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Dengetal. Collagen and Leather (2024) 6:14
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Collagen and Leather
Injectable dECM-enhanced hyaluronic
microgels withspatiotemporal release
ofcartilage-specic molecules toimprove
osteoarthritic chondrocyte’s function
Siyan Deng1,2, Hongfu Cao1,2, Yan Lu1,2, Wenqing Shi1,2, Manyu Chen1,2, Xiaolin Cui3,4, Jie Liang1,2,5,
Yujiang Fan1,2*, Qiguang Wang1,2* and Xingdong Zhang1,2
Abstract
The interior environment of articular cartilage in osteoarthritis (OA) presents substantial hurdles, leading to the mal-
function of chondrocytes and the breakdown of collagen II-enriched hyaline cartilage matrix. Despite this, most
clinical treatments primarily provide temporary relief from OA discomfort without arresting OA progression. This study
aimed to alleviate OA by developing intra-articular injectable dECM-enhanced hyaluronic (HE) microgels. The HE
hydrogel was engineered and shaped into uniformly sized microgels using microfluidics and photopolymerization
techniques. These microgels provided a spatiotemporal cascade effect, facilitating the rapid release of growth factors
and a slower release of ECM macromolecules and proteins. This process assisted in the recovery of OA chondrocytes’
function, promoting cell proliferation, matrix synthesis, and cartilage-specific gene expression in vitro. It also effec-
tively aided repair of the collagen II-enriched hyaline cartilage and significantly reduced the severity of OA, as dem-
onstrated by radiological observation, gross appearance, histological/immunohistochemical staining, and analysis
in an OA rat model in vivo. Collectively, the HE injectable microgels with spatiotemporal release of cartilage-specific
molecules have shown promise as a potential candidate for a cell-free OA therapy approach.
Keywords Osteoarthritis, Decellularized cartilage matrix, Hyaluronic acid, Spatiotemporal release, Intra-articular
injection
*Correspondence:
Yujiang Fan
fan_yujiang@scu.edu.cn
Qiguang Wang
wqgwang@126.com
Full list of author information is available at the end of the article
Page 2 of 17
Dengetal. Collagen and Leather (2024) 6:14
Graphical Abstract
1 Introduction
Osteoarthritis (OA) is a prevalent chronic disease that
afflicts hundreds of millions of people worldwide each
year [1]. It is characterized by pain, an inflammatory
response, joint destabilization, and the loss of colla-
gen II-enriched hyaline cartilage, ultimately affecting
the entire joint. Early treatment of OA often involves
the use of pharmacological interventions such as non-
steroidal anti-inflammatory drugs (NSAIDs), corticos-
teroids, and analgesics to alleviate pain [2, 3]. However,
these treatments frequently fail to halt further deterio-
ration of the joint structure and microenvironment and
are associated with adverse effects [4, 5]. Once the dis-
ease advances to a severe stage, surgical joint replace-
ment becomes the only viable treatment option. us,
it is crucial to develop effective intervention programs
that target the early stages of OA.
Intra-articular hyaluronic acid (HA) injections have
been extensively used in OA treatment, providing
effective pain relief and symptomatic therapy [6]. HA, a
glycosaminoglycan found abundantly in synovial fluid,
plays a crucial role in protecting articular cartilage and
promoting cartilage regeneration due to its inherent
properties, particularly viscoelasticity [7]. High molec-
ular weight HA, which is often reduced in OA joints,
maintains the rheological homeostasis of the synovial
fluid and helps dampen shock, lubricate, and nourish
joint tissues [8, 9]. erefore, intra-articular HA injec-
tions have been shown to function as viscosupplemen-
tation, leading to significant functional improvement
in OA joints. However, most HA preparations have a
short half-life in the joints due to rapid clearance and
swift degradation in vivo by enzymatic or hydrolytic
reactions, remaining in the joints for only a few hours
to a couple of days [6, 10, 11]. Consequently, achiev-
ing effective treatment results often requires multiple
injections, which can lead to difficulties in ensuring
patient compliance and an increased risk of side effects.
Page 3 of 17
Dengetal. Collagen and Leather (2024) 6:14
erefore, it is essential to optimize biomaterials for
intra-articular injections to aid in OA treatment.
Injectable hydrogels have been widely investigated for
use in OA arthrocentesis due to their slow degradation,
enhanced stability, and strong adhesion, which allows
for prolonged retention [12, 13]. However, the irregular
shape and large size of hydrogels can lead to high and
uneven injection forces during injection [14], potentially
damaging healthy tissues in the affected area. Compared
to block hydrogels, hydrogel microspheres, also known
as microgels, with their uniform size and spherical shape,
provide improved injectability [15, 16]. Among the vari-
ous production methods, microfluidics offers significant
advantages such as the rapid generation of microgels
with precise and consistent sizes [17]. Consequently,
HA-based microgels have been extensively studied and
have shown to effectively extend the degradation of HA,
provide lubrication, and offer biocompatibility. How-
ever, within the complex intra-articular joint environ-
ment, a single HA component has limited capacity to
replace the cartilaginous extracellular matrix and offer
multifunctionality.
