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 form 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 hematopoietic 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 extensively 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 fibres respectively, Type II collagen present in hyaline and elastic cartilages does not form fibres. It forms very thin fibrils which are disposed as a loose mesh that strongly interacts with the ground 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 hypoxia-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)-1β, Tumor necrosis factor (TNF)-α, 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-1β 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 continuous 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 (Hüsing 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 (Hüsing 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 substitute 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 significant 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 < 0.032) (NHS, 2006).

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 fibroblast-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 specific 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 fibrous tissue expressing collagen type I that does not have appropriate mechanical properties, which may lead to degradation 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 today's 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. confirms 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 (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-β1 (TGF-β1) increases, basic fibroblast growth factor (bFGF) can stimulate cell proliferation and/or biosynthesis in chondrocytes which were cultured under a variety of conditions reviewed by Darling and Athanasiou (Darling and Athanasiou, 2005).

A lot of physical stimulations have been found to be effective in stimulating chondrocytes proliferation and biosynthesis, including mechanical (Lee and Bader, 1997; Mauck et al., 2000), electrical (Brighton et al., 1984; Armstrong et al., 1988; Brighton et al., 1989; MacGinitie et al., 1994; Wang et al., 2004; Brighton et al., 2006), ultrasound (Parvizi et al., 1999; Noriega et al., 2007) and even laser (Torricelli et al., 2001) stimulations. 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).

Chapter 5 Applications of MSC in ACI

5.1 Potential cell resources for ACI

Although chondrocytes have been widely used in cartilage repair, there are conspicuous limitations for chondrocytes as the cell resource: their instability in monolayer culture and the rareness of donor tissues. The limitation of the transplant volume is always a major problem in autograft to be overcome (Kitaoka et al., 2001; Vinatier et. al, 2009). The OA chondrocytes synthesized significantly lower amount of collagen than was generated by chondrocytes from ACI donors without arthritis (Tallheden et. al, 2005). In the case of older OA patients, ACI is probably impossible in the clinic setting, because of the lack of autologous donor tissue (Kafienah et. al, 2007). As a result, potential cell sources are widely studied for the 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). Chondrocytes from costal cartilage were studied as a potential substitute. Costal cartilage 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). Human adult nasal chondrocytes proliferated approximately four times faster than human articular chondrocytes in monolayer culture, and had a markedly higher chondrogenic capacity showed by the mRNA and protein analysis of in vitro tissue engineered constructs (Kafienah 2002). Human embryonic germ cells were reported to be homogenously differentiated into hyaline cartilage. 3D tissue formation is achieved by encapsulating cells in synthetic hydrogels poly (ethylene glycol diacrylate) (PEGDA) (Varghese et al., 2006).

5.2 MSC as a potential cell resource for ACI

The use of MSC as the cell source has advantages in ACI because (1) easy access and availability of the source cells which incurs limited or reduced donor-site morbidity in cell acquisition; (2) a capacity of the source cells to differentiate readily into cell lineages of interest upon instructive differentiation cues; and (3) MSCs have a greater self-renewal or proliferative capacity than chondrocytes to generate sufficient quantity; (4) a lack of or minimal immunogenic or tumorigenic ability of the source cells (Song et al., 2004).

MSCs can be distinguished from hematopoietic stem cells (HSCs) in terms of cell-surface antigens and adhesion capability. They were originally isolated and identified from BM aspirate based on their tendency to adhere to tissue culture plastic (Song et al., 2004).

In vitro MSCs require a stimulus to differentiate into chondrocytes. This stimulus can be achieved with a large variety of different growth and differentiation factors, hormones or cytokines. Any growth factor of TGF-β subtypes (TGF- β1, TGF- β2, TGF- β3) is an active chondrogenic factor. Members of the BMP family like BMP-2, BMP-4, BMP-6 and BMP-7 act synergistically to TGF- β and enhance matrix production; they are themselves not sufficient to drive in vitro chondrogenesis of human. The same applies for IGF-1 and FGF-1 whilst FGF-2 and PTHrP suppress TGF- β-driven chondrogenesis, reviewed by Richter (Richter, 2009) and Raghunath (Raghunath et al., 2005).

In animal models, MSCs were implanted on 3D scaffolds. Successful formation of cartilage-like tissue was observed in parts of the defect (Chen et al., 2005; Wakitani et al., 1994; Meinel et al., 2004; Liu et al., 2006). Clinical trials on human patients using MSCs for articular cartilage repair are scarce (Wakitani, 2007; Wakitani et al., 2002). Wakitani et al. performed a study on 24 human patients. Autologous MSCs were obtained from the patients' bone marrow, expanded in monolayer culture, seeded onto a collagen type I membrane and transplanted into the cartilage defect. 12 patients served as the control group and received cell-free implants. 42 weeks after implantation, the arthroscopic and histological grading was better in the cell-transplanted group than the control group, although the clinical results were not significantly different (Wakitani et al., 2002). In a case report on two patients with patella defects, cultured MSCs were embedded in collagen gel, placed in defects and covered with autologous periosteum. 2 years after transplantation, clinical results showed that symptoms had improved. But histological experiment showed the repaired tissue consisted of fibrocartilage (Wakitani et al., 2004). Animal and clinical experiments results were reviewed by Pelttari (Pelttari et al., 2008) and Csaki (Csaki et al., 2008).

4.3 Hurdles in tissue-engineering cartilage from MSCs

A major hurdle in cartilage tissue engineering with MSCs is the aging of the cells in tissue engineered constructs. During in vitro chondrogenesis, MSCs not only up-regulate chondrogeneic markers as collagen type II and aggrecan, but also markers for hypertrophic chondrocytes such as collagen type X and alkaline phosphatase (ALP) which expressed in the deep area of normal cartilage (Johnstone et al., 1998).

The inclusion of parathyroid hormone-related protein (PTHrP) significantly suppressed the risk of hypertrophy which is indicated by type X collagen mRNA expression, in 3D tissue engineered constructs using BMSCs from patients with OA. mRNA and biochemical analyses of the constructs showed extensive synthesis of proteoglycan and type II collagen at the same time only low levels of type I collagen (Kafienah 2007).

Chapter 6 Cartilage Immunology and Transplantation

6.1 Immune tolerance

T cells discriminate self and non-self by several mechanisms. The forefront is central tolerance in the thymus. Central tolerance takes place during T cell development in the thymus. Positive selection makes sure only thymocytes whose receptors interacting with self-peptide:self-MHC complexes can survive and mature. T cells are positively selected for high affinity for self MHC molecules and expression of CD4 or CD8 molecules. Positive selection of T cells is mediated by thymical cortical epithelial cells (cTECs). T cells are also negatively selected (deleted) that react strongly with ubiquitous self antigens. After positive selection, thymocytes migrate to medulla and interact with a medullary thymic epithelial cells (mTECs) expressing costimulatory molecules and self-tissue-restricted antigens (TRA), the latter regulated in part by Aire, a transcription factor defective in the autoimmune disease (Rossi et al., 2007). T cells were negatively selected during the interactions with TRA. Positive selection guarantees T cells' function of recognising antigen peptides bound to MHC molecules of APC. Negative selection prevents T cells from overreacting with self-antigens. Central tolerance of T cells was reviewed by Murphy et al. (Murphy et al., 2008a).

Peripheral tolerance to self antigens has several main mechanisms: (1) self-reactive T cells in the circulation may ignore self antigens, for example when the antigens are in tissues sequestered from the circulation; (2) their response to a self antigen may be suppressed if the antigen is present in a privileged site; (3) self-reactive cells may under certain conditions be deleted or rendered anergic and unable to respond finally a state of tolerance to self antigens, (4) can also be maintained by regulatory T cells (Male et al., 2006a).

Tolerance is also imposed on B cells. B cell deletion takes place in both bone marrow and peripheral lymphoid organs. Differentiating B cells that express surface immunoglobulin receptors with high binding affinity for self membrane-bound antigens will be deleted soon after their generation in the bone marrow (Male et al., 2006b).

6.2 Immune privilege sites

Tissue grafts in some sites of the body that do not elicit immune responses are termed immunologically privileged sites. The privileged sites were long thought to be locations where adaptive immune responses are so dangerous that the immune system is not allowed entry, destroyed upon arrival, or prevented from functioning. However, subsequent studies showed that antigens do leave these sites and do interact with T cells. Instead of eliciting a destructive immune response, however, they induce tolerance or a response that is not destructive to the tissue (Male et al., 2006c).

All immune privilege tissues are involved in several of the following similar mechanisms: (1) mechanical barrier which prevents inflow of large serum proteins (IgG, complement, etc.), (2) low levels of MHC molecule expression and costimulatory molecules result in inefficient antigen presentation, (3) no conventional lymphatic drainage system to local lymph nodes, (4) low levels of leukocyte traffic, immunosuppressive cytokines (e.g. TGF-β and IL-10 secreted by Treg) (Male et al., 2006c), (5) the expression of Fas ligand by the tissues of immunologicaly privileged sites may provide a further level of protection by inducing the apoptosis of Fas-bearing lymphocytes that enter these sites (Takada et al., 2002).

6.3 Role of histocompatibility antigens

Histocompatibility antigens are the targets for rejection. In human, there are three MHC class I α-chain genes, called HLA-A,-B,and -C. There are also three pairs of MHC class II α- and β-chain genes, called HLA-DR, -DP, and -DQ. In mouse, class I molecules are termed as H-2K, H-2D, and H-2L. H-2A and E are class II molecules (Rammensee et al., 1995).

