(BQ) Part 2 book “Articular cartilage” has contents: Tissue engineering of articular cartilage, methods for evaluating articular cartilage quality, perspectives on the translational aspects of articular cartilage biology, ec for generation and evaluation of articular cartilage.
Tissue Engineering of Articular Cartilage • Need for in vitro tissue engineering • Cell source • Biomaterials and scaffold design • Bioactive molecules • Bioreactors and mechanical stimuli • Convergence of stimuli I n this chapter, we discuss the strategies employed by researchers striving to repair or regenerate articular cartilage through biological means While methods to repair cartilage using surgery exist (e.g., debridement, microfracture, and mosaicplasty), tissue engineering holds the promise of complete regeneration Furthermore, engineered constructs can be designed that are mechanically functional from day 1, potentially decreasing recovery time for the patient Focus has been placed on the three main pillars of tissue engineering: cell source, scaffold design, and external stimulation through the use of bioactive molecules and mechanical bioreactors (Figure 4.1) Cell sources that are discussed include primary chondrocytes as well as 257 Articular Cartilage Scaffolds Porous meshes Hydrogels Weaved fibers Composites Cells Scaffolds provide substrate for cell growth and mechanical integrity for postsurgical implantation Autologous chondrocytes Mesenchymal stem cells (marrow derived or adipose derived) Cartilage tissue engineering Scaffolds coated with bioactive molecules act as drug delivery systems for improved repair in vivo Bioactive molecules induce differentiation, proliferation, and metabolic activity of cells Bioactive molecules IGF TGF-β BMP PRP-derived cytokines Figure 4.1 Traditional paradigm of tissue engineering consisting of the three components of scaffolds, cells, and bioactive molecules (From Daher, R J et al., Nat Rev Rheumatol 5(11): 599-607, 2009 With permission.) stem and progenitor cells Natural, synthetic, and hybrid biomaterials have all been used for cartilage engineering, with the latest approaches building upon previous findings to create new and innovative scaffolds suitable for long-term repair Growth factors and other bioactive molecules are critical components to rapid, complete regeneration of tissues in the body, and those with proven roles in cartilage repair are reviewed here Finally, a comprehensive discussion of bioreactors and mechanical stimulation is included Incorporating sufficient mechanical integrity in engineered cartilage (neocartilage) is crucial for its success in the mechanically demanding joint environment Bioreactors that mimic cartilage’s physiological environment are described, along with reported experimental findings using each approach In all subsections, discussions of specific studies that have taken place in the past couple of 258 Tissue Engineering of Articular Cartilage decades are used to illustrate the current state of cartilage engineering, as well as future directions in this highly active field of research For decades, hyaline articular cartilage has been a primary target for tissue engineering efforts due to the lack of functional regeneration intrinsically within the joint In addition to focal defects, pathologies such as osteoarthritis can destroy the entire cartilage surface, resulting in loss of function and persistent pain This chapter highlights both the seminal tissue engineering studies focused on articular cartilage and the latest approaches that incorporate bioreactors, bioactive molecules, and specialized biomaterials Tissue engineering, in its classical sense, involves the manipulation of a complex interplay among biomaterials, growth factors, and cell populations (Mikos et al 2006) to achieve functional improvement or restoration Articular cartilage has been a high priority for tissue engineers since it does not naturally regenerate after injury Furthermore, the annual health care costs associated with musculoskeletal diseases and injuries are extremely large (estimated at $874 billion per year), and an effective reparative solution would not only reduce costs but also improve the quality of life for millions (U.S Bone and Joint Initiative 2014) The average age for patients undergoing arthroscopy who exhibit cartilage defects in the knee is 43, and, combined with the demographical data on adolescent cartilage injuries, as discussed in Chapter 3, the need to create a repair tissue that can last several decades is a major goal (Curl et al 1997) The earliest attempts at cartilage regeneration involved transplanting either minced cartilage tissue or dissociated chondrocytes (Chesterman and Smith 1968) Surgical solutions to cartilage defects typically include surface abrasion, microfracture, and debridement, all of which can reduce symptoms However, the repair tissue formed in response to these procedures is fibrocartilage, which, as discussed elsewhere in this book, has biomechanical properties that are markedly different from those of normal cartilage (Curl et al 1997) Fibrocartilage does not have the 259 Articular Cartilage biochemical composition or structural organization to provide proper mechanical function within the joint environment and will degrade over time because of insufficient load-bearing capacity (Hunziker 2002; Smith et al 2005) Because of this, current research is striving to produce a tissue that is hyaline-like in its biochemical composition and biomechanical properties The first part of this chapter focuses on in vitro tissue engineering approaches Attempts to tissue engineer within the in vivo environment are discussed; germane immunological considerations are presented in Section 6.2 It is natural to think that the in vivo environment probably comprises all conditions necessary to effect successful regeneration That is, the in vivo environment must contain the proper growth factors and mechanical stimuli, delivered in a well-sequenced manner through autocrine and paracrine signaling, to effect proper healing, the major missing component being metabolically active chondrocytes at the defect site Initial efforts at delivering mechanical stimuli in vitro attempted to emulate these signals, the natural thought being that the objective of bioreactors ought to be the creation of signals reminiscent of the native environment, for example, the Hz pace of walking and the low oxygen tension of the joint Unfortunately, the physiological conditions have not been shown to result in cartilage repair in vivo, and the act of mimicking these conditions in vitro should be questioned It may not be that physiological conditions are not required, but just that physiological conditions of a different developmental period may be more beneficial in generating functional cartilage To investigate this latter case, in vitro tissue engineering has been employed to recapitulate developmental conditions 4.1 The Need for In Vitro Tissue Engineering The primary advantage of in vitro tissue engineering is proposed to be immediate functionality The idea is that a tissue replacement that is mechanically and biologically functional before implantation will have a higher probability for success This is especially true 260 Tissue Engineering of Articular Cartilage for mechanically arduous environments, such as articulating joints Without the requisite mechanical characteristics, a tissue-engineered construct would be quickly destroyed by the high stresses that are part of normal loading of an ambulatory patient In contrast, a construct that possesses material properties comparable to those of the native