Healing Pattern of Bone Regeneration in Membrane-Protected Defects

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Healing Pattern of Bone Regeneration in Membrane-Protected Defects: A Histologic Study in the Canine Mandible Robert K. Schenk, MD, Prof Dr Med/Daniel Buser, DDS, PD Dr Med Dent/ W. Ross Hardwick,MS/Christer Dahlin, DDS, PhD

In this study, standard and prototype reinforced e-PIFE membranes were used to evaluate the pattern of bone regeneration in membrane-protected defects in the mandibles of four dogs. Following a healing period of 2 and 4 months, control sites without membranes exhibited incomplete osseous healing with a persisting defect. Test sites with membranes demonstrated significantly better bone healing, although bone regeneration was not yet completed at 4 months. The histologic evaluation showed that bone regeneration, once activated, progresses in a programmed sequence through a series of maturation steps, which closely resemble the pattern of bone development and growth. (INT J O RAL MAXILLOFAC IMPLANTS 1994;9:13-29.) Key words: bone defects, bone healing, guided bone regeneration, membranes

In the 1980s, the use of osseointegrated dental implants, anchored in the jawbone with direct bone-to-implant contact, became an increasingly important treatment modality for the replacement of missing teeth in completely and partially edentulous patients.1,2 One of the prerequisites for predicting a long-term prognosis for osseointegrated implants is a sufficient volume of healthy jawbone at possible recipient sites. Clinical experience has clearly demonstrated that the long-term prognosis of dental implants is compromised if the buccal bone plate is missing at the time of implant placement.3-5 However, a sufficient amount of bone volume is frequently lacking as a result of trauma or infectious diseases such as advanced periodontitis. For these situations, reconstructive surgical techniques have been developed to restore missing bone to allow the placement of dental implants in either a simultaneous or a staged approach. One possible technique is based on the application of barrier membranes first described in 1959 by Hurley et al6 for the treatment of experimental spinal fusion. In the 1960s, the research teams of Bassett 7-9 and Boyne10-12 tested microporous cellulose acetate laboratory (Millipore) filters for the healing of cortical defects in long bones and osseous facial reconstruction, respectively. The authors used the filters to establish a suitable environment for osteogenesis by excluding connective tissue cells from bone defects. In the early 1980s, this principle was tested in a number of systematic experimental studies for the regeneration of lost periodontal tissues, and the phrase "guided tissue 13



regeneration" was created.13 In the past 5 years, this concept has been tested again in a number of experimental studies for the regeneration of different types of bone defects.14-24 In these studies, barrier membranes were placed over bone defects and closely adapted to the surrounding bone surface, creating a secluded space between the bone and the membrane. With the placement of a barrier membrane, preference was given to osteoprogenitor cells originating from adjacent bone to populate and regenerate these defects with bone. Again, the membrane barrier was used to prevent the invasion of competing soft tissue cells from the mucosa into the defects. Uncovered control sites demonstrated incomplete bone regeneration and the presence of soft tissue within the defects. However, these studies gave only minor information about the sequence and pattern of bone regeneration in jaw defects. The purpose of the present study was to characterize the pattern of bone regeneration in membrane-protected mandibular defects in foxhound dogs. In addition, two different types of barrier membranes, made of the same basic material but with different stiffness properties, were compared.

Materials and Methods Surgical Procedure. Four adult male foxhound dogs, each weighing more than 25 kg, were used in this study. The protocol of the study was approved by the Flagstaff Animal Care and use Committee, and the animals received humane treatment and protection during the investigation. Oral prophylaxis was performed 2 weeks prior to tooth extraction and again 2 weeks prior to the membrane surgery. The mandibular second, third, and fourth premolars of each dog were extracted bilaterally, creating an edentulous mesiodistal space of approximately 50 mm. All surgical procedures were performed under general anesthesia, accomplished by pre-anesthesia sedation (atropine sulfate, 0.05 mg/kg, SQ) and anesthetic induction ([Telazol, A.H. Robbins Inc], 3.0 mg/kg, IV) followed by intubation and maintenance with halothane gas for the duration of the surgical procedure. Approximately 4 months after tooth extraction, the membrane surgery was performed. Prophylactic antibiotic treatment (trimethyl sulfate [Di-Trim, Syntex Animal Health Inc], 30 mg/kg BID) was begun the day before surgery and continued for 7 days postoperatively. Following a lateral, poncho-like incision of the buccal mucosa approximately 3 to 5 mm from the mucogingival junction, a supraperiosteal Rap preparation was raised to the mucogingival junction, where the periosteum was horizontally cut. In addition, vertical relieving incisions were made to the lingual aspect. Subsequently, a combined split-thickness/full-thickness Rap was carefully elevated to the lingual aspect using fine tissue elevators. The buccal periosteal Rap was also elevated to gain access to the buccal aspect of the alveolar ridge. Two rectangular bone defects to include the ridge crest were surgically created bilaterally in the area of the previously extracted second and fourth premolars using



