Immune Privilege of Tissue Engineered Articular Cartilage

Immune Privilege of Tissue Engineered Articular Cartilage The immune privilege of tissue engineered articular cartilage derived from mouse adult mesenchymal stem cells and the potential of tissue engineered cartilage as a gene delivery method Chapter 1 Stem cell biology 1.1 Categorization of stem cells Stem cells are generally defined as cells possessing the following 3 characteristics: (1) self-renewal, (2) the ability to produce all cell types made in that tissue, and (3) the ability to do so for a significant portion of the life of the host (Alberts et al., 1989; Reya et al., 2001), while progenitor cells are capable only of multi-lineage differentiation without self-renewal (Weissman, 2000). Stem cells can be classified by their ability to differentiate. The most primitive, totipotent stem cells have the ability to divide and produce all the differentiated cells in an organism, including both the embryonic and extraembryonic tissues of an organism. Totipotent stem cells include the fertilized egg and the cells produced by the initial divisions of it. In mammals, these cell divisions result in an implant in the uterus called the blastocyst. The blastocyst contains an outer sphere of trophoblast cells. Trophoblast cells are capable of implanting into the uterus and helping the form of placenta which provides nutrients to the embryo. Within the blastocyst are 10 to 20 pluripotent cells called the inner cell mass. In mammalian uterus, these inner mass cells will participate in the production of all tissues and organs of the developing embryo, then fetus, then born organism. Such pluripotent cells can produce any differentiated cells in the body, but are usually unable to for m the trophoblast cells. The best-known pluripotent stem cell is the embryonic stem (ES) cell, which are obtained from the inner cell mass of the blastocyst and exist for only a brief stage of embryonic development. The last major class of stem cells, multipotent stem cells, gives rise to a limited number of cell types which are responsible for organ growth and renewal such as neural stem cells, skin stem cells and haematopoietic stem cells (HSCs) (Cheshier et al., 2009). 1.2 Selected milestones of stem cell research In 1981, Martin isolated a pluripotent stem cell line from early mouse embryos (Martin, 1981). Wilmut in 1996 first cloned a mammal, a lamb named Dolly by transferring nuclear from the adult mammary gland cell to an enucleated unfertilized egg (Wilmut et al., 1997). In 1998, Thomson obtained the first human embryonic stem cell line from human blastocysts (Thomson et al., 1998). In 2001, President Bush banned scientists from using federal funds to study stem cells from sources other than those that had already been grown because of the ethical concerns. To avoid ethical dispute over the use of human embryonic cells for research purposes, many efforts have been taken on obtaining pluripotent stem cells from differentiated donor cells. In 2006, Yamanaka find a way to obtain pluripotent cells by reprogramming the nucleus of adult mice skin cells (Takahashi and Yamanaka, 2006). Such cells are now known as induced pluripotent stem (iPS) cells. 1.3 A brief introduction of several types of multipotent stem cell The best known multipotent stem cells are haematopoietic stem cells (HSCs), that give rise to all the blood cell types including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells), and lymphoid lineages (T-cells, B-cells, NK-cells). HSCs are vital elements in bone-marrow transplantation, which has already been used extensively in therapeutic settings (Reya et al., 2001). In the long-term culture systems, human and rodent Central Neural System (CNS) cells maintain the capacity to produce the three main mature cell classes of the CNS: neurons, astrocytes, and oligodendrocytes, which suggest stem cells and/or progenitors exist and can survive in the culture medium (Weiss et al., 1996; Carpenter et al., 1999). In 2000, Human CNS stem cells (hCNS-SCs) have been successfully isolated by FACs (Uchida et al., 2000). Cancer stem cell hypothesis was proposed by Reya 2001 (Reya et al., 2001). This hypothesis consists of 2 components. The first component postulates that normal tissue stem cells are the target for transforming mutations and successive mutations result in the formation of a tumor. The second component is that within every cancer a specific subset of cancer stem cells continuously gives rise to all the other cancer cells and only these cells within a tumor possess the ability to self-renew, continuously proliferate. Conflicting to the first component of the hypothesis, evidences indicate cancer stem cells can also arise from mutated progenitor cells rather than stem cells (Cheshier et al., 2009). In addition, mature cells such as Lymphocytes can lead to mouse T cell leukemia independently from HSCs (Yuan et al., 2006). For the latter component of cancer stem cell hypothesis, it is likely that the cancer stem cell hypothesis is applicable to some tumors but not to others. In hematopoiet ic and some solid malignancies, only 1 in 100 to 1 in 10 000 primary tumor cells are capable of reproducing the tumor in vivo, such as human breast cancer, human neuroepithelial tumors, head and neck squamous cell carcinomas, and colon cancer. But in melanoma, nearly 1 in 4 cells possessed the ability of proliferation and developing into cancer (Cheshier et al., 2009). Cancer stem cells and CNS stem cells were reviewed by Cheshier et al. (Cheshier et al., 2009). 1.4 Mesenchymal stem cells (MSCs) and their differentiation potential Bone marrow is composed of two main systems of cell, hematopoietic cells and the supporting stromal cells (Bianco et al., 2001). MSCs reside within the marrow, maintain a level of self-renewal, and give rise to progenitor cells that can differentiate into various lineages of tissue, including chondrocytes, osteoblasts, adipocytes, fibroblasts, marrow stroma, and other tissues of mesenchymal origin. The traditional opinion about the multipotent differentiation potential of MSCs was challenged by further studies. Interestingly, MSCs reside in a diverse host of tissues throughout the adult organism and possess the ability to ‘regenerate cell types specific for local tissues e.g. adipose, periosteum, synovial membrane, muscle, dermis, pericytes, blood, bone marrow, and most recently trabecular bone, reviewed by Tuan et al. (Tuan et al., 2003). Furthermore, in 2002, Jiang et al. reported a rare cell within human bone marrow mesenchymal stem cell cultures that can be expanded extensi vely without obvious senescence. This cell population can differentiate, not only into mesenchymal cells, but also cells with visceral mesoderm, neuroectoderm and endoderm characteristics in vitro. Most somatic cell types could be derived after this population of cells was injected into an early blastocyst (Jiang et al., 2002). These studies suggest mesenchymal stem cells maintained pluripotent properties. Chapter 2 Features of Articular Cartilage 2.1 Introduction Joint cartilage formed highly sophisticated structure during the evolutionary development. 