Umbilical Cord Mesenchymal Stromal Cells for Cartilage Regeneration Applications

Chondropathies are increasing worldwide, but effective treatments are currently lacking. Mesenchymal stromal cell (MSCs) transplantation represents a promising approach to counteract the degenerative and inflammatory environment characterizing those pathologies, such as osteoarthritis (OA) and rheumatoid arthritis (RA). Umbilical cord- (UC-) MSCs gained increasing interest due to their multilineage differentiation potential, immunomodulatory, and anti-inflammatory properties as well as higher proliferation rates, abundant supply along with no risks for the donor compared to adult MSCs. In addition, UC-MSCs are physiologically adapted to survive in an ischemic and nutrient-poor environment as well as to produce an extracellular matrix (ECM) similar to that of the cartilage. All these characteristics make UC-MSCs a pivotal source for a stem cell-based treatment of chondropathies. In this review, the regenerative potential of UC-MSCs for the treatment of cartilage diseases will be discussed focusing on in vitro, in vivo, and clinical studies.


Introduction
Chondropathies are a group of cartilage diseases that deviate from or interrupt the normal structure and function of cartilage, including osteoarthritis (OA), achondroplasia, spinal disc herniation (SDH), relapsing polychondritis, cartilage tumor (CT), and chondrocalcinosis [1]. There are over 100 types of arthritis. The most common forms are OA (degenerative joint disease) and rheumatoid arthritis (RA, autoimmune form of arthritis). OA is the most well-known chondropathy in the world, affecting the health of 343 million of people, while RA affects 14 million of people [2]. OA is a multifactorial and complex degenerative joint disease characterized by agerelated "wear and tear," chondrocytes' poor response to growth factors, altered biomechanical properties of articular cartilage, mitochondrial dysfunction, oxidative stress, and inflammation [3]. The degenerative lesions in cartilage are secondary to inflammation associated with hyperplasia and chondrocyte apoptosis [4]. Increasing age is linked to a reduction in subchondral blood vessels resulting in cartilage-related physiological and biochemical anomalies [5]. This pathological process results in secondary joint fibrosis, stiffness, pain, and decreased function, leading to a poor quality of life. Risk factors for chondropathies include trauma, genetics, age, sex, obesity, and degenerative pathology. The biological mechanisms of chondropathies remain largely unknown, and there is no effective way to treat the cartilage damage because of its nature.
Cartilage is a supportive connective tissue, and it has a dense and highly organized extracellular matrix (ECM) embedding chondrocytes [6]. Three types of cartilage tissue There are two main concepts for UC-MSCs' contribution to cartilage repair: preventing the degradation of cartilage, through the secretion of bioactive factors, and/or the differentiative potential of UC-MSCs to become chondrocytes. Several in vitro and in vivo studies indicated that UC-MSCs can play crucial roles in cartilage repair and regeneration by several mechanisms including (i) migration and homing, (ii) adaptation to cartilage hypoxic and nutrient-poor environment, (iii) chondrogenesis differentiation potential and promotion of survival, proliferation and differentiation of endogenous MSCs, (iv) synthesis and prevention of cartilage ECM degradation, and (v) anti-inflammatory and immunomodulatory properties.

Homing and Migration.
