J Periodontol • May 2014

Demineralization of the Contacting Surfaces in Autologous Onlay Bone Grafts Improves Bone Formation and Bone Consolidation Maria L. Rezende,* Alberto Consolaro,† Adriana C. Sant’Ana,* Carla A. Damante,* Sebastia ˜ o L. Greghi,* and Euloir Passanezi*

Background: Autologous bone grafts are usually well consolidated after 4 to 5 months but can be incompletely interlocked with the native bone. This study investigated the effect of acid demineralization of the graft–bed interface on graft consolidation. Methods: Onlay bone grafts were performed on the calvaria of 36 guinea pigs. Half of the animals had the graft– bed contacting surfaces demineralized with 50% citric acid (pH 1.0) for 3 minutes (test group). The other half received no demineralization (control group). The bone grafts were immobilized by a resorbable membrane glued to the recipient bed with cyanoacrylate. After 7, 30, and 90 days, specimens (n = 6) were obtained for light microscopy. Data from qualitative analysis and computerized histomorphometry were statistically processed at a significance level of 5%. Results: Osteogenesis was not seen at the interface after 7 days. After 30 days, the test group showed 34.39% – 13.4% of the interface area filled with mineralized tissue, compared to 17.14% – 8.6% in the control group (P = 0.026). After 90 days, the mean percentages of mineralized tissue at the interface in the test and control specimens were 54.00% – 11.23% and 38.65% – 7.76% (P = 0.041), respectively. Within groups, a higher percentage of the area filled with mineralized tissue was seen at 90 days compared to 30 days (P = 0.004 for control and 0.041 for test). Conclusions: Demineralization of the contacting surfaces between autologous bone graft and bone bed improved new bone formation and bone consolidation. These data need to be confirmed in humans. J Periodontol 2014;85: e121-e129. KEY WORDS Bone transplantation; citric acid; guided tissue regeneration. * Division of Periodontics, Department of Prosthodontics, Faculty of Odontology of Bauru, University of Sa˜o Paulo, Bauru, Sa˜o Paulo, Brazil. † Department of Stomatology, Division of Pathology, Faculty of Odontology of Bauru, University of Sa˜o Paulo.

A

utologous onlay bone grafts are considered the gold standard to provide sufficient bone volume for implant placement in severely resorbed alveolar ridges.1 Although these grafts usually take 4 to 5 months to become consolidated,2 clinicians have experienced detachment of the grafts during drilling and/or implant placement,3 suggesting that bone grafts may be incompletely interlocked with the native bone.2 The long-term outcome of onlay grafts may be influenced by the following: 1) embryologic origin of the grafted tissue;4,5 2) extent of its revascularization;4 3) architectural features of the graft;6 4) method for graft fixation;7 and 5) treatments applied to the graft–bed interface.8 Graft incorporation seems to be enhanced when the bone marrow is exposed by perforating or decorticating the host bed.9-11 Perforations can provide access for blood vessels and progenitor cells from the endosteal compartment to the grafted area12 and can also strengthen the physical bond between grafted bone and recipient surface.9 Nevertheless, decortication and/or perforations present disadvantages: 1) longer operating time; 2) impaired visibility as a result of extra bleeding; 3) additional surgical trauma by overheating generated by the drilling doi: 10.1902/jop.2013.130298

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Acid Demineralization Improves Bone Graft Outcome

