International Journal of

Molecular Sciences

Review
Strategies to Convert Cells into Hyaline Cartilage: Magic Spells
for Adult Stem Cells
Anastasiia D. Kurenkova 1 , Irina A. Romanova 2 , Pavel D. Kibirskiy 1 , Peter Timashev 1,2
and Ekaterina V. Medvedeva 1, *

1 Institute for Regenerative Medicine, Sechenov First Moscow State Medical University (Sechenov University),
119991 Moscow, Russia
2 World-Class Research Center “Digital Biodesign and Personalized Healthcare”, Sechenov First Moscow State
Medical University (Sechenov University), 119991 Moscow, Russia
* Correspondence: [email protected]

Abstract: Damaged hyaline cartilage gradually decreases joint function and growing pain signif-
icantly reduces the quality of a patient’s life. The clinically approved procedure of autologous
chondrocyte implantation (ACI) for treating knee cartilage lesions has several limits, including the
absence of healthy articular cartilage tissues for cell isolation and difficulties related to the chondro-
cyte expansion in vitro. Today, various ACI modifications are being developed using autologous
chondrocytes from alternative sources, such as the auricles, nose and ribs. Adult stem cells from
different tissues are also of great interest due to their less traumatic material extraction and their
innate abilities of active proliferation and chondrogenic differentiation. According to the different
adult stem cell types and their origin, various strategies have been proposed for stem cell expansion
and initiation of their chondrogenic differentiation. The current review presents the diversity in
developing applied techniques based on autologous adult stem cell differentiation to hyaline cartilage
Citation: Kurenkova, A.D.; tissue and targeted to articular cartilage damage therapy.
Romanova, I.A.; Kibirskiy, P.D.;
Timashev, P.; Medvedeva, E.V. Keywords: adult stem cells; chondrogenic differentiation; tissue markers; osteoarthritis; articular
Strategies to Convert Cells into cartilage; autologous chondrocytes; bone marrow; synovium; adipose tissue; dental pulp; periosteum;
Hyaline Cartilage: Magic Spells for perichondrium; extracellular vesicles
Adult Stem Cells. Int. J. Mol. Sci.
2022, 23, 11169. https://doi.org/
10.3390/ijms231911169

Academic Editors: Magali 1. Introduction

Cucchiarini and Henning Madry Articular cartilage has a complex structural tissue consisting of several layers, which
Received: 24 August 2022
differ by their composition in the collagen-type content, collagen fiber orientation and
Accepted: 19 September 2022
cell morphology and density [1] (Figure 1b). Such a complex hierarchical structure is
Published: 22 September 2022
fundamental to the unique biomechanical properties of articular cartilage [1] and, simulta-
neously, a challenge for regenerative medicine. However, finding a solution is essential
Publisher’s Note: MDPI stays neutral
since degenerative joint diseases are widespread. Osteoarthritis (OA) is the most common
with regard to jurisdictional claims in
joint disease, with progressive degeneration of articular cartilage and subsequent slow joint
published maps and institutional affil-
space narrowing, pathological remodeling of all joint tissues, including bone, and chronic
iations.
inflammatory processes accompanied by movement limitation [2–4]. Clinical remediation
is urgently required, with a growing list of developing drug treatments for OA being
unsuccessful in clinical trials [5].
Copyright: © 2022 by the authors.
Unfortunately, there is no better method for patients with severe knee articular car-
Licensee MDPI, Basel, Switzerland. tilage damage than total joint arthroplasty. Prevalent clinically available cartilage repair
This article is an open access article surgical techniques undertaken for minor osteochondral defects (<4 cm2 ) [6,7] are mi-
distributed under the terms and crofracture and mosaicplasty, leading to fibrocartilage neo-tissue formation. Successful
conditions of the Creative Commons restoration depends on the age and size of any cartilage defects, with the data reporting that
Attribution (CC BY) license (https:// mosaicplasty is the more durable of the two applied methods [8]. Currently, ACI method
creativecommons.org/licenses/by/ and its modification, known as the matrix-induced ACI (MACI) method, are used in clinical
4.0/). practice [6,7]. Twelve years of investigation have confirmed that MACI might be suitable for

Int. J. Mol. Sci. 2022, 23, 11169. https://doi.org/10.3390/ijms231911169 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 2 of 29

Int. J. Mol. Sci. 2022, 23, 11169 2 of 28

and its modification, known as the matrix-induced ACI (MACI) method, are used in clin-
ical practice [6,7]. Twelve years of investigation have confirmed that MACI might be suit-
able for medium-term
medium-term cartilage cartilage
repair [9].repair [9]. However,
However, all mentioned
all mentioned treatmentstreatments have
have limited lim-
capacity
ited capacity to induce long-term cartilage regeneration, resulting in mechanically inferior
to induce long-term cartilage regeneration, resulting in mechanically inferior fibrocartilage
fibrocartilage
degraded with degraded with time [6,7].
time [6,7].

Figure
Figure 1.
1. List
List of
of markers
markers for
for hyaline
hyaline cartilage
cartilage chondrocytes,
chondrocytes,hypertrophic
hypertrophicchondrocytes
chondrocytesand andfibro-
fibro-
cartilage.
cartilage. (a)
(a)Histological
Histologicalimages
imagesare
are photos
photosofof human
human knee
knee articular
articular cartilage
cartilage sections
sections stained
stained by
by
Safranin-O/Fast green; (b) scheme of the human articular cartilage structure.
Safranin-O/Fast green; (b) scheme of the human articular cartilage structure.

The
The demand of reconstructing
reconstructingthe thehighly
highlyorganized
organizedhyaline
hyaline cartilage
cartilage promotes
promotes fur-
further
ther tissue
tissue engineering
engineering research
research based
based on on different
different adult
adult stem
stem cellcell populations,
populations, whichdiffer
which dif-
fer significantly
significantly in their
in their proliferative
proliferative properties
properties andand chondrogenic
chondrogenic differentiation
differentiation ability
ability [10].
[10].
As a As a result,
result, the development
the development of approaches
of approaches to stimulate
to stimulate stem stem
cells’ cells’ differentiation
differentiation from
diverse
from sources
diverse will vary
sources will (Figure 2). 2).
vary (Figure

Figure 2. Schematic summary of the current review discussing distinct approaches of autologous
adult stem cell chondrogenic differentiation to repair hyaline cartilage damage.
Int. J. Mol. Sci. 2022, 23, 11169 3 of 28

2. Markers for Hyaline Cartilage Chondrocytes, Hypertrophic Chondrocytes and

Fibrocartilage
Hyaline cartilage is the tissue of clinical interest. On the contrary, the hypertrophic and
fibrocartilage chondrocyte phenotypes are associated with OA development [5]. During
the late stage of OA disease progression, chondrocytes change their phenotype [11] to
the hypertrophic and fibrocartilage chondrocytes that catalyze the destruction of articular
cartilage tissues [12]. For further discussion, it is necessary to note which markers (Figure 1a)
allow the distinction of hyaline cartilage chondrocytes from fibrocartilage cells, as well as
from hypertrophic chondrocytes, which tend to be replaced by bone [13].
The fate of cartilage existence depends upon the induction of some genes and suppres-
sion of others. Chondroprogenitors differentiate into mature hyaline cartilage chondrocytes
producing cartilage-specific matrix proteins, such as type II collagen (COL2) and aggre-
can (ACAN). Other hyaline cartilage ECM proteins are collagen types IX (COL9) and XI
(COL11), but they are less abundant than COL2 and ACAN [13,14].
Chondrocytes undergo hypertrophic differentiation characterized by the secretion
of specific ECM molecules [15], such as collagen type X (COL10), the most well-known
hypertrophy marker [16]. In vitro analyses demonstrate direct regulation of COL10 gene
(Col10a1) expression by myocyte-enhancer factor 2C (MEF2C) [17]. Genetic deletion of
Mef2C impairs hypertrophy, cartilage angiogenesis and cartilage following endochondral
ossification, preventing longitudinal bone growth in mice [17]. MEF2C seems to regulate an-
other hypertrophic marker belonging to the RUNX family of transcription factors, RUNX2
(also known as CBFA1). RUNX2 and another family member, RUNX3, are necessary for
chondrocyte hypertrophy, matrix mineralization and osteoblast differentiation [15,18,19].
On the contrary, RUNX1 suppresses the hypertrophic differentiation of chondro-
cytes [20], enhancing hyaline cartilage matrix production in cooperation with SOX-family
molecules [20,21], which regulate chondrogenesis [21] and are conserved across vertebrate
taxa [22,23]. Chondrocytes and chondroprogenitors express SOX9, a major chondrogenic
transcription factor, and its cofactors SOX5 and SOX6, which stimulate ECM gene expres-
sion, including genes of COL2 and ACAN [14,21]. Another highly conserved mammalian
molecule, a transmembrane chondroitin sulfate proteoglycan, named secondary ossification
center associated regulator of chondrocyte maturation (Snorc), was described as a mature
chondrocyte phenotype marker and restricted to hyaline cartilage structures [24]. Snorc
gene expression is mediated through the SOX9 enhancer in vitro [25] and in vivo [14]. One
more protein regulated directly by SOX9 is a ubiquitin ligase, WW domain-containing
protein 2 (WWP2) [26]. The sequence of the Wwp2 gene includes the gene of microRNA-140
(miR-140). Both WWP2 and miR-140 are abundantly and specifically expressed in cartilage
tissues [26,27] and maintain cartilage homeostasis via RUNX2 poly-ubiquitination and
degradation [28].
The fibrocartilage chondrocyte phenotype is mainly found in joints in the late stages of
OA. It is characterized by increased expression of collagen types I (COL1) and III (COL3),
as well as α-smooth muscle actin (αSMA), fibroblast-specific protein 1 (FSP1 or S100A4),
tissue inhibitor of metalloproteinase-1 (TIMP1) and cell migration inducing hyaluronidase-1
(CEMIP) (Figure 2) [29].
In most cases, it is enough to demonstrate the absence of the COL1 protein to exclude
the presence of fibrocartilage. A classical set of molecules confirming chondrocytes’ hyaline
cartilage state are SOX9, COL2 and ACAN. COL10, MEF2C and RUNX2 presence are
usually analyzed to reveal hypertrophy. Other markers are less commonly reported in
publications.

