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. 2023 Oct 26;24(21):15617. doi: 10.3390/ijms242115617

Table 1.

Summary of the results of the included studies.

Cells/Tissue In Vitro Model of Senescence Evaluations Main Results Ref.
NP cells
from healthy SD rats
(6–8 wks old)		 Group 1: cells;
Group 2: cells + TNFα (20 ng/mL) and IL1β (20 ng/mL)		 Cell proliferation (CCK8 assay);
Cell senescence (SA-β-Gal staining);
Cell cycle (Flow cytometry);
TE activity (ELISA);
ROS content (ROS assay);
AGG, COLL II, p53, p16,
MMP3, MMP13, ADAMTS4
gene expression (RT-PCR);
AGG, COLL II proteins (ICC);
p16, p53 proteins (WB)		 Group 2: ↑ ROS content, SA-β-Gal, G0/G1 phase, p16, p53, MMP3, MMP13 and ADAMTS4 expression; ↓ cell viability, TE activity, S phase, AGG, and COLL II proteins
than group 1		 Li et al., 2019 [19]
NP cells and discs
from healthy SD rats
(6–8 wks old)		 Group 1: cells;
Group 2: cells + TNFα (10 ng/mL);
Group 3: discs;
Group 4: discs + TNFα (200 ng/mL)		 Cell proliferation (CCK8 assay);
Cell senescence (SA-β-Gal staining);
Cell cycle (Flow cytometry);
TE activity, ROS content (ELISA);
p16, p53, AGG, COLL II
gene expression (RT-PCR);
AGG, COLL II, ERα,
ERβ proteins (ICC);
p16, p53, AGG, COLL II,
NF-κB p65, p-NF-κB p65, ERα,
ERβ proteins (WB);
GAG, HYP content;
AGG, COLL II proteins (IHC)		 Groups 2, 4: ↓ cell viability,
TE activity, AGG and
COLL II proteins; ↑ SA-β-Gal, ROS, and p53 and p16 proteins
than groups 1, 3		 Li et al., 2017 [20]
NP cells
from healthy SD rats
(6–8 wks old)		 Group 1: cells;
Group 2: cells + TNFα
(10, 20, 40 ng/mL);
Group 3: cells + TNFα (10 ng/mL)
+ recovery in normal
culture conditions;
Group 4: cells + TNFα (10 ng/mL) without recovery		 Cell proliferation (CCK8 assay);
Cell viability (LIVE/DEAD);
Cell senescence (SA-β-Gal staining);
Cell cycle (Flow cytometry);
TE activity (ELISA);
p53, p16, AGG, COLL II,
AKT gene expression (RT-PCR);
GAG content (DMMB assay);
AGG, COLL II proteins (ICC)		 Groups 2, 4: ↓ cell proliferation,
TE activity, AGG and
COLL II proteins; ↑ p53, p16, and AKT expression than group 1 in a dose-dependent manner.
Group 3: similar results
than group 4		 Li et al., 2017 [21]
NP cells
from healthy SD rats
(4 wks old);
BMSCs
from healthy SD rats
(4–5 days old)		 Group 1: cells + blank calcium
alginate gel balls;
Group 2: cells treated with
TNFα (20 ng/mL) + blank calcium
alginate gel balls;
Group 3: cells treated with
TNFα (20 ng/mL) + calcium alginate gel balls with BMSCs;
Group 4: cells treated with
TNFα (20 ng/mL) + calcium alginate gel balls with NP cells		 Cell viability (CCK8 assay);
Cell senescence (SA-β-Gal staining);
Cell cycle (Flow cytometry);
p16, p21, p53
gene expression (RT-PCR);
COLL II, MMP9, ZMPSTE24,
RelA proteins (IF)		 Groups 2, 4: ↑ SA-β-Gal than groups 1, 3.
