Arwa Alsayed Sukumaran Anil John A. Jansen Jeroen J. J. P. van den Beucken
Comparative evaluation of the combined application of titanium implants and calcium phosphate bone substitutes in a rabbit model
Authors’ affiliations: Arwa Alsayed, John A. Jansen, Jeroen J. J. P. van den Beucken, Department of Biomaterials (309), Radboudumc, Nijmegen, The Netherlands Arwa Alsayed, Department of Dentistry, Prince Sultan Military Medical City, Ministry of Defence, Riyadh, Saudi Arabia Sukumaran Anil, Department of Periodontics and Community Dentistry, College of Dentistry, King Saud University, Riyadh, Saudi Arabia
Key words: biphasic
calcium
phosphate,
bone
grafting,
bone
substitute,
histology,
Corresponding author: Jeroen JJP van den Beucken, PhD Department of Biomaterials (309), Radboudumc PO Box 9101, 6500 HB Nijmegen,The Netherlands Tel.:+31 24 3667305 Fax: +31 24 3614657 e-mail: [email protected]
rabbits. Following a randomization protocol, implants were alternately installed in one condyle
histomorphometry, intrabony defects Abstract Objectives: To study the healing of defects around titanium implants filled with biphasic calcium phosphates (BCP). Material and methods: Forty custom-made, titanium implants (Ti) with a diameter of 5 mm, and length of 8 mm, with two-sided gaps, were fabricated and installed in the femoral condyle of 20 without BCP bone substitute material (Ti) in the gaps and in the contralateral condyle gaps were filled with BCP bone substitute material (Ti+BCP). The implants were retrieved after 4 and 12 weeks of healing, after which histological and histomorphometrical analyses were done to assess the percentage of bone implant contact (BIC), the percentage of bone area (BA) and the percentage of particle area (PA) within the region of interest (ROI); the rectangular area joining the two arms of the L-shaped implant was considered as the ROI. Results: After 4 and 12 weeks of healing, Ti+BCP showed significantly higher BIC and BA values compared to Ti. Further, the BCP particles showed a significant decrease from 4 to 12 weeks of healing. The BCP particles (PA) showed a significant reduction from 31.6 11.0% at 4 weeks to 21.0 7.2% at 12 weeks. Conclusion: The addition of BCP bone substitute to fill peri-implant gaps significantly enhanced both bone formation (~2.5-fold) and bone to implant contact (>2-fold) for the custom-made titanium implants with two-sided gaps.
Date: Accepted 17 May 2014 To cite this article: Alsayed A, Anil S, Jansen JA, van den Beucken JJJP. Comparative evaluation of the combined application of titanium implants and calcium phosphate bone substitutes in a rabbit model. Clin. Oral Impl. Res. 26, 2015, 1215–1221 doi: 10.1111/clr.12435
Dental implants have revolutionized contemporary dental treatment for the replacement of missing teeth. With over 0.5 million annually placed dental implants in the United States (ADA 2011), dental implant therapy has become a widely accepted treatment modality for the replacement of missing teeth (Jung et al. 2008). The long-term success of dental implants largely depends on successful healing with safe integration into the jaw bone, which requires a suitable bone bed and appropriate implant properties (Chuang et al. 2002; Abrahamsson et al. 2009). Tooth extraction or loss and subsequent healing of the socket commonly results in osseous deformities of the alveolar ridge, including reduction in height and width. Since the quality of the bone bed is of utmost importance for a successful outcome of implant therapy (Mecall & Rosenfeld 1991), implant-supported restoration is challenged in those conditions, in which a suboptimal
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quantity and quality of bone is present in the bone bed. Particularly, a 25% decrease in alveolar bone width is observed during the first year after tooth loss and an overall 4 mm decrease in height over the next few years. Additionally, the bone resorption process on the alveolar ridge varies in the maxilla and mandible and resorption is faster in the labial and buccal parts of the alveolar ridge (Kingsmill 1999). Ridge preservation strategies involve modulation of the physiologic modeling process that occurs following tooth extraction (Bianchi & Sanfilippo 2002). The objective of these strategies is to increase or maintain bone volume, thereby promoting osseointegration and improving implant stability upon implant placement. Sufficient alveolar bone volume and favorable architecture of the alveolar ridge are essential to achieve ideal functional and esthetic prosthetic reconstruction following implant therapy. The peri-implant gap
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following immediate implant installation require filling with bone graft material, particularly when not all four bony walls have been preserved during extraction (Becker 2003). The delayed implant placement can be preceded by bone augmentation procedures, aimed to rebuild the natural contours of the bone tissue using bone graft materials (Horowitz et al. 2012). Osseous defects in the oral cavity have been successfully managed with a variety of biological and synthetic materials, including autografts, allografts, xenografts and alloplastic materials. Although autografts are unequivocally accepted as the gold standard, donor site morbidity and the limitations on the quantity of bone that can be harvested, demands from clinicians to look for alternatives (Moy et al. 1993). In view of immunological and disease transfer risks from allogeneic bone, research has focused extensively on developing alloplastic bone substitutes that are predominantly based on ceramics (e.g. calcium phosphates CaP, calcium sulfates, and bioactive glasses) (Zamet et al. 1997). In general, these ceramic materials are renowned for their osteoconductive and bioactive properties (Ignatius et al. 2001). The most commonly used ceramics are the CaP-based ceramics hydroxyapatite (HA) and beta tricalcium phosphate (b-TCP)(Knabe et al. 2000). b-TCP is more rapidly replaced by bone than the hardly degradable HA, owing to its higher solubility (Petrov et al. 2001). Upon degradation of b-TCP through a process of dissolution and absorption, a normal bone structure in the regenerated bone can be achieved (Cutright et al. 1972; von Arx et al. 2001). The development of biphasic calcium phosphate (BCP) ceramics (i.e. containing both HA and b -TCP) has provided materials in which bioactivity and degradation are controlled based on the ratios of the components (Simion et al. 1998; LeGeros et al. 2003). Several studies have shown that BCP granules have excellent biocompatibility and bioactivity and lead to new bone formation and degradation of the biomaterial (Piattelli et al. 1996; Gauthier et al. 1998; Valimaki et al. 2005). The rate of degradation or resorption of HA/TCP ceramics can be accelerated by increasing the amount of the more soluble phase, TCP (Daculsi 1998). Studies have shown that BCPs with higher b-TCP ratio are expected to yield more replacement of biomaterial by new bone (Daculsi et al. 1990; Gauthier et al. 1998). BCP compounds containing approximately 60% of HA and 40% of b-TCP seemed to provide the optimal bone
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conductive properties (Ellinger et al. 1986; Daculsi et al. 1990; Rouvillain et al. 2009). Nery et al. (1992) reported optimal bone regeneration in surgically created bone defects with BCP containing HA/b-TCP ratio of 85/15. However recently BCP products containing higher b-TCP ratios (BCP 60/40 and BCP 20/80) have been studied in prospect of its enhanced osteoconductive effect (Lee et al. 2013; Yang et al. 2014). Because simultaneous placement of bone grafts and implants shortens the treatment time without increasing complications or reducing the success rate (Boronat et al. 2010), the aim of this study was to comparatively evaluate the contribution of BCP bone graft to bone ingrowth and bone-to-implant contact upon simultaneous placement of BCP and titanium implants customized to present a reliable gap model. The experimental procedure involved implantation of the custom-made titanium implants with or without BCP graft material in the femoral condyle of rabbits. After implantation periods of 4 and 12 weeks, histological and histomorphometrical analyses (i.e. bone area, bone graft area, and bone-to-implant contact) were performed for comparison between grafted and non-grafted implants.
