Received: 25 November 2020 Revised: 10 January 2021
DOI: 10.1002/JPER.20-0832
ORIGINAL ARTICLE
Effects of hyaluronic acid and deproteinized bovine bone
mineral with 10% collagen for ridge preservation in
compromised extraction sockets
Jun-Beom Lee1 Seoyoon Chu1
Min-Jung Son1 Jungwon Lee2
In-Chul Rhyu1
1Department of Periodontology and
Dental research Institute, School of
Dentistry, Seoul National University,
Seoul, Republic of Korea
2 One-Stop Specialty Center, Seoul
National University Dental Hospital,
Seoul, Republic of Korea
3Department of Health Policy and
Management, College of Health Science &
Department of Health Care Sciences,
Graduate School & BK21 FOUR R&E
Center for Learning Health Systems,
Korea University, Seoul, Republic of
Korea
Correspondence
Ki-Tae Koo, DDS, MS, PhD, Department of
Periodontology and Dental Research Insti-
tute, School of Dentistry, Seoul National
University, 101, Daehak-ro, Jongno-gu,
Seoul 110-749, Korea.
Jun-Beom Lee and Seoyoon Chu con-
tributed equally to this study and should be
acknowledged as co-first authors.
J Periodontol. 2021;1–12.
Accepted: 11 January 2021
Heithem Ben Amara1 Hyun-Young Song1
Hae-Young Kim3 Ki-Tae Koo1
Abstract
Background: The aim of this study was to evaluate the efficacy of deproteinized
bovine bone mineral with 10% collagen (DBBM-C) soaked with hyaluronic acid
(HA) for ridge preservation in compromised extraction sockets.
Methods: Bilateral third, fourth premolars and first molar were hemisected,
distal roots were extracted, and then combined endodontic periodontal lesion
was induced in the remaining mesial roots. After 4 months, the mesial roots
were extracted and the following four treatments were randomly performed:
Absorbable collagen sponge (ACS), ACS soaked with HA (ACS+HA), ridge
preservation with DBBM-C covered with a collagen membrane (RP), ridge
preservation with DBBM-C mixed with HA and covered with a collagen mem-
brane (RP+HA). Animals were sacrificed at 1 and 3 months following treatment.
Ridge dimensional changes and bone formation were examined using micro-
computed tomography, histology, and histomorphometry.
Results: At 1 month, ridge width was significantly higher in the RP and RP+HA
groups than in the ACS and ACS+HA groups, while the highest proportion of
mineralized bone was observed in ACS+HA group. At 3 months, ridge width
remained significantly higher in the RP and RP+HA groups than in the ACS and
ACS+HA groups. ACS+HA and RP+HA treatments featured the highest pro-
portion of mineralized bone and bone volume density compared with the other
groups. No statistical difference was observed between ACS+HA and RP+HA
treatments.
Conclusions: Ridge preservation with the mixture DBBM-C/HA prevented
dimensional shrinkage and improved bone formation in compromised extrac-
tion sockets at 1 and 3 months.
KEYWORDS
alveolar bone grafting, bone regeneration, growth factors, hyaluronic acid, tooth extraction,
tooth socket
wileyonlinelibrary.com/journal/jper © 2021 American Academy of Periodontology 1
2 LEE et al.
1 INTRODUCTION
Many researchers have extensively investigated tooth
extraction sockets with regard to the healing events
and dimensional changes in conjunction with vari-
ous ridge preservation/augmentation techniques to limit
the changes through histologic, histomorphometric, and
radiographic analysis.1–7 The extraction socket heals fol-
lowing a series of events from the formation of blood
clot to the establishment of highly vascularized provi-
sional matrix to the osteogenesis.5 A systematic review
reported that horizontal bone loss of 29% to 63% and ver-
tical bone loss of 11% to 22% occurred after 6 months fol-
lowing tooth extraction through human re-entry studies.7
The resorption of sockets makes implant surgery prob-
lematic and challenging, and mandates ridge preserva-
tion/augmentation procedure to preserve the ridge con-
tour to secure implant placement.4,6
Most studies, to date, focused solely on fresh extrac-
tion sockets which do not highly reflect clinical reality
where most of the extractions are performed due to peri-
odontal disease or endodontic failures.8 Extraction sock-
ets of periodontally/endodontically compromised teeth
were reported to show different clinical and biological
manifestations compared with fresh sockets.9–11 The pres-
ence of periodontal and endodontic pathology can delay
extraction socket healing by affecting bone formation and
remodeling.10 In addition, compromised extraction sock-
ets may be more prone to show erratic healing resulting
in fibrous scar formation rather than bone formation after
healing period exceeding 12 weeks.12 The chronic pathol-
ogy may provoke marked resorption of hard and soft tissue,
thereafter making implant surgery more challenging, and
necessitating ancillary augmentation procedures.13
Ridge preservation/augmentation with deproteinized
bovine bone mineral (DBBM) and its collagenated form,
namely, DBBM with 10% collagen (DBBM-C) has been
reported to be successful in preserving ridge dimensions in