In many people, the HLA-DR cluster contains an extra β-chain gene (Gaur and Nepom, 1996). This means the three sets of genes can give rise to four types of MHC class II molecules. As individual express two alleles at a single gene locus, and both gene products being able to present antigen to T cells, a person typically expresses six different MHC class I molecules and eight different MHC class II molecules on his or her cells. For the MHC class II genes, the number of different MHC molecules may be increased further by the combination of α and β chains encoded by different chromosomes (Murphy et al., 2008b).

Graft rejection is an immunological response mediated primarily by T cells. CD4 and CD8 T cells recognize peptides bound to two different classes of MHC molecules. Recognition of non-self MHC molecules is a major determinant of graft rejection. When donor and recipient differ at the MHC, an alloreactive immune response is directed at the nonself, allogeneic MHC molecule or molecules present on the graft. In most tissues these will be predominantly MHC class I antigens (Arakelov and Lakkis, 2000). Mixed lymphocyte reaction studies have shown roughly 1-10% of all T cells in an individual will respond to stimulation by allogeneic cells (Murphy et al., 2008b).

Transplanted organs are rapidly rejected owing to large numbers of T cells react to nonself MHC molecules. T cells undergo a central selection when developing in the thymus that favours the survival of cells whose T-cell receptors interact weakly with the self-MHC molecules that are expressed in the thymus. It is thought that T-cell receptor interacts with one type of MHC molecule increases the likelihood that they will cross-react with other nonself MHC variants. The interaction between T-cell receptors and non-self-peptide:nonself MHC complexes is influenced by the bound peptide as well as by the MHC molecule, which means T cells can either only interact with the bound peptide, or interact only with the MHC molecule, or both. This phenomenon largely increases the chance of recognition (Murphy et al., 2008b).

As a result, MHC matching is important for preventing rejection. It is very important in bone marrow transplantation and kidney transplantation. However, in corneal transplantation, there is no benefit of HLA matching. In blood transfusion, MHC matching is not necessary as red blood cells and platelets express only small amounts of MHC class I molecules and do not express MHC II class at all; thus, they are not targets for the T cells of the recipient. Blood must be matched for ABO and Rh blood group antigens to avoid the rapid destruction of mismatched red blood cells by antibodies in the recipient (Murphy et al., 2008c).

6.4 Antigen present on chondrocytes

Normal chondrocytes were MHC class I HLA A, B, and C positive and class II negative. Expression of MHC class II was demonstrated in human articular cartilage chondrtocytes from OA and RA patients (Moskalewski et al., 2002). Most of the studies found chondrocyte from normal cartilage is immunogenic. Researchers found autoreactivity was induced by the injection of syngeneic chondrocytes in rats (Langer et al., 1972). Proliferative response of rat lymphocytes was also observed in mixed lymphocyte-chondrocyte cultures not only by allogeneic but also by syngeneic chondrocytes (Gertzbein and Lance, 1976; Gertzbein et al., 1977; Hyc et al., 1997). The syngeneic activation was confirmed with rabbit (Tiku et al., 1985), human, bovine (Glant and Mikecz, 1986) and rat chondrocytes (Lance, 1989). However, arguments were raised that stimulation of lymphocytes by chondrocytes could be caused by the remanants of enzymes used for isolation, and not by cartilage-specific antigen (Elves, 1983). The antigens present on chondrocytes were reviewed by Moskaleewski et al. (Moskalewski et al., 2002).

6.5 Antigens present in cartilage matrix

Numerous components of cartilage matrix, such as collagen type II, IX, and XI (Bujia et al., 1994a; Takagi and Jasin, 1992), core protein of proteoglycans (Dayer et al., 1990; Glant et al., 1998; Goodstone et al., 1996), or link proteins (Doran et al., 1995) have antigenic properties and could be involved in transplant rejection. The major components collagen II and PG are highly immunogenic (Buzas et al., 2005). Collagen II and PG induced autoimmune arthritis are all well established in mouse models (Brand et al., 2007; Rosloniec et al., 2001; Glant et al., 2005 ). These experiments prove matrix antigens induced immune response can breach self-tolerance leading to arthritis. In contrast, local non-specific inflammation alone is insufficient to breach self-tolerance (Nickdel et al., 2008).

However, when cartilage tissue is intact, chondrocytes are protected and separated from contact with both natural killer and T cells by the ECM. This effect together with the avascular and the alymphotic nature led to an efferent and afferent block to the immune system (Heyner, 1969; Bolano and Kopta, 1991).

6.6 Clinical results of osteochondral allograft transplantation

Osteochondral allograft transplantation involves transplanting a bilayer bone-cartilage graft. Clinical results of osteochondral allograft transplantation have demonstrated a high success rate (52%-95%) as defined by graft survival and good/excellent patient evaluations. The results were reviewed by Görtz et al. and Revell et al. (Görtz et al., 2006; Revell and Athanasiou, 2009).

6.7 Results of animal studies on allogeneic articular cartilage transplantation

Transplants of allogeneic cartilage tissue usually resist rejection (Craigmyle, 1958; Langer and Gross, 1974; Aston and Bentley, 1986; Glenn et al., 2006). Conflicting results were reported by Stevenson et al., antigen-matched allografts contained significantly less articular cartilage than antigen-mismatched allografts 11 months after transplantion in a dog model (Stevenson et al., 1989).

Isolated allogeneic chondrocytes producing cartilage after transplantation evoked strong reaction leading to cartilage resorption by infiltrating cells in mice and rabbits. (Heyner, 1969; Moskaleewski et al., 1966; Ksiazek T et al., 1983; Malejczyk and Moskalewski 1988; Malejczyk et al., 1991). Conflictingly, no clear sign of rejection was observed in rabbit models several weeks after transplantation (Chesterman and Smith, 1968; Bentley and Greer 1971; Bentley et al., 1978; Wakitani et al., 1989; Rahfoth et al., 1998). The lack of immune rejection in isolated allogeneic chondrocytes transplantation might be caused by the mechanical barrier formed by broken lamellae during the modelling of cartilage defects (Moskaleewski et al., 2002). It is still hard to say the exact reason why in so many reports rejection of cartilage produced by allogeneic chondrocytes was absent. It is also difficult to predict the outcome of such transplants in man (Moskaleewski et al., 2002).

No signs of rejection were observed in allogeneic chondrocytes transplantation after culture for several weeks in-vitro before transplantation (Itay et al., 1987; Wakitani et al., 1989; Noguchi et al., 1994; Freed et al., 1994; Sams and Nixon, 1995; Sams et al., 1995; Frenkel et al., 1997; Kawamura et al., 1998; Rahfoth et al., 1998; Fragonas et al., 2000; Tanaka et al., 2005; Masuoka et al., 2005; Shangkai et al., 2007). Conflicting results found by Kawabe and Yoshinao et al. Thin growth plate chondrocytes formed disks after culture for 10 days were transplanted into defects of allogeneic rabbits. Both cell-mediated toxicity and humoral response were observed after transplanted for two or three weeks (Kawabe and Yoshinao, 1991). The immune privilege of cartilage tissue was explained by the sequestration of chondrocytes by the matrix (Bacsich and Wyburn, 1947; Heyner, 1969, 1973). Chondrocytes could deposit matrix which ensures a lack of rejection if cultured in vitro for several weeks prior to transplantation (Freed et al., 1994; Moskaleewski et al., 2002).

6.9 Immune homeostasis balance and its break in articular cartilage

Chondrocytes are protected and separated from contact with both natural killer and T cells by the mechanical barrier ECM. This effect together with the unconventional avascular and the alymphotic nature led to an efferent and afferent block to the immune system (Hyner, 1969; Langer, 1974; Bolano and Kopta, 1991). As a conclusion of the above observations, the intact nature of ECM may act as the main factor to determine the results of allogeneic cartilage graft transplantations.

There are other potential mechanisms protect cartilage tissue away from the attacking of immune system. The negative central tolerance was explained in Chapter 5. T cells are negatively selected during interactions with medullary thymic epithelial cells (mTECs) expressing self-tissue-restricted antigens (TRA) which is regulated by Aire, a transcription factor. Campbell et al. reported Aire dependent expression of type II collagen antigens occurs in medullary thymic epithelial cells (mTECs), implying that there is central tolerance to self antigens found in articular cartilage, which limits spontaneous autoimmunity. Meanwhile, Aire-deficient mice are more susceptible to the induction of autoimmune arthritis (Campbell et al., 2009).

In the anterior chamber of the eye, both corneal endothelial cells and lens epithelial cells express little or no MHC class I Ags; as a result, these two cell types are highly vulnerable to lysis by NK cells. An immunosuppressive protein-macrophage migration inhibitory factor (MIF), present in the aqueous humor (AH), inhibits NK cell-mediated cytotoxicity by

preventing the release of cytolytic perforin granules (Apte et al., 1998). MIF exerts its function by directly abolishing the actions of macrophages and natural killer (NK) cells. MIF was found positively expressed on chondrocytes cultured in Poly-L-Lactic Acid (PLLA) scaffolds both in vitro and in vivo (Fujihara et al., 2009).

Fas ligand in immuneprivileged organs such as the testis (Bellgrau et al., 1995) and the anterior chamber of the eye (Griffith et al., 1995) has been proven to act by inducing apoptosis of invading Fas-bearing activated T cells. Fas ligand was also reported existing on intervertebral disc cells (Takada et al., 2002). But no Fas ligand was found on OA or normal chondrocytes (Hashimoto et al., 1997).

