**2. Classical tissue engineering approaches**

decreased 2,67%, even though waiting lines increased during the same year. Such decrease

It is clear that alternatives to organ transplantation need to be developed as soon as possible.

*Tissue engineering.* Tissue engineering refers to an "interdisciplinary field that applies the principles of engineering and the life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function" [3]. The term was first coined by Dr. Fung, from California University, which suggested this name during the National Science Foundation Meeting, in 1987 [2]. The first official definition dates to 1988, though, when Skalak and Fox published it after the "Tissue engineering Meeting" held in Lake Tahoe, USA during

In 1993, Langer and Vacanti described three strategies for the creation of new tissue in vitro [3]. **1.** *Isolated cells or substitutes*. The concept of treating injured tissues with isolated cells is currently regarded as cell therapy. Infusion of cells, e.g. stem cells, has presented several promising results, and have already been approved for human use for specific applications [21] but in some cases, is hindered by the lack of fixation of cells in the site of lesion. When injected systemically, stem cells are attracted to injured tissues, but are

**2.** *Tissue inducing substances.* At the time, tissue inducing substances included growth factors, small molecules, and other classes of molecules which, if delivered in the organism, would promote several effects on cells, such as growth [58], survival [58], migration [57] and neo

**3.** *Cells placed on or within matrices*. Associated cells and substrates provide the injured tissue with continuity, and promotes cell attachment and fixation. In this context, scaffolds may be associated with inducing substances, providing means to combine all the aforemen‐ tioned strategies. The combination of cells and matrices, in addition to inducing substances or not, is currently the main strategy for tissue engineering, as depicted in

Currently, tissue engineering focuses mainly of associating cells with supports (also called biomaterials or scaffolds), in order to: i. promote cell attachment and restrict their distribution in the tissue, ii. direct cell distribution and differentiation, iii. sustain large tissue losses while

Since its early days, tissue engineering has significantly evolved in each of its pillars – Cells, signaling molecules and scaffolds. This evolution covered both conceptual aspects - as evidenced above – as well as practical aspects, mainly reflected in the achievements of the field (for more information, go to conclusion section). Unfortunately, even though cells and signaling molecules platforms have evolved during the past decades, leading to major field evolution, the degree of success of tissue engineering methods is still highly dependent on the properties of the scaffold. Therefore, this study focuses on the main Achille`s Hill of tissue

occurred mainly due to a reduction in number of recovered organs [18].

also found in several organs such as lungs, liver and spleen [56].

new tissue is formed, and ultimately, to iv. lead to new tissue formation.

engineering: production of scaffolds for biological applications.

That`s where tissue engineering comes into picture.

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that year [1].

tissue formation.

Figure 1.

Association of cells to classic biodegradable solid/porous biomaterial represents a dominat‐ ing conceptual framework in tissue engineering. Actually, men have used biomaterials (alone, not associated to cells) in order to substitute eventual tissue loss since ancient civili‐ zations [11]. In the early days, all kinds of materials derived from natural and manufactured sources were used as biomaterials. Natural materials included wood and shells, and manu‐ factured materials comprised metals such as iron, gold and zinc. The host responses to these materials were extremely varied, and only after the concept of sterility, biomaterial implants began to achieve consistent safety. During the last 30 years, further progress has been made in understanding the interactions between the tissues and the materials.

Since then, tissue reconstitution evolved to a more sophisticated approach, in which the regen‐ eration of the tissue/organ was clearly viewed as the ideal way of treating injuries, compared to biomaterial science, which simply reconstitutes tissue structure, without restoring tissue func‐ tion in most situations. This paradigm evolution led to the emergence of tissue engineering.

Tissue engineering based on biomaterials relies on four main classes of materials: i. Poly‐ mers, ii. Ceramics, iii. Metals and iv. Composites (blends and combinations of the aforemen‐ tioned materials). Biomaterials may derive from natural or synthetic sources [12].

