bioengineering
Article
Comparative Analysis of Bone Regeneration According to
Particle Type and Barrier Membrane for Octacalcium Phosphate
Grafted into Rabbit Calvarial Defects
Se-Wook Pyo 1,† , Jeong-Won Paik 2,† , Da-Na Lee 2 , Young-Wook Seo 2 , Jin-Young Park 2 , Sunjai Kim 1
and Seong-Ho Choi 2, *
Citation: Pyo, S.-W.; Paik, J.-W.; Lee,
D.-N.; Seo, Y.-W.; Park, J.-Y.; Kim, S.;
Choi, S.-H. Comparative Analysis of
Bone Regeneration According to
Particle Type and Barrier Membrane
for Octacalcium Phosphate Grafted
into Rabbit Calvarial Defects.
Bioengineering 2024, 11, 215.
https://doi.org/10.3390/
bioengineering11030215
Academic Editor: Stuart Goodman
Received: 8 January 2024
Revised: 13 February 2024
Accepted: 21 February 2024
Published: 24 February 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Bioengineering 2024, 11, 215. https://doi.org/10.3390/bioengineering11030215
1 Department of Prosthodontics, Gangnam Severance Dental Hospital, Yonsei University College of Dentistry,
Seoul 06273, Republic of Korea; dentipyo@yuhs.ac (S.-W.P.); sunjai@yuhs.ac (S.K.)
2 Department of Periodontology, Research Institute for Periodontal Regeneration, Yonsei University College of
Dentistry, Seoul 03722, Republic of Korea; jpaik@yuhs.ac (J.-W.P.); danalee@yuhs.ac (D.-N.L.);
ahkupechia07@gmail.com (Y.-W.S.); jypark87@yuhs.ac (J.-Y.P.)
* Correspondence: shchoi726@yuhs.ac; Tel.: +82-2-2228-3189
† These authors contributed equally to this work.
Abstract: This animal study was aimed to evaluate the efficacy of new bone formation and volume
maintenance according to the particle type and the collagen membrane function for grafted octa-
calcium phosphate (OCP) in rabbit calvarial defects. The synthetic bone substitutes were prepared
in powder form with 90% OCP and granular form with 76% OCP, respectively. The calvarial de-
fects were divided into four groups according to the particle type and the membrane application.
All specimens were acquired 2 weeks (n = 5) and 8 weeks (n = 5) after surgery. According to the
micro-CT results, the new bone volume increased at 2 weeks in the 76% OCP groups compared to
the 90% OCP groups, and the bone volume ratio was significantly lower in the 90% OCP group after
2 weeks. The histomorphometric analysis results indicated that the new bone area and its ratio in
all experimental groups were increased at 8 weeks except for the group with 90% OCP without a
membrane. Furthermore, the residual bone graft area and its ratio in the 90% OCP groups were
decreased at 8 weeks. In conclusion, all types of OCP could be applied as biocompatible bone graft
materials regardless of its density and membrane application. Neither the OCP concentration nor
the membrane application had a significant effect on new bone formation in the defect area, but the
higher the OCP concentration, the less graft volume maintenance was needed.
Keywords: bone regeneration; octacalcium phosphate; concentration; collagen membrane
1. Introduction
Bone grafts are divided into autograft, allograft, xenograft, and alloplast according to
the origin of their material, and each is used individually or in combination depending on
the purpose in the field of bone regeneration [1]. Many studies related to synthetic bone
graft materials, which have various advantages such as mass production, controllability
of properties, and no risk of cross-infection, are continuously being conducted, and new
materials are being developed [2]. It is essential for bone substitute materials to provide a
variety of shapes and sizes with mechanical strength and biocompatibility to facilitate their
use in the regeneration of bone defects [3]. In particular, several studies have been reported
on hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP), which are calcium phosphate-
based biomaterials that are typically used as synthetic bone materials [4]. Sintered HA
bone is used as a scaffold material because it does not dissolve and maintains its shape
in a long-term bone defect. Compared with HA, β-TCP is a resorbable bone substitute
with inherent solubility in physiological environments. For this reason, biphasic calcium
phosphate (BCP), which consists of HA and β-TCP phases in various ratios, is mostly
https://www.mdpi.com/journal/bioengineering
Bioengineering 2024, 11, 215 2 of 14

capable of controlling the resorption rate [5]. MBCP+TM (Biomatlante SAS, 88 Édouard
Belin, Vigneux de Bretagne, France) is a synthetic bone graft substitute under the BCP
category, featuring a 20:80 ratio of HA to β-TCP. It possesses a micro- and macro-porous
structure that closely mirrors the natural architecture of human bone [6,7].
Octacalcium phosphate (OCP; Ca8 H2 [PO4 ]6 ·5H2 O), recently developed as a new
synthetic bone substitute, is a direct precursor of biological apatite [8]. The OCP shows
outstanding osteoconductive and osteoinductive properties owing to its unique physic-
ochemical properties. The OCP crystal has a water layer between two apatite layers, so
that the water layer is removed from the OCP in a physiological environment, and the
two apatite layers are combined to form HA crystals [9]. The OCP could be irreversibly
converted into sustainable biological apatite under physiological conditions, which would
be biodegradable at a bone defect site with a neutral pH [10,11]. Therefore, OCP has
properties as a biodegradable substance while also facilitating bone development through
osteoblast activity. There are several reports that biodegradation through direct resorp-
tion of osteoclast-like cells was observed at the OCP-grafted site in animal bone defect
models [12–14]. Despite this OCP biodegradability, most granules remain intact because
the degradation rate of OCP depends on granule size [15]. The resorption of bone sub-
stitutes is influenced by several factors. These factors include the size of particles, their
porosity, chemical and crystallographic properties (composition, Ca/P ratio, phase, and
crystallinity), and compatibility with body fluids, as indicated by pH [16]. Resorption of
substitutes is usually faster with smaller particle size, higher porosity, lower crystallinity,
and a higher non-stoichiometric ratio [17]. The rapid rate of resorption of OCP is an
essential consideration for clinicians.
In addition, a key principle of guided bone regeneration (GBR) involves creating a
secluded area for new bone formation by obstructing the encroachment of soft tissues [18].
In order to provide space for the stability of various bone substitutes, it is necessary to
use a barrier membrane that maintains structural integrity during the periods of cell
proliferation and maturation inside the wound. Particularly for resorbable membranes, it
is ideal for the rate of biodegradation to match the rate of new tissue formation without
leaving any residual material [19]. Collagen membranes can be explained by their well-
established scientific background and extensively validated clinical use [20]. Nevertheless,
to our knowledge, there has been no study on a GBR with different OCP concentrations or
collagen membrane application.
Therefore, the purpose of this animal study was to investigate the efficacy of new bone
formation and volume maintenance according to the OCP concentration and the collagen
membrane function in the GBR of the rabbit calvarium.

2. Materials and Methods

2.1. Material Preparation
Two types of synthetic bone substitutes, in powder and granular form respectively,
were prepared. The relative content of OCP was found to be 90% in powder and 76% in
granules. OCP powder was synthesized using a meticulously controlled process. The pro-
cedure involved dispersing dicalcium phosphate dihydrate (DCPD; Junsei Co., Ltd., Tokyo,
Japan), a carefully selected precursor, into deionized water to create a finely dispersed
suspension with a concentration of 12 g/L. Since the pH of the calcium phosphate solution
increases as the basic calcium phosphate hydrate is mixed with deionized water, an acidic
solution was added when the pH increased to adjust the pH of the calcium phosphate
solution to between 5 and 6 in the initial stage. Thereafter, the calcium phosphate solution
was heated to a temperature of 90◦ C or lower, and as a result, evaporation of the solvent
from the calcium phosphate solution was prevented, thereby suppressing a decrease in
the stability of the calcium phosphate solution. In addition, by adding a heterogeneous
nucleus containing at least one form of OCP and DCPD to the heated calcium phosphate
solution, OCP crystals were able to grow at a rapid rate centered on the heterogeneous
nucleus; that is, the crystal nucleus. As acidic OCP crystallizes in the calcium phosphate
Bioengineering 2024, 11, 215 3 of 14

solution, the pH of the calcium phosphate solution decreases to less than 5. Even in the
later stages, a basic solution such as ammonia water is added to maintain the pH of the
calcium phosphate solution. And then the solution was passed through filter paper. Since
the residue left on the filter paper included OCP crystals precipitated from the calcium
phosphate solution, it was washed with a solvent to remove impurities other than the OCP
crystals contained in the residue. The washed residue was then dried at a temperature of
70 ◦ C to 90 ◦ C, preferably 80 ◦ C, for about 24 h, and as a result, OCP crystals with high
purity were obtained. Two sizes of crystals with non-uniform size can be obtained from
the OCP prepared by this method; one crystal with a length of 10–15 µm and a width of
2–3 µm, and a second crystal with a length of 1–10 µm and a width of 0.5–3 µm. This
method of mechanically grinding a mixture of relatively small and large OCP crystals
allows the OCP crystals to be sized to the desired size. In this study, a meticulous particle
formation process was employed within a controlled environment, utilizing advanced
granulation techniques. OCP powder was prepared by hydrating DCPD as the raw ma-
terial with water in a pH-controlled system using acetic acid. The OCP granule was also
prepared by cementation of a mixture of α-tricalcium phosphate (α-TCP) and sodium
phosphate. The Ca/P ratio of the paste that was poured into the rectangular mold was
1.00. The dried sample was ground and sieved to a size of 500–850 µm [21]. Throughout
the entire manufacturing process, which adhered to a biomimetic approach, the temper-
ature was carefully maintained below 100 ◦ C to preserve the intrinsic properties of the
material. Moreover, the OCP material employed in this study underwent rigorous gamma
irradiation sterilization (25–40 kGy) to ensure the highest standards of safety and quality.
The OCP material was inserted into sterilized conical tubes (1.5 mL; 509-GRD-SC, Quality
Scientific Plastics, San Diego, CA, USA) and then packaged in Fisherbrand instant-sealing
sterile pouches (9 cm × 13 cm; Fisher Scientific, Pittsburgh, PA, USA). Then, the packaged
OCPs were sterilized by gamma irradiation into a ready-to-use form. The OCP granules
mounted by a sample holder or the OCP powders ground using an agate mortar and pestle
were characterized using an X-ray diffractometer (XRD, X’pert MPD-PRO; Panalytical,
Almelo, Netherlands).

2.2. Material Characterization

Phase analysis was conducted using X-ray diffraction (XRD; AERIS Malvern Panalyti-
cal Co., Ltd., Malvern, UK) with a Cu-Kα radiation source at a scan speed of 1.0◦ /min and
a step size of 0.022◦ . The reference database was based on the inorganic crystal structure
database (ICSD) [22]. The references for each substance are OCP (04-016-3473), HA (00-009-
0432), β-TCP (00-009-0169), DCPD (00-009-0077). Surface morphology was observed using
field-emission scanning electron microscopy (FE-SEM, S-4700 Hitachi, Tokyo, Japan).

2.3. Animals and Surgical Protocol

This study used ten male New Zealand white rabbits, each weighing between 2.8
and 3.2 kg. They were individually housed in separate cages under controlled laboratory
conditions and fed a standard diet. Every step, from the selection and care of the animals to
their preparation and the surgical procedures, was conducted in accordance with a protocol
sanctioned by the Institutional Animal Care and Use Committee, Yonsei Medical Center,
Seoul, Korea (approval number 2021-0237). All the protocols followed the ARRIVE (Animal
Research: Reporting of In vivo Experiments) guidelines for the study design [23–25].

