**8. Cartilage tissue engineering and low scaffold successful**

Many advances in the field of cartilage tissue engineering have been closely connected to the improved performance of biomaterials. Successful cartilage tissue engineering relies on four specific criteria: (1) cells, (2) signaling molecules, (3) biomaterials, and the (4) mechanical environment. Furthermore, they should be biocompatible, non-toxic, bioresorbable and highly permeable to facilitate mass transport [139].

The use of scaffolds to support replication of chondrocytes for production of cartilage in vitro has been the most common approach for tissue engineering of cartilage, however, despite the


**7. New proposals for repairing articular cartilage**

380 Regenerative Medicine and Tissue Engineering

ensure healthy cartilage tissue-engineered the [126-128].

permeable to facilitate mass transport [139].

In recent years they have sought new strategies for cartilage repair, with technological advances have currently been proposed the use of scaffolding or matrix on which cells can grow. Among the scaffolds used in the clinic (Table 2) are those that are based on collagen, hyaluronic acid and fibrin as these provide a substrate normally found in the structure of native articular cartilage. Collagen is a major extracellular matrix protein, exists to provide strength and stability to the connective tissues. At the clinic is used collagen I-III as scaffolds for growing chondrocytes in order to improve the structural and biological properties of the graft [116, 117] this is used as a sponge, foam, gel and membrane form, all these are subject to enzymatic degradation. Hyaluronic acid is another important component of articular cartilage matrix and is a glycosaminoglycan that is involved in homeostasis [118, 119] provides viscoelasticity to synovial fluid, is credited as a lubricant and shock absorbing properties, is essential for the correct structure of proteoglycans in articular cartilage. Between scaffolds containing hyalur‐ onic acid is the Hylaff-11, which is an esterified derivative of hyaluronic acid and is used for growing chondrocytes in three dimensions, has been shown that when using this type of scaffold maintaining the chondrocyte phenotype, so that chondrocytes are capable of produc‐ ing the proteins and molecules characteristic of a hyaline cartilage [120-122]. Fibrin is a protein involved in blood coagulation, is regarded as a biomaterial for cartilage repair, as can be found in gel form, having an adhesive function that is also biocompatible and biodegradable [123]. However in vivo studies in animals have shown to have low mechanical stability and can also trigger an immune response [124, 125], fibrin because this has only been used clinically to

Based on the foregoing and which is being used in the clinic and according to results obtained in patients who have been treated with different biomaterials has been observed that although there is a suitable biomaterial that contributes to the production of extracellular matrix to provide the right conditions for chondrocyte cell differentiation. So it is necessary to propose new biomaterials that help produce extracellular matrix, capable of activating a cascade of signaling that can form a cartilage which has structural properties suitable for tissue repair, as

Many advances in the field of cartilage tissue engineering have been closely connected to the improved performance of biomaterials. Successful cartilage tissue engineering relies on four specific criteria: (1) cells, (2) signaling molecules, (3) biomaterials, and the (4) mechanical environment. Furthermore, they should be biocompatible, non-toxic, bioresorbable and highly

The use of scaffolds to support replication of chondrocytes for production of cartilage in vitro has been the most common approach for tissue engineering of cartilage, however, despite the

well as having viscoelastic properties and to provide mechanical stability.

**8. Cartilage tissue engineering and low scaffold successful**

**Table 2.** Biomaterials most used in the clinic, with different components for the repair of articular cartilage by autologous transplantation method of chondrocytes from second and third generation.

apparent simplicity of cartilage, to our knowledge, tissue engineered cartilage has not been successfully reached so far [140-142].

In theory, a scaffold for tissue engineering should have a three dimensional porous structure forming an interconnected porous network. These structures should be made of biocompatible and biodegradable materials capable to provide mechanical strength, support cells ingrowth, promote cells adhesion, uniform cell spreading, and conserve phenotypes and functional characteristics of transplanted cells [143,144]. Unfortunately, this list of requirements looks too long and hard to accomplish. Probably this is one of the main reasons of why the advances in cartilage engineering have been too slow. But also we should rethink these concepts in order to find shorter and easier pathways to find more efficient and effective tissue engineering methods.

The vast majority of scaffolds used in tissue engineering are solid sponge-like porous struc‐ tures that are seeded with cells in a culture media. Analyzing this approach from the basic principles for the design of biomaterials, the biomimetism, easily we can find out that this process lacks of this basic concept. In natural tissues, cells grow in a physiological environment which is more like a gel medium than a porous scaffold, they do not form tissues by populating porous structures, but they do it by creating their own ECM starting from a gel-like environ‐ ment. Following this line, many researchers are proposing the encapsulation of cells in hydrogels instead of using porous scaffolds, looking to improve the biomimetic environment for cells [145,146,147,148].

