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Bone Regeneration in Membrane-Protected Defects

Dieter D. Bosshardt, MSc, PhD | Simon S. Jensen, DDS, Dr odont | Daniel Buser, DDS, Dr med dent

A sufficient amount of living bone is required for both the esthetic outcome and the long-term success of dental implant therapy. In about 50% of implant sites, however, there is a need for a procedure that predictably generates enough bone volume for the placement of a dental implant. There are several options for the enhancement of bone formation, including (1) osteoinduction by autogenous bone grafts or the addition of growth factors; (2) osteoconduction provided by autogenous bone grafts or bone substitutes that serve as a scaffold for new bone formation; (3) transfer of stem cells or progenitor cells that differentiate into osteoblasts; (4) distraction osteogenesis; and (5) guided bone regeneration (GBR) using barrier membranes. Regardless of the method used, there is always an underlying basic biologic mechanism of bone healing.

Bone demonstrates a unique potential for regeneration, which is probably best illustrated by fracture repair. Bone is able to heal fractures or local defects with regenerated tissue of equally high structural organization, without leaving a scar. The mechanism of this healing pattern is often considered as a recapitulation of embryonic osteogenesis and growth. Because bone has a unique spontaneous healing capacity, the trick in reconstructive surgery is to harness this great regenerative potential to enhance bone formation for clinical applications. Thus, adequate bone augmentation or treatment of any bone defect requires a profound understanding of bone development and morphogenesis at the cellular and molecular levels. This chapter summarizes the development, structure, function, and regeneration of bone and discusses the pros and cons of the various biomaterials used for GBR to provide the biologic rationale for selecting appropriate biomaterial combinations for successful bone augmentation around dental implants in the long term.

Development and Structure of Bone

Functions

Bone certainly represents a great achievement in the evolution of supporting tissues. However, it has many additional functions. These include (1) mechanical body support, motion, and locomotion; (2) support of teeth for biting and crushing of food; (3) support and protection of the brain, spinal cord, and internal organs; (4) housing of bone marrow, which is the source of hematopoietic cells; and (5) calcium homeostasis. It is probably because these functions are of vital importance that bone possesses an exceptional capacity for self-healing, repair, and regeneration.

Bone types and structural organization

The mammalian skeleton consists of long bones and flat bones. Based on the orientation of the collagen fibrils, three types of bone tissue can be identified: woven bone, lamellar bone, and an intermediate type—the primary parallel-fibered bone.

Woven bone is formed predominantly in embryos and growing children and is replaced later by lamellar bone. In adults, woven bone reappears when accelerated bone formation is required, as in the bony callus during fracture repair and in pathologic conditions like Paget disease of bone, renal osteodystrophy, hyperparathyroidism, or fluorosis. In woven bone, the collagen fibrils are oriented in a random manner, and the interfibrillar spaces are comparatively wide.¹ Besides having intertwined collagen fibrils, woven bone is characterized by a high number of large osteocytes and a high mineral density (Fig 2-1).

Fig 2-1 Light microscopic view of woven bone. This type of bone forms struts and ridges, which are always closely associated with blood vessels (BV) (Goldner trichrome stain).

Lamellar bone possesses a much more complex structure, characterized by matrix layers that consist of a parallel collagen fibril arrangement. One lamellar unit is about 3 to 5 µm wide, and the orientation of the fibrils changes from one lamella to another (Fig 2-2). Thus, lamellar bone may be regarded as a complex, plywood-like structure.²

Fig 2-2 Primary and secondary osteons in equine cortical bone. In polarized light, secondary osteons (asterisk) reveal a clear lamellar pattern. The wall of primary osteons consists of primary parallel-fibered bone, which is less birefringent.

Primary parallel-fibered bone is deposited in the early stages of bone formation, as well as during periosteal and endosteal bone apposition. Its collagen fibrils run parallel to the bone surface but lack lamellar organization (Fig 2-3). Primary parallel-fibered bone shares most physiologic properties with woven bone.

Fig 2-3 Light microscopic micrograph showing reinforcement of woven bone (asterisks) by parallel-fibered bone (toluidine blue surface stain).

Mature bone consists of cortical (compact) and cancellous (trabecular or spongy) bone. Based on the orientation of the lamellae, cortical bone matrix is subdivided into different compartments. The basic structural units are the osteons or Haversian systems—longitudinally oriented cylindrical structures with vascular (Haversian) canals in the center. In secondary osteons, the wall consists of concentric lamellae, whereas primary osteons are characterized by a more primitive parallel-fibered bone matrix (see Fig 2-2). Along the periosteal and endosteal surfaces, appositional growth often results in packets of circumferential lamellae (Fig 2-4). Remnants of circumferential lamellae and of earlier generations of osteons occupy the remaining space in the form of interstitial lamellae. The osteocytes within these remnants of cortical remodeling activity are often cut off from their vascular supply and die³ (Fig 2-5).

Fig 2-4 Polarized light micrograph of cortical bone from a rabbit tibia. Osteons built around Haversian canals are sandwiched between circumferential lamellae at the periosteal (top) and endosteal (bottom) surfaces.

Fig 2-5 Osteons are metabolic units. Staining of osteocytes demonstrates the lacunar-canalicular system. Necrotic fragment of an osteon after obliteration of the Haversian canal (asterisk) (undecalcified ground section; basic fuchsin stain).

The trabeculae of cancellous bone are also composed of bone structural units, ie, packets or walls, separated or glued together by cement lines. They also reflect local remodeling in earlier periods of growth and cancellous bone turnover.⁴

Bone cells

Bone formation, maintenance, and repair are regulated by mesenchymal and bone-marrow-derived cells. Osteoblasts, osteocytes, and bone-lining cells are of mesenchymal origin, whereas osteoclasts belong to the monocyte/macrophage lineage and thus originate from bone marrow. Osteal macrophages are resident cells in bone tissue and have key functions in bone formation and remodeling.⁵ Osteoblasts, bone-lining cells, and osteoclasts cover bone surfaces, whereas osteocytes are found in the interior of the bone matrix, and osteal macrophages are found in bone marrow cavities.

Osteoblasts are large cuboidal cells that form a single layer covering all periosteal or endosteal surfaces where bone formation is active.⁶ They are polarized cells that secrete osteoid unidirectionally toward the bone surface. The nucleus of an osteoblast is ovoid, and its cytoplasm is filled with abundant rough endoplasmic reticulum and a prominent Golgi complex (Fig 2-6). Heterogeneity among osteoblasts seems to exist and may reflect differences between types of bone and/or anatomical sites.⁶ The osteoblast is responsible for synthesis, assembly, and mineralization of the bone matrix. Osteoblasts originate from mesenchymal stem cells in bone marrow.⁷ Differentiation of cells of osteoblastic lineage is controlled by multiple transcription factors at various stages of their development. Runt-related transcription factor 2 (Runx2), also known as core-binding factor α1 (Cbfa1), and osterix (Osx), downstream from Runx2, are master switches for osteoblast differentiation.⁸ The expression of Runx2 is not restricted to cells of the osteogenic and chondrogenic lineage,^9,10 and the expression of Runx2 in fully differentiated cells suggests additional roles in osteoblast function.

Fig 2-6 (a) Light micrograph showing a single layer of osteoblasts (OB) lining the bone matrix (toluidine blue stain). (b) Transmission electron micrograph illustrating osteoblasts with abundant rough endoplasmic reticulum (rER) lining the osteoid or prebone (PB).

Some osteoblasts become osteocytes by inversion of matrix secretion or by entrapment through neighboring osteoblasts.¹¹ The speed of matrix deposition may determine the number of embedded osteocytes.¹² This is exemplified in woven bone, which is formed much more quickly than any other type of bone and possesses a high number of embedded osteocytes.¹³ The osteocyte is trapped in the bone matrix in a lacuna (Fig 2-7), and neighboring osteocytes are interconnected by tiny cytoplasmic processes extending through a dense canalicular system. This lacunar-canalicular system allows for diffusion of nutrients, waste products, and signaling molecules for cell communication with neighboring osteocytes, osteoblasts, bone-lining cells, osteoclasts, and macrophages. It is indispensable for osteocyte survival because diffusion of nutrients and waste products through the heavily mineralized bone matrix is almost impossible. However, the transport capacity of this system also has limitations. In mammals, the critical transport distance to keep osteocytes alive is approximately 100 µm.¹⁴ This explains why the wall thickness of both osteons and packets in trabecular bone rarely exceeds 100 µm. Healthy osteocytes are necessary for proper functioning of bone.¹⁵ Osteocytes are more than passive cells buried in the bone matrix; they may actively participate in bone homeostasis through their involvement in bone remodeling, ion exchange, and sensing of mechanical signals.^15,16 Importantly, osteocytes synthesize sclerostin, a negative regulator of bone formation,¹⁷ and are the main source of receptor activator of nuclear factor-κB ligand (RANKL), which is required for osteoclast differentiation and activity.¹⁸

Fig 2-7 Transmission electron micrograph showing an osteocyte embedded in its lacuna next to a cement line (CL). Longitudinally and transversely cut canaliculi (C) containing cytoplasmic processes (CP) are visible close to the osteocyte.

