Temporal dynamics of healing in rabbit cranial defects using guided bone regeneration

J Oral MaxillofacSurg 53:167-174, 1995

Temporal Dynamics of Healing in Rabbit Cranial Defects Using Guided Bone Regeneration CHRISTOPH H.F. H,~,MMERLE, DMD,* JORG SCHMID, DMD,* NIKLAUS P. LANG, DMD, MS, PHD,t AND ATILLA J. OLAH, MD, PHD:I: Purpose: The objective of this study was to histologically evaluate the early stages of bone regeneration using rabbit calvaria defects in conjunction with guided tissue regeneration. Materials: A semilunar cutaneous-periosteal flap was raised on the forehead of four rabbits exposing the top of the skull. A standardized transosseous skull defect (-> 15 mm in diameter) was made in the area of the right parietal bone with a rotating round bur. Care was taken not to damage the underlying dura. A flat expanded polytetrafluoroethylene (ePTFE) membrane was placed to cover the defect. The membrane was tightly adapted, extending at least 4 mm onto intact bone, and the flap was sutured. One, 2, 3, and 5 weeks later, the specimens were removed and processed using standard, undecalcified, hardtissue histologic techniques. Contact radiographs were also taken. Results: Bone growth increased with time, starting at the borders of the defect. At 1 week, trabeculae of woven bone grew into the highly vascularized loose connective tissue occupying the defect. Two weeks postsurgery, isolated islands of new bone were detected in this connective tissue. Subsequently, neighboring small islands merged to form large islands. In later stages, the primary trabeculae of woven bone were reinforced by layers of regularly deposited lamellar bone. Conclusion: Rabbit calvaria defects treated by guided tissue regeneration heal by ingrowth of woven bone from the defect margins and by formation of bony islands within the defect area. Bone healing showed the histophysiological characteristics of intramembranous bone.

attachment and alveolar bone could be achieved when cells from the gingival connective tissue and oral mucosal epithelium were excluded from the wound, v3 It was concluded that cells that have access and proliferate into a wound determine the type of tissue regenerating in that wound. As a result of these studies, the technique termed guided tissue regeneration (GTR) was developed. 4-8 GTR has also found applications in other areas, including the regeneration of bone tissue. 9 As a result of animal experiments ~°'H and clinical applications in humans, ~244 GTR has become a clinically accepted method for augmenting bone in situations with an inadequate volume for the placement of endosseous dental implants. The formation of new bone in conjunction with the placement of dental implants also is a clinically well documented and successful procedure. ~2'~5-~7

The method of guided tissue regeneration was developed to regenerate tissues lost as the result of periodontal disease. It was shown in a series of animal experiments that the regeneration of the periodontal tissue

Received from the University of Bern, Bern, Switzerland. * Assistant Professor, School of Dental Medicine. t Professor and Chairman, School of Dental Medicine. :) Full Professor, Institute of Anatomy. This study was partly funded by the Swiss National Foundation for the Promotion of Scientific Research Grant no. 3200-037618, and the Clinical Research Foundation (CRF), University of Bern, Switzerland. Address correspondence and reprint requests to Dr H~immerle: School of Dental Medicine, University of Bern, Freiburgstrasse 7, CH-3010 Bern, Switzerland. © 1995 American Association of Oral and Maxillofacial Surgeons 0278-2391/95/5302-0011 $3.00/0

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BONE REGENERATION OF CRANIAL DEFECTS

