Histologic Components

On a gross level, all bones are composed of two basic components: cortical (compact) bone and cancellous (trabecular) bone. Cortical bone is a dense, solid mass, except for its microscopic channels, and contains parallel stacks of curved sheets called lamellae, which are separated by bands of interlamellar cement. Regularly spaced throughout the lamellae are small cavities, or lacunae . Lacunae are interconnected by thin, tubular channels called canaliculi. Entrapped bone cells (osteocytes) are located in the lacunae, and their long, cytoplasmic processes occupy canaliculi. The cell processes within canaliculi communicate by gap junctions, with processes of osteocytes lying in adjacent lacunae. Canaliculi open to extracellular fluid at bone surfaces, thus forming an anastomosing network for the nutrition and metabolic activity of the osteocytes. Cortical bone possesses a volume fraction of pores less than 30% and has an apparent density of up to about 2 g/mL. Its compressive strength is approximately 10-fold that for a similar volume of cancellous bone.

Cancellous bone is porous and appears as a lattice of rods, plates, and arches individually known as trabeculae. It has a greater surface area and can be readily influenced by adjacent bone marrow cells. Because of this structural difference, cancellous bone has a higher metabolic activity and responds more readily to changes in mechanical loads.

Cortical and cancellous bone may consist of woven (primary) or lamellar (secondary) bone. Woven bone forms the embryonic skeleton and is then resorbed and replaced by mature bone as the skeleton develops. In the adult, woven bone is found only in pathologic conditions, such as fracture healing and in tumors. Woven and lamellar bones differ in formation, composition, organization, and mechanical properties. Woven bone has an irregular pattern of collagen fibers, contains approximately four times as many osteocytes per unit volume, and has a rapid rate of deposition and turnover. The osteocytes of woven bone vary in orientation, and the mineralization of woven bone follows an irregular pattern in which mineral deposits vary in size and in their relationship to collagen fibrils. In contrast, the osteocytes of lamellar bone are relatively uniform, with their principle axis oriented parallel to that of other cells and to the collagen fibrils of the matrix. The collagen fibrils of lamellar bone lie in tightly organized, parallel sheets, with uniform distribution of mineral within the matrix.

The irregular structure of woven bone makes it more flexible, more easily deformed, and weaker than lamellar bone. For these reasons the restoration of normal mechanical properties to bone tissue at the site of a healing fracture requires eventual replacement of the woven bone of the fracture callus with mature lamellar bone.

Incorporation of Bone Graft

Bone graft incorporation is a prolonged process with a sequence of complicated steps involving the interrelationship of the graft and host. This ultimately leads to the envelopment of a complex of necrotic old bone with viable new bone. The complex develops through resorption of the necrotic old bone with viable new bone being laid down. The incorporation of the bone graft is a dynamic process involving the following processes: osteoinduction, osteoconduction along with the availability of osteogenic cells, and the structural integrity, which provides mechanical support. This ultimately leads to the replacement of the graft by host bone in a predictable pattern under the influences of load bearing.

At the beginning, the inflammatory response at the host-graft interface results in migration of inflammatory cells and fibroblasts into the bone graft. In addition, the developing hematoma enhances the release of both cytokines and growth factors. Osteoinduction is the process whereby a tissue is influenced to form osteogenic elements through chemotaxis, mitosis, and differentiation of the host osteoprogenitor cells. Induction requires an inducing stimulus, such as a piece of bone or an osteogenic cell, and an environment favorable for osteogenesis. Osteoconduction is the process by which capillaries, perivascular tissue, and osteoprogenitor cells from the recipient bed grow into the graft. It can occur within a framework of nonbiologic materials or nonviable biologic materials. In viable bone grafts, osteoconduction is facilitated by osteoinductive processes and therefore occurs more rapidly than in nonviable or nonbiologic materials. Ultimately, this process results in the resorption of the original graft tissue and replacement with new host bone. Remodeling is a response to weight bearing.

Biomechanics of Graft Incorporation

Porosity is a dominant factor in determining the material properties of bone. It is directly related to the stiffness of the tissue and yield of strength. Therefore, any change in porosity results in important effects on the bone graft material properties. Cortical bone grafts initially may have as little as 5% to 10% porosity, whereas cancellous grafts may be as high as 70% to 80%. This explains the material strength of cancellous graft, which is roughly equivalent to 4% of that of cortical bone.

Cancellous grafts are incorporated by an early appositional phase. New bone formation onto the necrotic trabeculae of the graft tissue leads to an early increase in graft strength. It has been shown that necrotic bone maintains its mechanical strength. Cancellous grafts therefore initially strengthen with the addition of new bone. As the necrotic cores are resorbed, the mechanical strength of the graft area normalizes.

