Bone healing around nanocrystalline hydroxyapatite, deproteinized bovine bone mineral, biphasic calcium phosphate, and autogenous bone in mandibular bone defects Nina Broggini,1,2 Dieter D. Bosshardt,1,3 Simon S. Jensen,1,4 Michael M. Bornstein,1 Chun-Cheng Wang,5 Daniel Buser1 1
Department of Oral Surgery and Stomatology, School of Dental Medicine, University of Bern, Bern, Switzerland Private Practice, Studio Borsa Broggini Lanfranchini, Via Stazione 1, Balerna, Switzerland 3 Robert K. Schenk Laboratory of Oral Histology, School of Dental Medicine, University of Bern, Bern, Switzerland 4 Department of Oral & Maxillofacial Surgery, Copenhagen University Hospital (Rigshospitalet), Copenhagen, Denmark 5 Division of Periodontology, Department of Dentistry, National Taiwan University Hospital, Taipei, Taiwan 2
Received 31 July 2014; revised 25 September 2014; accepted 18 October 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33319 Abstract: The individual healing profile of a given bone substitute with respect to osteogenic potential and substitution rate must be considered when selecting adjunctive grafting materials for bone regeneration procedures. In this study, standardized mandibular defects in minipigs were filled with nanocrystalline hydroxyapatite (HA-SiO), deproteinized bovine bone mineral (DBBM), biphasic calcium phosphate (BCP) with a 60/40% HA/b-TCP (BCP 60/40) ratio, or particulate autogenous bone (A) for histological and histomorphometric analysis. At 2 weeks, percent filler amongst the test groups (DBBM (35.65%), HA-SiO (34.47%), followed by BCP 60/40 (23.64%)) was significantly higher than the more rapidly substituted autogenous bone (17.1%). Autogenous bone yielded significantly more new bone (21.81%) over all test groups (4.91%–7.74%) and significantly more osteoid (5.53%) than BCP 60/40 (3%) and DBBM (2.25%). At 8 weeks, percent filler amongst the test groups
(DBBM (31.6%), HA-SiO (31.23%), followed by BCP 60/40 (23.65%)) demonstrated a similar pattern and was again significantly higher as compared to autogenous bone (9.29%). Autogenous bone again exhibited statistically significantly greater new bone (55.13%) over HA-SiO (40.62%), BCP 60/40 (40.21%), and DBBM (36.35%). These results suggest that the osteogenic potential of HA-SiO and BCP is inferior when compared to autogenous bone. However, in instances where a low substitution rate is desired to maintain the volume stability of augmented sites, particularly in the esthetic zone, HA-SiO and C 2014 Wiley Periodicals, Inc. J Biomed DBBM may be favored. V Mater Res Part B: Appl Biomater 00B: 000–000, 2014.
Key Words: guided bone regeneration, unsintered nanocrystalline hydroxyapatite, deproteinized bovine bone mineral, biphasic calcium phosphate, autogenous bone graft
How to cite this article: Broggini N, Bosshardt DD, Jensen SS, Bornstein MM, Wang C-C, Buser D 2014. Bone healing around nanocrystalline hydroxyapatite, deproteinized bovine bone mineral, biphasic calcium phosphate, and autogenous bone in mandibular bone defects. J Biomed Mater Res Part B 2014:00B:000–000.
INTRODUCTION
The ideal bone substitute should not only stimulate new bone formation and promote neovascularization, but should also have a low substitution rate in order to maintain volume stability during on-going remodeling over time. The best documented xenogenic bone substitute is deproteinized bovine bone mineral (DBBM). Essentially considered a hydroxyapatite (HA) ceramic1 after the removal of its organic components, the substitution rate of DBBM is low resulting in the observation of residual particles several years following augmentation procedures.2,3 Alternatively, synthetic calcium phosphate bioceramics are favored for their biocompatibility, osteoconductivity, and non-immunogenicity. By varying the HA/tricalcium phosphate (TCP) ratio in alloplastic biphasic calcium phos-
phate (BCP), it is possible to influence substitution rate and bioactivity,4 such as the commercially available BCP with a HA/TCP ratio of 60/40% ((BCP 60/40) and C/P ratio: 1.6) which appears to mimic human cancellous bone structure.5 Alloplastic bone substitutes are often exposed to high sintering temperatures, resulting in low microporosity, and perhaps osteoconductivity.6 Non-sintered 76% nanocrystalline HA, embedded in 24% nanostructured silica gel matrix with interconnecting pores (HA-SiO), possesses a larger surface area (84 m2/g)7,8 than DBBM (79.7 m2/g).9 Granule porosity exists between 60% and 80%10 with pore sizes of 10–20 nm.7 A greater relative surface presumably leads to greater adsorption of biologically active non-collagenous proteins (i.e., fibronectin,
No benefit of any kind was received either directly or indirectly by the author(s). Correspondence to: N. Broggini; e-mail: [email protected] Contract grant sponsor: Department Funds of the University of Bern
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osteopontin, and bone sialoprotein)11 or bone marker molecules (i.e., alkaline phosphatase, bone morphogenetic protein 2, and osteocalcin).6,12 Adsorption would occur not only on the periphery, but internally due to high porosity, possibly expediting the healing cascade: cell colonization, cell spreading, cell differentiation, and bone matrix deposition.11 The aim of the present study was to compare the osteogenic potential and substitution rate of different bone substitutes in vivo relative to autogenous bone. MATERIALS AND METHODS
This study conformed to the “ARRIVE guidelines” (Animal Research: Reporting of In Vivo Experiments) which were developed to improve the design, analysis, and reporting of research using animals—maximizing information published and minimizing unnecessary studies (www.nc3rs.org.uk/ ARRIVE), as well as the “STROBE guidelines” which stands for an international, collaborative initiative of epidemiologists, methodologists, statisticians, researchers, and journal editors involved in the conduct and dissemination of observational studies, with the common aim of “STrengthening the Reporting of OBservational studies in Epidemiology” (www.strobe-statement.org). Surgical procedure Following sedation (ketamine (20 mg/kg)/xylazine (2 mg/ kg) I.M., and atropine (0.05 mg/kg)/midazolam (0.5 mg/kg) I.V.), general anesthesia was induced (isoflurane 1–1.5%) in eight adult G€ ottingen miniature pigs (mean weight: 60 6 8 kg (6SD); Surgical Research Unit (ESI)), University of Bern, Switzerland (ethical approval no. 99/08)). Preoperatively, 1 g amoxicillin I.M. was administered. Postoperatively, 1 g metamizolum, 150,000 IU procaine penicillin, and 150,000 IU benzathine penicillin I.V. were administered. Using a trephine, four standardized, non-critically sized, intraosseous defects (7 mm 3 4 mm) were created at the right lateral mandibular body and ramus [Figure 1(A)] and filled with either: 1. nanocrystalline HA embedded in a matrix of silica gel R 0.6 mm, Artoss GmbH, Rostock, (“HA-SiO”) (NanoBoneV Germany) or R 0.25–1 mm, Geistlich Pharma, Wolhu2. “DBBM” (Bio-OssV sen, Switzerland) or 3. synthetic BCP with a 60/40% HA/b-TCP ratio (“BCP 60/ R Bone Ceramic 90% porous 0.5–1 mm, 40”) (StraumannV Institut Straumann AG, Basel, Switzerland), or 4. particulated autogenous bone (“A”) (ground 1:1 corticocancellous bone 1–2 mm (milled cortico-cancellous bone (R. Quetin, Leimen, Germany)) as positive control [Figure 1(B)]. and mixed with blood prior to placement. Assignment was randomized and rotated in a cyclic permutation (www. randomization.com, seed: 17078). Two expanded polytetrafluoroethylene (ePTFE) membranes (GT9, W. L. Gore & Associates, Newark, DE) were used to cover the defects and fixed with titanium screws (Modus 1.5, Medartis AG, Basel, Swit-
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FIGURE 1. An example of the surgical site before (A) and after (B) bone grafting. In order of left to right in the second photograph (B): autogenous bone, HA-SiO, BCP 60/40, and DBBM. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
zerland). Primary closure was achieved in multiple layers using resorbable sutures (Vicryl 4-0, Ethicon, Norderstedt, Germany). The contralateral side was grafted identically 6 weeks later. Sacrifice after an additional 2 weeks created 2 and 8 week healing time points within each animal. Histological preparation and histomorphometric analysis As previously described,13 non-decalcified sections (80 mm) were glued to plexiglass with acrylic cement and stained superficially with toluidine blue. For qualitative histology, three regions of interest (ROI) were defined: (1) bottom of the defect, (2) center of the defect, and (3) top of the defect immediately under the barrier membrane (Figure 2). Three most central sections per defect were analyzed by a blinded, experienced examiner (B.H.).13 Percentages of newly-formed bone (osteoid, woven, parallel-fibered, and lamellar), residual grafting material, and soft tissue/marrow space were determined by point counting, using an integrative eyepiece with a square grid (distance between 6 3 6 test lines, 255 lm) at a magnification of 363.14 Statistical analysis Box plots were plotted for the distribution of bone, osteoid, filler material, and soft tissue for the two time periods.
