Types of Crosslinkers and Their Applications in Biomaterials and Biomembranes

Abstract

Biomaterials and biomembranes play a crucial role in a variety of applications, particularly in the medical field due to their ability to mimic natural biological structures and functions. Crosslinkers play also an important role in enhancing the structural integrity and functionality of biomaterials and in the design of biomembranes. This review article explores the fundamentals of biomaterials and biomembranes, with a particular focus on the role of crosslinkers in biology, chemistry and medicine. We explore the various types of crosslinkers commonly used in biomaterials synthesis, examining their chemical structure, classification, and synthesis methods. Additionally, we analyze the biological properties of crosslinkers and their interactions, highlighting their biological impact, particularly in cellular behavior and cytotoxicity. This article further emphasizes recent advances and innovation, particularly in tissue engineering, drug delivery, and wound healing. Finally, we conclude by addressing current challenges and suggesting potential futures directions for research in this field.

1. Introduction

Biomaterials and biomembranes are materials that can be used in the human body to help heal or replace tissues and organs, or to aid in restoring normal bodily functions. Although closely interconnected, biomaterials and biomembranes serve distinct functions, each playing an important role in various biomedical applications. Biomaterials can be natural or synthetic designed to interact with biological systems for clinical or medical purposes. These materials are commonly used in applications like implants [1,2], tissue engineering [3], prosthetics [4], drug delivery systems [5,6], regenerative medicine [7], and medical devices [8]. The primary characteristic of biomaterials is to support biological functions in the human body while minimizing adverse reactions. Biomaterials include metals, such as titanium and its alloys, stainless steel and cobalt–chromium alloys [9], various natural and synthetic polymers [10], ceramics [11], composites [12], biodegradable and bioactive materials [13], elastomers and rubbers [14], micro and nanoparticles [15], and hydrogels [16].

On the other hand, biomembranes are thin layers of specialized engineered structures designed to mimic or support the function of natural biological membranes. These membranes can separate or protect biological systems from the surrounding environment [17,18]. In biological systems, membranes consist of lipid bilayers, proteins, and carbohydrates that enclose cells and other organelles [19], playing a crucial role in (1) signal transduction through proteins on the plasma membrane which receive external signals, create pathways for signal transduction through lipid rafts where signaling molecules are concentrated, mediate cross-talk between various signaling pathways within the same cell, and aid in cell–cell communication [20]; (2) maintaining cellular integrity by acting as a barrier between the cell’s interior and the external environment, providing structural and mechanical support to the cell that helps maintain the shape and integrity of the cell and resist deformation [21]; (3) communication by enabling interaction between different cells and their surroundings through receptors on the plasma membrane by means of ion channels and thus creating membrane potential, membrane-bound proteins that can facilitate signaling between cells, and junctions that allow cells to exchange signals and information [22]; and (4) energy production, as they are critical in photosynthesis (chloroplast membranes), cellular respiration (mitochondrial membranes), and ion transport (plasma membrane and ion gradients), all of which are essential processes for generating and storing energy [23]. Given their functional versatility and ability to mimic or support natural biological membranes, biomembranes have a wide range of applications, such as in controlled or targeted drug delivery systems [24], tissue engineering [25], regenerative medicine [19] and artificial organs [26], medical implants [27], and diagnostic devices [28].

For biomaterials and biomembranes to perform optimally, crosslinkers must be used as they can dramatically influence the stability, functionality, and mechanical properties of these materials. Crosslinking occurs when polymer chains or molecules chemically bond together, significantly affecting the materials’ properties [29]. This process is critical in tissue engineering, drug delivery, and wound healing [30]. There are two major types of crosslinkers: chemical crosslinkers and physicals crosslinkers. Chemical crosslinkers include formaldehyde and glutaraldehyde (used to crosslink proteins or tissues) [31], ethylene glycol dimethylacrylate or EGDMA (to crosslink hydrogels) [32], isocyanates (for polymer-based biomaterials) [33], and genipin (a biodegradable crosslinker used in tissue engineering) [34]. Physical crosslinkers include ultraviolet (UV) light to crosslink certain polymers [35] or ionic interactions between anionic and cationic groups which create crosslinks in polyelectrolyte-based materials like hydrogels [36].

Therefore, the selection of crosslinker is consequently of high importance for the design of biomaterials and biomembranes since it affects the desired target properties. The crosslinkers enhance the mechanical strength of the crosslinked material, making them more resistant to deformation and tailored to have specific rigidity to exactly mimic natural tissue [37]. Crosslinkers improve material stability against changes such as in temperature or moisture and can be designed to degrade at a controlled rate [38]. They also offer the advantage of controlled and sustained delivery of drugs in response to specific stimuli (like temperature) or deliver to certain target locations under specific conditions only (like the pH level or the presence of a certain enzyme) [39] and are mostly biodegradable or bioabsorbable, which minimizes their long-term toxicity, especially in medical applications [40].

In this article, we review the fundamental properties of biomaterials and biomembranes and how they can enhance human bodily functions. We also investigate crosslinked materials, the biological and chemical properties of crosslinkers, and the various medical applications of crosslinked biomaterials and biomembranes. We investigate the mechanism of crosslinking, the recent advances in crosslinking techniques, and the emergence of new biomaterials and biomembranes.

2. Fundamentals of Biomaterials

Biomaterials are materials designed to interact with biological systems [41]. Biomaterials include a wide variety of compounds, such as metals including titanium and its alloys, stainless steel, and cobalt–chromium alloys, various natural and synthetic polymers, ceramics, composites, biodegradable and bioactive materials, elastomers and rubbers, micro and nanoparticles, and hydrogels. Table 1 summarizes the typical usage of various biomaterials.

Table 1. Typical usage of various biomaterials.

	Metals	Polymers	Ceramics	Composites	Biodegradable and Bioactive Materials	Elastomers and Rubbers	Microparticles and Nanoparticles	Hydrogels
Implants	✔	✔	✔	✔	✔			✔
Prosthetics	✔	✔	✔	✔	✔	✔
Drug Delivery Systems		✔		✔	✔		✔	✔
Tissue Engineering		✔	✔	✔			✔	✔
Diagnostic Devices		✔	✔		✔		✔

Table 1. Typical usage of various biomaterials.

	Metals	Polymers	Ceramics	Composites	Biodegradable and Bioactive Materials	Elastomers and Rubbers	Microparticles and Nanoparticles	Hydrogels
Implants	✔	✔	✔	✔	✔			✔
Prosthetics	✔	✔	✔	✔	✔	✔
Drug Delivery Systems		✔		✔	✔		✔	✔
Tissue Engineering		✔	✔	✔			✔	✔
Diagnostic Devices		✔	✔		✔		✔

Natural polymers are natural substances composed of large macromolecules formed by the binding of small identical or different molecules called monomers. These polymers can be produced via biological processes like protein and nucleic acid synthesis. Synthetic polymers are produced via artificial chemical reactions to produce a desirable product of industrial importance, e.g., polystyrene, polyvinyl chloride, nylon, silicon, etc. [42]. Polymers are widely used in our normal lives today; however, many polymers are resistant to degradation and contribute to the accumulation of polymer waste. This waste, as it continues to accumulate, becomes a serious threat to the environment and remains undecomposed for a long period. Due to these problems, scientists created biodegradable polymers that are easily degraded by microorganisms, such as aliphatic polyesters, which result in the reduced accumulation of waste and cause less harm and pollution to the environment [43,44] (Figure 1).

Synthetic aliphatic polyesters, derived from lactide, glycolide, caprolactone, and their copolymers, are among the most widely used degradable polymeric biomaterials for medical applications, including implantable devices, drug delivery systems [45], and tissue engineering materials. Despite its biocompatibility, polylactide (PLA) does not meet the optimal properties needed to replace soft tissues like ligaments, tendons, cartilage, or blood vessels [46].

In an attempt to form temporary implantable biomaterials, PLA was modified, improving its mechanical properties by means of crosslinking. This technique al-lows for the formation of structures with identical vulcanized rubbers [47]. The main drawbacks of these materials are that they are difficult to design with complex shapes due to the impossibility of remolding by heating or solvent casting.

In order to improve specific mechanical properties, PLA-based block polymers containing F 127 and Tetronic 1107 were synthesized, and their short-term degradation was studied. The results showed that the modification of the crystallinity of PLA blocks, along with the initial global molecular weight, can lead to a possible modulation of different degradation properties. In fact, an important decrease in molecular weight was detected with 200 kg.mol⁻¹ copolymers with the conservation of high mechanical integrity. In addition, a lower strain at failure was demonstrated in the case of copolymers based on PLA 96 compared to those containing PLA 94. These 200 kg.mol⁻¹ star and linear PLA co-polymers are considered to be promising materials for temporary biomedical applications requiring cell cultures due to their good biocompatibility and degradation rates [48].

On the other hand, many aspects of modern medicine have been affected by the use of smart biomaterials due to their ability to respond to modifications in physiological parameters. In fact, a study of the uses of smart biomaterials in tissue engineering was carried out. This recent study showed the impact of these smart biomaterials in many other medical fields, such as medical devices, drug delivery, and immune engineering, leading to promising therapies for the treatment of many debilitating diseases [49].

A vascular scaffold fabricated by electrospinning poly(ε-caprolactone) (PCL) and collagen was designed, leading to an important adhesion substrate for vascular cells and adequate structural support. However, the small-sized pores were responsible for the decrease in the efficacy of smooth muscle cell (SMC) seeding due to their inability to infiltrate into the scaffolds. The development of a bilayered scaffold that improves EC adhesion onto the luminal surface and the homogenous infiltration of SMCs into the outer layer was studied in order to overcome this challenge. This advanced scaffold was found to be biodegradable and biocompatible, allowing a good ability for infiltration and cell adhesion support. In addition, sufficient mechanical support, along with physiological vascular conditions, were provided. This recent study demonstrated that vessel tissue function could be improved by using these bilayered scaffolds, facilitating endothelialization and smooth muscle maturation [50].