2.4. Study Design

Four circular defects measuring 8 mm in diameter were created in the calvarium of
each rabbit using the same method as in previous studies [26,27]. Two different types of
synthetic bone substitutes, in powder and granular form, respectively, were applied to the
calvarial defects. In the groups using a barrier membrane, a resorbable collagen membrane
(Bio-Gide® , Geistlich Pharma AG, Wolhusen, Switzerland; LOT number 82100857) was cut
into the size of 10 mm × 10 mm and placed over each defect. As a result, the defects on
 synthetic
Bioengineering 2024, 11, x FOR bone
PEER REVIEW substitutes, in powder and granular form, respectively, were 4 ofapplied
15 t
calvarial defects. In the groups using a barrier membrane, a resorbable collagen m
brane (Bio-Gide®, Geistlich Pharma AG, Wolhusen, Switzerland; LOT number 82100
Four circular defects measuring 8 mm in diameter were created in the calvarium of
Bioengineering 2024, 11, 215 was cut into the size of 10 mm × 10 mm and placed over each defect. As a result, 14 the de
4 of of
each rabbit using the same method as in previous studies [26,27]. Two different types
on thesynthetic
calvarium were randomly assigned to one of the following
bone substitutes, in powder and granular form, respectively, were applied to the four experim
groups; (1) 90m: 90%InOCP
calvarial defects. with ausing
the groups membrane; (2) 90n: 90%
a barrier membrane, OCP without
a resorbable collagen a membran
mem-
the calvarium
brane (Bio-Gidewere randomly
®, Geistlich assigned
Pharma AG, toWolhusen,
one of the Switzerland;
following four LOT experimental
number groups;
82100857)
76m: 76%
(1)
OCP with a membrane; and (4) 76n: 76% OCP without a membrane. The
was90m: 90%the
cut into OCP
sizewith
of 10ammmembrane;
× 10 mm and(2) 90n:
placed90% OCP
over eachwithout
defect. aAsmembrane;
a result, the(3) 76m:
defects
five rabbits
76% OCP
on the
werewithsacrificed
calvariuma membrane; at and
were randomly
2 weeks
(4) 76n:(n
assigned76%=to5)
OCPand the other
without
one of
five
a membrane.
the following
atThe
8 weeks
first five(n = 5)
four experimental
surgical procedure.
rabbits
groups; were TheOCP
sacrificed
(1) 90m: 90% sample
at 2 with size
weeks (n = was calculated
5) and
a membrane; the other five
(2) 90n: 90% based
at 8 on a previous
weeks
OCP without (n = 5) after study
surgical
a membrane; (3) (Figu
procedure.
76m: 76% OCPThe sample size was calculated
with a membrane; based76%
and (4) 76n: on aOCP
previous study
without (Figure 1). The first
a membrane.
five rabbits were sacrificed at 2 weeks (n = 5) and the other five at 8 weeks (n = 5) after
surgical procedure. The sample size was calculated based on a previous study (Figure 1).

Figure 1. Schematic diagram of this study.

Figure 1. Schematic diagram of this study.
Figure 1. Schematic diagram of this study.
2.5. Surgical Protocol
rabbits
rabbits were
TheProtocol
2.5. Surgical wereanesthetized
anesthetizedwith
withintramuscular injections
intramuscular of zoletil
injections (15 (15
of zoletil mg/kg) and
mg/kg)
rompun
and (5 mg/kg)
rompun maintained
(5 mg/kg) with with
maintained inhalations of isoflurane
inhalations (2–2.5%).
of isoflurane The surgical
(2–2.5%). sites
The surgical
The
sites
wererabbits
were
shaven were
shaven
and thenanesthetized
and then draped
draped withalcohol
with with
alcoholintramuscular
and povidone
and povidone injections
iodine,
iodine, ofby
zoletil
followed
followed (15 mg/kg
by anes-
local local
rompun (5 mg/kg) maintained with inhalations of isoflurane (2–2.5%). Thethe
anesthesia
thesia with with
2% 2% lidocaine.
lidocaine. An An incision
incision was was
made made
along along
the the sagittal
sagittal midlinemidline
from from
the frontal
surgical
frontal
bone tobone to the occipital
the occipital bone. Abone. A full-thickness
full-thickness flap
flap was was elevated
elevated to expose
to expose the cranial
the cranial bone.
were shaven and then draped with alcohol and povidone iodine, followed by local a
bone. Four circular
Four circular defectsdefects with a diameter
with a diameter of 8 mmofwere
8 mm were using
created created using a trephine
a trephine bur
bur of corre-
thesia of
sponding size under copious saline irrigation, and randomly allocated to the four study the fr
with 2% lidocaine.
corresponding size An
under incision
copious was
saline made along
irrigation, and the sagittal
randomly midline
allocated to thefrom
four
bone to thegroups
study
groups occipital
(Figure(Figurebone.
2). After A full-thickness
2). After surgery,
surgery, flap
the animals
the animals were was
were elevated
managed
managed to expose
by administering
by administering the cranial b
antibi-
antibiotics
otics subcutaneously
subcutaneously (Baytril,
(Baytril, Bayer,Bayer, Leverkusen,
Leverkusen, Germany)
Germany) and and
were
Four circular defects with a diameter of 8 mm were created using a trephine bur of c were closely
closely monitored
monitored clini-
clinically during
cally during the entire
the entire periodperiod of healing.
of healing. The animals
The animals were sacrificed
were sacrificed eitherat2 either 2 or
sponding size under
8postoperatively.
copious
weeks postoperatively.
saline irrigation, and randomlyatallocated or 8 weeks
to the four s
groups (Figure 2). After surgery, the animals were managed by administering antibi
subcutaneously (Baytril, Bayer, Leverkusen, Germany) and were closely monitored
cally during the entire period of healing. The animals were sacrificed at either 2 or 8 w
postoperatively.

Figure 2. Surgical procedure. (A) Four circular defects measuring 8 mm in diameter were created
using a trephine bur. (B) The defects were allocated to the four study groups. Clockwise from bottom
to left: 90% OCP with a membrane (90m), 90% OCP without a membrane (90n), 76% OCP without a
membrane (76n), 76% OCP with a membrane (76m).
Bioengineering 2024, 11, 215 5 of 14

2.6. Evaluation
− Clinical observations: Animals were meticulously monitored for a duration of 2 to
8 weeks post-surgery, assessing for potential complications such as inflammation,
allergic reactions, postoperative bleeding, and infections around the surgical site.
− Micro-computed tomography (micro-CT) analysis: After the animals were sacrificed,
the surgical defects and adjacent tissues were removed in a single piece. These samples
were rinsed in sterile saline and preserved in 10% buffered formaldehyde solution for
10 days. After rinsing in water, all specimens were scanned using a micro-CT scanner
(SkyScan1173;Bruker-CT, Kartuizersweg 3B 2550 Kontich, Belgium). The samples
were mounted on a jig using parafilm for precise micro-CT analysis, during which
800 images were captured at a setting of 130 kV tube voltage, 60 µA current, and
through a 1.0 mm aluminum filter. Image reconstruction was performed using NRecon
software (Ver. 1.7.0.4, Bruker, Kontich, Belgium), with alignment of the cross-sectional
images for each specimen achieved through DataViewer software (Ver. 1.5.1.2, Bruker,
Kontich, Belgium), and quantitative analysis conducted using CTAn software (Ver.
1.17.7.2, Bruker, Kontich, Belgium). For data analysis, the defect was set as the region
of interest, and the threshold was set to 45–90 to analyze the following parameters:
• Total tissue volume (TV; mm3 ): total augmented volume of the defects.
• New bone volume (BV; mm3 ): volumetric measurements of the new bone within
the defects.
• Bone volume ratio (BV/TV; %): new bone volume to total augmented volume.
− Histologic and histomophometric analysis: Following micro-CT scanning, the spec-
imens underwent a 14-day decalcification process in 5% formic acid and were sub-
sequently embedded in paraffin. Serial 5 µm thick sections were obtained from the
central region of each calvarial defect. The central sections from each block were
subjected to hematoxylin and eosin (H&E) and Masson trichrome staining for his-
tological and histometric analyses. One blinded examiner conducted examinations
using a microscope (DM LB, Leica Microsystems, Wetzlar, Germany) equipped with a
camera (DC300F, Leica Microsystems). The slide images were saved as digital files,
and computer-aided histometric measurements were performed using an automated
image analysis system (Image-Pro Plus; Media Cybernetics, Silver Spring, MD, USA).
The assessment of bone healing and regeneration involved measuring the following
parameters in each histological section corresponding to the defect areas:
• Total augmented area (TA; mm2 ): Total sum of the area of new bone, residual
particles, connective tissue, adipose tissue, blood vessels within the defect area.
• New bone area (NB; mm2 ) and its ratio (%NB): Area of the newly formed bone
within the defect, and ratio of NB to TA.
• Residual bone graft area (RG; mm2 ) and its ratio (%RG): Area of the residual
material within the defect, and ratio of RG to TA.

2.7. Statistical Analysis

A power calculation was conducted using a power analysis program (G*Power 3.1
software, University of Kiel, Kiel, Germany), focusing on new bone area (NB) as the
primary outcome. Following the findings of Sohn et al., a sample size of 10 subjects was
recommended (assumed mean and standard deviation: 2 weeks, 1.88 ± 0.67 mm2 ; 8 weeks,
3.19 ± 0.27 mm2 ; power: 0.80; α < 0.05) [28]. The descriptive data from radiographic and
histometric results were expressed as median values, and difference between the maximum
and minimum values. Data were analyzed using SAS version 9.4 (SAS Institute, Cary, NC,
USA). The significance between four different groups at each time point were determined
by the Kruskal–Wallis test followed by the Mann–Whiney test. The Mann–Whiney test was
also used for comparisons between the two healing periods within the same experimental
group. Statistical significance was determined at the p < 0.05 level.
 same experimental group. Statistical significance was determined at the p < 0.05 level.

3. Results
3.1. Phase Analysis Using X-ray Diffration
Bioengineering 2024, 11, 215 6 of 14

The OCP synthetic powder and granules, produced through biomimetic process,
demonstrated the absence
3. Results of residual DCPD. Notably, X-ray diffraction analysis con-
ducted within the 2θ3.1.range of 3–60°
Phase Analysis revealed
Using the distinct presence of an OCP diffraction
X-ray Diffration
The OCP synthetic powder
peak (100), prominently observed at approximately and granules, produced3A).
4–5° (Figure through biomimetic process,
In distinction to the
demonstrated the absence of residual DCPD. Notably, X-ray diffraction analysis conducted
OCP synthesized immediately after production, the OCP derived through the granulation
within the 2θ range of 3–60◦ revealed the distinct presence of an OCP diffraction peak
process demonstrated the
(100), emergence
prominently of minute
observed quantities
at approximately of amorphous
4–5◦ (Figure calcium
3A). In distinction phos-
to the OCP
phate (ACP) ceramicssynthesized immediately
under humid after production,
conditions. the OCP
However, derived
it can through thethat
be observed granulation
the in-
process demonstrated the emergence of minute quantities of amorphous calcium phosphate
herent XRD pattern of OCP is well-preserved, indicating its structural stability throughout
(ACP) ceramics under humid conditions. However, it can be observed that the inherent
the granulation process. Phaseofanalysis
XRD pattern using XRD indicating
OCP is well-preserved, showed itsthat OCP stability
structural is the main constit-
throughout the
uent of both materials, regardless of size.
granulation process. Phase analysis using XRD showed that OCP is the main constituent of
both materials, regardless of size.