tissue would not fracture or degrade Because of this, many researchers believe articular cartilage engineering should place emphasis on biomimetic construct development in vitro Since the tissue resides in a mechanically demanding environment, the implanted construct needs to be developed to a point that it can withstand or respond to these mechanical loads Constructs possessing insufficient integrity will collapse in the articular defect, which not only prevents regeneration but also could accelerate degradation of the tissues surrounding it Also critical in this effort is the issue of integration of the construct with the surrounding tissues, discussed in more detail in Section 6.1.1 Efforts to heal large defects in vivo could fail without some means of protecting the structure of newly developed tissues By growing neocartilage in a laboratory, the culture environment can be carefully controlled with respect to nutrient supply, biological stimuli, and mechanical loading For a tissue like articular cartilage, possible treatments often depend on the type of damage to the joint For example, an osteochondral defect that reaches down into the subchondral bone introduces blood into the defect This influx of blood and marrow brings a variety of signals and cells to the injury site However, fibrocartilage will form in the defects if left untreated, filling the site with a disordered mass of fibrous tissue that possesses inferior mechanical functionality Another type of damage in cartilage is a chondral defect that does not extend through the depth of the tissue to reach vascularity In this case, some of the chemical and biological factors associated with osteochondral defects are not present Unfortunately, the mechanical functionality of the articular cartilage is still compromised due to disruption of the tissue’s surface Both osteochondral and chondral defects can be considered focal defects if the damage is localized to a single region 261 Articular Cartilage The most difficult type of cartilage injury to treat is a breakdown of the articulating surface caused by diseases, such as osteoarthritis and osteochondritis dissecans Most tissue engineering approaches create small constructs that can be fit into focal defect sites in the cartilage However, this would be insufficient for injuries affecting entire joint compartments or larger, since there would be no functional tissue in which to anchor the new constructs As yet, there are no successful approaches to treating osteoarthritis using tissue engineering Researchers are continually investigating alternative approaches, such as engineering a replacement tissue or scaffold that can completely resurface the joint (Moutos et al 2007) (Figure 4.2) Other possibilities include gene therapy or pharmaceuticals, which might have more success in treating systemic degeneration of articular cartilage Articular cartilage growth and development are affected by both biomechanical and biological stimuli On the mechanical side, loading is a required part of the normal joint environment As seen in previous chapters, while excessive forces can damage cartilage, some stimulation is necessary to promote chondrogenesis (Darling and Athanasiou 2003) Articular cartilage will atrophy in a mechanically static environment (Vanwanseele et al 2002), so researchers are currently evaluating a variety of loading approaches to prevent this while promoting the regeneration process An important factor to consider prior to mechanical loading is the choice of scaffold for the engineered construct The scaffold material not only affects how cells sense mechanical loads but also may provide an environment conducive to cell attachment and matrix synthesis In addition to mechanical stimuli, articular cartilage responds dramatically to growth factors that are naturally present in the joint environment or exogenously added The transforming growth factor β (TGF-β) superfamily includes growth factors that are present in developing bone and articular cartilage, as described in Chapter These molecules play an integral role in the natural development process, and can induce large changes on the growth of musculoskeletal tissues in vitro 262 Tissue Engineering of Articular Cartilage Figure 4.2 Whole joint scaffold using a mold of a sheep femur to shape a fiberbased scaffold This could then be coated in a cartilage-derived matrix and seeded with cells to form a cap of neocartilage that could be sutured in place (From Dr F Moutos, Duke With permission.) This section illustrates the importance of the in vitro culture environment on the growth, development, and functionality of native and engineered articular cartilage Following the paradigm for functional tissue engineering, four main categories are reviewed: cell sources, biomaterials, bioactive molecules, and bioreactors (Guilak 2002) 4.1.1 Characteristics of the In Vivo System Articular cartilage is a specialized tissue, with physiological characteristics that help retain tissue functionality for decades of rigorous use The composition and structure of articular cartilage, as well as its ambient mechanical microenvironment, are discussed in detail in Chapter 1 263 Articular Cartilage While these characteristics support the role of articular cartilage as a load-bearing surface, they become problematic if the tissue is damaged Articular cartilage is avascular, alymphatic, and aneural (Revell and Athanasiou 2009) The lack of a blood supply prevents the influx of cells (e.g., stem cells) that can repopulate a site and begin repair (Pridie 1959; Mankin 1982; Hunziker and Rosenberg 1996) How the absence of lymphatics and nerves may influence healing in articular cartilage is not clear They are all potential areas for future exploration For instance, the perception of pain in articular cartilage is paradoxical given the absence of nerves in this tissue, and it is unclear from where pain can arise It is likely that the perception of pain is due to the associated tissues of the joint and subchondral bone The mechanical microenvironment might be the largest hurdle to successful cartilage repair While it is necessary for normal tissue homeostasis, it also acts as a major driver to progressive deterioration if damage already exists The characteristics of the in vivo system present many difficulties when pursuing articular cartilage repair strategies However, the joint environment may provide some beneficial aspects as well For instance, it has long been thought that articular cartilage is immunoprivileged, meaning transplanted cells and tissues are less likely to induce a sustained immune response from the body This is directly related to the avascular and alymphatic nature of articular cartilage Some amount of immune response is still expected, since almost all procedures will require surgical access to the joint through the capsule or otherwise exposing the synovial space While the acute inflammation response to surgery or implantation can be severe, it can be limited by the use of arthroscopic or minimally invasive procedures The immune characteristics of the joint space have allowed for successful outcomes using allograft tissue, for example, osteochondral grafts and anterior cruciate ligament transplants (Barber et al 2010; Bedi et al 2010; Bhumiratana et al 2014) However, it needs to be demonstrated conclusively how xenogeneic tissues fare in a diarthrodial joint environment A discussion about immunogenicity in response to xenogeneic and allogeneic materials is provided in greater depth in Section 6.2 264 Tissue Engineering of Articular Cartilage In vivo studies of articular cartilage regeneration take