Colloids and Surfaces B: Biointerfaces 208 (2021) 112116

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Colloids and Surfaces B: Biointerfaces

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Protocols

Nano-hybrid gradient scaffold for articular repair

Gan Xu a, b, Yao Zhao b, Yusheng Geng b, Shujun Cao a, Panpan Pan a, Jianhua Wang b,
Jingdi Chen a, *
a
Marine College, Shandong University, Weihai 264209, PR China
b
College of Biological Science and Engineering, Fuzhou University, Fuzhou 350002, PR China

A R T I C L E I N F O A B S T R A C T

Keywords: Osteoarthritis disease can easily lead to articular cartilage degeneration and subchondral bone damage, so the
Osteoarthritis disease demand for suitable articular substitutes is gradually increasing. In order to simulate the complex environment of
Continuous scaffold different layers in natural joint, we fabricate the continuous one-phase gradient scaffold. In the study, CS (chi­
Nano hydroxyapatite
tosan) was modified with SH (sodium hyaluronate) and GO (graphene oxide) to form the whole scaffold. nHAP
Articular repair
(Nano-hydroxyapatite) was in situ generated with gradient distribution in the scaffold. Continuous interface can
better imitate the combination style of cartilage and subchondral bone at joint. The diverseness of scaffold’s
different layer in water absorption/retention rate and mechanical property is similar to the difference of articular
cartilage and subchondral bone. Meanwhile, the cell experiments demonstrated that the bionic scaffold can well
promote the proliferation of bone marrow mesenchymal stem cell. Articular defect model further confirmed that
the scaffold can better induce articular regeneration. Herein, the prepared scaffold might be an excellent
candidate for endogenous articular repair.

1. Introduction bone induction ability[17–19]. It plays an important role in inducing

Natural joints are constituted of cartilage and subchondral bone and
connected by a dense calcified cartilage layer [1,2]. Scaffold with
calcified cartilage layer can play an active role in promoting the
regeneration and repair of cartilage tissue, which is related to the me­
chanical properties and biological properties of different layers [3,4]. In
joint repair, designing a scaffold with continuous gradients in material
composition, structure, mechanical properties, and biological properties
is important [5].
Joint injuries generally involve damage to cartilage and subchondral
bone [6,7]. Cartilage is a highly specialized tissue that has no blood
supply and is difficult to regenerate spontaneously once injured [8,9].
The lowest layer of cartilage is calcified cartilage and part of the signal
molecules from subchondral bone can pass through it [10–12]. The
existence of calcified cartilage layer and the repair of subchondral bone
are conducive to the formation of cartilage [13,14]. In natural joints, the
main inorganic component of subchondral bone and cartilage calcifi­
cation layer is nano-hydroxyapatite [15]. It presents a gradient distri­
bution from calcified cartilage to subchondral bone and the content of
nHAP in subchondral bone is the highest [16]. nHAP has good
biocompatibility, proper biodegradability, high bone conductivity and
BMSCs to differentiate into osteoblasts, accelerating the formation of
subchondral bone, etc. [20–22]. GO has been shown to enhance the
biomineralization ability of bone scaffolds and has a potential role in
promoting the differentiation of BMSCs and bone formation [23–25].
GO modified chitosan can be used to accelerate biomineralization and
bone formation [26,27]. For the lack of self-repair ability, the repair of
cartilage layer relies on cells in subchondral bone migrating to the
cartilage layer to complete repair [28]. Cartilage materials simulating
the chondral environment are beneficial to the formation of cartilage
[29]. Hyaluronic acid is abundant in extracellular matrix (ECM) of
connective tissue[30]. For its high water retention, biocompatibility,
biodegradability and viscoelasticity, hyaluronic acid has good applica­
tion prospects in cartilage repair [31,32]. Sodium hyaluronate modified
chitosan has large pore structure, good moisture retention and degra­
dation [33–35]. In related reports, hyaluronic acid can enhance the
material’s adsorbability to chondrocytes and promote the proliferation
of chondrocytes [30,36]. Norimasa Iwasaki et al. [33] added hyaluronic
acid to chitosan to form a fundamental material for cartilage regenera­
tion and the cartilage analogue was get when cell was cultured in the
scaffold.
To prepare a continuous scaffold with cartilaginous layer, calcified

* Corresponding author.
E-mail address: jdchen@sdu.edu.cn (J. Chen).

https://doi.org/10.1016/j.colsurfb.2021.112116
Received 8 August 2021; Received in revised form 7 September 2021; Accepted 13 September 2021
Available online 16 September 2021
0927-7765/© 2021 Elsevier B.V. All rights reserved.
G. Xu et al.
cartilaginous layer, and subchondral bone layer, we prepared a bone/
cartilage nHAP gradient scaffold using secondary freeze-drying method.
When graphene oxide modified chitosan containing Ca2+ was added into
sodium hyaluronate modified chitosan, combining gravity and freezing
technology, graphene oxide modified chitosan finally focused on the
lower layer and sodium hyaluronic acid modified chitosan are mainly
concentrated in the upper. The middle layer is the mixed solution of
sodium hyaluronate modified chitosan and graphene oxide modified
chitosan. For the GO modified chitosan containing Ca2+ was mainly
concentrated in the lower layer and the strong adsorption of graphene
oxide on Ca2+, when the freeze-dried scaffolds were immersed in lye,
nHAP was generated in situ with gradient distribution. The porosity,
water retention, mechanical properties, degradability and nHAP
gradient of the prepared scaffold was tested. The experimental results
show that, compared with the lower layer, the upper layer has large pore
structure, high water retention, higher degradability and lower me­
chanical strength mechanical. SEM and EDS spectrum showed that
nHAP mainly generated in the middle and lower layer and presented
gradient distribution. In vitro mineralization experiments showed that
the lower layer of the prepared scaffold had good mineralization per­
formance. Biocompatibility experiments showed that the scaffold can
promote the proliferation and differentiation of bone marrow mesen­
chymal stem cells, and the rabbit joint defect experiment further proved
that the scaffold had a potential application prospect in promoting the
repair of joint defects.
