Accepted: 2 July 2017 DOI: 10.1111/jre.12490
ORIGINAL ARTICLE
Attributes of Bio-Oss® and Moa-Bone® graft materials in a pilot study using the sheep maxillary sinus model M. M. Smith | W. J. Duncan | D. E. Coates Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago, Dunedin, New Zealand Correspondence Dawn Coates, Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago, Dunedin, New Zealand. Email: [email protected] Funding information This study was funded by a University of Otago Fuller grant. Moa-Bone® was provided free of charge by Molteno® Ophthalmic Ltd.
Background and Objective: The aim of this pilot study was to characterize surface morphology and to evaluate resorption and osseous healing of two deproteinated bovine bone graft materials after sinus grafting in a large animal model. Material and Methods: Surfaces of a novel particulate bovine bone graft, Moa-Bone® were compared with Bio-Oss® using scanning electron microscopy. Six sheep then had maxillary sinus grafting bilaterally, covered with BioGide®. Grafted maxillae were harvested after 4, 6 and 12 weeks. Healing was described for half of each site using resin-embedded ground sections. For the other half, paraffin-embedded sections were examined using tartrate resistant acid phosphatase staining for osteoclast activity, runt-related transcription factor 2 immunohistochemistry for pre-osteoblasts and osteoblasts and proliferating cell nuclear antigen for proliferative cells. Results: Moa-Bone® had a smoother, more porous fibrous structure with minimal globular particles compared with Bio-Oss®. After 4 weeks, woven bone formed on both grafts and the Moa-Bone® particles also showed signs of resorption. After 12 weeks, Moa-Bone® continued to be resorbed, however Bio-Oss® did not; both grafts were surrounded by maturing lamellar bone. Moa-Bone® was associated with earlier evidence of runt-related transcription factor 2-positive cells. Moa-Bone® but not Bio-Oss® was associated with strong tartrate resistant acid phosphatase-positive osteoclasts on the graft surface within resorption lacunae at both 4 and 6 weeks post-grafting. Conclusion: Both materials supported osseous healing and maturation without inflammation. Moa-Bone® showed marked osteoclast activity after 4 and 6 weeks and demonstrated positive attributes for grafting, if complete remodeling of the graft within the site is desired. Further optimization of Moa-Bone® for maxillofacial applications is warranted. KEYWORDS
animal model, biomaterial, bone grafting, sinus lift
1 | INTRODUCTION
and the resultant edentulous space is insufficient height for implant placement. 4
The anatomy of the posterior maxilla, and bony remodeling follow-
Sinus augmentation procedures have been developed to improve
ing extraction of teeth often results in a lack of suitable bone for
bone volumes, and a variety of materials may be used for this. Bone
placement of dental implants.1-3 Furthermore, in the region of the
autografts have been identified as the gold standard for bone augmen-
maxillary sinus the quality of the residual maxillary bone is often poor
tation because of the reduced risk of disease transmission and the low
J Periodont Res. 2017;1–11.
wileyonlinelibrary.com/journal/jre © 2017 John Wiley & Sons A/S. | 1 Published by John Wiley & Sons Ltd
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SMITH et al.
2
(A)
(B)
(C)
(D)
F I G U R E 1 Surgical procedure on the maxillary sinus of sheep. (A) Arrow marks the surgical site. (B) Graft material in place before placement of the Bio-Gide® membrane. (C) Representative radiograph of the graft material and osteotomy site before sectioning (MB at 6 wk). Scale bar = 1.5 mm. (D) Diagram of the site retrieved for analysis. BO, Bio-Oss®; MB, Moa- Bone®
antigenic nature of the transplant. Autografts are characterized clini-
materials.20-25 In a series of studies in the sheep maxillary sinus model,
cally by complete remodeling of the graft material over time. The use
implants placed into sites augmented with DBB were characterized by
of autografts is, however, limited by the morbidity experienced at pro-
significantly higher “pull out” forces than non-grafted sites,26-28 under-
curement and the ability to harvest sufficient material. Deproteinated
lining the utility of DBB material and the sheep sinus model. According
bovine bone (DBB) has been used for a number of years in dentoalveo-
to Haas, pigs are unsuited to use as a model due to excessive thickness
lar grafting. DBB is normally produced from bovine bone using combi-
of the cortical bone, and dogs are unsuitable because the nasal sinus
nations of heat, organic solvents and strong chemicals such as sodium
lacks a Schneiderian membrane and does not undergo appropriate
hypochlorite, and the product sterilized using gamma radiation and/or
pneumatization.29
heat and pressure. The removal of all organic material is important to
The aim of the current pilot study was to characterize the surface
reduce the transmission of diseases, in particular bovine spongiform
features of MB and Bio-Oss® graft materials and to evaluate the os-
encephalopathy.5
teogenesis and bone remodeling associated with both graft materials
There are a number of bovine-derived xenograft products used for
in a sheep maxillary sinus model.
oral bone replacement grafting procedures, with published evidence supporting their use in animal preclinical models6-9 and human clinical trials.10 Bio-Oss® is a xenograft that is widely used in clinical practice, for which
2 | MATERIAL AND METHODS
there is a considerable body of published data.11 Bio-Oss® is processed using low temperatures (approximately 300°C) and organic solvents.12
This study conformed to the ARRIVE guidelines for pre-clinical ani-
It is reported to be highly porous, on a macro-and microscopic level,13
mal studies. Ethical approval was obtained from the University of
and is frequently used in clinical trials and compared to new or similar
Otago Animal Ethics Committee (AEC 50-80). After characterization
products.14,15 Moa-Bone® (MB) is another DBB product, sourced from
of the biomaterials, animal surgery was performed using six healthy
disease-free cattle in New Zealand and used for ophthalmic grafting. It
Romney-cross female sheep (ewes) in the weight range 70-90 kg and
is initially processed under pressure at high temperature, followed by
18-24 months of age. Sinus grafting was performed bilaterally on each
bleaching and centrifugation, with final sterilization by autoclaving. MB
animal, with the two treatments, MB or Bio-Oss®, randomly allocated
16
is light, porous and friable.
Particulate MB is a by-product arising from
to the left or right sinus. The grafted maxillary sinuses were harvested
the milling of the M-Sphere®, a spherical bone prosthesis used during
after animal killing at 4, 6 and 12 weeks post-grafting. For this pilot
reconstruction of an enucleated human orbit. The M-Sphere® is well tol-
study, two animals were used per time period, which provided suf-
erated in humans, causing no long-term inflammatory reaction.17 To our
ficient information regarding the responses to the two test grafts; sta-
knowledge, there are no studies of the potential uses of MB particulates
tistical comparisons were not required.
for maxillofacial applications, such as grafting into the maxillary sinus. Studies have described the suitability of sheep for biomaterial testing in cancellous bone as excellent.18,19 The utility of the sheep
2.1 | Biomaterials
sinus for implant research has been demonstrated by a number of
MB was supplied in the form of a sterile M- Sphere® (Molteno®
groups in studies involving both mesenchymal stem cells and implant
Ophthalmic Ltd., Dunedin, New Zealand) and crushed through a series
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SMITH et al.
of sieves with mesh apertures of 1.68, 1.18 and .25 mm (Endecotts
Animals were recovered to the Otago University farm and were con-
Ltd, London, UK) within a laminar flow hood, to achieve a size pro-
tinuously monitored and fed a normal diet. Animals were killed as pre-
file of between .25 and 1.00 mm. This was consistent with the par-
viously described
®
31
after 4, 6 and 12 weeks, by anesthetic overdose
ticle sizes of the commercially available Bio-Oss . The MB particles
followed by carotid artery perfusion with 5000 iu of heparin in 1 liter
were steam sterilized in an autoclave (Mercer 7, Mercer Medical, New
of saline followed by 2 liters of 10% neutral buffered formalin (BioLab
®
Zealand) for a standard cycle of 4 minutes at 134°C. Bio-Oss was
Ltd, Auckland, New Zealand).
supplied with a particle size of .25-1.00 mm in a sterile vial (Geistlich Pharma Australia and New Zealand, Chatswood, NSW, Australia).
2.2 | Scanning electron microscopy characterization
2.4 | Tissue collection and processing Tissue sections were resected en bloc using a hacksaw and trimmed with a custom-made guillotine before being immersed in 10% neutral
Representative samples of MB and Bio-Oss® particles were mounted
buffered formalin for 48 hours at 4°C with gentle rotation. Specimens
on 10 mm aluminum stubs and coated with a 10 nm layer of gold/pal-
were radiographed (Figure 1C) before being cut vertically through the
ladium using an Emitech K575X high-resolution sputter with carbon
graft site and separated into two halves for either resin or paraffin
coater attachment (EM Technologies Ltd, London, UK). High-power
embedding (Figure 1D). Evaluation of the samples was conducted by
scanning electron microscopy (SEM) images of the surface architec-
investigators blinded to the groups.
ture were obtained using a JEOL JSM-6700F field emission SEM (JEOL Ltd, Tokyo, Japan) at an acceleration voltage of 3.0 kV.
