Properties of Cartilage Tissue

Properties of Cartilage Tissue Chapter 1 1.1 Introduction Joint cartilage is highly sophisticated and has been optimised by evolution. There have been considerable research interests related to the cartilage cells, chondrocytes. In the last decades these studies made cartilage the first and very successful tissue engineering treatment. (Brittberg et al. 1994) 1.2 Categorization of cartilage tissue Cartilage tissue are categorised in three major types by their different biochemical composition and structure of their extracellular matrix (ECM). Elastic cartilage has a few cells, a small concentration of proteoglycans (PGs), and a relatively high proportion of elastin fibres. It is found in the epiglottis, small laryngeal, the external ear, auditory tube, and the small bronchi, where it is generally required to resist bending forces. Fibrocartilage also contains a small concentration of PGs, but far less elastin. The meniscus in the knee joint is made of fibrocartilaginous tissue. The third and most widespread cartilage in the human body is hyaline. It is resistant to compression or tensile forces due to the network organisation of type II collagen fibres associated with a high concentration of PGs. Hyaline cartilage can be found in the nose, the trachea, bronchi, and synovial joints. In the latter case, it is termed as articular cartilage, representing a unique type of connectiv e tissue. Its outwards thin layer covers the articulating joint surfaces and belies a specific structure with unique mechanical properties. These two layers acting as a covering material, is fibricated by the viscous synovial fluid. The joint capsule encloses the entire joint and retains the synovial fluid. (Schulz and Bader, 2006) 1.3 Composition of articular cartilage Articular cartilage is composed of chondrocytes and an extracellular matrix that consists of proteoglycans, collagens and water. (Darling and Athanasiou 2005) Chondrocytes contribute only between 5% of the tissue volume; the remaining 95% being composed of extracellular matrix (ECM), which is synthesised by the chondrocytes. (Mollenhauer, 2008; Buckwalter et al. 1988) The ECM of articular cartilage consists of about 60-85% water and dissolved electrolytes. The solid framework is composed of collagens (10-20%), PGs (3-10%), noncollagenous proteins and glycoproteins. (Buckwalter et al. 1997; Buckwalter et al. 1990) In articular cartilage, 95% of collagen in the ECM is comprised of collagen type II fibrils. The rest other collagen types are collagen type IX and XI and a small fraction of types III, VI, XII and XIV. (Eyre 2002) Type-I collagen forms thick fibres. Type-III forms thin  ¬Ã‚ bres. Unlike these two collagens, Type-II collagen which is present in hyaline and elastic cartila ges does not form  ¬Ã‚ bres, and its very thin  ¬Ã‚ brils are disposed as a loose mesh that strongly interacts with the ground substance. (Montes, 1996) This collagen component in articular cartilage provides tensile stiffness and strength to the tissue and opposes the swelling capacity generated by highly negatively charged glycosaminoglycans (GAGs) of the proteoglycans (PGs). The majority (50-85%) of the overall PG content in this tissue type were presented by large molecule aggrecan. This consist of a protein backbone, the core protein, to which unbranched GAGs side chains of chondroitin sulphate (CS) and keratan sulfate (KS) are covalently attached. ( 1.1) (Watanabe et al. 1998; Schulz and Bader, 2006) 1.1. Illustration of the extracellular matrix (ECM) organization of articular cartilage (Left) and the schematic sketches (Right) of the most relevant polysaccharides of proteoglycans (PGs) in articular cartilage. The PGs consist of a strand of hyaluronic acid (HA), to which a core protein is non-covalently attached. On the core protein, glycosaminoglycans (GAGs) such as keratan sulphate (KS) and chondroitin sulfate (CS) are covalently bound in a bottle brush fashion. (Modified from Schulz and Bader, 2006 and Mow and Wang, 1999) 1.4 Low capacity of self-repair The aneural and avascular nature of articular cartilage, coupled with its low cellularity, contribute to both the limited rate and incomplete nature of the repair process following damage. (Heywood et al., 2004) In addition, the low mitotic potential of chondrocytes in vivo also contributes to its poor ability to undergo self-repair. (Kuroda et al., 2006) Indeed, in experimental studies on adult animals, full-thickness