Cell Senescence: A Challenge in Cartilage Engineering and Regeneration

Abstract

Cartilage defects, most commonly caused by aging and degenerative disease, have become the primary target of cartilage tissue engineering due to a lack of effective treatments and limited regenerative abilities. The limited success of autologous chondrocyte implantation necessitates the development of alternative cell sources. Adult mesenchymal stem cells (MSCs) with multiple lineage differentiation potentials from various sources can supplement the shortage of human autologous chondrocytes. However, cell senescence presents a big challenge for large-scale ex vivo expansion and maintenance of MSC stemness. In this review, we will summarize some potential factors resulting in cell senescence during cartilage tissue engineering, including ex vivo expansion, donor age, and degenerative diseases, and the challenge in the identification of senescent cells. The presence of senescence-associated β-galacotosidase and DNA damage, accumulation of reactive oxygen species, the decline of DNA replication and telomerase activity, and shortened telomere length is indicative of senescence, but none of them are specific. To some extent, growth factors, antioxidants, serum deprivation, or platelet-rich plasma treatment as well as low oxygen have been successful in retarding cell senescence. Recently, decellurized extracellular matrix, especially decellularized stem cell matrix, has emerged as a more promising tool in retaining cells in a younger state. Some potential signaling pathways in cell senescence will also be discussed for their potential involvement in cartilage regeneration despite the fact that comprehensive mechanisms are still under investigation.

Introduction

Cartilage defects, which are becoming an exceedingly common problem associated with advancing age and degenerative diseases, are characterized by lack of spontaneous healing due to the limited regenerative capacity of adult chondrocytes. This characteristic makes cartilage an ideal model for tissue engineering, as it has no blood vessels or nerves. Autologous chondrocyte implantation (ACI), since it was first described,¹ has provided satisfying results for most patients over several years.^2–7 However, the success of ACI is restricted due to multiple factors, such as the shortage of autologous chondrocytes, dedifferentiation during monolayer expansion, and periosteum-related chondrocyte hypertrophy,⁸ as well as whether ACI could offer significant advantages over the simpler surgical technique of microfracture.^9,10 Scaling up tissue-engineering therapies for large numbers of patients suffering from cartilage defects will ideally involve the expansion of autologous cells through multiple population doublings without senescing or transforming. These cells can then differentiate into chondrocytes, produce the appropriate matrix, and survive in vivo without immune rejection.

Due to the shortage of autologous chondrocytes, adult mesenchymal stem cells (MSCs) with multiple lineage differentiation potentials derived from bone marrow (BMSCs), adipose (ASCs), synovium (SDSCs), and periosteum (PDSCs) have become more popular sources for cartilage repair and hold greater promise. BMSCs have proved to have strong chondrogenic differentiation capacity and, since first isolated, they are the most widely used stem cells in cartilage tissue engineering.^11–13 ASCs, due to easy accessibility and availability, have been widely used in regenerative medicine and tissue engineering.^14–18 As a tissue-specific MSC, SDSCs have been regarded as a promising therapeutic cell source for joint tissue regeneration due to higher proliferation and superiority in chondrogenesis over other MSCs.^19–24 Other chondrogenitor cells that have also been investigated in cartilage regeneration include cells derived from nucleus pulposus (NP) and meniscus.^25–27

Despite encouraging preliminary results, the approaches just mentioned have not yet been routinely applied in clinical practice except in young patients.²⁸ One critical point is the challenge presented by identifying and preventing cell senescence in order to maintain a large enough number of differentiated chondrocytes and undifferentiated MSCs, as MSCs represent a rare population. In general, during monolayer expansion, human chondrocytes tend to undergo spontaneous dedifferentiation,²⁹ while MSCs become senescent and differentiate into osteoblasts.³⁰ Cell senescence could be troublesome when trying to achieve success in cartilage engineering and regeneration. However, we were unable to find a published review on cell senescence in cartilage engineering and regeneration. The purpose of this review article is to provide a general background that links cell senescence and cartilage regeneration and emphasizes the importance of preventing cell senescence in the successful engineering of high-quality cartilage tissue. In this review, we summarize the pivotal aspects that could contribute to cell senescence during cartilage engineering and regeneration and evaluate methods that identify senescent markers in vivo and ex vivo; current efforts in preventing cell senescence in the cartilage regeneration field are also reviewed, and the involved signaling pathways are discussed (Fig. 1).

FIG. 1.

A diagram of review article structure. Color images available online at www.liebertonline.com/teb

Cell Senescence in Cartilage Regeneration

It is known that intrinsic and extrinsic factors are involved in the process of cell senescence. The intrinsic mechanism is called the Hayflick limit.³¹ Most somatic cell types reach cell-cycle arrest after a characteristic number of population doublings, which is also referred to as “cellular or replicative senescence.” In humans, cell-cycle arrest is typically reached after 20 to 100 cell population doublings. It prevents cells from immortalization and suppresses oncogenesis. However, it also limits the goal of large-scale ex vivo cell expansion as required for cartilage regeneration and tissue engineering applications. Although it is possible to overcome the Hayflick limit by genetically modifying cells, for instance, transfecting human articular chondrocytes with the human telomerase gene,³² such manipulations are regarded as potentially dangerous in the context of tissue engineering. On the other hand, extrinsic factor-associated senescence, also called stress-induced senescence, is considered more premature, as it can halt cell growth before the Hayflick limit is reached. Theoretically, through modifying the culture conditions and minimizing the stress in ex vivo culture, growth potentials can be regained. Recent concepts about cell senescence are similar to a stress-responsive, adaptive phenotype that develops through multiple stages during the development of degenerative diseases, which can spread from cell to cell and occur at any point in life.³³ In other words, senescence could be an alternative cell fate that develops in response to injury or metabolic dysfunction and might occur in nondividing as well as dividing cells.³⁰ We will briefly review the cell number limitations and loss of phenotype during ex vivo expansion, donor-site morbidity, and age-related decline in chondrogenic capacities; all these elements lead to cell senescence during cartilage tissue engineering. Critical obstacles for researchers and clinical therapists will also be presented.

