REVIEW

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Unfolding the Mystery Behind the Onset of Chondrocyte

Hypertrophy during Chondrogenesis: Toward Designing
Advanced Permanent Cartilage-mimetic Biomaterials
Nilotpal Majumder and Sourabh Ghosh*

plethora of factors is responsible for the

Successful recapitulation of the anatomical microarchitecture and impairment in articular cartilage function
biomechanics of the native articular cartilage under in vitro culture conditions that eventually leads to Osteoarthritis (OA).
is still an elusive topic of research. The major roadblock lies in maintaining the Aging, traumatic injury, excessive physical
stable chondrogenic phenotype in vivo or under long-term in vitro conditions. exercise,[2] obesity,[3] hyperlipidaemia,[4] el-
evation in blood sugar level as well as estro-
Tissue engineers worldwide has coined this aberrant loss of permanent
gen deficiency (menopause women),[5] syn-
cartilage characteristics to transient cartilage form as “chondrocyte ovial inflammation and osteophyte devel-
hypertrophy”. Although the following has been validated through the opment are considered as the key risk el-
expression of a few known markers but very little is understood regarding the ements that accelerate the degeneration of
molecular mechanism that dwells underneath. This review summarizes the healthy functioning articular cartilage. For
instance, during obesity, a wide variety of
precise aetiology behind the development and progression of the hypertrophic
pro-inflammatory cytokines and adipokines
phenotype in chondrocytes under in vitro chondrogenic conditions. Based on are released in the adipose tissue, which has
the current literature survey, it is deciphered that the type of cell utilized a direct contribution in orchestrating the
(chondrocytes or stem cells), the chondrogenic culture conditions (growth inflammatory cascade of OA phenotype,[6]
factors/biochemical mediators) and the culture microenvironment (oxygen whereas the impairment in estrogen avail-
tension, mechanical loading) during chondrogenesis have a direct correlation ability in post-menopausal women inter-
rupts the glycosaminoglycan and collagen
with the dysregulated activity of the chondrogenic signaling pathways
synthesis.[7] The clinical manifestation of
corroborating the onset of hypertrophic maturation of chondrocytes. OA are alarming pain and swelling in the
Furthermore, it is critically analyzed whether to completely inhibit these joint region with temporary to permanent
hypertrophy-inducing signaling pathways or apply a brake in terms of mechanical stiffness in certain cases.[8]
time-dependent dose due to their functional duality role in chondrogenesis. Clinicians worldwide are majorly fo-
cused on pain management therapy by
prescribing various non-steroidal anti-
inflammatory drugs (NSAIDs),[9] visco-
supplementation,[10] steroidal injections and physiotherapy[11]
1. Introduction as few available non-surgical therapeutic approaches to contain
The articular cartilage is a highly specialized connective tissue the symptoms of OA. The surgical treatment strategy in such
constituting articular chondrocytes as the main cell-type, 15–20% cases (Total knee arthroplasty or Unicompartmental knee arthro-
of type-II collagen (COL-II) arranged in an isotropic fashion, 4– plasty) further aggravates the condition of the patient with ad-
7% of different sized proteoglycans, without any vasculature and ditional problems of post-operative pain, joint stiffness and in
neural tissue.[1] The spatial arrangement of chondrocytes and situ infections.[12,13] This compelled the researchers to adopt tis-
collagen fibrils varies concerning the different anatomical lay- sue engineering-based strategies to develop phenotypically sta-
ers of the articular cartilage. When damaged, the extracellular ble articular cartilage construct as a long-term symptomatic re-
matrix (ECM)-trapped chondrocytes initiate repair, by clustering lief to OA manifestation by replacing the damaged tissue. Var-
and producing enhanced pericellular matrix components. But a ious research groups have deployed a wide array of biomateri-
als (silk,[14] chitosan,[15] collagen,[16] alginate,[17] PEG[18] ) and cell
source (Bone marrow -derived Mesenchymal Stem Cells,[19] Adi-
N. Majumder, S. Ghosh pose -derived Stem Cells,[20] articular chondrocytes,[21 induced
Department of Textile and Fibre Engineering
Indian Institute of Technology Delhi Pluripotent Stem Cells[22] ) combination to fabricate this complex
New Delhi 110016, India tissue phenotype using exogenous addition of pro-chondrogenic
E-mail: [email protected] growth factors like Transforming Growth Factor -𝛽 (TGF-𝛽),[23]
Bone Morphogenetic Protein (BMP),[24] Growth Differentiation
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/adfm.202300651
Factor-5 (GDF-5),[25] and Fibroblast Growth Factor -2 (FGF-
2)[25] . Despite all these extensive efforts in replicating the native
DOI: 10.1002/adfm.202300651

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Figure 1. Histology of a) Healthy cartilage and b) Osteoarthritic cartilage. Dense Safranin O staining in healthy cartilage envisages the presence of
enormous glycosaminoglycan content, whereas negligible positive staining is observed in the case of osteoarthritic cartilage samples. The fibrillation of
the superficial layer and cellular aggregation clearly distinguishes the hypertrophic cartilage from the healthy cartilage marked by an intact superficial
layer and homogenous cellular distribution.

articular cartilage microarchitecture and physiological function, phenomenon in cartilage tissue engineering. Specifically, we
preserving the stable chondrogenic phenotype post-implantation aimed to raise the following questions:
in vivo still remains the biggest challenge in cartilage tissue en-
gineering. 1) Are all articular chondrocytes or chondrogenically differenti-
The main reason for this failure lies in the traditional carti- ated MSCs destined to undergo hypertrophic differentiation
lage tissue engineering protocol ultimately leading to an unde- following de-differentiation or embryonic development when
sirable conversion of the newly formed articular cartilage to a exposed to chondrogenic stimulus in vitro?
“transient” phenotype through the process of “chondrocyte hy- 2) Does the exogenous addition of the signaling molecules and
pertrophy”, rather than re-establishment of “permanent” articu- morphogens or mimicking the biomechanical condition of
lar cartilage. Thus, post implantation, the cartilage construct in native cartilage tissue for chondrogenic differentiation trig-
the defect site eventually loses its chondrocyte phenotype and ger unwanted activation of the signaling pathways leading to
initiate hypertrophic traits like vascular sprouting[26] and ma- hypertrophy phenotype of chondrocytes?
trix calcification.[27] The former has been justified by the car- 3) Should we completely “block” or apply a “brake” to cartilage
tilage biologists as the inability of the resident cells to inhibit hypertrophy to achieve a stable chondrocyte phenotype?
the activation of permanent-to-transient cartilage transformation
signaling pathways, thereby reducing the efficiency of tissue- These questions will be answered by formulating logical hy-
engineered cartilage in pre-clinical studies. Moreover, the differ- potheses based on existing literature. Understanding this ratio-
ences in the differentiation of permanent articular cartilage and nale will enable researchers and clinicians worldwide to devise
transient cartilage during embryonic development is not compre- more efficient regenerative strategies to engineer articular carti-
hensively studied or understood by the researchers. Therefore, a lage in vitro while maintaining its stable phenotype and physio-
major research gap between identifying the challenge of main- logical function in vivo (Figure 1).
taining a stable chondrogenic phenotype in tissue-engineered
cartilage and developing OA models to screen anti-OA drugs lies 2. The Molecular and Cellular Level Differences in
in understanding the underlying mechanism governing the hy- Permanent Articular Cartilage and Transient
pertrophic differentiation of chondrocytes. Taken together, the Cartilage
onset of chondrocyte hypertrophy is getting identified as a poten-
tial barrier during the clinical translation of the tissue-engineered 2.1. Articular Cartilage Development
cartilage. There are attempted strategies to attenuate the expres-
sion of hypertrophy markers, but only a handful of research has The articular cartilage development commences with the mes-
directly pointed out “how” and “why” such terminal differentia- enchymal condensation step, where the pre-chondrocyte mes-
tion of chondrocytes occur. enchymal stem cells (MSCs) increase their volume occupancy
Thus, in our present review, we tried to understand the without increasing their proliferation rate with increased ex-
aetiology of chondrocyte hypertrophy as a routine barrier- pression SOX9 and COL-II.[28] The cellular aggregation due to

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cell-cell interaction causes deposition of different collagen tinct precursor cell populations. However, when the researchers
matrices (COL-II, Collagen-IX, and Collagen-XI), proteogly- have deployed a combination of EdU/BrdU pulse-chase analysis
cans (decorin, biglycan, and aggrecan) and downregulation of and COL-II mRNA lineage tracing of the proliferating chondro-
COL-I secretion followed by the induction of chondrogenic cyte, they successfully demonstrated that both the articular car-
differentiation.[29] The interzone formation marks the initiation tilage and transient cartilage are developed from a shared pool
of continuous cartilage template segmentation. The cells present of actively proliferating cells present in the cartilage analgen.[42]
in the three-layered interzone assume a flattened morphology There is further evidence that the cells originating from the inter-
with a redundant expression of COL-II and SOX9 along with en- zone contribute to the formation of transient cartilage, which de-
hanced expression of GDF-5, Autotaxin (ATX) and Chordin in marcates formation of independent cartilage element. The cells
the surrounding chondrogenic layers. The interzone is also a har- differentiates into the respective cartilage lineage depending on
bour of Wnt ligands (Wnt 9a and Wnt 4) that essentially promotes the zone-specific ectopic activation of BMP and Wnt signaling
the expression of articular cartilage specific genes (GDF5, ATX, pathways, respectively.[43] The cells that are restricted within the
and Chordin) in the interzone cells.[30,31] The permanent articu- region of Wnt signaling are dedicated toward permanent articular
lar cartilage differentiation originates from the cells near prox- cartilage fate insulated by a Noggin barrier,[42] whereas the cells
imity of the interzone. A wide constellation of transcription fac- present within the zone of BMP signaling proceed toward tran-
tors aid in the chondrogenic differentiation of the mesenchymal sient cartilage differentiation. The interzonal cells at the periph-
cells among which C-1-1 and OSR1/2 plays a significant role in ery transform to chondrocyte lineage with an increase in proteo-
stimulating Wnt ligand secretion and further blocking transient glycan deposition.[44] Thus, interzone formation by tissue engi-
cartilage differentiation.[32,33] A transcription factor c-Jun is also neering should be considered as a major intermediate milestone
known to promote articular cartilage differentiation by promot- for permanent articular cartilage segmentation (Figure 2).
ing the transcription of Wnt 9a and Wnt 4 ligands.[34] There are
also evidences of presence of GATA3 gene in the articular carti-
lage tissue that significantly regulated the expression of perma- 3. Are we Close Enough to Mimic the Native
nent cartilage specific genes (ATX and SERP2). [35,30] Articular Cartilage Differentiation Process in vitro?
Researchers globally have explored a wide array of 2D and 3D-
2.2. Transient Cartilage Development based approaches using vivid combinations of cell types, bioma-
terials, and biochemical mediators to simulate the tissue architec-
The transient cartilage differentiation, unlike the permanent ar-
ture, mechanical rigidity and physiological function of the native
ticular cartilage, originates from the center of the cartilage tem-
articular cartilage. When compared, the 3D culture model of ar-
plate constituting a pool of COL-II expressing cells located at
ticular chondrocytes revealed an advantageous role in enhanced
a far distance from the region of permanent articular cartilage
chondrogenic biomarker expression and ECM content deposi-
differentiation. The latter being the major source of Parathyroid
tion than the 2D model.[45] Although these 3D culture systems
Hormone Related Peptide (PTHrP) that inhibits transient carti- have demonstrated chondrogenic differentiation, their ambiva-
lage differentiation. During the growth of the cartilage template lent character has been equally exposed through the stable ex-
(also known as cartilage analgen), the cells evade the influence pression of terminal differentiation biomarkers like COL-I and
of PTHrP signaling cascade and immediately activate Indian COL-X. Thus, the in vitro chondrogenic stimulus to the BM-
Hedgehog (IHH) pathway initiating the hypertrophic differen- SCs and primary chondrocytes led to the formation of mechani-
tiation process.[36,37,38] The involvement of BMP signaling path-
cally inferior “fibrocartilage” instead of “hyaline cartilage”, more
way in endochondral ossification[39] is well explained by Biswas
prominently in monolayer than 3D culture conditions. This led
et al. where the BMP induced activation of ROS scavenging en-
the progressive group of tissue engineers to completely shift their
zyme Prdx1 significantly upregulated the IHH expression in the
paradigm to alternative 3D culture systems with a hope to achieve
differentiation chondrocytes.[30] Furthermore, the expression of
stable cartilage phenotype.
osteoblast actin assembly gene Dspyl3 in the hypertrophic chon-
drocytes is activated by the BMP signaling pathway.[40] The ma-
trix remodeling and vascular sprouting regulated by the ma-
3.1. 3D Aggregate-based or Scaffold-based Models
trix metalloproteases[41] and Vascular Endothelial Growth Factors
(VEGF) respectively is immediately followed by osteogenic differ-
3.1.1. 3D Aggregate
entiation with a remarkable increase in the expression of Runt-
related transcription factor-2 (RUNX2).[30] Thus, the transient
A 3D scaffold-free approach in the form of spheroid or pellet
cartilage with an elevated level of Collagen-X (COL-X), Collagen-I
culture seemed feasible for the investigators to alleviate the on-
(COL-I), and RUNX2 expression attains a bony tissue-like pheno-
set of hypertrophy. In a dynamic culture system withADSCs,
type hugely different from permanent hyaline cartilage.
Yoon et al. and Tu et al. demonstrated impressive chondrogenic
traits in spheroids independent of the presence of TGF-𝛽3.[46,47]
2.3. Common Point of Origin for Permanent and Transient A comparison between the ADSC spheroids and chondrocyte
Cartilage spheroid also revealed a higher level of ECM deposition in ADSC
spheroids than in chondrocytes, while the expression profile of
Evidence from the existing literatures propose that permanent COL-II followed a reverse order.[48] Such ace characteristics of the
articular cartilage and transient cartilage originates from two dis- spheroid culture system has additional advantage of increased