Cortical bone grafts first undergo osteoclastic bone resorption, which significantly increases graft porosity and thus decreases the graft strength. In the canine model of autogenous cortical transplant, the greatest compromise in mechanical strength occurs at 12 weeks ( Fig. 28-1 ). The strength returns to normal between 1 and 2 years after transplantation.

Graph illustrating the quantitative temporal interrelationships between the physical integrity and the biologic processes of repair within a segmental autogenous cortical bone transplant. The initial persistence of strength (0 to 4 weeks after transplantation) indicates the subsequent loss was caused by reparative processes rather than any intrinsic weakness in the material. The sudden loss in strength at 6 weeks is caused by the increased internal porosity. From 6 to 12 weeks, the decrease in mechanical strength is reduced by 50%. The level of porosity continues to increase until week 12 because of the temporal lag in the apposition of new bone formation. At 24 weeks, there is no significant improvement in strength, despite the beginning reduction in the porosity of the transplant and maturation of the callus. At 48 weeks, however, the physical integrity of the transplant has returned toward normal, primarily as the result of decreased material porosity, as the amount of callus has not increased. By 2 years, the physical integrity of the transplant has returned toward normal, primarily as a result of decreased material porosity, because the amount of callus has not increased. By 2 years, the physical integrity of the transplant and the internal porosity of the remaining transplanted material are normal. The biologic completeness of repair (i.e., approximately 50% of the graft is viable) is not significant, because mechanical strength has been retained. The admixture of necrotic and viable bone remains for the life of the individual’s skeletal metabolic activity.

(From Burchardt H: Biology of cortical bone graft incorporation. In Friedlaender GE, Mankin HJ, Sell KW, editors: Osteochondral allografts: biology, banking, and clinical applications, Boston, 1983, Little, Brown, p 55.)

Temporal Profile of Graft Incorporation

During the first week after grafting, both cancellous and cortical grafts have similar histologic features. Both are surrounded by coagulated blood, and the graft is the focus of a tissue response characterized by vascular buds infiltrating the grafted bed. By the second week, fibrous granulation tissue becomes increasingly dominant in the graft bed, the number of inflammatory cells decrease, and osteoclastic activity increases. Within the confines of the graft, osteocytic autolysis proceeds, resulting from anoxia and injury by surgery, with necrosis delineated by vacant lacunae. Some cells, however, survive by diffusion of nutrients from surrounding host tissues. Creeping substitution of cortical bone grafts progresses transversely and parallel to the long axis of the transplanted segment. Thus, the repair is found to be greater at the graft-host junctions.

A study done in rabbits showed the sequence of events during the process of dorsolateral intertransverse fusion. Three phases were identified. Phase 1 represents the early reparative phase (1 to 3 weeks) . It consists of hematoma formation and granulation tissue. There is minimal ossification. Phase 2 represents the middle reparative phase (4 to 5 weeks) , when the fusion solidifies. Finally, phase 3 represents the late remodeling phase (6 to 10 weeks) .

Both membranous and enchondral ossification play a role in the fusion process. Membranous ossification is the predominant mechanism that begins at the termini of the fusion mass and emanates from the decorticated transverse process. The central portion of the fusion mass, where the vascular supply is poorer and movement is greater, heals by cartilage formation and enchondral ossification.

Host Response and Incorporation of Autograft and Allograft

Autograft remains the gold standard in most fusion applications. In certain situations in which available autologous bone is insufficient or when large structural grafts are needed, allograft fusion rates can approach or equal those of autograft rates, without donor site morbidity. A successful spine fusion requires a sufficient area of decorticated host bone, ample graft material, minimal motion at the fusion site, and a rich vascular supply.

Histocompatibility matching has an important influence on the process of incorporation. Allograft that is mismatched for major histocompatibility complex antigens functions poorly compared with autogenous grafts. Bone cells display class I and class II histocompatibility antigens, and there are both cellular and humoral responses to bone allografts. Syngeneic grafts are the most successful. Grafts with major histocompatibility mismatch have delayed and incomplete revascularization, compared with syngeneic grafts. In addition, marked resorption of bone often occurs, resulting in almost complete loss of graft. Freezing the graft, followed by thawing, disrupts and kills the cells. It mutes the antigenicity in major mismatches and thus enhances incorporation of such grafts. However, the killing of cells also diminishes the biologic activity of the graft. It is the osteoinductive component that is mainly affected. The function of an allograft as an osteoconductive system seems virtually unimpaired.