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). 2.2 Categorization of cartilage tissues Cartilage tissue is categorised in three major types by different biochemical compositions and structures of their extracellular matrix (ECM). Elastic cartilage has a small concentration of proteoglycans (PGs), and a relatively high proportion of elastin fibres. It exists 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 possesses a small concentration of PGs, but far less elastins. The meniscus in the knee joint is made of fibrocartilage. Hyaline is the most widespread cartilage in the human body. It is resistant to compressive or tensile forces due to its special type II collagen fibril mesh filled with a high concentration of PGs. Hyaline cartilage can be found in the nose, trachea, bronchi, and synovial joints. In the latter case, it is termed as articular cartilage (Schulz and Bader, 2007). 2.3 Compositions of articular cartilage Chondrocytes contribute to only 1%- 5% of the tissue volume; the remaining 95%-99% being extracellular matrix (ECM). Chondrocytes sense and synthesize all necessary ECM components (Mollenhauer, 2008; Schulz and Bader, 2007). 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. In articular cartilage, 95% of collagen in the ECM is type II collagen fibrils. The rest other types are collagen type IX and XI and a small fraction of types III, VI, XII and XIV. Normal articular cartilage does not present type I collagen, which is concerned with fibrous tissue. Unlike Type I and Type III collagens which form thick fibres and thin  ¬Ã‚ bres respectively, Type II collagen present in hyaline and elastic cartilages does not form  ¬Ã‚ bres. It forms very thin  ¬Ã‚ brils which are disposed as a loose mesh that strongly interacts with the groun d substance. Type II collagen provides tensile stiffness and strength to articular cartilage and constrains the swelling capacity generated by highly negatively charged glycosaminoglycans (GAGs) of the proteoglycans (PGs). The majority (50-85%) of the PG content in articular cartilage were presented by large molecule aggrecan. It consists of a protein backbone, the core protein, to which unbranched GAGs side chains of chondroitin sulphate (CS) and keratan sulfate (KS) are covalently attached (Figure 1.1). The composition of articular cartilage was extensively reviewed by Schulz and Bader (Schulz and Bader, 2007). Figure 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, 2007 and Mow and Wang, 1999). 2.4 Low capacity of self-repair in articular cartilage 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). The low mitotic potential of chondrocytes in vivo also contributes to its poor ability to undergo self-repair (Kuroda et al., 2007). Some researchers believe that cartilage lesions less than 3mm in diameter self-repair with normal hyaline-like cartilage (Revell and Athanasiou, 2009; Schulz and Bader, 2007). In animal studies, full thickness cartilage defects, extending into the subchondral bone, have been reported to heal with the formation of fibrous tissue, which contains relatively low amount of type II collagen and aggrecan, but a relatively high concentration of type I collagen which is not present in normal adult articular cartilage and accordingly exhibits impaired mechanical properties (Hjertquist et al., 1971). 2.5 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 which acts as a pumping function (Mollenhauer, 2008). Within synovial joints, oxygen supply to articular chondrocytes is very limited, from 7.5% at the superficial zone down to 1% oxygen tension at the deep zone. It is supposed to be even further decreased under pathological conditions, such as osteoarthritis (OA) or rheumatoid arthritis (RA). 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. Nevertheless, changes in O2 tension have profound effects on cell metabolism, phenotype, gene expression, and morphology, as well as response to, and production of, cytokines (Pfander and Gelse, 2007; Gibson et al., 2008). The most important component of this hypoxic response is mediated by transcription factor hypo xia-inducible factor-1 (HIF-1), which is present in most hypoxia inducible genes (Pfander and Gelse, 2007; Gibson et al., 2008). 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 (Mollenhauer, 2008). Chapter 3 Osteoarthritis (OA) 3.1 Prevalence Osteoarthritis (OA) is the most common form of arthritis. More than 40 million US American citizens (approximately 15% of the overall population of the USA) suffer from arthritis (Schulz and Bader, 2007). OA can occur in any joint but is most common in certain joints of the hand, knee, foot and hip. OA is the most common reason for total hip- and knee-joint replacement (Wieland et al., 2005). Among US adults 30 years of age or older, symptomatic disease in the knee occurs in approximately 6% and symptomatic hip osteoarthritis in roughly 3% (Felson and Zhang, 1998). 3.2 The symptoms and diagnosis The symptoms of OA include pain, stiffness and loss of function. OA can be monitored by radiography, magnetic resonance imaging (MRI), and arthroscopy, but radiographs are still considered the gold standard (Wieland et al., 2005). 