Reduced Contraction of Skin Equivalent Engineered using Cell Sheets Cultured in 3D Matrices

Full Text available at following link:
http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TWB-4K18VTF-2&_coverDate=09%2F30%2F2006&_alid=481170456&_rdoc=1&_fmt=&_orig=search&_qd=1&_cdi=5558&_sort=d&view=c&_acct=C000059607&_version=1&_urlVersion=0&_userid=4420&md5=36f4a4e1bea9fa127e704ef716f0eb78

The main objective of the experiment described by this paper was to improve the mechanical properties of cell sheets used to generate tissue engineered dermal equivalents. Cell sheets have been used for a variety of tissue engineering purposes that range from artificial blood vessels to myocardial patches and artificial skin. While they have the advantage that the cells on the sheet can be used to engineer a natural neo-tissue, a major shortcoming of cell sheets is that they contract when removed from culture surfaces, causing graft sizes to be reduced. Cell sheets are also very fragile and difficult to handle. This paper describes a technique for resolving these shortcomings in which human fibroblasts sheets are cultured in combination with three-dimensional matrices to form a contiguous dermal construct that does not contract over time.

The group in this study hypothesized that fibroblast sheets grown in conjunction with three-dimensional matrices will maintain their regenerative capacity and result in reduced wound contraction. To test their hypothesis they used their technique with two kinds of 3D matrices: a PLGA scaffold, and a collagen sponge cross linked with hyaluronic acid (CHA). I won’t go into to much of the details of their materials and methods, but I will note that the 3D matrices and cell sheets were fabricated separately, at which point cell sheets were peeled off the Petri dishes on which they were grown and then folded over the matrices to from the 3D dermal equivalent. Human keratinocytes were then seeded on top of each dermal equivalent and allowed to culture for seven days. The constructs were then either raised in an air-water interface for four weeks to induce cell stratification, or they were transplanted onto full-thickness wounds in nude rats for four weeks.

In the end, both PLGA and HCA constructs to varying degrees came to resemble human skin with mechanical properties far more stable than those inherent to cell sheets alone. Each possessed a stratified keratinocyte layer that resembled native epidermis. In addition, cell migration of the fibroblasts from the sheet into the 3D scaffold was observed for both PLGA and HCA, creating a 3D matrix of fibroblasts resembling that of the dermis. However, following transplantation, it was found that the CHA sponges sloughed off the wound within two weeks. The PLGA scaffolds, in contrast, took after 1 week post-transplantation and registered a take rate of 100%. Wounds re-epithelialized 3 weeks after transplantation. This demonstrates both the successes and failures of the experiment. While both constructs exhibited properties that mimicked those of human skin, in the end it was found that the CHA matrix did not take due to its higher than normal rigidity, which prevented it from conforming to changing wound topography as the animals moved.

I chose to post this paper because of the similarity of its objectives with those of our class project as applied to tissue engineered skin, in addition to the fact that many of the methods that were used to characterize their 3D constructs were similar to or the same as the techniques that we have learned in this course. Like our project, the objective of the experiment described by this paper was to create a 3-dimmensional skin substitute that is easy to work with and has mechanical properties comparable to those of the dermis. To accomplish this, they had to characterize their construct by observing the cells using microscopy and ensure that the fibroblasts were correctly distributed in their 3D matrix. In addition, like we have done in class, they used immunostaining techniques to verify that their cells were continuing to express their proteins of interest and generating a matrix that resembled that of human skin.