low-speed rotary and hand instruments. Copious sterile saline irrigation was used during preparation of the bone defects. Although individual differences in the alveolar ridge dimensions did not allow a perfect standardization of the defects, a careful effort was made to keep the defect dimensions reasonably constant in size. Defect dimensions were approximately 8 mm apicocoronal, 12 mm mesiodistal, and 10 mm buccolingual at the bottom of the defects (± 1.0 mm in each dimension). Each dog received (a) one standard expanded polytetrafluoroethylene (e-PTFE) membrane (GoreTex Augmentation Material [GTAM], WL, Gore, Flagstaff, AZ); ( b) two prototype reinforced e-PTFE membranes (r-GTAM, WL Gore), each of which had been preformed into an arch shape; and (c) one nonmembrane control site. Treatments were randomly assigned. Each membrane was trimmed to shape and draped over the ridge so that the membranes completely covered the defects and extended beyond the defect margins by at least 2 to 3 mm (Fig 1). This created a space defined by the three bone walls of the defect as well as the shape and position of the membrane. The membranes were secured to the buccal bone by fixation screws made of stainless steel (Memfix System, Institut Straumann, Waldenburg, Switzerland). The screws also served as landmarks for locating the defect areas radiographically during the harvesting and sectioning procedures. Intravenously aspirated blood was injected in all sites underneath the membrane with a syringe to remove the air and to ensure the formation of a stable blood clot in the secluded space. Subsequently, primary wound closure was achieved with vertical mattress and interrupted e-PTFE sutures (Gore-Tex Sutures, WL Gore). Two randomly assigned dogs were killed at 2 months and the remaining two were killed at 4 months following surgery. Nonstandardized radiographs were taken within 10 days after surgery. Antiinflammatory (ibuprofen, 200 mg QID) and analgesic (Talwin-V, Winthrop Vet Sterling. Animal Health Inc, 30 mg/kg, IM) medications were given for 3 days following surgery to reduce postoperative swelling and pain. Oral prophylaxis was performed every 2 weeks. Chlorhexidine rinse was used twice per week. The dogs were maintained on a soft diet for the duration of the study. Sutures were removed 10 days after surgery. The animals were killed by induction of deep anesthesia with subsequent intravenous sodium pentobarbital overdose. Radiographs of each site were obtained immediately prior to sacrifice and again after removal of the blocks. Before the blocks were harvested, the soft tissues overlying the experimental sites of one of the 4-months dogs were removed to visualize the treated sites. The remaining sites were harvested with the soft tissues intact. Histologic Preparation. Block sections containing the test and control sites were removed and placed in 4% formalin/l% CaCl2 fixative. A radiograph was made of each specimen prior to histologic processing. The specimens were dehydrated and embedded in methyl methacrylate resin. Undecalcified sections approximately 500 µm thick were obtained using a low-speed diamond saw with coolant.



Approximately 12 orofacial sections could be obtained from each site. Subsequently, the sections were glued with acrylic cement to opaque Plexiglas, ground to a final thickness of about 80 µm, and stained superficially with toluidine blue or toluidine blue combined with basic fuchsin. In addition, 5-µm thin sections were cut with a microtome and stained with Goldner's trichrome or with von Kossa/McNeal's tetrachrome.25 Histomorphometric Analysis. Serial sections, taken at intervals of 1.2 mm, were photographed and enlarged to a final magnification of 10×. First, the defect size and the total bone volume formed were measured by point counting. The calculated defect served as a reference. It was determined by measuring the defect area in the mesial and distal wall, and multiplying their average by the length of the defect. The measured volume represented the actual size of the defect underneath the membrane. In addition, various components of the regenerated tissue were determined in photographs at a magnification of 40×. The results are calculated as percent of the measured defect volume.

Results Clinical Observations. Healing progressed uneventfully in three of the four animals. One dog in the 2-month group showed dehiscence of the suture line at the distobuccal comer of the flap in one quadrant 10 days following surgery. The membrane located at this site was not visibly exposed. The flap was resutured and appeared to heal without complications. At 7 weeks postoperatively, a small abscess was observed in the region of the previously noted dehiscence and over the corner of the membrane. The dog was placed on antibiotics (amoxicillin [Clavamox]) for the remaining week. The abscess subsequently decreased and there was no fistula formation. The animal was killed according to schedule. Healing of Control Defects. Bone regeneration in the four control defects was limited to the formation of a cap that sealed the surgically created openings of the marrow space. Clinically and radiographically, a deep indentation persisted in the contour of the alveolar crest, which was only partially filled by the collapsed mucosa (Figs 2 and 3). The structure of the bony cap was clearly seen in orofacial sections through the middle portion of the defect (Fig 4, D to H). Toward the mesial and distal walls of the defect, this cover was tangentially cut and could be mistaken as a bony filling (Fig 4, B and C). The openings of the marrow space were completely closed by 2 months. At 4 months there was some increase in bone density, but no further reduction of the defect size was noted. The periosteal bone surface was separated from the oral epithelium by rather dense fibrous tissue, which could not be delineated microscopically from the lamina propria. Defect Healing Under Barrier Membranes: 2 Months. Placement of the membrane created two compartments within the defect area. The inner compartment was initially filled by a blood clot and was almost completely separated from the outer compartment, which consisted of the repositioned tissue of the mucoperiosteal



