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COMPARATIVE STUDY
A Comparative Evaluation of Decalcified Freeze Dried Bone
Allograft, Hydroxyapatite and Their Combination in Osseous
Defects of the Jaws
Sonal Mishra •R. K. Singh •Shadab Mohammad •
R. Pradhan •U. S. Pal
Received: 4 July 2009 / Accepted: 11 October 2010 / Published online: 20 November 2010
ÓAssociation of Oral and Maxillofacial Surgeons of India 2010
Abstract
Objectives To evaluate decalcified freeze dried allograft
or hydroxyapatite and a combination of both as bone
autograft substitutes in the healing of osseous jaw defects.
Materials and Methods 24 patients participated in the
study which involved the filling of osseous defects in the
maxilla/mandible with decalcified freeze dried bone allo-
graft (DFDBA) or hydroxyapatite (HA) or a combined
graft composed of these two in equal proportions.
Results Bone formation occurred as early as 4 weeks in
the DFDBA and combination groups and 12 weeks in the
HA group which was verified by radiographs, Dentascans
(DentaScanÒSoftware Program, General Electric, USA)
and bone scintigraphy.
Conclusion Both these materials can be used as bone
graft substitutes in smaller defects although their suitability
in large defects is yet to be studied.
Keywords Bone Demineralised Freeze dried
Hydroxyapatite Bone Grafts
Introduction
Bone is a dynamic living tissue that shows marked struc-
tural alteration in response to injury, changes of stress and
vascular, endocrine, genetic and nutritional influences. It is
one of the few human organs that can undergo regeneration
rather than repair with formation of scar tissue. Bone is a
specialized connective tissue that provides support and
protection for the delicate and vital organs of the body and
also allows for locomotion.
The development of bone graft material to replace bone
remains a formidable challenge in maxillofacial surgery.
Although autogenous bone is the best material, however,
the advantage of an autograft is offset by the limited supply
of such bone and morbidity associated with surgery to
harvest the graft.
This article aims to study and compare the efficacy of
two such bone substitutes in the healing of osseous defects
of the jaws.
Materials and Methods
The present study comprised of 24 patients who attended the
outpatient department of Oral and Maxillofacial Surgery, U.P.
King George University ofDental Sciences, and Lucknowfor
jaw defect corrections between January and December 2005.
An informed consent of each patient was taken to par-
ticipate in the study.
And the patients were randomized in three groups:
Group A—where osseous defects were filled with
decalcified freeze dried bone matrix allograft (DFDBA).
Group B—where osseous defects were filled with porous
HA granules.
S. Mishra (&)
Department of Oral and Maxillofacial Surgery,
Rama Dental College, Hospital & Research Centre,
R-7, Medical College Campus, Kanpur 208002, U.P., India
e-mail: drycmishra@gmail.com
R. K. Singh S. Mohammad U. S. Pal
Department of Oral and Maxillofacial Surgery,
K.G.M.U., Lucknow, India
R. Pradhan
Department of Oral and Maxillofacial Surgery,
U.P. Dental College, Lucknow, India
123
J. Maxillofac. Oral Surg. (July-Sept 2010) 9(3):236–240
DOI 10.1007/s12663-010-0080-1
Group C—combination of DFDBA and HA granules in
equal proportions was used. The bone graft–alloplast
combination was packed tightly in the osseous defect.
The defects of bone were mostly as a result of cyst
enucleation and other such benign lesions larger than 1 cm
diameter (Fig. 1). Third molar sockets and locally aggres-
sive lesions were excluded.
The allograft material was prepared by the following
procedure:
1. Procurement of healthy bone: Healthy femur heads,
from the Department of Orthopaedic Surgery, King
George Medical University, Lucknow and kept in an
autoclaved container containing distilled water and
stored in the freezer.
2. Defatting of bone: Cortico-cancellous bone was then
cut by sterilized mallet and chisel into small pieces. The
graft material was then immersed in 100% ethyl
alcohol for 1 hour to remove fat from the bone marrow.
3. Decalcification of allograft material [1]: The bone was
then decalcified with 0.6 N HCl to remove the
calcium. The graft was then washed in sodium
phosphate buffer to remove the residual acid. The
demineralised bone was re-frozen for 2 weeks.
4. Freeze drying of bone: The cortico-cancellous bone
was then pulverized in an autoclaved kitchen mixer
especially kept for this purpose, in the presence of
liquid nitrogen at a temperature of -196°C and sieved
to almost powder form.
5. Sterilization of allograft material: Prepared allograft
material was stored in an air tight glass container and
sterilized by the c-radiation of 2.5 Mrad at Central
Institute for Medicinal and Aromatic Plants, Lucknow
and stored at 2–3°C until the allograft was used.
