IMPLANT DENTISTRY / VOLUME 23, NUMBER 2 2014
195
Evaluation of Simvastatin Grafting Around Immediate Dental Implants in Dogs Ghada Mansour, PhD,* Adham Al Ashwah, PhD,† and Azza Koura, PhD‡
he discrepancy between the diameters of the socket and that of the implant generates a gap between the walls of the socket and the fixture. In large bony defects, this void can be colonized by epithelial cells inducing fibrointegration, and implant failure.1 Guided bone regeneration (GBR), by the use of barrier membranes alone or in association with bone replacement graft, eliminates this problem and gives satisfactory results to achieve implant osseointegration.2 Nevertheless, none of these materials have shown to be ideal, and new materials are sought.3 The statins are commonly prescribed drugs used to reduce serum cholesterol concentrations and the risk of heart attack.4 Simvastatin, a member of statins, has been reported to promote osteoblastic differentiation in the human bone marrow stromal cells. It stimulates the alkaline phosphatase activity and enhances the expression of osteocalcin, which are early and late osteoblastic differentiation markers, respectively.5 Furthermore, simvastatin stimulates the expression of BMP-2,
T
*Professor, Department of Oral Diagnostic Sciences, Faculty of Dentistry, King Abdulaziz University, Jeddah, Saudi Arabia; Professor, Department of Oral Medicine, Periodontology, Oral Diagnosis and Oral Radiology, Faculty of Dentistry, Alexandria University, Alexandria, Egypt. †Associate Professor, Department of Oral Surgery, Faculty of Dentistry, Alexandria University, Alexandria, Egypt. ‡Associate Professor, Department of Oral Biology, Faculty of Dentistry, Alexandria University, Alexandria, Egypt.
Reprint requests and correspondence to: Ghada Mansour, PhD, Department of Oral Basic and Clinical Sciences, Faculty of Dentistry, King Abdulaziz University, 80209, Jeddah 21589, Saudi Arabia, Phone: +966-2-6403443, Fax: +966-2-6403316, E-mail: [email protected] ISSN 1056-6163/14/02302-195 Implant Dentistry Volume 23 Number 2 Copyright © 2014 by Lippincott Williams & Wilkins DOI: 10.1097/ID.0000000000000051
Introduction: Simvastatin (Zocor; MSD), a cholesterol-lowering drug, is used systemically in treatment of osteoporosis due to its boneforming potential. The present study was conducted to evaluate the regenerative potential of an optimized simvastatin formulation as a grafting material around immediate dental implants in experimental animals. Materials and Methods: The drug was formulated as granules in cellulosic polymeric matrix. Surgical extractions of left and right mandibular third premolars were performed in 10 dogs. The left side of the mandible was the study group, where Microdent (Implant Microdent System S.L-Comapedrosa, Barcelona, Spain) implants were immediately seated and simvastatin
granules were packed. The right side constituted the control group where only implants were placed. Five dogs were killed at 1 month and 5 at 3 months. The implants were removed and specimens were processed and stained with hematoxylin and eosin and trichrome stains. Results: Healing occurred in both groups, with better findings in simvastatin-filled defects, as evidenced by bone regeneration, with neovascularization. Conclusions: Simvastatin granules allowed for osteogenesis around immediate implants, resulting in their osseointegration. (Implant Dent 2014;23:195–199) Key Words: dental implants, statins, osseointegration, bone substitute
which causes mesenchymal cells to differentiate into bone or cartilage forming cells.6 In addition, simvastatin may have effects on the mevalonate pathway, leading to inhibition of osteoclast activity.7 The incorporation of simvastatin into hydrophobic matrices should be beneficial for local slow release at sites where new bone growth is required.8 However, injection of simvastatin into human-like periodontal defects produced unfavorable results, possibly because of the viscosity of the carrier or lack of space creation for new bone growth.9 The lack of an ideal carrier for simvastatin has markedly limited its topical use.10 To the best of our knowledge, there are no published data concerning the
use of simvastatin topically as an adjunctive material to immediate implants. Therefore, the present study was carried out to evaluate the regenerative potential of a recently developed optimized delivery device, using a single dose of simvastatin granules in cellulose polymeric matrix, as a grafting material around immediate dental implants in experimental animals, and to determine if it could enhance osseointegration of these implants.
MATERIALS
AND
METHODS
Experimental Animals
Ten adult systemically healthy mongrel dogs (Canis familiaris) were included in this study. Their age ranged from 18 to 24 months and their weight
196
EVALUATION
OF
SIMVASTATIN GRAFTING
from 12 to 15 kg. A split mouth design was used. The experimental sites were divided into 2 groups: study and control groups. Group 1 (study group) included 10 freshly extracted sockets at left mandibular third premolars, where immediate implants were placed with adjunctive topical application of (2.2 mg) simvastatin granules. Group 2 (control group) included 10 freshly extracted sockets at right mandibular third premolars, where only immediate implants were placed. All animal procedures were approved by the Animal Ethics Committee at the University of Alexandria.
