Chondrocyte Transplantation and Selection

5.514.

Chondrocyte Transplantation and Selection

A Lindahl, University of Gothenburg, Gothenburg, Sweden ã 2011 Elsevier Ltd. All rights reserved.

5.514.1. 5.514.2. 5.514.3. 5.514.4. 5.514.5. 5.514.6. 5.514.7. 5.514.8. 5.514.8.1. 5.514.8.2. 5.514.8.3. 5.514.9. 5.514.9.1. 5.514.9.2. 5.514.10. References

Introduction Cell Therapy and Cartilage Regeneration: State of the Art Cell Therapy Concepts in Cartilage Disease and Osteoarthritis Molecular Control Mechanisms in the Knee Joint – Implications for Cartilage Repair and OA The Uniqueness of Hyaline Cartilage Chondrocytes Adult Stem Cells and Stem Cell Niches Improvements in ACT Cell Technology – Alternative Cell Sources Autologous Use of Cells Osteoarthritic Cells Bone Marrow Stromal Cells or Mesenchymal Stem Cells Fat Stromal Cells and Cartilage Regeneration Universal Donor Cell Lines for Cartilage Repair Derivation of Chondrocytes from Human Embryonic Stem Cells Derivation of Chondrocytes from Umbilical Cord Cells and Umbilical Cord Blood Cells Conclusion

Glossary Articular cartilage A fibrous tissue comprising extracellular matrix proteins (mostly collagen and proteogylcans) produced by the cells (chondrocytes), and functioning to create a viscoelastic tissue with high biomechanical stability and low friction. It has a very limited ability for self-repair, mandating interventional approaches for repair. Autologous chondrocyte transplantation (ACT) A regenerative medicine and multistep surgical procedure used to treat traumatic cartilage damage or osteochondrosis dissecans, but not degenerative arthritis in adults by harvest of cartilage from the patient, expansion of the chondrocyte progenitor cells, harvest, and transplant of these cells back into the patient’s articulating cartilage site.

Abbreviations ACI ACT ATSC BMP BMPr1a BMSC COMP DIO2 FGF FRZB GDF5

Autologous chondrocyte implantation Autologous chondrocyte transplantation Adipose derived stromal cells Bone morphogenetic protein Bone morphogenetic protein receptor 1a Bone marrow stromal cell Cartilage oligomeric protein Iodothyronine deiodinase enzyme type 2 Fibroblast growth factor Frizzle related protein B Growth differentiation factor 5

190 190 190 191 192 193 193 194 194 194 195 195 195 197 197 198

Chondrocyte Specialized musculoskeletal cell type producing type II collagens and specialized proteoglycans responsible for the structure and unique viscoelastic propeties of cartilage tissue. The only cell found in cartilage. Chondroprogenitor donor cell-lines Cells representing chondrocyte precursors, capable of harvest and repeated culture to retain chondrocyte phenotypes, self-renewal, and tissue-regenerating properties of mature chondrocytes. Hyaline cartilage Specialized articular cartilage found in synovial joint surfaces distinct in composition, structure and function from fibrillar cartilage Osteoarthritis (OA) Cartilage degeneration characterized by cartilage degradation, the formation of osteophytes, and subchondral sclerosis, pain, and joint dysfunction.

GPR22 hES cells HLH HMGB-1 HSC ID1 IL-1 MSC OA SCN WJCs

G protein coupled receptor protein 22 Human embryonic stem cells Helix loop helix High mobility chromosomal box protein-1 Hematopoetic stem cell Inhibitor of differentiation 1 Interleukin 1 Mesenchymal stem cell Osteoarthritis Stem cell niche Wharton’s jelly stem cells

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5.514.1.

Introduction

Over 250 years ago, the famous anatomist William Hunter stated “ulcerated cartilage is a troublesome thing and once destroyed, is not repaired,” a statement that is still valid today.1 Adult articular cartilage consists mainly of extracellular matrix proteins produced by the cells (chondrocytes), which have the function of creating a visco elastic tissue with high biomechanical stability and low friction (Figure 1). Even though the cartilage is exposed to continuous mechanical wear, the cellular and extracellular matrix turnover is low, which could be a reason for the inability of adult articular cartilage to respond to injuries and subsequently repair lesions. The damage to cartilage could lead to the end-stage disease osteoarthritis (OA) that is characterized by cartilage degradation, the formation of osteophytes, and subchondral sclerosis. OA is one of the most common forms of musculoskeletal disease throughout the world with millions of affected individuals. The OA disease causes a burden to the society and health care system but despite the increase in knowledge in medical science no drug-based disease-modifying therapy exists basically due to the fact that (i) the disease process stretches over decades, (ii) no surrogate markers exist that can be used to monitor progression of OA, and (iii) no specific drug target and disease mechanism have been identified.2 The limited repair capacity of articular cartilage defects after trauma is the major contributing factor to early OA with 5% of the population between 35 and 54 years having radiographic signs of OA.3,4

