Experimental Evaluation of the Effects of Ankaferd Blood Stopper and Collagenated Heterologous Bone Graft on Bone Healing in Sinus Floor Augmentation Merve Cakir, DDS1/İnci Rana Karaca, Prof Dr Med Dent2/Ayşegül Firat, Dr Med3/ Figen Kaymaz, Prof Dr Med4/Suleyman Bozkaya, DDS5 Purpose: The aim of this study was to evaluate the effect of collagenated heterologous bone graft (CHBG) and Ankaferd Blood Stopper (ABS), a plant extract, on bone healing after sinus floor augmentation. Materials and Methods: Thirty-six New Zealand rabbits were used. Bilateral sinus augmentation was performed, and 72 bone defects were created. The maxillary sinuses were grafted with four different biomaterials: blood clot (control group), CHBG (Apatos Mix, OsteoBiol, Tecnoss) (graft group), ABS (ABS group), and ABS + CHBG (ABS+graft group). The rabbits were sacrificed at 1, 4, and 8 weeks after surgery. Histochemical and immunohistochemical examinations were performed on all samples. Staining with hematoxylin-eosin and Masson trichrome was performed, and bone marker activity was evaluated. Results: Lymphocyte infiltration was high at the first week in all groups and decreased from 1 to 8 weeks. All materials were biocompatible. Osteoclast numbers increased in the control group from 1 to 8 weeks and decreased in the other groups. There was no new bone formation in week 1 in all groups. New bone formation increased in all groups from 1 to 8 weeks, and at the fourth week, new bone formation was greater in the ABS and ABS+graft groups than in the other groups. There were osteoclasts around the bone graft materials, but degeneration of the graft was seen only in the ABS+graft group at week 8. Conclusion: ABS accelerates bone healing in sinus augmentation procedures and can be used alone or with CHBG. CHBG has osteoconductive properties, and ABS can accelerate bone graft degeneration. Int J Oral Maxillofac Implants 2015;30:279–285. doi: 10.11607/jomi.3793 Key words: Ankaferd Blood Stopper, bone healing, collagenated heterologous bone graft, sinus augmentation, tartrate-resistant acid phosphatase
M
issing teeth may result in functional and cosmetic deficiencies and traditionally have been treated with dentures, either complete or partial.1 Dental
1 Assistant
Professor, Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Istanbul Yeni Yuzyil University, Istanbul, Turkey. 2 Assistant Professor, Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Gazi University, Ankara, Turkey. 3Medical Doctor, Department of Anatomy, Faculty of Medicine, Hacettepe University, Ankara, Turkey. 4Department of Histology and Embryology, Faculty of Medicine, Hacettepe University, Ankara, Turkey. 5Assistant Professor, Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Gazi University, Ankara, Turkey. This material was presented as poster material at the AAOMS 96th Annual Meeting, September 8–13, 2014, Honolulu, Hawaii, USA. Correspondence to: Dr Merve Cakir, Faculty of Dentistry, Istanbul Yeni Yuzyil University, Dr. Azmi Ofluoglu Yerleskesi Yılanlı Ayazma Cd. No:26 Cevizlibag, Istanbul, Turkey. Email: [email protected] ©2015 by Quintessence Publishing Co Inc.
implants offer an alternative and have been used successfully to treat both partially and completely edentulous patients.1,2 However, placement of dental implants in edentulous arches is frequently limited by insufficient bone volume.3 The use of dental implants is more difficult in the edentulous maxilla than in the edentulous mandible because of various complicating factors. These include limited bone volume as a result of maxillary sinus pneumatization and alveolar resorption after tooth loss, poor bone quality (thin cortical bone and low-density trabecular bone), difficulty in accessing the region, and limited visibility.4–7 Many therapeutic options are available for the reconstruction of the edentulous posterior maxilla. Distal cantilevers, short implants, tilted implants, zygomatic implants, and maxillary sinus augmentation are some of them.5,6,8,9 Maxillary sinus augmentation has frequently been proposed as the best option for attaining sufficient bone height and volume for implant placement in the posterior maxilla.10–12 Since Boyne and James13 first introduced the procedure, several grafting materials have been used for augmentation, including autogenous grafts, allografts, The International Journal of Oral & Maxillofacial Implants 279
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xenografts, bone substitutes, or combinations of any of these.12,14,15 Autogenous grafts are often reported as the “gold standard” because they have osteogenic, osteoinductive, and osteoconductive properties.16,17 However, autogenous grafts have some disadvantages, including limited availability, a tendency to resorb, the need for additional surgery, and increased morbidity.17–19 These disadvantages have led researchers to seek out alternative graft materials.20 Recent studies showed higher implant survival/success rates with xenografts than with autogenous grafts.17 Deproteinized xenografts are safe and biocompatible and have osteoconductive properties.20 The most frequently used deproteinized bone graft is bovine bone. Its slower resorption has a beneficial effect on long-term preservation of the volume of regenerated bone, especially in large defects such as pneumatized maxillary sinuses.21,22 In addition to deproteinized bovine bone, a collagenated heterologous bone graft (CHBG) has been used recently in bone regeneration procedures.17,18,21,23 It is similar to human bone in that it is osteoconductive and integrates well at host sites.17 Because the bone-forming capacity of bone substitutes is limited, researchers have sought means to enhance their osteoconductivity.21 Ankaferd Blood Stopper (ABS) (Ankaferd İlaç Kozmetik) is a medicinal plant extract that has been used as a hemostatic agent.24 ABS is a standardized extract of the plants Thymus vulgaris, Glycyrrhiza glabra, Vitis vinifera, Alpinia officinarum, and Urtica dioica. Each of these plants has some effect on the endothelium, blood cells, angiogenesis, cellular proliferation, vascular dynamics, and cell mediators.24–26 Recent studies investigated the effect of ABS on bone healing26 and found that ABS has positive effects on bone healing and inflammation. The aim of the present study was to evaluate the effect of CHBG and ABS alone or together on bone healing in sinus augmentation procedures. To the authors’ knowledge, this is the first study of the effects of ABS on bone healing in sinus augmentation procedures.