Figure 3. (A) The XRD spectra of the OCP powder (top) and granules (bottom). The OCP peaks are
Figure 3. (A) The XRDindicated
spectrabyofasterisks
the OCP powder (top) and granules (bottom). The OCP peaks are
(*). (B) Representative high- and low-magnification FE-SEM images of the OCP
indicated by asterisks power
(*). (B)
andRepresentative
granule. high- and low-magnification FE-SEM images of the
OCP power and granule.
In order to investigate the surface properties of the bone graft material, scanning
electron microscopy (SEM) analysis was employed (Figure 3B). The OCP particles exhibited
In order to investigate
a delicatethe surface
ribbon-like properties
structure, of the
displaying bone
a wide graft
range material,
of sizes scanning
from a few elec-
micrometers to
tron microscopy (SEM) analysis
several was employed
tens of micrometers. (Figure 3B).
The agglomeration Theribbon-shaped
of these OCP particles OCPexhibited
particles wasa
observed, resulting in the formation of intricate micro-pores.
delicate ribbon-like structure, displaying a wide range of sizes from a few micrometers In contrast, the granulated
to
form exhibited comparatively smaller particles, likely attributable to additional processes.
several tens of micrometers. The agglomeration of these ribbon-shaped OCP particles was
3.2.the
observed, resulting in Clinical Findings of intricate micro-pores. In contrast, the granulated
formation
No significant
form exhibited comparatively smallerpostoperative
particles,complications, such as excessive
likely attributable bleedingprocesses.
to additional or swelling,
were observed. During the healing period, no specific inflammation, complications, or
abnormal findings were observed in any animal. Upon sacrifice and sample collection, it
3.2. Clinical Findingswas verified that the grafted materials remained intact within the defects.
No significant postoperative complications, such as excessive bleeding or swelling,
3.3. Micro-CT Analysis
were observed. During the The healing period,
new bone tissue no to
tended specific inflammation,
grow toward complications,
the center of the or ab-
defect region. Although
normal findings were observed in any animal. Upon sacrifice and sample collection, itwere
all groups showed new bone formation in the bone defects, none of the defects was
completely closed after 2 or 8 weeks (Figure 4).
verified that the grafted materials remained intact within the defects.

3.3. Micro-CT Analysis

 pletely closed after 2 or 8 weeks (Figure 4).

The new bone tissue tended to grow toward the center of the defect region. Although
all groups
Bioengineering showed new bone formation in the bone defects, none of the defects were7 com-
2024, 11, 215 of 14
pletely closed after 2 or 8 weeks (Figure 4).

Figure 4. Micro-CT images obtained 2 and 8 weeks after surgery. New bone formation occurred
mainly around the periphery of defect, and significantly increased at 8 weeks compared to 2 weeks
in all groups. The red circle represents the border of the defect, and the red arrow indicates the
direction of presumed new bone formation. (A,E) 76% OCP with a membrane group; (B,F) 76% OCP
without
Figurea 4.
membrane
Micro-CTgroup;
images (C,G)
Figure 4. obtained90%images
Micro-CT 2OCP with
andobtained a 2membrane
8 weeks and 8 weeks
after group;
after
surgery. (D,H)
surgery.
New New 90%
bonebone OCP without
formation
formation a
occurred
occurred
membrane group; the
mainly around (rightmainly around
top andofdown)
periphery the periphery of defect,
defect,three-dimensional and significantly increased
reconstruction
and significantly increased at 8 weeks
images.
at 8 weeks compared to 2 weeks
compared to 2 weeks in
all groups. The red circle represents the border of the defect, and the red arrow indicates the direction
in all groups. The red circle represents the border of the defect, and the red arrow indicates the
of presumed new bone formation. (A,E) 76% OCP with a membrane group; (B,F) 76% OCP without a
The TV
direction results membrane
of presumed were similar
new bone group; in all
formation.
(C,G) 90%groups
(A,E)
OCP with76% awithout
OCP with
membrane significant
a membrane
group; (D,H) 90% difference,
group;
OCP aand
(B,F)
without 76%de-
membraneOCP
without a membrane
creased at 8 weeks compared group; (C,G)
group; (rightto
top2and90%
weeks OCP with a membrane
with a statistically
down) three-dimensional group;images. difference only in a
significant
reconstruction (D,H) 90% OCP without
themembrane
76n group. group; (right
The BV top and
results were down) three-dimensional
significantly greater in reconstruction
the 76% groups images.
than in the 90%
The TV results were similar in all groups without significant difference, and decreased
groups at 2 weeks, and the difference
at 8 weeks compared to 2between
weeks withthe groups significant
a statistically decreased at 8 weeks.
difference The
only in the 76n
The TV results
BV/TV results also tended were similar
to BV
group. The be resultsin
greater all groups
wereinsignificantly without
the 76% greater
groups significant
than
in the 76% in difference,
the than
groups 90%ingroups andat 2de-
the 90% groups
creased
weeks, butatthey
8 weeks atcompared
showed similar to
2 weeks, and 2 weeks
the difference
results with
in all a statistically
between
groups significant
the groups decreased
at 8 weeks. 8 difference
As aatresult,
weeks. The BV/TVonly in
significant
results also tended to be greater in the 76% groups than in the 90% groups at 2 weeks,
the 76n were
increases group. The BV results
observed in the were significantly
BV/TV results greater in the
between 76% groups than
90% in the 90%
but they showed similar results in all groups2atand 8 weeks
8 weeks. in the
As a result, groups,
significant increases
butgroups at 2 weeks,
no differences were and
were the difference
observed
observed in in
the the
BV/TV between
76% OCP
results the groups
groups.
between 2 andThe decreased
results
8 weeks at
the8groups,
in theof90% weeks.
micro-CT The
but no
BV/TVare
analysis results alsodifferences
summarized tended to beobserved
were
in Figure 5greater
and in in 76%
the
Table the 76%groups.
1. OCP groups than in
The results themicro-CT
of the 90% groups at 2
analysis are
summarized in Figure 5 and Table 1.
weeks, but they showed similar results in all groups at 8 weeks. As a result, significant
increases were observed in the BV/TV results between 2 and 8 weeks in the 90% groups,
but no differences were observed in the 76% OCP groups. The results of the micro-CT
analysis are summarized in Figure 5 and Table 1.

Figure 5. Distribution of experimental groups and statistical significance within groups according
Figure 5. Distribution of experimental groups and statistical significance within groups according
to micro-CT analysis results. Small and capital letters of the same alphabet indicate significant
to micro-CT analysis results. Small and capital letters of the same alphabet indicate significant
differences between 2-week and 8-week results compared in the same experimental group. Even
differences between 2-week thoughandthere8-week resultsdifference,
is no significant compared in the same
if a distinction experimental
is needed, a prime markgroup.
is used Even
next to the
though there is no significant difference,
letter. The if a distinction
asterisk * indicates is needed,
statistical significance. (A)aTotal
prime
tissuemark is (B)
volume, usednewnext
bone to the
volume,
letter. The5.asterisk
Figure * indicates
Distribution (C)ofbone statistical significance.
volume ration.
experimental groups and(A) Total tissue
statistical volume,
significance (B) new
within boneaccording
groups vol-
ume, (C) bone volume
to micro-CT analysisration.
results. Small and capital letters of the same alphabet indicate significant
differences between 2-week and 8-week results compared in the same experimental group. Even
Table 1. Median
though with
there is minimum and
no significant maximum
difference, if a values
distinctionof allisgroups
needed, measured
a prime by micro-CT
mark is usedanalysis.
next to the
letter. The asterisk * indicates statistical significance. (A) Total tissue volume, (B) new bone vol-
ume, (C) bone volume ration.

Table 1. Median with minimum and maximum values of all groups measured by micro-CT analysis.
Bioengineering 2024, 11, 215 8 of 14

Table 1. Median with minimum and maximum values of all groups measured by micro-CT analysis.
Week Group TV BV BV/TV
Week Group TV Week BVGroup
Week Group Week TVBV/TV
Group
TV BV BVTV BVBV/TV
BV/TV BV/TV
Week Group TV 90m
BV
192.563 (144.331–219.075) 21.038 (10.813–33.831) a 12.004 (7.051–17.569) a
BV/TV
90m 192.563 (144.331–219.075) 90m a 90m
21.038 (10.813–33.831) 192.563 (144.331–219.075)
12.004 (7.051–17.569)
192.563 90m
a
(144.331–219.075) 21.03821.038
(10.813–33.831) a
(10.813–33.831)
192.563 a
(144.331–219.075) 12.004
21.038 (7.051–17.569)
12.004
a a
(7.051–17.569)
(10.813–33.831) a 12.004 (7.051–17.569) a
90m 192.563 (144.331–219.075) 2 21.038 (10.813–33.831) a 12.004 (7.051–17.569) a
90n 209.308 (136.747–252.565) 26.084 (13.647–32.505) a′ 12.787 (7.859–19.074) a′
2 2 2
90n 209.308 (136.747–252.565) 26.084 (13.647–32.505)
90n a′
209.3082(136.747–252.565)
12.787 (7.859–19.074) a′
26.08426.084 ′
a(13.647–32.505)
(13.647–32.505) a′ 12.787 (7.859–19.074) * **
a′ a′
a′
2 weeks 90n
weeks 209.308 (136.747–252.565)weeks
weeks weeks76m
90n
26.084209.308 (136.747–252.565)
(13.647–32.505)
90n
* *52.035
209.308 (136.747–252.565)
*
a′
26.084 12.787 (7.859–19.074)
12.787 (7.859–19.074)
(13.647–32.505) a′

* ** ** *
12.787 (7.859–19.074) a′

* 208.007
weeks(179.307–281.868) * (29.927–70.808) b 21.899 (14.640–31.131) b′ * **
(n = 5) 76m
(n=5)
76m 208.007 (179.307–281.868)
208.007 (179.307–281.868)
(n=5)
52.035 (29.927–70.808) 76m
76m
b 21.899
208.007
(14.640–31.131)
* 52.035 (29.927–70.808)
208.007
b′
(179.307–281.868)
(179.307–281.868) 52.035 52.035 b
(29.927–70.808)
(29.927–70.808) b *b * * 21.899 *
21.899 (14.640–31.131)
21.899 (14.640–31.131)
(14.640–31.131) b′ b′ b′
(n=5) (n=5)
(n=5)
76m 208.007 (179.307–281.868) * * 52.035 (29.927–70.808) b
*
21.899 (14.640–31.131) b′

76n
39.112 (31.501–74.109) a
229.541 (184.888–334.893) a
b ′
39.112 (31.501–74.109) b′ 21.154 (14.334–25.276) b
b
76n 76n 229.541 (184.888–334.893)
229.541 (184.888–334.893)
a

76n 76n
b′

39.112
* 229.541 (31.501–74.109)
21.154 (14.334–25.276)
(184.888–334.893)
76n
a
b
229.541 (184.888–334.893) a
39.11239.112 (31.501–74.109)
(31.501–74.109) b′
229.541 (184.888–334.893)
b′
a
* 21.154
21.154
*39.112
21.154 (14.334–25.276)
(14.334–25.276)
(14.334–25.276)
(31.501–74.109) b′
b b
21.154 (14.334–25.276) b
* *
Overall 208.657 (136.747–334.893) 32.401 Overall
(10.813–74.109) 208.65715.688
(136.747–334.893)
(7.051–31.131) 32.401 (10.813–74.109) 15.688 (7.051–31.131)
Overall 208.657 (136.747–334.893)
Overall Overall
32.401 (10.813–74.109)
208.657 (136.747–334.893)
208.657 (136.747–334.893)
Overall 32.40132.401 (10.813–74.109)
(10.813–74.109)
208.657 (136.747–334.893) 15.688
32.401 15.688 (7.051–31.131)
15.688 (7.051–31.131)
(7.051–31.131)
(10.813–74.109) 15.688 (7.051–31.131)
90m 161.167 (144.422–199.540) 90m
30.565 (28.767–38.564) 161.167 (144.422–199.540)
19.327 (17.849–22.715) A 30.565 (28.767–38.564) 19.327 (17.849–22.715) A
90m 161.167 (144.422–199.540) 90m
90m 161.167 (144.422–199.540)
30.565 (28.767–38.564)
161.167 (144.422–199.540)
30.565 (28.767–38.564)
30.565 (28.767–38.564)
19.327 (17.849–22.715)
19.327
A