2. Materials and methods
2.1. Materials
Chitosan (CS) (95% deacetylated) was purchased from Shanghai
BioScience & Technology Co., Ltd. Graphite powder was obtained from
Sigma-Aldrich (St. Louis, MO, USA). Sodium hyaluronate (SH) was
supplied by Sigma-Aldrich (St. Louis, MO, USA). The 1-ethyl-3-(3-dime­
thylaminopropyl) (EDC) and N-Hydroxysuccinimide (NHS) were sup­
ported by GL Biochem. (Shanghai) Co., Ltd. Calcium chloride and
dipotassium hydrogen phosphate trihydrate were acquired from Sigma-
Aldrich (St. Louis, MO, USA). CCK-8 and AO/EB staining reagents were
acquired from Nanjing Keygen Biotech. Co., Ltd. Fetal bovine serum
(FBS), Dulbecco’s minimum Eagle’s medium (DMEM), penicillin/
streptomycin, trypsin and other materials for cell experiments were
purchased from HyClone Co., Ltd (USA).
2.2. Fabrication of SH/GO/CS/nHAP scaffolds
2.2.1. Preparation of upper layer of cartilage
The details are as follows: SH (Sodium hyaluronate) (0, 50, 100, 150
mg) was added into 10 mL deionized water and stirred until fully dis­
solved. Chitosan solution was obtained by dissolving chitosan powder in
2% acetic acid liquid. Under the condition of magnetic stirring at 37 ℃,
prepared solutions above were mixed, and the crosslinking agent EDC/
NHS (molar ratio 2:1) was added to form a uniform mixture. After cross-
linking at room temperature for 4–6 h, the precursor solution of cartilage
layer was obtained.
2.2.2. Preparation of the subchondral bone layer
The GO was prepared by the improved Hummer’s method [37]. The
details are provided in the Supplementary Data. The CS/GO composite
was fabricated by published method [38]. Chitosan solution was ob­
tained by dissolving CS powder in 2% acetic acid liquid. GO was ultra­
sound in 10 mL deionized water to form a uniform GO dispersion. Under
the condition of magnetic stirring, GO was slowly added into the chi­
tosan solution and then fully stirred to form a uniform mixture. Subse­
quently, at the ratio of 1.67, calcium and phosphorus salt solutions were
added to the above mixture and stirred thoroughly to make it entirely
mixed. Then, cross-linking agent was added and cross-linked at room
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Colloids and Surfaces B: Biointerfaces 208 (2021) 112116
temperature for 4–6 h to obtain the precursor solution of subchondral
bone layer.
2.2.3. Preparation of integrated bone-cartilage scaffolds
The obtained cartilage layer precursor solution was added to the
preformed mold. Then the solution of subchondral bone layer was
slowly added to the cartilage layer. The above liquid was refrigerated at
4 ℃, − 20 ℃ and − 80 ℃. After being freeze-dried, the preliminary
scaffold was soaked in the alkaline solution (2.5% NaOH), and then kept
in a constant temperature shaker for continuous reaction for 8 h. Later
the scaffold was washed to neutral and freezed drying again. After that,
an integrated scaffold with two kinds of structure was obtained. S-
0 (control), S-1, S-2, S-3 represent prepared scaffolds by adjusting SH
content (0, 50, 100, 150 mg) in the experiment.
2.3. Physicochemical characterization of the scaffolds
The microscopic structure and elementary composition of scaffold
were tested by scanning electron microscopy (Nova Nano SEM 230, FEI,
USA) and energy-dispersive spectrometry (EDS). The crystalline phases
of the scaffolds were characterized by X′ Pert MPD Pro, PANalytical
(Netherlands). Fourier transform infrared spectroscopy (FT-IR) (Nicolet
Nexus spectrometer) was used to study functional groups of organic
components in scaffold. Renishaw invia microscope system (Invia Re­
flex) using laser source with wavelength of 532 nm detected Raman
spectrum of composite scaffold. X-ray photoelectron spectroscopy (XPS,
Manchester, England) were used to analyze binding energy state.
2.4. Porosity test
Liquid displacement method was used to assess the porosity of
scaffolds [39]. The dry material weighing Wd was soaked in the test tube
containing enough ethanol and was soaked for 5 min. The test tube was
placed in the glass desiccants under the vacuum state to allow ethanol
into the scaffold space. The excess ethanol was removed from the surface
of the scaffold and weighed (Ww). 5 parallel samples were set in each
group. Formula (1) was used to calculate the porosity of the cartilage
layer, bony layer and overall scaffold (P).
P = (Ww− Wd)/ρ×π×h× (D⁄2)2 × 100% (1)
ρ represents the density of ethanol (0.789 g/cm3). π is 3.14. D is the
scaffold diameter and h is the height.
The surface density (A) is calculated by formula:
A =Wd/π×h× (D⁄2)2× 100% (2)
π is 3.14. D is the scaffold diameter and h was the height.
2.5. Swelling ratio measurement
The dried scaffold (W1) was immersed in 15 mL PBS at 37 ℃ and
then taken out at the time points of 12, 24, 36, 48 and 60 h respectively.
The excess PBS liquid was removed with filter paper and the weight of
the scaffold was measured (W2). Each group had 5 parallel samples. The
swelling ratio (S) was calculated by formula (3):
S = (W2− W1)/W1× 100% (3)
2.6. Water uptake and retention ratio measurement
Water uptake and retention ratio measurement followed the process:
after drying, the scaffolds weighing Wd with different components were
immersed in distilled water for 24 h. The residual water on the surface of
the material was removed through dry filter paper, and then weighed
again (Ww1) to determine the water absorption rate. The soaked
G. Xu et al.
Fig. 1. (A) The fabrication process of SH/GO/CS/nHAP biphysic scaffold and the chemical construction of upper section, transition interface and lower section. (B)
EDS analysis of scaffold’s element distribution.
scaffold was centrifuged at 500 rpm for 3 min, weighed (Ww2), and the
water retention rate was calculated. The water absorption rate WA and
water retention rate WR of the support are calculated by the formulas (4)
and (5) respectively:
WA = (Ww1− Wd)/Wd× 100%
WR = (Ww2− Wd)/Wd× 100%
2.7. Analysis of mechanical properties
A universal material testing machine (INSTRON Model 1185, Shi­
madzu) was used to detect mechanical properties of scaffolds at upper,
lower layers and the whole two-phase at a speed of 1.0 mm /min. The
change of compressive stress was recorded, and the elastic modulus of
the scaffold was calculated by 30% deformation. Five parallel samples
were used in the measurement.