Ex vivo expansion

Chondrocyte senescence developing during ex vivo expansion is commonly characterized by accumulation of reactive oxygen species (ROS) and advanced glycation end products, increased expression of senescence-associated β-galactosidase (SA-β-gal), nuclear and mitochondrial DNA damage, and decreased mitochondrial function. Average articular chondrocyte telomere erosion rate in vivo is about 30 bp per year.³⁴ During an 8–10-fold cellular ex vivo expansion, telomere length is impaired as long as 900 bp due to loss of the telomerase activity of chondrocytes.³⁵

Similar to chondrocytes, MSC senescence results in cell proliferation arrest, characterized by flat shape, circumscribed nuclei, increased lysosome compartment, shortened telomere, and endogenous SA-β-gal activity.^36,37 All the characteristics just mentioned develop during MSC long-term ex vivo culture.³⁷ BMSCs isolated from fresh bone marrow aspirates underwent senescence with a change in morphology and shape after 38 population doublings and largely lost their ex vivo differentiation capacities at or around the sixth passage,^38,39 but there is also evidence of adverse changes as early as the first or second passage.^40,41 Surprisingly, the osteogenic differentiation potential, including alkaline phosphatase (ALP) expression and bone nodule formation ex vivo, appeared to be retained despite replicative senescence.⁴² Banfi et al. observed loss of osteogenic differentiation along with proliferation capacities in BMSCs passaged at around 22 cell doublings.⁴⁰ Consistent results were obtained from Muraglia et al., who investigated differentiation in a BMSC population derived from a single cell.⁴³ What the cases just mentioned have in common is the initial loss of adipogenic differentiation capacity. Overall, ex vivo expansion leads to a progressive decrease of proliferative abilities and differentiation potentials. Similarly, Li et al. found that human placenta-derived MSCs underwent aging and spontaneous osteogenic differentiation during regular culture expansion, with down-regulation of human telomerase reverse transcriptase and up-regulation of the osteogenic gene runt-related transcription factor 2 (Runx2) and ALP expression. Stem cell self-renewal associated genes octamer-binding transcription factor 4 (Oct4) and SRY (sex determining region Y)-box 2 (Sox2) expression declined progressively.³⁰

In addition, researchers have investigated the effect of seeding density during MSC ex vivo expansion. Despite sporadic evidence suggesting that plating density is not critical for maintaining a multipotent MSC population, time in culture does affect MSC characteristics in general; loss of adipogenic and chondrogenic differentiation abilities was observed in the higher density group.⁴⁴ More studies have shown that high-density plating produced a higher percentage of flattened human BMSCs with characteristics of cellular senescence and the loss of ability to differentiate, while low-density plating resulted in a higher proliferation rate as well as more multi-potent cells.^45–47 A recent study from our group also suggested that low-density plating promotes human SDSC proliferative ability and maintains multi-lineage differentiation potential, especially chondrogenic differentiation (unpublished data). The evidence just provided suggests that seeding density could be an influential factor for developing senescence; low-density seeding could be useful when selecting the homogenous subpopulations of MSCs with higher proliferation and differentiation potentials.^48,49

Donor age

Another parameter that should be considered is aging, as decreased proliferation and the propensity toward senescence were observed in aged donors. Chondrocytes obtained from aged individuals (older than 40 years) have a much lower ability to repair cartilage damage than those obtained from younger patients.⁵⁰ Pestka et al. recently revealed the difference in chondrocyte quality during ex vivo expansion in cells collected from 252 ACI patients.⁵¹ Results suggested that no specific parameters other than age could be identified as influencing the quality of cells.⁵¹ A more dramatic change in chondrogenic potentials of human chondrocytes from juveniles (<13 years old) and adults (13 years and older) is also documented by Adkisson et al.⁵²

More attention has been paid to multi-potent cells in cartilage tissue engineering. Differences in MSCs from aged donors have been found in proliferation, cell attachment, and senescence in both animal and human cells.^53–58 However, there have been conflicting reports about changes in MSC differentiation potentials attributed to donor age. Several previous studies have shown no difference attributable to donor age in human BMSC differentiation potential.^59–62 In recent years, Kretlow et al. observed different chondrogenic capacities of murine BMSCs from animals of different ages.⁶³ Zheng et al. demonstrated the impaired chondrogenic differentiation in aged rat BMSCs.⁶⁴ Microarray analysis indicated significant age-related differences in the expression of genes that influence cartilage extracellular matrix (ECM) formation.⁶⁴ Age-related mechanical properties and collagen content changes were also noted in bovine BMSCs.⁶⁵ Inconsistent results were obtained from human BMSCs as well. Scharstuhl et al. isolated BMSCs from the femoral shafts of 98 patients and investigated the relationship between chondrogenic differentiation capacities and age or osteoarthritis (OA).⁶² Surprisingly, no correlation from either factor was observed. However, Payne et al. found an age-related decline in chondrogenic differentiation in BMSCs even with transforming growth factor beta1 (TGF-β1) stimulation, though only in men.⁶⁶ Since 1999, donor age has been identified as an important factor in PDSC ex vivo chondrogenic differentiation.⁶⁷ Later, De Bari et al. demonstrated that adult PDSCs could undergo chondrogenesis regardless of donor age.⁶⁸ Our recent study indicated that human fetal SDSCs exhibited a decrease in proliferation and an increase in chondrogenic differentiation during passaging, while adult SDSCs displayed a decrease of both proliferation ability and chondrogenic potential during passaging (unpublished data).