Groups 2, 4: ↑ SA-β-Gal, MMP9 protein, p16, p21, and p53 expression; ↓ ZMPSTE24 protein than group 3		 Li et al., 2019 [22]
Discs of healthy pigs
(3–4 mo old)		 Group 1: discs;
Group 2: discs + intermittent
compression
(0.1 MP at 1.0 Hz for 2 hrs) +
perfusion-culture;
Group 3: discs + intermittent
compression
(1.3 MP at 1.0 Hz for 2 hrs) +
perfusion-culture		 Cell senescence (SA-β-Gal staining);
PG distribution
(Alcian Blue staining);
AGG, COLL II, TIMP1, TIMP3, ADAMTS4, MMP3, p16, p53
gene expression (RT-PCR);
p38, p-p38, p16, p53 proteins (WB);
AGG, COLL II, PCNA
proteins (IHC);
GAG, HYP content
(Biochemical measurement)		 Group 3: ↑ SA-β-Gal, p38, p-p38, p16, p21, p53, ADAMTS4, and MMP3 proteins; ↓ AGG, COLL II, GAG, HYP, and PCNA proteins, TIMP3 expression than groups 1, 2 Pang et al., 2017 [23]
NP cells
from healthy SD rats
(7–8 wks old),
suspended in collagen solution
and seeded into DBM		 Group 1: cells;
Group 2: cells + 20% dynamic
compression (1.0 Hz for 6 hrs/day)		 Cell proliferation (CCK8 assay);
Cell senescence (SA-β-Gal staining);
Cell cycle (Flow cytometry);
TE activity, ROS content (ELISA);
p16, p53 gene expression (RT-PCR);
p16, p53, NF-κB p65,
p-NF-κB p65 proteins (WB)		 Group 2: ↓ cell proliferation;
↑ SA-β-Gal, p53, p16, and NF-κB proteins, ROS content than group 1		 Jiang et al., 2018 [24]
Discs
from healthy pigs
(12–13 mo old)		 Group 1: discs;
Group 2: discs + static compression (0.4 MP at 1.0 Hz for 4 hrs/day);
Group 3: discs + dynamic
compression
(0.4 MP at 1.0 Hz for 4 hrs/day)		 Cells senescence (SA-β-Gal staining);
TE activity (ELISA);
AGG, COLL II, p16, p53
gene expression (RT-PCR);
COLL II, AGG proteins (IHC);
p53, p16 proteins (WB);
GAG, HYP content
(Biochemical measurement)		 Group 2: ↑ SA-β-Gal, p16 and
p53 proteins; ↓ TE activity, AGG,
COLL II, GAG, and HYP proteins
than groups 1, 3.
Group 3: ↓ SA-β-Gal, p16 and
p53 proteins; ↑ TE activity, AGG,
COLL II, GAG, and HYP proteins
than groups 1, 2		 Shi et al., 2018 [25]
AF cells
from healthy SD rats
(3 mo old)		 Group 1: cells;
Group 2: cells + 5% elongation
(1 Hz for 8 hrs/day);
Group 3: cells + 20% elongation
(1 Hz for 8 hrs/day)		 Cell proliferation (CCK8 assay);
Cell cycle (Flow cytometry);
TE activity (ELISA);
AGG, COLL II, BCL1, LC3,
p53, p16 gene expression (RT-PCR);
p16, p53, AGG, COLL II,
BCL1, LC3 proteins (WB)		 Group 1: similar results
than group 2;
Group 3: ↓ cell proliferation, TE activity, S phase, AGG, COLL II, BCL1, and LC3 proteins; ↑ G0/G1 phase, p16, and p53 proteins than groups 1, 2		 Zhao et al., 2019 [26]
NP cells
from healthy SD rats
(8 wks old)		 Group 1: cells;
Group 2: cells + H2O2
(0.05–0.15 mM)		 Cell morphology
(H/E, toluidine blue staining);
Cell proliferation (CCK8 assay);
Cell senescence (SA-β-Gal staining);
Nuclear morphology
(Hoechst staining);
ROS level;
Cell cycle, apoptosis
(Flow cytometry);
TNFα, IL1β, IL6, IL8
gene expression (RT-PCR);
p53, p21, p16 proteins (WB)		 Group 2: ↑ SA-β-Gal, p53, p21,
and p16 proteins, TNFα, IL1β, IL6, and IL8 expression; ↓cell viability and proliferation than group 1		 He et al., 2019 [27]
NP cells
from healthy subjects
(nr)		 Group 1: cells;
Group 2: cells + H2O2
(0–2 mM)		 ROS kinetics (DCFH-DA assay);
Cell viability (MTT assay);
Cell proliferation
(DNA synthesis assay);
Cell cycle (Flow cytometry);
Cell senescence (SA-β-gal staining);
Telomere length;
p16, p21 Catalase, SOD,
COX2, ADAMTS4, ADAMTS5, MMP1, MMP2, MMP9, MMP13, TIMP1, TIMP2, TIMP3, ACAN,
Biglycan, Decorin, Versican
gene expression (RT-PCR);
PARP, p-p38, p38, p-SAPK/JNK, SAPK/JNK, p-AKT, AKT, p-Chk2,
p-p53, p-ERK1/2, panERK, p21,
p-ATM, p53, p16,
pRb proteins (WB);
p-H2A.X (IF)		 Group 2: ↑ ROS; ↓ cell proliferation than group 1.