Materials and methods Implants and bone substitute materials
Forty custom-made titanium implants were used for this study (Fig. 1). The implants had a cylindrical shape with a diameter of 5 mm and a length of 8 mm. The implants were grit-blasted, cleaned and sterilized before installation. Ceramic biphasic calcium phosphate (BCP; 60% HA, 40% TCP) bone substitute material was obtained from CAM Bioceramics BV (Leiden, the Netherlands). This porous
(a)
granulate consisted of particles with dimensions of 425–500 lm and a porosity of 46%. Animals
The study was conducted during September 2013. A total of twenty, skeletally mature New Zealand white rabbits (male; 6 months old; weight 4–5 kg) were used for this study. The rabbits were sourced from the central facility of the King Saud University experimental animal research. The maintenance and health monitoring of the rabbits were done as per the international recommendations (Nicklas et al. 2002). The rabbits were housed in separate cages under laboratory conditions and fed a standard pelleted rabbit diet and access to water was ad libitum. The study was approved by the Ethical Committee of the Prince Sultan Military Medical City, Saudi Arabia. The surgical procedure was based on a well-established bilateral femoral implant model in rabbits with modifications (Felix Lanao et al. 2011; Alfarraj Aldosari et al. 2014). The randomization and distribution of the implants is shown in Table 1. All rabbits were identified with a microchip implanted in the body. Surgical procedure
Surgeries were performed using aseptic routines, and under general anesthesia by intramuscular injections of a combination dose of 35 mg/kg ketamine and a dose of 5 mg/kg xylazine. The surgery was performed by two researchers (AA, SA). The surgical areas were shaved, disinfected with iodine, and then isolated with sterile drapes. Infiltration anesthesia was performed at the experimental sites. The distal femoral condyles on both sides were accessed using incisions through the skin and fascia, and the medial bone surfaces of the femoral condyles were exposed using a periosteal elevator. A drill hole (5 mm
(b)
Fig. 1. (a) Schematic illustration of the implant used in the study. Custom-made Ti implant with a diameter of 5 mm and a length of 8 mm. (b) Actual implant presentation with a grit-blasted surface.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Alsayed et al Titanium implants and calcium phosphate bone substitutes
Table 1. The randomization and the implant installation scheme used in the study Animal no.
Right side
Left side
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Ti+BCP Ti Ti+BCP Ti Ti+BCP Ti Ti+BCP Ti Ti+BCP Ti Ti+BCP Ti Ti+BCP Ti Ti+BCP Ti Ti+BCP Ti Ti+BCP Ti
Ti Ti+BCP Ti Ti+BCP Ti Ti+BCP Ti Ti+BCP Ti Ti+BCP Ti Ti+BCP Ti Ti+BCP Ti Ti+BCP Ti Ti+BCP Ti Ti+BCP
(a)
(b)
(c)
(d)
4 Weeks
12 Weeks
diameter, 8 mm depth) was created in the center of the condyle using a dental drill (KAVO, Intrasept 905, KAVO Nederland BV, Vianen, The Netherlands). A 2 mm drill bit was first used to establish a pilot hole. This defect was gradually enlarged using a series of drills with diameters of 2.8, 3.6 and 4.2 mm till the final drill bit with a diameter of 5 mm (Nobel Biocare AB, Goteborg, Sweden) under continuous cooling with saline irrigation. All debris was removed from the defect by irrigation with saline. Following a randomization protocol, implants were alternately installed on one side without BCP bone substitute material in the gaps (group: Ti) and on the contralateral side gaps were filled with BCP bone substitute material (group: Ti+ BCP; Fig. 2). The wounds were closed and allowed to heal for 4 and 12 weeks. Post-surgery pain was controlled by the administration of Fynadyneâ (1.1 mg/kg body weight; Schering-Plough Laboratories, Saint Claire, France) for 2 days. To reduce the post-operative infection risk, a single dose of enrofloxacin (Baytrilâ, 10 mg/kg body weight; Bayvet Division, Chemagro Ltd, Etobicoke, ON, Canada) was administered. The animals were euthanized after 4 and 12 weeks (intravenous injection of pentobarbital; CEVA Sante Animale, Libourne, France) and the femoral condyles were harvested for histology. After retrieval of the femoral head the samples were numbered by a coding. The coding was not revealed to the researcher who performed the analysis (JB). Three sections were obtained from each femoral head and used for further analyses.
Fig. 2. Surgical procedure. (a) Incision through skin and fascia at the medial surface of the femoral condyle. (b) The drill hole (5 mm diameter, 8 mm depth) in the condyle. (c) The implant in position without filling of the gaps (Ti). (d) The implant in position with BCP-filled gaps (Ti+BCP).
Histology
After harvesting, all specimens were cleaned from adhering soft tissues. The femoral condyles including the implants were fixed in 10% formaldehyde. After fixation, the specimens were reduced in size and then dehydrated in increasing ethanol concentrations (70–100%). Finally, the specimens were embedded (non-decalcified) in methylmethacrylate (MMA) for 5 days (mixture of 300 ml MMA, 30 ml dibutylphthalate, and 5 g 2,20 azabisisobutyronitrile 98%). After polymerization in MMA, thin sections (10 lm) were prepared in longitudinal direction to the axis of the implant using an inner circular saw microtome (Leica RM 1600, Leica Microsystems, Wetzlar, Germany). Sections (3 per specimen) were stained with methylene blue and basic fuchsin and used for light microscopic assessment and histomorphometrical analysis.
Cambridge, UK). Three quantitative parameters were determined for two sides of each implant and for three sections per specimen: bone-to-implant contact (% of implant surface length; BIC), bone area (% of ROI; BA) and BCP area (% of ROI, BCP-A). BIC was analyzed along the total length of the implant (L-shaped at each side of the implant) and defined as the percentage of the implant surface in direct contact with bone without an intervening fibrous tissue layer. BA was analyzed in a ROI, using the Lshaped implant structure to set a rectangular ROI. BA was defined as the relative bone area within the ROI. BCP-A was analyzed in the same ROI and defined as the relative BCP area within the ROI. All measurements were performed using both color and morphological discrimination manually by the examiner as identification tools. Statistical analysis
Histology and histomorphometry
Histological evaluation was performed using an automated Zeiss Z1 Axio Imager microscope (Carl Zeiss Micro Imaging GmbH, G€ ottingen, Germany). Histomorphometry was performed using digital image analysis software (Leicaâ Qwin Pro-image analysis, Leica Microsystems Imaging Solutions Ltd,
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
All data are presented as mean standard deviation. Statistical analysis was performed with GraphPadâ Prism software (GraphPad Software Inc., San Diego, CA, USA), version 6.0. Gaussian distribution of the values was tested using the D’Agostino-Pearson omnibus normality test. A Student’s t-test (paired data for comparison of intra-animal data, i.e.
