In this section, we’ll be taking you through a short anatomy lesson on the human skin!  This is so that we can better understand what is minimally required of an artificial skin construct. We’ll also be introducing the design principles behind our live skin construct and our automated production process. 🙂

 

The Human Skin

The skin mainly consists of 3 layers — the epidermis, dermis and the deeper subcutaneous tissue. This is as illustrated in Figure 1.

Fig 1. The 3 main layers of the skin and their respective components and functions.

Various Degrees of Burns

The layers of the skin are affected to varying extents depending upon the severity of the burn. The severity of the burns is often classified into 3 degrees. In essence, first degree burns mainly affect the epidermis layer, second degree burns affect both the epidermis and dermis, while third degree burns affect till the subcutaneous tissue. Their characteristics are summarised in Fig 2 below. The various degrees of burns may require different treatment methods in order to repair the affected layers. In this project, we aim to develop artificial skin constructs that can protect second degree burn patients from wound infections and dehydration by covering the wound. We also hope that these live skin constructs can mimic the functions of the epidermis and dermis, thereby accelerating the wound healing process.

 

Two Must-Have Cells in the Skin

In order to mimic the functions of the epidermis and dermis, we must first understand the essential constituents of these layers. An important cell type in the epidermis is the keratinocyte cell type. These keratinocytes provide an impenetrable barrier to pathogens and play crucial roles in cell signalling. Another essential cell type would be the fibroblasts found in the dermis layer. The fibroblasts are responsible for the secretion of constituents of the extracellular matrix like collagen, growth factors, fibronectin and glycosaminoglycans which aid in wound healing. Studies have shown that keratinocytes stimulate the production of growth factors by fibroblasts. These growth factors, in turn, enhance keratinocyte proliferation. With mutually enhanced growth of keratinocytes and fibroblasts,  the wound healing process is accelerated. As such, these are the two essential cells that we will be incorporating into our live skin construct.

sth about replacing collagen with gelatin

For the fibroblasts to grow optimally in culture, they have to be submerged in cell culture medium. However, for the fibroblasts, they have to be first submerged until confluency is reached. Following that, they need to be exposed to air in order to differentiate(?)

 

https://www.omicsonline.org/open-access/cellular-approaches-to-tissueengineering-of-skin-a-review-2157-7552-1000150.php?aid=49358&view=mobile

Fibroblasts residing in the dermal region produce collagen, growth factors, glycosaminoglycans (GAGs), and fibronectin to initiate wound healing process. 

The presence of both keratinocytes and fibroblasts within the epidermal-dermal skin substitutes leads to the production of a variety of growth factors and cytokines which expedite wound healing

The epidermal-dermal skin substitutes are around 2.5mm thickness on average and delayed vascularization in such thick tissue-engineered skin construct remains a critical bottleneck in skin tissue engineering [85]. 

Keratinocytes found in epidermis (the outermost layer of the skin) form an impermeable barrier to pathogens and play an important role in cell signaling within the extracellular matrix.

https://www.ncbi.nlm.nih.gov/pubmed/17435785

 

 

Structure of Our Live Skin Construct

The skin construct should consists of a layer containing the fibroblasts and another layer consisting of the keratinocytes on top of it. For the construct to grow into a piece of skin, it would have to meet several conditions. Firstly, the construct must be able to keep both the fibroblasts and keratinocytes alive long enough for the stratification process of the epidermal layer to complete so that the skin can be formed. Secondly, the fibroblasts and keratinocytes must be able to adhere to their respective layers. This is crucial as it has been shown that NiH-3t3 cells, which are also the fibroblasts used in our experiments, if not adhered to the extracellular matrix, would not be able to progress pass the G1 stage of the cell cycle {Guadagno, 1991 #221}. The cell cycle arrest would prevent the fibroblasts from undergoing cell division, reducing the number of fibroblasts within the construct over time. This would prevent the formation of the skin as fibroblasts, as mentioned earlier, are required for the secretion of growth factors to promote keratinocyte proliferation and also the secretion of the extracellular matrix molecules which is an essential component of the dermal layer. For keratinocytes, various research over the years, as summarised by {Watt, 2002 #223}, has also shown that when the keratinocytes are not adhered to the extracellular matrix, they tend to exit the cell cycle and start differentiating. This would prevent keratinocytes from undergoing cell division, which is essential to provide a continuous source of keratinocytes for the stratification process to form the epidermal layer. For the formation of the epidermal layer, it is necessary for there to be one layer of keratinocyte stem cells which remains undifferentiated and adhered to the basement membrane such that it retains its ability to proliferate, producing more keratinocytes which, when not attached to the basement membrane, differentiates and migrates outwards forming the stratified layers. Thirdly, this particular construct must be easily removed from the plate which it is cultured on and also applied to the person. As a result, this construct must remain relatively solid at the normal body temperature of around 37 degree celcius.

For a novel and successful product, our live skin construct should be able to overcome the limitations of currently available treatment methods. As such, the following are some criteria that we considered in the design of our live skin construct and the features that match these criteria.

 

 

References:

1. Guadagno, T.M. and R.K. Assoian, G1/S control of anchorage-independent growth in the fibroblast cell cycle. The Journal of Cell Biology, 1991. 115(5): p. 1419.

2. Watt, F.M., Role of integrins in regulating epidermal adhesion, growth and differentiation. The EMBO Journal, 2002. 21(15): p. 3919-3926.