2.5 | Embedding of the tissue samples Resin embedding with methyl methacrylate (Sigma-Aldrich, St. Louis,
2.3 | Surgery
MO, USA) was conducted as previously described.31,32 Briefly, tis-
The anesthetic and surgical procedures have previously been de-
sue specimens were dehydrated in ascending grades of alcohol, im-
scribed in detail,24,25 and are modifications of the procedure originally
mersed in xylene for 2 days (two changes), followed by methacrylate
described by Haas and co-workers.26,29,30 Under general anesthetic
and methacrylate with plasticizer and setting agent for 2 days each,
the wool of the face was shorn, and the maxilla bone overlying the
and then embedded in polymerized methacrylate in a sealed glass jar,
sinus was exposed by a 6 cm extra-oral sagittal skin incision located
which was kept cool in the dark. Sequential 500 μm sections were
caudal to the facial tuberosity, followed by separation of the malar
cut from each resin-embedded specimen using a Struers precision
muscle and blunt dissection and partial detachment of the masse-
tabletop cut- off machine (Accutom; Struers GmbH, Birmensdorf,
ter muscle (Figure 1A). A 1 cm circular bony window was created
Switzerland), and press mounted on to an opaque acrylic base plate
through the lateral wall of the maxillary sinus using a Piezotome™
using a cyanoacrylate glue. Sections were then ground, using a ro-
(SL2 tip, Satalec, Acteon, Merignac, France), cooled by saline irriga-
tating grinding machine (Tegra- Pol; Struers GmbH, ZNL Schweiz,
tion. The antral Schneiderian membrane was elevated from the buc-
Birmensdorf, Germany). Final polished specimens had a thickness of
cal bony wall using a sinus elevation kit (Sinus Kit, Osstem Implants,
between 80 and 100 μm and were surface-stained with MacNeal’s
Seoul, Korea), and displaced dorso-cranially before placement of the
tetrachrome and toluidine blue.33
®
graft material. MB or Bio-Oss (.25 g) were mixed with 1 mL of blood
Tissue for paraffin embedding was decalcified with 10% EDTA as
from the surgical field and placed in the resulting pouch within the
previously described and the end-point for decalcification determined
sinus (Figure 1B).
by an oxylate test.34 After embedding, 4 μm serial sections were cut
Two pieces of sterile porcine collagen membrane (Bio- Gide®, Geistlich, Switzerland) measuring 12.5×12.5 mm were placed over
and mounted on 3- aminopropyltriethoxysilane- coated slides (Lab Scientific Inc, Highlands, NJ, USA).
each other in a bi-laminar style to secure the biomaterials within each site. The fascia and subcutis were closed in layers using resorbable sutures (Vicryl 3/0, Ethicon; Vicryl (Ethicon); Johnson & Johnson,
2.6 | Tartrate resistant acid phosphatase staining
Oraltec NZ Limited, Auckland, New Zealand) and the skin closed using
Tartrate resistant acid phosphatase (TRAP) staining was conducted
non- resorbable sutures (Maxon 2/0, Syneture; Maxon (Covidien),
using an Acid Phosphatase Leukocyte Kit (387A; Sigma-Aldrich) as
Tyco Healthcare Ltd., Auckland, New Zealand). After surgery, a long-
per the manufacturer’s instructions. However, the final incubation in
acting Bupivacaine HCl anesthetic (Marcaine .5%; AstraZeneca,
TRAP solution was reduced to 1 hour at 37°C in the dark.34
Amtech Medical Ltd., Wanganui, New Zealand) was administered to control pain in the site. Postoperatively animals were given the anti- inflammatory carprofen 4 mg/kg sc (Norbrook New Zealand Ltd,
2.7 | Immunohistochemistry
Auckland, New Zealand), as well as intramuscular antibiotic admin-
Sections were de-waxed in xylene before rehydrating through graded
istration of Strepcin containing 250 000 IU/mL procaine penicillin
alcohols to phosphate-buffered saline (PBS). Heat-based antigen re-
G with 250 mg/mL dihydrostreptomycin, 5 mL/animal (Stockguard
trieval was conducted for 10 minutes at 80°C in a bath of .01 mol/L
Laboratories Ltd, Hamilton, New Zealand), each day for 3 days.
sodium citrate buffer (pH 6). Sections were then blocked to reduce
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SMITH et al.
4
(A)
(B)
F I G U R E 2 Scanning electron microscopy images of the surface of the implant materials. (A) Moa-Bone® and (B) Bio-Oss® non-specific antibody binding with 20% goat serum for runt-related transcription factor 2 (RUNX2) or 20% rabbit serum for proliferating
3.2 | Surgery The surgery was uneventful and no sinus membrane perforations
cell nuclear antigen (PCNA) in 1% bovine serum albumin/PBS. RUNX2 immunohistochemistry was conducted with a rabbit antihuman polyclonal RUNX2 antibody (NBP1- 01004, Novus Bio,
were observed. No animal was observed to have any adverse short- or long-term events following surgery.
Littleton, CO, USA) and control sections treated with rabbit IgG (sc2027 Santa Cruz Biotechnology, Santa Cruz, USA), both at a concentration of 10 μg/mL. PCNA immunostaining was conducted with
3.3 | Histology of resin-embedded tissue
5.25 μg/mL of mouse antirat PCNA monoclonal antibody (Clone PC-
The histology of the sinus implant material was examined at 4, 6 and
10, M0879; Dako, Glostrup, Denmark) and mouse IgG (sc2025; Santa
12 weeks post-implantation. The region of interest for imaging was
Cruz Biotechnology) as a control. The primary antibodies and control
the site with grafted material immediately adjacent to the native maxil-
IgG solutions were left to incubate at 4°C overnight.
lary bone. At 4 weeks, some woven bone partially enveloped the MB.
Sections were then washed three times with PBS containing 1%
Sharply delineated semi-circular pits on the surfaces of the particles,
Tween-20 and 1% non-fat milk powder. The biotinylated second-
resembling resorption lacunae, were evident (Figure 3A). Connective tis-
ary antibodies were goat antirabbit H&L- F(ab)2 (ab6012; Abcam,
sue was highly evident. Bio-Oss® was also associated with woven bone
Cambridge, UK) for RUNX2, and a rabbit antimouse H&L- F(ab)2
and an irregular fibrous pattern of connective tissue filled the spaces. No
(ab5761; Abcam, Cambridge, UK) for PCNA; both at 2 μg/mL and
resorption lacunae were seen on the Bio-Oss®. At 6 weeks, the amount
incubated for 1 hour at room temperature. Endogenous peroxidase
of woven bone associated with both graft materials had increased
was quenched using .3% hydrogen peroxide in methanol for 10 min-
(Figure 3C,D). Resorption-like lacunae were most evident where the con-
utes, followed by treatment with Strep horseradish peroxidase
nective tissue abutted the MB (Figure 3C). Twelve weeks post-grafting,
(Vectastain Elite ABC; Vector Laboratories, Inc, Burlingame, CA, USA).
the MB was almost entirely encased by lamellar bone and the graft par-
Development was performed using 3,3′ diaminobenzidine (D3939;
ticles appeared smaller than previously observed. Bio-Oss® particles ap-
Sigma-Aldrich). Sections were counterstained with hematoxylin for
peared to be unchanged in size compared to the 4 week time point and
5 seconds followed by dehydration and coverslipped using Entellan
®
were predominantly surrounded by lamellar bone (Figure 3E,F).
(Merck, Darmstadt, Germany). Light microscopy was used to examine all sections and images were digitized using a JVC TK 1281 color video camera and SPOT® digital imaging software. Semiquantitative analysis was also conducted using
3.4 | Immunohistochemistry of biomaterials within the sheep sinus
the following scores for the presence of resorption lacunae and TRAP-
Decalcified paraffin- embedded sections of sheep sinuses containing
positive cells: 0 = no positive cells; 1 = occasional positive cells; 2 =
the implant materials were examined. Control IgG staining showed only
many positive cells; and 3 = large numbers of positive cells.
background staining (not shown). Table 1 gives an overview of the results from all time points for TRAP staining, resorption lacunae and the pres-
3 | RESULTS
ence of bone types and connective tissue adjacent to the graft material.
3.1 | Characterization of the biomaterials
3.5 | Four weeks post-implantation
MB was more difficult to handle compared to Bio-Oss®, as it was
The implant materials were surrounded by woven bone and connec-
more hydrophobic and electrostatic in nature. SEM revealed different
tive tissue (Figure 4A,B). Very strong TRAP staining of osteoclasts
surface morphologies; MB at high magnification showed evidence of
within the sheep sinus was noted at the 4 week time point for MB
a porous structure and retained a fibrous-like organization (Figure 2A).
(Figure 4C). The incubation time was reduced for one block as the
In contrast, Bio-Oss® had clusters of globular-like particles evenly dis-
staining completely swamped the slide. TRAP staining was associated
tributed over the surface of the xenograft (Figure 2B).
with large multinuclear osteoclast-like cells inside resorption lacunae
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SMITH et al.
