Keywords bovine - deproteinized - demineralized - osteoconductive - osteoinductive - bone filler
- innovation
Introduction
The demand for bone graft substitutes in implant treatment has increased a great deal
for the past decades. Human bone-derived particles such as freeze-dried bone allograft
(FDBA) have been used as osteoconductive particles while demineralized FDBA (DFDBA) has been applied due to its osteoinductive properties.[1 ] However, human bone products processed by tissue bank has typical problem of lack
in donor supply. Furthermore, allogenic bone grafts have been associated with risk
of disease transmission from donors to recipients.[2 ] Consequently, bone substitutes materials have been increasingly popular and, in
this respect, deproteinized bovine bone mineral (DBBM) particles is the most widely used bone substitute in implant and periodontal
surgeries.[3 ]
The DBBM particle is purely inorganic that is considered osteoconductive and exhibits
extremely slow degradation which may reduce the bone regeneration capacity and bone-implant
contact area.[4 ]
[5 ] Limitation of DBBM particle has made way to the utilization of other forms of bovine
bone particles, that is, freeze-dried bovine bone particles (FDBX) and demineralized
freeze-dried bovine bone particle (DFDBX) recapitulating the processing in allograft
production.[6 ] Despite concerns over the potential xenogeneic response upon its application, these
organic forms of xenograft material is proven to be nontoxic and nonimmunogenic in
in vitro and in vivo studies.[7 ]
[8 ]
[9 ] Much similar to human FDBA, the bovine FDBX retains inorganic and organic components
which make it osteoconductive and have some amount of osteogenic growth factors. Furthermore,
it undergoes complete degradation to support bone regeneration.[10 ] On the other hand, DFDBX is purely organic believed to have osteoinductive properties
as it release various osteogenic growth factors such as bone morphogenetic proteins
(BMPs) and transforming growth factor (TGF)-β. However, DFDBX particle has poor structural
strength as it is devoid of inorganic components.[11 ]
Taking into consideration the different biological and mechanical properties of the
three types of bovine bone particles, we assume that FDBX particles should have bone
healing capacity comparable with that of combined DBBM-DFDBX particles when utilized
for filling of bone defect. This animal study aims to compare the osteogenic processes
and bone forming capacity of mineralized FDBX and combined DBBM-DFDBX particles in
a nonspacemaking, critical size mandibular bone defect.
Material and Methods
This posttest control group design study uses New Zealand white rabbit as the experiment
subject. The animal research procedure was approved by research ethics committee,
Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia (022/HRECC.FODM/I/2019).
The inclusion criteria were 6-month-old, male rabbit weighing 3 to 3.5 kg. Animal
who suffered from wound infection or died before the experiment termination were excluded
from this study. The experimental research was conducted in the Animal Research Laboratory,
Stem Cells Research and Development Center, Universitas Airlangga, Surabaya, Indonesia.
Graft material used in this study is lyophilized (freeze-dried) bovine bone xenograft
(FDBX), DFDBX, and DBBM with particle size of 150 to 300 μm ([Fig. 1A ]). They were all processed by Tissue Bank, Dr. Soetomo Hospital, Surabaya, Indonesia.
Fig. 1 Bone grafting of critical size defect in rabbit mandible. (A ) the freeze-dried bovine bone xenograft (FDBX), deproteinized bovine bone mineral
(DBBM), and demineralized bovine bone xenograft (DFDBX) particle of 150–300 μm; (B ) location of the critical-size-defect at the ascending ramus of the rabbit mandible;
(C ) the bicortical, 10-mm, round-shaped defect created in the ramus of rabbit mandible
prior to grafting and (D ) after grafting with composite particle; (E ) the picture of rabbit mandible after euthanasia showing the margins of specimen
collection at the ramus bone (rectangular line delineating margins of resection).