3.3 The pathology of OA The pathologic characteristics of OA are the slowly developing degenerative breakdown of cartilage; the pathological changes in the bone, including osteophyte formation and thickening of the subchondral plate; the changes in the synovium such as inflammatory infiltrates; ligaments, which are often lax; and bridging muscle, which becomes weak. Many people with pathologic and radiographic evidence of osteoarthritis have no symptoms (Martel-Pelletier, 1999; Felson et al., 2000). A protease family of matrix metalloproteases (MMP) is responsible for the initial occurrence of cartilage matrix digestion. Of this family, collagenases, the stromelysins and the gelatinases are identified as being elevated in OA. Another group of MMP is localized at the cell membrane surface and is thus named membrane type MMP (MT-MMP) (Martel-Pelletier, 1999). Proinflamatory cytokines such as interleukin (IL)-1ÃŽ ², Tumor necrosis factor (TNF)-ÃŽ ±, IL-6, leukemic inhibitor factor (LIF) and IL-17 are first produced by the synovial membrane and then diffuse into the cartilage through the synovial fluid, where they activate the chondrocytes to produce proinflammatory cytokines. These proinflamatory cytokines are considered responsible for the catabolic pathological process (Martel-Pelletier, 1999). In OA cartilage, an increased level of an inducible form of nitric oxide synthase (iNOS) leads to a large amount of nitric oxide (NO) production (Pelletier et al., 2001). NO can inhibit the synthesis of cartilage matrix macromolecules such as aggrecans and can enhance MMP activity (Taskiran et al., 1994; Murrell et al., 1995). It is well stablished that proinflammatory cytokines such as IL-1ÃŽ ² act as the key mediators of cartilage breakdown and stimulate the release of inflammatory products (NO) and prostaglandin (PG)E2, via induction of iNOS and cyclo-oxygenase (COX)-2 enzymes (Chowdhury et al., 2008). 3.4 Risk factors Osteoarthritis is considered to be a systemic disease although severe joint injury may be sufficient to cause osteoarthritis. There are several systemic risk factors related to OA. (1) Age: Osteoarthritis increases with ages, the incidence and prevalence of disease increased 2- to 10-fold from 30 to 65 years of age and increased further thereafter in a community-based survey (Oliveria et al., 1995). (2) Hormonal status and bone density: women taking estrogen have a decreased prevalence of radiographic osteoarthritis (Nevitt et al., 1996). Before 50 years of age, the prevalence of osteoarthritis in most joints is higher in men than in women. After about age 50 years, women are more often affected with hand, foot, and knee osteoarthritis than men. In most studies, hip osteoarthritis is more frequent in men (van Saase et al., 1989). Evidence suggests an inverse relationship between osteoarthritis and osteoporosis (Felson et al., 2000). (3) Nutritional factors: evidence indicates that co ntinuous exposure to oxidants contributes to the development of many common age-related diseases, including osteoarthritis. McAlindon et al. reported a threefold reduction in risk for progressive radiographic osteoarthritis was observed in persons in the middle and highest tertile of vitamin C intake compared with those whose intake was in the lowest tertile (McAlindon et al., 1996a). Vitamin D intake was observed associated with the progression of OA although not associated with risk for new-onset radiographic osteoarthritis (McAlindon et al., 1996b; Lane et al., 1999). (4) Genetics: genetic factors account for at least 50% of cases of osteoarthritis in the hands and hips and a smaller percentage in the knees (Spector et al., 1996). Candidate genes for common forms of osteoarthritis include the vitamin D receptor gene, insulin-like growth factor I genes, cartilage oligomeric protein genes, and the HLA region (Felson et al., 2000). Local mechanical factors include the body weight and the pathological alterations of the mechanical environment of the joint. Persons who are overweight have a high prevalence of knee osteoarthritis (Felson et al., 1997). OA is also considered to be related to alterations in joint mechanical environments such as knee laxity, the displacement or rotation of the tibia with respect to the femur; proprioception, the conscious and unconscious perception of joint position and movement; knee alignment , knee position in reference to the hip and ankle (Felson et al., 2000). In addition, joint dysplasias, fractures of articular surfaces, and tears of menisci and ligaments that increase joint instability precede the development of osteoarthritis in a high percentage of affected joints. Risk factors for posttraumatic osteoarthritis include high body mass, high level of activity, residual joint instability or malalignment, and persistent articular surface incongruity (Buckwalter et al., 1997; Honkonen 1995). 3.5 Treatments The medicine treatment of OA was dominated by COX2 inhibitors (Flower 2003). The other medicines include glucosamine, chondroitin (McAlindon et al., 2000), and hyaluronic acid (Lo et al., 2003). In addition, both aerobic walking and muscle strengthening exercise reduce pain and disability from osteoarthritis (Roddy et al., 2005). Articular cartilage lesions, both of traumatic or pathological origin, do not heal spontaneously and often undergo progressive degeneration towards osteoarthritis (OA). The most frequently used treatments include the artificial joint replacement, mosaicplasty, marrow stimulation, and autologous condrocyte implantation (ACI) (Steinwachs et al., 2008). Total joint replacement is most commonly performed in people over 60 years of age. (NHS 2006; Brittberg et al., 1994) 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, 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 stabilised 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). The ACI tissue engineering treatment will be discussed in the next chapter. Chapter 4 Tissue engineering and autologous chondrocyte implantation (ACI) 4.1 Overview of tissue engineering technologies Tissue engineering is defined as ‘‘the application of the principles and methods of engineering and the life sciences toward the fundamental understanding of structure-function relationships in normal and pathological mammalian tissues and the development of biological substitutes to restore, maintain, or improve tissue function† (Langer and Vacanti, 1993). Three factors are considered as the principles of tissue engineering, including the utilization of biocompatible and mechanically suitable scaffolds, an appropriate cell source, and bioactive molecules to promote the differentiation and maturation of the cell type of interest (Song et al., 2004). Potential applications of tissue engineering are involved in the following fields: skin, cartilage, bone, cardiovascular diseases, organs (e.g. liver, pancreas, bladder, trachea and breast), central nervous system (e.g. spinal cord), and miscellaneous (e.g. soft tissue, ligaments). Although research is being carried out in all these fields, only few products have already entered the market. The most successful products up to now are: tissue engineered skin which is mainly used for wound cover, autologous chondrocyte implantation (ACI), and artificial bone graft (Hà ¼sing et al., 2003). 