flap. The size of the space created during the surgery varied, depending on the stiffness of the membrane used, the width of the ridge, and the accuracy of the surgical procedure. In Table 1, the calculated defect volume serves as a reference and represents the space available under optimal conditions, i.e., without any deformation of the covering membrane. At 2 months, the measured defect volume underneath the standard e-PTFE membranes was considerably smaller than the calculated one. This was partially the result of the less stiff membrane but was also related to the surgical procedure. Compared to the 4-month specimens, all defects of the 2-month group were considerably larger, mainly in the apicocoronal direction ( Figs 5 to 7). As a consequence, the membrane did not sufficiently overlap the bottom margins and became detached. This occurred in the two sites with standard e-PTFE membranes and resulted in folding and partial collapse of the membranes. In the four test sites of the 2-month group that were covered with a reinforced membrane, the protected space created during surgery was almost entirely maintained (Table 1). Pattern of Bone Regeneration. Bone formation began on the exposed surfaces of the defect. At 2 months, three newly formed bony hills were visible in the radiographs and appeared to grow toward the middle portion of the defect (Fig 5). Histologically, bone formation spread out throughout the whole extent of the defect and followed a rather uniform pattern (Fig 6). Similar to the control defect, it first sealed the opening of the marrow cavity. This was clearly seen at the bottom of the defect (Fig 6, F). Identical dome-shaped caps formed on the mesial and distal openings of the marrow space. Since their base was formed by the cortical bone bordering the defect wall, the first orofacial sections often revealed a cortical layer around a marrow cavity (Fig 6, A and B). In the following sections, the roof of the mesial dome was tangentially cut (Fig 6, D). As it grew farther into the middle portion, it overlaid the bottom cover (Fig 6, E and F) and finally fused with the bottom dome and its counterpart originating from the distal wall (Fig 6, G and H). Formation of the Primary Spongiosa. The outer circumference of the regenerate runs parallel to the inner surface of the membrane but was always separated from it by interposed granulation tissue (Fig 8a). In the middle portion of the defect, remnants of the original blood clot were still present within this layer. They were especially prominent in the approximately 80-µm-thick ground sections. In thin microtome sections, these remnants of the blood clot were thoroughly invaded by granulation tissue (Fig 8b). Along their borderline, the initial stages of intramembranous or direct bone formation were still active. A network of small blood vessels preceded the outgrowth of woven bone, which initially formed thin, bifurcating plates, first consisting of osteoid, followed by rapid mineralization (Figs 8c and 8d). These plate-like trabeculae fused and then confined 200- to 500- µm-wide intertrabecular spaces, which were filled by a well-vascularized, primitive fibrous bone marrow. Surfaces of the newly formed trabeculae were lined by a continuous layer of osteoblasts, and ongoing bone deposition further increased



their diameter. The result of this bone formation was a primary spongework or primary spongiosa. It formed a scaffold of randomly oriented trabeculae with diameters of 60 to 100 µm, resulting in an overall bone density of about 35% (Table 2). The intertrabecular spaces were extremely well vascularized, in contrast to the peripheral fibrous tissue compartment underlying the membrane. Defect Healing Under Barrier Membranes: 4 Months. In this series, all experimental sites were successful, regardless of whether standard or reinforced e-PTFE membranes were used. Accordingly, the results were quite homogeneous, as documented by the representative sequential sections. As seen in the radiograph (Fig 9) and in the serial sections (Fig 10), bone regeneration was not completed at 4 months. There was a definite gradient from the marginal portion toward the middle zone, where the formation of the primary spongiosa was still going on, particularly in the roof of the defect (Fig 10, E and F). Superficially, the net amount of bone volume within the membrane-protected space does not appear to differ much from the 2-month specimens. Morphometry, however, showed that the relative volume of the regenerated bone had increased in the second observation period (Table 2). At 2 months, bone occupied approximately 36% of the defect volume; at 4 months, this was nearly 55%. However, the bone volume, calculated in mm3, was smaller at 4 months (327 to 401 mm3), since the defects created at surgery were also considerably smaller in size (665 to 1,196 mm3), mainly because of the reduction in their apicocoronal depth (Figs 11 and 12). Compared to the situation at 2 months, the primary spongiosa had organized into a cortical layer and a secondary spongiosa, and the restoration of the marrow cavity was the most prominent event achieved in the third and fourth months of healing. As the morphometry shows (Table 2), less than 1% of the regenerate consisted of cortical bone at 2 months; whereas at 4 months, the cortical layer occupied 38%. Simultaneously, the density of the spongiosa dropped from 36% to 15%. Again, this differentiation started close to the border of the defect, i.e., at the bottom and at the mesial and distal wails. The formation of a cortical layer ("corticalization") is mainly the result of continuing bone deposition, whereas the secondary spongiosa results from remodeling consisting of substitution by resorption, followed by formation of new lamellar bone. Corticalization of the Primary Spongiosa. At 2 months, the primary spongiosa consisted mainly of trabeculae made of woven bone, most trabeculae being covered by osteoid seams, superficially lined by a continuous layer of osteoblasts (Fig 13a). Continuing bone formation resulted in a filling of the intertrabecular spaces, which were finally narrowed to the diameter of cortical vascular canals (Figs 13b to 13d). During the course of this bone deposition, both the structure and the quality of the newly formed bone matrix matured. The original woven bone was reinforced by parallel-fibered and finally by lamellar bone. Discrimination of these three bone matrix compartments was rather difficult in undecalcified microtome sections (Fig



