Stem Cell Biology and Tissue Engineering in Dental Sciences

By Paul Sharpe and Murugan Ramalingam

Stem Cell Biology and Tissue Engineering in Dental Sciences bridges the gap left by many tissue engineering and stem cell biology titles to highlight the significance of translational research in this field in the medical sciences. It compiles basic developmental biology with keen focus on cell and matrix biology, stem cells with relevance to tissue engineering biomaterials including nanotechnology and current applications in various disciplines of dental sciences; viz., periodontology, endodontics, oral & craniofacial surgery, dental implantology, orthodontics & dentofacial orthopedics, organ engineering and transplant medicine. In addition, it covers research ethics, laws and industrial pitfalls that are of particular importance for the future production of tissue constructs.

Tissue Engineering is an interdisciplinary field of biomedical research, which combines life, engineering and materials sciences, to progress the maintenance, repair and replacement of diseased and damaged tissues. This ever-emerging area of research applies an understanding of normal tissue physiology to develop novel biomaterial, acellular and cell-based technologies for clinical and non-clinical applications. As evident in numerous medical disciplines, tissue engineering strategies are now being increasingly developed and evaluated as potential routine therapies for oral and craniofacial tissue repair and regeneration.

• Diligently covers all the aspects related to stem cell biology and tissue engineering in dental sciences: basic science, research, clinical application and commercialization
• Provides detailed descriptions of new, modern technologies, fabrication techniques employed in the fields of stem cells, biomaterials and tissue engineering research including details of latest advances in nanotechnology
• Includes a description of stem cell biology with details focused on oral and craniofacial stem cells and their potential research application throughout medicine
• Print book is available and black and white, and the ebook is in full color

• Language: English
• Publisher: Academic Press
• Release date: Nov 5, 2014
• ISBN: 9780123977786

Book preview

2009.

Part I

Developmental Biology: A Blueprint for Tissue Engineering

Chapter 2

Developmentally Inspired Regenerative Organ Engineering

Tooth as a Model

Basma Hashmi¹,²; Tadanori Mammoto³; Donald E. Ingber¹,²,³    ¹ Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA

² Harvard School of Engineering and Applied Sciences, Cambridge, MA, USA

³ Vascular Biology Program, Boston Children’s Hospital and Harvard Medical School, Boston, MA, USA

Abstract

Due to rising demands and increasing shortages in organ transplantation, tissue engineers continue to actively investigate methods that could potentially induce organ regeneration in the future. Most engineering approaches attempt to recreate lost organs by using scaffolds that mimic the structure of the adult organ. However, tooth organ formation in the embryo results from complex interactions between adjacent epithelial and mesenchymal cells that produce whole teeth through sequential induction steps and progressive remodeling of increasing complex three-dimensional tissue structures. Using the tooth as a model and blueprint for regenerative organ engineering, this chapter reviews the key role that epithelial-mesenchymal interactions, associated mesenchymal condensation, and mechanical forces play in odontogenesis in the embryo. We also discuss dental engineering strategies currently under development that are inspired by this induction mechanism, which employ extracellular matrix proteins and mechanically active polymer scaffolds to induce tooth formation in vitro and in vivo.

Keywords

Tooth

Odontogenesis

Regeneration

Tissue engineering

Organ engineering

Polymer scaffold

Mesenchymal condensation

Epithelial-mesenchymal interactions

Chapter Contents

2.1 Introduction   17

2.2 Understanding Generation for Regeneration Strategies: A Tooth Model   17

2.3 Epithelial-Mesenchymal Interactions During Odontogenesis   18

2.4 ECM and Mechanical Forces as Regulators of Organogenesis   20

2.5 Engineering Approaches for Tooth Organ Regeneration   20

2.6 Conclusion   22

References   23

2.1 Introduction

Organ transplantation continues to pose a major problem worldwide [1]. More than 100,000 patients require organ transplantation every year in the United States alone; however, due to a supply-demand imbalance, close to 20 humans die every day while waiting for organ transplants. For this reason, the ultimate goal in the fields of Tissue Engineering and Regenerative Medicine is to regenerate whole organs in order to restore lost physiological and structural functions. Existing regenerative engineering approaches commonly rely on the use of tissue-specific cells from adult tissues or multipotent stem cells, either alone or in combination with three-dimensional (3D) adhesive scaffolds that mimic the microstructure of the organ that is to be replaced or repaired [2,3]. Significant progress has been made in producing biomaterials to repair simple tissues (e.g., skin, cartilage, or bone) [4,5]; however, these approaches still remain limited in achieving complete organ regeneration. One of the major challenges in this field is that existing tissue engineering approaches are focused on rebuilding adult tissues rather than recapitulating the way in which organs initially form (i.e., in the embryo). Therefore, it is important to identify the key factors and control processes that govern the embryonic organ, and to leverage them to develop more effective design criteria for organ engineering strategies.

2.2 Understanding Generation for Regeneration Strategies: A Tooth Model

One of the simplest model systems for studying mammalian organ formation is the tooth, which is an organ responsible for mastication. The tooth, like other organs, is comprised of epithelium, connective tissue, nerves, blood vessels, and ligaments, as well as specialized extracellular matrix (ECM), in this case, the hard, bone-like covering tissues of the tooth dentin and enamel. However, the simplicity of this organ makes it an excellent model for studying and understanding organ regeneration. As in the development of many other epithelial organs, the tooth forces through reciprocal interactions between the epithelium and mesenchyme that lead to condensation of the mesenchyme and subsequent budding and differentiation of the overlying epithelium to form the complex structure of the organ [6–9]. However, the simplicity of the budding process makes tooth especially amenable to experimental analysis. If we could uncover the principles necessary to regenerate a fully functionally tooth, it would likely also have important implications for engineering other epithelial organs that utilize similar developmental processes, such as bone, cartilage, kidney, pancreas, and heart.

Apart from understanding and attempting to engineer organ regeneration in the laboratory, tooth regeneration is of critical importance for dental medicine. Teeth ailments can range from simple dental caries to more serious genetic defects, such as agenesis (the failure to form teeth), the effects of which can be physically and mentally debilitating [10]. In fact, missing teeth are one of the most common developmental problems in children who are not eligible for dental implants. This is because their jawbones are immature and actively growing; as a result, 20% of children aged 9 to 11 have one or more missing permanent teeth in the United States [11]. Thus, development of a tissue engineering approach that could effectively regenerate teeth could have a significant positive impact on clinical dentistry.