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Ongoing studies by the present authors are pointing to a favorable behavior of precursor bone cells cultured on demineralized calvaria of guinea pigs. Simultaneously, attempts are being made to identify BMPs that may result after in situ demineralization of rats’ calvaria. In this way, the aim of the present histologic and histomorphometric study is to evaluate the effect of demineralization of the contacting surfaces between the bone graft and bone bed on the pattern of bone formation and graft consolidation. MATERIALS AND METHODS This study was approved by the Institutional Review Board for Animal Experimentation Figure 1. of the Faculty of Odontology of Surgical procedure. A) Drilling of the parietal bone using a 6.3-mm trephine bur. B) Autologous bone block transplanted to an adjacent area. C) Demineralization of the recipient bed (test group). D) Bauru, University of Sa˜o Paulo, Demineralization of the inner surface of the graft (test group). E) Collagen membrane used to immobilize Sa˜o Paulo, Brazil (CEEPA-05/ the grafts. F) Trimmed membrane covering the graft and glued to the recipient bed with cyanoacrylate at 2006). Thirty-six male guinea its edges (arrows). pigs (Cavia porcellus) weighing 500 to 600 g were used. General anesthesia was induced by intramuscular bur;13 4) bone loss substance; and 5) loss of volume injections of ketamine hydrochloride (25 mg/kg) related to bed resorption.9,10 and xylazine hydrochloride (5 mg/kg). A flap was Treatments applied to the bone graft–bone bed raised at the frontoparietal region to expose the interface, such as interposition of platelet-rich plasunderlying bone. A bicortical bone block was harma,8 bone morphogenetic proteins (BMPs),14 and vested from the parietal bone using a 6.3-mm low-intensity laser,15 have been reported with the trephine bur under low speed and copious saline goal of producing stimuli to cellular activity reirrigation. In 18 animals, the graft was transplanted sulting in greater bone deposition, bone volume to an adjacent area (control group). In the other 18 maintenance, and reduced time for graft conanimals (test group), before transplantation, the solidation. contacting surfaces between the host bed and graft Bone tissue is composed of an osteoconductive were demineralized with 50% citric acid gel (pH 1.0) mineral phase, but the osteogenic/osteoinductive (prepared at the Department of Biochemistry of the factors are contained within the organic phase.16 A Faculty of Odontology) during 3 minutes. The acid series of experiments coordinated by Urist and colwas profusely washed with sterile saline. The bone leagues17-19 demonstrated that demineralized fragblocks were immobilized by covering with a rements of bone or dentin induce bone formation sorbable bovine collagen membrane‡ exceeding subcutaneously and intramuscularly in rats. It was the dimensions of the graft to 5 mm. The demonstrated that the demineralization process remembrane edges were glued to the recipient bed leases BMPs, which induce stromal cells from the with ethyl cyanoacrylate,§ taking care to prevent adjacent tissues to differentiate into osteoblasts. the glue from reaching the graft–recipient bed Although demineralization of the bone matrix has interface (Fig. 1). The flap was sutured with 4-0 been demonstrated to be successful in increasing 15,20 black silk. The animals received oral analgesic the osteoinductive potential of bone grafts, in (acetaminophen, 200 mg,1 drop/kg) on the situ demineralization of the contacting surfaces between the bone graft and bone bed has never been used to improve or anticipate the graft con‡ Gem-Derm, Baumer, Sa˜o Paulo, Brazil. solidation. § Loctite Super Bonder Flex Gel, Henkel, Sa˜o Paulo, Brazil e122