Critical-size defect was created in the ascending ramus of mandible ([Fig. 1B ]) of 45 rabbits which were randomly divided into three groups. In the FDBX group
the bone defects were filled with FDBX particles, in the composite group with mixture
of DBBM and DFDBX particles at 1:1 weight ratio while in the Control group the defects
were left unfilled. The surgery was blindly performed by three operators. Two, 4,
and 8 weeks after bone grafting surgery five rabbits from each group were sacrificed
to collect specimens for histology and immunohistochemistry analysis.
Processing of Bovine Bone Particles
The bovine femoral bone was washed in pressurized lavage with distilled water to remove
blood and marrow. Pasteurization was done in water bath shaker at 60°C for 5 hours
followed by dissection to remove residual fat and soft tissue. The bone was cut into
smaller pieces and then underwent second cleaning, started with distilled water followed
by hydrogen peroxide solution until the bone turned white. Following washing with
distilled water, the bone was immersed in hexane solution to remove the residual fat
from marrow cavity. The bone chips were then kept in deep freezer for 24 hours followed
by dry heating until the water content was less than 10%. The bone chips were placed
in sterile vials and kept in sealed plastic pouch for sterilization process.
In order to have demineralized graft particles (DFDBX) a demineralization procedure
is performed by immersing the bone chips in 0.1% hydrogen chloride solution until
soft consistency was achieved, thereafter the bone chips were again washed with normal
saline solution until the pH of 7 was reached. The soft bone pieces were then cut
up into smaller chips, ground, and filtered to obtain particle size 150 to 300 μm.
The freeze-drying procedure was performed as previously described above. Finally,
particles were placed in sterile vials and kept in double-layer plastic packaging
prior to sterilization by gamma ray irradiation.
Surgical Procedure
Critical size defect model used in this study was a bicortical, 10-mm round-shaped
hole created with trephine bur at the angle of rabbit's mandible as shown above ([Fig. 1B ]). The rabbits were anesthetized by intramuscular injection of ketamine HCl at a
dose of 20 mg per kg body weight followed with xylazine 3 mg per 3 kg body weight.
After hair shaving and disinfection over the right mandible, skin incision and dissection
of the subcutaneous tissue were done until inferior border of the right mandible was
reached. Under copious saline irrigation a round shape defect was created with trephine
bur as described above ([Fig. 1C ]). The drilling was started from the buccal cortical bone further advanced medially
and stopped once the lingual cortical plate was breached in order to keep the lingual
periosteum intact. The defects in the treatment groups were randomly filled up with
FDBX or composite (mixed DBBM/DFDBX) particles ([Fig. 1D ]), whereas in the control group, the defects were not filled with any bone graft
materials. The buccal periosteum was then repositioned and stitched firmly to the
lingual periosteum to support the stability of the graft particles in the bony defects.
The overlying tissue was closed layer-by-layer with absorbable 4-0 suture material
and the skin with silk 3-0 suture.
At the end of second, fourth, and eighth week post-graft implantation, five rabbits
from each group were sacrificed to collect specimens for tissue analysis. The procedure
of specimen collection was conducted as follows. The rabbits were euthanized with
xylazine injection followed by exsanguination. The animal's death was confirmed with
cessation of circulation. The skin overlying the mandible is dissected and the mandible
removed from its articulation with the skull. Bicortical resection of the respective
alveolar bone was carried out along with 1 to 2 mm surrounding bone ([Fig. 1E ]). The specimens were soaked in 10% buffered formaldehyde solution for at least 3
days before further processing procedure.
Histology Examination
The specimens were decalcified with 10% ethylenediaminetetraacetic acid until decalcification
completion was confirmed with prick test. The decalcified specimens were embedded
into paraffin block, slicing done at 4 μm thick, which then deparaffinized with xylene,
rehydrated in 100% alcohol, and washed in distilled water. The slides were then stained
with hematoxylin and eosin.[12 ]
The findings from histology examination were used for quantitative assessment of osteoclast
and osteoblast, semiquantitative assessment of bone healing, and histomorphometry
of trabecular area. The osteoblasts and osteoclasts assessment were randomly performed
around the graft throughout the defect over 10 observation fields. Osteoblasts and
osteoclasts were designated as hexagonal-shaped cells and multinucleated cells, respectively.
