Background

All over the globe, burn injuries are a huge problem in the health sector, as previously mentioned it represents the third cause of infant mortality in Mexico and around 120 thousand people per year suffer a burn injury accident, based in the public health sector information. However, Mexico doesn't have the required infrastructure to treat third-degree burns, which causes a high mortality rate (NTX, 2017).

Furthermore, the rate of child mortality from burn injuries is 7 times higher in areas with low socioeconomic income. Each burn injury case could hit a cost of 141,750 USD due to the lack of specialized equipment and staff required to attend these delicate wounds. This problem has been ignored due to the following implications: healing process, high costs and the difficulties providing the specialized treatment. We concluded that accelerating this the process would have many advantages, not only in a faster skin regeneration but also it would have a psychological impact and reduce the economic cost.

The solution

Thus, we created TecTissue, a novel treatment that is capable of reducing wound healing time, preventing infections and consequently, decreasing mortality rate. We achieved this with synthetic biology, incorporating a growth factor and a scaffold.

TecTissue’s main objective is to provide families with a simple way solution that will help in the reduction of medical bills and hospitalization time, making the recovery process easier and also creating a chance of better life quality for the people that suffered from burn injuries. We used recombinant proteins like collagen, composed by non-canonical amino acids. We also implemented a growth factor produced by humans called Leptin. This growth factor has been reported to be essential in different skin regeneration processes. There were some other molecules used in our project such as tenascin and heparin. Both of them, bonded with collagen, make part of our scaffold which is encapsulated with leptin in nanoparticles of chitosan, in order to have an efficient delivery in the skin.

We tested the development of TecTissue in fibroblast cell line L-929 and human mesenchymal cells. To demonstrate our project it was necessary to elaborate a Mini Skin Simulator were the cell lines were provided with the conditions they required in order to get a positive proliferation. This Mini Skin Simulator was automatized to create a friendly device for the manipulation of cells in a culture and to reduce the possible contamination.

Extracellular matrix

The extracellular matrix (ECM) is a non-cellular structure found on the outside of the cell. Among other features, the organization of the cells, tissue, organs, and protection are the most important. It can be used as a biological material for damaged organs or tissue regeneration.

In order to generate an extracellular matrix, four molecules were used: a collagenous peptide, Tenascin type III-like, heparin and chitosan.

Collagen V was shown to bind heparan sulfate proteoglycans through its heparin binding site (HepV) with higher affinity than other collagens. A recombinant fragment of the α1(V) chain (Ile824 to Pro950) binding site binds heparin and heparan sulfate by electrostatic interactions [4]. The purpose of including this domain was to promote the binding of heparin, which then interacts with tenascin C fibronectin type III domain V (TNCIII5)[xaxa], forming a matrix that boosts cell adhesion.

We supplemented the scaffold with a recombinant human leptin β (LepB) to accelerate cell proliferation, as we validated that it could achieve a controlled drug delivery when encapsulated. Due to its wide use as a biomaterial, chitosan nanoencapsulation was employed. The efficacy of the system was evaluated with MTT proliferation assay performed in 96-well plates.

Components

Collagen

Collagen is the most abundant type of ECM used in experimental procedures to provide the cells with a scaffold for the reconstruction of multiple tissues.

A typical molecule of collagen has a fibrillar structure that consists of a helix made of three α chains. Most of the known types of collagen have a similar primary structure: Gly-X-Y, where X and Y can represent any amino acid, which generally are proline and hydroxyproline (Hyp), respectively. The amino acids occupying X and Y sites have been shown to change the collagen function. Furthermore, proline hydroxylation plays a crucial role by providing the triple helix with improved thermostability and structural integrity. Despite, with a specific set of conditions, exogenous Hyp has been shown to be activated by Pro-tRNA synthetase when supplied in the culture medium. So, Hyp is successfully incorporated in protein synthesis.

Tenascin

Tenascin C or TNC is a protein that is located in the extracellular matrix forming a disulfide-bonded hexabrachion[xexe]. This protein has a positive regulation in the regeneration and tissue remodeling, playing an important role in the regulation of this process. The TNC presence stimulates the migration of fibroblasts, furthermore inhibits the contraction of the fibrin-fibronectin matrix in order to prevent the premature contraction of the matrix before the adequate deposition of collagen. The third domain of TNC consists of a series of up to 15 fibronectin type III-like repeats (TNCIII)[xixi]. The subdomain TNCIII5 has the responsibility of the binding with heparin. The binding between heparin and tenascin is involved in affinity with many growth factors, specifically with Fibroblast Growth Factor (FGF) as well as TGF-β and IGF-BP. The high affinity of TNC for an extensive range of growth factors is mediated mainly by the TNCIII5 domain and that, despite being a high-end affinity, a promiscuous binding occurs[xoxo].