Group 2 at 0.5 mM: ↑ p-38, H2A.X, and p53 proteins, SA-β-gal, ROS,
and ECM-degrading enzymes; ↓ ECM structural components		 Dimozi et al., 2015 [28]
NP and AF cells of
healthy Wistar rats
(2 mo old);
NP and AF cells
from healthy
Wistar rats
(2 and 22 mo old);
NP and AF cells
from pz with disc herniation
(nr);
NP and AF cells
from healthy subjects
(nr)		 Group 1: cells from pz;
Group 2: cells from pz + γ-ray
(60Co gamma, 2 Gy/min);
Group 3: cells from young rats +
γ-ray (60Co gamma, 2 Gy/min);
Group 4: cells from old rats + γ-ray (60Co gamma, 2 Gy/min);
Group 5: cells from healthy
individuals + γ-ray
(60Co gamma, 2 Gy/min)		 Cell proliferation
(BrdU incorporation assay);
Cell senescence (SA-β-gal staining);
GL13 protein (ICC);
p16, GL13 proteins (IHC)		 Group 2: ↓ cell proliferation;
↑ SA-β-gal, GL13 proteins
than group 1.
Group 4: ↑ SA-β-gal, GL13 protein than group 3.
Group 2: ↑ p16, GL13 proteins
than group 5		 Veroutis et al., 2021 [29]
NP cells
from healthy bovine
(nr)		 Group 1: cells;
Group 2: cells + 10% FBS, 4.5 mg/mL glucose, 300 mOsm/kg H2O, 20% O2 + γ-ray
(60Co gamma, 2.5 Gy/min);
Group 3: cells + serum-free,
0.9 mg/mL glucose,
400 mOsm/kg H2O, 2% O2 + γ-ray
(60Co gamma, 2.5 Gy/min)		 Cell proliferation
(BrdU incorporation);
Cell senescence (SA-β-Gal staining);
p16, p21, ICAM1, MMP1,
MMP2, MMP3, ADAMTS4, ADAMTS5, AGG, COLL II
gene expression (RT-PCR)		 Group 2: ↓ cell proliferation,
↑ SA-β-Gal than group 1.
Groups 2, 3: ↑ p16, p21, ICAM1, MMP1, MMP2, MMP3, AGG,
and MMP1 expressions; ↓ COLL II expression than group 1		 Kouroumalis et al., 2019 [30]
Purchased human Chons Group 1: cells + monoenergetic
C-ions (75–95 MeV/mm);
Group 2: cells + X-ray
(225 kV, 13 mA);
Group 3: cells in collagen scaffold
+ monoenergetic C-ions
(75–95 MeV/mm);
Group 4: cells in collagen scaffold
+ X-ray (225 kV, 13 mA)		 Cell proliferation (Flow cytometry);
Cell toxicity
(Bioluminescent cytotoxicity assay);
Cell senescence (SA-β-gal staining);
Cell apoptosis (Flow cytometry);
COX2, p21, Caspase-3 proteins (WB)		 Group 1: ↓ cell viability
than group 2.