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Figure 2. Graphical illustration demonstrating the single point of origin of permanent and transient cartilage regulated by a wide array of transcription
factors and signaling molecules.

cell-cell communication and establishing a hypoxic microen- 3.2. 3D Bioprinted Models

vironment during development of articular cartilage tissue.[49]
However, Zhang et al. underlined the overall advantage of micro- 3.2.1. 3D Bioprinted Dispersed Cells
mass culture over pellet culture using BMSCs in achieving artic-
ular cartilage specific COL-II expression with diminished fibro- Earlier research from our laboratory has extensively elucidated
cartilage marker expressions.[50] Researchers have also reported the role of silk-based biomaterial and 3D bioprinting strate-
the use of minimally expanded human articular chondrocytes gies in instructing chondrogenic differentiation of BMSCs and
in developing spheroid-based microcartilage models depicting articular chondrocytes while firmly resisting the induction of
enhanced secretion of COL-II and glycoscaminoglycans (GAG) hypertrophy. Chameettachal et al.[14] utilized the same SF-G
along with a sturdy expression of hypertrophic marker COL-X.[51] bioink in developing a cartilaginous tissue construct using BM-
However, challenges about forming the necrotic core at the cen- SCs and articular chondrocytes in a 3D system. The results de-
ter of the spheroid, non-uniformity in ECM production and the picted the advantage of using primary chondrocytes over BM-
stunted proliferation rate of the involved cells led tissue engineers SCs in 3D bioprinted constructs in mitigating chondrocyte hy-
to contrive an alternative method to induce a stable phenotype in pertrophy while promoting chondrogenesis. The advantages of
long-term culture conditions. spatio-temporal control on the deposition of the desired bioink
and controlled porosity resulted due to 3D bioprinting, along
with the chondrogenic role of silk fibroin biomaterial, have
3.1.2. 3D Scaffold-based been exploited by Chawla et al.[19] They used a combination
of silk fibroin-gelatin (SF-G) blend and BMSCs to fabricate a
When primary chondrocytes, human BMSCs, human ADSCs, tissue-engineered cartilaginous construct and assessed the ad-
ESC (H9, WiCell Research Institute), and ESC-derived mes- ditional role of growth factor TGF-𝛽1 in chondrogenesis. Their
enchymal stem cells (developed by two different protocols, ED1 investigation demonstrated the upper hand of using a combina-
and ED2) were differentiated in silk and chitosan scaffolds with tion of silk bioink-based 3D bioprinting technique in develop-
and without BMP-6 in chondrogenic media, BMP-6-modified silk ing cartilage constructs owing to their advantage in activating
scaffolds containing ED1 MSCs demonstrated the best potential pro-chondrogenic Wnt/𝛽-catenin signaling cascade and spatio-
for cartilage regeneration. However, both the ED1 and ED2 cells temporal control over cell encapsulated biomaterial deposition
in the scaffolds depicted maximum hypertrophic features.[52] The and morphogen bioavailability.[19,54] Investigations have been car-
challenge of using primary chondrocytes is that only the last- ried out by several other researchers using 3D bioprinting on
stage human chondrocytes is accessible for research purposes a similar line, for instance., using a combination of nanocellu-
extracted during the cartilage reconstructive surgery. Such OA ar- lose and alginate embedded with human chondrocytes (Mark-
ticular chondrocytes displayed an altered phenotype compared to stedt et al.),[55] collagen hydrogel and human naso-septal chon-
normal chondrocytes, akin to growth plate chondrocytes. Thus, drocytes (Lan et al.),[56] and MSCs encapsulated in norbornene
findings from their research clearly demonstrated the onset of hy- conjugated hyaluronic acid hydrogel for in situ photo crosslink-
pertrophic maturation in the chondrocytes.[53] Therefore, adopt- ing (Galarraga et al.).[57] Despite such tremendous efforts, the
ing a 3D culture system (scaffold-based and scaffold-free) could fabricated tissue constructs fail to maintain the chondrogenic
not yet solve the problem with chondrocyte hypertrophy and property when cultured for a longer period under chondrogenic
achieve stable 3D cartilage tissue. stimulus.

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3.2.2. 3D Bioprinted Spheroids clinical phenotype.[62] However, when the underlying cellular and
molecular events in disease are considered in characterizing the
To overcome this challenge on chondrocyte hypertrophy, tissue specific tissue-type, such as the presence of various downstream
engineers have adopted an advanced strategy of directly 3D bio- signaling targets governing the ectopic activation of respective
printing the spheroids. This method was rightly adopted by Melo cellular signaling cascades, it is termed an endotype.[63,64] The ex-
et al., employing fibrin encapsulated BMSC spheroids bioprinted pression of a single hypertrophic marker can be observed in dif-
over a PEG-alginate bath. The spheroids successfully maintained ferent degrees governed by different underlying pathways signi-
the chondrogenic phenotype without interfering with the me- fying different stages of disease progression. Furthermore, due
chanical properties of the developed hydrogel system.[58] Moor et to the multi-layer anatomy of articular cartilage (Figure 1), the
al. also demonstrated the superior chondrogenic characteristics gene expression patterns might also vary with different layers
(COL-II and GAG secretion) of 3D bioprinted BMSC spheroid based on their biochemical and molecular composition. Grogan
encapsulated gelatin methacrylate (GelMA) constructs as com- et al. have extensively studied these gene expression profiles at
pared to dispersed cells in the hydrogel. The authors assumed different zones of articular cartilage.[65] Their research indeed
the mechanical robustness of the already secreted ECM of the revealed the varied expression of several markers like aggrecan
spheroids in vitro to be the primary reason behind maintain- (ACAN) and COL-II, exhibiting minimum expression in the su-
ing such increased cell viability and chondrogenicity while with- perficial zone but extensively present in the mid-zone.[65] Sev-
standing high shear stress during 3D printing.[59] On a similar eral other scientists have also deciphered different biomarkers
note, Nguyen et al.[22] and Scalzone et al.[60] used a coculture at both clinical and molecular levels governing the formation
system of induced pluripotent stem cells (iPSCs) as well as BM- of tissue-engineered cartilage in vitro from a wide range of cell
SCs with chondrocytes (spheroid and dispersed) encapsulated in sources.[66,67] Therefore, there is a dire need to refine the proto-
respective alginate and chitosan-based bioink to develop a 3D col for molecular characterization of the tissue-engineered carti-
bioprinted in vitro model of articular cartilage. However, results lage to compare with the articular cartilage tissue formed during
from all these attempts have clearly indicated the simultaneous embryonic development in vivo.
hypertrophic maturation of the developed neo-cartilage, even uti-
lizing the most advanced technique of tissue engineering.
Nonetheless, the efforts put forward by the cartilage tissue en- 4.1. Commonly Analyzed Biomarkers
gineers in collaboration with clinicians and cell biologists led
to the formation of engineered cartilage tissue in vitro, the on- The tissue-engineered cartilage developed using various scaffold-
set of further transdifferentiation could not be avoided in ev- based, or scaffold-free approaches are validated through mRNA
ery single culture system adopted (monolayer, 3D spheroids, and expressions of specific biomarkers. Among them, COL-II, SOX-
3D bioprinted). The reason for such hypertrophic conjecture of 9, and ACAN are universally used in evaluating the development
BMSCs or primary chondrocytes during chondrogenic differen- of hyaline articular cartilage. SOX9 protein during neo-cartilage
tiation in vitro is still a riddle to be resolved. Therefore, there formation is known to aid in the maintenance of cell morphol-
exists a paradoxical relationship where exogenous addition of ogy required during the mesenchymal condensation stage, be-
the pro-chondrogenic cytokines or small molecule inhibitors of sides regulating the expression of other pro-chondrogenic mark-
bone-forming signaling cascades in chondrogenic differentiation ers like ACAN and COL-II. The deposition of proteoglycans and
medium, although significantly induced chondrogenesis of the collagen matrices during the chondrogenic differentiation phase
cultured mesenchymal stem cells or primary chondrocytes but of embryonic development is marked by the expression of ACAN
did not attenuate their transient cartilage differentiation.[14,61] and COL-II, whereas the hypertrophic differentiation of the de-
Thus, it can be postulated that under the influence of chondro- veloped chondrocyte phenotype is generally confirmed by the se-
genic stimulus in vitro, the MSCs and chondrocytes (monolayer, cretion of mechanically inferior collagen matrices (COL-X and
3D spheroid or 3D scaffold) demonstrate an intermediate un- COL-I) as well as the matrix metalloproteases (MMP-1/13). The
stable chondrogenic phenotype that eventually undergoes hyper- expressions of these biomarkers have been extensively quanti-
trophic differentiation to finally transform into a transient carti- fied using RT-PCR by various research groups as the only bench-
lage. Henceforth, the current available cartilage tissue engineer- marks for successful chondrogenesis.[68,69] However, one cannot
ing strategies have expedited the development of an inconsistent fully validate successful chondrogenic differentiation without ex-
or “pseudo-chondrogenic” status of newly formed chondrogenic amining several associated biomarkers that has a significant role
tissue during in vitro chondrogenic differentiation. in the physiological functioning of the articular cartilage.

4. A Dire Need to Refine the Molecular 4.2. Specialized Biomarkers

Characterization of the Tissue-Engineered
Cartilage Proteoglycan 4 (PRG4) or lubricin is a superficial zone-based
marker of articular cartilage (also known as superficial zone
The biomarkers that characterize a tissue-engineered cartilage protein) composed of mucin-rich glycoproteins that aids in the
can be broadly categorized as a “clinical phenotype” and an “en- friction-free movement of the gliding joints by providing smooth
dotype”. When various observable attributes like the causative lubrication. At the same time, it arrests the progression of OA
agents and their associated risk factors distinguish a particular in the developing cartilage by limiting the synoviocytes prolif-
subpopulation of a cell (chondrocytes) in a tissue, it is termed as a eration. The chondroprotective role of PRG4 has been listed in