3. Autologous Chondrocytes from Different Sources

ACI and MACI procedures are approved for clinical application. However, these
therapies have significant drawbacks. In the late stages of OA, extraction of the autologous
chondrocytes is no longer an available option. Another widespread problem is that the
Int. J. Mol. Sci. 2022, 23, 11169 4 of 28

obtained chondrocytes are inclined to lose their chondrogenic features (de-differentiate) in

monolayers [30].

3.1. Chondrons, the Functional Units of Cartilage Tissue

To avoid chondrocyte de-differentiation in vitro, it was suggested to isolate chondrons
composed of chondrocytes surrounded by a pericellular matrix. A milieu of chondrocytes
play an essential role in the maintenance of their chondrogenic state and utilization of
chondrons could retard chondrocyte de-differentiation processes in vitro [31]. A recent
study demonstrated that cultured chondrons expressed higher levels of proteoglycan and
COL2 than chondrocyte culture [32]. Surprisingly, a mixture of chondrons and chondrocytes
significantly elevated the production of ECM in culture even more than the chondrons
alone. Implantation of alginate spheres, including chondrons and chondrocytes, accelerated
the regeneration of injured knee cartilage in rabbits [32].
The extraction of chondrons from cartilage is not more difficult than chondrocyte
isolation [32,33]. However, X-ray micro-computed tomography (µCT) methods showed
that the morphology of chondrons is affected by OA progression [34]. The limitations of
using autologous chondrons to repair a cartilage defect are the same as for autologous
chondrocytes.

3.2. Chondroprogenitors from Superficial Zone of Articular Cartilage

One way to avoid chondrocyte de-differentiation during expansion is to use chondro-
genic progenitors, which are cells with an incomplete differentiation cycle but a higher
ability to proliferate. A population of endogenous, slowly dividing progenitor cells was
discovered in the superficial zone of adult articular cartilage [35,36]. Recently, it was
confirmed that these articular cartilage progenitor cells (ACPCs) in the superficial zone
were responsible for the turnover of articular cartilage tissue in the postnatal period in
mice [36,37].
Since ACPC cultures have not been scrupulously investigated yet, there are no unique
cell markers for the selective isolation protocol, especially for human ACPCs. Different un-
successful efforts have been made [38,39] to set apart the ACPCs and mature chondrocytes;
however, the method of ACPC isolation, which is predominant in publications but has low
specificity, relies on adhesion to a fibronectin coating (owing to the high cell expression of
the fibronectin receptors integrin-α5 and -β1) [35].
To determine the optimal chondrogenic conditions, bovine ACPCs were cultured as
pellets (a high-cell-density scaffold-free culture compressed by centrifugation) for 21 days in
the presence of known chondrogenic factors, such as transforming growth factor-β (TGFβ)
1 to 3, bone morphogenetic proteins (BMP) 2 and 9, synthetic glucocorticoid dexametha-
sone, C-natriuretic peptide, dibutyryl-cAMP, concanavalin-A, ethanol and chelerythrine
chloride [40]. Histological analysis, immunohistochemical (IHC) labeling, polymerase
chain reaction (PCR) and atomic force microscopy measurements revealed BMP9 as a
promising chondrogenic factor for ACPC differentiation in vitro. However, the concentra-
tion of BMP9 in the culture medium should not exceed 100 ng/mL to avoid the production
of the hypertrophic marker COL10 [40].
Replacing the routinely used growth supplement fetal bovine serum (FBS) with human
platelet lysate in the culturing protocol led to faster doublings and increased expression
of ACAN and COL2, as well as COL10 and COL1, in ACPC monolayer cultures [41].
Compared to monolayers, the technique of ACPC expansion on commercial macroporous
gelatin-coated microcarrier beads in the presence of TGFβ1 resulted in a significant en-
hancement in ACPC proliferation, maintaining the ability to produce an ECM abundant
with COL2 and glycosaminoglycans (GAGs) [42].
The articular cartilage chondrocytes are well-specialized cells that are sensitive to
mechanical stimuli, which are a complex combination of compression, stretching, shear
stress and hydrostatic pressure. Mechanical loading increased SOX9 IHC labeling in the
superficial zone of bovine cartilage cultured as cylindrical osteochondral plugs [43]. The
Int. J. Mol. Sci. 2022, 23, 11169 5 of 28

application of bioreactor-generated joint-like movements demonstrated the mechanosen-

sitivity of ACPC cultures [44]. Intermittent hydrostatic pressure (IHP) applied to ACPCs
seeded within alginate beads for four weeks stimulated more significant production of
cartilage ECM components and the expression of chondrocyte-related genes compared to
cultures of adipose-derived stem cells (ADSCs) or even chondrocytes [45]. Mechanical stim-
ulation is assumed to be more relevant to chondroprogenitors than chondrocyte cultures.
During in vivo studies, a construct consisting of goat ACPCs seeded on a COL1/COL3
membrane (Chondro-Gide® ) was transplanted into a knee defect. Analysis showed accept-
able integration with the surrounding cartilage and COL2-positive staining of neo-tissue.
However, no difference was found in the final results between ACPC- and chondrocyte-
treated defects [46]. During a clinical trial, human CD146-positive cells collected from the
upper zone of articular cartilage were differentiated to a chondrogenic lineage in a culture
medium containing TGFβ3 and BMP4 [47] and surgically implanted on a COL1/COL3
membrane into knee cartilage defects. Subsequent magnetic resonance imaging (MRI) and
arthroscopy showed complete attachment of the construct and filling of the defect, with
histology and IHC estimation of neo-tissue confirming a good repair [47]. However, the
significant regeneration was believed to have resulted from the young age of the patients
treated (the average age was 25 years).

3.3. Non-Articular Autologous Chondrocytes

To exclude the donor-site morbidity of a joint during autologous articular chondrocyte
(AC) isolation, it is suggested that non-articular chondrocytes be used for joint defect
treatment. The efficacy of replacing hyaline cartilage cells with non-articular chondrocytes
remains debatable.

