Tissue Engineering of Articular Cartilage: A Mini-Review

Corresponding Author: Radoslav Zamborsky, Department of Traumatology, Faculty of Medicine, Slovak Medical University, Bratislava, Slovakia Email: radozamborsky@gmail.com Abstract: Damage of articular cartilage due to congenital anomaly, injury or pathological process may lead in decreasing of life quality of affected patients. In many cases, conventional therapeutical approaches may not bring expected results. Tissue engineering by the combination of material technology and cell-based therapy may represent hope for these patients. The main goal of this review article is to summarize current knowledge about biological characteristics of somatic stem cells, chondro-inductive substances and materials in respect to regeneration of articular cartilage.


Introduction
Recently, despite the advances in medicine, damaged articular cartilage as a consequence of inborn defects, injury or pathological process still represent serious medical problem. It may lead in gradual imobilization and decreasing of life quality in affected individuals. Mentioned is mainly contributed to low self-healing potential of mature articular cartilage due to intrinsic properties. Articular cartilage is aneural and avascular tissue with low density of cells. Moreover, high level of protease inhibitors inhibit tissue repair (Zhang et al., 2009a).
Small defects are usually regenerated by the migration of chondrocytes to cartilage lesions. They synthesize new ECM components of cartilage. In case of large scale and deep defects, this process is complicated by low cell density, by low mitotic potential of chondrocytes as well as by high level of protease inhibitors. It results in formation of the biomechanically insufficient fibrillar cartilage (Mobasheri et al., 2009). Unfortunately, current treatment techniques for cartilage reparation are insufficient and it is not possible to obtain expected results.
The tissue engineering offers new concept to solve this serious problem. The first cell-based therapy for articular cartilage treatment was introduced by Brittberg et al. (1994) who injected autologous chondrocytes into the lesion covered by periosteal flap in 23 people with deep cartilage defects in the knee.
This lead into formation of the hyaline-like cartilage. However, this approach showed some disadvantages, including reacquisition of chondrocyte phenotype during in vitro expansion and non-uniform distribution of cells due to gravitational force.
More recently, it was shown that somatic stem cell undergo the process of chondrogenic differentiation under propper conditions, both in vitro and in vivo. They should be obtained from different tissue sources and easily expended in vitro (Danisovic et al., 2011). Therefore, the great hope is addressed on their utilization in relation to repairing damaged articular cartilage.
The main goal of the present article is to review the current status and advances of the cartilage tissue engineering with respect to their potential application in orthopaedic surgery and traumatology.

Histology of Articular Cartilage
Articular cartilage is a type of hyaline cartilage ( Fig. 1) and belongs to connective tissues. It is composed of abundant Extracellular Matrix (ECM) which contains predominantly collagen type II, chondroitine sulphate and proteoglycans. Articular cartilage is characterized by zonality. Different organization of collagen fibbers and cross-linking to other components influence its biomechanical properties (Sophia Fox et al., 2009).
The ECM is very poor for cells-chondrocytes. On the periphery they have elliptic morphology. Chondrocytes localized inside ECM are of round morphology with average size of 10-30 µm. They formed isogenous cell groups in cavities, called cartilage lacunae (Mescher, 2013). They are responsible for production of ECM components. Moreover, they are involved in the maintaining and remodelling of the articular cartilage (Cucchiarini et al., 2012;Danisovic et al., 2013).

Cell Sources for Articular Cartilage Tissue Engineering
Chondrocytes are the cells of first choice for cartilage tissue engineering, because they occurs within articular cartilage in vivo. They have been used in all current Autologous Chondrocyte Implantation (ACI) procedures. Chondrocytes have been isolated from different sources, including low load-bearing area of knee cartilage and auricular cartilage (Beris et al., 2012;Malicev et al., 2009). However, this technique has some limits due to low mitotic potential and senescence of chondrocytes. Moreover, they undergo dedifferentiation process when cultured in vitro, gradually changing their morphology to a fibroblast-like shape and the production of type II collagen is replaced by the production of collagen type I. This problem may be overlapped by adding specific growth factors.
Somatic Stem Cells (SSCs) because of their biological characteristics represent another promising tool for cartilage tissue engineering. SSCs are undifferentiated cells with unique potential of selfrenewing and plasticity (Danisovic et al., 2011). This combination did not work in human. Moreover, utilization of c-myc led to malignant transformation (Shimizu et al., 2010). The first human IPSCs have been prepared by two independent research groups in USA (University of Wisconsin-Madison) and Japan (Kyoto University) from fibroblasts by using of Oct4, Sox2, Nanog a LIN28 (Yu et al., 2007;Takahashi et al., 2007). IPSCs displayed similar morphological and biological properties as embryonic stem cells, including expression of specific antigens, increased proliferation and telomerase activity and pluripotency. Moreover, they were able to produce embryoid bodies and teratomas (Yu et al., 2007). Since these discoveries, IPSCs belong to most promising tools of regenerative and personalized medicine. For overview of their biological properties, possibilities of preparation and utilization in biomedicine check article by (Csobonyeiova et al., 2013).
IPSCs were also studied in context of cartilage tissue engineering. Diekman et al. (2012) fabricated artificial cartilage tissue from IPSCs using micromass culture for purification of chondrogenic cells and pellet culture system with TGF-β3 to induce chondrogenic differentiation in vitro. Their results proved increased expression of collagen type II and aggrecan. More recently, Ko et al. (2014) demonstrated successful chondrogenesis and regeneration of damaged cartilage with human iPSCs. Chondrogenic differentiation was induced by using alginate hydrogel culture system. Afterwards, micro aggregates of alginate constructs were implanted in osteochondral defects created on the patellar groove of immunosuppressed rats. After 21 days, they observed greater glycosaminoglycan contents and better chondrocytic features including lacuna and abundant matrix formation. However, further studies are necessary for translation of IPSCs into clinical practice, mainly focused on their biological safety.