The cell counting was done blindly by two randomly assigned examiners using digital-aid
cell counting.
The semiquantitative assessment of bone healing at 4 and 8 weeks were performed with
the use of modified histological scoring system.[13 ] Briefly, the scoring system ranging from “1” to “4” were designated to the proportion
of various callus tissue which were fibrous tissue, hyaline cartilage, woven bone,
and trabecular bone found in a sample. The distribution of scores in each group at
one time point was summed and divided by 5 to obtain mean score of bone healing in
each group. Finally, histomorphometry assessment of bone formation was conducted by
measuring the trabecular area in the defect at 4 and 8 weeks. All of the examination
was performed with light microscope at magnification of ×40 and ×100, while the cell
counting and measurements were done manually by two blinded persons aided by image
software ImageRaster version 3.
Immunohistochemistry Examination
Samples for immunohistochemical staining were incubated in 3% peroxide acid for 30 minutes
to block endogenous peroxidase, soaked in 0.025% trypsin-phosphate buffer saline for
6 minutes, and finally washed with distilled water three times for 2 minutes. Samples
were stained for 30 minutes with mouse monoclonal anti-rabbit Runx2, receptor activator
of NF-kB ligand (RANKL), and osteoprotegerin (OPG) antibodies (Santa Cruz Biotechnology
Inc., USA), mouse monoclonal anti-rabbit osteocalcin, collagen-I, and alkaline phosphatase
(ALP) (Novus Biological, USA). The samples were then immersed in secondary antibody
Polytek HRP Anti-Rabbit Polymerized (Syntec Laboratories, USA) for 30 minutes at room temperature. Finally, the samples
were soaked in substrate DAB Chromogen ACB002 mixed with DAB Substrate High Contrast ACU005 for 5 minutes. After washing three times in phosphate-buffered saline, bluing reagent (BRT 125) was applied for 5 minutes which was directly washed in distilled water.
Finally, the slides were cleaned with xylene and followed by mounting for examination with light microscope (BX-41 model, Olympus, Japan) using digital
camera (DP-70 model, Olympus). The positive intracellular protein expression of collagen-I,
ALP, osteocalcin, RANKL, and OPG were indicated by brown-stained cytoplasm of osteoblast,
except for Runx2 expression which was shown by brown staining of osteoblast's nuclei.
The data obtained from this method was the total number of osteoblast positively stained
with the respective antibodies. The counting was done manually on 20 visual fields
in each sample performed by two randomly assigned examiners. The data was presented
as mean number of positively stained cells from five replications. The data of osteoblastic
expressions of Runx2, collagen-I, ALP, and osteocalcin were used to represent the
osteogenic differentiation and osteoblastic maturation processes, while osteoblast
expression of RANKL and OPG evaluate the process of osteoclastic differentiation during
bone healing.
Statistical Analysis
Statistical analysis was performed with software package SPSS version 17 (IBM Inc.).
Data from each experiment group was statistically analyzed with the assumption of
homogeneity of variances and normal distribution of errors being tested for the variables
examined. One-way analysis of variance followed by Tukey's Honest Significant Difference
multiple comparison test and Kruskal–Wallis followed by Dunn's post-test were used
for quantitative and semiquantitative data, respectively, with significance set at
p -value < 0.05.
Results
Microscopy of Bone Defect Healing at 2 and 4 Weeks Post-Bone Grafting
The microscopy of histology at 2 weeks showed that the defects in the control group
were filled with dense connective tissue and hyaline cartilaginous tissue, whereas
in the FDBX and composite groups, graft materials in central zones were surrounded
by dense connective tissue across the defect. At 4 weeks postimplantation, the control
group showed bone trabeculae along the bone edges with dense connective tissue in
the central zone of the defect. The central zone in the FDBX group showed residual
graft materials which were gradually replaced by woven bone. The composite group,
on the other hand, revealed that the residual graft materials were not replaced by
bone tissue, rather, they were surrounded by mineralized tissue in the form of woven
bone ([Fig. 2 ]).
