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Comparative Histomorphometric Analysis of Maxillary Sinus Augmentation With Absorbable Collagen Membrane and Osteoinductive Replaceable Bony Window in Rabbits Yong-Suk Moon, PhD,* Dong-Seok Sohn, DDS, PhD,† Jee-Won Moon, DDS, MSD,‡ Ju-Hyoung Lee, DDS, MSD,‡ In-Sook Park, DDS, PhD,‡ and Jung-Kwang Lee, DDS§
he placement of dental implants on edentulous posterior maxillae is a challenging procedure when insufficient and poor bone quantity and quality are present. Lateral window approaches with or without bone grafting are the most common surgical techniques to overcome vertical deficiency of atrophic posterior maxillae.1–4 To access the sinus cavity, various preparation methods of lateral windows have been reported. The complete osteotomy with a bone island, the hinge osteotomy, and the complete osteotomy with removal of the bony window in the lateral wall of sinus cavity were reported to access sinus cavities.5–7 Various bone grafting materials, such as autografts, allografts, xenografts, alloplasts, or combinations of different bone grafts,
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*Professor, Department of Anatomy, School of Medicine, Catholic University of Daegu, Daegu, Republic of Korea. †Professor, Department of Dentistry and Oral and Maxillofacial Surgery, Catholic University Hospital of Daegu, Daegu, Republic of Korea. ‡Assistant Professor, Department of Dentistry and Oral and Maxillofacial Surgery, Catholic University Hospital of Daegu, Daegu, Republic of Korea. §Clinical Instructor, Department of Dentistry and Oral and Maxillofacial Surgery, Catholic University Hospital of Daegu, Daegu, Republic of Korea.
Reprint requests and correspondence to: Dong-Seok Sohn, DDS, PhD, Department of Oral and Maxillofacial Surgery, Catholic University Hospital of Daegu, 3056-6 Daemyung-4 Dong, Namgu, Daegu 705-718, Republic of Korea, Phone: +82 53-6504288, Fax: +82 536227067, E-mail: [email protected] ISSN 1056-6163/14/02301-029 Implant Dentistry Volume 23 Number 1 Copyright © 2014 by Lippincott Williams & Wilkins DOI: 10.1097/ID.0000000000000031
Purpose: The aim of this animal study was to evaluate, through histomorphometric evaluation, bone regeneration in rabbit maxillary sinuses with absorbable collagen membranes and osteoinductive replaceable bony windows over bone grafts. Materials and Methods: Bilateral sinus augmentation procedures were performed in 16 adult male rabbits. The rabbits were randomly assigned to 4 groups of 4 rabbits each. Rectangular replaceable bony windows were made with a piezoelectric thin saw insert. In control group, grafted b-tricalcium phosphate (b-TCP) was covered by absorbable collagen membranes. In experimental groups, b-TCP was grafted and covered by replaceable bony windows. The rabbits were killed at 1, 2, 4, and 8 weeks postoperatively. The augmented sinuses were stained with
hematoxylin-eosin and Masson trichrome stains and examined, under light microscopy, for newly formed bone and soft tissue changes in the maxillary sinuses. Results: Histologically, significantly higher and faster new bone formation was observed in the augmented sinuses of the experimental groups, receiving homologous replaceable bony windows than in those of the control group receiving collagen membranes. Conclusion: This study demonstrates that, for augmentation, the use of the homologous replaceable bony window over bone graft material on the maxillary sinus accelerates bone regeneration. (Implant Dent 2014;23:29–36) Key Words: maxillary sinus augmentation, replaceable bony window, piezoelectric bone surgery
have been used to fill in the new compartment under the elevated sinus mucosa.2,8,9 A nonabsorbable or absorbable barrier membrane over the graft site was needed during vital bone regeneration to prevent fibrous connective tissue invagination to the bone graft site and to create higher new bone formation in the sinus.10,11 Sinus augmenta-
tion through the lateral approach with a barrier membrane has been proven to create higher vital bone regeneration than sinus augmentation without using barrier membranes.12 Recently, the benefits of using homologous replaceable bony windows, with and without bone grafts, through the lateral approach sinus augmentation, have
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COMPARATIVE HISTOMORPHOMETRIC ANALYSIS
been reported.13–18 Replaceable bony windows showed osteoinductive effects on bone regeneration and much higher new bone formation in the sinus compared with absorbable collagen membranes over the sinus bone graft.13,16 In addition, the replaceable bony window, as the homologous barrier, is free from cross-contamination of animal or human origin. The aim of this study was to compare the effect, on new bone formation, of homologous replaceable bony windows and absorbable collagen membranes over bone grafts in rabbit maxillary sinuses.
MATERIALS
AND
METHODS
Surgical Procedures
This study used 16 adult male New Zealand white rabbits that weighed between 2.8 and 3.2 kg (average, 3.0 kg) as experimental animals. This study was approved by the Animal Care and Use Committee at Catholic University Medical Center of Daegu. The 16 rabbits were randomly assigned to 4 groups of 4 rabbits each. All rabbits received the same surgical procedures. General anesthesia was induced with a combination of 30 mg/kg ketamine (Ketalar; Yuhan Co., Seoul, Korea) and 10 mg/kg xylazine (Rompun; Bayer Korea, Seoul, Korea) intramuscularly and 0.5 mL of lidocaine with 1:100,000 epinephrine was injected subcutaneously along the midline of the nasal dorsum. Each rabbit was stabilized on the surgical table, and skin and periosteal incisions were made at the middle of the nasal dorsum to expose nasal bone and the nasoincisional suture line. A rectangular replaceable bony window, about 5 3 10 mm, was made with a thin saw insert (S-Saw; Bukbu Dental Co., Daegu, Korea) connected with a piezoelectric device (Surgybone; Silfradent S.R.L., Sofia, Italy). Two windows were made at both nasal bones, and windows were located about 20 mm anterior to the nasofrontal suture line and 5 mm away from the middle suture line. The sinus mucosa was elevated anteroventrally with a blunt-ended curette, carefully avoiding membrane perforation, to accommodate the bone graft. In the control group, b-TCP (Cerasorb M;
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Curasan, Kleinostheim, Germany) was grafted into the new compartments under the elevated sinus membrane, and collagen membrane (BioSorb; IMTEC Co., Ardmore, OK) covered the bone grafts in a technique similar to conventional sinus augmentation procedures. In the experimental groups, b-TCP was grafted into the new compartments under the elevated sinus membrane, and the replaceable bony windows were replaced after careful elevation of the sinus mucosa (Fig. l). The muscular tissue was sutured with nylon sutures 4-0 (Blue nylon; Ailee Co., Busan, Korea). All animals were administered antibiotics intramuscularly with Gentamicin (Gentamicin; Donghwa Co., Seoul, Korea), 20 mg/kg for 3 days postoperatively. Tissue Preparation
The rabbits were killed at 1, 2, 4, and 8 weeks postoperatively under general anesthesia after intramuscular injection of Urethan (Junsei Chemical Co., Ltd., Tokyo, Japan). The rabbits were exsanguinated and perfused through the jugular veins with a fixative of 4% paraformaldehyde. The augmented sinus was segmented with a microsaw from the cranium and fixed with 4% paraformaldehyde for 24 hours and washed with 0.1 M phosphate buffer solution and decalcified with 10% EDTA-2Na solution for 2 weeks. The specimens were embedded in paraffin (Paraplast; Kendall, Mansfield, MA) and sliced coronally into serial sections approximately 5 mm thick. Both augmented sinuses were included in the specimens to compare at the same
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time. The specimens were stained with hematoxylin-eosin (H&E) and Masson trichrome (MT) stains, and examined, under light microscopy, for newly formed bone and soft tissue changes in the new compartments of the maxillary sinuses. Tartrate-Resistant Acid Phosphatase Stain
Tartrate-resistant acid phosphatase (TRAP) activity was detected using the recommended procedure with an acid phosphatase kit (SigmaAldrich, St. Louis, MO). The paraffinembedded sections in the control and experimental groups were cleared, dehydrated, and washed with distilled water. The sections were incubated 1 hour at 37°C in the dark with a mixture of naphthol AS-BI phosphate 25 mg as substrate, fast garnet GBC salt 15 mg, and 27 mmol tartaric acid diluted in 0.1 mol acetate buffer (pH 5.2). The sections were counterstained with acid hematoxylin and then mounted. Histomorphometric Analysis
Five randomly selected fields from each group were photographed using the AxioCam MRc5 (Carl Zeiss, Jena, Germany) interfaced with the Axiophot Photomicroscope (Carl Zeiss), and the AxioVision AC (Carl Zeiss) program was used for analysis. The following histomorphometric measurements were made: total augmented area, newly formed bone area, b-TCP particle area, and mature lamellar bone area. The total augmented area included newly formed bone, b-TCP particles, fibrous tissue, and vascular tissue within the new
Fig. 1. Beta-TCP was grafted into the new compartments under the elevated sinus membrane in experimental and control groups (A). The collagen membrane was covered over the bone grafts in control groups. In the experimental groups, the replaceable bony window was replaced over the bone graft (B). CM indicates collagen membrane; rB, replaceable bone.
