Materials and Design 203 (2021) 109621

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Materials and Design

journal homepage: www.elsevier.com/locate/matdes

3D bioprinting modified autologous matrix-induced chondrogenesis

(AMIC) technique for repair of cartilage defects
Yang Zhou 1, Ran Qin 1, Tong Chen, Kaibin Zhang, Jianchao Gui ⁎
Department of Sports Medicine and Joint Surgery, Nanjing First Hospital, Nanjing Medical University, No. 68 Changle Road, Nanjing, Jiangsu 210006, PR China

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Modified autologous matrix-induced

chondrogenesis to repair cartilage
defects.
• 3D printed biofilms combined
chondrogenic progenitor cells.
• Fibronectin was added to improve
the biological function of composite
hydrogel.
• A stable postoperative microenviron-
ment for bone mesenchymal stem cells.
• Reproducing layered structure similar
to natural cartilage.

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

Article history: Three-dimensional (3D) printing technology, has achieved good results in articular cartilage damage repair, yet
Received 21 October 2020 regeneration of hyaline cartilage similar to native cartilage and effectively performing clinical transformation re-
Received in revised form 26 January 2021 main challenging. In this study, we used 3D bioprinting technology to add chondrogenic progenitor cells (CPCs)
Accepted 27 February 2021
and fibronectin (FN) to an alginate/gelatin/hyaluronic acid (Alg/Gel/HA) composite hydrogel. We used this hy-
Available online 5 March 2021
drogel to prepare an active biofilm with uniform pores using modified autologous matrix-induced chondrogen-
Keywords:
esis (AMIC) technology to effectively repair cartilage defects. The Alg/Gel/HA composite hydrogel combined with
AMIC FN promoted the chondrogenic differentiation of CPCs by upregulating their gene expression of collagen II, SOX9,
3D bioprinting and especially PRG4. Adjacent biofilm provided adequate mechanical support and architectural integrity, offering
Cartilage repair a stable postoperative microenvironment for bone mesenchymal stem cells (BMSCs) released from subchondral
Chondrogenic progenitor cells bone, and ensured that a laminar structure similar to natural hyaline cartilage was regenerated in the rat cartilage
Fibronectin defect model. Characteristic cartilage-like lacuna structures and a gradient structure were observed in the AMIC
Gradient structure +FN + CPCs group. We developed an effective method to regenerate full-thickness cartilage defects, this
biofunctionalized cell-laden biofilm has great potential for application as a supplement to traditional AMIC tech-
nology to improve the quality of cartilage regeneration in a relatively feasible way.
© 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).

Abbreviations: Three-dimensional, 3D; Autologous matrix induced chondrogenesis, AMIC; Chondrogenic progenitor cells, CPCs; Fibronectin, FN; Sodium alginate, Alg; Gelatin, Gel;
Hyaluronic acid, HA; Proteoglycan 4, PRG4; COL2, Type 2 collagen; Bone mesenchymal stem cells, BMSCs; Osteoarthritis, OA; Autologous chondrocyte implantation, ACI; Microfracture,
MF; Hydroxyapatite, HAP; Extracellular matrix, ECM; Scanning electron microscopy, SEM; ANOVA, Analysis of variance; Interpenetrating Polymer Network, IPN; Enzyme linked
immunosorbent assay, ELISA.
⁎ Corresponding author at: Department of Sports Medicine and Joint Surgery, Nanjing First Hospital, Nanjing Medical University, Nanjing 210006, PR China.
E-mail address: gui1997@126.com (J. Gui).
1
These authors contributed equally to this work and should be considered co-first authors.