The bone-lining cell is the third cell type belonging to the osteoblast family. It is regarded as an inactive osteoblast covering the bone surface. Bone-lining cells are flat and have a reduced armamentarium of cytoplasmic organelles, which is indicative of low activity of both cell metabolism and protein synthesis. They are therefore also called inactive or resting osteoblasts. Bone-lining cells may participate in the initiation of resorption by release of osteoclast activation factors and by active contraction, which is thought to expose the bone surface for the attachment of osteoclasts.¹⁹

The osteoclast is a large, multinucleated cell. Osteoclasts differ morphologically from other giant cells, especially from foreign-body giant cells, and are conventionally identified by their location in a resorption cavity, the Howship lacuna (Fig 2-8). Their size varies from 30 to 100 µm, and the number of nuclei varies roughly from 3 to 30. Their primary function is to degrade bone matrix, a process called bone resorption. The cytoplasm is acidophilic and often contains vacuoles (Fig 2-9a). The marginal area of the osteoclast adheres to the mineralized surface and seals off the actual resorption chamber by the so-called sealing zone (clear zone) (see Fig 2-9a). In the central part of this chamber, the cell surface is enlarged by numerous cytoplasmic undulations forming a ruffled border (Fig 2-9b). Through the enlarged cell membrane, hydrogen ions and proteolytic enzymes are released to dissolve the mineral crystals and to degrade the organic bone matrix. Osteoclasts originate from hematopoietic stem cells and develop from the self-fusion of macrophages.²⁰ Because of their origin, it is not surprising that many growth factors and transcription factors that are involved in hematopoietic differentiation of cells other than osteoclasts also affect osteoclast differentiation.⁸ Marker proteins expressed by osteoclasts include tartrate-resistant acid phosphatase (TRAP),²¹ cathepsin K, vitronectin, calcitonin, macrophage colony-stimulating factor (M-CSF), and receptor activator of nuclear factor κB (RANK).

Fig 2-8 Light micrograph showing osteoclasts (OC) in Howship lacunae formed in the alveolar bone (fuchsin and toluidine blue stain).

Fig 2-9 (a) Transmission electron micrograph showing an osteoclast with the ruffled border (RB), the sealing zone (SZ), numerous mitochondria, vesicles, and vacuoles, but only a single nuclear profile (N). The ruffled border represents the site of bone matrix (BM) dissolution and degradation (undecalcified ultrathin section). (b) Enlarged transmission electron micrograph of the ruffled border (RB) and bone matrix (BM) (decalcified ultrathin section).

Macrophages in bone (osteal macrophages), another type of cells of hematopoietic origin, are also called osteomacs. These resident cells play substantial roles in bone biology, with key functions in regulating bone formation and remodeling. Furthermore, they are among the first cells that come in contact with implanted biomaterials used for GBR, and they can differentiate toward classic M1 or M2 macrophages or subsequently fuse into osteoclasts or other multinucleated giant cells.⁵

Bone matrix formation and mineralization

The osteoblast synthesizes a mixture of macromolecules that are secreted into the extracellular milieu to form the bone matrix—the osteoid or prebone—which consists of water, mineral, collagens, and noncollagenous macromolecules, the latter usually called noncollagenous proteins. The biochemical composition of bone has previously been reviewed,^22–27 and collagens play structural and morphogenic roles.²⁸ In mineralized tissues, they interact with various noncollagenous proteins and provide a scaffold for the accommodation of the mineral crystals.²⁹ The noncollagenous proteins of bone can roughly be classified into glycoproteins, proteoglycans, plasma-derived proteins, growth factors, and other macromolecules. In addition to its structural function, the bone matrix harbors molecules that play roles in biomineralization and matrix-cell interactions, and it also serves as a reservoir for growth factors and cytokines that may be released while osteoclasts resorb the bone matrix.

At a certain distance from the osteoblast, at the mineralization front, the osteoid converts into mineralized bone. The mineralization of woven bone is initiated by matrix vesicles. In contrast, matrix vesicles are rarely seen in the osteoid of mineralizing lamellar bone. However, the first mineral to appear among the collagen fibrils may be found at small discrete foci that are distributed within the osteoid and accumulate at the mineralization front (Fig 2-10). The colocalization of noncollagenous bone proteins such as osteopontin (Fig 2-11) and bone sialoprotein with these small mineralization foci and with amorphous gray or reticular patches of mineralized bone indicates the association of these proteins with the mineralization process. Bone acidic glycoprotein 75 and osteocalcin, on the other hand, show a diffuse distribution pattern throughout the mineralized bone matrix.²²

Fig 2-10 Transmission electron micrograph illustrating an osteoblast, the osteoid or prebone (PB), the mineralization front (MF), and the mineralized bone matrix (MB). Mineralization foci (arrows) and “gray patches” (arrowheads) are visible in the osteoid and in the mineralized bone matrix, respectively.

Fig 2-11 High-resolution immunocytochemistry showing an association between gold particle labeling for osteopontin and mineralization foci in the osteoid.

Bone Modeling and Remodeling

Structural aspects

Throughout life, the skeleton undergoes continuous physiologic remodeling, which serves the purpose of repair and mechanical adaptation. The terms modeling and remodeling often cause confusion. Modeling indicates a change in shape, whereas remodeling refers to tissue replacement or substitution without a change in architecture. Cortical bone remodeling may be distinguished from trabecular bone remodeling.

Cortical bone

The fundamental structural unit of cortical bone remodeling is the osteon (see Fig 2-2). Primary osteons are formed during appositional growth, whereas secondary osteons are the result of matrix substitution. The remodeling sequence is illustrated in transverse sections in Fig 2-12. First, a resorption canal is formed by osteoclasts. Later, osteoblasts appear and start refilling the canal with concentric lamellae. In compact human bone, completed secondary osteons have an outer diameter of 200 to 250 µm, with the central vascular canal—the Haversian canal—measuring 50 to 80 µm.³⁰ As a coherent cylindrical structure, secondary osteons rarely measure more than 2 to 3 mm in length. At intervals of 0.5 to 1.0 mm, they are interconnected by transverse vascular channels called Volkmann canals. Longitudinal sections of newly formed osteons have shown that bone resorption and deposition are coupled in time and space and occur in discrete remodeling sites called bone metabolizing units (BMUs). At the advancing front of a resorption canal, osteoclasts are assembled in a cutting cone (Fig 2-13). While the osteoclasts advance longitudinally, they widen the resorption canal up to its final diameter. The tip of a vessel loop follows immediately behind the osteoclasts. This loop lies in the center of the canal and is surrounded by perivascular cells thought to include osteoblast precursor cells. In the reversal phase (ie, between bone resorption and bone matrix deposition), the wall of the canal is lined by mononuclear cells. Further in the back of the cutting cone, osteoblasts appear and deposit the lamellar bone matrix, which will later mineralize. Depending on the species, completion of the osteon requires 2 to 4 months. These measurements and calculations are based on sequential fluorochrome labeling (Fig 2-14) and also allow an accurate determination of the resorption rate of the osteoclast in longitudinal sections, which amounts to 50 to 60 µm per day in dogs.^31–33

Fig 2-12 Cortical bone remodeling in the humerus of an adult man. (a) Resorption canal with osteoclasts (arrows). (b) Concentric lamellae deposited by osteoblasts. (c) Completed secondary osteon, bound by a cement line (arrow) (transverse sections through evolving secondary osteons; toluidine blue surface stain).

Fig 2-13 Longitudinal section through the tip of an evolving secondary osteon (bone metabolizing unit) during fracture repair in a canine radius. The arrows show the osteoclastic cutting cone. VL, vascular loop; OB, osteoblasts; PB, osteoid or prebone seam (microtome section, Goldner trichrome stain).

Fig 2-14 Sequential polychrome labeling of a bone metabolizing unit at weekly intervals (arrowheads) to measure the daily osteoclastic resorption rate.

Cancellous bone

While the original architecture of the trabecular framework is determined by the growth pattern, modeling in the spongiosa specifically changes the architecture of cancellous bone throughout life. The trabecular network undergoes profound changes that result in a structural adaptation to the prevailing functional load, or, as often stated, “according to Wolff’s law.”³⁴ This adaptation enables bone to withstand a given stress with a minimum amount of material. The mechanism of functional adaptation is not fully understood, and at present Wolff’s law just offers a convenient way to accept it as a fact, without being forced to look for other explanations.

Bone remodeling improves the quality of the tissue with regard to both its mechanical and its metabolic properties. Remodeling of trabecular bone replaces discrete portions (or packets) with new lamellar bone (Fig 2-15). Formation of a new packet begins with local recruitment of osteoclasts that form a cavity on the trabecular surface. The mean depth of these cavities is around 50 µm, and it rarely exceeds 70 µm. At the end of this resorptive phase, and after a short intermission or reversal phase, osteoblasts start depositing new bone matrix during the formative phase. In analogy to the osteons, the new packet is considered a bone structural unit (BSU), and the cell populations involved in its formation are BMUs. Considering the extent of the trabecular surface in the human skeleton, the control and dynamics of cancellous bone remodeling play an important role in the pathogenesis of metabolic bone disorders, particularly in osteoporosis.

Fig 2-15 Trabecular bone remodeling in a human iliac crest biopsy. (a) Resorptive phase (Von Kossa-McNeal stain). (b) Early formative phase (Von Kossa-McNeal stain). (c) Newly formed packet (bone structural unit [BSU]), clearly delineated by a reversal or cement line (arrows) (toluidine blue surface stain).