The use of carbon dots (CDs) as biosensors and bioimaging agents due to their particular properties was an important challenge for scientists when first evaluating the osteoconductivity of CDs in poly (ε-caprolactone) (PCL)/polyvinyl alcohol (PVA) [PCL/PVA] nanofibrous scaffolds. In fact, after several studies on the evaluation of eggshell-derived calcium phosphate (TCP3) and its cellular responses, an important proliferation and osteogenic differentiation was observed with these derivatives compared to other scaffolds. As a result, the implementation of CDs and novel eggshell-derived calcium phosphate showed high importance for the fabrication of bioscaffolds used for bone tissue engineering [51].

More research should be implemented to develop the economic and environmental aspects related to the application of these block copolymers for designing nanostructured materials [52].

Figure 1. Examples of the natural polymers and derivatives used for biomaterials design [53].

Figure 1. Examples of the natural polymers and derivatives used for biomaterials design [53].

3. Fundamentals of Biomembranes

Defining a biomembrane comprehensively is difficult because of its wide range of characteristics and functionalities. Biomembranes are a specific type of biomaterials used to form membranes that separate two regions and selectively regulate the movement of substances between them. While biomaterials are typically used for structural applications, biomembranes are designed to regulate the movement of substances across them, making them essential for applications such as filtration and drug delivery. Biomembranes can have different forms, such as being homogeneous (the plasma membrane of some bacteria) or heterogeneous (the plasma membrane of some animal cells), symmetrical (lipid bilayers created for model membranes) or asymmetrical (the plasma membrane of eucaryotic cells), solid (lipid rafts) or liquid (the plasma membrane of most cells), and neutral (the outer leaflet of the plasma membrane) or charged (positive, negative, or both) (the inner left of the plasma membrane) [54]. Regardless of the type, all biomembranes share the fundamental property of controlling the passage of substances in a specific way [55]. Biomembranes are versatile, as their properties can be “tailored” or customized to suit specific separation tasks, making them adaptable for different applications (e.g., tissue engineering, drug delivery systems, biosensors, and biomedical devices, etc.).

Biomembrane materials can be broadly classified into three main categories: inorganic (ceramic), synthetic polymers, and natural polymers [56]. Additionally, there are mixed membranes, inorganic/polymeric or polymeric/polymeric materials, used to enhance the properties of the membrane, offering a balance of durability and selectivity for specific applications. Inorganic membranes, such as alumina (Al₂O₃) and zirconia (ZrO₂), are widely used for their thermal and chemical stability. However, their higher production costs can be a limiting factor compared to polymeric and natural membranes [57]. This has led to a growing interest in low-cost raw materials such as clays, ash, and waste products to develop more affordable ceramic membranes. These membranes offer significant advantages, including lower costs and reduced environmental impact. However, the design and fabrication process is complex, requiring careful attention to material composition, additives, and safety concerns, particularly regarding the presence of heavy metals and toxic compounds [58].

Polymers can be divided into two types based on their origin: synthetic polymers, which are artificially created from petroleum-based sources, and natural polymers, which are derived from plants or animals [59,60]. Noticeably, biopolymer production is sustainable, carbon neutral, and renewable because biopolymers are made from sea or land plant materials [61,62].

Chitin is the second most abundant natural polysaccharide, commonly found in crustacean shells, insects, molluscan organs, and fungi. Chitosan is an eco-friendly polymer derived from chitin through N-deacetylation and consists of glucosamine and N-acetylglucosamine [63]. Its amino and hydroxyl groups enable diverse applications in pharmaceuticals, wastewater treatment, fuel cells, and biodegradable food packaging due to their antimicrobial and hydrophilic properties. Chitosan is available as membranes, films, and fibrous mats. Despite its advantages, chitosan has drawbacks that limit its application. Chitosan has low mechanical strength and low electrical conductivity. It is also very brittle due to its high glass transition temperature. These drawbacks are mitigated by blending with other polymers, adding inorganic fillers, or chemical modifications.

Alginate, a water-soluble polysaccharide from brown seaweed, is composed of mannuronic and guluronic acid units [64]. Its advantages include biocompatibility, non-toxicity, biodegradability, and ease of combination with divalent cations, making it useful in fields like drug delivery, tissue engineering, and textiles (Figure 2). Alginate can be manufactured into a variety of forms, such as film, microspheres, and fibers, because of its reversible solubility. However, like chitosan, alginates have certain limits to their applications due to several weaknesses, which include high water solubility and low mechanical strength. Alginate’s six-membered ring backbone makes it challenging to enhance rigidity or compactness, resulting in larger void volumes that allow significant water absorption. Excessive water uptake leads to membrane swelling, reducing selectivity (Table 2). Striking a balance between membrane permeability and selectivity for water or gas is essential. To overcome this limitation, alginate has been improved through methods like covalent crosslinking, ionic crosslinking, and non-bond interactions, which enhance its structural stability and functional performance [65].

Figure 2. SEM images for different biomembranes made using different biomaterials (chitosan, cellulose, alginate, gelatin, etc.) and crosslinked by various cross-linkers (genipin, glutaraldehyde, etc.) to highlight the microstructure organization after the crosslinking process (adapted from [66,67,68,69]). Red arrow indicates interconnected pores. Each yellow scale bar represents 100 μm.

Figure 2. SEM images for different biomembranes made using different biomaterials (chitosan, cellulose, alginate, gelatin, etc.) and crosslinked by various cross-linkers (genipin, glutaraldehyde, etc.) to highlight the microstructure organization after the crosslinking process (adapted from [66,67,68,69]). Red arrow indicates interconnected pores. Each yellow scale bar represents 100 μm.

Cellulose stands out as the most abundant natural polymer and is extensively documented in the literature for membrane fabrication. Its extensive use is due to its abundance and remarkable properties. Cellulose, a prevalent polysaccharide, is valued for its abundance, low cost, high hydrophilicity, and biocompatibility, making it ideal for industrial applications like paper, textiles, membranes, and pharmaceuticals. It is found in plants, trees, fungi, algae, and bacteria, with plant fibers being the primary source. Structurally, cellulose is a linear polymer with repeating units of D-glucopyranose [56]. Phase inversion via immersion precipitation is the most commonly used method for fabricating cellulose-based composite membranes. In this process, cellulose or a cellulose-containing polymer mixture is dissolved in a suitable solvent, and the resulting solution is cast onto an appropriate support layer. The cast layer is then submerged in a coagulation bath, often containing pure water as a non-solvent, which leads to the formation of the membrane. Another widely used technique for producing well-interconnected porous membranes is thermally induced phase separation (TIPS) [70]. TIPS involves lowering the temperature to induce phase separation in the dope solution, followed by solvent removal through extraction, evaporation, or freeze-drying. Interfacial polymerization is the predominant technique for fabricating thin-film composite (TFC) membranes [71], which involves coating cellulose-based polymers onto a polymeric substrate. Recent advancements in nanotechnology, coupled with the limitations of traditional methods, have led to the adoption of electrospinning for the production of nanoscale fibers with exceptional properties. Nanofibrous polymeric networks produced through electrospinning offer large surface areas, easy functionalization, and superior mechanical properties. This technique uses an electric field to create highly porous nanofibrous membranes from polymer fibers. Compared to membranes produced by traditional phase inversion methods, cellulose-based nanofibrous membranes exhibit higher flux, reduced fouling, and a nanoscale porous structure (Figure 3).

Figure 3. Summary of natural and synthetic biomembranes [72] (republished from [72] under license number 5998461306754).

Figure 3. Summary of natural and synthetic biomembranes [72] (republished from [72] under license number 5998461306754).

Polymer blending is a versatile technique used to enhance material performance. Natural polymers such as cellulose (cellulose acetate, CA) can be blended with synthetic polymers like polyether sulfone (PES) to improve membrane properties [73]. Additionally, blending natural polymers with ceramics, such as adding inorganic fillers like TiO₂ [74], Fe₃O₄, and alumina [75] into CA and CAP (cellulose acetate phthalate), has shown significant improvements in selectivity, hydrophilicity, surface area, and porosity, even with small amounts of filler materials. However, optimizing filler loading is a key challenge, as excessive loading can lead to poor dispersion, causing aggregation and mechanical instability. The toxicity of certain inorganic nanoparticles is also a concern, as their leakage into the aqueous environment can pose health and environmental risks. To address these issues, further purification stages are necessary.

There is a growing interest in blends composed entirely of bio-based materials. For instance, combining cellulose with biopolymers like chitosan has led to materials with unique properties, including antibacterial activity [76], metal ion adsorption, and self-healing characteristics [77].

Other natural polymers have been used in research, though they are not as widely applied as the ones mentioned earlier. One such polymer is poly(lactic acid) (PLA), a thermoplastic aliphatic polyester derived from renewable sources such as corn, sugar beets, or rice. Silk fibroin (SF), another natural biopolymer, has also gained attention due to its non-toxicity, excellent biocompatibility, and biodegradability. Silk fibroin is typically extracted from silkworm cocoons by removing the outer sericin layer. The polymer’s structure, which contains numerous amino (–NH₂) and carboxyl (–COOH) groups, makes it an effective adsorbent for heavy metal ions [78].

Table 2. Comparison of membrane types: origin, properties, applications, and limitations.