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ROI 2: Center of the defect. In all four groups, a cell-rich soft tissue, which did not resemble bone marrow, was present after 2 weeks [Figure 5(a,c,e,g)]. However, new bone was observed only in association with autogenous bone [Figure 5(a)] and HA-SiO [Figure 5(g)]. With autogenous bone, new bone formation was more abundant and mature, interconnecting autogenous bone particles [Figure 5(a)]. After 8 weeks, new bone was most mature with autogenous bone [Figure 5(b)]. While DBBM [Figure 5(d)] and HASiO [Figure 5(h)] demonstrated a comparable bone maturation grade, new bone formation with BCP 60/40 was less abundant and mature [Figure 5(f)]. Bone marrow was most abundant and mature with autogenous bone [Figure 5(b)] and lacking with BCP 60/40 [Figure 5(f)]. FIGURE 2. Representative example illustrating the grafted bone defect, which is laterally and at the bottom bordered by new bone, after 8 weeks of healing. For the descriptive analysis, three regions of interest were defined: one at the bottom of the defect, one in the center of the defect, and one at the top of the defect just underneath the barrier membrane. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Nonparametric repeated ANOVA measurements (Brunner–Langer models) with time (2 or 8 weeks) and a treatment modality factor were used for each tissue fraction separately. In case of a significant interaction between time and treatment in the model, Wilcoxon rank sum tests were used to further detect statistically significant differences between the four treatment groups with the autogenous bone serving as positive control. Due to the explorative nature of this study, no p-value corrections were performed. The Brunner–Langer models were applied using SAS 9.1.3 (SAS Institute Inc., Cary, NC). All other analyses were performed using R 2.9.2 software package (http://www.r-project.org). RESULTS
Qualitative histology All bone fillers were evenly distributed over the defects and provided mechanical support for the ePTFE barrier membranes during both observation periods (Figure 3). ROI 1: Bottom of the defect. New bone formation was observed close to the defect margins in conjunction with all fillers at 2 weeks. Woven bone trabeculae appeared to emerge from the defect walls and interconnected bone filler particles (Figure 4). New bone was most dense and mature with autogenous bone [Figure 4(a)] with already some conversion to parallel-fibered bone formation. In contrast, new bone formed in the presence of all three bone substitutes was less abundant and mature [Figure 4(c,e,g)]. Active bone formation was indicated by the presence of osteoid lined by osteoblasts. Mature bone marrow was not observed at this time. After 8 weeks, dense and mature bone was observed in all groups, of which autogenous bone was the most advanced [Figure 4(b)], followed by DBBM [Figure 4(d)] and HA-SiO [Figure 4(h)]. BCP 60/40 was the least mature [Figure 4(f)].
ROI 3: Top of the defect. After 2 weeks, new bone in close vicinity to the barrier membrane was only seen with autogenous bone [Figure 6(a)]: new tiny woven bone trabeculae interspersed among a cell-rich soft tissue and contacting autogenous bone particles. Mature bone marrow was not evident. After 8 weeks, new bone close to, or in contact with the barrier membrane was found in all groups [Figure 6(b,d,f,h)]. New bone formation was spars and immature for BCP 60/40 [Figure 6(f)]. Bone marrow was most mature with autogenous bone [Figure 6(b)] and lacking for BCP 60/40 [Figure 6(f)]. For HA-SiO, there was more osteoid and initial mineralization further away from the defect margins than in the other two bone substitute groups. Large multinucleated cells resembling osteoclasts were observed on the surface of all filler materials after 2 weeks. After 8 weeks, they were still present on DBBM, BCP 60/40, and HA-SiO surfaces, but not on autogenous bone particles. Histomorphometric analysis Significant time and treatment effects were found for all variables (bone/osteoid, filler, and soft tissue) (Table I). Bone values generally increased and filler values decreased over the time period analyzed. Autogenous bone clearly exhibited the highest values of new bone formation, followed by HA-SiO and BCP 60/40, and with the lowest values for DBBM. Autogenous bone clearly had the lowest filler values, followed by BCP 60/40, HA-SiO, and DBBM for both time points analyzed. Two weeks after surgery, the autogenous bone group presented with a statistically significant higher percentage of bone than the other three groups (p < 0.0002; Figure 7). Additionally, autogenous bone had significantly more osteoid than the DBBM and BCP 60/40. The soft tissue percentages were similar for the autogenous bone, HA-SiO, and DBBM; only BCP 60/40 presented significantly more soft tissue when compared to autogenous bone (Table I). Eight weeks after surgery, autogenous bone still presented with a statistically significant higher percentage of newly-formed bone than the other groups (p < 0.0002 for DBBM and p < 0.001 for HA-SiO and BCP 60/40, Figure 7), and an increase from a mean value of 21.8% bone after 2 weeks to 55.1% after 8 weeks. The grafted autogenous
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FIGURE 3. Histological overview sections showing bone defects grafted with (a,b) autogenous bone, (c,d) DBBM, (e,f) BCP 60/40, and (g,h) HA-SiO, after (a,c,e,g) 2 and (b,d,f,h) 8 weeks of healing. In all defects, the bone fillers provide sufficient mechanical support for the barrier membrane. For all grafting materials, bone formation starts at the defect margins. However, penetration of the defect area with new bone occurs at different rates. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
bone particles were mostly resorbed, and only accounted for 9.3%—statistically significant lower than the filler materials in the other groups (Figure 8). The lowest percentage of soft tissue was exhibited by HA-SiO (Table I), but there was no statistically significant difference when compared to autogenous bone.