19.327 (17.849–22.715)
(17.849–22.715) A A
* * 90m 161.167 (144.422–199.540) 30.565 (28.767–38.564) 19.327 (17.849–22.715) A
8
90n 155.934 (136.747–173.027) 8 30.685 (23.884–42.150) C * 155.934
21.449 (14.324–25.173) A′
′
90n* C
8 90n (136.747–173.027) 30.685 (23.884–42.150) C
21.449 (14.324–25.173) A′ A′ A
8 weeks 90n
weeks 155.934 (136.747–173.027) 8
weeks 90n 30.685 (23.884–42.150)
155.934 (136.747–173.027)
155.9348(136.747–173.027)
90n
* 30.685 (23.884–42.150) C
30.685 (23.884–42.150) C
155.934 (136.747–173.027) 21.449
30.685
21.449 (14.324–25.173)
21.449 (14.324–25.173)
(14.324–25.173)
(23.884–42.150) C
A′
21.449 (14.324–25.173) A′
weeks * *
76m 76m 173.027 (145.817–236.518)
173.027 (145.817–236.518)weeks 38.200 (33.107–44.354) D 20.279 (18.388–26.197)
38.200 (33.107–44.354) D * 20.279 (18.388–26.197)
(n = 5) (n=5)
(n=5)
76m
(n=5)
76m
76m 173.027 (145.817–236.518)
weeks
173.027 (145.817–236.518)
173.027 (145.817–236.518)
38.200 (33.107–44.354) D
38.200 (33.107–44.354) D
38.200 (33.107–44.354)
*
20.279 (18.388–26.197)
20.279
20.279
(18.388–26.197)
(18.388–26.197) *
173.027′(145.817–236.518)
D

A 76m 38.200 (33.107–44.354) D

20.279 (18.388–26.197)
76n 76n 181.749 (161.864–219.773)
181.749 (161.864–219.773)
A (n=5) 39.009 (32.037–52.048) D′
39.009
(n=5)
(32.037–52.048)
20.592 (14.577–31.214) D 20.592 (14.577–31.214)
76n 76n 181.749 (161.864–219.773)
181.749
A
(161.864–219.773) A 39.009 (32.037–52.048) D′
39.009 (32.037–52.048) D′ 20.592 (14.577–31.214)
20.592 (14.577–31.214)
76n 181.749 (161.864–219.773)
76n
A 39.009 181.749
(32.037–52.048) D′
(161.864–219.773) A 20.592
39.009 (14.577–31.214)
(32.037–52.048) D′
20.592 (14.577–31.214)
Overall 166.051 (136.747–236.518) 34.318 (23.884–52.048) 20.533 (14.324–31.214)
Overall 166.051 (136.747–236.518)
Overall Overall 34.318 (23.884–52.048)
166.051 166.051
(136.747–236.518)
34.31834.318
(23.884–52.048)
(136.747–236.518)
(23.884–52.048) 20.533 20.533 (14.324–31.214)
(14.324–31.214)
20.533 (14.324–31.214)
TV: Total tissue volume, BV: New bone volume, BV/TV: ratio
Overall of BV
166.051 toOverall
TV. Significant differences
(136.747–236.518) (p
34.318 (23.884–52.048)
166.051 (136.747–236.518) 20.533
34.318 (14.324–31.214)
(23.884–52.048) 20.533 (14.324–31.214)
< 0.05) between the four experimental groups are indicated by TV: TV: tissue
Total
different Total
smalltissue
volume,
letters volume,
at 2BV: BV:and
New
weeks New
bonebone volume,
volume, BV/TV:
BV/TV: ratio
ratio of BV
of BV to to
TV.TV. Significantdifferences
Significant differences(p (p
TV: Total tissue volume, BV: New bone volume, BV/TV: ratio of BV to TV. Significant differences (p < 0.05) between
TV:
by different capital letters at 8 weeks. Small and capital letters ofTotal
the
< 0.05) tissue
<between
0.05)
same volume,
between
alphabet
the fourthe BV: New
four
indicate bone
experimental
signifi-
experimental TV: volume,
groups
Total areBV/TV:
groups
tissue are
indicated
volume, ratio
byof
indicated
BV: by BV to
different
different
New bone TV. Significant
small
small atdifferences
letters
letters
volume, BV/TV:at2 2weeks
weeks (p
and
and
ratio of BV to TV. Significant differences (p
the four experimental groups are indicated by different small letters at 2 weeks and by different capital letters at
cant differences between 2-week and 8-week results compared <by inby
0.05) different
the
between
different same
capitalcapital
lettersletters
experimental
the four at 8< weeks.
at 8group.
experimental
weeks. Small
0.05) Small
groups areand
and
between thecapital
indicated
capital letters of the
by different
letters
four of the
experimental samesame
small alphabet
letters
alphabet
groups atindicate
2 weeks
indicate
are indicated signifi-
and
signifi-
by different small letters at 2 weeks and
Even though there is no significant difference, if a distinction by isdifferent
needed, acapital
prime
cant differences mark
lettersisat
betweenused next
2-week
8 weeks. and
Small 8-week
and results
capital compared
letters in inthethe same experimental group.
cant differences between 2-week and
by
8 weeks. Small and capital letters of the same alphabet indicate significant differences between 2-week and 8-week 8-week
different results
capital letters at of
compared the
8 weeks. same alphabet
same
Small indicate
experimental
and capital signifi-
group.
letters of the same alphabet indicate signifi-
to the letter. The asterisk indicates statistical significance. cant differences
Even though there
Even though there is no issignificant
between no significant
2-week and
cant
difference,
8-week
difference,
differences
ifdistinction
results a distinction
compared
if a between isinneeded,
the same
is needed,
2-week and
a prime
8-week
markisisused
experimental
a prime mark
results
used next
group.
compared next in the same experimental group.
results compared in the same experimental group. EvenEven
3.4. Histological Analysis
Even to the
to thethough letter.
letter. Thethere The though
asterisk there
is no significant
asterisk is nois
indicates no significant
statistical
difference,
indicates statistical
though there difference, if adifference,
significance. if a adistinction
if a distinction is needed, a prime mark is used next
significance.
significant distinction is needed, is next
prime mark is used
to the letter. The asterisk indicatestostatistical significance.
the letter. The asterisk indicates statistical significance.
needed,
The defectaspaces
prime were mark
occupiedis
by used next
the underlying to3.4.
brainthe letter.
Histological
tissues, The the asterisk
Analysis
and either over- indicates statistical significance.
3.4. Histological Analysis
lying periosteum or collagen membrane collapsed to fill theHistological
3.4. vacant Thespace. Thespaces
defect
Analysis grafted ma-occupied by the underlying brain tissues, and either the over-
were
The defectmatrixspaces werecom- 3.4. Histological
occupied Analysis brain tissues, and either the over-
by the underlying
terials were surrounded by the connective tissue of the extracellular
lying periosteum (ECM)
or collagen membrane
lying The defect
periosteum spaces
or were
collagen occupied
membrane thecollapsed
Thebydefect underlying
collapsed
spaces
to fill the tissues,
fill brain
to were
vacant space.
theoccupied byand
vacant space.
The grafted
either
theThe the over-
grafted
underlying
ma-
ma-
brain tissues, and either the over-
3.4. Histological Analysis
prising a loose collagen network. After 2 weeks, the infiltration
cells and proliferation of blood vessels were observed.lying
terials
Minimal were
of were
terials
periosteum
new
chronic
surrounded
bone
inflammatory
surrounded
orformation
collagenby
by the connective tissue of the extracellular matrix (ECM) com-
membrane
the
was connectivecollapsed
periosteumtissue or to
of fill
the
collagen the vacant space.
extracellular
membrane The (ECM)
matrix
collapsed grafted
to ma-
com-
prising a loose collagen network. After 2 weeks, the infiltration of chronic inflammatoryvacant space. The grafted ma-
lying fill the
terials
prising
observed around the grafted particles in the margins adjacent were
a the
to surrounded
loose collagen by the
network. connective
After
terials were tissue
2 weeks,
surrounded of the extracellular
the infiltration
byMinimal ofnew matrix
chronic
the connective (ECM)
inflammatory
tissue com-
of the was
extracellular matrix (ECM) com-
cells anddefect. The 76%
proliferation ofOCP
blood vessels were observed. bone formation
The defect spaces were occupied by the underlying brain tissues, and either the
groups showed a stable volumetric change in total tissue prising a loose
cells volume,
and
observed andcollagen
around ofnetwork.
the formation
proliferation the blood After
ofvessels
prising
grafted
2 weeks,
a were
loose
particles in the
the infiltration
observed.
collagen Minimal
marginsnetwork.
adjacent to
of chronic
new
thebone
After defect.
inflammatory
formation
2 weeks, Thethe76% was
infiltration
OCP of chronic inflammatory
new bone around the defect margin, and grafted particles cells and proliferation
were
observed observed
around due
the of the
to blood
grafted sup-vessels
particles
cells and were
in the observed.
margins
proliferation Minimal
adjacent
of blood tonew
the
vessels bone
defect.
were formation
The 76%
observed. was
OCP
Minimal new bone formation was
overlying periosteum or collagen membrane collapsed to fill the vacant space. The grafted
observed
port of relatively large particles. In contrast, in the groups
groups
groups showed a stable volumetric change in total tissue volume, and the formation of
with
new around
90% OCP,
showed athe
bone around grafted
as
stable a result particles
of
thevolumetric
observed
defect inaround
the
change
margin, andmargins
in total
grafted adjacent
tissue
the grafted
particles to the
volume,
particles
were defect.
and
in the
observedthe The 76%
formation
margins
due OCP of to the defect. The 76% OCP
adjacent
to the sup-
groups
new bone showed
around a stable
the defectvolumetric
groupschange
margin, and in atotal
grafted tissue
inparticles volume,
were and OCP,
observed theinformation
due astoathe sup-ofofvolume, and the formation of
materials were surrounded new by
port bone
the large
around
of relatively
connective
the defect
port
margin,
particles.
newInboneandtissue
of relatively
grafted
contrast,
around
of the
large
particles
in the defect were
groups
extracellular
particles.
withobserved
margin,90%
showed
In
due
andOCP,
contrast,
asto
grafted the sup-
aparticles
result
matrix (ECM)
stable
the volumetric
groups with change
90%
ofwere observed due to the sup-
total tissue
result

comprising a loose collagenport

network. After 2port weeks,
of relatively large particles. Inofcontrast,
the
in large
relatively infiltration
the groups withIn90%
particles. of chronic
OCP, in
contrast, as the
a result
inflammatory
of with 90% OCP, as a result of
groups

cells and proliferation of blood vessels were observed. Minimal new bone formation was
observed around the grafted particles in the margins adjacent to the defect. The 76% OCP
groups showed a stable volumetric change in total tissue volume, and the formation of new
bone around the defect margin, and grafted particles were observed due to the support
of relatively large particles. In contrast, in the groups with 90% OCP, as a result of being
strongly pressed by soft tissue and dura mater in the center of the defect, a thin, flat,
concave, disc-shaped tissue was observed. In the 8-weeks group, larger quantities of new
bone and osteoblasts were noted, predominantly originating from the periphery of the
defects. New bone was observed not only growing from the marginal area but also around
the centrally located remaining particles. The collagen membrane maintained a uniform
shape, but was absorbed compared to the findings at 2 weeks, and the amount of chronic
inflammatory cells around the membrane also decreased. ECM exhibited increased density
compared to the appearance observed after 2 weeks, resulting in almost complete closure
along the outer surface of the defect (Figure 6).

