**5. Conclusion**

apical and basal organization in vivo. So when placed back in 3D environment after being cultured as 2D monolayers they tend to regain their typical in vivo morphology [9, 74, 114], but the essence of the environment also has to be considered since it could provoke different morphology [12]. Moreover, both the mechanical and the structural state of the environment have to be taken into consideration. Recent studies have shown that gene expression patterns, fibroblast morphology, as well as organization of the extracellular environment differ not only between particular healthy tissues but also compared to tumor stroma [85, 115, 116]. Further‐ more it is currently evident that cells do feel and respond in different ways to the environment – fibroblast cells differentiate to myofibroblasts due to changes in the substrate stiffness [86] and begin to remodel the extracellular matrix. Stem cells have the ability to differentiate to osteogenic, myoblastic and neuronal lineages based just on the change on substrate stiffness [13]. Tumor associated stroma alone was shown to be able to induce desmoplastic stroma fibroblast differentiation [85] as well as at later stages of tumor progression, to be more permissive for epithelial invasion [117]. Taking into consideration these differences it is not surprising that the compliance of the substrate and its topography and mechanical features can control cellular behavior. Therefore the transplantation of cells into bioengineered scaffolds for the purposes of regenerative medicine or tissue engineering has to be precisely assessed and executed, since even the small differences in the substrate's composition, organization or stiffness have the potential to alter the donor cells gene expression [85, 118] or

Despite the drawbacks revealed about each type of bioengineered scaffolds they have already found use in regenerative medicine. Fibrin and collagen grafts for example were among the first used in burn patients for skin reconstruction. Study by Chua et al. has shown that use of skin tissue constructs has reduced mortality in patients with 60% of total body area burns from 100% in 1952 to 41,4% in 2003 [120]. The advance in the skin tissue regeneration is accentuated by the high number of currently available off-the-shelf bioengineered skin grafts [46], that

**•** Biomaterial – biological (autologus, allogenic and xenogenic) and synthetic (biodegradable,

Together with this immense success in the field of burn wounds and the increase in survival rate though, patients are faced with new obstacles. More often than not wounds in surviving patients develop severe fibrosis after healing. The resulting hypertrophic scars present a major discomfort in survivor's life by possibly limiting the range of motion of joints, also resulting in impaired thermal regulation and not at the least a disturbed visual appearance [121].

In spite of the advances made in skin tissue engineering and without undervaluing the accomplished results there is still a lot of progress to be made towards the prefect reconstruc‐ tion of the skin. There still isn't an available option that makes possible the regeneration of

**•** Duration of the graft – permanent, semi-permanent, temporary

**•** Composition regarding cell content – cellularized or acellular

**•** Anatomical structure – epidermal, dermal, composite

to promote tumorigenic transformation [119].

234 Regenerative Medicine and Tissue Engineering

could be grouped by several criteria:

non-biodegradable)

The evolution of the cell culturing method has led to the sophistication of culturing procedures with one main goal – to approximate in vitro conditions to in vivo. The advancement of threedimensional culturing models has allowed for an in depth understanding of cellular behavior and especially the role of the cell's environment has emerged as an important modulator of cellular functions. Current data has made it clear that future development and use of superior three-dimensional cultures should focus not just on the dimensionality of the environment per se, but mainly on its characteristics – biochemical composition, production source and possible unwanted cell-cueing signals, mechanical properties, etc. thus making 3D culturing, along with regenerative medicine multidisciplinary fields. However, as science advances, more and more questions emerge and await answers in order to confirm the long term safety of the applied methods for human therapy.

**Figure 2.** General strategy for design of a "perfect" 3D system. The specific set of ECM characteristics, typical for se‐ lected normal tissue can be determined. Based on these studies, a scaffold with matching parameters and suitable cells can be identified and combined in vitro for development of particular, tailored made 3D culture, meeting the needs of a specific research or specific therapy.

During the resolution of the moral issues concerning the use of human embryonic stem cells or donor organs the remaining fields continue to advance rapidly. It appears that isolation of autologus cells, their expansion in vitro and probably stimulation to produce extracellular matrix with the required dimensionality, biochemical and mechanical properties would represent the optimal tissue engineered scaffold for tissue and organ reconstruction. Further advance in the field of regenerative medicine and tissue engineering would possibly see the development of 4D model systems, incorporating time as the forth dimension. Such models would represent not just single stages of organ development or disease, but the chronology of their maturation/progression, thus providing an opportunity to transfer developmental processes to in vitro conditions.