MANSOUR
ET AL
Fig. 1. Immediate implant in place surrounded by simvastatin granules packed around the gap at the coronal portion of the socket.
Preparation of the Grafting Material
It was formulated as granules in cellulosic polymeric matrix: hydroxy propyl methyl cellulose (HPMC 4000 CP, Methocel; Alexandria Pharmaceutical Co., Alexandria, Egypt) in the Department of Pharmaceutics, Faculty of Pharmacy, Alexandria University. Granules were prepared to contain 2.2 mg of simvastatin per 150 mg total weight. The reported local simvastatin dose was 2.2 mg,11 and a preliminary clinical study revealed that 150-mg granules were suitable for surgical grafting.12 Weighed simvastatin/polymer blends were accurately mixed and wetted, forming a mass to be forced through a sieve. The obtained granules were sized, dried in a circulating air oven at 60°C, then subjected to vacuum, and stored in a desiccator until use. The wetting system was a 4:1 isopropyl alcohol/water mixture. The dried granules were subjected to particle size reduction by triturating and then sieved. The particle size ranging from 800 to 1000 mm was collected.
side, the implant was placed without simvastatin. The healing cap was secured on top of the fixture. The flap was sutured using 3-0 black silk interrupted sutures. Intramuscular antibiotic treatment using 15 mg/kg of tetracycline hydrochloride was continued for 48 hours after surgery and then mixed with dogs’ food for 7 days. A painkiller was also given the first day postoperatively. Animals were checked daily and fed with a soft diet postoperatively. Normal diet was resumed after 2 weeks. Five animals were humanely killed after 1 month and the other 5 at 3 months by iv injection of overdosed thiopental sodium. Sample Preparation
Fig. 2. Photomicrograph of a study group case after 1 month revealing a layer of connective tissue covering particles of simvastatin granules (arrows) (hematoxylin and eosin 3100).
bed with the long axis congruent to the long axis of the root being replaced, after drilling 3 to 4 mm beyond the apex. In the left mandibular premolars, simvastatin granules mixed with saliva were packed in direct contact with the implant (Fig. 1). On the contralateral
Tissue blocks, including bone, implants and surrounding soft tissues, were obtained after the animals were killed. These were fixed in 10% buffered neutral formalin for 2 weeks. They were then decalcified in 5% trichloroacetic acid for 4 to 6 weeks; the implants were removed, and blocks were processed to prepare paraffin sections. Serial sections (7 mm) were cut in a buccolingual direction. Every 14th section 100 mm apart was stained using hematoxylin and eosin and an adjacent section with Gomori trichrome stain. Sections were examined for evidence of bone regeneration, new attachment apparatus, and foreign body reaction.
RESULTS The histologic analysis evaluated the presence, quality, and quantity
Table 1. Histologic Features of Specimens Harvested at 3 Months From Study and Control Groups
Operative Procedure
Full-thickness buccal and lingual flaps were raised under general anesthesia. After interradicular sectioning of third premolar, each root was elevated, then atraumatically extracted, and the socket was debrided. Solid cylindrical titanium, sand-blasted, acid-etched, large-grit Microdent screw immediate implant (Implant Microdent System S.L-Comapedrosa) 3.5 mm in diameter and 10 mm long was manually inserted in the implant
Newly Formed Bone
Case No. 1 2 3 4 5
Neovascularization
Bone Notches (Implant Serrations)
Study Group (Left Side)
Control Group (Right Side)
Study Group (Left Side)
Control Group (Right Side)
Study Group (Left Side)
Control Group (Right Side)
+++ ++ ++ +++ +++
++ + + + +
++ +++ ++ +++ ++
+ + 6 + 6
+++ ++ ++ ++ +++
− − − − −
(+++), large amount; (++), good amount; (+), few amount; (6), very few amount; (−), absence.
IMPLANT DENTISTRY / VOLUME 23, NUMBER 2 2014
Fig. 3. A, Photomicrograph of a control group case at 3 months illustrating a continuous layer of new bone (NB) attached to the host bone (H) with a clear resting line (arrow), dilated blood vessels, and osteocytes. Connective tissue is found growing toward the site of the implant (hematoxylin and eosin 3400). B, Photomicrograph of a control group case at 3 months showing fine spicules of new bone (arrows), extravasated red blood cells (arrowheads) and dense collagen bundles lining host bone (H) (trichrome stain 3100).
Fig. 4. A, Photomicrograph of a study group case at 3 months revealing a layer of new bone attached to the host. Dilated and numerous blood vessels within the area of the attachment (arrows), numerous osteocytes, and dense collagen on the newly formed bone are seen. The interface of the implant on the bone is well seen (arrowheads) (hematoxylin and eosin 3100). B, At higher magnification; inset revealing a notch resulting from one of the serrations of the removed implant and a large number of osteocytes (hematoxylin and eosin 3400).
of the newly formed bone, residual simvastatin particles, neovascularization, and osseointegration in study and control sites. In the present study, the healing around endosteal implants differed in both groups, with enhancement of bone regeneration in simvastatintreated defects throughout the study period. After 1 Month
The control group showed a uniform layer of proliferating connective tissue around the host bone without any sign of new bone formation, in addition to few dilated blood vessels in all 5 specimens. As for the study group, simvastatin granules were found within the proliferating well-organized connective tissue with initiation of new bone formation in the form of thin bone trabecule in 4 of 5 cases (Fig. 2). All simvastatin-filled defects revealed dilated blood capillaries.