3.5. Histomorphometric Analysis

Except for a significant difference between the 90m (11.755 mm2 ) and 76n
(16.720 mm2 ) groups at 8 weeks, most of the TA results were measured consistently between
the experimental groups as well as between the findings at 2 and 8 weeks. The overall
median values representing TA in all groups were 14.890 mm2 for 2 weeks and 14.315 mm2
for 8 weeks. Statistically significant increases in NB and %NB were observed at 8 weeks
compared to 2 weeks in all groups except for the 90n group. In all groups, the overall
median values of NB increased from 0.975 mm2 for 2 weeks to 2.290 mm2 for 8 weeks, and
the values of %NB, the ratio of NB to TA, increased from 5.985% for 2 weeks to 16.128% for
8 weeks. However, there was no significant difference between the experimental groups
within the same period. On the other hand, RBG and %RBG showed a tendency to decrease
more at 8 weeks than at 2 weeks, including statistical significance only in the 90n group.
The overall median values of RBG and %RBG decreased from 2.405 mm2 and 15.3% for
2 weeks to 0.800 mm2 and 6.034% for 8 weeks, respectively. At 8 weeks, the RBG and %RBG
of the 76m group (3.580 mm2 /24.323%) were higher than those of the 90% OCP groups
with a significant difference. The 76n group (3.615 mm2 /21.968%) also showed higher
RBG and %RBG than the 90% OCP groups, but there was no statistical significance due to
 being strongly pressed by soft tissue and dura mater in the center of the defect, a thin, flat,
concave, disc-shaped tissue was observed. In the 8-weeks group, larger quantities of new
bone and osteoblasts were noted, predominantly originating from the periphery of the
Bioengineering 2024, 11, 215 defects. New bone was observed not only growing from the marginal area but also around 9 of 14
the centrally located remaining particles. The collagen membrane maintained a uniform
shape, but was absorbed compared to the findings at 2 weeks, and the amount of chronic
inflammatory cells around the membrane also decreased. ECM exhibited increased den-
large differences
sity compared between
to the animals.
appearance Theafter
observed results fromresulting
2 weeks, the histomorphometrical
in almost complete analysis
clo- are
summarized in Figure 7 and Table 2.
sure along the outer surface of the defect (Figure 6).

Bioengineering 2024, 11, x FOR PEER REVIEW 10 of 15

weeks. Statistically significant increases in NB and %NB were observed at 8 weeks com-
pared to 2 weeks in all groups except for the 90n group. In all groups, the overall median
values of NB increased from 0.975 mm2 for 2 weeks to 2.290 mm2 for 8 weeks, and the
values of %NB, the ratio of NB to TA, increased from 5.985% for 2 weeks to 16.128% for 8
weeks. However, there was no significant difference between the experimental groups
within the same period. On the other hand, RBG and %RBG showed a tendency to de-
crease more at 8 weeks than at 2 weeks, including statistical significance only in the 90n
group. The overall median values of RBG and %RBG decreased from 2.405 mm2 and 15.3%
for 2 weeks
Figure to 0.800 mm
6. Representative
2 and 6.034% for 8 weeks, respectively. At 8 weeks, the RBG
histological images of each experimental groups after 2 or 8 weeks. (HE
Figure 6. Representative histological images of each experimental groups after 2 or 8 weeks. (HE
and %RBG
stain, bar = 1of
mm)theAt76m group
2 weeks, (3.580
the new bonemm 2/24.323%)
formation were higher
was observed aroundthan thoseparticles
the grafted of the 90%
in OCP
stain, bar = 1margin
the adjacent mm) At of 2the
weeks,
defect the new
(blue bone At
arrows). formation
8 weeks, was observed
however, new around
bone wasthe graftednot
observed particles in
groups with a significant difference. The 76n group (3.615 mm 2/21.968%) also showed
the
onlyadjacent
growingmargin
from the of marginal
the defect (blue
area butarrows). At 8the
also around weeks, however,
centrally locatednew bone was
remaining observed not
particles
higher RBG and %RBG than the 90% OCP groups, but there was no statistical significance
(green
only arrows).from
growing The border of the defect
the marginal is indicated
area but by black
also around the dotted lines,
centrally and the
located direction of
remaining new (green
particles
due
bonetoformation
large differences
is indicatedbetween animals. The results from the histomorphometrical anal-
by black arrows.
arrows). The border of the defect is indicated by black dotted lines, and the direction of new bone
ysis are summarized in Figure 7 and Table 2.
formation is indicated by black arrows.
3.5. Histomorphometric Analysis
Except for a significant difference between the 90m (11.755 mm2) and 76n (16.720 mm2)
groups at 8 weeks, most of the TA results were measured consistently between the exper-
imental groups as well as between the findings at 2 and 8 weeks. The overall median val-
ues representing TA in all groups were 14.890 mm2 for 2 weeks and 14.315 mm2 for 8

Distributionof
Figure7.7.Distribution
Figure of experimental
experimental groups
groups and
and statistical
statistical significance
significancewithin
withingroups
groupsaccording
accordingto
histomorphometric analysis results. Small and capital letters of the same alphabet
to histomorphometric analysis results. Small and capital letters of the same alphabet indicate indicate significant
signif-
icant differences
differences between
between 2-week
2-week and 8-week
and 8-week results
results compared
compared in theinsame
the same experimental
experimental group. group.
Even
Even though
though therethere
is noissignificant
no significant difference,
difference, if a distinction
if a distinction is needed,
is needed, a prime
a prime markmark is used
is used next
next to the
toletter.
the letter. The asterisk
The asterisk indicates
indicates statistical
statistical significance.
significance. (A) Total
(A) Total augmented
augmented area,area, (B) new
(B) new bonebone
area,
area, (C) residual
(C) residual bonebone
graftgraft
area.area.

Table 2. Median with minimum and maximum values of all groups measured by histomorphomet-
ric analysis.
Bioengineering 2024, 11, 215 10 of 14

Bioengineering 2024, 11, x FOR PEER REVIEW 11 of 15

Bioengineering 2024, 11, x FOR PEER REVIEW 2024, 11, x FOR PEER REVIEW
Bioengineering Bioengineering 2024, 11, x FOR PEER REVIEW
Bioengineering 2024, 11, x FOR PEER REVIEW
Bioengineering 2024, 11, x FOR PEER REVIEW 11 of 15 11 of 15 11 of 15 11 15
of 15 11 of 15
Table 2. Median with minimum and maximum values of all groups measured
Bioengineering 2024, 11, x FOR PEER REVIEW
by histomorphometric analysis. 11 of

Week Group TA NB %NB RBG %RBG

Week Group Week Group
TA Week TA
Group NB
TA Week NB
Group NB%NB TA
Week
Week
Group
Group
RBG
%NB NB
TA %NB
TA Week RBG
%RBG
Group %NB NB
NB TA%RBG RBG %NB%NB RBG
NB RBG
%RBG
RBG %NB %RBG
%RBG RBG %RBG %RBG

90m 15.090 (11.450–16.170) 0.960 (0.420–1.240) a 5.937 (3.368–8.217) a 2.150 (1.470–3.990) a 14.515a (12.838–26.441) a
90m 90m
15.090 (11.450–16.170) 15.090 (11.450–16.170)
90m 0.960 (0.420–1.240)
15.090 a
(11.450–16.170)
0.960 (0.420–1.240) 90m 0.9605.937 a
(3.368–8.217)
(0.420–1.240) a
a15.090 (11.450–16.170) 90m 2.150
90m
5.937(1.470–3.990)
0.960a(0.420–1.240)
(3.368–8.217) a
15.090 (11.450–16.170)
5.937 (3.368–8.217)
15.090 (11.450–16.170)
14.515 (12.838–26.441)
2.150 (1.470–3.990)90m a
a
a5.937 (3.368–8.217) a
0.960 (0.420–1.240)
0.960 (0.420–1.240) a
a 14.515
15.090 2.150 (1.470–3.990)
(12.838–26.441)
(11.450–16.170)
a
5.937 a
(3.368–8.217)
2.150 (1.470–3.990)
5.937 (3.368–8.217) a
a
0.960 (0.420–1.240)2.150
a 2.150 a
14.515(1.470–3.990)
(12.838–26.441)
(1.470–3.990) a
a a
5.937 (3.368–8.217) a 14.515 (12.838–26.441)
14.515
14.515 (12.838–26.441)
a

(12.838–26.441)
2.150a (1.470–3.990) a a
14.515 (12.838–26.441) a

′ a′
1.340 (0.920–10.430) a
90n 17.010 (11.240–19.170) 0.200 (0.060–2.840) 1.7790.200
(0.495–15.477) 1.340 (0.920–10.430) a′ 11.744 (7.302–54.408) a′

2 weeks 2 weeks
90n 17.010
2 weeks (11.240–19.170)
90n 17.010 (11.240–19.170)
2 weeks 90n 17.010 0.200
(0.060–2.840)
2 weeks(0.060–2.840)
(11.240–19.170) 90n 0.2001.779 (0.495–15.477)
(0.060–2.840) 17.010 (11.240–19.170)
2 weeks 90n 90n 1.779 (0.495–15.477)
2 weeks
0.2001.779
1.340 (0.920–10.430) a′
(0.060–2.840) (0.495–15.477)
17.010 (11.240–19.170)
17.010 (11.240–19.170)
11.744 (7.302–54.408)
1.340 (0.920–10.430)
2 weeks
1.779 0.200
90n (0.495–15.477)
a′ a′
(0.060–2.840)
0.200 (0.060–2.840)
11.744
17.010 1.340 (0.920–10.430)
(7.302–54.408)
(11.240–19.170)
a′
1.779 a′
(0.495–15.477)
1.779 (0.495–15.477)
11.744(0.920–10.430)
0.200 (0.060–2.840)1.3401.340 (7.302–54.408)a′ a′ 1.779 (0.495–15.477)
(0.920–10.430) a′ 11.744 (7.302–54.408)
11.744 (7.302–54.408)
1.340
11.744 (7.302–54.408)
a′
a′ (0.920–10.430) a′ 11.744 (7.302–54.408) a′

(n=5)
76m (n=5)
14.690 (11.680–16.160) (n=5) (n=5) a′ 14.690 (n=5)
* *
′
a(0.030–1.470) 7.612 4.400
(0.257–10.007) (2.050–6.720) 29.952(2.050–6.720) (14.012–43.664)
(n = 5) 76m 14.690 (11.680–16.160) 1.160 (0.030–1.470) 7.6121.160
1.160
(0.257–10.007)
*
(0.030–1.470)4.400 (2.050–6.720)
* 7.612
29.952
(0.257–10.007)
1.160(14.012–43.664)
* * *(11.680–16.160) * ** 4.400 (2.050–6.720)
7.612 (0.257–10.007) * 29.95229.952
76m a′
14.690 (11.680–16.160) (0.030–1.470)a′a′
1.1607.612 (0.257–10.007)
(n=5)(11.680–16.160) 76m 4.400
a′
7.612(2.050–6.720) (n=5) 29.952 (14.012–43.664)
*
76m 14.690 (11.680–16.160) 76m (0.030–1.470) a′ (0.257–10.007) (0.030–1.470)a′ 4.400
14.690 (11.680–16.160) (2.050–6.720)
a′ 7.612 1.160
(0.257–10.007) a′ a′ 29.952 4.400 (2.050–6.720)
(14.012–43.664)
7.612 (0.257–10.007) a′ 29.952(2.050–6.720)
4.400 (14.012–43.664) (14.012–43.664)
76m 14.690 1.160 (0.030–1.470)
* * 4.400 29.952 (14.012–43.664)
a′ a′
76m 14.690 (11.680–16.160) 1.160 (0.030–1.470) a′ a′ (14.012–43.664)

