CHAPTER 1: Extracellular Matrix Scaffolds for Tissue Engineering and Biological Research

The regulation of cell functions is still one of the major challenges for the medical, pharmaceutical, and tissue engineering fields. There are many efforts to regulate cell functions; for example, genetic modification to alter intracellular signaling¹  and stimulation with soluble factors, including growth factors and hormones.^2,3  Matrices with wide diversity being adjacent to cells are also important for regulating cell functions, which are useful as cell regulating cues in a variety of fields. Many scaffolds have been proposed so far, such as metallic,⁴  inorganic,⁵  and synthetic and natural polymeric ones.⁶  These scaffolds regulated the cell functions to some extent. However, the function of these scaffolds was not as good as that of the native extracellular microenvironments. In the in vivo extracellular microenvironments, the cells are supported by the extracellular matrix (ECM) supplying the substrates. The ECM also regulates many cell functions through various modes. Thus, it is a major challenge to develop substrates that mimic the native ECM. Decellularized ECM (dECM), prepared by a technique for the specific removal of cellular components (the decellularization technique), is a promising approach for preparing substrates that mimic the native ECM. In this chapter, we summarize the roles of the ECM in the regulation of cell functions, and we also introduce trials to prepare substrates that mimic the native ECM for medical, pharmaceutical, and tissue engineering applications. The substrates mimicking the native ECM can also be used for comprehensive research into the roles of ECM as well as the above applications.

The ECM is composed of various proteins and carbohydrates. The number of ECM protein types is speculated to be approximately 300.⁷  These ECM proteins are categorized into four types: collagens, elastin, proteoglycans, and glycoproteins.⁸  Collagens provide the structural strength of the ECM in the strong fibers of tendons, the organic parts of bones and cartilage, the basement membranes, the viscous matrix of the vitreous humor, the dermis, and the capsules around organs.⁸  Elastin is one of the major structural proteins found in the native ECM. It determines the elasticity of connective tissues and various organs, including vascular walls, ligaments, skin, and so on.^9,10  Elastin is chemically inert and highly insoluble and possess many covalently cross-linked structures, which are formed between the lysyl residues and make the soluble tropoelastin into insoluble elastin.¹⁰  In addition, elastin was known to be the origin of matrikines, peptides originating from the fragmentation of matrix proteins and presenting biological activities, as described later (see Section 1.2.3.4)^11,12  Proteoglycans are composed of two parts: core proteins and glycosaminoglycans (GAGs). Additionally, free GAGs, such as hyaluronan, can exist in the ECM. GAGs are repeating polymers of disaccharides with carboxyl and sulfate groups; thus, proteoglycans with GAGs can have high negative charges. Due to these negative charges, GAGs chains can be extended and they contain a high water content, leading to space-filling and lubricating functions.⁸  Additionally, GAGs can bind to many growth factors to store them in the ECM and to regulate their availability and activity, as explained below.^13,14 

In addition to these collagens, elastins, and proteoglycans, there are approximately 200 types of glycoproteins, such as fibronectin and laminin.⁸  Many of these glycoproteins can interact with the cells to regulate their functions as addressed in detail below.

The ECM is composed of these molecules with many variations. Moreover, the composition of the ECM differs among different tissues and organs.¹⁵  For example, type I collagen exists in cancellous bone but not in articular cartilage. Instead of type I collagen, type II collagen exists in articular cartilage. Additionally, the composition of the ECM changes according to developmental stages and pathological states to precisely regulate cell functions.^16–19  Due to this compositional and structural complexity, it is difficult to prepare substrates mimicking the native ECM by conventional chemical and physical methods.

The ECM molecules described above can be assembled to form special structures. Several ECM molecules can interact with the same and/or other ECM molecules to form ECM structures.^20–22  For the cells in epithelial tissues, the ECM molecules are assembled into a sheet form called the basement membrane.²³  There are four fundamental components in the basement membrane: type IV collagen, laminins, nidogen, and perlecan.²³  Both type IV collagen and laminin form their own mesh structures, and these two mesh structures are bound via nidogen.²⁴  Other basement membrane molecules, including perlecan, are in this type IV collagen–laminin double mesh structure. Similar to epithelial tissues, the cells in muscles, nerves, and blood vessels are also supported by the basement membrane. In stromal tissues, the cells are surrounded by the ECM, which is a gel-like structure. This stromal ECM is composed of molecules including type I collagen, fibronectin, and elastin.

