Induction of fiber-like aggregation and gelation of collagen by ultraviolet irradiation at low temperature
Introduction
Collagen is a major component of the extracellular matrix, providing mechanical integrity and aiding the biological function of connective tissue. It is widely used as a basic building block for biological materials due to its well-known advantages, including good biocompatibility, acceptable biodegradability, and low immunogenicity [1,2]. In vivo, collagen is usually present in the form of highly ordered collagen fibers and confers various tissue-specific functions. For example, collagen fibers provide excellent mechanical properties, such as flexibility, strength, and resilience, as well as thermal stability for connective tissues such as ligaments and tendons [3]. In vitro, by applying suitable conditions, such as pH and temperature, collagen molecules also spontaneously and in orderly fashion form fibrous aggregates, i.e. fiber-like aggregation. Studies have shown that the microenvironment composed of such aggregates is more conducive to cell growth. Research of fiber reorganization as an important molecular behavioral characteristic of natural collagen has run through all aspects of collagen from the molecular behavior to the creation of new materials [4].
Fibrous protein self-assembly of natural collagen is a process of molecular interactions and ordered rearrangement. It is based on excluding water molecules from the surface and enhancing the hydrophobic interactions between collagen molecules [[5], [6], [7]]. Therefore, collagen self-assembly is an energy-consuming process. It can be achieved by increasing the temperature (typically to about 37 °C for mammalian collagen and to about 30 °C for fish collagen), which is referred to as temperature induction [8,9]. If the ambient temperature is below the threshold temperature for fibrillation and self-assembly, a collagen-based material having a fibrous structure is generally not achievable. However, for the preparation of biological materials that require low temperature conditions, such as embedding, loading, and targeted delivery of heat sensitive drugs and functional factors, temperature induction of collagen self-assembly is not an option. It is therefore critical to develop low-temperature induction methods for collagen fiber self-assembly.
UV irradiation is a commonly used method for cross-linking and product sterilization of collagen-based biomaterials that avoids the potential cytotoxicity of chemical cross-linking agents or bactericides [10,11]. Studies have shown that UV irradiation has three different effects on collagen molecules including the induction of intermolecular cross-links, the destruction of tyrosyl and phenylalanyl residues, and the degradation of polypeptide chains leading to conformational changes [[11], [12], [13]]. The wavelength and dose of UV radiation, as well as the physical and chemical environment of the collagen molecules are key factors in determining the effects of the irradiation. By comparing the sensitivity of natural collagen and heat-denatured collagen to UV irradiation, Rabotyagova et al. found that the degradation of collagen molecules induced by UV irradiation was stepwise [12]. UV irradiation first leads to the release of the bound water (intrachain and interchain) of the collagen α chain, which relaxes the conformation of the collagen triple helix, thereby allowing more photosensitive residues (such as tyrosine and phenylalanine) to be fully exposed. This subsequently leads to a photodegradation reaction. Analogous to temperature-induced changes in the molecular structure of collagen, it may be possible to use the relaxation of the triple-helical conformation upon UV irradiation to induce the self-assembly behavior of collagen molecules at low temperatures.
Studies on weakly acidic collagen solutions have shown that UV irradiation causes an increase in the viscosity of the solution at first, followed by a decrease [14]. The increase in viscosity is usually attributed to the cross-linking of the collagen molecules, while studies on the fiber-like aggregation behavior induced by UV irradiation under low temperature or on differential analysis of the properties of the gelation product have not been reported. Therefore, in this study a neutral collagen solution was induced to gel by UV irradiation under low temperature conditions, and compared to the gelation process that was temperature-induced at 37 °C as a control. A turbidity experiment was used to monitor the gelation process. The gelation products induced by UV irradiation were analyzed by hydroxyproline content analysis, transmission electron microscopy (TEM), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier-transform infrared spectroscopy (FTIR). These results led us to propose a mechanism for the UV irradiation-induced gelation process. Subsequently, thermal stability analysis, texture analysis, and cell growth assay were used to characterize differences in performance between the UV irradiation-induced gelation product and the temperature-induced gelation product.
Section snippets
Materials
Type I collagen extracted from bovine tendons was purchased from Kaolisen Biotechnology Co., Ltd. (Hebei, China). The other reagents used in the experiments were of analytical grade and were purchased from Shanghai Colloid Chemical Plant (Shanghai, China) and were used as received.
Different methods of inducing collagen gelation
Collagen was dissolved in 0.1 mol/L acetic acid at a concentration of 2 mg/mL. The solution was neutralized by dialysis against PBS (pH = 7.4, containing 0.15 mol/L NaCl), and stored at 4 °C until use.
