Bone biology

Bone is a complex tissue-organ system integrating multiple components in hierarchical layers of molecular cues, cellular communities, and networking highways. Bone moves through space and time in a dynamic manner modulated by homeostatic mechanisms nuanced through a coordinated intercalation of biological and biomechanical rhythms. The price we vertebrate species pay for maintaining this magnificently orchestrated tissue-organ is daunting.

Bone is the most metabolically expensive tissue in the human body. For every ounce of bone, a pound of soft tissue is required for maintenance [1]. Moreover, the human skeletal system must be rugged in order to handle years of cyclic loading at high forces on the order of kilonewtons, and highly sensitive to the calibrated kinetics of calcium and phosphate release in order to maintain meticulously modulated ion levels [2]. Consequently, the intrinsic design of bone and the dynamics that sustain it are an instructional core for regenerative bone therapeutics.

In this chapter we will introduce the profoundly compelling biodynamic structural marvel that gives shape to the amorphous mass in which it is wrapped and provides the fulcrums and pulleys that propel our anatomy along the avenues and boulevards of our towns. We will probe the blueprint of bone as a defining mold that guides and mentors attempts in the laboratory to design and develop compositions to repair and regenerate this structural tour de force.

Enamel formation

Enamel is the outermost covering of vertebrate teeth and the hardest tissue in the vertebrate body. During tooth development, ectoderm-derived ameloblast cells create enamel by synthesizing a complex protein mixture into the extracellular space where the proteins self-assemble to form a matrix that patterns the woven hydroxyapatite (Wang et al., 2007; Zhu et al., 2006). During enamel biomineralization, the assembly of the protein matrix precedes mineral replacement. The predominant protein of mammalian enamel is amelogenin, which is secreted from ameloblasts. It is a hydrophobic protein that self-assembles to form nanospheres that in turn influence the crystal type, organization, and packing of the crystallites (Du et al., 2005). In contrast to the mesenchyme-controlled biomineralization of bone, which uses collagen and remodels both the organic and the inorganic phases over a lifetime, enamel contains no collagen and does not remodel. The mature enamel composite contains hardly any protein (Smith et al., 1998) and is a tough, crack-tolerant, and abrasion-resistant tissue (White et al., 2001).

The process of enamel formation, termed amelogenesis, is the end product of a series of complex, dynamic, and programmed cellular, chemical and physiological events (Simmer et al., 2010). Enamel formation can be categorized into three distinct stages – the secretory, transition, and maturation stages. The secretory stage is characterized by active protein synthesis and secretion by the ameloblasts. Ameloblasts also deposit enamel crystals at oblique angles while they are moving in the direction of the future cusps to accommodate expansion of the enamel surface. The transition stage is of short duration and is characterized by extensive apoptosis and shortening of ameloblasts in preparation for their transition into the maturation stage. The maturation stage is characterized by removal of enamel organic materials and growth of hydroxyapatite crystals in thickness, as well as by regulated movement of ions into and out of the enamel matrix (Lu et al., 2008; Smith et al., 1998). Besides their involvement in crystal formation, ions also contribute to the color of the teeth (Yanagawa et al., 2004). Enamel in humans is characterized by a large diversity of color variations.

Introduction

The delivery of nucleic acids is a powerful technique to regulate cellular processes that has profound implications for regenerative medicine. Nucleic acid delivery is most widely thought of as delivery of a gene to induce expression of a specific factor. This approach is highly versatile, insofar as it can readily target secreted factors (e.g. growth factors), membranous proteins (e.g. receptors), or intracellular proteins (e.g. transcription factors). Expression can potentially be modulated through the promoter, with control provided by soluble, inductive factors, or be restricted to specific cell populations. The alternative to delivering nucleic acids to induce expression of target genes is to reduce or block expression. The discovery of RNA interference (RNAi) has identified a powerful mechanism for nucleic acid delivery to catalyze the degradation of target mRNA. Similarly, oligonucleotides can bind to a complementary strand of mRNA (antisense) to terminate translation or act as decoys that bind to transcription factors and limit their ability to influence transcription. The delivery of nucleic acids has the potential to either promote or inhibit virtually any cell process; however, a major challenge to capitalizing on this potential lies in the need for effective delivery systems.