(A)
(B)
(C)
(D)
(E)
(F)
F I G U R E 3 Resin sections of sinus implant regions containing MB (left-hand side) or BO (right-hand side). Sections are from weeks post-biomaterial implant surgery with (A, B) at 4 wk; (C, D) at 6 wk; (E, F) at 12 wk. BO, Bio-Oss®; CT, connective tissue; arrows, resorption like lucunae; LB, lamellar bone; MB, Moa- Bone®; WB, woven bone; scale bar = 100 μm T A B L E 1 Osteoclasts and bone formation around implant materials 4 wk Moa Bone Resorption lacunae
6 wk ®
3
Bio-Oss
®
0
Moa Bone
12 wk ®
3
Bio-Oss
®
0
Moa Bone®
Bio-Oss®
0
0
TRAP-positive osteoclasts
3
1
3
1
1
1
Tissue around graft
WB/CT
WB/CT
LB/WB/CT
LB/WB/CT
LB
LB/CT
0 = no positive cells/lacunae; 1 = occasional positive cells/lacunae; 2 = many positive cells/lacunae; 3 = large numbers of positive cells/lacunae. CT, connective tissue; LB, lamella bone; WB, woven bone.
on the MB (Figure 5A,C,E). Diffuse alkaline phosphatase staining was also localized to the connective tissue. Osteoclasts were also evident
3.6 | Six weeks post-grafting
within areas containing new woven bone. Around the Bio-Oss® par-
Residual graft material was visible at 6 weeks post-grafting within
ticles, very few TRAP-positive osteoclasts were detected in the sinus,
the sheep sinuses, typically surrounded by woven or lamella bone;
and were mostly associated with woven bone (Figures 4D and 5D).
however, in some areas the particles were still in contact with con-
RUNX2, a transcription factor associated with osteogenesis, posi-
nective tissue (Figure 6A,B). Strong TRAP staining was associated
tively stained multiple cells in the connective tissue associated with
with osteoclast-like cells on the MB and pits were evident that were
the MB; as well as being evident in some cells around the Bio-Oss®
consistent with resorption lacunae (Figure 6C). Alkaline phosphatase
with the morphology and location consistent with being osteoblasts
was also evident in the connective tissue around the MB. Only very
(Figures 4E,F and 5E,F). Discrete intracellular staining of PCNA+ve cells
occasionally were lightly stained TRAP cells seen in association with
was occasionally seen in the connective tissue associated with MB but
Bio-Oss® (Figure 6D). RUNX2 was evident in the cuboidal osteoblasts
®
not for Bio-Oss (Figures 4G,H and 5G,H).
associated with the areas of active osteogenesis around the graft
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SMITH et al.
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(A)
(B)
(C)
(D)
(E)
(F)
(G)
F I G U R E 4 MB (left-hand side) and BO (right-hand side) within a sheep sinus at 4 wk post-surgery. All decalcified paraffin-embedded sections have been counterstained with hematoxylin. (A, B) Hematoxylin and eosin-stained sections. (C, D) TRAP staining (brown) to identify osteoclasts. (E, F) Immunohistochemistry for RUNX2 (brown). (G, H) Immunohistochemistry for PCNA-positive proliferating cells (brown). BO, Bio-Oss®; CT, connective tissue; red arrowheads, examples of TRAP-positive osteoclasts; LB, lamellar bone; MB, Moa-Bone®; WB, woven bone; scale bar = 100 μm
(H)
materials (Figure 6E,F). PCNA-positive cells were detected in the os-
evidence of resorption lacunae on the surface of Bio-Oss® particles
teoblast layer and were more evident in connective tissue associated
in association with these TRAP-positive cells (Figure 7D). RUNX2
with MB (Figure 6G) than Bio-Oss® (Figure 6H).
was associated with the osteoblasts lining the bone-forming areas (Figure 7E,F). PCNA-positive cells were detected in the osteoblast
3.7 | Twelve weeks post-grafting
layer and connective tissue for both MB (Figure 7G) and Bio-Oss® (Figure 7H).
MB particles were noticeably reduced in size in the sections examined at 12 weeks (Figure 7A,C,E,G). In most cases, the MB particles were surrounded by lamella bone. Bio- Oss® particles were also
4 | DISCUSSION
surrounded by lamella bone; however, at times areas of connective tissue abutted, and in some of these regions, TRAP-positive
In the current pilot study, we used an animal model of sinus augmen-
cells were evident on the surface of the material. There was no
tation, independent of implant placement. We investigated two DBB
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SMITH et al.
F I G U R E 5 High magnification images of MB (left-hand side) and BO (right-hand side) within a sheep sinus at 4 wk post- surgery. All decalcified paraffin-embedded sections have been counterstained with hematoxylin. (A, B) Hematoxylin and eosin-stained sections. (C, D) TRAP staining (brown) to identify osteoclasts. (E, F) Immunohistochemistry for RUNX2 (brown). (G, H) Immunohistochemistry for PCNA- positive proliferating cells, which are only evident in G. BO, Bio-Oss®; MB, Moa- Bone®; red arrowheads are examples of multinuclear osteoclasts; black arrows are examples of positively stained cells; scale bars = 50 μm
(A)
(B)
(C)
(D)
(E)
(F)
(G)
(H)
materials, MB and Bio-Oss®, and found that they have different profiles of healing when used to augment the maxillary sinus.
The osseous response within the sheep maxillary sinus differed considerably between the MB and the Bio-Oss® grafted sites over
The two DBB materials differed considerably in their ease of han-
the 12 week period examined. There was a clear gradient of osseous
dling. MB was difficult to use clinically, demonstrating electrostatic
envelopment around the particles, with the graft materials closest to
effects and strong hydrophobicity. SEM analysis indicated that the
pre-existing bone surrounded by new bone, which became lamella
surface morphologies were quite different. MB was a smoother and
earlier, compared with the graft material that was at a distance from
more porous, fibrous structure with minimal globular particles. Bio-
the pre-existing bone. This gradient is similar to that reported by oth-
Oss® was an almost entirely globular surface, consistent with previous
ers.37 Inflammatory responses to Bio-Oss® have not been typically
The differences in the microscopic appearance of
observed38-40 and we found no evidence of a strong inflammatory
the two materials were consistent with previous research that indi-
reaction to either material. There was little difference between the
cated the micro-porosity of hydroxyapatite decreases with increasing
two graft materials with regard to their encapsulation in woven bone
processing temperatures.36
and production of lamella bone during the early osseous response;
investigations.
6,35
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SMITH et al.
8
(A)
(B)
(C)
(D)
(E)
(F)
(G)
F I G U R E 6 MB (left-hand side) and BO (right-hand side) within a sheep sinus at 6 wk post-surgery. All decalcified paraffin-embedded sections have been counterstained with hematoxylin. (A, B) Hematoxylin and eosin-stained sections. (C, D) TRAP staining (brown) to identify osteoclasts. (E, F) Immunohistochemistry for RUNX2 (brown). (G, H) Immunohistochemistry for PCNA-positive proliferating cells (brown). BO, Bio-Oss®; CT, connective tissue; LB, lamellar bone; MB, Moa-Bone®; WB, woven bone; red arrowheads, examples of TRAP-positive osteoclasts; black arrows, RUNX2-positive osteoblasts; scale bars A,B = 200 μm; C-H = 100 μm
(H)
however, Bio-Oss® was more likely to be in contact with connective
Zaffe et al38 who noted that Bio-Oss® was typically surrounded with
tissue at 12 weeks when compared to any remaining MB at the same
connective tissue and who noted marked resorption of the Bio-Oss®.
®
time point. Bio-Oss has been reported by others to be associated with
Others, however, have noted good osseointegration associated with
connective tissue and linked with delayed bone formation.41 Lindhe
Bio-Oss® both in experimental and clinical settings.39,43-46
42
et al
®
compared Bio-Oss versus non-grafted controls in human sub®
RUNX2, also known as core-binding factor α-1, is essential for os-
jects and found that the Bio-Oss particles were not resorbed but in-
teoblast maturation during both intramembranous and endochondrial
stead became surrounded by less mineralized bone and more fibrous
ossification.47,48 In previous studies of sinus grafting, RUNX2 has been
®
tissue than the control sites. These authors suggested that the Bio-Oss
detected in pre-osteoblasts situated in the connective tissue, and in
particles were resistant to resorption and delayed osseous healing. The
association with osteoblasts and young osteocytes, but not with ma-
MB test material in our study behaved differently, resorbing more rap-
ture osteocytes, which were negative.49 We noted a similar pattern of
®
idly than the Bio-Oss . The formation of bone in direct contact with
staining pattern for RUNX2 in our study. Of particular note was the
the Bio-Oss®, at the earliest time period of 4 weeks, was in contrast to
RUNX2 staining at 4 weeks within a more organized connective tissue
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F I G U R E 7 MB (left-hand side) and BO (right-hand side) within a sheep sinus at 12 wk post-surgery. All decalcified paraffin-embedded sections have been counterstained with hematoxylin. (A, B) Hematoxylin and eosin-stained sections. (C, D) TRAP staining (brown) to identify osteoclasts. (E, F) Immunohistochemistry for RUNX2 (brown). (G, H) Immunohistochemistry for PCNA-positive proliferating cells (brown). BO, Bio- Oss®; CT, connective tissue; LB, lamellar bone; MB, Moa-Bone®; red arrowheads, examples of TRAP-positive osteoclasts; scale bars A,B = 200 μm; C-H = 100 μm
(A)
(B)
(C)
(D)
(E)
(F)
(G)
(H)
matrix associated with MB, as compared to the more fibrous disorga®
trials; however, there is conflicting data on the rate of resorption. In
nized matrix associated with Bio-Oss . This may indicate the early acti-
a model of mandibular bone grafts, also in sheep, Bio-Oss® was iden-
vation of pre-osteoblasts in association with MB. More PCNA-positive
tified at 16 weeks post-grafting but particles were noted as having a
proliferative cells were also noted in association with the MB at the
rough surface consistent with the presence of resorption lacunae.50 A
earlier time points. At 6 and 12 weeks, staining of RUNX2 within the
study in dogs showed no histological signs of Bio-Oss® resorption 3
osteoblast layer was clearly evident and this staining was consistent
or 6 months after grafting while bone ingrowth was consistent with
with bone formation in association with both of the graft materials.
osteogenic regeneration of the defects.46 Studies on human sinus
Osteoclastic resorption of graft material and remodeling of bone ®
grafts showed varying results; Zaffe and co-workers38 reported that
was investigated using TRAP staining. We found that Bio-Oss was
19 weeks after implantation, very few patients had residual Bio-Oss®
associated with very few osteoclasts and there was little evidence of
present, large numbers of osteoclasts were apparent and resorp-
resorption lacunae even at 12 weeks post-surgery. A slow resorption
tion lacunae were highly evident. Others noted abundant Bio-Oss®
®
rate of Bio-Oss has been reported in both large animal and human
particles 3 months after implantation and reported the presence
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SMITH et al.
