that occurs in insect structural proteins. The product demonstrated light-controllable gelation, good adhesive properties, tissue regeneration promotion, and minimal inflammation⁸³.

Additionally, these bioadhesives have been employed to coat biologic scaffolds used for Achilles tendon or hernia repair. In the first case, a MAP-based tissue adhesive was used as a coating to secure surgical graft material, resulting in a construct that surpassed the adhesive strengths of constructs coated with commercially available bioglues⁶⁰. In the second, MAPs were combined with PEG and PCL to form a film used to coat a hernia repair biologic mesh, which was further tested for adhesiveness, mechanical strength, and biocompatibility. When compared to fibrin glue, this adhesive-coated construct demonstrated enhanced adhesive strength⁶².

Orthopedic medicine might also be benefited by mussel-inspired bioadhesive research. Inspired by the shell dwelling marine worm P. californica glue proteins, researchers developed a phosphate-based adhesive intended for fractured bone repair, with promising results that warrant extended research on the topic⁷⁵. In another case, a two-component bone glue was prepared by conjugating L-Dopa onto oxidized dextran and then mixing with chitosan in aqueous media. The system crosslinked via Schiff’s base formation, which also helped the glue to bind to exposed amino groups in fractured bone. The product displayed a bonding strength three times higher than that of fibrin glue and exhibited high biocompatibility⁷².

Surgical applications of mussel-inspired bioadhesives seem to be increasing and consolidating. Even though a universal tissue adhesive is far from existence, bioglues for specific uses are in development. However, the most promissory of these research efforts still need to be translated to clinical practice, to benefit patients and healthcare systems.

4.2.2. Tissue engineering

In this field, the applications are mostly directed towards tissue regeneration, in which case the bioadhesive may also work as a carrier of biomolecules to induce cell growth. For example, an adhesive mussel-inspired hydrogel was prepared by dopamine polymerization followed by complexation with sodium alginate (SA) in polyacrylamide (PAM) networks. It exhibited good adhesion to porcine skin, with an adhesive strength of 24.5 kPa. Additionally, the product demonstrated appropriate cell proliferation, attachment, and spreading, as well as functional expression of skin fibroblasts and keratinocytes, making it a potential material for skin tissue engineering applications ⁸⁹.

Similarly, to prevent scar formation, a bioadhesive based on recombinant collagen-binding MAPs and glycosaminoglycans was developed and tested. It was targeted to bind type I collagen and to regulate fibrillogenesis. Excisional skin wound animal models showed that the material was able to promote reepithelialization and neovascularization, as well as accelerated collagen synthesis. The new collagen demonstrated uniform fiber size and regular packing, improved wound healing rate, and effective scar inhibition, thus making this bioglue a promissory alternative for wound healing applications ⁹⁰.

In another case, to attain tissue repair and reconstruction, a mussel-inspired enzymatically biodegradable hydrogel composed of a DOPA-mimetic modified PEG linked with an alanine (Ala) dipeptide substrate was prepared. The material was designed with elastase-sensitive Ala-Ala links to degrade as a response to the enzyme secretion from neutrophils during the inflammatory phase, and rheological analysis demonstrated that the hydrogel was adequate for soft tissue contact. The adhesive capacity was measured by lap shear strength, obtaining a value of 30.4 ± 3.39 kPa. In addition, animal models demonstrated that the product biodegradation proceeded over a period of several months ⁸⁴.

Lastly, aiming for growth-factor-free cartilage regeneration, researchers developed a mussel-inspired pDA–chondroitin sulphate–polyacrylamide hydrogel with enhanced adhesive and mechanical properties91. The catechol-enriched hydrogel demonstrated good cell affinity and promoted higher cell adhesion and tissue integration as compared to chondroitin sulphate hydrogel. Additionally, the product exhibited improved mechanical properties to meet the demanding requirements of cartilage repair ⁹¹.