Introduction

An emerging trend in tissue engineering and regenerative medicine is the design of scaffolds that recapitulate a stem cell’s extracellular microenvironment, or “niche” (Cushing and Anseth 2007; Tibbitt and Anseth 2009). While the exact constituents of a stem cell niche are not well characterized, it is generally believed that morphogens, immobilized extracellular matrix (ECM) components, cell–cell interactions, and matrix elasticity are the major determinants that direct stem cell self-renewal or differentiation (Lutolf and Hubbell 2005). Research over the years has accumulated fundamental knowledge about stem cell biology in vitro. Most of the results were obtained from experiments conducting on flat, rigid, and two-dimensional (2D) tissue culture polystyrene (TCPS). These 2D environments, however, might not truly reproduce what a stem cell experiences in vivo, where relevant biological and biophysical cues are produced in a far more complex and spatially and temporally regulated manner. In fact, results from numerous studies have demonstrated that, compared with cell culture on TCPS, stem cells behave differently when cultured in three-dimensional (3D) matrices (Liu and Roy 2005; Ingber et al. 2006; Lee et al. 2008b; Lund et al. 2009).

Artificial matrices of synthetic or natural origins have been developed for culturing stem cells in 3D (Lee et al. 2008b; Lund et al. 2009). While natural ECM components, such as collagen and laminin, provide intrinsic biological recognition sites for cell attachment and migration, these natural matrices are ill-defined and poorly controlled with respect to their compositions and mechanical properties, respectively. Furthermore, one cannot afford to overlook the immunogenicity problems associated with allogenic or xenogenic materials, since this significantly limits the clinical translatability/relevance of stem-cell-based therapy. Matrices fabricated from synthetic polymers, on the other hand, provide well-defined and user-controllable chemical and physical properties (Nuttelman et al. 2008). The use of synthetic matrices, however, is not without challenges, insofar as synthetic polymers usually lack biological motifs for cellular recognition. Hence, significant efforts have been dedicated to fabricating synthetic polymeric scaffolds functionalized with biomimetic motifs that permit cellular interaction.

Introduction

The need for replacement organs and tissue substitutes is on the rise. At present, there is an insufficient amount of tissue replacements for failed or damaged organs, due to the lack of donors. In the USA alone, over twenty million patients per year suffer from some form of tissue- and/or organ-related malady, and are awaiting a replacement. The financial cost of health care for these patients has been estimated to be over $400 billion annually [1, 2]. Computer-aided tissue engineered substitutes are one of the most promising applications of tissue replacements to address this issue. These tissue arrays are fabricated using techniques from a variety of science and engineering disciplines to create the optimum tissue replacement (in terms of the targeted functionality). Additionally, these tissue constructs play a vital role as pre-formed extracellular matrices to which cells can readily attach, whereupon they can rapidly multiply and form new tissue [3, 4]. In recent decades, scientists have proven that these fields are evolving into one of the most promising therapeutic approaches in regenerative medicine [5–9].

Three-dimensional tissue scaffolds are designed with a preferred internal architecture, wherein porosity and material connectivity provide the required structural integrity, mass transport, and comprehensive microenvironment for cell and tissue growth. A literature survey has shown that cell survival and proliferation within the tissue scaffold are dependent on oxygen, vital molecules, and the micro-architecture of the scaffolds [10, 11]. The complexity of tissue scaffolds requires novel approaches and computational algorithms to match the desired criteria for internal architecture, permeability, pore size, and connectivity. The dynamics of a tissue scaffold is governed by its structural and topological configuration defined by porosity, pore interconnectivity, tortuosity, and scaffold material permeability and diffusivity [12–14]. The scaffold tortuosity characterizes the diffusion path length of fluid molecules through the scaffold, which shapes the internal architecture of the scaffold and plays a major role in tissue growth and proliferation [15–19]. Many cells respond more favorably to a three-dimensional (3D) microenvironment (than to a two-dimensional (2D) microenvironment) with intricate intracellular architectures where the cell’s morphological shape, behavior, and gene expression are richer, more robust, and more similar to in-vivo responses [20–22].

