Biodegradable Scaffolds for Cartilage Tissue Engineering

Being a connective tissue, the cartilage is present in almost all parts of the body like the rib cage, joints, nose, and ear. Its essential function in body is to serve as a cushion between the joints and prevent the bones friction against each other. In some areas like the rib cage, the cartilage keeps the bones together and creates a shockproof area. Osteoarthritis and traumatic rupture of the cartilage are among the related diseases. Damaged cartilage tissue can be only limitedly repaired because of the low density of chondrocyte and slow metabolism in the tissue. Previous studies achieved different outcomes for the joint-preserving treatment programs such as debridement, mosaicplasty, and perichondrium transplantation; however, the average long-term result is still unsatisfactory. The restriction of clinical success is mainly attributed to the long time required in most treatments for the regeneration of new cartilage at the site of defect. The mechanical conditions of these sites makes the repair process unflavored of the original damaged cartilage. Such problems can be permanently treated by using tissue engineered cartilage. Hence, the limitations can be defeated by using appropriate scaffolds, cell sources, and growth factors. This review dealt with the advances in cartilage tissue engineering, with the focus on cell sources, scaffold materials and growth factors used in cartilage tissue engineering.[ GMJ.2017;6(2):70-80] DOI: 10.22086/GMJ.V6I2.696


Introduction
C artilage is an avascular connective tis- sue with various significant functions like keeping some bones together, making the joint part shockproof and preventing the bones from rubbing against each other.The constituents of cartilage are chondrocytes specialized cells.These cells create the cartilaginous matrix, which is mainly made of collagen and proteoglycans.Development of cartilage occurs quite slowly.The chondro-cytes are fixed in a small space called lacunae, which do not allow their migration to the damaged areas.Any blood vessels do not supply the cartilage tissues; instead, the cartilage compression induces a pumping action leading to a diffusion that feeds the chondrocytes.Since the cartilage tissue has the low intrinsic regenerative ability, its self-heal is restricted, and the trauma-or disease-induced lesions tend to progressively degrade [1,2].Improper function or loss of cartilage causes diseases, namely, osteoarthritis and achondroplasia.
Despite the different levels of success achieved through various clinical therapeutic methods such as microfracture, mosaicplasty, and autologous chondrocytes injection, the durable outcomes are not good enough usual [3].A common reason for the failure of all the treatment mentioned above strategies is that compared with the natural cartilage, the newly-formed tissue has neither the structural organization of the cartilage nor any mechanical feature in common [4].Therefore, it is strongly essential to find the solution to these problems.The emerging technology of tissue engineering speeds up the repair of the damaged tissue if the self-heal fails.The studies dedicated to this field of research mainly aim to develop a replacement tissue with native cartilage organization, composition, and similar mechanical property, which can absolutely restore the joint functionality.Forming the building blocks of tissue engineering, the scaffolds, cells and growth factors are known as the tissue engineering triad.Some features of cartilage allow its reconstruction through tissue engineering.One of them is the simplicity of this tissue which is made of a single type of cell (chondrocytes).Moreover, its nutrition and excretion of wastes are done through diffusion instead of a vascular network; thus, the cell-scaffold constructs do not require being neovascularized.The present review elaborates on the issues related to the methods of engineering the cartilage by using a composite polymeric scaffold, chondroprogenitor cells, and various growth factors.The debate goes on with the paramount factors for cartilage tissue engineering such as cell source, scaffolds, and mechanical stimulation.The current condition of cartilage tissue engineering will also be discussed.The study concludes with mentioning the common limitations and accommodating recommendations for further approaches to cartilage engineering.

Search Strategies
This study was a structured literature review of articles published from 1998 to 2016.The keywords were searched on PubMed, Scopus, and Wiley Inter Science databases.The search was limited to English-language publications.Searching the keywords yielded 92 articles.To be included in the study, exact relation to the keywords was required for the publications.The editorials and manufacturer-supported publications were excluded from the review process.Finally, 73 citations remained as the basis for this review.