Cement lines

A cement line is a very characteristic structural entity of bone. It delineates and demarcates the interface between new and old bone. Two types of cement lines are distinguished: resting lines and reversal lines. Resting lines are smooth and strictly parallel to the lamellae. They are formed when bone formation is arrested and, after a resting period, resumes again. Reversal lines are formed during the reversal phase, ie, they constitute the matrix that is deposited directly against a bone surface previously resorbed by osteoclasts (Fig 2-16). Resting lines, which are produced by osteoblasts at sites not exposed to osteoclastic resorption, show structure and composition similar to reversal lines.

Fig 2-16 Transmission electron micrograph showing a cement line (arrows) at the interface between old and new bone.

Secondary osteons are always separated from or connected to the surrounding older bone matrix by a cement line. Likewise, the new packet formed after the completion of trabecular bone remodeling is separated from the older bone matrix by a cement line. Howship lacunae left behind by the osteoclasts give the cement line a crenated appearance (see Figs 2-12c and 2-15c). The number of cement lines—both resting and reversal—indicates the intensity of matrix turnover.

Molecular aspects

Normal bone remodeling and thus bone mass maintenance depends on a delicate balance between bone formation and resorption involving a variety of cell types, including at least osteoclasts, osteoblasts, osteocytes, bone lining cells, macrophages, and blood vessels, but also cells from the immune system, which communicate with each other via signaling molecules (ie, cytokines and growth factors). The consequences of an imbalance in the expression of signaling molecules are metabolic bone disorders or diseases like Paget disease of bone, osteopetrosis, osteoporosis, arthritis, or bone loss in periodontitis.^35,36 Regulation of bone remodeling is under both systemic and local control. Local factors are operative in a paracrine and autocrine fashion, and osteoblasts, osteoclasts, osteocytes, and inflammatory/immune cells function as both sources and targets of signaling molecules. Numerous cytokines and growth factors have anabolic and/or catabolic effects on bone formation.^37,38 Among these bone-regulatory molecules are parathyroid hormone; parathyroid hormone-related peptide; calcitonin; calcitriol (the active form of vitamin D); prostaglandin E₂; growth hormone; thyroid hormone; sex steroids (estrogen and testosterone); leptin; statins; interferon γ; tumor necrosis factor α (TNF-α); transforming growth factor α (TGF-α); TGF-β; bone morphogenetic proteins (BMPs); fibroblast growth factor (FGF); insulin-like growth factor 1 (IGF-1); platelet-derived growth factors (PDGF); interleukins (IL) 1, 6, 11, and 17; and sclerostin.

A breakthrough in bone and immunology research was the detection of the RANKL/RANK/OPG system, which was first identified in the late 1990s as a pivotal regulator of bone remodeling. Bone resorption is regulated by this system, consisting of RANK and its ligand RANKL (which are members of the TNF ligand and receptor families) and osteoprotegerin (OPG). RANKL is expressed mainly by osteocytes, but also by bone marrow stromal cells, osteoblasts, and certain fibroblasts, whereas RANK is expressed by osteoclast precursors and mature osteoclasts. The binding of RANK to RANKL induces osteoclast differentiation and activity and regulates osteoclast survival. OPG, however, is produced by osteoblasts, bone marrow stromal cells, and other cell types and is a soluble decoy receptor for RANKL that competes for this binding. Thus, OPG is a natural inhibitor of osteoclast differentiation and activation.

Any interference with this system can shift the balance between bone apposition and resorption. The expression of M-CSF plays an essential role in this regulatory system. Furthermore, it has been shown that a number of proinflammatory cytokines and growth factors, in particular IL-1 and TNF-α, regulate the expression of RANKL and OPG (Fig 2-17). The immune system modifies the balance between bone formation and resorption in a complex process involving T and B lymphocytes, dendritic cells, and cytokines. By the expression of RANKL on B cells, T cells, and marrow stromal cells, and the expression of RANK on osteoclast precursors, mature osteoclasts, T lymphocytes, B lymphocytes, and dendritic cells, these cells can directly influence bone resorption.^39–41

Fig 2-17 RANKL/RANK/OPG system. PTH, parathyroid hormone; 1α,25[OH]2D3, 1α,25-dihydroxyvitamin D3; PGE2, prostaglandin E2; IL-1, interleukin 1; IL-6, interleukin 6; IL-11, interleukin 11; IL-17, interleukin 17; TNF-α, tumor necrosis factor α; M-CSF, macrophage colony-stimulating factor; c-Fms, M-CSF receptor; RANK, receptor activator of nuclear factor-κB; RANK-L, RANK ligand; IFN-γ, interferon γ; IFNR, IFN-γ receptor; TGF-β, transforming growth factor β; OPG, osteoprotegerin.

The discovery of this important regulatory system, which links bone biology with immune cell biology, has opened the possibility of new therapeutic strategies. Attempts using recombinant OPG were successful in preventing bone resorption and loss. However, production of significant antibody titers in a patient given OPG brought this development to an early end. Another way of blocking RANK signaling is through the use of denosumab, a fully human monoclonal antibody against RANKL. Prolia (Amgen) is used in patients with osteoporosis, whereas Xgeva (Amgen) is used for the treatment of multiple myeloma, bone metastases from solid tumors, bone giant cell tumors, and hypercalcemia of malignancy refractory to bisphosphonate therapy. Like bisphosphonates, Prolia and Xgeva can cause osteonecrosis of the jaw. These findings have led to the new term medication-related osteonecrosis of the jaw (MRONJ), which replaces the older term bisphosphonate-related osteonecrosis of the jaw (BRONJ). To help prevent MRONJ, clinicians have to ask their patients if they take antiresorptive medication. MRONJ has also been described as a complication of cancer therapies that target angiogenesis. However, this association is more controversial. Use of antiangiogenic agents as a risk factor for MRONJ among patients receiving osteoclast inhibitors for cancer is more clearly established.

Biology of Bone Regeneration

Physiologic versus reparative regeneration

Regeneration is commonly understood as replacement of vanishing or lost components in the body by tissues or organs of equally high structural organization so that structure and function are completely restored. Physiologic regeneration is distinguished from reparative regeneration. Many tissues or organ systems undergo physiologic regeneration, ie, continuous replacement of cells or tissue elements. Remodeling of cortical and trabecular bone also represents regeneration; both cells and extracellular matrix are replaced. Reparative regeneration takes place when tissues are lost because of injury or disease. Bone has the unique potential to completely restore its original architecture, but there are certain limitations. The reconstruction of the original level of tissue organization occurs sequentially and closely repeats the pattern of bone formation occurring during development and growth. Likewise, some basic conditions have to be fulfilled, such as ample blood supply and mechanical stability provided by a solid base.

Activation of bone regeneration

Any bone lesion (fracture, defect, insertion of an implant, interruption of blood supply) activates wound healing and local bone regeneration by the release and local production of growth factors and other signaling molecules, as discussed in chapter 3 of this textbook. Bone is one of the richest sources of growth factors and other signaling molecules. Osteoinduction in its classic concept implies initiation of heterotopic (ectopic) bone formation; that is, bone formation at sites where bone physiologically does not normally exist. The term osteoinduction, however, is just as frequently applied if ossification is activated in contact with existing bones; that is, in the case of orthotopic bone induction. To avoid confusion, the term bone activation is preferred for orthotopic bone formation.

In heterotopic bone formation, inducible osteoprogenitor cells are found far from bone. These mesenchymal cells are abundant in subcutaneous connective tissue, skeletal muscles, and the spleen and kidney capsule. Their response to inductive stimuli, such as BMPs, is more complex than in orthotopic bone formation, and in fact mimics endochondral bone formation.⁴²

After subcutaneous implantation of BMPs in rats, proliferation of mesenchymal cells starts after 3 to 4 days. From days 5 to 8 onward, cartilage develops, and within 1 day it starts to mineralize. Vascular invasion and bony substitution of the calcifying cartilage follows from days 10 to 11 and onward. Intermediate cartilage differentiation seems to be mandatory if induction acts on inducible osteoprogenitor cells. Importantly, the response is always indirect bone formation.

In orthotopic bone formation, osteoprogenitor cells are found in tissues in direct proximity to bone, such as in bone marrow stroma, periosteum, endosteum, and intracortical canals. These cells respond to inductive signals with proliferation and differentiation directly into osteoblasts. Thus, in orthotopic bone induction, the inducing agents act on determined osteoprogenitor cells, and the cellular response is direct bone formation. The lag phase is short—seldom longer than 1 to 3 days—and the newly formed bone is laid down on preexisting bone surfaces.

Repair of bone defects

The repair of experimental bone defects is a good model for the study of bone regeneration. In contrast to fractures, defects are less subject to mechanical factors and to obstructions of the blood supply. Johner⁴³ examined the healing of bore holes with diameters of 0.1 to 1.0 mm in the rabbit tibia. Bone formation within these holes starts within a couple of days, without preceding osteoclastic resorption, and reveals a clear-cut size dependency. Holes with a diameter in the range of osteons (0.2 mm) are concentrically filled with lamellar bone. In larger holes, a scaffold of woven bone is formed first, and then lamellar bone is deposited in the newly formed intertrabecular spaces, which have a corresponding diameter of 150 to 200 µm. As in appositional growth, the matrix deposition rate of lamellar bone is restricted to a couple of microns per day, whereas woven bone rapidly bridges larger defects. After 4 weeks, both the small and the larger defects are completely filled with compact bone.