2.8. In vitro degradation
The weighing W1 sample was immersed in phosphate buffer solution
(PBS) containing lysozyme (0.5 mg/mL) to test the degradation
behavior of the composite scaffold. PBS is replaced every three days. At
1, 4, 7, 14 and 28 days, the scaffolds were taken out and weighed (W2),
Colloids and Surfaces B: Biointerfaces 208 (2021) 112116
and 5 parallel samples were set in each group. The degradation rate (D)
was calculated by formula (6):
Weight loss ratio = (W1-W2)/W1×100% (6)
(4)
(5) 2.9. Biomineralization study in vitro
The scaffolds were immersed in a centrifuge tube containing 15 mL
SBF and incubated in oscillator at 37 ◦ C for 1 and 3 days. The biomimetic
mineralization of scaffolds was analyzed by SEM and XRD. pH value of
SBF liquid was detected by pH meter (S20K, Mettler-Toledo,
Switzerland) for 5 weeks. At the same time, the weight gain rate of
each sample was calculated. Each group of scaffolds were removed from
SBF solution at 1, 3, 5, 7, 14, 21 and 28 days respectively and rinsed with
ultra-pure water for 3–5 times. Inductively coupled plasma-Atomic
Emission spectrometer, Liberty 200 (*/iCAP7400, USA), was used to
determine the content of calcium ions in immersion solution. The par­
allel samples were set to 5.
2.10. Cell viability and proliferation assay
AO and EB reagent dye were used in Live & Dead staining. The
proliferation of cell in scaffolds was investigated by Cell Counting Kit-8
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G. Xu et al.
Fig. 2. (A) Macro view of prepared scaffold and (B) SEM of cartilage layer (a-c) and subchondral bone layer (d-f) at magnification of 200 × , 1000 × and 10,000 × .
(C) The diagram of pore distribution. (D) Porosity analysis of different component scaffolds and (E) porosity measurement of S-2 scaffold’s different section. (F)
Apparent density of S-2 scaffold’s different section. (Statistic level of P*< 0.05, p** < 0.01 and p*** < 0.001).
(CCK-8) assay. Bone marrow mesenchyml stem cells were extracted
from the bone marrow of SD rat’s tibia (one month old). The detailed
information is included in the Supplementary Data.
2.11. Rabbit articular bone-cartilage model
18 healthy New Zealand Rabbit weighing 2.1 kg were bought from
the Animal Experiment Center and were divided into 3 groups. In the
knee patellar, defect model (Φ5mm and 4 mm high) was built. 8, 12 and
16 weeks were as three stages to evaluate the repair degree and the
relevant surgery procedure is described in the Fig. S3. CT was used to
evaluate bone regeneration and 3d visual modeling directly showed the
regeneration situation. Surgical steps and histological assessments were
descripted in the Supplementary Data. All the experiments meet the
local ethical committee and laboratory animal administration rules of
China.
2.12. Statistical analysis
Independent Student’s t-test was adopted to detect the difference of
different groups. The numerical values represent the mean ± standard
deviation. The different statistic level of P*< 0.05, p** < 0.01 and p***
< 0.001 was considered. Three replicates were set at least in each group.
3. Result and discussion
3.1. Fabrication mechanism of the scaffolds
Fig. 1A shows the biomimetic construction process of scaffolds and
its component of different layers. Firstly, amine groups (–NH2) of CS and
negative carboxyl radical (–COOH) of GO (Fig. S2A) and SH formed the
amide linkage (–NHCO–) (Fig. S2) under the crosslinking of EDC and
NHS to get different layers solution. Secondly, the precursor ions
including Ca2+ and PO43- were added into the subchondral bone layer
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Colloids and Surfaces B: Biointerfaces 208 (2021) 112116
and then slowly poured into the cartilage layer to form a one-phase
integration structure by gravity precipitation. After soaking in lye, it
can be seen that nHAP was generated on freeze-dried scaffold (Fig. S2C).
The distribution of main elements of the scaffold was further studied.
Through analysis of Fig. 1B, the distribution of element P and Ca has an
increasing gradient from top to bottom, while the distribution of
element C and O is uniform. The element distribution fully confirmed
that the distribution of nHAP is similar to the distribution of normal
human bone-cartilage inorganic components [1,40,41]. It can also be
observed from XPS test (Fig.S2.D).
3.2. Analysis of porosity and surface density
From the optical image in Fig. 2A, the composite scaffold has two-
phase structure. Fig. 2B(a–c) is the SEM image of the cartilage layer of
scaffold. It can also be found that there was denser pore structure on the
subchondral bone layer (Fig. 2B (d–f)). The further enlarged image of
the surface shows that the surface of the scaffold is relatively rough,
which is beneficial to cell adhesion, proliferation and differentiation in
scaffolds [42,43]. At the same time, large number of nHAP was found on
the surface of the subchondral bone layer at high magnification, indi­
cating that bone-like apatite had been successfully prepared by in-situ
process. The distribution of nHAP in the cross-section was relatively
uniform and the amount of nHAP on lower layer was more than the
upper layer. The pore structure of natural bone-cartilage showed the
difference of large pore in cartilage layer and small pore in bone layer
[5]. After observation, it can also be found that the pore size of the upper
layer (40–200 µm) is larger than that of the lower layer (30–100 µm),
and the pore wall of the upper layer is relatively thin. The difference in
pore structure has great influence on water absorption and mechanical
properties of different layers [44]. The large and loose pore structure
indicates that the scaffold has relatively low strength and high water
absorption. Besides, the pore structure of different layers determines the
heat conduction and material transfer efficiency [41]. As shown in
G. Xu et al. Colloids and Surfaces B: Biointerfaces 208 (2021) 112116

Fig. 3. (A) Hydrophilic diagram of scaffold’s different part in aqueous solution (B) Swelling ratio of different component scaffolds and (C) swelling ratio of S-2
scaffold’s different section. (D) Water uptake and (E) water retention ratio of scaffolds immersed in distilled water. (Statistic level of P*< 0.05, p** < 0.01 and p***
< 0.001).