MATERIALS AND METHODS Study Design
Thirty-six mature New Zealand white rabbits (aged 3 to 3.5 months) were used for the experiments. The animal research was approved by the Gazi University Local Ethics Committee for Animal Experiments (G.Ü. B.30.2. GÜN.0.05.06.00/111-9172). Four experimental groups were designated according to the sinus graft materials: blood clot (control group), CHBG (Apatos Mix, Osteo Biol, Tecnoss) (graft group), ABS (ABS group), and ABS + CHBG (ABS+graft group). Both sinuses in each rabbit
were augmented with two different materials; 72 sinuses in 36 animals were augmented. Twelve rabbits each were sacrificed at 1, 4, and 8 weeks after surgery.
Surgical Procedures
The rabbits were anesthetized with 50 mg/kg ketamine hydrochloride (Ketasol 10%, Richter Pharma) and 5 mg/kg xylazine hydrochloride (Rompun 2%, Bayer). In addition, 0.5 mL articaine hydrochloride (Ultracain D-S, Sanofi Aventis) was injected subcutaneously into each surgical field to provide local anesthesia. The surgical area was shaved and disinfected with iodine. The nasal bone and nasoincisal suture line were exposed via a horizontal incision, and a mucoperiosteal flap was elevated. Two ovoid windows were created bilaterally with round steel and diamond burs with sterile saline solution cooling. The sinus membrane was carefully raised from the floor and the lateral walls of the sinus with special sinus elevators. The sinus space was augmented with blood clot, CHBG, ABS, or ABS+CHBG. Material selection was done according to the blocked randomization method. Equal doses of graft materials were used, and mixed homogenously. The bone windows were covered with resorbable collagen membrane (Evolution, OsteoBiol, Tecnoss). The wounds were sutured with 4/0 resorbable material (Pegasorb, Doğsan). Antibiotics (Baytril-K 5%, Bayer) were injected intramuscularly for 3 days to prevent postsurgical infection. The sinus augmentation procedures were done as follows. In the control group, about 3 mL of blood was taken from an ear vein, coagulated, and packed into the sinus space. In the ABS group, ABS and blood were mixed in equal doses and packed into the sinus space. In the graft group, the CHBG was mixed with blood and packed into the sinus space. For the ABS+graft group, CHBG, blood, and ABS were mixed in equal amounts and packed into the sinus space.
Tissue Preparation
The rabbits were sacrificed with intramuscular ketamine hydrochloride injection. Samples were dissected free, fixed in formaldehyde for 48 to 72 hours, and decalcified with 10% ethylenediaminetetraacetic acid for up to 3 weeks. The specimens were embedded in paraffin and sliced into sections approximately 5 μm thick. Each sample was stained with hematoxylin-eosin (H&E) and Masson trichrome. In addition, assays for tartrate-resistant acid phosphatase (TRAP) and osteocalcin were performed to determine osteoclast and osteoblast activity. Lymphocyte and osteoblast density was graded on a four-point scale (grade 0 = no cells; grade 1 ≤ 15 cells/field; grade 2 = 15 to 50 cells/field; grade 3 ≥ 50 cells/field) according to the protocol of Hirshberg et
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Cakir et al
KT
500 µm
Fig 1 Control group, week 8. Blood (arrows) and new bone trabeculae (KT) are apparent (H&E; ×25, HE).
50 µm
Fig 2 Control group, week 4. Osteoclasts (arrows) are present (TRAP; ×400).
Fig 3 (left) ABS group, week 8. New bone trabeculae (KT) are forming (H&E; ×25). KT
Fig 4 (below) ABS group, week 4. Osteoblasts (arrows) (indirect immunoperoxidase; ×630).
KT
500 µm
al.27 New bone length was measured with Image J (U.S. National Institutes of Health) in sections that had been stained with Masson trichrome.
Statistical Analysis
Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS, IBM) Statistics 20 Data Editor program. The nonparametric KruskalWallis test was performed to evaluate differences in the amount of new bone and number of osteoclasts. The significance of any differences in new bone and osteoclast numbers between groups was further evaluated using the nonparametric Mann-Whitney U test for paired comparison. A P value < .05 was considered to indicate statistical significance for the KruskalWallis test, and P < .0083 was considered statistically significant for the Mann-Whitney U test according to Bonferroni correction.
RESULTS The materials used in this study were biocompatible and did not elicit any foreign-body reaction. There was no new bone formation at the first week in any group.
20 µm
New bone formation started at the fourth week adjacent to the cortical bone walls, and by the eighth week it was seen in the center of the cavity in all groups. There were osteoclasts around the bone graft materials, but degeneration of the graft was seen only in the ABS+graft group at 4 and 8 weeks. • Control group: There was severe lymphocyte infiltration at the first week; by 4 and 8 weeks lymphocyte infiltration was slight. There was no bone formation at the first week; it increased gradually to 8 weeks (Fig 1). Osteoblast density increased between 1 and 8 weeks, and osteoclast numbers were high at 1 and 8 weeks (Fig 2). • ABS group: There was severe lymphocyte infiltration at the first week, but it decreased to 8 weeks. There was no bone formation in the first week; this increased gradually to 8 weeks (Fig 3). Osteoblast density was low in the first week; it increased at 4 weeks (Fig 4) and was similar at 4 and 8 weeks. Osteoclast numbers were high at 1 and 8 weeks but decreased at 4 weeks (Fig 5). • Graft group: There was severe lymphocyte infiltration during the first week; it decreased to 8 weeks. There was no bone formation at the first week; this The International Journal of Oral & Maxillofacial Implants 281
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KT
Fig 5 (left) ABS group, week 4. Osteoclasts (arrow) are also apparent (TRAP; ×400). Fig 6 (right) Graft group, week 8. Specimen shows new bone trabeculae (KT), graft material (stars), and new bone around graft particles (arrows) (H&E; ×25).