Bone formation after trauma is dependent on the size of the defect. In cortical bone, circular defects of less than 200 #m have the potential to heal with concentrical formation of lamellar bone. 18'19 In larger defects of 200 to 500 #m, bone healing is characterized by formation of a trabecular network of woven bone bridging the defect. Subsequently, the spaces between the trabeculae are filled with lamellar bone. However, in defects of 500 # m and larger, bridging by direct formation of bone does not occur. After 3 weeks of healing, such defects exhibit a central area characterized by the presence of connective tissue. Where new bone formation occurs, it appears only at a low rate. Similar findings have been described for the healing of long bones in dogs. 19 In these studies, a gap width of 150 # m healed by lamellar bone formation, whereas a defect of 400 # m showed healing consisting of trabeculae of woven and lamellar bone. The rodent cranial defect model has been used extensively to study the effect of different surgical techniques, 2° various bone substitute materials, 2v24 and compounds aimed at enhancing bone healingY -27 It has been demonstrated that extensive bone defects prepared in the calvaria of rodents can be successfully closed by new bone formation using GTR. 28 These results were confirmed by other investigators using slightly modified experimental designs. 29'3° This animal model is useful to study the healing events during guided bone regeneration. Despite the fact that GTR is widely used in experimental studies and in clinical practice to obtain increased bone volumes, information about the sequence of events during guided bone regeneration is scarce. The aim of this study was to determine the temporal dynamics of healing in critical size cranial defects in rabbits treated by guided tissue regeneration.

Materials and Methods Four rabbits were anesthetized with Nembutal (Abbott, Chicago, IL) and the site of surgery was infiltrated with Neo-Lidocaton 2% (Pharmaton, Lugano, Switzerland). The top of the head and the base of the ears were shaved and disinfected. A semilunar incision was made over the skull and a cutaneous flap was raised on top of the forehead and reflected posteriorly. Similarly, the periosteum was cut and reflected posteriorly exposing the top of the cranial bone. To prevent drying, the skin and periosteum were covered with sterile gauze moistened with saline. A transosseous skull defect exceeding 15 mm in diameter was produced in the area of the right parietal bone by means of a dental handpiece using a #8 round burr and copious irrigation with sterile saline. Care was taken to avoid involvement of the sagittal and

coronal sutures, as well as not to damage the underlying dura. A flat, expanded polytetrafluoroethylene (ePTFE) membrane (GORE-Tex, W.L. Gore and Associates, Flagstaff, AZ) was placed over the defect in all animals. The membrane was tightly adapted, extending at least 4 to 5 mm onto intact bone. The periosteum was then sutured over the membrane with a resorbable suture material (Vicryl, Johnson & Johnson, Spreitenbach, Switzerland), and the cutaneous flap was adapted and sutured with silk sutures. The histologic identification of the original edges of the defect is difficult in advanced stages of bony healing. 29 Therefore, the animals assigned for 3- and 5-week regeneration periods received intraperitoneal doses of tetracycline (25 mg/kg body weight) for 7 days before the surgery to label the pre-existing bone. One, 2, 3, and 5 weeks later, the rabbits were killed with an injection of Vetanarcol (Veterinaria, Ztirich, Switzerland). HISTOLOGIC PREPARATION

The calvaria were removed and fixed in 4% neutral formalin. After contact radiography (Faxitron[Hewlett Packard, McMinnville, OR], Mammorgraphy Film Type S/3M/, 40kV, 1 sec), the specimens were trimmed, dehydrated, and embedded in methylmethacrylate. Consecutive, undecalcified sections (4 #m) were cut in the frontal plane through the entire calvarial defect, alternating with approximately 200-#m thick sawed sections (Accutome-2, Struers Tech, Rodovre/ Copenhagen, Denmark). The microtome sections were stained with Goldner's trichrome, von Kossa/McNeal stain, or remained unstained for observation under ultraviolet light. The sawed sections were ground to a thickness of 100 to 120 #m, polished, and stained with toluidine blue. 31 QUANTITATIVE ANALYSIS OF RADIOGRAPHS

The amount of newly formed bone was estimated on the radiographs using computer-assisted analysis and calculated as the percentage fraction of the original defect size. Variation coefficients of measurements performed by three observers, and of repeated determinations by the same observer on different days, were, on the average 13.1% and 6.7%, respectively.

Results All the animals recovered from the anesthesia without complications and behaved normally during the length of the study.