13a). However, in surface-stained ground sections, they could be clearly delineated ( Figs 13c and 13d). Corticalization ended with the formation of rather compact bone, consisting of remnants of the original woven bone scaffold, and the filled-in primary osteons with narrow vascular canals, surrounded by a wall made of parallel-fibered or lamellar bone (Fig 13d). The last step in corticalization was remodeling, i.e., the substitution of primary osteons by secondary osteons. In the 4-month specimens, cortical remodeling had just started, particularly adjacent to the borders of the defects (Fig 14a). These specimens clearly demonstrated secondary osteons by the presence of cement lines ( Fig 14b). Formation of the Secondary Spongiosa. Comparison of the central part of orofacial sections at 2 and 4 months (Figs 11 and 12) clearly showed the transformation of the spongiosa from a primary to a secondary type. It was accompanied by a reduction of overall bone density and widening of intertrabecular spaces, and led to a certain anisotropy in the orientation of the trabeculae. Originally, the primary spongiosa consisted of woven and parallel-fibered bone (Fig 15a). As a result of the remodeling, the primary scaffold disappeared and was replaced by somewhat thicker trabeculae. These trabeculae were composed of packets of lamellar bone formed by osteoclastic resorption, followed by lamellar bone deposition (Fig 15b) . The net result of cancellous bone remodeling is a reduction in bone density. This apparent bone loss, however, is more than compensated by the increase in bone mass resulting from corticalization (Table 2). Modeling of the External Shape. With the formation of a cortical layer, periosteal and endosteal surfaces lined by the respective periosteal and endosteal envelopes were also restored. These envelopes were the site of further bone formation or bone resorption, which changed the external shape of the regenerated bone as well as the configuration of the marrow cavity. Microscopic inspection of the periosteal surface at 4 months revealed resting inactive areas, sites of continuing appositional bone formation, and localized foci of osteoclastic bone resorption, all of which are reliable signs of ongoing shape-deforming modeling activities. Soft Tissue Compartments. Cell occlusive barrier membranes are designed for the separation of different tissue compartments. In the case of alveolar crest reconstruction, the membrane is effective as a barrier against tissue ingrowth from the masticatory and oral mucosa. At 4 months, the microscopic appearance of the outer and inner soft tissue layer was different. In the outer compartment, the connective tissue was composed of coarse collagen fibers (Figs 16a and 16b). The diameter of these fibers increased toward the membrane. In contrast, the inner compartment contained loose connective tissue with thin collagen fibers. Its cell population consisted mainly of fibroblasts. Histiocytes and blood-borne cells were extremely rare. This tissue was well vascularized, especially in the region of the periosteal envelope.



Both connective tissue compartments established contact with the e-PTFE membranes. Along this interface, there were no changes in the cellularity, and no signs of any foreign-body reaction could be found. The structure of the membranes varied somewhat. The standard (nonreinforced) membrane consisted of an inner cell-occlusive portion and an outer more porous portion with interstitial spaces averaging 25 µm. The reinforced prototype membranes were characterized by a porous structure similar to the outer portion of the standard membranes. The interstices of the porous parts were invaded by cells and small capillaries, originating mainly from the inner compartment (Fig 16c). In most cases, connective tissue was interposed between the newly formed bone and the membrane. But in some sites, bone had established contact with the inner surface of the membrane (Fig 16b). The interstices were then almost completely filled with bone formed by osteoblasts that had invaded the membrane (Fig 16d) . Bone deposition into the pores was also seen in sites where the membrane was pressed directly upon the periosteum by miniscrews. Bone marrow represents a third soft tissue compartment. At 2 months, it could be characterized as an extremely well-vascularized fibrous bone marrow (Fig 15a). At 4 months, it had matured and closely resembled marrow in the deeper part of the mandible (Fig 15b).

Discussion In the past 5 years, barrier membranes have been extensively tested in experimental studies for the regeneration of bone defects.14-24 At present, the terms "guided bone regeneration" (GBR) and "osteopromotion" are mainly used in the literature for this membrane technique. The results of these studies have demonstrated that the placement of a membrane promotes the osseous healing of bone defects, since competing nonosteogenic soft tissue cells are excluded from defect healing by the presence of the physical barrier. This observation was confirmed in the present study examining bone repair of large mandibular defects in foxhound dogs. Each of the four nonmembrane control sites demonstrated incomplete bone regeneration and a persisting defect within the time period of the investigation. In addition, the present study provided detailed information about the pattern of bone regeneration in membrane-protected defects. It is important to note that the membranes in the present study were placed over freshly created defects. Bone formation is activated by the release of growth factors and bone-inducing substances. Bone matrix is considered to be one of the richest sources for growth factors and related compounds, produced either by osteoblasts themselves or by other bone related cells.26-28 Bone matrix is directly exposed by any mechanical lesion, such as fractures, osteotomies, implant placement, or simply by creation of defects. Activators are released or made available from bone surfaces and are able to interact with the surrounding cell population. The result of this activation becomes apparent within a couple of days. Histologically, this activation