4.2 Autologous chondrocyte implantation (ACI) In 1984, a study in rabbits reported successful treatment of focal patellar defects with the use of ACI. One year after transplantation, newly formed cartilage-like tissue typically covered about 70 percent of the defect (Grande et al. 1989). In 1987, Brittberg firstly performed ACI in 23 people with deep cartilage defects in the knee. ACI is described as the following 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 (Figure 2; Brittberg et al., 1994). Genzyme Biosurgery with its product Carticel ® was the first company which introduced ACI into market and is the market leader in USA. Carticel ® is a classic ACI procedure using the periosteal cover (Hà ¼sing 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 tri-dimensional (3D), biodegradable scaffold. This kind of scaffold, cut to the required size, is fixed into the lesion by anchoring stitches or its sticky nature. The 3D cell seeded scaffold eliminates the using of cover, thus simplifies the surgery procedure, saves the surgery time, and opens up the possibility of an arthroscopic surgery instead of the open surgery which causes more tissue damage. HYALOGRAFT from Italy is one of the European market leaders. It is a cartilage substit ute made of autologous chondrocytes delivered on a biocompatible 3D matrix, entirely composed of a derivative of hyaluronic acid (Marcacci et al. 2005). 4.3 Clinical results 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 (Brittberg et al., 2003). Since 1997 the year FDA approved ACI, this method has been widely performed in more than 20,000 patients all over the world. It has been reported to be effective in relieving clinical symptoms, such as pain and function (Wakitani et al., 2008). In a randomised controlled study, Knutsen et al. studied 80 patients who needed local cartilage repair with lesions on the femoral condyles of 2-10 cm2. There were no signi ¬Ã‚ cant differences in clinical 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 Clinical results of ACI were reviewed by Gikas 2009 (Gikas et al., 2009). Generally speaking, the outcomes of ACI treatment have been encouraging. However, most randomised controlled studies showed no significant difference between ACI and traditional treatments. 4.4 Limitations of ACI Microfracture is a very simple and low-cost procedure whereas ACI costs about $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 to be blamed for the limitations of the traditional ACI procedure. The cell source in ACI is the cartilage tissue derived via an arthroscopic procedure from the low load-bearing area on the upper medial femoral condyle of the damaged knee. However, Wiseman et al. found the chondrocytes isolated from the low loaded area of the knee joint respond to mechanical stimulations 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 and an altered phenotype. Type II collagen, the major protein produced by chondrocytes in articular cartilage, are down-regulated in the culture, while collagen types I and III are increased (Glowacki et al., 1983; 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 origninal 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 appropriate 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 (Watt, 1988) and seeding in 3D structure (Benya and Shaffer, 1982) can maintain their differentiated phenotype much longer compared to cells spread in monolayer cultures. 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 more comprehensive tissue engineered strategies which incorporates cells, scaffold materials and potentially biochemical, biomechanical and/or physical stimulation in a controlled bioreactor environment. 4.5 Tissue engineering strategies for ACI 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 as described above. In addition, the limitation of the transplant volume is always a major problem in autograft to be overcome (Kitaoka et al., 2001; Vinatier et. al, 2009). Accordingly, potential cell sources are widely studied for the future improvement of ACI approach, which will be discussed in Chapter 4. Seeding in 3D structures (Benya and Shaffer, 1982) can maintain chondrocytes differentiated phenotype. Ideally, cell scaffolds for tissue engineering should meet several design criteria: (1) The surface should permit cell adhension and growth, (2) neither the polymer nor its degradation products should provoke inflammation or toxicity when implanted in vivo, (3) the material should be reproducibly processable into three dimensional structures, (4) the porosity should be at least 90% in order to provide a high surface area for cell-polymer interactions, sufficient space of extracellular matrix regeneration, and minimal diffusional constraints during in vitro culture, (5) the scaffold should resorb once it has served its purpose of providing a template for the regenerating tissue, since foreign materials carry a permanent risk of inflammation, and (6) the scaffold degradation rate should be adjustable to match the rate of tissue regeneration by the cell type of interest (Freed et al., 1994). Synthetic materials such as 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 extracellular matrix (ECM) (Barnewitz et al., 2006; Kaps et al., 2006). Moreover, PLGA is used as a scaffold material for matrix-based autologous chondrocyte transplantation clinically (Ossendorf et al., 2007). Natural materials have also been investigated in the application of tissue engineering scaffolds in ACI. 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 ACI products have used collagenous membraneas as 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 type I collagen with GAG in scaffolds had a positive effect on chondrocyte phenotype (van Susante et al., 2001). Hyaluronic acid is a non-sulphated GAG that makes up a large proportion of cartilage extracellular matrix (Schulz and Bader, 2007). Matrices composed of hyaluronan have been frequently used as carriers for chondrocytes. Facchini et al. con ¬Ã‚ rms the hyaluronan derivative scaffold Hyaff  ®11 as a suitable scaff old both for chondrocytes and mesenchymal stem cells for the treatment of articular cartilage defects in their study (Facchini et al., 2006). 