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several in vitro and in vivo investigations by Chawla et al.[19] specifically focused on the patient’s clinical characterization, di-
Alquaraini et al.[70] and Lefebvre et al.[71] correlated with the versity of symptoms, severity, degree of expression of biomarkers
expression profiles of other pro-chondrogenic genes. Another and their specific genetic traits.
definitive articular cartilage differentiation marker is Autotaxin
(ATX) present in chondrocyte’s plasma membrane that regulates 5. The Mystery Behind the Futile Cartilage Tissue
the production of lyso phosphatidic acid (LPA), which in turn Engineering Strategies: Chondrocyte Hypertrophy
is a potent regulator of fibrocartilage formation through COL-
I expression.[72,73] The downregulation of such ATX expression The terminally differentiated chondrocytes display an increase in
post 21 days culture of BMSCs in chondrogenic media has been cell volume due to osmotic swelling.[77] Such change in osmo-
reported by Chawla et al., necessitating the chondro-regulatory larity is attributed to the decrease in extracellular concentration
role of ATX in articular cartilage development.[19] Additionally, or increase in cytoplasmic concentration, increase in cellular or-
Singh et al have reported two new transcription factors, NFIA ganelle and ECM degradation around the periphery of the cell.[78]
and GATA3, as a confirmatory marker for articular cartilage dif- The alteration in osmolarity causes intracellular movement of
ferentiation. The study revealed the hypertrophy inhibiting role water through aquaporins and aid in bone tissue formation.[79]
of NFIA and the chondro-stimulating role of GATA3 in articu- However, it is still poorly understood whether the increase in cel-
lar cartilage tissue.[35] Hence, evaluating the potential mRNA ex- lular volume a biomarker for terminal chondrocyte differentia-
pressions of PRG4, ATX, NFIA, GATA3, etc in the newly fabri- tion is or vice versa. The articular chondrocytes eventually lose its
cated cartilage tissue is an apposite strategy to authenticate the differentiated trait and acquires a terminally differentiated state
complete recapitulation of anatomy and physiology of the native marked by unrestricted proliferation rate and gradually converts
articular cartilage tissue. into longitudinal bone. This state marks the expression of major
chondrocyte hypertrophy markers like COL-X,[80] IHH,[81] MMP-
13,[82] VEGF,[83] ALP, RUNX2, OPN, and OCN which largely
4.3. Signaling Pathway-Specific Biomarkers overlaps with the gene expression profile related to osteogenic
differentiation.[53,84]
Any embryonic tissue development process is regulated by a The end stage of hypertrophic chondrocytes is marked by ma-
stringent mechanistic crosstalk between several cellular signal- trix mineralization, where the ECM loses its viscoelastic prop-
ing pathways and the positive and negative expressions of their erty and transforms into a calcified matrix. Enhanced deposition
respective downstream targets that controls the fate of the neo- of hydroxyapatite crystals are carried out by the matrix vesicles
tissue. However, this portion is generally overlooked owing to its that leads to endochondral ossification.[85] The degradation of the
complex unsolved molecular level understandings. Our group mineralized matrix and the vascular invasion from the under-
has been instrumental in deciphering the regulatory role of neath subchondral bone is tightly regulated by the secreted ma-
chondrogenic signaling pathway downstream targets (𝛽-catenin, trix metalloproteinases by specialized group of cells termed as
SMAD4) with enhanced expression of 𝛽-catenin and catastrophic “chondroclasts”. For instance, MMP-13 interacts with the matrix
decrease in SMAD4 expression during chondrogenic differen- vesicle membrane to release the angiogenic stimulating factor
tiation of BMSCs and primary chondrocytes respectively.[14,19] VEGF during hypertrophic maturation and enhance the vascu-
Moreover, we are the first group to unfold the pro-Wnt character- lar sprouting over the calcified cartilage matrix.[86,87] It is also re-
istics of silk fibroin biomaterial in stimulating in vitro chondro- ported that cellular senescence or apoptosis is also a key end-stage
genesis by enhanced expression of 𝛽-catenin, as assessed from biomarker of hypertrophically matured chondrocytes. The syner-
detailed proteomic analysis.[54] Silk-gelatin bioink also upregu- gistic functioning of inorganic phosphate ions released during
lated several anabolic chondrogenic signaling pathways (Notch, calcified matrix degradation and intrinsic caspase molecule reg-
integrin signaling pathways, HIF-1) and inhibited catabolic path- ulate the process of apoptosis.[88] However, it would not be right
ways NF-𝜅B signaling. Hypoxia through its transcription factors to infer that cell death or apoptosis is the only determined fate of
Hypoxia inducible factor 1 alpha (HIF-1𝛼) and Histone deacety- terminally differentiated chondrocytes. There are investigations
lase (HDAC4), has been a crucial regulator of vascular invasion that also suggests that the terminally differentiated chondrocytes
and sprouting (angiogenesis) during the hypertrophic matura- can transform into osteogenic lineage under the influence of var-
tion of chondrocytes[74,75] whereas IHH is consistently expressed ious osteogenic signaling morphogens indicating the formation
by the hypertrophic chondrocytes during transdifferentiation of “transient” and not “terminally differentiated” chondrocytes
process.[76] The absence of such vascular invasion and consecu- during hypertrophic maturation.
tive IHH expression has been extensively examined with BMSCs
and primary chondrocytes under the presence of chondrogenic 6. Unfolding the Mystery: The Underlying
stimulus to develop permanent cartilage phenotype.[14,19] Mechanisms Governing Chondrocyte Hypertrophy
Thus, studying the expression profiles of these signaling tar-
gets and the additional biomarkers would substantially lead to the Numerous research has been carried out to decipher the molec-
development of anatomical relevant engineered tissue, but also ular symptoms and the associated regulators of hypertrophy but
corroborate the physiological function and phenotypic stability- the aetiology and the underlying mechanism governing the pro-
a key requisite in distinguishing the formation of “hyaline” and cess still remains an unsolved puzzle. To unfold this mystery,
“fibrocartilage” during in vitro chondrogenesis. Identifying such we have postulated the following hypotheses (Summarized in
markers at molecular level can aid in the progression of person- Table 1) to understand and elucidate the anticipated reasons be-
alized medicine, i.e., patient-specific cartilage grafts that will be hind the occurrence of this pleiotropic phenomenon.

Adv. Funct. Mater. 2023, 33, 2300651 2300651 (6 of 20) © 2023 Wiley-VCH GmbH
 Table 1. Summary of the postulated hypotheses regarding the onset of chondrocyte hypertrophy.

Serial no. Broad hypothesis Key observations Probable underlying mechanism Reference

1. Dedifferentiation of primary Stressed actin fibres with vimentin spawning over the Actin polymerization and presence of nuclear myocardin [173,174]
chondrocytes during in vitro cytoplasm in 2D chondrocyte culture system related transcription factor a (MRTFa) in

Adv. Funct. Mater. 2023, 33, 2300651

expansion protocol Enhanced expression of 𝛼-SMA in higher passage chondrocytes de-differentiated chondrocytes.
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resulting in biomechanically inferior tissue.

Downregulated expression of chondrogenic marker genes Mitogen associated protein kinase pathway (MAPK), p38, [98,99,100,101,102]
(COL-II, COMP-1, SOX9, ACAN, chondromodulin). extracellular signal related kinase pathway (ERK) and
Stimulated the secretion of COL-I with elevated expression of phosphatidylinsolitol-3-kinase pathway (PI3K) in the
hypertrophy markers ALP, RUNX2, COL-X process of cellular dedifferentiation

2. The natural tendency of stem cells A 21 days TGF𝛽-1 exposure to BMSCs demonstrated enhanced The variation in concentration and time of exposure of [19,175,109,110,112]
to undergo hypertrophic expression of hypertrophic markers COL-X and IHH. TGF𝛽-1 during chondrogenic differentiation of stem
maturation: dysregulation in the Mouse limb MSCs when exposed to TGF𝛽-1 caused enlarged cells.
chondrogenic pathways cell morphology, extensive mineralization followed by The initiation of Smad 1/5/8 pathway through ALK-1
abundant expression of VEGF, COL-X and Osteocalcin post activation by TGF𝛽 and non-canonical MAPK pathway
21 days of culture.
Media supplementation with BMP-2 protein during the The difference in time-point of addition of BMP protein [176,115,177,117,118]
chondrogenic differentiation of MSCs and ADSCs displayed during chondrogenesis, the variant of BMP
prominent hypertrophic traits marked by elevated expression (BMP-2/4/6/7/9) used as a media supplement or the
of COL-X, COL-I, ALP, IHH, RUNX2 and Osteopontin. concentration and exposure time.

2300651 (7 of 20)
The aberrant crosstalk between TGF𝛽 and BMP
signaling pathway followed by dysregulated activation
of RUNX2 transcription via Smad-1/5/8 canonical
pathway
Wnt proteins (Wnt 3a, 8c and 9c) and agonists (Melatonin and The ligand mediated activation of hypertrophic canonical [119,124,123,178]
BIO) demonstrated significant upregulation of hypertrophic Wnt signaling cascade or the intracellular signaling
maturation gene expressions (COL-X, RUNX2, ALP, crosstalk between several hypertrophy inducing
ADAMTS5) along with enhanced matrix calcification signaling pathways
Dexamethasone addition to the chondrogenic differentiation Concentration and exposure time. [179,180]
medium caused significant increase in ALP activity and
suppressed proteoglycan synthesis and accumulation in the
surrounding ECM
Exogenous treatment of BMSCs with Platelet rich plasma A disbalance in the exposure time and concentration [20,181]
during chondrogenesis elevated the expression of COL-I and during in vitro chondrogenic differentiation of stem
Osteocalcin cells.
(Continued)
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Table 1. (Continued).

Serial no. Broad hypothesis Key observations Probable underlying mechanism Reference

IHH signaling pathway independently or in association with The innate role of IHH [76,134]
several other osteogenic signaling cascade (BMP) depicted
elevated hypertrophic traits marked by overexpression of
matrix degrading enzymes (MMP-13) and COL-X.
3. The ubiquitous role of hypoxia HIF-1𝛼 promoted the autophagy of chondrocytes by supressing Through the post-translational modification of secreted [137,138]
during chondrogenic the activity of Bcl-2 and caspase-8 protein, a key trait collagen during chondrogenesis.
differentiation observed in hypertrophic chondrocytes
HIF-1𝛼 also elevated the secretion of IL-1𝛽 in the surrounding The activation of pro-inflammatory NF-𝜅B pathway [139]
cellular microenvironment causing ECM degradation.
HIF-2𝛼 is a potent transactivator of several endochondral The transcription of pro-hypertrophic marker genes [140,141]
ossification pathway genes like COL-10, MMP-13, and VEGF. through the activation of C/EBP𝛽 pathway

2300651 (8 of 20)
The expression of HIF-2𝛼 gene is highly promoted by the Hydrostatic pressure promotes cartilage matrix [142,143]
presence of hydrostatic pressure due to the higher degeneration through HIF-2𝛼 mediated secretion of
proportion of water content in the cartilage tissue. Aggrecanase and matrix degrading proteases along
with HIF-2𝛼 independent activation of NF-𝜅B pathway.
4. The imbalance in mechanical Excessive mechanical compression, oscillatory hydrostatic Activation of TNF-𝛼, IL-1𝛽, NF-𝜅B, Wnt and TGF-𝛽 and
loading during in vitro pressure, shear force and tensile loading enhanced the their mechanistic crosstalk
chondrogenesis expression of catabolic matrix degrading enzymes (MMP-13
and ADAMTS5) along with suppression of pro-chondrogenic
gene expressions.
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Figure 3. H&E staining of healthy and hypertrophic cartilage demonstrates increased cell volume and cell clustering in the hypertrophic cartilage. The
possible reason can be attributed to the osmotic influx of water through the aquaporin channels due to the change in osmolarity of the hypertrophic
microenvironment. A pertinent question is whether the increase in chondrocyte volume and clustering arise due to hypertrophic maturation or whether
this chondrocyte swelling is associated with abnormal (neo)matrix production and directly/indirectly causes cartilage graft failure. Moreover, when the
isolated chondrocytes are expanded in vitro in 2D, they lose their round morphology and assume a fibroblastic characteristic marked with an increased
expression of COL-I instead of COL-II through a process termed ‘cellular de-differentiation’. When these cells are cultured in 3D systems (embedded in
a hydrogel), the round morphology of the chondrocyte is restored with an expression of articular cartilage-specific markers. However, the ambiguity lies
in the fact that if the “cellular de-differentiation” phenomenon can be reversed in 3D culture models, what could be the possible source of chondrocyte
hypertrophy when the same chondrocyte-laden tissue constructs are implanted in vivo?

6.1. The tendency of the Articular Chondrocytes to Undergo 1 and superficial zone protein lubricin also diminished during
de-differentiation During Chondrogenesis the proliferative stage of chondrocytes.[96,97] There are reports that
also showed the elevated expression of several transient cartilage
For tissue engineering-based approaches, obtaining a high cell biomarkers like ALP and RUNX2 during the monolayer expan-
density is a critical variable to ensure homogenous secretion sion of chondrocytes.[98]
of ECM component and expression of enough cell-surface re- Researchers have tried to investigate the underlying mech-
ceptors to activate downstream signaling cascades. During the anism behind the occurrence of such unusual dedifferenti-
monolayer expansion of chondrocytes (irrespective of the origin), ation phenomenon and concluded the involvement of sev-
they inevitably loose their native cellular morphology along with eral cell signaling pathways like Mitogen-activated Protein Ki-
their physiological function.[89] The expansion phase of chon- nase (MAPK),[99] p38,[100] Extracellular Signal-regulated Kinase
drocytes results in a more fibroblastic morphology of chondro- (ERK),[101] and phosphoinositide 3-kinase (PI3K)[102] governing
cytes instead of a round one with a significant increase in cell the entire process of chondrocyte dedifferentiation. As already
volume[90,91] (Figure 3). The critical interplay between the altered discussed in section 3.2 that an increase in cellular volume
morphology of chondrocytes and the alteration of cytoskeleton followed by a catastrophic decrease in chondrogenic biomark-
assembly with increased expression of focal adhesion complex ers (COL-II, SOX9, and ACAN) with an increased expression
and thick F-actin fibres have been duly reported as the molec- of bone-related markers (RUNX2 and Alkaline Phospatase) are
ular changes underneath.[90–92] A significant downregulation of the key phenotypic alterations in a hypertrophic chondrocyte.
COL-II secretion and aggrecan[93,94] is marked by an equivocal in- Thus, it can be rightly postulated that the acquired dedifferenti-
crease in COL-I secretion and small sized proteoglycans (decorin ated phenotype of primary articular chondrocytes during chon-
and biglycan)[95] thereby altering the overall biomechanical prop- drogenic differentiation can be attributed to one of the possi-
erty of the surrounding matrix. The expression of several other ble reasons behind the hypertrophic maturation of the cultured
pro-chondrogenic markers like SOX9, chondromodulin, COMP- chondrocytes.

