Assessment of Healing in Calvarial Bone Defect by Allogenic Demineralized Bone Matrix and Adipose Derived Stem Cells

INTRODUCTION
The Craniomaxillofacial surgeons are facing
frequently critical sized calvarial bone defects that
present a challenge for reconstruction [1]. It can
result secondary to congenital or acquired causes
like; infection, trauma, post tumor excision or
deforming skeletal diseases [2].
Multiple treatment options are available to solve
this problem. It can vary from bone grafts, bone
substitutes or bone transport methods [2]. But till
now the golden standard is the autograft. They
offer minimum immunological rejection, complete
histocompatibility and can provide the best osteoinductive,
osteoconductive and osteogenic prop-
353
erties [3]. The limitations in using autografts are
the limited graft availability, bone resorption and
the need for an additional surgery with resulting
donor site morbidity [4].
Bone allograft is another option. It is obtained
from a cadaver of the same species. It has osteoinductive
ability by releasing bone morphogenic
proteins (BMPs). It has also osteoconductive properties
but lacks the osteogenic properties due to
the absence of viable cells [5]. Despite its wide
availability in various shapes and sizes without
sacrificing host structures and extra donor morbidity.
It fails to meet the reconstruction needs due to
the difficult preservation and possible infection
transmission [6,7].
A further step forward in bone reconstruction
was the bone tissue engineering (BTE). Tissue
engineering is to restore damaged or degenerated
tissues with a functional living construct from cell
development [8]. It is a new field in the bone
reconstruction armamentarium that builds up a
new thinking for bone replacement without extraneed
for the traditional bone grafting surgeries
with its complications [9].
Basically, a triad of stem cells, scaffold and
growth factors should interact to regenerate a new
bone reconstruct [9,10]. Multiple studies in this
field were greatly described to synthetize a novel
tissue engineered bone with multiple designs by
seeding diverse types of stem cells on different
scaffolds.
PATIENTS AND METHODS
This study was done between October 2014
and December 2017. It was conducted at The
Medical Research Center associated with Ain
Shams' Faculty of Medicine and approved from
the Research Ethics Committee (REC) of Faculty
of Medicine, Ain Shams University (No: FMASU
1969/2014).
A total of 42 Male albino rats were used after
approval of CARE (Committee on Animal Research
and Ethics), Ain Shams University, Faculty of
Medicine. It was divided into two models: Model
1: (Donor rats for adipose derived stem cells); 10
rats of young (5-6 weeks) male albino rats were
used. Model 2: 32 adult (6-7 months) male albino
rats weighing 260-340 grams were divided into
four groups; Group I (n=8): Control group where
the surgically created calvarial bone defect will be
left without repair; Group II (n=8): Reconstructed
by allogenic DBM without cell seeding; Group III
(n=8): Allogenic DBM seeded by ASCs; Group IV
(n=8): Reconstructed by prolene mesh seeded by
ASCs.
- Preparation of prolene mesh Scaffold:
A prolene mesh sized 30x30cm was cut into
small, symmetrical, circular Segments (scaffolds)
of 8mm diameter by using 8mm biopsy punch (Fig.
1). Each segment was separately sterilized and
stored.
- Preparation of DBM Scaffold:
Rats were anaesthetized using intramuscular
injection of ketamine (1-2 mg/kg) which was maintained
as required. Under sterile conditions, the
surgical incision site (Right parietal area) was
shaved with razor and lubricant then scrubbed by
povidone iodine and 70% alcohol and the rat was
draped by a disposable waterproof drape. A 2cm
incision was made along the sagittal suture, the
skin; musculature and periosteum were dissected
to expose the Right parietal bone. A circular defect
measuring 8mm diameter was then made using the
8mm biopsy punch with care to avoid injury to the
dura matter. The bone harvested from Group I
(control) and Group IV (reconstructed by prolene
mesh and ADSCs) were demineralized and used
as a donor for Group II (reconstructed by DBM
without cell seeding) and III (reconstructed by
DBM with cell seeding) respectively. Demineralization
was done by immediate bone transfer into
a solution of (0.6 N HCL) for 72 hours at 4ºc. The
HCL was changed every 24 hours. The acid was
then removed by washing the bone segments with
distilled water for 8 hours with continuous stirring
[23]. Each segment will be separately stored in
small plastic tubes at minus 70ºc while still immersed
in alcohol (Fig. 2).