Fig. 5. Photomicrograph of a study group case at 3 months revealing a larger area of new bone attached to host (H) and large dilated blood vessels (arrows). Dense layer of collagen bundles are growing and filling the space around the implant. Serrated border of the implant is seen (arrowheads) (trichrome stain 3100).
After 3 Months
The control group showed a layer of new woven bone attached to the host bone, some dilated blood vessels in 3 of the 5 studied cases, and numerous
197
osteocytes (Table 1). Tiny bony trabecule, unmineralized areas of osteoid tissue, and collagen bundle formation were apparent in all cases. The newly formed bone did not fill the whole space between host bone and the site of implant because no notches were apparent opposite the new bone in any of the studied specimens (Fig. 3, A and B). In the study group, peri-implant tissue revealed a uniform layer of new bone attached to the host with numerous dilated blood vessels and extravasated red blood cells at the site of formation, in addition to osteocytes in all studied specimens. Notches that occurred by the implant serrations were seen in all cases on the side of new bone where the implant was fixed, indicating almost bone fill between host bone and implant. However, active dense collagen bundle formation was still seen growing toward the implant side in some specimens, interposing between the newly formed bone and titanium fixture (Figs. 4 and 5).
DISCUSSION In contrast to delayed implantation, the immediate placement of a fixture after tooth extraction may help to preserve the bone alveolar dimension, allowing placement of longer and wider implants and improving the crownimplant ratio. As a result, the boneimplant contact surface area increases, giving a better chance of success and reduction of healing period.3 These benefits are accompanied by a major drawback due to the lack of adaptation of the alveolar bone in the cervical region of the implant. This peri-implant space is comparable with a circumferential vertical defect as it alters the immediate stability of the implant and can later be occupied by soft tissues and thus jeopardizes osseointegration.13 To overcome these problems, the application of GBR, with or without bone grafting material, was proposed; yet none of these materials have shown to be ideal. An alternative may be the local employment of bone-modulating drugs used to treat systemic bone diseases, such as bisphosphonates, calcitonin, vitamin D analogs, and estrogen; they are bone resorption inhibitors that
198
EVALUATION
OF
SIMVASTATIN GRAFTING
reduce the number and/or the activity of osteoclasts, without increments of true bone mass.14 In contrast, anabolic agents, such as statins, have been postulated to stimulate bone formation by inducing osteoblast activity and increasing bone mass. Statins have been systemically administered, causing increased trabecular bone volumes and bone formation rates in animal models.5 The bone-forming potential of simvastatin has been attributed to a variety of factors. These include antagonizing tumor necrosis factor-a inhibition of BMP-2–induced osteoblast differentiation,15 building up osteoblasts, and bringing them into maturity, enhancing alkaline phosphatase activity and mineralization, increasing sialoprotein, osteocalcin, and type I collagen, and decreasing production of PGE2, interleukin-1 and -6, with bone resorbing activity.16,17 Furthermore, simvastatin acts as an osteoclast inhibitor and suppresses RANKL-induced osteoclastogenesis through inhibition of reactive oxygen species–induced signaling pathways.18 The delivery system for simvastatin seems to be crucial to achieve clinically relevant bone growth in the oral/facial region. A local formulation of simvastatin granules in HPMC matrix was prepared for the present study to be tested around immediate implants as adjunct grafting material. The local dose used was 2.2 mg based on a previous study.11 In vivo results showed the suitability of this formulation as an osteogenic agent.12 Moreover, 50% cumulative simvastatin was reported to be released from HPMC granules.19 Using simvastatin granules as grafting material may also overcome problems encountered in its use as injection, as granules may act as a scaffold allowing space for new bone growth. The ideal graft material to induce osseointegration should have osseoinductive and osseoconductive properties.20 Simvastatin was chosen to be tested in this study because it is a potent bone growth stimulator and its use has led to osseoinduction in a previous research.21 In the present work, we used the largest possible diameter implant for
MANSOUR
ET AL