KT 50 µm
500 µm
50 µm
Fig 7 Graft group, week 4. Osteoclasts (arrows) (TRAP; ×400).
200 µm
Fig 8 ABS+graft group, week 8. New bone (stars) has formed at the border of the defect (arrows) (H&E; ×25).
increased gradually to 8 weeks (Fig 6). Osteoblast density increased between 1 and 8 weeks. Osteoclast numbers were high at the first week and declined thereafter (Fig 7). • ABS+graft group: There was severe lymphocyte infiltration at 1 and 4 weeks, and at 8 weeks, lymphocyte infiltration was slight. There was no bone formation at the first week; it increased gradually 1 to 8 weeks (Fig 8). Osteoblast density increased 1 to 8 weeks. Osteoclast numbers were high in the first week and declined to 8 weeks (Fig 9).
Specimen Content
New Bone At 1 and 8 weeks, there was no statistically significant difference between the control, ABS, graft, and ABS+graft groups. At the fourth week, there were statistically significant differences between groups (P = .013). The Mann-Whitney U test was performed for pairwise comparison, and a difference was found between the graft and ABS groups (P = .008): more new bone had formed in the ABS group than in the graft group. Number of Osteoclasts . At 1, 4, and 8 weeks, there were no statistically significant differences in the control, ABS, graft, and ABS+graft groups. ABS accelerated degeneration of the graft material, so the osteoclast numbers were higher in the ABS+graft group than in the other groups.
50 µm
Fig 9 ABS+graft group, week 4. Osteoclasts (arrows) (TRAP; ×400).
DISCUSSION Treatment of partially or totally edentulous patients with dental implants is done routinely all over the world.28 A sufficient amount of bone is needed to place dental implants. In the posterior maxilla, this may be complicated owing to insufficient bone volume and the presence of the maxillary sinuses.10 Several procedures and materials have been used to overcome this problem.5,8,9,14 Sinus floor augmentation has frequently been proposed as the best treatment option for achieving sufficient bone height and volume in the posterior maxilla.10–12 Several grafting materials have been used for augmentation of the maxillary sinus.12,14,15 In this study, CHBG was used. According to the literature, the hemostatic agent ABS has antimicrobial and antitumoral properties,25,29–31 and it also has positive effects on bone and soft tissue healing.26 In this study, the authors used ABS to determine its effects on bone healing in sinus augmentation procedures, both alone and with CHBG. In experimental studies, the species of animal used is important.32 Goats, dogs, and monkeys have been used for sinus floor augmentation experiments.12,32 However, the use of these animals is expensive, inasmuch as multiple experiments are needed for statistical analysis.32 Rabbits can also be used for experimental studies of sinus floor augmentation.20,32,33
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They have a maxillary sinus with a well-defined ostium similar to that of humans. The rabbit antral membrane has many serous glands, which are useful for the detection of membrane perforation; the rabbit’s metabolic rate is faster than that of humans; and healing in rabbits is three times faster than in humans, while physiologic bone healing is similar. Among the other benefits of rabbits are their lower cost and their simple breeding and anesthesia.20,32–38 Because of these advantages, rabbits were used in this experimental study. In experimental studies of sinus floor augmentation, different healing intervals have been employed.32–34,37,39 A 1-week postsurgery interval was chosen to observe the early effects of the employed materials on bone healing and inflammation. In previous studies, significant differences were seen in bone healing from 4 to 8 weeks, so the 4-week interval was chosen in this study as the second healing period. In two-stage sinus floor augmentation procedures, implant placement is often performed 6 to 8 months after the first operation.9,40–43 Eight weeks in rabbits is comparable to 6 to 8 months in humans35,38; therefore, this study was terminated at 8 weeks. Host reactions following implantation of a biomaterial, prosthesis, or medical device are similar to reactions after injury of organs or tissues.44,45 These reactions are: blood-biomaterial interactions, provisional matrix formation, acute inflammation, chronic inflammation, granulation tissue development, foreign-body reactions, and fibrosis. Acute inflammation usually ends in 1 week. Chronic inflammation may then begin and is characterized by lymphocytes and mononuclear cells. Chronic inflammation also has been used to describe foreign-body reactions. With biocompatible materials, both chronic and acute inflammation will end in a short time.44 In this study, lymphocyte density was used as a measure of inflammation and biocompatibility. In all groups, severe lymphocyte infiltration was seen after 1 week. This is because the acute inflammation reaction had not ended by the first week and is consistent with the results of Xu et al20 and Nannmark and Sennerby.46 From 1 to 8 weeks, lymphocyte infiltration was reduced in all groups, but at the end of the 8-week test period, there was still mild to moderate lymphocyte infiltration, which suggested that the materials used have a minimal irritative potential. In previous studies, ABS reduced inflammation,26 but in this study, ABS did not reduce lymphocyte infiltration; this could be the result of differences in surgical procedures and in the surgical area. The biomaterials used in this study did not cause any foreign-body reaction, in agreement with previous clinical and experimental studies.17,18,20,26,33,39,46