Introduction

The dentin–pulp complex is the principal inner component of the tooth beneath the superficial enamel layer in the tooth crown, and comprises the entire tooth root outlined with a thin cementum layer. The highly mineralized dentin confers structural integrity and insulative properties to the tooth and surrounds the pulp chamber and canals, which confer vitality to the tooth and whose neurovascular supplies exit through constricted foramina at the root apices. The pulp also has reparative mechanisms, activated by insults to the overlying dentin by noxious stimuli such as attrition, trauma, and caries. Together, the dentin–pulp complex plays a crucial role in tooth health.

The aforementioned noxious stimuli may lead to dentinal damage, as well as pulpal inflammation or necrosis. Such external damage to the dentin renders the pulp vulnerable to external invasion if the extent of the insult extends throughout the thickness of the dentin layer in question. Given the pulp’s solely apical blood supply and limited self-healing capacity, recovery from insult to pulp tissue is difficult, and the resulting inflammation is often irreversible. Currently, complete pulpal resection (root canal therapy) is the default treatment for necrosed or irreversibly inflamed pulp of a tooth that is otherwise restorable. Such teeth are restored first by obturating the pulp canals with an inert material, usually Gutta-Percha; then, direct restorative materials (such as silver amalgam or resin-based composites) and/or full-coverage crowns (metal/porcelain/combination) are used to restore the remainder of the tooth. Although these traditional restorative materials and methods have proven to be adequately effective in conserving teeth, they may render the remaining natural tooth structure mechanically compromised [1], and are incapable of repairing the tissue exposed to harmful stimuli [1, 2].

Introduction

Orthopedic injuries and diseases commonly affect soft tissues, including cartilage, which line the surface of articulating joints, as well as ligaments and tendons, which connect bone to bone and muscle to bone, respectively. Continued developments in tissue engineering have led to advancements in the regeneration of these tissues, while recently emphasis has been placed on the regeneration of the interfaces or insertion sites that connect these soft tissues to bone, which are characterized by a gradient of structural and mechanical properties [1]. The integrity of these regions is essential to facilitating synchronized joint motion, mediating load transfer between distinct tissue types, and sustaining heterotypic cellular communications necessary for interface function and homeostasis [2–4]. These critical junctions are also prone to injury, and healing is typically incomplete after surgical repair. The need for functional interface regeneration is highlighted by the fact that failure to restore the intricate tissue-to-tissue interface has been reported to compromise graft stability and long-term clinical outcome [5, 6].

Fundamentally, tissue engineering involves the use of cells, growth factors, and/or biomaterial scaffolds in a variety of ways to engineer tissues in vitro and in vivo. The principles of tissue engineering have been applied for the successful formation of connective tissues, including bone, cartilage, ligament, and tendon. Recently the focus in the field has shifted from tissue formation to tissue function [7], specifically to imparting physiologically relevant functionality to tissue-engineered grafts. One of the most significant challenges to clinical application is achieving biological fixation of musculoskeletal grafts with each other as well as to the native host environment [8].

Preimplantation embryo development sets the stage for pluripotency

Regenerative medicine has the potential to revolutionize health care by offering the promise of replacement cells, tissues, and organs to combat injury, disease, and aging. In an ideal setting, stem cell therapies would begin with a pluripotent cell that by definition is able to give rise to any cell formed in the embryo. Additionally this would most likely require that the stem cells could self-renew or were able to divide and give rise to either more pluripotent stem cells or progressively more differentiated cells under the control of extrinsic cues. Stem cells are biological cells found in multicellular organisms, that can mitotically divide and differentiate into specialized cell types and can self-renew to produce more stem cells. There are two broad types of stem cells: embryonic stem cells and adult stem cells. Embryonic stem cells originate from the inner cell mass of the preimplantation embryo and are considered pluripotent whereas in situ adult stem cells are considered multipotent. Embryonic stem cells (ESCs) possess characteristics that make them a potentially outstanding starting material for use in regenerative medicine. They are unique among cultured cells because they have an apparently limitless capacity to self-renew in vitro, as well as being pluripotent. Because of these extraordinary properties, ESCs have been an intense focus of research for more than 30 years.