Many of the genes and chemical cues that mediate tissue and organ development have been identified; however, these signals alone are not sufficient to explain how tissues and organs are constructed so that they exhibit their unique material properties and three-dimensional forms. It is becoming clear that organ development is a mechanochemical process in which masses of cells are shaped into functional organs through reciprocal physical and chemical interactions between epithelial and mesenchymal tissues, and this is particularly evident in the key role that mesenchymal cell compaction plays during tooth organ induction [6,7,12]. Recently a new class of multifunctional biomaterials was developed with unique mechanical actuation capabilities that can recapitulate key developmental biological events that occur during the mesenchymal condensation response, which are required for induction of tooth tissue and organ formation in the embryo [8]. This biologically inspired engineering approach recapitulates key developmental processes synthetically. Thus, in this chapter, we will discuss the how an increased understanding of developmental biology is being leveraged to develop entirely new biomaterials and engineering approaches for regenerative dental medicine.

2.3 Epithelial-Mesenchymal Interactions During Odontogenesis

Embryonic organ formation is a mechanochemical process in which masses of cells are shaped into functional organs through reciprocal interactions controlled by mechanical as well as chemical cues [6,7]. This is evident in the inductive tissue interactions between apposed epithelial and mesenchymal tissue layers that are responsible for directing formation of many organs during vertebrate development [9]. For example, during the formation of many epithelial organs (e.g., tooth, lung, pancreas, kidney, heart valve, breast, salivary gland, bone, cartilage, and hair follicle), instructive signals provided by the epithelium are transferred to the mesenchyme through key morphogenetic movements that result in a dense packing of mesenchymal cells, or what is known as mesenchymal condensation. Classic and recent embryological studies have shown that this process is crucial for the formation of many organs [9,13–16].

Odontogenesis, or tooth organ formation, provides one of the simplest examples of how this fundamental development control mechanism works. Physical compaction of the early dental mesenchymal cells shifts the inductive capacity for odontogenesis from the dental epithelium to the mesenchyme in vitro [12]. Specifically, during embryonic days 10 to 12 (E10-12) in the mouse, the initial potential for tooth formation resides within the dental epithelium, as heterotopic recombination of this epithelium with undifferentiated embryonic mesenchyme or with adult bone marrow-derived mesenchymal stem cells (aMSCs) results in formation of a differentiated tooth containing roots, dentin, and enamel when the tissue recombinant is implanted into living mice [17,18]. However, by E13 the dental epithelium’s inductive power is transferred to the previously undifferentiated dental mesenchyme, which is then capable of stimulating adjacent undifferentiated epithelium to form a tooth [19]. Moreover, this inductive mesenchyme also appears to contain all of the information necessary to induce formation of a whole tooth even when combined with epithelium isolated from non-dental buccal regions from adult mouse implanted in vivo [12].

As epithelial-mesenchymal interactions are crucial for tooth organ formation, it is critical to understand how this process works in the embryo in order to apply these principles to organ engineering. Studies carried out 50 years ago analyzing tooth development first identified the odontogenic developmental capabilities of the epithelium and mesenchyme, and these investigators even attempted to regenerate a tooth in vitro [20–25]. For example, when the oral epithelium and mesenchyme from day 20 gestation white rabbits were reconstituted in the highly vascularized chick chorio-allantoic membrane, formation of dentin-enamel junctions resulted [25]. However, it wasn’t until nearly two decades later that the inductive capability of the oral embryonic epithelium was first identified by demonstrating the ability of mandibular epithelium isolated from mouse embryos to stimulate an odontogenic response in non-dental mesenchyme [17].

Interestingly, the dental epithelium starts losing this inductive ability after E12, and there is a concomitant increase in the ability of the dental mesenchyme to induce whole tooth formation when recombined with non-dental epithelium and implanted in vivo [19]. These findings suggest that the early (< E13) DE programs undifferentiated non-dental mesenchymal cells to pursue the odontogenic lineage, and that the inductive ability, which resides primarily in early epithelium, is subsequently transferred to the underlying mesenchyme at ~ E13, which then drives subsequent stages of tooth development. For example, the embryonic epithelium has been shown to reprogram non-dental mesenchymal stem cells to express odontogenic genes [18]. Moreover, once the mesenchymal cells are programmed to become inductive (i.e., mimic the shift of inductive capacity normally observed on E13 in the embryo), they are able to induce full tooth differentiation when combined with embryonic dental epithelium an implanted under the kidney capsule in an adult mouse. The key genes that are expressed in the inductive mesenchyme at E13 have been identified as Pax9, Msx1, and Bmp4, among others [9,12,19,26–28]. This development process inspired additional studies in which the oral epithelium and mesenchyme were physically separated, then recombined in vitro, implanted under the kidney capsule to form a tooth rudiment, and finally this was transplanted to the oral cavity resulting in the formation of a functional tooth in a mouse (Figure 2.1) [29].

Figure 2.1 Mechanical control of odontogenic transcription factors during tooth development. (a) Phase contrast micrographs (left) showing mesenchymal cells cultured for 16 hours on microfabricated circular fibronectin (FN) islands (500 μm diameter) in vitro at low, medium, or high plating cell density (0.2, 1.2 or 2.4 × 10 ⁵ cells/cm ² , respectively), and graphs at right showing corresponding cell densities and projected cell areas (bars, 50 μm). (b) Graph showing Pax9 induction in mesenchymal cells cultured for 16 hours on the circular FN islands (500 μm diameter) at low or high plating density with or without Fgf8 (150 ng/ml) and/or Sema3f (150 ng/ml). (c) Graph showing induction of additional odontogenic factors (Egr1, Lhx6, Lhx8, Msx1, and BMP4) in mesenchymal cells cultured under the same conditions as in (b). (d) Freshly isolated E10 mesenchyme from first pharyngeal arch was physically compressed (1 kPa) for 16 hours using a mechanical compressor composed of two pieces of PDMS polymer that are overlaid with a metal weight. (e) Macroscopic images of mesenchyme that was cultured ex vivo for 16 hours in the absence (E10 Mes) or presence of compression (E10 Mes + C) (bar, 500 μm). (f) Graph showing expression of Pax9, Msx1 and BMP4 mRNAs in control (E10 Mes) versus compressed mesenchyme (E10 Mes + C) and expression of Pax9 in control mesenchyme versus mesenchyme treated with soluble Fgf8 (150 ng/ml) for 16 hours. (In all figures, *, p < 0.01.) Figure reproduced with permission from ref. 12.