Osteoclasts contain TRAP, which is used as a histochemical and biochemical marker for osteoclasts and bone resorption.47,48 In this study, TRAP was used as a marker of bone resorption (ie, osteoclast activity). In contrast to the results of Xu et al,33 osteoclast numbers increased from 1 to 8 weeks in the control group. A lack of stable vital bone and graft material in the sinus cavity, which resists positive air pressure, might have led to increased osteoclast numbers. This result was consistent with those of Asai et al.49 In the ABS, graft, and ABS+graft groups, the numbers of osteoclasts decreased from 1 to 8 weeks, consistent with the studies of Xu et al.33,37 Ideal bone grafts should eventually be absorbed, encourage new bone formation, and be replaced by newly regenerated bone.20,50 Slow resorption of the biomaterials used in sinus floor augmentation may play a role in space maintenance, and rapidly progressing degradation would endanger implant stability, so degeneration of the biomaterials should take an appropriate amount of time.51,52 In the present study, slow degeneration of the CHBG particles was observed in the graft-only group. On the other hand, in the ABS+graft group, resorption of the graft particles was seen at 8 weeks, which was similar to the results of studies in which autogenous bone and xenograft were used together.10,46,53 This result suggests that ABS may accelerate graft degeneration to the appropriate pace. Osteoblasts are bone-forming cells that are responsible for the synthesis, transport, and regulation of the organic matrix.54 In the present study, osteoblast density was evaluated by scoring, and the amount of the new bone was measured in microns. There are few studies26,55 of the effects of ABS on bone healing. One study26 concluded that ABS has positive effects on bone healing, whereas another study55 reported that ABS had no effects on bone healing. In the present study, in the ABS and the ABS+graft groups, new bone formation was rapid from 1 to 4 weeks, but by the end of the eighth week, new bone formation was similar in all groups. In all groups, new bone formation was increased from 1 to 8 weeks. According to these results, it was concluded that ABS may accelerate bone healing. In previous studies, newly formed bone was first seen adjacent to cortical bone walls. Over time, newly formed bone was observed in the center of the cavity20,21,39,56 and osteoblasts were localized with new woven bone around the graft particles and under the sinus membrane.10,18,23,32,33 The present results correlate with these findings.10,18,20,21,23,32,33,39,56
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CONCLUSION To the authors’ knowledge, the present study is the first to examine the effects of Ankaferd Blood Stopper (ABS) on bone healing in sinus augmentation procedures. It is concluded that ABS and collagenated heterologous bone graft are biocompatible. Collagenated heterologous bone graft exhibits osteoconductive properties, both with ABS and alone. ABS accelerates bone healing and can be used alone or with collagenated heterologous bone graft in sinus floor augmentation procedures. Further investigations are needed to better understand effects of ABS on bone healing.
ACKNOWLEDGMENTS This study was supported by the Gazi University Research Projects Unit (no. 03/2011-21). The statistical analysis was performed by Dinçer Göksüllük. The authors reported no conflicts of interest related to this study.
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35. Choi Y, Yun JH, Kim CS, Choi SH, Chai JK, Jung UW. Sinus augmentation using absorbable collagen sponge loaded with Escherichia coli-expressed recombinant human bone morphogenetic protein 2 in a standardized rabbit sinus model: A radiographic and histologic analysis. Clin Oral Implants Res 2012;23:682–689. 36. Wang X, Mabrey JD, Agrawal CM. An interspecies comparison of bone fracture properties. Biomedical Mater Eng 1998;8:1–9. 37. Xu H, Shimizu Y, Asai S, Ooya K. Grafting of deproteinized bone particles inhibits bone resorption after maxillary sinus floor elevation. Clin Oral Implants Res 2004;15:126–133. 38. Pripatnanont P, Nuntanaranont T, Vongvatcharanon S. Proportion of deproteinized bovine bone and autogenous bone affects bone formation in the treatment of calvarial defects in rabbits. Int J Oral Maxillofac Surg 2009;38:356–362. 39. Xu H, Shimizu Y, Asai S, Ooya K. Experimental sinus grafting with the use of deproteinized bone particles of different sizes. Clin Oral Implants Res 2003;14:548–555. 40. Katsuyama H, Jensen SS. ITI Treatment Guide: Sinüs Tabanı Yükseltme Işlemleri. Berlin: Quintessence, 2012. 41. van den Bergh JP, ten Bruggenkate CM, Disch FJ, Tuinzing DB. Anatomical aspects of sinus floor elevations. Clin Oral Implants Res 2000;11:256–265. 42. Woo I, Le BT. Maxillary sinus floor elevation: Review of anatomy and two techniques. Implant Dent 2004;13:28–32. 43. Zitzmann NU, Scharer P. Sinus elevation procedures in the resorbed posterior maxilla. Comparison of the crestal and lateral approaches. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1998;85:8–17. 44. Anderson JM, Rodriguez A, Chang DT. Foreign body reaction to biomaterials. Semin Immunol 2008;20:86–100. 45. Anderson MJ. Biological responses to materials. Annu Rev Mater Res 2001;31:81–110. 46. Nannmark U, Sennerby L. The bone tissue responses to prehydrated and collagenated cortico-cancellous porcine bone grafts: A study in rabbit maxillary defects. Clin Implant Dent Relate Res 2008;10:264–270.
47. Ek-Rylander B, Bill P, Norgard M, Nilsson S, Andersson G. Cloning, sequence, and developmental expression of a type 5, tartrateresistant, acid phosphatase of rat bone. J Biol Chem 1991;266: 24684–24689. 48. Fernandez-Tresguerres-Hernandez-Gil I, Alobera-Gracia MA, delCanto-Pingarron M, Blanco-Jerez L. Physiological bases of bone regeneration I. Histology and physiology of bone tissue. Med Oral Patol Oral Cirug Bucal 2006;11:E47–51. 49. 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. 50. Yazdi FK, Mostaghni E, Moghadam SA, Faghihi S, Monabati A, Amid R. A comparison of the healing capabilities of various grafting materials in critical-size defects in Guinea pig calvaria. Int J Oral Maxillofac Implants 2013;28:1370–1376. 51. Kim YS, Kim SH, Kim KH, et al. Rabbit maxillary sinus augmentation model with simultaneous implant placement: Differential responses to the graft materials. J Periodontal Implant Sci 2012;42:204–211. 52. Yildirim M, Spiekermann H, Biesterfeld S, Edelhoff D. Maxillary sinus augmentation using xenogenic bone substitute material Bio-Oss in combination with venous blood. A histologic and histomorphometric study in humans. Clin Oral Implants Res 2000;11:217–229. 53. Tadjoedin ES, de Lange GL, Bronckers AL, Lyaruu DM, Burger EH. Deproteinized cancellous bovine bone (Bio-Oss) as bone substitute for sinus floor elevation. A retrospective, histomorphometrical study of five cases. J Clin Periodontol 2003;30:261–270. 54. Gartner LP, Hiatt J. Color Textbook of Histology, ed 3. Philadelphia: Saunders, 2007. 55. Günay M, Amanvermez R, Keleş G. Ankaferd Blood Stopper: Does it have a role in fracture healing? Turk J Med Sci 2013;43:733–738. 56. Okazaki K, Shimizu Y, Xu H, Ooya K. Blood-filled spaces with and without deproteinized bone grafts in guided bone regeneration. A histomorphometric study of the rabbit skull using non-resorbable membrane. Clin Oral Implants Res 2005;16:236–243.
