1-s2.0-S1991790221000209

Journal of Dental Sciences 16 (2021) 1214e1221
Available online at www.sciencedirect.com
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journal homepage: www.e-jds.com
Original Article
Bone formation with functionalized 3D

printed poly-ε-caprolactone scaffold with
plasma-rich-fibrin implanted in critical-sized
calvaria defect of rat
Min-Chia Chen a,1, Hsien-Chung Chiu a,1, Po-Jan Kuo a,
Cheng-Yang Chiang a, Martin M. Fu a, Earl Fu b*
a
Department of Periodontology, School of Dentistry, National Defense Medical Center and Tri-Service
General Hospital, Taipei, Taiwan, ROC
b
Department of Dentistry, Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, New Taipei
City, Taiwan, ROC
Received 16 November 2020; Final revision received 29 January 2021

Available online 13 March 2021
KEYWORDS Abstract Background/purpose: Space-making is one of the essential factors for bone regen-
Bone tissue eration in severe bony defect. To test the hypothesis that an appropriately designed scaffold
engineering; may be beneficial for the bone formation in defect, the new bone formed in the critical-size
3-D printing scaffold; calvarial defect of rat was examined after implanted with a 3D-printed poly-3-caprolactone
Polycaprolactone; (PCL) scaffold, retaining with and without plasma rich fibrin (PRF).
Rat calvaria defect; Materials and methods: Thirty-two rats were divided into four groups (control, PCL, PRF, and
Platelet-rich fibrin PCL-plus-PRF). A custom-made 3D-printed PCL scaffold, 900 mm in pore size, retaining with and
without PRF, was implanted into a critical-sized calvarial defect, 6 mm in diameter. Animals
were sacrificed at week-4 or 8 after implantation for assessing the new bone formation by
dental radiography, micro-computed tomography (m-CT), and histology.
Results: By radiography and m-CT, significantly greater mineralization areas/volumes were
observed in defects with 3D-printed scaffold groups compared to that without the scaffold
in both two-time points. However, no advantage was found by adding PRF. Histology showed
that bone tissues grew into the central zone of the critical defect when 3D-printed PCL scaf-
fold was present. In contrast, for the groups without the scaffolds, new bones were formed
mostly along defect borders, and the central zones of the defects were collapsed and healed
with thin connective tissue.
Conclusion: Our results suggest that the use of a 900 mm pore size 3D-printed PCL scaffold may
have the potential in facilitating the new bone formation.
* Corresponding author.
E-mail address: fuearl@gmail.com (E. Fu).
1
Drs. Min-Chia Chen and Hsien-Chung Chiu contributed equally to this study.
https://doi.org/10.1016/j.jds.2021.01.015
1991-7902/ª 2021 Association for Dental Sciences of the Republic of China. Publishing services by Elsevier B.V. This is an open access article under
the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Downloaded for Anonymous User (n/a) at Egyptian Knowledge Bank from ClinicalKey.com by Elsevier on September 15,
2023. For personal use only. No other uses without permission. Copyright ©2023. Elsevier Inc. All rights reserved.
ª 2021 Association for Dental Sciences of the Republic of China. Publishing services by Elsevier
B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.
org/licenses/by-nc-nd/4.0/).
Introduction computer-aided-designed software (SolidWorks corp., Das-