10
of well-organized lamella bone in contact with the Bio-Oss®.40 In patients grafted with Bio-Oss
®
alone or mixed with a cell-binding
peptide (PepGen P-15), abundant Bio-Oss® was found 3-20 months after surgery and although reduced, some particles were still present
CO NFL I C T O F I NT ER ES T The authors declare no financial interest in Molteno® Ophthalmic Ltd or Geistlich Pharma AG.
after 7-8 years, typically encapsulated in bone.39,44 Biopsies collected 6-8 months after Bio-Oss® grafting in five patients also showed the presence of new bone around significant quantities of Bio-Oss®.45 Thus, it appears that some resorption of Bio-Oss® by osteoclasts may occur; however, once encapsulated in bone the bovine bone parti-
O RC I D D. E. Coates
http://orcid.org/0000-0003-4242-6846
cles may persist for long periods of time.51 The slow resorption rate
REFERENCES
has been viewed by some as beneficial for maintaining graft volume;
1. Browaeys H, Bouvry P, de Bruyn H. A literature review on biomaterials in sinus augmentation procedures. Clin Implant Dent Relat Res. 2007;9:166‐177. 2. Tong DC, Rioux K, Drangsholt M, Beirne OR. A review of survival rates for implants placed in grafted maxillary sinuses using meta-analysis. Int J Oral Maxillofac Implants. 1998;13:175‐182. 3. Pjetursson BE, Tan WC, Zwahlen M, Lang NP. A systematic review of the success of sinus floor elevation and survival of implants inserted in combination with sinus floor elevation: part I: lateral approach. J Clin Periodontol. 2008;35:216‐240. 4. Ten Bruggenkate CM, Van Den Bergh JP. Maxillary sinus floor elevation: a valuable pre-prosthetic procedure. Periodontol. 2000;1998(17):176‐182. 5. Wenz B, Oesch B, Horst M. Analysis of the risk of transmitting bovine spongiform encephalopathy through bone grafts derived from bovine bone. Biomaterials. 2001;22:1599‐1606. 6. Accorsi-Mendonça T, Conz MB, Barros TC, de Sena LÁ, Soares GdA, Granjeiro JM. Physicochemical characterization of two deproteinized bovine xenografts. Braz Oral Res. 2008;22:5‐10. 7. Liu J, Schmidlin PR, Philipp A, Hild N, Tawse-Smith A, Duncan W. Novel bone substitute material in alveolar bone healing following tooth extraction: an experimental study in sheep. Clin Oral Implants Res. 2016;27:762‐770. 8. Tovar N, Jimbo R, Gangolli R, et al. Evaluation of bone response to various anorganic bovine bone xenografts: an experimental calvaria defect study. Int J Oral Maxillofac Surg. 2014;43:251‐260. 9. Ramírez-Fernández MP, Calvo-Guirado JL, Delgado-Ruiz RA, MatéSánchez del Val JE, Vicente-Ortega V, Meseguer-Olmos L. Bone response to hydroxyapatites with open porosity of animal origin (porcine [OsteoBiol®mp3] and bovine [Endobon®]): a radiological and histomorphometric study. Clin Oral Implants Res. 2011;22:767‐773. 10. Barone A, Todisco M, Ludovichetti M, et al. A prospective, randomized, controlled, multicenter evaluation of extraction socket preservation comparing two bovine xenografts: clinical and histologic outcomes. Int J Periodontics Restorative Dent. 2013;33:795‐802. 11. Schneider OD, Weber F, Brunner TJ, et al. In vivo and in vitro evaluation of flexible, cottonwool-like nanocomposites as bone substitute material for complex defects. Acta Biomater. 2009;5:1775‐1784. 12. Schwartz Z, Weesner T, van Dijk S, et al. Ability of deproteinized cancellous bovine bone to induce new bone formation. J Periodontol. 2000;71:1258‐1269. 13. Figueiredo M, Henriques J, Martins G, Guerra F, Judas F, Figueiredo H. Physicochemical characterization of biomaterials commonly used in dentistry as bone substitutes—Comparison with human bone. J Biomed Mater Res B Appl Biomater. 2010;92:409‐419. 14. Lee JS, Shin HK, Yun JH, Cho KS. Randomized Clinical Trial of Maxillary Sinus Grafting using Deproteinized Porcine and Bovine Bone Mineral. Clin Implant Dent Relat Res. 2017;19:140‐150. 15. Zahid ZM, Rahman SA, Alam MK, et al. Prospective 3D assessment of CORAGRAF and Bio-Oss as bone substitutes in maxillary sinus augmentation for implant placement. J Hard Tissue Biol. 2015;24:43‐48. 16. Jordan DR, Ahuja N, Gilberg S, Bouchard R. Behavior of various orbital implants under axial compression. Ophthal Plast Reconstr Surg. 2005;21:225‐229.
however, graft resorption may result in a more natural autogenous bone.42,52 In our study, MB was associated with a strong activation of osteoclasts. TRAP staining was strongest at 4 and 6 weeks. The connective tissue had strong TRAP staining of isolated cells as well as diffusely within the tissue. In one section, this staining was so strong at 4 weeks the incubation time had to be reduced to visualize the section. The strongest TRAP staining was localized to large cells on the surface of the MB consistent with being osteoclasts and typically located in resorption lacunae. Osteoclasts were observed on the surface of implant material only when connective tissue was adjacent to the graft material. This suggests that the migration of the monocyte- derived osteoclast precursor cells to the graft material may require the presence of connective tissue and may explain the lack of TRAP- positive cells in the MB by 12 weeks, as it was typically encapsulated in bone. To the best of our knowledge, this is the first study to use MB for osseous sinus grafting. Compared to Bio-Oss® it showed a similar rate of osseointegration at 12 weeks and did not induce an inflammatory response. MB was rapidly resorbed and only small amounts were left in what was an almost entirely autologous bone graft by week 12. MB thus has some attributes that are consistent with potential utility as a graft material, particularly where complete remodeling of the graft site is desired. This behavior is similar to that of autogenous bone. This pilot preclinical study suggested that MB has the potential for use in sinus grafting; however, definitive statements would require statistically relevant samples sizes. Further optimization of MB for maxillofacial applications is warranted, particularly with respect to its handling properties. Further experimental work is required with sufficient animals per time period to permit robust statistical analysis, to demonstrate the efficacy of the MB graft when compared with commercially available materials such as Bio-Oss®; ideally, such future experimental work should also include analysis of the response of the grafted sites after restoration with loaded dental implants, as this is the clinical rationale for the use of bovine bone grafting materials in the maxillary sinus.
ACKNOWLE DGE ME N TS The assistance of the veterinary staff of the University of Otago Hercus Taieri Resource Unit is gratefully acknowledged.
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17. Suter AJ, Molteno AC, Bevin TH, Fulton JD, Herbison P. Long term follow up of bone derived hydroxyapatite orbital implants. Br J Ophthalmol. 2002;86:1287‐1292. 18. Stübinger S, Nuss K, Bürki A, et al. Osseointegration of titanium implants functionalised with phosphoserine- tethered poly(epsilon- lysine) dendrons: a comparative study with traditional surface treatments in sheep. J Mater Sci Mater Med. 2015;26:87‐98. 19. Nuss KM, Auer JA, Boos A, Von Rechenberg B. An animal model in sheep for biocompatibility testing of biomaterials in cancellous bones. BMC Musculoskelet Disord. 2006;7:67‐80. 20. Ardjomandi N, Duttenhoefer F, Xavier S, Oshima T, Kuenz A, Sauerbier S. In vivo comparison of hard tissue regeneration with ovine mesenchymal stem cells processed with either the FICOLL method or the BMAC method. J Craniomaxillofac Surg. 2015;43:1177‐1183. 21. Oshima T, Duttenhoefer F, Xavier S, Nelson K, Sauerbier S. Can mesenchymal stem cells and novel gabapentin-lactam enhance maxillary bone formation? J Oral Maxillofac Surg. 2014;72:485‐495. 22. Sauerbier S, Stubbe K, Maglione M, et al. Mesenchymal stem cells and bovine bone mineral in sinus lift procedures—an experimental study in sheep. Tissue Eng Part C Methods. 2010;16:1033‐1039. 23. Gutwald R, Haberstroh J, Kuschnierz J, et al. Mesenchymal stem cells and inorganic bovine bone mineral in sinus augmentation: comparison with augmentation by autologous bone in adult sheep. Br J Oral Maxillofac Surg. 2010;48:285‐290. 24. Duncan WJ, Gay JH, Lee MH, Bae TS, Lee SJ, Loch C. The effect of hydrothermal spark discharge anodization in the early integration of implants in sheep sinuses. Clin Oral Implants Res. 2016:975‐980. 25. Philipp A, Duncan W, Roos M, Hämmerle CH, Attin T, Schmidlin PR. Comparison of SLA® or SLActive® implants placed in the maxillary sinus with or without synthetic bone graft materials—An animal study in sheep. Clin Oral Implants Res. 2014;25:1142‐1148. 26. Haas R, Haidvogl D, Donath K, Watzek G. Freeze-dried homogeneous and heterogeneous bone for sinus augmentation in sheep Part I: histological findings. Clin Oral Implants Res. 2002;13:396‐404. 27. Haas R, Baron M, Zechner W, Mailath-Pokorny G. Porous hydroxyapatite for grafting the maxillary sinus in sheep: comparative pullout study of dental implants. Int J Oral Maxillofac Implants. 2003;18:691‐696. 28. Haas R, Mailath G, Dörtbudak O, Watzek G. Bovine hydroxyapatite for maxillary sinus augmentation: analysis of interfacial bond strength of dental implants using pull-out tests. Clin Oral Implants Res. 1998;9:117‐122. 29. Haas R, Donath K, Födinger M, Watzek G. Bovine hydroxyapatite for maxillary sinus grafting: comparative histomorphometric findings in sheep. Clin Oral Implants Res. 1998;9:107‐116. 30. Haas R, Baron M, Donath K, Zechner W, Watzek G. Porous hydroxyapatite for grafting the maxillary sinus: a comparative histomorphometric study in sheep. Int J Oral Maxillofac Implants. 2002;17:337‐346. 31. Duncan WJ. Sheep Mandibular Animal Models for Dental Implantology Research [Ph.D. Thesis]. 2005. 32. Duncan WJ, Lee MH, Dovban AS, Hendra N, Ershadi S, Rumende H. Anodization increases early integration of Osstem implants in sheep femurs. Ann R Australas Coll Dent Surg. 2008;19:152‐156. 33. Eurell JAC, Sterchi DL. Microwaveable toluidine blue stain for surface staining of undecalcified bone sections. J Histotechnol. 1994;17:357‐359. 34. Baharuddin NA, Coates DE, Cullinan M, Seymour G, Duncan W. Localization of RANK, RANKL and osteoprotegerin during healing of surgically created periodontal defects in sheep. J Periodontal Res. 2015;50:211‐219. 35. Hiller JC, Thompson TJ, Evison MP, Chamberlain AT, Wess TJ. Bone mineral change during experimental heating: an X-ray scattering investigation. Biomaterials. 2003;24:5091‐5097. 36. Wang C, Duan Y, Markovic B, et al. Proliferation and bone-related gene expression of osteoblasts grown on hydroxyapatite ceramics sintered at different temperature. Biomaterials. 2004;25:2949‐2956.