e extracellular matrix (ECM) serves as a natural
structure for cell adhesion, proliferation, migration, and
differentiation, in addition to providing biomechanical
capabilities. e decellularized ECM (dECM), derived
by eliminating cellular components and genetic material
from the ECM of human or animal tissues using decel-
lularization techniques, reduces immunogenicity and is
a promising biomaterial highly regarded in regenerative
tissue engineering applications, such as skin, cornea,
and nerve [18, 19]. Cartilage tissue-derived dECM has
been demonstrated to provide structural and functional
proteins that support cell attachment, growth, and dif-
ferentiation specific to cartilage tissue [20]. e dECM
retains most cartilage-specific matrix macromolecules,
including primarily collagen II and proteoglycans, along
with less abundant components such as fibulin and elas-
tin. ese contribute to the production and maintenance
of ECM, enhance chondrocyte morphology and pheno-
type, ensure cartilage homeostasis, and regulate newborn
cartilage tissues through various cytokines, enzymes,
and signaling pathways [21–24]. Moreover, it contains
numerous growth factors involved in chondrocyte func-
tion, including transforming growth factor-β (TGF-β),
insulin-like growth factor-1 (IGF-1), bone morphoge-
netic protein (BMP), fibroblast growth factor (FGF), and
epidermal growth factor (EGF) [25]. Members of the
TGF-β superfamily play a pivotal role in cartilage devel-
opment and repair [26, 27]; IGF-1 stimulates the anabolic
activity of chondrocytes and inhibits chondrocyte apop-
tosis [28, 29]; BMPs, particularly BMP-2 and BMP-7, pro-
mote chondrogenesis in MSCs and enhance chondrocyte
matrix production [30, 31]. In addition to growth factors,
erefore, cartilage-derived dECM, due to its similarity
to the natural microenvironment and the bioactivity of
cartilage formation, exhibits beneficial effects on tissue
morphogenesis, differentiation, homeostasis, and repair.
is study aimed to enhance the HA-based hydrogel
formulation, referred to HE, by incorporating a solution
derived from cartilage dECM to augment the limited
bio-functionality of HA and improve the injectability of
dECM. e HE microgels were fabricated using micro-
fluidics and photopolymerization, ensuring their uniform
size and mechanical strength for effective lubrication
and durability under extensive joint movements. It was
hypothesized that the HE microgels facilitate a spati-
otemporal cascade effect, with cartilage-specific growth
factors being released in the short term to prompt the
swift restoration of OA chondrocyte function. Concur-
rently, the extended release of structural ECM macromol-
ecules and collagens offers a sustained supply of essential
components, which are vital for cartilage regeneration
and the maintenance of homeostasis as the HE microgels
degrade. is combined process was anticipated to pro-
duce synergistic and enduring effects across the various
stages of OA chondrocyte recovery and cartilage regener-
ation, including improved viability of OA chondrocytes,
enhanced matrix secretion, collagen II synthesis, and
the preservation of cellular function and cartilage matrix
integrity, potentially offering a therapeutic approach for
OA. To validate this hypothesis, the effects of HE micro-
gels on OA chondrocyte proliferation, morphology, and
matrix secretion were investigated invitro. e findings
were further corroborated through intra-articular injec-
tion in a rat OA model invivo (Scheme1).
2 Materials andmethods
2.1 Materials
Hyaluronic acid (HA, Mw = 0.94 MDa) was purchased
from Florida Biochemistry Limited (Shandong, China).
Methacrylic anhydride (MA) was obtained from Chengdu
Chemical Xia Reagent Co. Liquid paraffin and Span
80 were purchased from Cologne Chemical (Chengdu,
China). 3-[(3-cholamidopropyl) dimethylamino] pro-
panesulfonic acid internal salt (CHAPS) were bought
from Sigma-Aldrich (USA). Hyaluronidase, type I col-
lagenase and DNase I were obtained from Solarbio
(Beijing, China). α-MEM medium was bought from Ser-
vicebio (Wuhan, China). Penicillin–streptomycin and
parenzyme were purchased from Hyclone (USA). Fetal
bovine serum (FBS, Gibco, KEEL) was obtained from
Life Technologies Corporation (USA). Fluorescein diac-
etate (FDA) and propidium iodide (PI) were bought from
Sigma-Aldrich (USA).
Page 4 of 17
Dengetal. Collagen and Leather (2024) 6:14
2.2 Hyaluronic acid methacryloyl (HAMA) synthesis
HA powder (2.5g) was dissolved in ultrapure water and
stirred to dissolve. Subsequently, the pH value of the
solution was adjusted to 8.5 with 1M NaOH under ice
bath conditions. en, MA (5.6mL) was added dropwise
controlling the pH value at 7.5–8.5 for a duration of 4h
in an ice bath. e reaction system was stirred at room
temperature overnight. After that, the solution was trans-
ferred to dialysis bags (Mw = 8000–14000kDa) and dia-
lyzed in ultrapure water for 3days at room temperature
to remove unreacted methacrylate groups. e dialysis
solution was frozen and lyophilized to obtain HAMA.
e chemical structure of HAMA was confirmed by H1
NMR (400MHz, Bruker AMX-400, USA) with D2O as
the solvent.