sault Systemes, Waltham, MA, USA), the scaffold with
Severe bony defect in the dental, oral, and maxillofacial 1.8 mm in height was created, while the shape were trim-
region is a common clinical challenge for clinicians. In order med by a 6 mm diameter hole puncher (Fig. 1A). The
to restore the lost anatomy and function, alveolar bone customized PCL scaffold presented interconnection 300 mm
augmentation is often required.1e3 During the augmenta- PCL struts with the interval space of 900 mm pore size
tion procedure, the space-making is one of the essential (Fig. 1). All scaffolds were soaked in alcohol for 24 h and
factors for bone growth.4 Particulate bone graft is one of exposed to UV radiation light (Philips, Eindhoven,
the materials frequently being used; however, due to its Netherlands) for 4 h under ambient conditions before use.32
spotted nature, it is hard to maintain it in a specific shape
at the augmentation site and to provide a stable volume Preparation of platelet-rich fibrin
during the healing period.5e7 Block bone graft is better in
preserving the anatomic morphology but still delicate in In this study, PRF was prepared as per the protocol
shaping during surgery.8 described by Dohan et al.24 Blood samples were collected
Three-dimensional-printed polycaprolactone (PCL) scaf- without anticoagulants from a volunteer in 10 ml tubes and
fold has the advantages in customizing macro-structural ar- split into 1.5 ml Eppendorf tubes where 6 mm PCL scaffolds
chitecture, interconnected porosity, and channel size for were added to the tubes and allowed to mix with the
nutrient diffusion.9e13 The 3D scaffold has excellent blood.33 All tubes were then immediately centrifuged at
biocompatibility,9,14,15 and may be able to provides the 3000 rpm, with 1670 g force, for 10 min (CN-1040, Hsiangtai,
necessary support and definite shape during the healing New Taipei City, Taiwan). After processing, the PRF and the
period.7 However, previous critical size defect studies using PCL-PRF complex scaffolds were incubated on ice until
200e750 mm pore size PCL scaffold was not able to achieve further required, whereas the deposits, including red blood
more than 20% of the bone fill in the defect without adding cells, were discarded.
mediators, bioactive agents, cells, or membrane.16e23
Plasma rich fibrin (PRF) is a biomaterial which can be
easily obtained from patient’s own blood after a process of
Animal experiment
centrifugation. PRF belongs to platelet concentrates that
involve a simplified preparation.24 Studies have shown Thirty two male SpragueeDawley rats, eight week-old,
many cytokines, and growth factors enmeshed into this were selected. Animals received the general anesthesia
fibrin network that potentially enhances bone regenera- with intraperitoneal atropine (0.05 mg/kg), titetamme/
tion.25 There are several advantages compare to platelet- zolazepam mixture (Zoletil 50, Virbac, Carros, French)
rich plasma in its natural structure and intact circulating (1.1 ml/100 g), and xylazine hydrochloride (Rompun, Bayer,
cells (platelets, leukocyte, and other cells).24 It was shown Leverkusen, Germany) (0.05 ml/100 g, IP). After incision, a
that the PRF clot stimulates proliferation and differentia- full-thickness flap was elevated, and the parietal bones
tion of osteoblasts in vitro26e28 and bone regeneration in were then aseptically exposed. Two circular defects were
several clinical trials.7,29e31 The present study therefore surgically created for each animal with a trephine (Komet,
aimed to test the bone formation from a 900 mm pore size Hannover, Germany). Animals were divided into four defect
3D printed PCL scaffold with plasma-rich-fibrin implanted in groups, including defect control, PRF, PCL, and PCL-plus-
critical-sized calvaria defect of the rat without adding any PRF (PRF þ PCL) groups (Fig. 1). During the surgery, the PCL
bone graft or membrane. and PRF þ PCL scaffolds could be all precisely placed within
the calvarial defects. In the PRF group, fibrin gel was
placed as described in a previous study.34 On weeks 4 and 8,
Materials and methods half of the animals were sacrificed by carbon dioxide
inhalation, and the skulls were excised and fixed in 4%
Fabrication of customized 3D-scaffolds paraformaldehyde. The specimens were prepared for