The prospects for an increased use of mussel-inspired bioadhesives in relation to tissue engineering are ample. Not only can they function as promoters of tissue adhesion but also as carriers of biomolecules to enhance cell responses. Hopefully, future research in tissue repair and regeneration will be enriched by the application of these versatile biomaterials.

4.2.3. Drug delivery

Bioadhesive-based drug delivery systems employ adhesive hydrogels in combination with therapeutic agents to reach specific body regions and deliver drugs for an extended period. These systems have been improved using mussel-inspired bioadhesive polymers ³. For instance, a muco-resistant platform based on mussel-inspired adhesive was developed as an oral mucosa drug delivery system. Hydro caffeic acid, a carboxyl-terminated catechol, was conjugated onto the backbone of chitosan. The product, dubbed Chi-C, showed a high potential to serve as a drug delivery carrier and to allow nanoparticle functionalization. Ex vivo lap-shear adhesion tests on porcine tongue and labial mucosa demonstrated a detachment stress of 10.3 ± 6.1 kPa. Additionally, the regeneration of oral mucosa was tested in animal models using triamcinolone acetonide as a drug model carried in a Chi-C oral patch. The results were positive, exhibiting a reconstructed mucosa within 7 days and suggesting the potential use of this material as a mucoadhesive drug delivery system⁹².

Another material for mucous delivery was developed to treat the chronic inflammation of the large intestine. One of the therapeutic approaches to ulcerative colitis includes the rectal administration of sulfasalazine (SSZ), but its therapeutic efficiency is reduced due to its limited residence time. Researchers hypothesized that the use of a mussel-inspired bioadhesive hydrogel composed of chitosan functionalized with catechols and loaded with SSZ (SSZ-Cat-Chitosan) might be helpful to surpass this limitation. The material was evaluated in animal models of ulcerative colitis and compared against oral treatment with SSZ, showing an increased delivery efficacy and therapeutic efficiency. These results also point to the potential use of enhanced mucoadhesive rectal drug delivery to improve the outcomes of ulcerative colitis, colon cancer, or related diseases⁹³.

In a similar fashion, catechol-containing tannic acid (TAc) and gelatin were combined by a Michael addition reaction under oxidizing conditions. The product called Gel-TAc was tested for adhesive strength, cytocompatibility, and antimicrobial and antifungal properties. The lap shear strength for Gel-TAc hydrogels crosslinked by silver nitrate were about 4 times that of fibrin glue, and the cytocompatibility tests were positive. The antimicrobial and antifungal studies suggest a strong biocidal activity, pointing to a potential use of Gel-TAc for wound closure and drug delivery ⁹⁴.

Drug delivery system design is taking advantage of mussel-inspired bioadhesives. Among the most relevant results are increased delivery efficiencies, extended retention time, and possible reduction in therapeutic doses. These developments in drug delivery present an opportunity for translational efforts that may result in reduced costs and expanded therapeutic alternatives for the public.

4.2.4. Cellular engineering

Cellular engineering employs bioadhesives for different purposes, from controlling the cell division process to improving cell-surface interactions. For example, with the objective of developing an improved version of a current hyaluronic acid (HA) based adhesive hydrogel for cell therapy, researchers conjugated dopamine onto HA using EDC-NHS chemistry. The hydrogel was formed by oxidative crosslinking and was used for injection-free of different cell types into various organs in animal models. Study results showed an improved adhesiveness of the cell-containing hydrogel on wet or beating organic surfaces, allowing the material to be applied as paint materials. Additionally, cell biocompatibility was enhanced as compared to HA-alone bioadhesives and the feasibility of this tissue adhesive for minimally invasive cell therapy was demonstrated⁶⁹. In another case, researchers investigated the feasibility of protecting living cells and controlling their division process by encapsulating them with pDA⁹⁵. They employed yeast cells as a model of encapsulation, which was found to be an efficient way not only of protecting and stabilizing the cells but also of fabricating artificial spores. It was demonstrated that this technique provided the cells with resistance against harsh environments while allowing control of the cell cycle. Additionally, due to the catecholic nature of the encapsulating agent, the cell surfaces can be easily functionalized⁹⁵.