2.3 Preparation andcharacterization ofdECM
Fresh porcine knee joints were collected, and the joint
cavity was exposed to extract transparent cartilage pieces
while avoiding calcified tissue. After ultrasonic clean-
ing in phosphate-buffered saline (PBS, Sigma Aldrich,
USA), the cartilage pieces were lyophilized. Subse-
quently, the cartilage pieces were ground into particles
using a cryogenic grinder at -20 ℃, 60 Hz and 3000 r/
min. e obtained pellets were mixed with PBS, filtered
through a 175μm sieve, and then decellularized as the
following steps reported previously [32, 33]. Firstly, the
cartilage particles were alternately placed in hypotonic
solution (10 mM Tris-HCI, pH = 8) and hypertonic
solution (50mM Tris-HCI, 1M NaCl, pH = 8), repe ated
three times and performing five freeze–thaw cycles
(liquid nitrogen and 37 °C water bath). After that, 1%
CHAPS solution treatment for 5 h was applied to the
cartilage ECM. en, the obtained ECM was treated
with nuclease solution (200 U/mL DNase I dissolved in
10mM Tris-HCI, pH = 7.5) for 9h at 37°C with agita-
tion, with a new solution replaced every 3h. e ECM
was next treated with 0.1% Triton-100 solution with agi-
tation and washed several times with distilled water to
remove residual detergent. Finally, the ECM powder was
obtained by centrifugation and lyophilized. e powder
was ground again through a 50 μm sieve and digested
with 0.1M acetic acid solution containing pepsin (pep-
sin: decellularized matrix = 1:10) for 2days. e digestion
was terminated by adjusting the pH to neutral to obtain
the dECM solution stored at 4 ℃.
e typical components retained in the dECM were
characterized as follows: 10mg decellularized tissues was
placed in 1mL papain working solution and treated with
a water bath at 65 ℃ overnight. After that, the superna-
tant was collected by centrifugation for the quantifica-
tion of DNA and GAGs content by a Quant-iT PicoGreen
dsDNA (B1302, Sigma) and the Blyscan glycosaminogly-
can (GAG) assay kit (B100, Biocolor) in accordance with
the outlined protocol. e dECM solution was applied to
slides, air-dried for 30min and then stained with hema-
toxylin–eosin (H&E) and toluidine blue (TB) according
to the Hematoxylin–Eosin Staining Kit (G1120, Solarbio)
Scheme1. Development of injectable dECM-enhanced hyaluronic microgels
Page 5 of 17
Dengetal. Collagen and Leather (2024) 6:14
and the Toluidine Blue Staining Kit (G2543, Solarbio),
respectively. e samples were then observed under
a light microscope and photographed for subsequent
analysis. e microstructure of decellularized tissues was
observed by imaging using a scanning electron micros-
copy (SEM, Japan).
2.4 Preparation andcharacterization ofHE hydrogel
HAMA (10 mg/mL) and dECM solution were mixed at
a mass ratio of 1:2 to obtain the experimental group HE.
en the solution supplemented with photo-initiator was
immediately injected into a homemade ring mold under
light protection. Afterwards, the liquid crosslinked to
form a gel under UV light (450nm), and a series of mate-
rial characterization was carried out in the subsequent
process. e pure HAMA solution was formed into a gel
in the same way as a control.
e lyophilized hydrogel was soaked in liquid nitrogen
for a few seconds, followed by fracturing in a brittle man-
ner to expose the cross-section, and the internal struc-
ture was observed by a scanning electron microscopy
after gold coating treatment. e SEM images were ana-
lyzed using ImageJ software to obtain the porosity and
pore size of the hydrogels.
e disintegration rate of the hydrogels was measured
using hyaluronidase and type I collagenase solutions to
simulate physiological conditions. e weight of fresh
hydrogel was recorded as W0. en the samples were
placed in PBS solutions containing type I collagenase (5
U/mL) and hyaluronidase (5 U/mL), respectively. Simul-
taneously they were placed in a thermostatic shaker at
90rpm and 37 ℃. e fresh enzyme solution was replaced
daily. e hydrogel was removed at certain time intervals,
and the water on the surface was carefully wiped with
filter paper to weigh(W1). e weight measurement was
repeated three times for each sample. e disintegration
rate was calculated using the following formula:
Hydrogel swelling properties were measured as follows.
Briefly, the initial weight of the lyophilized hydrogel was
noted as W0. e samples were immersed in PBS buffer
and placed in a constant temperature shaker at 90rpm
and 37°C. Finally, the hydrogels were periodically taken
out and weighed noted as Ws. e weight measurement
was repeated three times for each sample. Until the
hydrogel swelling reached equilibrium, the swelling ratio
was calculated using the following equation:
In addition, fresh hydrogels were tested at room tem-
perature using a Dynamic Mechanical Analyzer (DMA,
WeightLoss
=
(W0
−
W1)/W0
∗
100%
SwellingRatio
=
(Ws
−
W0)/W0
∗
100%
TA-Q800, USA) under multi-frequency mode (a fixed
frequency of 1–10Hz, an amplitude of 40μm, a preload
force of 0.002 N and a force track of 105%) to test the
energy storage modulus (G’) and loss modulus (G’’). Each
sample was tested three times in parallel.
2.5 Preparation andcharacterization ofmicrogels
e microfluidics device was utilized to prepare micro-
gels with uniform size. Briefly, liquid paraffin oil was used
as the continuous phase and Span 80 (5 wt% concentra-
tion) served as the surfactant to stabilize the droplets.
e dispersed phase was composed of either a mixture
of HAMA (1 wt% final concentration) and dECM (2 wt%
final concentration), or a pure HAMA solution with a
photo-initiator. Afterwards, the two phases were individ-
ually injected into different miro-channels of the device
and the flow rates were adjusted by microfluidic pumps.
e change in flow rates resulted in the size of micro-
gels. An ultraviolet (UV) lamp placed above the channels
ensured that microdroplets generated due to the shear
stress of the continuous phase liquid formed solid hydro-
gels by photo-cross-linking at a constant rate within
channels. e microgels were collected in a centrifuge
tube at the outlet of the channel and purified by washing
with acetone once and with PBS repeatedly to obtain HE
and HA microgels.
e purified microgels were photographed under
an optical microscope and the microgel particle size
was analyzed using ImageJ software. SEM was used to
observe the surface morphology of microgels. A certain
amount of microgels was incubated in a PBS solution
placed in a constant temperature shaker at 37°C. At spe-
cific time points, an appropriate volume of the superna-
tant was collected to determine the release profile of each
component from the microgels. Specifically, the collagen
content was quantified using a hydroxyproline assay kit
(Solarbio, BC0250), while IGF-1 (Bioswamp, Wuhan,
PR40070), TGF-β1 (Bioswamp, Wuhan, PR40065), and
BMP-2 (Bioswamp, Wuhan, PR40118) were measured
using enzyme-linked immunosorbent assay (ELISA) kits
to determine the levels of growth factors. Additionally,
the GAG content was assessed using the Blyscan glycosa-
minoglycan (GAG) assay kit (B100, Biocolor).