one of several forms Most commonly, cells or engineered constructs are implanted directly into a cartilage defect (Brittberg et al 1996) These experiments allow for assessment of tissue growth, integration, and longevity in its targeted biological environment Other approaches have used the in vivo environment in a manner similar to that of a bioreactor, implanting engineered constructs subcutaneously (Emans et al 2010; Responte et al 2012) This approach exposes the construct to a complex array of biological factors that would otherwise be difficult to apply individually Since it is difficult to induce vascularization in vitro, osteochondral constructs (Figure 4.3) that require vascularization of the engineered bone are often cultured in vivo (Emans et al 2005; Jin et al 2011; Qu et al. 2011) Other important differences between the in vivo environment and standard cell culture conditions include lower oxygen concentrations (discussed in more detail in Section 4.5.6), lower temperatures, and limited nutrient and waste diffusion In vitro conditions typically use ambient air oxygen tensions (~20%), standard body temperature (37°C), and an excess of culture medium that is changed regularly Compared with physiological conditions, these in vitro conditions are quite different; physiologically, oxygen tension is estimated to be ~7-10% at the surface and ~1% at the subchondral bone (Silver 1975; Malda et al 2003; Fermor et al 2007), knee joint temperature is ~35°C (Zaffagnini et al 1996), and Figure 4.3 In vivo culture of polymer (polyvinyl alcohol/gelatin-nano-hydroxyapatite/ polyamide [PVA-n-HA/PA6]) osteochondral constructs in an intramuscular pocket for weeks induces vascularization of the construct Left: Osteochondral PVA-n-HA/ PA6 cylinder; left side is n-HA6 and right side is PVA Implanted in the intramuscular pocket (center) and after weeks (right) (From Qu, D et al., J Biomed Mater Res B Appl Biomater 96(1): 9-15, 2011 With permission.) 265 Articular Cartilage diffusion of nutrients from the synovial fluid is restricted (Guilak et al 2000) The influence of these differences continues to be investigated, and it is not yet clear whether creating a true physiological environment is the best means of growing new articular cartilage Major drawbacks to in vivo approaches include a limited set of evaluation techniques, as well as the maintenance of well-controlled experiments The in vivo environment contains a multitude of chemical and mechanical signals that are in continuous flux and can be vastly different from one subject to another (Liu et al 2010; Qu et al 2011) Growth factors and cytokines circulating in the blood and present in the extracellular matrix can stimulate changes in the implanted cells Likewise, mechanical loading (see Section 1.2), such as compression, tension, hydrostatic pressure, and shear, can induce changes in the biological response of implanted cells and the structure of implanted constructs (see Section 4.5) Controlling these variables is often difficult and is challenging for studies The in vitro environment allows for precise control of growth factor levels, mechanical stimulation, and other experimental factors (Bobick et al 2009), but does not replicate the complexity of the physiological system Because of this, research typically progresses from in vitro studies to in vivo studies Ethical and cost considerations also play a role when determining whether a study should be done in vitro or in vivo For example, a dose-response study of a novel chemical would require a large number of samples, which can be accomplished in a more cost-effective manner in vitro than in vivo While there may not be a direct transfer of findings from in vitro to in vivo, the benefits of each system help researchers investigate a wide range of experimental conditions important for understanding cartilage regeneration 4.2 Cell Source Cells are one of the key components of tissue engineering Studies have shown that including cells in an engineered construct accelerates regeneration (Tatebe et al 2005) Furthermore, these implanted cells have been shown to remain in the tissue without being replaced by host cells 266 642 invasive methods, 406–407 noninvasive modalities, 397–406 overview, 396–397, 396f, 397f quantitative assessment techniques, 419–425 cells, 419–421 collagen, 421–422 proteoglycans and glycosaminoglycans, 422–425 Exercise, 208, 467 Exocytosis, definition, 622 Exogenous term, definition, 622 Experimental protocols animal protocols, 615 biomechanical properties, 606–615 compression, 606–611 friction coefficient measurement, 613–615 strain-to-failure tensile test, 612–613, 613f gross morphology documentation, 545–546, 546f India ink, 546–547, 547f histology, 547–555 Alcian blue, 554–555 clearing paraffin slides, 549–550 fixing cryosectioned slides, 548–549 hematoxylin and eosin (H&E), 552–554, 553f overview, 547–548 picrosirius red, 551–552 safranin O with fast green, 550–551, 550f immunohistochemistry, 555–559 antibody detection, 557–559 antigen retrieval, 556–557 overview, 555–556 insoluble matrix component, 581–590 DNA quantification by PicoGreen, 588–590, 589f glycosaminoglycan content assay, 583–585 lyophilization, 581–582 papain digestion, 582–583 total collagen content assay, 585–588, 586f protein analysis, 560–580 antibody-based detection methods, 568–580 overview, 560 protein extraction, 560–564 protein quantification, 564–568 RNA extraction, 590–606 2D cultures, 592–594 3D cultures and tissue, 595–596 gel electrophoresis for DNA and RNA, 597–599, 597f overview, 590–591 polymerase chain reaction (PCR), 599–601, 600f quantitative reverse transcription PCR (qRT-PCR), 601–606 Index RNAse/DNAse-Free DEPC water, 591–592 tissue and cell culture, 522–545 2D cultures, 532–536 3D cultures, 536–545 cell isolation, 528–530 cryopreservation of cells, 530–532 harvesting cartilage and production of explants, 523–527, 525f, 528f overview, 522–523 Extracellular matrix (ECM), 14, 19, 26, 26f, 44, 305f Ex vivo term, definition, 622 F 42 USC § 262, section 351, 497 510(k) clearances, 495, 499f FACE (Fluorophore-assisted carbohydrate electrophoresis), 424 FACS (Fluorescence-activated cell sorting), 411–413, 413f F-actin, 16 Factor 13A (FXIIIA), 112 Fast Green, 417, 550–551, 550f Fast spin echo, 400, 622 Father of chemistry, 520 Fatigue testing, 432–433, 433f FBN1, 171 FDA, see U.S Food and Drug Administration (FDA) FDA-2891 (Registration of Device Establishment), 494 FDA-2892 (Medical Device Listing), 494 FDA Modernization Act of 1997, 492 Federal Food, Drug and Cosmetic Act of 1938, 490, 497 FGF, see Fibroblast growth factors (FGF) Fibril collagens, 34 Fibrillation, 165, 186, 622 Fibrin, as scaffold, 279–280, 280f Fibroblast growth factors (FGF), 86–87 FGF1, 86 FGF2, 86, 87, 97, 118, 130, 201, 270f, 297, 298 FGF4, 96, 97 FGF7, 96 FGF10, 97 Fibrocartilage, 7f, 8, 9–10 Fibromodulin, 48 Fibronectin, 15, 93–94, 305 Fibula/tibia, Fidia, 502 Fin-Ceramica, 504t Finkelstein, Harry, 392 Fissures, 165, 186 Fixed charge density, 44 Flagella, 18 Index Flow cytometry, 411–413, 421, 578f, 578–580 Fluid shear, 331–336, 332f Fluorescence-activated cell sorting (FACS), 411–413, 413f Fluorophore-assisted carbohydrate electrophoresis (FACE), 424 Focal adhesion, 20, 357f, 622 Footwear, 467 Form 356h (Application to Market a New Drug, Biologic, or An Antibiotic Drug For Human Use), 493 Fra2, 103t Fragmentation, 400, 620 Frequency-dependent tests, 426 Friction characteristics, 60–62, 61f coefficient measurement, 613–615 definition, 622 testing, 431–432, 431f Frizzled cell surface receptor, 88, 98, 119 F-spondin, 192 G G-actin, 16 GAPDH (Glyceraldehyde-3-phosphate