Principles of bone biology and regeneration

Introduction

Bone is a dynamic tissue sensitive to a variety of factors with an inherent capacity that allows the translation of mechanical stimuli into biochemical signals, which therefore enhances its ability to adapt and sustain the physiological needs of the osseous structure. This adaptive potential is the result of tightly regulated and synergistic anabolic and catabolic events that lead to proper metabolic and skeletal structural homeostasis. Multiple factors exert an effect in this system (e.g., biochemical, hormonal, cellular, biomechanical) that will collectively determine bone quality. Clinically, bone quality is perceived as an important feature that dictates the mechanical properties of bone over time. Within the skeleton, such characteristics vary from one area to another and are determined by, among many things, cellular density and connectivity, bone density, bone macro- and microarchitecture, and the proportions of organic and inorganic matrix. Therefore, the success of implant therapy is influenced by the understanding of the basic biological and physiological principles of bone, as it will aid the surgeon in selecting the appropriate techniques to enhance the peri-implant bone homeostasis. Thus, the purpose of this chapter is to provide the clinician with foundational knowledge of bone development, composition, metabolism, and regeneration that serves as a primer for implant site development.

Bone development

During embryogenesis, the skeleton forms by either a direct or indirect ossification process. In the case of the mandible and the maxilla, mesenchymal progenitor cells condensate and undergo direct differentiation intoosteoblasts, a process known as intramembranous osteogenesis. In contrast, in the mandibular condyle, the long bones and vertebrae form initially through a cartilage template, which serves as an anlage that is graduallyreplaced by bone. The cartilage-dependent bone formation and growth process is known as endochondral osteogenesis (Fig. 1). Alveolar bone lost as a result of an injury, disease, or trauma undergoes a repair process that is essentially a combination of endochondral and intramembranous complementary osteogenic processes . A similar process occurs in most of the bone-related implant site development techniques, where osteoconduction, osteoinduction, and osteogenesis are exploited.

Fig. 1 During intramembranous osteogenesis, an ossification center develops through mesenchymal condensation. As the collagen-rich extracellular matrix develops and matures, osteoprogenitor cells undergo further osteoblastic differentiation. A subpopulation of osteoblasts becomes embedded in the mineralizing matrix and gives rise to the osteocyte lacunocanalicular network. Within the craniofacial complex, most bones develop and grow through this mechanism. On the other hand, long bones within the skeleton and the mandibular condyle are initially developed through the formation of a cartilaginous template that mineralizes and is later resorbed by osteoclasts and replaced by bone that is laid down afterward. The endochondral bone growth process leads to the formation of primary and secondary ossification centers that are separated by a cartilaginous structure known as the growth plate. As bone develops and matures through these two processes, structural distinct areas of compact bone and trabecular bone are formed and maintained through similar bone remodeling mechanisms.

Bone cellsWithin bone, different cellular components can be identified. The distinct cell populations include osteogenicprecursor cells, osteoblasts, osteoclasts, osteocytes, and hematopoietic elements of bone marrow. The content of this article will focus on the three main functional cells that are ultimately responsible for the proper skeletal homeostasis. Osteoblasts are ultimately the cells responsible for bone formation. They synthesize the organic matrix components and mediate the mineralization of the matrix (Fig. 2).

Fig 2 Osteoblasts are derived from bone marrow osteoprogenitor cells and are responsible for the synthesis of the immature bone matrix known as osteoid.n (A) The arrow depicts a group of osteoblasts that are lining the mature bone that contains embedded cells within the mineralized matrix. (B) Further detail of the osteoblasts lining the mature bone is clearly visualized through transmission electron microscopy (TEM). The abundant endoplasmic reticulum and Golgim apparatus within these cells reflects their high metabolic activity. Below the osteoblast layer, a collagen-rich unmineralized matrix is clearly depicted and comprises the osteoid. As the collagen mineralization occurs, a clear mineral front develops and a number of areas of crystal nucleation becomes visible. As the mineral propagates over the collagen fibers, a stable and mature bone matrix is accentuated. (C) This panel shows a higher magnification of the extracellular matrix adjacent to osteoblasts. A cross-sectional image of the collagen fibers is shown in close proximity to the cell. The arrows are pointing to a number of higher electron-dense vesicles that have detached from the osteoblast cytoplasm. These structures have been proposed to assist in the mineralization process by facilitating mineral crystal nucleation within as it occurs in cartilage.

Osteoblasts are located on bone surfaces exhibiting active matrix deposition, and as their bone forming activity nears completion, some osteoblasts further differentiate into osteocytes, while others remain in the periosteal or endosteal surface of bone as lining cells. Bone lining cells are elongated cells that cover a surface of bone tissue and exhibit no synthetic activity. Osteocytes are stellate-shaped cells that are trapped within the mineralized bone matrix in spaces known as lacunae. They maintain a network of cytoplasmic processesknown as dendrites. These osteocyte cytoplasmic projections extend through cylindrical encased compartmentscommonly referred to as canaliculi. They reach to different areas and contact blood vessels and other osteocytes. The osteocyte network is therefore an extracellular and intracellular communication channel that is sensitive at the membrane level to shear stress caused by the direction of fluid within the canaliculi space as the result of mechanical stimuli and bone deformation (Fig. 3).

Fig. 3 The osteocyte can be defined as the orchestrator of the remodeling process within bone. (A) As bone matrix is synthesized, a number of osteoblasts become embedded within the osteoid, which later mineralize and reside in the mature matrix as osteocytes, as shown in this backscatter scanning electron microscopy (SEM) image treated with osmium to allow the visualization of the cell. (B) As shown in this confocal image, the embedded osteocytes interconnect, forming a network throughout the bone that enables these cells in the mechanosensoring capacity important in tuning up the remodeling needs. (C) The SEM image of a casted lacunocanalicular network allows the visualization of the degree of connectivity between two osteocytes and the regular diameter of the canalicular structures. (D) The high degree of mineralization of the matrix surrounding the osteocyte is clearly depicted by transmission electron microscopy. Although these cells appeared “dormant,” they are metabolically active and secrete a number of factors that allows them to modify their microenvironment. (E) A transmission electron image of a dendrite within a canaliculus allows the visualization of the space through which fluid flows and stimulates by shear stress the surface of the osteocyte cell membrane. This unique biological architectural characteristic of the osteocyte and the lacunocanalicular network represents the foundation that allows the conversion of mechanical stimuli into biochemical signals necessary for proper bone homeostasis.

The mechanical signals are translated into biochemical mediators that will assist with the orchestration of anabolic and catabolic events within bone. This arrangement allows osteocytes to (i) participate in the regulation of blood calcium homeostasis and (ii) to sense mechanical loading and to transmit this information to other cells within the bone to further orchestrate osteoblast and osteoclast function . The bone formation activity is consistently coupled to bone resorption that is initiated and maintained by osteoclasts. Osteoclasts are specialized multinucleated cells that originate from the monocyte/macrophage hematopoietic lineage (Fig. 4). These cells have the capacity to develop and adhere to bone matrix to then secrete acid and lytic enzymes that degrade and break down the mineral and organic components of bone and calcified cartilage. The matrix degradation process results in the formation of a specialized extracellular compartment known as Howship’s lacuna .

Fig. 4 Osteoclasts are derived from cells of the macrophage/monocyte lineage and represent the bone resorbing units within the skeleton. (A) Histologically,osteoclasts can be depicted morphologically as multinucleated cells attached to bone matrix or through special staining such as the tartrate-resistant acid phosphatase (TRAP) stain highlighted in red and emphasized by the arrows. (B) A transmission electron microscopy image of a multinucleated osteoclast attached to the mineralized bone matrix is depicted in this panel. (C)The black arrow depicts the ruffled border at the resorbing end of the cells. This cytoplasm is rich in proton pumps and chloride channels and delineates a highly acidic extracellular space. This space is often referred to as the “Howship’s lacuna” and is sealed by the cell through the formation of an actin ring. The increased traffic of enzymes, protons, and chloride ions from the cell toward this space through the ruffled border acidifies this compartment, allowing the organic and inorganic components of the bone to be broken down.

Composition of bone

Fig. 5 Bone is initially laid down as a purely organic matrix rich in collagen as well as in other noncollagenous molecules. (A) The transition between a purely organic matrix to a mineralized matrix is clearly depicted in this transmission electron micrograph as an osteocyte becomes embedded within the mature matrix. (B) As the matrix matures, mineral nucleation and propagation is mediated by the organic components available in the extracellular

matrix. The figure shows aggregation of mineral crystals, forming circular structures. As the mineral propagates along the collagen fibrils, a clear mineralization front forms and clearly demarcates the transition between the osteoid area and the mature bone. (C) Chemical analysis of bone by Raman spectroscopy clearly highlights the organic counterpart in the matrix that is rich in collagen and noncollagenous proteins. (D) Collagen type I is the most abundant protein in bone and is homogeneously distributed and maintained within the mature matrix as depicted by immunohistochemistry in this figure. (E) Other molecules such as DMP1 will tend to have a more specific spatiotemporal distribution. DMP1 is highly expressed by osteocytes and has been proposed to play an important role in bone mineral metabolism.

Fig 6. (A) Backscatter scanning electron imagines highlight mineral by a bright signal. Notice the different degrees of mineralization that is depicted within the mature bone. (B) Specific elements within the mineral can be further identified by energy-dispersive X-ray spectroscopy (EDS). In this figure, characteristic peaks of calcium and phosphorus are significantly pronounced in bone as expected due to their high content within the hydroxyapatite crystals. (C). Notice the punctate mineral distribution from the mineralization front to a more evenly mineralized matrix in the more mature lamenar bone. (D and E) EDS maps of calcium and phosphorus distribution surrounding an osteocyte close to the mineralization front (darker area closer to the lower left corner of the panels).

Bone is a specialized connective tissue that is composed of organic and inorganic elements (Figs. 5 and 6). The organic matrix of bone makes up approximately 30–35% of the total bone weight and is formed of 90% collagen type I and the remaining 10% is composed of noncollagenous proteins, proteoglycans, glycoproteins, carbohydrates, and lipids. The organic matrix is synthesized by osteoblasts, and while it is still unmineralized is known as osteoid. Within the collagen fibers, mineral nucleation occurs in the form of ions of calcium and phosphate that are laid down and ultimately form hydroxyapatite crystals. Noncollagenous proteins along the surface of the collagen fibers assist in the propagation of the mineral and the complete mineralization of thematrix. The initiation of the mineral nucleation within osteoid typically occurs within a few days of secretion,but the completion and maturation through the propagation of the hydroxyapatite crystals is completed over a course of several months and as new matrix is synthesized, a mineralization front is clearly formed and is easily depicted with the use of bone fluorochrome labels (Fig. 7). In addition to providing the bone with its

strength and rigidity to resist load and protect highly sensitive organs, the mineralization of the osteoid allows the storage of minerals that contribute to systemic homeostasis.

Fig. 7 The bone formation rate is commonly analyzed by the use of bone fluorochromes. Calcein (in green) and alizarin red signals depict the mineralization front when administered at different intervals, as shown in this figure.

Bone metabolismCalcium homeostasis is of major importance for many physiological processes necessary to maintain health. The balance of serum ionized calcium blood concentrations results from a complex interaction between parathyroid hormone (PTH), vitamin D, and calcitonin. Figure .8 reflects how input from the diet and from the bones and excretion via the gastrointestinal tract and urine maintain homeostasis. Vitamin D is involved in the absorption of calcium, while PTH stimulates calcium release from the bone, reduces its excretion from the kidney and assists in the conversion of vitamin D into its biologically active form (1,25-dihydr oxycholecalciferol). Decreased intake of

calcium and vitamin D and estrogen deficiency may also contribute to calcium deficiency (Lips, 2006). These hormonal factors have a major impact on the rate of bone resorption; lack of estrogen increases bone resorption as well as decreases the formation of new bone. Osteocyte apoptosis has also been documented in estrogen deficiency. In addition to estrogen, calcium metabolism plays a significant role in bone turnover, and deficiency of calcium and vitamin D leads to impaired bone deposition. It is also well known that the parathyroid glands react to low calcium levels by secreting PTH, which increases bone resorption to ensure sufficient calcium in the blood.

Fig.8 Calcium balance is optimized and regulated by a process that involves different systems. The red and green arrows reflect how input from the diet, the skeleton, and excretion via the gastrointestinal tract and urine maintain its homeostasis. Different hormonal signals modulate this process. Vitamin D is involved in the absorption of calcium, while PTH stimulates calcium release from the bone, reduces its excretion from the kidney, and assists in the activation of vitamin D. Decreased intake of calcium and vitamin D and estrogen deficiency may also contribute to calcium deficiency.

Modeling and remodeling

Fig. 9 Bone remodeling. The bone remodeling cycle involves a complex series of sequential steps that are highly regulated. The “activation” phase of remodeling is dependent on the effects of local and systemic factors on mesenchymal cells of the osteoblast lineage. These cells interact with hematopoietic precursors to form osteoclasts in the “resorption” phase. Subsequently, there is a “reversal” phase, during which mononuclear cells are present on the bone surface. They may complete the resorption process and produce the signals that initiate formation. Finally, successive waves of mesenchymal cells differentiate into functional osteoblasts, which lay down matrix in the “formation” phase.

Bone is a highly dynamic tissue that has the capacity to adapt based on physiological needs. Hence, bone adjusts its mechanical properties according to metabolic and mechanical requirements . The skeletal adaptation mechanism is primarily executed by processes of bone resorption and bone formation and referred to collectively as bone remodeling (Fig.9). Bone is resorbed by osteoclasts, after which new bone is deposited by osteoblastic cells. From the perspective of bone remodeling, it has been proposed that osteoclasts recognize and are targeted to skeletal sites of compromised mechanical integrity and initiate the bone remodeling process for the purpose of inducing the generation of new bone that is mechanically competent. The remodeling process takes place in bone multicellular units (BMUs) (Fig. 10). A BMU is composed of (i) a front of osteoclasts residing on a surface of newly resorbed bone referred to as the resorption front; (ii) a compartment containing vessels and pericytes; and (iii) a layer of osteoblasts present on a newly formed organic matrix known as the deposition front. In Fig. 10, the resorption front is clearly visualized by the cells stained for tartrate-resistant acid phosphatase (TRAP). The number of new and active BMUs is regulated by a variety of hormones and cytokines, which dictates the spatiotemporal synchronization and coupling of the anabolic and catabolic remodeling events. One of the best studied remodeling coupling mechanisms is the receptor activator of nuclear factor κB ligand (RANKL)-mediated activation of osteoclasts (Fig. 11). RANKL is a cytokine produced by osteoblasts and other cells (e.g. lymphocytes) and resides on the surface of osteoblast-like cells. The cells produce RANKL in response to systemic hormones (e.g., 1,25- dihydroxyvitamin D3) and cytokines (e.g., interleukin [IL]-6). Cell contact between cells expressing RANKL and osteoclast precursors expressing receptor activator of nuclear factor κB (RANK) induces osteoclast differentiation, fusion, and activation. Modulation of

this coupling mechanism occurs through a molecule known as osteoprotegerin (OPG). OPG binds RANKL before it has an opportunity to bind to RANK. Therefore, OPG suppresses the capacity to increase bone resorption. OPG\ has been proposed as an attractive therapeutic agent for the treatment of bone disorders, particularly osteoporosis conditions, as it represents a safe and controlled mechanism to modify the bone remodeling rate.

Fig. 10 Bone multicellular units (BMUs). Bone remodeling occurs in local groups of osteoblasts and osteoclasts called BMUs; each unit is organized into an osteoclasts reabsorbing front, followed by a trail of osteoblasts reforming the bone to fill the defect left by osteoclasts. The red staining (tartrate acid phosphatase) highlights the resorption front. Notice the increased number of multinucleated osteoclasts in this area.

Fig. 11 Bone formation/resorption coupling. Bone formation and resorption processes are mutually and intimately linked. The osteoblastic/stromal cells provide an osteoclastogenic microenvironment by the presentation of RANKL to the osteoclast precursor, triggering their further differentiation and fusion that lead to the formation of multinucleated and active osteoclasts. This process is modulated by inhibitors of these interactions such as the osteoprotegerin (OPG) molecule. In addition, the bone formation by osteoblasts depends on the preceding resorption by osteoclasts.

Bone healingHealing of an injured tissue usually leads to the formation of a tissue that differs in morphology or functionfrom the original tissue. This type of healing is called repair. Tissue regeneration, on the other hand, is a termused to describe a healing that leads to complete restoration of morphology and function. The healing of bone tissue includes both regeneration and repair phenomena depending on the character of the injury. For example, a properly stabilized, narrow bone fracture (e.g., green stick fracture) will heal by regeneration, while a larger defect in the bone will often heal with repair. There are certain factors that may interfere with the bone tissue formation following injury, such as1. Failure of vessels to proliferate into the wound2. Improper stabilization of the coagulum and granulation tissue in the defect3. Ingrowth of “nonosseous” tissues with a high proliferative activity4. Bacterial contaminationThe healing of a wound includes four phases:1. Blood clotting2. Wound cleansing3. Tissue formation4. Tissue modeling and remodelingThese phases occur in an orderly sequence but, in a given site, may overlap in such a way that in some

areas of the wound, tissue formation may be in progress, while in other areas tissue modeling is the dominatingevent. Although bone tissue exhibits a large regeneration potential and may restore its original structure and function completely, bony defects may often fail to heal with bone tissue. In order to facilitate and/or promote healing, bone grafting materials have been placed into bony defects. It is generally accepted that the biological mechanisms forming the basis for bone grafting include three basic processes: osteogenesis, osteoconduction, and osteoinduction.Osteogenesis occurs when viable osteoblasts and precursor osteoblasts are transplanted with the grafting material into the defects, where they may establish centers of bone formation. Autogenous iliac bone and marrow grafts are examples of transplants with osteogenic properties.Osteoconduction occurs when nonvital implant material serves as a scaffold for the ingrowth of precursor osteoblasts into the defect. This process is usually followed by a gradual resorption of the implant material.Autogenous cortical bone or banked bone allografts may be examples of grafting materials with osteoconductive properties. Such grafting materials, as well as bonederived or synthetic bone substitutes with or without growth factors, have similar osteoconductive properties (examples shown in Table 1). However, degradation and substitution by viable bone is often poor. If the implanted material is not resorbable, which is the case for most porous hydroxyapatite implants, the incorporation is restricted to bone apposition to the material surface, but no substitution occurs during the remodeling phase.Osteoinduction involves new bone formation by the differentiation of local uncommitted connective tissue cells into bone-forming cells under the influence of one or more inducing agents. Demineralized bone matrix(DBM) or bone morphogenetic proteins (BMPs) are examples of such grafting materials.It often occurs that all three basic bone-forming mechanisms are involved in bone regeneration. In fact, osteogenesis without osteoconduction and osteoinduction is unlikely to occur since almost none of the transmitted cells of autogenous cancellous bone grafts survive the transplantation. Thus, the grafting material predominantly functions as a scaffold for invading cells of the host. In addition, the osteoblasts and osteocytes of the surrounding bone lack the ability to migrate and divide, which in turn means that the transplant is invaded by uncommitted mesenchymal cells that later differentiate into osteoblasts. On that basis, it is appropriate to define three basic conditions as prerequisites for bone regeneration: 1. The supply of bone forming cells or cells with the capacity to differentiate into bone forming cells2. The presence of osteoinductive stimuli to initiate the differentiation of mesenchymal cells into osteoblasts3. The presence of an osteoconductive environment forming a scaffold upon which invading tissue can proliferate and in which the stimulated osteoprogenitor cells can differentiate into osteoblasts and form boneThe placement of bone grafting materials to favor healing in osseous defects or to augment atrophic alveolar ridges to allow dental implant installation has been evaluated in a number of experimental and clinical studies. It has been found that the osteogenic environment that will maximize the bone regenerative potential of traditional advanced grafting procedures is often affected by local as well as systemic factors. In some cases, the incorporation of the bone graft in the recipient site may be partially or completely impaired, and in turn there may be bone resorption and bone loss associated with the donor grafting material. As a consequence, much of the intended volume is lost, and frequently, the defects heal with fibrous connective tissue

instead of bone. Currently, a number of biologically active products are available clinically to overcome this potential limitation by enhancing cell proliferation and differentiation and by allowing a more rapid and predictable bone anabolic signaling process between the graft and the host tissue.

Table 1 Scaffold materials for periodontal/craniofacial repair

The role of growth factors used in oral bone repair: implications for implant site development

Following elevation of a mucoperiosteal flap for alveolar bone reparative procedures, a blood coagulum is formed at the wound site releasing tissue growth factors locally such as platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-β) from degranulating platelets. These mitogens attract mesenchymal stem cells (MSCs) and fibroblasts to migrate into the osseous wound site and stimulate proliferation and differentiation of osteoblastic precursors. The process of tissue neogenesis as described earlier in this chapter is followed by the formation of granulation tissue as a source for future connective tissue cells such as osteoblasts, periodontal ligament (PDL) fibroblasts, or cementoblasts . For alveolar bone regeneration, mesenchymal cells are transformed into osteoprogenitor cells by locally expressed BMPs Wound healing approaches using growth factors to target restoration of tooth-supporting bone, PDL, and cementum have advanced the field of oral and periodontal regenerative medicine. A major focus of oral and craniofacial reconstruction has evaluated the impact of growth factor delivery strategies using growth factorproducing cells, proteins, or genes encoding growth factors (Fig.12) .Table 1.2 highlights various

delivery systems, bone replacement graft biomaterials and growth factors used clinically. Advances in molecular cloning have made available unlimited quantities of recombinant growth factors for applications in tissue engineering of the craniofacial complex includingalveolar bone. Recombinant growth factors known to promote skin and bone wound healing, such as PDGFs, insulinlike growth factors (IGFs), fibroblast growth factors (FGFs) and BMPs have been used in preclinical and clinical trials for the treatment of large periodontal osseous defects, as well as around dental implants. BMP-2 is currently U.S. Food and Drug Administration (FDA)-approved for the repair of large tooth-extraction socket defects associated with local alveolar ridge repair and sinus floor augmentation procedures, while PDGF-BB is approved for therepair of large osseous defects associated with periodontally involved teeth and for soft tissue repair in recessiontype defects.

Fig. 12 Regenerative medicine approaches for cell, protein, and gene delivery to the craniofacial complex. DNA and growth factors can be delivered to cells through different mechanisms, including direct injection to an in vivo site, transport to a site via a carrier matrix, or introduced ex vivo prior to cell transplantation. Genetic material can be transferred into cells using different vectors, the most common of which are plasmids, retroviruses, adenoviruses, and adenoassociated viruses. Growth factors delivery by gene therapy strategies aim to modulate cell proliferation, migration, matrix synthesis, and differentiation.

Table 2: Effects of growth factors in the different phases of wound healing