In this study, an 8mm biopsy punch is preferred
than trephine burr due to many reasons. It is easier,
354 Vol. 42, No. 2 / Assessment of Healing in Calvarial Bone Defect
accurate, disposable and less expensive especially
when compared with the trephine burr. It also
preserves the extracted bone as a block that can
be demineralized and reused as a scaffold with the
same defect dimension so, no need for hardware
fixation.
- Harvesting inguinal pad of fat:
Two bilateral inguinal folds Incisions were
done. The inguinal bad of fat were carefully dissected,
isolated and immediately transferred to a
sterile petri dish. The wounds are closed with a
4.0 absorbable suture (vicryl) (Fig. 3).
- Tissue processing:
The harvested fat was washed with phosphate
buffered saline (PBS) and cut into pieces of approximately
1-2mm diameter inside a laminar flow.
The tissue will then be rinsed three times in PBS
for 5 minutes.
Isolation of ADSCs:
The minced fat was digested by adding a 0.2%
collagenase type I and vigorous constant shaking
in a water bath shaker for 40 minutes at 37ºC. The
collagenase effect is neutralized by equal volume
of complete culture medium (CCM). The CCM is
reconstructed by adding 500mL DMEM, 65mL
FBS (final conc. 13%), and 1.5% antibiotic and
anti-mycotic mixture Penicillin G (10,000 units
/mL), streptomycin sulfate (10,000mg/mL) and
Amphotericin B (25mcg/mL) in a solution of 0.85%
NaCL (modified by medical research center team
instead of 10% FBS & 1% antibiotic/antimycotic
as described by Lu et al., 2008) [24]. CCM is filtered
through 0.22mm sterile filter unit then divided into
aliquots and stored at 4ºC. Before the experiment,
the aliquots were warmed to 37ºC. The cell suspension
was centrifuged. The formed cell pellet
was re-suspended in a 10ml of CCM to lyse the
red blood cells.
Culture:
Cell pellet will be cultured in a culture flask
25cm2 with CCM in the CO2 Incubator at 37ºc,
5% CO2 and 100% RH. The medium was replaced
every 72 hours. The non-adherent cells were discarded,
and the adherent cells were washed by
PBS.
Expansion:
The cell expansion was followed by the inverted
microscope. The cells were harvested at 80 to 90%
confluence at the 12th day from passage zero (Fig.
4).
Egypt, J. Plast. Reconstr. Surg., July 2018 355
The cultured cells were detached from the
culture flasks with 0.25% trypsin-EDTA and microscrubber.
- In vivo implantation:
The scaffolds (DBM and prolene mesh) wet by
DMEM solution were seeded by a 3x106 cells.
Group III with surgically created calvarial bone
defect was repaired by allogenic DBM with cell
seeding. The dimensions of the defect and the
DBM are equal so, press fit is enough for fixation.
Group IV was reconstructed by prolene mesh seeded
with ADSCs (Fig. 5).
Postoperatively, Analgesics and antibiotics were
administered. Wounds were followed daily for
signs of inflammation, disruption, hemorrhage and
exudation. Euthanasia was done 8 weeks post
implantation.
- Biopsy harvest:
The biopsies were taken as blocks containing
the reconstructed defect and a rim of the surrounding
calvarium.
- Evaluation:
In this study, 3-objectively based analytical
steps was used to evaluate the results; Gross evaluation
at autopsy, Radiological assessment by 3D
CT scans with software analysis for the surface
area of the newly formed bone (ImageJ 1.47v,
National Institute of Health, USA) and histological
evaluation.