dehydrogenase), 601 GAG, see Glycoaminoglycans (GAG) GAIT (Glucosamine/Chondroitin Arthritis Intervention Trial), 209 Gait modification, 208 Gal alpha(1,3)gal antigen (alpha-gal), 445, 484, 485f, 488 Galen, GAP (GTPase activating proteins), 133 Gas-controlled bioreactors, 351–354 GDF, see Growth differentiation factor (GDF) GDF1, 122t, 124t GDF2 (BMP9), 122t GDF3 (Vgr2), 122t GDF5 (CDPM1), 86, 108, 111, 122t, 124t, 131, 300 GDF6 (BMP13/CDMP2), 122t, 300 GDF7 (BMP12), 122t, 300 GDF8, 123t, 124t GDF9, 123t, 124t GDF9b (BMP15), 123t, 124t GDF10 (Sumitomo-BIP/BMP3b), 122t GDF11 (BMP11), 123t, 124t GDF15 (MIC1/PLAB/PTGFB/PDF), 123t GEF, see Guanine nucleotide exchange factors (GEF) Geistlich, 504t, 506t Gel electrophoresis, 597–599, 597f GelrinC, 501, 504t Genetic linkage, 170, 622 643 Genipin, 280 Genzyme Corporation, 215, 502, 506t Germany, articular cartilage repair products, 504t, 505t, 506t, 507t Giant Kelp (Macrocystis pyrifera), 276f Gli2, 104t Gli3, 104t Gli family, 104t Globular domains, 42, 44f Glossary of terms, 617, 631 Glucosamine, 209 Glucosamine/Chondroitin Arthritis Intervention Trial (GAIT), 209 Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 601 Glycerol, 531 Glycine, 34 Glycine-proline-hydroxyproline (GXY) peptide, 34 Glycoaminoglycans (GAG), 42, 43t Glycogen synthase kinase (GSK3), 88 Glycosaminoglycans assays, 583–585 Biocolor assay, 584–585, 586f DMMB assay, 583–584 quantitative assessment techniques, 422–425 sulfated, 417–419 Glypican, 47 Goats, 267, 293, 329f, 443, 484, 523 Gomphosis, Gout, 174 G protein-coupled receptors (GPCR), 622 Gradient echo, definition, 622–623 Growth appositional cells reponsible, 32 definition, 617 maturation and, 112–117 aging and disease, 114–115 cellular changes, 115–117, 116f matrix changes, 113–114, 113f postnatal through adolescence, 112–113 plate, see Epiphyseal plate Growth differentiation factor (GDF), 300 GDF1, 122t, 124t GDF2 (BMP9), 122t GDF3 (Vgr2), 122t GDF5 (CDPM1), 86, 108, 111, 122t, 124t, 131, 300 GDF6 (BMP13/CDMP2), 122t, 300 GDF7 (BMP12), 122t, 300 GDF8, 123t, 124t GDF9, 123t, 124t GDF9b (BMP15), 123t, 124t GDF10 (Sumitomo-BIP/BMP3b), 122t GDF11 (BMP11), 123t, 124t 644 GDF15 (MIC1/PLAB/PTGFB/PDF), 123t Growth factor receptors, mechanotransduction, 23 Growth factors, 296–304 Growth plate, see Epiphyseal plate Glycogen synthase kinase (GSK3), 88 GTPase activating proteins (GAP), 133 Guanidine, definition, 623 Guanine nucleotide binding protein (G protein), definition, 622 Guanine nucleotide exchange factors (GEF), 128, 133, 623 H Hands, 166f, 170, 186 Haptotaxis, 623 HCT/P, see Human cells, tissues, and cell- and tissue-based products (HCT/P) HDE (Humanitarian device exemption), 496 Heart, decellularized, 281, 282f Hematoxylin and eosin (H&E), 552–554, 553f Hemopexin domain, 623 Hemopexin-like domain, 49f, 50 Heparin affinity chromatography, 623 Heparin binding domain, 49f, 50 Heparin sulfate, 48 Heterotrimer, definition, 623 HGD, 171 HIF (Hypoxia-inducible factor), 314 Hips, 53, 164f, 170, 184, 186 Histological/Histochemical Grading System, 197 Histology constructs, 345f decellularization studies, 486 definition, 623 elastic cartilage, 7f evaluation of cartilage quality, 407–419 cells, 410–413 collagen, 413–416 overview, 407–410, 409f sulfated glycosaminoglycans, 417–419 experimental protocols, 547–555 Alcian blue, 554–555 clearing paraffin slides, 549–550 fixing cryosectioned slides, 548–549 hematoxylin and eosin (H&E), 552–554, 553f overview, 547–548 picrosirius red, 551–552 safranin O with fast green, 550–551, 550f fibrocartilage, 7f gap junctions, 15f hyaline cartilage, 7f osteoarthritis, 199f Index zonal architectures, 13f Histomorphology, definition, 623 Histopathology, definition, 623 Homeobox protein b8 (Hoxb8), 96, 99f Homeostasis cartilage homeostasis, 137–139, 137f definition, 137 mechanotransduction, 18–25, 23f, 24f Homogentisate 1,2-dioxygenase, 170 Horses, 443, 523 Hox, 101t Hoxa2, 101t Hoxa11, 101t Hoxa13, 101t Hoxb8 (Homeobox protein b8), 96, 99f Hoxd11, 101t, 108 Hoxd12, 101t, 108 Hoxd13, 101t, 108 Hox genes, 98f Human cells, tissues, and cell- and tissue-based products (HCT/P), 497–498 Human osteogenic protein (OP-1), 98, 111, 112, 118, 122t, 124t, 213, 300 Human osteogenic protein (OP-2), 122t Human osteogenic protein (OP-3), 122t Humoral immunity, 478f Hunter, William, 82 Hyaff-11, 222, 502 Hyaline cartilage collagen present, 35t, 38 description, 4, fibrocartilage and, histology, 7f symphysis joints, 10 synchondrosis joints, 10 tissue engineering and, 259 Hyalofast, 504t Hyalograft C, 502, 506t Hyaluronan (Hyaluronic acid) aggrecan and, 44, 46f, aggrecan molecules and, 44 allogeneic mesenchymal stem cells implantation, 483–484 amount in synovial fluid, 28 as biological marker of arthritis, 193t description, 42, 43t Hyaff-11, 222, 502 hydrogel encapsulation, 303f localization, 25 MACI and, 216f maturation and aging, 114 as scaffold, 222, 280, 505t Hybrid bioreactors, 349–351 Hydraulic permeability, 47, 54, 344, 628 Hydrolytic scission, 284, 286, 623 Index Hydrophilicity, 288f, 623 Hydrophobicity, 288f, 623 Hydrostatic pressure, 21–22 bioreactors and, 323–329 definition, 623–624 as mechanical stimuli, 120 normal, 323, 324f Hydroxylation, 34, 121, 624 Hydroxylysyl pyridinoline, 40 Hydroxyproline, 34, 401, 421–422, 585–588, 585f Hynes, Richard, 92 Hyperplasia, 172f, 173, 624 Hypoxia-inducible factor (HIF), 314 Hypoxia-inducible factor 1α (HIF-1α), 121 Hysteresis, 53, 624 HYTOP, 505t I Ibuprofen, 210 ICRS Cartilage Injury Evaluation Package, 197, 198f IDE, see Investigational device exemption (IDE) Idiopathic term, definition, 624 Idiopathic osteoarthritis, 169, 208, 467 IGD (Interglobular domain), 42, 44f IGF-1, see Insulin-like growth factor (IGF-1) Ihh (Indian hedgehog), 22, 87 Imaging techniques, 396–407 invasive methods, 406–407 noninvasive modalities, 397–406 overview, 396f, 396–397, 397f Immune responses, 476–480, 477f, 478f, 479f Immunoblot, see Western blot Immunohistochemistry antibody detection, 557–559 antigen retrieval, 556–557 cells, 410–413 collagen, 413–416 overview, 407–410, 409f overview, 555–556 sulfated glycosaminoglycans, 417–419 Impact injuries, 201–202, 202f India ink, 195, 199, 546 Indian hedgehog (Ihh), 22, 87 Indication, definition, 624 Inhibin A, 122t, 124t Inhibin B, 122t Injuries causes, 200–203, 202f cellular responses to, 203–205 classification, 195–200 cartilage microfractures, 199 chondral defects, 195, 199–200 International Cartilage Repair Society (ICRS), 197, 198f, 199f 645 osteochondral defects, 195, 200 Outerbridge classification, 196–197, 196f costs, 205–206 response to, 404–405, 404f silent, 202, 202f therapy, see Therapies torsional, 203 Injurious compression, 201–202, 202f Injurious impact loading, 201 Innate immunity, 479f Inorganic salts, 28 In situ hybridization techniques, 410 Institutional review board (IRB), 496 Instruct, 503, 506t Insulin-like growth factor (IGF-1), 118, 298, 300, 304, 321, 331, 334f Integration complexities, new with existing cartilage, 469–472, 470f, 472f Integrins α10β1 integrin, 90t α1β1 integrin, 90t, 112, 304, 307 α 2β1 integrin, 90t, 304, 310 α 3β1 integrin, 90t, 304, 310 α 4β1 integrin, 90t, 304 α 5β1 integrin, 24, 90t, 138, 304, 307, 310 α 6β1 integrin, 95 α Vβ integrin, 90t, 304, 307, 310 α Vβ integrin, 90t, 304, 307, 310 definition, 92, 92f , 624 heterodimers, 14 Interface testing, 470, 470f Interglobular domain (IGD), 42, 44f Interleukin (IL-1), 190 Intermediate filaments, 16f, 17 International Cartilage Repair Society (ICRS), 197, 198f, 199f Interstitial fluid, 29, 47, 54, 62, 624 Interterritorial matrix, 26f, 27, 48 Intervertebral disc, fibrocartilage, Introduction to Continuum