Effect of Levan on the Mechanical and Transport Properties of Silk–Levan Hydrogels for Tissue Engineering

Abstract

Osteoarthritis is a multifactorial degenerative disease characterized by the breakdown of articular cartilage (AC), affecting hundreds of millions of people worldwide. Among many treatment options, hydrogels have emerged as promising candidates for cartilage replacement, due to their structural resemblance to the native tissue. Hydrogels can be made from various materials, and this study focuses on silk fibroin-based hydrogels, which offer tunable mechanical performance and biocompatibility.
Tissue engineering, however, faces a persistent challenge: the trade-off between mechanical stiffness and permeability, as denser structures offer better mechanical properties but poorer fluid transport, which is essential for cartilage function. This study's approach to solving this problem is to develop a hybrid material incorporating levan, a recently characterized fructose-based polysaccharide. It was hypothesized that levan would act as a mesoscale structural modifier, partially decoupling these competing properties in acid-fibrillated silk networks.
Levan exhibits concentration-dependent self-assembly: below 0.1 mg/mL, it forms fibrils; above this concentration, it forms micelles. A factorial design comprising 30 samples was used to systematically assess the effects of silk concentration (20 and 40 mg/mL), levan concentration (0–1.5 mg/mL), and NaCl concentration (0–150 mM) on mechanical and transport properties. Characterization was performed through fluorescence kinetics (ThT), unconfined compression testing, bioimpedance spectroscopy, drying and rehydrating the samples, and FTIR spectroscopy. In this concentration range, levan showed no significant effect, with silk concentration dominating all measured responses.
A subsequent experiment was performed to explore higher levan concentrations and achieve a 1:1 silk-to-levan ratio. Samples were prepared at 40 mg/mL and 20 mg/mL; the 40 mg/mL preparation encountered several challenges, resulting in imprecise concentrations. The 20 mg/mL samples, in turn, had a smooth preparation and exhibited promising results, especially the 20 mg/mL levan sample, which showed an increase in the swelling ratio and a non-linear improvement in the mechanical moduli, suggesting a critical concentration threshold.
It is proposed that at high concentrations, levan micelles act as molecular templates, concentrating β-sheet formation around them and generating a more structured porous scaffold. Upon drying and rehydration, levan dissolves, leaving an open-pore network with enhanced fluid-uptake capacity. These findings indicate that levan is not effective as an additive (at low concentrations), but rather as a structural component of the hydrogel when present in sufficient amounts, simultaneously improving mechanical performance and swelling capacity. Further work is required to confirm the micellar template mechanism and optimize composition ratios.

Popular Abstract

One of the most widespread diseases is osteoarthritis. In this cartilage, the tissue between the bones, is damaged and lost throughout life, since it cannot self-repair. When cartilage is lost, it causes severe pain and related diseases, necessitating the development of effective treatment options. This study addresses the challenge of identifying a material to replace this essential natural tissue.
The study began with silk hydrogels, which have a consistency similar to natural tissue but face an important challenge: making them strong enough often closes their internal channels, reducing their permeability. Since cartilage needs to be both strong and permeable, this study introduced a second ingredient: levan, a natural sugar-like molecule produced by certain bacteria, which has a curious property: depending on how much of it is dissolved in water, it organizes itself in completely different ways.
The approach was to mix silk and levan to determine whether any effect would be achieved. The first round of experiments tested a range of low levan concentrations alongside varying concentrations of silk and NaCl. The results showed that levan had almost no effect on the tested properties. The conclusion was that at low levels, levan was essentially invisible to the network, and silk dominated the performance.
In the second round of experiments, however, higher levan concentrations were tested. The gels became mechanically stronger and noticeably more absorbent, especially in their rehydration abilities. The best results were achieved at a 1:1 silk-to-levan ratio, and the effect appeared to kick in suddenly at a threshold rather than build gradually, suggesting that micelles only begin to influence the gel's architecture once there are enough of them to make a difference.
The hypothesis is that the micelles act as a temporary template for the silk's gelation: they take up space during gel formation, forcing the silk to organize around them into a more structured, porous network. When the gel is dried and then rehydrated, the levan dissolves, leaving open pores that can fill with fluid. The findings open a new strategy to tissue engineering: levan, at the right concentration, guides the gelation to produce the desired structure for cartilage replacements.
As initial work on the combination of silk and levan continues, there is still much to study in this area. Exploring how levans' effects develop at higher concentrations and optimizing the composition are opportunities for future work to advance the development of an ideal cartilage replacement.

• author: de Oliveira Fernandes, Débora
• supervisor: Cedric Dicko
• organization: Pure and Applied Biochemistry
• course: KBKM01 20261
• year: 2026
• type: H2 - Master's Degree (Two Years)
• subject: Chemistry
• keywords: Silk fibroin, Levan, Hydrogel, Articular cartilage, Tissue engineering, Mechanical properties, Permeability, β-sheet, Swelling, Micellar template, Biotechnology
• language: English
• id: 9230019
• date added to LUP: 2026-06-10 14:30:16
• date last changed: 2026-06-10 14:30:16