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M
issing teeth may result in functional and cosmetic deficiencies and traditionally have been treated with dentures, either complete or partial.1 Dental
1 Assistant
Professor, Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Istanbul Yeni Yuzyil University, Istanbul, Turkey. 2 Assistant Professor, Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Gazi University, Ankara, Turkey. 3Medical Doctor, Department of Anatomy, Faculty of Medicine, Hacettepe University, Ankara, Turkey. 4Department of Histology and Embryology, Faculty of Medicine, Hacettepe University, Ankara, Turkey. 5Assistant Professor, Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Gazi University, Ankara, Turkey. This material was presented as poster material at the AAOMS 96th Annual Meeting, September 8–13, 2014, Honolulu, Hawaii, USA. Correspondence to: Dr Merve Cakir, Faculty of Dentistry, Istanbul Yeni Yuzyil University, Dr. Azmi Ofluoglu Yerleskesi Yılanlı Ayazma Cd. No:26 Cevizlibag, Istanbul, Turkey. Email: [email protected] ©2015 by Quintessence Publishing Co Inc.
implants offer an alternative and have been used successfully to treat both partially and completely edentulous patients.1,2 However, placement of dental implants in edentulous arches is frequently limited by insufficient bone volume.3 The use of dental implants is more difficult in the edentulous maxilla than in the edentulous mandible because of various complicating factors. These include limited bone volume as a result of maxillary sinus pneumatization and alveolar resorption after tooth loss, poor bone quality (thin cortical bone and low-density trabecular bone), difficulty in accessing the region, and limited visibility.4–7 Many therapeutic options are available for the reconstruction of the edentulous posterior maxilla. Distal cantilevers, short implants, tilted implants, zygomatic implants, and maxillary sinus augmentation are some of them.5,6,8,9 Maxillary sinus augmentation has frequently been proposed as the best option for attaining sufficient bone height and volume for implant placement in the posterior maxilla.10–12 Since Boyne and James13 first introduced the procedure, several grafting materials have been used for augmentation, including autogenous grafts, allografts, The International Journal of Oral & Maxillofacial Implants 279
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Cakir et al
xenografts, bone substitutes, or combinations of any of these.12,14,15 Autogenous grafts are often reported as the “gold standard” because they have osteogenic, osteoinductive, and osteoconductive properties.16,17 However, autogenous grafts have some disadvantages, including limited availability, a tendency to resorb, the need for additional surgery, and increased morbidity.17–19 These disadvantages have led researchers to seek out alternative graft materials.20 Recent studies showed higher implant survival/success rates with xenografts than with autogenous grafts.17 Deproteinized xenografts are safe and biocompatible and have osteoconductive properties.20 The most frequently used deproteinized bone graft is bovine bone. Its slower resorption has a beneficial effect on long-term preservation of the volume of regenerated bone, especially in large defects such as pneumatized maxillary sinuses.21,22 In addition to deproteinized bovine bone, a collagenated heterologous bone graft (CHBG) has been used recently in bone regeneration procedures.17,18,21,23 It is similar to human bone in that it is osteoconductive and integrates well at host sites.17 Because the bone-forming capacity of bone substitutes is limited, researchers have sought means to enhance their osteoconductivity.21 Ankaferd Blood Stopper (ABS) (Ankaferd İlaç Kozmetik) is a medicinal plant extract that has been used as a hemostatic agent.24 ABS is a standardized extract of the plants Thymus vulgaris, Glycyrrhiza glabra, Vitis vinifera, Alpinia officinarum, and Urtica dioica. Each of these plants has some effect on the endothelium, blood cells, angiogenesis, cellular proliferation, vascular dynamics, and cell mediators.24–26 Recent studies investigated the effect of ABS on bone healing26 and found that ABS has positive effects on bone healing and inflammation. The aim of the present study was to evaluate the effect of CHBG and ABS alone or together on bone healing in sinus augmentation procedures. To the authors’ knowledge, this is the first study of the effects of ABS on bone healing in sinus augmentation procedures.
MATERIALS AND METHODS Study Design
Thirty-six mature New Zealand white rabbits (aged 3 to 3.5 months) were used for the experiments. The animal research was approved by the Gazi University Local Ethics Committee for Animal Experiments (G.Ü. B.30.2. GÜN.0.05.06.00/111-9172). Four experimental groups were designated according to the sinus graft materials: blood clot (control group), CHBG (Apatos Mix, Osteo Biol, Tecnoss) (graft group), ABS (ABS group), and ABS + CHBG (ABS+graft group). Both sinuses in each rabbit
were augmented with two different materials; 72 sinuses in 36 animals were augmented. Twelve rabbits each were sacrificed at 1, 4, and 8 weeks after surgery.