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6.2. The natural Propensity of Stem Cells to Attain Transient signaling cascade can be hypothesized to be one of the key rea-
Phenotype under the Influence of Different Chondrogenic Media sons for the hypertrophy inducing role of TGF-𝛽.
Supplementation: Dysregulated Activation of Chondrogenic
Signaling Pathways
6.2.2. Bone Morphogenetic Proteins (BMP)
The stem cells possess excellent proliferation and differentiation
capability unlike autologous chondrocytes making them a per- Exogenous addition of BMP molecule to the chondrogenic dif-
fect fit for tissue engineering applications.[103] The BMSCs and ferentiation media of stem cells have been reported by numer-
ADSCs have been widely explored by the tissue engineers both ous researchers to stimulate the collagen and proteoglycan syn-
in pellet form as well as embedded in the 3D scaffolds for chon- thesis during chondrogenesis by activating the BMP signaling
drogenesis due to their easy isolation procedures besides bear- cascade.[24,115] However, akin TGF-𝛽 cascade, BMP also functions
ing the general properties of other stem cells.[104] BMSCs cul- via the canonical Smad 1/5/8 phosphorylation route activating
tured in 3D bioprinted SF-G construct under the continuous the transcription of RUNX2 responsive hypertrophic genes ALP,
chondrogenic stimulus for 28 days demonstrated excellent chon- COL-X, and MMP-13[116,117] confirming the functional duality
drogenic potential[54] whereas the same chondrogenic stimulus of BMPs in chondrogenesis.[24] A similar role of BMP-2 in in-
for 21 days caused the resurgence of hypertrophic traits.[19] Sim- ducing chondrocyte hypertrophy have been explained by Chawla
ilarly, ADSCs are one such cell source that is widely isolated et al. where the minimally expanded human articular chondro-
from the infrapatellar pad (IFP) or abdomen tissue that intrin- cytes when treated with BMP-2 molecule significantly upregu-
sically express SOX-9 and exhibit higher proliferation rate than lated the COL-X expression along with consistent GAG and COL-
BMSCs.[105,106] However, these cells also express osteogenic dif- II secretion.[51] Furthermore, both in vivo and in vitro investi-
ferentiation marker RUNX2 marking their inclination toward hy- gations have established the mechanistic crosstalk between the
pertrophic maturation.[107] When compared with BMSCs under TGF-𝛽 and BMP signaling cascade through the expression of
the influence of chondrogenic morphogens, ADSCs displayed each other’s respective downstream targets that regulate the ec-
an increased tendency toward hypertrophic maturation with el- topic hypertrophic maturation of chondrocytes.[14,118] Therefore,
evated levels of COL-X and COL-I expression but the tendency we can presume that the exogenous addition of BMP molecule
was reported to be less than BMSCs[107] . The commonly used dif- (dose, exposure time and time of addition) as a pro-chondrogenic
ferentiating agents for the chondrogenic differentiation of stem factor during chondrogenesis caused an aberrant dominance of
cells are ascorbic acid, insulin, TGF-𝛽1/3, Wnt ligands and BMP hypertrophic trait over the chondrogenic phenotype thereby pro-
proteins. The exogenous addition of these chondrogenic signal- moting endochondral ossification over permanent cartilage dif-
ing agents during in vitro chondrogenesis often activates cer- ferentiation.
tain transient cartilage-forming signaling cascades resulting in
the transforming the permanent cartilage phenotype to a hyper-
trophic state. 6.2.3. Wingless Protein (Wnt ligands)

Researchers have examined the differential significance of vari-

ous Wnt superfamily members like Wnt 3a,[119] Wnt 4,[120] Wnt
6.2.1. Transforming Growth Factor Beta Isoform (TGF-𝛽) 5a,[121] Wnt 9a[122] in endorsing the different stages of carti-
lage development and further delaying chondrocyte hypertrophy.
The TGF-𝛽 superfamily morphogens exhibits a functional du- However, the Wnt signaling pathway exhibits an ambiguous role
ality role during chondrogenesis by activating both chondro- by simultaneously promoting chondrogenesis as well as hyper-
genic and hypertrophic differentiation route.[23,108] The Smad- trophic differentiation of chondrocytes. This was confirmed by
dependent route or the canonical pathway of TGF-𝛽 mainly oper- Huang et al. in their detailed investigation by using Wnt signal-
ates via the ALK-5 phosphorylation-mediated activation of their ing inducer (BIO) and inhibitor (PKF-118-310) as a media sup-
downstream Smad targets (Smad2/3) forming a ternary com- plement in the chondrogenic differentiation media of MSCs. Re-
plex with Smad4 and inhibit the hypertrophic differentiation of sults from their study revealed a catastrophic downregulation
chondrocytes.[109] However, there are reports that suggests that of chondrogenic biomarker expressions with an increase in Ag-
TGF-𝛽 can also function via the phosphorylation of ALK-1 recep- grecanase (ADMATS5) production in the Wnt-activator group
tor activating Smad1/5/8 signaling pathway independent of ALK- whereas enhanced levels of cartilage-specific gene expression was
5 activation.[110] The RUNX2-Smad1 complex formed, highly es- observed when the signaling pathway was inhibited by PKF118-
calates the function of the RUNX2 protein thereby promoting 310.[123] A similar line of results have been demonstrated by sev-
the hypertrophic maturation of the chondrocytes toward an os- eral other research groups using different variants of Wnt pro-
teogenic lineage.[111] The TGF-𝛽 also activates the hypertrophic teins (over expression of Wnt8c and Wnt9c in chondrocytes[124]
marker expression, RUNX2 in mesenchymal cells by initiating or Wnt3a sequestration in human cartilage[125] ) and Wnt activa-
the non-canonical MAPK signaling pathway with JunB as the up- tors (Melatonin)[126] underlying the ambivalent role of Wnt path-
stream signaling target.[108] These findings clearly augment the way in chondrogenesis. Therefore, we can suspect that the pro-
findings by Chawla et al,[19] Futrega et al.,[112] Narcisi et al.[113] and chondrogenic Wnt signaling pathway under chondrogenic stim-
Pelttari et al.[114] where hypertrophic differentiation of chondro- ulus may transition to its hypertrophy-inducing role either due to
genically differentiated stem cells have been observed. Thus, the their dysregulated ligand-mediated activation mechanism or as a
unwanted activation of Smad1/5/8 and MAPK dependent TGF-𝛽 part of the mechanistic crosstalk with several other hypertrophic

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maturation signaling cascades (BMP and IHH) as evident from A possible mechanism for this pro-apoptotic role of HIF-1𝛼
the existing reports.[127,128] can be through the post translational modification of the se-
creted collagen by the differentiating chondrocytes as elucidated
by Yao et al.[138] Hypoxia also stimulated the production of IL-
6.2.4. Other Chondrogenic Mediators and Activated Hypertrophic 1𝛽 thereby creating an inflammatory niche further aggravating
Signaling Cascades the condition of the already degenerated cartilage matrix during
hypertrophy.[139]
Insulin and ascorbic acid are two vital media supplements dur- HIF-2𝛼, a family member of hypoxia-related proteins has
ing in vitro chondrogenic differentiation assisting in chondrocyte prominently depicted the pro-hypertrophic role during in vitro
proliferation and collagen synthesis respectively.[129,130] However, chondrogenesis by escalating the transcription of COL-X, MMP-
both these biochemical mediators induce profound hypertrophic 13, and VEGF.[140] The in vivo analysis by Yang et al. also
differentiation following the endochondral ossification route fol- corroborated the deleterious activity of HIF-2𝛼 in promoting
lowing the activation of ERK 1/2 mitogenic signal transduction transient cartilage differentiation when administered via aden-
pathway.[129,131] Enhancing the transcription process of the down- ovirus or overexpression of HIF-2𝛼 gene.[141] Moreover, a fasci-
stream effectors of ERK signaling cascade (COL-X, RUNX2, ALP, nating observation by Inoue et al. demonstrated that the hydro-
and MMP-13) makes these signaling molecules a positive regu- static pressure enhanced the expression of HIF-2𝛼 gene promot-
lator of chondrocyte hypertrophy.[108] Platelet rich plasma (PRP), ing cartilage degeneration, an eminent marker of chondrocyte
a commonly used chondrogenic inducer of BMSCs also demon- hypertrophy.[142] The presence of such hydrostatic pressure cor-
strated an upregulated expression of hypertrophy marker COL- responds to high water content (≈70%) in articular cartilage tis-
I.[132] A strong correlation between BMP and IHH signaling sue that acts as a negative stimulus to chondrocytes causing fur-
targets has been elucidated by Minina et al. where the expres- ther matrix degradation and apoptosis.[143] Scientists have also
sion levels of IHH has been tightly controlled by BMP protein described the derogatory function of hypoxia in chondrocyte hy-
suggesting a synergistic relationship in stimulating chondrocyte pertrophy by accelerating the production of pro-inflammatory
hypertrophy.[133] An increased expression of IHH in OA chon- molecules prostaglandin E-2 and cyclooxygenase-2, creating a
drocytes have been associated with enhanced expression level of suitable niche for OA development in the hypertrophic chondro-
hypertrophic biomarkers (MMP-13, COL-X) as elucidated by Wei cytes. Thus, we can presume that the so-called ‘ideal hypoxic en-
et al.[134] vironment for chondrogenesis can be manoeuvred during chon-
The embryonic articular cartilage development is ultimately drogenic differentiation leading to an undesirable hypertrophic
followed by an endochondral ossification route to finally undergo phenotype. Such manipulation can be either due to the endoge-
hypertrophic differentiation, a distinct strategy of bone devel- nous switch from pro-chondrogenic HIF-1𝛼 to pro-hypertrophic
opment besides via intramembranous ossification. Thus, dur- HIF-2𝛼 under hypoxic condition or are a result of the crosstalk be-
ing in vitro chondrogenic differentiation under the influence tween different dysregulated activation/suppression of chondro-
of the pro-chondrogenic signaling morphogens, the stem cells genic signaling molecules (described in the next section) supple-
display a pro-hypertrophic phenotype despite adequate chondro- mented in media in vitro. Furthermore, HIF-1𝛼 and HIF-2𝛼 pro-
genesis demonstrating their innate tendency following the em- motes the process of neo-vascularization or vascular sprouting
bryonic cartilage development pathway.[135] Furthermore, we can in the cartilage matrix from the underlying subchondral bone,
postulate that under the influence of chondrogenic media sup- a hallmark of transdifferentiated chondrocytes. Thus, the pro-
plements during the entire period of in vitro chondrogenesis, angiogenic property of these hypoxia-induced transcription fac-
stem cells display an ectopic dysfunctioning of several chondro- tors equally contributes to the hypertrophic maturation of the
genic signal transduction pathways. Such dysregulation can be chondrocytes when cultured in vitro (Figure 4)
attributed to the time of exposure and concentration of the bio-
chemical mediators deployed during the chondrogenic differenti-
ation phase. These aberrant signaling cascades enable the perma- 6.4. The Imbalanced Mechanical Loading During in vitro
nent cartilage to shed off its native stable phenotype and acquire Chondrogenesis
an osteogenic phenotype, an unanticipated outcome of chondro-
genic differentiation (Figure 4). Native articular cartilage of the knee joint is exposed to a constant
mechanical loading that plays a vital role in the physiological
homeostasis of the tissue. This mechanical loading comprises
6.3. The Ambiguous Role of Hypoxia Generated Factors of shear forces (parallel to the articulating surface), compression
forces (constant human locomotion) and hydrostatic pressure
Low oxygen tension or hypoxia is considered as an ideal cul- (synovial fluid) that renders the mechano-active nature of the
ture condition for chondrogenic differentiation in vitro. How- cartilage tissue. Thus, mimicking the native biomechanical con-
ever, studies have also addressed the cynical role of hypoxia dition during in vitro articular cartilage development is essential
during chondrogenesis with an upregulated expression of cru- to establish a stable hyaline cartilage phenotype. Investigation by
cial hypertrophic marker COL-X when BMSCs were cultured Nazempour et al. illustrated the chondro-stimulant role of com-
in Hyaluronic acid and collagen scaffolds.[136] HIF-1𝛼 which is bined hydrostatic pressure and shear forces in enhancing the
chondroprotective in nature also reported to promote autophagy secretion of GAG and collagen under in vitro conditions with fur-
and chondrocyte apoptosis by regulating the functioning of au- ther chondroprotective effects marked by decreased expression
tophagy and apoptosis-related proteins Bcl-2 and Caspase-8.[137] of COL-X.[144] Frank et al. also described the utility of sinusoidal

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Figure 4. The dysfunctioning of the pro-chondrogenic signaling pathways and their mechanistic crosstalk ensued from the vast array of chondrogenic
media supplements and the surrounding in vitro biomimetic microenvironment.

macroscopic shear deformation in elevating the proteoglycan have a neutral influence on COL-II mRNA expression.[153] Addi-
synthesis in the cartilage ECM.[145] A similar line of reports tionally, Pigeot et al also demonstrated the advantage of perfusion
envisaging the functional importance of biomechanical loading bioreactor-based dynamic culture system in promoting concomi-
in increasing the ECM deposition as well as enhancing the me- tant ECM deposition in genetically modified BMSCs.[154] Thus,
chanical stiffness of the engineered cartilage construct have been the differential behavior of static and dynamic loading toward
published by Kisiday et al.,[146] Jin et al.,[147] and Lee et al.[148] chondrogenesis can be attributed to a higher rate of matrix defor-
However, Ochetta et al. have demonstrated that a 30% confined mation, increased water exudation followed by a change in colla-
mechanical compression was sufficient enough to establish hy- gen fiber orientation of the cartilage ECM under static loading
pertrophic maturation in cultured human chondrocytes in a OA- conditions inhibiting chondrogenic maturation whereas a negli-
in-chip model.[149] Ge et al. also showed that a dynamic compres- gible matrix deformation and water exudation allow stable chon-
sion at an early time point significantly downregulated the chon- drogenesis in dynamic loading conditions.[155]
drogenic marker expressions compared to their static control.[150] The pro-hypertrophic role of hypertensile loading and hydro-
A comparative analysis of cyclic dynamic loading versus static static pressure on human and animal cartilage samples has been
loading on the viability of chondrocytes within the bovine carti- extensively studied by Chang et al.[156] Results from their in-
lage explants revealed enhanced cell death within 3 h of repetitive vestigations revealed the role of Gremlin mediated activation of
static loading condition, whereas the cell death was delayed to 6 NF-𝜅B signaling pathway that intensified the production of ma-
hours in dynamic cyclic loading.[151] Moreover, dynamic loading trix degrading enzymes (ADAMTS5 and MMP-13), a key hall-
contributed to a 20% enhancement in GAG synthesis compared mark feature of terminally differentiated chondrocytes . A de-
to an 84 kPa of static loading condition.[152] Furthermore, stud- tailed analysis of the underlying signaling cascades behind such
ies have clearly shown that dynamic compression positively reg- ambiguous function of mechanical loading in chondrogenesis
ulated the GAG metabolism in human articular chondrocytes but revealed the mechanistic crosstalk between a constellation of

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Table 2. Key signaling pathway inhibitors and their role in mitigating chondrocyte hypertrophy.

