Chinese Journal of Mechanical Engineering: Additive Manufacturing Frontiers 1 (2022) 100021

Contents lists available at ScienceDirect

Chinese Journal of Mechanical Engineering: Additive

Manufacturing Frontiers
journal homepage: www.elsevier.com/locate/cjmeamf

Additive Manufacturing of Hydroxyapatite Bioceramic Scaffolds with

Projection Based 3D Printing
Huifeng Shao a,b,c,d,∗, Zhiheng Nian a, Zhuoluo Jing a, Tao Zhang a, Jiahua Zhu a, Xiang Li b,c,
Youping Gong a, Yong He b,c,∗
a
School of Mechanical Engineering, Hangzhou Dianzi University, Hangzhou, 310018, China
b
State Key Laboratory of Fluid Power and Mechatronic Systems, School of Mechanical Engineering, Zhejiang University, Hangzhou, 310027, China
c
Key Laboratory of 3D Printing Process and Equipment of Zhejiang Province, School of Mechanical Engineering, Zhejiang University, Hangzhou, 310027, China
d
Jiangsu Key Laboratory of 3D Printing Equipment and Manufacturing, Nanjing Normal University, Nanjing, 210023, China

a r t i c l e i n f o a b s t r a c t

Keywords: Hydroxyapatite (HA) bioceramics have garnered considerable attention owing to their applications in the field of
Projection based 3D printing bone repair and excellent biocompatibility. Compared to extrusion-based 3D printing, projection-based 3D print-
Hydroxyapatite ing (3DPP) can fabricate parts with complex geometry, high accuracy, and efficiency, which is very promising for
Scaffold
bioceramic scaffolds. However, conventional 3DPP using a paste with low viscosity will cause severe shrinkage
Biosafety
of the parts after sintering, which makes it unsuitable for bioceramic scaffolds, and a system investigation of the
Bioceramic
printing process remains insufficient. In this study, we proposed a 3DPP device suitable for bioceramic scaffolds
and investigated the additive manufacturing of HA scaffolds. Ceramic paste properties and process parameters of
curing, debinding, and sintering were initially examined. The mechanical properties, shrinkage, and biocompati-
bility in vitro of the sintered samples were further investigated. The obtained results indicate that HA bioceramics
with uniform morphology, complex structure, and high accuracy can be manufactured using the 3DPP equip-
ment. HA scaffolds have the mechanical strength of human cancellous bone, while HA scaffolds cultured with
osteoblast precursor cells possess strong biocompatibility and can promote osteoblast adhesion, proliferation,
and differentiation. These results suggest a promising application of the 3DPP technique in the preparation of
bioceramic scaffolds, and the HA scaffolds fabricated using the 3DPP technique exhibit promising potential in
fulfilling a constructive role in the biomedical field of human bone regeneration repair.

1. Introduction ufacturing techniques offer unparalleled flexibility in achieving a con-

trolled geometric shape, uniform pore structure, complexity, and low
The demand for synthetic materials to repair or regenerate lost, in- cost compared to conventional methods [14–20]. Therefore, the shape
jured, or diseased bone is expected to increase because of the increase of the porous ceramic scaffold can be matched exactly to the shape of
in the occurrence of high-energy trauma in daily life, including sec- the bone defect [21].
ondary osteomyelitis and nonunion [1–3]. Hydroxyapatite (HA) bioce- The additive manufacturing technique has been adopted to fabri-
ramics have garnered considerable attention for their application in clin- cate porous HA scaffolds in the past few years [22–24]. These methods
ical bone repair over the past few years [4]. HA bioceramics are ideal are based on the fundamental principle of first slicing the 3D model,
materials for artificial bone scaffolds because of their high mechanical and then building the scaffold layers successively, according to the
strength, excellent biocompatibility, and ability to promote stem cell section, to fabricate porous scaffolds with regular shapes and control-
differentiation and proliferation [5,6]. Conventional fabrication meth- lable arrangements. However, the precision and efficiency of molding
ods for porous ceramic scaffolds include particulate leaching [7], gas cannot be guaranteed simultaneously. The point-by-point scanning 3D
foaming [8], organic foam impregnation [9], pore formation [10] and printing method requires a significant molding time [25]. However,
freeze-drying [11]. However, these conventional fabrication methods the projection-based 3D printing (3DPP) technique can theoretically
cannot efficiently control the pore size, structure, and porosity of porous avoid this disadvantage as it does not require a scanning path plan [26–
ceramic scaffolds, which are crucial for scaffolds [12,13]. Additive man- 28] and it has the advantages of high precision, complexity, and fast