Membrane Type	Origin	Properties	Applications	Limitations
Inorganic membrane [58]	Ceramics (alumina, zirconia, clay), metals	High mechanical strength Thermal resistance Chemical resistance	Gas separation Water treatment Pharmaceutical Filtration and desalination application	High production cost Fragility limited flexibility
Synthetic polymers (Petrochemical-derived) [79,80]	Polysulfone	Resistance to extreme pH, high temperatures Good mechanical properties	Microfiltration, ultrafiltration, reverse osmosis	Expensive Not eco-friendly Poor stability
	Poly (vinylidene fluoride) (PVDF)	High mechanical strength Chemical resistance Aging resistance Excellent thermal stability	Filtration, water treatment, pharmaceutical separations
Natural polymers (Plant or animal-derived) + their derivatives [61,62]	Chitosan (from crustacean shells, fungi, insects) acetylglucosamine [63]	Abundant Biocompatible Biodegradable Hydrophilic Less cost	Drug delivery Sensors Wound healing Tissue engineering Pharmaceutical applications water filtration	Low mechanical strength Low thermal stability
	Alginate (from seaweed) [64]
	Cellulose (from plants) [56]
Mixed matrix membranes	Combination of inorganic and polymeric materials	Enhanced properties, combining the best of both materials (e.g., durability, selectivity)	Water treatment Gas separation Industrial filtration	Challenges in achieving optimal dispersion of fillers, potential instability
Biopolymer blend	Chitosan–alginate blend Chitosan–cellulose blend Alginate–cellulose blend	Increased mechanical strength High stability Synergistic interactions	Drug delivery Water treatment Food packaging Biomedical applications	Complex preparation process Limited scalability potential instability under certain conditions

Table 2. Comparison of membrane types: origin, properties, applications, and limitations.

Membrane Type	Origin	Properties	Applications	Limitations
Inorganic membrane [58]	Ceramics (alumina, zirconia, clay), metals	High mechanical strength Thermal resistance Chemical resistance	Gas separation Water treatment Pharmaceutical Filtration and desalination application	High production cost Fragility limited flexibility
Synthetic polymers (Petrochemical-derived) [79,80]	Polysulfone	Resistance to extreme pH, high temperatures Good mechanical properties	Microfiltration, ultrafiltration, reverse osmosis	Expensive Not eco-friendly Poor stability
	Poly (vinylidene fluoride) (PVDF)	High mechanical strength Chemical resistance Aging resistance Excellent thermal stability	Filtration, water treatment, pharmaceutical separations
Natural polymers (Plant or animal-derived) + their derivatives [61,62]	Chitosan (from crustacean shells, fungi, insects) acetylglucosamine [63]	Abundant Biocompatible Biodegradable Hydrophilic Less cost	Drug delivery Sensors Wound healing Tissue engineering Pharmaceutical applications water filtration	Low mechanical strength Low thermal stability
	Alginate (from seaweed) [64]
	Cellulose (from plants) [56]
Mixed matrix membranes	Combination of inorganic and polymeric materials	Enhanced properties, combining the best of both materials (e.g., durability, selectivity)	Water treatment Gas separation Industrial filtration	Challenges in achieving optimal dispersion of fillers, potential instability
Biopolymer blend	Chitosan–alginate blend Chitosan–cellulose blend Alginate–cellulose blend	Increased mechanical strength High stability Synergistic interactions	Drug delivery Water treatment Food packaging Biomedical applications	Complex preparation process Limited scalability potential instability under certain conditions

4. Crosslinkers: Chemistry and Classification

Crosslinking is an important phenomenon in polymer chemistry; it enhances the properties of materials by forming connections between polymer chains. Crosslinkers create bonds between polymer chains, leading to a three-dimensional network structure. The nature of these bonds determines the properties and applications of the resulting material. Crosslinking can occur through different types of bonding: covalent, ionic, or hydrogen bonds.

4.1. Covalent Crosslinking

Covalent crosslinking is observed when stable covalent bonds are formed between polymer chains. These bonds are irreversible, creating durable materials with high mechanical strength and thermal stability. Covalent crosslinking is common in elastomers and hydrogels, where strong bonds are required to resist significant stress or deformation. Many covalent crosslinkers have been used: glutaraldehyde [81], formaldehyde, ethyleneglycol diglycidyl ether (EGDGE) [82], N,N’-methylenebisacrylamide (MBAA) [83], the isocyanate-based crosslinker [84], and others [85].

An example of covalent crosslinking is the use of glutaraldehyde as a crosslinker for collagen-based hydrogels. Glutaraldehyde forms covalent bonds with amino groups on collagen, resulting in a highly stable, crosslinked network used in biomedical applications such as tissue engineering and wound healing [86,87,88]. The general reaction for the covalent crosslinking process is as follows:

Collagen-NH₂ + Glutaraldehyde → Collagen-N=CH-R + H₂O

Collagen-NH₂ represents the amino group on collagen (usually from lysine).

Glutaraldehyde has two aldehyde groups, which can react with the amino groups.

Collagen-N=CH-R is the resulting Schiff base, where “R” represents the remaining part of the glutaraldehyde structure.

4.2. Ionic Crosslinking

Ionic crosslinking is observed when electrostatic interactions occur between oppositely charged groups on polymer chains. These bonds are weaker than covalent bonds, making these materials more flexible and responsive to environmental changes such as pH. Ionic crosslinkers are used in many applications, such as tissue engineering, wound healing, drug delivery systems, food and cosmetic industries, environmental and water treatments, etc. [89,90,91,92]. Many ionic crosslinkers have been used, like calcium ions (Ca²⁺) [93], magnesium ions (Mg²⁺) [94], zinc ions (Zn²⁺) [95], and polyvalent metal cations such as copper or iron [96]. An example of ionic crosslinking is the use of alginate, a naturally derived polysaccharide, which can be crosslinked using calcium ions to form a hydrogel. The divalent calcium ions bridge the negatively charged carboxylate groups on alginate chains, creating a gel matrix widely used in drug delivery systems. The general reaction for the ionic crosslinking process is as follows:

Alginate-COO⁻ + Ca²⁺ → Alginate-Ca²⁺(-COO)⁻ (hydrogel)

Alginate-COO⁻ represents the carboxylate group on the alginate chain.

Ca²⁺ is the calcium ion that crosslinks the chains.

Alginate-Ca²⁺(-COO)⁻ represents the crosslinked structure where calcium ions bridge the carboxylate groups.

4.3. Hydrogen Bonding

Hydrogen bonding crosslinking is observed when there are interactions between hydrogen atoms and electronegative atoms such as oxygen or nitrogen. These types of bonds are weaker than covalent and ionic bonds; they can provide a significant level of crosslinking, particularly in biological materials or polymers, enhancing their elasticity and self-healing properties.

Many hydrogen-bonding crosslinkers have been used such as polyvinyl alcohol (PVA) [97], urethan-based crosslinkers, polyethylene glycol (PEG) [98], carboxymethyl cellulose (CMC) [99], dextrin, starch, chitosan, and others [100]. An example of hydrogen bonding crosslinking is poly(vinyl alcohol) (PVA), which can be used as a hydrogen bonding crosslinker through the formation of intermolecular hydrogen bonds between the hydroxyl groups (-OH) present on the polymer chains or with other chemical species. This crosslinking method forms robust, biocompatible materials suitable for application in cartilage replacement and drug delivery [101]. The general reaction for the hydrogen bonding crosslinker is as follows:

PVA-OH⋅⋅⋅PVA-OH → Hydrogen Bond Formation

Each type of crosslinking covalent, ionic, and hydrogen bonding offers unique advantages that can be tailored to specific applications, from biomedical to industrial fields. The selection of crosslinking chemistry depends on the desired material properties, such as flexibility, strength, and responsiveness, providing a versatile toolkit for material science and engineering.

Here, we review some of the most common crosslinkers used in research and industry, including glutaraldehyde, genipin, EDC/NHS, and epoxies, each with unique mechanisms and applications. Each crosslinking agent has its advantages and limitations. The choice of crosslinker depends on the material requirements, desired properties, and application, providing a range of options for diverse scientific and industrial needs.

4.4. Glutaraldehyde

Glutaraldehyde is a widely used crosslinker, especially in the formation of covalent bonds with biomaterials containing amino groups such as chitosan, collagen, and gelatin [102]. Glutaraldehyde reacts with amine groups to form stable covalent linkages, making it ideal for applications that require durable and stable crosslinks. However, glutaraldehyde, like many other aldehydes, is toxic, limiting its use in some biomedical applications unless thorough post-processing is conducted (neutralization, washing, dehydration, etc.). Another strategy to overcome this toxicity issue is to add the glutaraldehyde in a limited quantity. Due to its high reactivity (crosslinking in a few seconds), the added glutaraldehyde quantity is totally consumed (forming the covalent linkages), which guarantees the complete consumption of the added crosslinker quantity. Generally, this strategy allows us to avoid the post-processing treatment or to minimize the necessary treatment time. Glutaraldehyde-crosslinked collagen is often used in tissue engineering and medical devices [103].

4.5. Genipin

Genipin, a natural crosslinker derived from the gardenia plant, is considered a safer alternative to synthetic agents like glutaraldehyde [104,105]. Genipin reacts with primary amines to form blue-colored crosslinked materials, making it especially useful in biomedical applications where biocompatibility is essential, such as tissue engineering and drug delivery. Genipin-crosslinked materials exhibit excellent biocompatibility and low cytotoxicity, making it an ideal crosslinker for hydrogels and collagen-based materials [106].

4.6. EDC/NHS

1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) is often used with N-hydroxysuccinimide (NHS) to activate carboxyl groups on polymers, allowing them to react with amine groups to form amide bonds [107]. The EDC/NHS system is widely used in biomaterial functionalization because it forms stable, covalent amide linkages without leaving residual toxic by-products. EDC/NHS crosslinking is commonly applied in the conjugation of peptides, proteins, and polysaccharides, particularly in drug delivery and tissue engineering applications where biocompatibility is critical [108].

4.7. Epoxies

Epoxy-based crosslinkers are synthetic agents commonly used for forming covalent bonds in polymers due to their high reactivity and ability to form durable, chemically resistant networks [109]. Epoxies are versatile, used in a variety of applications, from adhesives to coatings, and are particularly valued in the automotive, aerospace, and construction industries for their toughness and thermal stability. Epoxies are also applied in biomedical applications, though biocompatibility must be considered depending on the specific context [110].