cartilage defects extending into the subchondral bone, have been reported to heal with the formation of fibrous tissue, which contains relatively low amounts of type II collagen and aggrecan. It is also composed of a relatively high content present in type I collagen, not present in normal adult articular cartilage and accordingly exhibits impaired mechanical integrity. (Hjertquist et al., 1971; Eyre et al., 1992) 1.5 Metabolism of articular cartilage Joint cartilage is supplied with nutrients and oxygen by the synovial fluid diffusion facilitated by compressive cyclic loading during joint movements as a pumping function. (Mollenhauer, 2008) Chondrocytes are imbedded in ECM. Within synovial joints, oxygen supply to articular chondrocytes is very limited. The oxygen tensions are very low varying from around 6% at the joint surface to 1% in the deep layers of healthy articular cartilage. It is supposed to be even further decreased under pathological conditions, such as osteoarthritis or rheumatoid arthritis. The metabolism of chondrocytes is largely glycolytic. Oxygen-dependent energy generated by oxidative phosphorylation is just a minor contributor to the overall energy in chondrocytes. Articular chondrocytes appear to show a so-called negative Pasteur effect, whereby, glycolysis falls as O2 levels drop leading to the fall in ATP and matrix synthesis. (Gibson JS et al., 2008) A negative Pasteur effect would make chondrocytes parti cularly liable to suffer a shortage of energy under anoxic conditions. (Lee and Urban, 1997) Changes in O2 tension also have profound effects on cell phenotype, gene expression, and morphology, as well as response to, and production of, cytokines. Condrocytes live in hypoxic environments implies that speci ¬Ã‚ c factors are required to control certain genes that are responsible for glucose metabolism, energy metabolism, pH regulation, and other responses. The most important component of this hypoxic response is mediated by transcription factor hypoxia-inducible factor-1 (HIF-1), which is present in most hypoxia inducible genes. (Pfander and Gelse, 2007; Gibson JS et al., 2008) HIF-1a is necessary for anaerobic energy generation by upregulation of glycolytic enzymes and glucose transporters. (Yudoh et al. 2005) A previous study shows chondrocytes are not able to survive hypoxia in the absence of HIF-1. (Schipani et al. 2001) Moreover, the matrix turnover in articular cartilage is extremely slow. Proteoglycan turnover is up to 25 years. Collagen half-life is estimated to range from several decades up to 400 years. No immune-competent cells (macrophages, T-cells) enter the cartilage tissue. Thus chondrocytes have to defend themselves against hostile microorganisms, leading to its immunologically privileged. (Mollenhauer, 2008) 1.6 Mechanical conditions in vivo In vivo joint loading can result in high peak mechanical stresses (15-20 MPa) that occur over very short durations (1 s) causing cartilage compressive strains of only 1-3%. (Mollenhauer, 2008; Hodge et al., 1986) In contrast, sustained physiological stresses applied to knee joints for 5-30 min can cause compressive strains in certain knee cartilages as high as 40-45%. (Mollenhauer, 2008; Herberhold et al., 1999) A study of the response of articular cartilage from humans to impact load showed that articular cartilage could withstand impact loads of as much as 25 MPa at strain rates from 500 to 1000 s-1 without apparent damage. Impact loads exceeding this level caused chondrocyte death or fissure in the hip or knee. (Repo RU and Finlay JB, 1977) Chapter 2 Osteoarthritis and Treatments 2.1 Osteoarthritis, diagnosis and classification Most cartilage defects are due to direct trauma, but may also occur in avascular necrosis, osteochondritis dissecans, and a variety of cartilage disorders. The defect may be limited to the joint surface (chondral) or involve the underlying bone (osteochondral). (NHS guidance 2006) Articular cartilage defects can progress to osteoarthritis (OA) in some patients, which is a major health problem in developed countries. (Kuroda et al. 2006; Schulz and Bader, 2006; Buckwalter, 2002; Cicuttini 1996) Symptoms may include pain, catching, locking and swelling, and may lead to degenerative changes within the joint. (NHS guidance 2006) Arthroscopy has been used as the â€Å"gold standard† to confirmed cartilage defects. In a review of 31,516 knee arthroscopies of cartilage injury patients, the incidence of chondral lesions was 63%; the