Different Types of Cartilage
The human body has various types of cartilage including hyaline cartilage (e.g., tracheal and articular), elastic cartilage (e.g., ear) and fibrocartilage (e.g., meniscus and intervertebral disc) [5].Hyaline cartilage is located in the joints and facilitates their articulation.It is mostly composed of collagen type II fibers.The most flexible cartilage is elastic cartilage due to the more elastin fibers content.Its collagen content is mostly of type I collagen, but it also contains type II collagen.Fibrocartilage is found in the intervertebral discs.It attaches the tendons and ligaments to the bones.It is located in high-stress parts and guards the joints against shocks.Damaged hyaline cartilage is commonly taken over by fibrocartilage, whose rigidity does not let it bear the weight.

Cell Sources
The ideal cell source for cartilage tissue engineering should be easy to isolate and expand, and secretes abundant cartilage-specific extracellular matrix components.Being potential in cartilage tissue engineering has promoted the chondrocytes and stem cells to the most investigate cell sources [6].

Chondrocytes
Chondrocytes play the key role in the regeneration of cartilage.They can be taken from donor organs including cartilaginous tissues like the menisci of the knee joint, trachea, and nose.They have the capability to create, maintain and remodel the cartilage tissue in vitro.However, the autologous chondrocytes are scarcely accessible, and the cells gath-GMJ.2017;6(2):70-80www.gmj.irered from diseased joints are rather inactive.The expansion of chondrocyte in monolayer culture results in dedifferentiation and is presented as reduced proteoglycan synthesis and expression of the type II collagen, and overexpression of the type I collagen [7,8].
Chondrocytes from younger donors are more metabolically active in vitro; whereas, those taken from adult donors have higher chondrogenic potential and rapid expansion [9].Another drawback with the extracted articular chondrocytes is the morbidity at the donor site and loss of joint function.

Stem Cells
To overcome the limited supply of primary chondrocytes, it is suggested to use multipotent stem cells which are mainly isolated from the bone marrow, adipose and pre-implantation embryo tissue [10].The supplies of adult mesenchymal stem cells can be accessed in different tissues namely trabecular bone, bone marrow, deciduous teeth, periosteum, articular cartilage, adipose tissue, muscle and synovial membrane.Some specific signaling molecules (e.g., transforming growth factor-β (TGFβ) can inspire the differentiation of mesenchymal stem cells in diverse 3-dimensional (3D) culture environments [10][11][12].
The cartilage tissue engineering restrictedly benefit from the bone marrow extracted stem cells since the subsequent builds would have lower matrix accumulation and mechanical properties compared with the chondrocyte-seeded constructs [13,14].Adipose-isolated stem cells are capable of differentiating into chondrocytes in 3D culture systems in the presence of ascorbate, dexamethasone, and TGF-β [15].However, their chondrogenic potential is lower than that of the bone marrow-derived stem cells.Additional researches are needed to better comprehension the chondrogenic potential of these cells.Studies are also needed on other sources for cartilage tissue engineering like muscle, synovium, and periosteum, all of which have shown chondrogenic potential but still restrictedly compared to bone marrow-derived and/or adipose-derived stem cells [16].