https://doi.org/10.1016/j.matdes.2021.109621
0264-1275/© 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Y. Zhou, R. Qin, T. Chen et al.
1. Introduction
Osteoarthritis (OA) is one of the most common diseases in middle-
aged and elderly people. It is a chronic and progressive degenerative
disease characterized by the degeneration and destruction of articular
cartilage [1]. Articular hyaline cartilage maintains very low metabolic
activity, and lacks distribution of blood vessels, nerves, and lymph ves-
sels. Once damaged, it lacks the capacity to heal itself, and the damaged
cartilage will accelerate degeneration of the affected joints and the
onset of OA [2]. Therefore, effectively repairing early focal damage to ar-
ticular cartilage, controlling its degeneration, and rebuilding a durable
articular cartilage surface can effectively delay or prevent the occur-
rence of OA [3]. Traditional articular cartilage repair techniques include
osteochondral transplantation, autologous chondrocyte implantation
(ACI), osteotomy, microfracture technology (MF), and autologous
matrix-induced chondrogenesis (AMIC) technology [4,5]. However,
the repair effects are limited and lack long-term efficacy. Although
they can promote the formation of new articular surfaces, the newly
formed tissue is not necessarily hyaline cartilage [6].
The microfracture technique involves drilling a hole in the bone sur-
face so that blood from the bone marrow containing bone mesenchymal
stem cells (BMSCs) will ooze out to repair cartilage [7]. Because the local
microenvironment after surgery cannot recruit and guide the differenti-
ation of BMSCs, this results in regeneration of essentially fibrous carti-
lage, the strength of which is significantly lower than that of native
hyaline cartilage [8]. Therefore, improved microfracture technology
was proposed in the clinic, and it was named AMIC technology, which
provides a solution by combining microfracture with a collagen I/III ma-
trix applied to the cartilage defects. Compared with the microfracture
technique, this method can effectively and rapidly relieve pain, restore
joint function, successfully restore motor function, and has a relatively
long-term effect [9,10]. However, AMIC technology still cannot
completely repair cartilage defects, and regeneration of hyaline cartilage
with a gradient structure similar to native cartilage is still an urgent
problem to be solved [11].
In recent years, 3D printing technology has gradually spawned a
new generation of cartilage repair techniques. 3D printing technology
can accurately control the internal structure of the scaffold, construct a
cartilage-like structure, and control the internal pore size to achieve
customization according to cartilage defects. 3D bioprinting can also
print hydrogel embedded with cells or active cytokines, and provide a
new direction for constructing in vivo cartilage grafts [12]. In recent
years, there have been many advances in hydrogel-based 3D printing
for tissue engineering [13]. Recently, a direct coaxial 3D bioprinting sys-
tem was reported by Shao et al., who prepared a cell-laden structure
with effective vascularized nutrient delivery channels [14]. Dalya et al.
prepared spatially organized tissues by directing the growth of cellular
spheroids within 3D-printed polymeric microchambers to mimic the
development process of native cartilage [15]. As the most widely used
material for deposition 3D printing, hydrogel has many advantages,
such as a similar structure to human soft tissues and high water content
[16]. Commonly used natural hydrogel materials, such as sodium algi-
nate (Alg), a natural polysaccharide, have advantages such as a wide va-
riety of sources, low price, and good histocompatibility and
biodegradability [17]. It can quickly undergo ion exchange cross-
linking when encountering cations and has been widely used in carti-
lage tissue engineering [18]. However, Alg hydrogel does not contain
any RGD molecules due to its natural sources, and consequently it
lacks cell adhesion sites. In addition, its mechanical strength is poor,
and it is difficult to realize a complex three-dimensional structure.
Therefore, Alg-based hydrogels have been used, supplemented with
an ECM component such as hyaluronic acid (HA), hydroxyapatite
(HAP) or gelatin (Gel) [19–21] . Krishna C. R. Kolan et al. investigated
the alginate-gelatin composite bioink modified with bioactive glass
and the feasibility of the solvent-based 3D bioprinting technique for tis-
sue engineering applications [22]. Gel is taken from natural biological
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Materials and Design 203 (2021) 109621
collagen fibers and exhibits homology with collagen [23]. It can not
only replace the collagen component in the cartilage extracellular ma-
trix, but also enhance the hardness and viscosity of the Alg-based scaf-
fold and enhance its printability. HA is an acidic mucopolysaccharide
macromolecular substance and is widely present in connective tissues
of humans and animals. It can regulate various physiological functions
of cells as well as cell aggregation during tissue formation, and possesses
significant chondrogenic effects that show its application value in carti-
lage tissue engineering [24]. Meanwhile，HA is widely present in carti-
lage tissue and has a CD44 recognition site, which can specifically
recognize cartilage cells and maintain the chondrocyte phenotype
[25]. Fibrin-based bioinks also usually blend with other materials such
as gelatin, collagen, and alginate [26]. Therefore, in this study, we pre-
pared a composite hydrogel based on Alg, Gel, and HA, which improves
upon the defects of a single Alg hydrogel and has good printability. It can
be used for extrusion 3D printing to prepare active biofilms for modified
AMIC technology.
Although 3D bioprinting is expected to lead to a new generation of
cartilage regeneration therapy, it remains a huge challenge to regener-
ate hyaline cartilage similar to native cartilage and reconstruct a layered
structure with both mechanical and biological characteristics similar to
native cartilage [27]. The regeneration is driven by cells encapsulated
within the hydrogel. After implantation, through interaction with the
host's biomechanical and biochemical environment, cartilage regenera-
tion is achieved over time. Therefore, it is very important to use cells
with good regeneration ability in the bioink. Traditional seed cells
such as chondrocytes are associated with various problems such as lim-
ited availability, low proliferative ability, and dedifferentiation during
culture expansion [28]. Mesenchymal stem cells can be obtained from
a wide range of sources and can be induced to differentiate into
chondrocytes. They have strong reproductive capacity and good prolif-
eration and differentiation activity, but they also have problems such
as Chondrogenic progenitor cells (CPCs) are new seed cells for cartilage
tissue engineering. CPCs can be separated by differential adhesion to fi-
bronectin (FN) [29]. They have similar characteristics to the self-cloning
and proliferation of stem cells. They are primitive cells located in carti-
lage tissue and have the ability to self-proliferate as well as having the
potential to differentiate into chondrocytes by themselves [30,31]. FN
is a high molecular weight (450 kDa) glycoprotein that promotes cell–
cell and cell–substrate adhesion and cell migration, which are necessary
to maintain cell structure and function [32]. In our previous study, we
isolated CPCs through FN differential adhesion experiments and evalu-
ated their stemness, proving that FN can promote the proliferation, mi-
gration, and chondrogenesis of CPCs through integrin α5β1-dependent
signaling, thereby increasing the synthesis of both protein and RNA
[33,34]. However, little is known about the applicability of CPCs in car-
tilage tissue engineering, especially in combination with biomaterials
and 3D printing [35].
To address these issues, this research aimed to realize the co-
printing of FN, CPCs, and Alg/Gel/HA composite hydrogel through extru-
sion 3D bioprinting to prepare an active biofilm. This was then used to
modify AMIC technology, by using this active biofilm combined with
microfracture technology to improve the quality of regenerated carti-
lage for the repair of articular cartilage damage. First, the Alg/Gel/HA
composite hydrogel had a better cartilage-promoting effect compared
with AMIC technology using a single I/III collagen membrane. The uni-
form porosity and good structure of the active biofilm was conducive
to exchange of nutrients and communication between cells, effectively
promoting growth and proliferation, and providing certain mechanical
support before the generation of new cartilage [36]. The bioactive bio-
film effectively recruited BMSCs and provided a good microenviron-
ment to guide the BMSCs to differentiate into chondrocytes through
their interaction with biofilms [37,38]. FN can also promote the prolifer-
ation and differentiation of chondrogenic progenitor cells through
integrin α5β1-dependent signaling pathways. At the same time,
BMSCs stimulate CPCs through paracrine signals, promoting the
Y. Zhou, R. Qin, T. Chen et al.
proliferation of CPCs and the production of total chondrocyte extracellu-
lar matrix [15]. We evaluated the function of this biofilm in vitro and in
rat animal models. In general, this active biofilm combined the excellent
properties of Gel, Alg, and HA, had good biocompatibility, good mechan-
ical properties, and suitable uniform porosity. At the same time, the ad-
dition of CPCs and FN improved its ability to regenerate cartilage. The
biofilm offered a stable microenvironment for BMSCs and newly formed
tissue, possessing the typical layered structure characteristic of natural
hyaline cartilage, was regenerated in the rat cartilage defect. The find-
ings of this study can be used to improve AMIC technology to repair car-
tilage defects, providing a theoretical basis for future clinical
transformation [39].
2. Materials and methods
2.1. Materials
Alg from marine algae and FN were purchased from Sigma-Aldrich
(St Louis, MO, USA). Gel was purchased from Shanghai Aladdin Bio-
chemical Co. Ltd. (Shanghai, China). HA was purchased from Shanghai
Macklin Biochemical Co. Ltd. (Shanghai, China).
2.2. Preparation and characterization of Alg/Gel/HA composite hydrogels
To prepare the composite hydrogel, 2.5 g Gel, 1.25 g Alg and 0.1 g HA
were each added to 50 mL deionized water and heated and stirred at
37 °C until fully dissolved, forming a 5% Gel +2.5% Alg + 0.2% HA hydro-
gel precursor solution (w/v). Keeping other conditions constant, the
2.5 g Gel was replaced with 4 g and 5 g respectively, and the above
steps were repeated to obtain 8% Gel +2.5% Alg + 0.2% HA and 10%
