Description

Project source

Skin is the body's largest organ and natural barrier, which plays an important role in maintaining the stability of the internal environment and preventing microbial invasion. Since it is covered on the surface of the human body, it is vulnerable to get injured. Large area skin defects caused by burns and trauma lead to partial or complete loss of skin function, which brings unbearable and lasting physical and mental pain to patients. By now, due to limited medical technology, high cost and so on, autologous transplantation cannot meet the needs of patients. Furthermore, the treatment is quite time consuming, which affects the life quality of patients seriously. Therefore, the artificial skin and wound dressings for curing skin defects have been widely concerned by the society. In recent years, the development of regenerative medicine and 3D bioprinting has provided a new idea for skin transplantation and brought light to patients with skin diseases and skin-related industries.

Project background

3D bioprinting technology is based on 3D printing by using living cells as raw materials to print living tissues. It can print living skin cells as raw materials layer by layer with serious control. The constructed skin can be applied in vitro for skin disease research, medical treatment or skin topical drug development.Biological 3D printing provides a highly efficient, automated printing hierarchical structure. A well method of skin printing can control printing layer, area and cell density, and establish connections between cells. Furthermore, the printing method itself should not affect cell activity.
The most important part of 3D printed skin is the raw material used in 3D bioprinting, named bioink. Bioinks commonly use hydrogels made from collagen. With the continuous development of 3D printed skin technology, the materials for 3D printed skin are becoming more and more extensive. And by now, there are a large number of papers and patents on bioinks for 3D printed artificial skin world wide. However, the existing bioinks can not fully meet the needs of use.Most recently, the skin stem cell extraction technology and 3D scanning modeling technology developed (Reference 1), and fully customized skin can be printed in accordance with specific parts of the human body and specific 3D features through 3D scanning, which improves the fit and success rate of artificial skin and wounds. While, the problems of immune rejection and the incomplete differentiation of foreign stem cells are not taken into account in the extraction of stem cells.The artificial skin structure using collagen sponge mentioned in reference 2 can shorten the wound healing time and enhance the skin elasticity after wound healing, but there is bacterial contamination risk in the printing process.

Our project:

1. Why spider silk protein?

Spider silk protein, as a high performance biological material, has a very attractive application prospect in the field of tissue engineering, such as artificial tendon, ligament, organ and tissue repair. However, it is not practical to make use of natural spider silk due to the difficult breeding and the small silk production. Spiders are well known for their nature of occupying a large area and killing each other. Recently, scientists from all over the world have made a series of studies on the chemical composition, structure and gene composition of spider silk proteins., Now, the scientists have developed synthetic spider silk using gene technology. This provides us technical possibilities for the development of our project.

It has been reported that cell-loaded spider silk can be distributed by robot for printing without the need for cross-linking additives or thickening agents for mechanical stabilization. In such spider silk scaffolds, cells are able to adhere and proliferate with good viability for at least a week. The introduction of cell-binding motifs into spider silk proteins further achieved fine regulatory control of cell-material interactions. Therefore, spider silk hydrogels are considered a very attractive source for novel bioink production.

2. Why piriform silk (PySp) protein?

So far, PySp have been studied in the seven types of spider filaments. The natural silk is rarely produced and commonly mixed with other silk proteins in the attachment disc. In contrast to other spider silk family members, PySp1 may have evolved to possess special molecular properties that can be optimized for spinning into a rapidly solidifying liquid gelatinous substance.The abundant polar and charged amino acid residues embedded in its sequence seem to support this claim.This is important for building good 3D printed inks. In addition, given the higher hydrophilic residues embedded in the amino acid sequence of PySp1 than other spider silk family members, PySp1 could prove to be more suitable for developing expression systems to obtain large amounts of soluble protein.
In our project, we screened the DNA sequence encoding a repeat region (The repeat is named R.) of the pyriform silk of arachnid grandis. The project involved the following parts as shown in the figure1:
1. To obtain the coding sequence of recombinant piriform filament protein (PySp1) by using molecular biology techniques
2. To purify the (PySp1) protein and study the mechanical properties of different (PySp1) protein repeat modules
3. The protein was compared with other composite materials such as sodium alginate and natural skin such as fish skin.
4. The degradation/stabilization model of spider silk protein and spider silk protein composites was established.

Figure1: Work flow of our project

We plan to design 1-3 repeat modules (R-3R) (Due to workload and time constraints, 2-3R will be developed in future). Different repeat modules have different mechanical properties, and can be reasonably selected and proportioned according to the needs of printing products. In addition, the amino acid side chains of spider silk proteins can be modified and various cytokines can be added with various chemical modification methods and gene engineering fusion expression technology according to the needs. To achieve the purpose of changing the properties of scaffold materials and broaden the application field of spider silk proteins, bioinks for 3D printing should not only have good printing adaptability, but also have cytocompatibility. Since recombinant spider silk protein has low immunogenicity, good cytocompatibility and physical cross-linking ability. Our project is set to explore and evaluate its potential as a novel bioink raw materials.

References: