chapter 16 Bone Biology, Osseointegration, and Bone Grafting
Bone is a living tissue that serves two primary functions—structural support and calcium metabolism.1 It has a collagen protein matrix that is impregnated with mineral salts, including calcium phosphate (85%), calcium carbonate (10%), and small quantities of calcium fluoride and magnesium fluoride.2 The collagen protein fibers that form the bone matrix are extremely complex. To maintain normal bone structure, there must be a sufficient quantity of both proteins and minerals. The minerals found in bone are present primarily in the form of hydroxyapatites. Bone mass is an important concept because bone is a mass-efficient structure in which maximal strength is achieved with minimal mass owing to its architecture. Unnecessary bone is lost (e.g., atrophy and bone loss seen in paraplegic patients) through a net loss effect as bone remodeling occurs. Bone also contains small quantities of noncollagen proteins embedded in its mineral matrix. The important family of bone morphogenetic proteins (BMPs) is part of this group.
Three different types of cells—osteoblasts, osteocytes, and osteoclasts—are related to bone metabolism and physiology. The three are closely related to each other and are derived from similar precursors (Figure 16-1).
FIGURE 16-1 ▪ Osteoclasts resorb bone as it ages, while osteoblasts deposit new bone simultaneously and adjacently. Once the osteoblasts deposit bone and become encapsulated, they are known as osteocytes.
Osteoblasts, which are associated with the process of osteogenesis, are located in two general areas next to the bone surfaces where they deposit bone matrix. Therefore, they frequently are referred to as endosteal osteoblasts or periosteal osteoblasts. The cytoplasm of osteoblasts is intensely basophilic, which suggests the presence of ribonucleoproteins related to bone matrix protein component synthesis. Fine granules, which can be observed in the cytoplasm, are closely related to the site of active matrix deposit (Figure 16-2).
When osteoblasts become embedded in the bone matrix, they transform into osteocytes, which have a slightly basophilic cytoplasm. Prolongations of this cytoplasm extend from the osteocyte, through a network of fine canaliculi that emerge from the lacunae, to a specific distance. During bone formation, these prolongations extend beyond their normal limit, and a direct contiguity, or continuity, with adjacent osteocytes is evident. In mature bone, almost no extension of these prolongations is seen, but the canaliculi continue to function as a means of metabolic and biochemical messenger exchange between the blood system and the osteocytes.
The system of canaliculi connects the osteocyte lacunae with each other and the tissue spaces. Tissue fluid in these spaces mixes with fluid from the canaliculi, allowing a metabolic and biochemical messenger exchange between the bloodstream and the osteocytes. This mechanism allows the osteocytes to remain alive, regardless of the calcified intercellular substance that surrounds them. However, this duct system is not functional if it is located more than 0.5 mm from a capillary, which is why such an abundant blood supply is found in bone through capillaries that run through Haversian systems and Volkmann’s canals.
Osteoclasts are fused monocytes that appear histologically as multinucleated giant cells located in shallow excavations (Howship’s lacunae) along the mineralized surface.3 The cytoplasm of osteoclasts is slightly basophilic and granular, with characteristic vacuoles. Osteoclasts are responsible for bone resorption and form in response to parathyroid hormone. After the process of local bone resorption is complete, osteoclasts disappear, probably by degeneration.
Bone, the primary reservoir of calcium, has a tremendous turnover capability for responding to the metabolic needs of the body and is critical for maintaining a stable serum calcium level.1,2 Because calcium participates in many reactions, it has an essential life support function. It works in conjunction with the lungs and kidneys to help maintain the pH balance of the body through the production of additional phosphates and carbonates, as well as by electrical charge conduction in nerve and muscle, including cardiac muscle. In addition, the metabolic environment is an extremely important component of the biomechanical structure of bone. Bone undergoes continuous turnover in response to metabolic reactions, with the skull and jaws unquestionably affected by this turnover.
The structural integrity of bone may be compromised in times of normal metabolic calcium need and in disease states, thus altering bone structure and mass. This phenomenon can be noted in the bone structure of postmenopausal women, who experience a decrease in estrogen hormones. As bone mass is lost, the interconnections between bone trabeculae also are lost. Because normal interconnections play an important role in making bone a biomechanically rigid structure, this decrease leads to fragility.
Metabolic/hormonal interactions play an important role in the maintenance of bone structure, the most important of which is the linkage of bone resorption to new bone apposition through BMP in normal daily remodeling of bone. Approximately 0.7% of a human skeleton is resorbed daily and is replaced by new, healthy bone. Therefore, a turnover in the entire skeleton occurs approximately every 142 days. When osteoblasts lay down bone, they also secrete BMP into the mineral matrix. This acid-insoluble protein resides there until it is released by osteoclastic resorption. This acid insolubility is an evolutionary mechanism by which the pH of 1 created by osteoclasts is able to dissolve bone mineral without affecting BMP.4 Released BMP then is bound to the cell surface of undifferentiated mesenchymal stem cells, where it causes a membrane signal protein to become activated with high-energy phosphate bonds. This activation, in turn, affects the gene sequence in the nucleus, causing expression of osteoblast differentiation and stimulation of new bone production. A disturbance in this linkage may be the center point of osteoporosis.
Aging and metabolic disease states may reduce the normal turnover process, causing an increase in the mean age of the present bone. This increase may lead to fatigue, bone damage, compromised bone healing, failure to integrate implants, or loss of integration with an implant.5 Thus, it becomes very important for surgeons to realize that a compromised status may not be recognized until the clinician attempts to place implants, or until the implants have been in place for some time.
The macroscopic structure of bone is a continuum from dense cortical tissue to fine trabecular tissue (Figure 16-3). Between the two ends of this spectrum, no histological difference is seen in the type of bone, only in the relative amount of solid substance present and the geometrical fashion in which it is laid down (the size and number of spaces within it). In most cases, both cortical and trabecular tissues are found at every bone site, but the quantity and distribution of each may vary.
Cortical or compact bone is found in the diaphysis of long bones and on the external surface of flat bones (Figure 16-4). This tissue is organized in bony cylinders consolidated around a central blood vessel (referred to as a Haversian system). Trabecular, spongy, cancellous bone occupies substantial space within the bony tissue that constitutes the medullary cavity of the bone (Figure 16-5). The medullary cavities are filled with marrow: red marrow when there is active production of blood cells or a reserve population of mesenchymal stem cells, and yellow marrow when the cavity has been converted into a site for fat storage with age (Figures 16-6, 16-7).
FIGURE 16-4 ▪ Cortical bone is found on the external surfaces of bone. The tibia has a wide plateau region with a bump (Gerdy’s tubercle), which is the site of entry into the cancellous portion of the tibia. The bone is harvested across the wide portion of the tibia and inferiorly, avoiding thinning of the superior weight-bearing portion of the tibia.
FIGURE 16-5 ▪ Cancellous bone is found internally in bone. The cancellous bone structure is generally extremely porous.
FIGURE 16-6 ▪ Different stages of endochondreal ossification, from all cartilage to all bone with a minimal cartilage layer.
FIGURE 16-7 ▪ The periosteum, a connective tissue membrane surrounding cortical bone, should be repositioned carefully, so that its osteogenic potential after surgery can nurture the graft and/or underlying bone.
Except for the articular surfaces, the bone surface is covered with periosteum, which is composed of two layers of specialized connective tissue. The outer fibrous layers provide toughness to the periosteum because of its configuration of mainly dense collagenous fibers and fibroblasts. This layer is rich in nerve fibers and blood supply. The inner cellular layer, which is in direct contact with the bone, contains functioning osteoblasts and often is referred to as the cambium layer. The medullary cavities and spaces are covered by endosteum, which consists of a single layer of osteoblasts that form a very thin, delicate membrane. The endosteum is architecturally similar to the cellular cambium layer of the periosteum because of the presence of osteoprogenitor cells, osteoblasts, and osteoclasts.
At the microscopic level, four types of bone are present: woven, composite, lamellar, and bundle. Woven bone plays a principal role during healing. The ability of woven bone to form quickly (at a rate of approximately 30 to 60 m per day) is its main property. However, because woven bone is formed so rapidly, it develops in a disorganized fashion without lamellar architecture or Haversian systems and therefore is soft. As a result, woven bone has low biomechanical strength. Although it often is referred to in the literature as “embryonic bone,” this term is somewhat misleading because all adults have the ability to form this type of bone. Instead, woven bone is referred to as phase I bone during bone healing.6 Although woven bone (phase I bone) is laid down quickly, it normally does not last very long because it is not biomechanically sound. Obligatory resorption and replacement with more mature bone, termed phase II or lamellar bone, occurs.6–8 The term “composite bone” is used to describe the transitional state between woven bone (phase I bone) and lamellar bone (phase II bone). It is a woven bone lattice that is filled with lamellar bone.
Lamellar bone (phase II bone) is the principal, mature, load-bearing bone in the body, and this bone is extremely strong. Because it forms very slowly (at a rate of approximately 0.6 to 1.0 µm per day), it is well organized in its collagen structure and, thus, in its mineralized structure. Lamellar bone consists of multiple, oriented layers. Bundle bone is the principal bone found around ligaments and joints and consists of striated interconnections with ligaments.
At the molecular level, bone is a composite material. It is a cross-linked collagen matrix that consists of a three-dimensional multiple arrangement of matrix fibers. The orientation of collagen fibers determines the mineralization pattern. In this way, bone adapts to its biomechanical environment and projects maximal strength in the direction that is being loaded; this is the primary role of collagen fibers.
Intercellular bone substance has the homogenous aspect of an organized structure. The organic portion occupies 35% of the matrix and is formed primarily by osteocollagenous fibers, which are similar to collagen fibers in connective tissue. These fibers are joined by a cement-like substance, consisting mainly of glucosaminoglycan (protein-polysaccharide).
Sixty-five percent of bone weight corresponds to its inorganic component, localized only in the interfibrinous cement. Minerals are found predominantly in the form of calcium phosphate crystals with an apatite structure that corresponds to hydroxylapatite. These minerals form deposits of dense particles along the osteocollagenous fibers. The lacunae and ducts are covered by a thin layer of special organic cement that differs from the rest of the intercellular substance by its lack of fibers.
Calcified bone protein matrix consists of mineral components (65%), mainly hydroxylapatite, and nonmineral components (collagen [35%] and other proteins and peptides [5%]). These other proteins and peptides, such as BMP, regulate how bone is laid down and maintained. Bone matrix has the characteristic aspect of sequential layers that vary in thickness from 300 to 700 µm. These layers are the result of a rhythmical and uniform deposition of matrix. The fibers within each layer are parallel, with a spiral orientation that changes between layers, so that the fibers in one layer run perpendicular to those in the adjacent layer. This alternate disposition in fiber directions explains the division that occurs between layers.
Bone modeling is defined as any change in the form, size, or shape of bone. It can be an anabolic process with apposition of bone on the surface, or it can be a catabolic process with resorption of the surface. Because these two processes can occur separately on different surfaces, bone modeling is a surface-specific phenomenon that occurs during growth, as part of wound healing (e.g., during stabilization of an endosseous implant), and in response to bone loading. Modeling is an uncoupled process in which formation does not have to be preceded by resorption. Activation of cells to resorb or form bone can occur within the same bone on different surfaces. An example of this phenomenon is the orthodontic movement of a tooth, wherein the force applied results in bone resorption on the tooth surface and bone formation on the opposite surface, resulting in tooth movement with surrounding bone and not through the alveolus.
Bone modeling may be controlled by mechanical factors, as is the case with orthodontic tooth movement, or by growth factors, as is the case with bone healing, bone grafting, and osseointegration. Microstrain (ME) is a method of measuring the load applied to bone as percent deformation of tissue. For example, a load of 200 ME produces a deformation of 0.2% of the tissue. Between the range of 200 and 2,500 ME, there is normal functional response, in which strong bone is produced that is effective in facing increased loads. Atrophy occurs in cases in which the force is low (i.e., less than 200 ME). When the load is between 2,500 and 4,000 ME (i.e., a deformation of 0.25% to 0.4%), hypertrophy occurs, and there is a change in the size of the bone segment.9 The modeling that occurs during hypertrophy is lamellar bone formation. If the load exceeds 4,000 ME, there is a pathological overload, and the modeling that occurs is woven bone formation. In this situation, the bone responds as quickly as possible to meet the excessive load by producing the tissue that can be formed the fastest (i.e., woven bone, which has limited load-bearing capacity).9
The effects of biochemical control and growth factor influences can be seen in the two respective models of bone graft healing and osseointegration. For the purpose of a clear-cut understanding of the intricacies of bone formation and remodeling, the bone graft healing model will be discussed first.
Placement of a graft that consists of endosteal osteoblasts and stem cells (from a donor site such as the ileum or tibial head), and that is surrounded by a vascular and cellular tissue bed, creates a recipient site with a biochemistry that is hypoxic (O2 tensions, 3 to 10 mm Hg), acidotic (pH 4.0 to 6.0), and rich in lactate.10 The graft itself contains the osteocompetent cell populations, as well as islands of mineralized cancellous bone, fibrin from blood clotting, and platelets within the clot (Figure 16-8).
FIGURE 16-8 ▪ Bone graft material mixed with platelet rich plasma (PRP) in a syringe to be carried to the graft recipient site.
The endosteal osteoblasts and marrow stem cells survive the first 3 to 5 days largely because of their surface position and ability to absorb nutrients from recipient tissues. Osteocytes within the mineralized cancellous bone die as a result of their encasement in mineral, which serves as a nutritional barrier. Because the graft is inherently hypoxic (5 to 10 mm Hg) and the surrounding tissue is normoxic (50 to 55 mm Hg), an oxygen gradient that is greater than the 20 mm Hg (usually 35 to 50 mm Hg) required to stimulate macrophage chemotaxis is set up, and, in turn, the macrophages are stimulated to secrete macrophage-derived angiogenesis factor (MDAF) and macrophage-derived growth factor (MDGF).
Within the graft, the platelets entrapped within the clot degranulate within hours of graft placement, releasing platelet-derived growth factor (PDGF). Therefore, the inherent properties of the wound, particularly the oxygen gradient phenomenon and PDGF, initiate early angiogenesis from surrounding capillaries and mitogenesis of transferred osteocompetent cells11.
By day 3, capillary buds from existing capillaries outside of the graft can be seen. They penetrate the graft and proliferate within the cancellous bone network to form a complete network by day 10 to 14. As these capillaries respond to the oxygen gradient, MDAF messengers effectively reduce the oxygen gradient as they perfuse the graft, thus creating a shut-off mechanism to prevent over-angiogenesis.
Although PDGF seems to be the earliest messenger to stimulate early osteoid formation, PDGF probably is replaced by MDGF and other mesenchymal tissue stimulators from the transforming growth factor-beta family. During the first 3 to 7 days, stem cell populations and endosteal osteoblasts produce only a small amount of osteoid. After the vascular network has been established, osteoid production accelerates, presumably as a result of oxygen and nutrient availability. The initial osteoid that forms develops on the surface of the mineralized cancellous trabeculae from the endosteal osteoblasts. Shortly thereafter, individual osteoid islands develop between cancellous bone trabeculae, presumably from stem cells transferred within the graft. A third source of osteoid production develops from circulating stem cells, which also are attracted to the biochemical environment of the wound.12 These stem cells are postulated to seed the graft and proliferate, thereby contributing to osteoid production.
Throughout the first 3 to 4 weeks, this biochemical and cellular phase of bone regeneration proceeds to coalesce individual osteoid islands, surface osteoid on cancellous trabeculae, and host bone to clinically consolidate the graft. This process uses the fibrin network of the graft as a framework. Normally nonmotile cells, such as osteoblasts, may be somewhat motile via the process of endocytosis along a scaffold such as fibrin. The process of endocytosis is merely the transfer of the cell membrane from the retreating edge of the cell through the cytoplasm as a vesicle to the advancing edge to re-form a cell membrane. This process slowly advances the cell and allows it to secrete its product in the process. In this case, the product is osteoid on the fibrin network. This cellular regeneration phase often is referred to as phase I bone regeneration.6 By the time regeneration is nearly complete (4 to 6 weeks), sufficient osteoid production and mineralization have occurred to permit graft function. Bone at this stage has formed without going through a chondroblastic phase and histologically appears as random cellular bone, which a pathologist would refer to as woven bone.13
Because the amount of bone formed during phase I depends on osteocompetent cell density, donor sites with the highest cancellous trabecular bone areas are chosen. In rank order, it has been shown that the posterior and anterior ileum, tibial plateau, femoral head, and mandibular symphysis are potential donor sites with greater availability of cancellous bone than the calvarium, rib, or fibula.14 In addition, enhanced phase I bone yields are achieved by compacting the graft material. Technically, this enhancement often is accomplished with the use of a bone mill, followed by compaction in a syringe and then further compaction into the graft site using bone-packing instruments.