is manifested as a stimulation of neoangiogenesis, the recruitment of osteoblasts, and in the onset of bone matrix deposition, provided that the activators act upon committed responding cells. An influencing mechanism of membrane placement may be that stimulating growth factors are locally concentrated in the osseous wound at inductive doses, leading to osseous repair of such defects that normally would not heal spontaneously.29 The type of responding cells is important for the success of the GBR technique. Cell culture experiments have shown that two categories of osteoblast precursors exist30,31: determined (DOPC) and inducible (IOPC) osteogenic precursor cells. The IOPCs are found in fibrous tissue (i.e., subcutaneously, intramuscularly, or in the kidney capsule) and react to bone-inducing compounds with heterotopic, indirect, or endochondral ossification.32-34 The DOPCs, once activated, proliferate and then differentiate into preosteoblasts and osteoblasts. These cells start direct bone formation without an intermediate cartilaginous stage. The DOPCs exist in the periosteal, endosteal, and endocortical envelopes, as well as in bone marrow stroma cells.30,31 As shown in the present study, e-PTFE membranes confine a defect space that is only accessible from the adjacent marrow. The ingrowing blood vessels originate exclusively from the medullary system, and they are accompanied by perivascular cells that are derived mainly from bone marrow stroma cells. Accordingly, bone formation follows the direct pathway, and in none of the sites has any intermediate cartilage formation been detected. Besides the presence of osteoprecursor cells, bone formation has two basic prerequisites: ample blood supply and a solid base for bone deposition.35 The solid base is provided either by the preexisting bone surfaces or, in the case of indirect endochondral ossification, by the formation of calcified cartilage. This explains the pattern of bone regeneration in membrane-protected defects. It always starts on the bony surfaces bordering the walls of the defect and then expands toward the roof and into the middle portion of the secluded volume. The microscopic pattern of bone formation is of interest for the understanding of bone regeneration in a general sense. Three categories of bone tissue are discriminated: woven bone, parallel-fibered bone, and lamellar bone. This classification dates back to the 1930s,36 was later reactivated,37 but still is often overlooked. Woven bone is generally characterized by the random, felt-like orientation of its collagen fibrils and the great number of large, more spherical osteocytes. It mineralizes rapidly, and fluorochrome labeling results in a diffuse rather than in a band-like uptake of the dye. In the context of growth and bone repair, however, another property becomes important: its capacity to grow by forming a scaffold of rods and plates and thus spreading out into adjacent tissue compartments at a relatively rapid rate. The construction of this primary scaffold occurs parallel to or is even preceded by the elaboration of a vascular network. The resulting primary spongiosa is in fact a sponge-like structure, consisting of interlacing bony and



vascular elements. In the present study, the rapid formation of woven bone made it possible to occupy a considerable, and still increasing, share of the protected defect volume at 2 months by building up the primary spongiosa. Once the primary spongiosa has been established, parallel-fibered bone can be deposited on its surface. Seen in the electron microscope, its matrix consists of collagen fibrils that are deposited in a parallel fashion but not yet grouped into lamellae with alternating courses.38 The osteocytes are less numerous and more flattened, with the long axis running parallel to the fibrils. Deposition of parallel-fibered bone requires a smooth and solid surface. In growing bones, it forms the wall of the primary osteons that develop in periosteal and endosteal apposition sites. In the present study with membrane-protected defects, the deposition of parallel-fibered bone reinforced the trabeculae of the primary spongiosa and contributed substantially to its corticalization. Like lamellar bone, parallel-fibered bone reveals a band-like label after fluorochrome application. In polarized light, however, it is much less birefringent than lamellar bone. Lamellar bone represents the most mature and most familiar type of bone tissue. Its microscopic appearance needs no further explanation. In later stages of corticalization, lamellar bone is directly deposited on parallel-fibered matrix and often forms the innermost layer in the wall of primary osteons, yet the borderline between the two compartments is not clearly defined. All three types of bone tissue occur sequentially during physiologic growth. They also reappear during bone repair, both in healing cortical defects39-41 and during callus formation.42 Bone remodeling always results in lamellar bone deposition, both in cortical and cancellous bone. It is then delineated from the surrounding bone matrix by a cement line of the reversal type. Reversal lines mark the level where osteoclastic resorption comes to a standstill and bone formation starts. Bone remodeling occurs in discrete remodeling sites or "bone-metabolizing units" (BMUs).43,44 In cortical bone, they consist of a "cutter cone" of osteoclasts that form a resorption canal with a diameter of 100 to 200 µm. The osteoclasts are followed by a vascular loop and by osteoblasts, which deposit concentric lamellae upon the wall of the resorption canal and finally narrow it to the diameter of haversian canals (50 to 80 µm). The dynamics of cortical bone remodeling are of particular interest: At the cellular level, the daily linear osteoclastic resorption rate in dogs is 50 to 60 µm,45-47 and the average daily osteoblastic apposition amounts to 1.5 to 2.0 µm. Resorption and formation are slow, and their rates cannot be accelerated. Hence, the formation of a new osteon will take 5 to 6 weeks in dogs but 3 to 4 months in humans.43,44 An activation of remodeling, however, is possible by the recruitment of new remodeling units. A dramatic local activation follows any mechanical lesion of bone in the course of fractures or surgical interventions.42 A temporary interruption in the blood supply results in avascular or necrotic areas. Revascularization and remodeling then lead to the substitution of necrotic tissue by vital bone tissue.