Immune homeostasis balance in the privileged sites and any other tissues is controlled by co-stimulatory signals, dentritic cells, regulatory T cells (Male et al., 2006c). In induced autoimmune disease such as experimental autoimmune encephalomyelitis (EAE) and collagen induced arthritis (CIA), this balance is interrupted, leading to local inflammatory response. Lymphocyte surveillance phenomenon was observed by Bartholomaus et al. in EAE model. TMBP-GFP cells were effector T cells reactive against myelin basic protein (MBP) and had been retrovirally transduced to express green fluorescent protein (GFP). During the initiation process of EAE, diapedesis and scanning of the leptomeninges by the TMBP-GFP cells were the prelude to actual invasion of the CNS tissue. Finally, T cell surveillance turned into large scale lymphocyte invasion (Bartholomaus et al., 2009). Compared with the immune surveillance in brain, within cartilage tissue no immune-competent cells are considered (macrophages, T-cells) to access. It seems chondrocytes have to defend themselves against hostile microorganisms (Mollenhauer, 2008). Similar to the sensitization process happening in EAE model, when cartilage fragments were transplanted together with isolated chondrocytes subcutaneously, lymphocyte infiltrations were formed in the vicinity of the fragments. It seems allogeneic chondrocytes evoke immunization and may sensitize the immune response against allogeneic fragments (Moskalewski et al., 1966). In contrast, local non-specific inflammation alone is insufficient to breach self-tolerance (Nickdel et al., 2008), which suggests nonspecific inflammation cannot induce antigen specific attack.

Malejczy et al. studied the immune response of mouse against the syngeneic or the allogeneic chondrocytes intramuscular transplantation. Cartilage absorption and cell infiltrates were observed in both the syngeneic and the allogeneic group. Both cellular and humoral immune responses were observed in the allogeneic group by leukoagglutination test and indirect migration inhibition assay respectively (Malejczy et al., 1988; 1991).

Chapter 7 Immunomodulation of MSCs

7.1 Applications of MSC therapy

Currently, tissue engineering studies and commercialised products mainly focused on autologous cell sources, e.g. autologous chondrocytes or MSCs. However, due to the time costing nature of tissue engineering technologies, many therapies were delayed for a certain time. In addition, the obtaining processes of autologous cells normally require patients undergo small operations which incur hazards or pain. The revelation of immunomodulating properties of MSCs affirms the hope that allogeneic stem cell or tissue engineering therapies be provided as an alternative way of traditional organ or tissue transplantations, at the same time avoiding immune rejection. It also affirms the hope that ‘off the shelf' artificial tissues or organs be provided by allogeneic tissue engineering techniques instead of the expensive and time-costing case by case autogeneic tissue engineering techiniques.

MSCs may be used to engineer cartilage, bone, muscle, fat, tendon and neuronal cells as described in Chapter 3; they may serve as cell vehicles for gene therapy (Kanehira et al., 2007; Stoff-Khalili et al. 2007); they may enhance engraftment in haematopoietic stem cell transplants; and their immunoregulatory properties, if proven to be effective in vivo, could be of therapeutic use in autoimmune diseases (Tyndall et al., 2005). Direct mesenchymal stem cell therapy rather than tissue engineering has also been tested on variety of diseases. Gussoni et al. reported the partial restoration of dystrophin expression in the affected muscle of an animal model of Duchenne's muscular dystrophy after bone marrow transplantation (Gussoni et al., 1999). Stamm et al. injected autologous bone marrow cells into the infarct border zone in six patients. 3-9 months after surgery, all patients were alive and well, global left-ventricular function was enhanced in four patients, and infarct tissue perfusion had improved strikingly in five patients (Stamm et al. 2003). Le Blanc reported a striking immune suppressive effect of third party haploidentical MSCs on a case of severe acute graft-versus-host disease (Le Blanc et al., 2004).

7.2 Immunomodulation of MSC

The immunological surface marker of MSC is human leukocyte antigen (HLA) class I positive and HLA class II negative, and with no expression of the costimulatory molecules CD40, CD40L, CD80, or CD86 (Klyushnenkova et al., 2005; Le Blanc et al., 2003; Niemeyer et al., 2007). MHC molecules were controlled by cytokines, the expression of both MHC I and MHC II on the surface of MSCs can be increased after induction of interferon (IFN)-γ (Le Blanc et al., 2003).

Mixed lymphocyte culture (MLC) is a method to detect differences in HLA class II and reactivity in MLC is linked to risk of rejection and graft survival. MSCs fail to elicit a proliferative response from allogeneic lymphocytes in MLC and they also suppress the dentritic cell activated allogeneic lymphocytes (Le Blanc et al., 2003; Niemeyer et al., 2007).

MSCs can still escape recognition by alloreactive T-cells even after treatment with proinflamatory factor (IFN)-γ, cultured in differentiation medium (Le Blanc et al., 2003; Niemeyer et al., 2007) or transfected with co-stimulatory molecules (Klyushnenkova et al., 2005). MSCs also inhibit the differentiation of monocytes into immature dendritic cells (DCs) (Nauta et al., 2006). Furthermore, they escape lysis by cytotoxic T-cells and alloreactive killer inhibitory receptor mismatched natural killer cells (Rasmusson et al., 2003). However, the mechanism governing the suppressive effect is complex and not yet understood (Gotherstrom et al., 2007; Rasmusson et al., 2005). MSCs used for cellular cardiomyoplasty after an ischemic event revealed that paracrine action of MSC inhibited hypoxia induced apoptosis of cardiomyocytes during the acute phase of injury resolution (Gnecchi et al., 2005; 2006). Parekkadan et al. reported in Gal-N induced fulminant hepatic failure (FHF) mouse model, death rate was reduced after intravenous injections of human MSC conditioned medium (MSC-CM), by the mechanism of inhibiting panlobular leukocyte invasion, bile duct duplication, hepatocellular death and altering immune cell migration. Proteomic analysis of MSC-CM revealed a broad spectrum of molecules involved in immunomodulation and liver regeneration (Parekkadan et al., 2007). These studies indicate that MSCs can affect immunity and tissue cells by paracrine means.

It is also reported administration of MSCs before experimental autoimmune encephalomyelitis (EAE) onset or early during disease (before day 15 after EAE induction) successfully ameliorated severity of disease in pathological study. This effect was caused by inducing anergy of T cells but had no relation to CD4+CD69-CD25+Treg cells or T cell apoptosis. The T cell suppression effect was paralleled by a significant suppression of IFN-γ and TNF-α production by activated T cells, supporting a profound inhibition of the T helper 1 (Th1) response by MSCs (Zappia et al., 2005; Gerdoni et al., 2007). Kassis et al. reported survival rate and EAE score was improved after administration of MSCs (MSCs injection on day 10 after EAE induction) (Kassis et al., 2008).

The immunomodulatory effect of MSCs was reviewed by Gotherstrom (Gotherstrom et al., 2007) and Tyndall (Tyndall et al., 2005).

7.3 Immunomodulation of MSC after differentiation

Several studies investigated the immunomodulatory effect of MSCs and differentiated MSCs on mixed lymphocytes (Table 1). Le Blanc et al. found both undifferentiated human MSCs, and differentiated human MSCs which had been cultured respectively in adipogeneic, osteogeneic or chondrogeneic medium for 7 or 14 days, were unable to stimulate the proliferation of resting allogeneic human peripheral blood lymphocytes and also had immunosuppressive ability on the activated allogeneic hPBCs (Le Blanc et al., 2003). Niemeyer et al. confirmed the findings on MSCs and osteogeneic differentiated MSCs (Niemeyer et al., 2007). The above authors also found cell differentiation had no significant influence on the expression of immunological surface antigens, i.e. HLA-ABC-positive, Negative for HLA-2, B- and T-cell co-stimulatory molecules surface marker CD40, CD40L, CD80, CD86, and HLA-DR, DP, DQ by FACS or immunofluorescence microscopy. However, study by Chen et al. obtained conflicting results on xenogeneic mouse MSCs. After chondrogeneic differentiation, mouse MSCs could stimulate an 8 fold proliferation of hPBCs whereas the results are negative in undifferentiated MSCs or adipogeneic or osteogeneic differentiated MSCs. This immune response was reassured by cytotoxic study (Chen et al., 2007), which may suggest MSCs or chondrocytes have differences between species.


Stimulation Cells

Responder Cells

Methods of measuring response

Effect on resting lymphocytes

Effect on stimulated lymphocytes

Before Differentiation

After differentiation

Before Differentiation

After differentiation

Adipogeneic Differentiation

Osteogeneic Differentiation

Chondrogeneic Differentiation

Adipogeneic Differentiation

Osteogeneic Differentiation

Chondrogeneic Differentiation

Le Blanc et al., 2003

Allogeneic Human MSCs

Human peripheral blood lymphocytes

3-thymidine incorporation

No proliferation

No proliferation

No proliferation

No proliferation


Enhanced Suppression

Enhanced Suppression

Enhanced Suppression

Niemeyer et al., 2007

Allogeneic Human MSCs

Human peripheral blood mononuclear lymphocytes

3-thymidine incorporation

No proliferation

No proliferation



Allogeneic Human ASCs

Human peripheral blood mononuclear lymphocytes

3-thymidine incorporation

No proliferation

No proliferation



Chen et al., 2007

Xenogeneic Rat MSCs

Human peripheral blood lymphocytes

Biotrak ELISA

No proliferation

No proliferation

No proliferation

8 fold Proliferation

Xenogeneic Rat MSCs

Human peripheral blood lymphocytes eliminated DCs

Biotrak ELISA

No proliferation

Table 1. Immunomodulatory effect of MSCs and differentiated MSCs on mixed lymphocytes

Chapter 8 Encapsulated cell technology

Encapsulated cell technology is based on the immobilization of cells within a semipermeable membrane, to isolate cells from the attack of host immune system while allowing the bidirectional diffusion of therapeutic products, nutrients, oxygen and waste. It has many advantages as a drug delivery system. Compared with therapeutic products, it allows the delivery of the therapeutic product for a sustained and controlled long period of time as cells release the products continuously. Genetically modified cells can be immobilized to express any desired protein in vivo without the modification of the host's genome. The small size of the capsules allows their implantation in close contact to the blood stream to facilitate drug delivery and to maintain oxygen and nutrient supply into donor cells (Murua et al., 2008).