the clinical situation because good primary stability of implants helps in minimizing the distortional strains in the peri-implant tissues and improves bone regeneration.1 The current study revealed evidence of histologic healing in both groups around immediate implants. This may be attributed to several factors, including atraumatic extraction, implant primary stability, postoperative soft diet to reduce mastication forces, and daily postoperative observation. Schwartz-Arad et al22 theorized that better bone regeneration and improved results of bone-to-implant contact of immediate implants are due to the presence of periodontal cells at the coronal area and the existence of denser bone compared with edentulous areas where disuse atrophy occurs. In the present work, when comparing both studied groups, the simvastatinfilled defects revealed better bone regeneration than unfilled defects. After 1 month, both groups showed a delicate layer of connective tissue lining host bone, but the presence of simvastatin granules before their complete resorption in the study group acted as a scaffold for more granulation tissue and bone formation, in addition to its osteoinductive property.14 In the current study at 3 months, the test group showed more newly formed blood vessels indicating active bone formation, as well as larger areas of newly formed bone, almost filling the space between host bone and implant; whereas the control group showed larger areas of unmineralized connective tissue with scanty bony trabecule, suggesting a delay in bone healing. Additionally, there was an increasing number of osteocytes in the test group, revealing that the newly formed bone was healthy and well-functioning. The higher neovascularization revealed in the simvastatin group indicates an active metabolic state of tissues. This observation is in line with a study demonstrating that statins cause nitric oxide-mediated promotion of new blood vessel growth. These blood vessels may represent a source of monocytes, and later of osteoblasts needed for bone formation. Moreover, this rich vascularity may lead to the
development of nutritional supply lines that enhance bone formation.23 Maeda et al24 demonstrated in vitro that simvastatin increased the expression of vascular endothelial growth factor, a major angiogenic factor that regulates the growth of new capillaries. In the present work, the increased bone formation in the simvastatin group is in agreement with studies carried out on humans, where more clinical attachment gain and intrabony defect fill were seen at sites treated with scaling and root planing plus local simvastatin.12,25 Additionally, other researches have shown increased osteoblasts and boneforming surface in simvastatin injection sites in animals.26,27
CONCLUSIONS The current formula for simvastatin application around immediate implants has led to increased osteogenesis, angiogenesis, and osseointegration of implants when compared with controls. In a proper delivery system, simvastatin could be proposed for further clinical applications, such as socket filling for ridge preservation, preimplant reconstruction of osseous deficiencies, or sinus floor elevations. Future studies should include a longer follow-up and a larger sample size, to assess the longterm stability of improvement rendered by this treatment modality.
DISCLOSURE The authors claim to have no financial interest, either directly or indirectly, in the products or information listed in the article.
ACKNOWLEDGMENTS The authors would like to express their gratitude to Dr. Fatma Ismail, Department of Pharmaceutics, Faculty of Pharmacy, Alexandria University, for her help in preparing the drug used in this study.
REFERENCES 1. Covani U, Crespi R, Cornelini R, et al. Immediate implants supporting single crown restoration: A 4-year prospective study. J Periodontol. 2004;75:982–988.
IMPLANT DENTISTRY / VOLUME 23, NUMBER 2 2014 2. Jung RE, Windisch SI, Eggenschwiler AM, et al. A randomizedcontrolled clinical trial evaluating clinical and radiological outcomes after 3 and 5 years of dental implants placed in bone regenerated by means of GBR techniques with or without addition of BMP-2. Clin Oral Implants Res. 2009;20:660–666. 3. Ortega-Martinez J, Perez-Pascual T, Mareque-Bueno S, et al. Immediate implants following tooth extraction. A systematic review. Med Oral Patol Oral Cir Bucal. 2012;17:e251–e261. 4. Whang K, McDonald J, Khan A, et al. A novel osteotropic biomaterial OGPLG: Synthesis and in vitro release. J Biomed Mater Res. 2005;74:237–246. 5. Baek KH, Lee WY, Oh KW, et al. The effect of simvastatin on the proliferation and differentiation of human bone marrow stromal cells. J Korean Med Sci. 2005;20:438–444. 6. Wu Z, Liu C, Zang G, et al. The effect of simvastatin on remodeling of the alveolar bone following tooth extraction. Int J Oral Maxillofac Surg. 2008;37:170–176. 7. Edwards CJ, Spector TD. Statins as modulators of bone formation. Arthritis Res. 2002;4:151–153. 8. Junqueira JC, Mancini MNG, Carvalho YR, et al. Effects of simvastatin on bone regeneration in the mandibles of ovariectomized rats and blood cholesterol levels. J Oral Sci. 2002;44:117–124. 9. Morris MS, Lee Y, Lavin MT, et al. Injectable simvastatin in periodontal defects and alveolar ridges: Pilot studies. J Periodontol. 2008;79:1465–1473. 10. Maciel-Oliveira N, BradaschiaCorrea V, Arana-Chavez VE. Early alveolar bone regeneration in rats after topical administration of simvastatin. Oral Surg