* 0.990 (0.360–2.460) * * * * ′′ * * * * ′′ *
* (3.040–34.441) * 0.990 (0.360–2.460) 2.780 *
76n 13.560 76n(11.780–22.560) 0.990 (0.360–2.460) a
* 6.033 13.560(3.056–10.904)
* * (3.040–34.441) a
6.033 2.780
(3.056–10.904) (0.400–7.770) 20.501
(0.400–7.770) (3.040–34.441)
* * *20.501 ** 6.033 (3.056–10.904)* 20.50120.501
13.560 (11.780–22.560) 6.0330.990 (0.360–2.460) a′′
0.9906.033 (3.056–10.904)
13.560 (11.780–22.560) 76n 2.780
a′′
6.033(0.400–7.770) 20.501
* *
76n 13.560 (11.780–22.560) 76n (0.360–2.460) a′′ 0.990(3.040–34.441)
(3.056–10.904) (0.360–2.460)a′′ 2.780 (0.400–7.770)
(11.780–22.560) a′′ 6.033 0.990
(3.056–10.904)
(0.360–2.460) a′′ a′′ 2.780 (0.400–7.770)
6.033 (3.056–10.904) a′′ 20.501(0.400–7.770)
(3.040–34.441) (3.040–34.441)
* *
76n 13.560 (11.780–22.560) a′′ (3.056–10.904) a′′ 2.780 (0.400–7.770) 20.501
* *
76n 13.560 (11.780–22.560) 76n
0.990 (0.360–2.460) a′′ 13.560 (11.780–22.560) a′′ a′′
2.780 (0.400–7.770) a′′ 2.780
(3.040–34.441) 20.501 (3.040–34.441)

Overall 14.890 (11.240–22.560)

Overall 0.975 (0.030–2.840)
14.890 (11.240–22.560) Overall 0.9755.985 (0.257–15.477)
(0.030–2.840) Overall 2.405
14.890 (11.240–22.560) 5.985(0.400–10.430)
0.975 (0.030–2.840)
(0.257–15.477) 15.300 (3.040–54.408)
2.405 (0.400–10.430)
14.890 (11.240–22.560) 5.985 0.975
(0.257–15.477)
(0.030–2.840) 15.300 2.405 (0.400–10.430)
(3.040–54.408)
5.985 (0.257–15.477)0.975 (0.030–2.840) 15.300(0.400–10.430)
2.405 (3.040–54.408) 15.300 (3.040–54.408)
Overall Overall 14.890 (11.240–22.560)
14.890 (11.240–22.560) 0.975 (0.030–2.840) 0.975 (0.030–2.840)
5.985 (0.257–15.477) 2.405 (0.400–10.430) Overall 5.985 (0.257–15.477)
14.890 (11.240–22.560)
15.300 (3.040–54.408) Overall
0.975 (0.030–2.840) 14.890 (11.240–22.560)
2.405 (0.400–10.430)
5.985 (0.257–15.477) 2.405 (0.400–10.430) 5.985 (0.257–15.477) 15.300 (3.040–54.408) 15.300 (3.040–54.408)
2.405 (0.400–10.430) 15.300 (3.040–54.408)

* 90m* *90m 90m * 1.196*(0.000–9.796) *0.155 (0.000–1.390)

90m 11.755 (10.790–16.770) 2.245 (1.480–3.920)
* 2.24519.840 (12.726–27.625) 0.155
* (12.726–27.625)
(0.000–1.390)
(0.000–1.390)*A
A A A11.755 A A
2.245A (1.480–3.920)A A
* 0.155* (0.000–1.390) A
90m 11.755 (10.790–16.770) A (1.480–3.920) (10.790–16.770) A 19.840 2.245 (1.480–3.920)
A A
A0.155 (0.000–1.390)
11.755 (10.790–16.770) A A19.840 (12.726–27.625) 1.196 (0.000–9.796) A A
A 19.840 (12.726–27.625)
A 1.196(0.000–1.390)
0.155 (0.000–9.796)AA 19.840 (12.726–27.625) A 1.1961.196 (0.000–9.796) A
A * A * A
90m 11.755 (10.790–16.770) (12.726–27.625) A 2.245 (1.480–3.920)0.155 (0.000–9.796) 0.155
1.196 (0.000–9.796) A
(0.000–1.390) A 1.196 (0.000–9.796) A
A

*
11.755 (10.790–16.770) 2.245 (1.480–3.920) A 19.840 A

90m
90m 11.755 (10.790–16.770)
11.755 (10.790–16.770) A 2.245 (1.480–3.920) A
2.245 (1.480–3.920)
19.840 (12.726–27.625) A 0.155 (0.000–1.390) A
19.840
1.196 (0.000–9.796)(12.726–27.625) A

90n* ′ A′A′
90n 14.735 (9.970–17.130)
*
2.015 (1.160–3.620) 2.01515.691 (8.033–21.133)
8 weeks*90n 90n 15.691
0.180 (0.000–1.010)
* 1.087*(0.000–6.994) *(0.000–6.994) A′(0.000–6.994)
A′ A′

* * 1.08715.691 A*
90n 14.735 (9.970–17.130) (1.160–3.620) 14.735 (9.970–17.130) 2.015 (1.160–3.620)
(8.033–21.133) 0.180 (0.000–1.010)
14.735 (9.970–17.130) 15.6912.015
(8.033–21.133)
A′
(1.160–3.620) 1.087 0.180 (0.000–1.010)
A′ A′
15.691 (8.033–21.133) 1.087(0.000–1.010)
0.180 (0.000–6.994) 1.087 (0.000–6.994) A′

*
8 weeks
90n 14.735 (9.970–17.130) 8 weeks
2.015 (1.160–3.620)
8 weeks
(0.000–6.994)* (8.033–21.133)
90n 14.735 (9.970–17.130)
0.180* (0.000–1.010)
15.691 (8.033–21.133) 2.015 (1.160–3.620)0.180 (0.000–1.010) A′ 15.691 (8.033–21.133) 1.087 (0.000–6.994) 0.180 (0.000–1.010) A′ 1.087 A′

* 1.087* (0.000–6.994)
14.735 (9.970–17.130) 2.015 (1.160–3.620) A′

* * ** * * *
8 weeks
8 weeks 8 weeks 90n 14.735 (9.970–17.130)
*
2.015 (1.160–3.620)
* * 15.691 (8.033–21.133) *
0.180 (0.000–1.010)
8 weeks A′
* * A′
* * * *
*
(n=5) (n=5) (n=5)
A′13.070
(n=5)
*76m 76m 3.580 *(0.170–4.580) A′ 3.580 (0.170–4.580) B 24.323B
76m 13.070 (9.940–16.450)
76m 2.465 (1.310–5.030)
13.070 (9.940–16.450) A′
76m 2.46516.479 (11.313–50.604)
(1.310–5.030) (n=5)(9.940–16.450)
A′ (0.170–4.580)
16.479 A′
2.465 (1.310–5.030)
(11.313–50.604) B
3.580(n=5)
13.070 (9.940–16.450) A′ 24.323 (1.468–32.068)
A′ 16.4792.465
(11.313–50.604)
B
(1.310–5.030) B A′ A′ 24.323 3.580 (0.170–4.580)
(1.468–32.068) B B
16.479 (11.313–50.604) A′ 24.323(0.170–4.580)
3.580 (1.468–32.068)
B B
24.323 (1.468–32.068) B

(n = 5) (n=5) 76m
76m
13.070 (9.940–16.450)
13.070 (9.940–16.450) 2.465 (1.310–5.030) A′ 2.465 (1.310–5.030)
16.479 (11.313–50.604) 3.580 (0.170–4.580)
A′ B 16.479 (11.313–50.604)
13.070 (9.940–16.450)
24.323 (1.468–32.068)
76m
2.465 (1.310–5.030)
B
A′ 13.070 (9.940–16.450)
16.479 (11.313–50.604) 2.465 (1.310–5.030)3.580 (0.170–4.580)
A′ A′ B 16.479 (11.313–50.604) A′
24.323 (1.468–32.068) (1.468–32.068)
3.580 (0.170–4.580)
24.323 (1.468–32.068) B B B