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). Growth factors are proved to be able to promote the formation of new cartilage tissue in both explants and engineered constructs. Insulin-like growth factor-I (IGF-I), transforming growth factor-ÃŽ ²1 (TGF-ÃŽ ²1) increases, basic fibroblast growth factor (bFGF) can stimulate cell proliferation and/or biosynthesis in chondrocytes which were Immune Privilege of Tissue Engineered Articular Cartilage Immune Privilege of Tissue Engineered Articular Cartilage The immune privilege of tissue engineered articular cartilage derived from mouse adult mesenchymal stem cells and the potential of tissue engineered cartilage as a gene delivery method Chapter 1 Stem cell biology 1.1 Categorization of stem cells Stem cells are generally defined as cells possessing the following 3 characteristics: (1) self-renewal, (2) the ability to produce all cell types made in that tissue, and (3) the ability to do so for a significant portion of the life of the host (Alberts et al., 1989; Reya et al., 2001), while progenitor cells are capable only of multi-lineage differentiation without self-renewal (Weissman, 2000). Stem cells can be classified by their ability to differentiate. The most primitive, totipotent stem cells have the ability to divide and produce all the differentiated cells in an organism, including both the embryonic and extraembryonic tissues of an organism. Totipotent stem cells include the fertilized egg and the cells produced by the initial divisions of it. In mammals, these cell divisions result in an implant in the uterus called the blastocyst. The blastocyst contains an outer sphere of trophoblast cells. Trophoblast cells are capable of implanting into the uterus and helping the form of placenta which provides nutrients to the embryo. Within the blastocyst are 10 to 20 pluripotent cells called the inner cell mass. In mammalian uterus, these inner mass cells will participate in the production of all tissues and organs of the developing embryo, then fetus, then born organism. Such pluripotent cells can produce any differentiated cells in the body, but are usually unable to for m the trophoblast cells. The best-known pluripotent stem cell is the embryonic stem (ES) cell, which are obtained from the inner cell mass of the blastocyst and exist for only a brief stage of embryonic development. The last major class of stem cells, multipotent stem cells, gives rise to a limited number of cell types which are responsible for organ growth and renewal such as neural stem cells, skin stem cells and haematopoietic stem cells (HSCs) (Cheshier et al., 2009). 1.2 Selected milestones of stem cell research In 1981, Martin isolated a pluripotent stem cell line from early mouse embryos (Martin, 1981). Wilmut in 1996 first cloned a mammal, a lamb named Dolly by transferring nuclear from the adult mammary gland cell to an enucleated unfertilized egg (Wilmut et al., 1997). In 1998, Thomson obtained the first human embryonic stem cell line from human blastocysts (Thomson et al., 1998). In 2001, President Bush banned scientists from using federal funds to study stem cells from sources other than those that had already been grown because of the ethical concerns. To avoid ethical dispute over the use of human embryonic cells for research purposes, many efforts have been taken on obtaining pluripotent stem cells from differentiated donor cells. In 2006, Yamanaka find a way to obtain pluripotent cells by reprogramming the nucleus of adult mice skin cells (Takahashi and Yamanaka, 2006). Such cells are now known as induced pluripotent stem (iPS) cells. 1.3 A brief introduction of several types of multipotent stem cell The best known multipotent stem cells are haematopoietic stem cells (HSCs), that give rise to all the blood cell types including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells), and lymphoid lineages (T-cells, B-cells, NK-cells). HSCs are vital elements in bone-marrow transplantation, which has already been used extensively in therapeutic settings (Reya et al., 2001). In the long-term culture systems, human and rodent Central Neural System (CNS) cells maintain the capacity to produce the three main mature cell classes of the CNS: neurons, astrocytes, and oligodendrocytes, which suggest stem cells and/or progenitors exist and can survive in the culture medium (Weiss et al., 1996; Carpenter et al., 1999). In 2000, Human CNS stem cells (hCNS-SCs) have been successfully isolated by FACs (Uchida et al., 2000). Cancer stem cell hypothesis was proposed by Reya 2001 (Reya et al., 2001). This hypothesis consists of 2 components. The first component postulates that normal tissue stem cells are the target for transforming mutations and successive mutations result in the formation of a tumor. The second component is that within every cancer a specific subset of cancer stem cells continuously gives rise to all the other cancer cells and only these cells within a tumor possess the ability to self-renew, continuously proliferate. Conflicting to the first component of the hypothesis, evidences indicate cancer stem cells can also arise from mutated progenitor cells rather than stem cells (Cheshier et al., 2009). In addition, mature cells such as Lymphocytes can lead to mouse T cell leukemia independently from HSCs (Yuan et al., 2006). For the latter component of cancer stem cell hypothesis, it is likely that the cancer stem cell hypothesis is applicable to some tumors but not to others. In hematopoiet ic and some solid malignancies, only 1 in 100 to 1 in 10 000 primary tumor cells are capable of reproducing the tumor in vivo, such as human breast cancer, human neuroepithelial tumors, head and neck squamous cell carcinomas, and colon cancer. But in melanoma, nearly 1 in 4 cells possessed the ability of proliferation and developing into cancer (Cheshier et al., 2009). Cancer stem cells and CNS stem cells were reviewed by Cheshier et al. (Cheshier et al., 2009). 1.4 Mesenchymal stem cells (MSCs) and their differentiation potential Bone marrow is composed of two main systems of cell, hematopoietic cells and the supporting stromal cells (Bianco et al., 2001). MSCs reside within the marrow, maintain a level of self-renewal, and give rise to progenitor cells that can differentiate into various lineages of tissue, including chondrocytes, osteoblasts, adipocytes, fibroblasts, marrow stroma, and other tissues of mesenchymal origin. The traditional opinion about the multipotent differentiation potential of MSCs was challenged by further studies. Interestingly, MSCs reside in a diverse host of tissues throughout the adult organism and possess the ability to ‘regenerate cell types specific for local tissues e.g. adipose, periosteum, synovial membrane, muscle, dermis, pericytes, blood, bone marrow, and most recently trabecular bone, reviewed by Tuan et al. (Tuan et al., 2003). Furthermore, in 2002, Jiang et al. reported a rare cell within human bone marrow mesenchymal stem cell cultures that can be expanded extensi vely without obvious senescence. This cell population can differentiate, not only into mesenchymal cells, but also cells with visceral mesoderm, neuroectoderm and endoderm characteristics in vitro. Most somatic cell types could be derived after this population of cells was injected into an early blastocyst (Jiang et al., 2002). These studies suggest mesenchymal stem cells maintained pluripotent properties. Chapter 2 Features of Articular Cartilage 2.1 Introduction Joint cartilage formed highly sophisticated structure during the evolutionary development. 