The histologic events encountered during this study with membrane-protected defects reveal a rather impressive fact. Once activated, bone regeneration progresses in a programmed sequence through a series of maturation steps, which closely resemble the regular pattern of bone growth. This observation is consistent with the epigenetic model for the regulation of bone formation described by Turner.48 In this model, formation of bone and fracture callus is regulated by a positive feedback loop that stimulates osteoblasts to a state of higher individual activity. This process continues until a state is reached that turns the process off. At this point, homeostatic (negative feedback) processes take over and may regulate lamellar bone formation. Turner hypothesized that epigenetic regulation is important during the many phases of bone development and regeneration. In the present study, the first step was comparable to intramembranous ossification and ended at 2 months with the elaboration of a primary spongiosa, mainly consisting of woven bone. In the second period, this scaffold was transformed into an assembly of cortical and cancellous bone that closely corresponded to the original internal architecture of the mandible. At 4 months, regeneration was not yet completed. Shape-deforming modeling took place along the surfaces lined by the periosteal and endosteal envelopes. Remodeling had also started, first in the cancellous compartment and later in the cortical area. This observation justifies the assumption that bone maturation will continue for a longer period. Modeling and remodeling are subject to regulatory mechanisms that control their spatial distribution as well as the net balance of matrix turnover. Both can be activated by local factors such as surgical trauma or mechanical loading. With this background, the question arises as to how the placement of a dental implant into augmented bone influences the remodeling pattern in surrounding bone tissue. It can be anticipated that the surgical trauma would stimulate bone remodeling, since preparation of the recipient site leads again to the release of activators. Furthermore, the functional loading of an implant following the integration period should also activate bone remodeling around the implant. However, these details are not known at present and are currently under investigation in ongoing experimental studies. The present study also confirmed previous reports of the excellent biocompatibility of e-PTFE membranes,14,29 since no adverse reaction with foreign-body cells was found in the histologic sections. In addition, the safety of e-PTFE has been established by extensive biocompatibility testing and a long history of safe and effective use in vascular and soft tissue prostheses.49 However, acute inflammatory reactions can occur when the membrane becomes exposed through a soft tissue dehiscence resulting in bacterial contamination. This has been demonstrated in both experimental20-22 and clinical studies.50,51 Another important prerequisite for predictable results with the GBR technique is



the creation and maintenance of a secluded space. Clinical experience has shown that standard e-PTFE (GTAM) membranes can be partially compressed by the soft tissue cover during healing 50,52,53 This clinical observation has been confirmed in the present study, since the two standard e-PTFE membranes in the 2-month group demonstrated significant collapse, resulting in a smaller secluded space. One possible solution for avoiding this complication is the use of reinforced, stiffer membranes.24,54 The histomorphometric analysis used in this study showed that reinforced e-PTFE membranes maintained a larger space than standard e-PTFE (GTAM) membranes, especially in the 2-month group. However, clinically the use of stiffer membranes also has limitations, since increased stiffness can make precise adaptation of a membrane to the bone surface more difficult. Alternatives to reinforced membranes are membrane-supporting devices such as miniscrews and/or bone filling materials. The present study and clinical experience over 3 years demonstrated the clinical value of specially designed miniscrews (Memfix System). These miniscrews are clinically useful for reliable membrane fixation to provide a precise adaptation of the membrane to the surrounding bone.53,55 This is essential for sealing off the defect against soft tissue invasion from the mucosa. Miniscrews have also been successfully used as tent poles to support membranes.54,55 In addition, a variety of bone filling materials have been recommended for use in combination with barrier membranes. The use of appropriate bone fillers could not only support the membrane to maintain the created space but could also accelerate bone regeneration by their osteoconductive, or possibly osteoinductive properties. However, the biological behavior of such bone fillers in conjunction with membranes has not been systematically evaluated and needs further investigation.

Conclusions 1. This study confirms previous reports that the placement of barrier membranes promotes bone regeneration in alveolar ridge lesions. It is important to note that the surgically created defects were large and did not completely heal without membrane coverage. 2. Biologically, membrane coverage of bone defects creates a suitable environment for bone regeneration. Probable effects of membrane placement are fourfold: (1) physical support of the overlying soft tissue, thus preventing collapse of the space necessary for bone formation; (2) creation of a space filled with a blood clot into which osteogenic cells can migrate, and protection of the granulation tissue and the delicate vascular network during the organization of the hematoma; (3) exclusion of competing non-osteogenic cells originating from the overlying soft tissues, thus preventing scar tissue formation; and (4) possible local accumulation of growth factors and other bone-promoting substances. 3. Bone regeneration in membrane-protected defects closely follows the pattern of bone development and growth. The first stage corresponds to intramembranous (direct) ossification and results in the formation of primary spongiosa. In the



second stage, this scaffold is reinforced by parallel-fibered and lamellar bone. Simultaneously, a cortical layer and the secondary spongiosa are formed. The third stage is characterized by cancellous and cortical bone remodeling. 4. Bone regeneration was not completed within the 4-month time frame. Based on this information, a prolonged healing period may be required in larger defects prior to functional loading of the regenerated bone. 5. The reinforced membranes reliably preserved their original form throughout the healing period, whereas the standard e-PTFE membranes partially collapsed. Acknowledgments The authors are indebted to Mrs Maura Fahey and Kristin Dixon for technical support, and Chuck White for material engineering. The histologic preparation by Mr David Reist and the morphometric evaluation of the specimens by Mrs. Britt Hoffmann are highly appreciated. The study was financially supported by grants from W.L. Gore and Associates Inc, Flagstaff, Arizona, USA, and the Swiss Ao/ASIF Foundation, Davos, Switzerland.