4.8. Other Crosslinkers

Several other crosslinking agents, such as poly (ethylene glycol) diglycidyl ether (PEGDE) and formaldehyde, are also commonly used based on application-specific needs [111]. PEGDE, for example, is often used to create hydrogels with enhanced water retention and flexibility, making it ideal for soft tissue engineering and wound care applications [112]. Formaldehyde is used with caution due to its known toxicity and is primarily applied in industrial materials rather than biomedical contexts [113].

4.9. Mechanisms of Crosslinking: Reaction Pathways and Effects on Properties

Crosslinking in polymer chemistry involves various reaction pathways depending on the crosslinking agent and the type of bonds formed. Crosslinkers can bond through covalent, ionic, or hydrogen bonding mechanisms, each influencing the properties of the resulting polymer network. Here, we discuss several common crosslinking mechanisms.

4.9.1. Step-by-Step Mechanism of Crosslinking with Glutaraldehyde [114,115,116]

• Activation of the aldehyde group

Glutaraldehyde has two reactive aldehyde groups (-CHO) at each end of its molecule. Each aldehyde group can form a covalent bond with a nucleophilic site, such as an amino group (–NH₂), typically found on lysine residues in proteins.

• Nucleophilic attack by the amino group

The amino group of a protein or other nucleophilic molecules attack the carbonyl carbon of the glutaraldehyde aldehyde group. Carbonyl carbon is electrophilic, making it susceptible to nucleophilic attack. This reaction forms a hemiketal intermediate, which is unstable.

• Formation of a Schiff base (Imine bond)

The hemiketal intermediate undergoes dehydration (loss of water) to form a stable imine (Schiff base) linkage. This reaction involves the formation of a double bond between the nitrogen of the amino group and the carbon of the carbonyl group, resulting in the loss of water:

R-NH₂+ CHO−(CH₂)₃CHO → RNH−CH=(CH₂)₃CH=O + H₂O

where R-NH₂ represents an amino group (from a lysine residue or similar) and CHO−(CH₂)₃ CHO- represents glutaraldehyde.

• Crosslinking

The imine group formed in the previous step can further react with another aldehyde group from a second glutaraldehyde molecule or from another lysine residue, leading to the formation of a covalent crosslink between two molecules. This creates a three-dimensional network of crosslinked molecules.

This is a key step in biological sample fixation (e.g., tissues), as glutaraldehyde forms highly stable crosslinks between protein molecules, which helps to preserve their structure.

• Potential hydrolysis

The imine bond (Schiff base) formed between the lysine residue and glutaraldehyde is not always stable over time. It can undergo hydrolysis, especially in aqueous conditions, to regenerate the aldehyde group and the amine group. This can lead to some degree of “reversibility”, but under controlled conditions, the crosslinking can be quite stable.

• Hydrolysis reaction:

RNH=CH₂CH₂CHO + H₂O → RNH₂ + CHO−(CH₂)₃CH₂OH

This step is generally avoided or minimized by controlling the environmental conditions (e.g., reducing the pH or adding stabilizers) (Figure 4a).

4.9.2. Step-by-Step Mechanism of Enzymatic Crosslinking with Genipin [117,118,119,120]

• Genipin activation by transglutaminase (TGase)

The enzyme transglutaminase (TGase), which catalyzes the formation of covalent bonds between glutamine and lysine residues in proteins, plays a significant role in facilitating the activation of genipin. TGase activates the genipin molecule by catalyzing the hydrolysis of the epoxide ring, promoting the formation of reactive intermediates (such as a quinone methide or aldehyde group). These intermediates are highly electrophilic, are prone to nucleophilic attack, and can participate in crosslinking.

Genipin (C₉H₁₂O₅) + TGase + Enzyme → Reactive intermediate (aldehyde/quinone methide) + H₂O

• Crosslinking with amino groups

Once the genipin molecule is activated into a reactive intermediate, the electrophilic species (such as the quinone methide or aldehyde) can react with nucleophilic groups, like amine groups (–NH₂) found in proteins or collagen. This forms a covalent bond through a Schiff base formation or direct nucleophilic attack.

Protein-NH₂ + Reactive Genipin intermediate → Protein-NH-Genipin linkage + H₂O

The reaction typically forms a Schiff base (an imine bond, –C=N–), which stabilizes the linkage between the protein and the genipin molecule.

• Polymerization and crosslinking

If multiple reactive intermediates from genipin are present, they can further react with other amino groups (either from the same protein or other proteins), leading to the formation of a 3D-crosslinked network.

Protein-NH-Genipin linkage + Protein-NH₂ → 3D Crosslinked Protein Network

The final product is a stable, covalently crosslinked matrix, where proteins or biomolecules are connected via genipin-induced linkages, providing structural stability and mechanical strength to the material (Figure 4b) [121].

4.9.3. Step-by-Step Mechanism of Crosslinking Carbodiimide-Mediated with EDC/NHS [121,122,123,124]

The carbodiimide crosslinking system, using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) in the presence of N-hydroxysuccinimide (NHS), activates carboxyl groups, allowing them to react with primary amines to form covalent amide bonds. This reaction proceeds without leaving cytotoxic by-products, making it especially suitable for biological applications like drug delivery and tissue engineering applications [125].

• Activation of the carboxyl group by EDC

EDC is a carbodiimide compound that activates carboxyl groups by reacting with them. It interacts with the carboxyl group (–COOH) of a protein or other molecules containing a carboxyl functional group.

R-COOH + EDC → R-COO⋅EDC

The resulting intermediate is highly reactive but unstable.

• The formation of a more stable NHS-activated ester

N-Hydroxysuccinimide (NHS) is added to the reaction mixture. NHS reacts with the intermediate formed in the previous step, stabilizing it and converting it into an NHS ester.

R-COO⋅EDC + NHS →R-COO-NHS + EDC

The NHS ester is much more stable than the previous intermediate, allowing for easier handling and more controlled reaction conditions.

• Nucleophilic attack by amine group

The NHS ester, which is now reactive, can be attacked by nucleophilic amino groups (–NH₂) from another molecule (such as a protein or peptide). The nitrogen from the amine group performs a nucleophilic attack on the carbonyl carbon of the NHS ester, displacing the NHS group.

R-COO-NHS + R’-NH₂ → R-CONH-R’ + NHS

This results in the formation of a covalent amide bond between the carboxyl group and the amine group, and the NHS group is released.

• Crosslinking between two molecules

If both reactants (molecules with amine or carboxyl groups) are involved in the reaction, this can lead to crosslinking between the two biomolecules. The process can result in the formation of dimers, trimers, or higher-order crosslinked products depending on the concentration of the reagents and reaction conditions (Figure 4c).

4.9.4. Step-by-Step Mechanism of Epoxy Crosslinking [126,127,128,129,130]

Epoxy crosslinking is a process where epoxy resins (which contain epoxy groups, typically oxirane rings) are crosslinked with curing agents, such as polyamines, polyamides, or anhydrides, to form a three-dimensional polymer network bonded through covalent bonds. This reaction opens the epoxide ring, leading to a highly crosslinked polymer with excellent resistance to thermal and mechanical stress. Due to these properties, epoxy crosslinked materials are extensively used in coatings, adhesives, and structural applications, particularly in high-stress environments like automotive and aerospace industries [131]. Here is a step-by-step breakdown of the use of an amine group.

• Nucleophilic Attack

A diamine has two nucleophilic amine groups (-NH₂). One of the amines attacks the electrophilic carbon in the epoxide ring, opening the ring and forming a covalent bond between the epoxy and the amine group.

R-CH₂-O-CH₂-C + Diamine → R-CH₂-NH-CH₂-C

• Hydroxyl Group Formation

After the nucleophilic attack, the oxygen that was part of the epoxide ring now holds a hydrogen atom from the reaction. The oxygen is converted into a hydroxyl group (-OH).

• Crosslinking

The second amine group from diamine attacks another epoxide group, causing further crosslinking and forming a complex network.

R-CH₂-NH-CH₂-C → R-CH₂-NH-CH₂-C-NH-CH₂-C

• Curing and Hardening

As the crosslinking continues, a rigid structure is formed where the epoxy molecules are bound together by the curing agent.

4.9.5. Step-by-Step Mechanism of Ionic Crosslinking with Calcium Ions (e.g., Alginate) [132,133,134,135]

Ionic crosslinking occurs when divalent or multivalent cations, such as calcium ions, interact with negatively charged groups on polymers. For example, calcium ions can crosslink alginate, a natural polysaccharide derived from brown algae containing repeated units of M (mannuronic acid) and G (guluronic acid) that have carboxyl groups (-COOH). Calcium ions will bind to the carboxylate groups, forming an “egg-box” structure. This pathway produces materials that are flexible and reversible, making them suitable for applications such as controlled drug release [136].

• Interaction of Calcium Ions with Alginate

The ionic crosslinking process starts when calcium ions (Ca²⁺) are introduced to a solution of alginate. The calcium ions interact with the carboxyl groups on the alginate chains, particularly those located in the guluronic acid (G-block) regions. This is an electrostatic interaction, where the positively charged calcium ion interacts with the negatively charged carboxylate groups (-COO⁻) on the alginate.

R-COO⁻ + Ca²⁺ → R-COO^-Ca²⁺

This binding occurs because the calcium ion has a high charge density and can coordinate with two carboxylate groups from adjacent alginate chains.

• Formation of the “Egg-box” Structure

The key feature of the ionic crosslinking mechanism is the formation of the “egg-box” model. In this model, the calcium ion (Ca²⁺) forms a bridge between two alginate chains, specifically between the carboxylate groups of guluronic acid residues.

This creates a network where multiple chains of alginate are crosslinked through calcium ions, leading to the formation of a gel or a network structure.

(R-COO-Ca-COO-R) (egg-box structure)

This ionic crosslinking forms a gel that is stable as long as the calcium ions remain present.