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DISCUSSION
The osteogenic effect of an autogenous bone graft at the implant surface during simultaneous bone regeneration procedures may be regarded as important to ensure osseointegration, however its high substitution rate may not sustain longterm volume stability.15 Bone substitutes, although potentially
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FIGURE 4. Higher magnifications of the defect margins at the bottom of the defects grafted with (a,b) autogenous bone, (c,d) DBBM, (e,f) BCP 60/40, and (g,h) HA-SiO, after (a,c,e,g) 2 and (b,d,f,h) 8 weeks of healing. For all bone fillers, new bone (NB) is present in contact with old bone (OB) and interconnecting bone filler particles (*). Note that after 2 weeks, bone formation is most advanced for the autogenous bone group. In all groups, the soft tissue (ST) does not resemble a bone marrow (BM). Most new bone is woven bone. However, conversion from woven bone to parallel-fibered bone formation is first observed in the autogenous bone group. After 8 weeks, dense and mature bone (i.e., woven bone reinforced by parallel-fibered bone) is observed in all groups. However, maturation of the bone marrow is most advanced in the autogenous bone group, as indicated by the presence of large bone marrow cavities and trabecular bone (b), followed by the DBBM (d), the HA-SiO (h), and the BCP 60/40 (f) groups. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
osteoconductive and usually providing a scaffold upon which osteogenesis may take place, possess an inferior osteogenic potential as compared to autogenous bone.16–18
The rationale for clinical utilization, therefore, may be based primarily upon a low substitution rate, which maintains volume. The principle of guided bone regeneration is
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FIGURE 5. Higher magnifications of the central regions of the bone defects grafted with (a,b) autogenous bone, (c,d) DBBM, (e,f) BCP 60/40, and (g,h) HA-SiO, after (a,c,e,g) 2 and (b,d,f,h) 8 weeks of healing. After 2 weeks of healing, the cell-rich soft tissue (ST) present among new bone (NB) and filler particles (*) does not resemble bone marrow (BM) in any of the groups. New bone is seen in association with autogenous bone and HA-SiO particles only. In the autogenous bone group, the new bone is more abundant and more mature, interconnecting autogenous bone particles. In the HA-SiO group, tiny bone trabeculae and areas with osteoid containing mineralization foci are indicative of beginning mineralization. After 8 weeks, new bone was seen in all four groups (b,d,f,h), but was most mature with autogenous bone. While the DBBM and HA-SiO groups show comparable bone maturation, the new bone in the BCP 60/40 group is less abundant and less mature. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
to maintain a surgically created volume, thus excluding rapidly proliferating epithelial cells and fibroblasts, and permitting the growth of slower-growing bone cells and
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promoting angiogenesis. The actual biomaterials themselves may contribute to this principle by physically supporting the barrier membrane and preventing its collapse
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FIGURE 6. Higher magnifications of the defect margins underneath the barrier membrane covering the bone defects grafted with (a,b) autogenous bone, (c,d) DBBM, (e,f) BCP 60/40, and (g,h) HA-SiO, after (a,c,e,g) 2 and (b,d,f,h) 8 weeks of healing. After 2 weeks, new bone (NB) in the form of tiny woven bone trabeculae is only seen in the autogenous bone group. The woven bone trabeculae are surrounded by cell-rich soft tissue (ST) that does not resemble mature bone marrow (BM). In the bone substitute groups, the filler particles (*) are surrounded by soft tissue, which is cell rich, but distinctly different from that observed in the autogenous bone group. After 8 weeks, new bone close to or in contact with the barrier membrane is found in all groups. Immature and little new bone are found in the BCP 60/40 group. Bone marrow is most mature in the autogenous bone group and absent in the BCP 60/40 group. The two groups with DBBM and HA-SiO show small amounts of immature bone marrow. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
during initial wound healing. In the esthetic zone, a low substitution rate may be particularly important where the establishment of sufficient facial bone volume is critical to
maintain soft tissue dimensions.15 Stable long-term conditions have been documented clinically and radiographically using cone beam computed tomography (CBCT) after
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TABLE I. Effects of Autogenous Bone, HA-SiO, DBBM, and BCP-60/40 on New Bone Formation New Bone Formation 2 Weeks Autogenous bone HA-SiO DBBM BCP 60/40 8 Weeks Autogenous bone HA-SiO DBBM BCP 60/40 a b
21.81 7.21 4.91 7.74
(6.34)a (2.78)b (1.67)b (2.71)b
55.13 40.62 36.35 40.21
(7.53) (5.74)b (3.21)b (5.13)b
Osteoid 5.53 4.28 2.25 3
(1.48) (1.8) (1.41)b (1.31)b
Filler
Soft Tissue
17.1 34.47 35.65 23.64
(2.8) (3.15)b (2.61)b (2.77)b
55.56 54.03 57.19 65.63
(5.55) (3.62) (2.97) (3.62)b
9.29 31.23 31.60 23.65
(3.07) (3.65)b (3.44)b (1.91)b
35.58 28.15 32.05 36.14
(8.61) (6.45) (4.08) (5.41)
Results presented as the mean (S.D.); n 5 8 animals/group. Statistically significant difference (p < 0.05) as compared to autogenous bone.
contour augmentation in the esthetic zone using the guided bone regeneration (GBR) technique with a combination of locally harvested autogenous bone chips and DBBM particles.19,20 The goal of this study was to compare early osteogenic potential and percent filler of HA-SiO to well-documented bone substitutes, DBBM and BCP 60/40, relative to autogenous bone. Autogenous bone exhibited the significantly greatest amount of new bone formation and maturation, however, more rapid graft resorption. This is to be expected, since both osteogenic cells and osteoinductive factors are directly transferred.21 BCP 60/40 demonstrated the significantly highest soft tissue percentage over autogenous bone at 2 weeks (65.63%) and a low percent filler (23.64%), which may reflect a greater graft turnover compared to DBBM and HA-SiO. DBBM demonstrated a trend of lower
percentage of new bone formation (4.91% and 2.25% osteoid) with respect to HA-SiO and BCP 60/40, but a seemingly high percent filler (35.65%) at 2 weeks. These observations support previous investigations demonstrating the low substitution rate of DBBM.4,22 Alloplastic calcium phosphates, however, to which BCP 60/40 belongs, are influenced by the amount of TCP and could be expected to have a higher substitution rate4,16 as compared to DBBM.4 Hence, DBBM is often promoted for augmentation procedures where volume stability is important. Interestingly, HA-SiO performed similarly to autogenous bone with respect to early osteoid production, but was similar to DBBM with respect to percent filler, and similar to BCP 60/40 with respect to rapid new bone formation (Table I). Strikingly, this trend was again observed at 8 weeks.
FIGURE 7. Box plots of the histomorphometric data of percentages of new bone formation. The vertical boxes represent the range of data from minimum to maximum. The heavy horizontal bar within the box represents the median while the ends of the whiskers represent data within the 1.5 interquartile range (IQR) of the lower quartile and within the 1.5 IQR of the upper quartile. Any data not included between the whiskers is plotted as an outlier (small circle). AB 5 autogenous bone, DBBM 5 deproteinized bovine bone material, HA-SiO 5 synthetic nanocrystalline HA, BCP 60/40 5 synthetic BCP with a 60/40% HA/b-TCP ratio, 5 outliers.
FIGURE 8. Box plots of the histomorphometric data of percentages of remaining fillers. The vertical boxes represent the range of data from minimum to maximum. The heavy horizontal bar within the box represents the median while the ends of the whiskers represent data within the 1.5 interquartile range (IQR) of the lower quartile and within the 1.5 IQR of the upper quartile. Any data not included between the whiskers is plotted as an outlier (small circle). AB 5 autogenous bone, DBBM 5 deproteinized bovine bone material, HA-SiO 5 synthetic nanocrystalline HA, BCP 60/40 5 synthetic BCP with a 60/40% HA/b-TCP ratio, 5 outliers.