Growth Factors for Cartilage Regenertion
Growth factors are mainly the signaling molecules which trigger the differentiation of cells into certain phenotype.
The chondrocytes anabolic and catabolic processes are affected by some growth factors, cytokines, and hormones.
The most prominent factors that assist the regeneration of chondrocytes are polypeptide growth factor, TGF-β, basic fibroblast growth factor (FGF-2), insulin growth factor I (IGF-I), and bone morphogenetic growth factors (BMPs).They are employed either per se or in combination to improve the chondrogenesis.Polypeptide growth factors significantly affect the regulation of cell activities, like that of chondrocytes [5].Besides, it hinders the transcription of cartilage-specific matrix genes in longterm cultures [17].The growth and repair of cartilage is generally determined by the TGF-β superfamily members.The TGF-β1, 2 and 3 isoforms promote the proliferation of chondrocyte and the synthesis of extracellular matrix content by chondrocytes [18][19][20].The two isoforms of IGF are IGF-1 and IGF-2, the former of which is the most investigated form in cartilage restoration.It inspires the anabolic activity of chondrocytes and induces chondrogenesis in bone marrow-derived stem cells [21][22][23].The IGF-2 is an effective mitogen for articular chondrocytes and assists the differentiation of chondrocytes in 3D culture system.The FGF-18 is said to support the cartilage repair [24].Another kind of agents is a group of growth factors identified as bone morphogenetic proteins (BMPs), also known as cytokines or metabologens.Some BMPs are recognized as cartilage-derived morphogenetic proteins (CDMPs).So far, 20 BMPs have been recognized.The chief BMPs incorporating in cartilage repair are BMP-1, BMP-2, BMP-4, BMP-5, BMP-7, BMP-8a, BMP-9 and BMP-12.Proper cartilage formation highly depends on BMP activity [25][26][27].The BMPs are thoroughly involved in stages of chondrogenesis and directly control the expression of some chondrocyte definite genes.Hence, the chondrocyte proliferation and matrix synthesis are considerably influenced by this category of signaling molecules.The BMPs trigger the chondrogenic differentiation that is actually mediated through gap junction-mediated intercellular contact [28].In vitro addition of a combination of growth factors to chondrocyte and bone marrow-derived stem cell cultures is likely to increase their efficacy (Table -1).
The differentiation of the cartilage tissue is profoundly affected by the dosage of different growth factors.For instance, transient, rather than continuous, use of TGF-β3 yielded higher compressive properties and increased the glucose aminoglycan content of chondrocyte loaded hydrogels and bone marrow-derived stem cell-laden constructs [30,31].Almost all cartilage tissue engineering investigations applied 10 ng/ml of growth factors (e.g., TGF-β, FGF-2 and BMPs) [32].

Scaffolds
The scaffold is a 3D construction whereupon the cells can attach properly, and grow potentially.Different kinds of biomaterial are used for constructing the scaffolds.The ideal bio-material should be biocompatible, non-toxic, non-attractive, non-stimulatory of inflammatory cells, non-immunogenic.It should also have some particular features that aid adequate cell adhesion, proliferation, differentiation into specific phenotype like the mechanical support of the cartilage engineered tissue and having porosities that permit diffusion of nutrients and waste products.Moreover, these materials should be biodegradable and allow remodeling as the new cartilage forms and substitute the original build.They should be decay-resistant at physiological pH and body temperature [32].
The perfect scaffold for cartilage tissue engineering is the one with high porosity and pore-to-pore interconnectivity.High porosity (normally>90%) provides adequate space for in vitro cell adhesion, ingrowth, and restructuring of cells.Interconnected porous organization facilitates the cell migration, spread of physiological nutrients and gasses to the cells, and discharge of metabolic waste and side-products from cells [33].Mechanical stimulation can be certainly used for boosting the mechanical features of tissue-engineered cartilage.Bioreactors have been made to subject the cell-seeded constructs to mechanical loading regimes [34].The direct dynamic compression administered in chondrocyte-seeded scaffolds usually generate enhanced extracellular matrix production and/or proliferation and improves the compressive characteristics of the engineered tissue [35].

