Cell Sheet Engineering

An attractive alternative to current strategies, including TEC, is the use of cell sheets. Cell sheets have been used successfully for many years in the regeneration of two-dimensional tissues such as the epidermis67 and ocular surfaces.68 In recent years, tissue engineers have extended the use of cell sheets to produce three-dimensional (3D) neo-tissues. Using cell sheets has the advantage that an entirely natural neo-tissue assembled by the cells, with mature extracellular matrix (ECM), can be produced, thereby enhancing the biological potency of the constructs. L'Heureux et al.69 have successfully built a tissue engineered blood vessel by wrapping layers of fibroblasts and smooth muscle cells around a mandrel. This vessel exhibited a well defined 3-layered organization and had a burst-strength comparable to that of native human vessels. Advancement in the culture of cell sheets came with the development of culture dishes coated with temperature-responsive polymers, introduced by Okano's group. These culture surfaces become hydrophilic at temperatures below a critical point, and hydrophobic at temperatures above the critical point.70 As a result, cells attach and proliferate on this surface above the critical temperature, and easily detach when the temperature is reduced.71 It was shown that four layers of cardiomy-ocytes cultured using this technique formed electrically communicative and pulsatile myocardial tissue both in vitro and in vivo.12

However, although cell sheets are strong enough to allow careful manipulation in a laboratory to produce stacked or wrapped constructs, they contract extensively upon detachment from culture surfaces. Thus engineering large-size tissues, of specific shape and size, with cell sheets alone is a challenge, and external supports such as stainless steel rings and nondegradable polymeric membranes are therefore used.69'72 However, these supports will be removed at the point of application, upon which mechanical support for the constructs will be lost, resulting in the loss of size, shape and even biological functions which are dependent on mechanical stimuli. The aim of a group at NUS is to devise a novel technique to withstand the contraction of cell sheets in vitro, maintain the size of the desired graft, and apply it for tissue engineering of bone. The group hypothesized that those

Figure 2. Diagrammatic representation of cell-sheet and scaffold components I-III. A. shows cell sheet at the bottom of the culture plate; B. SEM picture showing PCL-CaP scaffold architecture; C. &D. cell sheet encapsulation of scaffold; E. SEM picture showing cell proliferation and ECM formation throughout the scaffold architecture (inside view); F. lateral view showing cell sheet attached around the outer part of the scaffold.

Figure 2. Diagrammatic representation of cell-sheet and scaffold components I-III. A. shows cell sheet at the bottom of the culture plate; B. SEM picture showing PCL-CaP scaffold architecture; C. &D. cell sheet encapsulation of scaffold; E. SEM picture showing cell proliferation and ECM formation throughout the scaffold architecture (inside view); F. lateral view showing cell sheet attached around the outer part of the scaffold.

high density cultures of osteogenically differentiated BMSCs, together with hybrid matrices, will produce mature TECs with the mineralized matrices integrated within. A diagrammatic representation of cell sheet and scaffold components is shown in Figs. 2 and 3.

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