Hydroxyapatite with average pore size 0.8 mm available
in ready to use in pre-sterilized (Gamma irradiated) gran-
ules/block form used in the study was HAG 3 manufac-
tured by Surgiwear Limited India.
The bone graft/alloplast/combination was packed tightly
but not too tightly in the osseous defect. Closure of wound was
achieved by interrupted sutures using 3-0 black silk (Fig. 2).
A pressure pack for half an hour was given to achieve
haemostasis. Patients were instructed to continue antibi-
otic, analgesics, vitamins for 5 days. Sutures were removed
on the fifth post-operative day.
Clinical evaluation was done for post-operative pain
(Visual Analogue Scale), post operative swelling, signs of
infection (persistent post-operative swelling, pus discharge,
pain) and signs of implant rejection (discharge, extrusion,
tissue dehiscence).
In all the post-operative cases intraoral periapical X-rays
were taken at immediate post-operative day 1, 4, 12, and
24 week intervals for radiographic evaluation. A measur-
ing grid of 1 91 mm size squares on a transparent sheet
was used for measurement of osseous defect by placing the
grid over the radiograph. The alveolar bone loss/gain was
measured by counting the number of squares overlapping
the defect at different follow ups. Squares which were half
filled with bone were not counted.
Percentage was calculated using following formula:
Fig. 1 Preoperative radiograph showing periapical pathology in 22
region Fig. 2 Enucleation of cyst followed by obliteration of the defect
with the graft (DFDBA and HA)
Sq:overlapping initial defect Sq:overlapping the defects at different follow ups
Sq:overlapping initial defect 100
J. Maxillofac. Oral Surg. (July-Sept 2010) 9(3):236–240 237
123
for, evidence of calcification (evident by increased radio-
opacity), bone formation (evident by formation of trabec-
ular pattern) and bridging of gap by normal bone was
calculated by using the above formula.
Three phase skeletal scintigraphy scan was done under
the gamma camera after intravenous infusion of
99m
Tc
MDP 4 weeks postoperatively.
Dentascan assessment was done between 4 and 6 weeks
postoperatively to assess new bone formation. Dentascan is
a specialized type of computed tomography study which is
performed on a conventional CT scanner used for imaging
all parts of the body. It uses thin 1 mm axial CT images of
the maxilla and mandible to reformat a series of multiple
cross-sectional and panoramic images of the jaw [2,3].
None of the patients agreed for histological examination
during post operative follow up, hence, it was not included
in the study.
Statistical Analysis
The following have been used in the present study to
analyse the data obtained.
1. Mean
2. Standard deviation
3. Student t test
4. Analysis of variance
5. Chi square (v
2
)(P\0.05 least significance, P\0.01
significant, P\0.001 highly significant.)
Results
Analysis of the three groups did not reveal any statistically
significant differences at any of the follow up periods except
for swelling and radiological evidence at four weeks [4].
Swelling was seen only in group C at fourth week follow
up. Also, there was moderate pain and pus discharge with
extrusion of the graft and tissue dehiscence. This difference
was statistically significant as compared to group A and B.
No signs of infection (persistent post operative swelling,
pain or pus discharge) were seen in any patient of group A
and B. In group C, however, all these three signs of
infection were seen in 2 (25%) patients at fourth week.
No signs of implant rejection (discharge, extrusion, tis-
sue dehiscence) were seen in any patient of group A and B.
In group C, however, all these three signs of implant
rejection were seen in 2 (25%) patients at fourth week. The
graft material was surgically removed at fourth week.
Radiological evidence of calcification, bone formation
and bridging of the gap with new bone as evident by for-
mation of irregular trabeculae of bone and appearance of
radio-opaque areas in the defect was evident from fourth
week onwards. It was seen in 62.5% cases of group A, in
no case of group B and 50% cases of group C; by this time
two grafts were rejected in group C. The difference
between group A and B was statistically significant
(P\0.01) at this point of time.
At 12 week follow up 62.5% cases each of group A and
B showed radiological evidence of calcification, bone
formation and bridging of the gap while 66.7% cases of
group C showed radiological evidence of calcification,
bone formation and bridging of the gap. At 24 weeks fol-
low up interval 75% cases of group A and B and 100% of
group C showed radiological evidence of bone formation
(Fig. 3).
The Dentascan and Bone Scintigraphic assessments
although performed in only a few representative cases after
4 weeks of graft placement supported and confirmed the
radiographic findings (Figs. 4,5).