Analysis of the gelation process
The gelation process of neutralized collagen solutions was monitored by a turbidity experiment as shown in Fig. 1. The absorbance at 310 nm during UI increased slowly with the increase of the UV irradiation dose, which shows that UV irradiation can induce collagen gelation. Furthermore, to rule out the possibility of induction of collagen gel by a rise in temperature caused by UV irradiation, we also assessed changes in turbidity of neutralized collagen solutions at 17 °C, the highest
Conclusion
The temperature-induced fiber-like aggregation and gelation of natural collagen are widely used in medicine, food production, and other fields. In this study, we found that UV irradiation causes formation of fiber-like aggregates and induces the gelation of collagen at low temperatures. The resulting gel displays improved thermal stability, mechanical properties, and cell growth compatibility compared with collagen gels that were temperature-induced at 37 °C. This study provides a novel method
CRediT authorship contribution statement
Chengzhi Xu: Conceptualization, Methodology, Writing - review & editing. Xu Wei: Writing - original draft. Feiyi Shu: Data curation. Xinxin Li: Investigation. Wenxin Wang: Resources. Ping Li: Formal analysis. Yuanyuan Li: Validation. Siman Li: Visualization. Juntao Zhang: Funding acquisition. Haibo Wang: Supervision, Project administration.
Declaration of competing interest
We declare that we do not have any commercial or associative interests that might represent a conflict of interest in connection with the work submitted.
Acknowledgements
This research was supported by the National Natural Science Foundation of China (Nos. 21676208, 21706201), Natural Science Foundation of Hubei Province (Nos. 2018CFA030, 2019CFB252), Foundation of Hubei Educational Commission (No. Q20181806), Wuhan Morning Light Plan of Youth Science and Technology (No. 2017050304010326), and Application Foundation Frontier Project of Wuhan Science and Technology Bureau (No. 2019020701011478).
References (40)
- et al.
Assembly of type I collagen fibrils de novo. between 37 and 41 °C the process is limited by micro-unfolding of monomers
J. Biol. Chem.
(1988) - et al.
Self-association of collagen triple helic peptides into higher order structures
J. Biol. Chem.
(2006) - et al.
Modification of layered atelocollagen by ultraviolet irradiation and chemical cross-linking: structure stability and mechanical properties
Biomaterials
(1994) Modification of collagen films by ultraviolet irradiation
Polym. Degrad. Stab.
(2000)- et al.
Collagen structural hierarchy and susceptibility to degradation by ultraviolet radiation
Mater. Sci. Eng. C
(2008) - et al.
Effects of ultraviolet irradiation on native and telopeptide-poor collagen
Biochimica et Biophysica Acta (BBA) - Protein Structure
(1971) - et al.
Insight into the role of grafting density in the self-assembly of acrylic acid-grafted-collagen
Int. J. Biol. Macromol.
(2019) - et al.
Effect of ultra-high pressure on molecular structure and properties of bullfrog skin collagen
Int. J. Biol. Macromol.
(2018) - et al.
Effect of crosslinking agents (basic chromium sulfate and formaldehyde) on the thermal and thermomechanical stability of rat tail tendon collagen fibre
Thermochim. Acta
(2000) - et al.
Collagen I self-assembly: revealing the developing structures that generate turbidity
Biophys. J.
(2014)
Effect of UV radiation on the infrared spectra of collagen
Polym. Degrad. Stab.
Effects of solar radiation on collagen and chitosan films
J. Photochem. Photobiol. B Biol.
Fourier transform infrared (FTIR) spectroscopic study of acid soluble collagen and gelatin from skins and bones of young and adult Nile perch (Lates niloticus)
Food Chem.
Thickening of the infarcted wall by collagen injection improves left ventricular function in rats
J. Am. Coll. Cardiol.
Role of pore size and morphology in musculo-skeletal tissue regeneration
Mater. Sci. Eng. C
Advanced collagen-based biomaterials for regenerative biomedicine
Adv. Funct. Mater.
The collagen suprafamily: from biosynthesis to advanced biomaterial development
Adv. Mater.
Structure, physiology, and biochemistry of collagens
Self-assembly of collagen-based biomaterials: preparation, characterizations and biomedical applications
J. Mater. Chem. B
Temperature-favoured assembly of collagen is driven by hydrophilic not hydrophobic interactions
Nat. Struct. Biol.
Cited by (19)
Ultrahigh pressure field: A friendly pathway for regulating the cellular adhesion and migration capacity of collagen
2024, International Journal of Biological MacromoleculesCollagen and gelatin: Structure, properties, and applications in food industry
2024, International Journal of Biological MacromoleculesPreparation of novel injectable photo-crosslinked collagen gels by a fast and simple method
2023, Materials LettersCollagen processing with mesoscale aggregates as templates and building blocks
2023, Biotechnology AdvancesCollagen and its derivatives: From structure and properties to their applications in food industry
2022, Food HydrocolloidsCitation Excerpt :The collagen gel network showed smaller fibril diameters and pore spaces with the increase of ambient temperature from 4 to 37 °C (Raub et al., 2007). C. Xu et al. (2020) recently developed a collagen gel induced using ultraviolet radiation at a low temperature. They found those collagen molecules were degraded and cross-linked in that gel formation process, which led to the collagen fibrils having more “branches” and promoting the intertwining of fibrils.