Oral Med Oral Pathol Oral Radiol Endod. 2011;112:170–179. 11. Thylin MR, Mc Connell JC, Schmid MJ, et al. Effects of simvastatin gels on murine calvarial bone. J Periodontol. 2002;73:1141–1148. 12. Galal A, Hommos A, Hussein S, et al. Evaluation of the local application of cholesterol lowering drug “Simvastatin” for the management of intrabony defects. Egypt Dent J. 2006;52:375–393. 13. Boix D, Gauthier O, Guicheux J, et al. Alveolar bone regeneration for immediate implant placement using an injectable bone substitute: An experimental study in dogs. J Periodontol. 2004;75: 663–671. 14. Hwang R, Lee EJ, Kim KH, et al. Calcylin, a Ca2+ ion-binding protein, contributes to the anabolic effects of Simvastatin on bone. J Biol Chem. 2004;279: 21239–21247. 15. Yamashita M, Otsuka F, Mukai T, et al. Simvastatin antagonizes tumor necrosis factor-a inhibition of bone morphogenetic proteins-2-induced osteoblast differentiation by regulating Smad signaling and Ras/Rhomitogen-activated protein kinase pathway. J Endocrinol. 2008;196:601–613. 16. Park JB. The use of simvastatin in bone regeneration. Med Oral Patol Oral Cir Bucal. 2009;14:e485–e488. 17. Nassar PO, Nassar CA, Guimaraes MR, et al. Simvastatin therapy in cyclosporine A-induced alveolar bone loss in rats. J Periodontal Res. 2009;44:479–488. 18. Moon HJ, Kim SE, Hun YP, et al. Simvastatin inhibits osteoclast differentiation by scavenging reactive oxygen species. Exp Mol Med. 2011;43:605–612. 19. Ismail FA. Design and in vitro evaluation of polymeric formulae of simvastatin
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for local bone induction. Drug Dev Ind Pharm. 2006;32:1199–1206. 20. Mavrogenis AF, Dimitriou R, Parvizi J, et al. Biology of implant osseointegration. J Musculoskelet Neuronal Interact. 2009;9: 61–71. 21. Lee Y, Schmid MJ, Marx DB, et al. The effect of local simvastatin delivery strategies on mandibular bone formation in vivo. Biomaterials. 2008;29:1940–1949. 22. Schwartz-Arad D, Yaniv Y, Levin L, et al. A radiographic evaluation of cervical bone loss associated with immediate and delayed implants placed for fixed restorations in edentulous jaws. J Periodontol. 2004;75:652–657. 23. Kureishi Y, Luo Z, Shiojima I, et al. The HMG-CoA reductase inhibitor simvastatin activates the protein kinase Akt and promotes angiogenesis in normocholesterolemic animals. Nat Med. 2000;6: 1004–1010. 24. Maeda T, Kawane T, Horiuchi N. Statins augment vascular endothelial growth factor expression in osteoblastic cells via inhibition of protein prenylation. Endocrinology. 2003;144:681–692. 25. Pradeep AR, Thorat MS. Clinical effect of subgingivally delivered simvastatin in the treatment of patients with chronic periodontitis: A randomized clinical trial. J Periodontol. 2010;81:214–222. 26. Lee Y, Liu X, Nashwad A, et al. Role of prostaglandin pathway and alendronate-based carriers to enhance statin-induced bone. Mol Pharm. 2011; 8:1035–1042. 27. Rutledge J, Schieber MD, Chamberlain JM, et al. Simvastatin application to augment facial jaw bone in a dog model: Pilot study. J Periodontol. 2011;82: 597–605.
195
Evaluation of Simvastatin Grafting Around Immediate Dental Implants in Dogs Ghada Mansour, PhD,* Adham Al Ashwah, PhD,† and Azza Koura, PhD‡
he discrepancy between the diameters of the socket and that of the implant generates a gap between the walls of the socket and the fixture. In large bony defects, this void can be colonized by epithelial cells inducing fibrointegration, and implant failure.1 Guided bone regeneration (GBR), by the use of barrier membranes alone or in association with bone replacement graft, eliminates this problem and gives satisfactory results to achieve implant osseointegration.2 Nevertheless, none of these materials have shown to be ideal, and new materials are sought.3 The statins are commonly prescribed drugs used to reduce serum cholesterol concentrations and the risk of heart attack.4 Simvastatin, a member of statins, has been reported to promote osteoblastic differentiation in the human bone marrow stromal cells. It stimulates the alkaline phosphatase activity and enhances the expression of osteocalcin, which are early and late osteoblastic differentiation markers, respectively.5 Furthermore, simvastatin stimulates the expression of BMP-2,
T
*Professor, Department of Oral Diagnostic Sciences, Faculty of Dentistry, King Abdulaziz University, Jeddah, Saudi Arabia; Professor, Department of Oral Medicine, Periodontology, Oral Diagnosis and Oral Radiology, Faculty of Dentistry, Alexandria University, Alexandria, Egypt. †Associate Professor, Department of Oral Surgery, Faculty of Dentistry, Alexandria University, Alexandria, Egypt. ‡Associate Professor, Department of Oral Biology, Faculty of Dentistry, Alexandria University, Alexandria, Egypt.
