Tough Gelatin Hydrogel for Tissue Engineering

Abstract Tough hydrogel has attracted considerable interest in various fields, however, due to poor biocompatibility, nondegradation, and pronounced compositional differences from natural tissues, it is difficult to be used for tissue regeneration. Here, a gelatin‐based tough hydrogel (GBTH) is proposed to fill this gap. Inspired by human exercise to improve muscle strength, the synergistic effect is utilized to generate highly functional crystalline domains for resisting crack propagation. The GBTH exhibits excellent tensile strength of 6.67 MPa (145‐fold that after untreated gelation). Furthermore, it is directly sutured to a ruptured tendon of adult rabbits due to its pronounced toughness and biocompatibility, self‐degradability in vivo, and similarity to natural tissue components. Ruptured tendons can compensate for mechanotransduction by GBTH and stimulate tendon differentiation to quickly return to the initial state, that is, within eight weeks. This strategy provides a new avenue for preparation of highly biocompatible tough hydrogel for tissue regeneration.


Relationship between fatigue threshold and molecular chain
In general, Young's modulus is not the same as stiffness.Young's modulus is a property of material composition and stiffness is a property of structure, namely, modulus is an internal property of a material.For example, for a material in tension or compression, its axial stiffness K = A•E/L, where A is the cross-sectional area, E is the Young's modulus, and L is the length of the material.

Relationship between the maximum elongation and molecular chain
The end distance is the distance from one end of the linear polymer chain to the other end.Because the end distance changes with different molecules and different times, there is no definite value and must be averaged, so the mean square end distance is often used 4, 5, 6 .The ratio of the mean square end distance of fully extended and freely rotating polymer chains determines the maximum elongation λ max of the polymer.The mean square radius of gyration is used for branching P. Suppose that the polymer chain contains many chain units, and the mass of each chain unit is m i .
Assuming that the distance from the center of massof the polymer chain to the unit of the i-th chain is s i , which is a vector, the average mass of s i For flexible molecules, the value of s 2 depends on the conformation of the chain.
The mean square radius of rotation  2 ̅̅̅ is obtained by averaging s 2 over all possible conformations of the molecular chain.It can be proved that for a Gaussian chain, when the molecular weight is very large, the following relationship exists between the undisturbed mean square terminal distance and the undisturbed mean square radius of rotation The end distance of a free-associating chain consisting of n bonds should be the vector sum of the individual bond lengths where f, j is the free spinning chain.The above results are brought into (4) For single-network hydrogels, the steric effect is not considered, the mean square end distance of fully extended is The mean square end distance of freely rotating is The maximum elongation λ max of the polymer is Due to Γ 0 ∼ √, λ max ~ n, Γ 0 and λ max all increase as n increases.Therefore, high fatigue threshold materials are often accompanied by high maximum elongation.To further demonstrate the synergistic effect of stretching and salt solution (SO 4 2- ), we stretched the same size hydrogels in air and immersed them in salt solution (controlling one variable and keeping the other conditions unchanged).It can be clearly found that the hydrogel stretched only in air has a significant increase in strain and a weaker increase in stress.In contrast, hydrogels immersed only in saline solution had a significant increase in stress and a weaker increase in strain.It was further confirmed that it was precisely because of the synergistic effect of stretching and salt solution that the stress and strain of the hydrogel were significantly increased.It can be clearly seen from the optical images that the hydrogel after training has excellent resistance to crack propagation, and the notch was arc-shaped, suggesting that the trained hydrogel has the ability to passivate cracks.However, conventional hydrogels do not have the ability to resist crack propagation, and the notch was sharp.From the FTIR results, it can be seen that the LMs may be anchored on the surface of the hydrogel, and the LMs particles fuse with each other to reduce the resistance during the stretching process.Therefore, LMTE still maintains stable deformation electrical responsiveness under large deformations.The left foot is the control group and the right foot is the experimental group.The right foot (severed sciatica) in the blank group can be clearly seen as atrophied and the left foot diastolic.
However, when we implanted the conductive scaffold into the left foot (severed sciatic nerve) for 2 months, we could find that the right foot was diastolic to a certain extent, indicating that the conductive scaffold had a certain repair effect.

Fatigue
threshold is estimated by Γ 0 = αψ √lJ/V 1, 2, 3 , where ψ is the volume fraction of polymer.Fatigue threshold Γ 0 is controlled by the chain length n of the same network, Γ 0 ∼ √.Traditional hydrogels are composed of water molecules and a flexible polymer network.Traditional hydrogels molecular chains are arranged in a disorderly manner.The molecular chain that actually participates in the deformation under the action of the axial force is only part of the length, so it cannot have high fatigue resistance.Here, we force the molecular chains of conventional hydrogels to align along the axial force direction by means of orientation training, making them into anisotropic structures.The length of its molecular chain along the axial force direction is increased, which in turn increases the breaking threshold several times compared to conventional hydrogels.

Figure S1 .
Figure S1.Mechanical properties of hydrogels.(a) Mechanical properties of hydrogel as a function of number of training times.(b) Mechanical properties of hydrogel as a function of salt concentration.

Figure S2 .
Figure S2.Mechanical properties of hydrogels with different treatment processes.

Figure S3 .
Figure S3.Crystal structure analysis of tough hydrogel.a XRD, b SAXS.The XRD and SAXS characterizations showed that the crystallinity of the hydrogel increased correspondingly with the increase of training times.And the results of XRD and SAXS characterization were roughly the same, which further proves the consistency of the data.This result was consistent with the DSC characterization results.

Figure S4 .
Figure S4.DSC measurement of hydrogel.According to the DSC characterization, the melting point of the hydrogel gradually increases with the increase of training times, which implies that the crystallinity of the hydrogel increases with the increase of training times.

Figure S8 .
Figure S8.H&E image of main organs of rats after implantation of tough hydrogel.

Figure S10 .
Figure S10.H&E and Masson staining of tendon tissues at two and six weeks.n = 3.All scale bars indicate 200 μm.

Figure S12 .
Figure S12.Polarized observation results of Sirius red staining at 2W and 6W.

Figure S13 .
Figure S13.Stained images of all remaining samples.

Figure S16 .
Figure S16.Mechanical properties of liquid metal based tough hydrogel electrons.The inset is the mechanical properties of liquid metal based initial hydrogel electrons.

Table S2 .
Primers for Quantitative Real-Time PCR.