Gel +2.5% Alg + 0.2% HA hydrogel precursor solutions (w /v).
2.2.1. Rheological characterization
The rheological properties of the bioink were analyzed using a rhe-
ometer (DHR-2, TA Instruments, New Castle, DE, USA) with a 20 mm
aluminum fixture. For the thermosensitive process, the oscillation
model was adopted. The test temperature range was 5–37 °C, the tem-
perature reduction rate was 5 °C/min, the strain was 1%, and the fre-
quency was 1 rad/s. The gel point was determined as the time when
the storage modulus (G') surpassed the loss modulus (G"). The mea-
surements of shear stress were performed by varying the shear rate
from 1 to 600 s−1 with rotational tests at 37 °C. A frequency sweep
was performed and by varying the angular frequency from 0.1 to
100 rad/s at 37 °C, the strain was 1%.
2.2.2. Mechanical tests
Mechanical properties of the Alg/Gel/HA composite hydrogels with
different concentration ratios were measured using a dynamic mechan-
ical analysis instrument (ElectroForce, TA Instruments). Briefly, the hy-
drogel was soaked in 4% CaCl2 solution for 10 min to undergo ionic
crosslinking into a round pie-like structure for compression testing.
Three groups were tested, each sample was placed between two com-
pression plates, and the compressive speed was set at 1 mm/min. The
compressive modulus was calculated according to the slope of the linear
region in the 5–10% strain range of the stress−strain curve.
2.3. Isolation and expansion of BMSCs and CPCs
BMSCs were harvested from 2-week-old male Sprague Dawley (SD)
rats. Specifically, bone marrow aspirates were collected from the femur
of each rat and mononuclear cells were separated by gradient centrifu-
gation and then plated into 100 mm dishes. Cells were cultured in
DMEM (Hyclone, Logan, UT, USA) containing 10% FBS and 1% penicil-
lin/streptomycin at 37 °C with 5% CO2. BMSCs grown to passage three
were used in the present study.
3
Materials and Design 203 (2021) 109621
CPCs were isolated in accordance with previously described method
[40]. Briefly, an aliquot of the freshly isolated cartilage cells was pelleted
by centrifugation, suspended in serum-free DMEM/F12, and plated into
FN-coated tissue culture plates, at a density of 500 cells cm−2. After
20 min, non-adherent cells were removed, and the attached cells were
cultured in DMEM containing 10% FBS and 1% penicillin/streptomycin
at 37 °C with 5% CO2. Cells were expanded until passage two before
being used for the study.
2.4. 3D bioprinting and characterization of cell encapsulated constructs
To prepare the cell-laden bio-ink for bioprinting, 2.5 g Gel, 1.25 g Alg,
and 0.1 g HA were each add to 30 mL DMEM basic medium and heated
and stirred at 37 °C until fully dissolved to form a 5% Gel +2.5%
Alg + 0.2% HA hydrogel precursor solution (w/v). Then, FN was added
to the solution to a final concentration of 20 μg/mL. Finally, the cell sus-
pension and the pre-prepared hydrogel solution were evenly mixed at a
volume ratio of 2:3 to obtain a cell-laden bio-ink with a cell concentration
of 106 cells/mL. Gel, Alg, HA, and CaC2l powder were disinfected with UV
for more than 2 h, and all operations were performed on a clean bench.
Biofilm scaffolds were fabricated using a pneumatic 3D Bioprinter
(EFL-BP-6601, Yongqinquan Intelligent Equipment Co., Ltd., Suzhou,
China) using a direct extrusion-based technique. Temperature control-
lers were placed around the syringe and nozzle to ensure a homoge-
neous plotting temperature, while a cooling system was also
implemented under the receiving platform to maintain a stable temper-
ature after the deposition of the cell-laden bioink. Cell-laden bioink was
loaded into the printing cartridge, and extruded onto the pre-cooling
platform. The entire printing system was contained within a plexiglass
box to prevent temperature fluctuations. The specific printing parame-
ters of hydrogels with different concentration ratios are given in
Table 1. Scaffolds for in vitro biological assessment had dimensions of
10 mm (length) × 10 mm (width) × 1 mm (height) (Fig. 1). Particularly
in animal experiments, biofilms were fabricated with a disk shape and a
diameter of 2 mm to maximize the covered area in the cartilage defect.
2.4.1. Scanning electron microscopy
The biofilm scaffolds were lyophilized, and the cross-section was ob-
served with a scanning electron microscope (JSM-IT100, Jeol Ltd.,
Tokyo, Japan).
2.4.2. Viability evaluation of cells
To evaluate the viability of CPCs in biofilm, a live/dead staining kit
(KeyGEN BioTECH Co., Ltd., Nanjing, China) was used according to the
manufacturer's instructions. After culturing the printed biofilm for 1,
7, and 14 days, the cells in biofilm were stained. Then, the cell-laden
scaffolds were observed and imaged under a confocal fluorescence mi-
croscope (Olympus FV3000, Tokyo, Japan). Calcein AM-labeled live
cells emit green fluorescence, and propidium iodide (PI)-labeled dead
cells emit red fluorescence. Under a confocal microscope, multi-layer
fluorescence images were captured, and the software Imaris x64 7.2.1
Table 1
Primers sequences used in qRT-PCR.
Gene Primer sequences
GAPDH F:5′-CAACTCCCTCAAGATTGTCAGCAA- 3′
R: 5′-GGCATGGACTGTGGTCATGA- 3′
Collagen II F: 5′-CAACACTGCCAACGTCCAGAT- 3′
R: 5′-TCTTGCAGTGGTAGGTGATGTTCT- 3′
PRG4 F: 5′-CACAATGAAGTCAAAGTGAGCA- 3′
R: 5′-CTAATGTTGGGCAGTGTTATCG- 3′
SOX9 F: 5′-CTTCCGCGACGTGGACAT- 3′
R: 5′-GTTGGGCGGCAGGTACTG- 3′
Acan F: 5′- AACTCAGTGGCCAAACATCC-3′
R: 5′-TCAGGAATCCCAGATGTTCC-3′
Y. Zhou, R. Qin, T. Chen et al.
Fig. 1. (A) Schematic diagram of the hydrogel biofilm model and specific dimensions. (B) Biofilm printing effects of three different ratio hydrogels. (C) Demonstration of 3D printability of
5% Gel +2.5% Alg + 0.2% HA hydrogel and photograph of the printed scaffolds with various shapes: branched vessel and meniscus.
was used for three-dimensional reconstruction. Cell viability was de-
tected at 1, 7 and 14 days, with 3 specimens at each time point. Image
J software was used to count the number of living/dead cells. For the
case where multiple cells overlap and affect the statistical accuracy,
the watershed method was adopted in this experiment to improve the
statistical accuracy. Click the statistical particles under the analysis to
get the number of live/dead cells, and 4 non-overlapping visual fields
were randomly selected for each experimental specimen for detection.
2.4.3. Cell morphological observations
Cells were seeded onto the biofilm and cultured for 1 and 14 days
and then rinsed using PBS and fixed in 4% paraformaldehyde solution
for 30 min. Subsequently, after washing three times with PBS, the cells
were permeabilized with 0.1% Triton X-100 for 10 min and stained
with TRITC phalloidin (Yeasen Biotech Co., Ltd., Shanghai, China) at a di-
lution of 1:1000 in PBS for 30 min in the dark. After washing three times
with PBS, DAPI (1:500) (Solarbio, Beijing, China) was applied to stain
nuclei for 10 min. Finally, the cell-laden scaffolds were washed with
PBS and imaged under a confocal fluorescence microscope (Olympus
FV3000), and the software Imaris x64 7.2.1 was used for three-
dimensional reconstruction.
2.4.4. Fibronectin release kinetics
To assess the release profile of FN from printed biofilm, a rat FN
ELISA kit was used (Cusabio, Wuhan, China). Briefly, printed biofilms
were placed into a 12-well plate, followed by addition of 200 μL of
PBS. The PBS was collected and replaced with fresh solution every
2 days for 2 weeks. The collected PBS was stored at −80 °C. Finally,
the content of FN was quantified using a rat-specific ELISA kit according
to the manufacturer's instructions.
2.5. In vitro biological assessment
2.5.1. Immunofluorescence and Alcian blue staining
To detect secretion of cartilage-specific extracellular matrix by em-
bedded CPCs, after culturing for 7 or 14 days, the bioprinted constructs
4
Materials and Design 203 (2021) 109621
were fixed in 4% paraformaldehyde at room temperature for 30 min and
then stained with tissue-specific markers. Briefly, after fixation and
blocking of non-specific antigen, bioprinted constructs were incubated
overnight at 4 °C with one of the following primary antibodies: rabbit
monoclonal anti-Lubrin/PRG4 (1:500, Abcam, Cambridge, UK) and
anti-SOX9 (1:500, Abcam), and rabbit polyclonal anti-Collagen II
(1:500, Bioss Antibodies, Woburn, MA, USA). After washing, they were
then incubated with a red fluorophore-labeled mouse anti-rabbit sec-
ondary antibody (1:500) for 2 h in the dark at room temperature. Fi-
nally, incubated sections were mounted in DAPI (Servicebio) and
pictures were taken with a scanning laser confocal microscope (Olym-
pus FV3000) within 24 h.
The 3D-printed biofilms were cultured in DMEM, and after 7 days of
culture, chondrogenic medium (Cyagen Biosciences Inc., Guangzhou,
China) was added for continuous induction and culture for 21 days. Sul-
fated glycosaminoglycans (GAGs) were stained with 0.5% Alcian blue
(Sigma, pH 1) for 10 min at room temperature.
2.6. qRT-PCR determination of gene expression levels in vitro
The gene expression levels of collagen II, PGR4 and SOX9 were ana-
lyzed by qRT-PCR. Briefly, biofilms were cultured for 3, 7, or 14 days,
then lysis solution containing 1.62% sodium citrate, 0.585% EDTA and
0.88% sodium chloride was used to lyse the biofilm to release cells.
Total RNA was extracted from cartilage using Trizol reagent (Invitrogen,
Carlsbad, CA, USA) and reverse-transcribed with PrimeScript RT Master
Mix (Perfect Real Time; TaKaRa Bio, Japan) following the manufacturer's
protocol. Quantitative real-time RT-PCR amplifications were performed
as described. The primers used in this study are listed in Table 2.
2.6.1. Effects of bioprinted biofilm on differentiation of BMSCs
An indirect contact co-culture model was applied to study the im-
pact of Alg/Gel/HA + CPCs+FN biofilm on differentiation of BMSCs re-
leased by microfracture, using Transwell culture plates (Corning, NY,
USA). In short, rat BMSCs were seeded at a density of 5 × 105 cells per
well into wells of 6-well culture plates. Then, Transwells culture plates
Y. Zhou, R. Qin, T. Chen et al.
Table 2
The specific printing parameters of hydrogels with different concentration ratios.
Printing speed Printing pressure Nozzle temperature Platform temperature Inter layer increment Nozzle diameter Inter filament distance
5%Gel+2.5%Alg+0.2%HA 360 mm/min 18 ~ 19kpa 37 °C
8%Gel+2.5%Alg+0.2%HA 360 mm/min 37 ~ 38kpa 37 °C
10%Gel+2.5%Alg+0.2% 360 mm/min 41 ~ 42kpa 37 °C
HA
loaded with printed biofilm were suspended over the wells seeded with
BMSCs. The BMSCs and biofilm were fed with 5 mL DMEM containing
10% FBS and 1% penicillin/streptomycin. BMSCs were seeded at the
same density in six-well culture plates without biofilm as the control
group.
To assess the impact of biofilm on chondrogenic differentiation of