Introduction

Normal blood vessels consist of three layers, including the tunica intima, the tunica media, and the tunica adventitia [1]. Each structural layer consists of distinct cell and matrix types. A monolayer of endothelial cells (ECs) lines the lumen of blood vessels to provide a continuous, selectively permeable, hemo-compatible blood-contacting surface. Meanwhile, ECs play a key role in various physiological and pathological processes including blood supply, metabolic homeostasis, immune cell trafficking, and inflammation [2]. Vascular smooth muscle cells (VSMCs) and pericytes cover the outside of the endothelium, protect the fragile channels from rupture, and contribute to the contraction and relaxation of the vessels [3]. The vascular wall extracellular matrix (ECM) is composed of structural proteins such as collagen and elastin and adhesion proteins such as fibronectin and laminin that determine mechanical strength, cell response, and ultimately hierarchical tissue organization [4]. An intact and functioning vasculature is crucial in order to maintain homeostasis and provides necessary nutrients and oxygen exchange to all parts of the body.

Diseases that affect the integrity of blood vessels lead to serious and often deadly outcomes. Vascular diseases are the major causes of morbidity around the world [5]. At present, approximately 12 million people in the USA are affected by peripheral vascular disease, but only approximately one in four of them has been diagnosed and is receiving treatment [6]. EC and SMC pathology has been implicated in various vascular diseases [7–10]. Current important therapeutic options for vascular disease incorporate the surgical implantation of stents or grafts and greatly reconstruct impaired vascular function to drain downstream tissues and organs. However, the implanted grafts may incompletely recover the functional integrity of the vasculature. In addition, these therapeutic methods neither provide long-lasting solutions nor prevent damage to downstream tissues and organs [11]. In this scenario, cell-based engineered vessel grafts may offer the opportunity to permanently and effectively treat many vascular diseases [11]. The regeneration of some or all of the vessel layers and their original properties may provide potentially functional vascular grafts. The use of autologous bypass grafts, including saphenous vein, internal mammary artery, and radial artery bypass grafts, remains an important therapeutic option for the treatment of coronary artery disease. However, many patients do not have a vessel suitable for use because of concomitant vascular disease, amputation, or previous harvest, and hence artificial grafts must also be used [12].

Gene therapy for regenerative medicine

Gene therapy refers to the delivery of genetic material that will activate, hinder, or modify the expression of specific genes to facilitate the natural cellular production of a therapeutic agent to treat disease [1, 2]. The concept has emerged as an effective method to control the course of a disease/disorder, modulate the host-response triggered by pathogen, or regenerate compromised biological tissues [2]. As such, the use of gene delivery technologies offers a novel approach for delivery of putative regenerative molecules to sites in the oral cavity and craniofacial complex [2]. Gene therapy is more advantageous than the traditional therapeutic delivery of compounds and proteins. A greater sustainability in comparison with a single dosage or several of a protein or compound is one of the primary advantages of therapeutic gene delivery. Although the half-lives of conventional pharmaceutical compounds or recombinant proteins range from hours to a few days, viral vector-gene delivery of the corresponding genes can lead to in-vivo expression lasting from weeks to years. Gene therapy also alleviates technical challenges that arise with protein expression and purification. Furthermore, gene delivery of an entire group of regenerative factors combined with existing tissue regeneration therapies could potentially replicate natural biological healing processes and allow engineering of complex three-dimensional (3D), multitissue structures (Figure 21.1) [2]. Therapeutic gene delivery for tissue regeneration is achieved through the use of both viral and non-viral vectors (Table 21.1).