humans14–18 and animals.1–3,19–21 Systematic reviews have
concluded that socket preservation procedures may help in
reducing the changes in bone dimensions following tooth
extraction, although a loss in width and height inevitably
occurs.4,6 Despite its benefit in attenuating dimensional
changes, the prolonged residence of DBBM was reported to
retard socket healing,22,23 thereby limiting the mechanical
stability of dental implants.24 The use of growth factors has
been suggested to enhance the healing process in extrac-
tion sockets with chronic pathology.25–29 Hyaluronic acid
(HA), a naturally occurring non-sulfated glycosaminogly-
can (GAG) with a high molecular weight, is a major com-
ponent in the extracellular matrix of periodontal tissues
including gingiva, periodontal ligament, alveolar bone,
and cementum.30 Our previous reports showed the pos-
itive effect of HA in infected sockets due to endodontic-
periodontal combined lesion in dogs.25,28,31 Through an
immunohistochemical analysis, the HA was shown to
increase the production of osteocalcin, a marker for bone
formation. However, the use of absorbable collagen sponge
(ACS), which is widely used as a carrier for growth fac-
tors, did not prevent ridge dimensional loss following tooth
extraction in our previous study.25 Therefore, the objec-
tive of the present study was to evaluate the efficacy of
HA with DBBM-C for ridge preservation in compromised
extraction sockets compared with HA with ACS in dogs via
histologic, histomorphometric, and microcomputed tomo-
graphic (micro-CT) analyses.
2 MATERIALS AND METHODS
2.1 Animals
The experiment was conducted from August 2018 to
August 2019. Eight beagle dogs (weight,10 to 15 kg; aged
12 to 24 months) were used in the experiment. During this
experiment, the dogs were kept in separate metal cages
(W900 × D800 × H800 mm) under ambient temperature
23◦ C ± 2◦ C, relative humidity 50% ± 10%, light/dark cycle
12/12, and air-conditioning 12 to 18 changes/hour. A stan-
dard pellet dog-food diet * was fed, and ad libitum access
to water was provided. The present study was performed
according to the Institute of Laboratory Animal Resources,
Seoul National University guidelines (#SNU-180723-5-1).
In addition, modified animal research: reporting in vivo
experiments (ARRIVE) guidelines were consulted in the
reporting of the present research.32
2.2 Surgical protocol
The flow diagram of the experiment is represented in Fig-
ure 1A. Eight beagle dogs were anesthetized intravenously
with 0.1 mg/kg of tiletamine/zolazepam, † 2.3 mg/kg of
xylazine, ‡ and 0.05 mg/kg of atropine sulfate. § To cre-
ate infected extraction sockets in the third, fourth pre-
molars, and the first molar (P3, P4, M1, respectively) in
the mandible, we followed the protocol according to our
previous study.24 Briefly, bilateral P3, P4, and M1 in the
mandible were hemisected, and distal roots were extracted.
In the remaining mesial roots, a #2 carbide round bur
was used to dig a notch in the mesial side of the mesial
* HappyRang, Seoulfeed, Gyeonggi-do, Korea
† Zoletil,Virbac, Carros, France
‡ Rompun, Bayer Korea, Ansan, Korea.
§ Jeil, Daegu, Korea
LEE et al.
F I G U R E 1 Timeline of the study (A) and surgical procedures: B) initial state before experiment; C) baseline, hemisection of teeth, extrac-
tion of distal roots and induction of a combined endodontic-periodontal lesion; D) chronic inflammatory states 4 months after the lesion-
inducing period; E) periapical radiographs showed periodontal and endodontic lesion; and F and G) extraction of mesial roots and treatment
according to groups
root and periodontal lesion was experimentally induced by
ligatures below the cemento-enamel junction using black
silk soaked in Porphyromonas gingivalis (Pg; ATCC 33277;
1 × 109 /mL). In addition, to provoke apical periodontitis
(endodontic lesions), the root pulp was extirpated using an
endodontic file and the canal was filled with absorbable
collagen sponge ** soaked in Pg (ATCC 33277; 1 × 109 /mL)
followed by the crown sealing†† (Figs. 1B and 1C). After
4 months (lesion-inducing period), both lesions were com-
municated in periapical radiographs in all beagle dogs, and
inflammatory signs were observed (Figs. 1D and 1E). Sub-
sequently, the extractions of the remaining mesial roots
in the mandible were performed and after meticulous
debridement, the extraction sockets were allocated to one
of the following treatments according to the block random-
ization design:
(1) ACS: absorbable collagen sponge †† ;
** Teruplug;
R Olympus Terumo Biomaterials Corporation, Tokyo, Japan
†† IRM, Dentsply, DE
3
(2) ACS+HA: ACS soaked with 1% of hyaluronic acid
gel ‡‡ (HA);
(3) RP: ridge preservation with DBBM-C §§ ;
(4) RP+HA: ridge preservation with DBBM-C mixed
with HA.
HA treatment consisted in a viscoelastic preparation of a
highly purified, high molecular weight fraction of sodium
hyaluronate (2.4 to 3.6 million daltons) in phosphate-
buffered saline. In sites where HA applied (ACS+HA
