Ian Yip Li Ma Nikos Mattheos Michel Dard Niklaus P. Lang
Authors’ affiliations: Ian Yip, Li Ma, Nikos Mattheos, Niklaus P. Lang, Oral Rehabilitation, Faculty of Dentistry, The University of Hong Kong, Hong Kong, Michel Dard, Department of Periodontology and Implant Dentistry, College of Dentistry, New York University, New York, NY, USA
Defect healing with various bone substitutes
Key words: animal study, biomaterials, bone substitutes Abstract Objective: Biphasic calcium phosphates (BCPs), mixture of hydroxyapatite (HA) and b-tricalcium phosphate (b-TCP) are synthetic bone substitutes, which are increasingly used in adjunctive implant and periodontal surgery. The aim of this study was to evaluate the rate and amount of bone
Corresponding author: Dr Ian Yip 4/F, Prince Philip Dental Hospital, 34 Hospital Road Sai Ying Pun Hong Kong Hong Kong Tel.: 28590315 Fax: 28590381 e-mail: [email protected]
regeneration in defects filled with three different BCPs in relation to deproteinized bovine bone mineral (DBBM). Method: Ten New Zealand rabbits were used in the experiment. Four defects of 6 mm in diameter were prepared in each rabbit, and they were filled with different biomaterials: BCP with HA/b-TCP ratio of 60/40 (BCPG1), ratio of 10/90 (BCPG2), a BCP with polylactide incorporated (moldable BCP) and DBBM. Group A (n = 5) rabbits were sacrificed after 3 months, and group B (n = 5) were sacrificed 6 months after surgery. Histological and histomorphometric analyses were performed. Mean percentages of mineralized new bone (%MNB), bone marrow (%BM), residual grafting material (%RG) and soft tissue (%ST) were calculated for each bone substitute. Results: Percentages of MNB in defects filled with the four bone substitutes were comparable after 3 months and 6 months. Amount of MNB regenerated for moldable BCP and DBBM after 6 months were significantly higher than after 3 months (P < 0.05), whereas those for BCPG1 and BCPG2 did not show significant change. Percentage RG was significantly higher in moldable BCP compared with BCPG1 (P < 0.05) after 3 months. Conclusion: After 3 months, the granules-form synthetic materials performed better than DBBM in terms of bone regeneration. The grafting materials performed similarly after 6 months of healing. Addition of polylactide in moldable BCP may slow down osteogenesis in grafted defects.
Introduction
Date: Accepted 11 March 2014 To cite this article: Yip I, Ma L, Mattheos N, Dard M, Lang NP. Healing of calvaria defect with various bone substitutes. Clin. Oral Impl. Res. 00, 2014, 1–9 doi: 10.1111/clr.12395
Oral implant therapies have become one of the main treatment options in replacement of missing teeth during the past decades. To deal with anatomic limitations, various adjunctive procedures for augmentation of the atrophic alveolar bone prior to implant placement have been advocated. These procedures include autogenous bone block grafting, sinus elevation techniques, guided bone regeneration and alveolar ridge preservation. Bone-grafting procedures are typically required in such alveolar ridge augmentations. The bone grafts could be obtained from the host (autografts), from other human donor (allografts), from other species (xenografts) or synthetic. Autogenous bone grafts, harvested extraorally or intra-orally, have been often considered as the gold standard of bone-grafting
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
materials for bone regeneration because viable bone cells could be obtained and therefore the graft is osteogenic. The presence of growth factors such as bone morphogenic proteins would also enhance bone formation (osteoinduction). However, obtaining a sizable autogenous graft would create donor site morbidity. The significant resorption often observed in autologous bone grafts can also jeopardize the outcomes (Johansson et al. 2001; Smolka et al. 2006; Sbordone et al. 2009, 2012, 2013). As the demand for minimally invasive dental implant procedures has increased, various bone graft substitutes had been developed and became commercially available. These bone-grafting materials could be used alone or being mixed with autogenous bone particles in an adjunctive procedure. The ideal properties of bone-grafting materials are as follows:
1
Yip et al Defect healing with various bone substitutes
• • • • • • • •
osteoinductive osteoconductive biocompatible totally replaced by bone appropriate resorption time in relation to bone formation maintain graft volume stability satisfactory mechanical properties no risk of disease transmission