∗
Corresponding authors at: School of Mechanical Engineering, Hangzhou Dianzi University, Hangzhou 310018, China. School of Mechanical Engineering, Zhejiang
University, Hangzhou 310027, China.
E-mail addresses: shaohf@hdu.edu.cn (H. Shao), yongqin@zju.edu.cn (Y. He).

https://doi.org/10.1016/j.cjmeam.2022.100021
Received 17 February 2022; Received in revised form 11 April 2022; Accepted 25 April 2022
Available online 16 June 2022
2772-6657/© 2022 The Author(s). Published by Elsevier Ltd on behalf of Chinese Mechanical Engineering Society (CMES). This is an open access article under the
CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)
H. Shao, Z. Nian, Z. Jing et al. Chinese Journal of Mechanical Engineering: Additive Manufacturing Frontiers 1 (2022) 100021

Fig. 1. Mechanical structure of 3DPP equipment.

Fig. 2. Fabrication process of 3DPP equip-

ment.

printing speed compared to other additive manufacturing methods. In 2. Materials and Methods
this study, 3DPP technology was applied to fabricate HA scaffolds to
ensure accuracy and significantly improve the molding efficiency. 2.1. Materials
The paste used for bioceramic scaffolds has the characteristics of
high viscosity and poor fluidity, and the conventional 3DPP that adopts HA powders with diameters of 20 nm and 12 𝜇m were purchased
a paste with low viscosity will cause severe shrinkage of the parts after from Shanghai Ruiyong Biotechnology Co. Ltd. The resin material was
sintering, which makes it unsuitable for bioceramic scaffolds. Therefore, obtained from Shanghai Guangyi Chemical Co., Ltd [29]. HA powder
a 3DPP equipment suitable for pastes with high viscosity was devel- and resin materials were ground in a planetary ball mill for 3 h, and a
oped. The improved rheological properties of the ceramic pastes were paste with a solid content of 10–50 wt% was prepared.
achieved using photosensitive resin and HA powder. The process pa-
rameters of 3DPP were analyzed and optimized to prepare complex HA 2.2. 3DPP equipment
ceramic structures with high precision. The biocompatibility of porous
HA scaffolds was tested in vitro. The 3DPP scaffold exhibits high mechan- A home-made 3DPP equipment was employed to fabricate the ce-
ical strength and a suitable pore structure for cell adhesion growth. The ramic samples, and its mechanical structure is illustrated in Fig. 1. The
porous HA scaffolds fabricated using the 3DPP technique demonstrated light source of the 3DPP equipment can emit ultraviolet light at a wave-
capabilities for application in the biomedical field of bone regeneration length of 405 nm. The dimensions of the experimental molding substrate
repair. were 45 mm × 65 mm, and the z axis accuracy was 10 𝜇m. Fig. 2 presents

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H. Shao, Z. Nian, Z. Jing et al. Chinese Journal of Mechanical Engineering: Additive Manufacturing Frontiers 1 (2022) 100021

Fig. 3. Characteristics of the printing process: (a) Influence of shear rate on paste viscosity with different solid content and powder size; (b) Influence of shear rate
on paste viscosity with different solid content (0, 10, 20, 30, 40, 50 wt%) for micron grade HA powder; (c) Curve between exposure time and single layer thickness;
(d) Fitting curve between exposure energy and layer thickness; (e) Top views of curing samples after different exposure times.

the printing principle of the 3DPP equipment. In the initial stage of print- scraper (Fig. 2(d)). Finally, repeating the steps from Fig. 2(a) to (d), the
ing, the distance between the printing platform or printing part and the part undergoes a layer-by-layer stacking formations until it is printed.
upper surface of the transparent membrane is the single-layer curing
thickness, as illustrated in Fig. 2(a). The light source then projects a 2.3. Preparation of ceramic samples
slice image of the model in that layer and solidifies the paste. When
the paste is completely solidified, the printing platform is lifted, and a The ceramic sample was designed using a 3D drawing software and
blank is formed at the rotating trough due to the poor fluidity of the exported to an STL format. Subsequently, the model was sliced and im-
high-viscosity paste (Fig. 2(c)). Next, the rotating trough rotates, and ported into the 3DPP equipment. The paste, which was poured into
the previous blank is filled with paste under the action of a stationary the rotating trough, was solidified with 2000 𝜇W/cm2 light intensity

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H. Shao, Z. Nian, Z. Jing et al. Chinese Journal of Mechanical Engineering: Additive Manufacturing Frontiers 1 (2022) 100021

Table 1
Characteristics of the paste with different powder sizes.

Size Advantage Disadvantage

Nanometer Good stability Low solid content

grade High printing accuracy High price
Micronmeter High solid content Easy to subside
grade Low price Low printing accuracy

(CKX53, Olympus Corporation, Japan). After culturing for one, three,

and five days, the samples were stained with calcein-AM and propid-
ium iodide. Fluorescent images of living and dead cells were obtained
using confocal fluorescence microscopy. The Cell Counting Kit-8 (CCK-
8; Dojindo, Kumamoto, Japan) assay was used to analyze cell viability.
The Alizarin Red S assay was used to detect mineralized bone nodular
structures after 21 days.

2.6. Statistical analysis

All data are expressed as the mean value ± standard deviation (SD).
Statistical analysis was performed using one-way analysis of variance
(ANOVA), and p < 0.05 was considered to be significant.

3. Results and Discussion

3.1. 3DPP scaffolds

It can be observed that the powder increase and decrease in powder

size resulted in the increase in the viscosity of the pastes (Fig. 3(a)).
The viscosity of all pastes decreased significantly with an increase in
Fig. 4. Stair-stepping of the 3DPP samples: (a) Formation mechanism of stair- the shear rate. When the solid content of the powder with a diameter of
stepping; (b) Side view of ceramic sample; (c) Top view of 3DPP sample and 20 nm reached 35 wt%, the paste completely lost fluidity. After setting
inset in (c) showing the 3D model of 3DPP sample. the pastes for one day, the paste with a powder size of 12 𝜇m slightly
subsided, while the other paste did not subside and still maintained op-
timal stability.
and the exposure time was set between 1 s and 300 s. In addition, the The characteristics of the two pastes are presented in Table 1. The
height of the layers was 50 𝜇m. The printed samples were then ultra- size of the powder affects the properties of the paste, such as its viscosity,
sonically cleaned in alcohol, and the residual paste was removed using stability, and printing accuracy. The paste with a higher viscosity cannot
high-pressure gas. Finally, thermal treatments including debinding and be paved layer-by-layer in the 3DPP process. The fluidity of the paste
sintering were performed to exhaust the resin and increase the density with a small amount of powder decreases rapidly when only a small
of the samples. amount of powder is added. The viscosity of the paste increased with
the solid content, and the upper limit of the solid content of the paste
2.4. Measurements and characterizations that met the printing requirements was reduced. The lower solid content
eventually triggers severe size shrinkage during the thermal treatment
Rheological measurements were performed using an AR-G2 rheome- process, which affects the molding of the prints. A paste with a large
ter (TA Instruments). The shrinkage of the samples was determined by powder will lead to serious settlement after standing for a period of
measuring the size of the samples before and after thermal treatment time. The 3DPP molding will last for several hours, causing an uneven
using a sliding caliper. Four samples were adopted to replicate this test. texture of the print and even failure of 3DPP molding. In addition, the
The compressive strength of the sintered samples was tested using a uni- scattering is intensified, which affects the printing accuracy in the X-Y
versal testing machine (Instron 5566, Germany) at a crosshead speed of plane direction. When the size of the powder is larger than the thickness
0.5 mm/min. Four samples were also adopted to replicate this test. The of the slice layer, the printing accuracy in the Z-direction is also affected.
surface morphologies of the sintered samples were observed using an Although there are large particles in the paste, the wear of the transpar-
optical microscope (Olympus). ent membrane is aggravated during the process of rotating and pressing
the forming platform. The solid content of the paste must be increased
2.5. Cell culture in vitro as much as possible to avoid defects during the size shrinkage. Hence, a
micrometer-grade HA powder was employed in this experiment.
Mouse preosteoblastic cells MC3T3-E1 were cultured in 𝛼-MEM sup- The rheological properties of different pastes (0, 10 wt%, 20 wt%,
plemented with 10% fetal bovine serum and 1% penicillin-streptomycin 30 wt%, 40 wt% and 50 wt%) with micron-grade ceramic powder are
in an incubator at 37 °C and 5% CO2 . The scaffolds were cut into 3– illustrated in Fig. 3(b). It can be observed that the viscosity of the pure
4 mm thin slices for the experiment, and a blank group was set as the photosensitive resin paste was the lowest and did not vary with the in-
control group, with each group containing six samples. Cells (5 × 104 ) crease in shear rate. The viscosity increased with increasing solid con-
were seeded on the surfaces of the samples. After a 24 h incubation pe- tent and decreased significantly with increasing shear rate. When the
riod, scaffold samples in a 48-hole plate were stained with calcein-AM solid content was higher than 20 wt%, the viscosity increased signifi-
for 20 min and washed twice with phosphate buffer solution (PBS). The cantly, and the paste exhibited no fluidity when it was 60 wt%. There-
cells were stained with 4’,6-diamidino-2-phenylindole (DAPI) dye solu- fore, a paste with a solids content of 50 wt% was employed in this ex-
tion for 30 min and observed under a confocal fluorescence microscope periment.

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H. Shao, Z. Nian, Z. Jing et al. Chinese Journal of Mechanical Engineering: Additive Manufacturing Frontiers 1 (2022) 100021

Fig. 5. Images of the printing samples af-

ter overexposure: (a) Top view of the print-
ing sample; (b) Distortion mechanism of the
side hole; (c) Side view of the normal side
hole; (d) Side view of the distorted side
hole. (For interpretation of the references
to color in this figure, the reader is referred
to the web version of this article.)

may be caused by insufficient exposure and low stiffness of the curing

sample. Between 2 s and 5 s, the curing samples exhibited a standard
shape and gradually increased stiffness. After 10 s, overexposure became
more significant. The curing layer thickness of the samples increased,
and the size of the cured samples exceeded the size of the model.
Light intensity, exposure time, and curing layer thickness influence
the printing effect. If the light intensity is insufficient, the paste is diffi-
cult to solidify or the strength of the curing part is insufficient. If the light
intensity is too strong, the scattering effect of the particles inside the
paste triggers overexposure. The evident layer structure was obtained
by solidifying the paste on the edge of the sample with scattered weak
light. In addition, deformation of the lateral pores of the sample is also
caused when some light penetrates the formed part. Insufficient expo-
sure time leads to insufficient stiffness of the sample, while excessive ex-
posure time can lead to overexposure. If the thickness of the slice layer
is negligible, the number of printing layers will increase significantly,
resulting in a reduction in printing efficiency. This also aggravates the
wear of the transparent membrane. In addition, owing to the character-
istics of the non-Newtonian fluid of the paste, the forming platform will
Fig. 6. Heating curve of sintering. be subjected to significant resistance when pressed, thereby resulting in
the bending deformation of the Z-axis, which reduces the printing accu-
racy in the Z direction. If the thickness of the slice layer is too large, the
The relationship between the single-layer thickness and exposure stair-stepping effect will occur, and a printing fault will occur because
time is presented in Fig. 3(c). The light intensity of the 3DPP equipment ultraviolet light cannot penetrate the paste.
was set to 2000 𝜇W/cm2 . Although thickness of a single curing layer Stair stepping and overexposure exert negative effects on printing ac-
increases with an increase in exposure time, the growth trend slows. A curacy. The formation mechanism of the stair-stepping process is illus-
possible reason is that the cured part weakens the transmission of ultra- trated in Fig. 4(a). The model was discretized by slices, and the printed
violet light, which hinders further increase in the curing layer thickness. parts were then formed by stacking layer-by-layer in the printing system.
Fig. 3(d) illustrates the relationship between the curing layer thickness The transition between layers was not completely smooth. The stair-
T and natural logarithm of the exposure E, according to a previous study stepping process is illustrated in Figs. 4(b) and (c).
[30]. The fitting equation was obtained using Origin Pro 9 software: Fig. 5 presents images of the printed samples after overexposure. Ex-
cessive exposure causes the paste outside the exposure zone to solidify.
𝑇 = 235.674 × ln𝐸 − 195.26
The size of the printed sample in the horizontal direction increased, and
The top views of the cured samples after different exposure times are the size of the pores decreased, as illustrated in Fig. 5(a). Excessive expo-
illustrated in Fig. 3(e). The cured sample produced a crimp after 1 s. This sure also causes shape distortion of the side holes of the printed sample.

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H. Shao, Z. Nian, Z. Jing et al. Chinese Journal of Mechanical Engineering: Additive Manufacturing Frontiers 1 (2022) 100021

Fig. 7. Ceramic structure.

Fig. 8. Ceramic samples: (a) 3D model of the sample; (b) Sample before and Fig. 10. Fluorescence test of MC3T3-E1 cells cultured with HA porous scaffolds
after thermal treatment. for 1 day.

Fig. 5(b) illustrates the distortion mechanism of side holes. When the printing accuracy, and the exposure time of a single layer was 5 s to
lower part of the side hole is printed layer by layer, the uncured paste ensure interlaminar bonding strength.
in the red area solidifies owing to the transmission effect of ultraviolet The thermal treatment parameters of the 3DPP samples are presented
light, thereby resulting in smaller side holes and shape distortion. The in Fig. 6. First, the heating rate was controlled at 1 °C/min, while the
distortion of the side hole could be improved by reducing the exposure sample was dehydrated at 80 °C and 110 °C for 30 min. Then, the heating
time (Figs. 5(c) and (d)). The layer thickness was set to 50 𝜇m to ensure rate was controlled at 0.5 °C/min, and the sample was debonded at 300

Fig. 9. Compressive test of porous samples: (a) Compressive test platform; (b) Curve between displacement and load.

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H. Shao, Z. Nian, Z. Jing et al. Chinese Journal of Mechanical Engineering: Additive Manufacturing Frontiers 1 (2022) 100021

Fig. 11. Staining of MC3T3-E1 cells cultured with HA scaffolds for 1,

3, and 5 days, respectively.

Table 2
Sample size for different forming stages.

Model Green body Sintered body

D (mm) 10 10.04 6.88

H (mm) 15 15.06 10.78

teroperability. The 3DPP equipment also has the advantages of small

size and low cost, and does not require expensive biological consum-
ables. However, the 3DPP sample shrank significantly during thermal
treatment owing to the low solid content of the paste. To avoid defects,
such as cracking, the thermal treatment process must be strictly con-
trolled. In addition, the penetration and scattering of light diminished
the printing precision of the 3DPP process. Because the viscosity of the
paste is excessive, some paste in the porous ceramic structure cannot be
cleaned out, which leads to blockage of some pores.