Finally, most recently, these findings were confirmed and, in addition, the early epithelium was shown to be able to induce adult bone marrow-derived stem cells (BMSCs) to undergo odontogenesis [12]. Importantly, this study also revealed that mechanical forces play a central role in this transfer of inductive capacity that accompanies mesenchymal condensation. Specifically, analysis of embryonic tooth formation in the mouse revealed that the early dental epithelium induces mesenchymal condensation by secreting two soluble morphogens, the motility-promoting factor Fgf8 and motility inhibitor Sema3f. A gradual gradient of Fgf8 attracts distant mesenchymal cells to move towards the epithelium, while a steep gradient of Sema3f prevents their subsequent movement and results in formation of a tightly packed mesenchymal cell mass. These studies also revealed that this physical cell compaction induces expression of Pax9 that drives odontogenesis in a RhoA-dependent manner. Most importantly, mechanical compression of mesenchymal cells alone was shown to be sufficient to trigger odontogenesis in vitro and induce tooth organ formation in vivo (Figure 2.2) [12]. Thus, it might be possible to develop tissue engineering approaches that harness this mechanical actuation to induce tooth organ formation, as will be described below.

Figure 2.2 Engraftment and occlusion of a bioengineered tooth unit in a tooth loss model. (a) Schematic representation of the protocol used to transplant a bioengineered tooth unit in a murine tooth loss model. (b) Photograph (upper) and sectional image (lower) of a calcein-labeled bioengineered tooth unit at 60 days post-transplantation in the subrenal capsule (bar, 200 mm). (c) Micro-computerized tomography (micro-CT) images of a bioengineered tooth unit (arrowhead) in cross-section (upper) and frontal section (first and second figures from the lower left) during the processes of bone remodeling and connection between the recipient jaw bone and alveolar bone of the tooth unit. Histological analysis of the engrafted bioengineered tooth unit at 40 days post-transplantation was also performed (bar, 500 mm and 100 mm in the third and fourth figure from the lower left (NT: natural tooth; BT: bioengineered tooth; AB: alveolar bone; PDL: periodontal ligament). (d) Sectional images of a calcein-labeled bioengineered tooth unit at 14, 30, and 40 days post-transplantation. The calcein-labeled bone of the bioengineered tooth units (arrowhead) was found to gradually decrease from the outside and finally disappear at 40 days post-transplantation (bar, 500 mm (upper), 50 mm (lower) (NT: natural tooth; BT: bioengineered tooth). (e) Oral photographs (upper) and micro-CT (lower) images showing occlusion of natural (left) and bioengineered teeth (right) (bar, 500 mm). (f) Knoop microhardn ess values of the enamel (upper) and dentin (lower) of a bioengineered tooth measured at 60 days post-transplantation in a subrenal capsule (SRC), and at 40 days post-transplantation in jawbone (TP) were compared with those of natural teeth in 11-week-old mice to assess the hardness of the bioengineered tooth. (Error bars indicate standard deviation; * P , 0.01.) Reproduced with permission from ref. [29].

2.4 ECM and Mechanical Forces as Regulators of Organogenesis

ECM scaffolds that support cell adhesion at the interface between interacting epithelium and mesenchyme undergo dynamic remodeling during organogenesis [30–34]. In addition to serving as an attachment scaffold, the ECM also modulates physical force distributions in cells and tissues based on its ability to resist and balance cell traction forces, and thereby control cell and cytoskeletal shape [35–37]. Importantly, changes in cell shape, in turn, modulate the sensitivity of cells to the chemical factors, and thereby govern cell fate switching [38,39]. In this manner, physical forces transmitted over ECM and to cells control development processes including growth, migration, differentiation, contractility, apoptosis, lineage specification, and cellular self-assembly [7,38,40–50]. ECM mechanics and the physical microenvironment are also critical determinants of stem cell fate. For example, ECM mechanics have been shown to direct mesenchymal stem cells (MSCs) along different stem lineages (e.g., bone, muscle, fat, nerve) based on the stiffness of the ECM substrate [44]. These effects are mediated by changes in the activity of the small GTPase RhoA, inside the cell, and in MSCs for example, inhibition and activation of RhoA stimulate adipogenic and osteogenic differentiation, respectively [49].

ECM also orients many growth factors, and some morphogens that play crucial roles in shifting the inductive capability from the dental epithelium to the mesenchyme in the embryo [51], such as Wnts and BMPs, also have been shown to associate with the ECM [52,53]. For example, using gene microarrays and proteomics combined with an informatics approach, multiple morphogens have been identified that exhibit increased expression during the critical phase of development when the inductive ability shifts from the dental epithelium to its mesenchyme. Wnts (Wnt4, Wnt6, Wnt7b, Wnt10a), Fgfs (Fgf4, Fgf8), and BMP4 all appear to play key roles in shifting the inductive ability from the epithelium to the mesenchyme in the tooth germ at E13-14, in addition to the transcription factors Pax9 and Msx1 that were previously known to mediate this process [12,51,54]. Furthermore, these studies revealed that mRNA levels for a number of ECM components (e.g., collagen I, III, IV, VI, emilin, fibulin, laminin, tenascin C, and versican) also increase in condensed dental mesenchyme at E13-14 compared to undifferentiated mesenchyme at E10 [12].

Further analysis of the condensing mesenchyme in the forming tooth revealed that it accumulates an ECM rich in type VI collagen during this induction process, and importantly, inhibition of collagen accumulation inhibits tooth differentiation in this model [12]. Thus, ECM appears to play a key role in tooth development during this early induction phase by sustaining differentiation processes that are triggered physically by mesenchymal cell compaction. Thus, as the physical and chemical properties of ECM are extremely important for tooth development, this knowledge must also be leveraged for organ engineering. Interestingly, whole adult organs (e.g., lung, heart, liver) have been successfully engrafted in vivo by first detergent-extracting the organs to isolate their natural ECM scaffolds and then repopulating them with progenitor cells [55–61]; however, this extraction method has not yet been carried out with tooth.

2.5 Engineering Approaches for Tooth Organ Regeneration

To recapitulate the endogenous regenerative capabilities of dental tissue, past tissue engineering approaches have largely focused on the importance of chemical factors and biomaterials for inducing tooth regeneration [62–64]. Synthetic scaffolds that have been explored for this purpose generally employ natural ECM components or synthetic polymers that exhibit high biocompatibility, low immunogenicity, high adhesive capacity, and controllable degradability in vivo. These scaffolds have been fabricated from collagen, hyaluronic acid, chitosan, alginate, fibrin, and silk among others [63,65–71]; however, they all are limited in their functionality and mechanical strength. Other synthetic biocompatible polymers that are stronger more robust, and are currently used for dental applications, such as polylactide-co-glycolide (PLGA), hydroxyapatite, polyglycolic acid (PGA), also have been explored for this purpose [62,72–74]. However, most of these synthetic polymers also have many limitations, including their inability to support cell adhesion without chemical modification, undesired induction of immunogenic responses, and release of toxic byproducts during degradation.