5.514.2. Cell Therapy and Cartilage Regeneration: State of the Art Articular cartilage has poor ability to self-repair5 and attempts of replacing the articular joint surface with osteochondral grafts were tested by Eric Lexer in 1908. Following that sporadic attempts were tried (see review by Aichroth6) but the variable methodology and clinical outcome did not justify wider clinical applications. Failures have generally been attributed to

Superficial layer Transitional layer

Deep radial layer

Calcified layer Figure 1 Normal healthy and osteoarthritic cartilage histology. Representive light micrographs of condylar cartilage obtained post mortem from joints with (a) normal healthy cartilage and (b) cartilage obtained at joint replacement surgery. Sections are stained with safranin-O fast green-iron haematoxylin. The different layers are an approximation and the calcified layer is indicated but not present in the sections. Figure modified from Arthritis Res. Ther. 2006, 8(1), R2.

insufficient bone fixation, and allografts have failed due to immunological rejections and autografts had a better success rate. Over the years treatment of smaller cartilage injuries have been mainly surgically based on the concept of recruiting bone marrow stromal cells (BMSCs) by drilling or microfracturing the subchondral bone, alternatively using osteochondral autografts. However, the resulting repair tissue is mostly fibrocartilaginous tissue lacking the biomechanical properties of the hyaline cartilage. Cellular attempts to regenerate cartilage were initiated in the 1960s when Green was able to isolate chondrocytes from cartilage and culture the cells.7 The pioneering work of Bentley in 19718 demonstrated the possibility that transplanted and isolated articular chondrocytes as well as epiphyseal chondrocytes taken from very young rabbits (6-weeks old) that were injected into defects drilled in the upper tibial articular surfaces could induce repair. The articular chondrocytes were mostly rejected but the epiphyseal chondrocytes survived in small numbers and laid down normal staining hyaline matrix which filled the defects completely. In subsequent experiments, it was demonstrated that frozen chondrocytes also were able to regenerate an osteochondral defect. The next step in cellular cartilage repair was taken with the autologous chondrocyte transplantation (ACT) technology developed by our group.9 ACT or autologous chondrocyte implantation (ACI) has in the last decade emerged as the first disease-modifying treatment with long-term excellent clinical result in patients with isolated cartilage injuries10,11 and osteochondral lesions in the knee and ankle.12,13 The good clinical outcome of the ACT technique for the treatment of cartilage injuries has resulted in a worldwide spread of the technology with over 35 000 patients treated, among those, 1500 have been treated by our group over the last 15 years. Four randomized controlled clinical trials have demonstrated treatment superiority for ACT over conventional treatment14–16 while one showed no difference, although ACT produced a better hyaline repair tissue.17 However, the latter study does not take into account the fact that inclusion criteria for ACT treatment have repeatedly failed previous conventional treatments as one-third of patients with cartilage injuries diagnosed for the first time improve spontaneously after debridement.18 Recent reports from our group with 15-year follow-up gives further support for the hypothesis that the ACT technology is a local diseasemodifying treatment where the graft has contributed to local cartilage regeneration.19 Furthermore, long-term follow-up with gadolinium enhancement demonstrated a hyaline repair tissue similar to surrounding cartilage.20

5.514.3. Cell Therapy Concepts in Cartilage Disease and Osteoarthritis We have to accept the fact that OA is a chronic disease where the time between initiation of disease and fully developed OA stretches over decades. The challenge for the development of new OA treatments based on cellular or pharmacological technological base is hampered by the absence of early diagnostic tools. Classical biochemical assays are often based on the leakage of components from damaged cells (compare liver damage and released liver enzymes). The OA assays that are

Chondrocyte Transplantation and Selection

available detect matrix components like cartilage oligomeric proteins (COMPs) inflammatory cytokines (interleukin-6 (IL-6) or high mobility chromosomal box protein-1 (HMGB-1). However, these markers are not sensitive enough for individual diagnostics at the early stage of the disease. The possibility of a regenerative approach to OA disease which could bridge the gap between the current stage of treatment (with the exception of traumatic defects which is not afflicted by OA disease) has changed given the recent advances in the knowledge of the regenerative capacity of different tissues (e.g., brain and heart) and the tissue ability to regenerate after injury.21 The paradigm shift has changed the focus toward understanding the mechanisms of the regeneration system within each tissue, that is, identification of specific stem cell niches (SCNs) that harbor and maintain the stem cells through an intricate interaction and control (Figure 2). The homeostatic control involves a wide range of secreted soluble and matrix interactive signals. These signals represent a conserved ‘developmental toolkit,’ which is also important for embryonic development and includes, but is not limited to, bone morphogenetic proteins (BMPs), Fibroblast growth factors (FGFs), Notch, Hedgehog, and Wnt signaling pathways,22,23 as well as the integrins and the cell surface proteoglycans. Particularly, the syndecans that represent a specific set of signaling molecules are able to modify intracellular events such as transcription. The signals are key players in disease mechanisms; for example, loss and gain of function studies in two Wnt coreceptors (Lrp5 and Lrp6) have drawn considerable attention to the importance of Wnt signaling pathways in skeletal biology.24 Breakdown of the self-renewal system is a key event in the development of cancer and regeneration in aged muscles can be enhanced by restoration of normal signaling in the Notch pathway.25 Continuous Wnt activation in the Klotho mice triggers accelerated cellular senescence of stem cells in several organs.26 Furthermore, increased Wnt signaling during aging alters muscle stem cell fate and increases fibrosis, a process accelerated by Wnt3A while antagonists to the same pathway regain muscle lineage.27 Future treatment of cartilage defects with cells and/or scaffolds have to adopt this new view of cartilage regeneration.