Serial no. Small molecule Targeted signaling pathway Inhibition mechanism Reference

1 Smurf 1/2 Bone morphogenetic protein (BMP) Degradation of Smad 1 and Smad 5 proteins [182,183]
2 Smad 6/7 Bone morphogenetic protein (BMP) Formation of heterodimers with Smad 4 via the [184]
MH2 domain
3 Noggin Bone morphogenetic protein (BMP) Blocking the ligand binding sites of both type-1 and [161,39]
type-2 BMP receptor
4 Chordin Bone morphogenetic protein (BMP) Competitive binding with BMP ligand for the type-I [185,186]
BMP receptor and inhibit BMP mediated ALP
activity
5 Dorsomorphin Bone morphogenetic protein (BMP) Inhibiting the phosphorylation of Smad 1/5/8 [187,188]
proteins
6 LDN193189 Bone morphogenetic protein (BMP) Inhibiting the phosphorylation of both Smad (Smad [61]
1/5/8) and non-Smad proteins (p38)
7 Matrilin-3 Bone morphogenetic protein (BMP) Interaction with the BMP-2 ligand and mitigating [189]
the Smad-1 activity
8 PKF118-130 Wnt/𝛽-catenin Preventing the translocation of 𝛽-catenin to the [123]
nucleus.
9 Rofecoxib Wnt/𝛽-catenin Blocking the production of Cox-2 [190]
10 XAV-939 Wnt/𝛽-catenin Phosphorylation mediated degradation of 𝛽-catenin [191]
through stabilization of AXIN2
11 Pyrvinium/C113 Wnt/𝛽-catenin Degradation of 𝛽-catenin through activation of [192]
casein kinase 1𝛼
12 SM04690 Wnt/𝛽-catenin Inhibition of protease mediated GAG degradation [164]
and secretion of inflammatory cytokines and NO.
13 Peptide-based inhibitor Wnt/𝛽-catenin Suppressing the Wnt mediated transcriptional [193]
activity by binding to the TCF docking site of
𝛽-catenin.
14 SAH-Bcl9/StAx-35R Wnt/𝛽-catenin Preventing the TCF/LEF promoter activity induced [194]
by Wnt 3a.
15 Dkk-1 Wnt/𝛽-catenin Inhibiting the translocation of 𝛽-catenin to the [195]
nucleus by internalization of LRP5/LRP6 complex
16 Taladegib Indian Hedgehog (IHH) Inhibiting the activation of Smoothened protein [196]
(Smo)
17 Curcumin Indian Hedgehog (IHH) and Notch Suppressing the expression of Gli2, NICD and Hey [197]
proteins
18 G141 Fibroblast Growth Factor (FGF) Competitively blocks the activation of FGF receptor [167]
through interaction with its non-ATP binding
pockets
19 Bevacizumab Vascular Endothelial Growth Factor Blocking the initiation of angiogenesis during [198]
(VEGF) hypertrophic maturation
20 Gefitinib Epidermal Growth Factor (EGF) Inhibiting the phosphorylation mediated activation [168]
of EGFR
21 Xanthotoxin p38 Mitogen Activated Protein Kinase Downregulating the phosphorylation of p38 and [199]
(MAPK) /Histone deacetylase 4 elevating the expression of HDAC4
(HDAC4)
22 Asiatic acid AMP-activated protein kinase (AMPK) Phosphorylation mediated activation of AMPK with [200]
/Phosphoinositide-3 kinase/Protein simultaneous decrease in phosphorylation level
kinase B (PI3K/AKT) of PI3K and AKT
23 U0126 Mitogen activated protein kinase kinase Inhibiting the activation of ERK 1/2 by supressing [201]
(MAPKK) /extracellular signal regulated the kinase activity of MEK.
kinase (ERK)
24 Cordycepin Phosphoinositide-3 kinase (PI3K)/Bapx1 Downregulating the expression of PI3K [202]
and Notch (hypertrophy suppression) and inhibiting nrf2
expression (chondrogenesis stimulation)
25 Kartogenin c-Jun N-terminal kinase (JNK)/ 𝛽-catenin Increasing the phosphorylation level of JNK while [203]
suppressing the 𝛽-catenin mediated transcription
activity.

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Figure 5. A comparison between the conventional cartilage tissue engineering approach and our adopted advanced strategy to achieve stable articular
cartilage phenotype post-implantation. The generic approach involves culturing the isolated BMSCs in 2D followed by mixing with the desired biomaterial,
allowing chondrogenic differentiation in the 3D scaffold and implanting them or direct injection of the BMSC-loaded hydrogel into the defect region.
However, challenges of bony tissue development post-implantation stand as a significant roadblock due to the hypertrophic differentiation of the BMSCs
either during in vitro chondrogenesis or post in vivo implantation in OA microenvironment. To mitigate the existing challenge, we have devised a
novel advanced strategy of targeting the signaling pathway responsible for this hypertrophic phenotype by using small molecule inhibitors/agonists
conjugated with our biomaterial. This will enable stable articular cartilage phenotype formation post-implantation by inhibiting the signaling pathway(s),
accelerating hypertrophy, and stimulating chondrogenesis. The question that still emerges is the optimum dose, exposure time and time of addition of
these small molecule mediators required to achieve this stable chondrogenic fate? Furthermore, whether inhibiting the hypertrophic maturation in vitro
can successfully mitigate the hypertrophy-related phenotypic drift under in vivo OA conditions remains an elusive topic to research further.

hypertrophy stimulating inflammatory and non-inflammatory 4 protein with the SMAD-1/5/8 BMP downstream target as evi-
signaling pathways like TNF-𝛼, IL-1𝛽, NF-𝜅B, Wnt, and TGF- dent from the downregulated the expression of hypertrophy in-
𝛽.[157] Thus, it can be rightly hypothesized that an improper bal- ducing genes IHH, COL-X and MMP-13 when incubated with an
ance in degree of application of mechanical loading as well as OA microcartilage model (Figure 6).[51,159] Several other natural
the time and point of application could possibly shift the carti- (Chordin,[160] Noggin[39,161] ) and synthetic (K02288[162,163] ) BMP
lage homeostasis paradigm from pro-chondrogenic to hypertro- inhibitors have also demonstrated impeding expression pattern
phy stimulating form, leading to the formation of a transient car- of IL-1𝛽 and BMP-2 protein with redundant ALP activity through
tilage phenotype or bone (Figure 4). competitive inhibitory binding mechanism with the BMP lig-
ands. Similarly, PKF118-310[123] and SM04690[164] targets the pro-
hypertrophic version of Wnt signaling pathway by mitigating the
7. Measures Adopted to Halt Chondrocyte expression of hypertrophy inducing targets of Wnt pathway be-
Hypertrophy: Should we Completely Block or sides promoting the expression of chondrogenic marker genes
Apply a Brake? (COL-II, ACAN, and SOX-9). The inhibition of IHH pathway
by Ipriflavone[165] and Cyclopamine[76,134] has been highly effec-
Small molecule regulators of pro-hypertrophic signaling path- tive in abrogating hypertrophic differentiation by reduced expres-
ways: A wide array of small molecules has been deployed by re- sion profile of IHH pathway associated genes SMO and Gli2 be-
searchers and clinicians worldwide to suppress the hypertrophic sides the redundant expression of osteogenic marker RUNX2,
maturation of chondrocytes with a common objective to develop MMP-13 and COL-X. A variety of other inhibitors have been re-
a phenotypically stable articular cartilage while supressing chon- ported like Bevacizumab[166] as an anti-VEGF antibody, G141[167]
drocyte hypertrophy.[158] One potent inhibitor of BMP signaling as a suppressor of Fibroblast growth factor (FGF) pathway and
pathway is LDN193189 that arrests the colocalization of SMAD- Gefitinib[168] as an inhibitor to Epidermal growth factor receptor

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Figure 6. Immunostaining of the developed OA microcartilage model using BMP-2 induction of human articular chondrocytes. The enhanced expres-
sion of chondrocyte hypertrophic marker MMP-13 and COL-X have been substantially reduced with the treatment of BMP signaling pathway inhibitor
LDN193189 and Compound A (developed by Novartis). Reproduced with permission.[51] Copyright 2020, The Company of Biologists.

(EGFR), significantly downregulated the overexpression of ma- ality in chondrogenesis where HIF-1𝛼 promotes chondrogenic
trix degradation components (MMP-1/13) along with enhanced differentiation and HIF-2𝛼 elevates hypertrophic maturation of
collagen and GAG synthesis in vitro details of which are included chondrocytes. The canonical Wnt signaling pathway is one of the
in Table 2. But the major challenge lies in the limited half-lives key regulators in the transition from a healthy chondrocyte to a
of such small molecules, for example, LDN193189 has a half- hypertrophic chondrocyte phenotype. In an animal model, the
life of 1.5 hours. Spatial tethering and controlled release strategy expression of Wnt 5a in was observed in the early chick perichon-
of such small molecules through polymeric biomaterial would drium whereas the same Wnt ligand further inhibited the hyper-
enhance their half-life, cell specificity, bioadhesivity, and active trophic transition.[121,171] Hence, instead of a long-term exposure
transportation to cellular receptors.[169] (Figure 5) to a particular biochemical mediator, an in vitro acceleration or
However, the critical question to raise is whether to com- retardation of this transition would result in direct stimulation
pletely inhibit these signaling cascades or draw a balance through or inhibition of endochondral ossification. Similarly, the unani-
their time-dependent dosage? Reports from different research mous Wnt14 expression both as the molecular marker for earliest
groups have shown the pro-chondrogenic activity of TGF-𝛽 in inducer of joint formation as well as in the MSCs of the cartilage
regulating the entire process of cartilage development right from tissue necessitates the development of stable cartilage tissue phe-
stimulating mesenchymal condensation to chondrogenic differ- notype simulating the in vivo anatomy and physiology.[172]
entiation and further inhibiting transient cartilage differentia- Due to the enigmatic role of these signaling pathways in regu-
tion. However, research from our group demonstrated a 21- lating stable chondrogenesis, a complete inhibition might com-
day exposure of TGF-𝛽1 to the BMSCs triggered hypertrophic promise the development of chondrogenic phenotype during the
differentiation.[112,170] A similar paradoxical relationship of BMP process of chondrogenic differentiation. Thus, drawing a brake
with chondrogenesis have been illustrated where BMP protein by applying stringent control over the exposure time and concen-
significantly enhanced the intracellular communication through tration of these signaling molecules can create a balance in up-
N-cadherin expression in one hand and the overexpression of regulation and downregulation of respective signaling cascades
chondrocyte hypertrophic marker genes (RUNX2, COL-I, COL-X, and thereby allow the development of stable articular cartilage
and ALP).[24,51] Hypoxia is also known to exhibit a dual function- phenotype.