Fig. 2D, the porosity of S-2 scaffold was higher than others, reaching
83%, while the control group approaches 60%. Therefore, comparing
with other groups, S-2 scaffold can provide a good physiological space
environment for bone cell infiltration, which is conducive to the adhe­
sion and proliferation of cells. However, because of the independence
and specialty of two layer structure, the experiment further investigated
the optimal S-2 scaffold. It can be seen from Fig. 2E that the porosity of
upper cartilage layer (96%) was much higher than that of subchondral
bone layer (74%), and the overall porosity is between the upper carti­
lage layer and subchondral bone layer. In addition, the surface density
and porosity were opposite (Fig. 2F). As a whole, the prepared scaffolds
have different porosity and pore size in different layers, which is similar
to the physiological structure of natural joint [45].

Fig. 4. (A) Load-distance curves, (B) compressive strength and elastic modulus of different component scaffolds. (C) Compressive strength and elastic modulus of S-2
scaffold’s different section. (D) Degradation in vitro of different scaffolds immersed in PBS containing lysozyme and (E) degradability of S-2 scaffold’s different
section. (Statistic level of P*< 0.05, p** < 0.01 and p*** < 0.001).

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G. Xu et al.
Fig. 5. (A) SEM images of control (a, c) and S-2 (b, d) scaffold after mineralization for 1 day and 3 days and element distribution analysis in S-2 scaffold with 3 days
mineralization. (B) XRD analysis of control and S-2 scaffolds immersed in SBF for 1 & 3 days. (C) Mass change of S-2 and control groups during biomineralization
process of 4 weeks. (D) pH value changes of SBF in S-2 and control groups during 5 weeks. (E) Analysis of compressive strength and elastic modulus of S-2 and control
group after mineralizing for 1 & 3 days. (F) The Ca2+ content of SBF in S-2 and control group for 1 & 3 days analyzed with ICP-OES. (Statistic level of P*< 0.05, p** <
0.01 and p*** < 0.001).
3.3. Swelling ratio, water uptake and water retention measurement
Low swelling rate is considered to be one of the factors of a good
scaffold. Fig. 3A displays the hydrophilia of different layers. As shown in
Fig. 3B, the variation of swelling rate of S-2 scaffold is relatively small
compared with other scaffolds, and the inflation equilibrium is relatively
long. Mainly due to the appropriate addition of SH, the effective cross­
linking points of SH/GO/CS/nHAP scaffold increased. In swelling pro­
cess, complete network structure is not easy to produce great changes
and the absorbable amount of liquid is limited. In view of the relatively
stable swelling rate of S-2 scaffold, each part was studied independently
in the experiment. As shown in Fig. 3C, the upper layer of the scaffold
showed a large swelling rate, while the lower layer changed small. The
difference of swelling rate indicates that the scaffold can better fit
osteochondral environment. Good water retention rate can not only
effectively avoid the accumulation of secretions, but also prevent tissue
wound dehydration [46]. The cartilage layer constructed in the study
was not significantly different from commonly used cartilage materials.
But compared with the commonly used bone repair materials, the water
retention of the cartilage layer was significantly enhanced. When two
layers were constructed through one-step process, there was no obvious
difference in water retention with common bone and cartilage material,
indicating that the scaffolds had good water retention. Fig. 3D shows
that, with the increase of SH content, the water absorption rate of the
scaffold also increases relatively. In particular, after being modified by
SH, the scaffold’s water absorption rate was higher than that of the
control group. At the same time, through further analysis of the water
retention rate, as shown in Fig. 3E, it was found that the water retention
rate of the scaffold modified with SH was also higher than that of the
scaffold without SH modification. In particular, the water retention rate
of S-2 group was the highest, reaching about 15%. The results show that
SH is beneficial to improve the absorbency and water retention of the
scaffold materials. The reason may be due to the high hydrophilicity of
SH molecule. To sum up, the S-2 scaffold has a higher water retention
performance and is more suitable for in vivo application.
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Colloids and Surfaces B: Biointerfaces 208 (2021) 112116
3.4. Mechanical property and degradation analysis
As a tissue replacement material, the scaffold should have enough
mechanical properties to support the defect and its surroundings orga­
nization. The good mechanical properties of the subchondral bone layer
can support the defect and the high water content and low strength of
the cartilage layer can reduce the damage caused by wear [47]. Fig. 4A
shows the load-distance curve of the composite scaffold. It can be seen
that the elastic deformation of the scaffold has a certain range, mainly
occurring in the range of 0–20%. Since the scaffold still has certain
bearing capacity at 30% deformation, the compressive strength of the
scaffold at 30% deformation was experimentally studied (Fig. 4B). It can
be found that the S-2 scaffold has the highest compressive strength
(0.28 ± 0.024mpa) and elastic modulus (0.272 ± 0.213mpa). When
appropriate SH was added, effective crosslink point was formed between
SH and CS, which enhanced the mechanical properties of the substrate
framework. Meanwhile, due to the effective doping of nHAP, the me­
chanical properties of the scaffold were greatly improved. According to
the analysis in Fig. 4C, the mechanical properties of the lower layer are
superior to those of the whole and the upper part, with the highest
compressive strength (0.38 ± 0.021Mpa) and elastic modulus
(0.369 ± 0.030Mpa), which is similar to the mechanical difference of
bone /cartilage. Meanwhile, implanted tissue replacement material
should be biodegradable and satisfy the needs of different tissue. The
degradation curve of the scaffold in PBS solution containing lysozyme
was shown in Fig. 4D. It can be seen that S-2 scaffolds have a relatively
fast degradation rate of 11.5% in about 14 days. However, the degra­
dation rates of the control group and S-3 changed significantly within 7
days, with the degradation rates of 12.1% and 15.1%, respectively. The
degradation rates of the S-1 group changed significantly in different
periods. Therefore, compared with other scaffolds, the degradation rate
of S-2 scaffolds is relatively low and stable in the same time. To analyze
each part and the overall degradation rate, as shown in Fig. 4E, it was
found that, at the time of day 1, the degradation rate of the upper part is
larger and the overall and lower layer at the time of 14 days to appear
relatively large degradation rate, which illustrated that the degradation
G. Xu et al.
Fig. 6. (A) Live & Dead staining (a-j) for the proliferation of rBMSCs cells cultured with extract liquid of scaffolds for 3 and 5 days. Live cells are stained in green and
dead cells in orange with AO/EB staining. (B) The quantitative analysis by CCK-8 assay for the proliferation of rBMSCs cells cultured with extract liquid of scaffolds
for different times. (Statistic level of P*< 0.05, p** < 0.01 and p*** < 0.001). (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
of the scaffold in different parts exist certain differences. The relatively
slow degradation process in lower layer is advantageous to the growing
and maturing of bone [48].