Groups 1, 3: ↑ SA-β-gal
than groups 2, 4.
Groups 3, 4: ↑ p21; ↓ COX2
than groups 1, 2		 Hamdi et al., 2015 [31]
Chons
from healthy horses
(4–7 yrs old);
Chons
from cadavers
(38–73 yrs old)		 Group 1: cells;
Group 2: cells + γ-ray (10 Gy);
Group 3: cells + TGFβ1 and bFGF;
Group 4: cells + γ-ray (10 Gy) + TGFβ1 and bFGF		 Cell senescence (SA-β-gal staining);
MMP13, IL6, IGFBP3, CCL2
gene expression (RT-PCR);
p16, γH2AX proteins (IF)		 Group 2: ↑ SA-β-gal, p16, γH2AX than group 1;
Group 4: ↑ SA-β-gal, γH2AX,
and p16 proteins, IGFBP3
and CCL2 gene expressions
than groups 2, 3		 Copp et al., 2021 [32]
BMSCs
from healthy SD rats
(3 wks old);
Chons
from healthy SD rats
(3 wks old)		 Group 1: BMSCs;
Group 2: Chons;
Group 3: BMSCs + Chons;
Group 4: Chons + γ-ray
(1.080 Gy/min for 10 min);
Group 5: BMCs + Chons + γ-ray (1.080 Gy/min for 10 min)		 Cell proliferation
(EdU incorporation assay);
Cell senescence (SA-β-Gal staining);
Bax, Bcl-2, CDKN2A, CDKN1A,
IL6, MMP1, MMP13, Sox9,
IL1β, Coll II, Nanog, Oct-4
gene expression (RT-PCR);
Caspase-3 protein (IHC);
COLL II, AGG proteins (WB)		 Group 4: ↑ SA-β-Gal, IL6
and MMP13 expression,
Caspase-3 protein; ↓ proliferation, Bax/Bcl2, IL1β and COLL II expression than group 2.
Group 5: ↓ proliferation, COLL II gene expression and protein; ↑ Caspase-3 protein, Bax/Bcl2, Oct4 and Nanog expression, SA-β-Gal,
AGG protein than groups 1, 3		 Cao et al., 2019 [33]
Chons
from healthy subjects
(28–59 yrs old)		 Group 1: cells;
Group 2: cells cultured
for 24 hrs + IL1β (10 ng/mL)		 Cell proliferation (CCK8 assay);
Cell apoptosis (Flow cytometry);
Cell senescence (SA-β-gal staining);
COLL II, ADAMTS5, MMP13
gene expression (RT-PCR);
GRP78 protein (WB)		 Group 2: ↓ COLL II expression;
↑ ADAMTS5 and MMP13 gene expressions, GRP78 protein
than group 1		 Liu et al., 2017 [34]
Chons
from NZW rabbits
(1 mo old)
and rats
(2 wks old)		 Group 1: rabbit cells +
serum-starved overnight conditions + DKK1 (100 ng/mL) +
IL1β (10 ng/mL);
Group 2: rabbit cells +
serum-starved overnight conditions + LiCl (20 mM) + IL1β (10 ng/mL);
Group 3: rabbit cells;
Group 4: rat cells + serum-starved overnight + Wnt-1 (10 ng/mL);
Group 5: rat cells;
Group 6: rat cells + serum-starved overnight + Wnt-1 (10 ng/mL) +
IL1β (10 ng/mL);
Group 7: rat cells + serum-starved overnight + Wnt-1 (10 ng/mL) + PBS		 Cell senescence (SA-β-gal staining);
MMP13, p53, 18s rRNA, MMP3
gene expression (RT-PCR);
β-catenin, MMP13, MMP3, p16,
p53, acetylated p53, SIRT-1
proteins (WB)		 Group 2: ↑ β-catenin, MMP13,
and acetylated p53, SIRT-1 proteins than group 1.