Fig. 12. CCK-8 results of MC3T3-E1 cells cultured with HA scaffolds for 1, 3, 3.2. Mechanical properties
and 5 days, respectively.
A porous scaffold sample with a diameter of 10 mm and altitude of
15 mm with a 1 mm hole diameter was designed (Fig. 8(a)). An image
°C and 400 °C for 120 min, and 500 °C for 60 min. After heating to 1100 of the sample before and after the thermal treatment is presented in
°C for 180 min at a heating rate of 3 °C/min, the furnace was naturally Fig. 8(b). It is deduced that the precision of the 3DPP sample is high
cooled to 25 °C naturally. Accordingly, the sintered ceramic sample was and the sizes of the samples at the different molding stages are presented
prepared. in Table 2. The shrinkage rate of the sample in the X-Y direction after
As illustrated in Fig. 7, a complex three-dimensional ceramic struc- thermal treatment was 31.47%, and the shrinkage rate of the sample
ture can also be fabricated. Compared to other additive manufacturing in the Z direction after sintering was 28.42%. The size of the sample
methods, 3DPP technology exhibits significant advantages in the fabri- decreased considerably after thermal treatment owing to the paste with
cation of ceramic structures. Owing to the surface exposure, a plan for a low solid content. The overall shrinkage of the samples was 66.39%.
the path of cross-section forming is not required, and its molding speed is After the thermal treatment, the porous scaffold sample was adopted
not influenced by the size of the sample in the horizontal direction. This for the compressive test (Fig. 9). At a deformation to 0.28 mm and load
can improve printing efficiency while ensuring manufacturing accuracy. of 229.9 N, the bottom layer of the sample was crushed. The compressive
The conventional UV curing 3D printing equipment is not suitable for strength and compressive modulus of the sample (porosity is 54.6%)
the fabrication of ceramic bone scaffolds because of the low solid con- were 6.18 MPa and 0.38 GPa, respectively. The compressive strength of
tent of the paste. The 3DPP equipment adopts the method of rotating the human cancellous bone ranges from 1.5 to 38 MPa, and its Young’s
paint, which is suitable for high-viscosity pastes, and can manufacture modulus range is 0.1–4.5 GPa. For the cortical bone, its compressive
artificial bone scaffolds with precision up to the micron level, complex strength ranges from 35 to 283 MPa, and its Young’s modulus range is
shape, controllable pore shape and structure, and controllable pore in- 5–23 GPa [29]. In the future, the mechanical properties of the sample

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H. Shao, Z. Nian, Z. Jing et al. Chinese Journal of Mechanical Engineering: Additive Manufacturing Frontiers 1 (2022) 100021

Fig. 13. Mineralization result of MC3T3-E1 cells cultured with HA scaffolds for 21 days. (For interpretation of the references to color in this figure, the reader is
referred to the web version of this article.)