37. Busenlechner D, Huber CD, Vasak C, Dobsak A, Gruber R, Watzek G. Sinus augmentation analysis revised: the gradient of graft consolidation. Clin Oral Implants Res. 2009;20:1078‐1083. 38. Zaffe D, Leghissa GC, Pradelli J, Botticelli AR. Histological study on sinus lift grafting by fisiograft and Bio-Oss. J Mater Sci Mater Med. 2005;16:789‐793. 39. Degidi M, Piattelli A, Perrotti V, Iezzi G. Histologic and histomorphometric evaluation of an implant retrieved 8 years after insertion in a sinus augmented with anorganic bovine bone and anorganic bovine matrix associated with a cell-binding peptide: a case report. Int J Periodontics Restorative Dent. 2012;32:451‐457. 40. Degidi M, Artese L, Rubini C, Perrotti V, Iezzi G, Piattelli A. Microvessel density in sinus augmentation procedures using anorganic bovine bone and autologous bone: 3 months results. Implant Dent. 2007;16:317‐325. 41. Stavropoulos A, Kostopoulos L, Mardas N, Nyengaard JR, Karring T. Deproteinized bovine bone used as an adjunct to guided bone augmentation: an experimental study in the rat. Clin Implant Dent Relat Res. 2001;3:156‐165. 42. Lindhe J, Cecchinato D, Donati M, Tomasi C, Liljenberg B. Ridge preservation with the use of deproteinized bovine bone mineral. Clin Oral Implants Res. 2014;25:786‐790. 43. Kim YK, Yun PY, Kim SG, Lim SC. Analysis of the healing process in sinus bone grafting using various grafting materials. Oral Surg Oral Med Oral Pathol Oral Radiol. 2009;107:204‐211. 44. Orsini G, Scarano A, Degidi M, Caputi S, Iezzi G, Piattelli A. Histological and ultrastructural evaluation of bone around Bio-Oss® particles in sinus augmentation. Oral Dis. 2007;13:586‐593. 45. Moon JW, Sohn DS, Heo JU, Kim JS. Comparison of two kinds of bovine bone in maxillary sinus augmentation: a histomorphometric study. Implant Dent. 2015;24:19‐24. 46. Schlegel KA, Fichtner G, Schultze-Mosgau S, Wiltfang J. Histologic findings in sinus augmentation with autogenous bone chips versus a bovine bone substitute. Int J Oral Maxillofac Implants. 2003;18: 53‐58. 47. Kidwai F, Edwards J, Zou L, Kaufman DS. Fibrinogen induces RUNX2 activity and osteogenic development from human pluripotent stem cells. Stem Cells. 2016;34:2079‐2089. 48. Ducy P. Cbfa1: a molecular switch in osteoblast biology. Dev Dyn. 2000;219:461‐471. 49. Zerbo IR, Bronckers AL, de Lange G, Burger EH. Localisation of osteogenic and osteoclastic cells in porous β-tricalcium phosphate particles used for human maxillary sinus floor elevation. Biomaterials. 2005;26:1445‐1451. 50. Adeyemo WL, Reuther T, Bloch W, et al. Healing of onlay mandibular bone grafts covered with collagen membrane or bovine bone substitutes: a microscopical and immunohistochemical study in the sheep. Int J Oral Maxillofac Surg. 2008;37:651‐659. 51. Galindo-Moreno P, Hernández-Cortés P, Mesa F, et al. Slow resorption of anorganic bovine bone by osteoclasts in maxillary sinus augmentation. Clin Implant Dent Relat Res. 2013;15:858‐866. 52. Lundgren S, Cricchio G, Hallman M, Jungner M, Rasmusson L, Sennerby L. Sinus floor elevation procedures to enable implant placement and integration: techniques, biological aspects and clinical outcomes. Periodontol. 2000;2017(73):103‐120.
How to cite this article: Smith MM , Duncan WJ, Coates DE. Attributes of Bio-Oss® and Moa-Bone® graft materials in a pilot study using the sheep maxillary sinus model. J Periodont Res. 2017;00:1–11. https://doi.org/10.1111/jre.12490
ORIGINAL ARTICLE
Attributes of Bio-Oss® and Moa-Bone® graft materials in a pilot study using the sheep maxillary sinus model M. M. Smith | W. J. Duncan | D. E. Coates Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago, Dunedin, New Zealand Correspondence Dawn Coates, Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago, Dunedin, New Zealand. Email: [email protected] Funding information This study was funded by a University of Otago Fuller grant. Moa-Bone® was provided free of charge by Molteno® Ophthalmic Ltd.
Background and Objective: The aim of this pilot study was to characterize surface morphology and to evaluate resorption and osseous healing of two deproteinated bovine bone graft materials after sinus grafting in a large animal model. Material and Methods: Surfaces of a novel particulate bovine bone graft, Moa-Bone® were compared with Bio-Oss® using scanning electron microscopy. Six sheep then had maxillary sinus grafting bilaterally, covered with BioGide®. Grafted maxillae were harvested after 4, 6 and 12 weeks. Healing was described for half of each site using resin-embedded ground sections. For the other half, paraffin-embedded sections were examined using tartrate resistant acid phosphatase staining for osteoclast activity, runt-related transcription factor 2 immunohistochemistry for pre-osteoblasts and osteoblasts and proliferating cell nuclear antigen for proliferative cells. Results: Moa-Bone® had a smoother, more porous fibrous structure with minimal globular particles compared with Bio-Oss®. After 4 weeks, woven bone formed on both grafts and the Moa-Bone® particles also showed signs of resorption. After 12 weeks, Moa-Bone® continued to be resorbed, however Bio-Oss® did not; both grafts were surrounded by maturing lamellar bone. Moa-Bone® was associated with earlier evidence of runt-related transcription factor 2-positive cells. Moa-Bone® but not Bio-Oss® was associated with strong tartrate resistant acid phosphatase-positive osteoclasts on the graft surface within resorption lacunae at both 4 and 6 weeks post-grafting. Conclusion: Both materials supported osseous healing and maturation without inflammation. Moa-Bone® showed marked osteoclast activity after 4 and 6 weeks and demonstrated positive attributes for grafting, if complete remodeling of the graft within the site is desired. Further optimization of Moa-Bone® for maxillofacial applications is warranted. KEYWORDS
animal model, biomaterial, bone grafting, sinus lift
1 | INTRODUCTION
and the resultant edentulous space is insufficient height for implant placement. 4
The anatomy of the posterior maxilla, and bony remodeling follow-
Sinus augmentation procedures have been developed to improve
ing extraction of teeth often results in a lack of suitable bone for
bone volumes, and a variety of materials may be used for this. Bone
placement of dental implants.1-3 Furthermore, in the region of the
autografts have been identified as the gold standard for bone augmen-
maxillary sinus the quality of the residual maxillary bone is often poor
tation because of the reduced risk of disease transmission and the low
J Periodont Res. 2017;1–11.
wileyonlinelibrary.com/journal/jre © 2017 John Wiley & Sons A/S. | 1 Published by John Wiley & Sons Ltd
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SMITH et al.
