Effect of Calcitonin on Bone Formation Around Titanium Implant. A Histometric Study in Rabbits
Alessandro Lourenço JANUÁRIO
Enilson Antonio SALLUM
Sérgio de TOLEDO
Antonio Wilson SALLUM
Francisco Humberto NOCITI Jr.
Department of Prosthesis and Periodontics, Dental School of Piracicaba, UNICAMP, Piracicaba, SP, Brazil
Correspondence: Prof. Francisco Humberto Nociti Junior, Faculdade de Odontologia de Piracicaba/Unicamp, Av. Limeira, 901, 13414-903 Piracicaba, SP, Brasil. Fax: +55-19-430-5218. e-mail: email@example.com
Braz Dent J (2001) 12(3): 158-162 ISSN 0103-6440
INTRODUCTION | MATERIAL AND METHODS | RESULTS | DISCUSSION | ACKNOWKEDGEMENT | RESUMO | REFERENCES
Bone healing around titanium implants has already been evaluated; however,
the effect of drugs such as calcitonin during the period of bone maturation
around titanium implants has not yet been investigated. The purpose of this
study was to evaluate the effects of calcitonin administration on the late
period of bone healing following titanium implant insertion. Twenty-seven
adult New Zealand rabbits received one implant in each femur. Thirteen animals
were randomly selected as the test group (2 IU/kg - calcitonin) and fourteen
animals served as control (saline). The animals were sacrificed 6, 8, 12
and 18 weeks after surgery. Endosteal/periosteal bone length (EB/PB), endosteal/periosteal
bone area (EBA/PBA) and total cortical length (TCL) around the implants were
analyzed. After 6, 8, 12 and 18 weeks, a positive time effect was strongly
observed (P<0.05). Considering the treatment factor, there was a positive
effect of calcitonin on EBA and EB variables at 12 weeks and TCL at 18 weeks.
In conclusion, the administration of salmon calcitonin to healthy animals
may improve bone mass at the later stages of bone healing following titanium
Key Words: bone healing, calcitonin, dental implants.
Edentulousness leads to impaired speech and masticatory ability and also may affect the psycho-social situation of the individual. Tooth loss can often be compensated by a conventional prosthesis; however, if this is not possible, a prosthesis supported by dental implants has been tried as an alternative (1-4). Because the success of osseointegration depends, in part, on the state of the host bone bed and its healing capacity, conditions such as osteoporosis (5-7), that may affect its quality and quantity, must be considered. Misch (8) reported that potential candidates for dental implants are generally older than 50 years, and may include a group with bone metabolism disorders (9).
Copp et al. (10) reported a substance involved in calcium metabolism, subsequently named calcitonin, that acted as an antagonist to parathyroid hormone (PTH), i.e., calcitonin would cause a decrease in serum calcium levels. Since its original discovery, calcitonin has been used to treat or prevent bone metabolism disorders, because of its capacity to inhibit bone resorption (11) and because of its analgesic properties (12). The effects of calcitonin in healthy individuals have been observed to be the same as those observed in bone metabolism disorders (13). Other studies have reported that calcitonin also promotes new bone formation
(14). A significant number of potential patients for dental implants might be taking calcitonin for bone metabolism disorders. In a pioneer study (15), it was suggested that the administration of calcitonin to healthy animals could possibly improve new bone formation around titanium implants at the later period of bone healing.
Thus, the purpose of the present investigation was to evaluate the effects of calcitonin administration on the maturation period of bone healing (16), following titanium implant insertion.
MATERIAL AND METHODS
Twenty-seven adult New Zealand rabbits aged 9-12 months (3000-3500 g) were used for this study. Each animal received an implant (AS Technology®, São José dos Campos, SP, Brazil) in both femurs. Thirteen animals were randomly selected as the test group and fourteen as the control group. The implants used were screw-shaped and rough-surfaced commercially pure titanium (99.4%), with an outer diameter of 2.6 mm and a total length of 7.0 mm.
Surgery was performed under general anesthesia produced by intravenous injection of 25% urethane (0.8 g/kg body weight; Sigma Chemical Co., St. Louis, MO, USA). The implants were inserted following the conventional osseointegration protocol (1). Skin was cleansed with iodine surgical soap. An incision approximately 3 cm in length was made and the bone surface of the femur surgically exposed by blunt dissection. Using an electric motor, unicortical implant beds were prepared. The implants were inserted until the screw thread had been completely introduced into the bone cortex. Finally, soft tissues were replaced and sutured.