IMPLANT DENTISTRY / VOLUME 23, NUMBER 1 2014 compartments. The percentage of newly formed bone or augmented b-TCP particles was analyzed as the ratio of the newly formed bone area or b-TCP particle area to the total augmented area in the control and the experimental groups. Only mature lamellar bone area was measured as the newly formed bone area. Mature lamellar bone was defined as a redcolored structure containing osteocytes in MT stain. The mean number of osteoclasts per square millimeter of total augmented area was measured manually at a magnification of 310, using an integrating eyepiece. The osteoclast was defined as a TRAPpositive cell (dark brown color) in TRAP stain. Statistical Analysis
Statistical analyses were performed with the SPSS program for Windows (version 19.0). In addition to standard descriptive statistical calculations (means and SD), analysis of variance (ANOVA) was used to determine statistical differences among and within groups. When ANOVA yielded significant results, indicating that the group was significantly different from the others, the Tukey test was also performed. The mean values were followed by 95% confidence intervals, and all the data were expressed as means 6 SD. The statistically significant levels were predetermined at P , 0.05.
RESULTS Histological Analysis
Beta-TCP particles were lightly stained and well differentiated from surrounding tissue in H&E and MT stains. Lamellar or host bone was stained red, and woven or newly formed bone was stained blue in MT stain. No signs of inflammation were shown in the control or experimental groups by H&E and MT stains under light microscopy. New bone formation increased in the new compartments under the elevated sinus membranes throughout the experimental period in both groups (Fig. 2). Control Group
One week postoperatively, most of the augmented sinus cavities were filled
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Fig. 2. Coronal sections of the nasal cavity in the rabbit at 1 week (A, E), 2 weeks (B, F), 4 weeks (C, G), and 8 weeks (D, H) after surgery. In each image, the left side shows the grafted sinus with b-TCP particles after maxillary sinus membrane elevation and covered collagen membrane, and the right side shows the grafted sinus with b-TCP particles and replaced bony window. CM indicates collagen membrane; rB, replaceable bone; NS, nasal septum (A–D: H&E stain, 312.5; E–H: MT stain, 312.5).
with blood clots, b-TCP particles, and soft tissue, without inflammation. Loose connective tissue, containing fibroblasts and blood cells, was observed under the collagen membranes, but newly formed bone was not seen around the b-TCP particles or under the collagen membranes (Fig. 3, A). Some newly formed bone was observed on the surface of the elevated sinus membranes and around the lateral wall of the maxillary sinuses (Fig. 4, A). At 2 weeks, newly formed bone was revealed partially on the surface and inside of the b-TCP particles, and osteoblasts were observed on the surface of newly formed bone. Dense connective tissue with fibroblasts and collagen fibers was observed under the collagen membranes, and numerous blood vessels had developed in the connective tissue (Fig. 3, B). More active new bone formation was observed along the b-TCP particles on the surface of the elevated sinus membranes and around the lateral wall of the maxillary sinuses (Fig. 4, B). Many osteoblasts were revealed on the surface of newly formed bone, and a few osteoclasts were also revealed. At 4 weeks, more soft tissue and blood vessels were evident around the b-TCP, and newly formed bone on the surface and inside of the b-TCP particles increased from that at 2 weeks. Dense connective tissue and blood vessels had developed under the collagen membranes, and around the b-TCP particles, under the collagen membranes, new bone formation was observed (Fig. 3, C). New bone formation highly
increased along the b-TCP particles on the surface of the elevated sinus membranes and around the lateral wall of the maxillary sinuses (Fig. 4, C). At 8 weeks, newly formed bone was thicker, and some mature lamellar bone with many osteocytes was revealed inside the new bone. The collagen membrane was merged with collagen fibers, and a lot of adipose tissue or bone marrow (BM) tissue was observed under the collagen membrane (Fig. 3, D). Soft tissue containing BM tissue and blood vessels had developed around the new bone. The size and the density of the b-TCP particles were less than that found at 4 weeks (Fig. 4, D). Experimental Groups
Central areas of the new compartments under the elevated sinus membranes were filled with blood clots, b-TCP particles, and soft tissue, without inflammation 1 week after surgery. New bone formation was revealed on the floor of replaced bony windows, and many osteoblasts were observed on the surface of the newly formed bone (Fig. 5, A). Some newly formed bone was observed under the surface of the elevated sinus membranes and around the lateral wall of the maxillary sinuses. No new bone formation was seen around the b-TCP particles (Fig. 6, A). At 2 weeks, newly formed bone was revealed partially on the surface and inside of the b-TCP particles, and soft tissue with osteoblasts and blood
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COMPARATIVE HISTOMORPHOMETRIC ANALYSIS
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Fig. 3. Photomicrograph showing the collagen membrane covered region at 1 week (A), 2 weeks (B), 4 weeks (C), and 8 weeks (D) after surgery. Loose connective tissue containing fibroblasts and blood cells were observed under the collagen membrane (A). Dense connective tissue with collagen fibers and numerous blood vessels (arrows) were observed under the collagen membrane (B). Dense connective tissue and blood vessels (arrows) were developed under the collagen membrane, and new bone formation was observed around the b-TCP particles (C). The collagen membrane was merged with collagen fibers, and adipose tissue was observed under the collagen membrane (D). CM indicates collagen membrane; N, newly formed bone; P, b-TCP particles. MT stain (A, B: 3100; C, D: 350).