Types of Scaffold
The scaffolds needed for cartilage repair has been made by using several types of materials with both natural and synthetic polymer basis in a variety of forms.Synthetic polymers are mainly favored because they are quite flexible in modifying the physical, mechanical and chemical properties; consequently, the ultimate scaffold can be simply processed into the desired form and dimensions.An enormous group of synthetic polymers has been already incorporated in cartilage tissue engineering successfully.The synthetic polymeric scaffolds used in cartilage tissue engineering are most frequently made of poly-α-hydroxy esters, especially polylactic acid (PLA) and polyglycolic acid (PGA).The reason is the biodegradability of these materials, besides being approved by the US Food and Drug Administration (FDA) for clinical applications [36].
A small number of synthetic polymers are beeing currently clinically evaluated for their potential use in cartilage repair.The main disadvantage which occurs by using the synthetic polymers is their cells frequently do not keep the chondrocyte phenotype and make an extracellular matrix with lower properties [37].
On the other side, the natural polymers are not only cost-effective, environment-friendly, highly biodegradable, less toxic, and renewable but also take low manufacturing and disposal costs [38].Furthermore, they have important controlling features that highly determine the success of cartilage tissue regeneration including remodeling, biological signaling, cell responsive degradation, and cell adhesion.
The most important drawback of natural polymers is their rapid degradation.Moreover, the scaffold making procedures might geopardize their biological properties.The threats of immune rejection and disease transmission demand strict monitoring and purification of the natural polymer [38,39].
The defects of natural and synthetic polymers can be compensated by using composite scaffolds made of two or more polymers, and functionalization of the polymers that provide proper conditions for cartilage regeneration.
Composites create and amalgamation of different features of various polymers to control the biodegradation, cell adhesion, proliferation, and differentiation [40].
A previous study reported the use of a composite scaffold composed of gelatin, hyaluronic acid, chondroitin-6-sulfate, and fibrin to improve the chondrogenesis, and the use of a composite scaffold of hydroxyapatite mixed with chitosan for the healing of osteochondral defects [41].Functionalization can create new functional groups in the polymer, which might supply particular cues to the cells for cartilage regeneration.Functionalized polymers are currently being employed to make up for the defects of natural and synthetic polymers.The integrin combining activity of adhesion proteins can be replicated by introducing short synthetic peptides, containing the Arg-Gly-Asp (RGD) or other like adhesion serieses in the polymer, which increases cell adhesion [42].Peptide ligands have modified several materials to support chondrogenesis.According to Hwang et al., human embryonic stem cell-derived cells can be enveloped in RGD-modified hydrogels and be used to enhance the cartilage creation [43].Attempts were made to improve in vitro construction of cartilage by rabbit chondrocytes through adding chitosan-alginate-hyaluronate complexes modified with RGD-containing proteins [44].In another study, RGD-coupled alginate hydrogels containing a co-transplantation of osteoblasts and chondrocytes, the formed growing tissues had structure and function similar to a growth plate cartilage [45].
A broad range of materials have been developed and are accessible in the form of injections, microspheres, and thermoreversible hydrogels, which can be applied for in situ tissue regeneration [46].Injectable biodegradable chitosan-hyaluronic acid-based hydrogels are also applicable for in situ cartilage tissue engineering [47].

Scaffold Architecture, Porosity, Stiffness and Biodegradability
The porosity, pore size and interconnectivity of scaffold materials can affect the cell migration and diffusion of nutrients, signaling molecules, oxygen and waste products [48].
For instance, inhomogeneous oxygen delivery from the periphery towards the center of the cell-seeded structure may result in cell death in the inner areas but not in the periphery [49].Moreover, when the material is porous, the mechanical interlocking developed between the implant and the surrounding natural cartilage creates a better mechanical stability at the interface.The proliferation and phenotype of chondrocytes are significantly impressed by the porosity and permeability features [50,51].The proper pore size for the better proliferation of scaffolds should be optimized between 100 and 500 μm [52].
The rigidity of scaffolds affects the mechanical properties of the seeded cell's surroundings, which can in turn impress the cell differentiation and tissue growth in culture.Enhancing the substrate rigidity affects the chondrocyte morphology; it can be transformed from a rounded shape to elongated shape on weaker substrates to a mainly flat morphology with actin stress fibers on more rigid substrates [53,54].Production and deposition of new tissue would be influenced by the 3D structure of the scaffold and its degradation rate.Optimal degradation kinetics guarantee the primary stability and shape of the scaffold.
Comparison of the rapid and slow degrading scaffolds revealed that the latter yields elevated and more homogeneous deposition of extracellular matrix [55].When the scaffold is degraded, the new tissue is permitted to integrate and reform into the adjacent cartilage after implantation.Ng et al. reported that supervised degradation of agarose scaffold via agarase enzyme improved the collagen content and dynamic mechanical characteristics related to control over time in culture.They attributed it to the facilitated transportation of nutrition and enlarged room for collagen fibril increase with time of culture [56][57][58][59].

Tissue Engineered Cartilage: Content, Structure and Functionality
The mechanical conditions of joints are quite challenging.The constituent material of tissue-engineered cartilage implant should be strong enough to survive or appropriately function under normal joint loading.An engineered tissue is not necessarily a precise duplication of the original natural tissue; it is rather likely to get and obtain features after implantation.