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The present data is consistent with human trephine cores taken after sinus floor elevation procedures with HA-SiO.23 At 3 months, mean new bone was 8%, residual HA-SiO 45%, and bone marrow 47% which roughly correlates to 2 weeks data in the current study: total new bone (new bone 1 osteoid) 11.5%, residual HA-SiO 34.5%, and soft tissue 54%. At 6 months, 48%, 28%, and 24%, respectively in humans, corresponded as well to our 8 weeks data: 41%, 31%, 28%, respectively. One possible explanation for HA-SiO’s performance is the geometry of not only a greater relative surface due to its nanoparticles, but also due to the interconnecting pores available for protein adsorption—a so-called nanotopography, as described by Ahmad et al.24 HA-SiO has also demonstrated superior in vitro cell proliferation of human osteoblasts over DBBM.25 Of noted interest, one study demonstrated osteoinductive potential of HA-SiO in the minipig subcutaneously after 5 weeks, and intramuscularly after 4 months.26 Moreover, the effect of silicon itself on bone regeneration may be significant8,27–29 and thus, Si-based calcium phosphates may be considered not simply as “bone graft substitutes” but instead, as true drug delivery devices of Si ion, whose in vivo release may positively influence cell activity.30 The concept of tailoring ion release has been studied and concluded that the rate of release of Si and Ca ions is more rapid for pore structures with larger modal pore diameters.31 Interestingly, the biological effects of exposed HA nanoparticles after degradation of the silica oxide gel has been shown to be concentration-dependent: promotion of mesenchymal stem cell growth when HA nanoparticle concentrations were lower than 20 mg/104 cells, whereas higher particle concentration would significantly inhibit cell growth.32 Therefore, only physical presence of either Si ion or HA nanoparticles is not adequate to automatically assure a positive outcome on bone formation. Mechanical testing or functional assessment studies along with cone beam computed tomography analysis is needed to confirm whether the apparent low substitution rate ensures long-term stability of implant placement with bone augmentation. Spectroscopic analysis would support an evaluation of the quality of new bone with filler material and more clearly identify differences of graft materials. Additionally, time zero data would permit a quantitative evaluation of graft turnover. Initial studies report that complete HA-SiO degradation may occur over the order of months in humans.8 This differs from DBBM which has shown the presence of bone substitute particles 4–10 years postoperatively in human biopsies after bone augmentation.2,3,22,33 Being that both HA-SiO and DBBM performed similarly in the current study with respect to graft stability up to 8 weeks, additional investigations are needed to determine the exact degradation sequence of HA-SiO as compared to DBBM in the long term, as well as ascertain the clinical implications of volume maintenance in bone augmentation procedures. ACKNOWLEDGMENTS
The authors thank B. Hofmann, D. Reist, and S. Owusu for specimen preparation; C. Moser for surgical assistance; and Dr. D.
Mettler, O. Beslac, and D. Zalokar for surgical facilities’ management. All authors declare no conflicts of interest. REFERENCES 1. Rumpel E, Wolf E, Kauschke E, Bienengraber V, Bayerlein T, Gedrange T, Proff P. The biodegradation of hydroxyapatite bone graft substitutes in vivo. Folia Morphol (Warsz) 2006;65:43–48. 2. Mordenfeld A, Hallman M, Johansson CB, Albrektsson T. Histological and histomorphometrical analyses of biopsies harvested 11 years after maxillary sinus floor augmentation with deproteinized bovine and autogenous bone. Clin Oral Implants Res 2010; 21:961–970. 3. Jensen SS, Bosshardt DD, Gruber R, Buser D. Long-term stability of contour augmentation in the esthetic zone. Histologic and histomorphometric evaluation of 12 human biopsies 14 to 80 months after augmentation. J Periodontol 2014;85:1549–1556. 4. Jensen SS, Bornstein MM, Dard M, Bosshardt DD, Buser D. Comparative study of biphasic calcium phosphates with different HA/ TCP ratios in mandibular bone defects. A long-term histomorphometric study in minipigs. J Biomed Mater Res B Appl Biomater 2009;90:171–181. 5. Jensen SS, Yeo A, Dard M, Hunziker E, Schenk R, Buser D. Evaluation of a novel biphasic calcium phosphate in standardized bone defects: A histologic and histomorphometric study in the mandibles of minipigs. Clin Oral Implants Res 2007;18:752–760. 6. Gerike W, Bienengraber V, Henkel KO, Bayerlein T, Proff P, Gedrange T, Gerber T. The manufacture of synthetic non-sintered and degradable bone grafting substitutes. Folia Morphol (Warsz) 2006;65:54–55. 7. Kirchhoff M, Bienengraber V, Lenz S, Gerber T, Henkel KO. A new synthetic bone replacement material with osteoinductive properties—In vivo investigations. BIOmaterialien 2006;80:7. 8. Gotz W, Gerber T, Michel B, Lossdorfer S, Henkel KO, Heinemann F. Immunohistochemical characterization of nanocrystalline hydroxyapatite silica gel (NanoBone(r)) osteogenesis: A study on biopsies from human jaws. Clin Oral Implants Res 2008;19:1016– 1026. € tz H, Duschner H, Wagner W. 9. Weibrich G, Trettin R, Gnoth SH, Go [Determining the size of the specific surface of bone substitutes with gas adsorption]. Mund Kiefer Gesichtschir 2000;4:148–152. 10. Werner J, Linner-Krcmar B, Friess W, Greil P. Mechanical properties and in vitro cell compatibility of hydroxyapatite ceramics with graded pore structure. Biomaterials 2002;23:4285–4294. 11. Jensen S, Bosshardt DD, Buser D. Bone grafts and bone substitute materials for GBR procedures. In: Buser D, editor. 20 Years of Guided Bone Regeneration in Implant Dentistry. Chicago, IL, USA: Quintessence; 2009. pp 71–96. € tz W, Bienengraber V, Henkel KO, 12. Gerber T, Holzh€ uter G, Go Rumpel E. Nanostructuring of biomaterials—A pathway to bone grafting substitute. Eur J Trauma 2006:132–140. 13. Schenk RK, Olah AJ, Herrmann W. Preparation of calcified tissues for light microscopy. In: Dickson GR, editor. Methods of Calcified Tissue Preparation. Amsterdam: Elsevier; 1984. pp 1–56. 14. Weibel ER. Stereological Methods, Vol. I: Practical Methods for Biological morphometry. New York: Academic Press; 1979. 15. Buser D, Chen ST, Weber HP, Belser UC. Early implant placement following single-tooth extraction in the esthetic zone: Biologic rationale and surgical procedures. Int J Periodontics Restorative Dent 2008;28:441–451. 16. Buser D, Hoffmann B, Bernard JP, Lussi A, Mettler D, Schenk RK. Evaluation of filling materials in membrane–protected bone defects. A comparative histomorphometric study in the mandible of miniature pigs. Clin Oral Implants Res 1998;9:137–150. 17. Broggini N, Hofstetter W, Hunziker E, Bosshardt DD, Bornstein MM, Seto I, Weibrich G, Buser D. The influence of PRP on early bone formation in membrane protected defects. A histological and histomorphometric study in the rabbit calvaria. Clin Implant Dent Relat Res 2011;13:1–12. 18. Jensen SS, Broggini N, Hjorting-Hansen E, Schenk R, Buser D. Bone healing and graft resorption of autograft, anorganic bovine bone and beta-tricalcium phosphate. A histologic and histomorphometric study in the mandibles of minipigs. Clin Oral Implants Res 2006;17:237–243.
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19. Buser D, Chappuis V, Bornstein MM, Wittneben JG, Frei M, Belser UC. Long-term stability of contour augmentation with early implant placement following single tooth extraction in the esthetic zone: A prospective, cross-sectional study in 41 patients with a 5- to 9-year follow-up. J Periodontol 2013;84:1517–1527. 20. Buser D, Chappuis V, Kuchler U, Bornstein MM, Wittneben JG, Buser R, Cavusoglu Y, Belser UC. Long-term stability of early implant placement with contour augmentation. J Dent Res 2013; 92(12 Suppl):176S–182S. 21. Burchardt H. The biology of bone graft repair. Clin Orthop Relat Res 1983:28–42. 22. Piattelli M, Favero GA, Scarano A, Orsini G, Piattelli A. Bone reactions to anorganic bovine bone (Bio-Oss) used in sinus augmentation procedures: A histologic long-term report of 20 cases in humans. Int J Oral Maxillofac Implants 1999;14:835–840. 23. Canullo L, Dellavia C. Sinus lift using a nanocrystalline hydroxyapatite silica gel in severely resorbed maxillae: Histological preliminary study. Clin Implant Dent Relat Res 2009;11 (Suppl 1):e7–e13. 24. Ahmad M, Jones JR, Hench LL. Fabricating sol–gel glass monoliths with controlled nanoporosity. Biomed Mater 2007;2:6–10. 25. Liu Q, Douglas T, Zamponi C, Becker ST, Sherry E, Sivananthan S, Warnke F, Wiltfang J, Warnke PH. Comparison of In Vitro BioR ) and BioOss(V R ) for Human Osteocompatibility of NanoBone(V blasts, Clin Oral Implants Res 2011;22:1259–1264. 26. Gotz W, Lenz S, Reichert C, Henkel KO, Bienengraber V, Pernicka L, Gundlach KK, Gredes T, Gerber T, Gedrange T, Heinemann F. A preliminary study in osteoinduction by a nano-crystalline
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27.