2
(A)
(B)
(C)
(D)
F I G U R E 1 Surgical procedure on the maxillary sinus of sheep. (A) Arrow marks the surgical site. (B) Graft material in place before placement of the Bio-Gide® membrane. (C) Representative radiograph of the graft material and osteotomy site before sectioning (MB at 6 wk). Scale bar = 1.5 mm. (D) Diagram of the site retrieved for analysis. BO, Bio-Oss®; MB, Moa- Bone®
antigenic nature of the transplant. Autografts are characterized clini-
materials.20-25 In a series of studies in the sheep maxillary sinus model,
cally by complete remodeling of the graft material over time. The use
implants placed into sites augmented with DBB were characterized by
of autografts is, however, limited by the morbidity experienced at pro-
significantly higher “pull out” forces than non-grafted sites,26-28 under-
curement and the ability to harvest sufficient material. Deproteinated
lining the utility of DBB material and the sheep sinus model. According
bovine bone (DBB) has been used for a number of years in dentoalveo-
to Haas, pigs are unsuited to use as a model due to excessive thickness
lar grafting. DBB is normally produced from bovine bone using combi-
of the cortical bone, and dogs are unsuitable because the nasal sinus
nations of heat, organic solvents and strong chemicals such as sodium
lacks a Schneiderian membrane and does not undergo appropriate
hypochlorite, and the product sterilized using gamma radiation and/or
pneumatization.29
heat and pressure. The removal of all organic material is important to
The aim of the current pilot study was to characterize the surface
reduce the transmission of diseases, in particular bovine spongiform
features of MB and Bio-Oss® graft materials and to evaluate the os-
encephalopathy.5
teogenesis and bone remodeling associated with both graft materials
There are a number of bovine-derived xenograft products used for
in a sheep maxillary sinus model.
oral bone replacement grafting procedures, with published evidence supporting their use in animal preclinical models6-9 and human clinical trials.10 Bio-Oss® is a xenograft that is widely used in clinical practice, for which
2 | MATERIAL AND METHODS
there is a considerable body of published data.11 Bio-Oss® is processed using low temperatures (approximately 300°C) and organic solvents.12
This study conformed to the ARRIVE guidelines for pre-clinical ani-
It is reported to be highly porous, on a macro-and microscopic level,13
mal studies. Ethical approval was obtained from the University of
and is frequently used in clinical trials and compared to new or similar
Otago Animal Ethics Committee (AEC 50-80). After characterization
products.14,15 Moa-Bone® (MB) is another DBB product, sourced from
of the biomaterials, animal surgery was performed using six healthy
disease-free cattle in New Zealand and used for ophthalmic grafting. It
Romney-cross female sheep (ewes) in the weight range 70-90 kg and
is initially processed under pressure at high temperature, followed by
18-24 months of age. Sinus grafting was performed bilaterally on each
bleaching and centrifugation, with final sterilization by autoclaving. MB
animal, with the two treatments, MB or Bio-Oss®, randomly allocated
16
is light, porous and friable.
Particulate MB is a by-product arising from
to the left or right sinus. The grafted maxillary sinuses were harvested
the milling of the M-Sphere®, a spherical bone prosthesis used during
after animal killing at 4, 6 and 12 weeks post-grafting. For this pilot
reconstruction of an enucleated human orbit. The M-Sphere® is well tol-
study, two animals were used per time period, which provided suf-
erated in humans, causing no long-term inflammatory reaction.17 To our
ficient information regarding the responses to the two test grafts; sta-
knowledge, there are no studies of the potential uses of MB particulates
tistical comparisons were not required.
for maxillofacial applications, such as grafting into the maxillary sinus. Studies have described the suitability of sheep for biomaterial testing in cancellous bone as excellent.18,19 The utility of the sheep
2.1 | Biomaterials
sinus for implant research has been demonstrated by a number of
MB was supplied in the form of a sterile M- Sphere® (Molteno®
groups in studies involving both mesenchymal stem cells and implant
Ophthalmic Ltd., Dunedin, New Zealand) and crushed through a series
|
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SMITH et al.
of sieves with mesh apertures of 1.68, 1.18 and .25 mm (Endecotts
Animals were recovered to the Otago University farm and were con-
Ltd, London, UK) within a laminar flow hood, to achieve a size pro-
tinuously monitored and fed a normal diet. Animals were killed as pre-
file of between .25 and 1.00 mm. This was consistent with the par-
viously described
®
31
after 4, 6 and 12 weeks, by anesthetic overdose
ticle sizes of the commercially available Bio-Oss . The MB particles
followed by carotid artery perfusion with 5000 iu of heparin in 1 liter
were steam sterilized in an autoclave (Mercer 7, Mercer Medical, New
of saline followed by 2 liters of 10% neutral buffered formalin (BioLab
®
Zealand) for a standard cycle of 4 minutes at 134°C. Bio-Oss was
Ltd, Auckland, New Zealand).
supplied with a particle size of .25-1.00 mm in a sterile vial (Geistlich Pharma Australia and New Zealand, Chatswood, NSW, Australia).
2.2 | Scanning electron microscopy characterization
2.4 | Tissue collection and processing Tissue sections were resected en bloc using a hacksaw and trimmed with a custom-made guillotine before being immersed in 10% neutral
Representative samples of MB and Bio-Oss® particles were mounted
buffered formalin for 48 hours at 4°C with gentle rotation. Specimens
on 10 mm aluminum stubs and coated with a 10 nm layer of gold/pal-
were radiographed (Figure 1C) before being cut vertically through the
ladium using an Emitech K575X high-resolution sputter with carbon
graft site and separated into two halves for either resin or paraffin
coater attachment (EM Technologies Ltd, London, UK). High-power
embedding (Figure 1D). Evaluation of the samples was conducted by
scanning electron microscopy (SEM) images of the surface architec-
investigators blinded to the groups.
ture were obtained using a JEOL JSM-6700F field emission SEM (JEOL Ltd, Tokyo, Japan) at an acceleration voltage of 3.0 kV.
2.5 | Embedding of the tissue samples Resin embedding with methyl methacrylate (Sigma-Aldrich, St. Louis,
2.3 | Surgery
MO, USA) was conducted as previously described.31,32 Briefly, tis-
The anesthetic and surgical procedures have previously been de-
sue specimens were dehydrated in ascending grades of alcohol, im-
scribed in detail,24,25 and are modifications of the procedure originally
mersed in xylene for 2 days (two changes), followed by methacrylate
described by Haas and co-workers.26,29,30 Under general anesthetic
and methacrylate with plasticizer and setting agent for 2 days each,
the wool of the face was shorn, and the maxilla bone overlying the
and then embedded in polymerized methacrylate in a sealed glass jar,
sinus was exposed by a 6 cm extra-oral sagittal skin incision located
which was kept cool in the dark. Sequential 500 μm sections were
caudal to the facial tuberosity, followed by separation of the malar
cut from each resin-embedded specimen using a Struers precision
muscle and blunt dissection and partial detachment of the masse-
tabletop cut- off machine (Accutom; Struers GmbH, Birmensdorf,
ter muscle (Figure 1A). A 1 cm circular bony window was created
Switzerland), and press mounted on to an opaque acrylic base plate
through the lateral wall of the maxillary sinus using a Piezotome™
using a cyanoacrylate glue. Sections were then ground, using a ro-
(SL2 tip, Satalec, Acteon, Merignac, France), cooled by saline irriga-
tating grinding machine (Tegra- Pol; Struers GmbH, ZNL Schweiz,
tion. The antral Schneiderian membrane was elevated from the buc-
Birmensdorf, Germany). Final polished specimens had a thickness of
cal bony wall using a sinus elevation kit (Sinus Kit, Osstem Implants,
between 80 and 100 μm and were surface-stained with MacNeal’s
Seoul, Korea), and displaced dorso-cranially before placement of the
tetrachrome and toluidine blue.33
®
graft material. MB or Bio-Oss (.25 g) were mixed with 1 mL of blood
Tissue for paraffin embedding was decalcified with 10% EDTA as
from the surgical field and placed in the resulting pouch within the
previously described and the end-point for decalcification determined
sinus (Figure 1B).
by an oxylate test.34 After embedding, 4 μm serial sections were cut
Two pieces of sterile porcine collagen membrane (Bio- Gide®, Geistlich, Switzerland) measuring 12.5×12.5 mm were placed over
and mounted on 3- aminopropyltriethoxysilane- coated slides (Lab Scientific Inc, Highlands, NJ, USA).
each other in a bi-laminar style to secure the biomaterials within each site. The fascia and subcutis were closed in layers using resorbable sutures (Vicryl 3/0, Ethicon; Vicryl (Ethicon); Johnson & Johnson,
2.6 | Tartrate resistant acid phosphatase staining
Oraltec NZ Limited, Auckland, New Zealand) and the skin closed using
Tartrate resistant acid phosphatase (TRAP) staining was conducted
non- resorbable sutures (Maxon 2/0, Syneture; Maxon (Covidien),
using an Acid Phosphatase Leukocyte Kit (387A; Sigma-Aldrich) as
Tyco Healthcare Ltd., Auckland, New Zealand). After surgery, a long-
per the manufacturer’s instructions. However, the final incubation in
acting Bupivacaine HCl anesthetic (Marcaine .5%; AstraZeneca,
TRAP solution was reduced to 1 hour at 37°C in the dark.34
Amtech Medical Ltd., Wanganui, New Zealand) was administered to control pain in the site. Postoperatively animals were given the anti- inflammatory carprofen 4 mg/kg sc (Norbrook New Zealand Ltd,
2.7 | Immunohistochemistry
Auckland, New Zealand), as well as intramuscular antibiotic admin-
Sections were de-waxed in xylene before rehydrating through graded
istration of Strepcin containing 250 000 IU/mL procaine penicillin
alcohols to phosphate-buffered saline (PBS). Heat-based antigen re-
G with 250 mg/mL dihydrostreptomycin, 5 mL/animal (Stockguard
trieval was conducted for 10 minutes at 80°C in a bath of .01 mol/L
Laboratories Ltd, Hamilton, New Zealand), each day for 3 days.
sodium citrate buffer (pH 6). Sections were then blocked to reduce
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SMITH et al.