Due to the above compositional complexity of the ECM, the ECM has various functions related to maintaining the homeostasis of living beings. The functions are classified into five categories: (1) scaffolding to maintain tissue and organ structures [Figure 1.1A, label (a)] (2) forming boundaries between the different tissues and organs [Figure 1.1A, label (b)], (3) transducing mechanical signals [Figure 1.1B, label (a)], (4) regulating the activity of soluble factors [Figure 1.1B, label (b)], and (5) directing signal transduction via interaction with the cells [Figure 1.1B, label (c)].

Scaffolding is the most fundamental role of the ECM. Cells can adhere to many ECM molecules, such as fibronectin, vitronectin, laminin, elastin, and collagen, via various receptors on the cell surface.^25–27  The cells adhering to the ECM molecules can be fixed onto the ECM scaffolds with tissue- or organ-specific structures. For example, epidermal keratinocytes are on the basement membrane of the skin.²³  When the skin is exposed to ultraviolet (UV) light, the basement membrane structure is disturbed. Finally, the number of wrinkles on the skin increases due to the disturbance of the basement membrane structure.²⁸  This example demonstrates that the scaffolding of the ECM is important for maintaining tissue and organ structures.

In addition to scaffolding, the ECM acts as a barrier to suppress abnormal cell migration and proliferation. For example, the epidermis and the dermis are divided by the basement membrane. It was reported that keratinocyte infiltration from the epidermis into the dermis was inhibited by type IV collagen.²⁹  Additionally, the basement membrane is degraded when carcinoma cells infiltrate into the stromal tissues and blood vessels.³⁰  Additionally, the ECM plays a role in inhibiting unregulated cell proliferation. For example, tumor cells degrade the ECM molecules that are around them to make space for proliferated cells. When tumor cells were cultured in a non-degradable collagen gel, the cells could not proliferate in the gel.³¹  As shown above, the ECM acts as a physical barrier to inhibit migration to incorrect tissues and to inhibit unexpected proliferation.

The stiffness of tissues and organs varies according to their type and is determined by the stiffness of cells, bodily fluids, and the ECM. This stiffness has been reported to influence various cell functions. For example, cells can migrate from soft to stiff elastic substrates, whereas they cannot migrate from stiff to soft elastic substrates. This phenomenon is known as durotaxis.³²  Additionally, the substrate stiffness can influence stem cell differentiation. When mesenchymal stem cells (MSCs) were cultured on the gel with three types of substrates (soft: 0.1–1 kPa, medium: 8–17 kPa, and stiff: 25–40 kPa), the cells exhibited different differentiation behaviors on these elastic substrates. MSCs on soft elastic substrates primarily differentiated into neurons, whereas the cells on medium elastic substrates primarily differentiated into myoblasts. Moreover, the cells on stiff elastic substrates primarily differentiated into osteoblasts but not into neurons or myoblasts.³³ 

Additionally, the cells are influenced by the topological properties of the substrates.^34,35  For example, the myoblasts matured into aligned myotubes on grooved substrates, and this aligned maturation did not occur on non-grooved substrates.³⁵  These examples demonstrate that the ECM can regulate cell functions via the transduction of ECM mechanical properties, such as stiffness, and topological properties.

As mentioned above, the ECM molecules can bind to many soluble factors, such as bone morphogenetic proteins (BMPs) and wingless and int-1-related (Wnt) proteins, to regulate their activity. Wang et al. reported that BMP signaling was regulated by the ECM in Drosophila.³⁶  Drosophila BMP signaling molecule, decapentaplegic (Dpp), was bound to type IV collagen to form a Dpp gradient pattern for the correct development of Drosophila embryos. Wnt proteins can bind to ECM proteins, such as proteoglycans, through their GAGs.³⁷  Wnt proteins can be locally concentrated by binding to the ECM proteins, leading to increased accessibility to the receptor on the cell surface.³⁸ 

Hepatocyte growth factor (HGF) is required for hepatocyte proliferation in liver regeneration. HGF is bound to the ECM in an intact liver. Once the liver is damaged, HGF is proteolytically released from the ECM by matrix metalloproteinase (MMP)-induced ECM degradation. However, the over-expression of tissue inhibitor of metalloproteinase 1 (Timp-1), an endogenous inhibitor of MMP, delayed hepatocyte proliferation. In contrast, the knockdown of Timp-1 promoted hepatocyte proliferation.^39,40  This example reveals that the ECM can act as a reservoir of growth factors.