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43. Frost HM. Bone Remodeling Dynamics. Springfield, IL: CC Thomas, 1963. 44. Frost HM: Bone Dynamics in Osteoporosis and Osteomalacia. Springfield, IL: CC Thomas, 1966. 45. Jaworski ZF, Duck B, Sekaly G. Kinetics of osteoclasts and their nuclei in evolving secondary Haversian systems. J Anat 1981;133:397-405. 46. Jaworski ZF, Lok E. The rate of osteoclastic bone erosion in Haversian remodeling sites of adult dog's rib. Calcif Tissue Res 1972;10:103-112. 47. Schenk RK: Cytodynamics and histodynamics of primary bone repair. In: Lane JM (ed). Fracture Healing. Edinburgh: Churchill Livingstone, 1987:23-32. 48. Turner CH. Functional determinants of bone structure: Beyond Wolff's law of bone transformation. Bone 1992;13:403-409. 49. Boyce B. Physical characteristics of expanded polytetrafluoroethylene grafts. In: Stanley JC (ed). Biologic and synthetic vascular prostheses. Philadelphia: Grune & Stratton, 1982:33. 50. Buser D, Brägger U, Lang NP, Nyman S. Regeneration and enlargement of jaw bone using guided tissue regeneration. Clin Oral Impl Res 1990;1:22. 51. Jovanovic S, Spiekermann H, Richter EJ. Bone regeneration around titanium dental implants in dehisced defect sites: A clinical study. Int J Oral Maxillofac Implants 1992;7:233-245. 52. Dahlin C, Andersson L, Linde A. Bone augmentation at fenestrated implants by an osteopromotive membrane technique. Clin Oral Impl Res 1991;2:159-165. 53. Buser D, Dula K, Belser U, Hirt HP, Berthold H. Localized ridge augmentation using guided bone regeneration. I. Surgical procedure in the maxilla. Int J Periodont Rest Dent 1993;13:29-45. 54. Tinti C, Vincenzi G, Cochetto R. Guided tissue regeneration in mucogingival surgery. J Periodontol 1993;64:1184-1191. 55. Buser D, Dula K, Hirt HP, Berthold H. Localized ridge augmentation using guided bone regeneration. In: Buser D, Dahlin C, Schenk RK (eds). Guided Bone Regeneration in Implant Dentistry. Chicago: Quintessence, 1994:189-233.







Fig. 1 Intrasurgical status demonstrating the created defects in the alveolar ridge of a dog. One defect is covered by a membrane stabilized with miniscrews; the uncovered defect serves as control.

Fig. 2 Clinical status 4 months after surgery. The test site (left) shows a normal alveolar ridge contour, whereas the control site (right) exhibits a pronounced deficit with soft tissue collapse.



Fig. 3 Radiograph of a control defect at 2 months. Bone formation is restricted to the margins of the defect.

Fig. 4 Serial sections (8 out of 9, site 2/L2) starting from the mesial margin (left in Fig 3) into the middle portion of a control defect at 2 months. Magnification ×2.5.



Fig. 5 Radiograph of a membrane-covered defect at 2 months. Three bony caps, originating from the walls of the defect, grow into the middle portion and have partially fused.

Fig. 6 Serial sections (8 out of 9, site 2/R1) of a membrane-covered defect, mounted in mesiodistal sequence. Magnification ×2.5.



Fig. 7 Serial sections (8 out of 9, site 2/L1) of an incompletely filled defect at 2 months. The membrane was not precisely adapted to the lingual wall of the defect and has partially collapsed. Magnification ×2.5.

Fig. 8a (Left) Section F of Fig 6. The bony cap sealing the marrow space at the bottom has not yet fused with the bone regenerate that originates from the mesial wall. Remnants of the hematoma (arrow) are still present underneath the lingual side of the membrane. Regarding the direction of vascular



ingrowth the intertrabecular spaces are longitudinally cut at the bottom cap but appear as cross sections in the mesial regenerate. Figs 8a to 8d Vascular invasion and woven

Fig. 8b (Right) Organization of the hematoma and intramembranous ossification. Remnants of the blood clot are recognized as accumulations of erythrocytes, penetrated by granulation tissue (arrows). The granulation tissue is invaded by blood vessels (arrowheads) and by woven bone, first consisting of osteoid (orange) and then undergoing rapid mineralization (green). Five-micron thin, undecalcified microtome section, Goldner's trichrome stain, Magnification ×45. Figs 8a to 8d Vascular invasion and woven



Fig. 8c (Left) Fusion of neighboring trabeculae around blood vessels confines the intertrabecular spaces. The mineralized bone is covered by osteoid seams and is lined by a continuous layer of osteoblasts. Goldner's trichrome stain, Magnification ×90. Figs 8a to 8d Vascular invasion and woven

Fig. 8d (Right) Border area of vascular ingrowth and bone formation. Erythrocytes, coming from the original blood clot are still scattered within



the granulation tissue. Bone formation resembles fetal intramembranous ossification. Trabeculae, consisting of osteoid (orange) grow out into loose fibrous tissue. Thereby some collagen fibrils are incorporated into the bone matrix. Vessels, mainly sinusoidal capillaries and thin walled veins, are tightly filled with erythrocytes. Goldner's trichrome stain, Magnification ×90. Figs 8a to 8d Vascular invasion and woven.

