British Journal of Oral and Maxillofacial Surgery 49 (2011) 3741

Available online at www.sciencedirect.com

Osteoconduction of different sizes of anorganic bone
particles in a model of guided bone regeneration
Xinwen Zhou
a
, Zhenting Zhang
a,
, Song Li
b
, Yuxing Bai
b
, Hui Xu
b,
a
Department of Prothodontics, School of Stomatology, Capital Medical University, Beijing, China
b
Department of Orthodontics, School of Stomatology, Capital Medical University, Beijing, China
Accepted 1 January 2010
Available online 27 January 2010
Abstract
The aimof this study was to evaluate the effect of two different sizes of anorganic bone particles (300500 and 8501000 m) on the formation
of new bone in a model of guided bone regeneration. In both groups, newly formed bone was seen histologically adjacent to the original
surface of the skull, and there were outgrowths to the centre of the secluded graft 4 weeks after implantation. Some particles near the surface
were in contact with the newly formed bone, and osteoconductive bone growth was present along their surface. Ten weeks after implantation
the area created by grafting with small particles seemed to have a denser structure than that created with large particles. Histomorphometric
analysis showed a higher density of newly formed bone in the small-particle group than in the large-particle group both 4 and 10 weeks
after implantation. The total contact length between newly formed bone and particles and the ratio of bone:space between the particles were
also signicantly higher in the small-particle group at both time points. We conclude that the size of grafted particles of bone and the spaces
between particles are important determinants of osteogenesis during guided bone regeneration.
2010 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
Keywords: Guided bone regeneration; Anorganic bone; Particle; Rabbit
Introduction
In recent years, a tissue regenerative technique, guided bone
regeneration (GBR), has been developed by which it is pos-
sible to regenerate resorbed alveolar bone and also close
small bony defects. These procedures require the use of
grafts to maintain the augmented space and to promote
osteogenesis.
14
Many biomaterials have been developed as
substitutes for bone, including ceramics, polymers, metals,
and organic or non-organic bone substitutes. Among them,
anorganic bone has been shown to be an excellent material for
bone grafts as it is biocompatible and osteoconductive, and
causes no immune reaction. It has facilitated the growth of

Corresponding authors. School of Stomatology, Capital Medical Uni-

versity, No. 4 of Tiantan xili, Chongwen District, Beijing 100050, China.
Tel.: +86 10 67099225; fax: +86 10 67099310.
E-mail addresses: zztdentist@hotmail.com (Z. Zhang),
huixudentist@hotmail.com (H. Xu).
new bone in numerous experimental studies, and has also
been used successfully in humans for the regeneration of
defects, augmentation of ridges, raising of the sinus oor,
and repair of periodontal defects.
5,6
The size of particles in the bone graft is an important
indicator of osteogenic activity.
7,8
However, the effect of
anorganic bone particles of different sizes in GBR has not
been examined in detail, so we have evaluated the osteocon-
ductive potential of different sizes of anorganic bone particles
in GBR histologically and histomorphometrically.
Material and methods
Preparation of bone grafts
The anorganic bone was prepared as described by Matsuda
et al.
9
The calcium:phosphorus ratio was 2:34, which is
close to that of human bone, and similar to that of commer-
0266-4356/$ see front matter 2010 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bjoms.2010.01.001
38 X. Zhou et al. / British Journal of Oral and Maxillofacial Surgery 49 (2011) 3741
cial anorganic bovine bone mineral.
10
The anorganic bone
was pulverised in a bone mill (TOM, Leibinger, Freiburg,
Germany) andsortedintoparticles of 300500 m(the small-
particle group) and 8501000 m (the large-particle group)
using a standard sieve (Tokyo Screen, Tokyo, Japan).
Surgical techniques
The study was approved by the Institutional Animal Care
and Use Committee of Capital Medical University. Twelve
adult male Japanese white rabbits (mean weight 3.0 kg) were
used, 6 rabbits being studied in each group. The rabbits were
anaesthetised with pentobarbitone sodium (0.5 mg/kg) given
intravenously, and 0.5 ml of 1% lignocaine with adrenaline
(1:100,000) was injected subcutaneously in each surgical
eld as local anaesthetic. The frontal bone was exposed via a
midsagittal incision through the skin and periosteum. A skin
and periosteal ap was raised to expose the skull on both sides
of the midline. Under generous irrigation with saline, 9 drill
holes were made into the experimental space using a round
bur to facilitate bleeding. Two titanium hemispherical domes
5 mmhigh, 0.5 mmthick, and 10 mmin diameter were placed
over the experimental area in each animal and anchored to
the bone surface with 2 miniscrews (Ti-SIS pins, SIS-System
Trade, Klagenfurt, Austria). Before placement the dome was
lled with either small or large particles. The periosteumwas
then sutured over the borders of the rim with non-resorbable
suture material.
Preparation of tissue
The rabbits were killed 4 and 10 weeks after opera-
tion. The experimental sites were resected, xed in 4%
paraformaldehyde, and demineralised in 10% EDTA at 4