Trauma and degenerative diseases

Trauma such as joint injuries is most commonly caused by mechanical factors, which promote human articular chondrocyte senescence by increasing oxidative stress, as characterized by cell-cycle arrest, senescent morphology, and increased SA-β-gal activity, possibly through accelerating telomere shortening.⁶⁹ Challenging human MSCs with oxidative stress results in similar characteristics, but manifests an increased tolerance regarding proliferation.⁷⁰ Intracellular ROS were found to correlate with articular chondroctye senescence through activation of p38 kinase, which further promotes ROS generation, forming a positive feedback loop. Hong et al. found that ionizing radiation induced chondrocyte senescence by negative post-transcriptional regulation of SIRT via ROS-dependent p38 kinase activation.⁷¹ Sirtuin 1, a mammalian Sir2 ortholog and nicotine adenine dinucleotide-dependent deacetylase, has reportedly been involved in cell aging pathways.⁷²

OA is characterized by degeneration of articular cartilage, limited intraarticular inflammation with synovitis, and changes in peri-articular and subchondral bone. Current pharmacological interventions that address chronic pain are insufficient, and no proven structure-modifying therapy is available.⁷³ Cell-based therapy and novel approaches using MSCs as an alternative cell source to chondrocytes are currently on trial.^74,75 Patients suffering from degenerative diseases such as OA are the main group that will benefit from successful cartilage regeneration.⁷⁶ Murphy et al. observed reduced chondrogenic differentiation capacities of BMSCs from patients with advanced OA.⁷⁷ Han et al. observed decreased proliferative and chondrogenic potentials of SDSCs collected from OA patients during ex vivo expansion; microarray analysis suggested late-passage cells overexpressed cell-cycle prolongation and cell aging-associated genes, while repressing expression of cell growth-related genes.⁷⁸ Additionally, aging changes in the joint tissue contribute to the development of OA, as cellular senescence results in the development of a senescent secretory phenotype, and aging changes the matrix with increased formation of advanced glycation end products that affect the mechanical properties of joint tissues.

Intervertebral disc (IVD) degeneration is another common disease associated with chondrocyte aging. Recently, the close link between cellular senescence and IVD degeneration has been revealed.^79,80 In vivo studies have shown that senescent NP cells accumulated or increased with aging and advancing IVD degeneration.^81,82 Senescent IVD cells behave abnormally; they may have deleterious effects on the disc matrix and contribute to the pathogenesis of degeneration. A decreased capacity to create or retain ECM components in response to mechanical loading of aged NP cells was also observed.⁸³ IVD cell senescence has also been linked to connective tissue degenerative diseases, such as OA. Thus, cells from these diseased tissues may not be ideal for use in cell therapy and tissue engineering.⁸⁰

Identification of Cell Senescence During Chondrogenic Differentiation

Senescent cells can be identified by several markers in vivo and ex vivo. Despite enlarged and flat cell morphology, SA-β-gal activity at an acidic pH becomes detectable, and p53, p21, and p16 levels also gradually increase as cells approach senescence.^84,85 In addition, senescent cells produce more ROS and have elevated levels of oxidative DNA damage.^86,87 To date, however, there have been no specific markers for cells found exclusively in the senescent state. The identification of senescent cells and a further understanding of the aging mechanisms would facilitate cartilage regeneration and the development of the tissue-engineering field in the future. Here, we summarize the most commonly used markers and their detection methods during cartilage tissue engineering.

SA-β-gal

SA-β-gal is the first and most widely used marker for the identification of senescent cells.⁸⁵ Its activity becomes detectable in cultured cells undergoing replicative or induced senescence but is not detectable in proliferating cells.⁸⁵ SA-β-gal activity has been detected in situ in senescent human articular cartilage and indicates chondrocyte replicative senescence both in vivo and ex vivo.⁶⁹ SA-β-gal has also been used as a marker for identifying the higher percentage of senescent MSCs in elderly donors.⁸⁸ However, the expression of SA-β-gal is not required for senescence.⁸⁹

The presence of SA-β-gal is usually detected using histochemical staining with chromogenic X-gal as a substrate. Recently, Debacq-Chainiaux et al. updated current protocols to detect SA-β-gal activity in senescent cells both ex vivo and in vivo; compared with the traditional cytochemical method, they described another fluorescent method using a fluorogenic substrate, 5-dodecanoylaminofluorescein di-beta-D-galactopyranoside, which is much faster, more quantitative, and more sensitive.⁹⁰

Over-loaded ROS

Besides playing important roles in many physiological processes, ROS also have the potential to cause oxidative damage to proteins, lipids, and DNA.⁹¹ The accumulation of ROS is associated with cell senescence^86,87,92 and has been identified in both chondrocytes^93,94 and MSCs.⁹⁵ Human articular chondrocytes actively produce ROS, and the accumulation of ROS was reported in the cartilage of old rats.^93,94,96,97 The imbalance of reduction–oxidation may be caused by the age-related decline in the activity and number of mitochondria, which plays a role in protecting cells from the harmful effects of ROS.⁹⁸ A consequence of increased oxidative stress is DNA damage and telomere shortening, leading to a decline in matrix production, chondrocyte and MSC senescence, and apoptosis.^70,99–101

Methods of detecting ROS are available through chemiluminescence, electron paramagnetic resonance spectroscopy, fluorescence, and enzymatic techniques.¹⁰² Different dyes, such as dichlorofluorescin diacetate and hydroehidine, are applied depending on the specific species that are to be detected.¹⁰²

DNA replication and DNA damage

The decline of DNA replication can be used as an index for the development of cell senescence. DNA replication can be measured by incorporation of thymidine analogous, such as 5-bromodeoxuridine or H-thymidine or 5-ethynyl-2-deoxyurdine, using autoradiography or flow cytometry.^103–105 The major limitation to the use of thymidine analogous is the inability to perform retrospective studies. Cell-cycle-restricted proteins, such as proliferating cell nuclear antigen and Ki-67, can be identified by antibodies through immunostaining or immunoblotting.^106–110 However, these DNA replication markers cannot be used to distinguish between senescent cells and quiescent or differentiated postmitotic cells.