5.514.4. Molecular Control Mechanisms in the Knee Joint – Implications for Cartilage Repair and OA In earlier studies, we found that the de- and redifferentiation process of human articular chondrocytes is similar to molecular programs found in fetal cartilage development.28 Given the fact that there are stem cell markers in the knee joint and a SCN in the surroundings, the Notch signaling pathway has been studied as it is involved in the regulation of joint development and growth29 and expressed in the surface layer of articular cartilage – laminae splendance.30 Since OA chondrocytes display an altered differentiation state compared to normal chondrocytes, it is expected that the expression of Notch markers in OA cartilage is altered. In healthy cartilage, Notch1, its ligand, Jagged1, and down stream effector gene hes5 (human version of the Drosophila gene, hairy and enhancer of split) are expressed in a few cells localized within the uppermost cell layers of the surface zone lamina splendence. This is in sharp contrast to the abundant expression of these markers detected in OA biopsies, where expression was extended to cells in deeper layers of the cartilage.31 A recent population study added a new susceptible locus on chromosome 7q22 with a strong correlation related to the G protein coupled receptor protein 22 (GPR22).32 Interestingly, the receptor could not be found in the articular cartilage but was localized to lamina splendence, thus colocalizing with the notch receptor in the superficial layer of articular cartilage. Primary OA has an estimated heritability of 40% for the knee, 60% for the hip, and 65% for the hand.2 To date, investigations of OA genetics have focused mainly on genome-wide linkage and candidate gene studies with controversial results due to lack of power and replication. Interestingly, two genes have been found to be consistently associated with OA in Caucasians: the growth differentiation factor 5 (GDF5)33 and iodothyronine deiodinase enzyme type 2 (DIO2),34 which are associated with SCN growth control.34,35 Furthermore, hip OA is associated, in females, with the frizzle related protein B (FRZB), which is an inhibitor of the Wnt signaling pathway and is also involved in the stem cell signaling36 (see Chapter 5.524,

Stem cell niche signalling in regeneration 1. Stem cell preservation

2. Progeny determination

3. Progeny migration

Regenerating synovial joint

Soluble signals TGF-b Cell signaling Notch–Delta Adhesion signaling integrins, syndecan, CD44, cadherins

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Matrix components: collagens, fibronectin, hyalurnan Matrix cellular receptors: syndecan, integrins

Figure 2 Schematic drawing of mechanisms in stem-cell-maintained tissue regeneration.

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Biomaterials for Replacement and Repair of the Meniscus and Annulus Fibrosus).

5.514.5. The Uniqueness of Hyaline Cartilage Chondrocytes When considering cartilage cell therapies it is necessary to recall the fundamental biology of cartilage and bone formation. Bone develops along two developmental pathways – either intramembraneous or by endochondral bone formation. Both processes are recognized by the process of cellular condensation where the endochondral bone formation has an intermediate cartilage step. Most of the skeletal bones are formed by endochondral formation and most of the skull bones are formed by intramebraneous formation. The endochondral bone formation is characterized by deposition of matrix into a cartilaginous rod, which is the template for the long bones with cartilaginous tissue in the epiphysis as well as the long bone shaft. Subsequently, the chondrocytes hypertrophy in the long bone area and this process is followed by capillary ingrowth and the start of the process of mineralization. Initially the epiphysis remains cartilaginous but a second ossification zone develops in the central part of the epiphysis. Between the ossification center and the long bone the epiphyseal cartilage is formed that is responsible for the longitudinal bone growth. The proximal cartilage causes endochondral ossification in both distal and proximal directions. Subsequently, the ossification toward the epiphysis ceases and a laminar organization of chondrocytes develops in the distal direction.37,38 Close to the bony epiphysis there is a uniformly distributed cell layer consisting of rarely dividing cells. This cell layer constitutes the stem cells of the growth plate and is usually called the