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hydroxyapatite in the mini pig. Folia Histochem Cytobiol 2010;48: 589–596. Patel N, Brooks RA, Clarke MT, Lee PM, Rushton N, Gibson IR, Best SM, Bonfield W. In vivo assessment of hydroxyapatite and silicate-substituted hydroxyapatite granules using an ovine defect model. J Mater Sci Mater Med 2005;16:429–440. Hing KA, Revell PA, Smith N, Buckland T. Effect of silicon level on rate, quality and progression of bone healing within silicatesubstituted porous hydroxyapatite scaffolds. Biomaterials 2006; 27:5014–5026. Xu S, Lin K, Wang Z, Chang J, Wang L, Lu J, Ning C. Reconstruction of calvarial defect of rabbits using porous calcium silicate bioactive ceramics. Biomaterials 2008;29:2588–2596. Bohner M. Silicon-substituted calcium phosphates—A critical view. Biomaterials 2009;30:6403–6406. Jones JR, Ehrenfried LM, Saravanapavan P, Hench LL. Controlling ion release from bioactive glass foam scaffolds with antibacterial properties. J Mater Sci Mater Med 2006;17:989–996. Liu Y, Wang G, Cai Y, Ji H, Zhou G, Zhao X, Tang R, Zhang M. In vitro effects of nanophase hydroxyapatite particles on proliferation and osteogenic differentiation of bone marrow-derived mesenchymal stem cells. J Biomed Mater Res A 2009;90:1083–10891. Sartori S, Silvestri M, Forni F, Icaro Cornaglia A, Tesei P, Cattaneo V. Ten-year follow-up in a maxillary sinus augmentation using anorganic bovine bone (Bio-Oss). A case report with histomorphometric evaluation. Clin Oral Implants Res 2003;14: 369–372.
BONE HEALING AROUND NANOCRYSTALLINE HYDROXAPATITE
Department of Oral Surgery and Stomatology, School of Dental Medicine, University of Bern, Bern, Switzerland Private Practice, Studio Borsa Broggini Lanfranchini, Via Stazione 1, Balerna, Switzerland 3 Robert K. Schenk Laboratory of Oral Histology, School of Dental Medicine, University of Bern, Bern, Switzerland 4 Department of Oral & Maxillofacial Surgery, Copenhagen University Hospital (Rigshospitalet), Copenhagen, Denmark 5 Division of Periodontology, Department of Dentistry, National Taiwan University Hospital, Taipei, Taiwan 2
Received 31 July 2014; revised 25 September 2014; accepted 18 October 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33319 Abstract: The individual healing profile of a given bone substitute with respect to osteogenic potential and substitution rate must be considered when selecting adjunctive grafting materials for bone regeneration procedures. In this study, standardized mandibular defects in minipigs were filled with nanocrystalline hydroxyapatite (HA-SiO), deproteinized bovine bone mineral (DBBM), biphasic calcium phosphate (BCP) with a 60/40% HA/b-TCP (BCP 60/40) ratio, or particulate autogenous bone (A) for histological and histomorphometric analysis. At 2 weeks, percent filler amongst the test groups (DBBM (35.65%), HA-SiO (34.47%), followed by BCP 60/40 (23.64%)) was significantly higher than the more rapidly substituted autogenous bone (17.1%). Autogenous bone yielded significantly more new bone (21.81%) over all test groups (4.91%–7.74%) and significantly more osteoid (5.53%) than BCP 60/40 (3%) and DBBM (2.25%). At 8 weeks, percent filler amongst the test groups
(DBBM (31.6%), HA-SiO (31.23%), followed by BCP 60/40 (23.65%)) demonstrated a similar pattern and was again significantly higher as compared to autogenous bone (9.29%). Autogenous bone again exhibited statistically significantly greater new bone (55.13%) over HA-SiO (40.62%), BCP 60/40 (40.21%), and DBBM (36.35%). These results suggest that the osteogenic potential of HA-SiO and BCP is inferior when compared to autogenous bone. However, in instances where a low substitution rate is desired to maintain the volume stability of augmented sites, particularly in the esthetic zone, HA-SiO and C 2014 Wiley Periodicals, Inc. J Biomed DBBM may be favored. V Mater Res Part B: Appl Biomater 00B: 000–000, 2014.
Key Words: guided bone regeneration, unsintered nanocrystalline hydroxyapatite, deproteinized bovine bone mineral, biphasic calcium phosphate, autogenous bone graft
How to cite this article: Broggini N, Bosshardt DD, Jensen SS, Bornstein MM, Wang C-C, Buser D 2014. Bone healing around nanocrystalline hydroxyapatite, deproteinized bovine bone mineral, biphasic calcium phosphate, and autogenous bone in mandibular bone defects. J Biomed Mater Res Part B 2014:00B:000–000.
INTRODUCTION
The ideal bone substitute should not only stimulate new bone formation and promote neovascularization, but should also have a low substitution rate in order to maintain volume stability during on-going remodeling over time. The best documented xenogenic bone substitute is deproteinized bovine bone mineral (DBBM). Essentially considered a hydroxyapatite (HA) ceramic1 after the removal of its organic components, the substitution rate of DBBM is low resulting in the observation of residual particles several years following augmentation procedures.2,3 Alternatively, synthetic calcium phosphate bioceramics are favored for their biocompatibility, osteoconductivity, and non-immunogenicity. By varying the HA/tricalcium phosphate (TCP) ratio in alloplastic biphasic calcium phos-
phate (BCP), it is possible to influence substitution rate and bioactivity,4 such as the commercially available BCP with a HA/TCP ratio of 60/40% ((BCP 60/40) and C/P ratio: 1.6) which appears to mimic human cancellous bone structure.5 Alloplastic bone substitutes are often exposed to high sintering temperatures, resulting in low microporosity, and perhaps osteoconductivity.6 Non-sintered 76% nanocrystalline HA, embedded in 24% nanostructured silica gel matrix with interconnecting pores (HA-SiO), possesses a larger surface area (84 m2/g)7,8 than DBBM (79.7 m2/g).9 Granule porosity exists between 60% and 80%10 with pore sizes of 10–20 nm.7 A greater relative surface presumably leads to greater adsorption of biologically active non-collagenous proteins (i.e., fibronectin,
No benefit of any kind was received either directly or indirectly by the author(s). Correspondence to: N. Broggini; e-mail: [email protected] Contract grant sponsor: Department Funds of the University of Bern
C 2014 WILEY PERIODICALS, INC. V
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osteopontin, and bone sialoprotein)11 or bone marker molecules (i.e., alkaline phosphatase, bone morphogenetic protein 2, and osteocalcin).6,12 Adsorption would occur not only on the periphery, but internally due to high porosity, possibly expediting the healing cascade: cell colonization, cell spreading, cell differentiation, and bone matrix deposition.11 The aim of the present study was to compare the osteogenic potential and substitution rate of different bone substitutes in vivo relative to autogenous bone. MATERIALS AND METHODS
This study conformed to the “ARRIVE guidelines” (Animal Research: Reporting of In Vivo Experiments) which were developed to improve the design, analysis, and reporting of research using animals—maximizing information published and minimizing unnecessary studies (www.nc3rs.org.uk/ ARRIVE), as well as the “STROBE guidelines” which stands for an international, collaborative initiative of epidemiologists, methodologists, statisticians, researchers, and journal editors involved in the conduct and dissemination of observational studies, with the common aim of “STrengthening the Reporting of OBservational studies in Epidemiology” (www.strobe-statement.org). Surgical procedure Following sedation (ketamine (20 mg/kg)/xylazine (2 mg/ kg) I.M., and atropine (0.05 mg/kg)/midazolam (0.5 mg/kg) I.V.), general anesthesia was induced (isoflurane 1–1.5%) in eight adult G€ ottingen miniature pigs (mean weight: 60 6 8 kg (6SD); Surgical Research Unit (ESI)), University of Bern, Switzerland (ethical approval no. 99/08)). Preoperatively, 1 g amoxicillin I.M. was administered. Postoperatively, 1 g metamizolum, 150,000 IU procaine penicillin, and 150,000 IU benzathine penicillin I.V. were administered. Using a trephine, four standardized, non-critically sized, intraosseous defects (7 mm 3 4 mm) were created at the right lateral mandibular body and ramus [Figure 1(A)] and filled with either: 1. nanocrystalline HA embedded in a matrix of silica gel R 0.6 mm, Artoss GmbH, Rostock, (“HA-SiO”) (NanoBoneV Germany) or R 0.25–1 mm, Geistlich Pharma, Wolhu2. “DBBM” (Bio-OssV sen, Switzerland) or 3. synthetic BCP with a 60/40% HA/b-TCP ratio (“BCP 60/ R Bone Ceramic 90% porous 0.5–1 mm, 40”) (StraumannV Institut Straumann AG, Basel, Switzerland), or 4. particulated autogenous bone (“A”) (ground 1:1 corticocancellous bone 1–2 mm (milled cortico-cancellous bone (R. Quetin, Leimen, Germany)) as positive control [Figure 1(B)]. and mixed with blood prior to placement. Assignment was randomized and rotated in a cyclic permutation (www. randomization.com, seed: 17078). Two expanded polytetrafluoroethylene (ePTFE) membranes (GT9, W. L. Gore & Associates, Newark, DE) were used to cover the defects and fixed with titanium screws (Modus 1.5, Medartis AG, Basel, Swit-