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8. Conclusion
[1] M. Pacifici, E. Koyama, M. Iwamoto, C. Gentili, Connect. Tissue Res.
The current review focused on comprehending the underlying 2000, 41, 175.
mechanism behind the occurrence of chondrocyte hypertrophy [2] F. Eckstein, M. Hudelmaier, R. Putz, J Anat 2006, 208, 491.
during the expansion and in vitro chondrogenesis of stem cells [3] L. K. King, L. March, A. Anandacoomarasamy, Indian J Med Res 2013,
(BMSCs, ADSCs) and primary chondrocytes. We formulated four 138, 185.
hypotheses to be the major causative reasons: a) the acquisition of [4] S. Nukala, S. R. Puvvada, E. Luvsannyam, D. Patel, P. Hamid, Cureus
de-differentiated phenotype by primary chondrocytes during ex- 2021, 13, e15999.
pansion as one of the contributors to hypertrophy phenotype, b) [5] D. T. Felson, M. C. Nevitt, Curr. Opin. Rheumatol. 1998, 10, 269.
the intrinsic property of stem cells to undergo hypertrophic mat- [6] H. Tilg, A. R. Moschen, Nat. Rev. Immunol. 2006, 6, 772.
uration under the influence of chondrogenic stimulants through [7] J. A. Roman-Blas, S. Castañeda, R. Largo, G. Herrero-Beaumont,
Arthritis Res. Ther. 2009, 11, 241.
the dysregulated activation of chondrogenic signaling pathways,
[8] D. J. Hunter, D. T. Felson, BMJ 2006, 332, 639.
c) the aberrant role of hypoxia, and d) the imbalance in mechan- [9] W. Hermann, S. Lambova, U. Muller-Ladner, Current Treatment Op-
ical loading conditions during in vitro chondrogenesis leading tions for Osteoarthritis 2018, 14, 108.
to the formation of a transient cartilage over a permanent one. [10] D. Webb, P. Naidoo, Orthop Res Rev 2018, 10, 73.
Literature-based evidence have been provided for each hypoth- [11] A. A. Abdel-Aziem, E. S. Soliman, D. M. Mosaad, A. H. Draz, J Phys
esis to elucidate the redundancy and their significant outcome Ther Sci 2018, 30, 307.
in causing hypertrophic maturation following chondrogenesis in [12] K. Rönn, N. Reischl, E. Gautier, M. Jacobi, Arthritis 2011, 2011,
vitro. But it is still a matter of comprehensive research whether 454873.
chondrocyte hypertrophy is an active driver of OA or a passive [13] J. N. Katz, B. E. Earp, A. H. Gomoll, Arthritis Care Res. (Hoboken).
consequence of OA. 2010, 62, 1220.
[14] S. Chameettachal, S. Midha, S. Ghosh, ACS Biomater. Sci. Eng. 2016,
Finally, while describing the different strategies in minimiz-
2, 1450.
ing chondrocyte hypertrophy, a question was raised as to whether [15] Y. Shen, Y. Xu, B. Yi, X. Wang, H. Tang, C. Chen, Y. Zhang, Biomacro-
completely inhibit the pathways leading to hypertrophy or apply a molecules 2021, 22, 2284.
strict checkpoint to ensure a proper balance between chondroge- [16] X. Ren, F. Wang, C. Chen, X. Gong, L. Yin, L. Yang, BMC Muscu-
nesis and hypertrophy. Such balance will successfully allow stable loskelet Disord 2016, 17, 301.
chondrogenesis without interfering in the embryonic endochon- [17] M. Lafuente-Merchan, S. Ruiz-Alonso, A. Espona-Noguera, P.
dral ossification route. The transient cartilage differentiation in Galvez-Martin, E. López-Ruiz, J. A. Marchal, M. L. López-Donaire,
the engineered or implanted cartilage would result in a mod- A. Zabala, J. Ciriza, L. Saenz-Del-Burgo, J. L. Pedraz, Mater. Sci. Eng.
ified tissue microenvironment (significantly different from na- C. Mater. Biol. Appl. 2021, 126, 112160.
tive articular cartilage), which blocks phenotypically stable artic- [18] X. Zhao, A. Papadopoulos, S. Ibusuki, D. A. Bichara, D. B. Saris, J.
Malda, K. S. Anseth, T. J. Gill, M. A. Randolph, BMC Musculoskelet
ular cartilage differentiation. In order to neutralize this transient
Disord 2016, 17, 245.
cartilage phenotype, a comprehensive tissue regeneration proto- [19] S. Chawla, A. Kumar, P. Admane, A. Bandyopadhyay, S. Ghosh, Int.
col/approach should be devised augmenting controlled release J. Bioprint. 2017, 7, 1.
of small molecules (e.g., zone-specific Wnt inducers or BMP in- [20] P. Van Pham, K. H.-T. Bui, D. Q. Ngo, N. B. Vu, N. H. Truong, N.
hibitors) from 3D Bioprinted cartilage construct to achieve simi- L.-C. Phan, D. M. Le, T. D. Duong, T. D. Nguyen, V. T. Le, N. K. Phan,
lar differentiation program akin native articular cartilage during Stem Cell Res Ther 2013, 4, 91.
embryonic development. The resultant phenotypically stable tis- [21] M. Bhattacharjee, S. Chameettachal, S. Pahwa, A. R. Ray, S. Ghosh,
sue engineered articular cartilage would circumvent all the prob- ACS Appl. Mater. Interfaces 2014, 6, 183.
lems associated to non-optimal consistency and can be used as a [22] D. Nguyen, D. A. Hägg, A. Forsman, J. Ekholm, P. Nimkingratana,
regenerative therapeutic strategy for the prevailing osteoarthritic C. Brantsing, T. Kalogeropoulos, S. Zaunz, S. Concaro, M. Brittberg,
A. Lindahl, P. Gatenholm, A. Enejder, S. Simonsson, Sci. Rep. 2017,
patients by the clinicians worldwide.
7, 658.
[23] K. Oka, S. Oka, T. Sasaki, Y. Ito, P. J. Bringas, K. Nonaka, Y. Chai, Dev
Acknowledgements Biol 2007, 303, 391.
The authors are thankful for funding from Department of Biotechnology, [24] N. Zhou, Q. Li, X. Lin, N. Hu, J.-Y. Liao, L.-B. Lin, C. Zhao, Z.-M.
India-Swiss project (BT/IN/Swiss/54/SG/2018-19), Government of India. Hu, X. Liang, W. Xu, H. Chen, W. Huang, Cell Tissue Res. 2016, 366,
101.
[25] B. Appel, J. Baumer, D. Eyrich, H. Sarhan, S. Toso, C. Englert, D.
Conflict of Interest Skodacek, S. Ratzinger, S. Grässel, A. Goepferich, T. Blunk, Os-
teoarthr. Cartil. 2009, 17, 1503.
The authors declare no conflict of interest.
[26] L. Pesesse, C. Sanchez, J.-P. Delcour, A. Bellahcène, C. Baudouin, P.
Msika, Y. Henrotin, Osteoarthr. Cartil. 2013, 21, 1913.
Keywords [27] M. R. Coe, T. A. Summers, S. J. Parsons, A. L. Boskey, G. Balian,
Bone Miner. 1992, 18, 91.
chondrocyte hypertrophy, chondrogenesis, de-differentiation, permanent [28] M. Y. Janners, R. L. Searls, Dev Biol 1970, 23, 136.
versus transient cartilage, stem cells [29] B. Zimmermann, J Anat 1984, 138, 351.
[30] T. Biswas, A. P. Jaswal, U. S. Yadav, A. Bandyopadhyay, Int. J. Dev.
Received: January 17, 2023 Biol. 2020, 64, 203.
Revised: March 13, 2023 [31] X. Guo, T. F. Day, X. Jiang, L. Garrett-Beal, L. Topol, Y. Yang, Genes
Published online: May 21, 2023 Dev. 2004, 18, 2404.

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[32] M. Iwamoto, Y. Higuchi, E. Koyama, M. Enomoto-Iwamoto, K. [58] B. A. G. De Melo, Y. A. Jodat, S. Mehrotra, M. A. Calabrese, T.
Kurisu, H. Yeh, W. R. Abrams, J. Rosenbloom, M. Pacifici, J. Cell Biol. Kamperman, B. B. Mandal, M. H. A. Santana, E. Alsberg, J. Leijten,
2000, 150, 27. 2019, 1906330, 1.
[33] M. Iwamoto, Y. Higuchi, M. Enomoto-Iwamoto, K. Kurisu, E. [59] L. De Moor, S. Fernandez, C. Vercruysse, L. Tytgat, M. Asadian, N.
Koyama, H. Yeh, J. Rosenbloom, M. Pacifici, Osteoarthr. Cartil. 2001, De Geyter, S. Van Vlierberghe, P. Dubruel, H. Declercq, Front Bioeng
9, S41. Biotechnol 2020, 8, 484.
[34] A. Kan, C. J. Tabin, Genes Dev. 2013, 27, 514. [60] A. Scalzone, A. M. Ferreira, C. Tonda-Turo, G. Ciardelli, K. Dalgarno,
[35] P. N. P. Singh, U. S. Yadav, K. Azad, P. Goswami, V. Kinare, A. P. Gentile, Sci. Rep. 2019, 9, 14630.
Bandyopadhyay, Development 2018, 145, dev156554. [61] R. A. G. Franco, E. McKenna, P. G. Robey, M. S. Shajib, R. W.
[36] B. Lanske, A. C. Karaplis, K. Lee, A. Luz, A. Vortkamp, A. Pirro, M. Crawford, M. R. Doran, K. Futrega, Stem Cell Rep. 2022, 17, 616.
Karperien, L. H. Defize, C. Ho, R. C. Mulligan, A. B. Abou-Samra, H. [62] A. Dell’Isola, R. Allan, S. L. Smith, S. S. P. Marreiros, M. Steultjens,
Jüppner, G. V Segre, H. M. Kronenberg, Science 1996, 273, 663. BMC Musculoskelet Disord 2016, 17, 425.
[37] M. Dentice, A. Bandyopadhyay, B. Gereben, I. Callebaut, M. A. [63] A. Mobasheri, W. E. van Spil, E. Budd, I. Uzieliene, E. Bernotiene,
Christoffolete, B. W. Kim, S. Nissim, J.-P. Mornon, A. M. Zavacki, A.-C. Bay-Jensen, J. Larkin, M. C. Levesque, O. Gualillo, Y. Henrotin,
A. Zeöld, L. P. Capelo, C. Curcio-Morelli, R. Ribeiro, J. W. Harney, C. Curr. Opin. Rheumatol. 2019, 31, 80.
J. Tabin, A. C. Bianco, Nat. Cell Biol. 2005, 7, 698. [64] W. E. Van Spil, O. Kubassova, M. Boesen, A.-C. Bay-Jensen, A.
[38] A. Vortkamp, K. Lee, B. Lanske, G. V Segre, H. M. Kronenberg, C. J. Mobasheri, Biochem. Pharmacol. 2019, 165, 41.
Tabin, Science 1996, 273, 613. [65] S. P. Grogan, S. F. Duffy, C. Pauli, J. A. Koziol, A. I. Su, D. D. D’Lima,
[39] J. Groppe, J. Greenwald, E. Wiater, J. Rodriguez-Leon, A. N. M. K. Lotz, Arthritis Rheum. 2013, 65, 418.
Economides, W. Kwiatkowski, K. Baban, M. Affolter, W. W. Vale, J. [66] S. P. Grogan, A. Barbero, J. Diaz-Romero, A.-M. Cleton-Jansen, S.
C. Izpisua Belmonte, S. Choe, J. Bone Joint Surg. Am. 2003, 85, 52. Soeder, R. Whiteside, P. C. W. Hogendoorn, J. Farhadi, T. Aigner, I.
[40] B. M. Abdallah, F. Figeac, K. H. Larsen, N. Ditzel, P. Keshari, A. Isa, Martin, P. Mainil-Varlet, Arthritis Rheum. 2007, 56, 586.
A. Jafari, T. L. Andersen, J.-M. Delaisse, Y. Goshima, T. Ohshima, M. [67] M. A. Asnaghi, L. Power, A. Barbero, M. Haug, R. Köppl, D. Wendt,
Kassem, J. bone Miner. Res. Off. J. Am. Soc. Bone Miner. Res. 2017, I. Martin, Front Bioeng Biotechnol 2020, 8, 283.
32, 913. [68] T. Xiao, W. Guo, M. Chen, C. Hao, S. Gao, J. Huang, Z. Yuan, Y.
[41] R. Kelwick, I. Desanlis, G. N. Wheeler, D. R. Edwards, Genome Biol. Zhang, M. Wang, P. Li, J. Peng, A. Wang, Y. Wang, X. Sui, L. Zhang,
2015, 16, 113. W. Xu, S. Lu, H. Yin, J. Yang, S. Liu, Q. Guo, Biomed Res. Int. 2017,
[42] A. Ray, P. N. P. Singh, M. L. Sohaskey, R. M. Harland, A. 2017.
Bandyopadhyay, Development 2015, 142, 1169. [69] Y. P. Singh, N. Bhardwaj, B. B. Mandal, ACS Appl. Mater. Interfaces
[43] M. Bhattacharjee, J. Coburn, M. Centola, S. Murab, A. Barbero, D. 2016, 8, 21236.
L. Kaplan, I. Martin, S. Ghosh, Adv. Drug Delivery Rev. 2015, 84, 107. [70] A. Alquraini, M. Jamal, L. Zhang, T. Schmidt, G. D. Jay, K. A. Elsaid,
[44] M. Pacifici, E. Koyama, M. Iwamoto, Birth Defects Res. Part C Embryo Arthritis Res. Ther. 2017, 19, 89.
Today Rev. 2005, 75, 237. [71] V. Lefebvre, P. Bhattaram, Prg4-expressing cells: articular stem cells
[45] M. M. J. Caron, P. J. Emans, M. M. E. Coolsen, L. Voss, D. A. M. or differentiated progeny in the articular chondrocyte lineage?, Vol.
Surtel, A. Cremers, L. W. van Rhijn, T. J. M. Welting, Osteoarthr. Cartil. 67, 2015, pp. 1151–1154.
2012, 20, 1170. [72] L. Wu, F. A. Petrigliano, K. Ba, S. Lee, J. Bogdanov, D. R. McAllister,
[46] H. H. Yoon, S. H. Bhang, J.-Y. Shin, J. Shin, B.-S. Kim, Tissue Eng., J. S. Adams, A. K. Rosenthal, B. Van Handel, G. M. Crooks, Y. Lin, D.
Part A 2012, 18, 1949. Evseenko, Osteoarthr. Cartil. 2015, 23, 308.
[47] T. Vy, H. Le, X. To, P. Nguyen, P. Huynh, T. Le, N. Vu, Biomed. Res. [73] K. Thirunavukkarasu, C. A. Swearingen, J. L. Oskins, C. Lin, H. H.
Ther. 2020, 7, 3697. Bui, S. B. Jones, L. A. Pfeifer, B. H. Norman, P. G. Mitchell, M. G.
[48] K. Tekhnologii, A. V Tsvetkova, I. V Vakhrushev, Y. B. Basok, A. M. Chambers, Osteoarthr. Cartil. 2017, 25, 935.
Grigor, L. A. Kirsanova, A. Y. Lupatov, V. I. Sevastianov, K. N. Yarygin, [74] M. M. Hickey, M. C. Simon, Curr. Top. Dev. Biol. 2006, 76, 217.
2021, 528. [75] E. Schipani, Semin Cell Dev Biol 2005, 16, 539.
[49] M. C. Stewart, K. M. Saunders, N. Burton-Wurster, J. N. Macleod, J. [76] K. K. Mak, H. M. Kronenberg, P.-T. Chuang, S. Mackem, Y. Yang,
bone Miner. Res. Off. J. Am. Soc. Bone Miner. Res. 2000, 15, 166. Development 2008, 135, 1947.
[50] L. Zhang, P. Su, C. Xu, J. Yang, W. Yu, D. Huang, Biotechnol. Lett. [77] P. G. Bush, C. A. Parisinos, A. C. Hall, J. Cell. Physiol. 2008, 214, 621.
2010, 32, 1339. [78] E. J. Mackie, L. Tatarczuch, M. Mirams, J. Endocrinol. 2011, 211, 109.
[51] S. Chawla, M. H. M. Berkelaar, B. Dasen, C. Halleux, S. Guth- [79] F. Wang, Y. Zhu, J Orthop Sci 2011, 16, 304.
Gundel, I. Kramer, S. Ghosh, I. Martin, A. Barbero, P. Occhetta, J. [80] M. A. Arnold, Y. Kim, M. P. Czubryt, D. Phan, J. McAnally, X. Qi, J.
Cell Sci. 2020, 133, jcs249094. M. Shelton, J. A. Richardson, R. Bassel-Duby, E. N. Olson, Dev. Cell
[52] R. Seda Tıǧlı, S. Ghosh, M. M. Laha, N. K. Shevde, L. Daheron, J. 2007, 12, 377.
Gimble, M. Gümüşderelioǧlu, D. L. Kaplan, J. Tissue Eng. Regener. [81] C. A. Yoshida, H. Yamamoto, T. Fujita, T. Furuichi, K. Ito, K. Inoue,
Med. 2009, 3, 348. K. Yamana, A. Zanma, K. Takada, Y. Ito, Genes Dev. 2004, 18, 952.
[53] P. M. van der Kraan, W. B. van den Berg, Osteoarthr. Cartil. 2012, 20, [82] D. Studer, C. Millan, E. Ozturk, K. Maniura-Weber, M. Zenobi-Wong,
223. Eur Cell Mater 2012, 24, 118.
[54] S. Chawla, G. Desando, E. Gabusi, A. Sharma, D. Trucco, J. [83] M. B. Mueller, R. S. Tuan, Arthritis Rheum. 2008, 58, 1377.
Chakraborty, C. Manferdini, M. Petretta, G. Lisignoli, S. Ghosh, J. [84] S. Chen, P. Fu, R. Cong, H. Wu, M. Pei, Genes Dis. 2015, 2, 76.
Mater. Res. 2021, 1. [85] J. P. Iannotti, S. Naidu, Y. Noguchi, R. M. Hunt, C. T. Brighton, Clin.
[55] K. Markstedt, A. Mantas, I. Tournier, H. Martínez Ávila, D. Hägg, P. Orthop. Relat. Res. 1994, 222.
Gatenholm, Biomacromolecules 2015, 16, 1489. [86] N. Ortega, D. J. Behonick, Z. Werb, Trends Cell Biol. 2004, 14, 86.
[56] X. Lan, Y. Liang, E. J. N. Erkut, M. Kunze, A. Mulet-Sierra, T. Gong, [87] N. Ortega, D. Behonick, D. Stickens, Z. Werb, Ann. N. Y. Acad. Sci.
M. Osswald, K. Ansari, H. Seikaly, Y. Boluk, A. B. Adesida, FASEB J. 2003, 995, 109.
2021, 35, e21191. [88] F. J. Blanco, R. Guitian, E. Vázquez-Martul, F. J. de Toro, F. Galdo,
[57] J. H. Galarraga, M. Y. Kwon, J. A. Burdick, Sci. Rep. 2019, 9, 19987. Arthritis Rheum. Off. J. Am. Coll. Rheumatol. 1998, 41, 284.