Senescence-associated heterochromatic foci (SAHFs) and senescence-associated DNA-damage foci can be identified in some senescent fibroblasts.^111–115 SAHFs are detected by the preferential binding of DNA dyes, such as 4,6-diamidino-2-phenylindole, and the presence of certain heterochromatin-associated histone modifications and proteins. However, the identification and investigation of SAHFs is not yet fully understood in chondrogenitor cells.

Telomere length and telomerase activity

Telomeres and telomerase play a key role in cell development, aging, and tumorigenesis. Telomerase adds DNA sequence repeats to telomeres and prevents the constant loss of important DNA from chromosome ends. Since most cell types lack telomerase expression, each round of replication ex vivo and in vivo will result in a loss of 50 to 200 bp of telomeric TTAGGG repeats.^116,117 Once telomere length decreases beyond a minimum critical length, it will trigger cellular senescence or apoptosis.^118,119 Replicative senescence is closely associated with telomere shortening and other factors, and human MSCs are no exception. The average telomere-shortening rate is 50 bp per population doubling.^120,121 Umbilical cord blood-derived MSCs, however, have slightly longer telomeres than other MSCs.¹²² Measurement of telomere length and detection of telomerase activity are applicable when investigating cell senescence during cartilage tissue engineering.

Cyclin-dependent kinase inhibitors

Senescence-related genes p53, p21, and p16 gradually increase in BMSC long-term culture. As a cell-cycle inhibitor, p21 is closely related with stem cell differentiation capacities;^37,123–126 it has been shown to increase in aging stem cells and contributes to an age-related decrease in regenerative potential. Caveolin 1, the principal structural component of caveolae, has been shown to be positively associated with articular chondrocyte senescence induced by oxidative stress or interleukin-1β.¹²⁷ Another important regulator of senescence is p16, which is widely used to identify senescent cells.¹²⁸ Many, but not all, senescent cells and some tumor cells express it.^129–131 Additional discussion can be found in the section of Senescence-associated signal transduction pathways.

Epigenetic changes

Overall, DNA methylation decreases as growth plate chondrocytes grow old in vivo.¹³² Despite a recent report suggesting that the down-regulation of aggrecan expression in aged or OA chondrocytes is not attributed to cytosine phosphate-guanine (CpG) methylation,¹³³ cartilage-degrading enzymes such as matrix metalloproteinases (MMPs) and aggrecanase-1 (ADAMTS-4) have been reported to be associated with CpG methylation in chondrocytes derived from OA patients.¹³⁴ Iliopoulos et al. also found that expression of leptin affects downstream MMP13 through epigenetic mechanisms.¹³⁵ Moreover, studies have shown changes in epigenetic alterations as well as gene expression profiles in senescent human MSCs. The dysregulation of histone H3 acetylation in K9 and K4, but not methylation of CpG islands, was found to be closely associated with gene expression, suggesting that the acetylation-related epigenetic changes also correlate with aging.³⁰ Recent reviews have summarized the techniques of DNA methylation and acetylation and discussed their advantages and disadvantages.^136,137 However, the comprehensive effect of epigenetic changes on stem cell aging is still under investigation.

Current Efforts in Preventing and Alleviating Cell Senescence During Cartilage Tissue Engineering

Growth factors

The application of growth factors could support and maintain proliferation and chondrogenesis activities⁵⁰ (Table 1). Evidence has shown that a medium supplemented during chondrocyte expansion with TGF-β1, fibroblast growth factor-2 (FGF-2), and platelet-derived growth factor-BB exhibited higher proliferation rates at any age and higher subsequent chondrogenic capacity in elderly donors.^50,138 However, none of the studies showed an accelerated rate of telomere shortening and a significantly increased amount of telomerase.¹³⁸

Table 1.

Current Efforts in Preventing and Alleviating Cell Senescence During Cartilage Tissue Engineering