and RP+HA), 0.85 mL of 1% HA and each carrier (ACS
and 0.5 g DBBM-C) were soaked for 15 minutes. Then,
the biomaterial was placed up to the level of the bone
crest and xenogeneic collagen membrane *** was sutured
equigingivally on top of the DBBM-C with a horizontal
mattress suture using resorbable sutures. ††† Full wound
‡‡ Healon, Pharmacia & Upjohn, Uppsala, Sweden
§§ Bio-Oss Collagen,
R Geistlich Pharma, Wolhusen, Switzerland
*** Mucograft
R Seal, Geistlich Pharma, Wolhusen, Switzerland
††† Vicryl,
R Ethicon, Somerville, NJ
4 LEE et al.
closure was obtained in all groups (Figs. 1F and 1G). The
beagle dogs were sacrificed at 1 and 3 months after the
induction of deep anesthesia followed by an overdose of
barbiturate and the mandibles were separated and fixed
immediately immersed in a fixative which consisted of
5% glutaraldehyde and 4% formaldehyde buffered to a
pH of 7.2.
2.3 Micro-CT analysis
After euthanizing the dogs, blocks of each site were pre-
pared and sliced. The blocks were scanned at 13.3-µm res-
olution, 60 kV, and 167 µA using a 0.5-mm aluminum filter
on a three-dimensional (3D) micro-CT instrument ‡‡‡ , and
acquired two-dimensional (2D) images were saved as bmp
files (2240 × 2240 pixels). The sagittal images were reori-
ented in their long axis using the image analysis software.
§§§ To evaluate the dimensional changes, the horizontal
ridge widths were measured according to Fickl et al.33
First, a horizontal line (HL) was drawn perpendicularly to
the long axis of the socket at the highest level between buc-
cal and lingual crest. Then, the horizontal width was mea-
sured at different levels 1, 2, and 3 mm below the HL on
the mesial and distal sockets, respectively. Considering the
size discrepancy of the extraction sockets of premolar and
molar, the width of sockets needed to be compared with
relative changes. For this purpose, the distal socket (spon-
taneously healed site) was considered a reference value
for mesial socket (experimental site). The relative changes
were calculated and expressed as percentages at 1-, 2-, and
3-mm below the HL, which were noted as RW1, RW2, and
RW3, respectively (Fig. 2B).
The 2D region-of-interest (ROI) was defined as a rectan-
gle positioned in the middle portion of the socket from the
highest coronally formed bone, with 2 mm in width and
3 mm in length (Fig. 2A) according to Barros et al.34 For
3D analysis, 2D ROI was drawn every five images on the
sagittal view, and all ROIs were collectively added up to
generate 3D-ROI. After binarization was performed with
the gray scale defined by a density of 50 to 100 for bone
and 100 to 255 for biomaterials,34 the following parame-
ters were calculated by the analyzing software: **** bone
volume/total volume (BV/TV) density and bone surface
(BS)/TV density.
‡‡‡ SkyScan
R 1172, Skyscan, Aartselaar, Belgium
§§§ DataViewer 1.5.2.4 64-bit version, Bruker microCT, Skyscan, Kontich,
Belgium
**** CTAn. v. 1.18.4.0, Bruker microCT, Skyscan, Kontich, Belgium
2.4 Histology and histomorphometric
analysis
The block samples were decalcified in 10% EDTA solution
†††† for 30 days and were routinely embedded in paraf-
fin solution. Subsequently, the paraffin specimens were
sectioned bucco-lingually with a tissue sliding microtome
‡‡‡‡ to obtain sections of 5-µm thickness. Then, a sec-
tion randomly selected per every five sections was stained
with hematoxylin and eosin (H&E) and Masson Trichrome
stain (MT). To assess bone regeneration, the area mea-
surements were performed within the 2D-ROI, same as
the micro-CT analysis (Figs. 2C and 2D), calculating the
percentages of the connective tissue (CT), total mineral-
ized bone (MB), newly formed bone (NFB) and residual
graft particles (RGP). NFB could be discriminated from old
bone by comparison of H&E-stained slides and MT-stained
ones.
2.5 Sample size calculation
The sample size was determined using G*Power software.
The probability of a type I error was set at 0.05 and the
probability of a type II error at 0.20. On the basis of the data
from our previous study on HA application in compro-
mised extraction sockets,31 a difference of 15.74% for mean
histomorphometric proportion of bone marrow with an SD
difference of 3.6% between HA and the control group was
set for the effect size calculation. Total of six samples per
group were obtained.
2.6 Statistical analysis
The Kruskal‒Wallis H test was performed to compare sta-
tistically the significant differences among the four groups
of treatment. If there was a significant difference among
the groups, the post-hoc Tukey method was applied to eval-
uate the intergroup statistical difference. Mann Whitney
U test was used to compare RGP between RP and RP+HA
groups. Finally, to compare all parameters between 1 and
3 months, Wilcoxon signed-rank test was performed. A P
value <0.05 was considered statistically significant. All sta-
tistical analyses were performed using SPSS 22.0. §§§§
†††† Sigma-Aldrich, St. Louis, MO
‡‡‡‡ HM315, Microme, Walldorf, Germany
§§§§ IBM Software, Armonk, NY
LEE et al. 5