Interestingly, several domains of ECM molecules can act in a free soluble form (matrikine or matricryptin) after proteolysis.^41–43  For example, laminins contain repeated epidermal growth factor (EGF)-like domains. These domains are released after proteolysis by MMP. Finally, this released EGF-like domain can interact with EGF receptors to activate a downstream signaling pathway.⁴²  Additionally, endostatin, which is located in the C-terminus of type XVIII collagen, can be released and can suppress angiogenesis.⁴³ 

In addition to mechanical signal transduction and the regulation of soluble factors' activity, ECM molecules themselves can activate intracellular signaling pathways by direct interaction with the cells. Such signal transduction is mainly activated by integrins, which are receptors on the cell membrane. Integrins are transmembrane heterodimers composed of α and β subunits. It has been reported that there are 18 α subunits and 8 β subunits that they form specific heterodimers. Twenty-four types of integrin heterodimers have been identified thus far.²⁵  When integrin heterodimers bind to ECM molecules at extracellular domains, the intracellular signaling molecules, such as talin, focal adhesion kinase (FAK), and paxillin, accumulate at the intracellular domain of the integrins, which leads to the activation of their downstream signaling pathway.^25,44,45  Finally, activated intracellular signals alter various cell functions (e.g., cell survival, proliferation, differentiation, migration, polarity, cytoskeletal assembly, and cell shape). Moreover, this intracellular signaling can cross-talk with the signaling activated by growth factors. Integrin signals can modulate the signaling activated by growth factors and their subsequent responses.^46,47  Signal transduction from integrins is very complicated but has been summarized in many reviews.^25,45,46  Thus, we will not further explain integrin signal transduction in this book.

As mentioned above, the ECM plays pivotal roles in the regulation of cell functions. Thus, ECM molecules have been used for many medical and pharmaceutical applications, but it is still necessary to develop more effective biomaterials that mimic the native ECM.

Isolated ECM molecules, such as collagens, fibronectin, and laminins, have been used for medical applications for a long time.⁴⁸  The effects of each molecule on cell functions have been compared for various types of cells and are still being investigated.^49,50  This simple approach allowed great progress for medical applications. However, the ECM is composed of many ECM molecules. It is expected that the combination of ECM molecules can induce stronger effects than single isolated ECM molecules. Thus, the combination of ECM molecules has also been attempted for use in regulating cell functions.⁵¹ 

As described above, the ECM is composed of many ECM molecules and ECM-bound soluble factors and regulates cell functions strongly and precisely. Thus, it has been required to reconstitute native ECM in vitro. Engelbreth–Holm–Swarm (EHS) murine sarcoma cells form tumor tissues in mice with abundant basement membrane components. Thus, basement membrane components can be extracted from EHS tumor tissue in a gel form (“Matrigel^®”), and this extracted gel can be used as an in vitro basement membrane model. Matrigel^® can induce cell functions more strongly than single isolated ECM molecules.^52–55  For example, primary hepatocytes can maintain their specific functions, such as albumin secretion, on Matrigel^® for a longer period of time than they canon type I collagen.⁵⁵  Due to the achievements using Matrigel^®, it is expected that the reconstitution of the native ECM in vitro will induce cell functions more strongly than single isolated ECM molecule-coated substrates.

Matrigel^® can induce cell function strongly. However, Matrigel^® has several restrictions. Matrigel^® is derived from murine tumors, although ECM derived from human normal tissues is often required. The hepatic functions of primary hepatocytes can be maintained at higher levels on a laminin-511-/521-containing normal basement membrane model than on a Matrigel^® model.⁵⁶  Additionally, Matrigel^® contains basement membrane components, although few or no basement membrane components are contained in the stromal ECM. Thus, Matrigel^® might not be suitable to mimic native stromal ECM. Accompanied by the recent progress of regenerative medicine and stem cell biology, the roles of ECM in regulating stem cell differentiation are being increasingly unveiled. The substrates mimicking native ECM for stem cell differentiation are required for regenerative medicine and stem cell-based engineering. For these reasons, alternatives to Matrigel^® became necessary.