• Gelation and Network Formation

As more calcium ions are added, more alginate chains are crosslinked, resulting in the formation of a three-dimensional network. The strength of the gel depends on the number of crosslinks formed between the alginate chains. High concentrations of calcium ions lead to more crosslinks, resulting in a more rigid gel. In contrast, low calcium ion concentrations produce weaker gels with fewer crosslinks.

• Reversibility and Stability

The formed gel is reversible and can be disassembled if the calcium ions are removed, typically by washing with a chelating agent like EDTA or by changing the pH. A chelating agent like EDTA can bind to the calcium ions and remove them from the alginate network, breaking the ionic bonds and leading to gel dissolution (Figure 4d).

R-COO-Ca-COO-R + EDTA → R-COOH + Ca-EDTA complex

Altering the pH of the solution can also influence the stability of the gel. At a low pH, the carboxylate groups on the alginate can protonate to form -COOH, reducing the ionic interaction with calcium ions and causing the gel to break apart. Each crosslinking mechanism offers distinct benefits and limitations, impacting the thermal stability, mechanical strength, elasticity, and biocompatibility of the resulting material. The choice of crosslinking chemistry is critical in tailoring material properties for specific applications, from biomedicine to industrial settings.

Figure 4. (a) The crosslinking process of glucose oxidase into the chitosan polymer using the glutaraldehyde crosslinker [100]. (b) The mechanism of chitosan and gelatin crosslinking using genipin [137]. (c) The mechanism of chitosan crosslinking using EDC/NHS [126]. (d) The crosslinking of sodium alginate with calcium chloride [138].

Figure 4. (a) The crosslinking process of glucose oxidase into the chitosan polymer using the glutaraldehyde crosslinker [100]. (b) The mechanism of chitosan and gelatin crosslinking using genipin [137]. (c) The mechanism of chitosan crosslinking using EDC/NHS [126]. (d) The crosslinking of sodium alginate with calcium chloride [138].

5. Biological Aspects of Crosslinkers

A wide selection of chemical crosslinkers, either natural or synthetic, has been already explored in various tissue engineering applications. Their biocompatibility and safety have been studied using both in vitro and in vivo methods. The choice of the crosslinker should be based on its ability to improve the properties of the scaffold without inducing any adverse effects on the tissues [139].

Glutaraldehyde (GTA) is one of the most frequently used synthetic crosslinkers in the generation of scaffold materials, including chitosan, gelatin, and collagen. It is highly stable, easily accessible, and inexpensive [140]. However, the presence of the aldehyde group has limited its use since it has induced cytotoxicity and inflammation [141,142,143,144]. To reduce these toxic effects, the aldehyde group can be washed off with a glycine or citric acid solution [145], and the concentration of GTA used could be limited to less than 8%, which has been determined to be biocompatible for the cells [146]. The use of GTA has been efficient in producing bioprostheses for heart valve replacement [145,147]. Several reports have developed GTA-crosslinked scaffolds and shown applications for their potential use in bone tissue engineering. These studies have been conducted on established cell lines, and some have been applied in vivo in mouse, rabbit, or rat models. Interestingly, a higher tensile strength and lower immunogenicity were shown in these GTA-crosslinked bone tissue scaffolds [139].

Other synthetic crosslinkers include carbodiimide agents. These have been used for generating scaffolds that show good biocompatibility for ophthalmic applications [148]. EDC/NHS crosslinked matrices are shown to be effective for bone and neural repair but in some cases induce low cell–ECM attachment [149,150,151]. Thus, it is highly advisable to reduce the concentration of EDC/NHS by up to 10-fold to obtain higher stability and mechanical strength [152]. Several studies have used EDC/NHS as crosslinker in the formation of bone matrices, with concentrations of 50 mM and 25 mM, respectively [139]. Dual crosslinking with GTA and EDC in decellularized human amniotic membranes and collagen scaffolds improves mechanical resistance with high biocompatibility, including stemness, adhesion, and survival of cells, leading to potential applications in tissue engineering [153,154].

Similar to GTA, epoxy compounds like BDDGE can exhibit some cytotoxicity. However, BDDGE is being used for skin regeneration by crosslinking gelatin nano-fibers with no apparent cytotoxicity [155]. In addition, collagen and chitosan crosslinked with epoxy compounds have demonstrated high mechanical and thermal stability in corneal progenitor cells and dermal cells [156,157].

To avoid the disadvantages of synthetic crosslinkers, the natural chemical genipin is being implemented in several studies for higher efficiency and biocompatibility. Compared to GTA, genipin has very low toxicity as demonstrated by MTT and proliferation assays, with 10000 times less cytotoxicity in 3T3 fibroblast cells [158,159]. With genipin, these cells can produce more collagen and have higher metabolic activity while modulating the growth and polarization of immune cells, leading to decreased immunogenicity [160]. The use of genipin has also shown good mechanical strength, higher cell uniformity, tissue growth, the secretion of glycosaminoglycans, and resistance to collagenase when compared to matrices crosslinked with GTA [161,162].

Genipin has been applied for the successful preparation of corneal lens implants [163], the repair of intervertebral discs using either fibrin hydrogel with silk scaffold or collagen scaffold [164,165], and for halting and repairing tendon tears [166,167]. Genipin crosslinked with elastin has also been effective in treating knee injuries in rabbit models [168].

Chitosan hydrogels crosslinked with genipin have shown high mechanical properties, improved swelling, and resistance to water. The biocompatibility of these hydrogels has been evaluated in vitro in 3T3 fibroblasts, macrophages, and dendritic cells and has revealed high cell viability and no cytotoxicity showing high potential for clinical applications [169,170,171]. Another study has used a chitosan–gelatin–polysaccharide scaffold crosslinked with genipin and has shown high neural cell proliferation and migration in vitro, indicating potential applications in neural regeneration to treat traumatic brain injuries [172]. Similarly, a spinal cord ECM matrix has been developed through genipin crosslinking for treating spinal cord injuries [173]. In addition, using genipin can increase stiffness in a collagen-based scaffold that is able to induce chondrogenesis [174]. Other studies have demonstrated the efficiency and biocompatibility of genipin-crosslinked scaffolds for applications in corneal tissue engineering [175], hepatic tissue engineering [176], and wound healing [177].

Genipin has also been used as a crosslinker in a hydroxyapatite–chitosan–L-arginine scaffold, leading to a 3-dimensional porous hydrogel that can be effectively applied for bone regeneration with high cell viability, as demonstrated by biocompatibility assays [178]. Various studies have evaluated the use of genipin-crosslinked scaffolds for applications in bone tissue engineering [139]. To ensure biocompatibility, data recommend keeping the dose below 0.5 mM for non-cytotoxic effects on cells [139,167,179]. Most of these scaffolds were based on chitosan, collagen, gelatin, and hydroxyapatite, and were tested on different types of established cell lines. Mouse and rabbit models have also been used to test the effectiveness of these scaffolds in vivo (reviewed in [139]). The concentrations of genipin used in these studies were variable, indicating that there is a need to optimize the concentration for tissue-specific applications. Together, the analysis of all data indicates that genipin use is considered effective and biocompatible when used at the optimal minimum concentration.

A range of other natural crosslinkers has been promising for use in tissue repair. Crosslinking using citric acid is able to promote the osteogenic differentiation of mesenchymal stem cells for bone regeneration [180] and improve mechanical strength and durability in methylcellulose hydrogel for applications in cell sheet engineering [181]. Tannic acid has been shown to be effective in wound healing applications with no adverse effects on cell function [182]. In addition, dual crosslinking of procyanidins with GTA has allowed the development of a vascular scaffold with higher mechanical strength and lower calcification for application in the tissue engineering of blood vessels [183]. The use of either cinnamaldehyde or epigallocatechin gallate crosslinkers increases the biomechanical strength of collagen scaffold in dental pulp cells, leading to improved cell proliferation, differentiation, and adhesion [184,185]. Epigallocatechin gallate has also been used to crosslink gelatin sponges, leading to improved bone formation [186].

In conclusion, with the continuous demand in regenerative medicine for optimal scaffolds for tissue engineering, it is important for scientists to keep on developing novel matrices based on crosslinked material that can match the microenvironment of the surrounding tissues while maintaining negligible cytotoxicity (Table 3).

Table 3. The main chemical crosslinkers used in tissue engineering applications, with some limitations and possible solutions.

Crosslinker	Limitations and Possible Solutions	Applications	References
Glutaraldehyde	- Cell toxicity and inflammation in vivo - Needs washing - Concentration used needs to be less than 8%	Bioprostheses for heart valve replacement Bone tissue engineering	[145,147] [139,145,147]
Carbodiimide	- Expensive - May induce low cell adhesion - Concentration reduced 10-fold for biocompatibility	Ophthalmic applications Bone tissue engineering Wound healing	[148] [139] [139,148,187]
Epoxy Compounds	- Cell toxicity	Bone tissue engineering Skin regeneration Ophthalmic applications Wound healing	[139] [155] [156] [139,155,156,157]
Genipin	- Expensive - Concentration used is advised to be less than 0.5 mM - Need to optimize concentration for specific tissue applications	Corneal tissue engineering In vertebral disc repair Bone tissue engineering Tendon tear repair Knee injury treatment Neural and spinal cord regeneration Chondrogenesis Hepatic tissue engineering wound healing	[163,175] [164,165] [139,178] [166,167] [168] [172,173] [174] [176] [139,163,164,165,166,167,168,172,173,174,175,176,177,178]
Citric Acid	- Might need catalyst - Might need high temperature for crosslinking	Bone tissue engineering Cell sheet engineering	[180] [181]
Tannic Acid	- Need to optimize concentration, pH, and temperature conditions	Wound healing	[182]

Table 3. The main chemical crosslinkers used in tissue engineering applications, with some limitations and possible solutions.

