Physical vs. chemical crosslinking methods

Updated: Jun 10

Crosslinking is a stabilization process in polymer chemistry that promotes the multidimensional interconnection of polymer chains, turning a liquid polymer into a solid or gel. This process is essential for polymers to become mechanically stronger and resistant to heat, wear or solvent attack, for example. Hydrogels are prevented from dissolving due to their crosslinked networks. Various crosslinking strategies can be applied to promote polymer network reticulation, which includes physical and chemical methods. 1 2

Figure 1: Synthesis of hydrogels by crosslinking polymer networks using physical or chemical methods. 3

Physically crosslinked hydrogels are usually stabilized by ionic/electrostatic interactions, hydrogen bonds or hydrophobic interactions. The main advantage of using physical crosslinking methods is biomedical safety due to the absence of chemical crosslinking agents, which avoids potential cytotoxicity. 1 2

Techniques to obtain chemical hydrogels include photopolymerization, enzyme-induced crosslinking, and Schiff base formation. Compared to physically crosslinked hydrogels, chemically crosslinked hydrogels generally exhibit greater stability under physiological conditions, excellent mechanical properties, and adjustable degradation behavior, due to the permanent covalent bonds formed. In addition, the links created are more substantial and more permanent, for example, permanent covalent bonds. 1 2

A wide spread bioink formulation is constituted by ionically crosslinked alginate hydrogel. In this case, crosslinking is usually mediated by calcium ions or other bivalent cations that promote electrostatic interactions between polymer chains. Cao et al (2012) carried out an experimental investigation on the influence of calcium ions on Schwann cell survival and proliferation during alginate hydrogel crosslinking in the biofabrication process. Ionically crosslinked alginate can provide a favorable environment for cell survival and proliferation. The study suggested that cell viability and proliferation in the hydrogel depends on alginate concentration and cell density, in addition to calcium concentration and time of exposure to calcium chloride solution. 4

In another context, using enzymatic chemical crosslinking, Broguiere et al. (2018) studied bacterial ligase Sortase A (SA) as a crosslinking enzyme for hydrogel-based tissue engineering. This study used hyaluronan (HA) as a model biopolymer and modified it with SA substrate peptides. The crosslinking kinetics, enzyme stability, cytocompatibility, and immunogenicity were studied, and the results were compared with controls of standard techniques. The team concluded that SA was remarkably more stable in solution than the control and could provide faster gelation. These results endorse the use of SA as a versatile enzymatic crosslinking strategy for 3D culture and tissue engineering applications. 5 6

Figure 2: Demonstration of underwater instant gelling with 1.5% HA-SA and phenol red for gel visualization. From left to right: the beginning of injection, end of injection, and after removing the water. 6

GelMA is an inexpensive protein-based polymer that can be cross-linked in the presence of a water-soluble photoinitiator and exposure to light, which leads to the formation of covalent bonds between acrylate groups. GelMA is commonly produced through the reaction of gelatin with methacrylic anhydride, allowing the chemical crosslinking of the polymer in addition to its thermal crosslinking, a physical type of crosslinking. Seeking to obtain a GelMA rigid enough to use in cartilage repair associated with mesenchymal stem cells derived from adipose tissue, Duchi et al. (2017) analyzed the crosslinking capacity of three different photoinitiators - LAP, Irgacure 2959 and VA086 - at a constant concentration of 0.1% w/v. Initially, the team's focus was to screen different photocuring conditions by studying the three photoinitiators and photocuring time. They found that reducing photoinitiator concentration and light intensity can further increase cell viability. Furthermore, LAP showed better results to provide adequate cell viability for the final 3D bioprinted constructs. 7 8


1. Hu, W., Wang, Z., Xiao, Y., Zhang, S. & Wang, J. Advances in crosslinking strategies of biomedical hydrogels. Biomater Sci 7, 843–855 (2019).

2. Maitra, J. & Shukla, V. K. Cross-linking in hydrogels-a review. Am. J. Polym. Sci 4, 25–31 (2014).

3. Bi, X. & Liang, A. In Situ‐Forming Cross‐linking Hydrogel Systems: Chemistry and Biomedical Applications. in Emerging Concepts in Analysis and Applications of Hydrogels (ed. Majee, S. B.) (IntechOpen, 2016).

4. Cao, N., Chen, X. B. & Schreyer, D. J. Influence of Calcium Ions on Cell Survival and Proliferation in the Context of an Alginate Hydrogel. ISRN Chemical Engineering 2012, (2012).

5. Nezhad-Mokhtari, P., Ghorbani, M., Roshangar, L. & Rad, J. S. Chemical gelling of hydrogels-based biological macromolecules for tissue engineering: Photo- and enzymatic-crosslinking methods. International Journal of Biological Macromolecules vol. 139 760–772 (2019).

6. Broguiere, N., Formica, F. A., Barreto, G. & Zenobi-Wong, M. Sortase A as a cross-linking enzyme in tissue engineering. Acta Biomater. 77, 182–190 (2018).

7. Duchi, S. et al. Handheld Co-Axial Bioprinting: Application to in situ surgical cartilage repair. Sci. Rep. 7, 5837 (2017).

8. GhavamiNejad, A., Ashammakhi, N., Wu, X. Y. & Khademhosseini, A. Crosslinking Strategies for 3D Bioprinting of Polymeric Hydrogels. Small vol. 16 2002931 (2020).

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