incidence of full-thickness articular cartilage lesions with exposed bone were 20% , with 5% of these occurring in patients under 40-years-old. (Marlovits, et al. 2008) Osteoarthritis (OA) severity is commonly graded from radiographic images in accordance with the Kellgren and Lawrence scale Bilateral. (Kellgren and Bier, 1956; Kellgren and Lawrence, 1957) Osteoporosis and erosions which included narrowing of joint space were recorded separately and graded as follows: 0 = no changes; 1 = doubtful joint space narrowing; 2 = minimal change, mostly characterized by osteophytes; 3 = moderate change, characterized by multiple osteophytes and/or definite joint space narrowing; and 4 = severe change, characterized by marked joint space narrowing with bone-on-bone contact with large osteophytes. (Kellgren and Bier, 1956; Husing et al. 2003) The radiologic grade of OA was inversely associated with the joint space width (JSW). (Agnesi et al. 2008) MRI is currently the standard method for cartilage evaluation, as it is a non-invasive, non-contact, multi-planar technique capable of producing high resolution, high contrast images in serial contiguous slices and it enables morphological assessment of the cartilage surface, thickness, volume and subchondral bone. The MRI classification of articular chondral defects are as follows: 1=Abnormal intrachondral signal with a normal chondral surface; 2=Mild surface irregularity and/or focal loss of less than 50% of the cartilage thickness; 3=Severe surface irregularity with focal loss of 50% to 100% of the cartilage thickness; 4=Complete loss of articular cartilage, with exposure of subchondral bone. (Marlovits et al. 2008) Agnesi et al. compared the radiologic grading of OA patients with the joint surface width measurements obtained from MRI images. (Agnesi et al. 2008) 2.2 Non-tissue engineering treatments Pain caused by osteoarthritis can be reduced through a number of methods. (Altman et al. 2006) These include: â€Å"Exercise programmes (strength and flexibility) and lifestyle changes Medication Dietary supplements Knee viscosupplementation Guidelines for viscosupplementation Other injections Custom foot orthotics Knee braces Other assisted devices (canes and walkers) Surgery† Total knee replacement is most commonly performed in people over 60 years of age. (NHS guidance, 2006; Altman et al., 2006; Brittberg et al., 1994) Besides that, the most frequently used treatments include the mosaicplasty, marrow stimulation, and autologous condrocyte implantation (ACI). (Steinwachs et al., 2008) Mosaicplasty is an autologous osteochondral transplantation method through which cylindrical periosteum grafts are taken from periphery of the patellofemoral area which bears less weight, and transplanted to defective areas. This transplantation can be done with various diameters of grafts. (Haklar et al., 2008; NHS guidance, 2006) Marrow stimulation methods include arthroscopic surgery to smooth the surface of the damaged cartilage area; microfracture, drilling, abrasion. All marrow stimulation methods base on the penetration of the subchondral bone plate at the bottom of the cartilage defect. The outflowing bone marrow blood contains the mesenchymal stem cells which are s tabilised by the clot formation in the defect. These pluripotent stem cells which are able to differentiate into fibrochondrocytes, result in fibrocartilage repair with varying amounts of type I, II and III collagen. (Steinwachs et al., 2008) 2.3 The tissue engineering treatment A 1984 study in rabbits reported successful treatment of focal patellar defects with the use of autologous condrocyte implantation (ACI). One year after transplantation, newly formed cartilage-like tissue typically covered about 70 percent of the defect. (Grande et al. 1989) In 1987, Mats Brittber et al. firstly performed ACI in 23 people with deep cartilage defects in the knee. (Brittberg et al., 1994) ACI is described as a three steps procedure: cartilage cells are taken from a minor load-bearing area on the upper medial femoral condyle of the damaged knee via an arthroscopic procedure, cultivated for four to six weeks in a laboratory and then, in open surgery, introduced back into the damaged area as a liquid or mesh-like transplant; at last, a periosteal flap sutured in place to secure the transplant. ( 2.1) (Husing et al., 2008) The cell density of the cultivated cell solution is required to be 30 x 106 cells/ml, or 2 x 106 cells per cm2 in a clinical setting today. (Brittberg