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FIGURE 1. An example of the surgical site before (A) and after (B) bone grafting. In order of left to right in the second photograph (B): autogenous bone, HA-SiO, BCP 60/40, and DBBM. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
zerland). Primary closure was achieved in multiple layers using resorbable sutures (Vicryl 4-0, Ethicon, Norderstedt, Germany). The contralateral side was grafted identically 6 weeks later. Sacrifice after an additional 2 weeks created 2 and 8 week healing time points within each animal. Histological preparation and histomorphometric analysis As previously described,13 non-decalcified sections (80 mm) were glued to plexiglass with acrylic cement and stained superficially with toluidine blue. For qualitative histology, three regions of interest (ROI) were defined: (1) bottom of the defect, (2) center of the defect, and (3) top of the defect immediately under the barrier membrane (Figure 2). Three most central sections per defect were analyzed by a blinded, experienced examiner (B.H.).13 Percentages of newly-formed bone (osteoid, woven, parallel-fibered, and lamellar), residual grafting material, and soft tissue/marrow space were determined by point counting, using an integrative eyepiece with a square grid (distance between 6 3 6 test lines, 255 lm) at a magnification of 363.14 Statistical analysis Box plots were plotted for the distribution of bone, osteoid, filler material, and soft tissue for the two time periods.
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ROI 2: Center of the defect. In all four groups, a cell-rich soft tissue, which did not resemble bone marrow, was present after 2 weeks [Figure 5(a,c,e,g)]. However, new bone was observed only in association with autogenous bone [Figure 5(a)] and HA-SiO [Figure 5(g)]. With autogenous bone, new bone formation was more abundant and mature, interconnecting autogenous bone particles [Figure 5(a)]. After 8 weeks, new bone was most mature with autogenous bone [Figure 5(b)]. While DBBM [Figure 5(d)] and HASiO [Figure 5(h)] demonstrated a comparable bone maturation grade, new bone formation with BCP 60/40 was less abundant and mature [Figure 5(f)]. Bone marrow was most abundant and mature with autogenous bone [Figure 5(b)] and lacking with BCP 60/40 [Figure 5(f)]. FIGURE 2. Representative example illustrating the grafted bone defect, which is laterally and at the bottom bordered by new bone, after 8 weeks of healing. For the descriptive analysis, three regions of interest were defined: one at the bottom of the defect, one in the center of the defect, and one at the top of the defect just underneath the barrier membrane. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Nonparametric repeated ANOVA measurements (Brunner–Langer models) with time (2 or 8 weeks) and a treatment modality factor were used for each tissue fraction separately. In case of a significant interaction between time and treatment in the model, Wilcoxon rank sum tests were used to further detect statistically significant differences between the four treatment groups with the autogenous bone serving as positive control. Due to the explorative nature of this study, no p-value corrections were performed. The Brunner–Langer models were applied using SAS 9.1.3 (SAS Institute Inc., Cary, NC). All other analyses were performed using R 2.9.2 software package (http://www.r-project.org). RESULTS
Qualitative histology All bone fillers were evenly distributed over the defects and provided mechanical support for the ePTFE barrier membranes during both observation periods (Figure 3). ROI 1: Bottom of the defect. New bone formation was observed close to the defect margins in conjunction with all fillers at 2 weeks. Woven bone trabeculae appeared to emerge from the defect walls and interconnected bone filler particles (Figure 4). New bone was most dense and mature with autogenous bone [Figure 4(a)] with already some conversion to parallel-fibered bone formation. In contrast, new bone formed in the presence of all three bone substitutes was less abundant and mature [Figure 4(c,e,g)]. Active bone formation was indicated by the presence of osteoid lined by osteoblasts. Mature bone marrow was not observed at this time. After 8 weeks, dense and mature bone was observed in all groups, of which autogenous bone was the most advanced [Figure 4(b)], followed by DBBM [Figure 4(d)] and HA-SiO [Figure 4(h)]. BCP 60/40 was the least mature [Figure 4(f)].
ROI 3: Top of the defect. After 2 weeks, new bone in close vicinity to the barrier membrane was only seen with autogenous bone [Figure 6(a)]: new tiny woven bone trabeculae interspersed among a cell-rich soft tissue and contacting autogenous bone particles. Mature bone marrow was not evident. After 8 weeks, new bone close to, or in contact with the barrier membrane was found in all groups [Figure 6(b,d,f,h)]. New bone formation was spars and immature for BCP 60/40 [Figure 6(f)]. Bone marrow was most mature with autogenous bone [Figure 6(b)] and lacking for BCP 60/40 [Figure 6(f)]. For HA-SiO, there was more osteoid and initial mineralization further away from the defect margins than in the other two bone substitute groups. Large multinucleated cells resembling osteoclasts were observed on the surface of all filler materials after 2 weeks. After 8 weeks, they were still present on DBBM, BCP 60/40, and HA-SiO surfaces, but not on autogenous bone particles. Histomorphometric analysis Significant time and treatment effects were found for all variables (bone/osteoid, filler, and soft tissue) (Table I). Bone values generally increased and filler values decreased over the time period analyzed. Autogenous bone clearly exhibited the highest values of new bone formation, followed by HA-SiO and BCP 60/40, and with the lowest values for DBBM. Autogenous bone clearly had the lowest filler values, followed by BCP 60/40, HA-SiO, and DBBM for both time points analyzed. Two weeks after surgery, the autogenous bone group presented with a statistically significant higher percentage of bone than the other three groups (p < 0.0002; Figure 7). Additionally, autogenous bone had significantly more osteoid than the DBBM and BCP 60/40. The soft tissue percentages were similar for the autogenous bone, HA-SiO, and DBBM; only BCP 60/40 presented significantly more soft tissue when compared to autogenous bone (Table I). Eight weeks after surgery, autogenous bone still presented with a statistically significant higher percentage of newly-formed bone than the other groups (p < 0.0002 for DBBM and p < 0.001 for HA-SiO and BCP 60/40, Figure 7), and an increase from a mean value of 21.8% bone after 2 weeks to 55.1% after 8 weeks. The grafted autogenous
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FIGURE 3. Histological overview sections showing bone defects grafted with (a,b) autogenous bone, (c,d) DBBM, (e,f) BCP 60/40, and (g,h) HA-SiO, after (a,c,e,g) 2 and (b,d,f,h) 8 weeks of healing. In all defects, the bone fillers provide sufficient mechanical support for the barrier membrane. For all grafting materials, bone formation starts at the defect margins. However, penetration of the defect area with new bone occurs at different rates. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
bone particles were mostly resorbed, and only accounted for 9.3%—statistically significant lower than the filler materials in the other groups (Figure 8). The lowest percentage of soft tissue was exhibited by HA-SiO (Table I), but there was no statistically significant difference when compared to autogenous bone.