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vessels was observed around the newly formed bone. Newly formed bone was expanded from the replaced bony windows to the center of new sinus cavities, and many osteoblasts, and a few osteoclasts, were observed there (Fig. 5, B). More active new bone formation was observed along the b-TCP particles on the surface of the elevated sinus membranes and around the lateral wall of the maxillary sinuses (Fig. 6, B). Four weeks after surgery, newly formed bone on the surface and inside of the b-TCP particles increased from that were seen at 2 weeks, and more soft tissue and blood vessels were evident around the b-TCP particles. New bone on the floor of the replaced bony windows was thicker, and some mature lamellar bone was revealed inside the newly formed bone (Fig. 5, C). BM, like soft tissue, was found along the new bone. Most of the b-TCP particles were seen in the bone formation, and new bone formation highly increased along the b-TCP particles on the surface of the elevated sinus membrane and around the lateral wall of the maxillary sinus (Fig. 6, C). At 8 weeks, the thickness and the density of new bone were highly increased, and abundant BM tissues were observed around the newly formed bone. Most of newly formed woven bone in the new compartments of the maxillary sinuses was replaced by mature lamellar bone at 8 weeks (Fig. 5, D). A lot of mature lamellar bone and BM were observed on the floor of the replaced bony windows (Fig. 6, D). The size and the density of b-TCP particles were less than that at 4 weeks. Histomorphometric Analysis
Fig. 4. Photomicrograph showing the bone formation of the control group at 1 week (A), 2 weeks (B), 4 weeks (C), and 8 weeks (D). Some newly formed bone was observed on the surface of the elevated sinus membrane, but no new bone formation was seen along the b-TCP particles (A). Newly formed bone was revealed partially on the surface and inside of the b-TCP particles, and lining osteoblasts (arrows) were observed on the surface of newly formed bone (B). New bone formation increased along the b-TCP particles around the lateral wall of the maxillary sinus and osteoblasts (arrows) were observed (C). Newly formed bone was thicker, and some mature lamellar bone was revealed inside the woven bone (D). N indicates newly formed bone; P, b-TCP particles; M, membrane; NB, nasal bone. MT stain (A: 3100; B–D: 350).
In the control group, the ratio of newly formed bone to the area of the augmented sinus at 1, 2, 4, and 8 weeks was 0.16 6 0.10%, 6.35 6 1.25%, 13.02 6 1.76%, and 21.74 6 2.40%, respectively. One-way ANOVA and post hoc comparisons showed that the new bone area at 2 weeks was significantly greater than at 1 week with P , 0.05, at 4 weeks was significantly greater than at 2 weeks with P , 0.05, and at 8 weeks was significantly greater than at 4 weeks with P , 0.05 (Fig. 7). In the control group, the ratio of the area
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Fig. 5. Photomicrograph showing the replaceable bony window replaced region at 1 week (A), 2 weeks (B), 4 weeks (C), and 8 weeks (D) after surgery. New bone formation was revealed the floor of replaced bony windows, and many osteoblasts (arrows) were observed on the surface of the newly formed bone (A). Newly formed bone was expanded from the replaced bony windows to the center of new sinus cavities (B). New bone on the floor of the replaced bony windows was thicker, and some mature lamellar bone was revealed inside the newly formed bone (C). A lot of mature lamellar bone and BM were observed on the floor of the replaced bony windows (D). rB indicates replaceable bone; N, newly formed bone; P, b-TCP particles; LB, lamellae bone; BM, bone marrow. MT stain (A: 3100; B–D: 350).
Fig. 6. Photomicrograph showing the bone formation of the experimental group at 1 week (A), 2 weeks (B), 4 weeks (C), and 8 weeks (D). Some newly formed bone and lining osteoblasts (arrows) were observed under the surface of the elevated sinus membranes; no new bone formation was seen around the b-TCP particles (A). Newly formed bone was revealed partially on the surface and inside of the b-TCP particles, and many osteoblasts (arrows) were observed around the newly formed bone (B). Newly formed bone around the b-TCP particles increased, and some lamellar bone was revealed inside the newly formed woven bone (C). Most of newly formed bone was replaced by mature lamellar bone, and abundant BM tissues were observed around the newly formed bone (D). N indicates newly formed bone; P, b-TCP particles; LB, lamellae bone; M, membrane; BM, bone marrow. MT stain (A: 3100; B–D: 350).
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of mature lamellar bone to the area of newly formed woven bone at 4 and 8 weeks was 3.69 6 1.25% and 13.02 6 3.69%, respectively, and at 8 weeks was significantly greater than at 4 weeks with P , 0.05 (Figs. 4, D and 8). The ratio of the area of augmented b-TCP to the area of the augmented sinus at 1, 2, 4, and 8 weeks was 55.13 6 5.41%, 47.15 6 4.12%, 35.50 6 4.83%, and 12.01 6 2.23%, respectively, at 2 weeks was not significantly different than at 1 week, at 4 weeks was significantly less than at 2 weeks, and at 8 weeks was significantly less than at 4 weeks with P , 0.05 (Fig. 9). In the experimental groups, the ratio of newly formed bone to the area of the augmented sinus at 1, 2, 4, and 8 weeks was 0.58 6 0.21%, 11.15 6 1.48%, 19.81 6 2.22%, and 31.28 6 3.51%, respectively. The new bone area at 2 weeks was significantly greater than at 1 week with P , 0.05, at 4 weeks was significantly greater than at 2 weeks with P , 0.05, and at 8 weeks was significantly greater than at 4 weeks with P , 0.05 (Fig. 7). The ratio of the area of mature lamellar bone to the area of newly formed woven bone at 4 and 8 weeks was 13.68 6 4.05% and 27.37 6 5.45%, respectively, and at 8 weeks was significantly greater than at 4 weeks with P , 0.05 (Figs. 5, D, 6, D, and 8). The ratio of the area of augmented b-TCP to the area of the augmented sinus at 1, 2, 4, and 8 weeks was 55.10 6 5.09%, 45.17 6 3.40%, 32.17 6 3.33%, and 7.59 6 1.29%, respectively, at 2 weeks was no significant different than at 1 week, at 4 weeks was significantly less than at 2 weeks, and at 8 weeks was significantly less than at 4 weeks with P , 0.05 (Fig. 9). The area of newly formed bone increased significantly in the experimental group from 2 to 8 weeks, compared with the control group (Fig. 7). The ratio of the area of mature lamellar bone to the area of newly formed woven bone increased significantly in the experimental group at 4 and 8 weeks, compared with the control group (Fig. 8). The area of augmented b-TCP decreased significantly in the experimental
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group at 8 weeks, compared with the control group (Fig. 9). Analysis of TRAP Stain
Fig. 7. Histomorphometric measurement of the area of newly formed bone to the area of the augmented sinus. *P , 0.05.
The number of TRAP-stained osteoclasts by l mm2 in the control group at 1, 2, 4, and 8 weeks was 8.50 6 5.93, 20.16 6 8.00, 26.17 6 7.12, and 22.10 6 6.27, respectively and, in the experimental groups, 10.61 6 7.14, 23.06 6 7.60, 28.38 6 7.79, and 18.55 6 6.15, respectively. The number of osteoclasts increased from 1 to 4 weeks in the control and the experimental groups, but there was no significant change. The number of osteoclasts decreased at 8 weeks for all groups compared with that at 4 weeks, but there was no significant change (Fig. 10). Control Group
Fig. 8. Histomorphometric measurement of the area of mature lamellar bone to the area of newly formed woven bone. *P , 0.05.