F I G U R E 2 Schematic images showing the region of interest and horizontal measurements. A, C, and D) The rectangle was positioned
in the middle region of the socket with dimension of 2 mm in width and 3 mm in length, from the highest point of the bone for the histomor-
phometric and microcomputed tomographic analyses. Histologic section stained with hematoxylin and eosin (C) and with Masson trichrome
(D) at the object magnification of 8×. B) After orientation of mid-sagittal images along the axis of socket, the horizontal line (HL) was drawn
perpendicularly to the axis at the highest level between buccal and lingual crest. Then, the horizontal ridge widths were measured on the
mesial socket (groups 1, 2, 3 and 4) and distal socket at the level of 1, 2, and 3 mm below the horizontal line. Subsequently, to overcome the size
discrepancy of the extraction sockets of premolar and molar, the relative changes of mesial sockets were calculated using the distal sockets as
a reference value, and were denoted as RW1, RW2, and RW3, respectively

3 RESULTS groups and there were no complications reported through-

out the whole experimental period.
3.1 Clinical results

Three months after baseline lesion induction, all remain- 3.2 Histologic findings
ing mesial roots (n = 48) of the mandibular P3, P4, and M1
exhibited typical signs of periodontal inflammation (gin- 3.2.1 At 1 month
gival swelling, redness, and deep pockets; Fig. 1D) and
endodontic infection (periapical radiolucency on periapi- A cortical cap at the entrance of socket in addition to
cal radiographs; Fig. 1E). After tooth extraction and ridge more abundant osteoid with parallel-oriented fibers was
preservation, the healing was achieved uneventfully in all observed in the ACS+HA group. The RP and RP+HA
6 LEE et al.

F I G U R E 3 Representative histologic sections at 1 month: A through D) ACS group at object magnification of 2×, 8×, 8×, and 30× from
the left. ACS group showed retarded healing process in socket. E through H) ACS+HA group at object magnification of 2×, 8×, 8×, and 30×
from the left. A cortical cap was observed at the entrance of the socket. I through L) RP group at object magnification of 2×, 8×, 8×, and 30×
from the left. Preservation of ridge dimension was observed but provisional matrix was filled inside the socket. with delayed healing process.
M through P) RP+HA group at object magnification of 2×, 8×, 8×, and 30× from the left. Preservation of ridge dimension was observed
and osteoid as well as provisional matrix was found inside the socket. The third column (C, G, K, O) was stained with Masson trichrome to
distinguish newly formed bone and other columns were stained with hematoxylin and eosin

group mainly composed of graft materials surrounded by
the provisional matrix. However, the ACS group mainly
featured fibrous granulation tissue (Fig. 3).
3.2.2 At 3 months
All groups showed a cortical cap at the entrance of the
sockets. Inside the sockets of all groups, osteons, NFB
and osteoid were observed. Specifically, the ACS+HA
group showed higher density of MB with more NFB
and osteoid inside the socket than the ACS group. In
the RP and RP+HA groups, NFB was formed around
RGP which seemed somewhat reduced. More osteoid
matrix was observed in the RP group than the RP+HA
group. In addition, highly interconnected trabecular pat-
tern was observed in the ACS+HA and RP+HA groups
(Figure 4).
LEE et al. 7

