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Journal of Dentistry
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A R T I C L E I N F O A B S T R A C T
Keywords: Objectives: The aim of this study was to investigate if a synthetic granular calcium phosphate compound (CPC)
Bone substitutes and a composite bisphosphonate-linked hyaluronic acid–calcium phosphate hydrogel (HABP·CaP) induced si-
Bone implant interactions milar or more amount of bone as bovine mineral in a modified sinus lift rabbit model.
Bone regeneration Material and methods: Eighteen adult male New Zeeland White rabbits, received randomly one of the two test
Ceramic granules
materials on a random side of the face, and bovine mineral as control on the contralateral side. In a sinus lift, the
Animal experiments
sinus mucosa was elevated and a titanium mini-implant was placed in the alveolar bone. Augmentation material
(CPC, HABP·CaP or bovine bone) was applied in the space around the implant. The rabbits were euthanized three
months after surgery and qualitative and histomorphometric evaluation were conducted. Histomorphometric
evaluation included three different regions of interest (ROIs) and the bone to implant contact on each installed
implant.
Results: Qualitative assessment (p = < .05), histomorphometric evaluations (p = < .01), and implant in-
corporation (p = < .05) showed that CPC and bovine mineral induced similar amount of bone and more than
the HABP·CaP hydrogel.
Conclusion: CPC induced similar amount of bone as bovine mineral and both materials induced more bone than
HABP·CaP hydrogel.
Clinical significance: The CPC is suggested as a synthetic alternative for augmentations in the maxillofacial area.
1. Introduction Since the 1980s, various synthetic calcium phosphate based mate-
rials have been in focus of research regarding biocompatibility and
Autologous bone grafting is used for augmentation in atrophic other properties, including osteoconductivity. Lately there has been a
edentulous jaw areas but is suboptimal because of unpredictable re- considerable interest in different injectable gels for tissue repair such as
sorption of the graft. To reduce patient morbidity, there is a need for hyaluronan gels combined with different bone inducing substances [4].
new replacement strategies with osteoconductive and osteoinductive Bone inducing substitute materials are advantageous over autologous
materials. There are several materials already on the market, which are bone grafts since graft site morbidity and severe patient concerns such
osteoconductive and are used successfully for this purpose [1]. Bovine as pain, postoperative bleeding, superficial skin sensory impairment,
bone mineral (hydroxyapatite) has been extensively explored during functional disorders (e.g. disturbed gait) are avoided [5–7]. Further,
the past decades in connection with implant placement [2,3]. Bovine replacing the bone substitute materials derived from animal origin
mineral is reported to be biocompatible and a well-functioning space eliminates the risk of zoonotic infections and results in less ethical,
maintainer but not resorbable at least during an 11-year timeframe [3]. cultural, and religious considerations for the patients. An optimal bone
⁎
Corresponding author.
E-mail addresses: amela.trbakovic@surgsci.uu.se (A. Trbakovic), patricia.hedenqvist@slu.se (P. Hedenqvist), torbjorn.mellgren@angstrom.uu.se (T. Mellgren),
cecilia.ley@slu.se (C. Ley), jons.hilborn@kemi.uu.se (J. Hilborn), stina.ekman@slu.se (S. Ekman), carina.johansson@biomaterials.gu.se (C.B. Johansson),
marianne.jensen-waern@slu.se (M. Jensen-Waern), andreas.thor@akademiska.se (A. Thor).
https://doi.org/10.1016/j.jdent.2017.12.009
Received 30 March 2017; Received in revised form 29 November 2017; Accepted 15 December 2017
0300-5712/ © 2017 Elsevier Ltd. All rights reserved.
A. Trbakovic et al. Journal of Dentistry 70 (2018) 31–39
substitute material should reduce grafting site morbidity, induce os- experiment and was approved by the Uppsala ethics committee for
teoconductivity or/and osteoinductivity, and ultimately keep a balance animal experiments (C 70/13). The animals were randomised in two
between volume and resorption working as a scaffold. groups (Microsoft Office Excel 2007, Microsoft, Kista, Sweden): Group
Studies show that several of the calcium phosphate materials have CPC Granule (n = 9) and Group HABP·CaP Granule (n = 9).
abilities for bone conduction, bone induction, and resorption of the Furthermore, the side for placement of the test material was rando-
material itself [8–10]. Hydroxyapatite, brushite and monetite are dif- mised to the maxillary sinus of one side and the control material, i.e.