2.6 Extraction andculture ofOA chondrocytes
e articular cartilage was cleaned with PBS several
times, cut into as thin and small pieces as possible and
digested with EDTA-free trypsase for 0.5 h. e pre-
vious solution was then replaced and incubated with
2mg/mL type II collagenase for 5h. e P0-generation
OA chondrocytes were obtained after passed through a
100μm strainer and cultured in α-MEM medium with
Page 6 of 17
Dengetal. Collagen and Leather (2024) 6:14
the addition of 1% double antibody, 20% KEL serum, and
50μg/mL VC. Once 90% confluence was reached, pas-
saging was accomplished in 24-well plates (P1) for subse-
quent cell experiments.
2.7 In vitroeects ofdierent microgels
2.7.1 Cell proliferation andmorphology
e in vitro effects of microgels on OA chondrocytes
were evaluated through a co-culture system within the
Transwell apparatus. Microgels, prepared at a concentra-
tion of 1mg/mL, were placed in the upper compartments
of a 24-well Transwell plate, with OA chondrocytes
seeded in the lower wells. e proliferation of OA chon-
drocytes, co-cultured with microgels for 3 and 7days,
was assessed using Live/dead cell staining and CCK-8. A
PBS solution containing FDA/PI was added to the well
plates and incubated in darkness for 5min. Samples were
then observed under a fluorescence microscope. Cell
proliferation was detected using the CCK-8 kit, with the
absorbance value of the incubation solution at 450nm
serving as the quantification metric. e cytoskeleton
was stained with a Rhodamine-phalloidin solution to
facilitate observation of cell morphology. Cells were
washed thrice with PBS and soaked in 4% paraformalde-
hyde for 30min at 4 ℃. Subsequently, cells were permea-
bilized with 0.1% Triton X-100 for 10min and cleaned
with PBS three times. ese samples were then stained
with a Rhodamine-phalloidin working solution (5 μg/
mL) overnight at 4 ℃. Finally, cells were washed with PBS
again and stained with a DAPI solution (10μg/mL) for
1 min before observation under a fluorescence micro-
scope. Photographs taken were analyzed by ImageJ soft-
ware to obtain semi-quantitative data on cell spreading
area and roundness.
2.7.2 Cellular matrix secretion
After culturing for 3 and 7days, cartilage-specific poly-
saccharides secreted by OA chondrocytes were assessed
by staining the cells with TB according to the Toluidine
Blue Staining Kit (G2543, Solarbio). Semi-quantitative
analysis of the staining images was done by ImageJ soft-
ware. For quantitative GAG determination, OA chondro-
cytes in the well plates were collected after 3 and 7days.
e DNA and GAG quantitation were performed as
mentioned in 2.4.1.
2.7.3 Chondrogenic phenotypes
To analyze RNA expression, OA chondrocytes after
5days of co-culture with microgels were collected in RNA
Later water and stored at -20 °C. e cellular RNA was
extracted with RNeasy Mini Kit (74,104, Qiagen) and the
concentration was detected by the microspectrophotom-
eter (ND1000, Nanodrop Technologies). Next, RNA was
reversed transcription (RT) extracted to cDNA using the
iScript™ cDNA Synthesis Kit (BIO-RAD). e polymer-
ase chain reaction (PCR) reaction was conducted on the
CFX96 Touch™ Real-Time Fluorescent PCR Detection Sys-
tem (BIO-RAD). e expression levels of cartilage matrix-
related genes (GAPDH, COL I, COL II, COL X, SOX9 and
Aggrecan) in OA cells were determined using GAPDH as
an internal reference. e primers are shown in Table S1.
2.8 In vivo eects ofdierent microgels
2.8.1 Establishment ofanOA rat model
e procedures adhered to the current guidelines for
the care of experimental animals. Male SD rats, aged
6–8 weeks and weighing approximately 250 g, were
selected for the establishment of the osteoarthritis
model. e rats were anesthetized with an intraperito-
neal injection of 2% sodium pentobarbital (2mL/kg), and
the anterior cruciate ligament in the knee joint cavity was
disrupted using previously reported methods, namely
anterior cruciate ligament transverse (ACLT) [34]. Four
weeks later, intra-articular injections of 100 μL of PBS, or
PBS mixed with a 10mg/mL concentration of HA or HE
microspheres ranging from 80 to 85μm, were randomly
administered to osteoarthritic rats. e meanings of the
group names are as follows: NA: as well as Native, healthy
rats; PBS: rats treated with PBS solution; HA: rats treated
with HA microgels; HE: rats treated with HE microgels.
2.8.2 Radiographic assessment
Four weeks post-injection, Micro-CT scans were per-
formed on the knee joints of the osteoarthritic rats, and
images were reconstructed based on the scanned data.
e knee width and joint space width were also semi-
quantitatively analyzed in each group.