Effort	Cell	Contribution	Reference
Growth factors
TGF-β1, PDGF-BB	Human chondrocyte	When combined with FGF-2, exhibited higher proliferation at all age groups and higher chondrogenic capacity in donors up to 40 years	⁵⁰
	Human chondrocyte	Higher proliferation rates, safe due to no accelerated telomere shortening and increased telomerase activity	¹³⁸
FGF-2	Rabbit BMSC	Improved chondrogenic repair in rabbit knee injury model through cell expansion	¹³⁹
	Human BMSC	Enhanced mitotic and chondrogenic potential	¹⁴⁰
	Human BMSC	Decreased senescence cell percentage	¹⁴¹
	Human aged BMSC (>50 year-old)	Effectively increased cell number even without serum	¹⁴³
IGF-I	Human aged BMSC (>50 year-old)	Increased proteoglycan content when applied with TGF-β2	¹⁴³
	Porcine SDSC	When combined with TGF-β1 and FGF-2, promoted proliferation; when combined with TGF-β1, enhanced chondrogenic differentiation	¹⁴⁴
	Horse BMSC	Pretreatment with TGF-β1 followed by IGF-I significantly increased formation of chondrocytic function markers, including proteoglycan content and procollagen type II mRNA production	²⁴⁴
Antioxidants
Nimesulide, 4-OH nimesulide	Human chondrocyte	Scavenged ROS and inhibited ROS production, protected cartilage from oxidative stress in chondrocytes	¹⁴⁵
N-acetylcysteine	Aged rat BMSC	Increased resistance to necrotic cell death and counteracted excess rate of proliferation	¹⁴⁶
Statin	Human chondrocyte from OA patients; in vivo mouse OA model	Inhibited IL-1β-induced cell senescence in vitro and cartilage matrix degrading enzyme production both in vitro and in vivo	¹⁴⁷
Melatonin	Porcine chondrocyte	Enhanced cartilage matrix synthesis in vitro	¹⁵⁰
Fullerene	Human chondrocyte from OA patients; in vivo rabbit OA model	Inhibited cartilage matrix degrading enzyme production, down-regulated matrix production, apoptosis, and premature senescence in vitro; significantly reduced cartilage degeneration in vivo	¹⁵¹
Nutrient-related treatment
Serum deprivation	Human chondrocyte	Primed proliferation and sustained proper commitment in chondrocytes from aged patients	¹⁵³
Platelet-rich plasma	Human chondrocyte and BMSC	Enhanced cell proliferation	¹⁵⁵
	Human BMSC	Enhanced BMSC proliferation and chondrogenic differentiation	¹⁵⁶
	Human BMSC	Improved BMSC expansion and retained multidifferentiation capacity	¹⁵⁸
Platelet lysates	Human BMSC	Maintained cell multidifferentiation properties and retained immunosuppressive activity	¹⁵⁷
Glucose reduction	Human BMSC	Exhibited greater self-renewal and anti-senescence abilities, while their differentiation potentials remained unaffected	¹⁵⁹
Hypoxia
	Human BMSC	Retained BMSCs in an undifferentiated and senescence-free state	^161,165
	Human ASC from IPFP of elderly patients with OA	Increased the synthesis and assembly of cartilage matrix	¹⁶³
	Porcine SDSC	Enhanced proliferation and chondrogenic abilities	¹⁶⁴
DECM
From native cartilage	Human ASC from subcutaneous fat tissue	Induced ASC chondrogenic differentiation	¹⁷⁹
	Human ASC and BMSC	Induced a chondrocytic phenotype in both cells	¹⁸⁰
	Dog BMSC	Supported BMSC adhesion, proliferation, and chondrogenic differentiation	¹⁸¹
Deposited by chondrocytes	Rabbit BMSC	Supported BMSC chondrogenic differentiation	¹⁸²
	Human BMSC	Induced BMSC chondrogenic differentiation	¹⁸³
	Porcine SDSC and chondrocyte	Enhanced both cell proliferation and chondrogenic differentiation	¹⁸⁴
DSCM		A concise review article	¹⁸⁵
Deposited by SDSCs	Porcine SDSC	Enhanced SDSC proliferation and chondrogenic potential	^184,186
	Porcine chondrocyte	Delayed expanded cell replicative senescence and enhanced cell proliferation and redifferentiation capacity, which is superior to DECM deposited by chondrocytes	^184,189
	Porcine ASC from IPFP	Enhanced ASC proliferation and chondrogenic potential	¹⁸⁷
	Porcine NP cell	Enhanced cell proliferation and redifferentiation capacity	¹⁹⁰
Deposited by BMSCs	Human BMSC	Rejuvenated and enhanced aged BMSC chondrogenic differentiation and hypertrophy	¹⁸⁸

BMSC, bone marrow-derived stem cell; ASC, adipose-derived stem cell; SDSC, synovium-derived stem cell; PDSC, periosteum-derived stem cell; IPFP, infrapatellar fat pad; NP, nucleus pulposus; ROS, reactive oxygen species; DECM, decellularized extracellular matrix; DSCM, decellularized stem cell matrix; TGF-β, transforming growth factor beta; FGF, fibroblast growth factor; OA, osteoarthritis; IGF-I, insulin-like growth factor-I.

Individually, 5 ng/mL FGF-2 has a mitogenic effect on MSCs in monolayer culture with or without a serum. In a rabbit knee injury model, Mizuta et al. found that FGF-2 could significantly improve the chondrogenic repair response through largely expanding the undifferentiated MSC proliferation.¹³⁹ Solchaga et al. proved the same mitotic and chondrogenic promoting effect of FGF-2 in human BMSCs.¹⁴⁰ Of the 358 transcripts regulated by FGF-2 treatment, 17% of the genes are related to proliferation. The largest percentage is associated with cellular signaling functions. Eighty percent of ECM-related genes are down-regulated, which is interpreted as a manifestation of the maintenance of an undifferentiated state due to FGF-2 treatment. Ito et al. also demonstrated that FGF-2 could decrease long-term culture-induced human MSC senescence and suppress G1 cell growth arrest by inhibiting p21, p53, and p16 expression levels, possibly through suppressing TGF-β2 mRNA expression.¹⁴¹

Five nanogram per milliliter of TGF-β2 is more effective in three-dimensional chondrogenesis. However, the expression of TGF-β2 and TGF-β3 declines with age, as do the TGF-β receptors, which results in a lack of TGF-β signaling response and, thus, reduces the level of proteoglycan synthesis and other chondrogenic markers.¹⁴² Insulin-like growth factor-I (IGF-I) at 100 ng/mL has an additional effect on TGF-β2 in increasing the proteoglycan production of human aged BMSCs.¹⁴³ Additionally, the application of IGF-I combined with TGF-β1 and FGF-2 stimulate porcine SDSC proliferation; a cocktail containing IGF-I and TGF-β1 enhances expanded SDSC chondrogenic differentiation.¹⁴⁴

Antioxidants

Nimesulide, the nonsteroidal anti-inflammatory drug commonly used in the treatment of OA, and its metabolite in human 4-hydroxynimesulide are demonstrated to function as antioxidants that protect cartilage from oxidative stress in chondrocytes.¹⁴⁵ N-acetylcysteine is also reported to protect aged rat MSCs from tumor necrosis factor-–induced death¹⁴⁶ and prevent chondrocytes from nitric oxide-induced apoptosis.¹⁰¹ Statins, HMG-CoA reductase inhibitors, have been reported to inhibit inflammatory arthritis through a protective effect against chondrocyte aging and degeneration of articular cartilage both in vivo and ex vivo.¹⁴⁷ Melatonin, known chemically as N-acetyl-5-methoxytryptamine, is a natural compound found in animals, plants, and microbes. It has a role as a pervasive and powerful antioxidant, thus protecting nuclear and mitochondrial DNA from oxidative damage.^148,149 It potentially exerts anti-aging effects due to its cytoprotective properties. A recent study from our group shows that melatonin is able to enhance cartilage matrix synthesis and regeneration ex vivo possibly through the TGF-β signaling pathway.¹⁵⁰ Another study from Yudoh et al. suggested that water-soluble C60 fullerene, a strong free radical scavenger, prevented the degeneration of OA articular cartilage by inhibiting the catabolic stress-induced production of matrix-degrading enzymes (MMPs 1, 3, and 13), down-regulation of matrix production, and apoptosis and premature senescence in human chondrocytes in vitro.¹⁵¹