There is, however, a threshold for this rapid bridging by woven bone. This threshold lies around 1 mm for bore holes in rabbit cortical bone.⁴³ Experimental studies on bony ingrowth into porous acetabular components in canine hip joint arthroplasty demonstrate similar results,⁴⁴ summarized in the often-quoted phrase osteogenic jumping distance. This term indicates that bone is not able to cross gaps wider than 1 mm in one single jump. In the case of implants, the situation becomes even more difficult because bridging of the defects starts from the bony side only. This does not mean that larger holes or gaps will stay open indefinitely, but filling takes longer, and there is no doubt that bore holes of 3 to 5 mm persist for several weeks, if not months, until repair is completed.

The bone healing around dental implants has been analyzed in relation to the distance between the implant surface and the surrounding bone,⁴⁵ in relation to the implant surface characteristics,^46,47 and in relation to implant materials and surface topographic and chemical modifications.⁴⁸

Guided Bone Regeneration

As outlined previously, bone tissue exhibits a remarkable regenerative potential and perfectly restores its original structure and mechanical properties. However, this capacity has limitations and may even fail if certain conditions are not fulfilled. Factors that impede or even prevent bone repair include (1) failure of vascular supply; (2) mechanical instability; (3) oversized defects; and (4) competing tissues of high proliferative activity. However, several options, alone or in combination, are available to promote and to support bone formation, including:

 Osteoinduction by growth factors

 Osteoconduction by autogenous bone grafts or substitutes

 Transfer of stem cells or progenitor cells that differentiate into osteoblasts

 Distraction osteogenesis

 GBR using barrier membranes

GBR, usually in combination with a bone grafting material, is the most widely used method of augmenting bone in routine dental practice. The use of barrier membranes for bone regeneration was adapted from studies on periodontal regeneration^49,50 and started in the mid 1980s with preclinical experiments.^51–53

The GBR principle

The key principle of GBR is to keep undesirable cells from nonosteogenic tissues from interfering with bone regeneration. To this end, a physical barrier membrane is placed between the region to be augmented with new bone and the adjacent soft tissue. Because bone is a relatively slow-growing tissue, both fibroblasts and epithelial cells have the opportunity to occupy available space more efficiently during wound healing and to build up a soft connective tissue much faster than bone is able to grow. If the occlusive barrier function lasts long enough and if the barrier membrane is not exposed to the oral cavity, optimal conditions exist for the ingrowth of blood vessels from the resident bone, allowing stem cells and osteoprogenitor cells to differentiate into osteoblasts, which produce the bone matrix. In other words, the barrier membrane creates a secluded space that allows bone to use its great, natural healing capacity in an undisturbed or protected manner.

It is noteworthy that the theory of total cell occlusion has been challenged by the awareness that nutrient transfer across the membrane may be important for successful bone regeneration.^54,55 Indeed, macroporous membranes were found to be more predictable GBR and for guided tissue regeneration (GTR) procedures and more conducive to uncomplicated clinical management than occlusive membranes.^56,57 Very recently, the function of a membrane as a mere tissue-separating barrier has been further questioned. There is some evidence suggesting that certain membranes have functions beyond the barrier role.⁵⁸ These emerging data suggest that the membrane contributes actively to the regeneration of underlying bone defects. It would not be surprising if the specific macrophage response to a biomaterial is involved in this contributing effect.

Biomaterials used for GBR

Barrier membranes

In practice, the barrier membrane is placed in direct contact with the outer surface of the bone surrounding the defect, and the mucoperiosteal flap is then repositioned and sutured. Clinicians have access to a wide range of membrane materials for GBR. To select the material best suited for a specific clinical application, it is necessary to understand the basic requirements for membrane materials used in these indications. These basic characteristics include:

 Biocompatibility

 Cell occlusion (occlusivity, occlusiveness)

 Space-making and space-maintaining ability

 Tissue integration

 Degradability

 Clinical handling

 Susceptibility to complications

While in the pioneering phase of GBR in the late 1980s and 1990s, the nonresorbable expanded polytetrafluoroethylene (ePTFE) membrane prevailed, resorbable membranes were evaluated later. Both nonresorbable and bioresorbable membranes have inherent advantages and disadvantages.

Nonresorbable membranes

The most widely used nonresorbable membrane type is made of PTFE. It was originally developed in the late 1960s, and marketing began in 1971. PTFE is hydrophobic in nature and biologically inert, which makes this biomaterial nonresorbable. Thus, the major advantage is its excellent barrier function, whereas the major disadvantage is the need for a second surgery to remove the membrane. The ePTFE membrane is produced by exposing PTFE to high tensile strain, resulting in expansion and formation of a porous microstructure. The most commonly used ePTFE membrane has a two-part design with a dense portion and a less dense portion. The inner (central) dense portion, located over the defect space, has a pore size of less than 8 µm to allow fluid exchange while preventing infiltration of cells (Fig 2-18a). In contrast, the microstructure of the outer portion, which is in contact with the bone at the defect margin, is less dense; it has pores 20 to 25 µm wide and a surface structure that encourages blood clot adhesion and soft connective tissue attachment to and invasion into the membrane, ultimately leading to tissue integration (Fig 2-18b). In dentistry, ePTFE membranes became the standard for guided tissue regeneration (GTR) and GBR procedures during the development phase of both techniques in the 1980s and early 1990s.⁵⁹

Fig 2-18 Soft and hard tissue compartments adjacent to an ePTFE barrier membrane (BM) (ground sections; toluidine blue and basic fuchsin surface stain). (a) The dense portion of the barrier membrane separates an outer, mucosal compartment from an inner, soft connective tissue compartment that is mainly accessible from the bone (B) compartment. There are no signs of a foreign-body reaction. (b) The porous part of the barrier membrane (asterisks) allows ingrowth of blood vessels and cells into the gaps in the membrane.

High-density PTFE (dPTFE) is different from ePTFE in that it possesses pores in the submicron scale (0.2 µm). Bacterial adhesion and infiltration is eliminated because of its high density and small pore size. Primary soft tissue closure is not required, allowing tissue healing with a barrier membrane being exposed to the oral cavity.

To overcome the problem of membrane collapse, the PTFE membrane can be mechanically stabilized with titanium, which is known as a titanium-reinforced PTFE. Alternatively, membranes made of metal meshes consisting of titanium or titanium alloy and cobalt-chromium alloy may be used.⁶⁰

As already mentioned, one big disadvantage of nonresorbable membranes is the need for their removal with an additional surgical intervention. Furthermore, complications associated with ePTFE membranes include difficult handling because of their hydrophobic nature, membrane collapse, and membrane exposure, often leading to infection and compromised regenerative outcome. These complications were the stimulus for the testing of another generation of biomaterials, the bioresorbable membranes.

Bioresorbable membranes

As the name indicates, bioresorbable or biodegradable membranes are broken down in the body and disappear over time. Thus, they have the advantage of eliminating the need for an additional surgery to remove the biomaterial. Two main categories of bioresorbable membranes exist: (1) synthetic polymers, and (2) polymers derived from various animal sources. Each membrane has distinct physicochemical properties and biologic effects. The degradation process has an important influence on the outcome, since (1) if degradation occurs too quickly, the biomaterial may not have a chance to fulfill its function as a barrier, and (2) products of degradation may contribute to unfavorable tissue responses, including foreign-body reactions, which may prevent tissue integration and bone formation and even result in bone resorption.⁶¹ Most clinically used bioresorbable membranes are made of processed collagen or aliphatic polyesters.

Synthetic membranes

Synthetic polyesters used as barrier membrane biomaterials are polyglycolides (PGAs), polylactides (PLAs), or copolymers thereof. Other aliphatic polyesters used are polydioxanones⁶² and trimethylene carbonates.⁶³ These synthetic biomaterials have both advantages and disadvantages. They can be made available in almost unlimited quantities. Another advantage is the ability of PGA, PLA, and their copolymers to completely biodegrade to carbon dioxide and water via the Krebs cycle.⁶⁴ Numerous factors are known to affect the degradation of bioresorbable polymers, such as their structure and chemical composition, molecular weight, shape, processing conditions, sterilizing processes, physicochemical factors, and mechanism of hydrolysis.^65,66 The use of these polymers as bone plates, screws, and delivery vehicles for drugs and growth factors has been associated with inflammatory and foreign-body reactions in orthopedic and maxillofacial surgery and implant dentistry (Fig 2-19). In certain cases, even surgical debridement and removal of the biomaterial may be required.^67–71

Fig 2-19 GLTC membrane at 12 weeks. Numerous large multinucleated giant cells (arrows) are interposed between an amorphous matrix (asterisks) that partly replaces the membrane material (undecalcified ground section, toluidine blue and basic fuchsin surface stain).