germinal cell layer. This cartilaginous structure is connected with the articular cartilage and the perichondral ring responsible for the circumferential bone growth. The articulating joint formation takes place in the cartilaginous rods where the formation of a small condensation zone named the digital ray is the first microscopic sign. This is followed by the formation of a three-layered interzone with two regions of high cell density with a region of lower density in between (Figure 3). The intermediate cell layer disappears through a process of apoptosis and thus forms the cavity of the joint while the surrounding high-density region forms the articulating surfaces.39 The surrounding joint capsule and the tendons are formed from cellular condensations laterally to the digital ray thus creating the full articulating joint. Little attention has been attributed to the cellular lineage of this structure since the concept has been that the joint is formed from the cartilage rod and thus similar to the hypertrophic cartilage, although the hypertrophic process is somewhat blocked. Several investigators have studied the factors responsible for cellular growth and structural patterning of the joint and in these studies the BMPs plays an important role. The first BMPs were isolated due to the remarkable capacity of ectopic bone formation. Subsequently many BMPs have been identified and studies of sequence homology have revealed that the BMPs form a subfamily within the TGF-b superfamily. GDF5 belongs to a new subgroup of the BMP family that includes GDFs 5, 6, and 7. GDF5 has been hypothesized to having multiple functions in the skeletogenesis, since exogenously injected GDF5 protein stimulated cartilage development and inhibited surrounding GDF5 expression and joint formation.40,41 Further evidence for a unique hyaline cartilage phenotype for articular cartilage was demonstrated in mice where cells expressing a member of the BMP family (GDF5) constitute a distinct cohort of progenitor

Developmental time (a)

(b)

(c)

(d)

(e)

(f)

Joint capsule

Proximal end

Interzone

Articular cartilage

?

Synovial cavity Distal end

Uninterrupted mesenchymal condensation

Joint site determination

Interzone formation + Chondrocyte differentiation

Cavitation

Morphogenesis

Joint formation

Mesenchymal cells Chondrocytes

Figure 3 Schematic overview of the localization of the BrdU-positive cells in the knee. Reproduced from J. Anat. 2009, 215(3), 355–363.

Chondrocyte Transplantation and Selection

cells responsible for limb joint formation including articular cartilage, ligaments, synovial lining, with limited contribution to the adjacent long bone cartilaginous shaft and growth plate formation.42 Congenital mutations in the GDF5 gene cause defects in digits, wrist, and ankle43 while deletion of antichondrogenic factors like Wnt9a causes limb and joint deformation.44 This gives an additional support for a lineage specificity for articular cartilage that has to be taken into account when discussing cell therapy models in the joint (see Chapter 5.538, Finger).

5.514.6.

Adult Stem Cells and Stem Cell Niches

The joint is a complex entity and it is still an important question if the joint and cartilage is a regenerating tissue with some repair capacity and in such case there has to exist a potential chondrogenic stem cell population within the joint. However, the poor capacity for self-repair of articular cartilage has been explained by cell senescence and the lack of chondroprogenitor cells. In contrast, human articular chondrocytes cultured in vitro display phenotypic plasticity with chondrogenic, adipogenic, and osteogenic potential28,45,46 although whether this is due to dedifferentiation of the cells or selection of a progenitor cell population is debatable. The anatomical location of the potential stem cells as well as the existence in a specific tissue in vivo is not well explored, particularly not in the joint. In recent years, it is becoming evident that stem cells are located in a special microenvironment termed stem cell niches (SCNs). The location and nature of these niches can vary depending on the tissue type but one common entity is that the signaling pathways are conserved.23 The bulge area of hair follicles where epithelial stem cells are resident and the intestinal SCN located near the crypt base has been extensively studied.47,48 An area of potential interest with regard to progenitor cells in the joint is the circumferential anatomical structure first described by Ranvier in 1873 and formerly named the perichondrial groove of Ranvier. This area is located at the periphery of the epiphyseal growth plate and has been demonstrated to contain proliferating cells.49 It has

also been suggested that perichondrial cells from the ring of LaCroix, which is a fibrous band that surrounds the groove of Ranvier and is continuous with the periosteum of the metaphysis, serve as a reservoir for precartilaginous cells in the germinal layer of the epiphyseal growth plate.50 The important role of an intact epiphyseal growth plate, and perichondrial zone, for longitudinal bone growth is well documented. Salter–Harris type VI fractures within the groove of Ranvier has demonstrated severe growth disturbances.51 An established method to identify stem cells within different tissues is the labeling of slow cycling cells corresponding to the stem cell population with bromodeoxyuridine (BrdU). Progenitor cells/stem cells can be identified and characterized due to expression of specific proteins, although no unique marker for these types of cells exists today. However, there are markers associated with stem cells in the literature, for example, Stro1, bone morphogenetic protein receptor 1a (BMPr1a), Patched, Notch1, Integrin b1, and N-cadherin. Indeed a SCN in the zone of Ranvier close to the knee joint has been demonstrated with the technique of long-term labeling with BrdU. The SCN expressed similar immunohistochemical markers as other SCNs, that is, b-catenin, Notch1, Noggin, and c-kit52 (Figure 4) and proliferating cells were present in the deeper areas of the articular cartilage. Although a significant decrease in the number of BrdU positive cells were detected at the later time points, some cells were still detected suggesting that progenitor cells might be present in the deeper layers of the cartilage. A similar SCN has also been identified in the intervertebral disk region53 The interesting question is, if these niches could potentially be involved in human joint and disk regeneration and disease – a positive answer would mean a paradigm shift for the concept of cellular treatment for cartilage defects and the OA disease mechanism as well as disk degeneration.