Serum deprivation, platelet-rich plasma, and low-glucose treatment

Serum contains many proteins, including cytokines and growth factors, all of which could have a profound influence on MSCs.¹⁵² Serum-free conditions have been shown to prime proliferation and help sustain chondrogenic potential of chondrocytes in aged patients.¹⁵³ However, serum deprivation significantly inhibited IVD cell proliferation and increased the positively stained SA-β-gal cells in both a monolayer and alginate culture system.¹⁵⁴ Interestingly, platelet-rich plasma, which is plasma enriched with platelets, has recently been reported to enhance chondrocyte and MSC proliferation and chondrogenic differentiation as well.^155–158 Similarly, glucose reduction treatment helps MSCs exhibit greater self-renewal and anti-senescence potential, evidenced by increased antioxidant defense response and oxygen consumption, increased electron transport chain complex expression, and decreased lactate production.¹⁵⁹

Hypoxia

An oxygen level of 21% has been used in a standard culture system initially to optimize the growth of fibroblasts; however, these conditions could be inherently stressful to other cell types. Since chondrocytes and MSCs reside in relatively low oxygen levels in vivo, they remain sensitive to the oxidative stress environment induced by a 21% oxygen level. Moussavi-Harami et al. found that a 21% oxygen level significantly attenuated the growth of human chondrocytes and MSCs and was associated with increased oxidant production.¹⁶⁰ MSCs cultured in hypoxia for a long time have better chondrogenic and adipogenic differentiation potentials, but not osteogenic potential.¹⁶¹ However, Schrobback et al. found that hypoxia during OA chondrocyte expansion and microcarrier bioreactor culture does not enhance intrinsic chondrogenic potential.¹⁶²

Meanwhile, lower oxygen tension enhanced the infrapatellar fat-pad-derived stem cells' chondrogenic differentiation through up-regulating hypoxia inductive factor 2α (HIF-2α) and increasing matrix synthesis and assembly.¹⁶³ SDSCs also benefited from low oxygen during ex vivo expansion and showed enhanced chondrogenic potential after incubation with an inductive medium for 14 days.¹⁶⁴ However, oxygen alone had no significant effect on human IVD cell proliferation or survival.¹⁵⁴ Hypoxia has been considered beneficial in preventing MSC senescence through down-regulation of extracellular signal-regulated kinases, inhibition of p16 gene up-regulation, and down-regulation of E2A-p21 by HIF-1 α -TWIST.¹⁶⁵

Decellularized ECM

Stem cells reside in “niches”—specific anatomic locations that control how they participate in tissue regeneration and maintenance. Their self-renewal potentials are balanced by niches that prevent stem cell depletion and overwhelming proliferation. ECM, known as a key component of the stem cell niche in vivo, is composed mostly of collagens, proteoglycans, and fibers. It functions as a reservoir for growth factors and provides natural and intrinsic cues that direct the remodeling process during cell differentiation.^166–169 Thanks to the highly conserved nature of ECM components between species,¹⁷⁰ decellularized ECM (DECM) is applicable in tissue-processing methods. In recent years, DECMs derived from different tissues or organs such as heart, lung, brain, liver, bladder, and adipose have been engineered through similar simple biochemical methods that function as a natural scaffold to support proliferation and differentiation of the recellularized cells.^171–177

As inspiring as Marshall Urist's work in the 1960s, demineralized bone matrix was able to induce ectopic bone formation due to the existence of the active osteoinductive ingredient bone morphogenetic protein (BMP).¹⁷⁸ Recently, Chen et al. demonstrated that components of native articular cartilage ECM can provide signals to drive ASCs toward chondrogenesis.^179,180 Cartilage-derived matrix can also induce chondrogenesis of BMSCs and can support neocartilage formation from chondrocytes without exogenous growth factors.^180,181 Cartilage-derived matrix is advantageous in serving as a scaffold for cell-based cartilage repair. DECM can also be deposited by chondrocytes.^182–184 Despite the fact that DECM deposited by cartilage and chondrocytes retains the cues for stem cell chondrogenesis, our recent study demonstrated that DECM deposited by stem cells was superior to chondrocyte-produced DECM in enhancing stem cell and chondrocyte proliferation and chondrogenic differentiation.¹⁸⁴

Decellularized stem cell matrix

Recent work in our laboratory demonstrates that decellularized stem cell matrix (DSCM) is a robust cell expansion system that enhances expanded stem cell proliferation and chondrogenic differentiation capacity.¹⁸⁵ DSCM deposited by SDSCs provides a tissue-specific microenvironment favoring expanded cell chondrogenesis.^164,186,187 DSCM deposited by human BMSCs provides a tissue-specific microenvironment favoring expanded BMSC endochondral bone formation.¹⁸⁸ A rejuvenated effect was also observed in chondrocytes¹⁸⁹ and NP cells¹⁹⁰ after expansion on DSCM deposited by SDSCs. Replicative senescence and dedifferentiation of chondrocytes and NP cells were delayed, and redifferentiation was enhanced, accompanied by increased expression of CD90, one of the MSC markers. In order to further investigate the rejuvenating mechanisms, both human fetal and adult SDSCs were used to generate DSCM. Intriguingly, fetal SDSC-derived DSCM has a superior effect over adult SDSCs in terms of enhancing proliferation and restoring chondrogenic differentiation potential (unpublished data). Similarly, Choi et al. reported that senescent human diploid fibroblasts could be restored to a more youthful state through interaction with ECM deposited by younger cells, possibly through delaying telomere shortening.¹⁹¹ Sun et al. also observed the rejuvenating effect of younger BMSC-derived DSCM both in vivo and in vitro.¹⁹² The literature just mentioned suggests that cellular senescence is profoundly influenced by cues from ECM and that DSCM can provide the necessary signals, at least partially, to reverse the senescing process.