dental radiography, m-CT, and histology. All the animal
protocols were approved by the Institutional Animal Care
A one-shot extruder (Filabot, Barre, Milwaukee, WI, USA)
and Use Committee, National Defense Medical Center,
was used to fabricate PCL monofilaments of a diameter
Taipei, Taiwan (IACUC-15-323).
1.70 0.08 mm from PCL pellets (Capa 6500, Perstorp
Specialty Chemicals AB, Perstorp, Sweden). The specific
filament diameter was required for use with a fused Dental radiography
deposition modeling 3D-printer (Logan, Eir Biotech, Taipei,
Taiwan). The average molecular weight was 50,000, and Using a computerized imaging system (Asahi Xspot; Asahi
the pellets were used as they were received. Using the Roentgen Ind. Co., LTD., Kyoto, Japan), all skull samples
1215
M.-C. Chen, H.-C. Chiu, P.-J. Kuo et al.
Figure 1 The computer-designed 3D-printed PCL scaffold customized for guided bone formation. (A. the scaffold, computer-
designed prototype; B and C. the scaffold, the cross-sectional and vertical views, by scanning electron microscopy; D. the
cartoon sculpture presenting a scaffold in the calvaria defect of the rat; E and F, the scaffolds implanted into the defects in rats)
(PCL: poly-caprolactone; PRF: plasma rich fibrin; and PCF þ PCL: PCL-plus-PRF).
were examined in a coronal view to determine the extent of were calculated. Among the four defect groups, the pa-
bone formation. The X-ray tube was operated at 70 kV with a rameters recorded in each of the defects and zones were
current of 6 mA for 0.12 s, at a distance 2 cm from source-to- compared.
sensor. Images were processed with the INFINITT Dental
PACS image system (INFINITT North America Inc., Phillips- Histology and histometry
burg, New Jersey, USA) and the Image J processing software
(National Institutes of Health, Bethesda, MD, USA). After being fixed in 10% formalin for 24 h, the parietal
The mean mineralized density threshold was set as a bones were then soaked in alcohol for sequential dehy-
radiopaque area. In each bony defect, two zones were dration before embedding in resin.35 Sixty four calvaria
defined: central (<4 mm in diameter) and peripheral zones specimens were cut into 80-mm-thick sections in cross-
(>4 mm in diameter). Radiopaque area (mm2) in each of sectional direction by a microtome (SP1600, Leica, Wet-
the defects (<6 mm in diameter) was measured and zlar, Germany). The slides were then stained with hema-
compared among the four defect groups. The areas from toxylin and eosin36 and the histology characteristics were
two zones of every defect were also measured and evaluated under light microscopy (DMI3000B, Leica, Wet-
compared. zlar, Germany). Under the microscope, the healed fibrous
tissues, bony tissues, and the residual PCL struts in the
Micro-computerized tomography defects were compared grossly and quantitatively (histo-
metry) among groups. The sections were cut from the
Using a high-resolution micro-tomography scanner (Sky- middle of defects in a longitudinal direction. The healed
scan1076, Aartselaar, Belgium), the specimens were tissue area was defined as the tissue space between the
scanned. The tube was operated at 50 kVp accelerated periosteum of the skin flap and the dura mater. The areas
potential, 200 mA beam current for 460 ms, 18 mm image of bone, connective tissue, and residual PCL were recor-
pixel size, and a 0.8-degree rotation step. Data was ded as the tissue areas in mm.2 Besides, the percentage of
collected and reconstructed with medical image process- each component in defect, including bone, connective
ing software (Medical image illustrator, Visualization and tissue, and residual PCL, in the total tissue area, were also
Interactive Media Laboratory, National Center for High- measured and recorded as a tissue area in %. The central
performance Computing, Taipei, Taiwan). After stan- zone was defined as the central 4 mm in diameter of the
dardization, the coronal, axial, and sagittal slices of each 6 mm diameter defect, whereas the peripheral zone was
defect were assessed. In the region of interest, the defined as the 2 mm ring space mesial to the defect margin
mineralized volume (MV) and its relative volume (MV/TV) (Fig. 1F).
1216
Statistical analysis Quantitatively, significant more MV/TV was found in the