Reprint requests and correspondence to: Ghada Mansour, PhD, Department of Oral Basic and Clinical Sciences, Faculty of Dentistry, King Abdulaziz University, 80209, Jeddah 21589, Saudi Arabia, Phone: +966-2-6403443, Fax: +966-2-6403316, E-mail: [email protected] ISSN 1056-6163/14/02302-195 Implant Dentistry Volume 23 Number 2 Copyright © 2014 by Lippincott Williams & Wilkins DOI: 10.1097/ID.0000000000000051
Introduction: Simvastatin (Zocor; MSD), a cholesterol-lowering drug, is used systemically in treatment of osteoporosis due to its boneforming potential. The present study was conducted to evaluate the regenerative potential of an optimized simvastatin formulation as a grafting material around immediate dental implants in experimental animals. Materials and Methods: The drug was formulated as granules in cellulosic polymeric matrix. Surgical extractions of left and right mandibular third premolars were performed in 10 dogs. The left side of the mandible was the study group, where Microdent (Implant Microdent System S.L-Comapedrosa, Barcelona, Spain) implants were immediately seated and simvastatin
granules were packed. The right side constituted the control group where only implants were placed. Five dogs were killed at 1 month and 5 at 3 months. The implants were removed and specimens were processed and stained with hematoxylin and eosin and trichrome stains. Results: Healing occurred in both groups, with better findings in simvastatin-filled defects, as evidenced by bone regeneration, with neovascularization. Conclusions: Simvastatin granules allowed for osteogenesis around immediate implants, resulting in their osseointegration. (Implant Dent 2014;23:195–199) Key Words: dental implants, statins, osseointegration, bone substitute
which causes mesenchymal cells to differentiate into bone or cartilage forming cells.6 In addition, simvastatin may have effects on the mevalonate pathway, leading to inhibition of osteoclast activity.7 The incorporation of simvastatin into hydrophobic matrices should be beneficial for local slow release at sites where new bone growth is required.8 However, injection of simvastatin into human-like periodontal defects produced unfavorable results, possibly because of the viscosity of the carrier or lack of space creation for new bone growth.9 The lack of an ideal carrier for simvastatin has markedly limited its topical use.10 To the best of our knowledge, there are no published data concerning the
use of simvastatin topically as an adjunctive material to immediate implants. Therefore, the present study was carried out to evaluate the regenerative potential of a recently developed optimized delivery device, using a single dose of simvastatin granules in cellulose polymeric matrix, as a grafting material around immediate dental implants in experimental animals, and to determine if it could enhance osseointegration of these implants.
MATERIALS
AND
METHODS
Experimental Animals
Ten adult systemically healthy mongrel dogs (Canis familiaris) were included in this study. Their age ranged from 18 to 24 months and their weight
196
EVALUATION
OF
SIMVASTATIN GRAFTING
from 12 to 15 kg. A split mouth design was used. The experimental sites were divided into 2 groups: study and control groups. Group 1 (study group) included 10 freshly extracted sockets at left mandibular third premolars, where immediate implants were placed with adjunctive topical application of (2.2 mg) simvastatin granules. Group 2 (control group) included 10 freshly extracted sockets at right mandibular third premolars, where only immediate implants were placed. All animal procedures were approved by the Animal Ethics Committee at the University of Alexandria.
MANSOUR
ET AL
Fig. 1. Immediate implant in place surrounded by simvastatin granules packed around the gap at the coronal portion of the socket.
Preparation of the Grafting Material
It was formulated as granules in cellulosic polymeric matrix: hydroxy propyl methyl cellulose (HPMC 4000 CP, Methocel; Alexandria Pharmaceutical Co., Alexandria, Egypt) in the Department of Pharmaceutics, Faculty of Pharmacy, Alexandria University. Granules were prepared to contain 2.2 mg of simvastatin per 150 mg total weight. The reported local simvastatin dose was 2.2 mg,11 and a preliminary clinical study revealed that 150-mg granules were suitable for surgical grafting.12 Weighed simvastatin/polymer blends were accurately mixed and wetted, forming a mass to be forced through a sieve. The obtained granules were sized, dried in a circulating air oven at 60°C, then subjected to vacuum, and stored in a desiccator until use. The wetting system was a 4:1 isopropyl alcohol/water mixture. The dried granules were subjected to particle size reduction by triturating and then sieved. The particle size ranging from 800 to 1000 mm was collected.
side, the implant was placed without simvastatin. The healing cap was secured on top of the fixture. The flap was sutured using 3-0 black silk interrupted sutures. Intramuscular antibiotic treatment using 15 mg/kg of tetracycline hydrochloride was continued for 48 hours after surgery and then mixed with dogs’ food for 7 days. A painkiller was also given the first day postoperatively. Animals were checked daily and fed with a soft diet postoperatively. Normal diet was resumed after 2 weeks. Five animals were humanely killed after 1 month and the other 5 at 3 months by iv injection of overdosed thiopental sodium. Sample Preparation
Fig. 2. Photomicrograph of a study group case after 1 month revealing a layer of connective tissue covering particles of simvastatin granules (arrows) (hematoxylin and eosin 3100).
bed with the long axis congruent to the long axis of the root being replaced, after drilling 3 to 4 mm beyond the apex. In the left mandibular premolars, simvastatin granules mixed with saliva were packed in direct contact with the implant (Fig. 1). On the contralateral
Tissue blocks, including bone, implants and surrounding soft tissues, were obtained after the animals were killed. These were fixed in 10% buffered neutral formalin for 2 weeks. They were then decalcified in 5% trichloroacetic acid for 4 to 6 weeks; the implants were removed, and blocks were processed to prepare paraffin sections. Serial sections (7 mm) were cut in a buccolingual direction. Every 14th section 100 mm apart was stained using hematoxylin and eosin and an adjacent section with Gomori trichrome stain. Sections were examined for evidence of bone regeneration, new attachment apparatus, and foreign body reaction.
