Louise E. Smith

A Comparison of Imaging Methodologies for 3D Tissue Engineering

Imaging of cells in two dimensions is routinely performed within cell biology and tissue engineering laboratories. When biology moves into three dimensions imaging becomes more challenging, especially when multiple cell types are used. This review compares imaging techniques used regularly in our laboratory in the culture of cells in both two and three dimensions. The techniques reviewed include phase contrast microscopy, fluorescent microscopy, confocal laser scanning microscopy, electron microscopy, and optical coherence tomography. We compare these techniques to the current “gold standard” for imaging three‐dimensional tissue engineered constructs, histology. Microsc. Res. Tech. 73:1123–1133, 2010.

State of the art in non‐invasive imaging of cutaneous melanoma

Background: This review focuses on looking at recent developments in the non‐invasive imaging of skin, in particular at how such imaging may be used at present or in the future to detect cutaneous melanoma.

The influence of substrate stiffness gradients on primary human dermal fibroblasts.

Materials mechanical properties are known to be an important regulator of cellular processes such as proliferation, differentiation and migration, and have seen increasing attention in recent years. At present, there are only few approaches where the mechanical properties of thin films can be controllably varied across an entire surface. In this work, we present a technique for controlled generation of gradients of surface elastic moduli involving a weak polyelectrolyte multilayer (PEM) system of approximately 100 nm thickness and time dependent immersion in a solution of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) as a crosslinking agent. Uniform surface chemistry across the gradient and wettability was provided by the addition of a 10 nm thick plasma polymer layer deposited from vapour of either allylamine or acrylic acid. We used the resultant stiffness gradients (0.5-110 MPa in hydrated state) to investigate the adhesion, morphology and proliferation on human dermal fibroblasts (HDFs). We show that substrate mechanical properties strongly influence HDF cell fate. We also found that in the experimental range of surface properties used in this study, the surface stiffness was a stronger driving force to cells fate compared to chemistry and wettability.

Production of Tissue-Engineered Skin and Oral Mucosa for Clinical and Experimental Use

Since the early 1990s, our understanding of how epithelial and stromal cells interact in 3D tissue-engineered constructs has led to tissue-engineered skin and oral mucosa models, which are beginning to deliver benefit in the clinic (usually in small-scale reconstructive surgery procedures) but have a great deal to offer for in vitro investigations. These 3D tissue-engineered models can be used for a wide variety of purposes such as dermato- and mucotoxicity, wound healing, examination of pigmentation and melanoma biology, and in particular, a recent development from this laboratory, as a model of bacterially infected skin. Models can also be used to investigate specific skin disease processes. In this chapter, we describe the basic methodology for producing 3D tissue-engineered skin and oral mucosa based on de-epidermised acellular human dermis, and we give examples of how these models can be used for a variety of applications.

Quantum Dot Superluminescent Diodes for Optical Coherence Tomography: Device Engineering

We present a 18 mW fiber-coupled single-mode superluminescent diode with 85 nm bandwidth for application in optical coherence tomography (OCT). First, we describe the effect of quantum dot (QD) growth temperature on optical spectrum and gain, highlighting the need for the optimization of epitaxy for broadband applications. Then, by incorporating this improved material into a multicontact device, we show how bandwidth and power can be controlled. We then go on to show how the spectral shape influences the autocorrelation function, which exhibits a coherence length of <;11 μm, and relative noise is found to be 10 dB lower than that of a thermal source. Finally, we apply the optimum device to OCT of in vivo skin and show the improvement that can be made with higher power, wider bandwidth, and lower noise, respectively.

Stem Cells for Cutaneous Wound Healing.