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). 2.2 Categorization of cartilage tissues Cartilage tissue is categorised in three major types by different biochemical compositions and structures of their extracellular matrix (ECM). Elastic cartilage has a small concentration of proteoglycans (PGs), and a relatively high proportion of elastin fibres. It exists 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 possesses a small concentration of PGs, but far less elastins. The meniscus in the knee joint is made of fibrocartilage. Hyaline is the most widespread cartilage in the human body. It is resistant to compressive or tensile forces due to its special type II collagen fibril mesh filled with a high concentration of PGs. Hyaline cartilage can be found in the nose, trachea, bronchi, and synovial joints. In the latter case, it is termed as articular cartilage (Schulz and Bader, 2007). 2.3 Compositions of articular cartilage Chondrocytes contribute to only 1%- 5% of the tissue volume; the remaining 95%-99% being extracellular matrix (ECM). Chondrocytes sense and synthesize all necessary ECM components (Mollenhauer, 2008; Schulz and Bader, 2007). 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. In articular cartilage, 95% of collagen in the ECM is type II collagen fibrils. The rest other types are collagen type IX and XI and a small fraction of types III, VI, XII and XIV. Normal articular cartilage does not present type I collagen, which is concerned with fibrous tissue. Unlike Type I and Type III collagens which form thick fibres and thin  ¬Ã‚ bres respectively, Type II collagen present in hyaline and elastic cartilages does not form  ¬Ã‚ bres. It forms very thin  ¬Ã‚ brils which are disposed as a loose mesh that strongly interacts with the groun d substance. Type II collagen provides tensile stiffness and strength to articular cartilage and constrains the swelling capacity generated by highly negatively charged glycosaminoglycans (GAGs) of the proteoglycans (PGs). The majority (50-85%) of the PG content in articular cartilage were presented by large molecule aggrecan. It consists of a protein backbone, the core protein, to which unbranched GAGs side chains of chondroitin sulphate (CS) and keratan sulfate (KS) are covalently attached (Figure 1.1). The composition of articular cartilage was extensively reviewed by Schulz and Bader (Schulz and Bader, 2007). Figure 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, 2007 and Mow and Wang, 1999). 2.4 Low capacity of self-repair in articular cartilage 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). The low mitotic potential of chondrocytes in vivo also contributes to its poor ability to undergo self-repair (Kuroda et al., 2007). Some researchers believe that cartilage lesions less than 3mm in diameter self-repair with normal hyaline-like cartilage (Revell and Athanasiou, 2009; Schulz and Bader, 2007). In animal studies, full thickness cartilage defects, extending into the subchondral bone, have been reported to heal with the formation of fibrous tissue, which contains relatively low amount of type II collagen and aggrecan, but a relatively high concentration of type I collagen which is not present in normal adult articular cartilage and accordingly exhibits impaired mechanical properties (Hjertquist et al., 1971). 2.5 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 which acts as a pumping function (Mollenhauer, 2008). Within synovial joints, oxygen supply to articular chondrocytes is very limited, from 7.5% at the superficial zone down to 1% oxygen tension at the deep zone. It is supposed to be even further decreased under pathological conditions, such as osteoarthritis (OA) or rheumatoid arthritis (RA). 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. Nevertheless, changes in O2 tension have profound effects on cell metabolism, phenotype, gene expression, and morphology, as well as response to, and production of, cytokines (Pfander and Gelse, 2007; Gibson et al., 2008). The most important component of this hypoxic response is mediated by transcription factor hypo xia-inducible factor-1 (HIF-1), which is present in most hypoxia inducible genes (Pfander and Gelse, 2007; Gibson et al., 2008). 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 (Mollenhauer, 2008). Chapter 3 Osteoarthritis (OA) 3.1 Prevalence Osteoarthritis (OA) is the most common form of arthritis. More than 40 million US American citizens (approximately 15% of the overall population of the USA) suffer from arthritis (Schulz and Bader, 2007). OA can occur in any joint but is most common in certain joints of the hand, knee, foot and hip. OA is the most common reason for total hip- and knee-joint replacement (Wieland et al., 2005). Among US adults 30 years of age or older, symptomatic disease in the knee occurs in approximately 6% and symptomatic hip osteoarthritis in roughly 3% (Felson and Zhang, 1998). 3.2 The symptoms and diagnosis The symptoms of OA include pain, stiffness and loss of function. OA can be monitored by radiography, magnetic resonance imaging (MRI), and arthroscopy, but radiographs are still considered the gold standard (Wieland et al., 2005). 