5.514.7. Improvements in ACT Cell Technology – Alternative Cell Sources OA patients and younger patients with small injuries are not subjected to ACT treatments due to the limited source of ‘normal’

Differentiated cells C

A Transit amplifying cells

B A

Figure 4 Development of bone and joint cartilage.

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Stem cells

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cartilage in an OA joint and the high costs of GMP-produced cells or grafts. This has raised the question if other cell types could replace the autologous chondrocytes (i.e., mesenchymal stem cells (MSCs), OA chondrocytes, human embryonic stem cells (hES cells), and umbilical cord or cord blood cells). When discussing different cell sources, there are requirements to be considered although some sources seem to be more easily accessible than others. Requirements for cells intended for hyaline cartilage repair is that they (i) are of the correct lineage and are able to differentiate toward hyaline cartilage. They should also be (ii) accessible and harvested with minimal (iii) morbidity for the patients. The requirement of multipotency and telomerase activity should be considered but more important is the (iv) capacity of cell expansion without loss of differentiation potential. Currently, the chondrocytes isolated from human articular cartilage are pluripotent with a unique differentiation capacity toward cartilage, bone and fat cells, and the cells are derived from the GDF5 lineage as earlier described. Alternative cell sources to consider would depend on the intended use. These could be divided into two distinct groups of treatments where one is autologous use and the second is universal donor use. The cell sources are discussed based upon these aspects as well as lineage specificity and expandability.

5.514.8.

Autologous Use of Cells

5.514.8.1. Osteoarthritic Cells The use of osteoarthritic cells could seem like a tempting source of cells which would mean that the doctor is able to harvest cells from a damaged knee for subsequent culture and expansion. Currently, autologous chondrocyte transplantation (ACT) is used to treat traumatic cartilage damage or osteochondrosis dissecans, but not degenerative arthritis. The original ACT technology is based on culture-expanded chondrocytes transplanted under a cover of periosteum. The main indication was treatment of small isolated lesions but gradually over the last decade the indication has been expanded to include lesions up to 20 cm2 in size. The implantation method has been followed by culture-expanded cells loaded on a membrane or into a biodegradable scaffold before implantation. Advantages of using scaffolds as cell carriers is that the cells can be positioned in the lesion arthroscopically and could potentially treat larger defects. This is especially interesting for young (under 60-years old) and active patients with developed OA, who at present lack an appropriate treatment alternative. Different scaffold materials have been used for this purpose, for example, different combinations of poly-Llactic acid and poly(lactic-co-glycolic acid)54 or materials obtained by esterification of hyaluronic acid.55 The etiology of OA has been suggested to contain a phenotypic alteration of the chondrocytes56 due to systematic, mechanical, or unknown reasons. Chondrocytes isolated from OA cartilage have been shown to be more metabolically active than cells isolated from non-OA regions in the same joint57and proteoglycan synthesis in OA cells have been demonstrated in comparable amounts to normal cartilage from ACT donors although collagen synthesis is significantly lower.58 Furthermore, chondrocytes isolated from less-severe grades of OA cartilage synthesize normal matrix components.59

When OA cells were cultured in a hyaluronan scaffold (Hyaff 11) only few genes were differentially expressed between OA and normal hyaline cartilage as determined by genome-wide oligonucleotide microarrays and the risk of differentiation into hypertrophic cartilage was not increased.60 In such systems the OA cells could fulfill the requirements for matrix-associated cartilage implantation and OA patients could likely benefit from the treatment. In another study the gene expression patterns of osteoarthritic (OA) chondrocytes after primary culture and subculture were compared to these with healthy chondrocytes and with articular chondrocytes expanded for treatment of patients by ACT. In those studies OA chondrocytes showed highly elevated levels of interleukine 1b (IL-1b) mRNA, but type I and II collagen levels were comparable to those of healthy chondrocytes. After primary culture, IL-1 b levels decreased to baseline levels, while the type II and type I collagen mRNA levels matched those found in chondrocytes used for ACT. OA chondrocytes generated type II collagen and proteoglycan-rich cartilage when transplanted in a SCID mice model.61 One conclusion from these studies could be that after expansion under suitable conditions, the cartilage of OA patients contains cells that are not significantly different from those from healthy donors prepared for ACT. OA chondrocytes supported by hyaluronan or collagen-based scaffolds could potentially be used for regenerating cartilage in humans although cells in single cell suspension are less suitable. Whether other types of scaffolds have similar capacity remains to be demonstrated.

