Advancing Tissue Engineering using Phenom Desktop SEM

Advancing Tissue Engineering using Phenom Desktop SEM

What is Tissue Engineering? 

Tissue engineering is an innovative and rapidly evolving field in medical science focused on creating biological substitutes to repair, replace, or enhance the function of damaged tissues and organs. It encompasses regenerative medicine, stem cell therapies, decellularised or engineered organs, and electrospun scaffolds. Applications of tissue engineering include treating burn injuries, diabetic wounds, and diseases that compromise tissue functionality.

The ultimate goal is to develop functional tissues that can mimic or even improve the normal operation of the damaged area. For example, stem cells extracted from a patient’s bone marrow can be grown and differentiated into cartilage cells (chondrocytes). These are then seeded onto an optimised scaffold matrix, designed to mimic the original tissue, and transplanted into the patient.

To achieve success, scientists must understand the tissue’s structure and properties, including its interaction with cellular adhesion, porosity, and biocompatibility.

By analysing scaffold structures with tools like the Phenom SEM, scientists are advancing the possibilities for tissue regeneration in humans. Detailed imaging helps refine scaffold designs and monitor cellular interactions, paving the way for more effective regenerative therapies.

This understanding ensures that engineered tissues can withstand normal functionality while promoting cellular interactions essential for repair and regeneration.

HOW CAN TISSUE ENGINEERING ASSIST WITH WOUND HEALING?

Wound healing is the process of replacement of destroyed or damaged tissue. While closing a wound might seem like the primary goal, it’s equally important to focus on the repair process occurring underneath.

There are different types of wound healing. Wound regeneration is the process of healing tissue fully restoring its normal function. Wound repair is the process of healing tissue without restoring it to its normal function, such as a scar where hair no longer grows.  

Within wound healing, there are three different stages including;

  • Inflammation: Prepares the wound for healing.
  • Proliferation: Generates new tissue.
  • Remodelling: Strengthens the new tissue.

Delays in any stage can lead to infections, which tissue-engineered scaffolds can prevent. These scaffolds promote regenerative healing by enhancing wound closure, providing nutrients, and creating an optimal environment for cellular interactions.

Optimising a tissue engineering scaffold is crucial for promoting cellular adhesion, growth, and regeneration. Phenom SEM’s advanced imaging capabilities provide detailed insights into scaffold porosity, fiber thickness, and structural integrity, ensuring these synthetic matrices mimic natural extracellular environments effectively.

Properties like porosity, thickness, and mechanical strength significantly influence the success of wound healing. 

HOW ARE TISSUES EXAMINED DURING WOUND HEALING?

Microscopy is an essential tool in wound healing research. Techniques like light, fluorescent, and confocal microscopy allow scientists to examine tissue samples, often stained with hematoxylin and eosin (H&E), to assess the cellular structure and observe changes in the skin’s layers.

Comparison of Microscopy Techniques in Wound Healing

Microscopy TechniqueResolutionSample PreparationApplications in Wound HealingKey Advantages
Light MicroscopyMicrometer scaleSimple staining (e.g., H&E)Examines overall tissue structure and cellular arrangementCost-effective and easy to use
Fluorescent MicroscopySub-micrometerFluorescent dyes or markersHighlights specific cell types or proteins in tissue samplesHigh specificity and contrast
Confocal MicroscopyNanometer scaleLaser scanning with dyesProvides 3D imaging and layer-by-layer analysis of wound tissuesHigh resolution and depth information
Evaluating the Applications of Light, Fluorescent, and Confocal Microscopy

WHY USE SEM IMAGING FOR TISSUE ENGINEERING? 

Scanning Electron Microscopy (SEM) provides high-resolution imaging at the nanometer scale, making it invaluable for studying tissue structures and morphologies, as discussed in biomedical research applications. Compared to optical microscopy, SEM offers finer details, a greater depth of field, and elemental analysis capabilities using an Energy Dispersive Spectroscopy (EDS) detector.

The Phenom desktop SEM series is particularly advantageous for tissue engineering offering high-resolution imaging of the tissues or synthetic tissue structures, the ability to observe the presence of cells and to analyse the surface topography.  

3D bioprinting combined with SEM imaging enables tissue engineers to examine microstructures and ensure that bioprinted tissues meet necessary specifications for cellular proliferation and mechanical strength.

From the entry-level SEM to a system with access to a larger sample compartment or field emission (FEG) source for ultra-high resolution, low kV imaging with STEM detector, all Phenom SEMs offer high speed, and ease of use with a small footprint.  They all come standard with x y stage movements, a digital optical microscope that stays with you throughout the entire time of imaging to help find different locations on your sample, a charge reduction mode that allows users to image samples without having to coat them with a gold or platinum layer, and as well as the ability to add on Python scripting. Different software options provide the ability to measure different parameters that can help promote wound healing, such as porosity measurements, fiber diameter measurements, and tensile strength measurements.

APPLICATIONS OF PHENOM SEM IN TISSUE ENGINEERING

1. Skin Grafts or Bioengineered Skin Substitutes  

Skin grafts or bioengineered skin substitutes are the standard of care for treating third-degree burns, where skin damage extends to the hypodermis and nerve endings are destroyed. Skin grafts can help prevent infections and can be autologous – where is skin taken from the same person and then transplanted to that damaged area, or allogeneic, when grafts from another person and transplanted to another patient. Before transplantation, tissues go through decellularisation – a method that removes the cells – to prevent patients from having an immune response that can lead to rejection of the tissue. Decellularisation usually leads to an extensive amount of tissue damage which compromises the success of a skin graft.  