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Alsayed et al Titanium implants and calcium phosphate bone substitutes
comparison of Ti vs. Ti-BCP at individual time points; unpaired data for comparison of inter-animal data, i.e. comparison of temporal effects) was used to analyze the data of bone-to-implant contact, bone area, and BCP area. Statistical significance was set at P < 0.05.
Table 2. The number of implants installed, retrieved, and used for analyses No. implants used for analyses
No. implants placed
No. implants retrieved
Experimental group
4 weeks
12 weeks
4 weeks
12 weeks
4 weeks
12 weeks
Ti Ti+BCP
10 10
10 10
8* 10
10 10
8 8†
10 10
* †
Implants were lost due to improper placement. Implants were lost due to processing errors.
Results General observations
(a)
All animals remained in good health during the implantation periods of 4 and 12 weeks, without clinical indications of inflammation or adverse tissue reactions. Table 2 represents the number of implants placed, retrieved, and used for analyses. From the total number of 40 implants placed, two were lost at retrieval due to improper placement and two were lost due to processing errors. The final number of implants used for analyses was 36, with eight implants per group at 4 weeks (n = 8) and 10 implants per group at 12 weeks of implantation (n = 10).
(b)
Histological evaluation
Representative histological sections after 4 weeks and 12 weeks implantation are shown in Figs 3 and 4, respectively. Gross evaluation of the histological sections after 4 weeks of implantation showed a largely empty gap for Ti with occasional bone to implant contact along the implant surface (Fig. 3a). In contrast, Ti+BCP showed bone ingrowth along the surface of BCP particles toward the implant surface (Fig. 3b). Filling of the gaps with BCP appeared incomplete with generally an area without BCP in the lower half of the gaps. At larger magnification, the newly formed bone had a trabecular appearance with embedded osteocytes. After 12 weeks of implantation, an increase in bone ingrowth with a trabecular appearance and a random localization within the gap was observed for Ti (Fig. 4a). For Ti+BCP, an increase in bone ingrowth was observed with a particular condensation of newly-formed bone tissue in the vicinity of the implant surface (Fig. 4b). Further, an apparent increase in bone to implant contact was observed. From high magnification micrographs (Fig. 5), direct bone bonding to both BCP particles and the titanium implant can be observed.
Fig. 3. Representative histological appearance of the implant bone interface after 4 weeks. (a) Ti showing occasional bone to implant contact along the implant surface. (b) Ti+BCP showing substantial bone ingrowth. At higher magnification (lower panels), limited bone to implant contact is apparent for Ti, while newly formed bone is shown clearly around the BCP particles for Ti+BCP. * indicates BCP particles (top panels, bar = 1 mm; lower panels, bar = 100 lm).
Bone to implant contact (BIC)
For Ti, bone to implant contact increased from 15.2 11.9% at 4 weeks to 24.9 17.4% at 12 weeks (P > 0.05; 95% CI: 4.30– 23.63). For Ti+BCP, significantly higher bone to implant contact values of 41.8 18.3% at 4 weeks (P = 0.0012; 95% CI: 12.09–41.08) and 54.0 22.9% at 12 weeks (P = 0.0049; 95% CI: 10.08–48.26) were observed. Similar as for Ti, Ti+BCP demonstrated no significant increase in bone to implant contact from 4 to 12 weeks (P > 0.05; 95% CI: 7.23–31.72).
Histomorphometrical analysis
The numerical histomorphometrical data on bone to implant contact, bone area, and particle area are presented in Table 3.
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Bone area (BA)
At 4 weeks, Ti+BCP showed a significantly higher bone area (P = 0.0042; 95%
CI: 4.46–19.97) of 20.5 9.3% compared to Ti with a bone area of 8.3 5.1%. Similarly at 12 weeks, Ti+BCP showed a significantly higher bone area (P = 0.0003; 95% CI: 8.80–24.20) of 27.5 8.5% compared to Ti with a bone area of 11.0 7.9%. For both Ti and Ti+BCP, no significant temporal increase (P > 0.05; respective 95% CI: 4.18–9.55 and 1.37–15.30) in bone area was observed from 4 to 12 weeks of healing. Particle area (PA)
For Ti+BCP, the BCP particle area showed a significant decrease (P = 0.0194; 95% CI: 19.41 to 1.93) from 31.6 11.0% at 4 weeks to 21.0 7.2% at 12 weeks.
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Alsayed et al Titanium implants and calcium phosphate bone substitutes
(a)
(b)
Fig. 4. Representative histologic appearance of the implant bone interface after 12 weeks. (a) Ti showing increased bone ingrowth with trabecular morphology randomly throughout the gap. (b) Ti+BCP showing increased bone ingrowth adjacent to BCP particles and condensation of new bone tissue in the vicinity of the implant surface. At higher magnification (lower panels), Ti shows apparent bone to implant contact, while Ti+BCP shows embedding of BCP particles within newly formed bone tissue. * indicates BCP particles (top panels, bar = 1 mm; lower panels, bar = 100 lm).
(a)
(b)
Fig. 5. High magnification histomicrographs showing bone formation around BCP particles (*) with direct contact between bone and particle after both 4 (a) and 12 (b) weeks of implantation. Additionally, direct bone contact with the titanium implant can be observed (bar = 200 lm).