3.5. Biomineralization in vitro
As shown in Fig. 5A, the lower layer of S-2 scaffold was immersed in
SBF liquid at 37 ℃ for 1 and 3 days to study the mineralization of nHAP
on the surface of scaffolds. According to the SEM figure after minerali­
zation, the content of nHAP on the surface of the scaffold increased with
the extension of incubation time. After 3 days of incubation, the distri­
bution of nHAP in the two groups increased significantly (Fig. 5A (c, d)).
It is worth noting that, compared with control group, the nHAP particle
size on the surface of S-2 scaffold was smaller during the same miner­
alization time and a complete and uniform apatite layer appeared. EDS
energy spectrum was used to analyze the surface elements of S-2
mineralization scaffold, and found that the main elements were Ca and P
(Fig. 5A). Meanwhile, the coating structure can induce the formation of
a large number of nHAP in physiological environment and improve the
biological activity of the material. XRD was used to analyze nHAP for­
mation on the surface of scaffolds in control and S-2 groups after
mineralization. As shown in Fig. 5B, by comparing the standard cards, it
was found that 2θ values respectively corresponded to (002), (102),
(211), (310), (222) and (322) crystal planes of nHAP [49]. It was also
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Colloids and Surfaces B: Biointerfaces 208 (2021) 112116
found that the peak intensity of XRD increased with culture time
increasing. However, at 3 days, 222 and 322 crystal plane peaks
appeared in the S-2 group, but not in the control group, indicating that
SH had some influence on the growth direction of nHAP, making it grow
mainly along the orientation of 222 and 322 crystal planes. In addition,
due to the formation of nHAP, the scaffold quality also changed
accordingly. As shown in Fig. 5C, comparing with control group, the
growth of nHAP in S-2 group has a continuous and slow process, which is
consistent with the Ca content test results (Fig. 5F). In addition, the mass
increase rate of group S-2 reached 36% at 21 days, indicating that there
was large amount of nHAP formation on its surface. As shown in Fig. 5D,
pH values of each group showed an upward trend in the first 7 days,
mainly caused by residual lye on the synthetic scaffold. Alkaline envi­
ronment is the main condition for in-situ synthesis of nHAP [50].
Therefore, the increase of pH in mineralization solution is conducive to
promoting the regrowth of bone-like apatite, which is consistent with
the results observed in SEM images after mineralization. After soaking
for 7 days, the pH value of the control group gradually decreased and
reached acidity at 35 days, which indicated that the scaffolds gradually
degraded. However, the pH value of the S-2 group reached the highest at
18 days, indicating that the environment of the S-2 group was conducive
to the massive generation of nHAP on the scaffold surface. Therefore, in
general, the environment made by S-2 scaffold has a relatively high pH
value at the same time point and can continuously promote the synthesis
G. Xu et al.
Fig. 7. (A) CT analysis of articular osteochondral repairs after 8, 12 and 16 weeks postimplantation. (B) Defect repairing images of articular osteochondral after 8, 12
and 16 weeks postimplantation.
of nHAP in simulated body fluids. At the same time, the mechanical
properties of the two scaffolds were evaluated after soaking in SBF liquid
for 1 and 3 days. As shown in Fig. 5E, the mechanical properties of the
scaffolds were time-dependent after soaking in the mineralized solution.
The compressive strength and elastic modulus of each scaffold increased
with the increase of time. Especially for the S-2 group, the compressive
strength (4.5mpa) and elastic modulus (3.9mpa) were about 3 times and
2 times higher than those of the control group at 3 days. At the same
time, due to the production of nHAP, the content of Ca2+ in the
mineralization liquid changed accordingly (Fig. 5F). The content of Ca2+
in the solution showed a decreasing trend with the extension of time. At
1 day, the content of Ca2+ in the SBF solution in S-2 group was
88.6 mg/kg which was higher than that in the control group
(44.4 mg/kg), indicating that the amount of nHAP on the surface of the
control group was relatively high. At 3 days after mineralization, the
content of calcium ions in SBF solution in the S-2 group was 63.6 mg/kg,
while the control group was still 42.2 mg/kg. The stable and slow for­
mation of nHAP is beneficial to its growth. The size of nHAP was rela­
tively small, which could be clearly identified by SEM images. In vivo
environment, because of the slow growth of nHAP, it is not easy to form
local aggregation. When the scaffold is implanted in vivo, the physio­
logical microenvironment will change dynamically due to the
biomineralization.
3.6. . In vitro cellular assays
In order to study the growth conditions of bone marrow mesen­
chymal stem cells in the contact with bone-cartilage scaffold. The rBMSC
cell was cultured in the bone-cartilage scaffold leaching solution for 3
and 5 days to study the cell growth state through dyeing. As shown in
figure Fig. 6A, AO/EB staining after 3 days in inverted fluorescence
microscope showed that the cells in each scaffold grow with good con­
dition and visible spindle shape can easily be found. At the same time,
the number of cells increased with the extension of incubation time in
the scaffold extracting solution. After 5 days of culture, the cells
8
Colloids and Surfaces B: Biointerfaces 208 (2021) 112116
basically covered the whole interface. Although there existed orange
spots, but the number was very small, indicating that the scaffold had
good cell compatibility and non-toxicity. At the same time, the quanti­
tative proliferation of rBMSCs cell co-cultured with bone-cartilage
scaffolds was also studied. As shown in Fig. 6B, it is worth noting that
OD values of all groups were significantly higher than the minimum
standard of non-toxic. With time prolongation, the corresponding OD
value also increased. Comparing with control group and blank group,
the proliferation of cells in the experimental group was not obvious on
the first day. At the 7th day, the OD values of each group increased
significantly and the OD values of the S-1, S-2 and S-3 groups were
significantly higher than those of other groups, indicating that the
addition of SH improved the cell proliferation ability. Therefore, CCK-8
experimental results showed that all two-phase integrated scaffolds had
good cell compatibility. The survival rate of S-2 component scaffold cells
was the highest.