Some TRAP-stained osteoclasts were seen along the elevated sinus membrane and around the lateral wall of the maxillary sinus at 1 week postoperatively (Fig. 11, A). Many osteoclasts were seen around the b-TCP particles, and some osteoclasts were seen along the elevated sinus membrane and around the lateral wall of the maxillary sinus at 2 weeks. At 4 and 8 weeks, many TRAP-stained osteoclasts were seen along the elevated sinus membrane, around the lateral wall of the maxillary sinus, and on the surface and inside of the b-TCP (Fig. 11, B and C). Experimental Groups
Fig. 9. Histomorphometric measurement of the area of b-TCP particles to the area of the augmented sinus. *P , 0.05.
TRAP-stained osteoclasts were seen on the replaced bony windows, the elevated sinus membranes, and the lateral wall of the maxillary sinuses at 1 week postoperatively (Fig. 11, D). Many osteoclasts were revealed on the surface of the new bone on the floor of replaced bony windows and around the b-TCP particles at 2 weeks. At 4 weeks, many TRAP-stained osteoclasts were seen in new bone and on the surface and inside of the b-TCP (Fig. 11, E). A few osteoclasts were seen around the newly formed bone and the BM and on the surface of the b-TCP at 8 weeks of experimentation (Fig. 11, F).
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Fig. 10. Histomorphometric measurement of the number of TRAP-stained osteoclasts by l mm2 of the augmented sinus.
Fig. 11. Photomicrograph shows TRAP-stained osteoclasts (arrows) in the control group at 1 week (A), 4 weeks (B), and 8 weeks (C), and in the experimental groups at 1 week (D), 4 weeks (E), and 8 weeks (F). N indicates newly formed bone; P, b-TCP particles; M, membrane (3100).
DISCUSSION Bone grafted maxillary sinus augmentation with and without bone grafting has been considered a highly predictable vertical augmentation procedure.2,13–15 To facilitate graft containment and prevent fibrous connective tissue invagination to the bone grafted sinus, a nonabsorbable or absorbable barrier over the graft site was needed during vital bone regeneration. Tarnow et al11 demonstrated that the placement of a nonabsorbable barrier membrane increases new bone formation in the grafted sinus and showed a positive effect on implant survival, compared with the nonbarrier membrane group.11
Wallace et al12 reported no significant difference of new bone formation in the sinus between a nonabsorbable or absorbable barrier membrane. However, the nonbarrier membrane group showed less new bone formation than did the barrier membrane group. As an alternative to the absorbable or nonabsorbable barrier membrane, a homologous bony window, detached from the lateral wall of the sinus cavity when preparing access to the sinus cavity, has been used to seal the lateral window of the sinus after sinus augmentation with/without bone grafting.3,4,13–17 Sohn et al13,16 first demonstrated that the replaceable bony window, unlike the
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collagen membrane, revealed osteoinductive efficacy during the healing period in the sinus, in animal studies. New bone formation was observed on the floor of the replaced bony window in the experimental group after 1 week. However, new bone formation was not observed under the collagen membrane in the control group after 1 week.13 The replaceable bony window, as a homologous barrier, revealed numerous positive effects on new bone formation in the sinus.13,16 The replaceable bony window prevented soft tissue ingrowth into the grafted sinus and, histologically, significantly higher and faster new bone formation was achieved in the experimental groups using the replaceable bony window than in the control group using the collagen membrane.13,16 In addition, the replaceable bony window has no risk of cross-contamination, unlike human- or animal-origin membranes. The results of this study correspond with those of previous studies.13,16 In this study, rabbits’ maxillary sinuses, known to be similar to humans’ sinuses, were used to verify the mechanism of new bone formation and to compare the effect on bone reformation between a replaceable bony window and a collagen membrane over the bone graft in the sinus.18 Many studies used rabbits’ maxillary sinuses to evaluate new bone formation using variable bone substitutes in the sinuses.19–22 In our study, when the homologous replaceable bony window was applied over the bone substitute, significantly higher new bone formation was revealed during the experimental period, compared with that in the control group receiving the collagen membrane. In addition, because of the osteoinductive function of the repositioned bony window, which showed new bone formation along the floor of the replaced bony window, the ratio of mature lamellar bone to the area of newly formed woven bone increased significantly in the experimental groups at 4 and 8 weeks, compared with that of the control group in which collagen membrane was used. The collagen membrane did not show any osteoinductive function during the experimental period.
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Bone remodeling is a synchronized and complex process of bone matrix formation by osteoblasts and absorption of bone osteoclasts.23 To evaluate osteoclast activity on bone remodeling, the number of TRAP-stained osteoclasts was calculated in this study. The presence of the enzyme, TRAP, is a generallyacknowledged cytochemical characteristic of osteoclasts and their mononuclear precursors.24,25 Many TRAP-stained osteoclasts were seen on the replaced bony windows, the elevated sinus membranes, and the lateral wall of the maxillary sinuses, revealed early in the healing period, indicating active new bone formation in the experimental groups. However, TRAP-stained osteoclasts were not seen along the collagen membrane, indicating no bone regeneration in the control group early in the healing period. Some were seen along the elevated sinus membrane and around the lateral wall of the maxillary sinuses early in the healing period. Elevated sinus membranes and the exposed lateral walls of the sinus cavities in both groups showed osteoinductive efficacy in this study, as in previous studies.13,16
CONCLUSION According to this study using histomorphometric analysis, we conclude that new bone formation is faster and denser in the experimental groups receiving the replaceable bony windows than in the control group receiving a collagen membrane over the bone graft. The replaceable bony window has osteoinductive potential, and accelerates new bone formation early in the healing period, compared with the collagen membrane over the bone graft.
DISCLOSURE The authors claim to have no financial interest, either directly or indirectly, in the products or information listed in the article.
ACKNOWLEDGMENTS This work was supported by the grant of Research Institute of Medical
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Science, Catholic University of Daegu (2008).
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13. Sohn DS, Kim WS, An KM, et al. Comparative histomorphometric analysis of maxillary sinus augmentation with and without bone grafting in rabbit. Implant Dent. 2010;19:259–270. 14. Sohn DS, Moon JW, Moon KN, et al. New bone formation in the maxillary sinus using only absorbable gelatin sponge. J Oral Maxillofac Surg. 2010;68: 1327–1333. 15. Sohn DS, Heo JU, Kwak DH, et al. Bone regeneration in the maxillary sinus using an autologous fibrin-rich block with concentrated growth factors alone. Implant Dent. 2011;20:389–395. 16. Sohn DS, Moon JW, Lee WH, et al. Comparison of new bone formation in the maxillary sinus with and without bone grafts: Immunochemical rabbit study. Int J Oral Maxillofac Implants. 2011;26: 1033–1042. 17. Moon JW, Sohn DS, Heo JU, et al. New bone formation in the maxillary sinus using peripheral venous blood alone. J Oral Maxillofac Surg. 2011;69:2357– 2367. 18. Kumlien J, Schiratzki H. The vascular arrangement of the sinus mucosa. A study in rabbits. Acta Otolaryngol. 1985;99:122–132. 19. Watanabe K, Niimi A, Ueda M. Autogenous bone grafts in the rabbit maxillary sinus. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 1999;88:26–32. 20. Wada K, Niimi A, Watanabe K, et al. Maxillary sinus floor augmentation in rabbits: A comparative histologichistomorphometric study between rhBMP-2 and autogenous bone. Int J Periodontics Restorative Dent. 2001;21:252–263. 21. Asai S, Shimizu Y, Ooya K. Maxillary sinus augmentation model in rabbits: Effect of occluded nasal ostium on new bone formation. Clin Oral Implants Res. 2002;13:405–409. 22. Albrektsson T. Repair of bone grafts. A vital microscopic and histological investigation in the rabbit. Scand J Plast Reconstr Surg. 1980;14:1–12. 23. Marks SC Jr, Popoff SN. Bone biology: The regulation of development, structure, and function in the skeleton. Am J Anat. 1988;183;41–44. 24. Minkin C. Bone acid phosphatase: Tartrate-resistant acid phosphatase as a marker of osteoclast function. Calcif Tissue Int. 1982;34:285–290. 25. Baron R, Neff L, Tran Van P, et al. Kinetic and cytochemical identification of osteoclast precursors and their differentiation into multinucleated osteoclasts. Am J Pathol. 1986;122:363–378.