Gross evaluation at autopsy:
The reconstructed defects were exposed and
examined. there no universal guidelines for data
analysis in a well settled rating scale. So, in this
current work, a proposal for a scoring system for
gross evaluation of healing at autopsy is offered.
It includes the four main parameters which is
subdivided into multiple rating points covering the
entire range or shades of healing possibility in a
numerical scale in which the minimum is 4; indicates
the worst healing and the maximum is 11;
the best. This score was extremely helpful in this
study (Table 1).
Table (1): Gross evaluation score for evaluation of bone
healing at autopsy.
Gross evaluation score at autopsy
123
12
123
123
Palpation:
No healing
Soft healing
Hard healing
Transillumination:
Trans-illuminant
Non-trans-illuminant
Mobility with pressure:
Mobile
Minimal mobility
No mobility
Integration with the surrounding bone:
Absent
Incomplete
Complete
Score: ( /11) Minimum 4
Maximum 11
Radiological Evaluation:
A 3D reconstructed CT Scanning was performed
for the heads of all study groups immediately after
euthanasia using a CT scanner (SOMATOM®
Definition Flash 128 Dual source, Siemens Medical
Solutions, Germany). The relation between the
surface area of the newly formed bone to the surface
area of the surgically created defect was analyzed
using a software (ImageJ 1.47v, National Institute
of Health, USA) (Fig. 6).
Histological evaluation:
The specimens were fixed in 10% formaldehyde
for 2 days, decalcified in 25% H2SO4 for 10 days
and mounted in paraffin. Serial sections 5mm-thick
were cut by a microtome in the sagittal plane and
including the defect and rim of the surrounding
calvarium then stained with hematoxylin and eosin
and examined by light microscopy to evaluate the
qualitative bone healing.
Fig. (1): The preparation of prolene mesh; Prolene mesh was cut by punch
into multiple small pieces (scaffolds).
356 Vol. 42, No. 2 / Assessment of Healing in Calvarial Bone Defect
Fig. (2): Harvest of an 8mm circular bone from rats calvaria; (a) Circular bone excised by 8mm biopsy punch. (b) Dura intact
after bone harvest. (c) Harvested bone. (d) The Demineralized bone scaffolds are immersed in alcohol.
Fig. (4): Cell Expansion (X100) under inverted microscope; (a) Passage zero; the cells appearance under the inverted microscope
(b) Day 3: The cells appeared as spindle shaped cells (c) Day 5: The cells are more confluent.
(A) (B) (C)
Fig. (3): Harvest of inguinal pad of fat: (a) Both inguinal folds are incised and dissected with isolation of
the inguinal pad of fat. (b) The harvested inguinal pad of fat collected in a petri dish.
(A) (B)
(A) (B)
(C) (D)
Egypt, J. Plast. Reconstr. Surg., July 2018 357
RESULTS
The rat's survival at all studied groups (Model
2) was uneventful apart from Four mortalities
during surgical creation of the critical size defects
(1 in group I, 1 in group II and 2 in group IV).
Theses rats were replaced.
Statistical methods:
The collected data were coded, tabulated, and
statistically analyzed using IBM SPSS statistics
(Statistical Package for Social Sciences) software
version 22.0, IBM Corp., Chicago, USA, 2013.
Descriptive statistics were done for quantitative
data as minimum & maximum of the range as well
as means (standard deviation) for quantitative
normally distributed data, while it was done for
qualitative data as number and percentage.
Inferential analyses were done for quantitative
variables using independent t-test in cases of two
independent groups with normally distributed data.
In qualitative data, inferential analyses for independent
variables were done using Fisher's Exact
test for differences between proportions with small
expected numbers. The level of significance was
taken at p-value <0.050 is significant, otherwise
is non-significant.
A- Gross evaluation at autopsy:
Gross evaluation score was highest in group
III (10.5/11), followed by Group II (10/11), then
group IV (9.3/11) and least in group I (2.1/11).
Group III was significantly higher than groups I&
VI (Table 2) (Fig. 7).