Biomechanics, 436 In utero term, Definition, 624 Investigational device exemption (IDE), 493f, 496, 501 Investigational new drug (IND), 492, 493f In Vitro Diagnostic Device Directive (IVDD), 492 In vitro system, 265, 266 defined, 624 In vivo system characteristics, 263–266 defined, 624 scaffold transplant, 219–222, 221f testing instruments, 425 IRB (Institutional review board), 496 Ion channels, mechanotransduction, 21–22 Isoforms, 86, 624 646 Isomers, 285, 625 stereo, 630 Isotropic organization, 39, 625 Israel, articular cartilage repair products, 504t ISTO/Zimmer, 503, 507t Italy, articular cartilage repair products, 504t IVDD (In Vitro Diagnostic Device Directive), 492 J Jabir ibn Hayyan, 520 JNK pathway, 127f, 131–132 Joints articulating, 60 ball-and-socket, 8f, 9f cartilage role, 8–11, 8f, 9f, 10f condyloid, 9f diarthrodial, 3, 10, 317 fibrous, 9–10 hinge, 9f large animal, 443, 524 pivot, 9f plane, 9f saddle, 9f schematic types, 9f shoulder, 8f small animal, 442, 525 symphysis, 10 synchondrosis, 10 synovial, 8f, 10–11, 10f, 106 Joy (cloned piglet), 485f Juvenile rheumatoid arthritis, 167 K Kellgren-Lawrence grade, 402, 625 Kelvin-Voigt model, 404 Kensey Nash, 504t Keratan sulfate (KS) aggrecan macromolecule complex, 46f description, 43t domain, 43, 44, 44f synthesis, 12 Kinases, definition, 625 Klf4, 272 Knees adduction moments, 177f bovine, 524–525, 525f compressive loading, during standing or walking, 317f fibrocartilage, joint normal illustration, 180f tearing of, 182f load experienced, 53 Index model skeleton, 8f osteoarthritis, 170, 184 peak forces during normal physiological loading, 200 synovial infiltration, 189f viscosupplementation, 210f zonal structure, 13f Korea, 507t KS, see Keratan sulfate (KS) L Lamina splendens, 31, 31f Laminins, 15, 25, 95 Laminin (LN-1), 95 Lap shear testing, 470f Larmor frequency, 398, 625 Lef1 (Lymphoid enhancer binding factor), 88, 103t Lefty1 (Lefty A/EBAF), 122t Lefty2 (Lefty B), 122t Leidy, Joseph, 82 Lifestyle changes, 208 Limb bud definition, 625 formation in mouse, 83 illustrated timeline, 97f initiation, 83 Limb field, 96, 625 Limb precursor cell origin, 88–105 cell adhesion molecules, 89–95 cadherins, 90t, 91, 91f integrin-extracellular matrix interactions, 93–95 integrins, 90t, 92–93, 92f, 93f other molecules, 95 overview, 89–90, 90t initiation of limb, 96–105 overview, 88–89, 89f LIN28, 272 Link protein, 25, 44, 46f, 188 Lithography, soft, 630 Little Leaguer’s elbow, 223 Live allograft cartilage, 482 Load-controlled testing, 606–609 Loading altered, 176–179, 177f biomechanics, injury, and, 175–184 compressive, see Compression injurious impact, 201 peak forces during normal physiological, knees, 200 tension, see Tension Loeys-Dietz syndrome, 171 Longitudinal magnetization, 399, 625 Long-SRY-box (L-Sox5), 100t, 135 Index Low-shear microgravity, 341–348 L-Sox5 (Long-SRY-box), 100t, 135 Lubricin, 49–51, 49f CACP and, 172f, 173 gene, 49, 172f, 173 in rheumatoid arthritis, 173 tribology and, 394 Lumican, 48 Lymphoid enhancer binding factor (Lef1), 88, 103t Lyophilization, 581–582, 625 Lysyl oxidase, 40, 314, 315f M MACI (Matrix-induced ACI), 216f, 506t Macrocystis pyrifera (Giant Kelp), 276f Macrophages, 472f, 478f, 479, 481, 484 Mad, 123 Magnetic bracelets, 209 Magnetic resonance imaging (MRI), 397–401, 398f arthroscopic evaluations and, 401 sodium, 402 Magnetic stimulation, 354 MaioRegen, 504t Mammalian target of rapamycin (mTOR), 116f MAPK, see Mitogen-activated protein kinase (MAPK) Marfan syndrome, 171 Marrow stimulation techniques, 212–213 Master transcriptional regulators, 272 Mater, Leo, 392 Mathematical models, articular cartilage mechanics, 434–437 Matrigel, 281 Matrilin, 170 Matrilin 1, 52t Matrilin (MATN3), 52t, 170 Matrix acellular dermal (ADM), 485 changes during arthritis, 186–190, 187f, 189f changes during growth and maturation, 113–114, 113f characteristics and organization, 27–51 fluid components, 28–30 overview, 27–28 zonal variations, 30–34 composition, 23 demineralized bone, 281, 496 experimental protocols, 581–590 DNA quantification by PicoGreen, 588–590, 589f glycosaminoglycan content assay, 583–585 lyophilization, 581–582 papain digestion, 582–583 total collagen content assay, 585–588, 586f 647 extracellular (ECM), 14, 19, 26, 26f, 44, 305f -induced ACI (MACI), 216f, 506t interterritorial, 26f, 27 metalloproteinases (MMP), 314–315 pericellular collagens localized, 35t, 38 description, 14–15, 15f, 25–27, 26f proteoglycans localized, 48 territorial, 26, 26f, 27 Matrix-GLA (glycineleucine-alanine) protein, 52t Matrix-induced ACI (MACI), 216f, 506t Matrix metalloproteinases (MMP), 314–315 MMP1, 193t MMP3, 193t MMP13, 138, 192, 193t Maxwell model, 435 MDD (Medical Devices Directive), 492 MDR (Medical Device Reporting), 494 Mechanical loading, 23–24, 120, 130, 262, 298, 314–315 Mechanical stimulation bioreactors and, 316–354 compression, 317–323 electrical and magnetic stimulation, 354 gas-controlled bioreactors, 351–354 hybrid bioreactors, 349–351 hydrostatic pressure, 323–329 overview, 316–317 shear, 329–348 tension, 348–349, 349f as signaling factor, 119–120 tissue hypertrophy and, 112 Mechanobiological force, definition, 625 Mechanoregulation, definition, 626 Mechanosensation, 21, 626 Mechanotransduction cytoskeletal components, 20–21 definition, 18, 356, 626 growth factor receptors, 23 homeostasis and, 18–25 ion channels, 21–22 matrix composition, 23–25 overview, 18–19, 19f phenotypes and, 14 primary cilium, 22–23, 23f, 24f in tissue engineering, 356–357, 357f homeostasis, 18–25, 23f, 24f Meckel’s cartilage, 112, 626 Medical Device Reporting (MDR), 494 Medical devices, 491, 494t, 494–496 Medical Device Listing (FDA-2892), 494 Medical Devices Directive (MDD), 492 Medical Metrics (M2) database, 206 Medipost, 507t Medtronic’s InFuse Bone Graft/LT-Cage, 496 648 Memory T cells, 480 Meniscal, 400 Meniscectomy, definition, 626 Mesenchymal stem cells (MSC), 269–271, 270f allogeneic , 483–484 directed differentiation, 420, 421, 421t markers for identifying, 421t Meshes, nonwoven, 284 Mesoderm, 88 MH2 domain, definition, 626 Mice age-dependent cartilage wear, 440f aggrecan deficiency, 45 collagen, helium ion scanning, 37f as donor, 523 hedgehog proteins, 87 noggin-lacked, 108 poly-L-lactide-ε-caprolactone planted, 286 PRG4 lack, 173 surgically-induced osteoarthritis, 192f type VI collagen and osteoarthritis, 441f Microfilaments, 16–17, 16f Microfracture, 212–213, 216f Micromass, 541, 541f Microparticles, 302–303 Micropatterning, 311–313, 313f, 626 Microspheres, 301, 301f Microtubules, 16f, 17–18 Middle zone (Transitional zone) Benninghoff model, 39, 39f cartilage intermediate-layer protein (CILP), 52t chrondrocyte morphology, 12–14, 13f, 15f description, 30f, 32, 33f MIS (Mullerian inhibitory substance), 123t MIS type II receptor (MIS RII), 124t Mitogen-activated protein kinase (MAPK) mechanical stimuli and, 120 signaling pathway, 126–134 ERK1/2, 127f, 128–130, 129f JNK, 127f, 131–132 overview, 126–128, 127f p38, 127f, 130–131 PI-3K, 132, 133f, 134f Mitosis, definition, 626 MMP1, 193t MMP3, 193t MMP13, 138, 192, 193t Moiety, definition, 626 Molecular Cloning: A Laboratory Manual, 521 Molecular pore space, definition, 626 Monosodium urate (MSU) crystals, 174, 174f, 203 Morphogenesis, definition, 85, 626 Morphogens bone morphogenetic proteins (BMP), 85–86 definition, 85, 626 Index fibroblast growth factors (FGF), 86–87; see also wingless and int-related proteins, 88 Mosaicplasty, 214, 215, 218, 626 Moscona, Aaron, 89 Mouse, see Mice MRI, see Magnetic resonance imaging (MRI) MSU, 203 Msx1, 103t Msx2, 103t MSX family, 103t mTOR (Mammalian target of rapamycin), 116f mTOR complex (mTORC1), 116f