The association of cells to biomaterials is called construct and is the base of current tissue engineering, as already stated. Construct-based classic tissue engineering platform derives from several basic assumptions, as described by Mironov et al., 2009 [31]: "1) cell growth is substrate attachment-dependent; cells need a solid substrate for attachment and prolifera‐ tion; 2) tissue constructs must have an organo-specific shape; a solid scaffold is essential to keep the desired shape; a tissue construct could not maintain its shape without a solid rigid scaffold; 3) the scaffold serves not only as an attachment substrate, but also as a source of inductive and instructive signals for cell differentiation, migration, proliferation and orienta‐ tion; 4) the porous structure of a solid scaffold will allow optimal cell seeding, tissue con‐ struct viability, and vascularization; and 5) mechanical properties initially provided by the rigid solid scaffold after its biodegradation will be maintained by controlled neomorphogen‐ esis of parenchymal and stromal tissue synthesized *in vitro* or *in vivo* in the tissue construct".

Considering these basic assumptions, currently, classic tissue engineering is made taking several aspects into account, which will be highlighted below.


**iii.** *Studying material tribology and surface topography* [19]. For several tissues, such as muscles and tendons, cell organization is paramount for optimal tissue function. The heart, for instance, works as a pumping organ, and needs to contract in a specific, synchronized way in order to actually eject blood into body and lungs. If muscle fibers are not synchronized, no pumping force is generated, and great deficit of function is witnessed by the patient [60]. Tendon is a specific connective tissue composed of parallel collagen fibers. Along with the heart, tendon constitutes another of several tissues which depend on specific cell organization for proper function. It has been thoroughly described in literature that tendon strength is directly linked to cell orientation, and, following injuries, the lack of orientation of scar tissue promotes tendon weakness, leading to repetitive lesion [61]. Tissue engineering approaches for tendon must promote cell alignment in order to achieve significant benefit for patients. cell alignment has been shown to be achievable and effective in promoting tissue organization and maturation [30], as shown in Figure 2.

Since then, tissue reconstitution evolved to a more sophisticated approach, in which the regen‐ eration of the tissue/organ was clearly viewed as the ideal way of treating injuries, compared to biomaterial science, which simply reconstitutes tissue structure, without restoring tissue func‐ tion in most situations. This paradigm evolution led to the emergence of tissue engineering.

Tissue engineering based on biomaterials relies on four main classes of materials: i. Poly‐ mers, ii. Ceramics, iii. Metals and iv. Composites (blends and combinations of the aforemen‐

The association of cells to biomaterials is called construct and is the base of current tissue engineering, as already stated. Construct-based classic tissue engineering platform derives from several basic assumptions, as described by Mironov et al., 2009 [31]: "1) cell growth is substrate attachment-dependent; cells need a solid substrate for attachment and prolifera‐ tion; 2) tissue constructs must have an organo-specific shape; a solid scaffold is essential to keep the desired shape; a tissue construct could not maintain its shape without a solid rigid scaffold; 3) the scaffold serves not only as an attachment substrate, but also as a source of inductive and instructive signals for cell differentiation, migration, proliferation and orienta‐ tion; 4) the porous structure of a solid scaffold will allow optimal cell seeding, tissue con‐ struct viability, and vascularization; and 5) mechanical properties initially provided by the rigid solid scaffold after its biodegradation will be maintained by controlled neomorphogen‐ esis of parenchymal and stromal tissue synthesized *in vitro* or *in vivo* in the tissue construct". Considering these basic assumptions, currently, classic tissue engineering is made taking

**1.** *Construct design.* Currently, scaffold design is a complex science, in which several aspects are carefully addressed in order to produce successful constructs. Those aspects include,

**i.** *Choosing the most suitable biomaterial for the envisioned application.* Organs and tissues

**ii.** *Customizing the material in order to promote cell colonization*. The ideal construct must

lysine-valine-alanine-valine (IKVAV) residue, derived from Laminin [28,29].

must also be thin, but most importantly, it must be transparent.

in the human body present different characteristics, therefore, the ideal biomaterial must reproduce as many as those features as possible. Such aspects include tissue resistance, elasticity, resilience, and chemical composition, among others, in addition to biocompatibility. For instance, a biomaterial designed to be used in a skin construct must be thin and elastic. A biomaterial for application in corneas, on the other hand,

promoterapidandequalcelladhesionandcolonization,thereforescaffoldsareusually porous and present a surface which is recognized directly or indirectly (by promoting protein adsorption) by the cell as a substrate for attachment. Usually, materials such as chitosan and collagen are used as biomaterials, due to their resemblance to the extracellularmatrixandtheirefficacyinpromotingcelladhesion[26,27].Incaseofother materials,suchasmetalsandceramics,biomaterialsurfacemaynotbeeasilyrecognized by cells. In order to improve cell contact and adhesion by cells, many strategies have beendeveloped, suchasblendingbiomaterials,recoveringthemwithother substances or still covering them with protein residues recognized by cells, such as isoleucine-

tioned materials). Biomaterials may derive from natural or synthetic sources [12].