F I G U R E 4 Representative histologic sections at 3 months: A through D) ACS group at object magnification of 2×, 8×, 8×, and 30× from
the left. Bone marrow was occupied almost in the ridge. E through H) ACS+HA group at object magnification of 2×, 8×, 8×, and 30× from
the left. Substantially increased mineralized bone was formed. I through L) RP group at object magnification of 2×, 8×, 8×, and 30× from the
left. Dimensional shrinkage was counteracted but only immature bone was observed inside the ridge. M through P) RP+HA group at object
magnification of 2×, 8×, 8×, and 30× from the left. Ridge dimension was not only preserved but there was rich amount of mineralized bone
inside the ridge. The third column (C, G, K, O) was stained with Masson trichrome to distinguish newly formed bone and other columns were
stained with hematoxylin and eosin

3.3 Histomorphometric analysis 3.3.1 At 1 month

The data are presented in Table 1 (for the comparison The ACS+HA group showed the highest proportion of MB
between 1 and 3 months see Figure S1 in online Journal compared with the other three groups with statistical sig-
of Periodontology. nificance (62.97% ± 4.39%, P < .05). In the ACS group,
8 LEE et al.

the ROI was mainly composed of CT (61.15% ± 24.36%).

The RP+HA group showed higher MB than the RP group
(46.10% ± 9.73% versus 43.58% ± 6.65%), but there was no
significant difference (P = .57). Similarly, higher NFB was
found in the RP+HA group than the RP group without sig-

2.96 ± 2.03
1.78 ± 0.78
2.75 ± 1.35

3.71 ± 1.39
RGP (%)

nificance (16.82% ± 6.84% versus 5.93% ± 2.46%, P = .24). In

0.456
0.225
addition, the ACS+HA group showed significantly higher
NFB than the ACS group (17.73% ± 10.36% versus 7.14% ±
1.84%, P = .043). The RP and RP+HA groups showed no
significant difference with respect to percentage of RGP.

3.3.2 At 3 months
35.05 ± 10.49a,b,c
61.15 ± 24.36a,b,c
33.18 ± 27.00a

The ACS+HA group showed significantly more MB than

44.34 ± 9.22b
33.08 ± 13.98

10.82 ± 4.96b
17.07 ± 6.79a

12.26 ± 5.55c

the ACS and the RP group (64.69% ± 3.98% versus 45.19%

CT (%)

± 3.06% versus 41.89% ± 5.03%, respectively and P = .001)

0.002
0.145

but not the RP+HA group (59.93% ± 5.44%, P = .405). The

ACS+HA group showed significantly higher NFB than the
ACS group (15.53% ± 2.41% versus 7.53% ± 2.19%, P < .05).
Similarly, the RP+HA group showed significantly more
NFB than the RP group (11.30% ± 3.06% versus 5.57%
± 1.44%, P = .021). When comparing, ACS+HA and the
RP+HA groups, no significant difference was observed
17.73 ± 10.36a,c

b,d

b,d

(P = .103). In terms of RGP, there was no significant dif-

a,c
c,d

5.57 ± 1.44c,d
7.53 ± 2.19a,b
7.14 ± 1.84a,b

16.82 ± 6.84

11.30 ± 3.06
15.53 ± 2.41
5.93 ± 2.46

ference between the RP and the RP+HA group (3.71% ±

NFB (%)

1.39% versus 2.96% ± 2.03%, P = .53).

0.008
0.033

Statistical significance between groups at each month and each column under post-hoc Tukey method.
MB, mineralized bone; NFB, newly formed bone; CT, connective tissue; RGP, residual graft particles.