Adv. Funct. Mater. 2023, 33, 2300651 2300651 (17 of 20) © 2023 Wiley-VCH GmbH
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[89] C. L. Murphy, J. M. Polak, J. Cell. Physiol. 2004, 199, 451. [122] D. Später, T. P. Hill, R. J. O’sullivan, M. Gruber, D. A. Conner, C.
[90] Y. Sasazaki, B. B. Seedhom, R. Shore, Rheumatology 2008, 47, Hartmann, Development 2006, 133, 3039.
1641. [123] X. Huang, L. Zhong, J. Hendriks, J. N. Post, M. Karperien, Int. J. Mol.
[91] G. Schulze-Tanzil, A. Mobasheri, P. de Souza, T. John, M. Shakibaei, Sci. 2018, 19, 561.
Osteoarthr. Cartil. 2004, 12, 448. [124] Y.-F. Dong, D. Y. Soung, E. M. Schwarz, R. J. O’Keefe, H. Drissi, J.
[92] G. M. Hoben, K. A. Athanasiou, Cell Tissue Res. 2008, 334, 469. Cell. Physiol. 2006, 208, 77.
[93] V. Graceffa, C. Vinatier, J. Guicheux, M. Stoddart, M. Alini, D. I. [125] J. Bertrand, T. Kräft, T. Gronau, J. Sherwood, F. Rutsch, F. Lioté,
Zeugolis, Biomaterials 2019, 192, 199. F. Dell’Accio, C. H. Lohmann, M. Bollmann, A. Held, T. Pap, Ann.
[94] Z. Lin, J. B. Fitzgerald, J. Xu, C. Willers, D. Wood, A. J. Grodzinsky, Rheum. Dis. 2020, 79, 975.
M. H. Zheng, J Orthop Res 2008, 26, 1230. [126] X. Wang, T. He, L. He, B. Yang, Z. Liu, M. Pang, P. Xie, L. Zhang, L.
[95] G. Schulze-Tanzil, Ann Anat 2009, 191, 325. Rong, Stem Cell Res Ther 2021, 12, 467.
[96] C. Cournil-Henrionnet, C. Huselstein, Y. Wang, L. Galois, D. [127] I. Papathanasiou, K. N. Malizos, A. Tsezou, Arthritis Res. Ther. 2012,
Mainard, V. Decot, P. Netter, J.-F. Stoltz, S. Muller, P. Gillet, Biorhe- 14, R82.
ology 2008, 45, 513. [128] X. Guo, K. K. Mak, M. M. Taketo, Y. Yang, PLoS One 2009, 4, e6067.
[97] F. Zaucke, R. Dinser, P. Maurer, M. Paulsson, Biochem. J. 2001, 358, [129] C. Phornphutkul, K.-Y. Wu, P. A. Gruppuso, Mol. Cell. Endocrinol.
17. 2006, 249, 107.
[98] D. G. Stokes, G. Liu, R. Dharmavaram, D. Hawkins, S. Piera- [130] F. M. Altaf, T. M. Hering, N. H. Kazmi, J. U. Yoo, B. Johnstone, Eur
Velazquez, S. A. Jimenez, Biochem. J. 2001, 360, 461. Cell Mater 2006, 12, 64.
[99] Y. Yao, C. Wang, NPJ Regen Med 2020, 5, 14. [131] T. M. Temu, K.-Y. Wu, P. A. Gruppuso, C. Phornphutkul, Am. J. Phys-
[100] D. H. Rosenzweig, S. J. Ou, T. M. Quinn, J. Cell. Mol. Med. 2013, 17, iol. Endocrinol. Metab. 2010, 299, E325.
508. [132] S.-Z. Wang, Q. Chang, X.-F. Kong, C. Wang, Stem Cells Int. 2015,
[101] S.-M. Yu, H. J. Yeo, S. Y. Choi, S. J. Kim, Int. J. Biochem. Cell Biol. 2015, 589124.
2016, 80, 10. [133] E. Minina, H. M. Wenzel, C. Kreschel, S. Karp, W. Gaffield, A. P.
[102] S.-M. Yu, S.-J. Kim, Int J Mol Med 2015, 35, 325. McMahon, A. Vortkamp, Development 2001, 128, 4523.
[103] M. Wang, Z. Yuan, N. Ma, C. Hao, W. Guo, G. Zou, Y. Zhang, M. [134] F. Wei, J. Zhou, X. Wei, J. Zhang, B. C. Fleming, R. Terek, M. Pei, Q.
Chen, S. Gao, J. Peng, A. Wang, Y. Wang, X. Sui, W. Xu, S. Lu, S. Liu, Chen, T. Liu, L. Wei, Osteoarthr. Cartil. 2012, 20, 755.
Q. Guo, Stem Cells Int. 2017, 2017, 4130607. [135] S. Chawla, A. Sharma, A. Bandyopadhyay, S. Ghosh, ACS Biomater.
[104] M. Borowiak, R. Maehr, S. Chen, A. E. Chen, W. Tang, J. L. Fox, S. L. Sci. Eng. 2018, 4, 3545.
Schreiber, D. A. Melton, Cell Stem Cell 2009, 4, 348. [136] T. D. Bornes, N. M. Jomha, A. Mulet-Sierra, A. B. Adesida, Stem Cell
[105] P. Tangchitphisut, N. Srikaew, S. Numhom, A. Tangprasittipap, Res Ther 2015, 6, 84.
P. Woratanarat, S. Wongsak, C. Kijkunasathian, S. Hongeng, I. R. [137] F. Zhang, J. Wang, J. Chu, C. Yang, H. Xiao, C. Zhao, Z. Sun, X. Gao,
Murray, T. Tawonsawatruk, Arthritis 2016, 2016, 4019873. G. Chen, Z. Han, W. Zou, T. Liu, Cell. Physiol. Biochem. Int. J. Exp.
[106] S. L. Francis, S. Duchi, C. Onofrillo, C. Di Bella, P. F. M. Choong, Cell. Physiol. Biochem. Pharmacol. 2015, 37, 1442.
Stem Cells Int. 2018, 2018, 8947548. [138] Q. Yao, M. Parvez-Khan, E. Schipani, Bone 2020, 140, 115572.
[107] B. O. Diekman, C. R. Rowland, D. P. Lennon, A. I. Caplan, F. Guilak, [139] C. C. Scholz, C. T. Taylor, Curr. Opin. Pharmacol. 2013, 13, 646.
Tissue Eng., Part A 2010, 16, 523. [140] T. Saito, A. Fukai, A. Mabuchi, T. Ikeda, F. Yano, S. Ohba, N. Nishida,
[108] T. F. Li, R. J. O’Keefe, D. Chen, Front Biosci 2005, 10, 681. T. Akune, N. Yoshimura, T. Nakagawa, Nat. Med. 2010, 16, 678.
[109] P. M. van der Kraan, E. N. Blaney Davidson, A. Blom, W. B. van den [141] S. Yang, J. Kim, J.-H. Ryu, H. Oh, C.-H. Chun, B. J. Kim, B. H. Min,
Berg, Osteoarthr. Cartil. 2009, 17, 1539. J.-S. Chun, Nat. Med. 2010, 16, 687.
[110] J. Pannu, S. Nakerakanti, E. Smith, P. ten Dijke, M. Trojanowska, J. [142] H. Inoue, Y. Arai, T. Kishida, R. Terauchi, K. Honjo, S. Nakagawa, S.
Biol. Chem. 2007, 282, 10405. Tsuchida, T. Matsuki, K. Ueshima, H. Fujiwara, O. Mazda, T. Kubo,
[111] A. Javed, J.-S. Bae, F. Afzal, S. Gutierrez, J. Pratap, S. K. Zaidi, Y. Lou, Int. J. Mol. Sci. 2015, 16, 1043.
A. J. Van Wijnen, J. L. Stein, G. S. Stein, J. Biol. Chem. 2008, 283, [143] K. Montagne, Y. Onuma, Y. Ito, Y. Aiki, K. S. Furukawa, T. Ushida,
8412. PLoS One 2017, 12, e0183226.
[112] K. Futrega, P. G. Robey, T. J. Klein, R. W. Crawford, M. R. Doran, [144] A. Nazempour, C. R. Quisenberry, N. I. Abu-Lail, B. J. Van Wie, Cell
Commun Biol 2021, 4, 29. Tissue Res. 2017, 370, 179.
[113] R. Narcisi, R. Quarto, V. Ulivi, A. Muraglia, L. Molfetta, P. Giannoni, [145] E. H. Frank, M. Jin, A. M. Loening, M. E. Levenston, A. J. Grodzinsky,
J. Cell. Physiol. 2012, 227, 3282. J. Biomech. 2000, 33, 1523.
[114] K. Pelttari, A. Winter, E. Steck, K. Goetzke, T. Hennig, B. G. Ochs, T. [146] J. D. Kisiday, M. Jin, M. A. DiMicco, B. Kurz, A. J. Grodzinsky, J.
Aigner, W. Richter, Arthritis Rheum. 2006, 54, 3254. Biomech. 2004, 37, 595.
[115] Y. Park, M. Sugimoto, A. Watrin, M. Chiquet, E. B. Hunziker, Os- [147] M. Jin, E. H. Frank, T. M. Quinn, E. B. Hunziker, A. J. Grodzinsky,
teoarthr. Cartil. 2005, 13, 527. Arch. Biochem. Biophys. 2001, 395, 41.
[116] B. S. Yoon, K. M. Lyons, J. Cell. Biochem. 2004, 93, 93. [148] D. A. Lee, T. Noguchi, S. P. Frean, P. Lees, D. L. Bader, Biorheology
[117] G. R. Gipson, E. J. Goebel, K. N. Hart, E. C. Kappes, C. Kattamuri, J. 2000, 37, 149.
C. McCoy, T. B. Thompson, Bone 2020, 140, 115549. [149] P. Occhetta, A. Mainardi, E. Votta, Q. Vallmajo-Martin, M. Ehrbar, I.
[118] B. Keller, T. Yang, Y. Chen, E. Munivez, T. Bertin, B. Zabel, B. Lee, Martin, A. Barbero, M. Rasponi, Nat. Biomed. Eng. 2019, 3, 545.
PLoS One 2011, 6, e16421. [150] Y. Ge, Y. Li, Z. Wang, L. Li, H. Teng, Q. Jiang, Effects of Mechani-
[119] G. Nalesso, J. Sherwood, J. Bertrand, T. Pap, M. Ramachandran, cal Compression on Chondrogenesis of Human Synovium-Derived Mes-
C. De Bari, C. Pitzalis, F. Dell’accio, J. Cell Biol. 2011, 193, enchymal Stem Cells in Agarose Hydrogel, 2021, Vol. 9.
551. [151] E. Lucchinetti, C. S. Adams, W. E. J. Horton, P. A. Torzilli, Osteoarthr.
[120] H.-H. Lee, R. R. Behringer, PLoS One 2007, 2, e450. Cartil. 2002, 10, 71.
[121] Y. Kawakami, N. Wada, S. I. Nishimatsu, T. Ishikawa, S. Noji, T. [152] K. Li, A. Williamson, A. Wang, R. Sah, Clin. Orthop. Relat. Res. 2001,
Nohno, Dev Growth Differ 1999, 41, 29. 391, S34.