Xenografts of animal origin had been a viable alternative material in bone regeneration. The absence of protein component in xenografts greatly reduced the risk of immunogenicity and disease transmission. Deproteinized bovine bone mineral (DBBM) was extensively investigated and became very popular due to the favorable clinical results when used as an adjunct in implant therapies. In an animal experiment conducted by Jensen et al. (2012), sinus grafts with increasing ratios of DBBM preserved volume of the grafts significantly better than autogenous grafts alone. One main drawback of the material was the slow and incomplete resorption, and thus a considerable volume of the graft would not be replaced by new bone (Valentini et al. 2000; Mordenfeld et al. 2010; Kolerman et al. 2012; Lee et al. 2012). Mordenfeld et al. (2010) obtained specimens from DBBM augmented sinuses 11 years following surgery and found that the mean percentage of residual particles was 17.3%. The utilization of synthetic bone graft materials was the next development. Calcium phosphates bone substitutes were among the most popular synthetic grafting materials in the past decades. Calcium and phosphates are building blocks of bone. In natural bone, 90–95% of inorganic phase consists of hydroxyapatite (HA). HA has a composition of Ca10(PO4)6(OH)2, which is a stable form of calcium phosphates. Synthetic HA and b-tricalcium phosphate (b-TCP), with a composition of Ca3(PO4)2, had been the most commonly used calcium phosphates in reconstructive surgery. They were biocompatible, bioactive and osteoconductive. However, there were limitations in the use of HA and b-TCP as bone substitutes. The relatively stable HA resorbed slowly and hence would limit the rate and amount of bone formation. On the other hand, because b-TCP resorbs relatively fast, the material may have been resorbed before adequate osteogenesis occurred. The function of being an osteoconductive scaffold may be compromised and hence the graft may loss some volume. In view of this, bone graft substitutes that consisted of mixtures of HA and b-TCP, known
2 |
Clin. Oral Impl. Res. 0, 2014 / 1–9
as biphasic calcium phosphates (BCP), were designed and evaluated. The concept of combing HA and b-TCP was developed by a group of researchers from the United States and France in the 1980s. The idea was to achieve an optimal balance between the two materials, so that the material became soluble but dissolved gradually. After implantation of BCP, the calcium and phosphate ions would be released during dissolution and seeding new bone formation (Daculsi et al. 2003). BCP with HA/b-TCP ratio of 60/40 (BCP 60/40) had been made commercially available as bone substitutes for adjunctive implant and periodontal therapy. The characteristics of bone formation of BCP 60/40 in relation to DBBM and other bone substitutes were evaluated in several animal and clinical studies. In a rabbit calvarial model, Rokn et al. (2011) showed that the amount of new bone formation and residual grafting materials were similar for both small and large BCP 60/40 particles and DBBM. Schwarz et al. (2007) compared the histological findings following guided bone regeneration around implants in dogs with BCP 60/40 and DBBM. Both grafting materials showed significant amount of bone regeneration compared with non-filled control. The remaining graft particles showed good biocompatibility and complete integration with no osteoclastic activities. The effectiveness of BCP 60/40 as a bone substitute was also evaluated in human studies. In two studies involving sinus grafting (Cordaro et al. 2008; Froum et al. 2008), BCP 60/40 and DBBM were compared histologically after healing periods of 6–8 months. The two studies did not show differences in vital bone formation between the two bone substitutes. Cordaro and coworkers found that the amounts of residual grafting materials between the two materials were similar. In contrast, Froum and colleagues reported lower amount of residual grafting materials for BCP 60/40 in relation to DBBM (26.6% vs. 37.7%). By varying the HA/b-TCP ratios, the characteristics of the grafting material could be altered. By reducing the HA/b-TCP ratio, the material would become more reactive. Jensen et al. (2009) compared three BCPs with varying HA/b-TCP ratios (20/80, 60/40 and 80/20) in mandibular defects of mini-pigs. Bone formation in defects with BCP 20/80 was similar to those grafted with autogenous bone, whereas those filled with BCP 60/40 and 80/ 20 were found to be similar to DBBM-filled defects. The amount of bone regeneration and resorption of the materials were inver-
sely proportional to HA/b-TCP ratios. The effect of further reducing the HA/b-TCP ratio on bone regeneration had not yet been investigated. Biphasic calcium phosphates were conventionally fabricated in blocks or powders. To improve clinical handling in some clinical situations, a newer formulation of BCPs was moldable. This BCP consists of granules, which are coated with bioresorbable polymers. After mixing with plasticizers, the material will harden. This may potentially eliminate the need of using a barrier membrane. Schmidlin et al. (2013) evaluated the properties of such moldable BCP in an animal study. The authors found that there were no differences in biocompatibility, bone formation and resorption between moldable BCP and DBBM after 16 weeks. On the other hand, the addition of polylactic acid to BCP was shown to delay bone formation in another animal study (Habibovic et al. 2008). As BCPs were developed into new formulations and mode of delivery, it is essential to evaluate the effectiveness of such grafting materials in animal models and also compare the outcomes with that of the currently accepted gold standards. The aim of this study is to compare the amount of bone regeneration and resorption of the bone substitutes in defects grafted with three different BCPs: BCPG1, BCPG2 and moldable BCP as compared to DBBM.