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Comparative Histomorphometric Analysis of Maxillary Sinus Augmentation With Absorbable Collagen Membrane and Osteoinductive Replaceable Bony Window in Rabbits Yong-Suk Moon, PhD,* Dong-Seok Sohn, DDS, PhD,† Jee-Won Moon, DDS, MSD,‡ Ju-Hyoung Lee, DDS, MSD,‡ In-Sook Park, DDS, PhD,‡ and Jung-Kwang Lee, DDS§
he placement of dental implants on edentulous posterior maxillae is a challenging procedure when insufficient and poor bone quantity and quality are present. Lateral window approaches with or without bone grafting are the most common surgical techniques to overcome vertical deficiency of atrophic posterior maxillae.1–4 To access the sinus cavity, various preparation methods of lateral windows have been reported. The complete osteotomy with a bone island, the hinge osteotomy, and the complete osteotomy with removal of the bony window in the lateral wall of sinus cavity were reported to access sinus cavities.5–7 Various bone grafting materials, such as autografts, allografts, xenografts, alloplasts, or combinations of different bone grafts,
T
*Professor, Department of Anatomy, School of Medicine, Catholic University of Daegu, Daegu, Republic of Korea. †Professor, Department of Dentistry and Oral and Maxillofacial Surgery, Catholic University Hospital of Daegu, Daegu, Republic of Korea. ‡Assistant Professor, Department of Dentistry and Oral and Maxillofacial Surgery, Catholic University Hospital of Daegu, Daegu, Republic of Korea. §Clinical Instructor, Department of Dentistry and Oral and Maxillofacial Surgery, Catholic University Hospital of Daegu, Daegu, Republic of Korea.
Reprint requests and correspondence to: Dong-Seok Sohn, DDS, PhD, Department of Oral and Maxillofacial Surgery, Catholic University Hospital of Daegu, 3056-6 Daemyung-4 Dong, Namgu, Daegu 705-718, Republic of Korea, Phone: +82 53-6504288, Fax: +82 536227067, E-mail: [email protected] ISSN 1056-6163/14/02301-029 Implant Dentistry Volume 23 Number 1 Copyright © 2014 by Lippincott Williams & Wilkins DOI: 10.1097/ID.0000000000000031
Purpose: The aim of this animal study was to evaluate, through histomorphometric evaluation, bone regeneration in rabbit maxillary sinuses with absorbable collagen membranes and osteoinductive replaceable bony windows over bone grafts. Materials and Methods: Bilateral sinus augmentation procedures were performed in 16 adult male rabbits. The rabbits were randomly assigned to 4 groups of 4 rabbits each. Rectangular replaceable bony windows were made with a piezoelectric thin saw insert. In control group, grafted b-tricalcium phosphate (b-TCP) was covered by absorbable collagen membranes. In experimental groups, b-TCP was grafted and covered by replaceable bony windows. The rabbits were killed at 1, 2, 4, and 8 weeks postoperatively. The augmented sinuses were stained with
hematoxylin-eosin and Masson trichrome stains and examined, under light microscopy, for newly formed bone and soft tissue changes in the maxillary sinuses. Results: Histologically, significantly higher and faster new bone formation was observed in the augmented sinuses of the experimental groups, receiving homologous replaceable bony windows than in those of the control group receiving collagen membranes. Conclusion: This study demonstrates that, for augmentation, the use of the homologous replaceable bony window over bone graft material on the maxillary sinus accelerates bone regeneration. (Implant Dent 2014;23:29–36) Key Words: maxillary sinus augmentation, replaceable bony window, piezoelectric bone surgery
have been used to fill in the new compartment under the elevated sinus mucosa.2,8,9 A nonabsorbable or absorbable barrier membrane over the graft site was needed during vital bone regeneration to prevent fibrous connective tissue invagination to the bone graft site and to create higher new bone formation in the sinus.10,11 Sinus augmenta-
tion through the lateral approach with a barrier membrane has been proven to create higher vital bone regeneration than sinus augmentation without using barrier membranes.12 Recently, the benefits of using homologous replaceable bony windows, with and without bone grafts, through the lateral approach sinus augmentation, have
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been reported.13–18 Replaceable bony windows showed osteoinductive effects on bone regeneration and much higher new bone formation in the sinus compared with absorbable collagen membranes over the sinus bone graft.13,16 In addition, the replaceable bony window, as the homologous barrier, is free from cross-contamination of animal or human origin. The aim of this study was to compare the effect, on new bone formation, of homologous replaceable bony windows and absorbable collagen membranes over bone grafts in rabbit maxillary sinuses.