In fresh cancellous allografts, the initial phase consisting of hemorrhage and necrosis is identical to that of the autograft. The fibrin clot and the same inflammatory response develop. However, in the allograft the fibrin clot breaks down, and the granulation tissue, which provides nutrition to the repair site, is invaded by chronic inflammatory cells rather than fibroblasts and blood vessel elements. The major portion of the delay appears to occur in osteoclastic resorption and new bone formation. Final graft incorporation remains incomplete.

In cortical allografts the length of time of creeping substitution is greatly prolonged. The invasion by host vessels and recruitment and differentiation of cellular elements to become osteoblastic and osteoclastic cells are greatly diminished. The proportion of necrotic graft bone to viable host bone is much greater in allogeneic grafts. In fact, the active process of graft substitution may last several years.

Modeling and Remodeling Associated with Spine Fusion

Bone modeling associated with spine fusion is extremely complex. Variables that may affect bone remodeling after graft insertion include (1) the design of implant, materials used, and methods of fixation; (2) the local bone, including its density and shape; and (3) the patient characteristics, including age, gender, hormonal balance, and activity. Osteoblasts and osteoclasts are influenced by the magnitude and state of strain imposed on them by load applied to the bone. Stresses or strains within a given range seem to be required to maintain a steady state remodeling of bone in which the rate of bone formation equals the rate of resorption. Stresses below the optimum are often associated with stress shielding, leading to bone resorption. Stresses and strains exceeding upper limits can also produce resorption of bone as a result of pressure necrosis. Cyclic stresses are required to maintain osseous homeostasis. Constant loads, even when within the desired range, can result in insufficient stimulus to maintain bone mass. Observations of strain-related electric potentials in bone, biopotentials, and electrical stimulation of osteogenesis suggest that bioelectric phenomena function as the regulators of adaptive remodeling of bone.

Growth Factors and Cytokines in Regulating Bone Remodeling

Bone cells carry out diverse functions and are mainly derived from two cell lines: mesenchymal and hematopoietic. The mesenchymal stem cell line consists of undifferentiated cells, or preosteoblasts, that differentiate into osteoblasts, bone-lining cells, and osteocytes. The hematopoietic stem cell line consists of circulating marrow monocytes that differentiate into preosteoclasts and osteoclasts. These cells are regulated by various cytokines.

Bone formation in spine fusion is a complex and regulated process. The cellular events involved in bone formation include chemotaxis of osteoblast precursors, proliferation of committed osteoblast precursors, and differentiation and expression of regulatory factors and structural proteins of bone and mineralization. These processes require tight regulatory control. They may be modulated by systemic hormones, such as parathyroid hormone, but predominant control is by local factors or cytokines. Cytokines are small proteins that serve as signaling agents for cells. Cytokines are classified based on their cellular origin and principal biologic activities. The main families include interleukins, tumor necrosis factors, growth factors, colony-stimulating factors, interferons, and chemokines.

Bone morphogenetic protein (BMP) is a member of the transforming growth factor beta (TGF-β) superfamily. The BMP constitutes a growth family of more than 12 proteins, 9 of which have been shown individually to induce ectopic bone formation. They are water soluble, noncollagenous substances found in the bone matrix with osteoinductive activity. BMPs 2, 4, and 7 are specially increased in the primitive mesenchymal and osteoprogenitor cells, fibroblasts, and proliferating chondrocytes present at the fracture site. During the phases of healing, the expression of BMPs 2, 4, and 7 is strongly present in undifferentiated mesenchymal cells during the inflammatory phase. During intramembranous ossification, these BMPs are strongly present in the proliferating osteoblasts. During chondrogenesis and endochondral ossification, BMPs 2 and 4 are found in proliferating chondrocytes and strongly in osteoblasts near the endochondral ossification front. BMP 7 is found in later stages of healing in proliferating chondrocytes and weakly in mature chondrocytes. BMPs also affect the expression of other growth factors that may mediate the effects of BMPs on bone formation.

They are the most widely investigated osteoinductive growth factor in spine fusion. Several animal studies have shown that recombinant human (rh) BMPs 2, 4, and 7 induce bone formation at an orthotopic site at which the integration with the preexisting bone is structurally sound. It has also been shown that BMP plus marrow yields the highest union rates (100%) and is three times better than autogenous cancellous graft.

However, other factors may play a role in the biologic response to rhBMP in vivo, including the time course activity of rhBMP on the bone formation process, its interaction with other growth factors, and the influence of delivery vehicles.