5.514.8.2. Bone Marrow Stromal Cells or Mesenchymal Stem Cells In the human bone marrow, a rare population of clonogenic nonhematopoietic stem cells (1/100 000 of the bone marrow mononuclear cells) resides and the cells were earlier denoted as bone marrow stromal cells (BMSCs). The function of these cells is to support hematopoetic stem cell (HSC) function. Furthermore, these cells have the capacity to differentiate into cells of various connective tissue cells such as bone,62,63 muscle,64 adipose tissue,65 cartilage,66 and tendon.67 Within the scientific literature, the acronym MSC has been used to represent (bone) marrow stromal cells,68 MSC, and multipotent mesenchymal stromal cells, and this sometimes causes confusion since MSCs can be found in various tissues. It is more correct to give the MSCs a prefix depending on the source of harvest although the cells have similar capacity; for example, MSC derived from bone marrow aspiration would therefore be described and termed BMSC. Other MSCs that are candidate for replacing chondrocytes in cell-based repair of cartilage lesions could be derived from many other sources besides the bone marrow, that is, from adipose tissue, where the cells are termed adipose derived stromal cells (ATSCs), from Wharton’s jelly in the umbilical cord (Wharton’s jelly stem cells, WJCs), from umbilical cord blood (umbilical cord blood stem cells), or from synovial tissues, which also have the capacity to differentiate into specialized cells in vitro (see Chapter 5.537, Adipose Tissue Engineering). However, it has not been clarified if MSCs can acquire the hyaline phenotype, and whether chondrocytes and MSCs show the same expression patterns of critical control genes in development. Several attempts have been made to achieve

Chondrocyte Transplantation and Selection regeneration of cartilage defects using MSC.66,69 One major drawback of using these cells in cartilage or intervertebral disk tissue engineering is that human MSCs express type X collagen, a marker of chondrocyte hypertrophy associated with endochondral ossification.70 In high-density culture systems the gene expression patterns were compared between articular chondrocytes and iliac crestderived MSCs. For articular cartilage, a significant decrease in expression of collagen type I was timed with increased expression of collagen types IIA and IIB during differentiation, thus indicating differentiation toward a hyaline phenotype. Chondrogenesis in MSCs on the other hand resulted in up-regulation of collagen types I, IIA, IIB, and X, demonstrating differentiation toward cartilage of a mixed phenotype. Expression of the helix loop helix (HLH) transcription factor 1 (HES1) increased significantly during chondrogenesis in chondrocytes while expression in MSCs was maintained at a low level. The HLH gene HES5 on the other hand was exclusively detected in chondrocytes. Expression of the HLH gene ID1 (inhibitor of differentiation 1) decreased significantly in chondrocytes while the opposite was seen in MSCs.71 These findings suggest that chondrocytes and MSCs formed different subtypes of cartilage, the hyaline and a mixed cartilage phenotype, respectively. These results are important when considering the use of MSCs in cartilage repair. The migration capacity of chondrocytes and mesenchymal stem cells (MSCs) is also important in cartilage regeneration and tissue engineering approach. A study analyzed growth factors and cytokines for their ability to induce migration of human articular chondrocytes and bone marrow-derived MSC in Boyden chamber assays. The platelet-derived growth factor was the most potent migrating agent but no differences between the two cell types could be detected.72 When stimulated with GDF5 (formerly named cartilage-derived morphogenetic protein-1 (CDMP-1))73 cartilaginous differentiation was demonstrated. In conclusion, the MSC are easily accessible from the iliac crest but the harvest causes some morbidity to the patients; it is possible for the cells to expand in monolayer cultures and have multipotency but the default pathway demonstrated is not hyaline cartilage but hypertrophic cartilage and bone formation. This could be due to the fact that the cells are not in the GDF5 lineage although MSCs are able to differentiate into chondrocytes but the tissue represents an unmineralized hypertrophic cartilage.

5.514.8.3. Fat Stromal Cells and Cartilage Regeneration Isolation procedure of adipose cells is well described and basically the liposuction material is thoroughly washed and digested by collagenase type I. The isolated cells are subsequently separated from adipose cells by centrifugation where superficial cells are the fat cells and the pellet contains the ATSCs. The differentiation capacities of these cells are similar to MSCs. The cell fraction has been evaluated in horses with cartilage defects and compared to treatment with MSC. Small differences were found although MSCs where more effective as treatment.74 The human ATSCs were examined regarding phenotypes and gene expression profile in the undifferentiated states and compared with that of BMSC. Flow cytometric analysis showed that ATSCs have a marker expression that is similar to that of BMSC. ATSCs were superior to BMSC in respect

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to functionality in maintenance of proliferating ability, and microarray analysis of gene expression revealed differentially expressed genes between ATSCs and BMSCs. The proliferating ability and differentiation potential of ATSCs were variable according to the culture condition75 including cartilage differentiation.76 ATSCs are abundantly available as source for differentiation for cartilage repair but the differentiation pathway is similar to MSCs and thus end in mineralized bone. Furthermore, the harvest of cells is connected to an additional operative procedure with risk of patient morbidity.