3.3.1. Nasal Chondrocytes

Both articular and nasal tissues are permanent hyaline cartilage throughout life but
have different origins during embryonic development. Nevertheless, in adults, nasal
cartilage tissue has a similar composition and structure to joint cartilage, but the calcified
zone is not present in nasal cartilage [48]. The monolayer of adult nasal chondrocytes (NCs)
has three-times faster proliferative activity and promising chondrogenic capacities [49]. In
an animal defect model, NCs within engineered grafts could generate hyaline cartilage
markers even more than ACs [50].
Engineered autologous human cartilage grafts obtained from a nasal septum biopsy
specimen were implanted into femoral full-thickness cartilage defects in a first-in-human
trial, including ten patients [51]. IHC characterization of the non-used portions of the
engineered cartilage constructs indicated positivity for hyaline cartilage markers, slight
positivity for COL1 and negativity for COL10. Twenty-four months after the surgery,
histological analysis of the biopsy at the engineered graft implantation site indicated het-
erogeneity in the repair tissue without the typical hyaline cartilage organization. However,
the high patient self-assessment scores and MRI analyses of the defect filling allowed the
authors to claim a satisfactory clinical outcome [51].
A pre-clinical study from 2021 showed that nasal-chondrocyte-based tissue-engineered
cartilage (NTEC) implanted into osteochondral cartilage defects of sheep prevented the
elevation of synovial fluid volumes and decreased concentrations of pro-inflammatory
cytokines induced by knee OA. Autologous NTEC graft implantation into a human was
evaluated in two patients with advanced OA. Fourteen months after the surgery, the pa-
tients confirmed improvements in pain, knee-joint function and quality of life. Accordingly,
the authors declared that future controlled trials on other joints would be initiated [52].

3.3.2. Costal Chondrocytes

Costal hyaline cartilage is another chondrocyte source for ACI and MACI therapies,
allowing for potential complications following additional joint surgery interventions to be
Int. J. Mol. Sci. 2022, 23, 11169 6 of 28

avoided. Moreover, autologous costal cartilage is already widely used as a source of grafts
in plastic surgery, demonstrating the procedure’s safety.
During culturing without growth factors, costal-derived chondrocytes (CCs) prolifer-
ate 9-fold faster than ACs. However, both the ACs and CCs gradually lose their phenotype
following the first four passages [53]. The addition of fibroblast growth factor 2 (FGF2)
in combination with commercial mesenchymal stem cell growth mediumTM (MSCGM)
induced stromal cell features with a significant content of COL2 and ACAN, with trace
amounts of COL1. At the same time, FGF2 and DMEM drove CCs to transition to fibroblast-
like cells [54]. In a rabbit osteochondral defect model, CCs expanded in the FGF2-containing
MSCGM, resulting in good defect healing [54].
A clinical test of autologous CC pellets with fibrin glue on seven patients with full-
thickness articular cartilage lesions included CCs expanded in MSCGM with FGF2 before
implantation [55,56]. The results showed significant improvements in all clinical scores
during the 5-year follow-up period confirmed by MRI scans. Nevertheless, hypertrophy
in implants or incomplete defect filling was observed in two patients [55]. According to
recently published data, co-culturing CCs with synovial membrane-derived mesenchy-
mal stem cells (SM-MSCs) could prevent hypertrophy and maintain the chondrogenic
phenotype of CCs in cartilage repair [57].

3.3.3. Growth Plate Chondrocytes

The epiphyseal/growth plate hyaline cartilage cells are responsible for the elongation
of long bones. The age of growth plate closure for humans is 17–25 years old [58]. Cell
harvesting could damage the immature growth plate and disrupt bone growth [59] and
it severely limits the use of growth plate chondrocytes for autologous transplantation.
However, porcine chondrocytes from proliferating (middle) zones of growth plate cartilage
have been isolated and characterized in vitro [60] as a more viable, proliferative and
preserved chondrogenic phenotype that lasts longer during passages compared to ACs [60].
It has been previously assumed that an avascular hyaline cartilage nature provides
“immune privilege”, inferring the potential of using the growth plate as a donor source in
the case of allogeneic (cadavers or amputated material) or xenogeneic transplantation. This
notion was disproved by researchers [61] when the rabbit model showed that, in contrast
to allogeneic transplantation, xenogeneic implants in joint cartilage provoked a marked
innate and adaptive immune response [61].
For obvious reasons, at present, studies about the application of growth plate chon-
drocytes to articular cartilage defect treatment are scarce.

3.3.4. Chondrocytes from the Auricle of an Ear

In contrast to joint cartilage, auricular cartilage is covered by a perichondrium, contains
elastic fibers and COL1 and does not show a defined zonality in its structure. Auricular
chondrocyte (AUC) yields per tissue volume are high and sufficient for cell therapy realiza-
tion. De-differentiation has been reported to proceed more rapidly in AUCs than in ACs,
which was closely related to their proliferative activity [62,63].
Wong et al. (2018) suggested converting the de-differentiated monolayer AUCs to
hyaline cartilage using ECM [64]. The data demonstrated that the COL-coated culture dish
promoted hyaline cartilage markers and suppressed auricular elastic cartilage markers
(such as COL1 and elastin) in rabbit AUCs compared to cultivation on plastic. Three months
after implantation into an osteochondral defect, AUCs cultured in COL2 scaffolds exhibited
successful integration with the articular cartilage, abundant proteoglycan syntheses and
intense staining for COL2 with traces of elastin staining [64]. It was noted that the AUCs
without the COL2 scaffold did not result in compatible healing outcomes.
Wang et al. (2018) suggested an original cell-free approach combining AUC sheets and
the microfracture technique [65]. Rabbit AUC sheets were gently decellularized, retaining
the native architecture and ECM composition. Decellularized AUC sheets tested in vitro
showed stimulation of potential migrating BM-MSCs, increasing SOX9 production and
Int. J. Mol. Sci. 2022, 23, 11169 7 of 28

decreasing COL10 production detected by RT-PCR and Western blots [65]. Agreeable
results for the application of decellularized AUC sheets in combination with microfracture
to osteochondral knee defect regeneration in rabbits were confirmed by macroscopic
observation, µCT imaging and histological analysis 12 weeks after the surgery [65].
Goat AUC sheets differentiated in a chondrogenic medium supplemented with TGFβ1,
insulin-like growth factor 1 (IGF1) and dexamethasone indicated a gradual increase in
hyaline-cartilage-specific ECM analyzed by histological and IHC staining [63]. However,
after subcutaneous implantation of AUC sheets in nude mice, a threat of AUC sheet tissue
ossification was demonstrated [63].
The clinically available growth factors FGF2 and IGF1 and the peptide hormone insulin
were proposed as optimal for AUC expansion in monolayer cultures [66]. Their effect
on the differentiation of three-dimensional (3D) cultures of AUCs was investigated [67].
Histological, biochemical and biomechanical analyses showed that IGF1 stimulated the
chondrogenesis of AUC 3D constructs in vitro better than the other molecules tested.
However, the chondrogenic effect was not confirmed after subcutaneous transplantation
into nude mice [67].
Easy access to the auricle cartilage makes this cell source too attractive for regenera-
tive medicine to give up trying to develop a strategy involving AUCs in cartilage defect
regeneration.

4. Bone-Marrow-Derived Mesenchymal Stem Cells

Beyond chondrocytes, BM-MSCs are a promising cell source for their proliferative
capacity and repair potentials for both cartilage and bone tissues. Historically, MSCs were
first explicitly described in the bone marrow [68,69]. In 2006, the first criteria for defining
MSCs were proposed by the International Society for Cellular Therapy (ISCT) and included
(a) proliferation ability and colony formation, (b) trilineage differentiation capacity and
(c) the presence of the cell surface markers CD271, CD44, CD105, CD73 and CD90 and
the complete absence of CD45, CD34, CD11b, CD19 and human leukocyte antigen-DR
isotype (HLA-DR) membrane proteins [70]. Since then, due to new knowledge about stem
cells, the criteria have been updated and become wider and less rigorous. According to
the new ISCT position statement, MSCs from different source tissues could have different
surface markers [71,72] and some negative markers should also be reconsidered, since some
populations are characterized, for example, by the expression of CD34 or HLA-DR [71]. In
addition, for the use of the term “stem” cells, it is important to confirm the self-renewal
of cells in vivo or in vitro [71]. Finally, the presence of immunomodulatory properties and
the characterization of trophic factors secreted by MSCs and some miRNAs have been
recommended to become mandatory criteria [71,73,74].
Despite the extensive study of bone cells, there is still no unique technique to isolate a
homogeneous population of BM-MSCs [75]. Cultivation of BM-MSCs of several passages
increases the homogeneity of a population, but not sufficiently. In addition to plastic
adhesion, negative selection of BM-MSCs was proposed; however, this increased the cost
of the procedure and the purity of the stem cell population was still low [76]. Currently,
selection using positive markers is preferable. Nonetheless, an isolated CD271-positive
population of BM-MSCs [77] included several BM-MSC subpopulations according to single-
cell RNA sequencing (scRNA-seq) analysis [78]. Thus, to date, BM-MSC heterogeneity has
made stem-cell-based therapy outcomes challenging to predict.