Native ECM exists in in vivo tissues and organs and its utilization as the source for the reconstitution of native ECM in vitro has been attempted. For this purpose, a decellularization technique has been applied. In the early stages of decellularization research and dECM development, several tissues (e.g., dermis, artery, aortic valve, peripheral nerve, small intestinal submucosa) are decellularized, and some of the human tissue- derived dECM are commercially available and used for tissue replacement or wound dressing.^57,58 

In this book, dECM is defined as “materials prepared from cell-ECM constructs by specific removal of cellular components”. “Cell–ECM constructs” include both three- and two-dimensional (i.e., sheet structured) constructs composed of cellular fractions and adjacent ECM, which can be constructed by in vitro cultured cells as well as in the living bodies as native tissues and organs. However, in this book, purified materials, like collagen and fibronectin, are not addressed as dECM because these purified materials are not expected to manipulate and improve cell functions similarly to the dECM defined as above. Currently, dECM defined as above has been prepared from various types of native tissues and organs and the cultured cell-reconstructed tissues have been used for a variety of medical and pharmaceutical applications in various forms (e.g., sheet, tubes, 3D scaffolds, and injectable gels). Examples of these trials will be addressed in the latter half of this book (Parts V and VI).

Characterization and functional analysis of dECM are usually performed based on histological, microscopic, and biological methods (Table 1.1).⁵⁹  For the confirmation of decellularization, detection of cell nuclear components (i.e., DNA) is necessary. The detection is performed by staining for cell nuclei (e.g., hematoxylin and Hoechst 33 258 staining) or by measuring DNA content. Additional intracellular components are often detected using histochemical methods (e.g., staining with fluorescently labeled phalloidin for fibrous actin detection and immunochemistry of cytosolic proteins).

For the compositional characterization of dECM, histological and immunochemical methods are always used. Eosin staining can detect non-nucleic components. Also, it is often required to examine specific ECM components. GAGs can be detected by alcian blue and toluidine blue staining. Collagens can be detected by Sirius red and Azan staining. For the detection of specific ECM molecules and glycosaminoglycans, labeling with specific antibodies or lectins are typically used. Recently, mass spectrometry has been started to be utilized for exhaustive dECM compositional analysis.

Structural analyses of dECM are performed using microscopic analyses, such as scanning electron microscopy (SEM) and transmission electron microscopy (TEM). In particular, TEM can be used to assess basement membrane formation because lamina densa is observed as an electron-dense region (see also Chapter 4). Furthermore, fast Fourier transform analysis can assess fibril alignment.⁶⁰  Recently, peptides have been developed to detect denatured collagen triple helix.^61,62 

Functional analyses of dECM have been performed by various methods. Examples of these analyses will be described in latter chapters.

As addressed above, the ECM can regulate various fundamental cell functions. Thus, isolated ECM molecules have been utilized for medical, pharmaceutical, and tissue engineering applications. Today, there has been substantial progress in these fields. It is required to regulate cell functions more strongly and precisely, which increases the importance of the ECM. The ECM is composed of many components that regulate cell functions. The reconstitution of native ECM has been expected to strongly induce cell functions. Despite many efforts, it is difficult to reconstitute native ECM in vitro by conventional chemical and physical methods due to its compositional and structural complexity. Thus, the decellularization technique is attractive for reconstituting native ECM for various applications.

Additionally, researchers in ECM biology have concentrated on clarifying the effects of single ECM molecules on cell functions. For this purpose, substrates coated with single isolated ECM molecules, gene engineering techniques (knocked-in/knocked-out animals and cells), etc. have been used.^63,64  However, it is difficult to comprehensively understand the roles of ECM from these studies focusing on single ECM molecules. In vitro ECM models mimicking native ECM will be a powerful tool. dECM is expected to be a suitable in vitro ECM model for comprehensive studies of the roles of ECM.⁶⁵  In this book, examples of the use of dECM for medical, pharmaceutical, and tissue engineering applications will be addressed after the explanation of its preparation (Part II), characterization (Part III), and modification and fabrication (Part IV). Additionally, some examples of dECM for ECM biology research will be introduced in Part V and Part VI.