4
(A)
(B)
F I G U R E 2 Scanning electron microscopy images of the surface of the implant materials. (A) Moa-Bone® and (B) Bio-Oss® non-specific antibody binding with 20% goat serum for runt-related transcription factor 2 (RUNX2) or 20% rabbit serum for proliferating
3.2 | Surgery The surgery was uneventful and no sinus membrane perforations
cell nuclear antigen (PCNA) in 1% bovine serum albumin/PBS. RUNX2 immunohistochemistry was conducted with a rabbit antihuman polyclonal RUNX2 antibody (NBP1- 01004, Novus Bio,
were observed. No animal was observed to have any adverse short- or long-term events following surgery.
Littleton, CO, USA) and control sections treated with rabbit IgG (sc2027 Santa Cruz Biotechnology, Santa Cruz, USA), both at a concentration of 10 μg/mL. PCNA immunostaining was conducted with
3.3 | Histology of resin-embedded tissue
5.25 μg/mL of mouse antirat PCNA monoclonal antibody (Clone PC-
The histology of the sinus implant material was examined at 4, 6 and
10, M0879; Dako, Glostrup, Denmark) and mouse IgG (sc2025; Santa
12 weeks post-implantation. The region of interest for imaging was
Cruz Biotechnology) as a control. The primary antibodies and control
the site with grafted material immediately adjacent to the native maxil-
IgG solutions were left to incubate at 4°C overnight.
lary bone. At 4 weeks, some woven bone partially enveloped the MB.
Sections were then washed three times with PBS containing 1%
Sharply delineated semi-circular pits on the surfaces of the particles,
Tween-20 and 1% non-fat milk powder. The biotinylated second-
resembling resorption lacunae, were evident (Figure 3A). Connective tis-
ary antibodies were goat antirabbit H&L- F(ab)2 (ab6012; Abcam,
sue was highly evident. Bio-Oss® was also associated with woven bone
Cambridge, UK) for RUNX2, and a rabbit antimouse H&L- F(ab)2
and an irregular fibrous pattern of connective tissue filled the spaces. No
(ab5761; Abcam, Cambridge, UK) for PCNA; both at 2 μg/mL and
resorption lacunae were seen on the Bio-Oss®. At 6 weeks, the amount
incubated for 1 hour at room temperature. Endogenous peroxidase
of woven bone associated with both graft materials had increased
was quenched using .3% hydrogen peroxide in methanol for 10 min-
(Figure 3C,D). Resorption-like lacunae were most evident where the con-
utes, followed by treatment with Strep horseradish peroxidase
nective tissue abutted the MB (Figure 3C). Twelve weeks post-grafting,
(Vectastain Elite ABC; Vector Laboratories, Inc, Burlingame, CA, USA).
the MB was almost entirely encased by lamellar bone and the graft par-
Development was performed using 3,3′ diaminobenzidine (D3939;
ticles appeared smaller than previously observed. Bio-Oss® particles ap-
Sigma-Aldrich). Sections were counterstained with hematoxylin for
peared to be unchanged in size compared to the 4 week time point and
5 seconds followed by dehydration and coverslipped using Entellan
®
were predominantly surrounded by lamellar bone (Figure 3E,F).
(Merck, Darmstadt, Germany). Light microscopy was used to examine all sections and images were digitized using a JVC TK 1281 color video camera and SPOT® digital imaging software. Semiquantitative analysis was also conducted using
3.4 | Immunohistochemistry of biomaterials within the sheep sinus
the following scores for the presence of resorption lacunae and TRAP-
Decalcified paraffin- embedded sections of sheep sinuses containing
positive cells: 0 = no positive cells; 1 = occasional positive cells; 2 =
the implant materials were examined. Control IgG staining showed only
many positive cells; and 3 = large numbers of positive cells.
background staining (not shown). Table 1 gives an overview of the results from all time points for TRAP staining, resorption lacunae and the pres-
3 | RESULTS
ence of bone types and connective tissue adjacent to the graft material.
3.1 | Characterization of the biomaterials
3.5 | Four weeks post-implantation
MB was more difficult to handle compared to Bio-Oss®, as it was
The implant materials were surrounded by woven bone and connec-
more hydrophobic and electrostatic in nature. SEM revealed different
tive tissue (Figure 4A,B). Very strong TRAP staining of osteoclasts
surface morphologies; MB at high magnification showed evidence of
within the sheep sinus was noted at the 4 week time point for MB
a porous structure and retained a fibrous-like organization (Figure 2A).
(Figure 4C). The incubation time was reduced for one block as the
In contrast, Bio-Oss® had clusters of globular-like particles evenly dis-
staining completely swamped the slide. TRAP staining was associated
tributed over the surface of the xenograft (Figure 2B).
with large multinuclear osteoclast-like cells inside resorption lacunae
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SMITH et al.
(A)
(B)
(C)
(D)
(E)
(F)
F I G U R E 3 Resin sections of sinus implant regions containing MB (left-hand side) or BO (right-hand side). Sections are from weeks post-biomaterial implant surgery with (A, B) at 4 wk; (C, D) at 6 wk; (E, F) at 12 wk. BO, Bio-Oss®; CT, connective tissue; arrows, resorption like lucunae; LB, lamellar bone; MB, Moa- Bone®; WB, woven bone; scale bar = 100 μm T A B L E 1 Osteoclasts and bone formation around implant materials 4 wk Moa Bone Resorption lacunae
6 wk ®
3
Bio-Oss
®
0
Moa Bone
12 wk ®
3
Bio-Oss
®
0
Moa Bone®
Bio-Oss®
0
0
TRAP-positive osteoclasts
3
1
3
1
1
1
Tissue around graft
WB/CT
WB/CT
LB/WB/CT
LB/WB/CT
LB
LB/CT
0 = no positive cells/lacunae; 1 = occasional positive cells/lacunae; 2 = many positive cells/lacunae; 3 = large numbers of positive cells/lacunae. CT, connective tissue; LB, lamella bone; WB, woven bone.
on the MB (Figure 5A,C,E). Diffuse alkaline phosphatase staining was also localized to the connective tissue. Osteoclasts were also evident
3.6 | Six weeks post-grafting
within areas containing new woven bone. Around the Bio-Oss® par-
Residual graft material was visible at 6 weeks post-grafting within
ticles, very few TRAP-positive osteoclasts were detected in the sinus,
the sheep sinuses, typically surrounded by woven or lamella bone;
and were mostly associated with woven bone (Figures 4D and 5D).
however, in some areas the particles were still in contact with con-
RUNX2, a transcription factor associated with osteogenesis, posi-
nective tissue (Figure 6A,B). Strong TRAP staining was associated
tively stained multiple cells in the connective tissue associated with
with osteoclast-like cells on the MB and pits were evident that were
the MB; as well as being evident in some cells around the Bio-Oss®
consistent with resorption lacunae (Figure 6C). Alkaline phosphatase
with the morphology and location consistent with being osteoblasts
was also evident in the connective tissue around the MB. Only very
(Figures 4E,F and 5E,F). Discrete intracellular staining of PCNA+ve cells
occasionally were lightly stained TRAP cells seen in association with
was occasionally seen in the connective tissue associated with MB but
Bio-Oss® (Figure 6D). RUNX2 was evident in the cuboidal osteoblasts
®
not for Bio-Oss (Figures 4G,H and 5G,H).
associated with the areas of active osteogenesis around the graft
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SMITH et al.
6
(A)
(B)
(C)
(D)
(E)
(F)
(G)
F I G U R E 4 MB (left-hand side) and BO (right-hand side) within a sheep sinus at 4 wk post-surgery. All decalcified paraffin-embedded sections have been counterstained with hematoxylin. (A, B) Hematoxylin and eosin-stained sections. (C, D) TRAP staining (brown) to identify osteoclasts. (E, F) Immunohistochemistry for RUNX2 (brown). (G, H) Immunohistochemistry for PCNA-positive proliferating cells (brown). BO, Bio-Oss®; CT, connective tissue; red arrowheads, examples of TRAP-positive osteoclasts; LB, lamellar bone; MB, Moa-Bone®; WB, woven bone; scale bar = 100 μm
(H)
materials (Figure 6E,F). PCNA-positive cells were detected in the os-
evidence of resorption lacunae on the surface of Bio-Oss® particles
teoblast layer and were more evident in connective tissue associated
in association with these TRAP-positive cells (Figure 7D). RUNX2
with MB (Figure 6G) than Bio-Oss® (Figure 6H).
was associated with the osteoblasts lining the bone-forming areas (Figure 7E,F). PCNA-positive cells were detected in the osteoblast
3.7 | Twelve weeks post-grafting
layer and connective tissue for both MB (Figure 7G) and Bio-Oss® (Figure 7H).
MB particles were noticeably reduced in size in the sections examined at 12 weeks (Figure 7A,C,E,G). In most cases, the MB particles were surrounded by lamella bone. Bio- Oss® particles were also
4 | DISCUSSION
surrounded by lamella bone; however, at times areas of connective tissue abutted, and in some of these regions, TRAP-positive
In the current pilot study, we used an animal model of sinus augmen-
cells were evident on the surface of the material. There was no
tation, independent of implant placement. We investigated two DBB
|
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SMITH et al.