MSCs exhibit certain capabilities of the homing of local mature leukocytes to inflammatory sites, such as rolling and adhesion [13]. Migration and homing ability into injured sites are considered as the primary steps for tissue repair in regenerative medicine. Different molecules mediate cell retention or mobilization such as adhesion molecules (E and P-selectin), integrins (particularly α4β1), stromal-derived factor 1 (SDF-1), and its receptor CXC chemokine receptor 4 (CXCR4) [13]. In addition, other factors play a key role in damaged joints homing such as CXCR1 and CXCR2, CC chemokine receptor 1 (CCR1), and monocyte chemoattractant protein 1 (MCP-1) through its receptor CCR2, vascular endothelial growth factor receptor 1 (Flt-1), plateletderived growth factor receptor α (PDGFR-α, CD140a), PDGFR-β (CD140b), and their respective ligands IL-8, macrophage inflammatory protein 1α (MIP-1α), placenta growth factor (PlGF), and PDGF [14]. In Table 2, we reported different proteins involved in homing and migration of UC-MSCs in comparison with those expressed by BM-and AT-MSCs. In particular, BM-and AT-MSCs show similar expression pattern. On the other hand, based on the quantitative data reported in literature, UC-MSCs express higher levels of different proteins than BM-and AT-MSCs such as HGF, IL-8, IL-1RA, IGF-1, ICAM-1, bFGF, MCP-1, MIP-1β, PDGF-AA, PDGF-AB, PDGF-R, CXCR4, CCR2, VEGF-A, and VEGF-1. Interestingly, unlike cells from BM-and AT-MSCs, UC-MSCs express integrin α4 and MIP-1β suggesting their strong ability in homing and migration. In addition, UC-MSCs show less hematopoietic effects than BM-and AT-MSCs (low levels of SDF-1 and VCAM-1).
Increased levels of SDF-1, MCP-1, IL-8, MIP-1 α, PlGF, and PDGF were found in synovial fluid of OA and RA patients [14]. Shen et al. demonstrated that UC-MSCs secrete growth factors and chemokines which may contribute to a chemoattractive environment such as SDF-1, MCP-1, hepatocyte growth factor (HGF), vascular cell adhesion protein-1 (VCAM-1), IL-8, insulin-like growth factor-1 (IGF-1), and vascular endothelial growth factor (VEGF) [15]. In addition, UC-MSCs express CXCR4, CCR2, and c-2 Stem Cells International    Stem Cells International met receptors. Therefore, UC-MSCs are able to migrate in vitro and in vivo via the SDF-1/CXCR4 and MCP-1/ CCR2 axes, and the secreted factors may induce the recruitment of cells from the surrounding tissues and promote regeneration of injured tissue [15]. To this regard, the SDF-1/CXCR4 axis has been shown to play a key role in endogenous and transplanted stem cell homing in the injured site promoting the regeneration of different tissues including cartilage [16,17].
One more ligand of integrin β1 is osteopontin (OPN), an osteogenic marker with several biological functions including migration, adhesion, and survival of MSCs [21]. On the other hand, OPN is also involved in regulation and propagation of inflammatory responses of macrophages, T-cells, and dendritic cells [22]. Notably, OPN is involved in different inflammatory pathologies including RA and OA pathogenesis [23,24]. In a study of Schneider et al., UC-MSCs showed similar osteogenic and migration abilities compared to BM-MSCs with the lesser expression of OPN and the major expression of matrix metalloproteinases (MMP)-1 and -2 [25].

Capacity of Adaptation to Cartilage Hypoxic Environment.
Because of the lack of vascularization, the physiological oxygen tension (physioxia) within human articular cartilage ranges between 2 and 5% [27]. Therefore, any MSC candidate for stem cell therapy of cartilage diseases should be able to adapt to a hypoxic environment with limited nutrient supply while maintaining its regenerative properties. Oxygen tension ranges from 1%-7% in bone marrow and from 10%-15% in adipose tissue [28,29]. Regarding perinatal tissues such as the UC, oxygen tension within the mammalian female reproductive tract is low, between 1.5% and 8%, and lasts throughout the fetal development with dissolved oxygen in the fetal circulation rarely exceeding 5% [30]. Moreover, the UC is supplied by only two arteries and one vein and lacking in capillaries or lymphatics suggesting that UC-MSCs are physiologically adapted to survive in a hypoxic environment. It has been shown that low oxygen tension increases UC-MSC proliferation potential and matrix production and enhanced  [31,32]. This increased chondrogenic differentiation can lead to hypoxiainducible factor-1 alpha (HIF-1α) and HIF-2α increased expression, NOTCH signaling activation, and the