central zones of the groups with PCL than those without
All data were analyzed using a statistical software package PCL.
(SPSS v.15.0, IBM, Chicago, IL, USA). Among the four groups,
the quantitative values obtained from dental radiography,
mCT, and histometry were compared by one-way ANOVA Histological observation and histometric analysis
with the post hoc analysis of Duncan’s test. A p-value of less
than 0.05 was set to indicate a significant difference. In general, the defects in the groups without PCL were
collapsed in central of the defect if compared to the groups
with PCL when examined by histology (Fig. 4). The new
Results bone found in the control and PRF groups were mainly
located along the defect margins, which was defined as the
Radiographic and m-computerized tomographic peripheral zone in the present study. In the groups with PCL
observation scaffold, however, new bone could be observed in the
spaces in between the residual PCL struts, but not inti-
Well-ordered row-by-row radiopaque squares, a waffle- mately contacting the PCL struts. Residual PCL struts,
lattice-like feature, were observed, in the defects observed as a circle or oval shape in cross-section, could be
implanted with PCL scaffold by dental radiography, well noticed in both PCL and PCL þ PRF groups.
despite the observation intervals (Fig. 2). The radiopaque Histometry confirmed the finding that significantly
areas among the four defect groups were statistically greater tissue area in the groups with PCL than those
indifferent if the whole defect or only the peripheral zone without PCL when either the whole defect or central zone
of the defect were measured (at both weeks of 4 and 8). of the defect was examined in both 4 and 8 weeks (Fig. 5A).
However, significantly more radiopaque areas in the two Similarly, higher percentage of bone or connective tissue
groups with PCL scaffold were noted within the central area was consistently observed within the whole defects
zones, if compared with the groups without PCL. On the and the central zones of defects in groups with PCL than
reconstructed images of m-CT, a similar finding of the those without PCL. Compared to the absolute tissue surface
waffle-like pattern was observed in the defect groups with area (in mm2), however, the results from tissue areas in
PCL (Fig. 3). In addition, the parallel radiolucent tunnels percentage were dissimilar (Fig. 5B). The bone and con-
running through the radiopaque squares were seen. nective tissue surface areas in percentage within whole
Figure 2 Radiographic evaluation of the mineralization for the 4 defect groups received with 3D-PCL scaffold and PRF. The top
images are presenting the mineralization of the four defect groups (the defect control, PRF, PCL, and the PCL þ PRF groups) by
dental radiography at the observation intervals of weeks 4 and 8. The lower plots are showing the quantitative comparison of the
radiopaque areas recorded among 4 defect groups from either the whole defects or from the divided central and peripheral zones
at the week 4 and 8, respectively (Data presented as mean and SD; and *significantly difference vs control at p < 0.05).
1217
Figure 3 Tomographic evaluation of the mineralization for the four defect groups received with 3D-PCL scaffold and PRF. The top
images, including the axial and coronal views, are presenting the mineralization grossly for the four defect groups (control, PRF,
PCL, and the PCL þ PRF) by m-CT at the observation intervals of week 4 and 8. In contrast, the half of reconstructed images, placed
in the third row, showing the mineralization at the red line cut through two images in the middle row at week-4 and -8, respec-
tively. The bottom plots are presenting the mineralization ratios (MV/TV) in whole defects and the central and peripheral zones
divided among groups (Data presented as mean and SD; and *significantly difference vs control at p < 0.05). (For interpretation of
the references to color in this figure legend, the reader is referred to the Web version of this article.)
defect were statistical indifferent among the four groups; scaffolds simplify the bone augmentation procedure by its
however, the higher bone areas in %, but the less connec- close fit design into the defect (Fig. 1). The 3D-customized
tive tissue areas in %, were observed within the central scaffold provides a more predictable way in surgery; this
zones of the PCL groups than those without PCL. In pe- advantage has been noticed in several case reports.7,37e40
ripheral zones, the fewer bone areas in %, but the higher In the present study, mineralized tissue could be easily
connective tissue areas in %, were noted in groups with PCL observed in between PCL struts with dental radiographs and
compared to groups without PCL (statistical difference was m-CT after 4 and 8 weeks of healing. Mineralization
obtained only at week 8). The residuals of PCL were appeared as the lattices and evenly distributed in defects
observed in defects of groups with PCL. In contrast, the of the scaffold groups. A particular mineralized pattern was