can be made closer to that of the human cortical bone by changing the will promote the biological behavior of bone formation [37]. The mi-
porosity of the sample. crostructures of porous scaffold can provide a place for cells to reside
and differentiate, and also adsorb biological factors that can promote
3.3. Cell culture analysis in vitro osteogenesis function in the medium, to provide a suitable microenvi-
ronment for cells, induce the expression of cell osteoblast genes, and
The activity and distribution of MC3T3-E1 cells in the scaffold after 1 ultimately promote cell ossification [38].
day of culture were examined by DAPI fluorescence staining (Fig. 10). In The in vitro cell culture results demonstrate that the porous HA scaf-
this experiment, the green part represents cells attached to the scaffold, folds exhibit favorable cytocompatibility. The material and the inherent
which is important for bone scaffolds [31]. Cell adhesion ability is gen- characteristics of the design and fabrication exert a significant influence
erally related to the properties, structural characteristics, and surface on cell attachment, proliferation, and differentiation.
morphology of the scaffold materials [32]. The surface of the porous
4. Conclusions
scaffold manufactured using 3DPP technology exhibits a certain rough-
ness, and there are several microstructures, such as protruding parti-
In summary, a 3DPP equipment suitable for manufacturing bioce-
cles and stair-stepping, which provide a significant number of adhesion
ramic scaffolds was developed, and hydroxyapatite ceramic structures
points for cells and facilitate cell grasping and adhesion [33]. In this
with high accuracy were successfully fabricated via the 3DPP technol-
current study, the porous HA scaffold was observed to undergo cell ad-
ogy. Micron HA powder and resin were utilized to prepare a paste with
hesion on both the inner and outer surfaces of the scaffold, which is
a solid content of 50 wt%, which was suitable for 3DPP. The opti-
beneficial to cell adhesion owing to its structure and material.
mized printing parameters based on the HA ceramic paste were iden-
Fig. 11 presents fluorescent images of living and dead cells in the
tified as a layer thickness, light intensity, and exposure time of 50 𝜇m,
scaffold after 1, 3, and 5 days of culture, respectively. The cells main-
2000 𝜇W/cm2 , and 5 s, respectively. The compressive strength and
tained a very high survival rate, which is consistent with the results of
compressive modulus of the HA porous scaffold were 6.18 MPa and
the cell adhesion experiment. After 5 days, the cell density increased
0.38 GPa, respectively. The in vitro cell culture results demonstrated
significantly. It exhibited the quantity proliferation in a 3D distribution.
that the HA porous scaffold exhibited no visible cytotoxicity and was
The results demonstrate that the HA scaffold exhibited optimal biocom-
beneficial to cell adhesion, proliferation, and differentiation owing to
patibility, and that the cells planted on the scaffolds survived and pro-
its material and structure. The obtained results also indicate that 3DPP
liferated continuously.
technology is ideally suitable for fabricating ceramic structures with
As illustrated in Fig. 12, the OD value of the HA groups increased
controlled sizes and shapes, which are required for patients in differ-
significantly compared to that of the control groups cultured for 1, 3,
ent repair areas.
and 5 days. The CCK-8 results indicated that the HA porous scaffold
promoted the proliferation of MC3T3-E1 cells and had no visible cyto- Declaration of Competing Interest
toxicity compared with the control group.
Fig. 13 demonstrates that several red areas, which are mineralized The authors declare that they have no known competing financial
nodules, appear on the scaffold. In addition to adhering to the surface interests or personal relationships that could have influenced the work
of the scaffold, the cells also underwent osteogenic differentiation dur- reported in this paper.
ing the culture period. The final feature of osteoblast differentiation is
the mineralization and production of calcium nodules, which is one of CRediT authorship contribution statement
the important features of biological ossification [34]. The obtained re-
sults indicate that HA scaffold can promote osteogenic differentiation Huifeng Shao: Conceptualization, writing review & editing. Zhi-
of MC3T3-E1 preosteoblasts, which is consistent with a previous study heng Nian: Writing review & editing. Zhuoluo Jing: Writing review
[35]. This is mainly because Ca2+ and phosphate can promote calcium & editing. Tao Zhang: Writing review & editing. Jiahua Zhu: Writ-
phosphate precipitation and provide a crystal nucleus for the calcifi- ing review & editing. Xiang Li: Writing review & editing. Youping
cation osteogenesis of bone cells [36]. The porous structure also has Gong: Writing review & editing. Yong He: Resources, funding acqui-
a more diversified mechanical stimulation on cell proliferation, which sition, writing review & editing.

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H. Shao, Z. Nian, Z. Jing et al. Chinese Journal of Mechanical Engineering: Additive Manufacturing Frontiers 1 (2022) 100021

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