Fig. (5): In vivo implantation; (a) Group III: Allogenic DBM seeded with ASCs; (b) Group IV: Prolene
mesh seeded with ADSCs.
(A) (B)
Fig. (6): A- 3D CT scan for group IV. B- The surface area marking of the newly formed bone by ImageJ
1.47v. C- The surface area marking of the defect by ImageJ 1.47v.
(A) (B) (C)
B- Radiological evaluation:
a- Radiological Extent of bone healing:
High grades of bone formation were most frequent
in group-III (62.5% healing of 75-100% of
the defect and 37.5% healing of 50-74%), followed
358 Vol. 42, No. 2 / Assessment of Healing in Calvarial Bone Defect
by group-IV (62.5% healing of 25-49% of the
defect and 37.5% healing of 50-74% of the defect),
then group-II (12.5% healing of 25-49% of the
defect) and least in group-I (no healing). Difference
were significant with all groups (Table 3) (Fig. 8).
Table (2): Comparison between study groups using a gross
evaluation score. Group III is significantly higher.
Findings
Mean ± SD
Range
Group-I
5.1±0.8
4.0-6.0
Group-II
10.0±0.8
9.0-11.0
Group-III
10.5±0.8
9.0-11.0
Group-IV
9.3±1.2
8.0-11.0
Comparison between group-III and other groups
p
Difference:
Mean ± SE
95% CI
Group-I
<0.001*
5.4±0.4
4.5-6.2
Group-II
0.207
0.5±0.4
–0.3-1.3
Group-IV
0.023*
1.3±0.5
0.2-2.3
#Independent t-test.
Difference: Group-III relative to other groups.
CI : Confidence interval.
SD: Standard Variation.
SE: Standard error.
Table (3): Comparison between study groups regarding radiological
extent of bone healing. Group III is significantly
higher.
Extent of Bone
formation
0-24%
25-49%
50-74%
75-100%
Group-I
8 (100.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
Group-II
7 (87.5%)
1 (12.5%)
0 (0.0%)
0 (0.0%)
Group-III
0 (0.0%)
0 (0.0%)
3 (37.5%)
5 (62.5%)
Group-IV
0 (0.0%)
5 (62.5%)
3 (37.5%)
0 (0.0%)
Comparison between group-III and other groups
p
RR (95% CI)
Group-I
<0.001*
–
Group-II
<0.001*
–
Group-IV
<0.006*
–
#Fisher's Exact test.
RR: Relative rate (group-III relative to other groups regarding 75-
100%).
CI: Confidence interval
Fig. (7): Comparison between study groups using a gross
evaluation score.
12
11
10
9
8
7
6
5
4
Gross evaluation score (4.0-11.0)
Group I Group II Group III Group IV
5.1
10.0 10.5
9.3
b- Radiological bridging:
High grades of radiological bridging the defect
length with new bone were most frequent
in group-III (75% entire length bridging and
25% partial length bridging), followed by group-
IV (37.5% partial length bridging and 62.5%
just formed bone over defect boarders), then
group-II (100% just formed bone over defect
boarders), and least in group-I (50% no healing
and 50% just formed bone over defect boarders).
Difference were significant with all groups (Table
4) (Fig. 9).
Fig. (8): Comparison between study groups regarding radiological
extent of bone healing.