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were found connected with the vessels of the opened bone marrow cavity of the diploe. Below the lower edge of the defect margin, dense connective tissue fiber bundles originating from the external surface of the dura mater ran towards the marginal zone of the defect and then spread out. The remainder of the defect contained loose connective tissue comprised of scarce collagen fibers without a preferential orientation (Fig 3D). Sparsely distributed cells, predominantly fibroblasts and macrophages, as well as a moderate number of wide capillaries, were seen. In addition, the surface of the membrane facing the defect was covered by a remarkably dense layer of inflammatory cells, which occasionally invaded the spaces between the lamellae of the membrane (Fig 3E). WEEK 2

FIGURE 1. Microradiographs of the defect area and surrounding skull bone at 1 (A), 2 (B), 3 (C) and 5 weeks (D) after surgery (original magnification ×4.5). A, Note discrete signs of new bone formation along parts of the defect margins (.arrows). B, The circumference of the gap exhibits a nearly continuous layer of new bone (arrows). Note small islands of radiopaque material within the defect isolated from the new marginal bone. C, Several new bony islands towards the center of the defect (arrows) appear. D, Multiple disseminated islands of new bone. Near the defect margin they tend to fuse forming larger particles (arrows).

At the end of the second week, a further gain of new bone deposited on the edges of the defect was observed radiographically. The fractional amount of this bone growth reached 35.9% _+ 2.6% (Figs IB, 2). The newly formed bone was distinguishable from the pre-existing bone by its lower degree of radiodensity. Histologic sections showed a considerable number of proliferating capillaries, which accompanied and even preceded the bone trabeculae growing towards the middle part of the gap (Fig 4A). In the course of bone apposition, surrounding connective tissue fibers be-

Percent of the original defect area

100% total [] border [] islands

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Radiopaque new bone:

WEEK 1

One week after surgery, regeneration of calcified tissue was already visible radiographically as a thin layer along the border of the defect (Fig 1A). It amounted to 23.1% + 3.3% of the original defect (Fig 2). Microscopic examination of the defect demonstrated new bone formation originating from the bony borders (Fig 3A-C). This new bone appeared as a scaffold of delicate trabeculae comprised of woven bone, from which several extensions were directed towards the center of the defect. All surfaces of the trabeculae were covered by osteoid seams lined by a dense layer of cuboidal osteoblasts. The trabeculae were embedded in a well-organized and vascularized granulation tissue in which wide capillaries could be observed adjacent to the trabeculae. Integration of collagen fiber bundles into the new bone matrix could be detected at various locations (Fig 3C). No signs of osteoclastic bone resorption were seen on the defect edges. Within the network of the trabecular scaffold numerous capillaries

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FIGURE 2. Histogram of the amount of radiopaque new bone within the defect area at 1, 2, 3, and 5 weeks after surgery. Open bars: total new bone, hatched bars: bone origination at the defect borders, and full bars: new bone as islands.

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BONE REGENERATION OF CRANIAL DEFECTS

FIGURE 3. Week 1 specimens. A, New bone formation originating from the defect margins (arrows) (original magnification ?<3.3). B, The new bone consists of irregularly shaped, delicate trabeculae (arrows) lined with osteoid seams and a layer of cuboidal osteoblasts (original magnification ×40). C, During generation of new trabeculae, collagen fiber bundles of the surrounding connective tissue become incorporated into the new bone matrix (arrows) resembling intramembranous bone formation (original magnification ×150). D, Enlargement from the central region of the defect interposed between the dura mater and the covering ePTFE membrane. Loose connective tissue containing scarce collagen fibers and a moderate number of blood capillaries are seen (arrows) (original magnification x65). E, The lower surface of the ePTFE membrane is lined by a dense cellular layer (arrowheads) invading the membrane spaces at several locations (original magnification ×33). (3A, B are polished ground sections, surface-stained with McNeal/toluidine blue and 3C, E represent undecalcified 5-#m microtome sections, using von Kossa stain.)