F I G U R E 5 High magnification images of MB (left-hand side) and BO (right-hand side) within a sheep sinus at 4 wk post- surgery. All decalcified paraffin-embedded sections have been counterstained with hematoxylin. (A, B) Hematoxylin and eosin-stained sections. (C, D) TRAP staining (brown) to identify osteoclasts. (E, F) Immunohistochemistry for RUNX2 (brown). (G, H) Immunohistochemistry for PCNA- positive proliferating cells, which are only evident in G. BO, Bio-Oss®; MB, Moa- Bone®; red arrowheads are examples of multinuclear osteoclasts; black arrows are examples of positively stained cells; scale bars = 50 μm
(A)
(B)
(C)
(D)
(E)
(F)
(G)
(H)
materials, MB and Bio-Oss®, and found that they have different profiles of healing when used to augment the maxillary sinus.
The osseous response within the sheep maxillary sinus differed considerably between the MB and the Bio-Oss® grafted sites over
The two DBB materials differed considerably in their ease of han-
the 12 week period examined. There was a clear gradient of osseous
dling. MB was difficult to use clinically, demonstrating electrostatic
envelopment around the particles, with the graft materials closest to
effects and strong hydrophobicity. SEM analysis indicated that the
pre-existing bone surrounded by new bone, which became lamella
surface morphologies were quite different. MB was a smoother and
earlier, compared with the graft material that was at a distance from
more porous, fibrous structure with minimal globular particles. Bio-
the pre-existing bone. This gradient is similar to that reported by oth-
Oss® was an almost entirely globular surface, consistent with previous
ers.37 Inflammatory responses to Bio-Oss® have not been typically
The differences in the microscopic appearance of
observed38-40 and we found no evidence of a strong inflammatory
the two materials were consistent with previous research that indi-
reaction to either material. There was little difference between the
cated the micro-porosity of hydroxyapatite decreases with increasing
two graft materials with regard to their encapsulation in woven bone
processing temperatures.36
and production of lamella bone during the early osseous response;
investigations.
6,35
|
SMITH et al.
8
(A)
(B)
(C)
(D)
(E)
(F)
(G)
F I G U R E 6 MB (left-hand side) and BO (right-hand side) within a sheep sinus at 6 wk post-surgery. All decalcified paraffin-embedded sections have been counterstained with hematoxylin. (A, B) Hematoxylin and eosin-stained sections. (C, D) TRAP staining (brown) to identify osteoclasts. (E, F) Immunohistochemistry for RUNX2 (brown). (G, H) Immunohistochemistry for PCNA-positive proliferating cells (brown). BO, Bio-Oss®; CT, connective tissue; LB, lamellar bone; MB, Moa-Bone®; WB, woven bone; red arrowheads, examples of TRAP-positive osteoclasts; black arrows, RUNX2-positive osteoblasts; scale bars A,B = 200 μm; C-H = 100 μm
(H)
however, Bio-Oss® was more likely to be in contact with connective
Zaffe et al38 who noted that Bio-Oss® was typically surrounded with
tissue at 12 weeks when compared to any remaining MB at the same
connective tissue and who noted marked resorption of the Bio-Oss®.
®
time point. Bio-Oss has been reported by others to be associated with
Others, however, have noted good osseointegration associated with
connective tissue and linked with delayed bone formation.41 Lindhe
Bio-Oss® both in experimental and clinical settings.39,43-46
42
et al
®
compared Bio-Oss versus non-grafted controls in human sub®
RUNX2, also known as core-binding factor α-1, is essential for os-
jects and found that the Bio-Oss particles were not resorbed but in-
teoblast maturation during both intramembranous and endochondrial
stead became surrounded by less mineralized bone and more fibrous
ossification.47,48 In previous studies of sinus grafting, RUNX2 has been
®
tissue than the control sites. These authors suggested that the Bio-Oss
detected in pre-osteoblasts situated in the connective tissue, and in
particles were resistant to resorption and delayed osseous healing. The
association with osteoblasts and young osteocytes, but not with ma-
MB test material in our study behaved differently, resorbing more rap-
ture osteocytes, which were negative.49 We noted a similar pattern of
®
idly than the Bio-Oss . The formation of bone in direct contact with
staining pattern for RUNX2 in our study. Of particular note was the
the Bio-Oss®, at the earliest time period of 4 weeks, was in contrast to
RUNX2 staining at 4 weeks within a more organized connective tissue
|
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SMITH et al.
F I G U R E 7 MB (left-hand side) and BO (right-hand side) within a sheep sinus at 12 wk post-surgery. All decalcified paraffin-embedded sections have been counterstained with hematoxylin. (A, B) Hematoxylin and eosin-stained sections. (C, D) TRAP staining (brown) to identify osteoclasts. (E, F) Immunohistochemistry for RUNX2 (brown). (G, H) Immunohistochemistry for PCNA-positive proliferating cells (brown). BO, Bio- Oss®; CT, connective tissue; LB, lamellar bone; MB, Moa-Bone®; red arrowheads, examples of TRAP-positive osteoclasts; scale bars A,B = 200 μm; C-H = 100 μm
(A)
(B)
(C)
(D)
(E)
(F)
(G)
(H)
matrix associated with MB, as compared to the more fibrous disorga®
trials; however, there is conflicting data on the rate of resorption. In
nized matrix associated with Bio-Oss . This may indicate the early acti-
a model of mandibular bone grafts, also in sheep, Bio-Oss® was iden-
vation of pre-osteoblasts in association with MB. More PCNA-positive
tified at 16 weeks post-grafting but particles were noted as having a
proliferative cells were also noted in association with the MB at the
rough surface consistent with the presence of resorption lacunae.50 A
earlier time points. At 6 and 12 weeks, staining of RUNX2 within the
study in dogs showed no histological signs of Bio-Oss® resorption 3
osteoblast layer was clearly evident and this staining was consistent
or 6 months after grafting while bone ingrowth was consistent with
with bone formation in association with both of the graft materials.
osteogenic regeneration of the defects.46 Studies on human sinus
Osteoclastic resorption of graft material and remodeling of bone ®
grafts showed varying results; Zaffe and co-workers38 reported that
was investigated using TRAP staining. We found that Bio-Oss was
19 weeks after implantation, very few patients had residual Bio-Oss®
associated with very few osteoclasts and there was little evidence of
present, large numbers of osteoclasts were apparent and resorp-
resorption lacunae even at 12 weeks post-surgery. A slow resorption
tion lacunae were highly evident. Others noted abundant Bio-Oss®
®
rate of Bio-Oss has been reported in both large animal and human
particles 3 months after implantation and reported the presence
|
SMITH et al.
10
of well-organized lamella bone in contact with the Bio-Oss®.40 In patients grafted with Bio-Oss
®
alone or mixed with a cell-binding
peptide (PepGen P-15), abundant Bio-Oss® was found 3-20 months after surgery and although reduced, some particles were still present
CO NFL I C T O F I NT ER ES T The authors declare no financial interest in Molteno® Ophthalmic Ltd or Geistlich Pharma AG.
after 7-8 years, typically encapsulated in bone.39,44 Biopsies collected 6-8 months after Bio-Oss® grafting in five patients also showed the presence of new bone around significant quantities of Bio-Oss®.45 Thus, it appears that some resorption of Bio-Oss® by osteoclasts may occur; however, once encapsulated in bone the bovine bone parti-
O RC I D D. E. Coates
http://orcid.org/0000-0003-4242-6846
cles may persist for long periods of time.51 The slow resorption rate
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however, graft resorption may result in a more natural autogenous bone.42,52 In our study, MB was associated with a strong activation of osteoclasts. TRAP staining was strongest at 4 and 6 weeks. The connective tissue had strong TRAP staining of isolated cells as well as diffusely within the tissue. In one section, this staining was so strong at 4 weeks the incubation time had to be reduced to visualize the section. The strongest TRAP staining was localized to large cells on the surface of the MB consistent with being osteoclasts and typically located in resorption lacunae. Osteoclasts were observed on the surface of implant material only when connective tissue was adjacent to the graft material. This suggests that the migration of the monocyte- derived osteoclast precursor cells to the graft material may require the presence of connective tissue and may explain the lack of TRAP- positive cells in the MB by 12 weeks, as it was typically encapsulated in bone. To the best of our knowledge, this is the first study to use MB for osseous sinus grafting. Compared to Bio-Oss® it showed a similar rate of osseointegration at 12 weeks and did not induce an inflammatory response. MB was rapidly resorbed and only small amounts were left in what was an almost entirely autologous bone graft by week 12. MB thus has some attributes that are consistent with potential utility as a graft material, particularly where complete remodeling of the graft site is desired. This behavior is similar to that of autogenous bone. This pilot preclinical study suggested that MB has the potential for use in sinus grafting; however, definitive statements would require statistically relevant samples sizes. Further optimization of MB for maxillofacial applications is warranted, particularly with respect to its handling properties. Further experimental work is required with sufficient animals per time period to permit robust statistical analysis, to demonstrate the efficacy of the MB graft when compared with commercially available materials such as Bio-Oss®; ideally, such future experimental work should also include analysis of the response of the grafted sites after restoration with loaded dental implants, as this is the clinical rationale for the use of bovine bone grafting materials in the maxillary sinus.
ACKNOWLE DGE ME N TS The assistance of the veterinary staff of the University of Otago Hercus Taieri Resource Unit is gratefully acknowledged.
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How to cite this article: Smith MM , Duncan WJ, Coates DE. Attributes of Bio-Oss® and Moa-Bone® graft materials in a pilot study using the sheep maxillary sinus model. J Periodont Res. 2017;00:1–11. https://doi.org/10.1111/jre.12490