4.1. Fundamental Growth Factors for BM-MSC Chondrogenic Differentiation

Currently, it is well established that the coordination of vertebrate limb development
during embryogenesis depends on the interaction of the WNT and FGF families of sig-
naling proteins [79]. The presence of FGF signals (FGF2 or FGF8) specifically countered
the irreversible blocking of SOX9 expression caused by WNT activity (WNT3A) in the
limb cells of embryos [79,80]. Human BM-MSCs (hBM-MSCs) cultured with a combination
of Wnt3a and FGF2 supported extensive cell expansion over multiple passages and im-
Int. J. Mol. Sci. 2022, 23, 11169 8 of 28

proved subsequent chondrogenic differentiation, increasing GAGs and COL2 content [81].
During late-stage chondrogenesis, active WNT signaling can promote osteogenesis [82].
Addition of the WNT inhibitor IWP2 for the last weeks of culturing reduced the content of
hypertrophic and osteogenic markers in differentiating BM-MSCs [81].
Another fundamental factor involved in skeletal development is a member of the
TGFβ superfamily, a pro-chondrogenic factor in embryogenesis [83] known as growth
and differentiation factor 5 (GDF5, also CDMP-1 or BMP14). It is recognized that all
mature joint components originate from Gdf5-expressing joint progenitors condensed in
the interzone during early joint development [84,85]. GDF5 overexpression in BM-MSCs
cultured for two weeks demonstrated significant chondrogenic effects, evidenced by SOX9
and COL2 expression, without changes for COL1 and COL10 [86]. A 24-week in vivo study
on a rabbit defect model showed that an implanted GDF5-conjugated scaffold supported
the chondrogenic differentiation of BM-MSCs and stimulated the complete filling of the
defect with a tissue comprising proteoglycans and COL2 [86]. Intra-articularly injected
GDF5-transfected autologous BM-MSCs demonstrated a similar effect according to the
high histological score assessment [87]. In contrast, another publication reported that GDF5
induced the tenogenic differentiation of BM-MSCs and a significant down-regulation of
non-tenogenic marker genes, such as SOX9 [88].
The pellets of BM-MSCs demonstrated high kinetic growth for at least ten passages
during culturing with members of the TGFβ superfamily, namely, TGFβ3 and BMP2 and
synthetic glucocorticoid dexamethasone [89]. Comparative data based on studies of hBM-
MSCs cultured with different glucocorticoids demonstrated that the glucocorticoid receptor
interaction enhanced TGFβ3-induced phosphorylation of Smad2 and Smad3 [90], leading
to subsequent mTORC1/AKT pathway activation and marked potentiation of transcrip-
tional gene activity for SOX9, COL2a1 and COL10a1 [91]. Under the same conditions,
dexamethasone demonstrated a substantially weaker chondrogenic effect in combination
with TGFβ3 than glucocorticoid fluocinolone acetonide [91].

4.2. External Stimuli Supporting Chondrogenic Differentiation of BM-MSCs

He et al. [92] cultured BM-MSCs for 12 weeks on a polyglycolic acid/polylactic acid
(PGA/PLA) scaffold in a serum-free chondrogenic medium containing TGFβ1, IGF1 and
dexamethasone, providing relatively mature hyaline cartilage-like tissue with superior
mechanical properties but a high content of COL1 and COL10. However, BM-MSCs
cultured on the PGA/PLA scaffold that were implanted into an osteochondral defect in a
swine model exhibited a higher cell density than the surrounding native cartilage, with
mature lacuna structures and satisfactory integration into both cartilage and subchondral
bone tissues [92]. The authors believe an in vitro step is needed to exclude the influence of
the OA environment of the joint during the construct maturation process. This hypothesis
is supported by a study where equine BM-MSCs overexpressing anti-inflammatory IL-10
did not prevent degradation of the ECM of cartilage explants in an in vitro OA model [93].
Within the articular cartilage microenvironment, endogenous growth factors, mechani-
cal stimulation and a hypoxic environment are favorable factors that support chondrogenic
cell properties. Chondrogenic differentiation of BM-MSCs induced by a combination of
dexamethasone and TGFβ3 under hypoxia leads to increased expression of chondrogenic
markers compared to normoxia. However, transplantation of hypoxic BM-MSC cultures
into ovine articular cartilage defects produced no benefits compared to normoxia [94].
BM-MSC micro-pellets cultured under hypoxia in a chondrogenic medium were stained for
GAG but showed elevated COL10 and RUNX2 levels compared to AC micro-pellets [95].
Generally, hypoxic conditions have pro-chondrogenic and anti-inflammatory impacts on
stem cell cultures, while negative effects on cell viability are not observed [96].
Physical stimuli, such as matrix stiffness and hydrostatic pressure, are fundamental
regulators of MSC fate. Cyclic pressure affects the composition of the ECM not only in
cultured chondrocytes but also in cultured BM-MSCs, apparently through similar calcium-
dependent mechanisms [97] and chondrogenic TGFβ3 stimulation of BM-MSC cultures
Int. J. Mol. Sci. 2022, 23, 11169 9 of 28

can be modulated by the timing of the applied mechanical stimulation [98]. Applying
hydrostatic pressure followed by shear stress without growth factor supplementation
increases the expression of Sox9, Acan and Col2 in cultured hBM-MSCs, compared with
TGFβ1 supplementation without mechanical stimuli [99].

4.3. BM-MSC Transplantation Clinical Trials for Cartilage Defect Treatment

In clinical trials, intra-articular injection of autologous BM-MSCs in patients with
end-stage OA was safe and showed statistically significant improvements in pain scores
and quality of life [100]. However, three months after the BM-MSC injection, the cartilage
catabolic biomarkers largely remained unchanged in the synovial fluid and no improve-
ments in cartilage morphology were observed, based on MRI, one year later [100].
A study comparing the long-term clinical outcomes of ACI versus BM-MSC applica-
tion was undertaken on seventy-two patients [101], where implantation of pre-expanded
BM-MSCs in α-MEM supplemented with autologous serum into knee chondral defects
resulted in equivalent clinical outcomes to first-generation ACI at up to 10 years [101].
Additionally, under arthroscopy, no significant differences were found between the ef-
ficacy of the transplantation of autologous BM-MSCs combined with microfracture and
microfracture alone [102].

5. Synovial Membrane-Derived MSCs

It is currently known that heterogeneous MSC subpopulations localize in different
joint regions, including the synovial membrane producing synovial fluid for lubrication
of articular cartilage and its nourishment through diffusion. De Bari et al. (2001) were the
first to demonstrate, on postmortem samples, that MSC-like cells isolated from human
knee joint synovium by enzymatic digestion were capable of three-lineage differentiation
in vitro [103,104].
Human synovial cell research, in most cases, involves synovial cells obtained during
surgeries from synovium inflamed as a consequence of trauma or destructive joint disease,
such as OA and rheumatoid arthritis (RA). It was identified that SM-MSCs of the OA
joint maintained the ability for chondrogenic differentiation in vitro, as was the case for
SM-MSCs from a healthy knee [105]. Kohno et al. (2017) found no significant differences
between SM-MSC pellets from RA and OA groups [106]; after chondrogenic induction, both
became transparent, intensively stained for Safranin O, showing positive immunostaining
for COL2 and had comparable mRNA levels of ACAN and COL2.

5.1. Factors for Chondrogenic Differentiation in SM-MSCs

Pellets of SM-MSCs cultured in DMEM supplemented with TGFβ3, BMP2 and syn-
thetic glucocorticoid dexamethasone demonstrated high kinetic growth for at least ten
passages compared to the pellets from the BM and periosteum [10] and they revealed a
synergistic dexamethasone-dependent (within 1–10 nM) chondrogenic effect estimated by
tissue volume, proteoglycan content and COL2 expression [89]. However, a dexamethasone
concentration over 100 nM promoted a minor level of adipocyte formation in SM-MSC
cultures [89].
Studies indicated that BMP2 alone could promote chondrogenic differentiation along
with hypotrophy and endochondral ossification [107,108]. Constitutive Smad7 expression
inhibited BMP2-induced chondrogenesis in stem cells [109] and could stimulate the ter-
minal maturation of chondrocytes [110]. In a BMP2-induced chondrogenic differentiation
experiment, inhibition of Smad7 expression with short interfering RNA (siRNA) led to
significantly increased expression of chondrogenic marker genes and lower expression of
COL10 and matrix metallopeptidase 13 (MMP13) in human SM-MSC cultures [108].
Among others, BMP7 supplementation to SM-MSC cultures demonstrated chon-
drogenic differentiation of cells, as assessed by the increase in COL2, SOX9 and ACAN
expression within two weeks [111]. After in vitro tests, BMP7-loaded fibrous biodegradable
scaffolds with SM-MSCs were implanted into rabbit osteochondral defects. Complete
Int. J. Mol. Sci. 2022, 23, 11169 10 of 28

healing with neocartilage tissue abundant with proteoglycans and COL2 was observed six
weeks after treatment [111].
A recent study showed that tetrahedral frame nucleic acids (tFNAs), a novel DNA
nanophase material, promoted the chondrogenic differentiation of SM-MSCs through
Smad2/3 phosphorylation in vitro [112], which affected SM-MSC proliferation by acti-
vating the Wnt/β-catenin pathway and enhanced the migration activity of SM-MSCs
in vitro [112]. Intra-articular injection of tFNAs in rabbits promoted more rapid cartilage
defect regeneration with less fibrous tissue than in the control group after 12 weeks. Such
outcomes suggest that tissue restoration was provided by the impact of tFNAs on the
number of BM-MSCs that migrated to the defect area [112].