Collagen membranes

Most natural resorbable membranes are made of collagen from animal tissues, although human sources exist as well. The collagen membranes have different tissue sources, including bovine tendon, bovine dermis, calf skin, porcine dermis, and human skin from cadavers.⁷² Collagen is the most abundant extracellular matrix protein in the body. Collagen has important structural functions, and it also supports cell attachment, cell differentiation, tissue repair, and tissue regeneration.^73,74 Collagen-based biomaterials are widely used in biomedical applications since they closely mimic the extracellular environment. Furthermore, collagen has low immunogenicity and is hemostatic.⁷⁵

On the other hand, collagen membranes have unfavorable mechanical properties⁷⁶ and may provide an inadequate barrier function because they biodegrade too quickly through enzymatic activities of macrophages and polymorphonuclear neutrophils.^77–79 Biodegradation of collagen membranes into carbon dioxide and water is caused by endogenous collagenases.⁷⁵ To prolong the barrier function, a number of cross-linking technologies, such as ultraviolet light, formaldehyde, glutaraldehyde, diphenylphosphoryl azide, and hexamethylene diisocyanate, have been used.^80,81 The glutaraldehyde technique was reported to leave cytotoxic residues.⁸² The degree of collagen cross-linking inversely affects the degradation rate.⁸³ Membrane ossification is a phenomenon observed in certain cross-linked collagen membranes. The modified collagen seems to trigger membrane ossification.^84,85

Non-cross-linked collagen membranes are currently the membrane of choice for most GBR procedures, whereas nonresorbable membranes made of dPTFE may be recommended for selected vertical augmentation procedures only. Figures 2-20 and 2-21 show two excellent regenerative outcomes when a non-cross-linked collagen membrane was used to cover a bone defect either created in the rabbit calvaria or lateral to a dental implant, respectively.

Fig 2-20 Light micrograph illustrating a non-cross-linked collagen membrane (Bio-Gide, Geistlich) (asterisks) over a bone defect 2 weeks after its creation in the rabbit calvaria. New bone (NB) has loosely filled the defect space and has even surpassed the height of the pristine old bone (OB) (undecalcified ground section; toluidine blue and basic fuchsin stain).

Fig 2-21 Light micrograph illustrating a buccal peri-implant bone defect 3 weeks after augmentation with autogenous bone chips (AB), a bone substitute (BS) (Bio-Oss, Geistlich), and a non-cross-linked barrier membrane (BM) (Bio-Gide). The double-layered barrier membrane perfectly covers the augmented region and blocks off from the oral mucosa (undecalcified ground section; toluidine blue and basic fuchsin stain).

Bone grafts and bone substitute materials

Since most bioresorbable membranes, and to a lesser degree ePTFE membranes, are largely incapable of maintaining defect space due to lack of sufficient rigidity, these membranes are often used in combination with autogenous bone grafts, bone substitute materials, or composite grafts. Bone fillers, however, serve many more purposes, including the following:

 Provide mechanical support to prevent membrane collapse

 Stabilize the blood clot

 Allow ingrowth of blood vessels

 Act as an osteoconductive scaffold for bone ingrowth

 May be osteoinductive

 May contain bone cells

 Become integrated in or replaced by bone

 Protect the augmented volume from resorption

The clinical indications for using bone filler materials range from grafting minor peri-implant bone defects to the regeneration of large continuity defects. Considering this wide range of purposes, it is to be expected that one single material cannot fulfill all requirements. Therefore, it will often be necessary to combine two or more materials to obtain a successful and predictable treatment outcome.

Bone filler materials can either be derived from the person being treated (autogenous bone grafts or autografts) or from an external source (bone substitute materials). The bone substitute materials are subdivided into allogeneic bone, xenogeneic bone, and alloplastic (synthetic) bone graft substitutes. Figure 2-22 shows a common classification of bone grafting materials. These materials possess different physical, chemical, and biologic characteristics. The biologic properties are commonly described as:

Fig 2-22 Classification of bone augmentation materials.

 Osteoconductive

 Osteoinductive

 Osteogenic

Osteoconductive materials possess a matrix that serves as a scaffold or framework. This matrix is used as a template and an enlarged solid base for bone deposition. Materials with osteoinductive properties contain proteins (BMPs) that stimulate and support proliferation and differentiation of uncommitted stem cells to become osteoblasts. Osteogenic means that the autogenous bone contains bone cells (bone-lining cells, osteoblasts, osteoblast uncommitted stem cells, and/or osteocytes) that are capable of directly or indirectly supporting bone formation at the transplantation site.

Autogenous bone grafts

Autogenous bone is a preferred bone graft material because it possesses osteoinductive, osteogenic, and osteoconductive properties. However, the harvesting of autogenous bone may require an additional surgical intervention, which increases the operative time, costs, intraoperative blood loss, pain, and recovery time. Moreover, it is associated with an increased risk of donor site morbidity (eg, increased postoperative pain, nerve injury, blood vessel injury, hematoma, infection, hernia formation, and cosmetic disadvantages). Finally, the supply of autogenous bone for grafting may be limited.

With transplantation of autogenous bone, bone-stimulating growth factors and viable osteogenic cells are brought to the recipient site.⁸⁶ The number of cells and the concentration of growth factors demonstrate great inter- and intraindividual variation and depend largely on patient’s age, presence of systemic diseases, and location of the donor site. The growth factors comprise BMPs, TGF-β, IGF, PDGF, and FGF. They are mainly present in the bone matrix and are released either passively or during resorption of the autografts. The higher the absolute surface area of the autograft, the faster the growth factors are set free. This means that blocks of cancellous bone release growth factors more readily than blocks of compact bone and that particulate autografts demonstrate faster release of bone-stimulating growth factors than do blocks.⁸⁷ More about growth factor release from autogenous bone grafts is discussed in chapter 3.

The cells of primary interest in GBR procedures are the osteogenic cells (ie, osteoblasts, bone-lining cells, preosteoblasts, and pluripotent stem cells). These cells are most numerous in trabecular bone and least numerous in compact bone. The osteogenic potential is greater in young healthy individuals than in elderly patients. This is mainly because of a decreased proliferative capacity of osteoprogenitor cells in older individuals, rather than due to compromised function of the osteoblasts.⁸⁸ The presence of living osteocytes in transplanted autogenous bone confirms that cells can survive the transplantation procedure, but whether transplanted osteocytes have a function in bone formation is as little known as what this function may be. As discussed previously, osteocytes play a very important role in bone physiology.

Autografts can be harvested at different intraoral or extraoral locations (Table 2-1) and can be used in different forms (Table 2-2).⁸⁹ Both autografts and bone substitute materials can be used as block grafts, which are beyond the scope of this chapter, or as particulate grafts.

Table 2-1 Donor sites for autografts

*More cortical than cancellous bone

^†More cancellous than cortical bone

+, Sufficient bone for augmenting a single-tooth gap; ++, sufficient bone for up to two sinus augmentations; +++, sufficient for major inlay and onlay augmentations and reconstruction of continuity defects.

Table 2-2 Characteristics of autografts and their indications

Autogenous particulate grafts

Bone particles are usually applied in an area where there is no need for mechanical strength, such as in peri-implant bone defects or in sinus floor elevation procedures. Particulate bone can be harvested either directly with a bone scraper (bone chips), with a piezoelectric device, with a bone collector (bone slurry or dust), or indirectly from a harvested bone block that is ground in a bone mill. The higher the relative surface area, the greater the osteoconductive and osteoinductive potential of autogenous bone. This is because the greater surface area allows more passive release of growth factors and more active liberation of growth factors by osteoclasts while they resorb the bone matrix.⁸⁷ While the osteopromotive potential of an autograft increases when it is particulated, the total number of intact osteogenic cells decreases through mechanical graft manipulation.⁹⁰ The smaller the particles, the more the stability of the graft decreases. In addition, the resorption rate increases considerably.⁹¹ In a recent preclinical study, bone chips, bone slurries, bone harvested with a piezoelectric device, and bone ground by a bone mill were tested for their ability to support bone healing in defects created in the mandible of minipigs and covered with an ePTFE membrane.⁹² The results showed that all bone defects healed with the same amount of new bone, regardless of the harvesting procedure and of the healing period (Fig 2-23). The highest number of osteoclasts was found after 1 to 2 weeks on the bone slurries, confirming that smaller particles trigger increased resorption (Fig 2-24).

Fig 2-23 Histologic sections illustrating standardized bone defects in the minipig mandible (undecalcified ground sections; toluidine blue stain). Old bone (OB) and an ePTFE membrane (asterisks) delineate the defects. The defects were filled with autogenous bone. (a) Particulate from corticocancellous bone with a bone mill. (b) Bone chips harvested with a bone scraper. (c) Bone particles harvested with a piezoelectric instrument. (d) Bone slurry from a bone trap filter. After 4 weeks of healing, all types of autogenous bone grafts are embedded in a trabecular network of new bone that completely fills the defect areas.

Fig 2-24 Histologic sections 1 week after implantation resorption of autogenous bone. (a) Particulate from corticocancellous bone with a bone mill (BM). (b) Bone chips harvested with a bone scraper (BS). (c) Bone particles harvested with a piezoelectric instrument (PI). (d) Bone slurry from a bone trap filter (BT). All harvesting techniques show numerous Howship lacunae (arrowheads) and osteoclasts (arrows). The lowest number of osteoclasts is found on BM particles, and the highest number is found on BT particles.

A vast body of scientific evidence from experimental and clinical studies documents the suitability of particulate autografts for bone augmentation procedures when a highly osteogenic graft is needed. Nevertheless, the advantages and disadvantages of the different harvesting procedures in the context of clinical applications need to be discussed.