Material and methods The study was approved by Committee of the Use of Live Animals for Teaching and Research, the University of Hong Kong. Ten New Zealand rabbits (6 months old, 3.5– 4 kg) were used. Pre-operatively, they were kept and taken care of by the Laboratory Animal Unit of the Faculty of Medicine, the University of Hong Kong under veterinary supervision. The 10 rabbits were divided into group A and group B with five rabbits in each group. The animals were sacrificed after 3 months (for group A) and 6 months (for group B) following the operation (Fig. 1).
Surgery Group A (5)* Group B (5)
Analyses Group A (5)
Beginning
3 months
Analyses Group B (5)
6 months
Fig. 1. Time-line for the animal experiments of group A and group B. *Number of samples in parentheses.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Yip et al Defect healing with various bone substitutes
Table 1. Particulars of the grafting materials tested in the animal experiment Bone substitute
Abbreviation
Granule size (mm)
Name of product
Manufacturer
Deproteinized bovine bone mineral Biphasic calcium phosphate 60% of HA and 40% of b- TCP Biphasic calcium phosphate 60% of HA and 40% of b-TCP + Polylactide coating (PLGA) Biphasic calcium phosphate 10% of HA and 90% of b-TCP
DBBM BCPG1
0.25–1 0.5–1
Bio-Ossâ Biphasic Calcium Phosphate G1
Geistlich Pharma AG, Wolhusen, Switzerland Cam Bioceramics B.V., Leiden, the Netherlands
Moldable BCP
0.45–1
Easy -GraftTM CRYSTAL
Degradable Solution AG, Schleiren, Switzerland
BCPG2
0.5–1
Biphasic Calcium Phosphate G2
Revisios B.V., Bilthoven, the Netherlands
Operation
The surgical methodology on rabbit calvaria described by Jung et al. (2006) was adopted in this study. Pre-emptive analgesics (buprenorphine 0.05 mg/kg, subcutaneously) were given to the animals pre-operatively. Ketamine (35 mg/kg), acepromazine (1 mg/kg) and xylazine (5 mg/kg) were given intramuscularly for the general anesthesia procedure. The hair over the rabbit cranium was shaved and disinfected by iodine solution. A sagittal incision was made from the nasal region to the mid-sagittal crest. A full thickness flap was raised to expose the frontal, parietal and occipital bone, and the flap was retracted. Under copious irrigation, four defects in the diameter of 6 mm were prepared with a trephine in each calvarium. The defect was finalized with preparation of inner table with round bur and care was taken not to damage the dura mater. The four defects in each animal were filled with four different bone substitutes. Three different BCPs and DBBM were used (Table 1). The two granules-form BCPs with HA/b-TCP ratio of 60/40, 10/90, and DBBM were packed into the defect after hydration with normal saline. An injectable and moldable form of BCP 60/40, in which polylactide coating (PLGA) was incorporated into the material, were injected into the defect after mixing of the liquid and powder phases (Fig. 2). The four defects were covered with a single resorbable barrier membrane (BioGideâ; Geistlich Pharma AG, Wolhusen, Switzerland). The scalp incision was then closed with silk sutures. In one animal in group A, one of the four defects was left unfilled but covered with resorbable membrane, to serve as a negative control. Post-operative care
Antibiotics (enrofloxacin 5–10 mg/kg for 3 days, then meloxicam 0.2 mg/kg thereafter for 14 days, subcutaneously) and analgesics (buprenorphine 0.05 mg/kg, subcutaneously) were given post-operatively. Sutures were removed 14 days after surgery. The condi-
Fig. 3. Areas (left, center and right) selected for histomorphometric analysis.
Fig. 2. The four surgically created calvarial defects were filled with various bone substitute.