Besides biomimetism, sponge-like scaffolds provides only a two dimensional surface for cell attachment, although their structure is 3D, cells attach to the walls of the scafflod, thus changing completely the way they are integrated into natural tissues. On the other hand hydrogels are capable to provide a real 3D environment when cells are seeded (encapsulated) into them [149].

#### **8.1. Hydrogels for articular cartilage tissue engineering**

Hydrogels are water-swollen, cross-linked polymeric structures [150] that possess unique mechanical and chemical properties that make them very attractive for a variety of biomedical applications; actually there are no other materials capable to display characteristics too close to natural tissues such as Hydrogels. Therefore hydrogels have been considered as a key material in the development of new biomaterials for tissue engineering and artificial organs fabrication.

Their particular properties come from their structure, composed of swollen randomly cross‐ linked networks of rod-like polymer chains with water filling the interstitial spaces.[151] Water commonly comprises more than 80% of the total volume. The physical properties of hydrogels are determined by the polymer composition and concentration, the cross-linking density between polymer chains [152], polymerization conditions [153], the addition of hydrophobic monomers which may create regions of more dense coiled or entangled chains, the introduc‐ tion of composite materials such as rubber or glass, the use of cross-linking agents such as glutaraldehyde, and the use of freeze-thawing procedures to induce partial crystallinity [154].

Hydrogels can be classified by the type of crosslinking: covalently or ionic cross-linked, physical gels, or entangled networks [155]. The two first are the most common gels. Physical gels are formed by non-covalent interactions, such as hydrogen bonding, and hydrophobic interactions [156]. Covalently or ionic cross linked gels are considerably more stable than physical gels and once they are formed they may not be re-melted again.

Hydrogels can be obtained from natural or synthetic polymers. Natural hydrogels come from proteins and polypeptides (commonly collagen and gelatin), polysaccharides (i.e. alginate, agarose, hyaluronic acid, fibrin, chitin and chitosan). On the other hand, synthetic polymers come from man-made materials such as polyester (i.e. poly L-lactic and polyglycolic acid, poly ε-caprolactone, polypropylene fumarate), polyethylene oxide, polyethylene glycol, polyvinyl alcohol, polyurethane, polydiol citrates, polyhydroxyethyl methacrylate, and many others polymers [157].

Although hydrogel scaffolding technologies plays a crucial role in cartilage tissue engineering, several studies has been shown low success cartilage tissue repair. They are unable to generate cartilaginous tissues with similar properties to native cartilage [141-142].

There are a number of reasons for scaffolds failure, we summarized some of them:


The vast majority of scaffolds used in tissue engineering are solid sponge-like porous struc‐ tures that are seeded with cells in a culture media. Analyzing this approach from the basic principles for the design of biomaterials, the biomimetism, easily we can find out that this process lacks of this basic concept. In natural tissues, cells grow in a physiological environment which is more like a gel medium than a porous scaffold, they do not form tissues by populating porous structures, but they do it by creating their own ECM starting from a gel-like environ‐ ment. Following this line, many researchers are proposing the encapsulation of cells in hydrogels instead of using porous scaffolds, looking to improve the biomimetic environment

Besides biomimetism, sponge-like scaffolds provides only a two dimensional surface for cell attachment, although their structure is 3D, cells attach to the walls of the scafflod, thus changing completely the way they are integrated into natural tissues. On the other hand hydrogels are capable to provide a real 3D environment when cells are seeded (encapsulated)

Hydrogels are water-swollen, cross-linked polymeric structures [150] that possess unique mechanical and chemical properties that make them very attractive for a variety of biomedical applications; actually there are no other materials capable to display characteristics too close to natural tissues such as Hydrogels. Therefore hydrogels have been considered as a key material in the development of new biomaterials for tissue engineering and artificial organs

Their particular properties come from their structure, composed of swollen randomly cross‐ linked networks of rod-like polymer chains with water filling the interstitial spaces.[151] Water commonly comprises more than 80% of the total volume. The physical properties of hydrogels are determined by the polymer composition and concentration, the cross-linking density between polymer chains [152], polymerization conditions [153], the addition of hydrophobic monomers which may create regions of more dense coiled or entangled chains, the introduc‐ tion of composite materials such as rubber or glass, the use of cross-linking agents such as glutaraldehyde, and the use of freeze-thawing procedures to induce partial crystallinity [154].

Hydrogels can be classified by the type of crosslinking: covalently or ionic cross-linked, physical gels, or entangled networks [155]. The two first are the most common gels. Physical gels are formed by non-covalent interactions, such as hydrogen bonding, and hydrophobic interactions [156]. Covalently or ionic cross linked gels are considerably more stable than

Hydrogels can be obtained from natural or synthetic polymers. Natural hydrogels come from proteins and polypeptides (commonly collagen and gelatin), polysaccharides (i.e. alginate, agarose, hyaluronic acid, fibrin, chitin and chitosan). On the other hand, synthetic polymers come from man-made materials such as polyester (i.e. poly L-lactic and polyglycolic acid, poly ε-caprolactone, polypropylene fumarate), polyethylene oxide, polyethylene glycol, polyvinyl

physical gels and once they are formed they may not be re-melted again.

for cells [145,146,147,148].