Group 4: ↑ cell senescence, p53,
and p16 proteins than group 5.
Group 6: ↑ MMP3 and MMP13 proteins than group 7		 Li et al., 2020 [35]
Chons
from cartilage of preserved (P-C) and severely damaged (D-C) areas of OA pz (nr)		 Group 1: Chons micromasses
from P-C;
Group 2: Chons micromasses
from D-C		 Cell proliferation (Flow cytometry);
Cell senescence (SA-β-gal staining);
Telomere length (TAT assay);
p16INK4a, p21, p53, COLL II,
SOX9, AGG, Ats4, Ats5, MMP12, MMP13, COLL I, COLL III, VCAN, IL6, IL8, p16, SA-β-gal
gene expression (RT-PCR);
p16INK4a, MMP13 proteins (IHC);
COLL II, p16INK4a proteins (IF);
SOX9, MMP13, p21, p53,
p16 proteins (WB);
GAG content
(Safranin O/Fast green staining)		 Group 2: ↓ cartilage matrix
deposition, telomere length,
COLL II, SOX9, and AGG expression; ↑ MMP13, p16, p21, and p53 proteins and expressions than group 1		 Wang et al., 2022 [36]
Purchased C2C12
(10th–12th in vitro passages)		 Group 1: myotubes;
Group 2: myotubes + 10%
of serum of fasted young donors;
Group 3: myotubes + 10%
of serum of old donors		 Desmin protein (ICC);
IgG2a monoclonal antipuromycin, p-mTOR, mTOR, p-Akt, Akt,
p-p70S6K1, p70S6K1, p-4EBP-1, 4EBP1, p-RPS-6, RPS-6, p-eEF2, eEF2, LC3A/B, Caspase-3,
MuRF-1, F-box (WB)		 Group 2: ↑ myotube diameter and NFI, Akt, p70S6K1, and eEF2 proteins than groups 1, 3.
Group 3: ↓ Myotube diameter than group 1		 Allen et al., 2021 [37]
Purchased C2C12 cells Group 1: cells + 5% plasma
from young subjects;
Group 2: cells + 5% plasma
from old subjects		 Cell proliferation (cell counter);
Scratch assay;
Cell diameter		 Group 1: ↓ scratch size; ↑ cell diameter, proliferation than group 2 Kalampouka et al., 2018 [2]
Purchased human myoblasts Group 1: cells at 1st passage +
simulated microgravity
(1 × 10−3 G; µG);
Group 2: cells at 1st passage +
normal gravity (1 G);
Group 3: cells at 5th passage +
normal gravity (1 G)		 Cell proliferation assay;
Cell senescence (SA-β-Gal activity);
Desmin, Myf5 proteins (ICC);
Cytoskeleton areas and
nuclei analysis;
Differentiation into myotubes
(IF for MHC)		 Groups 2, 3: ↑ MHC and
desmin proteins, myotubes
diameter, cell proliferation;
↓ Myf5 protein than group 1.