MATERIALS
AND
METHODS
Surgical Procedures
This study used 16 adult male New Zealand white rabbits that weighed between 2.8 and 3.2 kg (average, 3.0 kg) as experimental animals. This study was approved by the Animal Care and Use Committee at Catholic University Medical Center of Daegu. The 16 rabbits were randomly assigned to 4 groups of 4 rabbits each. All rabbits received the same surgical procedures. General anesthesia was induced with a combination of 30 mg/kg ketamine (Ketalar; Yuhan Co., Seoul, Korea) and 10 mg/kg xylazine (Rompun; Bayer Korea, Seoul, Korea) intramuscularly and 0.5 mL of lidocaine with 1:100,000 epinephrine was injected subcutaneously along the midline of the nasal dorsum. Each rabbit was stabilized on the surgical table, and skin and periosteal incisions were made at the middle of the nasal dorsum to expose nasal bone and the nasoincisional suture line. A rectangular replaceable bony window, about 5 3 10 mm, was made with a thin saw insert (S-Saw; Bukbu Dental Co., Daegu, Korea) connected with a piezoelectric device (Surgybone; Silfradent S.R.L., Sofia, Italy). Two windows were made at both nasal bones, and windows were located about 20 mm anterior to the nasofrontal suture line and 5 mm away from the middle suture line. The sinus mucosa was elevated anteroventrally with a blunt-ended curette, carefully avoiding membrane perforation, to accommodate the bone graft. In the control group, b-TCP (Cerasorb M;
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Curasan, Kleinostheim, Germany) was grafted into the new compartments under the elevated sinus membrane, and collagen membrane (BioSorb; IMTEC Co., Ardmore, OK) covered the bone grafts in a technique similar to conventional sinus augmentation procedures. In the experimental groups, b-TCP was grafted into the new compartments under the elevated sinus membrane, and the replaceable bony windows were replaced after careful elevation of the sinus mucosa (Fig. l). The muscular tissue was sutured with nylon sutures 4-0 (Blue nylon; Ailee Co., Busan, Korea). All animals were administered antibiotics intramuscularly with Gentamicin (Gentamicin; Donghwa Co., Seoul, Korea), 20 mg/kg for 3 days postoperatively. Tissue Preparation
The rabbits were killed at 1, 2, 4, and 8 weeks postoperatively under general anesthesia after intramuscular injection of Urethan (Junsei Chemical Co., Ltd., Tokyo, Japan). The rabbits were exsanguinated and perfused through the jugular veins with a fixative of 4% paraformaldehyde. The augmented sinus was segmented with a microsaw from the cranium and fixed with 4% paraformaldehyde for 24 hours and washed with 0.1 M phosphate buffer solution and decalcified with 10% EDTA-2Na solution for 2 weeks. The specimens were embedded in paraffin (Paraplast; Kendall, Mansfield, MA) and sliced coronally into serial sections approximately 5 mm thick. Both augmented sinuses were included in the specimens to compare at the same
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time. The specimens were stained with hematoxylin-eosin (H&E) and Masson trichrome (MT) stains, and examined, under light microscopy, for newly formed bone and soft tissue changes in the new compartments of the maxillary sinuses. Tartrate-Resistant Acid Phosphatase Stain
Tartrate-resistant acid phosphatase (TRAP) activity was detected using the recommended procedure with an acid phosphatase kit (SigmaAldrich, St. Louis, MO). The paraffinembedded sections in the control and experimental groups were cleared, dehydrated, and washed with distilled water. The sections were incubated 1 hour at 37°C in the dark with a mixture of naphthol AS-BI phosphate 25 mg as substrate, fast garnet GBC salt 15 mg, and 27 mmol tartaric acid diluted in 0.1 mol acetate buffer (pH 5.2). The sections were counterstained with acid hematoxylin and then mounted. Histomorphometric Analysis
Five randomly selected fields from each group were photographed using the AxioCam MRc5 (Carl Zeiss, Jena, Germany) interfaced with the Axiophot Photomicroscope (Carl Zeiss), and the AxioVision AC (Carl Zeiss) program was used for analysis. The following histomorphometric measurements were made: total augmented area, newly formed bone area, b-TCP particle area, and mature lamellar bone area. The total augmented area included newly formed bone, b-TCP particles, fibrous tissue, and vascular tissue within the new
Fig. 1. Beta-TCP was grafted into the new compartments under the elevated sinus membrane in experimental and control groups (A). The collagen membrane was covered over the bone grafts in control groups. In the experimental groups, the replaceable bony window was replaced over the bone graft (B). CM indicates collagen membrane; rB, replaceable bone.
IMPLANT DENTISTRY / VOLUME 23, NUMBER 1 2014 compartments. The percentage of newly formed bone or augmented b-TCP particles was analyzed as the ratio of the newly formed bone area or b-TCP particle area to the total augmented area in the control and the experimental groups. Only mature lamellar bone area was measured as the newly formed bone area. Mature lamellar bone was defined as a redcolored structure containing osteocytes in MT stain. The mean number of osteoclasts per square millimeter of total augmented area was measured manually at a magnification of 310, using an integrating eyepiece. The osteoclast was defined as a TRAPpositive cell (dark brown color) in TRAP stain. Statistical Analysis
Statistical analyses were performed with the SPSS program for Windows (version 19.0). In addition to standard descriptive statistical calculations (means and SD), analysis of variance (ANOVA) was used to determine statistical differences among and within groups. When ANOVA yielded significant results, indicating that the group was significantly different from the others, the Tukey test was also performed. The mean values were followed by 95% confidence intervals, and all the data were expressed as means 6 SD. The statistically significant levels were predetermined at P , 0.05.
RESULTS Histological Analysis
Beta-TCP particles were lightly stained and well differentiated from surrounding tissue in H&E and MT stains. Lamellar or host bone was stained red, and woven or newly formed bone was stained blue in MT stain. No signs of inflammation were shown in the control or experimental groups by H&E and MT stains under light microscopy. New bone formation increased in the new compartments under the elevated sinus membranes throughout the experimental period in both groups (Fig. 2). Control Group
One week postoperatively, most of the augmented sinus cavities were filled
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Fig. 2. Coronal sections of the nasal cavity in the rabbit at 1 week (A, E), 2 weeks (B, F), 4 weeks (C, G), and 8 weeks (D, H) after surgery. In each image, the left side shows the grafted sinus with b-TCP particles after maxillary sinus membrane elevation and covered collagen membrane, and the right side shows the grafted sinus with b-TCP particles and replaced bony window. CM indicates collagen membrane; rB, replaceable bone; NS, nasal septum (A–D: H&E stain, 312.5; E–H: MT stain, 312.5).
with blood clots, b-TCP particles, and soft tissue, without inflammation. Loose connective tissue, containing fibroblasts and blood cells, was observed under the collagen membranes, but newly formed bone was not seen around the b-TCP particles or under the collagen membranes (Fig. 3, A). Some newly formed bone was observed on the surface of the elevated sinus membranes and around the lateral wall of the maxillary sinuses (Fig. 4, A). At 2 weeks, newly formed bone was revealed partially on the surface and inside of the b-TCP particles, and osteoblasts were observed on the surface of newly formed bone. Dense connective tissue with fibroblasts and collagen fibers was observed under the collagen membranes, and numerous blood vessels had developed in the connective tissue (Fig. 3, B). More active new bone formation was observed along the b-TCP particles on the surface of the elevated sinus membranes and around the lateral wall of the maxillary sinuses (Fig. 4, B). Many osteoblasts were revealed on the surface of newly formed bone, and a few osteoclasts were also revealed. At 4 weeks, more soft tissue and blood vessels were evident around the b-TCP, and newly formed bone on the surface and inside of the b-TCP particles increased from that at 2 weeks. Dense connective tissue and blood vessels had developed under the collagen membranes, and around the b-TCP particles, under the collagen membranes, new bone formation was observed (Fig. 3, C). New bone formation highly
increased along the b-TCP particles on the surface of the elevated sinus membranes and around the lateral wall of the maxillary sinuses (Fig. 4, C). At 8 weeks, newly formed bone was thicker, and some mature lamellar bone with many osteocytes was revealed inside the new bone. The collagen membrane was merged with collagen fibers, and a lot of adipose tissue or bone marrow (BM) tissue was observed under the collagen membrane (Fig. 3, D). Soft tissue containing BM tissue and blood vessels had developed around the new bone. The size and the density of the b-TCP particles were less than that found at 4 weeks (Fig. 4, D). Experimental Groups
Central areas of the new compartments under the elevated sinus membranes were filled with blood clots, b-TCP particles, and soft tissue, without inflammation 1 week after surgery. New bone formation was revealed on the floor of replaced bony windows, and many osteoblasts were observed on the surface of the newly formed bone (Fig. 5, A). Some newly formed bone was observed under the surface of the elevated sinus membranes and around the lateral wall of the maxillary sinuses. No new bone formation was seen around the b-TCP particles (Fig. 6, A). At 2 weeks, newly formed bone was revealed partially on the surface and inside of the b-TCP particles, and soft tissue with osteoblasts and blood
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COMPARATIVE HISTOMORPHOMETRIC ANALYSIS
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Fig. 3. Photomicrograph showing the collagen membrane covered region at 1 week (A), 2 weeks (B), 4 weeks (C), and 8 weeks (D) after surgery. Loose connective tissue containing fibroblasts and blood cells were observed under the collagen membrane (A). Dense connective tissue with collagen fibers and numerous blood vessels (arrows) were observed under the collagen membrane (B). Dense connective tissue and blood vessels (arrows) were developed under the collagen membrane, and new bone formation was observed around the b-TCP particles (C). The collagen membrane was merged with collagen fibers, and adipose tissue was observed under the collagen membrane (D). CM indicates collagen membrane; N, newly formed bone; P, b-TCP particles. MT stain (A, B: 3100; C, D: 350).