Calcitonin (Miacalcic®, Sandoz A.G., Fertigung Schützenstrsse, Ravenburg, Germany) was diluted in 0.9% saline and administered im in single daily doses (2 IU/kg), as recommended (17), for 28 days. Antibiotics were not administered.
The animals were sacrificed by an overdose of urethane at 6, 8, 12 and 18 weeks after the surgical procedures. The femurs were dissected and fixed in 10% formal saline for 48 h. After that time, the specimens were decalcified with a 50% formic acid and 20% sodium citrate solution for 6 weeks. Eighteen days after the beginning of the decalcification process, the implant was removed from the bone tissue by an incision following the long axis of the implant. Following fixation and decalcification, the bone was embedded in paraffin, serial 6-µm sections were obtained and stained with H&E.
Histometrical analysis was carried out using an image analysis system (MOCHA, Jadel Scientific, San Rafael, CA, USA), and analyzed parameters (Figure 1) were endosteal bone length (EB) (mm), periosteal bone length (PB) (mm), endosteal bone area (EBA) (mm2), periosteal bone area (PBA) (mm2) and total cortical length (TCL) (mm). Measurements were obtained from 5 sections considering a 30-µm distance between each one.
The experimental design was factorial 2x4 (time x treatment) completely randomized. The degrees of freedom of the time factor were decomposed in orthogonal polynoms in order to obtain the best equation that fit the data. When the interaction between treatment and time was significant, the interactions of each level of the time and treatment factors were analyzed. The means were compared using the F test (a=5%). The following variables were transformed using the Box and Cox transformation to stabilize the variances: EB: (x)-0.5; PB: square root of x; EBA: (x)0.2; PBA: log 10(x); TCL: (x)-1.
Endosteal Bone Length (EB)
The F test showed a significant effect of the time factor and of the interaction between time and treatment factors (P<0.05) on endosteal bone length. The variable EB was transformed to x-0.5, therefore the results must be observed with an inverse effect. After separating the interaction time/treatment for the control group into fractions, a quadratic effect (P<0.05) was observed for the time factor in the control group, indicating the maximum endosteal bone length at 18 weeks. A quadratic effect (P<0.05) was also observed for the test group, indicating the maximum endosteal bone length at 12 weeks. Statistical differences between test and control groups at 6 and 8 weeks were not observed (P>0.05) (Table 1).
Periosteal Bone Length (PB)
The F test showed a significant effect of time on periosteal bone length (P<0.05). The regression analysis indicated a linear positive effect of time on the periosteal bone length, whereas statistical differences between test and control groups in terms of treatment were not observed (P>0.05) (Table 2).
Endosteal Bone Area (EBA)
The F test showed a significant effect of the time factor and of the interaction between time and treatment factors (P<0.05) on endosteal bone area. After separating the interaction time/treatment into fractions, a time linear positive effect (P<0.05) was observed for the control group whereas a quadratic effect (P<0.05) was observed for the treatment factor for the test group. Statistical analysis showed a greater endosteal bone area for the test group at 12 weeks and for the control group at 18 weeks (P<0.05), whereas at 6 and 8 weeks no difference between the groups was observed (P>0.05) (Table 3).
Periosteal Bone Area (PBA)
The F test detected a significant effect of time for periosteal bone area (P<0.05). The regression analysis indicated a linear positive effect of time on the periosteal bone area. No statistical differences between test and control groups in terms of treatment were observed (P>0.05) (Table 4).
Total Cortical Length (TCL)
The F test showed a significant effect of the time and treatment factors, and of the interaction between time and treatment factors (P<0.05). After separating the interaction time/treatment for the control and test groups into fractions, a quadratic effect (P<0.05) was observed for the time factor, indicating the maximum cortical length at 12 and 18 weeks, respectively. Statistical analysis showed a greater cortical length for the test group at 18 weeks (P<0.05) (Table 5).
The biocompatibility of modern implant materials, such as titanium, is so firmly established that it is not considered to be a significant factor in endosseous implant failure. An important feature of the osseointegration method is the emphasis put on efforts to minimize any damage to the host tissue during the surgical procedure. In the present study, the standard procedures described as prerequisites for long-term maintenance of the implant (1) were observed and resulted in a bone healing process similar to that reported previously (16).