Discussion The aim of the present study was to comparatively evaluate the contribution of BCP bone graft to bone ingrowth and bone-to-implant contact upon simultaneous placement of BCP and titanium implants customized to present a reliable gap model. Titanium implants with unfilled gaps served as controls. The implants were placed alternately in the left and right femoral condyles of rabbits for implantation periods of 4 and 12 weeks. Using histological and histomorphometrical
analyses of bone to implant contact, bone area, and BCP particle area, a significant effect of BCP bone graft on bone to implant contact and bone area were studied and compared to controls. Additionally, degradation of the BCP graft was observed, as demonstrated by a significant decrease in BCP particle area from 4 to 12 weeks of implantation. Earlier studies have shown that the rabbit femoral condyle can serve as a reliable model for studies on implant integration and bone regeneration (Felix Lanao et al. 2011; Liao et al. 2011; Bongio et al. 2012). In
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
comparison to other species, such as primates and some rodents, the rabbit has faster skeletal change and bone turnover (Gilsanz et al. 1988; Castaneda et al. 2006). Even though it may be difficult to extrapolate results from rabbit studies to the likely human clinical response, the contralateral design of the present study makes the data more reliable for comparative evaluations and increases the power of the statistical analyses (i.e. allowing for comparison of paired data for intra-animal comparisons between Ti vs. Ti+BCP at individual time points). Still, one needs to realize that the defect morphology (i.e. half cylinder shape per gap) in the current model is likely to be different from clinically encountered defect shapes. Hydroxyapatite (HA) and b-tricalcium phosphate (b-TCP) are the two most widely tested osteoconductive bioceramics. Even though both of these materials have excellent bone bonding capabilities, HA is less resorbable than b-TCP (Jensen et al. 2006; Habibovic et al. 2008). The combination of HA and b-TCP generates biphasic calcium phosphate (BCP), which possesses the reactivity of b-TCP and the stability of HA, hence providing an optimum regarding bioactivity, new bone growth, and resistance of the material to strain (Weiss et al. 2007; Hung et al. 2011). These BCP ceramics are biocompatible, osteoconductive, and degradable through a chemical and cellular process (Gauthier et al. 1998, 2005). The efficacy of BCP is based on the preferential dissolution of the b-TCP compared to HA, allowing the manipulation of bioactivity or biodegradation by adjusting the HA/b-TCP ratio (Daculsi et al. 1989). Through the combination of a balanced rate between a more stable phase (HA) and a more soluble one (b-TCP), it was possible to formulate a BCP with a controlled dissolution rate and different mechanical properties (LeGeros & Daculsi 1990). In the present study, the BCP particles showed a significant decrease from 4 to 12 weeks of healing. This can be attributed to the resorbability and replacement with newly formed bone. The sequential degradation guided by osteoclasts as part of the bone remodeling is postulated to be most desirable for new bone formation (Moradian-Oldak et al. 2006), although the contribution of osteoclasts to BCP particle degradation could not be confirmed using sections made from plasticembedded specimens. Jensen et al. (2007) observed a different pattern in appositional bone growth with BCP in an animal model. BCP with a 60 : 40 (HA:
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Table 3. Overview of the histomorphometrical data bone implant contact, bone area, and particle area Ti Parameter BIC (%)
Bone area (% of ROI)
Particle area (% of ROI)
Mean ( SD) Median 25% percentile 75% percentile Mean ( SD) Median 25% percentile 75% percentile Mean ( SD) Median 25% percentile 75% percentile
Ti+BCP
4 weeks
12 weeks
4 weeks
12 weeks
15.2 ( 11.9) 17.5 3.5 23.7 8.3 ( 5.1) 7 4.3 13.6 n.a. n.a. n.a. n.a.
24.9 ( 17.4) 25.8 10.9 38.7 11.0 ( 7.9) 9.8 3.6 15.9 n.a. n.a. n.a. n.a.
41.8 ( 18.3) 40.1 29.4 55.3 20.5 ( 9.3) 16.2 15.0 26.2 31.6 ( 11.0) 30.1 22.4 42.6
54.0 ( 22.9) 60.5 42.7 70.8 27.5 ( 8.5) 24.6 20.9 37.7 21.0 ( 7.2) 20.6 15.1 25.8
reported a 15–30 wt% of HA in BCP as optimal (Nery et al. 1992; Yamada et al. 1997; Arinzeh et al. 2005; Zhang et al. 2007; Hahn et al. 2009; Jensen et al. 2009). Consequently, the relationship between cellular activity and the percentage of b-TCP in BCP appears inconsistent. This might be explained by the presence of various admixtures in BCP samples used by various researchers, the differences in preparation, crystallinity, porosity and surface topography of the samples, as well as the models used in the studies (Dorozhkin 2012).
Conclusion TCP) ratio did not degrade during the 24 weeks of healing. However, when the proportion was altered (20 : 80) a continuous temporal reduction of area occupied by graft material was observed (Jensen et al. 2009). In the present study, we used BCP with the ratio 60 : 40 (HA:TCP) showed relatively
lower graft particles after 12 week compared to 4 week healing. This could be due to a species difference with the rabbit model in the present study compared to the mini-pig in other studies (Jensen et al. 2007, 2009). There is no consensus regarding the ratio of HA and TCP, as the majority of studies
The addition of BCP bone substitute to fill peri-implant gaps significantly enhances both bone formation (~2.5-fold) and bone to implant contact (>2-fold) for the custommade titanium implants with two-sided gaps.
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of porous beta-tricalcium phosphate/hydroxyapatite (60/40) composite as a bone graft extender using rat calvarial bone defect model. Scientific World Journal 2013: 481789. LeGeros, R. & Daculsi, G. (1990) In vivo transformation of biphasic calcium phosphate ceramics: ultrastructural and physicochemical characterizations. Handbook of bioactive ceramics 2: 17–28. LeGeros, R.Z., Lin, S., Rohanizadeh, R., Mijares, D. & LeGeros, J.P. (2003) Biphasic calcium phosphate bioceramics: preparation, properties and applications. Journal of Materials Science: Materials in Medicine 14: 201–209. Liao, H., Walboomers, X.F., Habraken, W.J., Zhang, Z., Li, Y., Grijpma, D.W., Mikos, A.G., Wolke, J.G. & Jansen, J.A. (2011) Injectable calcium phosphate cement with plga, gelatin and ptmc microspheres in a rabbit femoral defect. Acta biomaterialia 7: 1752–1759. Mecall, R.A. & Rosenfeld, A.L. (1991) Influence of residual ridge resorption patterns on implant fixture placement and tooth position. 1. The International Journal of Periodontics and Restorative Dentistry 11: 8–23. Moradian-Oldak, J., Wen, H.B., Schneider, G.B. & Stanford, C.M. (2006) Tissue engineering strategies for the future generation of dental implants. Periodontology 2000 41: 157–176. Moy, P.K., Lundgren, S. & Holmes, R.E. (1993) Maxillary sinus augmentation: histomorphometric analysis of graft materials for maxillary sinus floor augmentation. Journal of Oral and Maxillofacial Surgery 51: 857–862. Nery, E.B., LeGeros, R.Z., Lynch, K.L. & Lee, K. (1992) Tissue response to biphasic calcium phosphate ceramic with different ratios of ha/beta tcp in periodontal osseous defects. Journal of Periodontology 63: 729–735. Nicklas, W., Baneux, P., Boot, R., Decelle, T., Deeny, A.A., Fumanelli, M. & Illgen-Wilcke, B. & FELASA (Federation of European Laboratory Animal Science Associations Working Group on Health Monitoring of Rodent and Rabbit Colonies). (2002) Recommendations for the health monitoring of rodent and rabbit colonies in breeding and experimental units. Laboratory Animals 36: 20– 42. Petrov, O., Dyulgerova, E., Petrov, L. & Popova, R. (2001) Characterization of calcium phosphate phases obtained during the preparation of sintered biphase ca-p ceramics. Materials letters 48: 162– 167.