BMSCs, Alcian blue staining was performed on BMSCs after 7 days.
qRT-PCR was then utilized to test the gene expression of chondrogenic
differentiation markers (COLII, aggrecan (Acan), and SOX9) in the
BMSCs cultured with biofilm. After cultured for 7 or 14 days, total RNA
was extracted from the cell layer using Trizol reagent (Invitrogen, Carls-
bad, CA, USA) and reverse-transcribed with PrimeScript RT Master Mix
(Perfect Real Time; TaKaRa Bio Inc., Shiga, Japan) following the manu-
facturer's protocol. Quantitative RT-PCR amplifications were performed
as described above.
2.7. Articular cartilage defect models
SD rats were provided by the Animal Center of Nanjing Medical Uni-
versity. All animal protocols were approved by the animal ethics com-
mittee of Nanjing Medical University. In this experiment, 50 male SD
rats aged 3 months were utilized. A total of five groups were evaluated,
ten rats per group: microfracture only, AMIC+, AMIC+FN, AMIC+CPCs,
and AMIC+FN + CPCs. A full-thickness cylindrical cartilage defect
(2 mm in diameter and 1 mm in depth) was created on the trochlear
groove of each distal femur using a drill. In MF groups, a 0.5 mm K-
wire was used to drill three approximately 3 mm holes. In other four
AMIC-modified biofilm groups, 3D-printed Alg/Gel/HA biofilm only
(AMIC+) or containing FN (AMIC+FN), chondroprogenitor cells
(AMIC+CPCs), or FN and CPCs (AMIC+FN + CPCs), was placed over
the cartilage defect site and secured with fibrin glue (Guangzhou Beixiu
Biotechnology Co., Ltd., Guangzhou, China). After the operation, rats
were allowed to move freely in their cages and fed with standard food
and water. 6 or 12 weeks later, rats were sacrificed for further study.
After sacrifice, damaged cartilage repair site of femoral bottom was
collected and then general observation and photograph were per-
formed. The quality of cartilage repair was assessed by three blinded in-
dependent researchers using Wakitani's histological scoring standard.
All specimens were fixed with 4% paraformaldehyde, embedded in par-
affin and sectioned for histological and immunohistochemical analysis.
Sections were stained according to previously established methods
with hematoxylin and eosin (HE), Safranin-O, and anti-type II collagen
to evaluate the histological structure and cartilage ECM deposition in
the regenerated cartilage.
Besides, qRT-PCR was used to analyze the gene expression of specific
differentiation makers in each groups. Total RNA was extracted from
cartilage using Trizol reagent (Invitrogen) and reverse-transcribed
with PrimeScript RT Master Mix (Perfect Real Time; TaKaRa Bio) follow-
ing the manufacturer's protocol. Quantitative real-time RT-PCR amplifi-
cations were performed as described in Section 2.5.2.
2.8. Statistical analysis
All analyses were carried out using GraphPad Prism 6.0 software
(GraphPad Software, Inc., La Jolla, CA, USA). All data are shown as the
mean ± standard deviation (SD) and comparisons were carried out
Materials and Design 203 (2021) 109621
5 °C 0.18 mm*2 260 μm 1.1 mm
5 °C 0.18 mm*2 260 μm 1.1 mm
5 °C 0.18 mm*2 260 μm 1.1 mm
using Student's t-test or two-way ANOVA for comparing different
groups. Values of P < 0.05 were considered statistically significant.
3. Results and discussion
In this study, we developed a bioactive cell-laden biofilm combined
with FN to improve AMIC technology, accelerating cartilage regenera-
tion, and confirmed its repair effect of cartilage defects. Previous studies
have demonstrated that covering a microfractured area could result in
better cartilage regeneration [41]. Our combination AMIC technique
promoted cartilage regeneration and accumulation of the cartilage-
specific extracellular matrix, showing well integration with the sur-
rounding normal cartilage, as compared to the traditional MF and
AMIC technique. In the development and application of composite hy-
drogel scaffolds, based on 3D printing technology, it is of great signifi-
cance to integrate the complementary properties of materials and
have a good ability to induce cartilage differentiation. As a natural poly-
saccharide, Alg has good biocompatibility and biodegradability, and can
quickly gel by chelating with Ca2+. However, it lacks the corresponding
cell adhesion sites and the ability to induce cartilage differentiation [42].
Gel, as a degradation product of collagen widely present in cartilage tis-
sue, has a large number of RGD binding sites and has strong adhesion to
cells. Therefore, Alg/Gel-based hydrogels have been widely used in car-
tilage tissue engineering [43]. Hyaluronic acid is widely present in carti-
lage tissue and has been widely used in the treatment of osteoarthritis
in clinic.
3.1. Synthesis and characterization of composite hydrogel
To develop a suitable biofilm to replace collagen membrane and im-
prove the cartilage repair effect of AMIC technology, it is necessary to
develop a material which provides both a soft and stimulatory environ-
ment for cells (both CPCs and BMSCs) to maintain their chondrogenic
phenotype, while also providing a certain mechanical support before
neocartilage regeneration, after which the biofilm will be completely
degraded and eventually replaced by new cartilage. In this work, in
order to improve the biological activity and achieve a good cartilage re-
generation effect, HA was added to Alg/Gel-based hydrogel to prepare
an Alg/Gel/HA composite hydrogel. Using an Alg, Gel and HA
prepolymer mixture, a multimaterial Interpenetrating Polymer Net-
work (IPN) hydrogel was fabricated through temperature change and
Ca2+ mediated dual-crosslinking (Scheme 1). To optimize the polymer
blend for use as a bioink for depositional 3D bioprinting, Gel concentra-
tions of 5, 8, and 10% were investigated, while the Alg concentration was
fixed at 2.5% and the HA concentration was fixed at 0.2%. Both concen-
trations were previously reported as being ideal for the fabrication of
hydrogels with suitable toughness and enhanced promotion of
chondrogenic differentiation for cartilage engineering. Bioink was pre-
pared by compounding Alg, Gel and HA, which improved the disadvan-
tages of poor cell adhesion of Alg hydrogel and inhibited the rapid
degradation of HA to a certain extent. We tested the effect of different
Gel concentrations on the performance of the composite hydrogel, and
the results showed that the 3D-printed biofilm prepared from the hy-
drogel containing 5% Gel has the best formability.
In the process of cooling from 37 °C to 5 °C, the sample storage mod-
ulus (G′) was smaller than its loss modulus (G″) before the phase
5
Y. Zhou, R. Qin, T. Chen et al.
Scheme 1. Schematic illustration of 3D printing of chondrogenic progenitor cells (CPCs) and fibronectin (FN) encapsulated within biofilm for repair of cartilage defects. The experimental
process can be divided into three main steps: 1) The composition and preparation of cell-laden bioink; 2) Preparation of biofilm scaffolds by extrusion-based 3D bioprinting; 3) Validation
of the cartilage damage repair effect of biofilm on a rat model.
transition temperature, and it was in the sol state. After cooling down to
the phase transition temperature, the storage modulus (G′) suddenly
increased, and when it was greater than the loss modulus (G″), it ap-
peared as a gel state (Fig. 2A). It can be seen from Fig. 2A that the sol–
gel phase transition temperatures of the three hydrogels were 10.6 °C,
12.1 °C, and 14.8 °C, for Gel concentrations of 5, 8, and 10%, respectively.
As the concentration of Gel increased, the sol–gel phase transition tem-
perature of the composite hydrogel increased accordingly. Under condi-
tions of 1% strain, the angular frequency gradually increased from
0.1 rad/s to 100 rad/s, the average storage modulus (elastic modulus)
of 5, 8, and 10% Gel hydrogels were 7736 Pa, 8575 Pa, and 13,698 Pa, re-
spectively (Fig. 2B). Moreover, the three hydrogels showed low depen-
dence on frequency from 0.1–100 rad/s, indicating that the hydrogels
had relatively high elasticity. The initial viscosities of the three
hydrogels were 3.45 Pa.s, 4.59 Pa.s, and 8.76 Pa.s. The hydrogels of
three concentrations all showed shear thinning. As the shear rate in-
creased, the viscosity of the three composite hydrogels decreased and
gradually approached (Fig. 2C).
Creating a soft microenvironment that simulates chondrocyte pro-
duction of pericellular matrix and allows fast diffusion of nutrients is
fundamental to scaffolds used for repairing joint cartilage damage.
Meanwhile, it is also essential to provide good mechanical support in
the early stages of joint cartilage regeneration [44]. Fig. 2D and E sum-
marizes the mechanical properties of the three hydrogels with
different Gel concentrations. When the Alg/HA content was fixed, the
mechanical strength was slightly increased with the increment in the
content of Gel. And the compression modulus of the three different con-
centrations of hydrogel were 7.07 ± 1.30, 8.68 ± 1.52, and 11.20 ±
6
Materials and Design 203 (2021) 109621
0.40 kPa. The difference in elastic modulus between the 5% and 10%
Gel hydrogels was statistically significant.
The Alg/Gel/HA composite hydrogel has shear thinning and a suit-
able sol–gel phase transition temperature (Fig. 2A–C) [16]. A hydrogel
scaffold with a high degree of discrimination is prepared by extrusion-
based 3D printing. It has uniform porosity, a good structure (Fig. 2C)
and suitable mechanical properties (Fig. 2D, E). Consequently, 5%Gel
+2.5% Alg + 0.2%HA hydrogel was chosen for subsequent experiments.
This scaffold can provide cells with appropriate mechanical properties
in the early stage of cartilage regeneration to support the structure of
the microenvironment, but will degrade over time, eventually being re-
placed by newly formed cartilage tissue.
3.2. 3D bioprinting and evaluation of cell encapsulated biofilm
3D bioprinting is a kind of rapid prototyping technology, and indi-
vidual precision medical treatment can be performed according to the
shape of the patient's cartilage defect [43]. In this work, a pneumatic
extrusion-based 3D bioprinter was used to deposit the Alg/Gel/HA
bioinks and Table 1 shows the specific printing parameters of hydrogels
with different concentration ratios. A schematic diagram of hydrogel
biofilm model and specific dimensions are shown in Fig. 1A. The print-
ing effects of the three hydrogel biofilms are presented in Fig. 1B. The
pore width ranged from 200 to 400 μm, 150–300 μm, and 560–
740 μm. During the printing process, 8% Gel +2.5% Alg + 0.2% HA and
10% Gel +2.5% Alg + 0.2% HA hydrogels were prone to obvious filament
breakage and uneven filament production, while the biofilm prepared
form 5% Gel +2.5% Alg + 0.2% HA hydrogel had a clear grid structure,
Y. Zhou, R. Qin, T. Chen et al. Materials and Design 203 (2021) 109621