3.3 The pathology of OA The pathologic characteristics of OA are the slowly developing degenerative breakdown of cartilage; the pathological changes in the bone, including osteophyte formation and thickening of the subchondral plate; the changes in the synovium such as inflammatory infiltrates; ligaments, which are often lax; and bridging muscle, which becomes weak. Many people with pathologic and radiographic evidence of osteoarthritis have no symptoms (Martel-Pelletier, 1999; Felson et al., 2000). A protease family of matrix metalloproteases (MMP) is responsible for the initial occurrence of cartilage matrix digestion. Of this family, collagenases, the stromelysins and the gelatinases are identified as being elevated in OA. Another group of MMP is localized at the cell membrane surface and is thus named membrane type MMP (MT-MMP) (Martel-Pelletier, 1999). Proinflamatory cytokines such as interleukin (IL)-1ÃŽ ², Tumor necrosis factor (TNF)-ÃŽ ±, IL-6, leukemic inhibitor factor (LIF) and IL-17 are first produced by the synovial membrane and then diffuse into the cartilage through the synovial fluid, where they activate the chondrocytes to produce proinflammatory cytokines. These proinflamatory cytokines are considered responsible for the catabolic pathological process (Martel-Pelletier, 1999). In OA cartilage, an increased level of an inducible form of nitric oxide synthase (iNOS) leads to a large amount of nitric oxide (NO) production (Pelletier et al., 2001). NO can inhibit the synthesis of cartilage matrix macromolecules such as aggrecans and can enhance MMP activity (Taskiran et al., 1994; Murrell et al., 1995). It is well stablished that proinflammatory cytokines such as IL-1ÃŽ ² act as the key mediators of cartilage breakdown and stimulate the release of inflammatory products (NO) and prostaglandin (PG)E2, via induction of iNOS and cyclo-oxygenase (COX)-2 enzymes (Chowdhury et al., 2008). 3.4 Risk factors Osteoarthritis is considered to be a systemic disease although severe joint injury may be sufficient to cause osteoarthritis. There are several systemic risk factors related to OA. (1) Age: Osteoarthritis increases with ages, the incidence and prevalence of disease increased 2- to 10-fold from 30 to 65 years of age and increased further thereafter in a community-based survey (Oliveria et al., 1995). (2) Hormonal status and bone density: women taking estrogen have a decreased prevalence of radiographic osteoarthritis (Nevitt et al., 1996). Before 50 years of age, the prevalence of osteoarthritis in most joints is higher in men than in women. After about age 50 years, women are more often affected with hand, foot, and knee osteoarthritis than men. In most studies, hip osteoarthritis is more frequent in men (van Saase et al., 1989). Evidence suggests an inverse relationship between osteoarthritis and osteoporosis (Felson et al., 2000). (3) Nutritional factors: evidence indicates that co ntinuous exposure to oxidants contributes to the development of many common age-related diseases, including osteoarthritis. McAlindon et al. reported a threefold reduction in risk for progressive radiographic osteoarthritis was observed in persons in the middle and highest tertile of vitamin C intake compared with those whose intake was in the lowest tertile (McAlindon et al., 1996a). Vitamin D intake was observed associated with the progression of OA although not associated with risk for new-onset radiographic osteoarthritis (McAlindon et al., 1996b; Lane et al., 1999). (4) Genetics: genetic factors account for at least 50% of cases of osteoarthritis in the hands and hips and a smaller percentage in the knees (Spector et al., 1996). Candidate genes for common forms of osteoarthritis include the vitamin D receptor gene, insulin-like growth factor I genes, cartilage oligomeric protein genes, and the HLA region (Felson et al., 2000). Local mechanical factors include the body weight and the pathological alterations of the mechanical environment of the joint. Persons who are overweight have a high prevalence of knee osteoarthritis (Felson et al., 1997). OA is also considered to be related to alterations in joint mechanical environments such as knee laxity, the displacement or rotation of the tibia with respect to the femur; proprioception, the conscious and unconscious perception of joint position and movement; knee alignment , knee position in reference to the hip and ankle (Felson et al., 2000). In addition, joint dysplasias, fractures of articular surfaces, and tears of menisci and ligaments that increase joint instability precede the development of osteoarthritis in a high percentage of affected joints. Risk factors for posttraumatic osteoarthritis include high body mass, high level of activity, residual joint instability or malalignment, and persistent articular surface incongruity (Buckwalter et al., 1997; Honkonen 1995). 3.5 Treatments The medicine treatment of OA was dominated by COX2 inhibitors (Flower 2003). The other medicines include glucosamine, chondroitin (McAlindon et al., 2000), and hyaluronic acid (Lo et al., 2003). In addition, both aerobic walking and muscle strengthening exercise reduce pain and disability from osteoarthritis (Roddy et al., 2005). Articular cartilage lesions, both of traumatic or pathological origin, do not heal spontaneously and often undergo progressive degeneration towards osteoarthritis (OA). The most frequently used treatments include the artificial joint replacement, mosaicplasty, marrow stimulation, and autologous condrocyte implantation (ACI) (Steinwachs et al., 2008). Total joint replacement is most commonly performed in people over 60 years of age. (NHS 2006; Brittberg et al., 1994) 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, 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 stabilised 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). The ACI tissue engineering treatment will be discussed in the next chapter. Chapter 4 Tissue engineering and autologous chondrocyte implantation (ACI) 4.1 Overview of tissue engineering technologies Tissue engineering is defined as ‘‘the application of the principles and methods of engineering and the life sciences toward the fundamental understanding of structure-function relationships in normal and pathological mammalian tissues and the development of biological substitutes to restore, maintain, or improve tissue function† (Langer and Vacanti, 1993). Three factors are considered as the principles of tissue engineering, including the utilization of biocompatible and mechanically suitable scaffolds, an appropriate cell source, and bioactive molecules to promote the differentiation and maturation of the cell type of interest (Song et al., 2004). Potential applications of tissue engineering are involved in the following fields: skin, cartilage, bone, cardiovascular diseases, organs (e.g. liver, pancreas, bladder, trachea and breast), central nervous system (e.g. spinal cord), and miscellaneous (e.g. soft tissue, ligaments). Although research is being carried out in all these fields, only few products have already entered the market. The most successful products up to now are: tissue engineered skin which is mainly used for wound cover, autologous chondrocyte implantation (ACI), and artificial bone graft (Hà ¼sing et al., 2003). 