Fig. 2 Histology of bone defect healing 2 and 4 weeks after bone grafting. (A , D ) Control groups showing mineralized callus adjacent to bone edges of the defects.
(B , E ) Freeze-dried bovine bone xenograft (FDBX) group at 2 weeks showed grafts materials
surrounded by dense connective tissue which were replaced by marked bone formation
at 4 weeks. (C , F ) Composite groups at 2 weeks showed graft materials in the dense mesenchymal tissue,
at 4 weeks woven bone formed surrounding the residual graft materials (yellow arrow
pointing to residual graft material, black arrows to newly formed bone, hematoxylin
and eosin [H&E] staining, ×40 magnification).
Quantitative Assessment of Osteoclast and Osteoblast
The data from histology quantitative assessment show that the mean number of osteoblast
in the FDBX and composite group was statistically comparable to each other but they
were both significantly higher than the control group at 2, 4, and 8 weeks. The mean
number of osteoclast in the control group is, on the other hand, significantly higher
than the FDBX and composite group (p < 0.05). It was also shown that there were inverse relationships between osteoblast
and osteoclast count in all the groups at 2, 4, and 8 weeks. While mean osteoblast
number increase and osteoclast decrease in majority of groups, the osteoclast number
in the control groups was seen to increase with time ([Fig. 3 ]).
Fig. 3 Quantitative assessment of osteoclast and osteoblast at 2, 4, and 8 weeks (the arrows
in black point to osteoclast, red to osteoblast, mature bone is either bone edge [control
group] or residual graft materials [in grafted groups], hematoxylin and eosin [H&E]
staining, ×400 magnification). The mean number of osteoclast in the control group
are consistently higher than the freeze-dried bovine bone xenograft (FDBX) and composite
group, while the mean number of osteoblast in the FDBX and composite groups are significantly
higher than in the control group (p < 0.05). There exists an interdependent relationship between the number of osteoblast
and osteoclast at 2, 4, and 8 weeks. While osteoblast count is consistently increased
and osteoclast decreased from early to later stage of healing, the osteoclast count
in the control groups is seen to be consistently high at all time points.
Osteoblastic Differentiation and Maturation
The result of immunohistochemistry analysis of osteoblastic differentiation markers
shows that the mean expression of RUNX2, ALP, collagen-I, and osteocalcin were still
increasing until 8 weeks in all groups. It was also shown that all the osteoblastic
markers in the FDBX and composite groups were significantly higher (p = 0.001) than the control group at the three time points of observation. While all
osteoblastic differentiation and maturation markers in the composite group were consistently
higher than those in the FDBX group at all time points there was no significant difference
(p < 0.05) between the two groups ([Fig. 4 ]).
Fig. 4 Expression of osteoblastic differentiation and maturation markers. It is shown that
mean expression of (A ) RUNX2, (B ) collagen-type I, (C ) alkaline phosphatase, and (D ) osteocalcin constantly increase until 8 weeks in all groups. The mean expression
of those osteoblastic markers in the freeze-dried bovine bone xenograft (FDBX) and
composite group is significantly higher (p = 0.001) than the control group in all time points; however, no statistical difference
(p < 0.05) is found in all the above markers between the FDBX and composite groups throughout
the observation periods.
Osteoclastic Differentiation
The immunostaining results exhibit that mean osteoblastic RANKL expression in the
control group increases until 8 weeks of healing as opposed to those in the FDBX and
composite groups which gradually decrease with time. The mean expression of RANKL
in the control group was found significantly higher than the FDBX and composite group
at all time points. The mean expression of OPG tends to increase until 8 weeks, except
for the FDBX group which exhibits relatively constant expression. While the osteoblast
expression of OPG in the composite group was higher than the FDBX group specifically
in the last 4 weeks of healing, no significant difference was observed (p > 0.05) between the composite and FDBX groups ([Fig. 5 ]). The data also shows that OPG/RANKL expression ratio in the composite group was
consistently the highest among all groups and at all time points.