c a m e e m b e d d e d in the o s t e o i d and finally i n t e g r a t e d into the n e w b o n e . T h e c o n n e c t i v e tissue in the central part o f the d e f e c t a p p e a r e d d e n s e r b e c a u s e o f an inc r e a s e d fiber c o n t e n t as w e l l as to the c o n d e n s a t i o n o f

t h e s e fibers into bundles. In addition, the n u m b e r o f c a p i l l a r y v e s s e l s was c o n s i d e r a b l y a u g m e n t e d . S m a l l b o n e f o c i a r o s e w i t h i n this f i b r o v a s c u l a r tissue (Fig 4B). T h e i r t e x t u r e w a s c o n s i s t e n t w i t h that o f w o v e n

HAMMERLE ET AL

"171 bone, ie, irregular bundles of collagen fibers and extremely numerous, large osteocytes. These primitive ossification centers were located mostly in the neighborhood of blood vessels. They were covered by a dense layer of osteoblasts, with an underlying osteoid seam of approximately 8 to 10 # m in thickness. By means of radiographs and serial sections, they were identified as isolated bony islands without contact with the marginal bone. While the cellular layer along the lower surface of the membrane persisted, the internodular spaces now contained connective tissue of high cellularity and sporadic capillaries (Fig 4C). The preexisting bone adjacent to the defect margin exhibited intense remodeling, with bone formation clearly exceeding osteoclastic bone resorption.

WEEK 31 At 3 weeks, bony islands approached the central part of the defect (Fig 1C). Radiographic evaluation demonstrated that 55.5% _+ 4.2% of the original defect was occupied by new bone (Fig 2). The islands accounted for 11.4%, whereas the bone growing in from the borders amounted to 44.1%. As the individual islands grew, blood vessels lying in their immediate vicinity became incorporated into the new bone matrix (Fig 5A). Additionally, the primary trabecular scaffold underwent intense remodeling. Numerous osteoclasts arose and began to eliminate the primitive woven bone, whereas a new generation of osteoblasts deposited mature lamellar bone layers on the woven bone remnants (Fig 5B, C). New bone formation also could be observed in contact with the side of the membrane facing the defect, and occasionally even within the membrane spaces (Fig 5D). In other areas between the different layers of the membrane material, well-organized connective tissue with high cellularity and an abundance of blood vessels was seen. WEEK 5

FIGURE 4. Week2 specimens (undecalcified, 5-/~msections, using yon Kossa stain). A, Newly formed trabeculae originating from the bony defect borders are extending toward the defect center. Note proliferating capillaries (arrows) that accompanythe growing bone trabeculae (original magnification ×40). B, New bone islands arising isolated from the trabecular scaffold along the rim of the defect. Note the scalloped outlines of the islands and the high number of irregularly scattered, large osteocytes characterizing woven bone formation. Note cross-sectionof the ePTFE membraneon top (original magnification ×33). C, The internodular spaces of the ePTFE membrane (M) contain connective tissue of high cellularity and several blood vessels (arrows) (original magnification × 160).

The radiograph at 5 weeks showed further bone apposition at the margin (67.4% _+ 4.2%) and multiple islands of new bone scattered over the entire area (13.9% + 3.0%) occupying 81.3% _+ 2.3% of the original defect (Figs 1D, 2). Near the edges of the defect smaller islands tended to flow together forming larger islands, as well as merging with the marginal new bone. Within larger islands, osteoclastic bone resorption gave rise to !rregular holes in which bone marrow formed (Fig 6A). Newly formed and old bone could clearly be differentiated by ultraviolet microscopy because only the pre-existing bone contained tetracyline-fluorescent labels. As a consequence of the continuous remodeling of the primary bony network, most of the trabeculae now conrained only a small, intensely stained core of woven bone

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BONE REGENERATION OF CRANIAL DEFECTS