Heparin

Heparin is a highly sulfated glycosaminoglycan (GAG) which is known to be able to interact with multiple growth factors, improving its efficiency of function. Due to its capacity to increase the efficacy of growth factors: vascular endothelial growth factor (VEGF) and fibroblast growth factors 1 and 2 (FGF-1 and FGF-2). In addition, heparin has the main characteristic of being an anti-aging factor.

Chitosan

Chitosan (CS) is a biocompatible, biodegradable, and antimicrobial natural polymer. It is degraded into N-acetyl glucosamine by lysozymes and into carbon dioxide via glycoprotein pathway.

CS is water-insoluble except in acidic mediums (< 6.5 pH), where amino groups act as weak bases, being easily protonated. Furthermore, when positively charged, it’s able to interact with negative surfaces such as cell membranes, mucus lining, and anionic polymers. (Jose, Kunjanchan and Lammers, 2010). For CS to be employed as a delivery vehicle low isoelectric value proteins are better encapsulated at a pH greater than the pI value (pH>pI), this is because of the electrostatic interactions. (Quan and Wang, 2007) Nanoparticle formation is favored by CS ability to bind to polyanions due to the formation of complexes between both oppositely charged entities. The polyanion we chose to work with was sodium tripolyphosphate (TPP) under constant stirring.

Growth factor

Leptin is a regulator weight hormone (16 kDa) that controls the velocity for reducing fat. This hormone is produced by adipocytes and secreted into the bloodstream. Leptin is mainly synthesized in adipocytes, including subcutaneous adipocytes. However, the synthesis of leptin and its receptors has been detected in human and mice fibroblasts and keratinocytes. First of all, leptin signalling goes like this:

Leptin and its receptor are also expressed by human hair follicles. Several studies have been shown that human follicles papilla cell lines (not neonatal human dermal fibroblast) express leptin mRNA and produce significant amount of leptin in vitro. Also leptin induces STAT3-dependent signalling in human keratinocytes. This have shown that mice whose follicular keratinocyte and epidermal lack functional STAT3 are viable and display seemingly normal skin and hair follicle morphology, both, hair follicle cycling and wound healing are severely compromised. So, if STAT3 is disrupted or in deficit, skin regeneration and keratinocyte migration is retarded, just like their hair cycle progression.

The use of nanostructures for drug delivery is becoming widespread. Chitosan nanoencapsulation has been demonstrated to be an efficient method, because of its biocompatibility and biodegradability. Additionally, particle parameters can be easily modulated to achieve the desired size, shape, and release behavior. When the molecule thas is being carried requires to be gradually liberated, like leptin growth factor, a chitosan nanoparticle is an efficient vehicle that even partially protects proteins if external conditions change.

References

1. Laporte, L. et al. (2013) Tenascin C promiscuously Bind Growth Factors via Its Fifth Fibronectin Type III-Like Domain. doi:10.1371/journal.pone.0062076
2. Gilpin, S. (2017) Fibrillin-2 and Tenascin-C bridge the age gap in lung epitelial regeneration. doi: 10.1016/j.biomaterials.2017.06.027.
3. Midwood, K. et al. (2003) Tissue repair and the dynamics of the extracelular matrix. doi:10.1016/j.biocel.2003.12.003
4. Midwood. K. et al. (2016) Tenascin-C at a glance. doi:10.1242/jcs.190546
5. Gnanou, Y., Leibler, L., and Matyjaszewski, K. (2007). Macromolecular Engineering. Precise Synthesis, Materials Properties, Applications. Weinheim, Germany: WILEY-VCH Verlag GmbH & Co. KGaA
6. Bächinger, H. P., Mizuno, K., Vranka, J. A., & Boudko, S. P. (2010). Collagen formation and structure. In Comprehensive Natural Products II: Chemistry and Biology (Vol. 5, pp. 469-530). Elsevier Ltd.