5.514.9. Universal Donor Cell Lines for Cartilage Repair Although widely used, there are some inherent problems with the use of autologous chondrocytes. They show restricted proliferative capacity in culture and rapidly lose their functional properties in culture. Harvesting these cells is itself an additional injury to the joint surface, and even if the harvest site is located some distance from the lesion, this cartilage might be affected by the injury as well. An alternative method is the use of adult MSC. One drawback with these cells is that they tend to differentiate toward hypertrophic cartilage instead of hyaline cartilage, resulting in a tissue that is not adapted to the pressure and shear force that the joint is subjected to.66,69,70 Furthermore, the number of MSC, their proliferative capacity, as well as their synthetic abilities declines with age.77 The ultimate goal for human cartilage cell-based repair would be an off-the-shelf arthroscopic product based on a universal donor cell line combined with a suitable scaffold. For such cells their ethical aspects have to be acceptable and the immunogenic properties low as well as chondrogenic properties high. For this purpose cells have to be cultured in an industrial scale or within a hospital setting. For this type of cells, ATCS, umbilical cord stromal cells, and cell derived from hES cells fulfill the requirements to various extents.

5.514.9.1. Derivation of Chondrocytes from Human Embryonic Stem Cells Human embryonic stem (hES) cells, derived from the inner cell mass of the human blastocyst, may have significant potential to be used in cartilage tissue engineering. The most prominent advantage of using hES cells in cartilage regeneration is that they are immortal and could potentially provide unlimited numbers of chondrocytes or chondro-progenitors for transplantation. The majority of the published research concerning in vitro differentiation of embryonic stem (ES) cells into chondrocytes has been conducted on murine cells, using medium supplementation with members of the transforming growth factorbeta family (TGF-b) or bone morphogenic protein-2 and -4 (BMP-2, -4),78 or by coculturing with embryonic limb buds.79 The microenvironment of the culture has the capacity to influence cellular differentiation, and the ability to drive differentiation toward specific lineages in various coculture systems of embryonic tissues with stem/progenitor cells has been evaluated.80–82 These efforts have provided important knowledge and contribute to a better understanding of the mechanisms underlying chondrogenic differentiation, even though the use of differentiated cells for cartilage tissue engineering still remains a challenge.

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Conditioned media from cartilage progenitor cells did not induce increased chondrogenesis instead it supported the growth of a hES cell line that could be cultured without feeders (MFG-hESCs).83 When subjected to bone differentiation the cell line has high similarities with MSCs and has a higher expansion capacity and also retains its differentiation capacity at higher passages83 although with limited chondrogenic potential. In order to increase the chondrogenic differentiation of hES cell lines the cells have been cocultured with mitotically inactivated human articular chondrocytes in vitro. These cocultured

200 mm

(a)

progenitors demonstrated the best chondrogenic differentiation capacity so far from progenitors derived from hES cells.84 The system allows intimate contact between different cell types, thus resulting in more efficient transduction of molecular signals that induce chondrogenesis. The surface receptors of cocultured cells come into direct physical contact, and the autocrine and paracrine factors secreted by one cell type readily interact with the other cell type. In contrast to hES cells prior to the coculture and likewise chondrocytes, the cocultured hES cells were able to form and grow as pellets when cultured in 3D pellet mass

200 mm

(b) N

6

Bern score

5 4 3 2 1 0

Co-cultured hES cells

hES cells

(c)

Scaffold completely disintegrated after 2 weeks of culture

100 mm

(f)

100 mm

(e)

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100 mm

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Figure 5 Chondrogenic differentiation. Pellet mass cultures stained with Alcian blue van Gieson of hES cells prior to (a) and after (b) coculture. Chondrogenesis of the pellets evaluated according to the Bern scale (c). Alcian blue van Gieson staining of hES cells prior to (d) and after (e) coculture in scaffold. Colony formation in agarose suspension culture of hES cells prior to (f) and after (g) coculture. hES cell, human embryonic stem cells. Reproduced from Stem Cell. 2009, 27(8), 1812–1821.