3.7. In vivo bone repair of calvarial defects
As can be seen from Fig. 7B, compared with initial defect area, the
tissues of the three groups were all repaired and the surface was filled
with soft tissue. The depression degree of the control group in the defect
area slowed down and the sag of the S-2 group was further reduced, but
the defect area was not completely filled. At 12 weeks, the experimental
group was basically completely repaired. The surface was relatively
rough and only the new tissue and boundary circle existed, indicating
that the fusion of the new tissue and the interface still needed to further
matured. As can be seen from the 16-week repair diagram, the defect
area of the control and experimental group has been completely covered
by the new tissue. Compared with the 12-week repair effect, the surface
of the experimental group has a good fusion between the new tissue and
the host boundary. The contour line has basically disappeared and the
surface is relatively smooth and flat. At the same time, the repair process
of articular bone-cartilage defects in rabbits was tested by CT. The tissue
repair was analyzed by microscopic reconstruction at 8, 12 and 16
G. Xu et al.
Fig. 8. Histological analysis of defect area after 12 weeks (A) and 16 weeks (B) implantation. Blank defect group (a, d, g, j); CS/nHAP group (b, e, h, k); S-2 SH/GO/
CS/nHAP group(c, f, i, l). The yellow arrows are osteocytes and the red arrows are new blood vessels. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
weeks, respectively. As shown in Fig. 7A, through three-dimensional
reconstruction and comprehensive sagittal analysis, it was found that
the repair process of each group was enhanced with the passage of time,
and the repair effect of the S-2 group was better than that of the control
group. According to the 3D reconstruction, compared with other groups,
the articular cartilage defects in the 16-week S-2 repair group had been
completely repaired. The contour lines were basically disappeared and
the surface was smooth and flat, indicating the rapid growth of new
tissue. According to the 2d-x sectional analysis, it was found that the
central defect had been filled with new tissue. The background color and
bone density were consistent with the surrounding bone. The 2d-y sec­
tion also showed that the upper part of the defect was completely
overgrown with newborn tissue, which was close to the height of the
host on both sides. Further 2d-z examination revealed that the border
cortical bone was also largely closed and intact, with no fractures or
fractures. The results of CT analysis showed that the prepared integrated
scaffold could effectively repair the tissue defect after implantation in
the defect area for a certain period of time, realize the bidirectional
9
Colloids and Surfaces B: Biointerfaces 208 (2021) 112116
physiological requirements of articular cartilage and subchondral bone,
and effectively promote the growth and maturation of new tissue.
3.8. HE staining analysis
H&E staining was used to analyze the formation process of cartilage/
bone. The regenerated tissue could be identified by the morphology in
defect area. The blank group still had loose structure after 12 weeks
implantation (Fig. 8A (a, d)). Only a few osteocytes grow and bone
matrix formation was not obvious. Additionally, in the control group,
osteocyte increased significantly and calcium accumulation appeared. In
S-2 group (Fig. 8A (c, f)), calcium accumulated significantly and blood
vessels began to form. After 16 weeks implantation, mature collagen
could be observed in S-2 group and much more blood vessel formed in
regenerated bony layer (Fig. 8B (i, l)). The collagen fiber density and
calcium accumulation in blank (Fig. 8B (g, j)) and control (Fig. 8B (h, k))
group was significantly lower than the S-2 group. The results demon­
strated that S-2 scaffold can better promote the regeneration of joint.
G. Xu et al.
4. Conclusion
For the special physiological structure of joint, the experiment con­
structed a continuous gradient scaffold. The scaffold had different pore
size and porosity on different layers. And EDS spectrum showed that
nHAP generated with gradient distribution. Other relevant tests also
showed that the scaffold largely imitated the environment of different
layers in joint. In cell co-culture system, the bone marrow mesenchymal
stem cells showed high proliferation vigor. In rabbit defect experiment,
CT three-dimensional reconstruction and comprehensive sagittal anal­
ysis of each group showed that the tissue regeneration increased over
time and S-2 group was the best. HE staining further confirmed that S-2
scaffold can better promote joint repair. To sum up, the continuous
scaffold can better imitate the functions of different layers comparing
with traditional scaffold and is expected to be a proper candidate for
joint.
CRediT authorship contribution statement
Jingdi Chen conceived the project, and Gan Xu developed the
experimental plan, performed and evaluated the in vitro experiments.
Jingdi Chen gave the guidance on animal surgeries in vivo. Gan Xu and
Yao Zhao planned and established animals’ protocols. Yusheng Geng,
Shujun Cao and Jianhua Wang helped with the operation of animal
surgeries. Gan Xu recorded and analyzed all data of the in vitro and vivo
experiments. In addition, Gan Xu drafted the manuscript, Yao Zhao and
Panpan Pan helped to revise the manuscript, and all authors contributed
in discussion of the results.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgement
The research was supported by National Key Research and Devel­
opment Program of China (2017YFC1104102), National Natural Science
Foundation of China (31370958) and Key Program of Natural Science
Foundation of Fujian Province (2018Y0056).
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.colsurfb.2021.112116.
References
[1] H. Zhang, H. Huang, G. Hao, Y. Zhang, H. Ding, Z. Fan, L. Sun, 3D printing
hydrogel scaffolds with nanohydroxyapatite gradient to effectively repair
osteochondral defects in rats, Adv. Funct. Mater. 31 (2020) 2006697–2006709.
[2] C.D. Hoemann, C.H. Lafantaisie-Favreau, V. Lascau-Coman, G.P. Chen, J. Guzman-
Morales, The cartilage-bone interface, J. Knee Surg. 25 (2012) 85–97.
[3] Z. Liu, M.A. Meyers, Z. Zhang, R.O. Ritchie, Functional gradients and
heterogeneities in biological materials: design principles, functions, and
bioinspired applications, Prog. Mater. Sci. 88 (2017) 467–498.
[4] B.A. Harley, A.K. Lynn, Z. Wissner-Gross, W. Bonfield, I.V. Yannas, L.J. Gibson,
Design of a multiphase osteochondral scaffold III: fabrication of layered scaffolds
with continuous interfaces, J. Biomed. Mater. Res. A 92 (2010) 1078–1093.