e t al., 2003) Genzyme Biosurgery with its product Carticel ® was the first company which introduced ACT into the market and is market leader in USA. Carticel ® is a classic ACT procedure using the periosteal cover. (Husing et al., 2008) Today the periosteum is often replaced by an artificial resorbable cover such as collagen I/III and hyaluronan membrane, such as ChondroGide or Restore (De Puy, Warzaw, Indiana). (Gooding et al., 2006; Jones and Peterson, 2006) Another new method uses chondrocytes cultured on a three-dimensional, biodegradable scaffold. The scaffold, cut to the required size, is fixed into the lesion site with anchoring stitches. This method does not need the cover, thus simplifying the surgery and shorting the surgery time; opens up the possibility of arthroscopic surgery instead of open surgery which causes more tissue damage. HYALOGRAFT from Italy is one of the European market leaders. It is a cartilage substitute made of autologous chondrocytes delivered on a biocompatible tridimentsional matrix, entirely composed of a derivative of hyaluronic acid. (Marcacci et al. 2005) 2.4 Clinical follow-ups of ACI Brittberg studied the long-term durability of ACI-treated patients, 61 patients were followed for at least five years up to 11 years post-surgery (mean 7.4 years). After two years, 50 out of 61 patients were graded good-excellent. At the five to 11 years follow-up, 51 of the 61 were graded good-excellent. The total failure rate was 16% (10/61) at mean 7.4 years. (Brittberg et al., 2003) Since 1997 the year the FDA approved ACI, this method has been widely performed all over the world, in more than 20 000 patients. It has been reported to be effective in  improving clinical symptoms, such as pain and function. (Wakitani et al., 2008) 2.5 Randomised studies In 2004, Knutsen et al. studied 80 patients who needed local cartilage repair because of symptomatic lesions on the femoral condyles measuring 2-10cm2. The results showed there was no signi ¬Ã‚ cant difference in macroscopic or histological results between ACI and microfracture, and that there was no association between the histological  ¬Ã‚ ndings and the clinical outcome at the 2-year time point. (Knutsen et al., 2004) In the same series, there were no signi ¬Ã‚ cant differences in results at 5 years follow-up. (Knutsen et al., 2007) In another randomised controlled study that compared mosaicplasty with ACI, there was no significant difference in the number of patients who had an excellent or good clinical outcome at 1 year (69% [29/42] and 88% [51/58], respectively). In the subgroup of patients who had repairs to lesions of the medial femoral condyle, significantly more patients who had ACI had an excellent or good outcome (88% [21/24]) compared with those who had mosaicplasty (72% [21/29]) (p 2.6 The limitation of ACI The microfracture is a very simple and low-cost procedure whereas ACI costs almost $10 000 per patient. If ACI is not found to be more effective for improving articular cartilage repair than microfracture, the procedure will not be continued. (Wakitani et al., 2008) There are several possible reasons which should be blamed for the limitations of the traditional ACI procedure. The cell source in ACI is the cartilage tissue taken from a minor load-bearing area on the upper medial femoral condyle of the damaged knee via an arthroscopic procedure. However, Wiseman et al. found the chondrocytes isolated from the low loaded area of the knee joint respond in a distinct manner with the chondrocytes from the high loaded area, which suggests the traditional cell source of ACI may not provide enough mechanical response and may further lead to the insufficient mechanical properties of the repaired tissue. (Wiseman et al. 2003) As cultured in monolayer, chondrocytes undergo a process of dedifferentiation and adopt a more  ¬Ã‚ broblast-like morphology, which is accompanied by an increase in proliferation (Glowacki et al., 1983) and an altered phenotype. Type II collagen, the major protein produced by chondrocytes in articular cartilage, are down-regulated culture, while collagen types I and III are increased. (Stocks et al., 2002; Benya et al., 1978) The agregating proteoglycan aggrecan of articular cartilage, is down-regulated during dedifferentiation and replaced by proteoglycans not speci ¬Ã‚ c to cartilage, such as versican. (Glowacki et al., 1983; Stocks et al., 2002) Therefore, monolayer cultured chondrocytes do not express the true chondrocyte