subsequent Sox-9 induction [33]. In addition, the UC-MSCs cultured under hypoxic conditions showed increased expression of energy metabolism-associated genes including GLUT-1, LDH, and PDK1 suggesting a switching of cell metabolism from oxidative phosphorylation to anaerobic glycolysis [32]. The yield of lactate production from glucose, however, is significantly lower in UC-MSCs than it has been reported in BM-and AT-MSCs in both hypoxic and normoxic conditions [32]. This finding could be explained by our recent study [34]. We demonstrated that all the three UC-MSC populations (PV-, WJ-, and CL-MSCs) exhibit low levels of mitochondrial and glycolytic activities. Moreover, PV-, WJ-, and CL-MSCs showed comparable mitochondrial respiration parameters in both normal and oxygen and glucose deprivation followed by reperfusion (OGD/R) conditions maintaining their proliferation capacity. Interestingly, PV-MSCs showed the highest oxygen consumption rate and OGD/R affected their metabolism but not their viability suggesting a superior mitochondrial activity compared to the other UC-MSC populations. While CL-MSCs were the cells least affected suggesting their robust survival in ischemic environment. These evidences taken together suggest that UC-MSCs may be a pivotal source for stem cell-based therapy of ischemic pathologies including chondropathies, brain, heart, and lung diseases [35][36][37][38]. Further investigations are needed to better understand whether these slight but significant differences among the three UC-MSCs are due to the specific region's composition of different number of healthy mitochondria or improved adaptation of mitochondria to ischemic conditions.

Promotion of Survival, Proliferation, and Differentiation.
MSCs secrete growth factors that are involved in several biological processes such as homing and migration as well as promotion of survival, proliferation, and differentiation. Some of growth factors with a key role in cartilage repair are bone morphogenetic proteins (BMPs), epidermal growth factor (EGF), HGF, IGF, PDGF, VEGF, FGF, and TGF families and UC-MSCs are a rich source of them [39].
EGF is one of the ligands of EGF receptor (EGFR) that plays a key role in joint homeostasis. In particular, EGFR stimulates chondrocyte proliferation and survival as well as maintenance of cartilage in adulthood. On one hand, EGFR signaling promotes the lubrication of the articular surface by increasing the boundary lubricants Prg4 and HA from superficial chondrocytes [40]. On the other hand, EGFR signaling also can play a catabolic action by inhibiting the expression of the chondrogenic master transcription factor Sox9, thereby suppressing the synthesis of cartilage matrix proteins, such as type II collagen (Col II) and aggrecan, as well as by stimulating the expression of MMPs involved in cartilage degradation, such as MMP-13 [41]. Interestingly, Zhang et al. recently showed the UC-MSCs release EGFR ligands TGF-α and EGF attenuating OA progression via EGFR signaling pathway of cartilage superficial layer cells [42]. In addition, UC-MSCs inhibited the apoptosis of chondrocytes, increased the expres-sion of chondrogenesis-related genes (Col-2, Sox9), and reduced the expression of cartilage catabolism-related genes (MMP-13, ADAMTS-5) in vitro and in vivo [42].
HGF is a multifunctional growth factor that affects cell survival and proliferation, matrix metabolism, inflammatory response, and neurotrophic action playing an important role in normal bone and cartilage turnover [43]. In particular, HGF and VEGF can reduce tissue injury, inhibit fibrotic remodeling and apoptosis, promote angiogenesis, stimulate stem cell recruitment and proliferation, and reduce oxidative stress [44]. A recent comparative study showed that the secretion of HGF was three times higher in UC-MSCs compared to AT-MSCs and around nine times higher than in BM-MSCs [45]. In contrast, UC-MSCs secreted the lower levels of VEGF-A. This is probably due to the fact that VEGF-A and HGF signaling pathways reciprocally modulate each other [46]. IGF1 has been implicated in promotion of chondrogenesis and accumulation of cartilage-specific ECM molecules [47]. In addition, the synergy between TGF-β3 and IGF-1 promotes intervertebral disc regeneration [48]. WJ contains large amounts of IGF-I and IGF-I-binding proteins BP-3 and BP-1 suggesting a key role in stimulation of UC-MSCs to produce collagen and glycosaminoglycans (GAGs) in UC matrix as well as influencing the chondrogenic differentiation of these cells [49,50].