Chondrocyte Transplantation and Selection

culture system. Interestingly, a number of studies have reported that coculture with chondrocytes has a positive effect on the osteogenic differentiation of MSC while coculture of chondrocytes and embryonic stem cells direct the cells into the chondrogenic lineage or significantly decreases the osteogenic potential of the cells.81,85 MSC and hES cells thus respond differently to coculture with chondrocytes. The cellular morphology of the cocultured cells in pellet cultures were similar to that of articular chondrocytes cultured in pellet mass cultures (Figure 5). Significantly improved differentiation toward the chondrogenic lineage of the cocultured cells was demonstrated both by their ability to form colonies in agarose-suspension culture with a morphology very similar to that of chondrocytes cultured under the same conditions as well as significantly increased accumulation of a cartilaginous matrix in pellet masses.86 The only cell types with the ability to survive in agarose suspension culture are chondrocytes and tumor cells.87,88 However, the cells had low expression of stem cell markers (CD117, CD133, SSEA-4), indicating a poor capacity of tumor formation of these cells. Vats et al. recently demonstrated that coculture with chondrocytes from patients undergoing otorhinolaryngological– head and neck surgery and hES cells using well inserts for separating the cells displayed an increased accumulation of sulphated GAGs as well as expression of Sox9 and collagen type II.89 In this study, differentiation was assessed after 28 days of coculture. Furthermore, mouse ES cells can be programmed to differentiate into chondrocyte-like cells by coculture with progenitor cells from the limb buds of the developing embryo.79 In both of these studies, chondrocytes were present during the differentiation of the hES cells, either separated from the hES cells using a membrane or present in the micromass culture and eliminated after 4 days of culture. In high-density pellet mass cultures it is difficult to direct the differentiation of the hES cells into only one specific lineage. Human ES cells cultured as pellets did not produce any cartilage-like matrix and the only hES pellet staining positive for Alcian blue van Gieson also resembled embryoid bodies with structures of cells differentiating into other tissues than cartilage. After coculture, a more homogenous, likely lineage restricted, population of cells have been formed with an increased potential for chondrogenic differentiation. In accordance, no teratoma formation could be detected in the cocultured cells after injection under the kidney capsule in SCID mice model confirming phenotypical alteration of the cells and lack of pluripotency. In conclusion, derivation of chondrocytes progenitors from human ES cells could be possible but a lot of work remains. Derivation of xeno-free culture systems has to be combined with better differentiation protocol since the cartilage formed has more similarities with fetal cartilage than with adult, a finding also demonstrated in keratinocytes derived from hES cells.90

5.514.9.2. Derivation of Chondrocytes from Umbilical Cord Cells and Umbilical Cord Blood Cells The umbilical cord consists of umbilical veins and arteries and the supporting mesenchyme called Wharton’s jelly (Figure 6) where MSC-like cells can be isolated. These cells are denoted WJCs and are derived from the surrounding matrix between the umbilical blood vessels.91 The cells meet the criteria for

Placental vein

197

Wharton’s jelly

Allantois

Placental artery

Placental artery

Placental cord Figure 6 Wharton’s jelly.

stem cells as they have inherent self-renewal capacity and can be induced to differentiate into various cell types. The advantage of cells derived from the umbilical cord matrix is (i) the isolation of large numbers of cells without subsequent expansion in culture and (ii) that the cells are derived from fetal structures and could, like umbilical cord blood cells, thus have a better immunotolerance in the recipient patient. The question whether the different compartments of the umbilical cord have different MSCs has been addressed in experiments with cells isolated from umbilical cord blood, umbilical vein subendothelium, and the Wharton’s jelly. Within Wharton’s jelly, MSCs can been isolated from three indistinct regions: the perivascular zone, the intervascular zone, and the subamnion. It is not clear whether cells isolated from these different compartments of the umbilical cord represent distinctly different populations of MSCs. The WJCs express MSC surface markers, thus suggesting that they are of the MSC family; however, in experimental settings, WJCs differ from BMSCs because WJCs are slower in differentiating into adipocytes.92 Since many features are shared with MSCs such as poor ability to differentiate to adipocytes, shorter doubling times than BMSCs, and greater numbers of passages to senescence, it not clear whether the MSCs derived from umbilical cord blood are different from those found in Wharton’s jelly. For an extensive review, see Troyer and Weiss.93 However, the potential use of MSCs derived from umbilical cord or cord blood in the treatment of cartilage defects in humans remains to be established. Although the availability and immunogenicity could be of advantage, no scientific data today exist that support the notion that the cells are of lineage other than the bone default pathway.

5.514.10.

Conclusion

The development of human cell therapy for cartilage repair over the last decades has both changed the stage and the view of cartilage as a nonregenerating tissue. We have only started to understand the cartilage tissue regeneration and repair and the potential use of cells for curing patients suffering from cartilage defects and OA. The autologous chondrocytes harvest is so far the best cell source for cartilage repair although other potential sources for a more universal treatment might be around the corner.

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Tissue Engineering – Musculoskeletal, Cranial and Maxillofacial

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