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Piattelli, A., Scarano, A. & Mangano, C. (1996) Clinical and histologic aspects of biphasic calcium phosphate ceramic (bcp) used in connection with implant placement. Biomaterials 17: 1767–1770. Rouvillain, J.L., Lavalle, F., Pascal-Mousselard, H., Catonne, Y. & Daculsi, G. (2009) Clinical, radiological and histological evaluation of biphasic calcium phosphate bioceramic wedges filling medial high tibial valgisation osteotomies. The Knee 16: 392–397. Simion, M., Jovanovic, S.A., Trisi, P., Scarano, A. & Piattelli, A. (1998) Vertical ridge augmentation around dental implants using a membrane technique and autogenous bone or allografts in humans. The International Journal of Periodontics and Restorative Dentistry 18: 8–23. Valimaki, V.V., Yrjans, J.J., Vuorio, E. & Aro, H.T. (2005) Combined effect of bmp-2 gene transfer and bioactive glass microspheres on enhancement of new bone formation. Journal of biomedical materials research. Part A 75: 501–509. Weiss, P., Layrolle, P., Clergeau, L.P., Enckel, B., Pilet, P., Amouriq, Y., Daculsi, G. & Giumelli, B. (2007) The safety and efficacy of an injectable bone substitute in dental sockets demonstrated in a human clinical trial. Biomaterials 28: 3295– 3305. Yamada, S., Heymann, D., Bouler, J.M. & Daculsi, G. (1997) Osteoclastic resorption of calcium phosphate ceramics with different hydroxyapatite/ beta-tricalcium phosphate ratios. Biomaterials 18: 1037–1041. Yang, C., Unursaikhan, O., Lee, J.S., Jung, U.W., Kim, C.S. & Choi, S.H. (2014) Osteoconductivity and biodegradation of synthetic bone substitutes with different tricalcium phosphate contents in rabbits. Journal of biomedical materials research. Part B, Applied biomaterials 102: 80– 88. Zamet, J.S., Darbar, U.R., Griffiths, G.S., Bulman, J.S., Bragger, U., Burgin, W. & Newman, H.N. (1997) Particulate bioglass as a grafting material in the treatment of periodontal intrabony defects. Journal of Clinical Periodontology 24: 410–418. Zhang, Y., Yokogawa, Y. & Kameyama, T. (2007) Preparation of biphasic calcium phosphate porous ceramics prepared from fine powders with different particle size and its dissolution behavior in simulated body fluid. Key Engineering Materials 336: 1688–1691.
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Comparative evaluation of the combined application of titanium implants and calcium phosphate bone substitutes in a rabbit model
Authors’ affiliations: Arwa Alsayed, John A. Jansen, Jeroen J. J. P. van den Beucken, Department of Biomaterials (309), Radboudumc, Nijmegen, The Netherlands Arwa Alsayed, Department of Dentistry, Prince Sultan Military Medical City, Ministry of Defence, Riyadh, Saudi Arabia Sukumaran Anil, Department of Periodontics and Community Dentistry, College of Dentistry, King Saud University, Riyadh, Saudi Arabia
Key words: biphasic
calcium
phosphate,
bone
grafting,
bone
substitute,
histology,
Corresponding author: Jeroen JJP van den Beucken, PhD Department of Biomaterials (309), Radboudumc PO Box 9101, 6500 HB Nijmegen,The Netherlands Tel.:+31 24 3667305 Fax: +31 24 3614657 e-mail: [email protected]
rabbits. Following a randomization protocol, implants were alternately installed in one condyle
histomorphometry, intrabony defects Abstract Objectives: To study the healing of defects around titanium implants filled with biphasic calcium phosphates (BCP). Material and methods: Forty custom-made, titanium implants (Ti) with a diameter of 5 mm, and length of 8 mm, with two-sided gaps, were fabricated and installed in the femoral condyle of 20 without BCP bone substitute material (Ti) in the gaps and in the contralateral condyle gaps were filled with BCP bone substitute material (Ti+BCP). The implants were retrieved after 4 and 12 weeks of healing, after which histological and histomorphometrical analyses were done to assess the percentage of bone implant contact (BIC), the percentage of bone area (BA) and the percentage of particle area (PA) within the region of interest (ROI); the rectangular area joining the two arms of the L-shaped implant was considered as the ROI. Results: After 4 and 12 weeks of healing, Ti+BCP showed significantly higher BIC and BA values compared to Ti. Further, the BCP particles showed a significant decrease from 4 to 12 weeks of healing. The BCP particles (PA) showed a significant reduction from 31.6 11.0% at 4 weeks to 21.0 7.2% at 12 weeks. Conclusion: The addition of BCP bone substitute to fill peri-implant gaps significantly enhanced both bone formation (~2.5-fold) and bone to implant contact (>2-fold) for the custom-made titanium implants with two-sided gaps.
