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Journal of Dentistry 70 (2018) 31–39

Contents lists available at ScienceDirect

Journal of Dentistry
journal homepage: www.elsevier.com/locate/jdent

A new synthetic granular calcium phosphate compound induces new bone in T


a sinus lift rabbit model

Amela Trbakovica, , Patricia Hedenqvistb, Torbjörn Mellgrenc, Cecilia Leyd, Jöns Hilbornc,
Dmitri Ossipovc, Stina Ekmand, Carina B. Johanssone, Marianne Jensen-Waernb, Andreas Thora
a
Department of Surgical Sciences, Plastic & Oral and Maxillofacial Surgery, Uppsala University, 751 85 Uppsala, Sweden
b
Swedish University of Agricultural Sciences, Department of Clinical Sciences, PO Box 7054, 750 07 Uppsala, Sweden
c
Polymer Chemistry, Department of Chemistry, Ångströms Laboratory, Uppsala University, Box 538, 75121 Uppsala, Sweden
d
Swedish University of Agricultural Sciences, Department of Biomedical Sciences and Veterinary Public Health, Division of Pathology, PO Box 7028, 750 07 Uppsala,
Sweden
e
University of Gothenburg, The Sahlgrenska Academy, Institute of Odontology, Department of Prosthodontics, Dental Materials Science, P.O. Box 450, 405 30 Gothenburg,
Sweden

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

piezo-electric surgery (Mectron, Carasco, Italy) in the lateral wall of the


maxilla and removed. Great care was taken not to perforate the sinus
mucosa and to elevate it carefully. The periosteum was elevated and a
miniature dental titanium implant was installed in the edentulous
alveolar ridge. The implant was 2.2 mm × 6 mm, made by machined
commercially pure titanium Grade 4, and cleaned after fabrication in a
series of cleaning steps including detergent, butanol, and ethanol, (Enge
Mikroteknik, Vittsjö, Sweden). The fixture site was prepared using a
round burr of a diameter of 2.0 mm twist drill after making a mark
(Astra Tech system drills, DentsplySirona, Mölndal, Sweden). Space was
created in the sinus cavity, under the elevated sinus mucosa, allowing
the test material to be applied underneath. The materials (0.5–1.0 mL)
were packed as near the titanium implant as possible and the space was
filled out until the material reached the bone window. (Fig. 3a.b.c.)
Over the bone window, a 25 mm × 25 mm collagen membrane
(Geistlich Bio-Gide®, Geistlich Pharma AG, Wolhusen, Switzerland)
was placed over the grafted site to hold the grafting material in
place. The periosteum and muscle layer were sutured tightly with
non-woven Monocryl sutures 5-0 (Ethicon, Johnson & Johnson AB) to
Fig. 1. Representative scanning electron microscope (SEM) image of calcium phosphate secure the membrane in place, followed by skin sutures with the same
spheres (Leo 1550, Zeiss, Oberkochen, Germany). material. The surgery lasted 60–90 min and after that, the animals were
carefully monitored until fully recovered from anaesthesia. Pain relief
was administered s.c every 6 h with buprenorphine (0.01–0.05 mg/kg,
Temgesic®, 0.3 mg/ml, RB Pharmaceuticals, Slough, Berkshire, UK)
during the first 72 h. Additionally, half of the animals received
carprofen, and all animals ceftiofur, daily for four days.
During the first postoperative week, the animals were clinically
examined and weighed daily and after that weekly until the end of the
study.

2.3. Qualitative and quantitative analyses

Twelve weeks post-surgery, the rabbits were euthanized with pen-


tobarbital (Allfatal® vet. Omnidea AB, Stockholm, Sweden) i.v.
Examination of the implant area was performed by cone beam com-
puter tomograph (CBCT) (3D Accuitomo 170 J. Morita, Irvine, CA,
USA) for guidance of the exact excision of the maxillary bone blocks.
The parts of the maxilla containing the implants were removed using a
Fig. 2. Schematic illustration of formation of nanocomposite hydrogel through binding of
band saw (KT-400, Klaukkala, Finland). The surgical area was grossly
BP groups of the polymer to calcium phosphate nanoparticles and subsequent polymer-
nanoparticle networking, receiving the HABP·CaP as end result. examined and the oral cavity inspected.