C.
The specimens were then dehydrated in a graded series of
ethanol, embedded in parafn, and sliced into sections about
3 mthick. The sections were stained with haematoxylin and
eosin and examined under light microscopy.
Histomorphometric analysis
We examined 3 sections at least 30 m apart from each spec-
imen. For each section, four elds were chosen randomly and
evaluated under a light microscope at 10magnication and
a semiautomatic computer-assisted digitiser (Cadkey System
Corp., Tokyo, Japan). Areas and distances were marked by
hand with a mouse and calculated by the computer. The fol-
lowing histomorphometric measurements were made: area
of bone (percentage of newly formed bone to total mea-
sured area); total length of surface of particles; total length
of contact between newly formed bone and particles; degree
of contact between bone and particles (percentage of length
of contact between newly formed bone and particles to total
length of surface of particles); area of space between parti-
cles (percentage of space between particles to total measured
area); and ratio of bone:space between particles (percentage
of area of newly formed bone to area of space between par-
ticles). All measurements were made by the same author. To
test the reliability of measurement, 9 sections were also mea-
sured by a second examiner. Repeated measurements made
on the same images resulted in non-signicant differences
between the measurements.
Statistical analysis
The signicance of differences between groups and implan-
tation times was assessed by two-way analysis of variance
(ANOVA) using Tukeys method with the assistance of the
Statistical Package for the Social Sciences, version 14.0
(SPSS Inc., Chicago). Probabilities of less than 0.05 were
accepted as signicant. All data are expressed as mean
(SD).
Results
Histological ndings
4 weeks after implantation
In both groups, new bone had formed beneath the domes, the
rims of which seemed to have adapted well to the surface
of the skull, preventing growth of the surrounding connec-
tive tissue into the membrane-secluded space. Newly formed
bone was present adjacent to the original skull surface and
showed outgrowths to the centre of the secluded graft space,
in the top part of which the particles were surrounded by
brous connective tissue. There were no signs of inamma-
tion (Fig. 1A and B).
10 weeks after implantation
In both groups, the secluded graft space was occupied by
newly formed bone, connective tissue, and particles of anor-
ganic bone. The created area grafted with the small particles
seemed to contain more bone than the area grafted with large
particles. In the small-particle group, newly formed bone
showed many interconnections, in most parts of the spaces
(Fig. 2A). In the large-particle group, the newly formed bone
had limited intercommunications. Some central areas con-
tained brous tissue with no evidence of ossication. There
were no signs of inammation (Fig. 2B).
Histomorphometrical analysis
Area of bone
In both groups the area of bone increased with time. There
were signicant differences between 4 and 10 weeks after
implantation (p <0.001). There was signicantly more bone
area in the small-particle group than in the large-particle
group both 4 and 10 weeks after implantation (p <0.01)
(Table 1, Fig. 3).
X. Zhou et al. / British Journal of Oral and Maxillofacial Surgery 49 (2011) 3741 39
Fig. 1. Histological ndings 4 weeks after implantation. Newly formed bone
is present adjacent to the original skull. Some anorganic bone particles near
the skull are in contact with the bone tissue in both groups (SB: the original
skull bone; NB: newly formed bone; P: particles; F: brous connective tis-
sue). (A) Small-particle group and (B) large-particle group (haematoxylin
and eosin, original magnication 10).
Total length of surface of particles
The total length of the surface of small particles was signi-
cantly more than that of the large particles at 4 and 10 weeks
after implantation (p <0.001) (Table 1).
Total length of contact between newly formed bone and
particles
In both groups, the total length of contact between newly
formed bone and particles increased with time. There were
signicant differences in both the small-particle (p <0.01)
Fig. 2. Histological ndings 10 weeks after implantation. (A) In the small-
particle group, newly formed bone shows many interconnections and is
present in most parts of the spaces. (B) In the large-particle group, the newly
formed bone shows limited intercommunications and some central areas of
the secluded graft space contain brous connective tissue without ossi-
cation (NB: newly formed bone; P: particles; F: brous connective tissue)
(haematoxylin and eosin, original magnication 10).
and the large-particle (p <0.05) groups. The length of con-
tact between bone and particles in the small-particle group
was signicantly greater than that in the large-particle
group at 4 and 10 weeks after implantation (p <0.001)
(Table 1).
Degree of contact between bone and particles
In both groups, the degree of contact between bone and parti-
cle increased with time, and there were signicant differences
between 4 and 10 weeks after implantation (p <0.05). The
Table 1
Mean (SD) histomorphometric measurements after grafting with the different sizes of deproteinised bone particles.
Group
(particle size
m)
Time
(weeks)
Bone
area (%)
Total length of
surface of
particles (m)
Total length of contact
between bone and
particles (m)
Degree of
contact between
bone and
particles (%)
Space between
particles (%)
Ratio of
bone:space
between
particles (%)
300500 4 15.9 (2.6) 1491.1 (205.2) 566.8 (85.8) 39.2 (6.7) 50.3 (2.8) 34.6 (2.7)
10 26.3 (3.4) 1576.9 (157.6) 864.1 (105.4) 56.8 (7.9) 57.5 (4.3) 46.7 (4.1)
8501000 4 10.5 (2.1) 678.9 (98.3) 295.6 (34.5) 43.2 (5.3) 41.7 (3.5) 25.1 (3.0)
10 16.7 (1.5) 630.2 (65.7) 423.3 (76.6) 61.7 (8.1) 46.2 (2.8) 36.1 (2.2)
40 X. Zhou et al. / British Journal of Oral and Maxillofacial Surgery 49 (2011) 3741
Fig. 3. Diagram of differences between the formation of bone in the two
groups.
degree of contact did not differ signicantly between the
groups at any time (Table 1).
Area of space between particles
The area was signicantly greater in the small-particle group
both 4 and 10 weeks after implantation (p <0.01) (Table 1).
Ratio of bone:space between particles
In both groups, the ratio of bone:space between particles
signicantly increased with time (p <0.05). The ratio of
bone:space was signicantly higher in the small-particle