Date: Accepted 17 May 2014 To cite this article: Alsayed A, Anil S, Jansen JA, van den Beucken JJJP. Comparative evaluation of the combined application of titanium implants and calcium phosphate bone substitutes in a rabbit model. Clin. Oral Impl. Res. 26, 2015, 1215–1221 doi: 10.1111/clr.12435
Dental implants have revolutionized contemporary dental treatment for the replacement of missing teeth. With over 0.5 million annually placed dental implants in the United States (ADA 2011), dental implant therapy has become a widely accepted treatment modality for the replacement of missing teeth (Jung et al. 2008). The long-term success of dental implants largely depends on successful healing with safe integration into the jaw bone, which requires a suitable bone bed and appropriate implant properties (Chuang et al. 2002; Abrahamsson et al. 2009). Tooth extraction or loss and subsequent healing of the socket commonly results in osseous deformities of the alveolar ridge, including reduction in height and width. Since the quality of the bone bed is of utmost importance for a successful outcome of implant therapy (Mecall & Rosenfeld 1991), implant-supported restoration is challenged in those conditions, in which a suboptimal
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
quantity and quality of bone is present in the bone bed. Particularly, a 25% decrease in alveolar bone width is observed during the first year after tooth loss and an overall 4 mm decrease in height over the next few years. Additionally, the bone resorption process on the alveolar ridge varies in the maxilla and mandible and resorption is faster in the labial and buccal parts of the alveolar ridge (Kingsmill 1999). Ridge preservation strategies involve modulation of the physiologic modeling process that occurs following tooth extraction (Bianchi & Sanfilippo 2002). The objective of these strategies is to increase or maintain bone volume, thereby promoting osseointegration and improving implant stability upon implant placement. Sufficient alveolar bone volume and favorable architecture of the alveolar ridge are essential to achieve ideal functional and esthetic prosthetic reconstruction following implant therapy. The peri-implant gap
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following immediate implant installation require filling with bone graft material, particularly when not all four bony walls have been preserved during extraction (Becker 2003). The delayed implant placement can be preceded by bone augmentation procedures, aimed to rebuild the natural contours of the bone tissue using bone graft materials (Horowitz et al. 2012). Osseous defects in the oral cavity have been successfully managed with a variety of biological and synthetic materials, including autografts, allografts, xenografts and alloplastic materials. Although autografts are unequivocally accepted as the gold standard, donor site morbidity and the limitations on the quantity of bone that can be harvested, demands from clinicians to look for alternatives (Moy et al. 1993). In view of immunological and disease transfer risks from allogeneic bone, research has focused extensively on developing alloplastic bone substitutes that are predominantly based on ceramics (e.g. calcium phosphates CaP, calcium sulfates, and bioactive glasses) (Zamet et al. 1997). In general, these ceramic materials are renowned for their osteoconductive and bioactive properties (Ignatius et al. 2001). The most commonly used ceramics are the CaP-based ceramics hydroxyapatite (HA) and beta tricalcium phosphate (b-TCP)(Knabe et al. 2000). b-TCP is more rapidly replaced by bone than the hardly degradable HA, owing to its higher solubility (Petrov et al. 2001). Upon degradation of b-TCP through a process of dissolution and absorption, a normal bone structure in the regenerated bone can be achieved (Cutright et al. 1972; von Arx et al. 2001). The development of biphasic calcium phosphate (BCP) ceramics (i.e. containing both HA and b -TCP) has provided materials in which bioactivity and degradation are controlled based on the ratios of the components (Simion et al. 1998; LeGeros et al. 2003). Several studies have shown that BCP granules have excellent biocompatibility and bioactivity and lead to new bone formation and degradation of the biomaterial (Piattelli et al. 1996; Gauthier et al. 1998; Valimaki et al. 2005). The rate of degradation or resorption of HA/TCP ceramics can be accelerated by increasing the amount of the more soluble phase, TCP (Daculsi 1998). Studies have shown that BCPs with higher b-TCP ratio are expected to yield more replacement of biomaterial by new bone (Daculsi et al. 1990; Gauthier et al. 1998). BCP compounds containing approximately 60% of HA and 40% of b-TCP seemed to provide the optimal bone
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conductive properties (Ellinger et al. 1986; Daculsi et al. 1990; Rouvillain et al. 2009). Nery et al. (1992) reported optimal bone regeneration in surgically created bone defects with BCP containing HA/b-TCP ratio of 85/15. However recently BCP products containing higher b-TCP ratios (BCP 60/40 and BCP 20/80) have been studied in prospect of its enhanced osteoconductive effect (Lee et al. 2013; Yang et al. 2014). Because simultaneous placement of bone grafts and implants shortens the treatment time without increasing complications or reducing the success rate (Boronat et al. 2010), the aim of this study was to comparatively evaluate the contribution of BCP bone graft to bone ingrowth and bone-to-implant contact upon simultaneous placement of BCP and titanium implants customized to present a reliable gap model. The experimental procedure involved implantation of the custom-made titanium implants with or without BCP graft material in the femoral condyle of rabbits. After implantation periods of 4 and 12 weeks, histological and histomorphometrical analyses (i.e. bone area, bone graft area, and bone-to-implant contact) were performed for comparison between grafted and non-grafted implants.
Materials and methods Implants and bone substitute materials
Forty custom-made titanium implants were used for this study (Fig. 1). The implants had a cylindrical shape with a diameter of 5 mm and a length of 8 mm. The implants were grit-blasted, cleaned and sterilized before installation. Ceramic biphasic calcium phosphate (BCP; 60% HA, 40% TCP) bone substitute material was obtained from CAM Bioceramics BV (Leiden, the Netherlands). This porous
(a)
granulate consisted of particles with dimensions of 425–500 lm and a porosity of 46%. Animals
The study was conducted during September 2013. A total of twenty, skeletally mature New Zealand white rabbits (male; 6 months old; weight 4–5 kg) were used for this study. The rabbits were sourced from the central facility of the King Saud University experimental animal research. The maintenance and health monitoring of the rabbits were done as per the international recommendations (Nicklas et al. 2002). The rabbits were housed in separate cages under laboratory conditions and fed a standard pelleted rabbit diet and access to water was ad libitum. The study was approved by the Ethical Committee of the Prince Sultan Military Medical City, Saudi Arabia. The surgical procedure was based on a well-established bilateral femoral implant model in rabbits with modifications (Felix Lanao et al. 2011; Alfarraj Aldosari et al. 2014). The randomization and distribution of the implants is shown in Table 1. All rabbits were identified with a microchip implanted in the body. Surgical procedure
Surgeries were performed using aseptic routines, and under general anesthesia by intramuscular injections of a combination dose of 35 mg/kg ketamine and a dose of 5 mg/kg xylazine. The surgery was performed by two researchers (AA, SA). The surgical areas were shaved, disinfected with iodine, and then isolated with sterile drapes. Infiltration anesthesia was performed at the experimental sites. The distal femoral condyles on both sides were accessed using incisions through the skin and fascia, and the medial bone surfaces of the femoral condyles were exposed using a periosteal elevator. A drill hole (5 mm
(b)
Fig. 1. (a) Schematic illustration of the implant used in the study. Custom-made Ti implant with a diameter of 5 mm and a length of 8 mm. (b) Actual implant presentation with a grit-blasted surface.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Alsayed et al Titanium implants and calcium phosphate bone substitutes
Table 1. The randomization and the implant installation scheme used in the study Animal no.
Right side
Left side
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Ti+BCP Ti Ti+BCP Ti Ti+BCP Ti Ti+BCP Ti Ti+BCP Ti Ti+BCP Ti Ti+BCP Ti Ti+BCP Ti Ti+BCP Ti Ti+BCP Ti
Ti Ti+BCP Ti Ti+BCP Ti Ti+BCP Ti Ti+BCP Ti Ti+BCP Ti Ti+BCP Ti Ti+BCP Ti Ti+BCP Ti Ti+BCP Ti Ti+BCP
(a)
(b)
(c)
(d)
4 Weeks
12 Weeks
diameter, 8 mm depth) was created in the center of the condyle using a dental drill (KAVO, Intrasept 905, KAVO Nederland BV, Vianen, The Netherlands). A 2 mm drill bit was first used to establish a pilot hole. This defect was gradually enlarged using a series of drills with diameters of 2.8, 3.6 and 4.2 mm till the final drill bit with a diameter of 5 mm (Nobel Biocare AB, Goteborg, Sweden) under continuous cooling with saline irrigation. All debris was removed from the defect by irrigation with saline. Following a randomization protocol, implants were alternately installed on one side without BCP bone substitute material in the gaps (group: Ti) and on the contralateral side gaps were filled with BCP bone substitute material (group: Ti+ BCP; Fig. 2). The wounds were closed and allowed to heal for 4 and 12 weeks. Post-surgery pain was controlled by the administration of Fynadyneâ (1.1 mg/kg body weight; Schering-Plough Laboratories, Saint Claire, France) for 2 days. To reduce the post-operative infection risk, a single dose of enrofloxacin (Baytrilâ, 10 mg/kg body weight; Bayvet Division, Chemagro Ltd, Etobicoke, ON, Canada) was administered. The animals were euthanized after 4 and 12 weeks (intravenous injection of pentobarbital; CEVA Sante Animale, Libourne, France) and the femoral condyles were harvested for histology. After retrieval of the femoral head the samples were numbered by a coding. The coding was not revealed to the researcher who performed the analysis (JB). Three sections were obtained from each femoral head and used for further analyses.