Fig. 5 Expression of osteoclastic differentiation markers. (A ) The mean expression of osteoclast RANKL in freeze-dried bovine bone xenograft (FDBX)
and composite groups decreases from 2 through 8 weeks of healing as opposed to that
in the control groups which show gradual increase with time. The mean expression of
osteoclastic RANKL in the control groups is significantly higher (p < 0.05) than the FDBX and composite group at 4 and 8 weeks of healing. (B ) The mean osteoblast expression of osteoprotegerin (OPG) protein increases gradually
through the observation periods, no significant difference is observed in OPG expressions
between the composite and FDBX groups (p > 0.05) during the same periods of healing. (C ) The OPG/RANKL ratio in the composite groups is much higher, > twofold those in the
FDBX group specifically at 4 and 8 weeks indicating more osteoblastic activities in
the composite group.
Semiquantitative Assessment of Bone Healing
The histology comparative assessment of bone healing shows a clearly distinct level
of tissue maturation in the control group compared to the FDBX and composite group
at 4 weeks of healing. Fibrous tissue and hyaline cartilage were the major tissues
in the control group with small amount of bone tissue at the periphery of the defect,
while woven bone were more evident in the FDBX and composite group. At 8 weeks the
bone tissue in the control and FDBX group was more obvious yet still much less compared
with the composite group. Semiquantitative assessment of bone healing showed that
the bone healing scores in the composite and FDBX groups were significantly higher
than the control group at 4 and 8 weeks. In addition, the healing scores in the composite
group were significantly higher (p < 0.05) compared to the FDBX group at all time points ([Fig. 6 ]).
Fig. 6 Semiquantitative assessment of bone healing in the control, freeze-dried bovine bone
xenograft (FDBX), and composite group at 4 and 8 weeks of healing. The mean score
of bone healing in the composite and FDBX groups is consistently higher than the control
groups at 4 and 8 weeks; furthermore, the healing scores in the composite groups are
found to be significantly higher (p < 0.05) compared to the FDBX groups at those two time points (note: blue arrows pointing
to connective tissue, yellow to hyaline cartilage, red to woven bone, black to trabecular
bone, hematoxylin and eosin [H&E] staining with ×100 magnification).
Assessment of Trabecular Area (Histomorphometry)
The comparative histological assessment of healing at low magnification showed distinct
streaky pattern of bony tissue formation in the control and FDBX group compared to
large, interconnecting trabecular area seen in the composite group at 4 weeks and
specifically at 8 weeks. The result of histomorphometry revealed that the mean trabecular
area in the composite group increased from 4 to 8 weeks; whereas those in the control
and FDBX group remained constant with time. The mean trabecular areas in the composite
group were significantly higher (p < 0.05) than the FDBX and control groups at all time points ([Fig. 7 ]).
Fig. 7 Assessment of trabecular area at 4 and 8 weeks of healing. (Top ) Histology of bone healing at 8 weeks showing woven and trabecular area seen in (A ) the control and (B ) freeze-dried bovine bone xenograft (FDBX) groups, as opposed to interconnecting
trabecular area incorporating with the host bone in (C ) the composite groups (black arrows pointing to trabecular area, yellow arrows indicating
host bone, hematoxylin and eosin [H&E] staining, ×100 magnification). (Bottom ) The mean trabecular areas in the composite groups are significantly higher (p < 0.05) than the FDBX and control groups at 4 and 8 weeks. The trabecular area in
the composite groups increases from 4 to 8 weeks; whereas those in the control and
FDBX group are found to remain constant with time.
