Project Description

Background

Skin diseases are the fourth most common cause of all human illnesses, affecting nearly one-third of the world's population. As the largest organ in the human body, the skin is the first line of defense against the invasion of external pathogens. However, scald, burn and other accidents often lead to skin damage, large skin defects caused by trauma, resulting in partial or complete loss of skin function, to patients bring unbearable and lasting physical and mental pain and economic pressure.

We further searched found that there are still many aspects of skin injury repair need to be improved.

  1. Recovery time is long: Due to the large area of suppuration of tissue cells, tissues are in a state of hypoxia, and cytochrome oxidase is unable to reduce oxygen to water. Oxygen atoms will be robbed of an electron and become oxygen free radicals. Oxygen free radicals will carry out per-oxidation, increase the permeability of capillaries, aggravate the ischemia, edema and hypoxia of damaged sites and adjacent tissues, and destroy the structure and function of cell membranes. Destroy mitochondria, cut off cell energy, make cells necrotic, destroy lysosome, tissue lysis, cell autolysis.
  2. Easy to cause infection: wound is a good place for bacterial growth, appropriate temperature, humidity, and exudate nutrients are conducive to bacterial proliferation on the wound; The breakdown of the skin's defense barrier makes it easy for bacteria to invade deep tissues. And large area injury after immune dysfunction, systemic anti-infection ability is low, prone to infection. So far, wound and systemic infection are the most common complications and one of the main causes of death in skin injury patients, especially in large area burn patients. Traditional burn surgical anti-infection focuses on bacteria rather than the injured tissue and body itself. Although it has certain effects, it is not ideal. In addition, antibacterial drugs, wound dryness, and operation also damage the skin injury, leaving scars and even disabilities on the wound surface. This is the so-called palliative not cure the root cause, instead of the root cause and bid.
  3. Scar formation: The skin tissue is damaged and develops into blisters, which can ulcerate and form scars. Hypertrophic scars will be caused by deep second-degree burns or third-degree burns.
  4. Progressive necrosis: human skin has the characteristics of low heat conduction; skin temperature rises slowly after contact with heat source; Skin temperature also drops slowly after removal from heat source. Therefore, although the heat source has been removed after burn, the residual heat in the skin tissue, especially in the deep tissue, can still exist for a long time, resulting in sustained damage. But ideal measures are lacking. Finally, wound pain is the greatest physical and psychological pain of burn patients, and may even cause or aggravate shock. The skin is rich in nerve tissue, mainly in the form of free nerve endings distributed in the dermis in a network. (Wang Guangshun, n.d.)

The problem of skin damage repair needs to be solved urgently.

Our strategy

Epidermal Growth Factor (EGF) is useful in the healing of more serious skin injuries. For example, chronic skin diseases and large burns can be treated with EGF in an aesthetically pleasing and efficient manner. EGF is a powerful protein that, when applied to the skin, accelerates healing, and increases the rate of skin renewal on aging skin.

The application of biochemical and molecular biological approaches has produced considerable information concerning the structure of the growth factors and their individual receptors, their classification into families of related molecules, the relationship of receptors and growth factors to oncogene products, and the plethora of cellular events that constitute the mitogenic response. Also, some clues are available regarding the second messenger pathways that mediate biological responses to growth factors.

Wound healing is a localized process which involves inflammation, wound cell migration and mitosis, neo vascularization, and regeneration of the extracellular matrix. Recent data suggest the actions of wound cells may be regulated by local production of peptide growth factors which influence wound cells through autocrine and paracrine mechanisms. Two peptide growth factors which may play important roles in normal wound healing in tissues such as skin, cornea, and gastrointestinal tract are the structurally related peptides epidermal growth factor (EGF) and transforming growth factor alpha (TGF-α). EGF/TGF-α receptors are expressed by many types of cells including skin keratinocytes, fibroblasts, vascular endothelial cells, and epithelial cells of the GI tract. In addition, EGF or TGF-α are synthesized by several cells involved in wound healing including platelets, keratinocytes, and activated macrophages. Healing of a variety of wounds in animals and patients was enhanced by treatment with EGF or TGF-α.

Spider silk has amazing toughness, strength and malleability, and is also a biocompatible material (Gosline J. et al, 1999). In the past decades, research has focused on the bio-inspired material spider silk in the use of medical applications (Wang Y. et al., 2018). There have been many reports on the research of spider silk in skin injury repair. Due to its non-immunogenicity, easy degradation into non-toxic by-products, and strong toughness of spider silk, it has been applied to films, fibers, foams and hydrogels (Madaghiele M. et al, 2014; Dhaliwal K., & Lopez N., 2018). The research on spider silk has led us to choose it as the main material for solving skin damage in our project. 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. 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. We screened the DNA sequence encoding a repeat region of the pyriform silk of arachnid grandis, and the repeat region is named R.