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DISCUSSION
The osteogenic effect of an autogenous bone graft at the implant surface during simultaneous bone regeneration procedures may be regarded as important to ensure osseointegration, however its high substitution rate may not sustain longterm volume stability.15 Bone substitutes, although potentially
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ORIGINAL RESEARCH REPORT
FIGURE 4. Higher magnifications of the defect margins at the bottom of the defects grafted with (a,b) autogenous bone, (c,d) DBBM, (e,f) BCP 60/40, and (g,h) HA-SiO, after (a,c,e,g) 2 and (b,d,f,h) 8 weeks of healing. For all bone fillers, new bone (NB) is present in contact with old bone (OB) and interconnecting bone filler particles (*). Note that after 2 weeks, bone formation is most advanced for the autogenous bone group. In all groups, the soft tissue (ST) does not resemble a bone marrow (BM). Most new bone is woven bone. However, conversion from woven bone to parallel-fibered bone formation is first observed in the autogenous bone group. After 8 weeks, dense and mature bone (i.e., woven bone reinforced by parallel-fibered bone) is observed in all groups. However, maturation of the bone marrow is most advanced in the autogenous bone group, as indicated by the presence of large bone marrow cavities and trabecular bone (b), followed by the DBBM (d), the HA-SiO (h), and the BCP 60/40 (f) groups. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
osteoconductive and usually providing a scaffold upon which osteogenesis may take place, possess an inferior osteogenic potential as compared to autogenous bone.16–18
The rationale for clinical utilization, therefore, may be based primarily upon a low substitution rate, which maintains volume. The principle of guided bone regeneration is
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FIGURE 5. Higher magnifications of the central regions of the bone defects grafted with (a,b) autogenous bone, (c,d) DBBM, (e,f) BCP 60/40, and (g,h) HA-SiO, after (a,c,e,g) 2 and (b,d,f,h) 8 weeks of healing. After 2 weeks of healing, the cell-rich soft tissue (ST) present among new bone (NB) and filler particles (*) does not resemble bone marrow (BM) in any of the groups. New bone is seen in association with autogenous bone and HA-SiO particles only. In the autogenous bone group, the new bone is more abundant and more mature, interconnecting autogenous bone particles. In the HA-SiO group, tiny bone trabeculae and areas with osteoid containing mineralization foci are indicative of beginning mineralization. After 8 weeks, new bone was seen in all four groups (b,d,f,h), but was most mature with autogenous bone. While the DBBM and HA-SiO groups show comparable bone maturation, the new bone in the BCP 60/40 group is less abundant and less mature. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
to maintain a surgically created volume, thus excluding rapidly proliferating epithelial cells and fibroblasts, and permitting the growth of slower-growing bone cells and
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promoting angiogenesis. The actual biomaterials themselves may contribute to this principle by physically supporting the barrier membrane and preventing its collapse
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ORIGINAL RESEARCH REPORT
FIGURE 6. Higher magnifications of the defect margins underneath the barrier membrane covering the bone defects grafted with (a,b) autogenous bone, (c,d) DBBM, (e,f) BCP 60/40, and (g,h) HA-SiO, after (a,c,e,g) 2 and (b,d,f,h) 8 weeks of healing. After 2 weeks, new bone (NB) in the form of tiny woven bone trabeculae is only seen in the autogenous bone group. The woven bone trabeculae are surrounded by cell-rich soft tissue (ST) that does not resemble mature bone marrow (BM). In the bone substitute groups, the filler particles (*) are surrounded by soft tissue, which is cell rich, but distinctly different from that observed in the autogenous bone group. After 8 weeks, new bone close to or in contact with the barrier membrane is found in all groups. Immature and little new bone are found in the BCP 60/40 group. Bone marrow is most mature in the autogenous bone group and absent in the BCP 60/40 group. The two groups with DBBM and HA-SiO show small amounts of immature bone marrow. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
during initial wound healing. In the esthetic zone, a low substitution rate may be particularly important where the establishment of sufficient facial bone volume is critical to
maintain soft tissue dimensions.15 Stable long-term conditions have been documented clinically and radiographically using cone beam computed tomography (CBCT) after
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TABLE I. Effects of Autogenous Bone, HA-SiO, DBBM, and BCP-60/40 on New Bone Formation New Bone Formation 2 Weeks Autogenous bone HA-SiO DBBM BCP 60/40 8 Weeks Autogenous bone HA-SiO DBBM BCP 60/40 a b
21.81 7.21 4.91 7.74
(6.34)a (2.78)b (1.67)b (2.71)b
55.13 40.62 36.35 40.21
(7.53) (5.74)b (3.21)b (5.13)b
Osteoid 5.53 4.28 2.25 3
(1.48) (1.8) (1.41)b (1.31)b
Filler
Soft Tissue
17.1 34.47 35.65 23.64
(2.8) (3.15)b (2.61)b (2.77)b
55.56 54.03 57.19 65.63
(5.55) (3.62) (2.97) (3.62)b
9.29 31.23 31.60 23.65
(3.07) (3.65)b (3.44)b (1.91)b
35.58 28.15 32.05 36.14
(8.61) (6.45) (4.08) (5.41)
Results presented as the mean (S.D.); n 5 8 animals/group. Statistically significant difference (p < 0.05) as compared to autogenous bone.
contour augmentation in the esthetic zone using the guided bone regeneration (GBR) technique with a combination of locally harvested autogenous bone chips and DBBM particles.19,20 The goal of this study was to compare early osteogenic potential and percent filler of HA-SiO to well-documented bone substitutes, DBBM and BCP 60/40, relative to autogenous bone. Autogenous bone exhibited the significantly greatest amount of new bone formation and maturation, however, more rapid graft resorption. This is to be expected, since both osteogenic cells and osteoinductive factors are directly transferred.21 BCP 60/40 demonstrated the significantly highest soft tissue percentage over autogenous bone at 2 weeks (65.63%) and a low percent filler (23.64%), which may reflect a greater graft turnover compared to DBBM and HA-SiO. DBBM demonstrated a trend of lower
percentage of new bone formation (4.91% and 2.25% osteoid) with respect to HA-SiO and BCP 60/40, but a seemingly high percent filler (35.65%) at 2 weeks. These observations support previous investigations demonstrating the low substitution rate of DBBM.4,22 Alloplastic calcium phosphates, however, to which BCP 60/40 belongs, are influenced by the amount of TCP and could be expected to have a higher substitution rate4,16 as compared to DBBM.4 Hence, DBBM is often promoted for augmentation procedures where volume stability is important. Interestingly, HA-SiO performed similarly to autogenous bone with respect to early osteoid production, but was similar to DBBM with respect to percent filler, and similar to BCP 60/40 with respect to rapid new bone formation (Table I). Strikingly, this trend was again observed at 8 weeks.
FIGURE 7. Box plots of the histomorphometric data of percentages of new bone formation. The vertical boxes represent the range of data from minimum to maximum. The heavy horizontal bar within the box represents the median while the ends of the whiskers represent data within the 1.5 interquartile range (IQR) of the lower quartile and within the 1.5 IQR of the upper quartile. Any data not included between the whiskers is plotted as an outlier (small circle). AB 5 autogenous bone, DBBM 5 deproteinized bovine bone material, HA-SiO 5 synthetic nanocrystalline HA, BCP 60/40 5 synthetic BCP with a 60/40% HA/b-TCP ratio, 5 outliers.