Senescence-Associated Signal Transduction Pathways Involved in Chondrogenic Differentiation

The extensive proliferation and multi-differentiation potentials of MSCs have been revealed, making them an ideal source for tissue-engineering applications. Identification of senescence-related signaling pathways involved in chondrogenesis is becoming more important for developing strategies that overcome replicative senescence and attaining targeted differentiation. Among others, the FGF, TGF-β/Smads, Notch, Wnt/β-catenin, and CCN protein family and involved signaling pathways have been suspected to be involved not only in preventing senescence, but also in proliferation and multi-lineage differentiation of MSCs.

Notch and Wnt/β-catenin signaling pathways

Notch is a transmembrane protein involved in cell fate determination.¹⁹³ Notch 1 is only expressed temporarily and spatially in developing cartilage. When expressed and activated in the early stage of chondrogenesis, Notch signaling has a strong inhibitory effect on both proliferation and differentiation of chondrogenic cells. Expression of Notch decreases as the chondrogenic differentiation proceeds.¹⁹⁴ Notch signaling inhibits TGF-β3 induced chondrogenesis of human MSCs through binding to a SOX9 enhancer site, thus preventing subsequent Col 2AI activation.¹⁹⁵

The Wnt/β-catenin pathway is related to stem-cell self-renewal and proliferation. Maruyama et al. found that, besides directly regulating renewal and proliferation, activation of β-catenin also alters the lineage commitment of MSCs to differentiate into chondrocytes through cooperating with FGF receptor 1 (FGFR1) and balancing between BMP and FGF signaling pathways.¹⁹⁶

Increasing evidence indicates that Wnt and Notch signaling interact with each other synergistically and antagonistically. The interaction exists throughout development and continues in a homeostatic context in many systems.¹⁹⁷ Since the Wnt signaling is also involved in inhibiting adipogenesis of MSCs through β-catenin dependent and independent pathways,¹⁹⁸ tumor necrosis factor-alpha (the activator of the NF-kB pathway) inhibits adipogenesis by the β-catenin/TCF4-dependent pathway.¹⁹⁹ Interactions between NF-kB and β-catenin/Tcf pathways also play a part in regulating MSC proliferation and differentiation.²⁰⁰ However, balance among signaling pathways is more important in regulating MSCs through lineage differentiation, as well as maintaining stemness.

p53-p21-pRB/p16-pRB signaling pathways

Two tumor-suppressor proteins, p53 and pRB, are also involved in the p53-p21-pRB pathway. Senescence stimuli activate p53, which then induces pRB related senescence by activating p21, the transcriptional target of p53. This senescence is reversible on the subsequent inactivation of p53.¹²⁹ The p53-p21-pRB pathway has also been demonstrated as playing a more important role in senescent NP chondrocytes in vivo.⁸² In addition, the p53/p21 pathway is activated by excessive activation of Wnt/β-catenin signaling, which leads to MSC senescence as well.²⁰¹ The induction of caveolin 1 up-regulates p53 and p21 and down-regulates pRB. Caveolin overexpression also activates the p38 mitogen-activated protein kinase (MAPK) pathway, suggesting that both of these pathways are involved in mediating chondrocyte senescence.¹²⁷ Caveolin 1 overexpression has been shown to be responsible for loss of adipogenic differentiation abilities in senescent human MSCs.²⁰²

In the p16-pRB pathway, senescence stimuli induce p16, which then activates pRB. Once the pathway is activated, the senescence cannot be reversed by inactivating pRB or silencing p16.¹²⁹ The expression of the p16 gene is closely related with human MSC senescence. Knockdown of the p16 gene will rescue human MSCs from senescence and promote their proliferation.²⁰³

CCN protein family and involved signaling pathways

The CCN protein family contains six members, CCN1-6, which are small-secreted cysteine-rich proteins that function as modulator proteins during development. CCNs are widely and highly expressed in adult and embryonic tissues and can be induced by growth factors, cytokines, or cellular stress. In stem-cell differentiation, CCN1-4 have been reported to be involved with osteogenesis, adipogenesis, and chondrogenesis. Expression of CCN proteins has been reported to cooperate with other signaling pathways such as BMP, TGF, and Wnt.^204–206 CCN1 promotes human primary mesenchymal progenitor cell proliferation.²⁰⁷ CCN2, also named connective tissue growth factor, is involved in more than one step of chondrogenic differentiation, such as proliferation, maturation, and hypertrophic differentiation. Both the BMP and Wnt signaling pathways have been proved to be a mutual target of CCN2.²⁰⁸ CCN2 interacts with BMP2 through forming a complex that regulates the prehypertrophic and hypertrophic chondrocyte proliferation and differentiation.²⁰⁹ CCN2 modulates Wnt signaling through binding to the lipoprotein receptor-related protein 6 co-receptor.²¹⁰ The expression of CCN2 in chondrocytes is regulated by Rac1 and actin pathways mediated by TGF-β/Smad signaling.²¹¹ In addition, CCN2 and CCN3 exert the opposite effect on regulating proliferation and differentiation in chondrocytes.²¹² CCN4 is a target of the Wnt1 pathway and may function as a promoter of MSC proliferation and osteoblastic differentiation while inhibiting chondrogenic differentiation.^213,214