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vessels was observed around the newly formed bone. Newly formed bone was expanded from the replaced bony windows to the center of new sinus cavities, and many osteoblasts, and a few osteoclasts, were observed there (Fig. 5, B). More active new bone formation was observed along the b-TCP particles on the surface of the elevated sinus membranes and around the lateral wall of the maxillary sinuses (Fig. 6, B). Four weeks after surgery, newly formed bone on the surface and inside of the b-TCP particles increased from that were seen at 2 weeks, and more soft tissue and blood vessels were evident around the b-TCP particles. New bone on the floor of the replaced bony windows was thicker, and some mature lamellar bone was revealed inside the newly formed bone (Fig. 5, C). BM, like soft tissue, was found along the new bone. Most of the b-TCP particles were seen in the bone formation, and new bone formation highly increased along the b-TCP particles on the surface of the elevated sinus membrane and around the lateral wall of the maxillary sinus (Fig. 6, C). At 8 weeks, the thickness and the density of new bone were highly increased, and abundant BM tissues were observed around the newly formed bone. Most of newly formed woven bone in the new compartments of the maxillary sinuses was replaced by mature lamellar bone at 8 weeks (Fig. 5, D). A lot of mature lamellar bone and BM were observed on the floor of the replaced bony windows (Fig. 6, D). The size and the density of b-TCP particles were less than that at 4 weeks. Histomorphometric Analysis
Fig. 4. Photomicrograph showing the bone formation of the control group at 1 week (A), 2 weeks (B), 4 weeks (C), and 8 weeks (D). Some newly formed bone was observed on the surface of the elevated sinus membrane, but no new bone formation was seen along the b-TCP particles (A). Newly formed bone was revealed partially on the surface and inside of the b-TCP particles, and lining osteoblasts (arrows) were observed on the surface of newly formed bone (B). New bone formation increased along the b-TCP particles around the lateral wall of the maxillary sinus and osteoblasts (arrows) were observed (C). Newly formed bone was thicker, and some mature lamellar bone was revealed inside the woven bone (D). N indicates newly formed bone; P, b-TCP particles; M, membrane; NB, nasal bone. MT stain (A: 3100; B–D: 350).
In the control group, the ratio of newly formed bone to the area of the augmented sinus at 1, 2, 4, and 8 weeks was 0.16 6 0.10%, 6.35 6 1.25%, 13.02 6 1.76%, and 21.74 6 2.40%, respectively. One-way ANOVA and post hoc comparisons showed that the new bone area at 2 weeks was significantly greater than at 1 week with P , 0.05, at 4 weeks was significantly greater than at 2 weeks with P , 0.05, and at 8 weeks was significantly greater than at 4 weeks with P , 0.05 (Fig. 7). In the control group, the ratio of the area
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Fig. 5. Photomicrograph showing the replaceable bony window replaced region at 1 week (A), 2 weeks (B), 4 weeks (C), and 8 weeks (D) after surgery. New bone formation was revealed the floor of replaced bony windows, and many osteoblasts (arrows) were observed on the surface of the newly formed bone (A). Newly formed bone was expanded from the replaced bony windows to the center of new sinus cavities (B). New bone on the floor of the replaced bony windows was thicker, and some mature lamellar bone was revealed inside the newly formed bone (C). A lot of mature lamellar bone and BM were observed on the floor of the replaced bony windows (D). rB indicates replaceable bone; N, newly formed bone; P, b-TCP particles; LB, lamellae bone; BM, bone marrow. MT stain (A: 3100; B–D: 350).
Fig. 6. Photomicrograph showing the bone formation of the experimental group at 1 week (A), 2 weeks (B), 4 weeks (C), and 8 weeks (D). Some newly formed bone and lining osteoblasts (arrows) were observed under the surface of the elevated sinus membranes; no new bone formation was seen around the b-TCP particles (A). Newly formed bone was revealed partially on the surface and inside of the b-TCP particles, and many osteoblasts (arrows) were observed around the newly formed bone (B). Newly formed bone around the b-TCP particles increased, and some lamellar bone was revealed inside the newly formed woven bone (C). Most of newly formed bone was replaced by mature lamellar bone, and abundant BM tissues were observed around the newly formed bone (D). N indicates newly formed bone; P, b-TCP particles; LB, lamellae bone; M, membrane; BM, bone marrow. MT stain (A: 3100; B–D: 350).
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of mature lamellar bone to the area of newly formed woven bone at 4 and 8 weeks was 3.69 6 1.25% and 13.02 6 3.69%, respectively, and at 8 weeks was significantly greater than at 4 weeks with P , 0.05 (Figs. 4, D and 8). The ratio of the area of augmented b-TCP to the area of the augmented sinus at 1, 2, 4, and 8 weeks was 55.13 6 5.41%, 47.15 6 4.12%, 35.50 6 4.83%, and 12.01 6 2.23%, respectively, at 2 weeks was not significantly different than at 1 week, at 4 weeks was significantly less than at 2 weeks, and at 8 weeks was significantly less than at 4 weeks with P , 0.05 (Fig. 9). In the experimental groups, the ratio of newly formed bone to the area of the augmented sinus at 1, 2, 4, and 8 weeks was 0.58 6 0.21%, 11.15 6 1.48%, 19.81 6 2.22%, and 31.28 6 3.51%, respectively. The new bone area at 2 weeks was significantly greater than at 1 week with P , 0.05, at 4 weeks was significantly greater than at 2 weeks with P , 0.05, and at 8 weeks was significantly greater than at 4 weeks with P , 0.05 (Fig. 7). The ratio of the area of mature lamellar bone to the area of newly formed woven bone at 4 and 8 weeks was 13.68 6 4.05% and 27.37 6 5.45%, respectively, and at 8 weeks was significantly greater than at 4 weeks with P , 0.05 (Figs. 5, D, 6, D, and 8). The ratio of the area of augmented b-TCP to the area of the augmented sinus at 1, 2, 4, and 8 weeks was 55.10 6 5.09%, 45.17 6 3.40%, 32.17 6 3.33%, and 7.59 6 1.29%, respectively, at 2 weeks was no significant different than at 1 week, at 4 weeks was significantly less than at 2 weeks, and at 8 weeks was significantly less than at 4 weeks with P , 0.05 (Fig. 9). The area of newly formed bone increased significantly in the experimental group from 2 to 8 weeks, compared with the control group (Fig. 7). The ratio of the area of mature lamellar bone to the area of newly formed woven bone increased significantly in the experimental group at 4 and 8 weeks, compared with the control group (Fig. 8). The area of augmented b-TCP decreased significantly in the experimental
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COMPARATIVE HISTOMORPHOMETRIC ANALYSIS
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group at 8 weeks, compared with the control group (Fig. 9). Analysis of TRAP Stain
Fig. 7. Histomorphometric measurement of the area of newly formed bone to the area of the augmented sinus. *P , 0.05.