Fig. 9 Radiograph of a membrane-covered defect at 4 months. The outline of the former defect is still clearly demarcated because of the lower radiodensity of the bony regenerate. The bony filling is almost complete, except for a small area in the roof of the middle portion.



Fig. 10 Serial sections (8 out of 10, site 4/R2) of a membrane-covered defect. Sections A and H demonstrate the original bone structure in the distal and mesial wall of the defect. Magnification ×2.5.



Fig. 11 (Left) Orofacial section through the middle portion at 2 months (site 2/R2) to show primary spongiosa filling most of the space confined by the reinforced membrane. Ground section, surface staining with toluidine blue and basic fuchsin, Magnification ×4.



Fig. 12 (Right) Orofacial section through the distal portion at 4 months (site 4/R2, Fig 12C) showing the elaboration of a cortical layer around the secondary spongiosa in the marrow space. The vascular pattern is demonstrated by the densely packed erythrocytes in the veins (dark brown). Magnification ×4.

Fig. 13a At 2 months, the primary spongiosa consists of woven bone lined by osteoid seams (blue) and osteoblasts (dark blue). Well-vascularized, fibrous bone marrow occupies the intertrabecular space. Microtome section, 5 µm, stained with von Kossa and McNeals tetrachrome, Magnification ×90.



Fig. 13b Same, toluidine blue-stained ground section. Woven bone matrix contains numerous densely packed, spherical osteocytes. Magnification ×90.

Fig. 13c Woven bone, identifiable by the arrangement of the osteocytes, is covered by more regularly structured parallel-fibered bone and, at 2 months, in some places by lamellar bone. Bone deposition is still in progress. Magnification ×90.



Fig. 13d At 4 months, corticalization has led to formation of primary osteons. Their wall consists of parallel-fibered and lamellar bone. The original woven bone is still recognizable and fills the space between the primary osteons. Magnification ×90.

Fig. 14a Cortical bone remodeling begins with osteoclastic resorption, followed by deposition of lamellar bone into the resorption canals. Remodeling is more intense in the cortical area adjacent to the original cortex and leads to a temporary porosity. The asterisk marks an area of primary bone where remodeling has not yet started. Surface-stained ground section, Magnification ×30.



Fig. 14b Two evolving secondary osteons, confined by cement lines of the reversal type (arrows). Their wall consists of lamellar bone; the haversian canal is still lined by an osteoid seam and osteoblasts. Magnification ×90.

Fig. 15a Primary spongiosa at 2 months. Its trabeculae consist of woven bone, reinforced by parallel-fibered bone. Well-vascularized, fibrous bone marrow. The onset of remodeling is indicated in some locations by the presence of osteoclasts (arrowheads). Magnification ×90. Figs 15a and 15b Formation of the secondary spongiosa.



Fig. 15b Trabeculae of the secondary spongiosa are composed of various matrix compartments (packets), formed by several generations of remodeling units, and are always separated by cement lines. Two cement lines (arrows) confine a remnant of the primary spongiosa, now enclosed by more recently formed lamellar bone. Note the fat cells in the matured, still well-vascularized bone marrow. Magnification ×90. Figs 15a and 15b Formation of the secondary spongiosa.

Fig. 16a Coronal zone at 4 months. Along the periosteal surface (to the left), bone formation is still going on. The membrane clearly separates the loose fibrous tissue of the inner compartment from the mucosa in the outer compartment. Site 4/L1, Magnification ×30. Figs 16a to 16d Soft tissue compartments (ground sections, surface-stained with toluidine blue and basic fuchsin).



Fig. 16b Nonreinforced membrane at 4 months. In this location the bony regenerate has established contact with the inner surface of the cell-occlusive part of the membrane. The outer membrane surface is lined by concentric layers of dense fibrous tissue belonging to the lamina propria of the keratinized mucosa. Site 4/R1, Magnification ×35. Figs 16a to 16d Soft tissue compartments (ground sections, surface-stained with toluidine blue and basic fuchsin).

Fig. 16c Interstices of the porous part of a reinforced membrane are infiltrated by vessels and loose fibrous tissue derived from the inner compartment. Site 4/R2, Magnification ×45. Figs 16a to 16d Soft tissue compartments (ground sections, surface-stained with toluidine blue and basic fuchsin).



Fig. 16d In another location, bone has been deposited on the inner surface and invaded the porous part of the membrane, the interstices of which were almost completely filled by bone. Magnification ×45. Figs 16a to 16d Soft tissue compartments (ground sections, surface-stained with toluidine blue and basic fuchsin).

Healing Pattern of Bone Regeneration in Membrane-Protected Defects

Healing Pattern of Bone Regeneration in Membrane-Protected Defects

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