tions and well-beings of the animals were monitored before being sacrificed. The animals were euthanized with pentobarbital (150 mg/kg) at according to the time-line of the experiment. Histological and histomorphometric analysis
The calvaria that contained the grafting materials were harvested. The specimens were fixed with 10% paraformaldehyde solution, followed by decalcification with 14.5% EDTA acid. Histological sectioning with a thickness of 6 lm was cut and then stained with hematoxylin and eosin (H & E) stain. In each H & E stained slide series, 1 slide was selected for analysis. The slides were examined with light microscope (Nikonâ Eclipse VL100POL, Tokyo, Japan) incorporated to a digital video camera (Nikonâ Digital Sight DS-Ri1). The unit was connected to a computer with image analysis software (NISElements AR 3.00, Nikonâ Laboratory Imaging software, Japan). The defects were divided into three areas: left, center and right (Fig. 3). The center of each area was analyzed under a magnification of 2609. Histomorphometric analysis was performed using a modification of the methods described by Schroeder & M€ unzel-Pedrazzoli (1973). Square grids with 10 9 10 grid points were generated over the tissues in the middle of each area. Four tissue structures were
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
identified and measured at the intersection of each grid point. The structures include
• • • •
Mineralized new bone (MNB) Bone marrow (BM) Residual grafting material (RG) Soft tissue (ST)
The volumetric density of different structures in the defects was calculated (Fig. 4). The histomorphometric analysis was performed by a single investigator (IY). Intraexaminer reliability was determined by remeasurement of eight slides (two slides for each grafting material of group A) 1 week after the first examination. Statistical analysis
The data were entered and analyzed using IBM SPSS software (IBM Corporation, New York, NY, USA). The intra-examination reliability was evaluated with intra-class correlation coefficient (ICC). ANOVA test was used to determine the difference between the volumetric densities among the four grafting materials. Two-sample t-test was used to compare the volumetric densities of tissues for the same bone substitute between group A and B. The level of significance was set at a ≤ 0.05.
Results Forty defects in 10 rabbits were created surgically as planned. The defects were filled with the four biomaterials accordingly and were
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Clin. Oral Impl. Res. 0, 2014 / 1–9
Yip et al Defect healing with various bone substitutes
Fig. 4. Histomorphometric analysis with 10 9 10 grid points. MNB, mineralized new bone; BM, bone marrow; RG, residual grafting material; ST, soft tissue.
covered with bioresorbable membranes. The intra-operative and post-operative conditions of the animals were stable without any complications. By the time of animal sacrifice, there were no signs of wound dehiscence, infections nor membrane exposures. Histological observations
The morphology of the negative control defect without bone substitutes was depicted in Fig. 5a. Complete bridging of the defect was observed. However, a depression was present at the surface of the defect, which implied that the resorbable membrane was not well supported. The area underneath the membrane was mainly bone marrow, whereas MNB was present mainly in the deeper half of the defect. In contrast, the superficial part of the defects grafted with bone substitutes was well supported and MNB could be found in the full thickness of the grafted site (Fig. 5b). The residual grafting materials were observed throughout the defects. Mineralized new bone and bone marrow were more evenly distributed inside the defect. Histological morphology of defects filled with various bone graft materials is shown in Fig. 6. Grafting materials were identified, and each material took up different degree of H & E stain. Particles of BCPG1 were not identi-
fied; the reason could be the materials did not take up H & E stains. The shape of the particles, however, was assumed to be similar to the empty spaces left among different tissues (Fig. 6b). Particles of moldable BCP and BCPG2 were larger in size. DBBM particles appeared fragmented in residual graft spaces, and the BCPG1 particles were more slender. Moldable BCP particles were more rounded and BCPG2 were irregular in shape. Moldable BCP and BCPG2 particles appeared to have more interfacial contact with bone comparing with DBBM. When the samples from group A and group B were compared, the sizes of the grafting particles were smaller in group B. The grafting particles also appeared less irregular. No foreign body reactions adjacent to the four grafting materials could be identified in any of the samples. Histomorphometric analyses were carried out at the left, center and right side of the defects of each sample, and the three areas were analyzed as described. The mean percentages of MNB, bone marrow, residual grafting particles and soft tissues were calculated. Intra-examiner calibration
Intra-examiner reliability for the histomorphometric analysis was carried out with remeasurement of 10 slides 1 week after the first measurement using the samples from group A (two for DBBM, three for BCPG1, three for moldable BCP and two for BCPG2). When the first and second data sets were compared, the ICCs were high (>0.9 for all measurement parameters of % MNB, BM, RG and ST). This indicated the histological measurements were highly reproducible. Histomorphometric analyses of Group A