Discussion
Autogenous or allogeneic bone particulate remains the gold standard bone graft for
alveolar bone augmentation associated with dental implant treatment. Limitation in
source and availability of these bone grafts have led to the utilization of xenogeneic
bovine bone because of its similarity to human bone in terms of porosities, biological,
and physical properties. The most widely used bovine bone substitutes were DBBM particles
which has good osteoconductive properties and high mechanical stability.[14 ] However, it was associated with very slow degradation that might lower its bone
regeneration capacity.[4 ] In order to overcome DBBM limitation, mineralized FDBX and DFDBXs were developed
recapitulating the concept and processing of human FDBA and DFDBA.[15 ] Despite the concern over the potential xenogeneic response upon its clinical application,
this organic forms of xenograft material did not show abnormal immune response in
few in vitro
[7 ]
[16 ]
[17 ]
[18 ] and in vivo studies[7 ]
[9 ]
[19 ] as well as in a clinical study.[8 ]
The FDBX particles were composed of organic and inorganic structures in the form of
mineralized extracellular matrix which serve as osteoconductor and osteoinductor.
The chemical removal of fat and tissue as well as lyophilization procedures eliminate
the antigenicity and pathogenicity of FDBX particle; however, such treatment potentially
lower the osteogenic growth factors concentration thus reducing the osteoinductive
capability of this particle.[10 ]
The demineralized bovine bone, or DFDBX, particles were processed with acid decalcification
which eliminates the mineral phase while preserving the organic components. Much similar
to DFDBA, it was known to retain varying concentration of BMPs and TGF-β, the two
most important osteoinductive growth factors.[11 ] However, demineralized bone particles have limited osteoconductive properties and,
practically, have no mechanical strength.[20 ]
Based on the above description, we hypothesized that mineralized FDBX particles and
combined DBBM/DFDBX particles should have comparable bone regeneration capacity for
grafting of bone defects. We evaluate the cellular and biomolecular events in response
to grafting, the quality of bone healing, and the quantity of bone formation at various
bone regeneration and remodeling stages.
The graft materials, clearly seen either in the FDBX or composite groups in 2 weeks,
presented different histological features in 4 weeks of healing. The FDBX graft materials
were replaced with woven bone; however, the composite graft materials remained and
were surrounded by mineralized tissue ([Fig. 2 ]). There is a principle difference in bone healing events between mineralized bone
graft and hydroxyapatite (HA) crystalline particles upon implantation. Mineralized
graft undergo osteoclastic bone resorption followed by osteoblastic bone formation,
the process referred to as “creeping substitution” while the amount of residual HA
in DBBM phase of composite graft was more stable due to higher resistance to osteoclastic
resorption.[21 ] The finding in this study was consistent with an experiment in rabbit model showing
that residual material in defects filled with DBBM was significantly higher than that
observed in defects filled with biphasic calcium phosphate, indicating that DBBM resorption
is very slow.[22 ]
Quantitative assessment of osteoclast and osteoblast in this study were to evaluate
the bone formation and remodeling events in the defects regenerated with different
types of bovine bone particles. The consistently higher osteoclast number in the control
group ([Fig. 3A ]) reflects active resorption activities throughout the observed time points. This
finding was in accordance with the result of a study of fracture sheep model that
exhibited high osteoclasts number and density at various stages of fracture healing.
At the early stage, osteoclasts were active and absorbed the mineralized endosteal
bone to recanalize the medullary cavity and restore vascularity. At later stage, the
number of osteoclasts was still high as it was responsible for the absorption of woven
callus to form lamellar bone.[23 ] The early-onset osteoblastic bone formation in the grafted groups ([Fig. 3 ]) suppressed osteoclast activities through a modulative osteoblastic expression of
OPG and RANKL.[24 ]
Significant increase in osteoblast number was noted in the FDBX and composite groups;
however, there were no significant difference documented between the two groups. Both
mineralized FDBX and composite DBBM/DFDBX graft particles were considered to have
some amount of osteogenic growth factors contained within the extracellular matrix.