One potential way to circumvent these limitations would be to develop scaffolds inspired by development that recapitulate key features of the mesenchymal condensation process that drives tooth formation in the embryo [8]. Past studies that attempted to induce mesenchymal cell compaction by exposing the cells to the morphogens that drive these processes in the embryo did not prove successful [12]. Recently, a synthetic mechanically actuatable scaffold was developed that successfully stimulated compaction of embryonic dental mesenchymal cells, induced expression of key odontogenic genes, and stimulated tooth tissue formation in vitro and in vivo (Figure 2.3) [8]. In this study, undifferentiated embryonic mesenchymal cells from the mouse mandible were injected at room temperature within the pores of a thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm) hydrogel composed of 10% N-isopropylacrylamide (NIPAAm) monomer and 1% N,N′-methylenebisacrylamide (BIS), which was chemically modified with RGD-containing adhesive peptide. This gel autonomously contracts in three dimensions when warmed to body temperature (37 °C), which causes the mesenchymal cells to round and take on the morphology of a condensed mesenchyme in the embryo. This mechanical compaction response was shown to be sufficient to induce expression of the key odontogenic transcription factor Pax9 in vitro and to stimulate formation of mineralized tooth tissue in vivo [8].

Figure 2.3 Induction of mesenchymal condensation and tooth differentiation using a developmentally inspired, shrink-wrap, polymer gel. (a) Fluorescent micrographs of E10 dental mesenchymal cells grown overnight in swollen GRGDS-PNIPAAm hydrogel at 34 °C versus in contracted GRGDS-PNIPAAm hydrogel at 37 °C (bar, 50 μm). (b) Light micrographs showing hematoxylin and eosin (H&E) staining of the mesenchymal cells that appear spread in the swollen GRGDS-PNIPAAm hydrogel, whereas they are compact and rounded in the contracted GRGDS-PNIPAAm hydrogel at 37 °C (bar, 50 μm). (c) Graph showing the quantification of the corresponding projected cell areas; *** p  < 0.001. Reproduced with permission from ref. 8.

Exploration of the full potential of this new developmentally inspired approach to tooth engineering will require that similar studies be carried out in combination with epithelium, and both tissues (epithelium and induced mesenchyme) have been shown to be required for tooth formation in tissue recombination experiments [12,18,29]. Nevertheless, these findings provide the proof-of-principle for developing organ engineering approaches that recapitulate normal developmental process that are initially used to drive organ formation in the embryo. They also demonstrate the importance of focusing on mechanical design criteria, as well as chemical features, when devising new tissue engineering biomaterials.

2.6 Conclusion

While significant advances have been in the engineering of some tissues (e.g., skin, cartilage), there is still a major void between current capabilities and the field’s ultimate goal of regenerating whole human organs de novo. This is likely due in large part to our inability to effectively recapitulate the key features of the local tissue microenvironment that are crucial for organ formation. Most of the past focus in this field has been on creation of synthetic polymer scaffolds that mimic the architecture of adult tissue, addition of important soluble morphogens, and delivery of regenerative stem cells. While there have been some small successes in terms of stimulating tissue repair, there is still a great need for alternative approaches that are more effective at inducing true organ regeneration.

Developmentally inspired approaches to tooth organ engineering represent a potentially exciting new path to pursue in this area. The discovery that the physical compaction of undifferentiated mesenchymal cells is the key signal that mediates the shift of odontogenic inductive capability from the dental epithelium to the mesenchyme at E12-13 provides a fundamental new insight into how organs develop [12]. The level of tooth differentiation produced by these shrink wrap-like polymers also might be enhanced in the future by incorporating ECM components and growth factors that are important for sustaining tooth development in the embryo, and by combining epithelium with the condensed mesenchyme. If this can be demonstrated with adult MSCs or dental pulp stem cells, then this could represent a viable new approach to tissue engineering in vivo.

Importantly, the development of mechanically actuable polymers that induce tooth tissue formation by mimicking the mesenchymal condensation response represents a major departure from most biomaterial engineering strategies used for regenerative medicine, which primarily focus on design and optimization of scaffold chemistry and structure [75,76]. In the future, it also might be possible to manipulate the size and shape of teeth that are formed by altering the shape of the scaffolds given that the size and shape of the condensed cell mass have been shown to dictate the final three-dimensional form of the organ [15,77]. Moreover, as mesenchymal condensation is required for induction of many types of epithelial organs, this bioinspired mechanical actuation system could be broadly useful for control of stem cell function, tissue engineering and regenerative medicine.

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Chapter 3

Extracellular Matrix Molecules

Jasvir Kaur¹; Dieter P. Reinhardt¹,²    ¹ Department of Anatomy and Cell Biology, Faculty of Medicine, McGill University, Montreal, QC, Canada

² Faculty of Dentistry, McGill University, Montreal, QC, Canada

Abstract

The extracellular matrix (ECM) is a dynamic, heterogeneous, three-dimensional suprastructure that anchors and surrounds cellular compartments in tissues and organs. The ECM is essentially composed of four components: collagens; elastin; glycoproteins; and proteoglycans that undergo tissue-specific post-translational modifications and cell-mediated hierarchical assembly to generate macromolecular structures whose biomechanical and biophysical properties range from a pliable translucent hydrogel to rock-hard mineralized tissue.

The ECM molecules and their respective assemblies provide a structural and instructive scaffold during development and throughout life, that endows tissues with tensile strength, elasticity, hydration, and the ability to withstand mechanical forces. The coupling of ECM proteins to the intracellular cytoskeleton through cell surface receptors alters the cellular genetic machinery in response to external biomechanical cues. Moreover, the ECM molecules function as a repository for growth factors, cytokines, and proteinases. The spatio-temporal release of these soluble ECM components, and their subsequent cell signaling cascades, modulate ECM turnover and fundamental cellular processes including differentiation, proliferation, migration, and survival.

The ECM is a major determinant of cell fate and survival, and its importance is underscored by the multitude of acquired and hereditary disorders associated with the defects in synthesis, secretion, and/or assembly of ECM proteins. Alterations in the structural and functional properties of the ECM molecules are also underlying causes for derailed wound healing and angiogenesis, cancer metastasis, and other disease states.