100
90
80
70
60
50
40
30
20
10
0
Group I Group II Group III Group IV
100-75%
74-50%
49-25%
24-1%
100 87.5
12.5
37.5
62.5
62.5
37.5
%
Egypt, J. Plast. Reconstr. Surg., July 2018 359
C- Histological evaluation:
The new bone formation was evident in group-
III, where multiple Islands of new irregular bone
formation over dead necrotic bone that suggest the
presence of the DBM remnants. The bone cells are
seen inside their lacuanae resting over dead bone
with small amount of Fibrous Tissue. In group-IV,
a homogenous wide spaces resembling prolene
mesh was seen and surrounded by fibrous tissue
and newly formed bone. The amount of bone is
more than fibrous tissue. In group-II, 3 zones could
be identified; first zone is Dead bone with necrotic
bone lamellae and absent cells which is sugessting
the prescence of DBM; second zone is junctional
zone that shows a new bone formation where blood
vessels and osteocytes are seen inside their lacuanae;
third zone is the surrounding rim of the calverial
bone. The control group (group-1), showed
minimal islands of bone formation with greater
amount dense fibrous tissue (Fig. 10). Interestingly,
there were no abnormal cellular infiltration or
immune reaction rather than normal cells included
in normal healing process. This result is comparable
to the result reached by Khaled, 2008 [28] and is
considered another prove for the non–immunogenicity
of the allogenic ADSC and for DBM.
Table (4): Comparison between study groups regarding radiological
bridging. Group III is significantly higher.
Bridging
No bone
formation
Just formed
over defect
borders
Partial length
bridging
Entire length
bridging
Group-I
4 (50.0%)
4 (50.0%)
0 (0.0%)
0 (0.0%)
Group-II
0 (0.0%)
8 (100.0%)
0 (0.0%)
0 (0.0%)
Group-III
0 (0.0%)
0 (0.0%)
2 (25.0%)
6 (75.0%)
Group-IV
0 (0.0%)
5 (62.5%)
3 (37.5%)
0 (0.0%)
Comparison between group-III and different group
p
RR
(95% CI)
Group-I
<0.001*
5.000
(1.448-17.271)
Group-II
<0.001*
5.000
(1.448-17.271)
Group-IV
<0.003*
5.000
(1.448-17.271)
#Fisher's Exact test. CI : Confidence interval.
RR: Relative rate (group-III relative to other groups regarding).
Fig. (10): Histological evaluation
(H&E x 400). A- Group
I; showing island of bone formation
(NB) with osteocytes
seen inside lacuanae surrounded
by dense fibrous tissue (FT).
B- Group II; 3 zones; Dead
bone(DB) with necrotic bone
lamellae and absent cells; second
zone is junctional zone
with a new bone formation;
third zone is the surrounding
rim of the calverial bone (CB).
C- Group III; Islands of new
irregular bone formation over
dead necrotic bone. D- Group
IV; Homogenous wide spaces
resembling prolene mesh (PM)
and surrounde by fibrous tissue
and newly formed bone.
(A) (B)
(C) (D)
Fig. (9): Comparison between study groups regarding radiological
bridging.
Entire span
Partial
Borders
None
100
90
80
70
60
50
40
30
20
10
0
Group I Group II Group III Group IV
%
50
50
100 75
25
37.5
62.5
Bone tissue healing was most frequent in group-
III (62.5%), followed by group-IV (25%), fibrous
tissue healing was more evident in group-I (62.5%)
then in group-II (50%). Difference were significant
with groups I& II (Table 5) (Fig. 11).
360 Vol. 42, No. 2 / Assessment of Healing in Calvarial Bone Defect
methods [2]. The autograft is the golden standard,
but, its main drawback is limited donor, extramorbidity
and resorption [4].
Moreover, there is a high demand in reconstructive
field for an already made bone that is available
for urgent and extensive reconstruction where the
needs are more than the autografts can meet. So,
multiple studies were started to synthetize a novel
tissue engineered bone and Researchers in this
field tried to design a new bone tissue regenerate
by seeding diverse types of stem cells on different
scaffolds.
Though the lipoaspirate was considered as a
waste for many years, it is a very rich source for
ADSCS. This type of stem cells is clonogenic i.e.
can form colonies in culture conditions [11,12].
Also, it has an osteogenic potential and surprisingly
Its osteogenicity isn't greatly affected by age factor
in contrast to BMSCs [13]. Furthermore, every
processed 300mL of lipoaspirate can produce between
1x107 and 6x108 ADSCs [14-17] and every
1gm of surgically excised adipose tissue, yield
approximately 5x103 stem cells [18]. Its harvest
and expansion are greatly easier than BMSCs [19-
20]. Also, ADSCs are more superior to BMSCs in
bone regeneration as it could be maintained in
vitro for longer periods with a constant doubling,
more proliferative capacity and lower senescence
[11,12].