Fig. 2. Rheological properties and mechanical characterizations of Alg/Gel/HA composite hydrogels with different gelatin concentrations. (A) Effect of temperature on storage modulus (G′)
and loss modulus (G″). (B) Storage modulus (G′) and loss modulus (G″) through frequency sweep analysis. (C) Shear rate dependencies of viscosities of the three formulated hydrogels.
(D) Compressive stress−strain curves. (E) Compressive modulus of the three hydrogels.(F)SEM (scanning electron microscopy) image of biofilm scaffold made from 5% Gel +2.5%
Alg + 0.2% HA. (Whole and local enlarged view.)

uniform pores and uniform filament distance. Because of the nature of
the shear thinning and viscosity changes, 5% Gel +2.5% Alg + 0.2% HA
gel precursors achieved smooth extrusion and rapid covalent
crosslinking by Ca2+. The thermo-responsive properties of the compos-
ite hydrogel provided mechanical support during bioprinting before
Ca2+ covalent crosslinking, while at the same time, the ideal structure
(200–400 μm pore size) and certain mechanical strength (elastic mod-
ulus: 7.07 ± 1.30 kPa) make it an ideal bioink for printing scaffolds to
treat cartilage defects. Therefore, the 5% Gel +2.5% Alg + 0.2% HA hy-
drogel was chosen for subsequent experiments. As observed by SEM,
the 3D-printed biofilm scaffold reproduced the predesigned structures
very well (Fig. 2C).
To evaluate the compatibility of Alg/Gel/HA hydrogel and any ad-
verse effects that may have been caused by the printing process, cell vi-
ability and morphology were monitored. The cytocompatibility of
composite hydrogels was evaluated by the live/dead assay of CPCs.

7
Y. Zhou, R. Qin, T. Chen et al. Materials and Design 203 (2021) 109621

CPCs maintained a high survival rate (> 90%) after 1, 7, and 14 days of
culture (Fig. 3A,E). Three-dimensional reconstruction of live and dead
staining also revealed the even distribution of CPCs throughout the bio-
film (Fig. 3B). The cell growth process was continuously observed for
two weeks to monitor cell proliferation and morphology. Compared to
the first day, the number of cells increased significantly after two
weeks (Fig. 3C). It has been proven that when MSCs spread on stiff sub-
strates, and with high cytoskeletal tension, they express high levels of
osteogenic cell makers, while when they maintain a round shape,
chondrogenic differentiation is favored [45,46]. As shown in Fig. 3C,
CPCs remained round in shape in the biofilm instead of visibly
stretching during culture. These findings indicate that the composite
hydrogels provide an ideal chemical and mechanical microenvironment
for the chondrogenic differentiation of CPCs. In addition, the cells began
to connect with each other and some aggregates were found. According
to our research and previous studies, FN enhances proliferation, migra-
tion, and the chondrogenic differentiation capacity of CPCs through the
integrin α5β1-dependent signaling pathway, with a FN concentration
of 20 μg/mL having the best effect [33]. Therefore, we prepared a com-
posite hydrogel combined with FN (20 μg /mL) to improve the biological
function of the hydrogel. ELISA was performed to test the release kinet-
ics of FN; a burst release was observed within the first eight days, after
which the release of FN significantly slowed down. The results showed
that FN can be effectively retained at the site of a cartilage defect for a
period of time after implantation, and that this enhances the prolifera-
tion and migration of CPCs. At the same time, the slow release of FN

Fig. 3. (A) Fluorescent microscopic images of CPCs after the live/dead assay on days 1, 7, and 14. (B) Three-dimensional reconstruction of live and dead staining of CPCs used the software
Imaris x64 7.2.1. (C) Cytoskeletal staining of CPCs cultured in the biofilm scaffold for 1 and 14 days. (D) Local magnification of cytoskeletal staining of CPCs at day 14. (E) Quantitative
analysis of live/dead staining. (F) Cumulative fibronectin release from printed biofilm. Alcian blue staining of printed CPCs grown in normal or chondrogenic medium in biofilm after
21 days in culture. Representative images were captured using (G) inverted microscope (H) stereo microscope. (I\ \K) Relative quantification of mRNA levels of chondrogenic makers
(PRG4, COLII, and SOX9) expressed by CPCs in each group by qRT-PCR cultured for 3, 7, or 14 days. (* indicates P <0.05, ** indicates P < 0.01, *** indicates P < 0.001). (2D: two-
dimensional control culture of CPCs in Petri dish, 3D: three-dimensional culture of CPCs in biofilm, 3D + FN: three-dimensional culture of CPCs in biofilm combined with fibronectin).