The advances in cellular engineering are being enhanced by the inclusion of mussel-inspired bioadhesives. Their multifunctionality allows them to attach, functionalize, protect, and control cell interactions with the surroundings. As with tissue engineering applications, catechol-based adhesives may be expected to work as adjuvants to boost cell proliferation.

4.2.5. Other applications

Even if it sounds counterintuitive, mussel-inspired bioadhesives can also be used to suppress adhesion. For instance, to prevent protein adsorption from blood or bodily fluids onto the surface of titanium medical implants, a mussel-inspired antifouling coating was developed⁷¹. A catechol-grafted dextran with a high binding compatibility for inorganic materials was prepared. Its adhesive strength on metallic surfaces, serum protein adsorption rate, and degree of cell adhesion were investigated. It was demonstrated that the coating was effective in reducing albumin adsorption to only 1.8% of that of uncoated titanium, at neutral pH. Furthermore, cell adhesion was also minimized as compared to bare metal, with cells exhibiting a less extended morphology. These results suggest that the antifouling compound could be used to confer protein- and cell-resistive properties to metallic surfaces employed in healthcare and bioengineering⁷¹. Another coating to prevent uropathogen adherence on ureteral stents was prepared by conjugating methoxy polyethylene glycol-dihydroxyphenylalanine with PEG. The material was used to coat stents that in turn were positioned into the bladder of animal models of infection. Results indicated a reduction in bacterial content in urine and stent samples, an increased resistance to bacterial attachment, and an enhanced infection clearance as compared to that of uncoated stents⁷⁴.

On the other hand, to enhance adhesiveness and to overcome the drawbacks of current DNA immobilization techniques, a mussel-inspired bioglue was developed to bind DNA onto a variety of surfaces without altering its bioactivity⁹⁶. The material was based on a random copolymer of N-(3,4-dihydroxyphenethyl) methacrylamide and aminoethylmethacrylamide. Its efficiency was demonstrated by binding DNA onto different substrates and then hybridizing the biomolecule with a complimentary oligonucleotide sequence. This promissory methodology may be applied to biomolecular detection and analysis⁹⁶.

5. Conclusions and Future Perspectives

The discovery of the extraordinary adhesive properties of mussel foot proteins has prompted a cascade of research on mussel-inspired bioadhesives aimed to solve a myriad of problems in healthcare and bioengineering. The mussels have the innate ability to form strong bonds with almost any substrate, regardless of the presence of water. This remarkable ability has generated considerable research interest and effort in creating polymer-catechol adhesive systems via mimicking the underlying biochemistry. These mussel-inspired adhesives are extremely attractive in the field of medical adhesion and sealing, where catechol groups can increase adhesion to tissue surfaces. Currently, innovative DOPA chemistries are the focal point of mussel inspired research to develop tissue adhesives with potential to function as medical glues. However, these synthetic mimics still cannot achieve the actual mussel adhesive behavior in terms of tissue adhesion under hydrophilic conditions.

Catechol functionalization is the prevalent strategy when it comes to bioadhesives’ synthesis, since it has been demonstrated that catechols play a crucial role in wet adhesive performance. However, when compared to the natural complexity of the adhesive phenomena exhibited by some species, this biomimetic approach presents several limitations.

One of those limitations is the control of catechol oxidation. Currently, mussel inspired adhesives do not have the ability to control the oxidation of DOPA or catecholic side groups grafted on the polymer backbone, making these groups susceptible to uncontrollable oxidation and thus limiting their adhesiveness. There are some proposed ways to circumvent this issue by attempting to preserve the catechol adhesive activity either with antioxidant moieties, or hydrophobic or non-polar groups in close proximity with the catecholic grafts ^{20, 28}. One of these alternatives is the prevention of the formation of quinones, which may be attained by shielding the catechols with non-polar groups such as glycine or protecting them with hydrophobic moieties like tyrosine residues. Another one is the reduction or retardation of quinone formation by using borate or cysteine protection groups to prevent oxidation. These improvements may be able to shield DOPA or other catechols from uncontrollable oxidation, thus increasing the adhesive effect.