History

The concept that post-natal tissues self-renew, and do so by means of a stem/progenitor cell, is not a new one, being based on observations made around 1900, primarily in the field of hematology. While this concept was originally thought to apply to tissues with a high rate of turnover (blood, the gastrointestinal tract, epidermis), studies over the past few decades have demonstrated that virtually all tissues, including connective tissues, have the ability to self-renew, albeit at different rates, depending on the demands imposed upon them [1]. Methods to establish cell cultures in vitro were first attempted in the late 1800s and early 1900s by a number of investigators, namely Bernard, Roux, Harrison, and others [2], but it is Alexis Carrel who is credited with the methodology employed to establish and maintain connective tissue cells in vitro [3]. With subsequent refinements in the following years, connective tissue cell cultures rapidly became an essential tool in cell physiology and molecular biology. Perhaps the first real evidence of stem/progenitor cells within a population of connective tissue cells emerged in the late 1960s, when Alexander Friedenstein and co-workers isolated clones of rapidly adherent bone marrow stromal cells (BMSCs), derived from a single cell, a colony-forming unit-fibroblast (CFU-F). Approximately 10%–20% of single CFU-F-derived strains had the ability to form bone, hematopoiesis-supportive stroma, and marrow adipocytes upon in-vivo transplantation in open systems, and cartilage as well in closed systems. Friedenstein and his collaborator, Maureen Owen, later called these cells “multipotent bone marrow stromal stem cells” [4, 5]; more recently, the term “skeletal stem cell” has been coined [6].

Connective tissue stem/progenitor cells in tissue engineering and regenerative medicine

Over the last several decades, putative stem/progenitor cells have been isolated from a long list of connective tissues. These cells have been collectively termed “mesenchymal stem cells” or more recently “mesenchymal stromal cells” (“MSCs”), because of their adherence to tissue culture plastic, fibroblastic morphology, and expression of cell surface markers [7]. How similar or dissimilar “MSCs” are from different connective tissues, and whether they are true stem/progenitor cells, is not well known [8]. Nonetheless, these populations may be useful in the reconstruction of connective tissues.

Introduction

Global demand for bone grafts has been increasing rapidly in recent years. In particular, an increase in the elderly population worldwide at an annual rate of more than 5% has led to a rapid increase in the bone-diseased population because the elderly suffer easily from osteoporotic fractures, degenerative scoliosis, and degenerative spondylolisthesis [1].

Bone is a complex biomineralized organ with an intricate hierarchical microstructure assembled through the deposition of apatite minerals on collagenous matrix [2], and is able to self-heal a small defect. However, the self-healing is less effective for a large defect. For this, bone grafting is required in orthopaedic surgery.

Bone grafting is a surgical procedure that introduces new bone or a substitution material (bone graft) into defects in bone or around broken bone to help bone healing. Depending on the source of bone grafts, they may be roughly divided into autologous bone grafts (autografts), allografts and artificial (synthetic) bone grafts. These bone grafts have their own particular advantages and drawbacks. Theoretically, an autograft should be the best choice because the bone graft is the patient’s own bone, taken frequently from his/her hip bone or pelvis, which renders clear advantages such as a mechanical match with the damaged bone, excellent osteogenic potential, and immune safety [3]. Nevertheless, the principal disadvantage of using autograft bone is that the patient needs to bear additional chronic pain at the incision site, runs potential risks such as infection, additional blood loss, and morbidity, and must bear a longer operation duration and higher costs [4]. Therefore, the other approaches are becoming popular.

Regenerative medicine aims to regenerate tissues and organs for medical therapies by harnessing the regenerative potentials of various stem cells. Stem cells include embryonic stem cells, multipotent adult stem cells, tissue specific stem cells, and induced pluripotent stem cells. These stem cells are the driving force for regeneration. There is a growing recognition of the effect of the three-dimensional (3D) matrix microenvironment on the fate and function of stem cells. A key challenge facing regenerative medicine is to generate 3D microenvironments (matrix, signals, supporting cells etc.) that can recapitulate those in development or healing to maintain stemness, to accelerate proliferation, or to direct the stem cells to differentiate toward a specific therapeutic lineage. Biomaterials can serve as 3D matrices; they can play critical roles in creating the 3D microenvironments for stem cells to facilitate regeneration. As the interactions and the overlap of the fields of biomaterials, stem cells, and regenerative medicine are rapidly growing, there is an urgent need for understanding of, and technologies to utilize, the interactions between biomaterials and stem cells for regenerative medicine.