382 Regenerative Medicine and Tissue Engineering

**8.1. Hydrogels for articular cartilage tissue engineering**

into them [149].

fabrication.


#### **8.2. Trends in hydrogel-scaffolding cartilage repair**

However towards designing biomimetic native environments cartilage is still a challenge due articular cartilage is intricately organized and heterogeneous tissue. This tissue reveals a highly defined structural organization that can be subdivided into two domains, the cartilage zones and the organization of the extracellular matrix. In that sense the ECM of articular cartilage is a unique environment with complex heterogeneity and spatial conformation very difficult to mimic. One of the most notable variations in this tissue is the spatial organization of collagen network and cells arrangement. [141]. Moreover cartilage presents different morphology, gene expression, matrix spatial array between cultured populations isolated from distinct cartilage zones [142]. However, intensive researches have been focus on the development of an ideal scaffold material with versatile properties that actively contribute to cartilage repair [158]. In that regard, there have been several attempts trying to recreate the different zones in cartilage by different hydrogel fabrication technologies, giving as a result tridimensional homogeneous structures with little resemblance to the native organization in cartilage, so it is necessary to material scientists thinking in others design hydrogel-scaffolding strategies trying to biomi‐ metic hierarchical structures capable to deliver bioactive molecules such as growth factors with an ideal mechanical response and mediated by adhesive molecules in order to have an integration tissue [159].

Currently strategies in the design of biomimetic cartilage hydrogels are governed by the use of collagen Type I and derived from porcine small intestine submucosa implants. Although the chondrocytes typically lose their phenotype, the gene expression patterns changed when they are removed from their native environment, so give them a proper environment is necessary to keep its phenotype of chondrocytes in different populations to recreate the zonal organization [160]. In addition, biological trials *in vitro* be made taking into account the cell density for each zone [161,162].

According with reference [163] concentrations of 12-25 million cells/cm2 are needed to increase the matrix production and mechanical properties of human adult chondrocytes under static conditions. Nevertheless, material researches are focus on fabrication of three-dimensional artificial arrays in form of hydrogels using macromolecules present in the cartilage interterritorial matrix and trying to mimic the distinct cartilage zonal [160]; however, no substantial data of the formation of cartilage are reported.

Others approaches in cartilage tissue engineering are the use of hydrogel culture employed mesenchymal stem cells (MSCs) and the use of bioreactors in order to provide the necessary biochemical and biomechanical stimulations to enhance chondrogenesis [164,165]. Due to the many mentioned limitations related to chondrocyte sources, there is much effort to explore better alternative cell sources. Desirable characteristics for such sources include accessibility, availability, and chondrogenic capacity. Consequently, stem cells such as adult mesenchymal stem cells (MSCs) have emerged as promising cell sources for articular cartilage tissue engineering. Chondrogenic potentials of MSCs from different tissues have also been investi‐ gated and compared. Specifically, MSCs from bone marrow are the most popular considering they are easily harvested (via the iliac crest) and have good chondrogenic potential. Many in vitro and in vivo studies have revealed promising results of marrowderived MSCs combined with various biomaterials or growth factors for repairing cartilage defects [164,166]. Recently, Johnson et al. describe the discovery and characterization of kartogenin, a small molecule that induced stem cells to take on the characteristics of chondrocytes and improves joint function and promotes the regeneration of cartilage in vivo in two rodent models of chronic and acute joint [166].

Mechanical stresses are an important factor of chondrocyte function as they stimulate them to increase the synthesis of ECM components. In cartilage culturing processes the main types of mechanical forces currently being investigated are hydrostatic pressure, direct compression, shear environments [167, 168].

Finally, to better recapitulate the ECM environment for cartilage tissue engineering, research‐ ers have to introduce several biological signals, including chondroitin sulfate (CS), hyaluronic acid (HA), and collagen type I and II, into tissue-engineered scaffolds to encourage tissue specificity [169]. CS, hyaluronic acid, and collagen type II have been shown to promote or enhance chondrogenesis of mesenchymal stem cells (MSCs) in hydrogel-based culture systems. In addition to the physical cues of native matrix, cells are exposed to an array of biological cues throughout the ECM that direct cellular behavior.

Cells are constantly interacting with the surrounding ECM, which gives rise to a dynamic transfer of information between the extracellular and intracellular space. In addition, biological trials *in vitro* be made taking into account the cell density for each zone [169].

Tissue engineering should be the best way to achieve successful cartilage regeneration by combining novel biologically inspired scaffolds approaches, nanotechnology, cell sources such as stem cells, chondrogenic factors, and physical stimuli [165].