The mean percentages of various tissues in defects grafted with the four grafting materials for group A are listed in Table 2 and Fig. 7. Mineralized new bone was filled up to one-fourth to half of the defects, with defects filled with BCPG1 having greatest amount of
MNB regeneration (46.33%), followed by BCPG2 (38.42%). Moldable BCP had the least amount of bone regenerated (25%) after 3 months. However, there were no statistically significant differences in %MNB among the groups. The mean percentages of bone marrow were variable among the groups. The proportion was lowest in defects with DBBM (3%), and it was statistically lower than defects with moldable BCP and BCPG2 (P < 0.01) (Table 3). Residual grafting materials occupied at least a quarter of the defects after 3 months. Amount of residual grafting material were highest in defects filled with moldable BCP (54.8%) followed by DBBM (45.33%). Defects with BCPG1 had the lowest percentage of residual materials (26.67%), and it was significantly lower than that of moldable BCP (P < 0.05). Mean percentage of soft tissues in the defects were
Authors’ affiliations: Ian Yip, Li Ma, Nikos Mattheos, Niklaus P. Lang, Oral Rehabilitation, Faculty of Dentistry, The University of Hong Kong, Hong Kong, Michel Dard, Department of Periodontology and Implant Dentistry, College of Dentistry, New York University, New York, NY, USA
Defect healing with various bone substitutes
Key words: animal study, biomaterials, bone substitutes Abstract Objective: Biphasic calcium phosphates (BCPs), mixture of hydroxyapatite (HA) and b-tricalcium phosphate (b-TCP) are synthetic bone substitutes, which are increasingly used in adjunctive implant and periodontal surgery. The aim of this study was to evaluate the rate and amount of bone
Corresponding author: Dr Ian Yip 4/F, Prince Philip Dental Hospital, 34 Hospital Road Sai Ying Pun Hong Kong Hong Kong Tel.: 28590315 Fax: 28590381 e-mail: [email protected]
regeneration in defects filled with three different BCPs in relation to deproteinized bovine bone mineral (DBBM). Method: Ten New Zealand rabbits were used in the experiment. Four defects of 6 mm in diameter were prepared in each rabbit, and they were filled with different biomaterials: BCP with HA/b-TCP ratio of 60/40 (BCPG1), ratio of 10/90 (BCPG2), a BCP with polylactide incorporated (moldable BCP) and DBBM. Group A (n = 5) rabbits were sacrificed after 3 months, and group B (n = 5) were sacrificed 6 months after surgery. Histological and histomorphometric analyses were performed. Mean percentages of mineralized new bone (%MNB), bone marrow (%BM), residual grafting material (%RG) and soft tissue (%ST) were calculated for each bone substitute. Results: Percentages of MNB in defects filled with the four bone substitutes were comparable after 3 months and 6 months. Amount of MNB regenerated for moldable BCP and DBBM after 6 months were significantly higher than after 3 months (P < 0.05), whereas those for BCPG1 and BCPG2 did not show significant change. Percentage RG was significantly higher in moldable BCP compared with BCPG1 (P < 0.05) after 3 months. Conclusion: After 3 months, the granules-form synthetic materials performed better than DBBM in terms of bone regeneration. The grafting materials performed similarly after 6 months of healing. Addition of polylactide in moldable BCP may slow down osteogenesis in grafted defects.
Introduction
Date: Accepted 11 March 2014 To cite this article: Yip I, Ma L, Mattheos N, Dard M, Lang NP. Healing of calvaria defect with various bone substitutes. Clin. Oral Impl. Res. 00, 2014, 1–9 doi: 10.1111/clr.12395
Oral implant therapies have become one of the main treatment options in replacement of missing teeth during the past decades. To deal with anatomic limitations, various adjunctive procedures for augmentation of the atrophic alveolar bone prior to implant placement have been advocated. These procedures include autogenous bone block grafting, sinus elevation techniques, guided bone regeneration and alveolar ridge preservation. Bone-grafting procedures are typically required in such alveolar ridge augmentations. The bone grafts could be obtained from the host (autografts), from other human donor (allografts), from other species (xenografts) or synthetic. Autogenous bone grafts, harvested extraorally or intra-orally, have been often considered as the gold standard of bone-grafting
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
materials for bone regeneration because viable bone cells could be obtained and therefore the graft is osteogenic. The presence of growth factors such as bone morphogenic proteins would also enhance bone formation (osteoinduction). However, obtaining a sizable autogenous graft would create donor site morbidity. The significant resorption often observed in autologous bone grafts can also jeopardize the outcomes (Johansson et al. 2001; Smolka et al. 2006; Sbordone et al. 2009, 2012, 2013). As the demand for minimally invasive dental implant procedures has increased, various bone graft substitutes had been developed and became commercially available. These bone-grafting materials could be used alone or being mixed with autogenous bone particles in an adjunctive procedure. The ideal properties of bone-grafting materials are as follows:
1
Yip et al Defect healing with various bone substitutes
• • • • • • • •
osteoinductive osteoconductive biocompatible totally replaced by bone appropriate resorption time in relation to bone formation maintain graft volume stability satisfactory mechanical properties no risk of disease transmission