2.3.1. Sample processing


by weight were made and transferred into Eppendorf tubes.
The samples were first immersed in 10% neutral buffered formalin
for 30-31 days, followed by rinsing in tap water and subsequent dehy-
2.2.3. Deproteinized bovine bone mineral (DBBM) dration. They were then subjected to increasing concentrations of
As control material bovine mineral was chosen and commercially ethanol (70% – 100%), pre-infiltrated in diluted resins before infiltra-
attained (Geistlich, Bio-Oss®, Geistlich Pharma AG, Wolhusen, tion in pure resin and finally embedded in pure resin (Technovit 7200,
Switzerland). Particle size was 0.25–1 mm. The characteristics of this Kulzer, Hanau, Germany) and polymerized under UV light (ExaktR
biomaterial have been extensively described elsewhere [3,31]. Apparatebau, Norderstedt, Germany). Non-decalcified cut and ground
sections were prepared according to the Donath technique using the
2.2.3.1. Surgery. The surgical procedure was a modification of a ExaktR equipment [34]. In brief, the cured samples were divided in the
previous rabbit animal model by Kim et al. [32]. The rabbits were water-cooled band-saw and the surface of each block was ground par-
premedicated subcutaneously (s.c.) with the sedative medetomidine allel prior to be glued onto a supporting plexi-glass. An initial thick
(2 mg/kg, Domitor® Orion Pharma Animal Health, Sollentuna, Sweden) section was prepared which then was ground (using SiC wet grinding
and intramuscularly (i.m.). with the antibiotic ceftiofur (5 mg/kg, papers starting with 800 grit up to 1200 grit papers) in water-cooled
Exenel®, Orion Pharma AB, Animal Health, Sollentuna, Sweden). grinding equipment. No final polishing was used. The sections were
Anaesthesia was induced and maintained by continuous infusion of prepared to have a final thickness of about 15 μm and finally the sec-
sufentanil (Sufenta®, Jansen-Cilag, Sollentuna, Sweden) and midazolam tions were stained with a mixture of toluidine blue and pyronin [35].
(Midazolam Actavis, Actavis AB, Stockholm, Sweden) as described by
Hedenqvist et al. [33]. After skin preparation of the cheek area 2.3.2. Histological examination
(including shaving and washing with 2% Chlorhexidine), 4.5 mg Qualitative histology was performed as previously described by
(0.9 mL) of prilocain (Citanest®, 5 mg/ml, AstraZeneca, Sweden) was Hedenqvist et al. [26]. In the present study, we also included the pilot
administered sc. per side. A skin incision was made in the cheek area a rabbit in all evaluations. In brief, two veterinary pathologists (CL, SE),
few mm above the inferior border of the incisive bone and the maxilla. blinded to treatment evaluated the sections separately and then agreed
The subcutaneous tissue and masseter muscle were divided to expose on a consensus scoring. One section per animal from each side was
the periosteum. A bone window (10 mm × 10 mm) was made with examined for degree of implant incorporation in bone (rating as good,

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A. Trbakovic et al. Journal of Dentistry 70 (2018) 31–39

Fig. 3. Representative images showing three surgical


sites in the maxillary sinuses with implants installed
and the grafting materials applied in place: a.) CPC
granules, b.) HABP·CaP material and c.) bovine mi-
neral chips.

Histomorphometry was performed by one of the authors (ATr) on


the same samples as in the qualitative histology evaluation. All samples
were photographed in objectives of x1 and x4 with a Nikon E600 light
microscope with Nikon DXM 1200 camera (Nikon E600 microscope and
Nikon DXM 1200 camera, Nikon Instruments, Melville, NY, USA) con-
nected to a PC. All slides were photographed under same conditions for
exposure time, lamp intensity, and camera gain. The measurements and
analysis were done in NIS-elements imaging software (NIS-elements
Basic Research, Nikon, Tokyo, Japan). White balance adjustments, ca-
librations, and transformations of the photographs into binary images
were performed on each image. The amount of new bone was marked in
red, measured and presented as binary area fraction, e.g. (binary area
(red))/(the total region of interest (ROI) area) = binary area fraction.
Fig. 4. A schematic illustration of the three different regions of interests (ROIs): a) the
The new and old bone was interpreted visually by its colour, where
Free ROI that include whole material area and bone adjacent to it, marked with red
border. b) The Rectangular ROI, size area of 3.3 mm × 2.7 mm, placed were most of the “pale purple” colour was judged as old bone, while new bone had a
augmented material could be observed, and c) the Implant ROI that include the area of more “dark purple” appearance.
five grooves at both sides. The new bone is presented as area percentage of the different For the measurements of the new bone formation in contact to the
ROI areas in the different ROIs. (For interpretation of the references to colour in this materials, two different ROIs were used:
figure legend, the reader is referred to the web version of this article.)