In 1964, Chang proposed the idea of using microcapsules for the immunoprotection of transplanted cells (Chang 1964). In 1980, Lim and Sun designed microencapsulated islets corrected the induced diabetic state of rats for 2 to 3 weeks (Lim and Sun, 1980). Considerable progress has been made toward understanding the biological and technological requirements for successful transplantation of encapsulated cells in experimental animal models, reviewed by Orive et al. However, isolation of sufficiently good islets and development of a suitable biocompatible encapsulation material became the major barrier of this technique (Orive et al., 2003). Many different materials are employed to encapsulate cells. Among them, alginates are nowadays the most studied and characterized for cell encapsulation technology (Murua et al., 2008).

Genetic engineering has contributed to the development of modified cells which are capable of providing an improved supply of therapeutic products. In one study, recombinant mouse myoblasts enclosed in alginate-polylysine-alginate microcapsules, showed continuous expression of human Factor IX for at least 7 months in vivo (Hortelano et al., 1996). In another study, hybridoma cells enclosed in cellulose sulfate capsules showed detectable levels of antibody in mouse serum for as long as 4 months after transplantation (Pelegrin et al., 1998).

Cartilage tissue is considered to be immune privileged and semi-permeable. Tissue engineered (TE) cartilage tissue derived from human adult mesenchymal stem cell (hMSC) will be tested for its immune protection in current study. If successful, the TE cartilage can act as an encapsulation material, enclosed in which the differentiated MSC is planned to be genetically modified to provide therapeutic products in current study, since MSC has already been used as a gene delivery method in previous studies (Kanehira et al., 2007; Stoff-Khalili et al. 2007).

Chapter 9 Aims and Objectives

One of the most exciting areas of regenerative medicine is the use of stem cells, which promise to provide cures for diseases such as Parkinson's, spinal cord injury, diabetes, osteoarthritis and heart failure. However, the majority of existing cell therapies is based on autologous cells, which is very expensive because of its case-by-case cell culture. Previous work in our lab has successfully manufactured tissue engineered (TE) cartilage derived from human adult mesenchymal stem cells (Kafienah et al., 2007) and established a kit of assays to analysis the quality of tissue engineered cartilage (Hollander et al., 1994; Dickinson et al., 2005). The current project aims at providing safe, economically available, off-the-shelf allogeneic tissue engineering cartilage products and other allogeneic mesenchymal stem cell therapies in future. The objectives are as follows:

• To establish mouse mesenchymal stem cell (MSC) lines in our lab.

• To establish and optimize the manufacture of mouse TE cartilage derived from mouse MSC by adjusting the established methods based on human.

• To investigate the immunological reaction of TE cartilage by implanting TE cartilage in different strains of mice.

• To investigate the immunosuppressive effect and mechanisms of MSCs on antigen induced T cell proliferation and the therapeutic effect of MSCs on EAE mouse models.

• To investigate the potential of TE cartilage as a way of gene delivery method.


Alberts B, Bray D, Julian L, Raff M, Roberts K, Watson JD. Differentiated cells and the maintenance of tissues, in Molecular Biology of the Cell. 2nd edition. Edited by: Alberts B, Bray D, Julian L, Raff M, Roberts K, Watson JD. Garland Publishing, New York. 967-68. 1989.

Apte R, Sinha D, Mayhew E, Wistow G, Niederkorn J. Cutting edge: role of macrophage migration inhibitory factor in inhibiting NK cell activity and preserving immune privilege. J Immunol.160:5693-6. 1998.

Arakelov A, Lakkis F. The alloimmune response and effector mechanisms of allograft rejection. Semin Nephrol.20:95-102. 2000.

Armstrong P, Brighton C, Star A. Capacitively coupled electrical stimulation of bovine growth plate chondrocytes grown in pellet form. J Orthop Res.6:265-71. 1988.

Aston J, Bentley G. Repair of articular surfaces by allografts of articular and growth-plate cartilage. J Bone Joint Surg Br.68:29-35. 1986.

Bacsich P, Wyburn G. The significance of the mucoprotein content on the survival of homografts of cartilage and cornea. Proc R Soc Edinb Biol.62:321-7. 1947.

Barnewitz D, Endres M, Krüger I, Becker A, Zimmermann J, Wilke I, et al. Treatment of articular cartilage defects in horses with polymer-based cartilage tissue engineering grafts. Biomaterials.27:2882-9. 2006.

Bartholomäus I, Kawakami N, Odoardi F, Schläger C, Miljkovic D, Ellwart J, et al. Effector T cell interactions with meningeal vascular structures in nascent autoimmune CNS lesions. Nature.462:94-8. 2009.

Bellgrau D, Gold D, Selawry H, Moore J, Franzusoff A, Duke R. A role for CD95 ligand in preventing graft rejection. Nature.377:630-2. 1995.

Bellgrau D, Gold D, Selawry H, Moore J, Franzusoff A, Duke R. A role for CD95 ligand in preventing graft rejection. Nature.377:630-2. 1995.

Bentley G, Greer Rr. Homotransplantation of isolated epiphyseal and articular cartilage chondrocytes into joint surfaces of rabbits. Nature.230:385-8. 1971.

Bentley G, Smith A, Mukerjhee R. Isolated epiphyseal chondrocyte allografts into joint surfaces. An experimental study in rabbits. Ann Rheum Dis.37:449-58. 1978.

Benya P, Padilla S, Nimni M. Independent regulation of collagen types by chondrocytes during the loss of differentiated function in culture. Cell.15:1313-21. 1978.

Benya P, Shaffer J. Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels. Cell.30:215-24. 1982.

Bianco P, Riminucci M, Gronthos S, Robey P. Bone marrow stromal stem cells: nature, biology, and potential applications. Stem Cells.19:180-92. 2001.

Bolano L, Kopta J. The immunology of bone and cartilage transplantation. Orthopedics.14:987-96. 1991.

Bolano L, Kopta J. The immunology of bone and cartilage transplantation. Orthopedics.14:987-96. 1991.

Bolano L, Kopta J. The immunology of bone and cartilage transplantation. Orthopedics.14:987-96. 1991.

Brand D, Latham K, Rosloniec E. Collagen-induced arthritis. Nat Protoc.2:1269-75. 2007.

Brighton C, Jensen L, Pollack S, Tolin B, Clark C. Proliferative and synthetic response of bovine growth plate chondrocytes to various capacitively coupled electrical fields. J Orthop Res.7:759-65. 1989.

Brighton C, Unger A, Stambough J. In vitro growth of bovine articular cartilage chondrocytes in various capacitively coupled electrical fields. J Orthop Res.2:15-22. 1984.

Brighton C, Wang W, Clark C. Up-regulation of matrix in bovine articular cartilage explants by electric fields. Biochem Biophys Res Commun.342:556-61. 2006.

Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O, Peterson L. Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med.331:889-95. 1994.

Brittberg M, Peterson L, Sjögren-Jansson E, Tallheden T, Lindahl A. Articular cartilage engineering with autologous chondrocyte transplantation. A review of recent developments. J Bone Joint Surg Am.85-A Suppl 3:109-15. 2003.

Brodkin K, García A, Levenston M. Chondrocyte phenotypes on different extracellular matrix monolayers. Biomaterials.25:5929-38. 2004.

Buckwalter JA, Lane LE. Athletics and osteoarthritis. Am J Sports Med.25:873-81. 1997.

Bujia J, Alsalameh S, Naumann A, Wilmes E, Sittinger M, Burmester G. Humoral immune response against minor collagens type IX and XI in patients with cartilage graft resorption after reconstructive surgery. Ann Rheum Dis.53:229-34. 1994.

Buzás E, Végvári A, Murad Y, Finnegan A, Mikecz K, Glant T. T-cell recognition of differentially tolerated epitopes of cartilage proteoglycan aggrecan in arthritis. Cell Immunol.235:98-108. 2005.

Campbell I, Kinkel S, Drake S, van Nieuwenhuijze A, Hubert F, Tarlinton D, et al. Autoimmune regulator controls T cell help for pathogenetic autoantibody production in collagen-induced arthritis. Arthritis Rheum.60:1683-93. 2009.

Carpenter M, Cui X, Hu Z, Jackson J, Sherman S, Seiger A, et al. In vitro expansion of a multipotent population of human neural progenitor cells. Exp Neurol.158:265-78. 1999.


Chen J, Wang C, Lü S, Wu J, Guo X, Duan C, et al. In vivo chondrogenesis of adult bone-marrow-derived autologous mesenchymal stem cells. Cell Tissue Res.319:429-38. 2005.

Chen X, McClurg A, Zhou G, McCaigue M, Armstrong M, Li G. Chondrogenic differentiation alters the immunosuppressive property of bone marrow-derived mesenchymal stem cells, and the effect is partially due to the upregulated expression of B7 molecules. Stem Cells.25:364-70. 2007.