The difference was in the way by which growth factors were released from the graft
matrix. While DFDBX provide more ready-to-use molecules upon macrophages-mediated
matrix degradation, the growth factors in FDBX were embedded in mineralized matrix
which need more time to be utilized by the tissue.[25 ]
The biomolecular events in osteoblastic differentiation and maturation in the defect
were evaluated by immunohistochemistry. Significant increase in the expressions of
Runx2, collagen-I, ALP, and osteocalcin documented in the FDBX and composite group
were consistent with the significant increase in osteoblast number in both the grafted
groups. This increase in these markers may be associated with increased osteoblastic
differentiation of mesenchymal stem cells (MSCs) and osteoprogenitor cells populating
the defects. These undifferentiated cells were recruited either from circulation,
endothelium, or from the surrounding bone marrow by growth factors such as BMPs or
BMPs released by the organic matrix of the bone granules.[26 ]
Apart from chemokine effect, the release of BMP2 also increased RUNX2 transcription
activity, which induces gene expression related to osteoblast differentiation and
improves bone formation. Additionally, TGF-β promotes osteoprogenitor proliferation,
early differentiation, and relation between osteoblastic lineage through MAPK and
Smad2/3 selective pathway leading to induction of collagen-1 expression.[27 ]
In vitro study of MSC osteogenic differentiation reveals maximum ALP expression level until
day 14, followed by increase in osteocalcin and osteopontin expression leading to
calcium-phosphate deposition.[28 ]
This study also reveals that osteoclast number was interdependently related to osteoblast
number in all the groups. The high OPG/RANK expression ratio, specifically in the
composite group, at 4 and 8 weeks indicate that osteoblastic bone formation was the
dominating event during the remodeling phase in this group. This finding may be associated
with osteoblast-osteoclast interaction which occur during bone healing and bone remodeling
process. Many studies suggested that osteoblast modulate monocyte-to-osteoclast differentiation
through OPG and RANKL expressions.[29 ] In the RANKL/RANK/OPG pathway, RANKL binds to RANK as its receptor and eventually
leads to osteoclast precursor maturation. OPG was known as a decoy receptor for RANKL
which prevents RANKL-RANK binding and the following reactions.[30 ]
The bone healing score results in this study were found to be linear with that of
histomorphometry whereby the composite group showing higher amount of bone than the
FDBX group. This result was supported by one study comparing mineralized cancellous
allograft material to a 1:1 weight ratio combination of DFDBA and deproteinized mineralized
bovine bone in bilateral sinus grafts and concluded that resorption and replacement
by new bone occurred more rapidly in the mineralized cancellous allograft material
but that both groups resulted in successful new bone formation.[31 ] Similar study evaluating the effect of a combination of demineralized bone matrix
(DBM) and HA compared with HA alone on osteogenesis in vitro and in vivo concluded that DBM/HA putty showed better osteoinductivity and conductivity over
HA alone.[32 ]
Although FDBX was considered as having some level of osteoinductivity, composite DBBM/DFDBX
granules obviously had higher osteogenic induction capacity. In addition to biologic
activities through the release of BMPs from DFDBX granules, the DBBM granules also
had osteoinductive effect but through a different mechanism from the BMP pathway.
HA substrate increased ALP, collagen-I, and osteocalcin gene expressions in uncommitted
C3H10T1/2 culture without upregulation of core binding factor 1 (Cbfa1, Runx2).[33 ]
[34 ] As such, strategy of combining DBBM and DFDBX particles not only would increase
osteoconductivity but also osteoinductive properties through biphasic osteoblastic
differentiation effects.
From the result of this study it may be concluded that, within the limitation of this
study, combined DFDBX-DBBM granules had better bone regeneration capacity than mineralized
freeze-dried bovine bone (FDBX) granules for filling of bone defect. This might be
due to better osteoconductivity and osteoinductivity together with higher mechanical
stability. Further study is required to validate bone regenerative efficacy in larger
defects and with various ratios combination.