FIGURE 8. Box plots of the histomorphometric data of percentages of remaining fillers. The vertical boxes represent the range of data from minimum to maximum. The heavy horizontal bar within the box represents the median while the ends of the whiskers represent data within the 1.5 interquartile range (IQR) of the lower quartile and within the 1.5 IQR of the upper quartile. Any data not included between the whiskers is plotted as an outlier (small circle). AB 5 autogenous bone, DBBM 5 deproteinized bovine bone material, HA-SiO 5 synthetic nanocrystalline HA, BCP 60/40 5 synthetic BCP with a 60/40% HA/b-TCP ratio, 5 outliers.
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The present data is consistent with human trephine cores taken after sinus floor elevation procedures with HA-SiO.23 At 3 months, mean new bone was 8%, residual HA-SiO 45%, and bone marrow 47% which roughly correlates to 2 weeks data in the current study: total new bone (new bone 1 osteoid) 11.5%, residual HA-SiO 34.5%, and soft tissue 54%. At 6 months, 48%, 28%, and 24%, respectively in humans, corresponded as well to our 8 weeks data: 41%, 31%, 28%, respectively. One possible explanation for HA-SiO’s performance is the geometry of not only a greater relative surface due to its nanoparticles, but also due to the interconnecting pores available for protein adsorption—a so-called nanotopography, as described by Ahmad et al.24 HA-SiO has also demonstrated superior in vitro cell proliferation of human osteoblasts over DBBM.25 Of noted interest, one study demonstrated osteoinductive potential of HA-SiO in the minipig subcutaneously after 5 weeks, and intramuscularly after 4 months.26 Moreover, the effect of silicon itself on bone regeneration may be significant8,27–29 and thus, Si-based calcium phosphates may be considered not simply as “bone graft substitutes” but instead, as true drug delivery devices of Si ion, whose in vivo release may positively influence cell activity.30 The concept of tailoring ion release has been studied and concluded that the rate of release of Si and Ca ions is more rapid for pore structures with larger modal pore diameters.31 Interestingly, the biological effects of exposed HA nanoparticles after degradation of the silica oxide gel has been shown to be concentration-dependent: promotion of mesenchymal stem cell growth when HA nanoparticle concentrations were lower than 20 mg/104 cells, whereas higher particle concentration would significantly inhibit cell growth.32 Therefore, only physical presence of either Si ion or HA nanoparticles is not adequate to automatically assure a positive outcome on bone formation. Mechanical testing or functional assessment studies along with cone beam computed tomography analysis is needed to confirm whether the apparent low substitution rate ensures long-term stability of implant placement with bone augmentation. Spectroscopic analysis would support an evaluation of the quality of new bone with filler material and more clearly identify differences of graft materials. Additionally, time zero data would permit a quantitative evaluation of graft turnover. Initial studies report that complete HA-SiO degradation may occur over the order of months in humans.8 This differs from DBBM which has shown the presence of bone substitute particles 4–10 years postoperatively in human biopsies after bone augmentation.2,3,22,33 Being that both HA-SiO and DBBM performed similarly in the current study with respect to graft stability up to 8 weeks, additional investigations are needed to determine the exact degradation sequence of HA-SiO as compared to DBBM in the long term, as well as ascertain the clinical implications of volume maintenance in bone augmentation procedures. ACKNOWLEDGMENTS
The authors thank B. Hofmann, D. Reist, and S. Owusu for specimen preparation; C. Moser for surgical assistance; and Dr. D.
Mettler, O. Beslac, and D. Zalokar for surgical facilities’ management. All authors declare no conflicts of interest. REFERENCES 1. Rumpel E, Wolf E, Kauschke E, Bienengraber V, Bayerlein T, Gedrange T, Proff P. The biodegradation of hydroxyapatite bone graft substitutes in vivo. Folia Morphol (Warsz) 2006;65:43–48. 2. Mordenfeld A, Hallman M, Johansson CB, Albrektsson T. Histological and histomorphometrical analyses of biopsies harvested 11 years after maxillary sinus floor augmentation with deproteinized bovine and autogenous bone. Clin Oral Implants Res 2010; 21:961–970. 3. Jensen SS, Bosshardt DD, Gruber R, Buser D. Long-term stability of contour augmentation in the esthetic zone. Histologic and histomorphometric evaluation of 12 human biopsies 14 to 80 months after augmentation. J Periodontol 2014;85:1549–1556. 4. Jensen SS, Bornstein MM, Dard M, Bosshardt DD, Buser D. Comparative study of biphasic calcium phosphates with different HA/ TCP ratios in mandibular bone defects. A long-term histomorphometric study in minipigs. J Biomed Mater Res B Appl Biomater 2009;90:171–181. 5. Jensen SS, Yeo A, Dard M, Hunziker E, Schenk R, Buser D. Evaluation of a novel biphasic calcium phosphate in standardized bone defects: A histologic and histomorphometric study in the mandibles of minipigs. Clin Oral Implants Res 2007;18:752–760. 6. Gerike W, Bienengraber V, Henkel KO, Bayerlein T, Proff P, Gedrange T, Gerber T. The manufacture of synthetic non-sintered and degradable bone grafting substitutes. Folia Morphol (Warsz) 2006;65:54–55. 7. Kirchhoff M, Bienengraber V, Lenz S, Gerber T, Henkel KO. A new synthetic bone replacement material with osteoinductive properties—In vivo investigations. BIOmaterialien 2006;80:7. 8. Gotz W, Gerber T, Michel B, Lossdorfer S, Henkel KO, Heinemann F. Immunohistochemical characterization of nanocrystalline hydroxyapatite silica gel (NanoBone(r)) osteogenesis: A study on biopsies from human jaws. Clin Oral Implants Res 2008;19:1016– 1026. € tz H, Duschner H, Wagner W. 9. Weibrich G, Trettin R, Gnoth SH, Go [Determining the size of the specific surface of bone substitutes with gas adsorption]. Mund Kiefer Gesichtschir 2000;4:148–152. 10. Werner J, Linner-Krcmar B, Friess W, Greil P. Mechanical properties and in vitro cell compatibility of hydroxyapatite ceramics with graded pore structure. Biomaterials 2002;23:4285–4294. 11. Jensen S, Bosshardt DD, Buser D. Bone grafts and bone substitute materials for GBR procedures. In: Buser D, editor. 20 Years of Guided Bone Regeneration in Implant Dentistry. Chicago, IL, USA: Quintessence; 2009. pp 71–96. € tz W, Bienengraber V, Henkel KO, 12. Gerber T, Holzh€ uter G, Go Rumpel E. Nanostructuring of biomaterials—A pathway to bone grafting substitute. Eur J Trauma 2006:132–140. 13. Schenk RK, Olah AJ, Herrmann W. Preparation of calcified tissues for light microscopy. In: Dickson GR, editor. Methods of Calcified Tissue Preparation. Amsterdam: Elsevier; 1984. pp 1–56. 14. Weibel ER. Stereological Methods, Vol. I: Practical Methods for Biological morphometry. New York: Academic Press; 1979. 15. Buser D, Chen ST, Weber HP, Belser UC. Early implant placement following single-tooth extraction in the esthetic zone: Biologic rationale and surgical procedures. Int J Periodontics Restorative Dent 2008;28:441–451. 16. Buser D, Hoffmann B, Bernard JP, Lussi A, Mettler D, Schenk RK. Evaluation of filling materials in membrane–protected bone defects. A comparative histomorphometric study in the mandible of miniature pigs. Clin Oral Implants Res 1998;9:137–150. 17. Broggini N, Hofstetter W, Hunziker E, Bosshardt DD, Bornstein MM, Seto I, Weibrich G, Buser D. The influence of PRP on early bone formation in membrane protected defects. A histological and histomorphometric study in the rabbit calvaria. Clin Implant Dent Relat Res 2011;13:1–12. 18. Jensen SS, Broggini N, Hjorting-Hansen E, Schenk R, Buser D. Bone healing and graft resorption of autograft, anorganic bovine bone and beta-tricalcium phosphate. A histologic and histomorphometric study in the mandibles of minipigs. Clin Oral Implants Res 2006;17:237–243.
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BONE HEALING AROUND NANOCRYSTALLINE HYDROXAPATITE