Despite the diversity of multiple scaffolding in
the field of BTE, the DBM is considered an ideal
scaffold. It is osteoconductive and non–immunogenic
as the demineralization process exposes the
proteins and other various growth factors, which
are present in the extracellular matrix, to be available
for the host environment and it destroys the
antigenic surface structure of the bone [21]. The
DBM is also osteoconductive [22]. It could be
prepared and preserved easily. It is commercially
available in various ranges of shapes and forms as
such as morselized particles and struts.
In this study, the DBM scaffold is compared to
prolene mesh scaffold which was used by Khaled,
2008 [28] and seeded by BMSCs. The advantages
of this type of scaffolding are the availability, low
cost, resistance to infection, malleability, biocompatibility
and the ability for cell adherence and
support. But its main disadvantages are the nondegradability;
that might affect the mechanical
property of the new tissue regenerate. Also, it is
non osteoconductive nor osteogenic. On the other
hand, the DBM scaffold biocompatible, biodegradable,
non-immunogenic and at the same time,
Table (5): Histological evaluation; Comparison between study
groups regarding type of tissue healing.
Type of tissue
healing
Mostly fibrous
fibrous > bone
bone > fibrous
Group-I
5 (62.5%)
3 (37.5%)
0 (0.0%)
Group-II
4 (50.0%)
4 (50.0%)
0 (0.0%)
Group-III
0 (0.0%)
3 (37.5%)
5 (62.5%)
Group-IV
0 (0.0%)
6 (75.0%)
2 (25.0%)
Comparison between group-III and other group
p
RR
(95% CI)
Group-I
0.006*
3.667
(1.397-9.624)
Group-II
0.007*
3.667
(1.397-9.624)
Group-IV
0.315
2.333
(0.662-8.219)
#Fisher's Exact test. CI : Confidence interval.
RR: Relative rate (group-III relative to other groups regarding complete
filling).
Fig. (11): Histological evaluation; Comparison between study
groups regarding type of tissue healing.
100
90
80
70
60
50
40
30
20
10
0
Group I Group II Group III Group IV
%
Complete Partial None
37.5
62.5
50
50
62.5
37.5
25
75
DISCUSSION
The incidence of bone defects represents a
major burden for the individual, families and the
whole society. In USA, more than 1,600,000 bone
grafts are implanted per year and 6% from this
figure are craniofacial bone grafts [25]. Unfortunately,
in our country, A statistical description for
the magnitude of the critical size calvarial defects
and the need for reconstruction is lacking.
Many reconstructive options are available from
bone grafts, bone substitutes or bone transport
Egypt, J. Plast. Reconstr. Surg., July 2018 361
osteoconductive and to some extent osteogenic
26,27. So, seeding the DBM with ADSCs can cover
nearly all phases of bone healing i.e. The osteogenesis,
osteoconduction and osteoinduction.
The results obtained from this work advocates
the use of Allogenic Demineralized Bone Matrix
and Adipose Derived Stem Cells as a reconstructive
tool for bone regeneration. But further clinical
studies are needed to evaluate the rule of ADSCs
seeded over DBM in the unfavorable conditions
as this study and the previous studies were conducted
in normal healthy conditions, but the clinical
situation is a little bit different as many local factors
as osteomyelitis, osteoradionecrosis and soft tissue
scarring may exist or general factors like smoking,
diabetes, obesity and osteoporosis may also affect
healing. So, this work recommends Further clinical
studies to produce an evidence based clinical application
of bone tissue bioengineering in reconstruction
of craniomaxillofacial bone defects.
Conclusion:
This study presents a beneficial method for
reconstruction of critical size calvarial bone defects
by preparing an already made non-immunogenic
new bone by seeding DBM with ADSCs.