8
Y. Zhou, R. Qin, T. Chen et al.
from the biofilm can effectively recruit BMSCs released from
subchondral bone.
Extrusion-based 3D bioprinting can realize the co-printing of FN,
CPCs, and Alg/Gel/HA composite hydrogel to prepare a bioactive biofilm.
In this study, we used chondrogenic progenitor cells as seed cells in-
stead of traditional chondrocytes or mesenchymal stem cells, thus
avoiding the problems of dedifferentiation (chondrocytes), ossification,
and hypertrophy (MSCs) during the culture process. The introduction of
FN into a composite hydrogel improves the biological activity of the bio-
film. ELISA results confirmed the slow release of FN from the biofilm
after printing (Fig. 3F). FN retained within the biofilm can effectively en-
hance the proliferation, migration, and chondrogenic differentiation ca-
pacity of CPCs through the integrin α5β1-dependent signaling pathway.
Some of the FN released from the biofilm was able to effectively recruit
BMSCs and promote their proliferation and differentiation.
3.3. In vitro biological assessment of biofilm function
Due to the lack of cell adhesion sites and poor mechanical strength of
single-material hydrogel, in this study, we prepared an Alg/Gel/HA
composite hydrogel biofilm. HA has a CD44 recognition site, which
can specifically recognize chondrocytes and maintain the chondrocyte
phenotype. Gel, as a degradation product of collagen widely present in
cartilage tissue, has a large number of RGD binding sites, which can sig-
nificantly improve the ability of composite hydrogels to support cell ad-
hesion. To assess the chondrogenic differentiation of CPCs in biofilm
in vitro, the GAG secretion of CPCs cultured in biofilm was measured
using Alcian blue staining. The Alcian blue staining of the control
group and the induction group were both positive on biofilm, and the
experimental group was slightly darker (Fig. 3G, H). The results show
that CPCs embedded in biofilm prepared from Alg/Gel/HA composite
hydrogel possess the ability to spontaneously differentiate into
chondrocytes. The mRNA levels of COL2, PRG4, and SOX9 were evalu-
ated by qRT-PCR for 3,7, and 14 days (Fig. 3I–K). CPCs encapsulated
within the biofilm exhibited higher mRNA expression for every protein
expressed by native CPCs within 14 days. The addition of FN increased
the expression levels of chondrogenesis-related genes to different ex-
tents, in particular, the expression of PRG4. Interestingly, CPCs in biofilm
combined with FN had the highest expression of PRG4, a key factor in
joint lubrication [35], with the 3D + FN group displaying approximately
a 5.5-fold (day14) increase compared to the 3D group without FN (2.6-
fold). Furthermore, at day 14, CPCs in biofilm combined with FN re-
vealed higher mRNA levels with 2.4-fold increases in COLII, 5.5-fold in
PRG4, and 1.7-fold in SOX9, compared with CPCs cultured in plates
(Fig. 3K). The results indicated that Alg/Gel/HA composite hydrogel
stimulates the chondrogenic differentiation of CPCs.
Immunofluorescence analysis of COL2, PRG4 (cartilage-specific
markers) and SOX9 (a master regulatory gene of chondrogenesis),
showed a similar tendency (Fig. 4A). CPCs secrete cartilage-specific ex-
tracellular matrix under uninduced conditions. CPCs in the biofilm+FN
group showed a markedly enhanced green fluorescence signal for COL2,
compared the fluorescence signal of CPCs in the biofilm group. Specially,
strongly positive staining for PRG4 was present in the biofilm+FN
group after 14 days. The green signal from the stained chondrogenic
transcription factor SOX9 was detected in all groups.
The indirect contact co-culture model was applied to study the im-
pact of Alg/Gel/HA + CPCs+FN biofilm on chondrogenic differentiation
of BMSCs, simulating the BMSCs released by microfracture (Fig. 4B).
After 14 days, positive Alcian blue staining of BMSCs on the same well
below the Transwell was observed (Fig. 4B), and was increased com-
pared to that in BMSCs cultured on normal culture plates (control)
(Fig. 4B). This proved that the Alg/Gel/HA + CPCs+FN biofilm can pos-
itively stimulate chondrogenic differentiation of BMSCs released by
microfracture. Further qRT-PCR results confirmed that BMSCs co-
cultured with Alg/Gel/HA + CPCs+FN biofilm under the Transwell
9
Materials and Design 203 (2021) 109621
plates showed higher levels of COL2 and SOX9 than BMSCs cultured
under control environment (Fig. 4C-E).
The production of cartilage-specific extracellular matrix and in-
creased expression of chondrogenic genes (COL II, PRG4, and especially
SOX9) during culture was confirmed by immunofluorescence staining
(Fig. 4A) and qRT-PCR (Fig. 3I–K) . Previous studies always used TGF-β
throughout the culture period, which is costly. The Alcian blue staining
of the control group and the induction group were both positive on the
biofilm (Fig. 3G, H). These results suggest that exposure of CPCs to TGF-
β is not necessary in this designed biofilm and that CPCs embedded
within the biofilm possess the ability to spontaneously differentiate
into chondrocytes [15]. Therefore, this method can shorten the required
time in vitro, save costs, and avoid adverse effects caused by exposure to
TGF-β [45]. In addition, in cartilage defects, plenty of BMSCs are released
from sites of microfracture surrounding the biofilm. As the results
in vitro demonstrated, the bioactive biofilm encapsulated with CPCs
and FN stimulated BMSCs to undergo chondrogenic differentiation cul-
tured under the biofilm (Fig. 4) [47]. It's worth noting that the interac-
tions of BMSCs and CPCs can maintain the chondrogenic phenotype of
CPCs, which can promote hyaline cartilage formation [48]. Besides, the
BMSCs produce Multiple cytokines and thus induce CPCs to undergo
chondrogenic differentiation and proliferation through paracrine
signals.
3.4. In vivo assessment of cartilage defect repair
The ability of CPCs and FN encapsulated in Alg/Gel/HA biofilm for
cartilage damage repair was evaluated in a rat cartilage defect model.
The experiments were divided into five groups as follows: MF
(microfracture only), AMIC + (microfracture +3D-printed Alg/Gel/HA
biofilm), AMIC+FN (microfracture +3D-printed Alg/Gel/HA biofilm
combined with FN), AMIC+CPCs (microfracture +3D-printed Alg/Gel/
HA biofilm containing FN) and AMIC+FN + CPCs (microfracture
+3D-printed Alg/Gel/HA biofilm containing FN and CPCs). A full-
thickness cylindrical cartilage defect (2 mm in diameter) was created
on the trochlear groove of the distal femur, and then microfracture sur-
gery was performed and biofilms were implanted in the injured area
(Fig. 5A). General observation of the cartilage defects at 6 weeks and
12 weeks post-operation were executed to assess the cartilage regener-
ation ability of the biofilm. According to Wakitani's histological scoring
standard, the scores for each group are shown in Fig. 5C. 6 and
12 weeks after surgery, the scores were 14.5 and 12.5, 13.5 and 11, 11
and 7.75, 10.25 and 6.5, and 6.5 and 2.5 for the MF, AMIC+, AMIC
+FN, AMIC+CPCs, and AMIC+FN + CPCs groups, respectively. There
was no significant inflammatory response or synovitis in any of the
groups at any time point (Fig. 5B). At 6 weeks post-implantation, the
newly formed tissues had an irregular surface, and the interface be-
tween newly formed cartilage and normal cartilage was still noticeable.
The neocartilage in the AMIC+CPCs group was solid and well integrated
with the surrounding normal cartilage, especially in the AMIC
+FN + CPCs group, while the neocartilage in the AMIC+ and AMIC
+FN groups were baggy. In the MF group, only a small amount of regen-
erated tissue was observed in the defect and this resembled fibrous tis-
sue (Fig. 5B). The colour of new tissue was similar to the surrounding
tissue, indicating that the biofilm had been well degraded. At 8 weeks
post-implantation, the regenerated cartilage were still not well inte-
grated with the nearby native cartilage. Nonetheless, the newly formed
tissue in the AMIC+CPCs groups had become smoother and well inte-
grated with the surrounding normal cartilage. Especially, the interface
between neocartilage and normal cartilage could hardly be distin-
guished in the AMIC+FN + CPCs groups. It's worth noting that both
AMIC+CPCs and AMIC+FN + CPCs groups displayed pregnant cartilage
regeneration at 6 weeks and 12 weeks, compared with three other
groups. Especially, the AMIC+FN + CPCs groups exhibited the best
macroscopic cartilage regeneration covering the entire cartilage lesion
modeling area.
Y. Zhou, R. Qin, T. Chen et al. Materials and Design 203 (2021) 109621

Fig. 4. (A) COLII, PRG4, and SOX9 immunostaining of CPCs in each group. (green: COLII, PRG4, and SOX9, blue: nuclei) (B) Schematic diagram of evaluation method for investigating the
effects of Alg/Gel/HA + CPCs+FN biofilm on differentiation of BMSCs outside the construct. BMSCs were stained with Alcian blue. (C-E) Relative quantification of mRNA levels for COLII,
Acan and SOX9 expressed by BMSCs co-cultured with Alg/Gel/HA + CPCs+FN biofilm by qRT-PCR at 7 and 14 days (* indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001).