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

Implant healing and incorporation was evaluated by two different Table 1


measurements, Implant ROI and BIC (bone to implant contact). a) The assessment of degree of screw incorporation in bone was based on blinded sub-
jective assessment of: amount of bone surrounding screw, amount of bone in screw
Objectives of x4 were used for both of these ROIs. c) For “Implant ROI”,
grooves, and amount of direct contact between bone and screw. The assessment was
the binary area fraction of the new bone formed in five grooves at both graded: + well incorporated (all 3 parameters adequate), (+) satisfactory (1–2 para-
sides of the implant was measured, starting from the third thread from meters adequate) and – poor incorporation (no parameter adequate). b) Bone associated
the implant head. (Fig. 4c) BIC was measured in five implant grooves at to the material was graded by: yes (moderate or major agent association to bone) and no
both sides of the implant, starting three threads from the implant head. (minor or no association to bone).

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

3.1. Animal experiments

In all rabbits, anaesthesia and surgery were without complications.


The handling and insertion properties of the materials used in this study
were different. The CPC granules and the bovine mineral was efficiently
compressed around the implant through the lateral opening to the
maxillary sinuses whereas the HABP·CaP tended to be dislocated and
fall apart into smaller fragments in contact with blood when inserted
into the sinus cavity. All rabbits recovered well and resumed eating
within 12 h. One rabbit was reluctant to drink from the water bottle,
and water bowls were therefore introduced to all animals. The rabbits
lost a mean of 7% of body weight during the first week postoperatively
but had recovered the weight loss by day 21 postoperatively. There was
no difference in body weight loss between treatment groups. During the
three months of follow-up, all rabbits remained clinically healthy and
kept their body weight. At the end of the study, mean body
weight ± SD was 3.9 ± 0.3 kg.
Fig. 5. Subjective scoring (median) of new bone in New Zealand White rabbits three
3.2. Histological examination months after bilateral sinus lift and augmentation with either a granular calcium phos-
phate compound (CPC, n = 8) or hyaluronic acid bisphosphonate hydroxyapatite hy-
No differences in bone formation between rabbits treated with drogel (HABP·CaP, n = 9), and bovine mineral chips (control, n = 17) serving as a con-
carprofen or not were found [26]. Specimens from one rabbit (a CPC+ tralateral control. † p < .05 (Signed rank test). * p < .05 (Rank sum test).
control) were excluded from the qualitative examination because of
incorrect specimen slicing and could not be evaluated according to the incorporated with bone in 9/9 animals and the degree of screw implant
qualitative criteria in one slide but did not affect the quantitative incorporation in bone these samples was poor in 6/9 animals. (Fig. 5)
measurements when two specimen slides were combined. (Table 1) In 1/18 sections with bovine mineral, 0/8 sections with CPC
and 9/9 sections with HABP·CaP material an inflammatory reaction
3.3. Qualitative assessment with presence of macrophages, including multinucleated giant cells,
compatible with a foreign body reaction towards the material, was
Generally, because the sinus membrane was elevated by the apical present. (Fig. 6a, b). In contrast, this type of reaction was not seen in
free part of the micro-implant, old bone was only found in the cervical bone sections treated with CPC. However, in one animal treated with
part of the implant, i.e. around the first two-three implant threads and CPC, evidence of a mild suppurative sinusitis was found and in another
was not included in the measurements of the different ROIs. animal, a moderate suppurative sinusitis, associated with rupture of
Microscopically, bovine mineral appeared as “bone chips” in- sinus lining and dislocation of CPC test material, was observed.
corporated in newly formed bone in almost all (17/18) animals. The
degree of implant incorporation was scored as good or satisfactory in
28/35 sections (Table 1). Sections from bone treated with CPC dis- 3.4. Histomorphometry
played remaining clumps of pale brown granular material in varying
sizes. The CPC was associated with bone in 8/8 sections, and the degree There was no difference in new bone formation or amount of BIC
of implant incorporation was scored as good or satisfactory in all eight between the CPC and the control in any of the ROIs. There was a dif-
sections. Sections of bone treated with HABP·CaP showed multifocal ference in BIC between HABP·CaP and control
deposits of pale yellow-brown fine granular material and was poorly (p = .05) (Fig. 7). Further, there was a difference between CPC and

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A. Trbakovic et al. Journal of Dentistry 70 (2018) 31–39

Fig. 6. a.) Representative captured image of one of


the HABP·CaP histology samples with inflammatory
cells in close proximity to the test material. Black
arrows pointing out the inflammatory cells. b.) A
magnification of an area of the picture above where
arrows are pointing out two multinucleated giant
cells.

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.

36
A. Trbakovic et al. Journal of Dentistry 70 (2018) 31–39

Fig. 9. Representative captured images taken with a


4 x microscope objective of the specimens showing
cross section slides of the three different materials,
the colour purple is showing the new bone forma-
tion. From the top and down: a.) CPC, b.) HABP·CaP
and c.) bovine mineral (note that the bovine mineral
chips are stained light purple and lack cell nucleus).

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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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.
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Carprofen neither reduces postoperative facial expression scores in rabbits treated
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