ferent crystalline phases of calcium phosphate, similar in their chemical bovine mineral (Geistlich Bio-Oss®, Geistlich Pharma AG, Wolhusen,
construction but presenting quite different properties. Hydroxyapatite Switzerland) was placed in contralateral sinus. The rabbits were also
is a bone conductive material and shows low, or no resorption rate in subjected to an unrelated study on the effect of the non-steroidal anti-
vivo [3] whilst monetite demonstrate both bone inductive and con- inflammatory drug (NSAID) carprofen on bone formation [26]. Within
ductive properties as well as resorbability [11]. Brushite on the other each material group, rabbits were randomised to receive either 5 mg/
hand, is quite reactive and transforms quickly into hydroxyapatite but kg of carprofen (Norocarp vet, 50 mg/ml, N-Vet, Uppsala, Sweden) or
can also synthetically be made as monetite. In the presence of pyr- the equivalent volume of saline. The protocol ran for three months.
ophosphate, monetite and brushite have the tendency to stay in their
active form until resorbed [9]. Calcium phosphate resorption is medi- 2.2. Material preparation
ated by both a cellular processes and passive dissolution [12–15].
Pyrophosphate is produced by hydrolysis of extracellular ATP and is a 2.2.1. Calcium phosphate ceramic granules (CPC)
well-known and important component in bone mineralisation where it The preparation of the calcium phosphate compound was based on a
acts as an inhibitor and regulator of hydroxyapatite formation previously published composition [18]. The cement was prepared from
[9,16,17]. mono-calcium phosphate monohydrate (MCPM), β-tricalcium phos-
The calcium phosphate compound (CPC) used in this study has been phate (β-TCP), glycerol and deionized water. 0.4 mL of water and 4.5 g
shown to have a slow resorption rate and is now in use as a solid of glycerol were stirred into a homogenous liquid before it was added to
scaffold for large skull defects in humans [18–20]. a Falcon tube with a mixture of 11.2 g of MCPM and 13.8 g of β-TCP.
Bisphosphonates (BP) are chemically analogues to pyrophosphates The cement was first mixed in a cap vibrator (Ivoclar Vivadent AG,
with strong binding capacity to hydroxyapatite. They are used in Schaan, Liechtenstein) followed by a quick mixing with a mortar and
treatments of osteoporosis and are known to influence bone formation pestle. The cement paste was subsequently extruded through a 1 mm
by decreasing osteoclast activity, but also to some degree increase os- holed screen (Caleva bench-top screen extruder, Caleva process solu-
teoblast activity [21,22]. Nejadnik et al. recently showed that non- tions Ltd. UK, Dorset) and shaped into granules (Spheronizer 120, Ca-
covalent cross-linked Bisphosphonate-hyaluronic acid-calcium phos- leva process solutions Ltd. UK, Dorset).
phate nanocomposite hydrogel (HABP·CaP) could heal itself if ruptured. The granules were sieved to obtain granules of a size between
After implantation of this material in rat bone defects, formation of new 0.60 mm and 1.18 mm, after which they were cured in 100% humidity
trabecular-like bone was detected not only at the edges of the defect but at 37 °C for 24 h. The glycerol was removed by dissolution in a water
also in the middle of the grafted material after 4 weeks [23]. In the bath for 48 h. The granules were then dried at room temperature for
study by Hulsart-Billström et al. it was shown that BPs covalently linked 24 h in a laminar flow cabinet. The final step was to autoclave the
to hyaluronic acid (HA) hydrogel exhibited reduced release rate of the granules at 124 °C for 20 min. The phase composition of the granules
in situ encapsulated bone morphogenic protein (BMP) [24]. This effect was characterized by X-ray diffraction (XRD; D8 Advance, Bruker AXS
was believed to prolong the local activity of the morphogen, which in GmbH, Karlsruhe, Germany). The granules were ground by mortar and
combination with anti-resorptive activities of the immobilized BPs pestle prior to testing. A structural characterization with a 2-theta setup
should be helpful in increasing bone formation [4,21]. was used with a beam knife, a Nickel filter and a Cu-K irradiation
In the present study, the aim was to explore if the granular form of source. The resulting diffractogram was collected between 2θ of 5–60°
the CPC material is a potentially suitable grafting material in the at steps of 0.02° with 0.25 s per step and a rotation of 80 rpm. Phase
maxillofacial area e.g., in sinus augmentations. Further, we wanted to composition of CPC granules from XRD analysis was β-CPP 6.1 wt%, β
investigate the possible benefits that HABP·CaP may have on local bone –TCP 10.4 wt%, Brushite 0.5 wt% and Monetite 82.8 wt%, followed by
formation. The hypothesis was that the two test materials would be as Rietveld refinement. The different crystalline phases were quantified by
efficient with regards to bone formation as bovine bone in a rabbit sinus Rietveld refinement analysis, using BGMN software (www.bgmn.de)