Muir, Helen, 392 Mullerian inhibitory substance (MIS), 123t Multipotency, definition, 627 Muscle, 268f, 270f Myotome, definition, 627 N Nanog, 272 Nanomelia, 45 National Institute of Aging, 114 National Institutes of Health (NIH), 209 Natural killer (NK) cells, 479f Natural scaffolds, 275–281 agarose, 275, 276f, 277, 277f alginate, 275, 276f chitin, 278–279 collagens, 277–278 fibrin, 279–280, 280f hyaluronic acid, 280 protein mixtures, 281 reconstituted matrices, 281, 282f silk, 279 N-cadherin, 90t, 91, 95, 293, 294t NDA (New drug application), 492 Neocartilage, 627 Neoplastic tissues, definition, 627 Netherlands, articular cartilage repair products, 506t Neural cell adhesion molecule (N-CAM), 90t, 95 Neural crest, definition, 627 Neutrophils, 479 New drug application (NDA), 492 Newtonian (fluid), definition, 627 NIH (National Institutes of Health), 209 Noggin, 94, 108 Nonfibril collagens, 38 Nonsteroidal anti-inflammatory drugs (NSAID), 208 Nonsurgical Treatments, 208 Novocart 3D, 507t Index NSAID, see Nonsteroidal anti-inflammatory drugs (NSAID) Nusse, Roel, 88 O OARSI (Osteoarthritis Research Society International), 197, 209 OARSI Osteoarthritis Cartilage Histopathology Assessment System (OOCHAS), 197 Obesity, 179 Ochronosis, 170 Oct-3/4, 272 Office of Combination Products, 489, 496 Office of Device Evaluation, 492 OP-1 (Human osteogenic protein 1), 98, 111, 112, 118, 122t, 124t, 213, 300 OP-2 (Human osteogenic protein 2), 122t, 300 OP-3 (Human osteogenic protein 3), 122t, 300 Optical probe techniques, 407, 408 Orbital shakers, 332 Orthopedics, definition, 627 Osseofit, 505t Ossification, intramembranous, 109 Osteoarthritis, 164–166, 164f, 165f advanced glycation endproducts (AGE) and, 40 aggrecan degradation, 46 collagen VI, 441f confirmation of, 186 early development, 187f hand, 170 hip, 170 histology with ICRS grades, 199f idiopathic, 169, 208, 467 injury leading to, 180–183, 182f, 184f knee, 170 most common targets, 186 pain symptom, 185–186 PRG4 expression, 173 primary, 180–181, 252 radiographic incidence, 184 role of meniscus, 179–180, 180f secondary, 181, 208 surgically induced, 192f Osteoarthritis Research Society International (OARSI), 197, 209 Osteochondral allografts, 205 Osteochondral defects, 195, 200, 203–204 Osteochondral plugs, 214–215 Osteochondritis dissecans, 223 Osteochondrosis, 223 Osteogenesis, 109, 188, 299, 621, 627 Osteolysis, definition, 627 Osteophytes, 188–190, 192 Osterix, 104t 649 Outerbridge classification, 196, 196f Oxygen tension, 120–121 P P38 pathway, 127f, 130–131 P130Cas, 22 Palmitoylation, definition, 627 Papain digestion, 582–583 Parathyroid hormone-related protein, 112 Patched (PTC), 22, 87, 87f Patient-derived cell repair, 215–217 PAX, 102t Pax9, 102t PCL (Polycaprolactone), 283f, 286 PCR, see Polymerase chain reaction (PCR) PCNA (Proliferating cell nuclear antigen), 410 PD98059, 129 PDGF, see Platelet-derived growth factor (PDGF) PDP (Product development protocol), 495, 496 PEG, see Poly-ethylene glycol (PEG) Peptide inclusion, 308–313, 308f, 309f, 310f, 313f Perfusion bioreactors, 336–338 Perfusion shear, 336–340, 337f Pericellular matrix collagens localized, 35t, 38 description, 14–15, 15f, 25–27, 26f proteoglycans localized, 48 Perichondrium, 8, 109, 109f, 110, 110f, 627 Periodic acid-Schiff (PAS) reagent, 418 Periosteal flaps, 628 Peripheral blood, as source of stem cells, 268f Perlecan, 47, 48 Permeability, hydraulic, 47, 54, 344, 628 Perspectives, translational aspects business aspects and regulatory affairs, 488–497 overview, 488–489 pathways to market, 491–497 regulatory bodies, 489–491 challenges and opportunities, 466–475 complexities of integrating, 469–472, 470f, 472f manufacturing complex tissue-engineered products, 473–475, 473f, 475f overview, 466–469 immune responses and transplants, 476–488 allogeneic transplants, 480–483, 483f humoral and cellular immunity, 476–480 xenogeneic transplants, 483–488, 485f, 487f overview, 465 U.S statutes and guidelines, 497–508 current cartilage products, 501–503 emerging cartilage products, 503, 504t–505 650 emerging cellular therapies, 503, 505, 506t–507t, 507–508 overview, 497–501, 499f PGA, see Poly-glycolic acid (PGA) Phagophore, 116f, 628 Phase I trials, 492, 493f Phase II trials, 492, 493f Phase II trials, 492, 493f Phase III trials, 492, 493f Phase IV trials, 492, 493f Phase IV trials, 492, 493f Phenotypes, definition, 628 Phorbol esters, definition, 628 Phosphorylation, definition, 628 Phosphorylation activation loop, definition, 628 Photooxidation approach, 486 Physical therapies, 467 Physis, see Epiphyseal plate PI-3K pathway, 132, 133f, 134f, 628 PicoGreen, DNA quantification by, 588–590, 589f Picrosirius red, 413–414, 414f, 551–552 Pierce, 561 Pigs, 443, 445, 523 Pitx, 102t Pitx1, 96, 102t Pitx2, 102t PLA, see Poly-lactic acid (PLA) Placenta, as source of stem cells, 268f Plasmin, 50 Plasminogen activator inhibitor (PAI-1), 50 Platens, 318, 318f Platelet-derived growth factor (PDGF), 118, 270f, 298, 302 PLGA, see Poly-lactic-co-glycolic acid (PLGA) Pluripotency, definition, 628 Pluripotent stem cells, induced, 271–272 PMA (Pre-market approval), 493f, 495, 499f Polycaprolactone (PCL), 283f, 286 Poly-D,L-lactide, 285 Poly-D-lactide, 285 Poly-ethylene glycol (PEG), 287–288 Poly-glycolic acid (PGA), 283f, 284–285, 482 Polyglycolides, 283f Poly-lactic acid (PLA), 285 Poly-lactic-co-glycolic acid (PLGA), 285–286 Poly-L-lactide, 285 Poly-L-lactide-ε-caprolactone, 286, 287f Polymerase chain reaction (PCR), 599–601, 600f; see also Quantitative reverse transcription PCR (qRTPCR) Poly-meso-lactide, 285 Polysaccharide, definition, 628 Polyvinyl alcohol/gelatin-nano-hydroxyapatite/ polyamide (PVA-n-HA/PA6), 265f Polyvinylidene difluoride (PVDF), 573 Index Postnatal, 112 Preclinical damage, 396f, 397 Preclinical data, 395, 446 Preclinical phase, 493 Precocious arthropathy, definition, 628 PRELP (Proline-arginine-rich end leucine-rich repeat protein), 48 Preparative electrophoresis, definition, 628–629 Prestrain, definition, 629 PRG4, see Protein proteoglycan (PRG4) PRG4 gene, 49, 172f, 173 Pridie drilling, 212 Pridie, Kenneth Hampden, 162 Primary cilium, 22–23, 23f, 24f Probe, 407 Product development protocol (PDP), 495, 496 Progenitor cells, 32, 200, 204, 269–271, 270f Progress zone (PZ), 99f Prolargin, 48 Proliferating cell nuclear antigen (PCNA), 410 Proline, 34 Proline-arginine-rich end leucine-rich repeat protein (PRELP), 48 Proper labeling, 497 Proprioception, 178, 629 Prostaglandin E2 (PGE2), 22, 192, 235 Protein coating, 304–307 peptide inclusion, 308–313 as scaffold, 281 Protein analysis, 560–580 antibody-based detection methods, 568–580 overview, 560 protein extraction, 560–564 2D cultures, 561–562 3D cultures and liquid nitrogen pulverization, 562–564, 563f overview, 560–561 protein quantification, 564–568 bicinchoninic acid, 565–566 Coomassie blue assay, 566–568 overview, 564–565 Protein coating, 304, 308 Protein extraction, 560–564 2D cultures, 561–562 3D cultures and liquid nitrogen pulverization, 562–564, 563f overview, 560–561 Protein proteoglycan (PRG4), 49f, 49–51 CACP and, 172f, 173 gene, 49, 172f, 173 in rheumatoid arthritis, 173 tribology and, 394 Protein quantification, 564–568 bicinchoninic acid, 565–566 Index Coomassie blue assay, 566–568 overview, 564–565 Proteoglycans aggregations, 44–45, 45f, 46f in arthritis development, 187–188 cartilaginous, 42 chondroitin sulfate-rich, 26 definition, 42 minor, 47–48 molecules for structural integrity, 51, 52t present during chondrogenesis, 47 quantitative assessment techniques, 422–425 significance, 46–47 small leucine-rich repeat proteoglycans (SLRP), 48 superficial zone protein (SZP), 