Keywords

Collagen

Elastic fibers

Glycoproteins

Proteoglycans

Glycosaminoglycans

Basement membranes

Connective tissue disorders

Chapter Contents

3.1 Introduction   25

3.1.1 Overview   25

3.1.2 Extracellular Matrix Proteins   26

3.1.3 Crosslinking   27

3.2 Collagens   27

3.2.1 Collagen Biosynthesis and Processing   28

3.2.2 Fibril-Forming Collagens   28

3.2.2.1 Biomineralization   30

3.2.3 Fibril-Associated Collagens (FACITs)   30

3.2.4 Network-Forming Collagens   30

3.2.5 Anchoring Fibrils   31

3.2.6 Other Collagens   31

3.2.7 Collagenopathies   31

3.2.7.1 Osteogenesis Imperfecta (OI)   31

3.2.7.2 Ehlers-Danlos Syndrome   33

3.2.7.3 Skeletal Dysplasias and Chondrodysplasias   33

3.2.7.4 Other Collagenopathies   33

3.3 Glycoproteins   34

3.3.1 Fibronectin   34

3.3.2 Fibrillins and Latent TGF-β-Binding Proteins (LTBPs)   34

3.3.2.1 Structural and Functional Properties of Fibrillins and LTBPs   34

3.3.2.2 Fibrillinopathies   35

3.3.3 Fibulins   36

3.3.4 Other Glycoproteins   36

3.3.4.1 Tenascin   36

3.3.4.2 The Small Integrin-Binding Ligand N-Linked Glycoproteins (SIBLINGs)  36

3.3.4.3 Thrombospondins   37

3.4 Elastin and Elastic Fibers   37

3.4.1 Elastic Fiber Assembly   37

3.4.2 Elastin-Associated Pathologies   37

3.5 Basement Membranes   37

3.5.1 Laminins   38

3.5.2 Collagen Type IV   38

3.5.3 Basement Membrane Proteoglycans   38

3.5.4 Basement Membrane-Associated Pathologies   39

3.6 Proteoglycans and Glycosaminoglycans   40

3.6.1 Glycosaminoglycans   40

3.6.2 Proteoglycans   41

3.6.2.1 Cell Surface Proteoglycans   41

3.6.2.2 Modular Proteoglycans   41

3.6.2.3 Small Leucine Rich Proteoglycans (SLRPs)  41

3.7 Concluding Remarks   42

Acknowledgments   42

Abbreviations  42

References   43

3.1 Introduction

3.1.1 Overview

The extracellular matrix (ECM) is a dynamic three-dimensional structure that anchors and surrounds cellular compartments in tissues and organs. The ECM is composed of interconnected macromolecular assemblies that exist either as an interstitial matrix, as a special sheet-like structure known as a basement membrane, or as mineralized matrix. The ECM contributes to the structural integrity of organs and tissues during development and homeostasis. It is also a major determinant of cell fate and survival. There are four principal components of the extracellular matrix: (1) collagens; (2) elastin; (3) glycoproteins; and (4) proteoglycans. Collagens and elastin form insoluble crosslinked suprastructures that resist tensile forces and confer elasticity to tissues, respectively. Embedded within these suprastructures are proteoglycans and associated glycosaminoglycans that hydrate the matrix, provide turgor, and regulate the diffusion of nutrients, metabolites, and hormones. Glycoproteins such as fibronectin and laminins polymerize into multimeric assemblies and provide connections between cells and the ECM through their interactions with other suprastructures, growth factors, cytokines, and cell surface receptors.

The ECM was essential for the evolution and diversification of multicellular organisms [1]. During development, the strictly controlled expression and assembly of ECM proteins into multimolecular assemblies provides cells with a protective and instructive scaffold for tissue morphogenesis and organogenesis. The continuous turnover of ECM constituents in an adult organism maintains tissue homeostasis, and regulates key cellular processes such as adhesion, proliferation, differentiation, migration, and survival [2]. Defects in either synthesis, secretion, and/or assembly of ECM proteins result in a wide spectrum of heritable and acquired disorders that are often characterized by extensive inter- and intra-familial phenotypic variability [3].

Decades ago, the ECM was originally regarded as an inert mechanical scaffold. More recent research, however, clearly shows the ECM being a dynamic structure that is altered under pathological conditions such as cancer and wound healing, and is involved in preserving the self-renewing capacity of the stem cell niche in adult tissues [2,4]. Furthermore, ECM proteins act as reservoirs that regulate the activation, sequestration, diffusive range of growth factors and cytokines in a tissue-specific and spatiotemporal manner. Mechanical deformations and proteolytic cleavage lead to the release of these soluble components from ECM assemblies, and enable their interactions with cell surface receptors and activation of cell signaling cascades. Moreover, under both physiological and pathological conditions, proteolytic cleavage of various ECM proteins exposes cryptic sequences and generates bioactive fragments that possess biological functions distinct from their intact parent molecule [5].

The interaction between cells and the ECM is reciprocal, and is at the crux of developmental patterning and tissue homeostasis in metazoans. Cells secrete and direct the temporal and hierarchical assembly of ECM proteins into supramolecular assemblies. In turn, the four ECM composites and water determine the porosity, topology, and stiffness of tissues, which can range from hard mineralized tissue of the bone to a transparent soft gel consistency, such as in the vitreous body of the eye [6]. In addition, cells secrete soluble physiologically active components such as growth factors and cytokines whose activity is sequestered and modulated by ECM proteins in a strict spatiotemporal fashion. The extracellular cues from both structural and soluble components of the ECM are transduced by integrin and other cell surface receptors. These lead to rearrangement of the actin cytoskeleton, translocation of transcription factors into the nucleus, and changes in the cell’s expression profile [7]. The interplay between the cells and the ECM is illustrated schematically in Figure 3.1.

Figure 3.1 Overview of the ECM. This schematic displays the reciprocal relationship between cells and the ECM. Cells of primarily mesenchymal origin secrete structural components (collagens, elastin, glycoproteins, proteoglycans, glycosaminoglycans) and soluble components (growth factors and cytokines). The structural components provide an architectural framework that endows structural integrity to tissues and functions as a repository for the soluble physiologically active components. The ECM alters cell behavior by transducing biomechanical cues such as matrix stiffness through cell surface receptors, and through the regulated release and activation of soluble components that in turn trigger their respective cell signaling cascades. This reciprocal relationship is essential for development and tissue homeostasis.

3.1.2 Extracellular Matrix Proteins

Cells of mesenchymal origin, which include fibroblasts, osteoblasts, chondroblasts, and some smooth muscle cells, synthesize vast amounts of ECM proteins. There are ~ 300 proteins that bioinformaticians define as the core matrisome, including collagens, glycoproteins, and proteoglycans [8]. In addition, there are ECM-associated proteins including growth factors and enzymes involved in proteolytic cleavage and protein modification (e.g., crosslinking enzymes). Growth factors and cytokines in the ECM include, but are not limited to bone morphogenetic proteins (BMPs), transforming growth factor (TGF-α, -β), fibroblast growth factors (FGF), Wnt, and many more that functionalize the core matrisome proteins. These families of growth factors stimulate key limb patterning pathways (BMPs, Wnts), synthesis of ECM proteins (TGF-β), and other cellular processes. Proteolytic cleavage under physiological and pathological conditions results in the overall remodeling and turnover of the ECM, exposure of cryptic sites, and release of growth factors [9]. These functions are fulfilled primarily by three proteinase families: matrix metalloproteinases (MMPs); a disintegrin and metalloproteinases (ADAMs), and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) [10–12].