J Periodontol • May 2014

Rezende, Consolaro, Sant’Ana, Damante, Greghi, Passanezi

observing the following: 1) the presence of granulation tissue; 2) definition of boundaries of graft and bone bed; 3) presence of osteogenesis; 4) incorporation of graft and bed to the newly formed bone; and 5) intensity of inflammatory infiltrate, if present. The membrane edges were evaluated in relation to cyanoacrylate remnants and tissue reaction around the remnants. Quantitative analyses. Digital images of sections taken at ·10, ·20, and ·40 magnification were analyzed by a single examiner (MLR; k test = 0.99) masked to the experiment using a software for image analysis¶ according to the following parameters: 1) percentage of area filled with mineralized tissue at the graft–bed interface: the interface area was measured within the perimeter limited by the borders of the Figure 2. Bone tissue histologic analysis after 7 days. Microscopic views (original magnification ·10) of one of the graft facing the recipient bed, specimens from the control group (A) and one from the test group (B) at 7 days of evaluation in which no and the area corresponding to bone formation is observed at the interface of the bone graft–bone bed (if). The resorbable membrane is mineralized tissue was subidentifiable in A and B (arrows). C) Area outlined in A corresponding to the graft–bed interface (if) in which tracted from the interface area newly formed fibrin (black arrows) and remnants of the periosteum (blue arrow) can be seen (original and converted in percentage magnification ·20). D) Area outlined in B corresponding to the graft–bed interface (if) in which incipient granulation tissue (gt) is present (original magnification ·20). E) View of the grafted bone from one area; 2) linear length of graft specimen of the test group showing osteoplasts containing osteocytes (green arrows) (original and bone bed surfaces consolmagnification ·40). F) At the interface (if) of the bone graft (gr) and recipient bed (rb) of this idated to the newly formed specimen of the test group, the demineralized surfaces showed a thin layer of basophilic osteoid-like bone: the linear extent of each bone tissue (arrows) (original magnification ·40; Masson’s trichrome). if = interface; gr = bone graft; surface (graft and bed) was rb = recipient bone bed; gt = granulation tissue. measured, and the extent of these surfaces consolidated to the newly formed bone was subtracted from the first postoperative day and a single dose of total extent and converted to percentage. benzathine penicillin in a proportion of 16,000 IU intraperitoneally. The suture was removed after Statistical Analyses 7 days. Mann-Whitney U test was used for intergroup and The animals were sacrificed by anesthetic overintragroup comparison of mean area of mineralized dose at 7, 30, and 90 days postoperatively (n = 6). tissue at the interface and for linear measurements, Blocks containing the grafts and surrounding soft both at a significance level of 5%. tissues were removed and immersed in 10% formalin for at least 48 hours to be processed for light miRESULTS croscopy. Cross-sections with 4-mm thickness obQualitative Analyses tained from the center of each specimen were stained At 7 days. Osteogenesis or osteoclastic activity was by Masson’s trichrome. not present at the bone graft–recipient bed interface in control and test specimens (Figs. 2A and 2B). Mild, Microscopic Analyses diffuse, and non-specific inflammatory infiltrate was Qualitative analyses. The graft–bed interface was i Olympus CX2 Microscope, Olympus America, Center Valley, PA. analyzed by an examiner (AC; k test = 0.99) masked ¶ NIH ImageJ v.1.38x, Wayne Rasband, National Institutes of Health, to the experiment using a light microscopei and Bethesda, MD. e123

Acid Demineralization Improves Bone Graft Outcome

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but the limits of both graft and bed were still well defined (Fig. 3C). The newly formed bone at the interface was richly cellularized and randomly distributed (Fig. 3C). Osteoclastic activity or inflammatory infiltrate was not present at the interface In the test group, the graft– bed interface was predominantly filled with newly formed bone (Fig. 3B). The limits of the graft and bed were well defined with evidence of some remodeling process (Fig. 3D). At the interface, immature bone trabeculae emerging from bone surfaces were intersected to each other promoting structural union (Figs. 3B and 3D). Osteogenesis was moderate to intense, and newly formed bone was also seen at the extremities of the graft underneath the membrane (Fig. 3F). At the membrane edges the Figure 3. cyanoacrylate was associated Bone tissue histologic analysis after 30 days. Microscopic views of one of the specimens from the control group (A) and one from the test group (B) at 30 days of evaluation in which the interfaces (if) between the with foreign body granulomas graft (gr) and recipient bed (rb) are seen (original magnification ·10). C) Within the magnified area in both the test and control outlined in red of A, there is granulation tissue (gt) and richly cellularized new forming bone (black arrows) groups (Fig. 3E). eventually fused to the recipient bed (rb) whose boundaries can still be identified (white arrows) (original At 90 days. In the control magnification ·20). D) Magnification of the interface area outlined in green in B where intense new bone group, the graft–bed interface formation is shown with immature bone trabeculae (black arrows) promoting structural union between the graft (gr) and recipient bed (rb). The boundaries between mature and woven bone can be identified (white predominantly presented priarrows) with evidence of remodeling (original magnification ·20). E) Edge of the membrane (m) covering mary bone with organized marthe specimen shown in A where remnants of cyanoacrylate (ca) are seen surrounded by foreign body row spaces. This new bone was granulomas with multinucleated giant cells containing particles of the glue in the cytoplasms (arrows) structurally imbricated to the (original magnification ·40). F) Magnification of the area outlined in black in B where newly forming bone graft and to the recipient bed (arrows) is seen at the extremity of the graft (gr) in the space created underneath the membrane (m) (original magnification ·20; Masson’s trichrome). if = interface; gr = bone graft; rb = recipient bone bed; (Figs. 4A and 4C). No ingt = granulation tissue; m = membrane; ca = cyanoacrylate. flammatory infiltrate was present. Remnants of membrane associated with a network of newly formed fibrin in and cyanoacrylate were still present in both groups. the control group (Fig. 2C). Newly formed granulaIn the test group, the graft–bed interface was tion was present in the test group (Fig. 2D). In both almost entirely filled with new bone without ingroups, most osteoplasts of the grafted tissue conflammatory cells (Fig. 4B). The Graft and recipient tained morphologically viable osteocytes (Fig. 2E). bed shared the newly formed trabeculae and In the test group, the demineralized surfaces of the marrow spaces (Figs. 4B and 4D). Osteogenesis bone graft and recipient bed showed a thin layer of and remodeling process were intense in the grafted basophilic osteoid-like bone tissue (Fig. 2F). Cyabone. noacrylate was surrounded by macrophages and Cyanoacrylate was associated with foreign body multinucleated giant cells. granulomas in all specimens from both groups. At 30 days. In the control group, the graft–bed interface was predominantly filled with granulation tissue Quantitative Analyses with mild osteogenesis and eventual remnants of Mineralized tissue at the graft–bed interface. Table 1 periosteum (Fig. 3A). Trabeculae of woven bone shows the mean area of mineralized tissue at the coming from the recipient bed toward the graft and graft–bed interface. No mineralized tissue was vice versa were seen interconnecting both structures, present at 7 days in any specimen. At 30 days, the e124