2.8.3 Histologic evaluation
e rats were executed by over-anesthetized after four
weeks of sample injection, and the collected articular
joints were photographed to observe the width of the
articular synovial cartilage. e gross appearance was
scored according to the scoring criteria (Table S2) [35].
e knee joints were immersed in a 4% paraformalde-
hyde solution for 1week, decalcified with EDTA solu-
tion at 37 ℃, embedded in paraffin, and sectioned. e
sections underwent H&E staining, TB staining, Masson
staining, and Sirius Red staining to analyze the histologi-
cal characteristics of the tissues. e severity of osteoar-
thritis in each group of specimens was assessed based on
the staining results, with reference to the Mankin histo-
logical score (Table S3). Subsequently, the sections were
immunologically stained using antibodies against type I
collagen and type II collagen, following the protocols of
the antibody kits.
Page 7 of 17
Dengetal. Collagen and Leather (2024) 6:14
2.9 Statistical analysis
Statistical analysis was performed using GraphPad
Prism 8 software, and multiple t-tests were used to ana-
lyze inter- and intra-group differences. Data results
were expressed as mean ± standard error (SE) and
mean ± standard deviation (SD) for at least three samples
per test, with p values indicating statistically significant
results (*p < 0.05, **p < 0.01 and ***p < 0.001).
3 Results anddiscussion
3.1 Preparation andcharacterization ofdECM
e decellularization of fresh porcine cartilage slices
was prepared by optimizing the previously established
decellularized cartilage ECM preparation protocol. is
involved performing multiple grinding and sieving to
retain small-sized particles, thereby eliminating cells
and preserving tissue-specific components as much as
possible. As shown in Fig. 1A, for further preparation
of the composite hydrogel, the dECM particles were
digested into the solution by pepsin to be co-mingled
with HAMA to form a gel. DAPI, H&E, and TB stain-
ing, as well as the results of microstructural images and
semiquantitative analyses (Fig.1B, C) demonstrated that
there were virtually no cells in the dECM as compared
to fresh natural cartilage. e success of decellulariza-
tion was further confirmed through DNA quantitative
analysis, revealing the removal of approximately 98% of
immunogenic cellular components. e remaining DNA
concentration in dECM was 44.30 ± 0.51 ng/mg tissue
weight, which is below the critical decellularization crit-
erium of 50ng/mg (Fig.1D) [36]. Critical components of
the ECM were characterized before and after decellulari-
zation. Despite the decellularization process inevitably
results in the loss of ECM components, collagen, with its
Fig. 1 (A) Scheme of decellularized cartilage ECM preparation. (B) Representative DAPI, histologic staining of H&E staining, TB staining and SEM
images of native ECM and dECM. (C) Semi-quantitative determination of DAPI. (D) DNA, (E) Collagen, (F) GAG contents of native ECM and dECM.
Error bars represent SD (n ≥ 3). ∗ p < 0.05, ∗ ∗ p < 0.01, ∗ ∗ ∗ p < 0.001
Page 8 of 17
Dengetal. Collagen and Leather (2024) 6:14
large molecular size, intermolecular entanglement, and
relatively low solubility, exhibited a high retention rate
of approximately 80% after decellularization (Fig. 1E).
GAG quantification showed that after the decellulariza-
tion process, approximately 45% of GAG was retained
(Fig.1F).
3.2 Preparation andcharacterization ofHE hydrogels
HAMA was synthesized by grafting methacrylic anhy-
dride onto HA, and the chemical structure was deter-
mined by 1H NMR as shown in Fig. S1. Distinct new
resonant peaks at 5.8 and 6.2ppm in the HAMA spectra
confirmed the existence of the methacryloyl group sug-
gesting that the hyaluronic acid has been modified by
methacrylic anhydride. In combination of HA and car-
tilage-derived dECM, it formed HE hydrogels to mimic
the physiological extracellular matrix (Fig. 2A). e
microstructure of hydrogels was observed by scanning
electron microscopy (Fig. 2B, C). ey both presented
an interconnected porous structure, which facilitated
nutrient transfer and cell migration and proliferation.
e addition of dECM significantly decreased the pore
size and porosity of the HE hydrogels compared to the
control. DMA results showed that the storage and loss
modulus of the HE hydrogels were higher than those of
the HA group as the frequency increased. is indicated
a significant enhancement in mechanical properties due
to the incorporation of dECM (Fig.2D, E). e degrada-
tion behavior of hydrogels was evaluated by two enzyme
solutions (Fig.2F, G). In the hyaluronidase solution, the
rate of HA loss was higher, suggesting that dECM could
slow down the degradation rate of the material and effec-
tively alleviate the problem of short retention time of HA
in intra-articular injection therapy. In PBS buffer, the
hydrogels rapidly reached solubilization equilibrium and
exhibited excellent water absorption and retention prop-
erties (Fig. 2H). e dual-network hydrogel structure
formed by the addition of dECM to HA was conducive
to delaying the release of the active ingredients of dECM,
especially the large molecular weight components, such
as GAG and collage, realizing its sustained effect on
the recovery of chondrocyte phenotype and cartilage
reconstruction.