[5] J.M. Oliveira, V.P. Ribeiro, R.L. Reis, Advances on gradient scaffolds for
osteochondral tissue engineering, Prog. Biomed. Eng. 3 (2021) 033001–033022.
[6] C. Deng, J. Chang, C. Wu, Bioactive scaffolds for osteochondral regeneration,
J. Orthop. Translat. 17 (2019) 15–25.
[7] D. Yang, J. Xiao, B. Wang, L. Li, X. Kong, J. Liao, The immune reaction and
degradation fate of scaffold in cartilage/bone tissue engineering, Mater. Sci. Eng. C
Mater. Biol. Appl. 104 (2019) 109927–109943.
[8] R.A. Muzzarelli, F. Greco, A. Busilacchi, V. Sollazzo, A. Gigante, Chitosan,
hyaluronan and chondroitin sulfate in tissue engineering for cartilage regeneration:
a review, Carbohydr. Polym. 89 (2012) 723–739.
10
Colloids and Surfaces B: Biointerfaces 208 (2021) 112116
[9] M. Liu, X. Zeng, C. Ma, H. Yi, Z. Ali, X. Mou, S. Li, Y. Deng, N. He, Injectable
hydrogels for cartilage and bone tissue engineering, Bone Res. 5 (2017)
17014–17033.
[10] K.P. Arkill, C.P. Winlove, Solute transport in the deep and calcified zones of
articular cartilage, Osteoarthr. Cartil. 16 (2008) 708–714.
[11] H.X. Cai, P.L. Wang, Y. Xu, Y. Yao, J. Liu, T. Li, Y. Sun, J. Liang, Y.J. Fan, X.
D. Zhang, BMSCs-assisted injectable Col I hydrogel-regenerated cartilage defect by
reconstructing superficial and calcified cartilage, Regen. Biomater. 7 (2020) 35–46.
[12] W.D. Lee, M.B. Hurtig, R.M. Pilliar, W.L. Stanford, R.A. Kandel, Engineering of
hyaline cartilage with a calcified zone using bone marrow stromal cells,
Osteoarthr. Cartil. 23 (2015) 1307–1315.
[13] R. Longley, A.M. Ferreira, Recent approaches to the manufacturing of biomimetic
multi-phasic scaffolds for osteochondral regeneration, Int. J. Mol. Sci. 19 (2018)
1755–1771.
[14] C.H. Lee, J.L. Cook, A. Mendelson, E.K. Moioli, H. Yao, J.J. Mao, Regeneration of
the articular surface of the rabbit synovial joint by cell homing: a proof of concept
study, Lancet 376 (2010) 440–448.
[15] Y.Z. Zhang, J.R. Venugopal, A. El-Turki, S. Ramakrishna, B. Su, C.T. Lim,
Electrospun biomimetic nanocomposite nanofibers of hydroxyapatite/chitosan for
bone tissue engineering, Biomaterials 29 (2008) 4314–4322.
[16] A. Di Luca, C. Van Blitterswijk, L. Moroni, The Osteochondral Interface as a
Gradient Tissue: From Development to the Fabrication of Gradient Scaffolds for,
Regener. Med. Birth Defects Res. Part C-Embryo Today-Rev. 105 (2015) 34–52.
[17] J. Chen, P. Pan, Y. Zhang, S. Zhong, Q. Zhang, Preparation of chitosan/nano
hydroxyapatite organic-inorganic hybrid microspheres for bone repair, Colloids
Surf. B Biointerfaces 134 (2015) 401–407.
[18] Y.L. Li, C.S. Liu, Nanomaterial-based bone regeneration, Nanoscale 9 (2017)
4862–4874.
[19] G. Campi, F. Cristofaro, G. Pani, M. Fratini, B. Pascucci, P.A. Corsetto,
B. Weinhausen, A. Cedola, A.M. Rizzo, L. Visai, G. Rea, Heterogeneous and self-
organizing mineralization of bone matrix promoted by hydroxyapatite
nanoparticles, Nanoscale 9 (2017) 17274–17283.
[20] Z. Li, W. Mi, H. Wang, Y. Su, C. He, Nano-hydroxyapatite/polyacrylamide
composite hydrogels with high mechanical strengths and cell adhesion properties,
Colloids Surf. B Biointerfaces 123 (2014) 959–964.
[21] R. Ying, H. Wang, R. Sun, K. Chen, Preparation and properties of a highly dispersed
nano-hydroxyapatite colloid used as a reinforcing filler for chitosan, Mater. Sci.
Eng. C Mater. Biol. Appl. 110 (2020) 110689–110696.
[22] C. Koski, A.A. Vu, S. Bose, Effects of chitosan-loaded hydroxyapatite on osteoblasts
and osteosarcoma for chemopreventative applications, Mater. Sci. Eng. C Mater.
Biol. Appl. 115 (2020) 111041–111049.
[23] Y.W. Zhu, S. Murali, W.W. Cai, X.S. Li, J.W. Suk, J.R. Potts, R.S. Ruoff, Graphene
and graphene oxide: synthesis, properties, and applications, Adv. Mater. 22 (2010)
3906–3924.
[24] P. Yu, R.Y. Bao, X.J. Shi, W. Yang, M.B. Yang, Self-assembled high-strength
hydroxyapatite/graphene oxide/chitosan composite hydrogel for bone tissue
engineering, Carbohydr. Polym. 155 (2017) 507–515.
[25] G. Wei, C. Gong, K. Hu, Y. Wang, Y. Zhang, Biomimetic hydroxyapatite on
graphene supports for biomedical applications: a review, Nanomaterials 9 (2019)
1435–1454.
[26] Y. Zhao, J. Chen, L. Zou, G. Xu, Y. Geng, Facile one-step bioinspired mineralization
by chitosan functionalized with graphene oxide to activate bone endogenous
regeneration, Chem. Eng. J. 378 (2019) 122174–122187.
[27] M. Li, Y.B. Wang, Q. Liu, Q.H. Li, Y. Cheng, Y.F. Zheng, T.F. Xi, S.C. Wei, In situ
synthesis and biocompatibility of nano hydroxyapatite on pristine and chitosan
functionalized graphene oxide, J. Mater. Chem. B 1 (2013) 475–484.
[28] A.R. Poole, T. Kojima, T. Yasuda, F. Mwale, M. Kobayashi, S. Laverty, Composition
and structure of articular cartilage - a template for tissue repair, Clin. Orthop.