Fig. 2. Surgical procedure. (a) Incision through skin and fascia at the medial surface of the femoral condyle. (b) The drill hole (5 mm diameter, 8 mm depth) in the condyle. (c) The implant in position without filling of the gaps (Ti). (d) The implant in position with BCP-filled gaps (Ti+BCP).
Histology
After harvesting, all specimens were cleaned from adhering soft tissues. The femoral condyles including the implants were fixed in 10% formaldehyde. After fixation, the specimens were reduced in size and then dehydrated in increasing ethanol concentrations (70–100%). Finally, the specimens were embedded (non-decalcified) in methylmethacrylate (MMA) for 5 days (mixture of 300 ml MMA, 30 ml dibutylphthalate, and 5 g 2,20 azabisisobutyronitrile 98%). After polymerization in MMA, thin sections (10 lm) were prepared in longitudinal direction to the axis of the implant using an inner circular saw microtome (Leica RM 1600, Leica Microsystems, Wetzlar, Germany). Sections (3 per specimen) were stained with methylene blue and basic fuchsin and used for light microscopic assessment and histomorphometrical analysis.
Cambridge, UK). Three quantitative parameters were determined for two sides of each implant and for three sections per specimen: bone-to-implant contact (% of implant surface length; BIC), bone area (% of ROI; BA) and BCP area (% of ROI, BCP-A). BIC was analyzed along the total length of the implant (L-shaped at each side of the implant) and defined as the percentage of the implant surface in direct contact with bone without an intervening fibrous tissue layer. BA was analyzed in a ROI, using the Lshaped implant structure to set a rectangular ROI. BA was defined as the relative bone area within the ROI. BCP-A was analyzed in the same ROI and defined as the relative BCP area within the ROI. All measurements were performed using both color and morphological discrimination manually by the examiner as identification tools. Statistical analysis
Histology and histomorphometry
Histological evaluation was performed using an automated Zeiss Z1 Axio Imager microscope (Carl Zeiss Micro Imaging GmbH, G€ ottingen, Germany). Histomorphometry was performed using digital image analysis software (Leicaâ Qwin Pro-image analysis, Leica Microsystems Imaging Solutions Ltd,
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
All data are presented as mean standard deviation. Statistical analysis was performed with GraphPadâ Prism software (GraphPad Software Inc., San Diego, CA, USA), version 6.0. Gaussian distribution of the values was tested using the D’Agostino-Pearson omnibus normality test. A Student’s t-test (paired data for comparison of intra-animal data, i.e.
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Alsayed et al Titanium implants and calcium phosphate bone substitutes
comparison of Ti vs. Ti-BCP at individual time points; unpaired data for comparison of inter-animal data, i.e. comparison of temporal effects) was used to analyze the data of bone-to-implant contact, bone area, and BCP area. Statistical significance was set at P < 0.05.
Table 2. The number of implants installed, retrieved, and used for analyses No. implants used for analyses
No. implants placed
No. implants retrieved
Experimental group
4 weeks
12 weeks
4 weeks
12 weeks
4 weeks
12 weeks
Ti Ti+BCP
10 10
10 10
8* 10
10 10
8 8†
10 10
* †
Implants were lost due to improper placement. Implants were lost due to processing errors.
Results General observations
(a)
All animals remained in good health during the implantation periods of 4 and 12 weeks, without clinical indications of inflammation or adverse tissue reactions. Table 2 represents the number of implants placed, retrieved, and used for analyses. From the total number of 40 implants placed, two were lost at retrieval due to improper placement and two were lost due to processing errors. The final number of implants used for analyses was 36, with eight implants per group at 4 weeks (n = 8) and 10 implants per group at 12 weeks of implantation (n = 10).
(b)
Histological evaluation
Representative histological sections after 4 weeks and 12 weeks implantation are shown in Figs 3 and 4, respectively. Gross evaluation of the histological sections after 4 weeks of implantation showed a largely empty gap for Ti with occasional bone to implant contact along the implant surface (Fig. 3a). In contrast, Ti+BCP showed bone ingrowth along the surface of BCP particles toward the implant surface (Fig. 3b). Filling of the gaps with BCP appeared incomplete with generally an area without BCP in the lower half of the gaps. At larger magnification, the newly formed bone had a trabecular appearance with embedded osteocytes. After 12 weeks of implantation, an increase in bone ingrowth with a trabecular appearance and a random localization within the gap was observed for Ti (Fig. 4a). For Ti+BCP, an increase in bone ingrowth was observed with a particular condensation of newly-formed bone tissue in the vicinity of the implant surface (Fig. 4b). Further, an apparent increase in bone to implant contact was observed. From high magnification micrographs (Fig. 5), direct bone bonding to both BCP particles and the titanium implant can be observed.
Fig. 3. Representative histological appearance of the implant bone interface after 4 weeks. (a) Ti showing occasional bone to implant contact along the implant surface. (b) Ti+BCP showing substantial bone ingrowth. At higher magnification (lower panels), limited bone to implant contact is apparent for Ti, while newly formed bone is shown clearly around the BCP particles for Ti+BCP. * indicates BCP particles (top panels, bar = 1 mm; lower panels, bar = 100 lm).
Bone to implant contact (BIC)
For Ti, bone to implant contact increased from 15.2 11.9% at 4 weeks to 24.9 17.4% at 12 weeks (P > 0.05; 95% CI: 4.30– 23.63). For Ti+BCP, significantly higher bone to implant contact values of 41.8 18.3% at 4 weeks (P = 0.0012; 95% CI: 12.09–41.08) and 54.0 22.9% at 12 weeks (P = 0.0049; 95% CI: 10.08–48.26) were observed. Similar as for Ti, Ti+BCP demonstrated no significant increase in bone to implant contact from 4 to 12 weeks (P > 0.05; 95% CI: 7.23–31.72).
Histomorphometrical analysis
The numerical histomorphometrical data on bone to implant contact, bone area, and particle area are presented in Table 3.