5.2. Different Cell Subpopulations Labeled in Synovium

Synovium could be histologically classified into the lining layer, sub-lining layer and
perivascular region consisting of several types of cells [113,114]. An SM-MSC popula-
tion that was found was not homogeneous and MSC-like cells were detected in all three
regions. The obtained hSM-MSCs demonstrated different patterns of proliferation and
differentiation in vitro, depending on their location in the synovium [114].
Recently, specific markers to separate hSM-MSCs according to their location in the
synovium were identified [114]. After immunostaining human OA synovium sections, the
markers selected were CD55+ CD271− for synovial cells in the lining layer, CD55− CD271−
in the sub-lining layer and CD55− CD271+ CD90+ in the perivascular region [114]. The
cells from the different synovial regions were cultured in DMEM supplemented with
TGFβ3 and BMP2 and showed different proliferation and chondrogenic differentiation
patterns in vitro [114]. The perivascular synovium cells showed large-sized pellets and
higher expression levels of SOX9, ACAN and, unfortunately, COL10 mRNA at the end of
chondrogenic cultivation [114].
Fibroblast activation protein-α (FAPα) was recently confirmed as a biomarker of tissue
inflammation in the lining and sub-lining layers of the synovial membrane and to be
associated with a pathogenic synovial cell phenotype [115,116]. The FAPα-expressing
protein in the combination of podoplanin (PDPN) and CD90 (also known as thymus cell
antigen 1, THY1) enabled synovial cells in the sub-lining layer (FAPα+ PDPN+ THY1+ ) to be
distinguished from the synovial cells in the lining layer (FAPα+ PDPN+ THY1− ) in human
RA tissue [116]. Injecting PDPN+ FAPα+ THY1− or PDPN+ FAPα+ THY1+ cells into the
inflamed ankle joint of mice is a different method for investigating arthritis degeneration
processes. Injection of PDPN+ FAPα+ THY1+ cells resulted in more severe joint swelling
with higher immune cell infiltration, with little effect on joint tissue destruction. In contrast,
the injection of PDPN+ FAPα+ THY1− cells selectively mediated bone and cartilage damage,
with little effect on inflammation [116].

5.3. Clinical Trials of Autologous SM-MSC Transplantation into Cartilage Defects

Most in vivo research reported successful cartilage repair following SM-MSC trans-
plantation, despite the diversity of laboratory animals and cell harvesting techniques used,
strategies of cell delivery and methods for outcome estimation [117]. A 2015 clinical trial
of autologous SM-MSCs was undertaken [118] using SM-MSCs expanded in α-MEM con-
taining 10% autologous human serum for 14 days. The solution with expanded SM-MSCs
was syringed into an osteochondral defect under arthroscopic control. The Lysholm score
increased after treatment in each patient. Fibrous tissue was observed at the surface in
three of four biopsy specimens analyzed. The sequential MRI showed that at six months
and six years later, in contrast to the bone defect part, the cartilage defect was incompletely
filled with tissue [118].
In 2018, implantation of a scaffold-free tissue-engineered construct generated from
autologous SM-MSCs was performed on five patients in Japan [119]. SM-MSCs were
enzymatically extracted from arthroscopically taken synovium and cultured for 2–3 weeks
in monolayers with ascorbic acid 2-phosphate (Asc-2P), which significantly increased the
Int. J. Mol. Sci. 2022, 23, 11169 11 of 28

self-supporting mechanical properties of the cartilage-like construct without containing

artificial scaffolding [119,120]. The tissue-engineered construct was highly adherent to the
normal cartilage; therefore, implantation into the defect without using sutures or fixation
glue was performed. Forty-eight weeks post-surgery, according to MRI assessments and
biopsy specimen histology, defect repair with abundant proteoglycan tissue and without
hypertrophy occurred in all cases. The patients reported significant clinical improvement
at the final 24-month follow-up [119].

6. Adipose-Derived Stem Cells

The essential features of adipose tissue as a cell source are the excellent stem cell yield
from fat and, of course, the minimal risk for patient health while obtaining a relatively
large amount of tissue [121]. Adipose-derived stem cells (ADSCs) express a profile of
cell-surface molecules, similar but not identical to that of BM-MSCs [122], and ADSCs may
be differentiated into mesenchymal lineages [123]. A study on rabbits demonstrated that
the expansion properties and chondrogenic potential reduced with age for BM-MSCs, but
not for ADSCs [124].

6.1. Clinical Trials of ADSC Intra-Articular Injections for Cartilage Damage Treatment
The attractive availability of ADSCs has initiated many clinical studies of the regen-
erative possibilities of ADSCs injected into joints to restore articular cartilage tissue lost
due to OA. A meta-analysis in 2021 [125] demonstrated a statistically significant improve-
ment in WOMAC (Western Ontario and McMaster Universities Osteoarthritis) scores after
intra-articular injections of ADSCs in patients with knee OA [126], providing improved
joint function and pain reduction between two months and two years after treatment [125].
However, the therapeutic effect duration is still unknown due to a lack of long-term studies.
In most studies, intra-articular ADSC injections were performed on unspecified joint spaces.
Only six studies included a control group and only two conducted a second-look arthro-
scopic assessment and subsequent histological analysis [125]. Overall, this confidently
demonstrates the safety of ADSC therapy, rather than its effectiveness, in OA treatment.
A Phase IIa proof-of-concept randomized, single-blind clinical trial showed that after
six months, intra-articular injection of autologous ADSCs in combination with microfracture
and hyaluronic acid provided clinical improvement for patients with knee cartilage defects
compared to microfractures alone [127]. The study outcomes were supported by decreased
WOMAC total indexes and radiological, arthroscopic and histological evaluation during the
second-look arthroscopy [127]. Complete recovery of hyaline cartilage was not observed.
However, the results are sufficiently promising to encourage larger scales of randomized
clinical trials.

6.2. Practices to Improve the Chondrogenic Potential of ADSCs

To improve the chondrogenic potential in vitro, ADSCs encapsulated with fibrin and
thrombin in atelocollagen (COL1 fibers without antigenic active telopeptides) gel were
cultured in chondrogenic differentiation medium supplemented with dexamethasone,
insulin–transferrin–selenium (ITS) and TGFβ3 over 21 days [128]. Histological analysis
of the atelocollagen scaffolds seeded with ADSCs revealed lacunae-like structures and
glycosaminoglycan accumulation. Cartilage-matrix-related proteins were abundant and
hypertrophic markers were at low levels [128].
In most cases, strategies to demonstrate the ability of ADSCs to undergo chondrogenic
differentiation incorporate a high-cell-density culture supplemented with TGFβ1. Hypoxic
cell expansion is a widespread practice to facilitate chondrogenic differentiation too. ADSC
pellets differentiated in a chondrogenic medium with dexamethasone and TGFβ1 after
hypoxic expansion (1% of O2 ) increased the production of the chondrogenic markers (SOX9,
COL2) in vitro, but not to the extent that BM-MSC pellets did [129]. At the same time, the
hypoxic expanded ADSC- and BM-MSC-derived chondrogenic pellets showed COL10 gene
Int. J. Mol. Sci. 2022, 23, 11169 12 of 28

up-regulation, both in vitro and, after intra-articular injection, in an animal knee defect
model [129].
The culturing of ADSCs within slowly degraded gelatin scaffolds, organized like
hyaline cartilage lacunae [130,131], led to up-regulation of SOX9 and COL2 expression
and GAG production two weeks after chondrogenic induction compared to randomly
organized scaffolds. As a result, it was suggested that the scaffold geometry influenced the
chondrogenic differentiation of ADSCs [130]. Additionally, MRI inspection, histological
examinations and IHC staining of the ADSC-seeded organized scaffold samples implanted
subcutaneously into nude mice showed a high chondrogenecity potential in implants [130].