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[153] O. Démarteau, D. Wendt, A. Braccini, M. Jakob, D. Schäfer, M. [177] M. Knippenberg, M. N. Helder, B. Zandieh Doulabi, P. I. J. M.
Heberer, I. Martin, Biochem. Biophys. Res. Commun. 2003, 310, Wuisman, J. Klein-Nulend, Biochem. Biophys. Res. Commun. 2006,
580. 342, 902.
[154] S. Pigeot, T. Klein, F. Gullotta, S. J. Dupard, A. G Garcia, A. García- [178] X. Wang, T. He, L. He, B. Yang, Z. Liu, M. Pang, P. Xie, L. Zhang, L.
García, S. Prithiviraj, P. Lorenzo, M. Filippi, C. Jaquiery, L. Kouba, M. Rong, Stem Cell Res Ther 2021, 12, 1.
A. Asnaghi, D. B. Raina, B. Dasen, H. Isaksson, P. Önnerfjord, M. [179] H. A. Awad, Y.-D. C. Halvorsen, J. M. Gimble, F. Guilak, Tissue Eng.
Tägil, A. Bondanza, I. Martin, P. E. Bourgine, Adv. Mater. 2021, 33, 2003, 9, 1301.
2103737. [180] A. A. Stewart, C. R. Byron, H. C. Pondenis, M. C. Stewart, Am. J. Vet.
[155] D. Truhn, K. T. Zwingenberger, J. Schock, D. B. Abrar, K. L. Radke, Res. 2008, 69, 1013.
M. Post, K. Linka, M. Knobe, C. Kuhl, S. Nebelung, J. Mech. Behav. [181] F. Cialdai, A. Colciago, D. Pantalone, A. M. Rizzo, S. Zava, L.
Biomed. Mater. 2021, 120, 104558. Morbidelli, F. Celotti, D. Bani, M. Monici, Int. J. Mol. Sci. 2020, 21,
[156] S. H. Chang, D. Mori, H. Kobayashi, Y. Mori, H. Nakamoto, K. 407.
Okada, Y. Taniguchi, S. Sugita, F. Yano, U.-I. Chung, J.-R. Kim- [182] G. Murakami, T. Watabe, K. Takaoka, K. Miyazono, T. Imamura, Mol.
Kaneyama, M. Yanagita, A. Economides, E. Canalis, D. Chen, S. Biol. Cell 2003, 14, 2809.
Tanaka, T. Saito, Nat. Commun. 2019, 10, 1442. [183] Y. Zhang, C. Chang, D. J. Gehling, A. Hemmati-Brivanlou, R.
[157] T. Fang, X. Zhou, M. Jin, J. Nie, Xi. Li, Int Orthop 2021, 45, 1125. Derynck, Proc. Natl. Acad. Sci. USA 2001, 98, 974.
[158] S. Chawla, A. Mainardi, N. Majumder, L. Dönges, B. Kumar, P. [184] W. Ishida, T. Hamamoto, K. Kusanagi, K. Yagi, M. Kawabata, K.
Occhetta, I. Martin, C. Egloff, S. Ghosh, A. Bandyopadhyay, A. Takehara, T. K. Sampath, M. Kato, K. Miyazono, J. Biol. Chem. 2000,
Barbero, Cells 4034, 2022, 11. 275, 6075.
[159] J. H. Boergermann, J. Kopf, P. B. Yu, P. Knaus, Int. J. Biochem. Cell [185] H. Troilo, A. L. Barrett, A. P. Wohl, T. A. Jowitt, R. F. Collins, C. P.
Biol. 2010, 42, 1802. Bayley, A. V Zuk, G. Sengle, C. Baldock, Biochem. Soc. Trans. 2015,
[160] N. Nakayama, C. Han, L. Cam, J. Lee, J. Pretorius, S. Fisher, R. 43, 795.
Rosenfeld, S. Scully, R. Nishinakamura, D. Duryea, G. Van, B. Bolon, [186] E. Canalis, A. N. Economides, E. Gazzerro, Endocr Rev 2003, 24, 218.
T. Yokota, K. Zhang, Development 2004, 131, 229. [187] V. Dexheimer, J. Gabler, K. Bomans, T. Sims, G. Omlor, W. Richter,
[161] S.-Y. Chien, C.-H. Tsai, S.-C. Liu, C.-C. Huang, T.-H. Lin, Y.-Z. Yang, Sci. Rep. 2016, 6, 36655.
C.-H. Tang, Cells 2020, 9, 927. [188] E. Williams, A. N. Bullock, Bone 2018, 109, 251.
[162] C. E. Sanvitale, G. Kerr, A. Chaikuad, M. C. Ramel, A. H. Mohedas, [189] X. Yang, S. K. Trehan, Y. Guan, C. Sun, D. C. Moore, C. T. Jayasuriya,
S. Reichert, Y. Wang, J. T. Triffitt, G. D. Cuny, P. B. Yu, C. S. Hill, A. Q. Chen, J. Biol. Chem. 2014, 289, 34768.
N. Bullock, PLoS One e62721, 2013, 8. [190] Y. R. Yang, X. F. Yang, H. C. Duan, J. Q. Qiao, J Biol Regul Homeost
[163] A. H. Mohedas, Y. Wang, C. E. Sanvitale, P. Canning, S. Choi, X. Xing, Agents 2020, 34, 785.
A. N. Bullock, G. D. Cuny, P. B. Yu, J. Med. Chem. 2014, 57, 7900. [191] C. Lietman, B. Wu, S. Lechner, A. Shinar, M. Sehgal, E. Rossomacha,
[164] V. Deshmukh, H. Hu, C. Barroga, C. Bossard, S. KC, L. Dellamary, P. Datta, A. Sharma, R. Gandhi, M. Kapoor, P. P. Young, JCI Insight
J. Stewart, K. Chiu, M. Ibanez, M. Pedraza, T. Seo, L. Do, S. Cho, 2018, 3.
J. Cahiwat, B. Tam, J. R. S. Tambiah, J. Hood, N. E. Lane, Y. Yazici, [192] C. A. Thorne, A. J. Hanson, J. Schneider, E. Tahinci, D. Orton, C. S.
Osteoarthr. Cartil. 2018, 26, 18. Cselenyi, K. K. Jernigan, K. C. Meyers, B. I. Hang, A. G. Waterson, K.
[165] L. Guo, X. Wei, Z. Zhang, X. Wang, C. Wang, P. Li, C. Wang, L. Wei, Kim, B. Melancon, V. P. Ghidu, G. A. Sulikowski, B. LaFleur, A. Salic,
Arthritis Res. Ther. 2019, 21, 109. L. A. Lee, D. M. 3rd Miller, E. Lee, Nat. Chem. Biol. 2010, 6, 829.
[166] T. Nagai, M. Sato, M. Kobayashi, M. Yokoyama, Y. Tani, J. Mochida, [193] T. N. Grossmann, J. T.-H. Yeh, B. R. Bowman, Q. Chu, R. E.
Arthritis Res. Ther. 2014, 16, 427. Moellering, G. L. Verdine, Proc. Natl. Acad. Sci. USA 2012, 109,
[167] W. Xu, Y. Xie, Q. Wang, X. Wang, F. Luo, S. Zhou, Z. Wang, J. Huang, 17942.
Q. Tan, M. Jin, H. Qi, J. Tang, L. Chen, X. Du, C. Zhao, G. Liang, L. [194] A. Held, A. Glas, L. Dietrich, M. Bollmann, K. Brandstädter, T. N.
Chen, Sci. Rep. 2016, 6, 24042. Grossmann, C. H. Lohmann, T. Pap, J. Bertrand, Osteoarthr. Cartil.
[168] X. Zhang, V. A. Siclari, S. Lan, J. Zhu, E. Koyama, H. L. Dupuis, M. 2018, 26, 818.
Enomoto-Iwamoto, F. Beier, L. Qin, J. bone Miner. Res. Off. J. Am. [195] L. Zhong, X. Huang, E. D. Rodrigues, J. C. H. Leijten, T. Verrips,
Soc. Bone Miner. Res. 2011, 26, 2622. M. El Khattabi, M. Karperien, J. N. Post, Stem Cells Dev. 2016, 25,
[169] K. E. Uhrich, S. M. Cannizzaro, R. S. Langer, K. M. Shakesheff, Chem. 1808.
Rev. 1999, 99, 3181. [196] J. Luan, H. Tao, Y. Su, Am. J. Transl. Res. 2020, 12, 1985.
[170] Q. Yang, B.-H. Teng, L.-N. Wang, K. Li, C. Xu, X.-L. Ma, Y. Zhang, [197] Z. Cao, C. Dou, S. Dong, Chem. Pharm. Bull. 2017, 65, 762.
D.-L. Kong, L.-Y. Wang, Y.-H. Zhao, Int. J. Nanomed. 2017, 12, 6721. [198] G. Vadalà, L. Ambrosio, C. Cattani, R. Bernardini, A. Giacalone, R.
[171] X. Martineau, É. Abed, J. Martel-Pelletier, J.-P. Pelletier, D. Papalia, V. Denaro, J Clin Med 2021, 10, 2825.
Lajeunesse, PLoS One 2017, 12, e0180711. [199] Z. Cao, Y. Bai, C. Liu, C. Dou, J. Li, J. Xiang, C. Zhao, Z. Xie, Q. Xiang,
[172] C. Hartmann, C. Tabin, Cell 2001, 104, 341. S. Dong, Mol. Med. Rep. 2017, 16, 2740.
[173] B. Kinner, M. Spector, J. Orthop. Res. Off. Publ. Orthop. Res. Soc. [200] Z. Yang, L. Feng, J. Huang, X. Zhang, W. Lin, B. Wang, L. Cui, S. Lin,
2001, 19, 233. G. Li, Eur. J. Pharmacol. 2021, 907, 174265.
[174] J. Parreno, S. Raju, A. Jiang, K. Andrejevic, R. A. Kandel, Osteoarthr. [201] I. Prasadam, X. Mao, W. Shi, R. Crawford, Y. Xiao, J Mol Med 2013,
Cartil. 2014, 22, S167. 91, 369.
[175] X. Zhang, N. Ziran, J. J. Goater, E. M. Schwarz, J. E. Puzas, R. N. [202] Z. Cao, C. Dou, J. Li, X. Tang, J. Xiang, C. Zhao, L. Zhu, Y. Bai, Q.
Rosier, M. Zuscik, H. Drissi, R. J. O’Keefe, Bone 2004, 34, 809. Xiang, S. Dong, BMB Rep. 2016, 49, 548.
[176] A. F. Steinert, B. Proffen, M. Kunz, C. Hendrich, S. C. Ghivizzani, U. [203] H. Jing, X. Zhang, M. Gao, K. Luo, W. Fu, M. Yin, W. Wang, Z. Zhu,
Nöth, A. Rethwilm, J. Eulert, C. H. Evans, Arthritis Res. Ther. 2009, J. Zheng, X. He, FASEB J. Off. Publ. Fed. Am. Soc. Exp. Biol. 2019, 33,
11, R148. 5641.

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Nilotpal is a Ph.D. student at Department of Textile and Fibre Engineering, Indian Institute of Technol-
ogy Delhi, India. His research mainly focuses on devising advanced 3D bioprinting strategies while
modulating cell signaling pathways to study articular cartilage degeneration and regeneration. He
has completed his B.Tech-M.Tech dual degree in Biotechnology from Kalinga Institute of Industrial
Technology Deemed to be University (KIIT-DU), India.

Sourabh Ghosh is a Professor at Department of Textile and Fibre Engineering, Indian Institute of Tech-
nology Delhi, India. He is the head of Regenerative Engineering laboratory. His research focuses on
the interface between fundamental and applied research, by combining the principles of Textile Tech-
nology and Tissue Engineering, using silk and collagen proteins, bioprinting and establishment of in
vitro disease models.

Adv. Funct. Mater. 2023, 33, 2300651 2300651 (20 of 20) © 2023 Wiley-VCH GmbH