4.2 Autologous chondrocyte implantation (ACI) In 1984, a study in rabbits reported successful treatment of focal patellar defects with the use of ACI. One year after transplantation, newly formed cartilage-like tissue typically covered about 70 percent of the defect (Grande et al. 1989). In 1987, Brittberg firstly performed ACI in 23 people with deep cartilage defects in the knee. ACI is described as the following 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 (Figure 2; Brittberg et al., 1994). Genzyme Biosurgery with its product Carticel ® was the first company which introduced ACI into market and is the market leader in USA. Carticel ® is a classic ACI procedure using the periosteal cover (Hà ¼sing 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 tri-dimensional (3D), biodegradable scaffold. This kind of scaffold, cut to the required size, is fixed into the lesion by anchoring stitches or its sticky nature. The 3D cell seeded scaffold eliminates the using of cover, thus simplifies the surgery procedure, saves the surgery time, and opens up the possibility of an arthroscopic surgery instead of the open surgery which causes more tissue damage. HYALOGRAFT from Italy is one of the European market leaders. It is a cartilage substit ute made of autologous chondrocytes delivered on a biocompatible 3D matrix, entirely composed of a derivative of hyaluronic acid (Marcacci et al. 2005). 4.3 Clinical results 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 (Brittberg et al., 2003). Since 1997 the year FDA approved ACI, this method has been widely performed in more than 20,000 patients all over the world. It has been reported to be effective in relieving clinical symptoms, such as pain and function (Wakitani et al., 2008). In a randomised controlled study, Knutsen et al. studied 80 patients who needed local cartilage repair with lesions on the femoral condyles of 2-10 cm2. There were no signi ¬Ã‚ cant differences in clinical 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 Clinical results of ACI were reviewed by Gikas 2009 (Gikas et al., 2009). Generally speaking, the outcomes of ACI treatment have been encouraging. However, most randomised controlled studies showed no significant difference between ACI and traditional treatments. 4.4 Limitations of ACI Microfracture is a very simple and low-cost procedure whereas ACI costs about $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 to be blamed for the limitations of the traditional ACI procedure. The cell source in ACI is the cartilage tissue derived via an arthroscopic procedure from the low load-bearing area on the upper medial femoral condyle of the damaged knee. However, Wiseman et al. found the chondrocytes isolated from the low loaded area of the knee joint respond to mechanical stimulations 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 and an altered phenotype. Type II collagen, the major protein produced by chondrocytes in articular cartilage, are down-regulated in the culture, while collagen types I and III are increased (Glowacki et al., 1983; 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 origninal 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 appropriate 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 (Watt, 1988) and seeding in 3D structure (Benya and Shaffer, 1982) can maintain their differentiated phenotype much longer compared to cells spread in monolayer cultures. 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 more comprehensive tissue engineered strategies which incorporates cells, scaffold materials and potentially biochemical, biomechanical and/or physical stimulation in a controlled bioreactor environment. 4.5 Tissue engineering strategies for ACI 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 as described above. In addition, the limitation of the transplant volume is always a major problem in autograft to be overcome (Kitaoka et al., 2001; Vinatier et. al, 2009). Accordingly, potential cell sources are widely studied for the future improvement of ACI approach, which will be discussed in Chapter 4. Seeding in 3D structures (Benya and Shaffer, 1982) can maintain chondrocytes differentiated phenotype. Ideally, cell scaffolds for tissue engineering should meet several design criteria: (1) The surface should permit cell adhension and growth, (2) neither the polymer nor its degradation products should provoke inflammation or toxicity when implanted in vivo, (3) the material should be reproducibly processable into three dimensional structures, (4) the porosity should be at least 90% in order to provide a high surface area for cell-polymer interactions, sufficient space of extracellular matrix regeneration, and minimal diffusional constraints during in vitro culture, (5) the scaffold should resorb once it has served its purpose of providing a template for the regenerating tissue, since foreign materials carry a permanent risk of inflammation, and (6) the scaffold degradation rate should be adjustable to match the rate of tissue regeneration by the cell type of interest (Freed et al., 1994). Synthetic materials such as 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 extracellular matrix (ECM) (Barnewitz et al., 2006; Kaps et al., 2006). Moreover, PLGA is used as a scaffold material for matrix-based autologous chondrocyte transplantation clinically (Ossendorf et al., 2007). Natural materials have also been investigated in the application of tissue engineering scaffolds in ACI. 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 ACI products have used collagenous membraneas as 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 type I collagen with GAG in scaffolds had a positive effect on chondrocyte phenotype (van Susante et al., 2001). Hyaluronic acid is a non-sulphated GAG that makes up a large proportion of cartilage extracellular matrix (Schulz and Bader, 2007). Matrices composed of hyaluronan have been frequently used as carriers for chondrocytes. Facchini et al. con ¬Ã‚ rms the hyaluronan derivative scaffold Hyaff  ®11 as a suitable scaff old both for chondrocytes and mesenchymal stem cells for the treatment of articular cartilage defects in their study (Facchini et al., 2006). 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). Growth factors are proved to be able to promote the formation of new cartilage tissue in both explants and engineered constructs. Insulin-like growth factor-I (IGF-I), transforming growth factor-ÃŽ ²1 (TGF-ÃŽ ²1) increases, basic fibroblast growth factor (bFGF) can stimulate cell proliferation and/or biosynthesis in chondrocytes which were