6.3. Alternative Strategies for Treating Joint Defects with ADSCs

An alternative strategy for treating joint defects involved impeding the terminal hy-
pertrophic differentiation of ADSCs using the ECM protein matrilin-3 (MATN3) [132] as
a component binding to BMP2 and preventing BMP2 downstream signaling, required
for Col10 expression in chondrocytes [133]. ADSCs encapsulated with Matn3 in hydrogel
showed favorable effects by increasing the chondrogenic marker and decreasing the hy-
pertrophy marker mRNA and protein expression in vitro. In vivo, MATN3-loaded ADSC
spheroids prevented subchondral bone sclerosis in a chronic OA mouse model [132].
Recently, a strategy was proposed to simplify the cell therapy procedure and remove
steps associated with the enzymatic isolation, cell expansion and scaffold fabrication.
Autologous fractionated adipose tissue (so-called ECM/SVF-gel in the study), a gel-like
substance consisting of adipose tissues mechanically destroyed by simple mechanical
shifting between two syringes, was applied to cartilage injury repair in combination with
a microfracture in a rabbit model [134]. Twelve weeks after the ECM/SVF-gel injection,
the knee joints were evaluated using MRI, macroscopic observation, histology and IHC.
It was reported that the ECM/SVF-gel could improve articular cartilage regeneration,
integration with surrounding normal cartilage and the expression of COL2 compared to
the microfracture treatment alone [134].
Despite the achieved progress of ADSC applications in clinical studies, cell therapies
involving ADSCs require further method modifications to improve differentiation efficacy.

7. Periosteum- and Perichondrium-Derived Stem Cells

Skeletal long bone formation starts from chondrogenic cell condensation and is subse-
quently transformed into differentiated chondrocytes surrounded by a thin tissue sheath,
termed the perichondrium [135]. The terminal step of the chondrocyte differentiation
process is hypertrophy, after which the cartilage is replaced by bone tissue. The perios-
teum is the new sheath of flattened, elongated cells surrounding the bone region. The
perichondrium and periosteum are reported to have similar morphology, composed of
two layers, the outer fibroblastic layer and the inner cambial layer, containing a niche of
pluripotent stem cells with both chondrogenic and osteogenic capabilities of differentia-
tion [136,137]. The collagen fiber orientation in the perichondrium and periosteum concurs
with the assessed directions of tissue growth [138]. In situ hybridization analysis of genes
from embryonic-day-12 tissue revealed that the perichondrium and periosteum are distinct
tissues at the molecular level [139]. Both the perichondrium and periosteum participate in
limb growth regulation by serving as sources of signaling molecules involved in prolifer-
ation, differentiation and hypertrophy of the chondrocytes and respond to signals from
underlying cells [140].

7.1. Periosteum
7.1.1. Periosteal Adult Stem Cell Populations
The periosteum is a double-layer thin membrane that is well vascularized and in-
nervated. It envelopes nearly every bone in the body [141], except for the intra-articular
surfaces and sesamoid bones [142]. The inner cambium layer of the periosteum contains
a heterogeneous cell population, including fibroblasts, endothelial cells, mast cells, peri-
Int. J. Mol. Sci. 2022, 23, 11169 13 of 28

cytes and several specific periosteal stem cell (PSC) populations that have been recently
identified [143–146], each with distinct physiologic functions [144]. PSCs display clonal
multipotency and self-renewal [144,147]. Single-cell and bulk transcriptional profiling
shows that PSCs are distinct from other skeletal stem cells and mature mesenchymal
cells [144]. The periosteum plays the role of being a reservoir of osteoblasts and osteochon-
droprogenitors [144,148,149]. It is responsible for the radial bone growth and turnover of
the bone cortex throughout life and orchestrates the healing of bone fractures [143,144,146].
Notably, the osteoblastic potential of the periosteum differs with location [150], with the
calvarial periosteum demonstrating less osteogenic potential than the periosteum covering
the tibia [151].
Modern genetic engineering techniques allow for cell labeling in the periosteum for
detailed research of periosteal adult stem cell populations in vivo and in vitro. The lineage
tracing method demonstrated that the adult periosteum contains αSMA-positive cells that
are long-term, slow-cycling, self-renewing osteochondroprogenitors, identified by cell sort-
ing and scRNAseq methods as different mesenchymal populations to those present within
the bone marrow [149]. Using a fluorescent reporter adult animal model, most Prx1-labeled
cells of the periosteal cambium layer were reported to be periosteal osteochondroprogeni-
tor cell populations converting to osteogenic cells in response to mechanical stimulation,
directly sensed via the primary cilia [152]. Changes in the mechanical properties, such
as loss of the periosteum’s baseline stress, result in significantly increased crimping of
collagen in the periosteum’s fibrous layer and changes in the nucleus shape of cells in the
cambium layer. These signals influence the equilibrium of the periosteal stem cell niche
and change the fate of the periosteum-derived cells [153].

7.1.2. Periosteal Graft Transplantation

The efforts of periosteal graft transplantation began a relatively long time ago. In 1982,
a rabbit study investigated articular cartilage defects using periosteal grafts and, after one
year, the defects were filled with white glistening tissue, very similar to normal hyaline
cartilage [154]. Research on patients followed clinically through arthroscopic examination
and radiography showed similar outcomes [155]. Unfortunately, the long-term outcomes
(6–9 years) of periosteal transplantation deteriorated with time and the method failed to
produce hyaline cartilage and prevent the development of arthrosis [156]. The promising
results after periosteal grafting obtained in 1980 could be due to the young age of the
rabbits (6 months old) [154] and patients (16–34 years old) [155] and the lack of techniques
for estimating results that are available now. As a result, studies on rabbits by O’Driscoll
et al. [157] indicated better regeneration when the periosteal graft was sutured into the
defect with the cambium layer facing the joint space, not the subchondral bone. Under
continuous passive motion after transplantation, the periosteal grafts produced tissue
containing predominantly Col2 [157].
For the ACI method, during arthrotomy, the joint defect was covered with a periosteal
flap taken from the proximal medial tibia, under which chondrocytes were injected [158];
however, the results of transplantation with the periosteum were inconsistent. Additionally,
the periosteal tissue was too thin and fragile for surgical manipulations [159]. In the clinic
trials, the incidences of transplant hypertrophy were reported to be the highest in the
group of patients treated with periosteum-covered ACI compared to other ACI technique
variations [160].
Recently, an adult periosteum was reported that had a SOX9-positive skeletal pro-
genitor population capable of giving rise to osteoblasts, mature cortical osteocytes and
chondrocytes [161]. However, applying periosteum grafts for cartilage repair retains a risk
of subsequent ossification [162] through endochondral mechanisms, as it naturally happens
during long bone fracture healing [147]. To understand the tissue perfusion impact on the
periosteum graft ossification along with cartilage regeneration, a vascularized pedicled
periosteal flap and a non-vascularized periosteal graft were applied to a rabbit knee de-
fect model for eight weeks, with anti-ossification reagents, such as fulvestrant and IL1b,
Int. J. Mol. Sci. 2022, 23, 11169 14 of 28

injected every two weeks to prevent bone formation [163]. Improved defect regeneration
was observed in the vascularized periosteum flap group with a higher COL2 expression
and proteoglycan content compared to the non-vascularized periosteum group and the
groups with anti-ossification medication [163].
Theoretically, periosteal flap transplantation could be adapted so it can be performed
arthroscopically. However, the periosteum alone has been shown not to repair defective
hyaline cartilage. Adult human periosteum-derived cell cultures have a high proliferation
rate with stable retention of the TGFβ1-induced chondrogenic differentiation potential
along 15 passages independently of donor age [164]. Such in vitro characteristics have made
stem cells from the periosteum an interesting precursor cell source for tissue engineering
constructs. Key aspects of developing methods for repair of articular cartilage could
potentially be based on tissue engineering structures seeded with chondrocyte precursors
from the cambium layer.

7.2. Perichondrium
The perichondrium is a thin layer of dense connective tissue surrounding the nasal,
costal and tracheal hyaline cartilage and the auricular elastic cartilage in adults. It leaves
only the articular cartilage surfaces uncovered. The first investigations of the chondrogenic
regeneration potential of the adult perichondrium occurred during the 1970s [165–168].
At that time, costal perichondrium transplantation reconstruction had been used for ar-
ticular surfaces in small-finger joints damaged by infection or trauma. Decades later, an
observational study evaluating the long-term results was published [169]. The injured
finger joints restored by perichondrium transplants remained essentially pain free and
mostly well functioning without the need for additional surgeries up to 41 years after the
procedure [169], which encourages us today.