Surgical Procedures
The rabbits were anesthetized with 50 mg/kg ketamine hydrochloride (Ketasol 10%, Richter Pharma) and 5 mg/kg xylazine hydrochloride (Rompun 2%, Bayer). In addition, 0.5 mL articaine hydrochloride (Ultracain D-S, Sanofi Aventis) was injected subcutaneously into each surgical field to provide local anesthesia. The surgical area was shaved and disinfected with iodine. The nasal bone and nasoincisal suture line were exposed via a horizontal incision, and a mucoperiosteal flap was elevated. Two ovoid windows were created bilaterally with round steel and diamond burs with sterile saline solution cooling. The sinus membrane was carefully raised from the floor and the lateral walls of the sinus with special sinus elevators. The sinus space was augmented with blood clot, CHBG, ABS, or ABS+CHBG. Material selection was done according to the blocked randomization method. Equal doses of graft materials were used, and mixed homogenously. The bone windows were covered with resorbable collagen membrane (Evolution, OsteoBiol, Tecnoss). The wounds were sutured with 4/0 resorbable material (Pegasorb, Doğsan). Antibiotics (Baytril-K 5%, Bayer) were injected intramuscularly for 3 days to prevent postsurgical infection. The sinus augmentation procedures were done as follows. In the control group, about 3 mL of blood was taken from an ear vein, coagulated, and packed into the sinus space. In the ABS group, ABS and blood were mixed in equal doses and packed into the sinus space. In the graft group, the CHBG was mixed with blood and packed into the sinus space. For the ABS+graft group, CHBG, blood, and ABS were mixed in equal amounts and packed into the sinus space.
Tissue Preparation
The rabbits were sacrificed with intramuscular ketamine hydrochloride injection. Samples were dissected free, fixed in formaldehyde for 48 to 72 hours, and decalcified with 10% ethylenediaminetetraacetic acid for up to 3 weeks. The specimens were embedded in paraffin and sliced into sections approximately 5 μm thick. Each sample was stained with hematoxylin-eosin (H&E) and Masson trichrome. In addition, assays for tartrate-resistant acid phosphatase (TRAP) and osteocalcin were performed to determine osteoclast and osteoblast activity. Lymphocyte and osteoblast density was graded on a four-point scale (grade 0 = no cells; grade 1 ≤ 15 cells/field; grade 2 = 15 to 50 cells/field; grade 3 ≥ 50 cells/field) according to the protocol of Hirshberg et
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Cakir et al
KT
500 µm
Fig 1 Control group, week 8. Blood (arrows) and new bone trabeculae (KT) are apparent (H&E; ×25, HE).
50 µm
Fig 2 Control group, week 4. Osteoclasts (arrows) are present (TRAP; ×400).
Fig 3 (left) ABS group, week 8. New bone trabeculae (KT) are forming (H&E; ×25). KT
Fig 4 (below) ABS group, week 4. Osteoblasts (arrows) (indirect immunoperoxidase; ×630).
KT
500 µm
al.27 New bone length was measured with Image J (U.S. National Institutes of Health) in sections that had been stained with Masson trichrome.
Statistical Analysis
Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS, IBM) Statistics 20 Data Editor program. The nonparametric KruskalWallis test was performed to evaluate differences in the amount of new bone and number of osteoclasts. The significance of any differences in new bone and osteoclast numbers between groups was further evaluated using the nonparametric Mann-Whitney U test for paired comparison. A P value < .05 was considered to indicate statistical significance for the KruskalWallis test, and P < .0083 was considered statistically significant for the Mann-Whitney U test according to Bonferroni correction.
RESULTS The materials used in this study were biocompatible and did not elicit any foreign-body reaction. There was no new bone formation at the first week in any group.
20 µm
New bone formation started at the fourth week adjacent to the cortical bone walls, and by the eighth week it was seen in the center of the cavity in all groups. There were osteoclasts around the bone graft materials, but degeneration of the graft was seen only in the ABS+graft group at 4 and 8 weeks. • Control group: There was severe lymphocyte infiltration at the first week; by 4 and 8 weeks lymphocyte infiltration was slight. There was no bone formation at the first week; it increased gradually to 8 weeks (Fig 1). Osteoblast density increased between 1 and 8 weeks, and osteoclast numbers were high at 1 and 8 weeks (Fig 2). • ABS group: There was severe lymphocyte infiltration at the first week, but it decreased to 8 weeks. There was no bone formation in the first week; this increased gradually to 8 weeks (Fig 3). Osteoblast density was low in the first week; it increased at 4 weeks (Fig 4) and was similar at 4 and 8 weeks. Osteoclast numbers were high at 1 and 8 weeks but decreased at 4 weeks (Fig 5). • Graft group: There was severe lymphocyte infiltration during the first week; it decreased to 8 weeks. There was no bone formation at the first week; this The International Journal of Oral & Maxillofacial Implants 281
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Cakir et al
KT
Fig 5 (left) ABS group, week 4. Osteoclasts (arrow) are also apparent (TRAP; ×400). Fig 6 (right) Graft group, week 8. Specimen shows new bone trabeculae (KT), graft material (stars), and new bone around graft particles (arrows) (H&E; ×25).
KT 50 µm
500 µm
50 µm
Fig 7 Graft group, week 4. Osteoclasts (arrows) (TRAP; ×400).
200 µm
Fig 8 ABS+graft group, week 8. New bone (stars) has formed at the border of the defect (arrows) (H&E; ×25).
increased gradually to 8 weeks (Fig 6). Osteoblast density increased between 1 and 8 weeks. Osteoclast numbers were high at the first week and declined thereafter (Fig 7). • ABS+graft group: There was severe lymphocyte infiltration at 1 and 4 weeks, and at 8 weeks, lymphocyte infiltration was slight. There was no bone formation at the first week; it increased gradually 1 to 8 weeks (Fig 8). Osteoblast density increased 1 to 8 weeks. Osteoclast numbers were high in the first week and declined to 8 weeks (Fig 9).