Relat. Res. (2001) S26–S33.
[29] Y. Huang, D. Seitz, F. Konig, P.E. Muller, V. Jansson, R.M. Klar, Induction of
articular chondrogenesis by chitosan/hyaluronic-acid-based biomimetic matrices
using human adipose-derived stem cells, Int. J. Mol. Sci. 20 (2019) 4487–4512.
[30] H. Tan, C.R. Chu, K.A. Payne, K.G. Marra, Injectable in situ forming biodegradable
chitosan-hyaluronic acid based hydrogels for cartilage tissue engineering,
Biomaterials 30 (2009) 2499–2506.
[31] S. Yamane, N. Iwasaki, T. Majima, T. Funakoshi, T. Masuko, K. Harada, A. Minami,
K. Monde, S. Nishimura, Feasibility of chitosan-based hyaluronic acid hybrid
biomaterial for a novel scaffold in cartilage tissue engineering, Biomaterials 26
(2005) 611–619.
[32] H. Park, K.Y. Lee, Cartilage regeneration using biodegradable oxidized alginate/
hyaluronate hydrogels, J. Biomed. Mater. Res. Part A 102 (2014) 4519–4525.
[33] N. Iwasaki, Y. Kasahara, S. Yamane, T. Igarashi, A. Minami, Chitosan-based
hyaluronic acid hybrid polymer fibers as a scaffold biomaterial for cartilage tissue
engineering, Polymers 3 (2010) 100–113.
[34] T. Kutlusoy, B. Oktay, N.K. Apohan, M. Süleymanoğlu, S.E. Kuruca, Chitosan-co-
Hyaluronic acid porous cryogels and their application in tissue engineering, Int. J.
Biol. Macromol. 103 (2017) 366–378.
[35] N. Mohan, P.V. Mohanan, A. Sabareeswaran, P. Nair, Chitosan-hyaluronic acid
hydrogel for cartilage repair, Int. J. Biol. Macromol. 104 (2017) 1936–1945.
[36] A. Di Martino, M. Sittinger, M.V. Risbud, Chitosan: a versatile biopolymer for
orthopaedic tissue-engineering, Biomaterials 26 (2005) 5983–5990.
[37] D.C. Marcano, D.V. Kosynkin, J.M. Berlin, A. Sinitskii, Z.Z. Sun, A. Slesarev, L.
B. Alemany, W. Lu, J.M. Tour, Improved synthesis of graphene oxide, ACS Nano 4
(2010) 4806–4814.
[38] D. Depan, B. Girase, J.S. Shah, R.D. Misra, Structure-process-property relationship
of the polar graphene oxide-mediated cellular response and stimulated growth of
G. Xu et al.
osteoblasts on hybrid chitosan network structure nanocomposite scaffolds, Acta
Biomater. 7 (2011) 3432–3445.
[39] S. Liu, C. Zhou, S. Mou, J. Li, M. Zhou, Y. Zeng, C. Luo, J. Sun, Z. Wang, W. Xu,
Biocompatible graphene oxide-collagen composite aerogel for enhanced stiffness
and in situ bone regeneration, Mater. Sci. Eng. C Mater. Biol. Appl. 105 (2019)
110137–110147.
[40] P.A. Mouthuy, H. Ye, J. Triffitt, G. Oommen, Z. Cui, Physico-chemical
characterization of functional electrospun scaffolds for bone and cartilage tissue
engineering, Proc. Inst. Mech. Eng. Part H 224 (2010) 1401–1414.
[41] X. Miao, D. Sun, Graded/Gradient porous biomaterials, Materials 3 (2009) 26–47.
[42] C. Deng, R. Lin, M. Zhang, C. Qin, Q. Yao, L. Wang, J. Chang, C. Wu, Micro/
Nanometer-structured scaffolds for regeneration of both cartilage and subchondral
bone, Adv. Funct. Mater. 29 (2019) 1806068–1806082.
[43] D.D. Deligianni, N.D. Katsala, P.G. Koutsoukos, Y.F. Missirlis, Effect of surface
roughness of hydroxyapatite on human bone marrow cell adhesion, proliferation,
differentiation and detachment strength, Biomaterials 22 (2001) 87–96.
[44] B. Kreklau, M. Sittinger, M.B. Mensing, C. Voigt, G. Berger, G.R. Burmester,
R. Rahmanzadeh, U. Gross, Tissue engineering of biphasic joint cartilage
transplants, Biomaterials 20 (1999) 1743–1749.
11
Colloids and Surfaces B: Biointerfaces 208 (2021) 112116
[45] W.M. Groen, P. Diloksumpan, P.R. van Weeren, R. Levato, J. Malda, From intricate
to integrated: biofabrication of articulating joints, J. Orthop. Res. 35 (2017)
2089–2097.
[46] J. Ruan, X.S. Wang, Z. Yu, Z. Wang, Q. Xie, D.D. Zhang, Y.Z. Huang, H.F. Zhou, X.
P. Bi, C.W. Xiao, P. Gu, X.Q. Fan, Enhanced physiochemical and mechanical
performance of chitosan-grafted graphene oxide for superior osteoinductivity, Adv.
Funct. Mater. 26 (2016) 1085–1097.
[47] S. Nair, N.S. Remya, S. Remya, P.D. Nair, A biodegradable in situ injectable
hydrogel based on chitosan and oxidized hyaluronic acid for tissue engineering
applications, Carbohydr. Polym. 85 (2011) 838–844.
[48] B.Q. Zhang, H. Sun, L.N. Wu, L. Ma, F. Xing, Q.Q. Kong, Y.J. Fan, C.C. Zhou, X.
D. Zhang, 3D printing of calcium phosphate bioceramic with tailored
biodegradation rate for skull bone tissue reconstruction, Bio-Des. Manuf. 2 (2019)
161–171.
[49] T. Kokubo, H.M. Kim, M. Kawashita, Novel bioactive materials with different
mechanical properties, Biomaterials 24 (2003) 2161–2175.
[50] V.M. Rusu, C.H. Ng, M. Wilke, B. Tiersch, P. Fratzl, M.G. Peter, Size-controlled
hydroxyapatite nanoparticles as self-organized organic-in organic composite
materials, Biomaterials 26 (2005) 5414–5426.