The Phenom SEM can evaluate the success of decellularisation processes by assessing tissue porosity and cellular removal. Phenom’s integrated PoroMetric software calculates pore characteristics to analyse tissue damage caused by different decellularisation treatments. Research by Dr. Dominic Dominguez, Application Scientist Nanoscience Instruments, found normal skin had an average porosity of 43.41% compared to skin after Tonicity treatment having an average porosity of 46.2% and Triton X-100 having an average porosity of 62.88%. The results demonstrated that Tonicity treatment had similar per cent porosity compared to the original skin samples and thus preserved tissue structure better than Triton X-100. 

Porometric software also indicated that Tonicity treatment removed cells from the tissue, whereas Triton X-100 did not completely remove all the cells and caused more destruction to the tissue compared to using Tonicity. Overall, the Phenom SEM was able to help determine that the Triton X-100 treatment was not successful at removing cells and it actually caused more destruction to the tissue structure compared to the Tonicity treatment.

2. Electrospun Fibers Used as Synthetic Tissue Scaffolds

Electrospun fibers are used as synthetic tissues to mimic the extracellular matrix for cellular migration and proliferation. The needle-based technique applies a voltage to a polymer solution in a syringe which creates fibers that are spun onto a collector to create a scaffold with a specific structure, porosity, and thickness. Changing the reagents helps determine different biomimetic cues in the body. The presence of growth factors can be altered to increase the proliferation of cells, induce cellular migration, or even enhance wound healing. 

Phenom SEM imaging can be used to observe the process of electrospinning. Researchers studying lipid-infused electrospun scaffolds used the Phenom to confirm biocompatibility and measure fiber diameters. The Phenom FiberMetric software enabled automatic fiber diameter measurements, revealing that increased lipid concentration decreased fiber size without hindering cellular growth. The control had an average fiber size of 1.5 microns compared to the 10% treatment having an average fiber size of 0.93 microns. The FiberMetrics software can capture multiple measurements within seconds and provides reporting measurements to understand the mechanical properties of the tissue or the tissue’s replacement.

3. Tensile Strength Testing

During the scar formation after a wound is healed, did you know that only 70% of the normal tensile strength is recovered compared to the original skin? Being able to mimic or improve the original tissue’s mechanics is the goal of wound healing. Using an electrospun scaffold can aid with strengthening the mechanical properties, and there are various tools used to measure the mechanical properties as well, such as a mechanical tester or a rheometer. 

The Phenom XL with the tensile stage allows users to measure the tensile or compressive strength of a material, to view it live under SEM imaging and to record the force and distance in order to calculate the stress and strain of a material. Another useful feature in the Phenom XL is users can navigate to find the same area of interest previously visited so comparisons can easily be made quickly and easily and it’s not like finding a needle in a haystack.

Impact of Lipid Concentration on Fiber Thickness

WHY CHOOSE PHENOM SEM FOR TISSUE ENGINEERING?

Key features include:

  • Charge reduction mode for imaging nonconductive samples without coating.
  • Software tools like PoroMetric and FiberMetric for detailed pore and fiber analyses.
  • A tensile stage for live mechanical testing.
  • Versatile detector modes (BSD and SED) for capturing elemental and topographical details.

Phenom SEM combines high resolution at high magnification imaging with an intuitive, easy-to-use interface that allows even novices to quickly obtain their first high-quality results. It offers multiple detector modes including Backscatter detector (BSD) and Secondary Electron Detector (SED) modes as well as elemental identification using the integrated Energy Dispersive Spectrometer (EDS or X-ray) detector. Images can be captured with either the BSD, SED or have both detectors on simultaneously. This percentage can be adjusted to capture the elemental contrast from the backscatter detector and topography from the secondary electron detector. 

Phenom’s charge reduction mode and the ability for low kV imaging accommodates insulating and beam-sensitive samples with a resolution of 2.0 nm that reveals the finest details. When imaging non-conductive samples, many users will experience charging on samples, making it difficult to collect an image. One way to fix this problem is by coating samples with gold or platinum. The Phenom charge reduction sample holder and software also can adjust the vacuum settings (high to low) to capture images of nonconductive samples without coating and still run elemental analysis using the EDS detector.

The integrated PoroMetric software allows the user to gather data on the distribution of pores and pore parameters like pore size and aspect ratio. Similarly, Phenom FiberMetric Software enables measurement of micro- and nanofibers. Tensile Sample Holder for Phenom XL allows for tensile testing. By measuring the force required to elongate a specimen to the breaking point, material properties can be determined which will allow designers and quality managers to predict how materials and products will behave in their intended applications.

With the Phenom SEM, tissue engineers can analyse scaffold structures, optimise designs, and ensure successful tissue regeneration, paving the way for new advances in wound healing and tissue engineering.

Would you like to learn more about our Advancing Tissue Engineering analysis and solutions? Contact our team for expert consultation.

To learn more about these innovations and discover even more features of the Phenom SEM or Morphologi 4ID and Mastersizer 3000+, Contact us.