FIGURE 5. Week 3 specimens. A, Island of woven bone in the centraI part of the defect. Note that the blood vessels (arrows) have become incorporated into the new bone (original magnifcation ×50). B, Osteoclasts (Oc) are resorbing the primarily formed woven bone (Wb), whereas osteoblasts (Ob) deposit layers of mature lamellar bone on the remnants of the original trabecular scaffold (original magnification × 100). C, Remnants of the dark-stained, primary trabecular scaffold (arrows) are covered by new bone lamellae (original magnification ×25). D, Formation of bone spicules between the nodes of the ePTFE membrane (M) (original magnification x66). (Figures 5A, D are undecalcified, using von Kossa-stained microtome sections and 5B, C are ground sections which are surface-stained with McNeal/toluidine blue.) surrounded by thick bone layers of regular lamellar texture. The presence of osteoid seams with overlying osteoblasts indicated continuation of the osteogenic process. In the central part of the original bone defect, fiber bundles were oriented predominantly parallel to the surface of the membrane. Some of them deviated from this arrangement, however, running perpendicularly towards the membrane. They appeared to be incorporated within the cellular layer on the inner surface of the membrane or even anchored to the membrane itself (Fig 6B). Consistent with the results from the specimens at all the different time points was the finding of increased radiodensity of the skull adjacent to the defects. On microscopic examination, this phenomenon was confirmed by the finding o f higher bone density in the border area of the gap.

Discussion The present study was aimed at describing the temporal dynamics of the healing pattern of bone in cranial

defects treated by GTR. The results show that new bone was intramembranous in nature, originating in part from the bony borders of the defect. In addition, formation of new bone occurred in scattered islands isolated from the borders of the defect. Although the major part of the total new bone grew in from the defect margins at 5 weeks, 13.9% of the original defect area was occupied by bony islands. Therefore, osseous defect closure arising both from the margins of the bone defect and as islands m a y be a faster healing process than marginal bone formation alone. Earlier reports have shown that defects in the calvaria of rabbits of the size used in this study do not heal spontaneously with osseous tissue even after observation periods as long as 36 weeks. 26-3°'32However, several studies have shown that large defects in the calvaria o f rabbits heal with complete bony bridging when treated with nondegradable 28'29 or biodegradable membranes using the principles of GTR. 3° The formation o f new bone in the gap was detected

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173

FIGURE 6. Week 5 specimens.A, Bony island near the membrane surface showing large holes containing bone marrow (arrows). Ground section surface-stainedwith McNeal/toluidine blue (original magnification ×20). B, Bundles of collagen fibers (arrowheads) inserting into the dense, cellular layer covering the side of the membrane (M) facing the defect. Some fibers seem to insert directly into the membrane (arrowheads). Microtome section using Goldner stain (original magnification ×80). exclusively within the space between the membrane overlying the defect and the dura mater, ie, along the defect rim, in the fibrovascular tissue within the defect, and between the lamellae of the membrane. Hence, the primary source of osteogenic cells and nutrition by vessels was the diploe rather than the endocranium. These findings are in agreement with earlier reports on healing of skull defects in rabbits. 26'3° Using various concentrations of transforming growth factor-fl to enhance bone growth, the defect margins of the diploe rather than the periosteum were identified as the primary source of new bone formation. 26'27 In the present experiment, the periosteum was excluded from access to the wound by the ePTFE membrane; however, new bone formation was successfully achieved. In two recently published studies aimed at closing skull defects with GTR, it was shown that, despite excluding both the dural and the periosteal tissues from taking part in the healing process by membranes, bone regeneration was still successful. 28-3° These studies clearly demonstrated that the diploe has a sufficient regenerative capacity to achieve osseous defect closure. In general, new bone is only formed at locations that guarantee biomechanical stability and allow angiogenesis, ie, where pressure and tensile forces are excluded. 33 Otherwise, an intermediate tissue with appropriate mechanical properties arises before ossification. This tissue allows the unimpeded ingrowth of capillaries, which always precedes the formation of new bone tissue. The organized blood clot in the experimentally created defect of the rabbit calvarium fulfills the requirement of such an intermediate tissue. However,