ECM proteins are typically composed of multiple domains which can be unique to a protein family, or shared by other extracellular and even intracellular proteins. The fibronectin type III and epidermal growth factor (EGF)-like domains are prime examples of domains that predate evolution of the metazoan ECM and are expressed in multiple ECM proteins [13]. Each domain has distinct functions such as multimerization, mediating protein interactions or calcium-binding, and these properties are typically maintained even when this domain is excised from the full-length protein. This modular nature of ECM proteins contributes to the multifunctionality of a single ECM protein. The complexity and functional diversity of ECM proteins is enhanced by multiple alternatively spliced forms of a protein in which particular domains are rearranged, repeated, excised or included.

During synthesis, ECM proteins often undergo extensive post-translational modification such as proteolytic cleavage, hydroxylation, phosphorylation, disulfide bond formation (see Section 3.1.3) and glycosylation that alter the structural-functional properties of the protein. Glycosylation is a common post-translational modification where multiple carbohydrate moieties are added to amino acid side chains, and which alter, among other properties, protein solubilization. The most common types of glycosylation are N-glycosylation of asparagine residues, and O-glycosylation of serine and threonine residues; the N and O refer to nitrogen and oxygen that is glycosylated, respectively [14].

3.1.3 Crosslinking

Covalent intra- and intermolecular crosslinking catalyzed by the lysyl oxidase (LOX) and the transglutaminase family of crosslinking enzymes imparts the various macromolecular assemblies with tensile and resilient properties that maintain the architectural scaffold of the organs and tissues. LOX are copper-dependent amine oxidases with five family members including LOX and LOX-like proteins 1-4 (LOXL1-4) that catalyze crosslinking of collagen and elastin monomers through deamination of lysine and hydroxylysine residues into allysine and hydroxyallysine residues, respectively. These residues subsequently form spontaneous covalent interactions with either other allysine/hydroxyallysine or unmodified lysine/hydroxylysine residues. These divalent crosslinks mature into tri- and tetravalent crosslinks. Pyridinoline and pyrrole are trivalent crosslinks specific to collagens, while the elastin specific mature crosslinks are desmosine and isodesmosine [15,16]. Covalent crosslinking is a tissue-specific process, which explains why the skin dermis, bone, and cornea have different tensile properties despite being composed of the same collagen constituents [17]. Degraded collagens peptides containing crosslinking peptides are found in the blood and urine, and are being explored as biomarkers for diseases associated with altered bone remodeling, such as osteoporosis and osteoarthritis [17].

The second family of crosslinking enzymes is the transglutaminases (TG) that catalyze intra- and intermolecular crosslinking between glutamine and lysine residues in various extracellular matrix proteins. There are eight members in the TG family, localized in the nucleus (TG2), cytoplasm (FXIII, TG1, TG2, TG3), cell surface (TG1, TG2), ECM (TG2), and plasma (FXIII). Of particular importance is the ubiquitously distributed TG2, also known as tissue TG, which catalyzes the homo- and heteropolymerization of ECM proteins, and protects them from proteolytic degradation. Intracellularly, TG2 has GTPase enzymatic activity, and is implicated in cell signaling cascades. Collagens, fibronectin, fibrillins, microfibril-associated glycoprotein, SIBLINGs (see Section 3.3.4.2), and osteonectin/SPARC/BM-40 are substrates for transglutaminases. The Nε(γ-glutamyl) lysine crosslinking stabilizes the three-dimensional structure of ECM and contributes to its insolubility. TGs have pleiotropic functions, which are comprehensively discussed by Lorand et al. [18].

The formation of intra- and intermolecular disulfide bonds between two cysteine residues is essential for correct folding and stabilization of numerous ECM proteins, and is catalyzed by protein disulfide isomerase. Fibronectin, fibrillins, laminins, thrombospondin, and other ECM proteins attribute their structural stability to disulfide bonding [19].

While the above-mentioned crosslinking mechanisms are beneficial for tissue homeostasis, non-enzymatic age-related crosslinking such as glycation causes tissue stiffness and deteriorates the biomechanical properties of the connective tissue [20].

In the following sections, we will discuss the structural and functional properties of ECM proteins, and their roles in development, tissue homeostasis, growth factor regulation, and in acquired and inherited disorders.

3.2 Collagens

Collagens are the major structural component of the connective tissue and the most abundant protein family in animals, forming ~ 30% of the total protein mass [15]. The bulk of collagen molecules are synthesized and secreted by fibroblasts, but also by chondroblasts, odontoblasts, and osteoblasts. Collagens self-assemble into structurally and functionally diverse multimeric assemblies such as parallel bundles of fibrils in the tendon; orthogonal lattices in the cornea; and a concentric interlocking weave in bone and skin [14,21]. These diverse suprastructures impart their respective tissues with tensile strength and a scaffold that protects them from mechanical stress [22]. Collagens also form the scaffold for biomineralization in teeth and skeletal tissue (see Section 3.2.3). The interplay between collagens and cells mediate cell adhesion and motility during tissue morphogenesis, growth, and wound healing [23].

There are 28 collagen types that are numbered with roman numerals (I-XXVIII) based on their order of discovery. Collagen molecules are trimeric, and can be either homo- or heterotrimeric. The constituent polypeptide chains are called α-chains, and are denoted by Arabic numerals, so that the two α1 and α2 composite chains of collagen type I are denoted as α1(I)2α2(I). In humans, over 42 genes encoding α-chains have been identified for the 28 collagens [22].

The collagen superfamily is defined by its collagen domain (COL), which contains the repeating tripeptide sequence, (Glycine-X-Y)n, where X and Y can be any amino acids but are frequently proline and 4-hydroxyproline, respectively. The COL domain in each of the three constituent α-chains is intertwined into a right-handed triple-helix which forms a rope-like rod structure that contributes to the tensile properties of collagen. Glycine, being the smallest amino acid, occupies the center of the triple helix and is essential for the compactness and integrity of the molecule [22]. The amount of Glycine-X-Y repeats correlates with the rigidity of the molecules so that imperfections or interruptions in the Glycine-X-Y sequence lead to kinks and pliability of the assembled collagen [24]. Collagens also contain a number of noncollagenous (NC) domains that participate in a wide spectrum of interactions with other matrix proteins and cells [22].