Images of HE-stained tissue are shown in Fig. 6. At week 6, the most
obvious cartilage damage in the MF group was observed in all five
groups, where little tissue was regenerated and the neocartilage lacked
characteristic cartilage-like lacunae structures. Then improvement in
the cartilage layer was observed after implantation of biofilm, and a
little neocartilage regeneration was verified. However, the structures
of neocartilage were different from native cartilage and the articular
surface was irregular. For the defects filled with Alg/Gel/HA biofilm
combined with FN or CPCs, some cartilage regeneration was observed,
but the neocartilage was thinner than normal, and might not be well

Fig. 5. (A) Images of cartilage injury model during surgery. (B) Gross morphology of regenerated cartilage tissues at 6 and 12 weeks after surgery. (C) Histological scoring for the repaired
tissues in the different groups. * indicates that the histological scores in these groups were significantly lower than that in the MF group (P < 0.005).

10
Y. Zhou, R. Qin, T. Chen et al.
integrated with the surrounding normal cartilage tissue, as well as lack-
ing well-organized maturation layers. On the other hand, although the
cartilage matrix was unevenly distributed to a small extent, the
chondrocytes proliferated and were arranged neatly. Interestingly, the
defects filled with biofilm combined with FN and CPCs showed regener-
ated hyaline cartilage, similar to native cartilage. Furthermore, no
boundary could be observed between neocartilage and the adjacent na-
tive cartilage. At 12 weeks post-implantation, area of cartilage defects
were almost completely covered by the regenerated cartilage tissue. A
distinct boundary between the neocartilage and adjacent normal carti-
lage could still be observed in the MF group while it was almost invisible
in the other AMIC+ groups. The AMIC+FN + CPCs groups still
displayed the best performance in cartilage regeneration in all five
groups. They also had characteristic cartilage-like lacunae and gradient
structures similar to native cartilage tissue.
Moreover, the repair effect of cartilage defects was further evaluated
by Safranin O, which stained the proteoglycan of newly formed cartilage
red (Fig. 7). At 12 weeks postoperation, on the surface of the regener-
ated cartilage tissue, a light red-stained layer was observed in all groups.
There was almost on difference in neocartilage between AMIC
+FN + CPCs groups and the surrounding normal cartilage tissue, the
red-stained layer was the most compact, and the staining was the
most uniform. Compared to the MF group, although the biofilm im-
planted group stimulated cartilage formation, the structure of the re-
generated cartilage in the AMIC+FN + CPCs groups was more similar
to normal cartilage tissue. Likewise, a distinct boundary was seen be-
tween the neocartilage and surrounding native cartilage. By contrast,
the defects were filled with hyaline cartilage-like tissue in the AMIC
+FN + CPCs groups at 6 weeks, which was positively stained by
Safranin O.
The results of type II collagen immunohistochemical staining are
shown in Fig. 8A. All AMIC-modified groups showed significant in-
creases in type II collagen expression compared to the MF control. The
Fig. 6. HE staining images of the regenerated tissues in the different groups at 6 and 12 weeks after surgery.
11
Materials and Design 203 (2021) 109621
addition of CPCs or FN-functionalized biofilm further increased the ex-
pression of type II collagen in the regenerated cartilage tissue. Similar
to the results of HE and Safranin O staining, COL II was more positively
and uniformly stained in the AMIC+FN + CPCs group than any other
group, and the protein expressions were increased with time. It's
worth noticing that the newly formed cartilage tissue showed clustered
and spherical lacunae, that is, characteristic cartilage-like lacunae
structures.
To further quantitatively evaluate the repair efficacy for cartilage de-
fects in vivo, qRT-PCR was used to detected chondrogenic difference
gene expression in the newly formed tissue (Fig. 8B-C). Compared
with that in the MF group, the gene levels of SOX9 and COL2 were up-
regulated in the AMIC+CPCs groups at 6 weeks and 12 weeks and
were significantly increased after the synergies between CPCs and FN
in the AMIC+FN + CPCs group. The AMIC+ and AMIC+FN groups
also tended to promote expression of chondrogenic difference markers,
although the increase was not significant.
Because the local microenvironment after surgery cannot recruit
and guide the differentiation of BMSCs, this results in the regenera-
tion of essentially fibrous cartilage with significantly lower strength
than native cartilage. AMIC technology fixes the released cells onto
the cartilage defect using a collagen membrane and improves the
cartilage repair effect to a certain extent, but it still cannot regenerate
natural hyaline cartilage. To overcome the lack of chemical cues in
local microenvironment after microfracture surgery for the released
cellular component, we incorporated a CPC-laden biofilm based on
an Alg/Gel/HA composite hydrogel combined with FN into the defect
area [49]. When the biofilm was implanted in vivo, the cartilage de-
fects showed the best cartilage regeneration with a gradient struc-
ture among all groups in the AMIC+FN + CPCs group. The cell
morphology of the regenerated cartilage from the AMIC+FN + CPCs
group was similar to native hyaline cartilage with round
chondrocytes within lacunae (Figs. 6, 7).
Y. Zhou, R. Qin, T. Chen et al. Materials and Design 203 (2021) 109621

Fig. 7. Safranin-O staining of the regenerated tissues in the different groups at 6 and 12 weeks after surgery.

The traditional AMIC technology lacks a good postoperative micro-
environment, and cannot effectively induce the chondrogenic differen-
tiation of BMSCs released from subchondral bone. The regenerated
cartilage tissue is basically fibrocartilage instead of natural hyaline carti-
lage, and lacks long-term efficacy. In this study, a 3D printed active bio-
film was used to replace the collagen membrane, which effectively
adhered to cartilage defects. The active biofilm as a whole provides a
good microenvironment and can effectively induce BMSCs to differenti-
ate into cartilage. For one thing, this effect may benefit from the co-
culture of CPCs and BMSCs. According to the literature, when MSCs
are co-cultured with CPCs, interactions between the two cell types
maintained the chondrogenic phenotype of CPCs and stimulated the
MSCs to differentiate into chondrocytes, which contributed to chondro-
genesis [48,50]. On the other hand, thanks to the effective controlled re-
lease of FN in biofilms, BMSCs were effectively recruited. At the same
time, CPCs present in biofilms can spontaneously differentiate into
chondrocytes. The high expression of PRG4 and COLII in vitro and
in vivo experiments confirms the superficial localization of PRG4 and

Fig. 8. (A) Immunohistochemical staining for Type II collagen of the regenerated tissues in the different groups at 6 and 12 weeks after surgery. Expression levels of the chondrogenic-
specific genes COLII, PRG4 and SOX9, in newly formed cartilage with biofilm implantation for 6 (A) and 12 weeks (B). *,# indicate P < 0.05, **, ## indicate P < 0.01, and ***,### indicate
P < 0.001.