Specimen Content
New Bone At 1 and 8 weeks, there was no statistically significant difference between the control, ABS, graft, and ABS+graft groups. At the fourth week, there were statistically significant differences between groups (P = .013). The Mann-Whitney U test was performed for pairwise comparison, and a difference was found between the graft and ABS groups (P = .008): more new bone had formed in the ABS group than in the graft group. Number of Osteoclasts . At 1, 4, and 8 weeks, there were no statistically significant differences in the control, ABS, graft, and ABS+graft groups. ABS accelerated degeneration of the graft material, so the osteoclast numbers were higher in the ABS+graft group than in the other groups.
50 µm
Fig 9 ABS+graft group, week 4. Osteoclasts (arrows) (TRAP; ×400).
DISCUSSION Treatment of partially or totally edentulous patients with dental implants is done routinely all over the world.28 A sufficient amount of bone is needed to place dental implants. In the posterior maxilla, this may be complicated owing to insufficient bone volume and the presence of the maxillary sinuses.10 Several procedures and materials have been used to overcome this problem.5,8,9,14 Sinus floor augmentation has frequently been proposed as the best treatment option for achieving sufficient bone height and volume in the posterior maxilla.10–12 Several grafting materials have been used for augmentation of the maxillary sinus.12,14,15 In this study, CHBG was used. According to the literature, the hemostatic agent ABS has antimicrobial and antitumoral properties,25,29–31 and it also has positive effects on bone and soft tissue healing.26 In this study, the authors used ABS to determine its effects on bone healing in sinus augmentation procedures, both alone and with CHBG. In experimental studies, the species of animal used is important.32 Goats, dogs, and monkeys have been used for sinus floor augmentation experiments.12,32 However, the use of these animals is expensive, inasmuch as multiple experiments are needed for statistical analysis.32 Rabbits can also be used for experimental studies of sinus floor augmentation.20,32,33
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Cakir et al
They have a maxillary sinus with a well-defined ostium similar to that of humans. The rabbit antral membrane has many serous glands, which are useful for the detection of membrane perforation; the rabbit’s metabolic rate is faster than that of humans; and healing in rabbits is three times faster than in humans, while physiologic bone healing is similar. Among the other benefits of rabbits are their lower cost and their simple breeding and anesthesia.20,32–38 Because of these advantages, rabbits were used in this experimental study. In experimental studies of sinus floor augmentation, different healing intervals have been employed.32–34,37,39 A 1-week postsurgery interval was chosen to observe the early effects of the employed materials on bone healing and inflammation. In previous studies, significant differences were seen in bone healing from 4 to 8 weeks, so the 4-week interval was chosen in this study as the second healing period. In two-stage sinus floor augmentation procedures, implant placement is often performed 6 to 8 months after the first operation.9,40–43 Eight weeks in rabbits is comparable to 6 to 8 months in humans35,38; therefore, this study was terminated at 8 weeks. Host reactions following implantation of a biomaterial, prosthesis, or medical device are similar to reactions after injury of organs or tissues.44,45 These reactions are: blood-biomaterial interactions, provisional matrix formation, acute inflammation, chronic inflammation, granulation tissue development, foreign-body reactions, and fibrosis. Acute inflammation usually ends in 1 week. Chronic inflammation may then begin and is characterized by lymphocytes and mononuclear cells. Chronic inflammation also has been used to describe foreign-body reactions. With biocompatible materials, both chronic and acute inflammation will end in a short time.44 In this study, lymphocyte density was used as a measure of inflammation and biocompatibility. In all groups, severe lymphocyte infiltration was seen after 1 week. This is because the acute inflammation reaction had not ended by the first week and is consistent with the results of Xu et al20 and Nannmark and Sennerby.46 From 1 to 8 weeks, lymphocyte infiltration was reduced in all groups, but at the end of the 8-week test period, there was still mild to moderate lymphocyte infiltration, which suggested that the materials used have a minimal irritative potential. In previous studies, ABS reduced inflammation,26 but in this study, ABS did not reduce lymphocyte infiltration; this could be the result of differences in surgical procedures and in the surgical area. The biomaterials used in this study did not cause any foreign-body reaction, in agreement with previous clinical and experimental studies.17,18,20,26,33,39,46
Osteoclasts contain TRAP, which is used as a histochemical and biochemical marker for osteoclasts and bone resorption.47,48 In this study, TRAP was used as a marker of bone resorption (ie, osteoclast activity). In contrast to the results of Xu et al,33 osteoclast numbers increased from 1 to 8 weeks in the control group. A lack of stable vital bone and graft material in the sinus cavity, which resists positive air pressure, might have led to increased osteoclast numbers. This result was consistent with those of Asai et al.49 In the ABS, graft, and ABS+graft groups, the numbers of osteoclasts decreased from 1 to 8 weeks, consistent with the studies of Xu et al.33,37 Ideal bone grafts should eventually be absorbed, encourage new bone formation, and be replaced by newly regenerated bone.20,50 Slow resorption of the biomaterials used in sinus floor augmentation may play a role in space maintenance, and rapidly progressing degradation would endanger implant stability, so degeneration of the biomaterials should take an appropriate amount of time.51,52 In the present study, slow degeneration of the CHBG particles was observed in the graft-only group. On the other hand, in the ABS+graft group, resorption of the graft particles was seen at 8 weeks, which was similar to the results of studies in which autogenous bone and xenograft were used together.10,46,53 This result suggests that ABS may accelerate graft degeneration to the appropriate pace. Osteoblasts are bone-forming cells that are responsible for the synthesis, transport, and regulation of the organic matrix.54 In the present study, osteoblast density was evaluated by scoring, and the amount of the new bone was measured in microns. There are few studies26,55 of the effects of ABS on bone healing. One study26 concluded that ABS has positive effects on bone healing, whereas another study55 reported that ABS had no effects on bone healing. In the present study, in the ABS and the ABS+graft groups, new bone formation was rapid from 1 to 4 weeks, but by the end of the eighth week, new bone formation was similar in all groups. In all groups, new bone formation was increased from 1 to 8 weeks. According to these results, it was concluded that ABS may accelerate bone healing. In previous studies, newly formed bone was first seen adjacent to cortical bone walls. Over time, newly formed bone was observed in the center of the cavity20,21,39,56 and osteoblasts were localized with new woven bone around the graft particles and under the sinus membrane.10,18,23,32,33 The present results correlate with these findings.10,18,20,21,23,32,33,39,56
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Cakir et al
CONCLUSION To the authors’ knowledge, the present study is the first to examine the effects of Ankaferd Blood Stopper (ABS) on bone healing in sinus augmentation procedures. It is concluded that ABS and collagenated heterologous bone graft are biocompatible. Collagenated heterologous bone graft exhibits osteoconductive properties, both with ABS and alone. ABS accelerates bone healing and can be used alone or with collagenated heterologous bone graft in sinus floor augmentation procedures. Further investigations are needed to better understand effects of ABS on bone healing.