Cheshier S, Kalani M, Lim M, Ailles L, Huhn S, Weissman I. A neurosurgeon's guide to stem cells, cancer stem cells, and brain tumor stem cells. Neurosurgery.65:237-49; discussion 49-50; quiz N6. 2009.

Chesterman P, Smith A. Homotransplantation of articular cartilage and isolated chondrocytes. An experimental study in rabbits. J Bone Joint Surg Br.50:184-97. 1968.

Chowdhury T, Arghandawi S, Brand J, Akanji O, Bader D, Salter D, et al. Dynamic compression counteracts IL-1beta induced inducible nitric oxide synthase and cyclo-oxygenase-2 expression in chondrocyte/agarose constructs. Arthritis Res Ther.10:R35. 2008.

Cicuttini F, Baker J, Hart D, Spector T. Choosing the best method for radiological assessment of patellofemoral osteoarthritis. Ann Rheum Dis.55:134-6. 1996.

Cima L, Vacanti J, Vacanti C, Ingber D, Mooney D, Langer R. Tissue engineering by cell transplantation using degradable polymer substrates. J Biomech Eng.113:143-51. 1991.

CRAIGMYLE M. An autoradiographic and histochemical study of long-term cartilage grafts in the rabbit. J Anat.92:467-72. 1958.

Csaki C, Schneider P, Shakibaei M. Mesenchymal stem cells as a potential pool for cartilage tissue engineering. Ann Anat.190:395-412. 2008.

Darling E, Athanasiou K. Growth factor impact on articular cartilage subpopulations. Cell Tissue Res.322:463-73. 2005.

Dayer E, Mathai L, Glant T, Mikecz K, Poole A. Cartilage proteoglycan-induced arthritis in BALB/c mice. Antibodies that recognize human and mouse cartilage proteoglycan and can cause depletion of cartilage proteoglycan with little or no synovitis. Arthritis Rheum.33:1394-405. 1990.

Dickinson S, Sims T, Pittarello L, Soranzo C, Pavesio A, Hollander A. Quantitative outcome measures of cartilage repair in patients treated by tissue engineering. Tissue Eng.11:277-87.

Doran M, Goodstone N, Hobbs R, Ashton B. Cellular immunity to cartilage link protein in patients with inflammatory arthritis and non-arthritic controls. Ann Rheum Dis.54:466-70. 1995.

Elves MW. Immunology of cartilage, in Cartilage. vol. 3. Edited by Hall BK. Academic Press, New York, 229-65. 1983.

Facchini A, Lisignoli G, Cristino S, Roseti L, De Franceschi L, Marconi E, et al. Human chondrocytes and mesenchymal stem cells grown onto engineered scaffold. Biorheology.43:471-80. 2006.

Felson D, Lawrence R, Dieppe P, Hirsch R, Helmick C, Jordan J, et al. Osteoarthritis: new insights. Part 1: the disease and its risk factors. Ann Intern Med.133:635-46. 2000.

Felson D, Zhang Y. An update on the epidemiology of knee and hip osteoarthritis with a view to prevention. Arthritis Rheum.41:1343-55. 1998.

Felson D, Zhang Y, Hannan M, Naimark A, Weissman B, Aliabadi P, et al. Risk factors for incident radiographic knee osteoarthritis in the elderly: the Framingham Study. Arthritis Rheum.40:728-33. 1997.

Flower R. The development of COX2 inhibitors. Nat Rev Drug Discov.2:179-91. 2003.

Fragonas E, Valente M, Pozzi-Mucelli M, Toffanin R, Rizzo R, Silvestri F, et al. Articular cartilage repair in rabbits by using suspensions of allogenic chondrocytes in alginate. Biomaterials.21:795-801. 2000.

Freed L, Grande D, Lingbin Z, Emmanual J, Marquis J, Langer R. Joint resurfacing using allograft chondrocytes and synthetic biodegradable polymer scaffolds. J Biomed Mater Res.28:891-9. 1994.

Freed L, Vunjak-Novakovic G, Biron R, Eagles D, Lesnoy D, Barlow S, et al. Biodegradable polymer scaffolds for tissue engineering. Biotechnology (N Y).12:689-93. 1994.

Frenkel S, Toolan B, Menche D, Pitman M, Pachence J. Chondrocyte transplantation using a collagen bilayer matrix for cartilage repair. J Bone Joint Surg Br.79:831-6. 1997.

Fujihara Y, Asawa Y, Takato T, Hoshi K. Tissue reactions to engineered cartilage based on poly-L-lactic acid scaffolds. Tissue Eng Part A.15:1565-77. 2009.

Gaur L, Nepom G. Ancestral major histocompatibility complex DRB genes beget conserved patterns of localized polymorphisms. Proc Natl Acad Sci U S A.93:5380-3. 1996.

Gerdoni E, Gallo B, Casazza S, Musio S, Bonanni I, Pedemonte E, et al. Mesenchymal stem cells effectively modulate pathogenic immune response in experimental autoimmune encephalomyelitis. Ann Neurol.61:219-27. 2007.

Gertzbein S, Lance E. The stimulation of lymphocytes by chondrocytes in mixed cultures. Clin Exp Immunol.24:102-9. 1976.

Gertzbein S, Tait J, Devlin S, Argue S. The antigenicity of chondrocytes. Immunology.33:141-5. 1977.

Gibson J, Milner P, White R, Fairfax T, Wilkins R. Oxygen and reactive oxygen species in articular cartilage: modulators of ionic homeostasis. Pflugers Arch.455:563-73. 2008.

Gikas P, Bayliss L, Bentley G, Briggs T. An overview of autologous chondrocyte implantation. J Bone Joint Surg Br.91:997-1006. 2009.

Glant T, Buzás E, Finnegan A, Negroiu G, Cs-Szabó G, Mikecz K. Critical roles of glycosaminoglycan side chains of cartilage proteoglycan (aggrecan) in antigen recognition and presentation. J Immunol.160:3812-9. 1998.

Glant T, Buzás E, Finnegan A, Negroiu G, Cs-Szabó G, Mikecz K. Critical roles of glycosaminoglycan side chains of cartilage proteoglycan (aggrecan) in antigen recognition and presentation. J Immunol.160:3812-9. 1998.

Glant T, Mikecz K. Antigenic profiles of human, bovine and canine articular chondrocytes. Cell Tissue Res.244:359-69. 1986.

Glenn RJ, McCarty E, Potter H, Juliao S, Gordon J, Spindler K. Comparison of fresh osteochondral autografts and allografts: a canine model. Am J Sports Med.34:1084-93. 2006.

Glowacki J, Trepman E, Folkman J. Cell shape and phenotypic expression in chondrocytes. Proc Soc Exp Biol Med.172:93-8. 1983.

Gnecchi M, He H, Liang O, Melo L, Morello F, Mu H, et al. Paracrine action accounts for marked protection of ischemic heart by Akt-modified mesenchymal stem cells. Nat Med.11:367-8. 2005.

Gnecchi M, He H, Noiseux N, Liang O, Zhang L, Morello F, et al. Evidence supporting paracrine hypothesis for Akt-modified mesenchymal stem cell-mediated cardiac protection and functional improvement. FASEB J.20:661-9. 2006.

Gooding C, Bartlett W, Bentley G, Skinner J, Carrington R, Flanagan A. A prospective, randomised study comparing two techniques of autologous chondrocyte implantation for osteochondral defects in the knee: Periosteum covered versus type I/III collagen covered. Knee.13:203-10. 2006.

Goodstone N, Doran M, Hobbs R, Butler R, Dixey J, Ashton B. Cellular immunity to cartilage aggrecan core protein in patients with rheumatoid arthritis and non-arthritic controls. Ann Rheum Dis.55:40-6. 1996.

Görtz S, Bugbee W. Allografts in articular cartilage repair. J Bone Joint Surg Am.88:1374-84. 2006.

Götherström C. Immunomodulation by multipotent mesenchymal stromal cells. Transplantation.84:S35-7. 2007.

Griffith T, Brunner T, Fletcher S, Green D, Ferguson T. Fas ligand-induced apoptosis as a mechanism of immune privilege. Science.270:1189-92. 1995.

Gussoni E, Soneoka Y, Strickland C, Buzney E, Khan M, Flint A, et al. Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature.401:390-4. 1999.

Haklar U, Tuzuner T, Uygur I, Kocaoglu B, Guven O. The effect of overlapping on the primary stability of osteochondral grafts in mosaicplasty. Knee Surg Sports Traumatol Arthrosc.16:651-4. 2008.

Hashimoto S, Setareh M, Ochs R, Lotz M. Fas/Fas ligand expression and induction of apoptosis in chondrocytes. Arthritis Rheum.40:1749-55. 1997.

Heyner S. The significance of the intercellular matrix in the survival of cartilage allografts. Transplantation.8:666-77. 1969.

Heyner S. The antigenicity of cartilage grafts. Surg Gynecol Obstet.136:298-305. 1973.

Heywood H, Sembi P, Lee D, Bader D. Cellular utilization determines viability and matrix distribution profiles in chondrocyte-seeded alginate constructs. Tissue Eng.10:1467-79.

Hjertquist S, Lemperg R. Histological, autoradiographic and microchemical studies of spontaneously healing osteochondral articular defects in adult rabbits. Calcif Tissue Res.8:54-72. 1971.