76n 16.720 (13.190–20.550) B

76n 16.720 (13.190–20.550)
76n B 2.505 (1.120–3.320)
16.720 (13.190–20.550)
2.505
A′′
76n 2.50515.688
(1.120–3.320)
B
A′ 16.720
(1.120–3.320) ′ (13.190–20.550) 76n 3.615
(7.349–16.831) A′′
76n
(0.000–7.650)
15.688 A′′ B2.505 (1.120–3.320)
(7.349–16.831)
15.688 16.720 (13.190–20.550)
(7.349–16.831)
16.720 (13.190–20.550)
A′′ 21.968 (0.000–37.226)
3.615 (0.000–7.650)A′′15.6882.505
76n A ′′
(7.349–16.831)
(1.120–3.320)
B
2.505 (1.120–3.320)
B A′′
A′′ A′′ 21.968
16.720 3.615 (0.000–7.650)
(0.000–37.226)
(13.190–20.550) B15.688 (7.349–16.831)2.505 (1.120–3.320)
15.6883.615
(7.349–16.831) (0.000–7.650)
A′′
A′′
A′′ 21.968(0.000–7.650)
3.615 (0.000–37.226)
3.615 (0.000–7.650) 15.688 (7.349–16.831) A′′ 21.968 (0.000–37.226)
21.968 (0.000–37.226)
3.615 (0.000–7.650)
21.968 (0.000–37.226) 21.968 (0.000–37.226)
76n 16.720 (13.190–20.550) B 2.505 (1.120–3.320) A′′ 15.688 (7.349–16.831) A′′ 3.615 (0.000–7.650) 21.968 (0.000–37.226)
Overall 14.315 (9.940–20.550)
Overall 2.290 (1.120–5.030)
14.315 (9.940–20.550) Overall 2.29016.128 (7.349–50.604)
(1.120–5.030) Overall 0.800
14.315 (9.940–20.550) (0.000–7.650)
16.128 2.290 (1.120–5.030)
(7.349–50.604) 6.034 (0.000–37.226)
0.800 (0.000–7.650)
14.315 (9.940–20.550) 16.1282.290
(7.349–50.604)
(1.120–5.030) 6.034 0.800 (0.000–7.650)
(0.000–37.226)16.128 (7.349–50.604) 6.034(0.000–7.650)
0.800 (0.000–37.226) 6.034 (0.000–37.226)
Overall 14.315 (9.940–20.550) Overall
2.290 (1.120–5.030) 14.315 (9.940–20.550)
16.128 (7.349–50.604) 2.290 (1.120–5.030)0.800 (0.000–7.650) 16.128 (7.349–50.604) 6.034 (0.000–37.226)0.800 (0.000–7.650) 6.034 (0.000–37.226)
Overall Overall
14.315 (9.940–20.550)
14.315 (9.940–20.550) 2.290 (1.120–5.030)TA: Total augmented 16.128 NB:
2.290
area,(7.349–50.604)
TA: New
Totalbone
(1.120–5.030)
area, %NB:
augmented 0.800ratio
area, NB: of NBbone
New
(0.000–7.650) toTA:
TA, RBG:
Total
area, residual
ratio bone
augmented
%NB: ofarea,
16.128
NBgraft
6.034 NB:
to area,
TA,New
(0.000–37.226)
(7.349–50.604)
TA:%RBG:
RBG: bone
Total ratio%NB:
area,
residual of RBG
bone
augmented toarea,
ratio
graft
area,TA.
of NB
NB: Significant
to TA,
%RBG:
New differences
RBG:
ratio
bone ofresidual
RBG
area, %NB:
0.800
to(p<0.05)
bone
TA. graft
(0.000–7.650)
of NBarea,
ratioSignificant %RBG:
to TA, RBG:ratio
differences of RBG
(p<0.05)
residual to graft
bone TA. Significant differences
area, %RBG:
6.034 (0.000–37.226)
(p<0.05)
ratio of RBG to TA. Significant differences (p<0.05)
TA: Total augmented area, NB: New bone area, TA: Totalratio
%NB: augmented
of NB toarea, NB: New
TA, RBG: bonebone
residual area,graft
%NB: ratio
area, of NBratio
%RBG: to TA, ofRBG:
RBG to residual bone graftdifferences
TA. Significant area, %RBG: ratio of RBG to TA. Significant differences (p<0.05)
(p<0.05)
between the four experimental groups
between theare indicated
four by different
experimental groupssmall
between
are lettersthe
indicated at four
2by
weeks and small
by different
experimental
different groups
letters capital
are
at letters
indicated
2 the
weeks and at
by8different
by weeks. Small
differentsmall and capital
letters
capital letters
at 2atweeks
letters 8by byof
and
weeks. the
by
Small different
and capital
capital
between
between the fourfour experimental
experimental groups
groups are are indicated
between
indicated the different
four
different small
experimental
small letters
letters groups letters
atletters
2atweeks
2 are
weeks at 8and
and weeks.
of indicated
the byby Small and
different
different
by different capitalcapital
capital
small letters
letters
at 8atat
letters
letters 8of
weeks.the Small
2weeks.
weeks Small
byand
andand capital
different
capital letters
capital
letters of the at 8 weeks. Small and capital letters of the
letters
of the
TA: Total augmented area, NB: New bone area, %NB: ratio of NB to TA, RBG: residual bone graft area, %RBG: ratio of RBG to TA. Significant differences (p < 0.05) between the four
TA: Total augmented area, NB: Newsame
bone area, %NB:
alphabet ratio
indicate ofsame
NB alphabet
significant to TA, RBG:
differences residual
between
indicate bonedifferences
2-week
significant graft
and area,
8-week
same %RBG:
results
alphabet
between ratio of
compared
indicate
2-week RBG
in the to
significant
and 8-week
same
TA.
same
same Significant
experimental
differences
results
alphabet
alphabet between
compared differences
group.
in
indicate
indicate
Even
2-week
the (p<0.05)
though
and
same
significant
significant 8-weekthere
experimental
differences
differences
isbetween
results
same no
between
significant
compared
group.
alphabetEven
2-week
2-week in the
though
and
indicate
and same
there experimental
8-weekis no
significant
8-week results
resultsdifferences
comparedgroup.
significant
compared Even
in the
between
in the though
same
2-week
same there
and is nogroup.
experimental
8-week
experimental significant
group.
results Even though
compared
Even though there
inthere
the sameis significant
is no no significant group. Even though there is no significant
experimental
between the four experimental groups are indicated
difference, by different
if a distinction small
is difference,
needed, ifletters
a prime at 2isweeks
mark
a distinction used andtoby
next
is needed, different
a the letter.
difference,
prime markThe capital letters
isaasterisk
if distinction
used next atneeded,
indicates
toisthe8difference,
weeks.
statistical
letter. Small
a prime
The
difference, mark
asterisk
if aand
significance. capital
is used
indicates
distinction letters
next
is to of
the
statistical
needed, the
letter.
a prime The
significance. asterisk
mark is indicates
used next statistical
to the significance.
letter. The asterisk indicates statistical significance.
experimental groups are indicated by different small letters at 2 weeks and by different capital letters at 8 weeks. Small and capital letters of the same alphabet indicate significant
same alphabet indicate significant differences between 2-week and 8-week results compared in the same experimental group. Even though there is no significant
if a distinction is needed, a prime mark difference,
is usedif next
a distinction is needed,
to the letter. a prime
The asterisk mark isstatistical
indicates used nextsignificance.
to the letter. The asterisk indicates statistical significance.

differences
difference, between
if a distinction is needed, 2-week and
a prime mark 8-week
is used results
next to the letter. Thecompared instatistical
asterisk indicates the same experimental group. Even though
there is no significant difference, if a distinction is needed, a prime mark is used
significance.
next to the letter. The asterisk indicates statistical significance.
Bioengineering 2024, 11, 215 11 of 14

4. Discussion
OCP has been proven to be effective in new bone formation due to its high bone re-
generation ability and rapid bioabsorption compared to xenogeneic bone or other synthetic
bone [2]. Earlier investigations have been conducted to assess the efficacy of innovative
biomaterials derived from amorphous calcium phosphate (ACP), octacalcium phosphate
(OCP), and hydroxyapatite (HA) in addressing critical size defects in the rabbit skull.
Therefore, this study did not establish separate positive and negative controls [29].
According to the solubility isotherm, calcium phosphate materials demonstrate a
solubility sequence in the order of HA < TCP < OCP < DCPD (dicalcium phosphate
dehydrate) at pH 7.4. Although a high osteogenesis capacity is more important than
solubility, OCP, which has higher solubility than TCP, can be considered advantageous
for inducing new bone tissue [30]. In other words, OCP causes a rapid bone regeneration
compared to TCP because it has high biodegradability (rate of disappearance) through
cellular phagocytosis in vivo and at the same time has an excellent bone formation ability.
The basic theory for using BCP as a commonly used synthetic bone substitute is that new
bone formation and resorption can be regulated by the HA:TCP ratio [31]. For BCP, the
degree of solubility depends on the β-TCP/HA ratio, with higher ratios increasing the
solubility [32,33]. In the case of MBCP, it is suggested to mix β -TCP and HA in a ratio of
80:20% to form hard particles that are easy to handle for application to the defect [34].
OCP has been difficult to develop as a synthetic bone substitute because its original
crystal structure could be changed during the sintering process, making it brittle and
fragile, and unable to be formed into larger solid masses [35]. These limitations have
led most of studies on the commercialization of OCP-based bone substitutes to focus on
coating OCP onto DBBM or combining it with collagen [36,37]. Previous investigations on
OCP encompassed studies involving OCP granules and composites with biodegradable
polymers like collagen, gelatin, hyaluronic acid, alginate, polycaprolactone (PCL), chitosan,
silk fibroin, and polylactic-co-glycolic acid (PLGA) [38]. Recently, a novel synthesis method,
eliminating the need for sintering, has been devised to enable the large-scale production of
high-purity OCP with enhanced physical properties [3]. However, studies on the difference
in osteogenesis ability depending on the concentration of OCP are still difficult to find as
far as we know.
The usefulness of 8 mm defect formation in rabbit calvaria for new bone formation has
been fully validated in previous studies [28]. In this study, four defects with a diameter of
8 mm were formed in rabbit calvaria, and the new bone formation ability according to the
difference in OCP concentration and whether or not the collagen membrane was applied
was compared after 2 and 8 weeks, respectively. The two concentrations of commercial
OCP synthetic bone substitutes used in this study were prepared by mixing OCP and
HA in weight ratios of 90:10 and 76:24, respectively. In the micro-CT results, significantly
greater amounts of BV and BV/TV in the 76% groups than in the 90% groups at 2 weeks
demonstrated that osteogenesis was rapidly induced in the OCP group containing more
HA as a scaffold. Although significantly increased BV and BV/TV were observed at
76% OCP at week 2, this result does not necessarily mean rapid new bone formation,
which means that the grafted space is relatively sufficiently maintained. According to
the histomorphometric results, new bone formation indices such as NB and %NB were
greater at 8 weeks compared to 2 weeks, but there was no significant difference between
the experimental groups. However, the reduced differences in RBG and %RBG between the
2- and 8-week results were significantly greater in the 90% groups. In the 76% groups, the
graft material was not absorbed to a significant extent over time and was mostly maintained.
As a result, the indices related to the RBG showed significantly greater results in the 76%
groups than in the 90% groups at 8 weeks. A thin, flat, concave, disc-shaped tissue observed
in histological findings for the 90% groups at 2 weeks also showed that high concentrations
of OCP were related to rapid biodegradation.
A resorbable collagen membrane was also used in this study, considering the char-
acteristics of synthetic bone graft materials that lack rigidity in terms of maintaining the
Bioengineering 2024, 11, 215 12 of 14

defect space. As a result, it was observed that the two types of OCP maintained the graft
material stably, without any histologically specific complications or abnormal findings,
regardless of whether the collagen membrane was used or not. An ideal bone substitute
material should stabilize the space of the defect during the period of new bone formation,
which is closely related to the concentration of the bone graft material [39]. Regarding the
bone substitute material used in this study, 76% OCP containing 24% HA could be applied
as granules of relatively large particles, while 90% OCP containing 10% HA was difficult
to apply as a slurry type. Although there was no statistical significance, all of the indices
related to NB and RBG at 2 weeks showed greater values in the membrane-application
groups (90m, 76m) than in the non-application groups (90n, 76n) This fact suggests that
the membrane is effective in keeping the initial OCP volume stable. Therefore, despite the
faster bone regeneration effect in high-concentration OCP, it is difficult to continuously
maintain the augmented volume, so the use of a collagen membrane for long-term stability
is effective.
In all experimental groups in this study, a similar degree of new bone formation was
observed at 8 weeks, but it was confirmed that there were differences in the amount of re-
maining graft material and the rate of new bone formation depending on the concentration
of OCP. In general, bone regeneration in rabbits progresses at about three times the speed
of humans [40]. Therefore, 2 weeks of observation in rabbits corresponds to 1.5 months
in humans and 8 weeks of observation in rabbits corresponds to 6 months in humans. In
cases where OCP is applied for GBR, it is recommended to adjust the OCP concentration
ensuring that the resorption rate of the graft material is similar to the rate of new bone
formation. Compared to previous studies using OCP, this study has value as an in vivo
experiment that directly compares the results depending on the application method of OCP
bone substitute and whether a collagen membrane is applied and determines a clinically
efficient method.

5. Conclusions
In conclusion, OCP synthetic bone substitutes with two different compositions all
showed excellent osteoconduction efficacy, but there were differences in their space-
maintaining capabilities depending on the OCP concentration. Although it did not have a
significant effect on new bone formation, the use of collagen membranes could be effective
when using a high concentration of OCP, which lacks the capability of maintaining space.
Further research will be needed to determine the most suitable concentration for OCP to
exhibit osteoconductive efficacy while maintaining the defect space.