7.2.1. Perichondrium Transplantation

About twenty years ago, chondrogenic stimulation of perichondrium grafts in vitro
promoted by synovial fluid collected from non-inflammatory joints was shown. Then,
costal and auricular perichondria from adult rabbits were transplanted intra-articularly
and intra-abdominally for eight weeks [170]. The morphology analysis of graft sections
demonstrated that better chondrogenesis-like processes were found in rib perichondrium
specimens placed in the knee joint, in contrast to grafts placed intraperitoneally. However,
the scarcity of analysis methods in the 1990s meant that representative data for this outcome
could not be provided [170].
The costal perichondrium contains a unique niche housing pro-chondrogenic cells,
contributing to costal cartilage regeneration [171]. The rib perichondrium and periosteum
were extracted from a rat with green fluorescent protein (GFP) transgene expression and
transplanted into articular cartilage defects of regular laboratory rats [172]. Tracing of
the GFP cells showed newly formed tissues that originated from the perichondrial or
periosteal transplants, which acquired chondrocyte marker expression patterns at day 112
post-surgery. However, in contrast to the perichondrium, the transplanted periosteum
expressed COL1 throughout the experiment and lost SOX9-expressing cells two weeks
after the surgery [172].
In 2002, clinical applications of perichondrial grafts to isolated cartilage defects did
not show advantages compared to the drilling procedure in the 10 years of follow-up. Irreg-
ularities, such as calcifications of the graft, subchondral sclerosis or pain, were seen to the
same extent in both groups [173]. New reports regarding transplantation of perichondrium
flaps/grafts into articular cartilage defects have not yet emerged.

7.2.2. Perichondrium-Derived Adult Stem Cells

The perichondrium has been used as a source of stem cells for follow-up cell ther-
apy procedures. In vitro tests of auricular PchDCs obtained through fibronectin adhesion
showed cell expression of MSC-appropriate positive surface markers, high colony-forming
Int. J. Mol. Sci. 2022, 23, 11169 15 of 28

efficacy and proliferative ability and mesenchymal trilineage differentiation potential [174].
PchDCs isolated by collagenase digestion from auricular and tracheal perichondria demon-
strated high proliferation and hyaline cartilage-like matrix protein production according
to histology and IHC assessments [175]. Chondrogenic stimulation resulted in porcine
pellets of auricular or tracheal PchDCs returning high expression rates of COL2 and ACAN
without a trace of COL10 and elastic fibers [175].
Cells grown from explants of rabbit rib perichondria in culture media with TGFβ1
were shown to enhance proliferation rates and Col2 gene expression [176]. Similar results
were demonstrated for cultures of aged perichondrium-derived cells (PchDCs) [177].
Finally, another research strategy to consider is the transplantation of rat rib PchDCs
infected by adenoviral vectors containing sequences of BMP2 and/or IGF1 [178]. Cells
producing both growth factors placed into a joint defect generated repair tissue with a
hyaline morphology and high proteoglycan and COL2 contents. Transplantation of unin-
fected perichondral cells in the control group resulted in insufficient repair two weeks after
the surgery. However, transplantation of BMP2-producing cells could lead to osteophyte
formation if they partially dislocate to the joint margins [178].
Considering the above data, research undertaken with high caution and responsi-
bility may develop cell therapies for restoring hyaline cartilage based on periosteal cells.
Currently, the perichondrium has lost the interest of researchers as a cell source for regener-
ation therapies.

8. Dental Pulp Stem Cells

In the central cavity of each tooth, there is an unmineralized tissue, dental pulp, which
contains vast numbers of different cell types, including a stem cell niche [179]. Dental pulp
stem cells (DPSCs) originate from neural crest cells and demonstrate MSC features, such as
colony formation in vitro [180], a rapid proliferation rate, a strong capacity to self-renew
and multiple differentiation potentials [181,182]. DPSCs have the classical MSC surface
markers and lack CD11b, CD14, CD19, CD34, CD45, CD79a and HLA-DR transmembrane
proteins [182,183]. The DPSC harvesting method is minimally invasive [184–186]. DPSCs
are obtained from collected teeth by enzymatic digestion or by the explant method [187].
Additionally, DPSCs have impressive cryopreservation abilities and are suitable for banking
stem cells collected after routine teeth extraction [188].

8.1. Conversion of DPSCs to Chondrocytes

DPSCs have been applied in articular hyaline cartilage tissue regeneration and tissue
engineering [189]. It was demonstrated that human DPSCs suppressed OA macrophage
activation in a direct co-culture system in vitro and attenuated damage to the articular
cartilage in a rabbit knee OA model in vivo [190]. Local injection of DPSCs in a model
of rats with temporomandibular joint (TMJ) arthritis relieved hyperalgesia and synovial
inflammation and attenuated cartilage matrix degradation, according to histologic staining
and µCT scanning results [191].
Attempts to directly stimulate the conversion of DPSCs into chondrocytes have also
been made. Chondrogenic differentiation of DPSCs in vitro was achieved using a differen-
tiation medium with ITS and ascorbic acid. DPSCs acquired a more rounded morphology
and had higher amounts of COL2 and ACAN, with no expression of COL1. However, it
could not reach the same expression levels of ECM proteins obtained by primary articular
chondrocyte cultures in the same differentiation medium [192].
In a recent in vitro study, Longoni et al. cultured DPSC pellets for 21 days in a
chondrogenic medium with ITS and dexamethasone supplemented with different growth
factors, such as TGFβ1, TGFβ3, BMP2, BMP6, BMP7 and IGF1, in various combinations.
Nevertheless, cells in the pellets had a spindle fibroblast-like morphology and did not
form typical hyaline cartilage lacunae and the ECM mostly had COL1 without COL2 and
ACAN expression [181]. To improve the chondrogenic differentiation outcomes, human
DPSCs were cultured in a 3D organization based on 3% agarose wells in a commercial
Int. J. Mol. Sci. 2022, 23, 11169 16 of 28

chondrogenic medium supplemented with TI, TGFβ1 and a recombinant analog of IGF1.
The synthesis of ACAN, COL2 and less COL1 was then immunodetected in microtissues
formed by DPSCs [193].
Some peculiar efforts have been undertaken to improve the chondrogenic differentia-
tion of DPSC cultures. The chondrogenesis-related genes were significantly up-regulated
and GAG synthesis was increased in the DPSC monolayer on day 21 when the chondro-
genic medium was accompanied by taurine. Elastin expression in the culture was also
observed [194]. To improve the chondrogenic differentiation of the human DPSCs, an
extract of sonicated lactic acid bacteria was added to the chondrogenic medium, including
ITS, dexamethasone and TGFβ1 [195]. This extract enhanced the expression of early stage
chondrogenic marker genes and hypotrophic marker genes for the first week in DPSC
pellet cultures compared to the control group [195].

8.2. TGFβ3 as Chondrogenic Initiator for DPSC Cultures

More impressive results have arisen from research regarding the co-culturing of hu-
man costal chondrocytes (CCs) with human DPSCs in pellets [196]. Data demonstrated
that CCs could supply a chondro-inductive environment that promoted the chondrogenic
differentiation of DPSCs. However, CCs alone could not stop the mineralization of the
pellets, so the exogenous growth factor FGF9 was added to prevent hypertrophic differ-
entiation and to stabilize the permanent cartilage-like phenotype of the DPSC and CC
co-culture. The co-culture pellets with added FGF9 in a chondrogenic medium with TGFβ3
demonstrated more intense Alcian blue and COL2 IHC staining. In contrast, the DPSCs
cultured in regular DMEM formed only fibrous tissues and the DPSCs cultured in the
chondrogenic medium with TGFβ3 but without FGF9 showed the highest expression level
of COL10 [196]. The same study showed that biodegradable PGA scaffolds seeded with
DPSCs preliminarily co-cultured with CCs formed homogeneous cartilage-like tissue with
a similar structure and high matrix deposition eight weeks after transplantation into the
backs of nude mice [196].
Transduced human DPSCs with the highly expressed chondrogenic growth factor
TGFβ3 were seeded on a poly-L-lactic acid/polyethylene glycol electrospun fiber scaffold
and implanted under the skin in nude mice [197]. Under TGFβ3 influence, transplanted
DPSCs showed the presence of chondrogenic markers (COL2, SOX9 and ACAN), as verified
by IHC staining and mRNA level measurement. An in vivo study of human DPSCs seeded
onto a COL2/COL3 scaffold in a mini-pig knee defect model was conducted, but there
were no representative results [198].
DPSCs have potential as an alternative source of adult MSCs for joint cartilage regener-
ation. However, there are still no valuable pre-clinical or clinical trial results using DPSCs.