RESULTS The histologic analysis evaluated the presence, quality, and quantity
Table 1. Histologic Features of Specimens Harvested at 3 Months From Study and Control Groups
Operative Procedure
Full-thickness buccal and lingual flaps were raised under general anesthesia. After interradicular sectioning of third premolar, each root was elevated, then atraumatically extracted, and the socket was debrided. Solid cylindrical titanium, sand-blasted, acid-etched, large-grit Microdent screw immediate implant (Implant Microdent System S.L-Comapedrosa) 3.5 mm in diameter and 10 mm long was manually inserted in the implant
Newly Formed Bone
Case No. 1 2 3 4 5
Neovascularization
Bone Notches (Implant Serrations)
Study Group (Left Side)
Control Group (Right Side)
Study Group (Left Side)
Control Group (Right Side)
Study Group (Left Side)
Control Group (Right Side)
+++ ++ ++ +++ +++
++ + + + +
++ +++ ++ +++ ++
+ + 6 + 6
+++ ++ ++ ++ +++
− − − − −
(+++), large amount; (++), good amount; (+), few amount; (6), very few amount; (−), absence.
IMPLANT DENTISTRY / VOLUME 23, NUMBER 2 2014
Fig. 3. A, Photomicrograph of a control group case at 3 months illustrating a continuous layer of new bone (NB) attached to the host bone (H) with a clear resting line (arrow), dilated blood vessels, and osteocytes. Connective tissue is found growing toward the site of the implant (hematoxylin and eosin 3400). B, Photomicrograph of a control group case at 3 months showing fine spicules of new bone (arrows), extravasated red blood cells (arrowheads) and dense collagen bundles lining host bone (H) (trichrome stain 3100).
Fig. 4. A, Photomicrograph of a study group case at 3 months revealing a layer of new bone attached to the host. Dilated and numerous blood vessels within the area of the attachment (arrows), numerous osteocytes, and dense collagen on the newly formed bone are seen. The interface of the implant on the bone is well seen (arrowheads) (hematoxylin and eosin 3100). B, At higher magnification; inset revealing a notch resulting from one of the serrations of the removed implant and a large number of osteocytes (hematoxylin and eosin 3400).
of the newly formed bone, residual simvastatin particles, neovascularization, and osseointegration in study and control sites. In the present study, the healing around endosteal implants differed in both groups, with enhancement of bone regeneration in simvastatintreated defects throughout the study period. After 1 Month
The control group showed a uniform layer of proliferating connective tissue around the host bone without any sign of new bone formation, in addition to few dilated blood vessels in all 5 specimens. As for the study group, simvastatin granules were found within the proliferating well-organized connective tissue with initiation of new bone formation in the form of thin bone trabecule in 4 of 5 cases (Fig. 2). All simvastatin-filled defects revealed dilated blood capillaries.
Fig. 5. Photomicrograph of a study group case at 3 months revealing a larger area of new bone attached to host (H) and large dilated blood vessels (arrows). Dense layer of collagen bundles are growing and filling the space around the implant. Serrated border of the implant is seen (arrowheads) (trichrome stain 3100).
After 3 Months
The control group showed a layer of new woven bone attached to the host bone, some dilated blood vessels in 3 of the 5 studied cases, and numerous
197
osteocytes (Table 1). Tiny bony trabecule, unmineralized areas of osteoid tissue, and collagen bundle formation were apparent in all cases. The newly formed bone did not fill the whole space between host bone and the site of implant because no notches were apparent opposite the new bone in any of the studied specimens (Fig. 3, A and B). In the study group, peri-implant tissue revealed a uniform layer of new bone attached to the host with numerous dilated blood vessels and extravasated red blood cells at the site of formation, in addition to osteocytes in all studied specimens. Notches that occurred by the implant serrations were seen in all cases on the side of new bone where the implant was fixed, indicating almost bone fill between host bone and implant. However, active dense collagen bundle formation was still seen growing toward the implant side in some specimens, interposing between the newly formed bone and titanium fixture (Figs. 4 and 5).