3.3 Preparation andcharacterization ofHE microgels
To improve intra-articular injectability for the treatment
of osteoarthritis, the HE microgels were fabricated using
microfluidics devices. As shown in Fig.3A, the continu-
ous phase enters the channel from both sides, generating
and encapsulating the dispersed-phase droplets under
the shear forces of the continuous phase. ese droplets
are then cured by a UV light source above the collection
channel. Upon determining the material concentration,
the flow rate ratio of the continuous and dispersed phases
was adjusted by a syringe pump, resulting in a corre-
sponding change in the diameter of the microgels. In this
study, the flow rate ratio of the continuous and dispersed
phases was set at 10:1. is resulted in HE microgels with
a uniform size distribution and an average particle size
ranging from 80–85 μm. is ensured that the micro-
gels could pass through a 1mL syringe (with an inner
diameter of 0.25mm) without disrupting their morphol-
ogy, thereby ensuring good injectability (Fig.3B). Under
microscopic observation, the microgels were dispersed
in a regular spherical shape. For better observation, a
dye solution was added dropwise to the solution, reveal-
ing that the dECM was uniformly distributed within the
microgels. e microscopic morphology of the microgels
was further studied using scanning electron microscopy,
as shown Fig.3C. e incorporation of dECM enhanced
the composition of microgels with a diverse range of
growth factors, as well as GAG, and collagens. ese
components play crucial roles in the cellular behavior
of chondrocytes, such as proliferation, morphology and
matrix secretion. e release profiles of key growth fac-
tors, such as IGF-1, TGF-1β, and BMP-2, and the carti-
lage-specific structural macromolecules and proteins,
including GAG and collagen, in both microgels were
evaluated. e release curves demonstrated that approxi-
mately 60% of the small molecular weight growth factors
were released within 7days (Fig.3D-F), while the water-
soluble GAG exhibited a slower release trend due to the
formation of a network structure through HA cross-link-
ing (Fig.3G). Consequently, the structural collagen with
high molecular weight demonstrated the slowest release
rate (Fig.3H), and its releasing behavior in hyaluronidase
condition suggested that its gradual release is depend-
ent on the degradation of microgels (Fig.3I). e release
of growth factors, structural ECM macromolecules and
proteins showed a significant temporal correlation, indi-
cating that microgels possess biomimetic properties for
actively modulating the dynamic microenvironment dur-
ing cartilage formation.
3.4 HE microgels enhance proliferation andmorphology
ofOA chondrocytes
To observe the effects of both microgels on cell proliferation
and morphology, transwell chambers were utilized to co-
culture microgels with OA chondrocytes invitro (Fig.4A).
e viability and proliferation of OA chondrocytes were
assessed by live/dead staining result (Fig. 4B) and CCK-8
assay (Fig. 4C). e results collectively demonstrated
that the addition of HE microgels significantly increased
the proliferative activity of OA chondrocytes. Notably,
at 3days, the difference between the HE group and the
remaining groups was the most significant, accompanied
Page 9 of 17
Dengetal. Collagen and Leather (2024) 6:14
by a pronounced increase in cell number. is may be
attributed to the rapid release of growth factors such as
IGF-1, TGF-1β, and BMP-2 from HE hydrogels, which
recover OA chondrocytes’ function to boost proliferation.
OA chondrocytes exhibited irregular cell morphology and
hypertrophy in invitro culture. e ability of HE microgels
to promote morphological recovery of OA chondrocytes
with increasing co-culture time was indicated by rhoda-
mine/DAPI staining (Fig.4D), as well as semi-quantitatively
by spreading area (Fig.4E1) and cell roundness (Fig.4E2).
At 3days, with the rapid release of growth factors from the
HE microgels, the larger cell spreading area of OA chon-
drocytes corresponded to enhanced proliferation, consist-
ent with the CCK-8 data. e decrease in spreading area
and cell roundness of OA chondrocytes in the HE group
at 7days may indicate the synergetic effects of the continu-
ously released growth factors and the slow-released dECM
macromolecules on the recovery of cell morphology.
Fig. 2 (A) Scheme of hydrogel constituents. (B) Hydrogel microstructure (SEM). (C) Pore size and porosity of hydrogels. (D) Storage and E loss
modulus of hydrogels. (F-G) Degradation behavior of hydrogels in two enzyme solutions. (H) Solubilization properties of hydrogels. Error bars
represent SD (n ≥ 3). ∗ p < 0.05, ∗ ∗ p < 0.01, ∗ ∗ ∗ p < 0.001
Page 10 of 17
Dengetal. Collagen and Leather (2024) 6:14
Fig. 3 (A) Schematic diagram of the protocol for the preparation of microgels by microfluidics. (B) Particle size distribution of two kinds
of microgels. (C) SEM images as well as representative light microscopy images of the two microgels. Release curve of D IGF-1, E TGF-1β, F BMP-2,
G GAG, and H, I collagen from microgels
Page 11 of 17
Dengetal. Collagen and Leather (2024) 6:14
3.5 HE microgels promote cartilage matrix secretion ofOA
chondrocytes
GAG is a crucial component of cartilage ECM and serves
as an indicator for assessing the chondrocytes’ function
in cartilage regeneration and homeostasis. e ability of
HE microgels to regulate cartilage ECM secretion in OA
chondrocytes was further investigated. e HE group
exhibited superior pro-chondrogenic matrix secretion in
OA chondrocytes compared to the HA group (Fig.5A),
as evidenced by stronger positive staining from tolui-
dine blue (TB) staining after 3 and 7days of co-culture,
corroborated by semi-quantitative data (Fig. 5B). HE
microgels contributed to an increase in cell number
and enhancement of cartilage-specific polysaccharide
synthesis, as shown in the quantitative analysis of DNA