Groups 1, 3: ↑ SA-β-Gal than group 2		 Takahashi et al., 2021 [38]
BMSCs
from OA pz		 Group 1: cells at 10th passage;
Group 2: cells at 4th passage		 Tri-lineage differentiation;
CFU assay;
Cell proliferation
(Flow cytometry, MTT);
Cell senescence (SA-β-gal staining);
Telomere length (TAT assay);
p16, MMP13 proteins (IHC);
p16, p21, p53, COLL II,
SOX9, AGG, Ats4, Ats5,
MMP12, MMP13, COLL I, COLL III, VCAN, IL6, IL8, SA-β-gal
gene expression (RT-PCR);
COLL II, p16 proteins (IF);
SOX9, MMP13, p21, p53,
p16INK4a proteins (WB)		 Group 1: ↓ proliferation, differentiation potential, telomere length, COLL II, SOX9, and AGG expressions and proteins; ↑ p16, SA-β-gal, MMP13, p21, and p53 expressions and proteins than group 2 Wang et al., 2022 [36]
NPMSCs
from pz
(37–52 yrs old)		 Group 1: cells;
Group 2: cells + TNFα (10 ng/mL)		 Cell viability (Calcein-AM);
Cell senescence (SA-β-Gal staining);
Cell proliferation (CCK8 assay);
p16, p21, p53, IL6, MMP13, ADAMTS5, brachyury, FOXF1, Pax1, CA12, HIF1α, COLL Iα1,
COLL IIα1, SOX9, AGG, LPL,
PPAR2, ALP, Runx2
gene expression (RT-PCR);
COLL II, CA12 proteins (IF, WB);
GAG content (DMMB assay)		 Group 2: ↑ senescent cell morphology, SA-β-Gal, p16, p21, p53, IL6, MMP13, and ADAMTS5 expressions
than group 1		 Li et al., 2018 [39]

Abbreviations: Ref = references; NP = nucleus pulposus; SD = Sprague–Dawley; wks = weeks; TNFα = Tumor necrosis factor α; IL = interleukin; CCK8 = cell counting kit-8 reagents; Sa-β-Gal = Senescence-associated β-galactosidase; TE = telomerase; ELISA = enzyme-linked immunosorbent assay; ROS = reactive oxygen species; AGG = aggrecan; COLL = collagen; MMP = matrix metalloproteinase; ADAMTS = a disintegrin and metalloproteinase with thrombospondin motifs; RT-PCR = real time PCR; ICC = Immunocytochemistry; WB = Western blotting; GAG = glycosaminoglycan; HYP = hydroxyproline; IHC = Immunohistochemistry; ER = estrogen receptor; DMMB = dimethylmethylene blue; BMSCs = bone marrow mesenchymal stem cells; ZMPSTE24 = zinc metallopeptidase STE24; IF = immunofluorescence; mo = months; hrs = hours; PG = proteoglycan; TIMP = tissue inhibitor of metalloproteinase; PCNA = proliferating cell nuclear antigen; NF-κB = nuclear factor kappa-light-chain-enhancer of activated B cells; AF = Annulus Fibrosus; H2O2 = hydrogen peroxide; BrdU = 5-bromo-2′-deoxyuridine; Chons = chondrocytes; COX-2 = cyclooxygenase-2; yrs = years; bFGF = basic fibroblastic growth factor; TGFβ = transforming growth factor beta; IGFBP3 = insulin-like growth factor binding protein 3; CCL2 = C-C motif chemokine ligand 2/monocyte chemoattractant protein 1; GRP78 = 78-KDa glucose-regulated protein; NZW = New Zealand white; NPMSCs = nucleus pulposus mesenchymal stem cells; DBM = decalcified bone matrix; DCFH-DA = Dichloro-dihydro-fluorescein diacetate; ECM = extracellular matrix; pz = patients; ICAM1 = intercellular adhesion molecule 1; Oct-4 = octamer-binding transcription factor 4; CDKN = cyclin dependent kinase inhibitor; LiCl = Lithium chloride; PBS = Phosphate buffered saline; SIRT-1 = Sirtuin-1; DKK1 = Dickkopf-1; OA = osteoarthritis; TAT = telomere analysis technology; EEF2 = Eukaryotic Translation Elongation Factor 2; NFI = Nuclear Factor I; MuRF1 = Muscle RING-finger protein-1; RPS6 = Ribosomal Protein S6; EIF4EBP1 = Eukaryotic Translation Initiation Factor 4E Binding Protein 1; µG = microgravity; G = gravity; MYF5 = Myogenic Factor 5; MHC = Myosin heavy chain; CFU = colony-forming unit; ALP = alkaline phosphatase; RUNX2 = Runt-related transcription factor 2; PPAR = peroxisome proliferator-activated receptors; LPL = Lipoprotein lipase; HIF = Hypoxia-inducible factor; H/E= Hematoxylin/Eosin; CA12 = Carbonic anhydrase 12; nr = not reported; ↑ = increase; ↓ = decrease.