The skin is the ultimate barrier to the human body, protecting it from mechanical damage, radiation, extreme temperatures, and pathogens. The skin consists of three layers: the epidermis (outer layer), dermis (middle), and hypodermis (inner layer). Each layer serves a distinct purpose. The epidermis provides the primary barrier whereas the dermis and hypodermis contain hair follicles, sweat glands, connective tissue, and fat to provide mechanical strength and warmth to our bodies. During minor injuries, the skin can normally regenerate the damaged areas on its own. In cases of extensive injuries, such as through disease or burn wounds, the skin is unable to regenerate on its own which leads to scar tissue formation or in worst cases, chronic wounds that need medical attention.
Allogenic skin grafts for wound healing
Skin grafts are applied to severely damaged areas to aid in restoring skin to its normal function. When a severe wound is covering a large area of the body, doctors may not be able to use an autologous skin graft, which can be taken from another undamaged region of the patient’s body. Under such circumstances, allogenic skin grafts can be used which are typically harvested from cadavers.
Decellularization of allogenic skin grafts, which involves the complete removal of cellular material from the extracellular matrix (ECM), is critical for avoiding rejection once the graft is applied to a patient’s wound. Different techniques that rely on chemical agents, biologics, or mechanical force can be used to decellularize tissue. Tissue density, lipid content, and thickness can influence how well each approach will work for an individual skin graft. Oftentimes, the ECM can be damaged in the process. Therefore, it is important when developing a preparation protocol, that the tissue is adequately characterized to ensure the ECM morphology remains intact with minimal damage throughout the cell removal process.
Characterizing tissue morphology with SEM
Scanning electron microscopy (SEM) produces high-resolution (~1-10 nm) images of a sample by scanning the surface with a focused electron beam. SEM imaging offers a multi-modal approach to analyzing how the ECM responds to different decellularization methods, thus aiding in the preparation of high-quality allogenic skin grafts. Unlike staining histology which uses light microscopy, SEM makes it much easier to visualize at a higher resolution and quantify structural changes in complex structures like the ECM.
In the following case study, the Phenom XL Desktop SEM was used to evaluate the removal of cells in porcine skin tissue treated with two different decellularization agents. As seen in the native tissue sample, pores throughout the ECM are filled with skin cells. The pores are roughly 10-µm in diameter. Treatment 1 (Tonicity) resulted in complete removal of skin cells whereas Treatment 2 (Triton X-100) resulted in partial removal. Treatment 2 also resulted in noticeable degradation to the ECM, as seen by the smaller secondary pores throughout the sample, measuring about 1-µm in size. Therefore, the SEM images convey the effectiveness of each treatment, showing that Treatment 1 appears to be a more effective decellularization method that preserves the native structure of the ECM.
SEM images of native tissue compared to tissue prepared with different decellularization treatments.
Image analysis software can also be applied to quantify pore size distributions which can be used to estimate porosity of a decellularized ECM. For Phenom Desktop SEMs, porosity measurements made with PoroMetric image analysis software provide several size parameters of individual pores as well as reporting the overall pore area ratio, which can be used to estimate porosity. In these porcine skin tissue samples, the porosity ranges from 43-61% with about 2000 pores measured in each image.
SEM imaging can be used to visualize tissue morphology with a level of detail not attainable through conventional light microscopy techniques. SEM imaging provides detailed morphological analysis of decellularized tissue, allowing healthy ECM to be transferred to wounds and promote healing. However, wound healing encompasses more than just skin grafts. Tissue engineering addresses a critical drawback of allogenic skin grafts: the risk of incomplete cell removal and subsequent rejection by the patient. Biomimetic skin mimics the native ECM, morphologically and mechanically, while intrinsically having no trace of cellular material thus promoting healthy proliferation of host skin cells to promote wound healing. To learn more about how SEM can be a multi-modal tool for the analysis and optimization of engineered tissue, watch our webinar: Advancement of Tissue Engineering Using Desktop SEM.
Technology: Scanning Electron Microscopy