**4. Does a perfect 3D system exist?**

In vitro 3D models present the opportunity to investigate in depth the molecular mechanisms of the interactions between cells and the extracellular matrix. The increased number of research based on 3D scaffolds, mimicking specific physiological and developmental processes, as well as tissues, bridges the gap between laboratory investigation and unimpeded tissue regenera‐ tion. At the same time the increasing number of research has made it clear that variables such as cell source, the extracellular matrix's biochemical and mechanical properties, growth factor cues and developmental stage affect cell behavior and therapy outcome, imposing the need of even more careful reconsideration of yet the slightly interfering environmental factors for the successful outcome of regenerative therapies or tissue engineering.

Development of optimal bioengineered scaffolds requires indepth knowledge of physiology and understanding of cell–cell and cell–ECM interactions. The choice of inappropriate model system could have a negative effect on the study or the therapy outcome. Such an example could be any of the discussed 3D culturing systems. Sometimes even slight changes in experimental design like difference in cell count in collagen gels for example could lead to different migrational patterns [87] or as mentioned, the change of the substrate stiffness can induce different responses of cells to the environment [14]. Still, the obscured causes under‐ lying such different outcomes are yet to be defined. Another example could be the inappro‐ priate use of three-dimensional BME gels. In vivo basement membranes represent thin extracellular structures that underlie endothelial and epithelial structures. Basement mem‐ branes also surround muscle, fat and nerve cells. Moreover the composition and the amount of the basement membrane are specific and vary in different tissues and during particular stages of development. Even though introduction of cells to a 3D environment, based on basement membrane matrix, results in numerous morphological and physiological changes, there are a number of discrepancies to be considered and questions to be answered:

**•** Contact with the basement membrane is not typical for all cell types, but a lot of cells express receptors for components of the basement membrane matrix. Therefore what would happen with cells that accidentally get caught in such unnatural environment? Would they become apoptotic or would they transform into malignant cells?

Even though there are a lot of difficulties with the production of iPS cells, already a plethora of articles has demonstrated the ability of iPS cells to differentiate to numerous types of cells, thus providing the ability to direct the in vitro differentiation of iPS cells to the required cell type for the specific therapy. Moreover significant results have been accomplished, namely the generation of adult mice from iPS cells [110] thus confirming the vast capabilities of such cells. Therapeutically, iPS cells have been used together with gene therapy to correct genetic defects in mice, with two studies already showing promising results in the treatment of different types of anaemia [111, 112]. Future efforts in the field of iPS cells though have to be made before a successful iPSC therapy for human patients becomes a reality – research is currently targeted towards the development of methods of pluripotency induction, not relying on viral transduction, therefore lowering the possibility of cancer induction by the transplanted iPS cells as well as improvement of the efficacy of reprogramming. Furthermore the remaining questions of whether the donor cells are completely reprogrammed or retain a "memory" of

In vitro 3D models present the opportunity to investigate in depth the molecular mechanisms of the interactions between cells and the extracellular matrix. The increased number of research based on 3D scaffolds, mimicking specific physiological and developmental processes, as well as tissues, bridges the gap between laboratory investigation and unimpeded tissue regenera‐ tion. At the same time the increasing number of research has made it clear that variables such as cell source, the extracellular matrix's biochemical and mechanical properties, growth factor cues and developmental stage affect cell behavior and therapy outcome, imposing the need of even more careful reconsideration of yet the slightly interfering environmental factors for the

Development of optimal bioengineered scaffolds requires indepth knowledge of physiology and understanding of cell–cell and cell–ECM interactions. The choice of inappropriate model system could have a negative effect on the study or the therapy outcome. Such an example could be any of the discussed 3D culturing systems. Sometimes even slight changes in experimental design like difference in cell count in collagen gels for example could lead to different migrational patterns [87] or as mentioned, the change of the substrate stiffness can induce different responses of cells to the environment [14]. Still, the obscured causes under‐ lying such different outcomes are yet to be defined. Another example could be the inappro‐ priate use of three-dimensional BME gels. In vivo basement membranes represent thin extracellular structures that underlie endothelial and epithelial structures. Basement mem‐ branes also surround muscle, fat and nerve cells. Moreover the composition and the amount of the basement membrane are specific and vary in different tissues and during particular stages of development. Even though introduction of cells to a 3D environment, based on basement membrane matrix, results in numerous morphological and physiological changes,

there are a number of discrepancies to be considered and questions to be answered:

successful outcome of regenerative therapies or tissue engineering.

their differentiated state are still to be answered.