group both 4 and 10 weeks after implantation (p <0.01)
(Table 1).
Discussion
Guided bone regeneration is widely used to augment the
width of the alveolar ridge before implants are inserted. The
success or failure of treatment with osseointegrated implants
in edentulous patients depends on the volume and quality
of newly formed bone in the augmented space, as adequate
primary anchorage is often difcult in low density bone.
11,12
We have presented a model that replicates a clinical
one-wall defect. Corresponding preclinical models with
hemispheres that provide space for bone substitutes have been
established in rats and rabbits.
1315
In our model the forma-
tion of bone is supported by opening the marrow cavity with
small drills, which also permit blood to permeate into the
augmented area to provide a provisional extracellular matrix
for the immigration of repair cells.
16
Histologicallybothsizes of particles inducedbonygrowth,
which indicates that the anorganic bone particles have bone-
conductive properties. The particles acted as a scaffold to
facilitate the formation of bone. Scaffolding is a critical
component in tissue engineering because it provides the
three-dimensional clues for seeding, migration, and growth
of cells, and formation of newtissue.
17,18
Histomorphometric
analysis showed a higher density of newly formed bone in the
small-particle group than in the large-particle group. The total
length of the surface of the small particles was signicantly
greater than that of the large particles, and the contact length
between bone and particles in the small-particle group was
signicantly greater than that in the large-particle group, so it
may be that the size of the particles plays a part in osteogenic
activity. The small particles may provide a large surface area
around which more bone may form.
With porous graft materials, the size of the pores and the
porosity have critical roles in the formation of bone.
19,20
It
was claimed that a minimum pore size of 100 m was nec-
essary for bone to grow into the porous materials.
21
The size
of pore is comparable to the size of the spaces between parti-
cles, the spaces between the small particles were signicantly
larger than those between the large particles. The possible
reasons are that the anorganic bone particles were irregu-
larly shaped and were lightly packed into the secluded graft
space without compression during operation. The differences
in osteogenicity between the two groups may be linked to the
specic packing and the macroporosity that was created. The
small particles will have created a different macroporosity in
the tissue-engineered construct, and this could be favourable
for the formation of bone.
This requires a rich blood supply,
21
and the larger interpar-
ticular spaces may allow the ingrowth of capillaries, as such
ingrowth precedes the formation of new bone. The small-
particle group showed evidence of more and denser amounts
of new bone than the large-particle group. Histomorphomet-
ric analysis showed that the ratio of bone:space of the small
particles was signicantly greater than that of the large par-
ticles after implantation. We therefore suggest that the space
between the particles is an important factor for osteocon-
duction. The small particles might provide a better scaffold
on which tissue may inltrate and regenerate than the large
particles.
Acknowledgements
The study was supported by Beijing scientic and technolog-
ical new star program (2006B62) and the Scientic Research
Foundation for the Returned Overseas Chinese Scholars,
Ministry of Education of China.
References
1. Geurs NC, Korostoff JM, Vassilopoulos PJ, Kang TH, Jeffcoat M, Kel-
lar R, et al. Clinical and histologic assessment of lateral alveolar ridge
augmentation using a synthetic long-term bioabsorbable membrane and
an allograft. J Periodontol 2008;79:113340.
2. Dies F, Etienne D, Abboud NB, Ouhayoun JP. Bone regeneration in
extraction sites after immediate placement of an e-PTFEmembrane with
or without a biomaterial. A report on 12 consecutive cases. Clin Oral
Implants Res 1996;7:27785.
3. Okazaki K, Shimizu Y, Xu H, Ooya K. Blood-lled spaces with and
without deproteinized bone grafts in guided bone regeneration. A histo-
morphometric study of the rabbit skull using non-resorbable membrane.
Clin Oral Implants Res 2005;16:23643.
X. Zhou et al. / British Journal of Oral and Maxillofacial Surgery 49 (2011) 3741 41
4. Donos N, Kostopoulos L, Tonetti M, Karring T. Long-term stability
of autogenous bone grafts following combined application with guided
bone regeneration. Clin Oral Implants Res 2005;16:1339.
5. Hmmerle CH, Jung RE, Yaman D, Lang NP. Ridge augmentation
by applying bioresorbable membranes and deproteinized bovine bone
mineral: a report of twelve consecutive cases. Clin Oral Implants Res
2008;19:1925.
6. Berglundh T, Lindhe J. Healing around implants placed in bone defects
treated with Bio-Oss. An experimental study in the dog. Clin Oral
Implants Res 1997;8:11724.
7. Shapoff CA, Bowers GM, Levy B, Mellonig JT, Yukna RA. The
effect of particle size on the osteogenic activity of composite grafts
of allogeneic freeze-dried bone and autogenous marrow. J Periodontol
1980;51:62530.
8. Zaner DJ, Yukna RA. Particle size of periodontal bone graftingmaterials.
J Periodontol 1984;55:4069.
9. Matsuda M, Kita S, Takekawa M, Ohtsubo S, Tsuyama K. Scanning
electron and light microscopic observations on the healing process after
sintered bone implantation in rats. Histol Histopathol 1995;10:6739.
10. Xu H, Shimizu Y, Asai S, Ooya K. Experimental sinus grafting with the
use of deproteinized bone particles of different sizes. Clin Oral Implants
Res 2003;14:54855.
11. Sennerby L, Thomsen P, Ericson LE. A morphometric and biomechanic
comparison of titanium implants inserted in rabbit cortical and cancel-
lous bone. Int J Oral Maxillofac Implants 1992;7:6271.
12. Henry PJ, Tolman DE, Bolender C. The applicability of osseoin-
tegrated implants in the treatment of partially edentulous patients:
three-year results of a prospective multicenter study. Quintessence Int
1993;24:1239.
13. Slotte C, Lundgren D. Impact of cortical perforations of contiguous
donor bone in a guided bone augmentation procedure: an experimen-
tal study in the rabbit skull. Clin Implant Dent Relat Res 2002;4:
110.
14. Stavropoulos A, Kostopoulos L, Nyengaard JR, Karring T. Fate of bone
formed by guided tissue regeneration with or without grafting of Bio-
Oss