Autogenous bone from bone collectors. With this harvesting method, bone dust is collected through a filter that is connected to the suction device used during preparation of the implant bed. The philosophy is intriguing, because the acquisition of the autogenous bone graft causes no additional discomfort for the patient, but several disadvantages exist. Viable cells are clearly present in lower numbers than in other autografts,^90,93 the amount of bone that can be collected can only cover small peri-implant defects, and contamination of the graft with bacteria from the oral cavity occurs frequently.⁹⁴

Autogenous bone from bone mills. Particulate bone ground in a bone mill requires a bone block to be harvested from a donor site and involves an additional surgical intervention, causing morbidity. This procedure may provide more autograft volume than is available with bone dust from bone collectors. The milling process may reduce the number of viable cells.

Autogenous bone collected with a piezoelectric technique. There are not many data available on this harvesting technique. A recent preclinical study has shown that the dimensions of particulate autografts harvested with a piezoelectric device resemble those of bone chips collected with a bone scraper.⁹²

Autogenous bone from bone scrapers. This harvesting technique has been advocated over the past few years for minor bone regeneration procedures, such as extraction sockets and localized sinus augmentation procedures, or coverage of dehiscence-type defects, either alone or in combination with bone substitute materials.^95–97 With this technique, small particles of cortical bone are harvested by scraping the bone surface—a simple intraoral approach with which up to 5 cm³ of bone can be obtained.⁹⁵ Osteocytes have been shown to survive the grafting procedure, but because of the cortical nature of the graft, very few osteoblasts and osteoblast precursors are expected to be present. The resistance against resorption is presumably low because of the high surface area–to-volume ratio.⁹⁷

Autografts are still considered the gold standard in osseous reconstructive surgery.⁹⁸ However, significant drawbacks related to the use of autografts have intensified the search for alternatives. First, there is the unpredictable resorption of up to 60% of corticocancellous block grafts.⁹⁹ If uniform resorption could be expected, it would be unproblematic to perform a standardized overcompensation of the augmented volume. Second, there is donor site morbidity. This is most pronounced in relation to the extraoral donor sites,¹⁰⁰ but it may also be significant in intraoral grafting procedures.⁸⁹ Finally, it can be a problem that autogenous bone is not available in unlimited quantities. With the main goal of reducing or even eliminating the shortcomings of autografts, the search for appropriate bone substitute materials has been going on for the past 50 years.

Allografts

Allografts consist of bone obtained from a donor and used in a member of the same species. Transplantation of bone from one individual to another has been performed in orthopedic surgery for more than 130 years.¹⁰¹

Allografts are usually stored in bone banks, and they may be used as fresh frozen bone (FFB), freeze-dried bone allograft (FDBA), or demineralized freeze-dried bone allograft (DFDBA). FFB is rarely used in GBR procedures because of a high risk of immunologic rejection and disease transmission, whereas the freeze-drying of FDBA and DFDBA is reported to reduce the immunogenicity of the material, potentially improving the clinical outcome. Allografts are available as blocks or in particulate forms of both cortical and cancellous origin.^102,103

FDBA and DFDBA have been shown to be biocompatible and to contain osteoinductive molecules such as BMPs.¹⁰⁴ Demineralization of allografts is intended to expose the BMPs, additionally increasing the immediate osteoinductive potential. However, FDBA loses some of its mechanical stability during the demineralization process, and DFDBA should therefore be used in combination with a space-maintaining material if the bone defects are not self-contained. Different batches of DFDBA have been shown to contain very different concentrations of BMPs,¹⁰⁵ and the osteoinductivity can therefore be expected to vary correspondingly.

Histologic evidence from an experimental comparative study in the mandibles of minipigs showed that allografts decelerated new bone formation in comparison with autografts (positive control) and coagulum (negative control).¹⁰⁶ DFDBA showed osteoconductive properties, but osteoinductive potential could not be demonstrated.¹⁰⁶ Therefore, FFB, FDBA, and DFDBA indisputably contain osteoinductive molecules. However, it is still debatable whether the concentrations of these BMPs are sufficient to elicit clinically relevant osteoinductive potential and whether they are present in an active form.

Allografts are widely used in the United States, whereas local regulations in Europe restrict the collection of human bone. This has limited their popularity among clinicians. Compared with the limitations of autografts, donor site morbidity is not an issue, and allografts are available in abundant quantities. However, resorption is reported to take place, as is seen with autografts.¹⁰²

Xenografts

Xenografts, or xenogeneic bone substitutes, consist either of bone mineral derived from animals or bonelike minerals derived from calcifying corals or algae which have had the organic component removed to eliminate the risk of immunogenic reactions or transmission of diseases.

A few species of calcifying corals were found to have a calcium carbonate skeleton with a geometry similar to that of human cancellous bone, with interconnected macropores of 20 to 600 µm. The coralline calcium carbonate is transformed into hydroxyapatite (HA) by a hydrothermal exchange reaction with phosphorus. Experimental studies have demonstrated that the osteoconductive potential of coral-derived bone substitutes is less than that of other bone substitute materials.^106,107 Currently, coralline HA is seldom used for onlay grafts in GBR procedures because of a high rate of late complications.¹⁰⁸ When used as particulate, the granules tend to migrate, and the ones that are kept at the augmented site predominantly become encapsulated by fibrous tissue. The blocks, on the other hand, most often show bone formation throughout the augmented volume, but they are prone to develop late dehiscences.¹⁰⁹

There is also a group of marine algae that consist of a calcified exoskeleton made of calcium carbonate. The natural material is converted into fluorohydroxyapatite through an exchange reaction with ammonium phosphate at around 700°C. The morphologic structure is made up of pores arranged in parallel with a mean diameter of 10 µm and connected through microperforations. The pore configuration is thus not ideal for vascular ingrowth, but cellular invasion of the pores and bone deposition directly on the material surface have been documented.^110,111 Neovascularization is instead expected to take place in between the bone substitute particles. In contrast to coralline HAs, phycogenic fluorapatite undergoes slow resorption by enzymatic and cellular degradation, but at a lower rate than autografts.¹¹⁰

Most xenografts originate from natural bone sources in animals. In particular, cancellous bovine bone has been used as a source for these bone substitute materials because of its close similarity to cancellous human bone. The organic component is removed by heat treatment, by a chemical extraction method, or by a combination of the two, in order to eliminate the risk of immunologic reactions and disease transmission. Since the first reports of bovine spongiform encephalopathy, there has been a particular focus on the ability of these extraction methods to completely eliminate all proteins from the bovine bone source.^112,113 However, despite the hypothetical risk of organic remnants in bovine bone substitutes, there have been no reports of disease transmission with the use of these biomaterials. In contrast, a few cases of transmission of HIV and hepatitis related to allogeneic materials have been reported.¹¹⁴

Bovine-derived xenografts are generally biocompatible and osteoconductive, although the production methods have a strong impact on their biologic behavior. For instance, high-temperature treatment resulted in less bone formation and lower osteoconductivity.^107,111,115 This difference most likely reflects the production-related changes in surface characteristics. Temperatures exceeding 1000°C cause a sintering of the natural hydroxyapatite, with the apatite crystals growing and the intercrystalline spaces disappearing to a large extent.¹¹⁶ This reduces the microroughness and micro- and nanoporosity of the bone substitute and increases the crystallinity.

Alloplastic bone substitutes

Alloplastic or synthetic bone substitutes have the advantage of not bearing any risk of disease transmission and are available in large quantities. Furthermore, it is theoretically possible to specifically design these biomaterials with material characteristics that meet the requirements for a specific clinical indication. Chemistry, phase distribution between crystalline and amorphous material, crystal size, gross morphology and size, particle size, pore size and interconnectivity, macro- and nanoporosity, and surface roughness and texture at the macroscale and nanoscale can be tailored. A greater understanding of the importance of the different material characteristics has helped us to understand why materials with identical chemical compositions and macromorphologies have performed so different biologically in vivo.

The synthetic biomaterials currently on the market can be divided into three groups: calcium phosphates (CaPs), bioactive glasses, and polymers (see Fig 2-22). Of these, CaPs—and especially HA and tricalcium phosphate (TCP)—have been most intensively studied due to their composition, which closely resembles the inorganic phase of bone.¹¹⁷ In general, HA is considered to be osteoconductive and nonresorbable, whereas TCP also demonstrates osteoconductive properties but resorbs rapidly. Therefore, combinations of HA and TCP—biphasic calcium phosphates (BCPs)—have been investigated with the goal of benefitting from both the stable space-keeping properties of HA and the resorbable space-making properties of TCP.¹¹⁸ By varying the HA:TCP ratio, it was possible to modulate the substitution rate and bioactivity of these biomaterials.^119,120 BCPs are a valuable alternative for clinicians and patients who are not comfortable with implantation of material of human or animal origin. Future perspectives may involve the development of a selection of two to three BCPs to fit individual clinical indications. A very interesting and promising research field is the development of biomaterials, particularly BCPs, that are osteoinductive. This biomaterial-induced or intrinsic osteoinduction should not be confused with the biomaterial-free induction of undifferentiated inducible osteoprogenitor cells.¹²¹ Thus far, this intrinsic, biomaterial-dependent osteoinduction is considered unpredictable in various animal models and in humans. A mechanism for biomaterial-induced heterotopic ossification has been proposed, for example by Bohner and Miron.¹²¹

Bioglasses are silica-based materials that were first introduced in the early 1970s. These glasses exhibit bone bonding as a result of the surface reactive silica, calcium, and phosphate groups that are characteristic of these materials. Silica is believed to play a critical role in bioactivity. Bioactive glasses are very biocompatible materials. Some experimental data support their use in GBR procedures such as ridge preservation and sinus augmentation. However, there are inherent limitations on the currently available bioglass products. Because of their granular and nonporous nature, they cannot reliably serve as space-maintaining devices, although macroporous glass ceramics are now also available.¹²²

Bone healing in membrane-protected defects without addition of a bone filler

The histologic healing pattern of bone regeneration under a barrier membrane without addition of a bone filler was demonstrated by Schenk et al in a landmark experimental study.¹²³ Saddle-type defects were created in the mandibles of dogs two months after tooth extractions. The defects were covered with (test) or without (control) a barrier membrane (Fig 2-25). A standard ePTFE membrane and two different prototype ePTFE membranes reinforced with polypropylene mesh were used. The position of the membranes was secured by two miniscrews. No bone filler was used. However, intravenously aspirated blood was injected under each membrane. Histologic analysis was performed after healing periods of 2 and 4 months.