Branemark et al. (2) observed a process of cortical thickening which they called corticalization. They believed that this process would be indicative of a successive load-adapted bone remodeling. In both groups of the present study, following insertion of a biocompatible device into cortical bone, a bridging callus of woven bone formed at the endosteal and periosteal surfaces in agreement with Roberts (16). Since the initial callus is formed by immature woven bone, it is “replaced” by a well-organized and strong lamellar bone. In this study, the implants were not submitted to any load, and all of them showed a “corticalization” process. Thus, we consider that this bone response model constitutes just a step in the entire bone healing process even in the absence of load.
Nociti Jr. et al. (15) first evaluated the influence of salmon calcitonin administration on the initial period of bone healing after inserting titanium implants in the femur of healthy animals. Their results did not show significant benefits of calcitonin. However, the data observed for the 6-weeks group indicated that calcitonin administration could promote some improvement after the initial phase of bone healing. Therefore, the present study was carried out to investigate the effects of calcitonin administration on the later period of bone healing following titanium implant insertion.
The results of the present study demonstrated that at the end of 18 weeks the administration of salmon calcitonin led to a greater total cortical length than that observed for the control group. However, further investigations should be carried out if the greater extension observed histologically in the present study represents mechanical benefits to the implant. In addition, the possible benefits of administering calcitonin to osteoporosis/Paget’s disease/hyperparathyroidism individuals who will receive a titanium dental implant and also the events after dental implant placement in these same individuals who could have been taking calcitonin as a medicament remain to be investigated.
Certainly, in the present study, the differences observed between test and control groups occurred as a consequence of the calcitonin administration to the animals, because it was the unique variable between the groups. This observation is in agreement with Buclin et al. (18), who reported that calcitonin produces its biological effects in healthy animals, although Avioli (19) reported that the calcitonin effects could be observed only in individuals with bone metabolism disorders.
A neutralizing effect of antibodies has been reported for some cases of calcitonin administration (20). Although the investigation of the presence of antibodies was not the goal of the present study, antibodies may have not neutralized the effect of the calcitonin because there were significant differences for some parameters. The emergence of side effects as a consequence of the use of calcitonin has been reported for humans (13), however in the present study no such side effects were observed.
We conclude that the administration of salmon calcitonin to healthy animals following titanium implant insertion may cause improvement in bone mass around the implant.
This investigation was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Brazil, grant 97/06585-6). In addition, the authors would like to thank Maria Aparecida Santiago Varella for preparing the histological sections.
Januário AL, Sallum EA, De Toledo S, Sallum AW, Nociti Jr FH. Efeito da calcitonina sobre a formação óssea ao redor de implantes de titânio. Um estudo histométrico em coelhos. Braz Dent J 2001;12(3):158-162.
O reparo ósseo ao redor de implantes de titânio tem sido avaliado, entretanto o efeito de medicamentos, tais como calcitonina, durante o período de maturação óssea ao redor de implantes de titânio não têm sido investigado. A proposta deste estudo foi avaliar o efeito da administração da calcitonina no período tardio de reparo ósseo após a colocação de implantes de titânio. Vinte e sete coelhos Nova Zelândia adultos receberam um implante em cada fêmur. Treze animais foram escolhidos aleatoriamente para compor o grupo teste (calcitonina - 2 UI/kg) e quatorze o grupo controle (solução salina). Os animais foram sacrificados 6, 8, 12 e 18 semanas após o procedimento cirúrgico. Os parâmetros analisados foram o comprimento do osso endosteal/periosteal (EB/PB), a área do osso endosteal/periosteal (EBA/PBA) e o comprimento total da cortical (TCL) ao redor do implante. Um efeito positivo do tempo foi fortemente observado após 6, 8, 12 e 18 semanas (P<0.05). Em relação ao fator tratamento, foi observado um efeito positivo da calcitonina para as variáveis EBA e EB após 12 semanas e TCL após 18 semanas. Com base nos resultados do presente estudo, pode-se concluir que a administração de calcitonina de salmão em animais saudáveis pode melhorar a massa óssea nos estágios tardios de reparo ósseo após a colocação de implantes de titânio.
Unitermos: reparo ósseo, calcitonina, implantes dentais.
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Accepted February 5, 2001
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