with increasing defect size, the biomechanically stable zone becomes successively limited to the marginal area of the hole, whereas the central region is exposed to biomechanical forces presumably preventing bone formation. This is underscored by experimental and clinical observations which showed that, in large bone defects, bone formation is limited to the defect margins. 27-3°'32 In the central region of a large defect, a dense, membrane-like sheath of fibrous connective tissue persists, preventing complete bony healing. In a recent study, ePTFE membranes were successfully used to achieve bony closure of skull defects. 2~ Thus, it is conceivable that a relatively stiff membrane like the ePTFE membrane may protect the delicate tissue within the gap from biomechanical forces. Moreover, collagen fiber bundles anchored at the lower surface of the membrane, as seen in this experiment, may be interpreted as an additional protective structure. Under these conditions, unimpeded vascular ingrowth and subsequent osteogenesis can take place, as demonstrated by the formation of numerous bony islands isolated from the rim of the defect and the underlying dura mater. The presence of bony islands within the defect is in contrast to observations of previous studies in which the proliferation of new bone in this pattern did not occur unless a sutural growth area was given access to the defectY '32 Additional studies are needed to explain this finding. Acknowledgment The investigators thank G.R. Arpagaus, Swiss Red Cross, Blood Transfusion Service, Bern, Switzerland. The membranes were provided by W.L. Gore and Associates, Flagstaff, AZ.

174 References 1. Karring T, Nyman S, Lindhe J: Healing following implantation of periodontitis affected roots into bone tissue. J Clin Periodont 7:96, 1980 2. Nyman S, Karring T, Lindhe J, et al: Healing following implantation of periodontitis affected roots into gingival connective tissue. J Clin Periodont 7:394, 1980 3. Karring T, Isidor F, Nyman S, et al: New attachment formation on teeth with a reduced but healthy periodontal ligament. J Clin Periodont 12:51, 1985 4. Nyman S, Gottlow J, Karring T, et al: The regenerative potential of the periodontal ligament. An experimental study in the monkey. J Clin Periodontol 9:257, 1982 5. Pontoriero R, Lindhe J, Nyman S, et al: Guided tissue regeneration in degree II furcation-involved mandibular molars. A Clinical Study. J Periodontol 15:247, 1988 6. Becker W, Becker B, Berg L, et al: New attachment after treatment with root isolation procedures: Report for treated Class III and Class II furcations and vertical osseous defects. Int J Periodont Rest Dent 3:9, 1988 7. Pontoriero R, Lindhe J, Nyman S, et al: Guided tissue regeneration in degree II fucation involved mandibular molars: A clinical study of degree III involvements. J Clin Periodont 16:170, 1989 8. Caffesse RG, Dominiguez LE, Nasjleti CA, et al: Furcation defects in dogs treated by guided tissue regeneration (GTR). J Periodontol 61:45, 1990 9. Nyman S: Bone regeneration using the principle of guided tissue regeneration. J Clin Periodontol 18:494, 1991 10. Dahlin C, Linde A, Gottlow J, et al: Healing of bone defects by guided tissue regeneration. Plastic Reconstr Surg 81:672, 1988 11. Seibert J, Nyman S: Localized ridge augmentation in dogs: A pilot study using membranes and hydroxyapatite. J Periodontol 3:157, 1990 12. Nyman S, Lang NP, Buser D, et al: Bone regeneration adjacent to titanium dental implants using guided tissue regeneration. A report of 2 cases. Int J Oral Maxillofac Implants 5:9, 1990 13. Buser D, Br~igger U, Lang NP, et al: Regeneration and enlargement of jaw bone using guided tissue regeneration. Clin Oral Impl Res 1:22, 1991 14. Wachtel HC, Langford A, Bernimoulin JP, et al: Guided bone regeneration next to osseointegrated implants in humans. Int J Oral Maxillofac Implants 6:127, 1991 15. Lazzara RM: Immediate implant placement into extraction sites: Surgical and restorative advantages. Int J Periodont Rest Dent 9:333, 1989 16. Becker W, Becket B, Handelsman M, et al: Guided tissue regeneration for implants placed into extraction sockets: A study in dogs. J Periodontol 62:703, 1991

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