Design

As shown in Figure 1, We designed a prokaryotic expression system (BBa_K4865002), inserting R (one repetitive region of pyriform silk gene-PySp1, BBa_K4865001) sequence into a plasmid with EGF (BBa_K4865000). And, a pelB signal sequence is on the N-terminus (BBa_K4223000) which directs the protein of interest to the E. coli inner membrane. IPTG-induced production of EGF-R by E. coli BL21 resulted in a significant yield.

Figure 1. Constitution of T7 Promoter-RBS-PelB-EGF-R (Pysp1)-T7 Terminator gene circuits.

We conducted the following experiments with fusion proteins, as shown in Figure 2:

  1. Experiment on the proliferation effect of fusion protein on human skin fibroblasts (HSF).
  2. Preparation and characterization of spider silk protein composite materials.
  3. The degradation/stability model of spider silk protein composite was established.

Figure 2. Work Flow of Our Project

Goal

Spider silk protein has attracted much attention on account of its excellent mechanical properties, biodegradability, and bio-compatibility. As the main protein component of spider silk, spider fibroin plays important role in spider spinning under natural circumstances and bio-material application in medicine as well (J. Biomater. Appl. Et al, 2021). Our project is to address the application of spider silk protein in the medical field. By combining artificial spider silk fibers with EGF with the bio-engineering technology, it is possible to achieve the effect of promoting cell regeneration and tissue repairing. Due to the high extensibility and toughness of Spider fibroin, it can effectively assist in the healing of damaged skin. So, we plan to create a medical dressing using spider silk protein as the main raw material. In our goal, we expect this Spider fibroin medical dressing to be able to treat skin injury. Also, due to the material properties of Spider fibroin, and the need for a moist and breathable environment for the recovery of damaged skin (Charles K. Field MD. et al, 2004). We hope to present it in the form of a hydrogel around the surface of the skin for treatment. It can achieve antibacterial and anti-inflammatory effects through contact with the skin surface to accelerate the process of wound repair.

Reference:

Archer S.B., Henke A., Greenhalgh D.G., Warden G.D. (1998) The use of sheet autografts to cover extensive burns in patients. J Burn Care Rehabil., 19:33-8.

Charles K. Field MD 1, 1, The beneficial effects of a moist versus a dry wound environment include: prevention of tissue dehydration and cell death, Grinnell, F., Katz, M., Hermans, M., Leipziger, L., Cordts, P., Hutchinson, J., Winter, G., Hunt, T., Varghese, M., Horikoshi, T., Knighton, D., Wood, S., Lydon, M., Browse, N., Burnand, K., Falanga, V., … Mulder, G. (2004, March 18).

Cohen S. (1983). The epidermal growth factor (EGF). Cancer, 51(10), 1787-1791.

David G., Greenhalgh M.D. Management of Burns.

Dhaliwal K., & Lopez N. (2018). Hydrogel dressings and their application in burn wound care. Br J Community Nurs, 1;23(Sup9):S24-S27.

February 1988 Progress in Clinical and Biological Research 262(1):207-16 DOI:10.1146/annurev.bi.48.070179.001205

Gosline J., Guerette P., Ortlepp C., & Savage K. (1999). The mechanical design of spider silks: From fibroin sequence to mechanical function. The Journal of Experimental Biology, 202(23), 3295-3303.

Kreis R.W., Mackie D.P., Vloemans A.W., Hermans R.P., Hoekstra M.J. (1993) Widely expanded postage stamp skin grafts using a modified Meek technique in combination with an allograft overlay. Burns, 19: 142-5.

Madaghiele M., Demitri C., Sannino A. & Ambrosio L. (2014). Polymeric hydrogels for burn wound care: Advanced skin wound dressings and regenerative templates. Burns & Trauma, 2(4), 153-161.

Schultz G., Clark W., Rotatori D.S. (1991). EGF and TGF‐α in wound healing and repair. Journal of cellular biochemistry, 45(4), 346-352.

Wang Guangshun. (n.d.). The existing problems of traditional burn treatment methods. Retrieved May 30, 2023, from http://www.mebo.com/newmebo/kepu/jiangzuo/jz2.htm

Wang Y., Beekman J., Hew J., Jackson S., Issler-Fisher A., Parungao R., Maitz P. (2018). Burn injury: Challenges and advances in burn wound healing, infection, pain and scarring. Advanced Drug Delivery Reviews, 123, 3-17.

Zhang Y., Wang T., He J., Dong J. (2016). Growth factor therapy in patients with partial-thickness burns: a systematic review and meta-analysis. International wound journal, 13(3), 354-366.