Crosslinker	Limitations and Possible Solutions	Applications	References
Glutaraldehyde	- Cell toxicity and inflammation in vivo - Needs washing - Concentration used needs to be less than 8%	Bioprostheses for heart valve replacement Bone tissue engineering	[145,147] [139,145,147]
Carbodiimide	- Expensive - May induce low cell adhesion - Concentration reduced 10-fold for biocompatibility	Ophthalmic applications Bone tissue engineering Wound healing	[148] [139] [139,148,187]
Epoxy Compounds	- Cell toxicity	Bone tissue engineering Skin regeneration Ophthalmic applications Wound healing	[139] [155] [156] [139,155,156,157]
Genipin	- Expensive - Concentration used is advised to be less than 0.5 mM - Need to optimize concentration for specific tissue applications	Corneal tissue engineering In vertebral disc repair Bone tissue engineering Tendon tear repair Knee injury treatment Neural and spinal cord regeneration Chondrogenesis Hepatic tissue engineering wound healing	[163,175] [164,165] [139,178] [166,167] [168] [172,173] [174] [176] [139,163,164,165,166,167,168,172,173,174,175,176,177,178]
Citric Acid	- Might need catalyst - Might need high temperature for crosslinking	Bone tissue engineering Cell sheet engineering	[180] [181]
Tannic Acid	- Need to optimize concentration, pH, and temperature conditions	Wound healing	[182]

6. Chemical Aspects of Crosslinkers

The development and refinement of chemical crosslinkers have become the center of attention in materials science, polymer chemistry, and biomedical research. Recent examinations have introduced novel approaches to improve crosslinking effectiveness, strengthen structural durability, and encourage eco-friendly solutions (Figure 5). Many tools and methods are commonly used to measure crosslinking and crosslinking density in polymers: swelling tests, differential scanning calorimetry (DSC), infrared spectroscopy (FTIR), dynamic mechanical analysis (DMA), nuclear magnetic resonance (NMR), mechanical testing (tensile strength, compression set, etc.), and electron spin resonance (ESR). Often, a combination of techniques is used to obtain a comprehensive understanding of the material’s crosslinked network.

This section explores the major developments in sustainable crosslinking techniques, innovative hydrogel compositions, and advanced chromatographic materials while focusing on the strategies and optimization methods utilized.

A significant study in this area, conducted by Zhang and colleagues in 2024, explores advancements in sustainable dual-crosslinking polymer networks (DCPNs) [188]. The research explores the combination of dynamic covalent bonds, such as imine, disulfide, and ester bonds, with non-covalent forces, such as hydrogen bonding and coordination interactions, to enhance both mechanical performance and recyclability. The study optimizes crosslinking efficiency through molecular dynamics simulations and rheological assessments. Researchers were able to achieve a customizable balance between elasticity and rigidity by adjusting the proportion of covalent and non-covalent crosslinking elements, thus making these materials highly relevant for applications in flexible electronics and biomedical scaffolds.

The improvement of hydrogel technology has taken a significant leap forward with the introduction of formulations that do not require crosslinkers. A study by Alsaafeen et al. [189] in 2023 outlines a streamlined, single-step method for creating hydrogels using a combination of gelatin, chitosan, and glycerol. Instead of depending on conventional chemical crosslinkers, this approach exploits hydrogen bonding and hydrophobic interactions to achieve gel formation. Through techniques like differential scanning calorimetry (DSC) and rheometry, the researchers fine-tuned the gelation time and temperature, showing that a controlled cooling process strengthens the hydrogel network and enhances its mechanical properties. These insights are particularly valuable for biomedical fields such as tissue engineering and wound care, where eliminating synthetic crosslinkers reduces potential cytotoxic effects.

The effectiveness of linking proteins and polysaccharides depends significantly on the specific reaction conditions and type of crosslinking agents used. A study by Alavarse et al. [190] explored different crosslinking strategies, including enzymatic, chemical, and physical approaches. The researchers applied analytical techniques such as Fourier-transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR) spectroscopy to measure bond formation to evaluate crosslinking density. By adjusting factors like pH, ionic strength, and temperature, they enhanced gel strength and improved the resistance of protein–polysaccharide complexes to degradation. Such insights contribute to the advancement of biocompatible hydrogels for applications in drug delivery and tissue regeneration.

Achieving efficient crosslinking in chromatographic stationary phases is essential in the field of analytical chemistry. A study by Zhang et al. [191] investigated strategies to enhance the synthesis of hyper-crosslinked stationary phases tailored for ultra-fast, high-temperature, two-dimensional liquid chromatography. The work employed high-performance liquid chromatography (HPLC) to assess how varying crosslinker concentrations impact retention times and separation efficiency. By incorporating a modified Friedel–Crafts alkylation technique, they strengthened the polymer network, thus enabling it to function at elevated temperatures without deterioration. Adjustments to reaction durations and catalyst levels have further refined chromatographic resolution, highlighting its relevance for pharmaceutical and environmental applications.

In nanomedicine, researchers have made advancements in crosslinking techniques, where a recent study by Siddoway et al. (2023) introduced an innovative pentablock copolymer tailored for vaccine delivery [192]. The work utilized dynamic light scattering (DLS) and transmission electron microscopy (TEM) to analyze the size and structural characteristics of the nanoparticles. By employing a factorial experimental approach, the team optimized the balance between hydrophobic and hydrophilic segments, resulting in an enhanced immune response with minimal toxicity. The copolymer’s crosslinked core played a crucial role in prolonging antigen retention and enabled controlled release, thus representing a notable breakthrough in vaccine development.

In the field of tissue engineering, genipin, a naturally occurring compound known for its ability to facilitate crosslinking, has been extensively investigated. A study conducted by Pugliese et al. [193] in 2019 examined the formation of peptide-based hydrogels, utilizing genipin as a crosslinking agent. Through rheological assessments and analysis of the swelling behavior, the researchers refined the crosslinking parameters. By modifying the concentration of genipin and the duration of the reaction, they developed hydrogels with improved mechanical strength and controlled degradation rates. These optimized hydrogels demonstrated strong cell attachment and proliferation, making them promising for use in regenerative therapies and drug release systems. Recent developments and refinement of crosslinkers have led to pronounced enhancements in material durability, compatibility with biological systems, and eco-friendliness. Techniques such as computational modeling, experimental design strategies, and sophisticated spectroscopic methods have allowed for greater precision in regulating crosslinking mechanisms. Accordingly, research efforts should prioritize eco-friendly crosslinkers (i.e., the production of safe, biodegradable alternatives to conventional synthetic crosslinking agents), optimized multi-scale approaches (i.e., combining simulation techniques with experimental data to better predict and fine-tune crosslinking processes), and adaptive polymer networks (i.e., investigating crosslinkers that respond to environmental triggers like pH shifts, temperature variations, or external stimuli for versatile material applications). By promoting innovation through interdisciplinary research, scientists can continue to improve material performance and broaden the functional scope of next-generation polymer systems.

Crosslinkers are vital in shaping the mechanical properties, stability, and degradation patterns of polymers, especially in biomedical and structural contexts. By altering the concentration, form, and chemical composition of crosslinks, scientists can adjust the material’s durability, flexibility, and rate of degradation. Various studies have examined these influences, offering valuable knowledge for creating advanced materials with specific and desired performance traits.

Mechanical strength is a crucial characteristic of polymer networks since it defines their capacity to tolerate external stressors without deforming or failing. The mechanical behavior of crosslinked materials is primarily governed by factors such as crosslinker density, bond strength, and the structure of the polymer chains. In a study by Jiang et al. [194] in 2013, it was shown that varying crosslinking methods impacted the stiffness and toughness of polymer networks. Their work indicated that networks with a high degree of crosslinking exhibited greater stiffness due to a more compact polymer framework, which improved their ability to withstand more load. However, excessively dense crosslinking may lead to brittleness, making the material less capable of absorbing mechanical energy and more prone to cracking under stress. Further research emphasized that adjusting crosslinker density in hydrogels allows for fine-tuning of their mechanical properties. The work on degradable hydrogels revealed that increasing crosslink density boosted the material’s initial strength but could also slow down its degradation rate, which might affect its long-term effectiveness in biomedical applications [195,196].

Polymeric materials must retain their structural integrity over time, even when exposed to various mechanical, chemical, or environmental strains. A method to enhance the stability of chemically crosslinked hydrogels was developed by adjusting the chemistry of the crosslinkers. The findings revealed that hydrogels with covalent crosslinks resisted mechanical and hydrolytic breakdown more effectively, making them well suited for applications like tissue engineering scaffolds and drug delivery systems [197]. Similarly, Carberry et al. [198] explored how the chemical properties of crosslinkers influenced stability through their interactions with the surrounding environment. Hydrogels crosslinked with thioesters, for example, exhibited adjustable stability depending on factors such as thiol concentration and the pKa of the crosslinkers. These insights are valuable for designing biomaterials that should remain stable for a set period of time before undergoing controlled degradation.

The rate at which polymeric materials degrade plays a key role in biomedical fields, particularly in drug delivery systems and tissue regeneration. Ideally, degradation should occur in a controlled manner, thus ensuring that the material preserves structural integrity while gradually breaking down in response to specific environmental factors. In a 2007 study, the degradation behavior of chemically crosslinked hyaluronic acid hydrogels was examined, revealing that the choice of crosslinker significantly influences the degradation rate. The research highlighted that the breakdown of hydrogels can be regulated by selecting crosslinkers with certain chemical properties, such as a hydrolysable ester or disulfide bonds [199]. Likewise, another study developed an innovative biodegradable polymer crosslinker that enabled the independent regulation of stiffness, toughness, and the degradation rate. This finding facilitated the creation of materials that combined mechanical durability with predictable degradation, making them ideal for applications in soft tissue engineering and wound healing [200].

The research discussed above underlines the significant impact of crosslinker chemistry on the mechanical properties, stability, and degradation of polymeric materials. By carefully choosing the right crosslinkers and adjusting their concentrations, scientists can create materials with specific desired characteristics, making them suitable for use in fields like biomedical engineering, drug delivery, and advanced structural applications. The ability to adjust these properties provides new possibilities in the development of smart materials, where crosslinking methods are employed to design adaptable and responsive systems for specialized uses.