3.2.1 Collagen Biosynthesis and Processing

Studies to elucidate the mechanism of collagen fibril formation have mostly been performed in developing tendons, which are composed primarily of crosslinked bundles of collagen type I fibers [25]. Translation of mRNA encoding pre-pro-α-chains occurs on membrane-bound ribosomes in the endoplasmic reticulum. The N-terminal signal peptide on the pre-pro-α-chains regulates the selection, alignment, and translocation of appropriate pre-pro-α-chains into the endoplasmic reticulum lumen [26]. In the lumen, the signal peptide is cleaved, and the collagen pro-α-chains undergo a number of co- and post-translational modifications. Almost all proline and some lysine residues in the COL domain undergo enzymatic hydroxylation by prolyl and lysyl hydroxylases, respectively. Inhibition or insufficient proline hydroxylation leads to triple-helix instability and retention of the α-chains in the endoplasmic reticulum. A dietary deficiency in vitamin C (ascorbic acid), a co-factor for prolyl-4-hydroxylase, causes scurvy, characterized by decreased de novo collagen synthesis, reduction in connective tissue strength, insufficient wound healing, and teeth falling out of sockets [27].

Hydroxylation of lysine residues is a prerequisite for the attachment of galactose and glucosylgalactose residue on certain hydroxylysine residues. Glycosylation determines the compactness and viscosity of the collagen suprastructures. Following these modifications, the α-chains are intertwined into a super coiled triple-helix, stabilized by inter-chain hydrogen bonds. Triple-helix formation is initialized from the carboxyl terminus in fibrillar collagens and from the amino terminus in membrane collagens. Triple-helix formation precludes further post-translational modifications, and is dependent on the enzyme peptidyl prolyl cis-trans isomerase, which converts the conformation of proline peptide bonds from the cis to trans conformation [15]. As collagens have the propensity to spontaneously assemble into higher order fibrils, most collagen types are secreted into the extracellular environment as propeptides through cellular invaginations called fibropositors [25]. Primarily the tolloid family of metalloproteinase cleaves the C-terminal propeptides, while N-terminal propeptides are cleaved by proteinases of the ADAMTS family. Telopeptides are the short non-helical domains that remain after proteolytic cleavage, and contain the crosslinking residues that drive intermolecular crosslinking among homotypic and heterotypic collagen fibrils [28]. As mentioned above, pyridinoline and pyrrole are trivalent crosslinks specific to collagens, and are essential for the tensile properties of collagens.

Collagen fibril assembly is a cell-mediated process that is dependent on interactions with integrins, fibronectin, FACITs, proteoglycans, and the nucleating collagen type V and XI [29]. Longitudinal fusion and lateral growth of collagen fibrils via crosslinks forms collagen fibers with diameters in the range of 20-500 nm, depending on the developmental stage and tissue type. Fibers are further crosslinked by LOX and TGs to form bundles of collagen fibers, filaments, networks, and lattices [17,30].

The collagen superfamily is divided into subfamilies based on structural homology between the collagens and the type of scaffolding suprastructures formed. The domain structure and three-dimensional organization of collagen suprastructures is illustrated in Figure 3.2.

Figure 3.2 Collagen Assemblies. The collagen superfamily is divided into subfamilies based on structural homology and the three-dimensional suprastructures formed. (a) Fibril-forming collagens. The assembly of collagen type I is shown as an example. Proteolytically processed, fibril-forming collagen molecules are exclusively composed of an uninterrupted triple helical COL domain that spontaneously assembles in to a quarter staggered head-to-tail arrangement to form collagen fibrils (red lines). Subsequent and longitudinal fusion of fibrils give rise to collagen bundles that are crosslinked by TGs. (b) FACITs consist of tandem repeats of COL domains (red box) interrupted by NC domains (black line). FACITs decorate the surface of fibril-forming collagens (right). The domain arrangement of collagen type IX, a prototypic FACIT, is shown as an example. (c) Network-forming collagens. (i) Collagen type IV forms a chicken-wire network in basement membranes. The NC C-terminal domain forms dimers (grey circle), while the N-terminal 7S domain (black line) mediates tetramerization. Subsequent end-to-end and lateral interactions give rise to a three-dimensional network. (ii) Collagen type VI molecules form anti-parallel, disulfide-bonded staggered dimers that align laterally to form tetramers. End-to-end interactions of collagen type VI tetramers form a microfibril network. (iii) The NC domains (grey circle) of collagens type VIII and X multimerize in to hexagonal lattices. (d) Anchoring fibrils. Collagen type VII rivets the basement membrane to the underlying dermis through interactions with basement membrane components and the fibril-forming collagens in the dermis. Collagen type VII molecules form staggered antiparallel dimers through their C-terminal NC domain (grey circles). Subsequent lateral interactions, crosslinked by TGs, give rise to anchoring fibrils. (e) Other collagens. (i) Multiplexins are characterized by tandem repeats of COL domains interrupted by NC sequences. They possess GAG attachment sites in the N-terminal region, and have a unique C-terminus characterized by a trimerization domain, a hinge domain, which is proteolytically cleaved to release the endostatin domain. The domain structure of collagen type XVIII is exemplified for the multiplexin family. (ii) Transmembrane collagens typically possess a small intracellular domain, a hydrophobic transmembrane domain, and a large, C-terminal extracellular domain composed of multiple COL domains (red box) interrupted with NC domains (black line).

3.2.2 Fibril-Forming Collagens

Collagen types I, II, III, V, XI, XXIV, and XXVII are fibril-forming collagens characterized by a long uninterrupted COL domain flanked by small globular NC domains [15]. Collagens I, II, and III are the most abundant proteins in vertebrates, and are the major composite of the fibrillar collagen assemblies [15]. Fibril-forming collagens spontaneously self-assemble longitudinally in a head-to-tail quarter staggered arrangement with a periodicity (D) of 64-67 nm. When visualized by electron microscopy after negative staining, this arrangement creates a distinct banding pattern (Figure 3.2a). The interfibrillar space created by this staggered gap arrangement allows assembly of various non-collagenous ECM proteins, and is a template for biomineralization (see Section 3.2.2.1).

The quantitatively minor fibril collagen type V and XI nucleate collagen type I and II assembly, respectively [22,29,31]. This subclass is characterized by a partially processed N-propeptide that hinders co-assembly into the staggered arrangement of fibrils [32]. Instead, collagen type V and XI are present in the fibril core and project into the fibril surface through the staggered gaps to interact with cell surface receptors [33].

Fibril-forming collagens are composed of heterotypic fibers, and the gene dosage of major and minor fibril collagens types dictates the architecture and thus the biomechanical properties of the matrix. Non-cartilaginous connective tissues, such as the skin dermis, cornea, tendons, blood vessels, bone, and dentin are composed of heterotypic fibrils containing collagen types I, III, and V [30]. Collagen type II fibrils are found almost exclusively in cartilage and the vitreous humor of the eye, where it co-assembles with collagen type IX and XI fibrils. Collagen type IX, the prototypic FACIT (see Section 3.2.3), stabilizes collagen type II fibril assemblies in the cartilage [34]. Collagen type II also binds to the chondrogenic growth factors BMP-2 and TGF-β, and regulates both the mechanical stability and homeostasis of cartilage tissue [35].