Hollander A, Heathfield T, Webber C, Iwata Y, Bourne R, Rorabeck C, et al. Increased damage to type II collagen in osteoarthritic articular cartilage detected by a new immunoassay. J Clin Invest.93:1722-32. 1994.

Honkonen S. Degenerative arthritis after tibial plateau fractures. J Orthop Trauma.9:273-7. 1995.

Hortelano G, Al-Hendy A, Ofosu F, Chang P. Delivery of human factor IX in mice by encapsulated recombinant myoblasts: a novel approach towards allogeneic gene therapy of hemophilia B. Blood.87:5095-103. 1996.

Hüsing B, Bührlen B, Gaisser S. Human tissue engineered products today's markets and future products. 2003 13/08/2008

Hyc A, Malejczyk J, Osiecka A, Moskalewski S. Immunological response against allogeneic chondrocytes transplanted into joint surface defects in rats. Cell Transplant.6:119-24.

Itay S, Abramovici A, Nevo Z. Use of cultured embryonal chick epiphyseal chondrocytes as grafts for defects in chick articular cartilage. Clin Orthop Relat Res.284-303. 1987.

Jiang Y, Jahagirdar B, Reinhardt R, Schwartz R, Keene C, Ortiz-Gonzalez X, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature.418:41-9. 2002.

Johnstone B, Hering T, Caplan A, Goldberg V, Yoo J. In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res.238:265-72. 1998.

Jones D, Peterson L. Autologous chondrocyte implantation. J Bone Joint Surg Am.88:2502-20. 2006.

Jones D, Peterson L. Autologous chondrocyte implantation. J Bone Joint Surg Am.88:2502-20. 2006.

Kafienah W, Jakob M, Démarteau O, Frazer A, Barker M, Martin I, et al. Three-dimensional tissue engineering of hyaline cartilage: comparison of adult nasal and articular chondrocytes. Tissue Eng.8:817-26. 2002.

Kafienah W, Mistry S, Dickinson S, Sims T, Learmonth I, Hollander A. Three-dimensional cartilage tissue engineering using adult stem cells from osteoarthritis patients. Arthritis Rheum.56:177-87. 2007.

Kanehira M, Xin H, Hoshino K, Maemondo M, Mizuguchi H, Hayakawa T, et al. Targeted delivery of NK4 to multiple lung tumors by bone marrow-derived mesenchymal stem cells. Cancer Gene Ther.14:894-903. 2007.

Kaps C, Frauenschuh S, Endres M, Ringe J, Haisch A, Lauber J, et al. Gene expression profiling of human articular cartilage grafts generated by tissue engineering. Biomaterials.27:3617-30. 2006.

Kassis I, Grigoriadis N, Gowda-Kurkalli B, Mizrachi-Kol R, Ben-Hur T, Slavin S, et al. Neuroprotection and immunomodulation with mesenchymal stem cells in chronic experimental autoimmune encephalomyelitis. Arch Neurol.65:753-61. 2008.

Kawabe N, Yoshinao M. The repair of full-thickness articular cartilage defects. Immune responses to reparative tissue formed by allogeneic growth plate chondrocyte implants. Clin Orthop Relat Res.279-93. 1991.

Kawamura S, Wakitani S, Kimura T, Maeda A, Caplan A, Shino K, et al. Articular cartilage repair. Rabbit experiments with a collagen gel-biomatrix and chondrocytes cultured in it. Acta Orthop Scand.69:56-62. 1998.

Kitaoka E, Satomura K, Hayashi E, Yamanouchi K, Tobiume S, Kume K, et al. Establishment and characterization of chondrocyte cell lines from the costal cartilage of SV40 large T antigen transgenic mice. J Cell Biochem.81:571-82. 2001.

Kitaoka E, Satomura K, Hayashi E, Yamanouchi K, Tobiume S, Kume K, et al. Establishment and characterization of chondrocyte cell lines from the costal cartilage of SV40 large T antigen transgenic mice. J Cell Biochem.81:571-82. 2001.

Klyushnenkova E, Mosca J, Zernetkina V, Majumdar M, Beggs K, Simonetti D, et al. T cell responses to allogeneic human mesenchymal stem cells: immunogenicity, tolerance, and suppression. J Biomed Sci.12:47-57. 2005.

Knutsen G, Drogset J, Engebretsen L, Grøntvedt T, Isaksen V, Ludvigsen T, et al. A randomized trial comparing autologous chondrocyte implantation with microfracture. Findings at five years. J Bone Joint Surg Am.89:2105-12. 2007.

Ksiazek T, Moskalewski S. Studies on bone formation by cartilage reconstructed by isolated epiphyseal chondrocytes, transplanted syngeneically or across known histocompatibility barriers in mice. Clin Orthop Relat Res.233-42.

Kuroda R, Ishida K, Matsumoto T, Akisue T, Fujioka H, Mizuno K, et al. Treatment of a full-thickness articular cartilage defect in the femoral condyle of an athlete with autologous bone-marrow stromal cells. Osteoarthritis Cartilage.15:226-31. 2007.

Lance E. Immunological reactivity towards chondrocytes in rat and man: relevance to autoimmune arthritis. Immunol Lett.21:63-73. 1989.

Lane N, Gore L, Cummings S, Hochberg M, Scott J, Williams E, et al. Serum vitamin D levels and incident changes of radiographic hip osteoarthritis: a longitudinal study. Study of Osteoporotic Fractures Research Group. Arthritis Rheum.42:854-60. 1999.

Langer F, Gross A. Immunogenicity of allograft articular cartilage. J Bone Joint Surg Am.56:297-304. 1974.

Langer F, Gross A, Greaves M. The auto-immunogenicity of articular cartilage. Clin Exp Immunol.12:31-7. 1972.

Langer R, and Vacanti JP. Tissue engineering. Science. 260:920-26, 1993.

Le Blanc K, Rasmusson I, Sundberg B, Götherström C, Hassan M, Uzunel M, et al. Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet.363:1439-41. 2004.

Le Blanc K, Tammik C, Rosendahl K, Zetterberg E, Ringdén O. HLA expression and immunologic properties of differentiated and undifferentiated mesenchymal stem cells. Exp Hematol.31:890-6. 2003.

Lee D, Bader D. Compressive strains at physiological frequencies influence the metabolism of chondrocytes seeded in agarose. J Orthop Res.15:181-8. 1997.

Lim F, Sun A. Microencapsulated islets as bioartificial endocrine pancreas. Science.210:908-10. 1980.

Liu Y, Shu X, Prestwich G. Osteochondral defect repair with autologous bone marrow-derived mesenchymal stem cells in an injectable, in situ, cross-linked synthetic extracellular matrix. Tissue Eng.12:3405-16. 2006.

Lo G, LaValley M, McAlindon T, Felson D. Intra-articular hyaluronic acid in treatment of knee osteoarthritis: a meta-analysis. JAMA.290:3115-21. 2003.

Loewi G, Muir H. The antigenicity of chondromucoprotein. Immunology.9:119-27. 1965.

MacGinitie L, Gluzband Y, Grodzinsky A. Electric field stimulation can increase protein synthesis in articular cartilage explants. J Orthop Res.12:151-60. 1994.

Male D, Brostoff J, Roth DB, and Roitt I. A variety of mechanisms maintain tolerance in peripheral lymphoid organs, in Immunology. 7th Edition. Mosby Elsevier, Philadelphia, PA. 351-56. 2006a.

Male D, Brostoff J, Roth DB, and Roitt I. A variety of mechanisms maintain tolerance in peripheral lymphoid organs, in Immunology. 7th Edition. Mosby Elsevier, Philadelphia, PA. 357-60. 2006b.

Male D, Brostoff J, Roth DB, and Roitt I. A variety of mechanisms maintain tolerance in peripheral lymphoid organs, in Immunology. 7th Edition. Mosby Elsevier, Philadelphia, PA. 236-39. 2006c.

Malejczyk J, Moskalewski S. Effect of immunosuppression on survival and growth of cartilage produced by transplanted allogeneic epiphyseal chondrocytes. Clin Orthop Relat Res.292-303. 1988.

Malejczyk J, Osiecka A, Hyc A, Moskalewski S. Effect of immunosuppression on rejection of cartilage formed by transplanted allogeneic rib chondrocytes in mice. Clin Orthop Relat Res.266-73. 1991.

Marcacci M, Berruto M, Brocchetta D, Delcogliano A, Ghinelli D, Gobbi A, et al. Articular cartilage engineering with Hyalograft C: 3-year clinical results. Clin Orthop Relat Res.96-105. 2005.

Martel-Pelletier J. Pathophysiology of osteoarthritis. Osteoarthritis Cartilage.7:371-3. 1999.

Martin G. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci U S A.78:7634-8. 1981.

Masuoka K, Asazuma T, Ishihara M, Sato M, Hattori H, Yoshihara Y, et al. Tissue engineering of articular cartilage using an allograft of cultured chondrocytes in a membrane-sealed atelocollagen honeycomb-shaped scaffold (ACHMS scaffold). J Biomed Mater Res B Appl Biomater.75:177-84. 2005.

Mauck R, Soltz M, Wang C, Wong D, Chao P, Valhmu W, et al. Functional tissue engineering of articular cartilage through dynamic loading of chondrocyte-seeded agarose gels. J Biomech Eng.122:252-60. 2000.

McAlindon T, Jacques P, Zhang Y, Hannan M, Aliabadi P, Weissman B, et al. Do antioxidant micronutrients protect against the development and progression of knee osteoarthritis? Arthritis Rheum.39:648-56. 1996a.