Figure 5. Approaches of different crosslinking processes providing different mechanical strengths and properties (A) bonding groups: thermal gelation, hydrogen bonding and ionic crosslinking and (B) crosslinking via radiation, enzymes or photocrosslinking [201].

Figure 5. Approaches of different crosslinking processes providing different mechanical strengths and properties (A) bonding groups: thermal gelation, hydrogen bonding and ionic crosslinking and (B) crosslinking via radiation, enzymes or photocrosslinking [201].

7. Medical Applications of Crosslinked Biomaterials and Biomembranes

The use of chemical crosslinkers has gained immense interest in the development of suitable scaffolds for tissue engineering and regenerative medicine and has been significantly implemented in advanced drug delivery technologies. These applications are being explored to overcome the use of traditional organ transplantation techniques, helping in replacing damaged tissues due to diseases, aging, or accidental injuries. Crosslinkers help in enhancing the biomechanical strength, physical properties, and the structural integrity of the scaffold materials, leading to improved biocompatibility and biodegradation. Crosslinked biomaterials have been especially employed in the fields of obstetrics and gynecology. These materials enable localized, controlled, and sustained therapeutic delivery, exemplified by drug-releasing intrauterine devices (IUDs). These devices offer the long-term delivery of contraceptive hormones, anti-inflammatory drugs, or antibiotics, reducing the reliance on systemic treatments and improving adherence to therapy [202]. For instance, crosslinked polymer matrices have been pivotal in the prolonged release of levonorgestrel, a widely used contraceptive hormone, providing an effective and low-maintenance solution for years [203,204]. Current innovations are focusing on biodegradable crosslinked materials that dissolve after releasing their drug payload, eliminating the need for device removal and enhancing patient convenience (Table 4).

Crosslinked hydrogels are increasingly being used for targeted drug delivery in the treatment of uterine disorders such as endometriosis and adenomyosis. These systems deliver anti-inflammatory and anti-fibrotic drugs directly to the uterine lining, ensuring precise treatment with minimal systemic effects. In managing postpartum hemorrhage, crosslinked systems are utilized to deliver hemostatic agents directly to the source of bleeding, improving treatment effectiveness [205]. In gynecological oncology, crosslinked nanoparticles are emerging as precision drug carriers, particularly in ovarian and endometrial cancer treatments. These nanoparticles can target specific cancer cell receptors, allowing for highly specific drug delivery with reduced toxicity [206]. Stimulus-responsive systems, which release drugs based on environmental triggers like pH or temperature, further enhance the precision and control of drug delivery.

Microneedle patches made from crosslinked polymers are being developed as non-invasive solutions for the transdermal delivery of hormones and vaccines, including contraceptives and HPV vaccines, offering a more patient-friendly alternative to injections [207]. Additionally, crosslinked hydrogels have been used to deliver tocolytic agents locally at the cervix to prevent preterm labor, improving outcomes for both mothers and infants while minimizing systemic exposure [208,209].

On the other hand, crosslinked biomaterials play a crucial role in enhancing wound healing and tissue regeneration after obstetric and gynecological surgeries. Crosslinked hydrogels and membranes serve as effective wound dressings by maintaining moisture, promoting cell growth, and protecting against infections [210,211]. For example, hydrogels made from chitosan have demonstrated benefits in cesarean wound care by accelerating tissue repair, minimizing scarring, and providing antimicrobial properties [212,213].

Postoperative adhesion prevention is another critical application. Crosslinked biomembranes act as physical barriers to reduce adhesion formation, a common complication that can lead to chronic pain, infertility, or bowel issues. Biodegradable membranes, such as those made from hyaluronic acid, have shown excellent results in clinical use for minimizing adhesions [214,215].

In reconstructive surgeries, particularly after gynecological cancer treatments, crosslinked scaffolds like silk fibroin promote cell growth and vascularization, making them ideal for vaginal reconstruction [216,217]. Similarly, alginate-based systems support tissue regeneration by encouraging angiogenesis, providing solutions for extensive reconstructive needs [218]. For conditions like Asherman’s syndrome, bioengineered uterine scaffolds made from crosslinked biomaterials help regenerate tissue, restore uterine function, and prevent adhesions [219].

Crosslinked biomaterials have enabled the creation of advanced implants for gynecological use. Biodegradable vaginal stents made from crosslinked polymers are used following reconstructive or prolapse surgeries, offering improved comfort and eliminating the need for removal, unlike traditional silicone stents [220]. Pelvic organ prolapse treatments now incorporate crosslinked collagen scaffolds to reinforce weakened tissues, reducing complications like erosion and infection associated with synthetic meshes [221,222]. Injectable hydrogels with crosslinked structures provide minimally invasive options for stress urinary incontinence, offering structural support and long-lasting results [209].

Crosslinked polymer meshes, enhanced with antimicrobial agents or growth factors, improve biocompatibility and reduce inflammation in urogynecological surgeries. Crosslinked elastomers are also being explored for cervical cerclage devices, offering better mechanical support during high-risk pregnancies [223].

In fertility preservation, crosslinked hydrogels encapsulate ovarian tissue during cryopreservation to maintain viability [224,225]. Artificial oocyte creation using crosslinked matrices is another area of research with the potential to improve outcomes for individuals undergoing fertility-compromising treatments [226]. Additionally, hydrogels serve as carriers during embryo transfer, providing a protective environment and optimizing implantation success rates [225,227].

Crosslinked biomaterials are transforming in vitro fertilization (IVF) by addressing challenges like oocyte preservation, embryo culture, and implantation. Hydrogels protect oocytes and embryos during cryopreservation, preserving their structure and viability, especially for women undergoing fertility-threatening treatments [225,228]. Encapsulation techniques improve post-thaw survival and developmental potential [229].

Three-dimensional scaffolds made from crosslinked polymers replicate the uterine environment, enhancing embryo growth and implantation success rates compared to traditional culture methods [230,231]. Crosslinked hydrogels are also used to deliver hormones and growth factors to the uterine lining, creating a favorable environment for embryo implantation [232,233].

Bioengineered uterine scaffolds address implantation challenges, such as scarring, by mimicking healthy uterine tissue and promoting regeneration [234,235]. Localized hormone delivery via crosslinked hydrogels further supports implantation while minimizing side effects [206].

Research into artificial gametes and ovarian tissue preservation using crosslinked biomaterials offers new hope for patients with diminished ovarian reserves. These advancements represent a significant shift in reproductive medicine, providing innovative solutions to overcome infertility [236].

Table 4. Examples of crosslinked biomaterials in obstetrics and gynecology applications.

Application	Material	Function	References
Drug-Eluting IUDs	Crosslinked polymers	Sustained release of contraceptive hormones	[237]
Postpartum Hemorrhage Control	Crosslinked hydrogels	Localized delivery of hemostatic agents	[238,239]
Cesarean Section Wound Dressing	Chitosan hydrogels	Enhanced healing and antimicrobial activity	[211]
Adhesion Prevention Membranes	Hyaluronic acid membranes	Prevention of postoperative adhesions	[240]
Vaginal Stents	Crosslinked biodegradable polymers	Structural support post-surgery	[241]
Injectable Hydrogels for Incontinence	Crosslinked hydrogels	Minimally invasive structural support	[209]
Cancer Drug Delivery Systems	Crosslinked nanoparticles	Targeted therapy for gynecological cancers	[206]
Reconstructive Surgery Scaffolds	Crosslinked silk fibroin	Support tissue regeneration	[216,217]
Cervical Cerclage Devices	Crosslinked elastomers	Mechanical support during pregnancy	[241,242]

Table 4. Examples of crosslinked biomaterials in obstetrics and gynecology applications.

Application	Material	Function	References
Drug-Eluting IUDs	Crosslinked polymers	Sustained release of contraceptive hormones	[237]
Postpartum Hemorrhage Control	Crosslinked hydrogels	Localized delivery of hemostatic agents	[238,239]
Cesarean Section Wound Dressing	Chitosan hydrogels	Enhanced healing and antimicrobial activity	[211]
Adhesion Prevention Membranes	Hyaluronic acid membranes	Prevention of postoperative adhesions	[240]
Vaginal Stents	Crosslinked biodegradable polymers	Structural support post-surgery	[241]
Injectable Hydrogels for Incontinence	Crosslinked hydrogels	Minimally invasive structural support	[209]
Cancer Drug Delivery Systems	Crosslinked nanoparticles	Targeted therapy for gynecological cancers	[206]
Reconstructive Surgery Scaffolds	Crosslinked silk fibroin	Support tissue regeneration	[216,217]
Cervical Cerclage Devices	Crosslinked elastomers	Mechanical support during pregnancy	[241,242]

Many crosslinked materials have been FDA-approved, especially in the biomedical and drug delivery feed. The most important ones are as follows:

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Restasis (Cyclosporine A): A formulation for treating dry eye disease using hydrogel crosslinked based systems for sustained drug release.

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Vicryl (Polyglactin 910): A crosslinked polymer suture material used in both absorbable and non-absorbable forms.

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Monocryl (Poliglecaprone 25): A monofilament absorbable suture made from a crosslinked polymer, designed for fast absorption and minimal tissue irritation.

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Duramorph (morphine sulfate): A sustained-release form of morphine delivered via a crosslinked polymeric matrix used in certain pain management systems.

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Lupron Depot (leuprolide acetate): A crosslinked PLGA-based depot formulation for the controlled release of leuprolide in the treatment of prostate cancer and endometriosis.

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Depo-Provera (medroxyprogesterone acetate): An injectable contraceptive formulation using crosslinked PLGA for sustained release over months.

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Aquacel (hydrocolloid dressing): Made from crosslinked sodium carboxymethylcellulose, it helps in absorbing exudates and promoting healing in chronic wounds.

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Marqibo (vincristine sulfate liposome): A liposomal formulation of vincristine for cancer treatment.