phenotype, and their ability to regenerate damaged cartilage tissue is impaired. Upon implantation, dedifferentiated cells may form a  ¬Ã‚ brous tissue expressing collagen type I that does not have the proper mechanical properties, which may lead to deg radation and failure of the repair tissue. (Brodkin et al., 2004) Chondrocytes grown in conditions that support their round shape, such as plating in high-density monolayer (Kuettner et al., 1982; Watt, 1988) and seeding in 3-D gels (Benya et al., 1982) can maintain their differentiated phenotype much longer compared to cells spread in monolayer cultures. Chapter 3 Tissue engineering strategies for articular cartilage 3.1 Introduction Although ACI can still be considered to be one of commonly form of repair of cartilage defects, it does have a number of scientific limitations. Some of those can be resolved using a more comprehensive tissue engineered strategy which incorporates cells, scaffold materials and potentially biochemical, biomechanical and/or physical stimulation in a controlled bioreactor environment. 3.2 Cell sources For a conventional ACI approach, chondrocytes are derived from the low loading area and then cultured in a monolayer. However, chondrocytes derived from the low load bearing area of the knee joint respond in a distinct manner with the chondrocytes from the high loaded area. Chondrocytes cultured in monolayer have a dedifferentiation phenomenon (Described in the previous chapter). In addition, the limitation of the transplant volume is always a major problem in autograft to be overcome (Kitaoka et al., 2001). Thus, potential cell sources are widely studied for the future improvement of ACI approach. Chondrocytes from immature animals (approximately 1-6 weeks old) have been used widely in tissue engineering studies for their ability to increase matrix synthesis and to produce better mechanical properties (Darling and Athanasiou, 2005). Kitaoka et al. examined the possibility of using hyaline cartilage of costal cartilage as a substitute to the knee joint articular cartilage. Costal cartilage cells are derived from 8-week-old male SV40 large T-antigen transgenic mice. Three mouse chondrocyte cell lines (MCC-2, MCC-5, and MCC-35) were established using cloning cylinders, which is a kind of mold. These cell lines showed chondrocytic characteristics, including formation of cartilage nodules that could be stained with alcian blue, and mRNA expression for type II collagen, type XI collagen, ALPase, osteopontin, aggrecan, and link protein (Kitaoka et al., 2001). Animal-derived bone marrow cells, in particular from rabbit origin, have shown a highly variable chondrogenic potential (Solchaga et al., 1999). The establishment of some bone marrow stromal cell lines having the ability of diffrentiation to chondrocyte has been reported, as well as some other cell lines established from rat calvaria, mouse c-fos-induced cartilage tumor and mouse embryonic carcinoma, respectively. Each of the cell lines showed chondrocytic phenotypes (Kitaoka et al., 2001). LVEC cells derived from EBs of human embryonic germ cells were reported to be homogenously differentiated into hyaline cartilage. Three dimensional tissue formation is achieved by encapsulating cells in synthetic hydrogels poly (ethylene glycol diacrylate) (PEGDA) followed by incubation in chondrocyte-conditioned medium (for the recipe, please see the paper) (Varghese et al., 2006). Periosteum consists of two layers. Fibroblasts are from the fibrous layer and progenitor cells are from the cambium layer. Progenitor cells are supposed to be able to differentiate into chondrocytes. Emans et al. compared the chondrocyte and the periosteum cell as cell source for autologous chondrocyte implantation (ACI) on animals. The results turned out that the condrtocytes are much better for ACI procedure (Emans et al., 2006). 3.3 Scaffolds Biomaterial scaffolds provide a critical means of controlling engineered tissue architecture and mechanical properties; allow cells attach, grow in and proliferation; allow the cell signals travelling through (Freed et al., 2006). In many in vitro or in vivo approaches, cells are grown on biomaterial scaffolds for further research or just for implantation, where new functional tissue is formed, remodelled and integrated into the body. The biomaterials which compose scaffolds are required to satisfy several properties. At first, the material as a support structure must possess enough mechanical strength to protect the cells contained in. Secondly, the material must have some bioactivity to accommodate cells for attachment, growth, proliferation and migration. The material should act as a vehicle for gene, protein and oxygen delivery. Furthermore, the material should be biodegradable for the new cartilage to form and replace the original structure. In this regard, the material should be non-toxic, non-inflammatory active, and also non-immunogenic. Finally, the material should be easy to handle for surgery procedure (Stoop, 2008). 