Fig 2-25 Surgical procedure of GBR. Two defects were created in the exposed mandible of a dog 2 months after tooth extractions. The right defect is open, but the left one is covered with a nonresorbable ePTFE membrane. Miniscrews fix the membrane and mark the corners of the defect for radiography.

Healing without a barrier membrane

The control defects showed a consistent repair pattern in which bone formation was restricted to the defect margins, ie, mesial and distal walls of the defect and at the bottom of the defect (Fig 2-26a). Closure of the marrow space was completed after 2 months, but bone formation made no further progress. At 4 months, bone had slightly increased in density.

Fig 2-26 Buccolingual ground sections through the central portion of (a) a control site (without membrane) and (b) a test site (with a nonresorbable ePTFE membrane) after 2 months. While a low bony cover seals off the marrow space at the bottom of the control defect, the regenerated bone completely fills the secluded space beneath the barrier membrane at the test site (undecalcified ground section; toluidine blue and basic fuchsin stain).

Healing under a barrier membrane

Membrane protection resulted in a dramatic change in bone regeneration. The membrane maintained the space created during surgery and clearly separated the outer compartment, constituting the oral mucosa, from the inner space, which was mainly accessible from the marrow cavity (Fig 2-26b). This inner compartment was initially filled with a blood clot, and at 2 months, remnants of the coagulum could still be recognized in the middle portion of the defect (Figs 2-27 and 2-28).¹²³ However, the hematoma was completely penetrated by granulation tissue and blood vessels. The majority of the secluded volume now consisted of a spongeous bony regenerate that enclosed, between its trabeculae, a labyrinth of tiny, interdigitating marrow spaces filled with hypervascularized, loose, soft connective tissue. Both the vessels and the fibrous tissue were in continuity with the original bone marrow at the bottom of the defect. Bone formation started, as in the controls, from the margins of the defects, where it spread over the openings of the marrow cavity (see Fig 2-28).

Fig 2-27 Selected serial buccolingual sections, arranged in a mesiodistal sequence, of a membrane-covered defect after 2 months. (Reprinted with permission from Schenk et al.¹²³)

Fig 2-28 The fifth section from Fig 2-27 to show (1) the bony cover of the bottom marrow space and (2) a tangential section through the top of the bony hill originating from the mesial wall of the defect, as well as (3) the associated hematoma.

Thus, there were basically three centers of bone formation, and these formed a dome-shaped seal over the openings of the marrow cavity. From these bony covers, bone further expanded into the center of the membrane-bound space. Both radiographs (Fig 2-29) and serial ground sections (see Fig 2-27) demonstrated this characteristic pattern of bone healing.¹²³ See Fig 2-28 for an illustration of the third center of bone formation at the bottom of the defect.

Fig 2-29 Radiograph of a membrane-covered defect after 2 months shows bony ingrowth from the mesial and distal walls, as well as from the bottom of the defect. (Reproduced with permission from Schenk et al.¹²³)

Formation of the primary spongeous scaffold. The histology of bone formation in the membrane-protected space exhibited a remarkable similarity to that found during bone development and growth (Fig 2-30). The infiltration of the hematoma by granulation tissue followed the basic pattern of wound healing. The invading vascular sprouts were accompanied by cells originating from the bone marrow at the periphery of the defect, thus enabling mesenchymal stem cells to differentiate into osteoblasts.¹²⁴ From the cut cortical and trabecular surfaces, woven bone sprouted out, mostly in the shape of thin, bifurcating plates (Fig 2-31). A particular characteristic of this primary cancellous scaffold is the perfect interdigitation with the vascular plexus. As already mentioned, angiogenesis and an ample blood supply are mandatory for bone development and maintenance.

Fig 2-30 Organization of the hematoma and woven bone formation. Blood vessels and bone-forming cells invade the former hematoma (right) and construct a scaffold of woven bone (Goldner trichrome stain).

Fig 2-31 In the advancing ossification front, blood vessels (BV) and outgrowing trabeculae are tightly interconnected. Goldner trichrome stain stains osteoid seams in red and mineralized bone matrix in green.

Transformation into compact bone and a regular spongiosa. While the original primary trabeculae consisted exclusively of woven bone, it later served as a template for the apposition of parallel-fibered bone followed by lamellar bone, which eventually would constitute both compact bone and a regular spongiosa with mature bone marrow. These events occurred 3 to 4 months after surgery (Fig 2-32).

Fig 2-32 Transformation of the primary spongiosa spongework into cortical bone and cancellous bone. (a) After 2 months, the spongiosa is denser at the periphery than in the center of the bony regenerate. (b) Cortical bone and secondary spongiosa after 4 months. A compact bone layer in the periphery confines a cancellous bone in the center with well-defined trabeculae and regular bone marrow (surface staining with toluidine blue and basic fuchsin.)

Remodeling of cortical bone. During the fourth month, the cortical bone entered its last phase of maturation: Haversian remodeling.

Modeling of the bony regenerate. At the end of the fourth month, growth and modeling of the bone within the membrane-confined space continued, particularly in the central portion. With the formation of a cortical bone layer, the periosteal and endosteal envelopes were also restored. As long as modeling was occurring, the bone surface was locally lined by osteoblasts and an osteoid seam, or was covered by osteoclasts or Howship lacunae, as a sign of ongoing or past resorptive activity.

Bone healing in membrane-protected defects with addition of a bone filler

The healing pattern of bone under a barrier membrane with the addition of various particulate bone fillers was analyzed in numerous experimental animal studies. One standardized animal model was introduced by Buser et al.¹⁰⁶ In this model, which excludes the interference of the particular conditions in the oral cavity, standardized bone defects were created in the mandibles of minipigs with extraoral access. This study showed that particulate bone fillers have different biologic characteristics with regard to both bone formation potential and bone filler degradation dynamics. After 4 weeks, which was the earliest observation period in this study, significantly more new bone was formed when autogenous bone was used as a filler, as compared to DFDBA, a synthetic β-TCP biomaterial, and coral-derived HA (Fig 2-33).¹⁰⁶ After 12 and 24 weeks, there was still more bone formation in the autogenous bone group than in the groups with DFDBA and coral-derived HA, but most new bone was found in the TCP group. On the other hand, TCP showed the greatest degradation rate, meaning that the volume of all three other fillers was more stable. Another important finding was that the autograft was the most osteoconductive filler material over the entire observation period (Fig 2-34).¹⁰⁶ From this study, it was concluded that defects filled with autograft clearly demonstrated the best results in the early phase of healing and that the TCP biomaterial used showed a fast degradation and substitution rate.

Fig 2-33 Percentage of new bone in standardized bone defects in the mandibles of minipigs grafted with different materials. (Data from Buser et al.¹⁰⁶)

Fig 2-34 Percentage of grafting material surface covered with bone as an indicator of the osteoconductive potential of different grafting materials in standardized bone defects in the mandibles of minipigs. (Data from Buser et al.¹⁰⁶)

Based on these intriguing results, earlier observation periods down to 1 week were chosen, and other bone filler biomaterials were tested in subsequent studies.^{119,120,125,126} Collectively, these studies confirmed that the autograft caused most new bone formation—at least up to 4 weeks (Figs 2-35 to 2-37)—and was the most osteoconductive filler material (Fig 2-38). It was also confirmed that the β-TCP biomaterial lagged behind but was catching up with the autograft group. Furthermore, these studies demonstrated that use of synthetic β-TCP led to faster bone formation than use of synthetic HA, and that biphasic BCP with a 60:40 ratio of HA and TCP was somewhere in between (see Fig 2-36). Varying the ratio of HA and TCP showed that the higher the TCP content was, the more quickly bone formation occurred (see Fig 2-37).

Fig 2-35 Percentage of new bone in standardized bone defects in the mandibles of minipigs after grafting with different materials. (Data from Jensen et al.¹²⁵)

Fig 2-36 Percentage new bone in standardized bone defects in the mandibles of minipigs after grafting with different materials. The three bone substitute materials have identical material characteristics except for the chemical composition. In the early healing phases, more new bone formation is seen in defects grafted with TCP than with BCP, which resulted in more new bone formation than HA. (Data from Jensen et al.¹¹⁹)

Fig 2-37 Percentage of bone formation in standardized bone defects in the mandibles of minipigs after grafting. In the early healing phases, more new bone formation is seen in defects grafted with BCPs with high TCP content. (Data from Jensen et al.¹²⁰)

Fig 2-38 Percentage of grafting material surface covered with new bone in standardized bone defects in the mandibles of minipigs. (Data from Jensen et al.¹²⁰)

What has not been mentioned thus far is the HA of bovine origin. Various brands of bovine-derived bone substitutes are available on the market. However, one of them outclasses all other forms in terms of quantity of scientific data: Bio-Oss (Geistlich), also known as deproteinized bovine bone mineral (DBBM), bovine-derived bone mineral (BDBM), or anorganic bovine bone (ABB). Bio-Oss has been commercially available for regenerative dentistry for more than 30 years.