Cartilage Extracellular Matrix
Repair. UC-MSCs can increase the ECM synthesis and inhibit the cartilage ECM destruction supporting the tissue repair. UC stromal tissue shares a number of features with cartilage ECM: UC-MSCs are able to synthetize aggrecan, type II collagen, and express SOX-9 transcription factor [12]. The deposition of ECM molecules and regulation of MMPs and their inhibitors (TIMPs) are the main mechanisms involved in cartilage ECM synthesis. MSCs secrete high levels of TIMP-1 and TIMP-2, which inhibit MMP-9 and MMP-2, respectively, thus, suppressing cartilage ECM resorption [65]. UC-MSCs secrete MMP-2, -8, -9, and -13 as well as TIMP-1 and TIMP-2 suggesting a balance between protection of ECM and antifibrotic activity ( Table 2) [66][67][68]. In addition, UC-MSCs secrete growth factors such as HGF, IGF-1, and TGF-β superfamily members that stimulate cartilage ECM synthesis. In particular, HGF has been involved in inhibition of the fibrosis and apoptosis of chondrocytes and increase ECM synthesis [65]. IGF-I and IGF-I-BP-3 and -BP-1 stimulate UC-MSCs to produce collagen and glycosaminoglycans (GAGs) [49]. BMP-2 increases TIMP-1 expression while BMP-7 inhibits MMP-1 and MMP-13 suppressing the ECM degradation [63,64].

Anti-Inflammatory and Immunomodulatory Properties.
The microenvironment of damaged articular cartilage is particularly challenging, due to hypoxia, insufficient blood supply, and concurrent inflammation. The latter contributes to the degeneration of the joints because it hampers the proliferation of chondrocytes and the deposition of cartilage matrix, resulting in low efficiency of repair. Immunomodulatory and antiinflammatory properties of UC-MSCs have been widely described (Table 2) [69]. In particular, UC-MSCs express MHC class I (HLA-ABC) at low levels and lack MHC class II (HLA-DR, -DP, and -DQ). Moreover, they express other molecules belonging to noncanonical type I MHC such as HLA-G, HLA-E, and HLA-F [70][71][72]. Interestingly, HLA-G interacts with Ig-like transcript (ILT) receptors (ILT-2, ILT-3, and ILT-4), which are expressed by T and B lymphocytes, as well as natural killer (NK) cells and mononuclear phagocytes [69]. Through this interaction, HLA-G displays relevant immune functions which physiologically contribute to maternal-fetal immunotolerance. In addition, UC-MSCs lack CD40/CD40L, CD80, CD86, and B7 costimulatory antigens implicated in the activation of T and B cell responses and express coinhibitory molecules including B7-H3/CD276, CD73, Indolamine 2,3-dioxygenase-1 (IDO-1), Galectin-1 (Gal-1), and leukemia inhibitory factor (LIF) [73]. WJ-MSCs showed an immunosuppressive function by inhibiting the proliferative response of T helper cells (Th/CD4+) Type 1 (Th1) and Th17 and increasing Th2 and regulatory T cells (Tregs) [74]. UC-MSCs have been shown to be able to suppress the proliferation of both CD4 and CD8 cytotoxic T lymphocytes (Tc) and to decrease proinflammatory IFN-γ in activated peripheral blood mononuclear cells (PBMCs) [54,75]. Moreover, secreted factors such as HGF and TGF-β1 may function as mediators for T cell suppression [76,77]. UC-MSCs are also able to inhibit B-cells and natural killer (NK) cell proliferation as well as regulate monocyte/macrophage system by reducing the infiltration of macrophages in injured tissues and shifting macrophages toward a M2 antiinflammatory phenotype [78,79]. In the synovia of OA patients, various immune cells have been identified including M1 macrophages, T cells Th1, Th17 and Tc, and B cells, leading to chronic inflammation, exacerbation of arthritis, and tissue damage [80]. UC-MSCs have been shown to reduce synovial inflammatory cells infiltration, such as CD4+ T cells and macrophages, as well as significantly decrease the expression of interleukin-(IL-) 1β and tumor necrosis factor-α (TNF-α), while increasing antiinflammatory factors TNF-α-induced protein 6 (TSG-6) and IL-1 receptor antagonist (IL-1RA) in rat OA models induced by monosodium iodoacetate (MIA) [81,82]. In another study of MIA-induced OA in rabbits, UC-MSCs showed a prominent cartilage protective effect due to upregulation of growth factors FGF-2, TGF-β1, and IGF-1, secretion of ECM molecules (collagen type-I alpha-1 chain, collagen type-II alpha-1 chain, and aggrecan), reduction of the expression levels of proinflammatory cytokines Tnf-α, IL-1β, IL-6, and IL-17, and increase of anti-inflammatory cytokines TGF-β1, IL-10, and IL-1RA [83]. Interestingly, our group and others showed that UC-MSCs keep their hypoimmunogenic and immunomodulatory properties even when they had undergone in vitro chondrocyte differentiation [50,84].