lift model. with Profex interface (http://profex.doebelin.org). The reference
structures were PDF#04-008-8714 for β-TCP [27] PDF#04-009-3876
2. Materials and methods for brushite [28]. PDF#04-009-3755 for monetite [29] and PDF#04-
009-3876 for β-calcium pyrophosphate (β-CPP) [30]. (Fig. 1) All work
2.1. Animals and study design was performed using sterile technique.
Eighteen adult male New Zeeland White rabbits from a specific- 2.2.2. Bisphosphate-hyaluronic acid-calcium phosphate nanocomposite
pathogen-free colony (Lidköpings Kaninfarm, Lidköping, Sweden) were hydrogel (HABP·CaP)
used. The breeding colony was free from known rabbit pathogens ac- Physically cross-linked HABP·CaP nanocomposite hydrogel material
cording to recommended health monitoring [25] At the time of surgery, was prepared according to our previously published procedure [23].
the rabbits were 36 ± 4 weeks old and weighed 3.7 ± 0.2 kg Formation of the nanocomposite hydrogel is triggered by mixing of
(mean ± SD). The rabbits were housed individually in cages with a equal volumes of two aqueous solutions, 4% hyaluronan-bispho-
floor area of 0.42 m2, equipped with a shelf and a covered area. Stan- sphonate and 12% hydroxyapatite, 1.5 mL of 4% hyaluronan-bispho-
dard pelleted rabbit diet (Lactamin K3, Lantmännen, Stockholm, sphonate solution and 1.5 mL of 12% hydroxyapatite dispersion were
Sweden) and autoclaved hay were fed and rabbits had access to auto- taken into two separate 3 mL syringes, which were then connected
claved straw for bedding and water ad lib. The light – dark cycle was through a plastic connector (Fig. 2). Mixing of the components was
12:12 h with lights on at 07:00. Room temperature was 18 ± 3° C and achieved by passing the liquid precursors from one syringe to the other
humidity 55 + 10%. The animals were acclimatized for two weeks and several times. The nanocomposite hydrogel was formed and the nano-
accustomed to handling. On the day before surgery, the rabbits were composite finally collected in one of the syringes. The other syringe was
clinically examined. taken away and the nanocomposite was extruded through a needle in
The study was designed as a prospective, randomised, controlled portions onto a balance plate. Nine portions of approximately 325 mg
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A. Trbakovic et al. Journal of Dentistry 70 (2018) 31–39
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A. Trbakovic et al. Journal of Dentistry 70 (2018) 31–39
a) A “Free ROI” (objective of x1) where the external border was out-
satisfactory or poor) and graft material in association with bone (yes or lining the bone found in contact with the tested material. The fact
no). The degree of implant incorporation was evaluated according to that the material in different samples ended up in different areas
the following criteria: amount of bone surrounding the implant, amount around the implant resulted in different area sizes and locations of
of grooves containing bone and amount of bone in direct contact with the Free ROIs, 1,0 mm2–17,8 mm2 (mean area 10,8 mm2). (Fig. 4a)
the implant. If all three parameters were fulfilled; i.e. the implant was b) “Rectangular ROI” described the bone–material ratio and had con-
surrounded by moderate or abundant bone, and moderately-extensively sistent size area between the samples of 3.3 mm × 2.7 mm,
incorporated with regard to number of grooves filled with bone and (8.9 mm2). Due to the material location around test implants the site
presence of direct bone-implant contact, the incorporation was scored of the ROI varied in the samples to were most amount of the tested
as good. If only one or two of the criteria were adequate the in- material could be seen, resulting in same area size but different
corporation was satisfactory; otherwise poor. Also, sections were eval- locations in all the samples. (Fig. 4b)
uated for inflammatory changes, such as influx of inflammatory cells.
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A. Trbakovic et al. Journal of Dentistry 70 (2018) 31–39
a.
2.4. Statistical analyses Degree of screw incorporation + (+) –
Statistical analyses were performed with Sigma Plot 13.x (SYSTAT, CPC 4 4 0
HABP·CaP 2 1 6
Chicago, Illinois). Qualitative data was graded as ordinal data and
Control 9 8 1
measurements of implant incorporation was conducted by grading the
data starting with good = 2, followed by satisfactory = 1 and poor = 0.
b.