Fig. 3 24 weeks postoperative radiograph showing healed osseous
defect with residual hydroxyapatite granules
Fig. 4 Dentascan done 4 weeks postoperatively showing no evidence
of bone formation and radioopaque HA granules
238 J. Maxillofac. Oral Surg. (July-Sept 2010) 9(3):236–240
123
Discussion
The allograft, DFDBA and the alloplastic material, HA
were tried in this study due to their osteoinductive and
osteoconductive properties respectively and their avail-
ability in sufficient quantities as and when needed. There
have been numerous encouraging results with both these
bone substitutes.
Since both these materials were found to be useful, it
was thought to combine them together and to assess their
collective effectivity in bone formation and to see whether
these collectively offered any additional advantage as
opposed to their singular use.
The DFDBA was used because the very processes
involved in its preparation i.e. decalcification exposes on
its surface, the bone morphogenetic proteins (BMPs) which
are osteoinductive [5–11] that is, they induce differentia-
tion of mesenchymal cells into cartilage and bone [12].
BMPs are natural proteins which play important roles
during embryogenesis and mediate in specific aspects of
skeletal growth and development during later adult life
[13]. BMPs have been extracted from bone, dentin, and
osteosarcoma tissue[12].
Thus, in group A, the bone formation ensued immedi-
ately after filling DFDBA because of the BMPs which were
exposed during the decalcification process.
Also, the freeze drying at -196°C destroys the antige-
nicity of the DFDBA [14–17].
.
Porous HA is an abundantly available non toxic material
[18] that is bioactive and allows new bone to be formed
directly on its surface without any intervening layer of
fibrous tissue [19].
Synthetic HA possesses a similar composition to HA
crystals present in bone, enamel and dentin and exhibits
osteoconduction by acting as a scaffold for new bone to
grow through the implant material as long as there is
enough vital host bone surrounding it [20].
HA, when implanted does not evoke an inflammatory or
foreign body response [9,21,22] and has a good tissue
tolerance [23].
Thus, in group B, the initiation of bone formation is
different i.e. by osteoconduction alone as the HA granules
used merely act as a trellis for blood vessels and migration
of osteoblasts from surrounding healthy bone, a process
called ‘‘creeping substitution’’, whereby the HA is slowly
resorbed and replaced by bone.
HA when used with DFDBA as opposed to their single
use also showed good results as a synergistic effect of
osteoinduction and osteoinduction was observed [24–26].
The cause of failure in 2 patients of group C who
showed signs of infection along with extrusion of the grafts
and wound dehiscence in the fourth week was therefore
thought to be possible because of the presence of a sub-
clinical infection.
The response in group C was thus intermediate between
groups A and B in terms of timing of bone formation,
calcification and bridging of gap.
The study attempted to use CT (Dentascan) as a monitor
of bone remodeling and is in accordance with many reports
on the use of CT for subtle bone changes in the jaws. There
was however, no evidence of bone formation at 4 weeks in
the few CT scans that were taken.
The Scintigraphic assessment with
99m
Tc-MDP was
done in four patients between 2 and 4 months post-opera-
tively. The perfusion, blood pool and delayed images were
obtained in all patients. The delayed images revealed
increased tracer uptake indicating a well perfused graft
with no evidence of its necrosis. The sites showed
increased osteoblastic activity as a sign of reparative pro-
cess [27].
Conclusions
DFDBA when used for the obliteration of osseous defects
bypasses the phase of obligatory resorption and shows
early evidence of new bone formation. HA when used for
obliteration of osseous defects shows (a) delayed first
evidence of bone formation as compared to the decalcified
freeze dried bone matrix allograft and (b) it undergoes
resorption but takes a long period to resorb completely and
be replaced by bone. The radiograph assessment score over
time frame indicates early bone formation with DFDBA
and the combination of DFDBA and HA than HA alone.
Dentascan offers three-dimensional visualization of the jaw
bones. In cases where we did Dentascan, no evidence of
bone formation was seen although in one case appearance
Fig. 5 Scintigraphy done at 4 weeks after surgery showing increased
uptake of
m99
Tc at surgical site representing new bone formation at
grafted site
J. Maxillofac. Oral Surg. (July-Sept 2010) 9(3):236–240 239
123
of mild amount of trabeculae formation was seen, but a
statistical conclusion could not be drawn because of the
small sample size and high cost of the investigation. Bone
Scintigraphy assessment results showed increased osteo-
blastic activity and viability of the graft, however, both
Dentascan and Bone Scintigraphy are very expensive
investigative tools and not affordable by most patients who
seek treatment at our institution. More recent and newer
investigative tools were not used due to their unavailability
at my institution.
A prospective randomized controlled trial with a large
sample size and over a long period, is however, warranted
to validate these observations more conclusively.
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