Author Contributions: Conceptualization, J.-W.P. and S.-H.C.; methodology, J.-W.P.; software, S.-W.P.;
validation, S.K.; formal analysis, S.-W.P.; investigation, D.-N.L.; resources, Y.-W.S.; data curation,
J.-Y.P.; writing—original draft preparation, S.-W.P.; writing—review and editing, J.-W.P.; visualization,
S.-W.P.; supervision, S.K.; project administration, J.-W.P.; funding acquisition, S.-H.C. All authors
have read and agreed to the published version of the manuscript.
Funding: This research was supported by a grant of the Korea Health Technology R&D Project
through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health
& Welfare, Republic of Korea (grant number: HI20C2114).
Institutional Review Board Statement: The animal study protocol was approved by the Institutional
Animal Care and Use Committee, Yonsei Medical Center, Seoul, Korea (approval number 2021-0237).
Informed Consent Statement: Not applicable.
Data Availability Statement: The datasets generated or analyzed during the current study are
available from the corresponding author upon reasonable request.
Acknowledgments: The authors would like to thank Joosung Kim and Sukyoung Kim (HudensBio
Co., Ltd., Gwangju, Republic of Korea) for kindly providing information related to the preparation
and properties of the octacalcium phosphate material used in this study.
Conflicts of Interest: The authors declare no conflicts of interest.
Bioengineering 2024, 11, 215 13 of 14

References
1. Haugen, H.J.; Lyngstadaas, S.P.; Rossi, F.; Perale, G. Bone grafts: Which is the ideal biomaterial? J. Clin. Periodontol. 2019, 46
(Suppl. 21), 92–102. [CrossRef]
2. Kim, J.; Kim, S.; Song, I. Biomimetic Octacalcium Phosphate Bone Has Superior Bone Regeneration Ability Compared to
Xenogeneic or Synthetic Bone. Materials 2021, 14, 5300. [CrossRef] [PubMed]
3. Kim, J.-S.; Jang, T.-S.; Kim, S.-Y.; Lee, W.-P. Octacalcium Phosphate Bone Substitute (Bontree® ): From Basic Research to Clinical
Case Study. Appl. Sci. 2021, 11, 7921. [CrossRef]
4. Bucholz, R.W. Nonallograft osteoconductive bone graft substitutes. Clin. Orthop. Relat. Res. 2002, 395, 44–52. [CrossRef] [PubMed]
5. Bohner, M.; Santoni, B.L.G.; Döbelin, N. β-tricalcium phosphate for bone substitution: Synthesis and properties. Acta Biomater.
2020, 113, 23–41. [CrossRef] [PubMed]
6. Arinzeh, T.L.; Tran, T.; McAlary, J.; Daculsi, G. A comparative study of biphasic calcium phosphate ceramics for human
mesenchymal stem-cell-induced bone formation. Biomaterials 2005, 26, 3631–3638. [CrossRef] [PubMed]
7. Ebrahimi, M.; Botelho, M.G.; Dorozhkin, S.V. Biphasic calcium phosphates bioceramics (HA/TCP): Concept, physicochemical
properties and the impact of standardization of study protocols in biomaterials research. Mater. Sci. Eng. C Mater. Biol. Appl. 2017,
71, 1293–1312. [CrossRef] [PubMed]
8. Kawai, T.; Tanuma, Y.; Matsui, K.; Suzuki, O.; Takahashi, T.; Kamakura, S. Clinical safety and efficacy of implantation of
octacalcium phosphate collagen composites in tooth extraction sockets and cyst holes. J. Tissue Eng. 2016, 7, 2041731416670770.
[CrossRef]
9. Fowler, B.O.; Moreno, E.C.; Brown, W.E. Infra-red spectra of hydroxyapatite, octacalcium phosphate and pyrolysed octacalcium
phosphate. Arch. Oral. Biol. 1966, 11, 477–492. [CrossRef]
10. LeGeros, R.Z. Properties of osteoconductive biomaterials: Calcium phosphates. Clin. Orthop. Relat. Res. 2002, 395, 81–98.
[CrossRef]
11. Detsch, R.; Hagmeyer, D.; Neumann, M.; Schaefer, S.; Vortkamp, A.; Wuelling, M.; Ziegler, G.; Epple, M. The resorption of
nanocrystalline calcium phosphates by osteoclast-like cells. Acta Biomater. 2010, 6, 3223–3233. [CrossRef]
12. Honda, Y.; Anada, T.; Kamakura, S.; Morimoto, S.; Kuriyagawa, T.; Suzuki, O. The effect of microstructure of octacalcium
phosphate on the bone regenerative property. Tissue Eng. Part A 2009, 15, 1965–1973. [CrossRef] [PubMed]
13. Miyatake, N.; Kishimoto, K.N.; Anada, T.; Imaizumi, H.; Itoi, E.; Suzuki, O. Effect of partial hydrolysis of octacalcium phosphate
on its osteoconductive characteristics. Biomaterials 2009, 30, 1005–1014. [CrossRef] [PubMed]
14. Murakami, Y.; Honda, Y.; Anada, T.; Shimauchi, H.; Suzuki, O. Comparative study on bone regeneration by synthetic octacalcium
phosphate with various granule sizes. Acta Biomater. 2010, 6, 1542–1548. [CrossRef] [PubMed]
15. Tanuma, Y.; Anada, T.; Honda, Y.; Kawai, T.; Kamakura, S.; Echigo, S.; Suzuki, O. Granule size-dependent bone regenerative
capacity of octacalcium phosphate in collagen matrix. Tissue Eng. Part A 2012, 18, 546–557. [CrossRef] [PubMed]
16. Liu, H.; Webster, T.J. Nanomedicine for implants: A review of studies and necessary experimental tools. Biomaterials 2007, 28,
354–369. [CrossRef] [PubMed]
17. Eliaz, N.; Metoki, N. Calcium Phosphate Bioceramics: A Review of Their History, Structure, Properties, Coating Technologies and
Biomedical Applications. Materials 2017, 10, 334. [CrossRef]
18. Wang, H.L.; Boyapati, L. “PASS” principles for predictable bone regeneration. Implant. Dent. 2006, 15, 8–17. [CrossRef]
19. Sbricoli, L.; Guazzo, R.; Annunziata, M.; Gobbato, L.; Bressan, E.; Nastri, L. Selection of Collagen Membranes for Bone
Regeneration: A Literature Review. Materials 2020, 13, 786. [CrossRef]
20. Bunyaratavej, P.; Wang, H.L. Collagen membranes: A review. J. Periodontol. 2001, 72, 215–229. [CrossRef]
21. Jeong, C.H.; Kim, J.; Kim, H.S.; Lim, S.Y.; Han, D.; Huser, A.J.; Lee, S.B.; Gim, Y.; Ji, J.H.; Kim, D.; et al. Acceleration of bone
formation by octacalcium phosphate composite in a rat tibia critical-sized defect. J. Orthop. Transl. 2022, 37, 100–112. [CrossRef]
22. Bergerhoff, G.; Hundt, R.; Sievers, R.; Brown, I.D. The inorganic crystal structure data base. J. Chem. Inf. Comput. Sci. 1983, 23,
66–69. [CrossRef]
23. Kilkenny, C.; Browne, W.J.; Cuthill, I.C.; Emerson, M.; Altman, D.G. Improving bioscience research reporting: The arrive
guidelines for reporting animal research. J. Pharmacol. Pharmacother. 2010, 1, 94–99. [CrossRef] [PubMed]
24. Pae, H.C.; Kang, J.H.; Cha, J.K.; Lee, J.S.; Paik, J.W.; Jung, U.W.; Kim, B.H.; Choi, S.H. 3d-printed polycaprolactone scaffold mixed
with β-tricalcium phosphate as a bone regenerative material in rabbit calvarial defects. J. Biomed. Mater. Res. Part B Appl. Biomater.
2019, 107, 1254–1263. [CrossRef] [PubMed]
25. Kamal, M.; Andersson, L.; Al-Asfour, A.; Bartella, A.K.; Gremse, F.; Rosenhain, S.; Gabato, S.; Hölzle, F.; Kessler, P.; Lethaus, B.
Bone regeneration in rabbit calvarial critical-sized defects filled with composite in situ formed xenogenic dentin and biphasic
tricalcium phosphate/hyroxyapatite mixture. J. Biomed. Mater. Res. Part B Appl. Biomater. 2019, 107, 773–782. [CrossRef] [PubMed]
26. Xu, S.; Lin, K.; Wang, Z.; Chang, J.; Wang, L.; Lu, J.; Ning, C. Reconstruction of calvarial defect of rabbits using porous calcium
silicate bioactive ceramics. Biomaterials 2008, 29, 2588–2596. [CrossRef] [PubMed]
27. Kim, J.; McBride, S.; Fulmer, M.; Harten, R.; Garza, Z.; Dean, D.D.; Sylvia, V.L.; Doll, B.; Wolfgang, T.L.; Gruskin, E. Fiber-
reinforced calcium phosphate cement formulations for cranioplasty applications: A 52-week duration preclinical rabbit calvaria
study. J. Biomed. Mater. Res. Part B Appl. Biomater. 2012, 100, 1170–1178. [CrossRef] [PubMed]
28. Sohn, J.-Y.; Park, J.-C.; Um, Y.-J.; Jung, U.-W.; Kim, C.-S.; Cho, K.-S.; Choi, S.-H. Spontaneous healing capacity of rabbit cranial
defects of various sizes. J. Periodontal Implant. Sci. 2010, 40, 180–187. [CrossRef]
Bioengineering 2024, 11, 215 14 of 14

29. Guastaldi, F.P.S.; Matheus, H.R.; Faloni, A.P.D.S.; de Almeida-Filho, E.; Cominotte, M.A.; Moretti, L.A.C.; Verzola, M.H.A.;
Marcantonio, E., Jr.; de Almeida, J.M.; Guastaldi, A.C. A new multiphase calcium phosphate graft material improves bone
healing—An in vitro and in vivo analysis. J. Biomed. Mater. Res. Part B Appl. Biomater. 2022, 110, 2686–2704. [CrossRef]
30. Suzuki, O.; Shiwaku, Y.; Hamai, R. Octacalcium phosphate bone substitute materials: Comparison between properties of
biomaterials and other calcium phosphate materials. Dent. Mater. J. 2020, 39, 187–199. [CrossRef]
31. Daculsi, G. Biphasic calcium phosphate concept applied to artificial bone, implant coating and injectable bone substitute.
Biomaterials 1998, 19, 1473–1478. [CrossRef]
32. Daculsi, G.; LeGeros, R.Z.; Nery, E.; Lynch, K.; Kerebel, B. Transformation of biphasic calcium phosphate ceramics in vivo:
Ultrastructural and physicochemical characterization. J. Biomed. Mater. Res. 1989, 23, 883–894. [CrossRef]
33. Daculsi, G.; LeGeros, R.Z.; Mitre, D. Crystal dissolution of biological and ceramic apatites. Calcif. Tissue Int. 1989, 45, 95–103.
[CrossRef]
34. Gauthier, O.; Bouler, J.M.; Aguado, E.; Pilet, P.; Daculsi, G. Macroporous biphasic calcium phosphate ceramics: Influence of
macropore diameter and macroporosity percentage on bone ingrowth. Biomaterials 1998, 19, 133–139. [CrossRef] [PubMed]
35. Kamakura, S.; Sasaki, K.; Homma, T.; Honda, Y.; Anada, T.; Echigo, S.; Suzuki, O. The primacy of octacalcium phosphate collagen
composites in bone regeneration. J. Biomed. Mater. Res. A 2007, 83, 725–733. [CrossRef]
36. Jung, Y.; Kim, W.H.; Lee, S.H.; Ju, K.W.; Jang, E.H.; Kim, S.O.; Kim, B.; Lee, J.H. Evaluation of New Octacalcium Phosphate-Coated
Xenograft in Rats Calvarial Defect Model on Bone Regeneration. Materials 2020, 13, 4391. [CrossRef] [PubMed]
37. Kawai, T.; Matsui, K.; Ezoe, Y.; Kajii, F.; Suzuki, O.; Takahashi, T.; Kamakura, S. Efficacy of Octacalcium Phosphate Collagen
Composite for Titanium Dental Implants in Dogs. Materials 2018, 11, 229. [CrossRef]
38. Suzuki, O.; Hamai, R.; Sakai, S. The material design of octacalcium phosphate bone substitute: Increased dissolution and
osteogenecity. Acta Biomater. 2023, 158, 1–11. [CrossRef]
39. Zhao, R.; Yang, R.; Cooper, P.R.; Khurshid, Z.; Shavandi, A.; Ratnayake, J. Bone Grafts and Substitutes in Dentistry: A Review of
Current Trends and Developments. Molecules 2021, 26, 3007. [CrossRef] [PubMed]
40. Clark, C.L.; Strider, J.; Hall, C.; Ferguson, H.W.; Armstrong, K.L.; Runner, R.R.; Baur, D.A. Distraction osteogenesis in irradiated
rabbit mandibles with adjunctive hyperbaric oxygen therapy. J. Oral. Maxillofac. Surg. 2006, 64, 589–593. [CrossRef] [PubMed]

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.