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J Periodontol • May 2014

test specimens showed significantly higher interface area filled with mineralized tissue than the control specimens (P = 0.026). The same was observed at 90 days (P = 0.041). Within groups, a higher percentage of area filled with mineralized tissue was seen at 90 days compared to 30 days, regardless of demineralization (P = 0.041 and P = 0.0043 for test and control groups, respectively).

Consolidation of the graft and recipient bed to the newly formed bone. Table 2 shows the mean results for the length of surfaces consolidated to the newly formed bone at the graft–recipient bed interface. The consolidated surfaces increased with time in the control group (P = 0.041), but in the test group, the increase was not statistically significant (P = 0.132). The average length of consolidated surfaces was not significantly different between graft and recipient bone at 30 days (P = 0.349 for control and P = 0.937 for test) or 90 days (P = 0.409 for control and P = 0.588 for test). However, when the consolidation surfaces were considered as a whole (recipient bed plus graft), there were more consolidated surfaces at 30 days in the test group than in the control group (P = 0.026). The same was true at 90 days (P = 0.041).

Figure 4.

Bone tissue histologic analysis after 90 days. Microscopic views (original magnification ·10) of one of the specimens from the control group (A) and one from the test group (B) in which the original limits of the graft (gr) and recipient bed (rb) are not well defined (yellow arrows) and fused to the newly formed bone (nb) at some points of the interface. Remnants of the partially resorbed membrane (m) are still present; the red arrows point to newly formed bone at the extremities of the grafts (gr) underneath the membrane (m). C) Magnification of the area outlined in A (original magnification ·40) where newly formed bone (nb) is consolidated to the mature bone of the graft and recipient bed (arrows) in the remodeling process. D) Magnification of the area outlined in B (original magnification ·20) corresponding to the graft–bed interface where the new bone (nb) is consolidated to the mature bone of the graft and recipient bed (arrows) sharing a medullar space (me) (Masson’s trichrome). gr = bone graft; rb = recipient bone bed; m = membrane; nb = new bone; me = medullar space.

DISCUSSION The acid demineralization of the bone graft–bone bed interface resulted in significantly more area of newly formed bone and more extensive consolidation of this new bone to the graft and recipient bed compared to the standard procedure (without demineralization) at 30 and 90 days of healing. These data suggest that bone demineralization may have positive effects on osteogenesis. To the best of the authors’ knowledge, this is the first time that results from demineralization of graft– bed interface are reported. Even when bone marrow is exposed by perforating or

Table 1.

Average Percentage of Area Occupied by Mineralized Tissue at Graft–Bed Interface Control Group (%)

Test Group (%)

Mean – SD

n

Mean – SD

n

30

17.14 – 8.6a

6

34.39 – 13.4b

6

90

38.65 – 7.76c

6

54.00 – 11.23d

6

Observation Period (days)

Different letters at the same line or column represent a statistically significant difference (P

Demineralization of the contacting surfaces in autologous onlay bone grafts improves bone formation and bone consolidation.

Autologous bone grafts are usually well consolidated after 4 to 5 months but can be incompletely interlocked with the native bone. This study investig...
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