The number of TRAP-stained osteoclasts by l mm2 in the control group at 1, 2, 4, and 8 weeks was 8.50 6 5.93, 20.16 6 8.00, 26.17 6 7.12, and 22.10 6 6.27, respectively and, in the experimental groups, 10.61 6 7.14, 23.06 6 7.60, 28.38 6 7.79, and 18.55 6 6.15, respectively. The number of osteoclasts increased from 1 to 4 weeks in the control and the experimental groups, but there was no significant change. The number of osteoclasts decreased at 8 weeks for all groups compared with that at 4 weeks, but there was no significant change (Fig. 10). Control Group
Fig. 8. Histomorphometric measurement of the area of mature lamellar bone to the area of newly formed woven bone. *P , 0.05.
Some TRAP-stained osteoclasts were seen along the elevated sinus membrane and around the lateral wall of the maxillary sinus at 1 week postoperatively (Fig. 11, A). Many osteoclasts were seen around the b-TCP particles, and some osteoclasts were seen along the elevated sinus membrane and around the lateral wall of the maxillary sinus at 2 weeks. At 4 and 8 weeks, many TRAP-stained osteoclasts were seen along the elevated sinus membrane, around the lateral wall of the maxillary sinus, and on the surface and inside of the b-TCP (Fig. 11, B and C). Experimental Groups
Fig. 9. Histomorphometric measurement of the area of b-TCP particles to the area of the augmented sinus. *P , 0.05.
TRAP-stained osteoclasts were seen on the replaced bony windows, the elevated sinus membranes, and the lateral wall of the maxillary sinuses at 1 week postoperatively (Fig. 11, D). Many osteoclasts were revealed on the surface of the new bone on the floor of replaced bony windows and around the b-TCP particles at 2 weeks. At 4 weeks, many TRAP-stained osteoclasts were seen in new bone and on the surface and inside of the b-TCP (Fig. 11, E). A few osteoclasts were seen around the newly formed bone and the BM and on the surface of the b-TCP at 8 weeks of experimentation (Fig. 11, F).
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Fig. 10. Histomorphometric measurement of the number of TRAP-stained osteoclasts by l mm2 of the augmented sinus.
Fig. 11. Photomicrograph shows TRAP-stained osteoclasts (arrows) in the control group at 1 week (A), 4 weeks (B), and 8 weeks (C), and in the experimental groups at 1 week (D), 4 weeks (E), and 8 weeks (F). N indicates newly formed bone; P, b-TCP particles; M, membrane (3100).
DISCUSSION Bone grafted maxillary sinus augmentation with and without bone grafting has been considered a highly predictable vertical augmentation procedure.2,13–15 To facilitate graft containment and prevent fibrous connective tissue invagination to the bone grafted sinus, a nonabsorbable or absorbable barrier over the graft site was needed during vital bone regeneration. Tarnow et al11 demonstrated that the placement of a nonabsorbable barrier membrane increases new bone formation in the grafted sinus and showed a positive effect on implant survival, compared with the nonbarrier membrane group.11
Wallace et al12 reported no significant difference of new bone formation in the sinus between a nonabsorbable or absorbable barrier membrane. However, the nonbarrier membrane group showed less new bone formation than did the barrier membrane group. As an alternative to the absorbable or nonabsorbable barrier membrane, a homologous bony window, detached from the lateral wall of the sinus cavity when preparing access to the sinus cavity, has been used to seal the lateral window of the sinus after sinus augmentation with/without bone grafting.3,4,13–17 Sohn et al13,16 first demonstrated that the replaceable bony window, unlike the
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collagen membrane, revealed osteoinductive efficacy during the healing period in the sinus, in animal studies. New bone formation was observed on the floor of the replaced bony window in the experimental group after 1 week. However, new bone formation was not observed under the collagen membrane in the control group after 1 week.13 The replaceable bony window, as a homologous barrier, revealed numerous positive effects on new bone formation in the sinus.13,16 The replaceable bony window prevented soft tissue ingrowth into the grafted sinus and, histologically, significantly higher and faster new bone formation was achieved in the experimental groups using the replaceable bony window than in the control group using the collagen membrane.13,16 In addition, the replaceable bony window has no risk of cross-contamination, unlike human- or animal-origin membranes. The results of this study correspond with those of previous studies.13,16 In this study, rabbits’ maxillary sinuses, known to be similar to humans’ sinuses, were used to verify the mechanism of new bone formation and to compare the effect on bone reformation between a replaceable bony window and a collagen membrane over the bone graft in the sinus.18 Many studies used rabbits’ maxillary sinuses to evaluate new bone formation using variable bone substitutes in the sinuses.19–22 In our study, when the homologous replaceable bony window was applied over the bone substitute, significantly higher new bone formation was revealed during the experimental period, compared with that in the control group receiving the collagen membrane. In addition, because of the osteoinductive function of the repositioned bony window, which showed new bone formation along the floor of the replaced bony window, the ratio of mature lamellar bone to the area of newly formed woven bone increased significantly in the experimental groups at 4 and 8 weeks, compared with that of the control group in which collagen membrane was used. The collagen membrane did not show any osteoinductive function during the experimental period.
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COMPARATIVE HISTOMORPHOMETRIC ANALYSIS
Bone remodeling is a synchronized and complex process of bone matrix formation by osteoblasts and absorption of bone osteoclasts.23 To evaluate osteoclast activity on bone remodeling, the number of TRAP-stained osteoclasts was calculated in this study. The presence of the enzyme, TRAP, is a generallyacknowledged cytochemical characteristic of osteoclasts and their mononuclear precursors.24,25 Many TRAP-stained osteoclasts were seen on the replaced bony windows, the elevated sinus membranes, and the lateral wall of the maxillary sinuses, revealed early in the healing period, indicating active new bone formation in the experimental groups. However, TRAP-stained osteoclasts were not seen along the collagen membrane, indicating no bone regeneration in the control group early in the healing period. Some were seen along the elevated sinus membrane and around the lateral wall of the maxillary sinuses early in the healing period. Elevated sinus membranes and the exposed lateral walls of the sinus cavities in both groups showed osteoinductive efficacy in this study, as in previous studies.13,16
CONCLUSION According to this study using histomorphometric analysis, we conclude that new bone formation is faster and denser in the experimental groups receiving the replaceable bony windows than in the control group receiving a collagen membrane over the bone graft. The replaceable bony window has osteoinductive potential, and accelerates new bone formation early in the healing period, compared with the collagen membrane over the bone graft.
DISCLOSURE The authors claim to have no financial interest, either directly or indirectly, in the products or information listed in the article.
ACKNOWLEDGMENTS This work was supported by the grant of Research Institute of Medical
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Science, Catholic University of Daegu (2008).
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