49f, 49–51 Prx1, 102t Prx2, 103t PRX family, 102t–103t Pubic symphysis, 8, 10 Pulse sequences, definition, 629 Push-out testing, 470f PVA-n-HA/PA6 (Polyvinyl alcohol/gelatin-nanohydroxyapatite/polyamide 6), 265f PVDF, see Polyvinylidene difluoride (PVDF) Pytela, Robert, 92 PZ (Progress zone), 99f Q Quantitative reverse transcription PCR (qRT-PCR), 601–606 cDNA creation, 603–604 overview, 601–602 quantitative real-time PCR, 604–606 significance, 420 R Rabbits aggrecan half-life, 28 aging, 114 allogeneic transplants, 481, 484 articular cartilage, 525 cartilage stiffness, 114 as donor, 523 Marhall Urist discovery, 85 osteoarthritis, 165f, 169f, 446f scaffold made of PCl-HA, 286, 287f synovial infiltration, 189f zonal architecture, 13f Rac1, 111 Radiofrequency pulse, 398f, 399, 629 Radioimmunoprecipitation assay (RIPA), 560 Radius/ulna, 651 Raman microscopy, 415 Rats, 86, 114, 481, 523 Recombinant human bone morphogenetic protein (rhBMP), 496 Reconstituted matrices, as scaffold, 281, 282f Regentis, 501, 504t Registration of Device Establishment (FDA-2891), 494 Repetition time (TR), definition, 629 Responte, Donald J., 119 RGD, see Arginine-glycine-aspartate (RGD) peptide RhBMP (Recombinant human bone morphogenetic protein), 496 Rheumatoid arthritis, 166–167, 166f juvenile, 167 PRG4 expression, 173 ultrasound, 405f, 406 Rhodamine phalloidin, 410 Rho GTPase, signaling pathway, 133–134, 135f Rho GTPases, 133 RIPA (Radioimmunoprecipitation assay), 560 Ribonucleic acid (RNA) 18S ribosomal RNA, 597, 601 gel electrophoresis, 597f, 597–599 extraction, 590–606 2D cultures, 592–594 3D cultures and tissue, 595–596 gel electrophoresis for DNA and RNA, 597f, 597–599 overview, 590–591 polymerase chain reaction (PCR), 599–601, 600f quantitative reverse transcription PCR (qRT-PCR), 601–606 RNAse/DNAse-Free DEPC water, 591–592 Roche, 561 Rodents, see Mice; Rats Rotating bioreactors, 341–348, 341f, 345f, 348f Runx2, 103t, 136 S SA-CAT (Stretch-activated cation channel), 22 Sachs, Julius von, 341 Sacrificial layer mechanism, 62 Safranin O, 417–418, 417f, 423f, 441f, 550–551, 550f SaluCartilage, 501 SaluMedica, 501 Sanchez-Adams, Johannah, 522 Scaffoldless, 292–294, 292f, 294f Scaffolds articular cartilage repair products, 504t, 505t, 506t, 507t cell-seeded, 502 652 definition, 629 composite scaffolds, 289–291 natural scaffolds, 275–281 agarose, 275, 276f, 277, 277f alginate, 275, 276f chitin, 278–279 collagens, 277–278 fibrin, 279–280, 280f hyaluronic acid, 280 protein mixtures, 281 reconstituted matrices, 281, 282f silk, 279 synthetic elastin-like polypeptide (ELP), 289, 289f overview, 281, 283f, 284 polycaprolactone (PCL), 286 poly-ethylene glycol (PEG), 287–288 poly-glycolic acid (PGA), 284–285 poly-lactic acid (PLA), 285 poly-lactic-co-glycolic acid (PLGA), 285–286 poly-L-lactide-ε-caprolactone, 286, 287f Scaffolds Repetition time (TR), 629 Scaling, 475f Schwartz Biomedical, 501 Scleraxis, 103t Sclerotic process, definition, 629 Sclerotome, definition, 629 SDS (Sodium dodecyl sulfate), 282 SDS-PAGE (Sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel, 597 Second harmonic generation (SHG) microscopy, 414, 415f Self-assembling process, definition, 629 Semicrystalline structure, definition, 629 Serological characteristics, definition, 630 Sex-determining region Y (SRY)-box (Sox), 98, 100t Sox2, 272 Sox6, 100t, 135 Sox9, see Sex-determining region Y (SRY)-box-9 (Sox9) Shear, 329–348 characteristics, 59–60, 59f contact shear, 330–331, 331f fluid shear, 331–336, 332f low-shear microgravity, 341–348, 341f overview, 329–330 perfusion shear, 336–340, 337f testing, 430–431 Shearing, definition, 630 Sheep, 220, 267, 443, 486, 523 ShenzhenHornetcorn, 507t SHG microscopy, see Second harmonic generation (SHG) microscopy Short-tau inversion recovery (STIR), 400, 630 Shoulder, 8f, 53, 212, 224 Index Sigma, 561 Signal factors cascading, 121–134 BMP/TGF-β superfamily, 121–123 MAPK and Rho GTPase, 126–134 morphogen signaling, 121 receptors and Smads, 123–126 mechanical stimuli, 119–120 morphogens and growth factors, 117–119, 119f oxygen tension, 120–121 Silk, as scaffold, 279 Single cell biomechanics, 419–420, 420f Sirius red, see Picrosirius red SLRP (Small leucine-rich repeat proteoglycans), 35, 48 Sma, 123 Smad2, 125, 126 Smad3, 125, 126 Smad4, 125, 126 Smad6, 112, 126 Smad7, 126 Smads, 123 Small leucine-rich repeat proteoglycans (SLRP), 35, 48 Smith and Nephew, 505t Smoothened (Smo), 87, 87f Sodium dodecyl sulfate (SDS), 486 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel, 597 Sodium hyaluronate, 210f Sodium MRI, 402 Soft lithography, definition, 630 Somatomedin, definition, 630 Somatomedin B domain, 49f, 50, 94 Sonic hedgehog (Shh), 22, 87, 96, 97, 102, 104, 139 Sox, see Sex-determining region Y (SRY)-box (Sox) Sox2, 272 Sox6, 100t, 135 Sox9, see Sex-determining region Y (SRY)-box-9 (Sox9) SP(XKLF), 104t Spine, 186, 190 Spin-echo, 223f, 400, 622, 630 Spinner flask, 332–335 Spondyloepimetaphyseal dysplasia, 45 SRY, see Sex-determining region Y (SRY) SRY-box-9 (Sox9), 11, 45, 98, 100t, 135, 136f Stains Alcian blue, 417f, 418, 423f, 554–555 hematoxylin and eosin (H&E), 552–554, 553f picrosirius red, 413–414, 414f, 551–552 safranin O with fast green, 417–418, 417f, 423f, 441f, 550f, 550–551 toluidine blue, 417f, 418, 423f Staining, nuclear, 486 Index Standard ELISA, 569 Steinberg, Malcom, 89 Stem cells anatomical locations, 268f embryonic and induced pluripotent, 271–272 mesenchymal, 269–271, 270f products derived from, 498–500, 499f Stereoelectronic effect, definition, 630 Stereoisomers, definition, 630 Sterically hinder, definition, 630 Stickler syndrome, 171–172 Short-tau inversion recovery (STIR), 400, 630 STIR (Short-tau inversion recovery), 400, 630 Strain-to-failure tensile test, 612–613, 613f Stress relaxation definition, 631 compression test, 610–611, 610f Stretch-activated cation channel (SA-CAT), 22 Stribeck curve, 61f Stromelysin, 193t Structural integrity, 51 Stryker EMEA, 502 Subchondral bone Benninghoff model, 39f compression, 55, 56 defect, Outerbridge classification, 196f, 197 fibrillation and fissures, 165, 165f microfractures, 199 osteoarthritis, 188 synovial infiltration, in exposed, 189f Subchondral cysts, 188 Superficial zone (Tangential zone) Benninghoff model, 39, 39f chondrocyte morphology, 12–14, 13f, 15f collagen present, 35t; see also Collagen I compressive strains, 55, 295, 321 description, 30f, 32, 33f prevalent integrins, 304 significance, 203, 295 wear in, 183 Superficial zone protein (SZP), 49–51, 49f CACP and, 172f, 173 gene, 49, 172f, 173 in rheumatoid arthritis, 173 tribology and, 394 Surgical procedures, as cause of injury, 203 Switzerland, 504t, 506t SYBR Green, 602 Synchondroses, definition, 631 Syndecan, 47, 90t, 94, 95 Syndesmosis, 9, 631 Synergism, definition, 631 Synergy, 354–355, 355f Synovial joint, formation, 108–109, 109f Synovial membrane, 10f 653 Synovium, as source of stem cells, 268f Synthetic scaffolds elastin-like polypeptide (ELP), 289, 289f overview, 281, 283f, 284 polycaprolactone (PCL), 286 poly-ethylene glycol (PEG), 287–288 poly-glycolic acid (PGA), 284–285 poly-lactic acid (PLA), 285 poly-lactic-co-glycolic acid (PLGA), 285–286 poly-L-lactide-ε-caprolactone, 286, 287f SZP, see Superficial zone protein (SZP) T 21 CFR 50, 501 21 CFR 56, 501 21 CFR 300-460, 497 21 CFR 312, 501 21 CFR 600-680, 493, 497 21 CFR 800-898, 497 21 CFR 812, 501 21 CFR 820, 494 21 CFR 1271, 497, 498 21 USC §§ 351-360dd, 497 28S/18S ribosomal RNA ratio, 597 2D cultures, 532–536 chondrocytes dedifferentiation, 522–523 monolayer, 533–534 overview, 532–533, 533f passaging cells, 534–536 protein extraction, 561–562 RNA extraction, 592–594 3D