RA is a chronic inflammatory autoimmune disease characterized by chronic proliferation of synovial cells and progressive joint damage [85]. Fibroblast-like synoviocytes (FLS) play an important role in thickening of the synovium determining arthritis and cartilage degradation as well as inflammation and degradation of the joints. UC-MSCs inhibit Cadherin 11 expression in RA FLS by secreting IL-10. This event precludes the ability of FLS from RA patients to migrate and erode cartilage of other joints, thereby improving arthritis [86].

Preclinical and Clinical Studies of UC-MSCs for the Treatment of Cartilaginous Diseases
Thanks to their chondrogenic potential and immunomodulatory and anti-inflammatory properties, as well as their ability to promote endogenous repair mechanisms, UC-MSCs have been regarded as potential therapeutic agents against cartilage degradation. In particular, early evidences that emerged from in vitro studies on cell cultures (summarized in Table 3) have been confirmed in several in vivo animal 7 Stem Cells International          14 Stem Cells International  Table 4) and in recent clinical trials (reported in Table 5).
Preliminary in vitro studies investigated the chondrogenic potential of UC-MSCs, demonstrating their ability to achieve both hyaline, fibrous, and elastic cartilage phenotypes as well as nucleus pulposus-like cell differentiation capacity [87][88][89][90]. In addition, also the osteogenic, adipogenic, and myogenic differentiation potential have been reported suggesting UC-MSCs could be a pivotal stem cells source for tissue engineering applications in orthopaedics [91]. Comparative studies reported slightly differences in chondrogenesis between the UC-, BM-, and AT-MSCs. In particular, according to Danišovič et al., BM-MSCs showed the best chondrogenic potential while Hildner and coworkers showed that differentiated UC-MSCs present a more fibrous than hyaline cartilage phenotype compared to AT-MSCs suggesting their role in regeneration of fibrocartilage-like meniscus [87,92]. Moreover, the results of Wu and coworkers indicate that, although nucleus pulposus stem/progenitor cells (D-NP-MSCs) isolated from degenerated intervertebral disc (IVD) shared the MSCs characteristics with UC-MSCs, the latter showed better proliferation capacity and differentiation potential, suggesting that UC-MSCs as a suitable source for regenerative therapy of IVD degeneration [93]. Furthermore, UC-MSCs may be an attractive alternative to condylar cartilage cells for temporomandibular joint tissue engineering applications [94]. Interestingly, UC-MSCs displayed superior anti-inflammatory, immunomodulatory, and trophic effects compared to adult MSCs including articular cartilage (AC), Hoffa's fat pad (HFP), synovial membrane (SM), and maintain their immunomodulatory and antiinflammatory properties after differentiation [50,84,95]. Moreover, coculture experiments of UC-MSCs and articular cartilage cells (ACs), fibroblast-like synoviocytes (FLSs), and nucleus pulposus cells (NPCs) showed their suitability for the treatment of arthritis, synovitis, and IVD degeneration [61,[96][97][98]. Finally, there are several cartilage tissue engineering studies that demonstrated the osteochondral differentiation capacity of UC-MSCs in different scaffold constituted by acellular cartilage extracellular matrix (ACECM), alginate enriched in hyaluronic acid (Alg/HA), polycaprolactone/collagen (PCL/Coll), polyglycolic acid (PGA), poly L-lactide/D-lactide/glycolide (PLGA), poly-L-lactic acid (PLLA), polyvinyl alcohol-polycaprolactone (PVA-PCL), and silk fibroin/hyaluronic acid (SF/HA).