several aspects into account, which will be highlighted below.

but are not restricted to:

298 Advances in Biomaterials Science and Biomedical Applications

**iv.** *Optimizing biomaterial degradation rate.* Tissue engineering has been envisioned to promote tissue regeneration, therefore in this context, biomaterials should be biodegradable. Ideally, biomaterial should gradually degrade, at the same rate as neotissue is formed.

**Figure 2.** Cell orientation promotes major contractile strength of construct. In order to assess cell orientation impor‐ tance for construct function, Dr. Parker`s group engineered bidimensional cardiac muscles with different micropat‐ terned surfaces in order to promote degrees of cell orientation. Confluent unaligned isotropic (A), aligned anisotropic (B) and non confluent, 20μm spaced, parallel arrays of myocardial fibers (C) were build and studied *in vitro*. The cited work showed that contractile force increases with major sarcomere alignment, as measured by peak systolic stress in kPa (D). This panel was based on [60] and was kindly provided by Dr. Kevin Kit Parker, from Harvard University.


**Figure 3.** Importance of non-static cell seeding and culture for construct equal distribution of cell elements and viabili‐ ty of construct. As depicted, static culture seeding promotes cell accumulation in specific regions of the construct. As no media is perfused through the construct, nutrient distribution along the biomaterial depends on diffusion only, which leads to normoxic construct edges, colonized with cells and hypoxic center, which is not feasible to be colon‐ ized by cells. Nonstatic culture strategies, on the other hand, promote equal cell seeding and colonization of the con‐ struct, as it enhances nutrient diffusion. Even though nonstatic culture is not sufficient to promote viability of large constructs, it increases maximum size of constructs and is more adequate for tissue engineering purposes.

**4.** *Construct implant.* The ultimate function of a construct built *in vitro* is to be implanted and substitute/regenerate an injured organ or tissue. Construct implant must be performed in order to promote construct integration and viability. Several techniques have already been applied to promote construct long term viability, such as designing VEGF (vascular endothelial growth factor) releasing constructs; previously implanting the construct in an ectopic site, in order to promote *in vivo* vascularization, prior to implantation; and reducing the size of implants, among others.

In spite of its major advances, scaffold-based tissue engineering suffers from several limitations, as explored by Mironov et al., 2009 [31], and further explained here:

**1.** Vascularization of thick tissue constructs

**2.** *Cell seeding.* Cell seeding is paramount for construct optimization, and must be carefully planned. Usually, most cells have low capacity of invasion, therefore, cell seeding must be optimized to promote an equal distribution of cells along construct surface and

**3.** *Construct maintenance in vitro.* The maintenance of small constructs may be achievable in static cultures, but when it comes to larger constructs, static culture is hindered by the limitation of nutrient diffusion. Several strategies have been developed for large construct

maintenance *in vitro*, represented mainly by bioreactors, as depicted in Figure 3.

**Figure 3.** Importance of non-static cell seeding and culture for construct equal distribution of cell elements and viabili‐ ty of construct. As depicted, static culture seeding promotes cell accumulation in specific regions of the construct. As no media is perfused through the construct, nutrient distribution along the biomaterial depends on diffusion only, which leads to normoxic construct edges, colonized with cells and hypoxic center, which is not feasible to be colon‐ ized by cells. Nonstatic culture strategies, on the other hand, promote equal cell seeding and colonization of the con‐ struct, as it enhances nutrient diffusion. Even though nonstatic culture is not sufficient to promote viability of large

**4.** *Construct implant.* The ultimate function of a construct built *in vitro* is to be implanted and substitute/regenerate an injured organ or tissue. Construct implant must be performed in order to promote construct integration and viability. Several techniques have already been applied to promote construct long term viability, such as designing VEGF (vascular endothelial growth factor) releasing constructs; previously implanting the construct in an ectopic site, in order to promote *in vivo* vascularization, prior to implantation; and

constructs, it increases maximum size of constructs and is more adequate for tissue engineering purposes.

reducing the size of implants, among others.

interior [62].