3.3.1 Natural materials Collagen-based biomaterials are widely used in todays clinical practice (for example, haemostasis and cosmetic surgery). Collagen is also be commonly used as main components in tissue engineered skin products. Several commercial autologous chondrocyte transplantation (ACT) products have used collagenous membraneas the replacement for the periosteum to close the defect, such as ChondroGide or Restore (De Puy, Warzaw, Indiana) (Cicuttini et al., 1996; Jones and Peterson, 2006) The .combination of collagen with glycosaminoglycan (GAG) in scaffolds had a positive effect on chondrocyte phenotype. Condrocytes were cultured in porous type I collagen matrices in the presence and absence of covalently attached chondroitin sulfate (CS) up to 14 days in a study (van Susante et al., 2001). Hyaluronic acid is a non-sulphated GAG that makes up a large proportion of cartilage extracellular matrix. In its unmodified form, it has a high biocompatibility (Schulz and Bader, 2007). Matrices composed of hyaluronan have been frequently used as a carrier for chondrocytes. Facchini et al. con ¬Ã‚ rms the hyaluronan derivative scaffold Hyaff  ®11 as a suitable scaffold both for chondrocytes and mesenchymal stem cells for the treatment of articular cartilage defects in their study. HYALOGRAFT from Italy is one of the European market leaders for ACT. It is a cartilage substitute made of autologous chondrocytes delivered on a biocompatible tridimentsional matrix, entirely composed of a derivative of hyaluronic acid (Marcacci et al., 2005). Fibrin plays a major role in general wound healing and specially during healing of osteochondral defects. Fibrin glue is currently used for the fixation of other chondrocyte scaffold constructs in defects. Some investigators used fibrin glues as a matrix, but owing to the exceedingly high concentrations and protein densities involved, the glue impeded rather than facilitated cell invasion and did not support a healing response (Stoop, 2008). Susante et al. found fibrin glue does not offer enough biomechanical support as a three-dimensional scaffold (van Susante et al., 1999). Another study found fibrin and poly(lactic-co-glycolic acid) hybrid scaffold promotes early chondrogenesis of articular chondrocytes in vitro. They used the natural polymer fibrin to immobilize cells and to provide homogenous cells distribution in PLGA scaffolds (Shaban et al., 2008). Sugar-based natural polymers such as chitosan, alginate and agarose can be formulated as hydrogels and in some cases sponges or pads. Although these materials are extensively used in in vitro research, their role in in vivo cartilage reconstruction is still limited (Stoop, 2008). Alginate possesses a number of suitable properties as a scaffold material for cartilage tissue engineering. The mobility of alginate allows the ability of cells to be distributed throughout the scaffold before the gelling phase. Its well-characterized mechanical properties are suitable for the transmission of mechanical stimuli to cells. Furthermore, it has been proved its ability to reestablish and maintain the differentiated state of chondrocytes during long-term culture (Heywood et al., 2004). Agarose is a clear, thermoreversible hydrogel which has been applied in numerous studies in cartilage tissue engineering. This hydrogel is supportive of the chondrocyte phenotype and allows for the synthesis of a fu nctional extracellular matrix. Agarose is neutrally charged, and forms solid gels at room temperature. The initial strength of the gel is dependent on the rate of gelling, which in turn is dependent on the ambient temperature. Gel strength is also strongly dependent on the concentration of the gel in solution. Basic science studies involving agarose gel formation have demonstrated that rapid cooling leads to a decreased, more homogeneous pore size. Increasing the gel concentration additionally decreases gel pore size and permeability. A number of studies have used agarose for the investigation of chondrocyte growth and response to mechanical stimuli (Ho MMY et al., 2003). 