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Cypher (sirolimus-eluting stent): A drug-eluting stent that uses crosslinked polymer coatings to release sirolimus for the prevention of restenosis.

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Tisseel (fibrin sealant): A crosslinked fibrin-based adhesive used in surgery to help control bleeding and promote tissue healing.

8. Recent Advances and Innovations

The design and the development of biomaterials and biomembranes have seen significant advancements through innovative crosslinking techniques. Among these, light-induced and enzyme-mediated crosslinking techniques have gained more attention due to their precision, biocompatibility, and adaptability to diverse biomedical and bioengineering applications.

Recently, light-crosslinking techniques have been used to design hydrogels for cartilage regeneration applications because of their capacity to control the location of crosslinking and the timing under physiological environments [243]. Light is a clean energy source that can be used specifically with regard to wavelength (in the ultraviolet UV to infrared IR region) [244]. In this technique, two polymer chains are covalently bounded after exposure to light irradiation in the presence of photo-initiators. The light irradiation offers fast crosslinking within seconds to minutes, making it ideal for use in 3D-bioprinting, dental materials, and rapid polymerization. However, biological changes caused by UV radiation have long been considered harmful, such as in cases caused by chemical agents, but have also been recognized as a benefit [245,246]. Many studies reported in the literature have shown the potential dose–response cytotoxicity of some photoinitiators, and there are several biosafety concerns associated with the application of UV light when it comes to using living cells and tissues. Long exposure times and high light intensities damage the cell’s DNA and may generate reactive oxygen species that induce accelerated tissue aging and/or immunosuppression [247]. This challenge was overcome by the use of longer wavelengths, including visible light, in conjunction with a photoinitiator that can be activated by those wavelengths [248].

Recent efforts have focused on the search for mild crosslinking methods that can take place in the presence of cells by means of non-cytotoxic reactions. Enzymatic crosslinking is the most cytocompatible process that preserves cell viability and bioactivity since this technique relies on the use of a biocatalyst that drives the formation of covalent bonds between the biomolecules and non-exogenous reagents that are used. As enzymes naturally exist in the body, the biocompatibility of enzymatically crosslinked hydrogels is higher than that of chemical hydrogels crosslinked via other methods, such as photo, thermal, or chemical–catalyst crosslinking (Figure 6). Enzyme-mediated crosslinking offers a “greener” approach to covalent bond formation in biology and biotechnology. This could include the use of aqueous solvents in mild conditions, at room temperature, and with heavy metal free reagents, making it ideal for biomedical applications, food science, and tissue engineering [245,249].

Figure 6. Enzyme-mediated crosslinking processes using (a) Transglutaminase, (b) Tyrosinase, (c) Horseradish peroxidase and (d) Lysyl oxidase (reproduced from [243] under the license number 5998490451810).

Figure 6. Enzyme-mediated crosslinking processes using (a) Transglutaminase, (b) Tyrosinase, (c) Horseradish peroxidase and (d) Lysyl oxidase (reproduced from [243] under the license number 5998490451810).

Enzyme-mediated crosslinking using tyrosinases, transferases, or peroxidases have proven its efficiency and has attracted increasing attention for application in polymer hydrogel synthesis due to the environmentally friendly process and the possibility of obtaining biomaterials [250].

9. Challenges and Future Directions

The increasing demand for biomaterials, the development of new biomembranes, and the growth rate of their markets make it crucial to develop new crosslinkers that better suit the applications of advanced biomaterials and biomembranes while addressing the issues related to biocompatibility and production scalability. Among the list of new crosslinkers, click chemistry-based crosslinkers, photo-crosslinkable crosslinkers, enzyme-based crosslinkers, and self-healing crosslinkers are included.

Click chemistry-based crosslinkers are considered as highly efficient and sensitive crosslinkers [251]. They retained attention due to their specificity, minimizing by-products, and their ability to function under mild conditions. Key types of click chemistry-based reactions for crosslinking are as follows: copper-catalyzed azide–alkyne cycloaddition (CuAAC) [252], copper-free alkyne cycloaddition (SPAAC) [253,254], the Diels–Alder (DA) reaction [255], thiol–ene reactions [256], and tetrazine–norbornene ligation [257]. The main difference between these click chemistry-crosslinkers remains in the functions that are involved in the bonding and the formation of the new structure (the reaction between an azide group (−N₃) and an alkyne group (−C≡C) with or without a catalyst (copper), cycloaddition between a diene group and a dienophile, the reaction between a thiol group (−SH) and an ene group (C=C), and the reaction between tetrazines and strained norbornene derivatives). However, the main advantage includes the biocompatibility issue, particularly for those functioning as copper-free crosslinkers (without catalyst), making them tolerated in biological systems [258,259].

The photo-crosslinkable crosslinkers refer to chemicals that can be activated by ultraviolet (UV) light, leading to the formation of covalent bonds between molecules. Typically, this type of crosslinker requires an activator to initiate the bonding and is inactive without a light inducer [260]. The use of light eliminates the need for high temperatures or harsh chemicals, making the process of crosslinking safe and more environmentally friendly. Some types of chemical compounds are necessary to allow the covalent bonds formation, e.g., cinnamate groups [261], azide groups [262], diazirines, and benzoin derivates [263]. Depending on the target application, the chemical compound is selected to create the bonds while achieving mechanical strength and the required stability. Cinnamate groups are mainly used in creating photo-responsive hydrogels, coatings for medical devices, or controlled drug delivery systems [264], while azide-based crosslinkers are generally utilized in bioconjugation and biomaterial engineering. On the other hand, diazirine-based and benzoin derivatives are used for studying protein–ligan interactions [265], in creating photo-cross-linked biopolymers, and in dental materials and 3D-printing materials that can be cured layer-by-layer using light [266]. Finally, the main challenges and considerations that should be taken into account while using photo-crosslinkable crosslinkers are the following: First, the degree of light penetration, where the ability of crosslinking depends on the ability of light to penetrate in deep homogenous materials. Second, the precision of the crosslinking process to ensure uniform crosslinking is sometimes challenging in complex geometries and when dealing with biological systems. The last challenge is the toxicity of some compounds like azides or diazirines that require special attention when used in some applications.

Enzyme-based crosslinkers seem to be the safest in the list of crosslinkers since they rely on the use of natural biological agents, enzymes to catalyze the formation of covalent bonds between molecules [249]. The main interest in using this type of crosslinker is the high level of biocompatibility and the specificity of the bonding formation. In addition, enzyme-based crosslinking occurs at neutral pH in the absence of harsh chemicals (mild conditions) and produces fewer by-products than the typical chemical crosslinker process. The common enzyme-catalyzed crosslinking reaction includes three types of reactions: (1) oxidative crosslinking using oxidase enzymes (e.g., laccases, peroxidases, etc.) [267,268], (2) transamination using transaminases or aminotransferases (e.g., transglutaminases) [269,270], and (3) esterification or amide bond formation using esterases [271,272] or transglutaminases enzymes [269,273]. Based on the target application (biomaterials and tissue engineering, pharmaceutical applications, food industry [274], environmental remediation, etc.), the enzyme is selected to create the bonds while achieving mechanical strength and the required stability [275]. However, the main limitations of these processes are related to the loss of enzymatic activity at extreme conditions or in the presence of high concentrations of salts and the cost that can be generated for scalability while using purified enzymes in large-scale industrial applications.

Finally, self-healing crosslinkers refer to agents or materials that can restore their original structures and properties after damage by physical stress or environmental factors [276]. Even if they are relatively less used than other crosslinkers agents for biological applications, they may offer innovative solutions for enhancing the durability, longevity, and functionality of materials for industrial applications [277]. They usually work through a combination of dynamic covalent bonds, non-covalent interactions, or reversible chemical reactions [278,279]. Self-healing crosslinkers involve Diels–Alder reactions, ionic bonding in polymers, reversible hydrogen bonding, microencapsulation and vascular networks, and polymer networks with reversible crosslinks. On the other hand, it is challenging and complex to design materials with self-healing properties, especially in terms of achieving controlled and reproducible healing under various conditions. The cost of the self-healing materials can also be expensive due to the complexity of manufacturing, which takes this factor into consideration.

Future directions on the role of crosslinkers in biology, chemistry, and medicine will focus on improving the crosslinking efficiency and selectivity. At the same time, addressing biocompatibility issues will be important to develop more eco-friendly and less cytotoxic crosslinkers in Figure 7 [280]. This will enable the use of more biomaterials and the development of innovative bio-membranes while maintaining affordable fabrication and distribution costs.

Figure 7. Using click chemistry to crosslink refillable depots. Left to middle: azide–alginate strands are combined and injected into target tissues and crosslinked in situ. Middle to right: intravascular administration of cyclooctyne-conjugated therapeutics allows selective capture and display of drug at gel site (reproduced with permission from [280] under license number 5998491504493).

Figure 7. Using click chemistry to crosslink refillable depots. Left to middle: azide–alginate strands are combined and injected into target tissues and crosslinked in situ. Middle to right: intravascular administration of cyclooctyne-conjugated therapeutics allows selective capture and display of drug at gel site (reproduced with permission from [280] under license number 5998491504493).

10. Conclusions

In conclusion, biomaterials and biomembranes enhanced by the various types of crosslinkers continue to revolutionize different fields (chemistry, biology, and medicine). Crosslinkers play an important role in improving the structural integrity and functionality of biomaterials and in biomembranes design, leading to significant advancements in applications such as tissue engineering, drug delivery, and wound healing. Despite the promising progress, the toxicity of many crosslinkers and the need for more specificity in crosslinking mechanisms remain a challenging need in order to achieve a biocompatible and eco-friendly process. Future research should focus on developing more biocompatible crosslinkers, exploring more synthesis methods, and optimizing their interaction with biological systems to express their full potential. The integration of crosslinkers into biomaterials design will be key to achieving effective, personalized, and sustainable medical solutions.