Xenografts of animal origin had been a viable alternative material in bone regeneration. The absence of protein component in xenografts greatly reduced the risk of immunogenicity and disease transmission. Deproteinized bovine bone mineral (DBBM) was extensively investigated and became very popular due to the favorable clinical results when used as an adjunct in implant therapies. In an animal experiment conducted by Jensen et al. (2012), sinus grafts with increasing ratios of DBBM preserved volume of the grafts significantly better than autogenous grafts alone. One main drawback of the material was the slow and incomplete resorption, and thus a considerable volume of the graft would not be replaced by new bone (Valentini et al. 2000; Mordenfeld et al. 2010; Kolerman et al. 2012; Lee et al. 2012). Mordenfeld et al. (2010) obtained specimens from DBBM augmented sinuses 11 years following surgery and found that the mean percentage of residual particles was 17.3%. The utilization of synthetic bone graft materials was the next development. Calcium phosphates bone substitutes were among the most popular synthetic grafting materials in the past decades. Calcium and phosphates are building blocks of bone. In natural bone, 90–95% of inorganic phase consists of hydroxyapatite (HA). HA has a composition of Ca10(PO4)6(OH)2, which is a stable form of calcium phosphates. Synthetic HA and b-tricalcium phosphate (b-TCP), with a composition of Ca3(PO4)2, had been the most commonly used calcium phosphates in reconstructive surgery. They were biocompatible, bioactive and osteoconductive. However, there were limitations in the use of HA and b-TCP as bone substitutes. The relatively stable HA resorbed slowly and hence would limit the rate and amount of bone formation. On the other hand, because b-TCP resorbs relatively fast, the material may have been resorbed before adequate osteogenesis occurred. The function of being an osteoconductive scaffold may be compromised and hence the graft may loss some volume. In view of this, bone graft substitutes that consisted of mixtures of HA and b-TCP, known
2 |
Clin. Oral Impl. Res. 0, 2014 / 1–9
as biphasic calcium phosphates (BCP), were designed and evaluated. The concept of combing HA and b-TCP was developed by a group of researchers from the United States and France in the 1980s. The idea was to achieve an optimal balance between the two materials, so that the material became soluble but dissolved gradually. After implantation of BCP, the calcium and phosphate ions would be released during dissolution and seeding new bone formation (Daculsi et al. 2003). BCP with HA/b-TCP ratio of 60/40 (BCP 60/40) had been made commercially available as bone substitutes for adjunctive implant and periodontal therapy. The characteristics of bone formation of BCP 60/40 in relation to DBBM and other bone substitutes were evaluated in several animal and clinical studies. In a rabbit calvarial model, Rokn et al. (2011) showed that the amount of new bone formation and residual grafting materials were similar for both small and large BCP 60/40 particles and DBBM. Schwarz et al. (2007) compared the histological findings following guided bone regeneration around implants in dogs with BCP 60/40 and DBBM. Both grafting materials showed significant amount of bone regeneration compared with non-filled control. The remaining graft particles showed good biocompatibility and complete integration with no osteoclastic activities. The effectiveness of BCP 60/40 as a bone substitute was also evaluated in human studies. In two studies involving sinus grafting (Cordaro et al. 2008; Froum et al. 2008), BCP 60/40 and DBBM were compared histologically after healing periods of 6–8 months. The two studies did not show differences in vital bone formation between the two bone substitutes. Cordaro and coworkers found that the amounts of residual grafting materials between the two materials were similar. In contrast, Froum and colleagues reported lower amount of residual grafting materials for BCP 60/40 in relation to DBBM (26.6% vs. 37.7%). By varying the HA/b-TCP ratios, the characteristics of the grafting material could be altered. By reducing the HA/b-TCP ratio, the material would become more reactive. Jensen et al. (2009) compared three BCPs with varying HA/b-TCP ratios (20/80, 60/40 and 80/20) in mandibular defects of mini-pigs. Bone formation in defects with BCP 20/80 was similar to those grafted with autogenous bone, whereas those filled with BCP 60/40 and 80/ 20 were found to be similar to DBBM-filled defects. The amount of bone regeneration and resorption of the materials were inver-
sely proportional to HA/b-TCP ratios. The effect of further reducing the HA/b-TCP ratio on bone regeneration had not yet been investigated. Biphasic calcium phosphates were conventionally fabricated in blocks or powders. To improve clinical handling in some clinical situations, a newer formulation of BCPs was moldable. This BCP consists of granules, which are coated with bioresorbable polymers. After mixing with plasticizers, the material will harden. This may potentially eliminate the need of using a barrier membrane. Schmidlin et al. (2013) evaluated the properties of such moldable BCP in an animal study. The authors found that there were no differences in biocompatibility, bone formation and resorption between moldable BCP and DBBM after 16 weeks. On the other hand, the addition of polylactic acid to BCP was shown to delay bone formation in another animal study (Habibovic et al. 2008). As BCPs were developed into new formulations and mode of delivery, it is essential to evaluate the effectiveness of such grafting materials in animal models and also compare the outcomes with that of the currently accepted gold standards. The aim of this study is to compare the amount of bone regeneration and resorption of the bone substitutes in defects grafted with three different BCPs: BCPG1, BCPG2 and moldable BCP as compared to DBBM.