and GAG (Fig.5C-E). e regulatory effect of HE micro-
gels on cartilage matrix synthesis in OA chondrocytes
was not significantly evident at 3 days, suggesting that
the observed effect during this time period may be pri-
marily attributed to enhanced proliferation based on the
CCK-8 data. However, the GAG/DNA values of the HE
group significantly surpassed those of the other groups at
7days, providing further evidence for the enhancement
of matrix synthesis by growth factors and degradation
products. In addition, the qPCR assessed the expression
of various cartilage-specific genes in OA chondrocytes
after 5 days of co-culture with microgels (Fig.5F): the
expression of COL I
Fig. 4 (A) Schematic diagram of co-culture method assessing interactions between porous microgels and cells in vitro. (B) Live/dead staining
images of OA cells after 3 and 7 days of co-culture. (C) Measurement of OA cell proliferation by CCK-8 kit. (D) Cytoskeleton staining after 3
and 7 days of co-culture, cell spreading area (E1), cell circularity (E2). Error bars represent SD (n ≥ 3). ∗ p < 0.05, ∗ ∗ p < 0.01, ∗ ∗ ∗ p < 0.001
Page 12 of 17
Dengetal. Collagen and Leather (2024) 6:14
gene was associated with chondrogenic fibrosis, COL
II and AGG represented ECM synthesis, COL X gene was
expressed in chondrocyte hypertrophy, and SOX9 was
the essential cartilage-promoting factor, which can alle-
viate the progression of OA. HE microgels significantly
decreased the expression level of COL I and COL X genes
and significantly increased the expression of COL II, AGG,
and SOX9. is finding was further supported by the close
relationship among the three key growth factors and the
structural ECM macromolecules towards the chondro-
cyte’s phenotype, based on the gene interaction network
map generated using the STRING database (Fig.5G). In
summary, HE microgels demonstrated enhanced secre-
tion of cartilage matrix and expression of cartilage-specific
genes in OA chondrocytes invitro, as evidenced by his-
tological staining, GAG/DNA analysis, and gene expres-
sion levels. is can be attributed to the synergistic effect
of spatiotemporal release of cartilage-specific molecules,
which rapidly recover the function of OA chondrocytes
with the short-term release of cartilage-specific growth
factors, and continuously support the cartilage regen-
eration and homeostasis with the long-term release of
Fig. 5 (A) Toluidine blue staining images and B semi-quantitative results of OA chondrocytes after 3 and 7 days of co-culture. (C) GAG,
D DNA, and E GAG/DNA quantification. (F) Expression levels of cartilage matrix-related genes in OA chondrocytes co-cultured for 5 days. (G)
Plot of the relationship between ECM proteins genes, growth factors genes and cartilage phenotype related genes. Error bars represent SD
(n ≥ 3). ∗ p < 0.05, ∗ ∗ p < 0.01, ∗ ∗ ∗ p < 0.001
Page 13 of 17
Dengetal. Collagen and Leather (2024) 6:14
structural ECM macromolecules and proteins, such as
GAGs and collagens, as the HE microgels degrades.
3.6 In vivoevaluation ofHE microgels forOA alleviation
e therapeutic potential of HE microgels for OA alle-
viation was evaluated using a rat OA model established
by ACLT surgery. Different samples were injected into the
joint cavity four weeks post-surgery (Fig.6A), and the joints
were reconstructed using micro-computed tomography
(micro-CT) to assess the OA progression. e subchondral
bone treated with PBS was found to be severely deformed,
with a concave and uneven surface. In contrast, the joints
Fig. 6 (A) Scheme of evaluating the effect of microgels in OA rat models. (B) micro-CT, (C) joint space width and D knee width of samples.
(E) gross appearance of the keen joints four weeks after sample injection and F gross morphology score. Error bars represent SD
(n = 6). ∗ p < 0.05, ∗ ∗ p < 0.01, ∗ ∗ ∗ p < 0.001
Page 14 of 17
Dengetal. Collagen and Leather (2024) 6:14
treated with HE microgels more closely resembled the
morphology and structure of healthy rat joints (Fig. 6B).
According to semi-quantitative knee analysis (Fig.6C, D),
there was no statistical difference between the HE micro-
gel treatment and the native group (NA). A decrease in
joint space width reduction and maintenance of knee
width suggest that HE microgels attenuate osteoarthritic
degeneration after four weeks of injection [37]. e gross
view of the knee joint (Fig.6E) and the gross morphology
score (Fig.6F) were consistent with the joint visualization
results, suggesting the effectiveness of the treatment.
e H&E staining of the natural joints showed neat and
orderly chondrocyte arrangement, a homogeneous matrix,
and a smooth surface of the cartilage layer (Fig. 7A). In
Fig. 7 Four weeks after intra-articular injection, tissue sections of rat OA knee joints were stained for A H&E, B TB, C Masson, D Sirius Red, E COL II,
and F COL I. Semi-quantitative analysis of G TB, H COL II, and I COL I staining, J Mankin histologic scores for cartilage structure, chondrocyte, matrix
staining and tidal line integrity (n = 6). ∗ p < 0.05, ∗ ∗ p < 0.01, ∗ ∗ ∗ p < 0.001
Page 15 of 17
Dengetal. Collagen and Leather (2024) 6:14
contrast, the PBS group exhibited significant tissue fibro-
sis, disorganized cell arrangement, and an uneven carti-
lage layer. Despite the lubricating effect of HA leading to a
smoother surface, the HA-treated group displayed a reduc-
tion in thickness and lighter staining color matrix layer
along with irregularly arranged cells. is suggests that
HA’s ability to effectively repair damaged cartilage tissue
is limited. On the other hand, the HE group showed a sig-
nificant improvement in both cell arrangement and matrix,
which can be attributed to the combined lubricating prop-
erties of HA and the cartilage matrix synthesizing function
of dECM. Furthermore, the TB (Fig.7B), Masson (Fig.7C),
Sirius Red (Fig.7D) staining and semi-quantitative analysis
(Fig.7G) allowed for a general assessment of GAG levels.