Phosphatidylinositol 3-kinases/Akt and MAPK signaling pathways

The involvement of the phosphatidylinositol 3-kinases (PI3K)/Akt signaling pathway in chondrocyte senescence was proved by investigating the mechanisms of the role of oxidized low-density lipoprotein (ox-LDL) in cartilage degeneration. Zushi et al. showed that ox-LDL binding to lectin-like ox-LDL receptor 1 causes stress-induced premature senescence of chondrocytes and results in suppression of telomerase activity by inactivating the PI3K/Akt pathway.²¹⁵ Since caveolin-1 overexpression has been correlated with human MSC senescence,¹¹³ Yudoh et al. further found that the application of angiogenic growth factors, including vascular endothelial growth factor, FGF-2, or hepatocyte growth factor, can inhibit IL-1β-induced chondrocyte senescence by down-regulating caveolin-1 expression.²¹⁶ This effect may be mediated through the p42/44 MAP kinase and PI3K/Akt signaling pathways.²¹⁶

FGF and TGF-β/Smads signaling pathways

FGFs and related receptors (FGFRs) have long been known to play a pivotal role in cell proliferation and chondrogenesis during embryonic limb development. FGFR1-3 are expressed in a stage-dependent pattern during ex vivo chondrogenic differentiation and in vivo skeletogenesis. FGFR3 signaling is capable of inducing reversible chondrocyte senescence.²¹⁷

TGF-β signaling is the main initiator of chondrogenic differentiation in mesenchymal progenitor cells^218,219 and is also involved in all stages of chondrogenesis. TGF-β signals through both type I receptor activin receptor like kinase (ALK) 5 and ALK1. The balance of ALK5/ALK1 determines the overall effect of TGF-β signaling in chondrocytes. However, in aging and OA-related TGF-β signaling changes, Blaney Davidson et al. reported a significant loss of expression of the TGF-β type I receptor ALK5 and phosphorylation of Smad2/3 in murine articular cartilage.²²⁰ However, there was no significant difference observed in nonphosphorylated Smad2 and Smad3 in young and old animals.

IGF-I signaling pathway

IGF-I plays a critical role in regulating growth and tissue formation during both embryonic and postnatal development. The aberrant expression of IGF-I signaling is also involved in OA²²¹ and cell senescence/aging.^222–225 An age-related decline in response to IGF-I stimulation has been identified in both bone^226,227 and cartilage.^228–230 Boehm et al. suggested that IGF-I signals through phosphorylation and activation of the protein kinase Akt, which requires binding to heat shock protein 90 (Hsp90) for activation. The level of Hsp90 decreases with aging, which further influences the collagen II and MMP13 expression and results in the dysregulation of cartilage ECM.²³¹ Fortier et al. demonstrated that the decreased responsiveness of IGF-I in aging articular chondrocytes could be due to the decreased cell division cycle 42 protein,²³² a member of the Rho-subfamily of GTPase that regulates the organization of the actin skeleton and other cellular activities.²³³

Recently, the role of IGF-I has been characterized in stem cells, such as haematopoietic progenitor cells,^234,235 adult neural stem cells,²³⁶ and possibly embryonic stem cells.²³⁷ IGF-I has been demonstrated to induce chondrogenesis of MSCs derived from adipose, periosteal, and human umbilical cord.^238–241 When combined with other growth factors, such as TGF-β1 or FGF-2, IGF-I can induce SDSC and BMSC proliferation and chondrogenic differentiation.^21,242–244 However, there is another report suggesting that adenoviral mediated-transferred IGF-I did not have a chondrogenic effect on human MSCs.²⁴⁴

Mammalian target of rapamycin/Akt signaling pathways

Mammalian target of rapamycin (mTOR) is a highly conserved serine/threonine kinase²⁴⁵ involved in the integration of many environmental signals, such as oxygen, nutrient, and growth factor signaling.²⁴⁶ Akt has been found to be positively related with chondrocyte maturation, proliferation, cartilage matrix production, and cell growth during skeletal development. Recent studies have revealed the close relationship with mTOR and PI3K/Akt signaling pathways, suggesting that Akt activates mTOR and positively regulates the four cellular processes just mentioned.^247–250 mTOR is also reported to directly influence chondrocyte differentiation, possibly through regulating the Indian hedgehog signaling pathway.²⁵¹

Other regulators and related signaling pathways

Epidermal growth factor (EGF) receptor-1, which can be activated by the EGF family ligands such as heparin-binding EGF-like growth factor, is also reported to increase MSC proliferation and multi-differentiation potentials.²⁵² Toll-like receptors (TLRs) and Nod-like receptors (NLRs) are known for initiating innate immune response in vivo. TLRs have been shown to be involved in regulating BMSC or ASC proliferation, differentiation, migration, and immunomodulation.^253–255 Kim et al. also suggested that both TLR and NLR agonists could promote human umbilical cord blood-derived MSC chondrogenesis, though they have no effect on proliferation.²⁵⁶

Perspective

Better knowledge about aging has been discovered in the past few years. Given that aging is an irreversible process, researchers have started to think about how they can moderate it. Since cell senescence challenges us in every aspect of our lives, let alone the cartilage tissue-engineering field, slowing down the development of cell senescence would benefit millions of suffering patients. As investigators, identifying senescence in an earlier state, understanding deeper causal mechanisms, and exerting more effort searching for a safe and efficient method is what we hope to attain. Current efforts in applying growth factors, antioxidants, and modulating nutrients and oxygen factors have significantly improved proliferative abilities in both chondrocytes and MSCs. The promoted chondrogenic differentiation also alleviated progression to cell senescence to a lesser extent. However, efficiency and concerns about immune rejection as well as transformation of cells could be worrisome. Fortunately, the creation of the ex vivo microenvironment using DECM, especially DSCM, has given us hope. Through incubating cells in a more youthful and natural ex vivo niche, cell senescence could be slowed. However, things are never as easy as we want; the rejuvenating effect that ECM manifests still needs to be thoroughly evaluated and investigated. Hopefully, as we move along the right path with useful tools, we will overcome the challenge of cell senescence.

Footnotes

Acknowledgment

The authors thank Suzanne Smith for her help in editing the article.

Disclosure Statement

No competing financial interests exist.

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