Some controversy remains as to whether DBBM is truly resorbable.¹²⁷ It has been shown in vitro that osteoclast progenitor cells are able to proliferate on DBBM surfaces, and later, as osteoclast-like cells, produce resorption pits. Compared with native bovine bone, however, the osteoclasts are reduced in number and size, and the resorption pits are less pronounced.¹¹⁵ Experimental in vivo studies have also demonstrated multinucleated giant cells on DBBM surfaces that stain positive for TRAP^{119,120,128–130} and occasionally display a sealing zone, a ruffled border, and shallow Howship lacunae¹³⁰ (Fig 2-39). These findings suggest that these cells have osteoclast-like properties, but fail to resorb this biomaterial, at least at a fast pace. Also, in the mandibular defect model in the minipig, a significant reduction in volume over observation periods of up to 1 year was not seen.¹²⁰ Human biopsies after sinus augmentation confirm that particles of DBBM can still be found up to 20 years postoperatively¹³¹ (Fig 2-40). Thus, in daily practice, this particular xenograft must be considered very resistant to resorption.

Fig 2-39 Histologic sections of human biopsy specimens grafted with DBBM (thin sections of decalcified tissue). (a) The DBBM particles show good tissue integration. Newly formed bone (NB) covers part of the bone substitute surface and bridges neighboring DBBM particles (basic fuchsin and toluidine blue stain). (b) Osteoclast-like, multinucleated giant cells (arrows) are frequently seen lining the DBBM surface, which occasionally shows shallow resorption depressions (arrowheads) (toluidine blue stain alone). (c) Staining for tartrate-resistant acid phosphatase (TRAP) (red staining) identifies osteoclast-like, multinucleated giant cells on the surface of the DBBM particles (stained for TRAP and counterstained with toluidine blue).

Fig 2-40 Histologic section of a human biopsy specimen harvested 20 years after sinus floor elevation with DBBM. DBBM particles are interconnected by bone matrix (B) and surrounded by mature fatty bone marrow (BM) (thin paraffin section of decalcified tissue; toluidine blue and basic fuchsin stain).

In the studies by Jensen et al^120,125 and Broggini et al,¹²⁶ the amount of new bone in defects filled with DBBM increased over time, yet at a slower pace than around the autograft particles (see Figs 2-35 and 2-37). Likewise, osteoconductivity was lower with DBBM than with autograft particles (see Fig 2-38). This difference was clearly visible for up to 4 weeks after defect fill, and most pronounced after 1 to 2 weeks (Fig 2-41). Although bone regeneration started from the defect margins for all types of bone fillers, progression of bone formation and consequently defect fill with new bone was fastest when autogenous bone chips were used (Fig 2-42).

Fig 2-41 Histologic sections of standardized bone defects in the minipig mandible 2 weeks after GBR with an ePTFE membrane (asterisks) and autogenous bone chips (AB) (a) or DBBM (b) (undecalcified ground sections; toluidine blue and basic fuchsin stain). The defect margins are delineated by old bone (OB) and the membrane. Formation of new bone (arrowheads) begins from the defect margins. New bone formation is much more advanced (arrowheads) in defects filled with autogenous bone than with DBBM.

Fig 2-42 Histologic sections of standardized bone defects in the minipig mandible 1 and 2 weeks after GBR with an ePTFE membrane and autogenous bone chips (AB) (undecalcified ground sections; toluidine blue and basic fuchsin stain). Formation of new bone (NB) begins from the surface of old bone (OB) at the defect margins and progresses towards the center of the defect. New bone formation is already visible after 1 week (a) and much more advanced after 2 weeks (b).

These and other studies came to the conclusion that it would be advantageous to combine the best of two worlds: autogenous bone, with its excellent osteopromotive and osteoconductive properties, boosting bone formation in the early healing phase; and DBBM, with its low degradation rate, helping to keep the gained bone volume stable over the long term. Indeed, clinical and radiographic data from humans^132–135 have confirmed this biology-inspired concept, in which autogenous bone chips and DBBM are placed layer by layer to augment peri-implant bone defects. In this two-layer approach, the autograft particles are placed close to the implant surface to promote bone outgrowth from preexisting bone, and the DBBM particles form an external shield against resorption. Tiny biopsies harvested 14 to 80 months after contour augmentation in patients supplied histologic evidence of long-term stability when applying this concept.¹²⁹ While the histology has shown good integration of DBBM in newly formed bone (Fig 2-43), histomorphometry has demonstrated stable new bone volume and no signs of significant substitution of DBBM particles over time (Fig 2-44). Thus, earlier findings from preclinical studies were confirmed, and it was suggested that the low substitution rate of DBBM may be the reason for the clinically and radiographically documented long-term stability of contour augmentation, when a combination of autogenous bone chips, DBBM particles, and a collagen membrane is used. In a recent preclinical study, it was shown that the addition of autogenous bone chips and the presence of a collagen membrane increased bone formation around DBBM particles¹³⁶ (Figs 2-45 and 2-46).

Fig 2-43 Histologic section of a human biopsy specimen harvested 74 months after contour augmentation lateral to a dental implant with particulate autogenous bone, DBBM, and a collagen membrane (decalcified tissue; toluidine blue and basic fuchsin stain). Low (a) and high (b) magnifications demonstrate good tissue integration of DBBM particles. Newly formed bone (NB) covers a substantial portion of the DBBM surfaces and bridges neighboring DBBM particles. Adipocytes indicate presence of mature bone marrow.

Fig 2-44 Histograms for 12 human biopsy specimens harvested from 14 to 80 months after contour augmentation with particulate autologous bone, DBBM, and a collagen membrane. (a) Area fractions of new bone and DBBM. (b) Percentage of DBBM surface covered with new bone.

Fig 2-45 Light micrograph demonstrating perfect structural integrity of an augmented site 3 weeks after contour augmentation with autogenous bone chips (AB), a bone substitute (BS) (Bio-Oss), and a non-cross-linked barrier membrane (BM) (Bio-Gide) of a buccal peri-implant bone defect in a dog mandible. Residues of the double-layered barrier membrane are still clearly visible, and the oral mucosa (OM) is blocked off. New bone (NB) has formed in the inner portion of the augmented region (undecalcified ground section; toluidine blue and basic fuchsin stain).

Fig 2-46 Relative amounts of osteoid, new mineralized bone, DBBM, bone autograft, and soft tissue in the grafted region 3 and 12 weeks after contour augmentation in dog mandibles. Four combinations of 3 different materials were tested: (A) autogenous bone chips, (D) DBBM, and (M) collagen membrane. The addition of autogenous bone chips and the presence of the collagen membrane increased bone formation around DBBM particles.

Have we reached the limit with the contour augmentation technique using GBR? As we have seen, bone formation around bone substitutes lags behind bone formation around autogenous bone chips during early healing periods. Thus, it is tempting to find ways to improve the performance of bone substitutes. One possibility is to coat them with biologics like growth factors, a process called biofunctionalization. In particular, DBBM appears to be very suitable for this, since its high macro- and nanoporosity can absorb a lot of the patient’s own proteins and other macromolecules from the environment (Fig 2-47a) and can be precoated with biologically active molecules^137,138 (Fig 2-47b). In chapter 3 of this book, the scientific background and value of biofunctionalizing biomaterials with bone-conditioned medium (BCM)—ie, the patient’s own growth factors released from bone—is discussed in detail.

Fig 2-47 (a) Ultrathin section of a human biopsy specimen retrieved from a bony site augmented with DBBM and embedded in acrylic resin. High-resolution postembedding immunocytochemistry with an antibody against a typical bone-related noncollagenous protein demonstrates gold particle (black dots) labeling preferentially at the periphery of the DBBM particle. This finding indicates that DBBM takes up the patient’s own proteins from the wound environment after implantation. (b) High-resolution postembedding immunocytochemistry with an antibody against enamel matrix proteins demonstrates the ability of DBBM to be precoated with biologically active molecules prior to implantation. CT, soft connective tissue.

Conclusion

Bone has the unique capability to rebuild its original structure and function in response to a trauma. The trick in GBR is to harness this great regenerative potential to enhance bone formation around dental implants or at sites destined for implant placement. Under stable mechanical conditions, bone is formed directly or primarily, provided that two essential conditions are present: an ample blood supply and a solid base for bone deposition. The solid base is provided by the bony margins of a defect. Bone healing around dental implants is special in the sense that the implant surface in the defect region is devoid of bone, and thus bone has to grow into the defect from the walls of the preexisting bone. While the function of the barrier membrane is to keep away unwanted, fast-growing tissues, the functions of bone fillers are manifold. Since one type of bone filler cannot fulfill all requirements, the best of two worlds should be combined: autogenous bone with its great osteoconductive, osteoinductive, and osteogenic potential, boosting bone formation in the early healing phase; and a low-substitution filler that keeps the gained bone volume stable in the long term.

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