ACKNOWLEDGMENTS This study was supported by the Gazi University Research Projects Unit (no. 03/2011-21). The statistical analysis was performed by Dinçer Göksüllük. The authors reported no conflicts of interest related to this study.
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13. Boyne PJ, James RA. Grafting of the maxillary sinus floor with autogenous marrow and bone. J Oral Surg 1980;38:613–616. 14. Browaeys H, Bouvry P, De Bruyn H. A literature review on biomaterials in sinus augmentation procedures. Clin Implant Dent Relat Res 2007;9:166–177. 15. Emmerich D, Att W, Stappert C. Sinus floor elevation using osteotomes: A systematic review and meta-analysis. J Periodontol 2005; 76:1237–1251. 16. Giannoudis PV, Dinopoulos H, Tsiridis E. Bone substitutes: An update. Injury 2005;36(suppl 3):S20–27. 17. Scarano A, Piattelli A, Perrotti V, Manzon L, Iezzi G. Maxillary sinus augmentation in humans using cortical porcine bone: A histological and histomorphometrical evaluation after 4 and 6 months. Clin Implant Dent Relat Res 2011;13:13–18. 18. Orsini G, Scarano A, Piattelli M, Piccirilli M, Caputi S, Piattelli A. Histologic and ultrastructural analysis of regenerated bone in maxillary sinus augmentation using a porcine bone-derived biomaterial. J Periodontol 2006;77:1984–1990. 19. Pradel W, Eckelt U, Lauer G. Bone regeneration after enucleation of mandibular cysts: Comparing autogenous grafts from tissueengineered bone and iliac bone. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;101:285–290. 20. Xu H, Shimizu Y, Onodera K, Ooya K. Long-term outcome of augmentation of the maxillary sinus using deproteinised bone particles experimental study in rabbits. Br J Oral Maxillofac Surg 2005;43: 40–45. 21. Park JW, Ko HJ, Jang JH, Kang H, Suh JY. Increased new bone formation with a surface magnesium-incorporated deproteinized porcine bone substitute in rabbit calvarial defects. J Biomed Mater Res 2012; 100:834–840. 22. Stravropoulos A. Deproteinised bovine bone xenograft. In: Pietrzak WS (ed). Orthopedic Biology and Medicine: Musculoskeletal Tissue Regeneration, Biologic Materials and Methods. Totowa, NJ: Humana Press, 2008. 23. Scarano A, Piatelli A, Assenza B, et al. Porcine bone used in sinus augmentation procedures: A 5-year retrospective clinical evaluation. J Oral Maxillofac Surg 2010;68:1869–1873. 24. Goker H, Haznedaroglu IC, Ercetin S, et al. Haemostatic actions of the folkloric medicinal plant extract Ankaferd Blood Stopper. J Int Med Res 2008;36:163–170. 25. Fisgin N, Cayci Y, Coban AY, et al. Antimicrobial activity of plant extract Ankaferd Blood Stopper. Fitoterapia 2009;80:48–50. 26. Isler SC, Demircan S, Cakarer S, et al. Effects of folk medicinal plant extract Ankaferd Blood Stopper on early bone healing. J Appl Oral Sci 2010;18:409-414. 27. Hirshberg A, Lib M, Kozlovsky A, Kaplan I. The influence of inflammation on the polarization colors of collagen fibers in the wall of odontogenic keratocyst. Oral Oncol 2007;43:278–282. 28. Blomqvist JE, Alberius P, Isaksson S. Two-stage maxillary sinus reconstruction with endosseous implants: A prospective study. Int J Oral Maxillofac Implants 1998;13:758–766. 29. Akkoc N, Akcelik M, Haznedaroglu C, et al. In vitro antibacterial activities of Ankaferd Medical Plant Extract. J Med Sci 2009;29: 410-415. 30. Beyazit Y, Kurt M, Kekilli M, Goker H, Haznedaroglu IC. Evaluation of hemostatic effects of Ankaferd as an alternative medicine. Alt Med Rev 2010;15:329–336. 31. Tas A, Koklu S, Beyazit Y, et al. Percutaneous ankaferd injection to in vivo liver tissue in comparison to ethanol in an experimental rat model. Clin Res Hepatol Gastroenterol 2011;35:549–553. 32. 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. 33. Xu H, Shimizu Y, Ooya K. Histomorphometric study of the stability of newly formed bone after elevation of the floor of the maxillary sinus. Br J Oral Maxillofac Surg 2005;43:493–499. 34. Allegrini S Jr, Yoshimoto M, Salles MB, Konig B Jr. The effects of bovine BMP associated to HA in maxillary sinus lifting in rabbits. Ann Anat 2003;185:343–349.
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