McAlindon T, Felson D, Zhang Y, Hannan M, Aliabadi P, Weissman B, et al. Relation of dietary intake and serum levels of vitamin D to progression of osteoarthritis of the knee among participants in the Framingham Study. Ann Intern Med.125:353-9. 1996b.

McAlindon T, LaValley M, Gulin J, Felson D. Glucosamine and chondroitin for treatment of osteoarthritis: a systematic quality assessment and meta-analysis. JAMA.283:1469-75. 2000.

Meinel L, Hofmann S, Karageorgiou V, Zichner L, Langer R, Kaplan D, et al. Engineering cartilage-like tissue using human mesenchymal stem cells and silk protein scaffolds. Biotechnol Bioeng.88:379-91. 2004.

Mollenhauer J. Perspectives on articular cartilage biology and osteoarthritis. Injury.39 Suppl 1:S5-12. 2008.

Moskalewski S, Kawiak J, Rymaszewska T. Local cellular response evoked by cartilage formed after auto- and allogeneic transplantation of isolated chondrocytes. Transplantation.4:572-81. 1966.

Moskalewski S, Hyc A, Osiecka-Iwan A. Immune response by host after allogeneic chondrocyte transplant to the cartilage. Microsc Res Tech.58:3-13. 2002.

Mow V, Wang C. Some bioengineering considerations for tissue engineering of articular cartilage. Clin Orthop Relat Res.S204-23. 1999.

Murphy K, Travers P, Walport M. Positive and negative selection of T cells, in Janeway's Immunobiology. 7th edition. Edited by: Murphy KM, Travers P, Walport M. Garland Science Publishing, New York, Taylor and Francis Group.288-98. 2008a.

Murphy K, Travers P, Walport M. The major histocompatibility complex and its functions, in Janeway's Immunobiology. 7th edition. Edited by: Murphy KM, Travers P, Walport M. Garland Science Publishing, New York, Taylor and Francis Group. 196-12. 2008b.

Murphy K, Travers P, Walport M. The responses to alloantigens and transplant rejection. 7th edition. Edited by: Murphy KM, Travers P, Walport M. Garland Science Publishing, New York, Taylor and Francis Group. 637-48. 2008c.

Murrell G, Jang D, Williams R. Nitric oxide activates metalloprotease enzymes in articular cartilage. Biochem Biophys Res Commun.206:15-21. 1995.

Murua A, Portero A, Orive G, Hernández R, de Castro M, Pedraz J. Cell microencapsulation technology: towards clinical application. J Control Release.132:76-83. 2008.

Nauta A, Kruisselbrink A, Lurvink E, Willemze R, Fibbe W. Mesenchymal stem cells inhibit generation and function of both CD34+-derived and monocyte-derived dendritic cells. J Immunol.177:2080-7. 2006.

Nevitt M, Cummings S, Lane N, Hochberg M, Scott J, Pressman A, et al. Association of estrogen replacement therapy with the risk of osteoarthritis of the hip in elderly white women. Study of Osteoporotic Fractures Research Group. Arch Intern Med.156:2073-80. 1996.

NHS Mosaicplasty for knee cartilage defects. 2006 13/08/2008

Nickdel M, Conigliaro P, Valesini G, Hutchison S, Benson R, Bundick R, et al. Dissecting the contribution of innate and antigen-specific pathways to the breach of self-tolerance observed in a murine model of arthritis. Ann Rheum Dis.68:1059-66. 2009.

Niemeyer P, Kornacker M, Mehlhorn A, Seckinger A, Vohrer J, Schmal H, et al. Comparison of immunological properties of bone marrow stromal cells and adipose tissue-derived stem cells before and after osteogenic differentiation in vitro. Tissue Eng.13:111-21. 2007.

Noguchi T, Oka M, Fujino M, Neo M, Yamamuro T. Repair of osteochondral defects with grafts of cultured chondrocytes. Comparison of allografts and isografts. Clin Orthop Relat Res.251-8. 1994.

Noriega S, Mamedov T, Turner J, Subramanian A. Intermittent applications of continuous ultrasound on the viability, proliferation, morphology, and matrix production of chondrocytes in 3D matrices. Tissue Eng.13:611-8. 2007.

Okihana H, Shimomura Y. Effect of direct current on cultured growth cartilage cells in vitro. J Orthop Res.6:690-4. 1988.

Oliveria S, Felson D, Reed J, Cirillo P, Walker A. Incidence of symptomatic hand, hip, and knee osteoarthritis among patients in a health maintenance organization. Arthritis Rheum.38:1134-41. 1995.

Orive G, Hernández R, Gascón A, Calafiore R, Chang T, De Vos P, et al. Cell encapsulation: promise and progress. Nat Med.9:104-7. 2003.

Ossendorf C, Kaps C, Kreuz P, Burmester G, Sittinger M, Erggelet C. Treatment of posttraumatic and focal osteoarthritic cartilage defects of the knee with autologous polymer-based three-dimensional chondrocyte grafts: 2-year clinical results. Arthritis Res Ther.9:R41. 2007.

Parekkadan B, van Poll D, Suganuma K, Carter E, Berthiaume F, Tilles A, et al. Mesenchymal stem cell-derived molecules reverse fulminant hepatic failure. PLoS One.2:e941. 2007.

Parvizi J, Wu C, Lewallen D, Greenleaf J, Bolander M. Low-intensity ultrasound stimulates proteoglycan synthesis in rat chondrocytes by increasing aggrecan gene expression. J Orthop Res.17:488-94. 1999.

Pelegrin M, Marin M, Noël D, Del Rio M, Saller R, Stange J, et al. Systemic long-term delivery of antibodies in immunocompetent animals using cellulose sulphate capsules containing antibody-producing cells. Gene Ther.5:828-34. 1998.

Pelletier J, Martel-Pelletier J, Abramson S. Osteoarthritis, an inflammatory disease: potential implication for the selection of new therapeutic targets. Arthritis Rheum.44:1237-47. 2001.

Pelttari K, Steck E, Richter W. The use of mesenchymal stem cells for chondrogenesis. Injury.39 Suppl 1:S58-65. 2008.

Pfander D, Gelse K. Hypoxia and osteoarthritis: how chondrocytes survive hypoxic environments. Curr Opin Rheumatol.19:457-62. 2007.

Raghunath J, Salacinski H, Sales K, Butler P, Seifalian A. Advancing cartilage tissue engineering: the application of stem cell technology. Curr Opin Biotechnol.16:503-9. 2005.

Rahfoth B, Weisser J, Sternkopf F, Aigner T, von der Mark K, Bräuer R. Transplantation of allograft chondrocytes embedded in agarose gel into cartilage defects of rabbits. Osteoarthritis Cartilage.6:50-65. 1998.

Rahfoth B, Weisser J, Sternkopf F, Aigner T, von der Mark K, Bräuer R. Transplantation of allograft chondrocytes embedded in agarose gel into cartilage defects of rabbits. Osteoarthritis Cartilage.6:50-65. 1998.

Rammensee H, Friede T, Stevanoviíc S. MHC ligands and peptide motifs: first listing. Immunogenetics.41:178-228. 1995.

Rasmusson I, Ringdén O, Sundberg B, Le Blanc K. Mesenchymal stem cells inhibit the formation of cytotoxic T lymphocytes, but not activated cytotoxic T lymphocytes or natural killer cells. Transplantation.76:1208-13. 2003.

Rasmusson I, Ringdén O, Sundberg B, Le Blanc K. Mesenchymal stem cells inhibit lymphocyte proliferation by mitogens and alloantigens by different mechanisms. Exp Cell Res.305:33-41. 2005.

Revell C, Athanasiou K. Success rates and immunologic responses of autogenic, allogenic, and xenogenic treatments to repair articular cartilage defects. Tissue Eng Part B Rev.15:1-15. 2009.

Revell C, Athanasiou K. Success rates and immunologic responses of autogenic, allogenic, and xenogenic treatments to repair articular cartilage defects. Tissue Eng Part B Rev.15:1-15. 2009.

Reya T, Morrison S, Clarke M, Weissman I. Stem cells, cancer, and cancer stem cells. Nature.414:105-11. 2001.

Richter W. Mesenchymal stem cells and cartilage in situ regeneration. J Intern Med.266:390-405. 2009.

Roddy E, Zhang W, Doherty M. Aerobic walking or strengthening exercise for osteoarthritis of the knee? A systematic review. Ann Rheum Dis.64:544-8. 2005.

Romaniuk A, Malejczyk J, Kubicka U, Hyc A, Olszewski W, Moskalewski S. Rejection of cartilage formed by transplanted allogeneic chondrocytes: evaluation with monoclonal antibodies. Transpl Immunol.3:251-7. 1995.

Rosloniec E, Cremer M, Kang A, Myers L. Collagen-induced arthritis. Curr Protoc Immunol.Chapter 15:Unit 15.5. 2001.

Rossi S, Kim M, Leibbrandt A, Parnell S, Jenkinson W, Glanville S, et al. RANK signals from CD4(+)3(-) inducer cells regulate development of Aire-expressing epithelial cells in the thymic medulla. J Exp Med.204:1267-72. 2007.

Sams A, Minor R, Wootton J, Mohammed H, Nixon A. Local and remote matrix responses to chondrocyte-laden collagen scaffold implantation in extensive articular cartilage defects. Osteoarthritis Cartilage.3:61-70. 1995.

Sams A, Nixon A. Chondrocyte-laden collagen scaffolds for resurfacing extensive articular cartilage defects. Osteoarthritis Cartilage.3:47-59. 1995.