3.3.3 Comparison between 1 and 3 months

MB increased without significance in the ACS group and

RP+HA group during this period (30.56% and 30.03%,
respectively). However, there were no statistical differ-
*The P value among all groups analyzed by non-parametric Kruskal‒Wallis test.
62.97 ± 4.39a,b,c

b,d
45.19 ± 3.06a,b
a,c
Histomorphometric measurements at 1 and 3 months

41.89 ± 5.03c,d
34.61 ± 13.04a

ences for MB in the ACS+HA and RP group during

b

59.94 ± 5.44
64.69 ± 3.98
46.10 ± 9.73
43.58 ± 6.65

this period (2.73% and 3.88%, respectively). NFB slightly

MB (%)

decreased in the ACS+HA group and RP+HA group with-

0.002
0.024

out statistical difference (12.45% and 32.80%, respectively).

However, the ACS group and RP group were similar in NFB
between two periods. CT decreased in all groups but the
statistical differences were shown only in the ACS group
and ACS+HA group.
ACS+HA

ACS+HA
P value*

P value*
RP+HA

RP+HA

All data are presented as mean ± SD.

ACS

ACS

3.4 Micro-CT analysis

RP

RP

The data are shown in Table 2 (for the comparison

between 1 and 3 months see Figure S2 in online Journal of
Periodontology).
TA B L E 1

3 months
1 month

a,b,c,d
LEE et al. 9

TA B L E 2 Two-dimensional and three-dimensional microcomputed tomographic measurements at 1 and 3 months

BV/TV (%) BS/TV (%) RW1 (mm) RW2 (mm) RW3 (mm)
1 month ACS 24.69 ± 13.08a,b,c 16.67 ± 4.92a,b −4.05 ± 4.96a,b −4.13 ± 3.71a,b 0.27 ± 4.91
ACS+HA 47.54 ± 14.06 a,d
18.44 ± 3.10 c,d
−4.58 ± 5.65 c,d
−3.38 ± 5.46 c,d
−1.34 ± 7.37
RP 33.56 ± 3.49b,d 23.79 ± 2.03a,c 16.92 ± 1.80a,d 9.79 ± 6.57a,d 6.78 ± 6.16
RP+HA 42.43 ± 7.17c
21.33 ± 1.45 b,d
15.91 ± 7.67 b,c
6.15 ± 3.72b,c
8.58 ± 2.42
P value* 0.034 0.032 0.006 0.012 0.084
3 months ACS 36.04 ± 10.39a,b 18.62 ± 4.12a,b,c −1.92 ± 7.21a,c −2.02 ± 3.71a,b −1.59 ± 2.64
ACS+HA 53.29 ± 7.39a,c 22.73 ± 3.64a −3.74 ± 7.48b,d −0.31 ± 5.46c,d −0.70 ± 3.09
RP 38.23 ± 7.98 c
24.73 ± 5.25 b
11.09 ± 4.93 a,d
8.94 ± 8.48 a,c
3.91 ± 7.75
RP+HA 46.32 ± 13.01b 24.74 ± 2.32c 9.39 ± 9.27b,c 8.98 ± 7.25b,d 6.64 ± 8.15
P-value* 0.034 0.041 0.006 0.021 0.061
All data are presented as mean ± SD.
BV/TV, bone volume density; BS/TV, bone surface density; RW1, horizontal ridge width 1 mm below crest; RW2, horizontal ridge width 2 mm below crest; RW3,
horizontal ridge width 3 mm below crest.
a,b,c,d
Different letters indicate statistical significance between groups at each month and each column under post-hoc Tukey method.
*The P value among all groups analyzed by non-parametric Kruskal‒Wallis test.