Biomaterials for Cartilage Tissue Engineering
Cartilage tissue engineering employs many biomaterials of natural or synthetic origin (Table 1). They may be in form of hydrogel, sponges, fibrous meshes and nanofibres. The crucial characteristics are their non-toxicity and biocompatibility. Other characteristics, such as porosity (size and orientation of pores) and structural strenght also influnce their final utilization (Liu et al., 2013).
The most commonly used natural material is collagen which belongs to basic constituents of cartilage in vivo. The mechanical properties of collagen-based scaffolds may be easily controlled by chemical modifications . It was shown that chondrocytes cultured within collagen scaffolds maintain their original phenotype and production of Glycosaminoglycans (GAGs) under in vitro conditions. Moreover, several authors provide evidence of strong chondroinductive effect on SSCs (Zhang et al., 2012;Zheng et al., 2010). Hyaluronic Acid (HA) is another natural biopolymer studied in the context of cartilage tissue engineering. Ha also occurs in native cartilage and should be used itself or in combination with other biomaterials. It was shown that chondrocytes cultured within HA hydrogels are forced to produce collagen type II and aggrecan typical for hyaline cartilage. Furthermore, SSCs cultured within photo-cross-linked HA hydrogel undergo chondrogenic differentiation (Chung and Burdick, 2009).
Besides the above mentioned natural biopolymers, a variety of synthetic polymers may be applied in cartilage tissue engineering. When compared with natural biomaterials, they have several advantages, including highly controlled physical characteristics, consistency, uniformity and unlimited production (Yu et al., 2012).
The most widely used are Polylactic Acid (PLA) and Polyglycolic Acid (PGA) (and their co-polymer). Both of them belong to biodegradable polymers. It was demonstrated that they increase chondrocyte proliferation and GAGs production. Moreover, several authors provide evidence of their effect on SSCs proliferation and chondrogenic differentiation (Foldberg et al., 2012;Xue et al., 2012).
Poly (Ethylene Glycol) (PEG) and its derivates, mainly in form of hydrogel wer also evaluated in respect to cartilage regeneration. Hwang et al. (2010) demonstrated that chondrocytes cultured within PEG scaffold remain alive and underwent chondrogenic differentiation. More recently, Cui et al. (2014) prepared cartilage constructs by using 3D printing technology. They used PEG-based scaffold with chondrocytes and demonstrated their full viability and prominent production of collagen type II and GAGs.
There are a lot of other synthetic materials that have been studied in respect to cartilage tissue engineering, e.g., poly (α-hydroxy esters), poly (propylene fumarate), poly (urethane) (Yu et al., 2012).

Growth Factors
Growth factors play pivotal role in the process of chondogenesis. They represent a group of biologically active polypeptides that may affect cell proliferation and differentiation. In the hyaline cartilage, specific growth factors regulate homeostasis, integrity and development. The effect of growth factor on chondrogenic differentiation may differ depending on its dose, specific cell type and cell differentiation (Yu et al., 2012).
Most studied growth factors in respect to cartilage tissue engineering include members of Transforming Growth Factor-β (TGF-β) superfamily, Fibroblast Growth Factor (FGF) family and Insulin-like Growth Factor 1 (IGF-1).
TGF-β superfamily contains at least 20 members in vertebrates. The best candidates for cartilage tissue engineering are TGF-β1, TGF-β3, BMP-2, BMP-4, BMP-7 and CDMP-1 (also known as GDF-5). TGF-β1 promotes the synthetic activity of chondrocytes and decreases catabolic activity of IL-1 and MMPs in vivo. TGF-β3 enhances synthesis of sulphated GAGs. BMP-2 stimulates synthesis of cartilage-specific ECM. BMP-4 is essential for normal embryogenic development and exhibits osteogenic and chondrogenic potential in vivo. BMP-7 has significant anabolic activity by which protects cartilage against damage. GDF-5 increases proliferation of chondrocytes as well as play important roles during the development of skeleton and joints (Fortier et al., 2011).
FGF family contains at least 23 members in vertebrates. Mainly FGF-2, FGF-4, FGF-8 and FGF-18 were studied in the context of cartilage tissue engineering. It was shown, that FGF-2 promotes the proliferation of chondrocytes in vivo. FGF-2 with FGF-4 and FGF-8 are involved in the process of anabolic pathways activation which leads to decrease of aggrecanase effect after cartilage load. Furthermore, FGF-18 is involved in a variety of biological processes, including embryonic development, cell growth, morphogenesis and tissue repair (Ellman et al., 2013).
IGF-1 stimulates chondrocytes to synthesize cartilage-specific ECM and decreases catabolic responses. Moreover, it was demonstrated that IGF-1 has an additive effect on increase of cartilage matrix synthesis when acts with TGF-β1, BMP2 and BMP7 Gow et al., 2010).

Conclusion
Recently, cartilage tissue engineering provides new promising approach which should be used in healing patients with damaged articular cartilage. It combines different types of cells (chondrocytes and stem cells), various scaffolding materials and appropriate growth factors to prepare fully biologically active artificial cartilage tissue. However, prior to translation into clinical practice the further studies have to be carried out, mainly focused on safety of stem cells expanded under in vitro conditions. Considerable progress can be expected also in field of material technology, mainly in combination with 3D bioprinting.