Besides the control of oxidation, some other limitations are related to the wet conditions and the surface interactions. In those cases, tissue adhesion may also be increased by improving the dehydration of the substrate as well as the superficial electrostatic interactions. The first step in the wet adhesion process is related to the intimate contact between adhesive and substrate, which may be prevented or reduced by the presence of a hydration layer. Even though dopamine by itself may act on removing this layer, the close presence of cationic residues such as lysine or arginine result in an enhanced dehydration effect that increases the overall adhesiveness. Additionally, the removal of the hydration layer also results in improved catechol-substrate electrostatic interactions, such as the formation of hydrogen bonds, which also reflects on increased wet adhesion.

Furthermore, the optimization of the catechol and water content in the bioadhesive may also have a positive effect in the adhesive’s performance, since it has been demonstrated that an excess of catechol content does not reflect on increased adhesive strength, and that elevated water contents can negatively affect the performance ²⁸.

In addition to increased adhesiveness, current mussel inspired tissue adhesives also demand improvements in material properties that control their hydrophilicity, blood compatibility, biocompatibility, and biodegradability. For instance, UV irradiation, photo-initiators, and/or strong oxidizing agents are used in the curing process^{1, 97-101}, which may have potentially toxic effects on healthy cells around the application site. This fact may render the product suitable for topical use only. A gentler crosslinking performed at physiological conditions might be ideal, provided it would satisfy the requirements of wet adhesion and short curing times. Thus, research efforts that focus on employing milder crosslinking strategies could result in reduced cytotoxicity and enhanced biocompatibility for the internal use of mussel-inspired bioadhesives.

Likewise, the inclusion of non-catechol mediated chemistries represents an interesting strategy to increase adhesive performance. For instance, the naturally occurring self-healing of mussel’s byssal thread is attributed to the coordination of histidine, a non-catechol, and divalent cations such as Zn²⁺ or Cu²⁺. Since coordinated bonds differ from covalent bonds in that their rupture is not irreversible, they can be included as sacrificial bonds to promote self-healing and adhesiveness. Similarly, catechol analogues with chelating functionality such as tannic acid, or the oxidation-resistant 3-hydroxy-4-pyridinone, can be used in the bioadhesive formulation to control oxidation. The latter also providing the ability to create reversible coordinated bonds with divalent cations, similar to histidine. This approach could result in enhanced adhesive properties, since the combination of catecholic and non-catecholic interactions may present synergistic effects and allow the bonding of dissimilar surfaces^102-104.

As knowledge expands on the many different adhesive strategies that nature exhibits, the development of multifunctional bio glues with the ability to mimic a combination of nature strategies seems to be an attractive approach for future research. For instance, mussel-inspired chemistries have been combined with gecko-inspired physical interactions to create hybrid adhesives able to perform reversible attachment in wet or dry conditions. The product consisted of arrays of polymeric nanopillars to simulate gecko’s foot-hairs, or setae, coated with mussel-mimetic polymers⁷⁶. Moreover, research on the framework of setae is revealing the composition of the corneous beta proteins (CBPs) responsible for geckos’ dry adhesion on hydrophobic or hydrophilic substrates¹⁰⁵. Consequently, the combination of mussel- and gecko-inspired chemistries could result in hybrid bio glues able to work under various conditions. Similarly, the study of velvet worm¹⁰⁶, snail epiphragm¹⁰⁷, and other species’ adhesive phenomena may result in additional sources of materials for the development of combination bio glues.

In conclusion, there is plenty of room for improvement in mussel-inspired bioadhesives. The challenges to develop an adhesive that closely mimics mussels’ natural performance are many, but researchers continue discovering and developing strategies to narrow the gap. Additionally, the combination of nature-inspired strategies and materials represents an attractive line of research to develop multifunctional bioglues able to perform under various conditions and to adhere to various different substrates.