Material and methods The study was approved by Committee of the Use of Live Animals for Teaching and Research, the University of Hong Kong. Ten New Zealand rabbits (6 months old, 3.5– 4 kg) were used. Pre-operatively, they were kept and taken care of by the Laboratory Animal Unit of the Faculty of Medicine, the University of Hong Kong under veterinary supervision. The 10 rabbits were divided into group A and group B with five rabbits in each group. The animals were sacrificed after 3 months (for group A) and 6 months (for group B) following the operation (Fig. 1).
Surgery Group A (5)* Group B (5)
Analyses Group A (5)
Beginning
3 months
Analyses Group B (5)
6 months
Fig. 1. Time-line for the animal experiments of group A and group B. *Number of samples in parentheses.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Yip et al Defect healing with various bone substitutes
Table 1. Particulars of the grafting materials tested in the animal experiment Bone substitute
Abbreviation
Granule size (mm)
Name of product
Manufacturer
Deproteinized bovine bone mineral Biphasic calcium phosphate 60% of HA and 40% of b- TCP Biphasic calcium phosphate 60% of HA and 40% of b-TCP + Polylactide coating (PLGA) Biphasic calcium phosphate 10% of HA and 90% of b-TCP
DBBM BCPG1
0.25–1 0.5–1
Bio-Ossâ Biphasic Calcium Phosphate G1
Geistlich Pharma AG, Wolhusen, Switzerland Cam Bioceramics B.V., Leiden, the Netherlands
Moldable BCP
0.45–1
Easy -GraftTM CRYSTAL
Degradable Solution AG, Schleiren, Switzerland
BCPG2
0.5–1
Biphasic Calcium Phosphate G2
Revisios B.V., Bilthoven, the Netherlands
Operation
The surgical methodology on rabbit calvaria described by Jung et al. (2006) was adopted in this study. Pre-emptive analgesics (buprenorphine 0.05 mg/kg, subcutaneously) were given to the animals pre-operatively. Ketamine (35 mg/kg), acepromazine (1 mg/kg) and xylazine (5 mg/kg) were given intramuscularly for the general anesthesia procedure. The hair over the rabbit cranium was shaved and disinfected by iodine solution. A sagittal incision was made from the nasal region to the mid-sagittal crest. A full thickness flap was raised to expose the frontal, parietal and occipital bone, and the flap was retracted. Under copious irrigation, four defects in the diameter of 6 mm were prepared with a trephine in each calvarium. The defect was finalized with preparation of inner table with round bur and care was taken not to damage the dura mater. The four defects in each animal were filled with four different bone substitutes. Three different BCPs and DBBM were used (Table 1). The two granules-form BCPs with HA/b-TCP ratio of 60/40, 10/90, and DBBM were packed into the defect after hydration with normal saline. An injectable and moldable form of BCP 60/40, in which polylactide coating (PLGA) was incorporated into the material, were injected into the defect after mixing of the liquid and powder phases (Fig. 2). The four defects were covered with a single resorbable barrier membrane (BioGideâ; Geistlich Pharma AG, Wolhusen, Switzerland). The scalp incision was then closed with silk sutures. In one animal in group A, one of the four defects was left unfilled but covered with resorbable membrane, to serve as a negative control. Post-operative care
Antibiotics (enrofloxacin 5–10 mg/kg for 3 days, then meloxicam 0.2 mg/kg thereafter for 14 days, subcutaneously) and analgesics (buprenorphine 0.05 mg/kg, subcutaneously) were given post-operatively. Sutures were removed 14 days after surgery. The condi-
Fig. 3. Areas (left, center and right) selected for histomorphometric analysis.
Fig. 2. The four surgically created calvarial defects were filled with various bone substitute.