Schnabel M, Marlovits S, Eckhoff G, Fichtel I, Gotzen L, Vécsei V, et al. Dedifferentiation-associated changes in morphology and gene expression in primary human articular chondrocytes in cell culture. Osteoarthritis Cartilage.10:62-70. 2002.

Schulz R, Bader A. Cartilage tissue engineering and bioreactor systems for the cultivation and stimulation of chondrocytes. Eur Biophys J.36:539-68. 2007.

Shangkai C, Naohide T, Koji Y, Yasuji H, Masaaki N, Tomohiro T, et al. Transplantation of allogeneic chondrocytes cultured in fibroin sponge and stirring chamber to promote cartilage regeneration. Tissue Eng.13:483-92. 2007.

Song L, Baksh D, Tuan R. Mesenchymal stem cell-based cartilage tissue engineering: cells, scaffold and biology. Cytotherapy.6:596-601. 2004.

Spector T, Cicuttini F, Baker J, Loughlin J, Hart D. Genetic influences on osteoarthritis in women: a twin study. BMJ.312:940-3. 1996.

Stamm C, Westphal B, Kleine H, Petzsch M, Kittner C, Klinge H, et al. Autologous bone-marrow stem-cell transplantation for myocardial regeneration. Lancet.361:45-6. 2003.

Steinwachs M, Guggi T, Kreuz P. Marrow stimulation techniques. Injury.39 Suppl 1:S26-31. 2008.

Stevenson S, Dannucci G, Sharkey N, Pool R. The fate of articular cartilage after transplantation of fresh and cryopreserved tissue-antigen-matched and mismatched osteochondral allografts in dogs. J Bone Joint Surg Am.71:1297-307. 1989.

Stoff-Khalili M, Rivera A, Mathis J, Banerjee N, Moon A, Hess A, et al. Mesenchymal stem cells as a vehicle for targeted delivery of CRAds to lung metastases of breast carcinoma. Breast Cancer Res Treat.105:157-67. 2007.

Stokes D, Liu G, Coimbra I, Piera-Velazquez S, Crowl R, Jiménez S. Assessment of the gene expression profile of differentiated and dedifferentiated human fetal chondrocytes by microarray analysis. Arthritis Rheum.46:404-19. 2002.

Stoop R. Smart biomaterials for tissue engineering of cartilage. Injury.39 Suppl 1:S77-87. 2008.

Takada T, Nishida K, Doita M, Kurosaka M. Fas ligand exists on intervertebral disc cells: a potential molecular mechanism for immune privilege of the disc. Spine (Phila Pa 1976).27:1526-30. 2002.

Takada T, Nishida K, Doita M, Kurosaka M. Fas ligand exists on intervertebral disc cells: a potential molecular mechanism for immune privilege of the disc. Spine (Phila Pa 1976).27:1526-30. 2002.

Takagi T, Jasin H. Interactions between anticollagen antibodies and chondrocytes. Arthritis Rheum.35:224-30. 1992.

Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell.126:663-76. 2006.

Tallheden T, Bengtsson C, Brantsing C, Sjögren-Jansson E, Carlsson L, Peterson L, et al. Proliferation and differentiation potential of chondrocytes from osteoarthritic patients. Arthritis Res Ther.7:R560-8. 2005.

Tanaka T, Komaki H, Chazono M, Fujii K. Use of a biphasic graft constructed with chondrocytes overlying a beta-tricalcium phosphate block in the treatment of rabbit osteochondral defects. Tissue Eng.11:331-9.

Taskiran D, Stefanovic-Racic M, Georgescu H, Evans C. Nitric oxide mediates suppression of cartilage proteoglycan synthesis by interleukin-1. Biochem Biophys Res Commun.200:142-8. 1994.

Thomson J, Itskovitz-Eldor J, Shapiro S, Waknitz M, Swiergiel J, Marshall V, et al. Embryonic stem cell lines derived from human blastocysts. Science.282:1145-7. 1998.

Tiku M, Liu S, Weaver C, Teodorescu M, Skosey J. Class II histocompatibility antigen-mediated immunologic function of normal articular chondrocytes. J Immunol.135:2923-8. 1985.

Torricelli P, Giavaresi G, Fini M, Guzzardella G, Morrone G, Carpi A, et al. Laser biostimulation of cartilage: in vitro evaluation. Biomed Pharmacother.55:117-20. 2001.

Tuan R, Boland G, Tuli R. Adult mesenchymal stem cells and cell-based tissue engineering. Arthritis Res Ther.5:32-45. 2003.

Tyndall A, Walker U, Cope A, Dazzi F, De Bari C, Fibbe W, et al. Immunomodulatory properties of mesenchymal stem cells: a review based on an interdisciplinary meeting held at the Kennedy Institute of Rheumatology Division, London, UK, 31 October 2005. Arthritis Res Ther.9:301. 2007.

Uchida N, Buck D, He D, Reitsma M, Masek M, Phan T, et al. Direct isolation of human central nervous system stem cells. Proc Natl Acad Sci U S A.97:14720-5. 2000.

Vacanti C, Langer R, Schloo B, Vacanti J. Synthetic polymers seeded with chondrocytes provide a template for new cartilage formation. Plast Reconstr Surg.88:753-9. 1991.

van Saase J, van Romunde L, Cats A, Vandenbroucke J, Valkenburg H. Epidemiology of osteoarthritis: Zoetermeer survey. Comparison of radiological osteoarthritis in a Dutch population with that in 10 other populations. Ann Rheum Dis.48:271-80. 1989.

van Susante JLC, Pieper J, Buma P, van Kuppevelt T, van Beuningen H, van Der Kraan P, et al. Linkage of chondroitin-sulfate to type I collagen scaffolds stimulates the bioactivity of seeded chondrocytes in vitro. Biomaterials.22:2359-69. 2001.

Varghese S, Theprungsirikul P, Ferran A, Hwang N, Canver A, Elisseeff J. Chondrogenic differentiation of human embryonic germ cell derived cells in hydrogels. Conf Proc IEEE Eng Med Biol Soc.1:2643-6. 2006.

Vinatier C, Bouffi C, Merceron C, Gordeladze J, Brondello J, Jorgensen C, et al. Cartilage Tissue Engineering: Towards a Biomaterial-assisted Mesenchymal Stem Cell Therapy. Curr Stem Cell Res Ther. 2009.

Wagers A, Weissman I. Plasticity of adult stem cells. Cell.116:639-48. 2004.

Wakitani S. [Present status and perspective of articular cartilage regeneration]. Yakugaku Zasshi.127:857-63. 2007.

Wakitani S, Goto T, Pineda S, Young R, Mansour J, Caplan A, et al. Mesenchymal cell-based repair of large, full-thickness defects of articular cartilage. J Bone Joint Surg Am.76:579-92. 1994.

Wakitani S, Imoto K, Yamamoto T, Saito M, Murata N, Yoneda M. Human autologous culture expanded bone marrow mesenchymal cell transplantation for repair of cartilage defects in osteoarthritic knees. Osteoarthritis Cartilage.10:199-206. 2002.

Wakitani S, Kawaguchi A, Tokuhara Y, Takaoka K. Present status of and future direction for articular cartilage repair. J Bone Miner Metab.26:115-22. 2008.

Wakitani S, Kimura T, Hirooka A, Ochi T, Yoneda M, Yasui N, et al. Repair of rabbit articular surfaces with allograft chondrocytes embedded in collagen gel. J Bone Joint Surg Br.71:74-80. 1989.

Wakitani S, Mitsuoka T, Nakamura N, Toritsuka Y, Nakamura Y, Horibe S. Autologous bone marrow stromal cell transplantation for repair of full-thickness articular cartilage defects in human patellae: two case reports. Cell Transplant.13:595-600. 2004.

Wang W, Wang Z, Zhang G, Clark C, Brighton C. Up-regulation of chondrocyte matrix genes and products by electric fields. Clin Orthop Relat Res.S163-73. 2004.

Watt F, Dudhia J. Prolonged expression of differentiated phenotype by chondrocytes cultured at low density on a composite substrate of collagen and agarose that restricts cell spreading. Differentiation.38:140-7. 1988.

Weiss S, Reynolds B, Vescovi A, Morshead C, Craig C, van der Kooy D. Is there a neural stem cell in the mammalian forebrain? Trends Neurosci.19:387-93. 1996.

Weissman I. Translating stem and progenitor cell biology to the clinic: barriers and opportunities. Science.287:1442-6. 2000.

Wieland H, Michaelis M, Kirschbaum B, Rudolphi K. Osteoarthritis - an untreatable disease? Nat Rev Drug Discov.4:331-44. 2005.

Wilmut I, Schnieke A, McWhir J, Kind A, Campbell K. Viable offspring derived from fetal and adult mammalian cells. Nature.385:810-3. 1997.

Wiseman M, Henson F, Lee D, Bader D. Dynamic compressive strain inhibits nitric oxide synthesis by equine chondrocytes isolated from different areas of the cartilage surface. Equine Vet J.35:451-6. 2003.

Yuan Y, Yu H, Boyer M, Song X, Cao S, Shen H, et al. Hematopoietic stem cells are not the direct target of spontaneous leukemic transformation in p18(INK4C)-null reconstituted mice. Cancer Res.66:343-51. 2006.

Zappia E, Casazza S, Pedemonte E, Benvenuto F, Bonanni I, Gerdoni E, et al. Mesenchymal stem cells ameliorate experimental autoimmune encephalomyelitis inducing T-cell anergy. Blood.106:1755-61. 2005.