3.4.1 At 1 month 3.4.3 Comparison between 1 and 3 months

The BV/TV was significantly higher in the ACS+HA group
compared with the ACS group (47.53% ± 14.05% versus
24.69% ± 13.08%, respectively and P = .024) but BS/TV was
not statistically different between the two groups. (ACS
16.67% ± 4.92% versus ACS+HA 18.44% ± 3.10%, respec-
tively and P = .84). Similarly, the RP+HA group showed
more BV/TV than the RP group (42.43% ± 7.17% versus
33.56% ± 3.49%, respectively and P = .015) but BS/TV
was not statistically different between the two groups (RP
23.79% ± 2.03% versus RP+HA 21.33% ± 1.45%, respectively
and P = .64). RW1, RW2 were significantly higher in the RP
and RP+HA groups than the ACS and ACS+HA groups
(P = .001). RW3 did not show any statistical difference
among the groups.
3.4.2 At 3 months
The BV/TV was significantly higher in the ACS+HA group
compared with the ACS group (53.28% ± 10.39% versus
36.04% ± 10.39%, respectively and P = .039). RP+HA
group showed more BV/TV than the RP group (46.32% ±
13.02% versus 38.23% ± 7.98%, respectively) but there was
no statistical significance (P = .34). The ACS group showed
the lowest BS/TV (18.62% ± 4.13%) with statistical signif-
icance while the other three groups (ACS+HA, RP, and
RP+HA) did not show any statistical differences. RW1 was
significantly higher in the RP and RP+HA group com-
pared with the ACS and ACS+HA group (P = .001). RW2
was significantly higher in the RP+HA group than the ACS
and ACS+HA groups. RW3 did not show any statistically
significant differences among the groups.
The BV/TV and BS/TV increased without significance in
all groups. RW1 increased without significance in the ACS
group and ACS+HA group and decreased in the RP group
and RP+HA group. RW2 increased without significance in
the ACS group, ACS+HA group, and RP+HA group and
decreased in the RP group. RW3 decreased without signifi-
cance in the ACS group, RP group, and RP+HA group and
increased in the ACS+HA group.
4 DISCUSSION
The present study demonstrated in compromised sockets
at 1 month and 3 months following tooth extraction that:
1) ACS+HA treatment enhanced bone formation but was
not able to prevent ridge dimensional loss; 2) RP treatment
reduced ridge shrinkage but showed less bone formation;
3) RP+HA group featured benefits from both RP and HA,
consisting into a reduction of dimensional loss and promo-
tion of bone formation in extraction sockets.
Strong lines of evidence from preclinical and clini-
cal studies1–3,13–16,34 suggest that ridge preservation with
DBBM-C graft covered with collagen membrane is effec-
tive in attenuating the dimensional changes of extrac-
tion sockets. In support of the results from previous
studies,3,33,35 sockets grafted with DBBM-C and collagen
membrane in RP and in RP+HA showed less dimen-
sional resorption in comparison with control sites receiv-
ing ACS+HA or ACS only. Fickl et al. (2008) showed in
beagles dogs that the reduction of horizontal ridge width at
1 mm below the lingual crest was smaller in grafted than
in non-grafted sites (4.4 ± 0.3 mm versus 3.7 ± 0.3 mm),
10
highlighting that the use of DBBM-C was able to minimize
the degree of horizontal ridge changes.33
However, other studies suggested that DBBM-C might
impede de novo bone formation in comparison with unas-
sisted sockets23,36 eventually reducing the mechanical sta-
bility of endosseous implants. One reason might be the
additional space secured by DBBM-C graft, thus requir-
ing a longer time to be filled with new bone. Importantly,
extraction sockets of periodontally-diseased teeth feature
a delayed healing in comparison with non-compromised
sockets.9,10 As a matter of fact, Araujo et al. (2008) reported
58.1% ± 10.7% of MB 3 months following ridge preserva-
tion with DBBM-C in fresh sockets.2 In the present study,
the proportion of MB in RP did not exceed 41.89% ± 5.03%
of MB in the RP group thus illustrating the lower healing
potential of compromised sockets.
Herein, osteopromotive growth factors in combination
with DBBM-C can be indicated to enhance bone forma-
tion in ridge preservation treatment. In particular, HA
plays a crucial role in bone repair by enhancing cell migra-
tion, adhesion, and inducing proliferation and differenti-
ation of mesenchymal cells into osteoblastic cells.30,37,38
Using an animal model similar to the present study, the
production of osteocalcin, a marker of osteogenesis, in
compromised sockets treated with HA-loaded ACS was
similar to recombinant human bone morphogenetic
protein-2‒loaded ASC. Based on the above findings, HA
was assumed to promote bone formation when mixed with
DBBM-C for preservation treatment of compromised sock-
ets. This hypothesis was confirmed by the radiographic and
histological results of the present study. RP+HA showed
histologically more NFB compared with RP along with a
radiographically higher bone density than in RP, therefore
suggesting a promotion of bone formation in extraction
sites. Nonetheless, bone formation in RP+HA remained
smaller in comparison with ACS+HA. This could be
explained by the reduced HA-loading capacity of DBBM-
C compared with ACS. ACS has been used for delivery
of various growth factors in the extraction socket.25,39,40