measured areas in the two groups with PCL, including the observed in the 900 mm pore size PCL spaces. Similar de-
absolute and relative areas, were statistically indifferent. signs have been done in previous studies with smaller (i.e.
200e750 mm) pore size PCL scaffold,16e23 but minimal new
bone formation or a maximum of 20% mineralization vol-
Discussion umes could be obtained in all of those studies without
adding other materials such as cells, mediators, bone
In this study, 900 mm pore size 3D-printed PCL scaffolds with grafts, or membrane. In contrast, around 40% of the surface
plasma-rich-fibrin were implanted into rat calvarial defects area of the defect may be mineralized in our study using a
without adding any bone graft or membrane. After 4 and 8 900 mm pore size scaffold instead. The relatively high new
weeks, new bone could be found in around 40% of the bone formation rate in our study was consistent with the
surface area in the central part of the defect when PCL results from a previous study showing around 30% BV/TV in
scaffold was present. In contrast, mineralization only porcine calvaria using a pore size of 1000 um.18 We further
existed along the defect borders in those groups without demonstrated that the new bone could be formed evenly in
scaffold (control and PRF groups) (Figs. 2 and 3). both central and peripheral zones of the defects with the
In general, because 3D-printed PCL scaffold does not help of PCL scaffold (Fig. 5). Further study is definitely
require additional cutting or shaping during surgery, the needed to explore the necessity of using such large pore
1218
Figure 4 Histological evaluation of tissue healed in 4 defects groups received with the 3D-PCL scaffold and PRF. The histological
sections were presenting the bone formation in the four calvaria defect groups, including the control, PRF, PCL, and PCL þ PRF
groups, respectively.
size; however, the pore size needs to be small enough to platelet-derived growth factor, transforming growth factor,
prevent tissue from collapse, but at the same time large vascular endothelial growth factor, and epithelial growth
enough to provide space for bone growth. In the small pore factor (EGF). However, our result showed there is no sig-
size scaffold, the PCL struts were designed to support, but nificant benefit in healing rate or new bone formation ratio
they may further occupy the tissue spaces and let bone in 8 weeks. The details of the no significance are not
grow less. Therefore, an appropriate scaffold design with known; however, the concentrations of growth factors33
large enough pore size may be one of the key procedures in and the allogenic origins might not reach the effective-
using the scaffold for bone tissue engineering. ness. Finally, likewise the bone formation, connective tis-
In the present study, the histological finding of the newly sue growth into the defect space nearly to 60% of four
formed bone islands between the scaffold struts confirmed groups (Fig. 4), which means that a barrier to prevent soft
that the implanted scaffold could maintain the defect tissue invading during the early healing period may result in
volume well and might also present good compatibility with a better outcome.
surrounding tissue (Fig. 4). However, the new bone tissues Using a 3D-printed 900 mm pore size PCL scaffold, we
was not closely touching the strut but rather had a small demonstrated that a large pore size PCL scaffold was able
distance with connective tissue in between. This finding to provide an architecture for new bone formation without
suggests that the improvement in cell affinity with the PCL bone graft or membrane barrier in rat critical-size calvarial
scaffold is needed. defects. However, the beneficial effect of supplementary
When considering the effect of active loading agents, PRF was not able to be observed. Further research on the
PRF is believed to contain numerous growth factors, such as design modification of PCL and the necessity of adding
1219
Figure 5 Histometric analysis of the tissue area. The left plot A showing the tissue area (mm2) in the whole defects, as well as
the central (C) and the peripheral (P) zones; The right plot B presenting the tissue areas in % for the bone and connective tissue, as
well as the residual PCL, in the defects at the week 4 and 8. (White bar: control group, gray bar: PRF group, black bar: PCL group,
and dark bar: PRF þ PCL group) (*p < 0.05, vs control).
mediators or barrier membrane is still needed to find a Buddhist Tzu Chi Medical Foundation, Taiwan, ROC (TCRD-
better result of 3D-printed PCL on alveolar bone TPE-108-43).
augmentation.
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Journal of Dental Sciences 16 (2021) 1214e1221

Available online at www.sciencedirect.com

journal homepage: www.e-jds.com

Bone formation with functionalized 3D

Received 16 November 2020; Final revision received 29 January 2021

Introduction computer-aided-designed software (SolidWorks corp., Das-

Statistical analysis Quantitatively, significant more MV/TV was found in the

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