12
Y. Zhou, R. Qin, T. Chen et al.
COLII. Under the recruitment and action of the slowly released FN,
BMSCs adhering to the lower layer of the biofilm differentiated into hy-
pertrophic chondrocytes, and the abundant presence of type X collagen
in the deep zone was confirmed, indicative of regenerated superficial
zone articular cartilage and deep zone hypertrophic cartilage in newly
formed cartilage.
The layered structure of natural cartilage, such as arrangement of
collagenous fibers or density distribution of chondrocytes, also should
be taken into account in future studies to achieve hyaline cartilage re-
generation [46]. Although the thickness of cartilage in the rat limited
the possibility of applying a layered scaffold in this study, it is very sig-
nificative to evaluate the possibility of using layered cartilage scaffolds
in large animals. In addition, an intact subchondral bed is essential to
the treatment of the chondral lesion [48]. We also need to improve
the preparation process of the scaffold to enhance the resolution of
the printed structure. For example, low-viscosity hydrogels can be fab-
ricated with high resolution via model-support bioink interaction [51].
Consequently, regeneration of the osteochondral area as a whole need
be noticed instead of the articular cartilage alone [48]. Although some
cartilage repair results are promising, remodeling a consistent and sta-
ble osteochondral interface to facilitate good graft-to-bone attachment
is still an unsolved problem [52].
4. Conclusion
In summary, we generated a 3D-printed bioactive biofilm to modify
AMIC technology. Covering a defect with biofilm provides adequate me-
chanical support and architectural integrity, offering a stable microenvi-
ronment for BMSCs released from subchondral bone. We verified that a
laminar structure similar to natural hyaline cartilage was regenerated in
the rat cartilage defect model. By introducing FN into CPC-laden biofilm,
we showed that CPCs embedded in biofilm possess excellent potential
to undergo chondrogenic differentiation under uninduced conditions.
Alg/Gel/HA + CPCs+FN biofilm can positively stimulate chondrogenic
differentiation of BMSCs released by microfracture. It can provide cells
with certain mechanical properties in the early stage of cartilage regen-
eration to support the structure of the microenvironment, and will de-
grade over time, eventually being replaced by newly formed cartilage
tissue. With this bioactive biofilm, the AMIC outcome was significantly
enhanced by interaction between the CPC-laden biofilm and BMSCs re-
leased from bone marrow during the healing process. Replacing the tra-
ditional collagen membrane with active biofilm resulted in the
regeneration of a thicker and healthier cartilage. For clinical translation,
we could incorporate AMIC surgery and 3D bioprinting by performing
an arthroscopy procedure to replace the damaged cartilage with 3D-
printed biofilm after microfracture surgery.
Author statement
Yang Zhou: Methodology, Investigation, Writing - original draft. Ran
Qin: Investigation, Writing - original draft. Tong Chen: Methodology,
Validation. Kaibin Zhang: Formal analysis. Jianchao Gui: Conceptualiza-
tion, Resources, Supervision, Writing - review & editing
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
This work is financially supported by the National Natural Science
Foundation of China (No. 81601954, No. 81672210 and No.81673995).
13
Materials and Design 203 (2021) 109621
References
[1] D.J. Huey, J.C. Hu, K.A. Athanasiou, Unlike bone, cartilage regeneration remains elu-
sive, Science (New York, N.Y.) 338 (6109) (2012) 917–921, https://doi.org/10.1126/
science.1222454.
[2] S.C. Mastbergen, D.B. Saris, F.P. Lafeber, Functional articular cartilage repair: here,
near, or is the best approach not yet clear? Nat. Rev. Rheumatol. 9 (5) (2013)
277–290, https://doi.org/10.1038/nrrheum.2013.29.
[3] J. Martel-Pelletier, A.J. Barr, F.M. Cicuttini, P.G. Conaghan, C. Cooper, M.B. Goldring,
S.R. Goldring, G. Jones, A.J. Teichtahl, J.P. Pelletier, Osteoarthritis, Nat. Rev. Dis.
Primers 2 (2016) 16072, https://doi.org/10.1038/nrdp.2016.72.
[4] C. Becher, M.A. Malahias, M.M. Ali, N. Maffulli, H. Thermann, Arthroscopic
microfracture vs. arthroscopic autologous matrix-induced chondrogenesis for the
treatment of articular cartilage defects of the talus, Knee Surg. Sports Traumatol.
Arthrosc. 27 (9) (2019) 2731–2736, https://doi.org/10.1007/s00167-018-5278-7.
[5] D.L. Richter, R.C. Schenck Jr., D.C. Wascher, G. Treme, Knee articular cartilage repair
and restoration techniques: a review of the literature, Sports Health 8 (2) (2016)
153–160, https://doi.org/10.1177/1941738115611350.
[6] E.A. Makris, A.H. Gomoll, K.N. Malizos, J.C. Hu, K.A. Athanasiou, Repair and tissue en-
gineering techniques for articular cartilage, Nat. Rev. Rheumatol. 11 (1) (2015)
21–34, https://doi.org/10.1038/nrrheum.2014.157.
[7] J.M. Hurst, J.R. Steadman, L. O’Brien, W.G. Rodkey, K.K. Briggs, Rehabilitation follow-
ing microfracture for chondral injury in the knee, Clin. Sports Med. 29 (2) (2010)
257–265 , viii https://doi.org/10.1016/j.csm.2009.12.009.
[8] T. Guo, M. Noshin, H.B. Baker, E. Taskoy, S.J. Meredith, Q. Tang, J.P. Ringel, M.J.
Lerman, Y. Chen, J.D. Packer, J.P. Fisher, 3D printed biofunctionalized scaffolds for
microfracture repair of cartilage defects, Biomaterials 185 (2018) 219–231,
https://doi.org/10.1016/j.biomaterials.2018.09.022.
[9] L. Weigelt, R. Hartmann, C. Pfirrmann, N. Espinosa, S.H. Wirth, Autologous matrix-
induced Chondrogenesis for Osteochondral lesions of the talus: a clinical and radio-
logical 2- to 8-year follow-up study, Am. J. Sports Med. 47 (7) (2019) 1679–1686,
https://doi.org/10.1177/0363546519841574.
[10] A. Schiavone Panni, C. Del Regno, G. Mazzitelli, R. D’Apolito, K. Corona, M. Vasso,
Good clinical results with autologous matrix-induced chondrogenesis (Amic) tech-
nique in large knee chondral defects, Knee Surg. Sports Traumatol. Arthrosc. 26 (4)
(2018) 1130–1136, https://doi.org/10.1007/s00167-017-4503-0.
[11] L. de Girolamo, H. Schonhuber, M. Vigano, C. Bait, A. Quaglia, G. Thiebat, P. Volpi, Au-
tologous matrix-induced Chondrogenesis (AMIC) and AMIC enhanced by autolo-
gous concentrated bone marrow aspirate (BMAC) allow for stable clinical and
functional improvements at up to 9 years follow-up: results from a randomized
controlled study, J. Clin. Med. 8 (3) (2019)https://doi.org/10.3390/jcm8030392.
[12] L.G. Bracaglia, B.T. Smith, E. Watson, N. Arumugasaamy, A.G. Mikos, J.P. Fisher, 3D
printing for the design and fabrication of polymer-based gradient scaffolds, Acta
Biomater. 56 (2017) 3–13, https://doi.org/10.1016/j.actbio.2017.03.030.
[13] J. Yang, Y.S. Zhang, K. Yue, A. Khademhosseini, Cell-laden hydrogels for
osteochondral and cartilage tissue engineering, Acta Biomater. 57 (2017) 1–25,
https://doi.org/10.1016/j.actbio.2017.01.036.
[14] L. Shao, Q. Gao, C. Xie, J. Fu, M. Xiang, Y. He, Directly coaxial 3D bioprinting of large-
scale vascularized tissue constructs, Biofabrication 12 (3) (2020), 035014, https://
doi.org/10.1088/1758-5090/ab7e76.
[15] H. Rogan, F. Ilagan, X. Tong, C.R. Chu, F. Yang, Microribbon-hydrogel composite scaf-
fold accelerates cartilage regeneration in vivo with enhanced mechanical properties
using mixed stem cells and chondrocytes, Biomaterials 228 (2020) 119579, https://
doi.org/10.1016/j.biomaterials.2019.119579.
[16] Q. Gao, X. Niu, L. Shao, L. Zhou, Z. Lin, A. Sun, J. Fu, Z. Chen, J. Hu, Y. Liu, Y. He, 3D
printing of complex GelMA-based scaffolds with nanoclay, Biofabrication 11 (3)
(2019), 035006, https://doi.org/10.1088/1758-5090/ab0cf6.
[17] P. Rastogi, B. Kandasubramanian, Review of alginate-based hydrogel bioprinting for
application in tissue engineering, Biofabrication 11 (4) (2019), 042001, https://doi.
org/10.1088/1758-5090/ab331e.
[18] K. Markstedt, A. Mantas, I. Tournier, H. Martínez Ávila, D. H?gg, P.J.B. Gatenholm, 3D
Bioprinting Human Chondrocytes with Nanocellulose-Alginate Bioink for Cartilage
Tissue Engineering Applications, 16(5), 2015 1489–1496.
[19] J. Schiavi, L. Reppel, N. Charif, N. de Isla, D. Mainard, N. Benkirane-Jessel, J.F. Stoltz, R.
Rahouadj, C. Huselstein, Mechanical stimulations on human bone marrow mesen-
chymal stem cells enhance cells differentiation in a three-dimensional layered scaf-
fold, J. Tissue Eng. Regen. Med. 12 (2) (2018) 360–369, https://doi.org/10.1002/
term.2461.
[20] F. You, X. Chen, D.M.L. Cooper, T. Chang, B.F. Eames, Homogeneous hydroxyapatite/
alginate composite hydrogel promotes calcified cartilage matrix deposition with po-
tential for three-dimensional bioprinting, Biofabrication 11 (1) (2018) 015015,
https://doi.org/10.1088/1758-5090/aaf44a.
[21] C. Antich, J. de Vicente, G. Jimenez, C. Chocarro, E. Carrillo, E. Montanez, P. Galvez-
Martin, J.A. Marchal, Bio-inspired hydrogel composed of hyaluronic acid and algi-
nate as a potential bioink for 3D bioprinting of articular cartilage engineering con-
structs, Acta Biomater. 106 (2020) 114–123, https://doi.org/10.1016/j.actbio.2020.
01.046.
[22] K.C.R. Kolan, J.A. Semon, B. Bromet, D.E. Day, M.C. Leu, Bioprinting with human stem
cell-laden alginate-gelatin bioink and bioactive glass for tissue engineering, Int. J.
Bioprint. 5 (2.2) (2019) 204, https://doi.org/10.18063/ijb.v5i2.2.204.
[23] P. Dubruel, R. Unger, S.V. Vlierberghe, V. Cnudde, P.J. Jacobs, E. Schacht, C.J.
Kirkpatrick, Porous gelatin hydrogels: 2. In vitro cell interaction study,
Biomacromolecules 8 (2) (2007) 338–344, https://doi.org/10.1021/bm0606869.
[24] S.H. Jeong, M. Kim, T.Y. Kim, H. Kim, J.H. Ju, S.K. Hahn, Supramolecular injectable
hyaluronate hydrogels for cartilage tissue regeneration, ACS Appl. Bio Mater. 3 (8)
(2020) 5040–5047, https://doi.org/10.1021/acsabm.0c00537.
Y. Zhou, R. Qin, T. Chen et al.
[25] S. Stichler, T. Bock, N. Paxton, S. Bertlein, R. Levato, V. Schill, W. Smolan, J. Malda, J.
Tessmar, T. Blunk, J. Groll, Double printing of hyaluronic acid/poly(glycidol) hybrid
hydrogels with poly(epsilon-caprolactone) for MSC chondrogenesis, Biofabrication
9 (4) (2017), 044108, https://doi.org/10.1088/1758-5090/aa8cb7.
[26] A. Shpichka, D. Osipova, Y. Efremov, P. Bikmulina, N. Kosheleva, M. Lipina, E.A.
Bezrukov, R.B. Sukhanov, A.B. Solovieva, M. Vosough, P. Timashev, Fibrin-based
Bioinks: new tricks from an old dog, Int. J. Bioprint. 6 (3) (2020) 269, https://doi.
org/10.18063/ijb.v6i3.269.
[27] A.C. Daly, F.E. Freeman, T. Gonzalez-Fernandez, S.E. Critchley, J. Nulty, D.J. Kelly, 3D
bioprinting for cartilage and Osteochondral tissue engineering, Adv Healthc Mater
6 (22) (2017)https://doi.org/10.1002/adhm.201700298.
[28] D. Cinats, S. Miller, Z. Abusara, S.M. Heard, C. Hutchison, N. Schachar, S.
Timmermann, Evolution of a novel tissue preservation protocol to optimize
osteochondral transplantation outcomes, Cartilage (2018)https://doi.org/10.1177/
1947603518812557 1947603518812557.
[29] Y. Jiang, R.S. Tuan, Origin and function of cartilage stem/progenitor cells in osteoar-
thritis, Nat. Rev. Rheumatol. 11 (4) (2015) 206–212, https://doi.org/10.1038/
nrrheum.2014.200.
[30] K. Brady, S.C. Dickinson, A.P. Hollander, Changes in chondrogenic progenitor popu-
lations associated with aging and osteoarthritis, Cartilage 6 (2 Suppl) (2015)
https://doi.org/10.1177/1947603515574838 30s–5s.
[31] K. Xue, X. Zhang, Z. Gao, W. Xia, L. Qi, K. Liu, Cartilage progenitor cells combined
with PHBV in cartilage tissue engineering, J. Transl. Med. 17 (1) (2019) 104,
https://doi.org/10.1186/s12967-019-1855-x.
[32] P. Singh, C. Carraher, J.E. Schwarzbauer, Assembly of fibronectin extracellular matrix,
Annu. Rev. Cell Dev. Biol. 26 (2010) 397–419, https://doi.org/10.1146/annurev-
cellbio-100109-104020.
[33] T. Tao, Y. Li, C. Gui, Y. Ma, Y. Ge, H. Dai, K. Zhang, J. Du, Y. Guo, Y. Jiang, J. Gui, Fibro-
nectin enhances cartilage repair by activating progenitor cells through integrin
alpha5beta1 receptor, Tissue Eng Part A 24 (13–14) (2018) 1112–1124, https://
doi.org/10.1089/ten.TEA.2017.0322.
[34] Y. Li, J. Zhou, X. Yang, Y. Jiang, J. Gui, Intermittent hydrostatic pressure maintains and
enhances the chondrogenic differentiation of cartilage progenitor cells cultivated in
alginate beads, Develop. Growth Differ. 58 (2) (2016) 180–193, https://doi.org/10.
1111/dgd.12261.
[35] R. Levato, W.R. Webb, I.A. Otto, A. Mensinga, Y. Zhang, M. van Rijen, R. van Weeren,
I.M. Khan, J. Malda, The bio in the ink: cartilage regeneration with bioprintable
hydrogels and articular cartilage-derived progenitor cells, Acta Biomater. 61
(2017) 41–53, https://doi.org/10.1016/j.actbio.2017.08.005.
[36] L. Shao, Q. Gao, C. Xie, J. Fu, M. Xiang, Z. Liu, L. Xiang, Y. He, Sacrificial microgel-laden
bioink-enabled 3D bioprinting of mesoscale pore networks, Bio-Design Manufact. 3
(1) (2020) 30–39, https://doi.org/10.1007/s42242-020-00062-y.
[37] S. Jordahl, L. Solorio, D.B. Neale, S. McDermott, J.H. Jordahl, A. Fox, C. Dunlay, A. Xiao,
M. Brown, M. Wicha, G.D. Luker, J. Lahann, Engineered Fibrillar Fibronectin net-
works as three-dimensional tissue scaffolds, Adv. Mater. 31 (46) (2019),
e1904580, https://doi.org/10.1002/adma.201904580.
[38] J. Zhang, X. Ma, D. Lin, H. Shi, Y. Yuan, W. Tang, H. Zhou, H. Guo, J. Qian, C. Liu, Mag-
nesium modification of a calcium phosphate cement alters bone marrow stromal
cell behavior via an integrin-mediated mechanism, Biomaterials 53 (2015)
251–264, https://doi.org/10.1016/j.biomaterials.2015.02.097.
14
Materials and Design 203 (2021) 109621
[39] A. Dhawan, P.M. Kennedy, E.B. Rizk, I.T. Ozbolat, Three-dimensional bioprinting for
bone and cartilage restoration in Orthopaedic surgery, J Am Acad Orthop Surg 27
(5) (2019) e215–e226, https://doi.org/10.5435/JAAOS-D-17-00632.
[40] G.P. Dowthwaite, J.C. Bishop, S.N. Redman, I.M. Khan, P. Rooney, D.J. Evans, L.
Haughton, Z. Bayram, S. Boyer, B. Thomson, M.S. Wolfe, C.W. Archer, The surface
of articular cartilage contains a progenitor cell population, J. Cell Sci. 117 (Pt 6)
(2004) 889–897, https://doi.org/10.1242/jcs.00912.
[41] C. Erggelet, M. Endres, K. Neumann, L. Morawietz, J. Ringe, K. Haberstroh, M.
Sittinger, C. Kaps, Formation of cartilage repair tissue in articular cartilage defects
pretreated with microfracture and covered with cell-free polymer-based implants,
J. Orthop. Res. 27 (10) (2009) 1353–1360, https://doi.org/10.1002/jor.20879.
[42] E. Axpe, M.L. Oyen, Applications of alginate-based bioinks in 3D bioprinting, Int. J.
Mol. Sci. 17 (12) (2016)https://doi.org/10.3390/ijms17121976.
[43] J.H.Y. Chung, S. Naficy, Z. Yue, R. Kapsa, A. Quigley, S.E. Moulton, G.G. Wallace, Bio-
ink properties and printability for extrusion printing living cells, Biomater Sci 1
(7) (2013) 763–773, https://doi.org/10.1039/c3bm00012e.
[44] Y.A.J. Bruna, A.G. de Melo, Shreya Mehrotra, Michelle A. Calabrese, Tom Kamperman,
Biman B. Mandal, Maria H.A. Santana, Eben Alsberg, Jeroen Leijten*, Su Ryon Shin*,
3D printed cartilage-like tissue constructs with spatially controlled mechanical
properties %J advanced functional materials, Adv. Funct. Mater. 29 (51) (2019).
[45] M. Costantini, J. Idaszek, K. Szoke, J. Jaroszewicz, M. Dentini, A. Barbetta, J.E.
Brinchmann, W. Swieszkowski, 3D bioprinting of BM-MSCs-loaded ECM biomi-
metic hydrogels for in vitro neocartilage formation, Biofabrication 8 (3) (2016),
035002, https://doi.org/10.1088/1758-5090/8/3/035002.
[46] A.C. Daly, D.J. Kelly, Biofabrication of spatially organised tissues by directing the
growth of cellular spheroids within 3D printed polymeric microchambers, Biomate-
rials 197 (2019) 194–206, https://doi.org/10.1016/j.biomaterials.2018.12.028.
[47] C.D. Hoemann, J. Sun, M.D. McKee, A. Chevrier, E. Rossomacha, G.E. Rivard, M.
Hurtig, M.D. Buschmann, Chitosan-glycerol phosphate/blood implants elicit hyaline
cartilage repair integrated with porous subchondral bone in microdrilled rabbit de-
fects, Osteoarthr. Cartil. 15 (1) (2007) 78–89, https://doi.org/10.1016/j.joca.2006.
06.015.
[48] Y. Zhu, L. Kong, F. Farhadi, W. Xia, J. Chang, Y. He, H. Li, An injectable continuous
stratified structurally and functionally biomimetic construct for enhancing
osteochondral regeneration, Biomaterials 192 (2019) 149–158, https://doi.org/10.
1016/j.biomaterials.2018.11.017.
[49] W. Shi, M. Sun, X. Hu, B. Ren, J. Cheng, C. Li, X. Duan, X. Fu, J. Zhang, H. Chen, Y. Ao,
Structurally and functionally optimized silk-fibroin-gelatin scaffold using 3D print-
ing to repair cartilage injury in vitro and in vivo, Adv. Mater. 29 (29) (2017)
https://doi.org/10.1002/adma.201701089.
[50] V.V. Meretoja, R.L. Dahlin, F.K. Kasper, A.G. Mikos, Enhanced chondrogenesis in co-
cultures with articular chondrocytes and mesenchymal stem cells, Biomaterials 33
(27) (2012) 6362–6369, https://doi.org/10.1016/j.biomaterials.2012.05.042.
[51] E.Y. Tan, R. Suntornnond, W.Y.J.D. P, A. Yeong, Manufacturing, High-Resolution
Novel Indirect Bioprinting of Low-Viscosity Cell-Laden Hydrogels via Model-
Support Bioink Interaction, 2020.
[52] L. Chen, C. Deng, J. Li, Q. Yao, J. Chang, L. Wang, C. Wu, 3D printing of a lithium-
calcium-silicate crystal bioscaffold with dual bioactivities for osteochondral inter-
face reconstruction, Biomaterials 196 (2019) 138–150, https://doi.org/10.1016/j.
biomaterials.2018.04.005.