On one hand, the biocompatible and biodegradable col-
lagen structure is known to promote cellular penetration
and to form extracellular matrix.41 On the other hand, Guo
et al. found that the ACS used for hemostatic agents could
delay the early healing process of extraction sockets.42
They found that proliferation and osteogenic differentia-
tion of cells were delayed including the apoptosis of peri-
odontal ligament (PDL) cells. This might be attributed to
the compression of PDL during application, acidic pH,
and intense foreign body reaction. In line with these
observations, the ACS group showed significantly lower
NFB in comparison with the other treatments. There-
LEE et al.
fore, despite the prominent loading capacity of ACS, graft
materials may be suggested to replace ACS for delivery
of growth factors and simultaneous preservation of ridge
dimension.11,43
Compromised extraction sockets exhibit more dimen-
sional changes and an impaired healing as compared with
fresh sockets.10,12 Serving as a scaffold for tissue forma-
tion, DBBM-C enables additional 3D stability, which is crit-
ical for osteogenesis and for counteracting the dimensional
alterations of the ridge, particularly for the preservation
compromised sockets. Importantly, the matrix architecture
of DBBM-C provides inherent spaces that can be poten-
tially occupied by growth factors. A slight compression of
DBBM-C matrix during mixture with HA would secure a
proper infiltration of the growth factor and a subsequent
release in tissues following grafting.
HA was reported to show conflicting results according
to its molecular weight. Low molecular weight HA (LMW-
HA) was demonstrated to increase the expression of matrix
metalloproteinases, and stimulate a phagocytic activity of
neutrophils in vitro and in vivo.44,45 LMW-HA was also
found to be mediated in the stimulation on cPLA2 α and
cPLA2 α-derived lipid signaling in mouse macrophages and
human primary monocytes.46 On the other hand, high
molecular weight HA (HMW-HA) was shown to have a
protective effect by limiting the proinflammatory process.
The exact mechanisms are not known but it was suggested
that HMW-HA might obstruct LMW-HA-mediated Toll-
like receptor-2 signaling pathway.47 In this study 1% HMW-
HA was used not to exaggerate the inflammatory state of
compromised sockets. In addition, after tooth extraction,
the socket was meticulously debrided to resolve chronic
inflammation in all groups.
Shortcomings of this study include the difficulty to stan-
dardize the size and location of the defects during chronic
lesion induction. This is mainly due to the disease pro-
gression in periodontal tissues which varied among ani-
mals. Thus, attempts have been made to control the defect
shape and size by surgically creating standardized defects
of acute nature.11,48 Further studies are required to provide
relevant infection animal models featuring identical defect
size and location.
In this study, due to the size discrepancy of the extrac-
tion sockets of premolar and molar, the radiographic hori-
zontal measurements cannot be compared directly among
groups. Therefore, relative percentages were calculated
with distal extraction site (spontaneously healed) as a ref-
erence. However, a number of previous studies used a ref-
erence with a hemisected root which was not extracted
to limit the ridge alterations.3,35,49 Therefore, this aspect
could be another limitation of the present study.
LEE et al.
5 CONCLUSIONS
In this study, ridge preservation with DBBM-C and HA
improved the bone healing process and simultaneously
prevented dimensional shrinkage in compromised extrac-
tion sockets at 1 and 3 months indicating DBBM-C as a pos-
sible carrier for HA.
AC K N OW L E D G M E N T
The project (17-243) was supported by a grant from the
Osteology Foundation, Switzerland. The authors report no
conflicts of interest related to this study.
AU T H O R CO N T R I B U T I O N S
Heithem Ben Amara and Ki-Tae Koo contributed to the
conception, and design of the study. Heithem Ben Amara,
Seoyoon Chu, Hyun-Young Song, Min-Jung Son, and Jung-
won Lee have involved in data collection and investigation
of the study. Jun-Beom Lee, Jungwon Lee, and Hae-Young
Kim have been involved with data analysis and interpre-
tation. Jun-Beom Lee, Heithem Ben Amara, and Seoyoon
Chu drafted the article and are responsible for the integrity
of the data used in this study. Ki-Tae Koo and In-Cheol
Rhyu critically revised the article.
ORCID
Jun-Beom Lee https://orcid.org/0000-0002-5571-118X
Heithem Ben Amara https://orcid.org/0000-0002-7927-
9838
Jungwon Lee https://orcid.org/0000-0002-5508-442X
Ki-Tae Koo https://orcid.org/0000-0002-9809-2630
In-Chul Rhyu https://orcid.org/0000-0003-4110-6381
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S U P P O RT I N G I N F O R M AT I O N
Additional supporting information may be found online
in the Supporting Information section at the end of the
article.
How to cite this article: Lee J-B, Chu S, Amara
HB, et al. The effects of hyaluronic acid and
deproteinized bovine bone mineral with 10%
collagen for ridge preservation in compromised
extraction sockets. J Periodontol. 2021;1-12.
https://doi.org/10.1002/JPER.20-0832