9. Extracellular Vesicles (EVs) as a Tool for Cell Fate Manipulation

Another way to manipulate the fate of cell populations is to use extracellular vesicles
(EVs), first discovered in the last century [199,200]. EVs are a heterogeneous population
of lipid bilayer-enclosed particles produced by different cell types as an essential carrier
of cell-to-cell communication mediators [201], implemented through the transfer of a
variety of proteins, lipids and RNA cargos. Due to their unique biological roles, EVs
coordinate complex processes, such as development, inflammation, tumorigenesis and
even maintenance of a stem cell niche [201]. EVs release responses to external influences,
such as cyclic hydrostatic pressure [202], oxidative stress [203] and hypoxia [204]. Types
of EVs are distinguished for subclasses by their fundamental mechanisms of biogenesis,
diversity of their composition and size [205] and selective cargo loading [206,207]. The
major subclasses of EVs are composed of membrane-derived vesicles called microvesicles
(MVs) (100–1000 nm) as a result of the outward budding of the plasma membrane and
exosomes (50–150 nm) originating from endosomal multivesicular bodies [205,208].
Most commonly for research purposes, EVs are isolated from a culture medium using
centrifugation and filtration methods, enzyme-linked immunosorbent assay (ELISA) and
Int. J. Mol. Sci. 2022, 23, 11169 17 of 28

different exosome isolation kits that are commonly found. However, the homogeneity of the
isolated particles remains low and current methods cannot efficiently separate the different
types of EVs extracted [209]. Over the past two decades, EVs have attracted intense interest
regarding their capability to regulate stem cell self-renewal and differentiation [201]. It was
demonstrated that soluble mediators released by stem cells, rather than the cells themselves,
could provide significant therapeutic benefits [208,210] that open up great opportunities
for their application in regenerative medicine.

9.1. Chondroprotective Effects Mediated by EVs

The immunomodulatory capacity of EVs has been extensively recognized. Using
in vitro models of OA based on chondrocytes treated with pro-inflammatory factors, the
immunosuppressive properties of EVs derived from BM-MSCs [211,212], ADSCs [213]
and embryonic stem cell cultures [214] were shown to decrease the expression of catabolic
(ADAMTS5 and MMP13) and pro-inflammatory markers (COX2, iNOS and interleukins)
and increase levels of COL2 and ACAN. These outcomes were probably mediated by
the inhibition of the nuclear factor kappa-light-chain-enhancer of activated B (NF-kB)
pathway [215].
EVs derived from TGFβ3-pretreated BM-MSCs exerted chondroprotective and anti-
inflammatory functions in OA-like murine chondrocytes, confirmed by re-inducing the
expression of chondrocyte markers while inhibiting catabolic (MMP13, ADAMATS5) and
inflammatory (iNos) markers in vitro [210]. Additionally, intra-articular injection of BM-
MSC-derived EVs had a moderate protective effect on cartilage and bone from OA damage
in a collagen-induced arthritis model in mice [210].
Studies involving DPSC-derived EVs are much less common. A single intravenous
injection of conditioned medium (CM) from human DPSCs in a mouse model of RA
markedly attenuated tissue destruction of the ankle joint compared to the control and even
the group with intravenous injection of CM from BM-MSCs [216].

9.2. EV-Mediated Chondrogenesis

EVs can promote chondrogenesis through cell-to-cell transfers of EVs’ natural cargo.
Data obtained from a rabbit TMJ OA model also confirmed that human BM-MSC-derived
EVs could promote cartilage reconstruction mediated by increasing factors related to
cell proliferation (PCNA protein) and cartilage matrix formation (COL2, ACAN) [217].
Recently published proteomic analysis of EVs obtained from adipose tissue, BM and
synovium revealed their distinct protein profiles [218]. Simultaneously, BM-MSC culture
studies reported comparable chondrogenic effects implemented by EVs of different origins.
According to gene ontology (GO) analysis, the three types of EV proteomes differentially
regulate biological processes, such as ECM organization, cartilage condensation, cell growth
and cell migration. Additionally, there was no apparent difference among the three EV-
treated groups of BM-MSCs encapsulated in Matrigel with the chondrogenic factor TGFβ3
in a nude mouse subcutaneous transplantation model [218].

9.3. Exosomal microRNAs as Therapeutics

Mechanisms of EVs’ actions of various origins are not known for certain. However, an
important role is increasingly evident in the presence of various regulatory microRNAs,
which affect different signaling pathways of target cells. To date, it has become possible to
use genetically engineered or chemically modified exosomes, a subclass of EVs, for targeted
delivery of therapeutics to specific types of cells or tissues [219]. For instance, exosomes
derived from chondrocyte cultures induced chondrogenic differentiation of BM-MSCs by
transferring exosomal miR-8485 to regulate the Wnt/β-catenin pathway [220]. Equally,
exosomes isolated from rabbit chondrocytes demonstrated stimulation of chondrogenesis
with minimal hypertrophy in ACPCs harvested via differential adhesion to fibronectin and
implanted subcutaneously into mice [221]. In vitro treatment of hBM-MSCs with exosomes
Int. J. Mol. Sci. 2022, 23, 11169 18 of 28

derived from miR-95-5p-overexpressing human chondrocytes promoted cartilage-specific

gene expression in TGFβ3-induced chondrogenesis [222].
The response of human chondrocyte cultures to SM-MSC-Exo application was effective
proliferation and migration. However, the drawback was the striking inhibition of Sox9
and its downstream genes, including Acan and Col2, via the WNT5a and WNT5b factors
enriched in exosomes [223]. This side effect was blocked through exosomes isolated from
SM-MSCs transfected with miR-140-5p identified as a cartilage matrix modulator. SM-MSC-
Exos with miR-140-5p enhanced the proliferation and migration of chondrocytes without
damaging SOX9, ACAN and COL2 secretion in vitro. They mitigated the severity of the
damage to knee articular cartilage in a rat OA model in vivo [223].
The application of EVs yields a promising, cell-free field of regenerative medicine
without apparent adverse effects, such as immunogenicity or tumorigenicity. However,
in addition to the unclear molecular mechanism of EVs outcomes, the formal difficulties
associated with EV heterogeneity, lack of unified isolation protocol and method of quality
attestation need to be addressed.

10. Conclusions
Today, the idea of applying autologous adult stem cells for cartilage regeneration
is the most-developed concept in regenerative medicine. The different types of adult
stem cells obtained from variable tissue sources have different advantages in accessibility,
immunogenicity, proliferative and chondrogenic abilities. Clinical trials of intra-articular
injections of undifferentiated autologous stem cells did not provide the desired outcomes;
in most cases, there was no significant difference in efficiency between injected stem cells
and surgical practices, such as microfracturing.
Different stem cell populations respond to chondrogenic stimuli in various ways, so
strategies initiating chondrogenicity could vary from one adult stem cell population to
another. Very likely, there is no unique differentiation method suitable for all or several
stem cell populations of a patient. Chondrogenic factors, such as growth factors, regulatory
molecules, the geometry of the scaffolds used, cell density seeded and physical stimuli, can
independently control the fate of stem cells but can give a synergistic effect in combinations.
Heterogeneity within the adult stem cell population from one tissue source makes more
complicated the assessment of the chondrogenic efficiency of these factors. It is necessary to
determine the specific markers that separate the internal cell subsets of one type of stem cell
according to their chondrogenicity and obtain the optimal ones. Single-cell transcriptome
sequencing has become a technique helping to perform cell clustering based on specific
markers and compare cell subsets defined. The development of various external options to
guide the fate of stem cells enables the evolution of joint damage therapies and investigates
the mechanisms of cellular interactions.

Author Contributions: Writing—original draft preparation—E.V.M., A.D.K., I.A.R. and P.D.K.;

writing—review and editing, E.V.M., A.D.K., I.A.R. and P.D.K.; supervision, P.T. All authors have
read and agreed to the published version of the manuscript.
Funding: Supported by the Russian Science Foundation (Grant No. 21-75-10082 to E.V.M.).
Institutional Review Board Statement: This review paper does not require the ethical approval.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

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