or Biogran

: an experimental study in the rat. J Clin Periodontol

2004;31:309.
15. Yamada Y, Nanba K, Ito K. Effects of occlusiveness of a titaniumcap on
bone generation beyond the skeletal envelope in the rabbit calvarium.
Clin Oral Implants Res 2003;14:45563.
16. Modder UI, Khosla S. Skeletal stem/osteoprogenitor cells: cur-
rent concepts, alternate hypotheses, and relationship to the
bone remodeling compartment. J Cell Biochem 2008;103:
393400.
17. Thomson RC, Yaszemski MJ, Powers JM, Mikos AG. Fabrication of
biodegradable polymer scaffolds toengineer trabecular bone. J Biomater
Sci Polym Ed 1995;7:2338.
18. Ma PX, Choi JW. Biodegradable polymer scaffolds with well-
dened interconnected spherical pore network. Tissue Eng 2001;7:23
33.
19. Chang BS, Lee CK, Hong KS, Youn HJ, Ryu HS, Chung SS, et al. Osteo-
conduction at porous hydroxyapatite with various pore congurations.
Biomaterials 2000;21:12918.
20. Borden M, Attawia M, Khan Y, Laurencin CT. Tissue engineered
microsphere-based matrices for bone repair: design and evaluation. Bio-
materials 2002;23:5519.
21. Karageorgiou V, Kaplan D. Porosity of 3D biomaterial scaffolds and
osteogenesis. Biomaterials 2005;26:547491.