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Bone area (BA)
At 4 weeks, Ti+BCP showed a significantly higher bone area (P = 0.0042; 95%
CI: 4.46–19.97) of 20.5 9.3% compared to Ti with a bone area of 8.3 5.1%. Similarly at 12 weeks, Ti+BCP showed a significantly higher bone area (P = 0.0003; 95% CI: 8.80–24.20) of 27.5 8.5% compared to Ti with a bone area of 11.0 7.9%. For both Ti and Ti+BCP, no significant temporal increase (P > 0.05; respective 95% CI: 4.18–9.55 and 1.37–15.30) in bone area was observed from 4 to 12 weeks of healing. Particle area (PA)
For Ti+BCP, the BCP particle area showed a significant decrease (P = 0.0194; 95% CI: 19.41 to 1.93) from 31.6 11.0% at 4 weeks to 21.0 7.2% at 12 weeks.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Alsayed et al Titanium implants and calcium phosphate bone substitutes
(a)
(b)
Fig. 4. Representative histologic appearance of the implant bone interface after 12 weeks. (a) Ti showing increased bone ingrowth with trabecular morphology randomly throughout the gap. (b) Ti+BCP showing increased bone ingrowth adjacent to BCP particles and condensation of new bone tissue in the vicinity of the implant surface. At higher magnification (lower panels), Ti shows apparent bone to implant contact, while Ti+BCP shows embedding of BCP particles within newly formed bone tissue. * indicates BCP particles (top panels, bar = 1 mm; lower panels, bar = 100 lm).
(a)
(b)
Fig. 5. High magnification histomicrographs showing bone formation around BCP particles (*) with direct contact between bone and particle after both 4 (a) and 12 (b) weeks of implantation. Additionally, direct bone contact with the titanium implant can be observed (bar = 200 lm).
Discussion The aim of the present study was to comparatively evaluate the contribution of BCP bone graft to bone ingrowth and bone-to-implant contact upon simultaneous placement of BCP and titanium implants customized to present a reliable gap model. Titanium implants with unfilled gaps served as controls. The implants were placed alternately in the left and right femoral condyles of rabbits for implantation periods of 4 and 12 weeks. Using histological and histomorphometrical
analyses of bone to implant contact, bone area, and BCP particle area, a significant effect of BCP bone graft on bone to implant contact and bone area were studied and compared to controls. Additionally, degradation of the BCP graft was observed, as demonstrated by a significant decrease in BCP particle area from 4 to 12 weeks of implantation. Earlier studies have shown that the rabbit femoral condyle can serve as a reliable model for studies on implant integration and bone regeneration (Felix Lanao et al. 2011; Liao et al. 2011; Bongio et al. 2012). In
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
comparison to other species, such as primates and some rodents, the rabbit has faster skeletal change and bone turnover (Gilsanz et al. 1988; Castaneda et al. 2006). Even though it may be difficult to extrapolate results from rabbit studies to the likely human clinical response, the contralateral design of the present study makes the data more reliable for comparative evaluations and increases the power of the statistical analyses (i.e. allowing for comparison of paired data for intra-animal comparisons between Ti vs. Ti+BCP at individual time points). Still, one needs to realize that the defect morphology (i.e. half cylinder shape per gap) in the current model is likely to be different from clinically encountered defect shapes. Hydroxyapatite (HA) and b-tricalcium phosphate (b-TCP) are the two most widely tested osteoconductive bioceramics. Even though both of these materials have excellent bone bonding capabilities, HA is less resorbable than b-TCP (Jensen et al. 2006; Habibovic et al. 2008). The combination of HA and b-TCP generates biphasic calcium phosphate (BCP), which possesses the reactivity of b-TCP and the stability of HA, hence providing an optimum regarding bioactivity, new bone growth, and resistance of the material to strain (Weiss et al. 2007; Hung et al. 2011). These BCP ceramics are biocompatible, osteoconductive, and degradable through a chemical and cellular process (Gauthier et al. 1998, 2005). The efficacy of BCP is based on the preferential dissolution of the b-TCP compared to HA, allowing the manipulation of bioactivity or biodegradation by adjusting the HA/b-TCP ratio (Daculsi et al. 1989). Through the combination of a balanced rate between a more stable phase (HA) and a more soluble one (b-TCP), it was possible to formulate a BCP with a controlled dissolution rate and different mechanical properties (LeGeros & Daculsi 1990). In the present study, the BCP particles showed a significant decrease from 4 to 12 weeks of healing. This can be attributed to the resorbability and replacement with newly formed bone. The sequential degradation guided by osteoclasts as part of the bone remodeling is postulated to be most desirable for new bone formation (Moradian-Oldak et al. 2006), although the contribution of osteoclasts to BCP particle degradation could not be confirmed using sections made from plasticembedded specimens. Jensen et al. (2007) observed a different pattern in appositional bone growth with BCP in an animal model. BCP with a 60 : 40 (HA:
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Table 3. Overview of the histomorphometrical data bone implant contact, bone area, and particle area Ti Parameter BIC (%)
Bone area (% of ROI)
Particle area (% of ROI)
Mean ( SD) Median 25% percentile 75% percentile Mean ( SD) Median 25% percentile 75% percentile Mean ( SD) Median 25% percentile 75% percentile
Ti+BCP
4 weeks
12 weeks
4 weeks
12 weeks
15.2 ( 11.9) 17.5 3.5 23.7 8.3 ( 5.1) 7 4.3 13.6 n.a. n.a. n.a. n.a.
24.9 ( 17.4) 25.8 10.9 38.7 11.0 ( 7.9) 9.8 3.6 15.9 n.a. n.a. n.a. n.a.
41.8 ( 18.3) 40.1 29.4 55.3 20.5 ( 9.3) 16.2 15.0 26.2 31.6 ( 11.0) 30.1 22.4 42.6
54.0 ( 22.9) 60.5 42.7 70.8 27.5 ( 8.5) 24.6 20.9 37.7 21.0 ( 7.2) 20.6 15.1 25.8
reported a 15–30 wt% of HA in BCP as optimal (Nery et al. 1992; Yamada et al. 1997; Arinzeh et al. 2005; Zhang et al. 2007; Hahn et al. 2009; Jensen et al. 2009). Consequently, the relationship between cellular activity and the percentage of b-TCP in BCP appears inconsistent. This might be explained by the presence of various admixtures in BCP samples used by various researchers, the differences in preparation, crystallinity, porosity and surface topography of the samples, as well as the models used in the studies (Dorozhkin 2012).
Conclusion TCP) ratio did not degrade during the 24 weeks of healing. However, when the proportion was altered (20 : 80) a continuous temporal reduction of area occupied by graft material was observed (Jensen et al. 2009). In the present study, we used BCP with the ratio 60 : 40 (HA:TCP) showed relatively
lower graft particles after 12 week compared to 4 week healing. This could be due to a species difference with the rabbit model in the present study compared to the mini-pig in other studies (Jensen et al. 2007, 2009). There is no consensus regarding the ratio of HA and TCP, as the majority of studies
The addition of BCP bone substitute to fill peri-implant gaps significantly enhances both bone formation (~2.5-fold) and bone to implant contact (>2-fold) for the custommade titanium implants with two-sided gaps.
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