The presence of bone associated with the material was assessed as Agent associated to bone + –
yes = 1 or no = 0. For the qualitative assessment, Mann Whitney Rank
Sum test was used to compare the three different materials, and CPC 8 0
Wilcoxon Signed Rank test for comparison of the test materials with HABP·CaP 0 9
Control 17 1
controls. For the histomorphometric data the Student’s t-test was used to
compare the test materials and paired t-test for test material against
control. The significance level was set at p < .05.
3. Results
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A. Trbakovic et al. Journal of Dentistry 70 (2018) 31–39
Fig. 8. Percentage of new bone formation (mean, SD) at three different regions of interest
(ROI) in New Zealand White rabbits three months after bilateral sinus lift. The sinus space
around the implant was augmented with either a granular calcium phosphate compound
(CPC, n = 9) or hyaluronic acid bisphosphonate hydroxyapatite hydrogel (HABP·CaP,
Fig. 7. Percentage of new bone formation in contact with the implant in New Zealand n = 9). Bovine mineral chips (control, n = 18) served as a contralateral control. *
White rabbits three months after bilateral sinus lift. The sinus space around the implant p < .05, *** p < .001 (Rank sum test), † p < .05 (Signed rank test).
was augmented with either a granular calcium phosphate compound (CPC, n = 9) or
hyaluronic acid bisphosphonate hydroxyapatite hydrogel (HABP·CaP, n = 9). Bovine
mineral chips (control, n = 18) served as a contralateral control. † < 0.05 (Signed rank Evaluations by histology and the histomorphometric analysis in-
test). dicate that the CPC granules have bone conductive ability and integrate
well within the new bone. Marked bone formation was seen around the
HABP·CaP in regard to bone formation in the free ROI (p < .001), the bovine bone mineral. In contrast, limited amount of bone was found in
rectangular ROI (p < .001) and in the implant ROI (P = 0,004) with contact to HABP·CaP material, suggesting poor integration between
CPC showing more bone in all three. (Figs. 8 and 9a–c ). material and bone. New bone was mostly found distant from the
HABP·CaP. As known from previous studies, new bone formation can be
found even if no foreign material is applied in a sinus lift procedure, but
4. Discussion
from bleeding alone, which could explain the finding of bone at a dis-
tance from the HABP·CaP [36–38].
This study showed that augmentation with CPC granules results in
Further evaluation to establish bone inductive properties of CPC
the similar amount of new-formed bone as the treatment with bovine
granules in ectopic sites would be of interest. The amount of new bone
mineral in a rabbit sinus lift model. Augmentation with HABP·CaP
and absence of inflammation in the specimens treated with CPC in-
however, displayed less amount of bone formation. The handling
dicate that the material is biocompatible. The next logical step would be
properties of the materials during surgery differed. The CPC granules
a human trial with CPC granules in sinus lifts, or as onlay material in
and bovine mineral were unyielding and easy to handle, whereas the
atrophic alveolar jawbone defects before or simultaneously with im-
HABP·CaP had the consistency of a gel-foam. This plays a significant
plant placement. The material is currently being tested in a study of
role, especially in challenging grafting sites, in which the material
demanding cleft augmentation. Additionally, three patients have been
needs to be applied in an exact anatomical localization and the tissue
treated with CPC for sinus floor augmentation and are now being
space needs to be supported by the grafting material. It was difficult to
evaluated at this time.
properly apply HABP·CaP nanocomposite due to its much lower density
The rabbit is well established as a model for testing of bone implants
as compared to CPC granules and bovine mineral. It made it less ef-
[39]. Bone characteristics of rabbits are more similar to humans in
fective as space maintainer. The advantage of the HABP·CaP material on
comparison with rodents and compared to dogs and sheep: the rabbit is
the other hand is that it may be injected.