As was previously stated, the biochemistry of the recipient tissue and the graft itself is largely inherent. However, studies and experience with platelet-rich plasma (PRP) additions to the graft have shown early consolidation and graft mineralization in half the time, with a 15% to 30% improvement in trabecular bone density.12 The concept is that PRP, which is a fibrin clot (also called fibrin glue), is rich in platelets, which, in turn, release PDGF. It has been theorized that this enhanced quantity of PDGF initiates the osteocompetent cell activity more completely than what will inherently occur in the graft and clot milieu alone. Additionally, the enhanced fibrin network created by PRP is believed to enhance osteoconduction throughout the graft, supporting graft consolidation.
The cellular bone regeneration that occurs during phase I is disorganized woven bone that is structurally sound but is not structurally on par with mature bone. The random organization and hypercellular nature of this bone are similar to those seen in a fracture callus. This bone will undergo obligatory resorption and replacement remodeling. Eventually, it will be replaced by phase II bone, which is less cellular, more mineralized, and more structurally organized (Figure 16-9).6,13
FIGURE 16-9 ▪ There is a sequence of events that takes place over time for bone remodeling/formation.
As occurs with all bone remodeling, the replacement of phase I bone by phase II bone is initiated by osteoclasts, which are fused mononuclear cells that arrive at the graft site though the newly developed vascular network.3 It has been postulated that these osteoclasts resorb phase I bone in a normal remodeling-replacement cycle. BMP is released during resorption of both the newly formed phase I bone and the nonviable original cancellous trabecular bone. As with normal bone turnover, BMP acts as the link or couple between bone resorption and new bone apposition. Stem cells in the graft from the original transplantation and newly arrived stem cells from local tissues and the circulation respond by osteoblast differentiation and new bone formation. New bone forms as the jaw and graft function, developing in response to the demands placed on it. This bone develops into mature Haversian systems and lamellar bone that is capable of withstanding the normal shear forces placed on the jaw through opening and closing functions, and it tolerates impact compressive forces that are typical of denture-borne and implant-borne prosthetic functions. Histologically, such grafts are involved in long-term remodeling that is consistent with normal skeletal turnover. A periosteum and an endosteum develop as part of this long-term remodeling cycle. Radiographically, the graft takes on the morphology and cortical outlines of the mandible or maxilla over several years.
When a roughened surface implant is placed into the cancellous marrow space of the mandible or maxilla, only a small amount of bone from the trabecular bone within the marrow is in contact with the metal surface of the implant. The remaining surface of the implant is exposed to the fibrofatty marrow space. The initial response seen is a migration of osteoblasts and osteoid production to the implant surface. The source of these osteoblasts is surface endosteal osteoblasts of the trabecular bone and the inner surface of the buccal and lingual cortex. It is probable that these cells are responding to the release of BMP from surgical placement of the implant and the initial resorption of bone crushed against the metal surface. The osteophyllic phase lasts for approximately 1 month (Figure 16-10).
After contact, the bone cells spread along the metal surface (osteoconduction), laying down osteoid. The bone that is deposited is often a thin layer. This phase continues over the next 3 months as more bone is added to the total surface area of the implant. At this time (4 months after initial placement), the maximum surface area is covered by bone, and after this, no further increase in bone to the surface area is observed.
The final phase, the osteoadaptive phase, begins approximately 4 months after implant placement, at the same time that the osteoconductive phase ends. It is associated with a steady state (no gain or loss of bone against metal) resorption remodeling sequence that continues even after the implants are exposed and loaded (Figure 16-11).
One of the first definitions of osseointegration was a “direct functional and structural connection between living bone and the surface of a load-bearing implant.” However, perspectives on what constitutes osseointegration may vary.