Among the experimental groups, representative images of
the HE group demonstrated the superior results by best
preserving the GAGs and minimizing the fibrocartilage
production in the regenerated cartilage matrix. In addition,
COL I and II, classic indicators representing the quality of
regenerated cartilage, were assessed by immunohistochem-
ical staining (Fig.7E, F) and semiquantitative data (Fig.7H,
I) to further evaluate the quality of neocartilage tissues.
e HE group exhibited significantly higher production of
COL II with minimal level of COL I, indicating its supe-
riority in the regeneration of healthier hyaline cartilage.
Based on above observations, Mankin histological scores
(Fig.7J) were utilized to illustrate the differences between
the experimental groups and the natural group in terms of
cartilage structure, chondrocytes, matrix staining, and tide-
mark integrity. e results suggested that the HE groups
had achieved the significant improvements over the PBS
and HA groups in the overall Mankin score, with distinct
amelioration in OA cartilage structure and matrix stain-
ing. Collectively, HE microgels effectively promoted the
repair of articular cartilage and significantly alleviated the
degree of OA, evidenced by radiological observation, gross
appearance, histological/immunohistochemical staining,
and analysis in an OA rat model invivo.
4 Conclusion
is study successfully pioneered a cell-free OA therapy
approach, utilizing intra-articular injectable dECM-
enhanced HA microgels. ese microgels, characterized
by their uniform size and mechanical strength, were fab-
ricated using microfluidics and photopolymerization.
ey provided a spatiotemporal cascade effect, enabling
the rapid release of cartilage-specific growth factors such
as IGF-1, TGF-β, and BMP-2. is facilitated the swift
recovery of OA chondrocytes’ function. Simultaneously,
the slower release of cartilage structural ECM macromole-
cules and proteins, including GAGs and collagens, contin-
uously supplied essential elements for supporting cartilage
regeneration and homeostasis as the microgels degraded.
is enhancement was observed in terms of cell prolifera-
tion, morphology, matrix synthesis, and cartilage-specific
gene expression. Furthermore, HE microgels effectively
promoted the regeneration of collagen II-enriched hya-
line cartilage and significantly alleviated the degree of OA,
evidenced by radiological observation, gross appearance,
histological/immunohistochemical staining, and analysis
in an OA rat model invivo. Together, it underscores the
potential of HE microgels in dECM combined with HA as
a promising therapeutic approach for OA.
Abbreviations
OA Osteoarthritis
HE DECM-enhanced hyaluronic
NSAIDs Non-steroidal anti-inflammatory drugs
HA Hyaluronic acid
ECM Extracellular matrix
dECM Decellularized ECM
TGF-β Transforming growth factor-β
IGF-1 Insulin-like growth factor-1
BMP Bone morphogenetic protein
FGF Fibroblast growth factor
EGF Epidermal growth factor
MA Methacrylic anhydride
CHAPS 3-[(3-Cholamidopropyl) dimethylamino] propanesulfonic acid inter-
nal salt
HAMA Hyaluronic acid methacryloyl
GAG Glycosaminoglycan
H&E Hematoxylin–eosin
TB Toluidine blue
Supplementary Information
The online version contains supplementary material available at https:// doi.
org/ 10. 1186/ s42825- 024- 00158-6.
Supplementary Material 1.
Acknowledgements
We would like to thank Sichuan University Analysis & Testing Center for techni-
cal assistance with Micro-CT.
Authors’ contributions
SYD designed this project and revised the manuscript. SYD, HFC, YL and WQS
performed experiments and analyzed data. MYC and XLC performed experi-
ments and draw the scheme. JL, YJF, QGW and XDZ reviewed the manuscript.
All authors read and approved the final manuscript.
Funding
This work was supported by the National Key Research Program of China
(2023YFB4605800), the Natural Science Foundation of China (32071353) and
the 111 Project (B16033).
Availability of data and materials
All data generated or analyzed during this study are presented in this article.
Declarations
Ethics approval and consent to participate
Human OA articular cartilage tissue was obtained from total joint replacement
surgery under the ethical support of the Biomedical Ethics Review Committee
of West China Hospital, Sichuan University (2022200). The animal experiments
were approved by the Ethical Committee of Sichuan University (KS2020308).
All the animals were purchased from Laboratory Animal Center of Sichuan
University. The animal experiment guidance from the ethical committee and
Page 16 of 17
Dengetal. Collagen and Leather (2024) 6:14
the guide for care and use of laboratory animals of Sichuan University were
followed during the whole experiment course.
Competing interests
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.
Author details
1 National Engineering Research Center for Biomaterials, Sichuan University,
Chengdu, Sichuan 610065, China. 2 College of Biomedical Engineering,
Sichuan University, Chengdu, Sichuan 610065, China. 3 School of Medicine,
The Chinese University of Hong Kong, Shenzhen 518172, China. 4 Department
of Orthopedic Surgery & Musculoskeletal Medicine, Centre for Bioengineer-
ing & Nanomedicine, University of Otago, Christchurch 8011, New Zealand.
5 Sichuan Testing Center for Biomaterials and Medical Devices Co.Ltd, 29
Wangjiang Road, Chengdu, Sichuan, China.
Received: 18 January 2024 Revised: 21 March 2024 Accepted: 24 March
2024
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