**4. Does a perfect 3D system exist?**

232 Regenerative Medicine and Tissue Engineering


All these discrepancies and unanswered questions do not make the BME gels unsuitable model systems, but stress the importance of experimental design. Hakkinen et al. have shown that normal fibroblasts respond unexpectedly in 3D BME gels – they acquire a rounded morphol‐ ogy with short protrusions, possibly for environmental sensing and do not migrate [12] – behavior typical for non-malignant fibroblasts, thus supporting the notion that an adequate model should be chosen for each experimental design. Indeed BME gels provide scientists with a great tool to study malignant cell invasion through the basement membrane [70, 113], with numerous assays developed for investigation of the mechanisms of invasion. Moreover comparison between different three-dimensional scaffolds in terms of cell morphology, adhesion and migration and cytoskeletal organization concluded that indeed careful attention should be paid on the experimental design [12].

Use of other types of 3D gels faces researchers with similar problems. Neither collagen nor fibrin or hyaluronan gels incorporate other components of connective tissue. They are suitable for investigation of a specific response of a specific cell type towards distinctively modulated environments, but are still far away from the complex organization of the native extracellular environment. At the same time the provisional matrix of a blood clot is formed of disorganized fibrin mesh, just like the in vitro 3D gels. Cell-derived matrices on the other hand represent a complex, cell-organized structure based on cell-specific type of expressed matrix components. However, it is possible that in vitro generated cell derived matrices could also have different matrix composition, pore sizes, mechanical and biochemical characteristics than in vivo analogues because of the initially unnatural substrate cells were cultured on. Thus it is important of one to consider the specific characteristic of the tissue in vivo or the nature of the modeled process in order to be able to select an appropriate 3D matrix for in vitro research or for in vitro based preparation for therapies.

Besides looking at the ECM as a 3D structure one must not forget that in vivo cells do have a specific polarity that is usually lost during conventional 2D cultivation, and sometimes even in 3D, if an inappropriate environment has been selected. Fibroblasts for example, lack an 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 to promote tumorigenic transformation [119].

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 could be grouped by several criteria:


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 hair follicles and sebatious glands, thus allowing for rehabilitation of thermal regulation. Even though the perfect skin graft is still to be created, the available skin models have made a significant impact in in vitro studies regarding skin biology and skin disease progression: engineered skin of different aspects helps in producing more relevant results than 2D cultures, thus leading to reduced use of animals for experimentation. As discussed earlier investigation of cell–cell and cell–extracellular matrix interactions has evolved rapidly due to the use of skin tissue-mimicking models. Wound healing, skin contraction [122] and angiogenesis are all fields benefiting from the use of 3D skin equivalents. Investigation of skin diseases such as melanoma invasion [123, 124], psoriasis [125] and skin blistering disorders [126] is also making significant progress based on engineered skin.

Besides skin tissue engineering biological scaffolds have found use in other sections of tissue engineering. Promising results are obtained with decellularization and recellularization of heart, liver and lungs. After recellularization of a hearth-derived ECM, a macroscopic con‐ traction and pump function of the newly generated heart were observed in vitro [91] – indeed a promising result for the generation of a biological artificial heart. Recellularization of liver [92] and lungs [127] has gone even further and transplantation of the newly formed organs back to animals showed that they are able to sustain their natural functions. Probably one of the highest successes of tissue engineering for regenerative medicine is the successful trans‐ plant of trachea in a 30-year old patient [128] based on decellularized tracheal scaffold, reseeded with autologus epithelial and mesenchymal cells thus reducing the possibility of organ rejection and the need of strong immunosupressants. Despite the great success in the field the major disadvantage of these scaffolds still persists – the need of a donor organ for their preparation. Nevertheless the first successful transplantation of entirely bioengineered constructs, a completely new tracheobronchial tube in a patient suffering from tracheal cancer, is already a reality, partly due to the virtue to three-dimensional model systems [129].

Based on the review of decades of research it is currently evident that a perfect 3D system able to fulfill all scientific and medicinal requirements does not exist. On the contrary – a careful and differential approach towards appropriate 3D system selection, experimental or thera‐ peutical design and data interpretation is required for every particular case (Figure 2). Such approach would contribute to the optimal outcome of the specific therapy or the distinct experiment thus leading the way to unimpeded regenerative medicine.