Optimum healing of a cutaneous wound involves a well-orchestrated cascade of biological and molecular processes involving cell migration, proliferation, extracellular matrix deposition, and remodelling. When the normal biological process fails for any reason, this healing process can stall resulting in chronic wounds. Wounds are a growing clinical burden on healthcare systems and with an aging population as well as increasing incidences of obesity and diabetes, this problem is set to increase. Cell therapies may be the solution. A range of cell based approaches have begun to cross the rift from bench to bedside and the supporting data suggests that the appropriate administration of stem cells can accelerate wound healing. This review examines the main cell types explored for cutaneous wound healing with a focus on clinical use. The literature overwhelmingly suggests that cell therapies can help to heal cutaneous wounds when used appropriately but we are at risk of clinical use outpacing the evidence. There is a need, now more than ever, for standardised methods of cell characterisation and delivery, as well as randomised clinical trials.

Quantum Dot Superluminescent Diodes for Optical Coherence Tomography: Skin Imaging

We present a high-power (18 mW continuous wave exiting a single-mode fiber and 35 mW exiting the facet), broadband (85 nm full-width at half-maximum) quantum dot-based superluminescent diode, and apply it to a time-domain optical coherence tomography (OCT) setup. First, we test its performance with increasing optical feedback. Then we demonstrate its imaging properties on tissue-engineered (TE) skin and in vivo skin. OCT allows the tracking of epidermal development in TE skin, while the higher power source allows better sensitivity and depth penetration for imaging of in vivo skin layers.

Using swept-source optical coherence tomography to monitor the formation of neo-epidermis in tissue-engineered skin

There is an increasing need for a robust, simple to use, non‐invasive imaging technology to follow tissue‐engineered constructs as they develop. Our aim was to evaluate the use of swept‐source optical coherence tomography (SS‐OCT) to image tissue‐engineered skin as it developed over several weeks. Tissue‐engineered skin was produced using both de‐epithelialized acellular dermis (DED) and amorphous collagen gels. In both cases the epidermis could be readily distinguished from the neodermis, based on a comparison with standard destructive histology of samples. Constructs produced with DED showed more epidermal/dermal maturation than those produced using collagen. The development of tissue‐engineered skin based on DED was accurately monitored with SS‐OCT over 3 weeks and confirmed with conventional histology. Copyright

Evaluating the use of optical coherence tomography for the detection of epithelial cancers in vitro

Optical coherence tomography (OCT) is a noninvasive imaging methodology that is able to image tissue to depths of over 1 mm. Many epithelial conditions, such as melanoma and oral cancers, require an invasive biopsy for diagnosis. A noninvasive, real-time, point of care method of imaging depth-resolved epithelial structure could greatly improve early diagnosis and long-term monitoring in patients. Here, we have used tissue-engineered (TE) models of normal skin and oral mucosa to generate models of melanoma and oral cancer. We have used these to determine the ability of OCT to image epithelial differences in vitro. We report that while in vivo OCT gives reasonable depth information for both skin and oral mucosa, in vitro the information provided is less detailed but still useful. OCT can provide reassurance on the development of TE models of skin and oral mucosa as they develop in vitro. OCT was able to detect the gross alteration in the epithelium of skin and mucosal models generated with malignant cell lines but was less able to detect alteration in the epithelium of TE models that mimicked oral dysplasia or, in models where tumor cells had penetrated into the dermis.

Effect of Surface Chemical Functionalities on Collagen Deposition by Primary Human Dermal Fibroblasts

Surface modification has been identified as an important technique that could improve the response of the body to implanted medical devices. Collagen production by fibroblasts is known to play a vital role in wound healing and device fibrous encapsulation. However, how surface chemistry affects collagen I and III deposition by these cells has not been systematically studied. Here, we report how surface chemistry influences the deposition of collagen I and III by primary human dermal fibroblasts. Amine (NH3), carboxyl acid (COOH), and hydrocarbon (CH3) surfaces were generated by plasma deposition. This is a practically relevant tool to deposit a functional coating on any type of substrate material. We show that fibroblasts adhere better and proliferate faster on amine-rich surfaces. In addition, the initial collagen I and III production is greater on this type of coating. These data indicates that surface modification can be a promising route for modulating the rate and level of fibrous encapsulation and may be useful in informing the design of implantable biomedical devices to produce more predictable clinical outcomes.