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A. Trbakovic et al. Journal of Dentistry 70 (2018) 31–39
less expensive to use as a research animal making them useful for pre- osteoinduction [42–45]. Inductive properties of monetite have been
clinical studies [40]. The surgical procedure was in this study well evaluated in earlier studies in which importance of porosity, geometric
tolerated and the rabbits all recovered quickly; no intensive care or shape of the onlay and anatomical place was found to be important for
forced feeding was necessary, and no clinical infections occurred. bone formation [45,46]. Sheikh et al. showed in their study of vertical
The current study compliments few previously performed studies in monetite blocks in rabbit calvaria that more porous monetite blocks
rabbits in which maxillary sinus lift augmentation is combined with resulted in a higher amount of new bone and larger degree of resorption
placement of a mini implant as reviewed by Stübinger and Dard of the material. It was also noted that most new bone was formed in the
[32,39,41]. In the current study, some difficulties were encountered lateral parts of the monetite blocks, which again demonstrates the
during surgery and specimen preparation, e.g. obtaining the same angle importance of the geometric shape [45–47]. This was also seen in the
of insertion of the implant in every sinus and rabbit. Cone beam CT study by Engstrand et al., in which ceramic tiles with the same ceramic
images of the specimens were obtained to guide preparation of the blend as in the present study were used for cranial defects. By re-
samples prior to the sectioning technique. The different locations of the gistering osteoblastic activity with F18 PET/CT, the bone integration
tested materials in relation to the implant and the implants angulation was first noticed in the periphery of the defects in tiles next to the
complicated the evaluations. Nevertheless, both the subjective and the existing bone, possibly because of better blood and nutrient supply in
histomorphometric evaluations showed that treatments with CPC and this area. After 50 months of non-eventful function and healing,
bovine mineral resulted in higher amount of new bone and better de- bleeding bone could be seen in a biopsy also in the central tiles of the
gree of implant incorporation compared to treatment with HABP·CaP. defect. There was clinical evidence that the material did not give rise to
The individual variation in anatomy of the maxillary sinuses in rabbits any negative soft tissue reactions [18]. The authors stated that the key
resulted in difficulty of applying the exact same amount of the mate- substance in the ceramic compound is the β-calcium pyrophosphate
rials. The aim to overcome this was by applying as much material in (which inhibits the resorption of the other calcium phosphates) and that
each sinus compartment around the implant as possible and close the the resulting properties of the compound are highly dependent on the
bony window with the collagenous membrane. exact chemical composition of the four phases of calcium phosphates
Small crystalline pieces of non-bone associated CPC material were [18]. In the present study, we used a granular form of the CPC, which
observed histologically. These could possibly be discarded calcium may be preferred for augmentation in the sinus lift model or in major
particles that had been released when the material was dissolving. extraction alveoli, where the volume needs to be maintained during
Further studies with longer healing periods would be needed to healing, as well as in cases with alveolar bone defects. The size of the
evaluate the course of bone induction and possibly the materials’ final granules may also be an important factor for the result. Smaller gran-
and total transformation into bone. For cranial vault reconstructions, ules are harder to apply and may theoretically be resorbed faster and
the same compound but in a mosaic form has now been implanted since thereby have a shorter lifetime as space maintainer. The present results
six years, showing promising results with a high degree of biocompat- indicate that the CPC granules in the samples were well integrated with
ibility and new bone forming in a slow but steady pace between the bone. Further investigations need to be carried out investigating how
solid ceramic tiles [18,20]. the CPCs granular size affects bone formation. For this study, it would
Brushite and monetite have been evaluated as bone substitute ma- have been more advantageous to have had the exact same size of
terials and have both shown promising results acting in forms of onlays, granules and of the bovine mineral chips, since they both varied in size
granules and injectable cement, displaying desirable properties such as (Control (0.25 mm–1.0 mm); CPC (0.60 mm–1.18 mm)).
osseointegration in conjunction with implants, osteoconduction and The hyaluronic acid hydrogel was shown to be a promising and a
37
A. Trbakovic et al. Journal of Dentistry 70 (2018) 31–39
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Author contributions healing hybrid nanocomposites consisting of bisphosphonated hyaluronan and
calcium phosphate nanoparticles, Biomaterials 35 (25) (2014) 6918–6929.
Conceived and designed the experiments: ATC AT PH MWJ JH [24] G. Hulsart-Billström, P.K. Yuen, R. Marsell, J. Hilborn, S. Larsson, D. Ossipov,
Bisphosphonate-linked hyaluronic acid hydrogel sequesters and enzymatically re-
Performed the experiments: ATC AT PH MWJ leases active bone morphogenetic protein-2 for induction of osteogenic differ-
Analysed the data: ATC PH CL SE entiation, Biomacromolecules 14 (9) (2013) 3055–3063.
Contributed reagents/materials/analysis tools: DO TM CBJ [25] M. Mähler, M. Berard, R. Feinstein, A. Gallagher, B. Illgen-Wilcke, K. Pritchett-
Corning, M. Raspa, FELASA recommendations for the health monitoring of mouse,
Writing-original draft: ATC
rat, hamster, guinea pig and rabbit colonies in breeding and experimental units,
Writing- review & editing: ATC AT PH MWJ JH DO TM CL SE CBJ Lab. Anim. 48 (3) (2014) 178–192.
[26] P. Hedenqvist, A. Trbakovic, A. Thor, C. Ley, S. Ekman, M. Jensen-Waern,
Carprofen neither reduces postoperative facial expression scores in rabbits treated
Acknowledgements
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