3.3.2 Synthetic materials Potential synthetic material scaffolds for the tissue engineering of bone or cartilage include: PL (Polylactic acid) PGLA (Polyglycolicacid and copolymers) CF-PU-PLLA (Carbonfibre-Polyurethane-Poly(L-lactide)-Graft) CF-Polyester (Polyester-Carbonfibre) PU (Polyurethane) PLLA (Capralactone (Poly-L-Lactide/epsilon-Caprolactone) PLLA-PPD (Poly- L-Lactic Acid and Poly- p-Dioxanol) PVA-H (Polyvinylalcohol-Hydrogel) ß-TCP (Tricalcium phosphate) CDHA (Calcium-deficient hydroxyapatite) (Haasper et al., 2008) The major advantages of the synthetic polymers are their design flexibility and avoid of disease transmission. Synthetic polymers can be easily processed into highly porous 3-dimensional scaffolds, fibres, sheets, blocks or microspheres. However, there are also disadvantages of some synthetic polymers, such as the potential increase in local pH resulting from acidic degradation products, excessive inflammatory responses and poor clearance and chronic inflammation associated with high molecular weight polymer (Stoop, 2008). Poly(glycolic acid) (PGA), poly(lactic acid) (PLA), and poly(lactic-co-glycolic acid) (PLGA) have been investigated for use as cartilage tissue engineering scaffolds (Cima et al., 1991; Vacanti et al., 1991). Both, in vitro and in vivo studies have demonstrated these scaffold maintained the chondrocyte phenotype and the production of cartilage-speci ¬Ã‚ c extracellular matrix (ECM) (Barnewitz et al., 2006; Kaps et al., 2006). In addition, PLGA is used as a scaffold material for matrix-based autologous chondrocyte transplantation clinically for more than 3 years (Ossendorf et al., 2007). 3.4 Biomedical stimulation Growth factors are proved to be able to promote the formation of new cartilage tissue in both explants and engineered constructs (Darling and Athanasiou, 2005). Insulin-like growth factor-I (IGF-I) can dramatically increase biosynthesis level of choncroctyes, especially in the presence of mechanical stimulation (Bonassar et al. 2001; Jin et al. 2003). Transforming growth factor-ÃŽ ²1 (TGF-ÃŽ ²1) increases biosynthesis in engineered constructs and also stimulates the cellular proliferation (Blunk et al. 2002; van der Kraan et al. 1992). Basic fibroblast growth factor (bFGF) stimulates cell proliferation (Adolphe et al. 1984) and biosynthesis (Fujimoto et al. 1999) in chondrocytes which were cultured under a variety of conditions. 3.5 Mechanical stimulation ACI is considered a proper way for the repair of cartilage defect. However, one of the obstacles to the use of autologous chondrocytes is the limited in vitro proliferation rate of these cells. A lot of stimulations have been found to be effective in stimulating cell proliferation and ECM synthesis, including mechanical, electrical, ultrasound (Parvizi et al., 1999; Noriega et al., 2007) and even laser (Torricelli et al., 2001) stimulation. Mechanical forces due to body movement are experienced by articular cartilage every day. These forces include direct compression, tensile and shear forces, or the generation of hydrostatic pressure and electric gradients. Some level of these forces is beneficial to chondrocytes. (Schulz and Bader, 2007; Shieh and Athanasiou, 2007) There are many studies which have described the design of bioreactor systems, which can apply shear forces, perfusion, tension, hydrostatic pressure, static compression, dynamic compression on cartilage explants, monolayer cultured cells or tissue engineered constructs. (Schulz and Bader, 2007) Previous work on these bioreactor systems has demonstrated that chondrocytes are highly mechanosensitive. A summary of the key studies is provided in Table 3.1. Static compression leads to decreased levels of sulfate and proline incorporation (Sah et al., 1989; Ragan et al., 1999). Type II collagen and aggrecan gene expression increase transiently, but decrease when exposed to longer durations of static compression (Ragan et al., 1999). In contrast, dynamic compression of cartilage explants stimulates sulfate and proline incorporation, while chondrocytes em bedded in hydrogels produce more matrix and form robust constructs when cyclically compressed. (Lee and Bader, 1997; Mauck et al., 2000) Table 3.1. Influence of the different models of mechanical stimulation on the biochemical response of articular chondrocytes. References Type of m