Collagen
The low level of collagen content and the subsequent weak tensile characteristics are considered the main deficiency of tissue-engineered cartilage.The maximum collagen level gets up to 15-35% of the natural amount within five to twelve weeks [60,61].
The in vitro synthesis of collagen is highly affected by some culture features including the cell source, cell seeding density, growth factors, mechanical stimulation, and scaffold properties [62,63].The low level of collagen contents might be due to the rapid synthesis of glycosaminoglycans (GAGs) that prevents the increase of collagen.The self-assembly of collagen might be adjusted by modified transport of synthesized products or altered extracellular biochemical surroundings.Studies announced that in vitro degradartion of type I and II collagen was strain dependent [64,65].The tissue-engineered cartilage contains more GAGs than collagens; it has negative effects on the tensile characteristics of the tissue.
The plausible reason can be the higher or changed crosslinking, the larger size or altered direction of fibril [66,67].

Proteoglycan
The GAGs content and compressive characteristics enhanced with increasing the culture duration, densifying the cell seeding, adding  [69].Human polymer-based cartilage tissue engineering grafts prepared of human autologous fibrin, PGA and human chondrocytes were reported to be clinically helpful in regenerating the articular cartilage damages [70].A different investigation used chitosan hydrogels to repair a sheep articular cartilage damage, and the outcome was satisfactory successful [71].Since a few years ago, polymeric nanofibers have been employed by many scientists for cartilage regeneration [33].
Finding an ideal cell source is of paramount importance.Despite the optimal performance of primary native chondrocytes, their utilization is almost impractical due to inadequate availability.The main challenge of developing the chondrocytes is to be cautious not to lose the phenotype.Seemingly, the stem cells can be a promising substitute; however, their cartilage tissue has weaker properties than those of the chondrocytes.In the near future, studies will prove if stem cells are the optimal cell supply for cartilage tissue engineering.Quite little is known about the effects of sequences and concentrations of growth factors on the cartilage regeneration.Although cartilage regeneration is multifactorial and influenced by multiple growth factors, the majority of investigations engaged a single growth factor.A limited number of systems have been established with the privilege of the biphasic release of dual growth factors.Double growth factor-releasing alginate-based nanoparticle/ hydrogel system was just utilized to deliver BMP-7 and TGFβ-2 to improve chondrogenesis [72].Therefore, a study should be conducted to focusing on the release of several growth factors at a time, to favor the making of more natural cartilage tissue.The extracellular matrix content is undoubtedly so significant, but the extent to which the native matrix components should be reproduced is vague in pre-implantation of engineered cartilage implants.It is probable to acquire the GAGs is possible to reach its native amount in engineered cartilage, but the collagen content is almost always far less than the natural level.Future studies are recommended to investigate methods that enhance collagen content, which is highly essential for the proper mechanical functioning of the tissue.Meanwhile, most studies on cartilage tissue engineering take cells from young adults and even fetal animals, not from elderly osteoarthritis patients.There is a need for a comprehensive study on use of the cells from elderly osteoarthritis patients to broaden the outcomes for treating human cartilage defects.
The final and maybe most challenging issue is the translation of the findings of in vitro and animal studies to be marketed and employed in clinical conditions.Despite the development of numerous cartilage products and growth factor carrier materials, only a limited number are approved for clinical application.
It can be attributed to the cost of production and materials, manufacturing scale-up, sterility, and patent subjects.Besides, there are regulatory barriers including quality control and quality assurance for reliable manufacturing, comparability researches required for component and procedure changes, the establishment of shipping and storage states, and proper shelf life [73].

Conclusion
Currently, both the existing and new cartilage engineering products need to be approved by the corresponding organizations so that they can be available for clinical uses.To bring the obtained laboratory results into the use for treating human cartilage defects, cost-effective tissue engineered products needs to be produced.It will encourage the patients with cartilage defects to seek tissue engineering solution instead of other surgical options and prosthetics.Undoubtedly, despite the extensive progress achieved in regenerative medicine, developments are still highly required in cartilage tissue engineering to find the ideal economical states for cartilage regeneration.The authors wish that the progress in this field will find more great application in therapeutic strategies in regenerative medicine to solve the problem of the aging population of the world.

Table 1 .
The Growth Factors Evaluated For Their Effects on Chondrocyte Growth and Matrix Production