300 Advances in Biomaterials Science and Biomedical Applications

Cell survival requires continuous supply of nutrients and oxygen, as well as the removal of metabolites, which, if accumulated, might be toxic. Such demand is addressed mainly by osmosis, therefore, to facilitate nutrient and metabolite flow, cells must be kept near vessels and capilars. Actually, few cells are able to survive at more than 200um distance from the nearest blood vessel [8]. Cells cultured in tridimensional scaffolds also need to be maintained in homeostasis in order to survive. *In vitro*, several strategies have been developed in order to maintain construct viability prior to implantation, mainly through the development of bioreactors, as reviewed by Rauh et*. al.* [10]. *In vivo*, however, none of those strategies are applicable, and only through vascularization cells are kept alive, especially for modular organs, such as heart, kidney and liver, which are organized in functioning units and require their own vascular supply [9]. Studies indicate that vessels grow at a rate of <1mm a day [6], and although the effectiveness of an enhanced angiogenic response using various growth factors has been demonstrated in many tissue systems, the rate of angiogenesis hasn`t been accelerated so far [7]. Considering the relatively large sizes of constructs for humans, it is clear the urgent need to promote faster vascularization of tissue constructs, or to improve cell survival in scaffolds.

Actually, several attempts of increasing tissue vascularization are underway.

As previously mentioned, increasing cell survival is also an interesting strategy. Actually, recently, oxygen generating scaffolds have been developed and tested with encouraging results, even though no *in vivo* tests were performed [7].

**2.** Precise placing of different multiple cell types inside 3D porous scaffolds is technologi‐ cally challenging.

Modular organs, such as the heart, liver, kidneys and others, are complex structures of several types of cells, including stromal and parenchymal cells. They function as working units, such as muscle fibers, liver lobules and kidney nephrons. Modular organs also count on intrinsic vascular system for cell survival, constituting incredibly difficult organs to build *in vitro*, even though many papers have shown a significant capacity of self-organization of several cells [22,23,24]. For such organs, whole organ approaches are more suitable then partial reconstitution of those structures.

On the other hand, non-modular organs have witnessed several successful strategies, such as the construction of bladders [44] and the recellularization of tracheas [15], both of which have already been translated to the clinic.

**3.** Achieving organo-specific level of cell density in tissue constructs remains a big challenge

Currently, porous matrices are paramount to allow cell invasion and colonization of the matrix. Porous present in the matrices are usually optimized to have specific sizes and to be interconnected, in order to permit cell invasion. The size of the produced porous is usually large, though, and cells within the scaffold are not able to fully fill it and achieve cell density similar to natural tissues. Therefore, it is almost as if cells were still in twodimensional surfaces [31]. Actually, extracellular matrix molecules can be washed out from 3D porous scaffolds in the same way as in 2D cultures, and may not provide means for real tridimensional tissue formation.

**4.** Recent reports on the effect of matrix rigidity on (stem) cell differentiation can undermine the value of solid rigid biodegradable scaffolds at least for certain tissue applications [35-37]

Stem cell differentiation has traditionally employed cocktails of various growth factors, but recently, mechanobiological concepts have been described as important to cell fate decision. The mechanism underlying cellular response to tension comprises the force generated by myosin bundles sliding along actin filaments and transmission to the ECM. Transduction of these signals link the extracellular and intracellular worlds, ultimately affected by proteins such as Rho GTPases, which not only regulate contraction of stress fibers, but also regulate gene expression by acting over their effector target proteins, [45].

Actually, matrix rigidity has been involved in embryonic development, as well as adult stem cell differentiation. As expected, rigid surfaces facilitates adult stem cell differentiation into bone, and soft surfaces lead to differentiation of adult stem cells into soft tissues, such as fat or central nervous system (brain) [45].

**5.** Biodegradability of constructs

Even though it makes sense that biomaterials should be absorbed by the body in order to give space to neotissue formation, the same is not true when whole tissue engineering is planned. There is no use in spending efforts in order to build a construct, which will be invaded by inflammatory cells and vessels and disorganized, previously to being substituted by neotissue.

Therefore, even though current tissue engineering techniques are fairly successful in treating bone, skin and cartilage loss, they are extremely limited in treating large tissue loss, as well as in regenerating complex tissues, such as heart annd kidneys, among sev‐ eral other tissues and organs.