DISCUSSION In contrast to delayed implantation, the immediate placement of a fixture after tooth extraction may help to preserve the bone alveolar dimension, allowing placement of longer and wider implants and improving the crownimplant ratio. As a result, the boneimplant contact surface area increases, giving a better chance of success and reduction of healing period.3 These benefits are accompanied by a major drawback due to the lack of adaptation of the alveolar bone in the cervical region of the implant. This peri-implant space is comparable with a circumferential vertical defect as it alters the immediate stability of the implant and can later be occupied by soft tissues and thus jeopardizes osseointegration.13 To overcome these problems, the application of GBR, with or without bone grafting material, was proposed; yet none of these materials have shown to be ideal. An alternative may be the local employment of bone-modulating drugs used to treat systemic bone diseases, such as bisphosphonates, calcitonin, vitamin D analogs, and estrogen; they are bone resorption inhibitors that
198
EVALUATION
OF
SIMVASTATIN GRAFTING
reduce the number and/or the activity of osteoclasts, without increments of true bone mass.14 In contrast, anabolic agents, such as statins, have been postulated to stimulate bone formation by inducing osteoblast activity and increasing bone mass. Statins have been systemically administered, causing increased trabecular bone volumes and bone formation rates in animal models.5 The bone-forming potential of simvastatin has been attributed to a variety of factors. These include antagonizing tumor necrosis factor-a inhibition of BMP-2–induced osteoblast differentiation,15 building up osteoblasts, and bringing them into maturity, enhancing alkaline phosphatase activity and mineralization, increasing sialoprotein, osteocalcin, and type I collagen, and decreasing production of PGE2, interleukin-1 and -6, with bone resorbing activity.16,17 Furthermore, simvastatin acts as an osteoclast inhibitor and suppresses RANKL-induced osteoclastogenesis through inhibition of reactive oxygen species–induced signaling pathways.18 The delivery system for simvastatin seems to be crucial to achieve clinically relevant bone growth in the oral/facial region. A local formulation of simvastatin granules in HPMC matrix was prepared for the present study to be tested around immediate implants as adjunct grafting material. The local dose used was 2.2 mg based on a previous study.11 In vivo results showed the suitability of this formulation as an osteogenic agent.12 Moreover, 50% cumulative simvastatin was reported to be released from HPMC granules.19 Using simvastatin granules as grafting material may also overcome problems encountered in its use as injection, as granules may act as a scaffold allowing space for new bone growth. The ideal graft material to induce osseointegration should have osseoinductive and osseoconductive properties.20 Simvastatin was chosen to be tested in this study because it is a potent bone growth stimulator and its use has led to osseoinduction in a previous research.21 In the present work, we used the largest possible diameter implant for
MANSOUR
ET AL
the clinical situation because good primary stability of implants helps in minimizing the distortional strains in the peri-implant tissues and improves bone regeneration.1 The current study revealed evidence of histologic healing in both groups around immediate implants. This may be attributed to several factors, including atraumatic extraction, implant primary stability, postoperative soft diet to reduce mastication forces, and daily postoperative observation. Schwartz-Arad et al22 theorized that better bone regeneration and improved results of bone-to-implant contact of immediate implants are due to the presence of periodontal cells at the coronal area and the existence of denser bone compared with edentulous areas where disuse atrophy occurs. In the present work, when comparing both studied groups, the simvastatinfilled defects revealed better bone regeneration than unfilled defects. After 1 month, both groups showed a delicate layer of connective tissue lining host bone, but the presence of simvastatin granules before their complete resorption in the study group acted as a scaffold for more granulation tissue and bone formation, in addition to its osteoinductive property.14 In the current study at 3 months, the test group showed more newly formed blood vessels indicating active bone formation, as well as larger areas of newly formed bone, almost filling the space between host bone and implant; whereas the control group showed larger areas of unmineralized connective tissue with scanty bony trabecule, suggesting a delay in bone healing. Additionally, there was an increasing number of osteocytes in the test group, revealing that the newly formed bone was healthy and well-functioning. The higher neovascularization revealed in the simvastatin group indicates an active metabolic state of tissues. This observation is in line with a study demonstrating that statins cause nitric oxide-mediated promotion of new blood vessel growth. These blood vessels may represent a source of monocytes, and later of osteoblasts needed for bone formation. Moreover, this rich vascularity may lead to the
development of nutritional supply lines that enhance bone formation.23 Maeda et al24 demonstrated in vitro that simvastatin increased the expression of vascular endothelial growth factor, a major angiogenic factor that regulates the growth of new capillaries. In the present work, the increased bone formation in the simvastatin group is in agreement with studies carried out on humans, where more clinical attachment gain and intrabony defect fill were seen at sites treated with scaling and root planing plus local simvastatin.12,25 Additionally, other researches have shown increased osteoblasts and boneforming surface in simvastatin injection sites in animals.26,27
CONCLUSIONS The current formula for simvastatin application around immediate implants has led to increased osteogenesis, angiogenesis, and osseointegration of implants when compared with controls. In a proper delivery system, simvastatin could be proposed for further clinical applications, such as socket filling for ridge preservation, preimplant reconstruction of osseous deficiencies, or sinus floor elevations. Future studies should include a longer follow-up and a larger sample size, to assess the longterm stability of improvement rendered by this treatment modality.
DISCLOSURE The authors claim to have no financial interest, either directly or indirectly, in the products or information listed in the article.
ACKNOWLEDGMENTS The authors would like to express their gratitude to Dr. Fatma Ismail, Department of Pharmaceutics, Faculty of Pharmacy, Alexandria University, for her help in preparing the drug used in this study.
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