tions and well-beings of the animals were monitored before being sacrificed. The animals were euthanized with pentobarbital (150 mg/kg) at according to the time-line of the experiment. Histological and histomorphometric analysis
The calvaria that contained the grafting materials were harvested. The specimens were fixed with 10% paraformaldehyde solution, followed by decalcification with 14.5% EDTA acid. Histological sectioning with a thickness of 6 lm was cut and then stained with hematoxylin and eosin (H & E) stain. In each H & E stained slide series, 1 slide was selected for analysis. The slides were examined with light microscope (Nikonâ Eclipse VL100POL, Tokyo, Japan) incorporated to a digital video camera (Nikonâ Digital Sight DS-Ri1). The unit was connected to a computer with image analysis software (NISElements AR 3.00, Nikonâ Laboratory Imaging software, Japan). The defects were divided into three areas: left, center and right (Fig. 3). The center of each area was analyzed under a magnification of 2609. Histomorphometric analysis was performed using a modification of the methods described by Schroeder & M€ unzel-Pedrazzoli (1973). Square grids with 10 9 10 grid points were generated over the tissues in the middle of each area. Four tissue structures were
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
identified and measured at the intersection of each grid point. The structures include
• • • •
Mineralized new bone (MNB) Bone marrow (BM) Residual grafting material (RG) Soft tissue (ST)
The volumetric density of different structures in the defects was calculated (Fig. 4). The histomorphometric analysis was performed by a single investigator (IY). Intraexaminer reliability was determined by remeasurement of eight slides (two slides for each grafting material of group A) 1 week after the first examination. Statistical analysis
The data were entered and analyzed using IBM SPSS software (IBM Corporation, New York, NY, USA). The intra-examination reliability was evaluated with intra-class correlation coefficient (ICC). ANOVA test was used to determine the difference between the volumetric densities among the four grafting materials. Two-sample t-test was used to compare the volumetric densities of tissues for the same bone substitute between group A and B. The level of significance was set at a ≤ 0.05.
Results Forty defects in 10 rabbits were created surgically as planned. The defects were filled with the four biomaterials accordingly and were
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Yip et al Defect healing with various bone substitutes
Fig. 4. Histomorphometric analysis with 10 9 10 grid points. MNB, mineralized new bone; BM, bone marrow; RG, residual grafting material; ST, soft tissue.
covered with bioresorbable membranes. The intra-operative and post-operative conditions of the animals were stable without any complications. By the time of animal sacrifice, there were no signs of wound dehiscence, infections nor membrane exposures. Histological observations
The morphology of the negative control defect without bone substitutes was depicted in Fig. 5a. Complete bridging of the defect was observed. However, a depression was present at the surface of the defect, which implied that the resorbable membrane was not well supported. The area underneath the membrane was mainly bone marrow, whereas MNB was present mainly in the deeper half of the defect. In contrast, the superficial part of the defects grafted with bone substitutes was well supported and MNB could be found in the full thickness of the grafted site (Fig. 5b). The residual grafting materials were observed throughout the defects. Mineralized new bone and bone marrow were more evenly distributed inside the defect. Histological morphology of defects filled with various bone graft materials is shown in Fig. 6. Grafting materials were identified, and each material took up different degree of H & E stain. Particles of BCPG1 were not identi-
fied; the reason could be the materials did not take up H & E stains. The shape of the particles, however, was assumed to be similar to the empty spaces left among different tissues (Fig. 6b). Particles of moldable BCP and BCPG2 were larger in size. DBBM particles appeared fragmented in residual graft spaces, and the BCPG1 particles were more slender. Moldable BCP particles were more rounded and BCPG2 were irregular in shape. Moldable BCP and BCPG2 particles appeared to have more interfacial contact with bone comparing with DBBM. When the samples from group A and group B were compared, the sizes of the grafting particles were smaller in group B. The grafting particles also appeared less irregular. No foreign body reactions adjacent to the four grafting materials could be identified in any of the samples. Histomorphometric analyses were carried out at the left, center and right side of the defects of each sample, and the three areas were analyzed as described. The mean percentages of MNB, bone marrow, residual grafting particles and soft tissues were calculated. Intra-examiner calibration
Intra-examiner reliability for the histomorphometric analysis was carried out with remeasurement of 10 slides 1 week after the first measurement using the samples from group A (two for DBBM, three for BCPG1, three for moldable BCP and two for BCPG2). When the first and second data sets were compared, the ICCs were high (>0.9 for all measurement parameters of % MNB, BM, RG and ST). This indicated the histological measurements were highly reproducible. Histomorphometric analyses of Group A
The mean percentages of various tissues in defects grafted with the four grafting materials for group A are listed in Table 2 and Fig. 7. Mineralized new bone was filled up to one-fourth to half of the defects, with defects filled with BCPG1 having greatest amount of
MNB regeneration (46.33%), followed by BCPG2 (38.42%). Moldable BCP had the least amount of bone regenerated (25%) after 3 months. However, there were no statistically significant differences in %MNB among the groups. The mean percentages of bone marrow were variable among the groups. The proportion was lowest in defects with DBBM (3%), and it was statistically lower than defects with moldable BCP and BCPG2 (P < 0.01) (Table 3). Residual grafting materials occupied at least a quarter of the defects after 3 months. Amount of residual grafting material were highest in defects filled with moldable BCP (54.8%) followed by DBBM (45.33%). Defects with BCPG1 had the lowest percentage of residual materials (26.67%), and it was significantly lower than that of moldable BCP (P < 0.05). Mean percentage of soft tissues in the defects were