This book, Biomaterials and Regenerative Medicine, overviews the state-of-the-art knowledge on stem cells, interactions of biomaterials with stem cells, biomaterials design for regenerative medicine, and the animal models and clinical applications of biomaterials for regeneration. While providing a comprehensive overview of the field, the emphasis is on the design principles, fabrication technologies, physical characterization, and biological evaluation of the biomaterials for stem cell research and regenerative medicine.

Introduction

Traditionally tissue engineering entails the seeding and culturing of differentiated somatic cells onto biodegradable scaffolds, with subsequent implantation of the cell–scaffold constructs into the defective or damaged sites to regenerate tissues [1]. In this approach, the scaffold acts as a three-dimensional (3D) framework to provide physical support and accommodate cell growth and deposition of extracellular matrices, and its biodegradability allows the scaffold material to be resorbed in pace with new tissue formation. Despite some encouraging successes in clinical trials [2, 3], two key limitations with this approach include the limited source of exogenous donor cells and the lack of adequate vascularity to maintain vitality of the newly regenerated tissues. To address these limitations, current advanced tissue engineering techniques gear toward harnessing a biomimetic scaffold that provides a synthetic regenerative microenvironment to support natural tissue regeneration and angiogenesis [4]. In addition to providing physical support, the ideal biomimetic scaffold would preferably also deliver bioactive factors, which instruct endogenous stem cell recruitment and differentiation three-dimensionally and in a controlled manner [5] (Figure 20.1). Various bioactive factors, including growth factors [6–8], nucleic acids [9], and integrin-binding ligands [10], have successfully been delivered or presented on biodegradable scaffolds. Among these, growth factors are the most important soluble signals in the natural regenerative microenvironment, being actively involved in stem cell recruitment, proliferation, and differentiation, angiogenesis, and tissue morphogenesis. Although they are potent, growth factors are expensive and have short half-lives in vivo. Therefore, scaffolds with controlled-release capacity are desired in order to preserve growth factor bioactivity and to prolong their function at therapeutic levels over an extended time period. However, there remain significant challenges in delivering growth factors effectively from scaffolds, including the need to preserve the bioactivity of growth factors during the possibly harsh incorporation process, the control of their release over an extended period during tissue regeneration, and the need for release to be restricted locally so as to avoid toxic or unwanted systemic side effects. Additionally, each individual delivery strategy is related, and sometimes restricted, to the type of scaffold utilized.

Introduction

Some strategies in tissue engineering and regenerative medicine (TERM) rely on the use of an appropriate biomaterial to guide and foster tissue repair and regeneration. Collagen-based materials are perhaps the most widely investigated of these biomaterials because collagen is the primary structural protein responsible for tissue integrity in most tissues [1]. Collagen gels offer several advantages as a scaffolding material, including the ability to deliver a homogeneous distribution of entrapped cells into a specific geometry and excellent biocompatibility and transport properties [2]. However, collagen does have some disadvantages, including suppression of cell proliferation and protein synthesis [3, 4], issues that can limit the quality of the engineered tissue produced.

An alternative biopolymer that shares similar properties to collagen is fibrin. Fibrin is a natural biopolymer involved in the wound-healing process, and it forms the provisional matrix of a clot. It rapidly polymerizes to form a biocompatible and biodegradable fibrous scaffold that promotes cell proliferation and ECM synthesis. Another attractive property of fibrin is that its precursor (fibrinogen) can be extracted from the patient’s blood, making it an autologous material. In this chapter we will review the properties of fibrin and fibrin-based engineered tissues and how these materials are being incorporated into TERM technologies.