3.2.2.1 Biomineralization

Fibril-forming collagens have important roles in biomineralization (also referred to as calcification), a biological process that occurs in skeletal tissue and teeth to generate a hard connective tissue [36]. In skeletal tissue, the three main cell types, osteoblasts, osteocytes, and osteoclasts are involved in producing, assembling, and remodeling the matrix, whereas in teeth odontoblasts secrete the organic and inorganic components of these hard tissues. The organic components of hard connective tissue include collagens (collagen type I), noncollagenous proteins (ex. SIBLINGs), and proteoglycans (see Section 3.6). The inorganic matrix is comprised of hydrated calcium phosphate (Ca5(PO4)3OH) crystals, which are deposited within and around the staggered fibrillar collagens, which undergoes regulated nucleation and growth to generate skeletal structures and teeth. Bones and teeth in vertebrates derive their strength and rigidity from these hydroxyapatite crystals that are essential for skeletal support, protection to internal organs, locomotive functions, and serve as a reservoir for minerals and hematopoietic cells. Biomineralization is a highly regulated process which can otherwise have deleterious effects such as calcification in soft tissues (blood vessels, kidney stone formation) and in hard connective tissue, where it can cause dental dysplasia, dentinogenesis imperfecta, osteogenesis imperfecta, osteoporosis, hypophosphatemic rickets, and many other disease states.

3.2.3 Fibril-Associated Collagens (FACITs)

The fibril-associated collagens with interrupted triple helices (FACIT) family includes collagen type IX, XII, XIV, and XX, and is characterized by a tandem repeat of short COL domains interrupted by NC domains (Figure 3.2b). All FACITS include a FACIT domain, defined as a cysteine-containing motif, GXCXXXC. FACITs modulate the surface properties of the fibrillar collagens by incorporating in the interfibrillar space in staggered fibrils. Collagen type XVI, XIX, XXI and XXII are grouped as FACIT-like collagens, because even though they share certain structural homologies to FACITs they have distinct functional properties. FACIT-like collagens are localized either at the basement membrane or at junctions that separate different tissue types [22].

3.2.4 Network-Forming Collagens

Collagen type IV, VI, VIII, and X are classified as network-forming collagens [22]. Collagen type IV is the primary collagen found in basement membranes, and is discussed in detail in Section 3.5.2. Collagen type VI has a ubiquitous tissue distribution and possesses the propensity to assemble into various suprastructures including microfibrils, hexagonal lattices, and broad banded structures (Figure 3.2c). Collagen type VI assemblies anchor cells to the connective tissue through interactions with integrins, cell surface proteoglycans, and other suprastructures. Interactions with various ECM constituents in turn modulate the assembly of collagen type VI networks in various tissues [37]. Collagen type VIII and X are short chain collagens, that multimerize to form hexagonal lattices (Figure 3.2c). Collagen type VIII is localized to the subendothelium of blood vessels and the Descemet’s membrane, which separates the corneal epithelium from the stroma [38]. The distribution of Collagen type X is localized to hypertrophic cartilage and the hematopoietic stem cell niche at the chondro-osseous junction [39,40].

3.2.5 Anchoring Fibrils

Collagen type VII is the major component of anchoring fibrils, which connects and stabilizes the basement membrane to the underlying dermis [37]. Collagen type VII is synthesized as a homotrimer, with numerous Gly-X-Y imperfections, and assembles into a staggered anti-parallel dimer (Figure 3.2d). The protruding N-terminal NC domains on either end interact with laminin and collagen type IV networks in the basement membrane, and collagen fibrils in the underlying dermis [41].

3.2.6 Other Collagens

The transmembrane family of collagens includes collagens type XIII, XVII, XXIII, XXV, and XXVIII, and is defined by an N-terminal cytoplasmic domain, hydrophobic transmembrane domains, and a large C-terminal extracellular domain composed of multiple COL domains interrupted with NC domains (Figure 3.2e). The extracellular domains of transmembrane collagens can undergo shedding to produce soluble fragments that bind to other ECM components and modulate cell behavior [33]. Collagen type XIII is located at focal adhesion sites where it is implicated in cell adhesion and migration through interactions with fibronectin, integrins, perlecan, and nidogen/entactin. It is also implicated in bone remodeling and the coupling of bone mass regulation to mechanical use [42]. Expression of collagen type XXV and XXVIII is localized to neuronal tissue. Collagen type XVII is a component of the hemidesmosome anchoring complex, which rivets epithelial cells to the underlying basement membrane.

The multiplexin subfamily of collagens includes collagens XV and XVIII, and is characterized by a C-terminal domain that contains three functionally distinct subdomains: a trimerization domain; a hinge domain; and an endostatin domain (Figure 3.2e) [43]. Proteolytic cleavage of the hinge domain releases the endostatin domain into tissues and plasma, where it is attributed to have anti-angiogenic properties. Both collagens XV and XVIII are also structural heparan sulfate proteoglycans (see Sections 3.5.3 and 3.5.4) that are involved in basement membrane homeostasis. The proteoglycan form of collagen type XVIII interacts with FGFs, platelet-derived growth factors (PDGFs), vascular endothelial growth factors (VEGFs), and thus regulates cell adhesion, differentiation, and motility.

Matricryptins are bioactive fragments derived from the regulated cleavage of the NC C-terminal domain of basement membrane-associated collagens (IV, VIII, XV, and XVIII) [5]. These bioactive fragments possess functional properties that are distinct from the intact parent molecules. Cleavage of all six collagen type IV α-chains produces the following matricryptins: arrestin; canstatin; tumstatin; tetrastatins 1-3; pentastatins 1-3; and hexastatins-1 and -2. Cleavage of collagen type VIII, XV, and XVIII generates the bioactive fragments vastatin, endostatin XV/restin, and endostatin XVIII, respectively [43]. Matricryptins are associated with a spectrum of physio-pathological processes, and fragments possess anti-angiogenic and anti-tumor properties that are currently being investigated as potential cancer therapeutic targets.

3.2.7 Collagenopathies

Mutations in collagens manifest as heritable connective tissue disorders that are collectively termed collagenopathies. These disorders often have heterogeneous genetic etiologies that highlight the concerted role of collagens and other modifying genes involved in collagen synthesis and assembly in connective tissue synthesis. The major collagenopathies are osteogenesis imperfecta (OI), Ehlers-Danlos syndrome (EDS), and a spectrum of skeletal dysplasias and