In vivo studies in different animal models from mice to horses confirmed in vitro studies showing the feasibility of using UC-MSCs for the treatment of IVD degeneration, OA, RA, and cartilage defects repair. UC-MSC transplantation promotes chondrogenesis and improves the histology, cellularity, and ECM proteins content along with reduction of inflammation in a preclinical model of IVD degeneration. In the same way, UC-MSCs induce regeneration and repair of cartilage reducing its destruction, promote recovery from movement impairment, and reduce joint effusion and inflammation slowing down the progression of OA animal models. In addition, preclinical studies on RA treatment showed that UC-MSCs exerted the best therapeutic effect in reducing bone resorption, joint destruction, and inflammatory factor expression compared to BM-MSCs. Interest-ingly, several evidences support the regenerative potential of UC-MSCs in cartilage and osteochondral defects repair.
Following the promising in vitro and in vivo results, clinical applications have been attempted using UC-MSCs for the treatment of OA and RA (Table 5). In summary, clinical trials for the treatment of OA showed significant amelioration of pain and disability at 6 and 12 months of follow-up. No severe adverse events were reported. The main outcomes for RA patients treated with UC-MSCs were significant reduction of RA serological markers and improvement of health index and joint function index 1 year after treatment. No new or unexpected safety issues in 1-year follow-up. Despite the promising results of clinical trials, further basic and translational research investigations are needed to better understand the best stem cell candidate, scaffold materials, and/or best cellular derivatives which can be suitable for the different types of cartilage regeneration. In parallel, there is the need to increase the knowledge about underlying regenerative mechanisms. Finally, more research is needed to convert preclinical evidences obtained in animal models, to human-based clinical applications for cartilage regeneration. Consensus is still lacking in key points such as the methods to obtain the cell source, the use of scaffolds as well as bioactive molecules in parallel to the administration of stromal cells. As shown in the human studies reviewed so far, the achievement of amelioration of some parameters and confirmation of the safety of the overall procedure still needs more data generated on the interaction of the transplanted cells with the host tissue, their proper differentiation in vivo, as well as the long-term achievements of this cellular replacement strategy.

Conclusions
In conclusion, UC-MSCs represent a promising candidate for the therapy of chondropathies, as highlighted by the encouraging results emerged from in vitro and in vivo investigations and from the available results from clinical trials. UC-MSCs are characterized by several potential advantages such as a frank multilineage differentiation potential, immunomodulatory, and anti-inflammatory properties, as well as MSCs the ability to constitutively produce molecules that are involved in cartilage matrix biogenesis and in the trophic and reparative functions. In addition, UC-MSCs are able to migrate, home, and survive in an ischemic and nutrient-poor environment like cartilage as well as to produce an extracellular matrix (ECM) similar to that and induce endogenous repair mechanisms. We believe that these results warrant the need for further researches that can better define the criteria leading to the adoption of UC-MSCs in the stem cell-based therapy of cartilage diseases, as well as characterizing the mechanism of repair and increase the knowledge on the biomechanical properties of the regenerated cartilage tissue in vivo.

Conflicts of Interest
Prof. Giampiero La Rocca is member of the Scientific board of Auxocell Laboratories, Inc. The other authors report no conflicts. 16 Stem Cells International Stem Cells International