cultures, 536–545 cell-only techniques, 541–545 micromass, 541–542 pellet culture, 542–543, 543f self-assembly, 543–545, 544f chondrocytes dedifferentiation, 523 hydrogel encapsulation, 536–541 agarose, 537–539 alginate beads, 539–541, 540f overview, 536 protein extraction, 562–564, 563f RNA extraction, 595–596 T1ρ, 403f definition, 400 mapping, 400–401 T1 time, 399 T1 tissue, 631 T2 time, 399 T2 tissue, 631 Takeichi, Masatoshi, 91 T-Alk (BMP RII/ BRK3), 124t TBF Tissue Engineering, 506t T-box (Tbx), 96 654 Tbx, 101 Tbx4, 96, 101t, 102t Tbx5, 96, 101t Tbx family, 101t–102t T-cell transcription factors (TCF), 88, 103t TDT-mediated dUTP nick end labeling (TUNEL), 410, 411f TE, see Echo time (TE) Telomeres, definition, 631–632 TEM, see Transmission electron microscopy (TEM) Temporomandibular joint disc, Tenascin, 52t, 94 Tension bioreactors and, 348–349, 349f characteristics, 57–59, 58f definition, 631 oxygen, 120–121 strain-to-failure tensile test, 612–613, 613f testing, 428–430, 430f Teratoma, 272, 632 Territorial matrix, 26, 26f, 27 TeTec, 507t TEY (Threonine-Glutamate-Tyrosine) sequence, 128 TGF-β, see Transforming growth factor β (TGF-β) Therapies, 206–222 nonsurgical treatments, 208–210 aspirin, 210 chondroitin, 209 copper bracelets, 209 corticosteroids, 210 cyclooxygenase (COX2) inhibitors, 210 glucosamine, 209–210 ibuprofen, 210 lifestyle changes, 208 magnetic bracelets, 209 nonsteroidal anti-inflammatory drugs (NSAID), 208 viscosupplementation, 210, 210f physical, 467 surgical treatments, 211–222 adjunctive treatments, 217–219, 218f arthroscopic abrasion arthroplasty, 211–212, 216f autologous implants, 213–217, 216f complete joint replacement, 217 debridement, 211–212, 216f marrow stimulation techniques, 212–213 overview, 211 total hip joint replacement, 207, 207f in vivo scaffold transplant, 219–222, 221f Thomas, Hugh Owen, 162 Threonine-Glutamate-Tyrosine (TEY) sequence, 128 Index Threonine-Proline-Tyrosine (TPY) sequence, 131 Thyroxine, 112 Tidemark, 33–34, 35t, 38 in arthritis development, 187 Benninghoff model, 39, 39f Time constant, 400, 632; see also T1ρ; T1 time; T2 time Tissue engineering bioactive molecules, 308–313, 308f, 309f, 310f, 313f catabolic and other structure modifying factors, 313–315 growth factors, 296–304 peptide inclusion, 308–313 protein coating, 304–307 biomaterials and scaffold design, 272–296 composite scaffolds, 289–291 mimicking the zonal structural of articular cartilage, 294–296 natural scaffolds, 275–281 overview, 272–274, 274f scaffoldless, 292–293, 294f synthetic scaffolds, 281, 283f, 284–289 bioreactors and mechanical stimulation, 316–354 compression, 317–323 electrical and magnetic stimulation, 354 gas-controlled bioreactors, 351–354 hybrid bioreactors, 349–351 hydrostatic pressure, 323–329 overview, 316–317 shear, 329–348 tension, 348–349, 349f cell sources, 266–272 embryonic stem cells and induced pluripotent stem cells, 271–272 mesenchymal stem cells and progenitor cells, 269–271, 270f need for alternative, 267–269, 268f overview, 266–267 convergence of stimuli, 354–357 potential mechanisms, 356–357, 357f synergism versus additive effects, 354–355, 355f endochondral ossification and, 111 hyaline cartilage, 259 main pillars, 257–259, 258f manufacturing complex products, 473f, 473–475, 475f motivation, 222–225, 223f need for in vitro, 260–266, 263f, 265f significance, 259–260 in vivo environment and, 260 Tissue hypertrophy and ossification, 109–112, 110f Index Tissue inhibitors of metalloproteinases (TIMP), 193t T lymphocytes, 480 Toluidine blue, 417f, 418, 423f Tooth, Total hip joint replacement, 207, 207f TPY (Threonine-Proline-Tyrosine) sequence, 131 Toynbee, Joseph, 82 TR, see Repetition time (TR) Transcription factors, 100t–104t, 134 c-fos, 22 limb bud development, 96–98, 99f, 105 in signaling pathways and maintenance, 134–136, 136f temporal expression during bone formation, 105f Transfection, 272, 301 Transforming growth factor β (TGF-β) superfamily signaling pathways, 125–126, 125f ligands, known receptors, 123t–124t encapsulation within hydrogel, 303 TGF-β1, 118, 121, 122t, 130, 192, 293, 297, 298, 299, 303, 303f, 321 TGF-β1/Smad3, 111 TGF-β3, 122t, 123, 321 TGFBR2, 171 TGF-β RII, 124t Transmission electron microscopy (TEM), 408, 409f, 411, 416 Transplants allogeneic, 480–483, 483f xenogeneic, 483–488 Transverse magnetization, 399, 632 TRB Chemedica, 505t Tribology, 60 Tributyl phosphate (TnBP), 486 Triton X-100, 282, 486 Trufit, 501, 505t T suppressor cells, 480 Tubercle, definition, 632 Tubulin, 17–18 TUNEL, see TDT-mediated dUTP nick end labeling (TUNEL) U U0126, 129 Ultrafiltrate, 10, 28, 632 Ultrasound, 394, 405, 405f, 406, 407 Umbilical cord blood, 268f, 505 United States articular cartilage repair products, 504t, 505t, 506t 655 cell-based products, 502 costs of arthritis, 194 epidemiology arthritis, 184 osteoarthritis, 164 rheumatoid arthritis, 167 food and medicine regulatory body, see U.S Food and Drug Administration most commonly orthopedic procedure, 406 statutes and guidelines, 497–508 Urist, Marshall, 85, 281 U.S Food and Drug Administration (FDA), 491, 492, 395 V Vascular endothelial growth factor (VEGF), 110, 121 Vascularity, 29 Vascularization, 107f, 110, 186, 265, 265f, 632 VEGF, see Vascular endothelial growth factor (VEGF) Verhoeff-Van Gieson methods, 418 Vgr1/DVR6 (BMP6), 111, 122t, 124t Viscoelasticity definition, 632 measurements, 426 simple models, 435 Viscosupplementation, 210, 210f Vitamin C (Ascorbic acid), 34 Vitronectin, 15, 50, 94–95, 307 Voltage-sensitive calcium channel (VSCC), L-type, 22 W Watanabe, Masaki, 392 Water, 28 Wear characteristics, 60–62, 61f definition, 632 Weight loss, 208 Western blot, 419, 573, 573–578, 573f Wharton’s jelly, as source of stem cells, 268f Wiley and Springer, 521 Wnt2b, 97 Wnt5, 108 Wnt7a, 118 Wnt8c, 97 Wnt9a, 108, 118 Wnt14, 108 Wnts, 118–119, 119f Wolff, Julius, 18 Wolff’s law, 18 656 X Xenogeneic transplants, 483–488 definition, 632 X-weighted term, 399 Y Young’s modulus; see also Mathematical models compressive, 54, 200, 290, 428 deep zone (radial zone), 13 growth factors and mechnical stimuli, 298 middle zone (transitional zone), 13 shear, 59, 60 superficial zone (tangential zone), 13 tensile, 57–58, 429–430, 612 Z Zones articulating surface, 31–32, 31f deep zone (radial zone) Benninghoff model, 39, 39f chondrocyte morphology, 12–14, 13f, 15f Index compressive strains, 55, 295, 321 description, 30f, 32–33 integrins prevalent, 304 proteoglycan and collagen contents, 33f middle zone (transitional zone) Benninghoff model, 39, 39f cartilage intermediate-layer protein (CILP), 52t chrondrocyte morphology, 12–14, 13f, 15f description, 30f, 32, 33f mimicking, 294–296 overview, 30–31, 30f superficial zone (tangential zone) Benninghoff model, 39, 39f chondrocyte morphology, 12–14, 13f, 15f collagen present, 35t; see also Collagen I compressive strains, 55, 295, 321 description, 30f, 32, 33f prevalent integrins, 304 significance, 203, 295 wear in, 183 tidemark and calcified zone, 33–34 Zone of polarizing activity (ZPA), 96, 99f ... (Lee et al 20 10) 28 6 Tissue Engineering of Articular Cartilage (a) (b) (e) Top Side 10 11 12. 43 12. 50 (c) 6.00 (square) (d) 43 12 5.09 1. 62 Front 10 .23 3.85 11.79 6.00 400 µm 7.87 2. 5 2. 00 4.55... heart (scale bars 50 μm) (From Ott, H C et al., Nat Med 14 (2) : 21 3 -22 1, 20 08 With permission.) 28 2 Tissue Engineering of Articular Cartilage H H O CH3 OH O O H n OH O O Polylactide H O OH n n... al 20 02; Sekiya et al 20 02; Deng et al 20 07; Kurth et al 20 07; Sanchez-Adams and Athanasiou 20 12) For the past two decades, research has focused on inducing stem and progenitor cells to form cartilagenous