The objective of this study was to evaluate the long-term effects of an autonomous RUNX2-supressing gene circuit on cartilage matrix accumulation and compressive mechanics in the tissues produced by MSC-derived chondrocytes (MdChs). Human mesenchymal stem cells (MSCs) were differentiated into chondrocytes and genetically modified using a lentiviral short hairpin RNA (shRNA) targeting RUNX2 (shRUNX2). Two configurations, low and high levels of shRUNX2 expression, were implemented. The influence on RUNX2 suppression, matrix protein secretion, and compressive mechanics was evaluated. Both low and high shRUNX2 suppressed RUNX2 expression, showing significant improvement in cartilage matrix accumulation with increased collagen type II and aggrecan deposition. High shRUNX2 more effectively suppresses RUNX2 gene expression and enhances sulfated glycosaminoglycan and matrix accumulation compared with the control and low shRUNX2 group. This study demonstrates the long-term effect of autonomous RUNX2 suppression in enhancing cartilage matrix accumulation and mechanical properties, providing a promising approach for advancing cartilage tissue engineering and repair strategies.
Get full access to this article
View all access options for this article.
References
1.
Sophia FoxAJ, BediA, RodeoSA. The basic science of articular cartilage: Structure, composition, and function. Sports Health, 2009; 1(6):461–468; doi: 10.1177/1941738109350438
2.
KuivaniemiH, TrompG, ProckopDJ. HUMU 750 MUTATION UPDATE Mutations in Fibrillar Collagens (Types I, II, III, and XI), Fibril-Associated Collagen (Type IX), and network-forming collagen (Type X) Cause a spectrum of diseases of bone, cartilage, and blood vessels. Hum Mutat, 1997; 9(4):300–315; doi: 10.1002/(SICI)1098-1004(1997)9:4<300::AID-HUMU2>3.3.CO;2-8
VincentTL, McClurgO, TroebergL. The extracellular matrix of articular cartilage controls the bioavailability of pericellular matrix-bound growth factors to drive tissue homeostasis and repair. Int J Mol Sci, 2022; 23(11):6003; doi: 10.3390/ijms23116003
5.
HolyoakDT, TianYF, van der MeulenMCH, et al.Osteoarthritis: Pathology, mouse models, and nanoparticle injectable systems for targeted treatment. Ann Biomed Eng, 2016; 44(6):2062–2075; doi: 10.1007/S10439-016-1600-Z
6.
HinoiE, BialekP, ChenYT, et al.Runx2 inhibits chondrocyte proliferation and hypertrophy through its expression in the perichondrium. Genes Dev, 2006; 20(21):2937–2942; doi: 10.1101/GAD.1482906
7.
JiangY, TuanRS. Origin and function of cartilage stem/progenitor cells in osteoarthritis. Nat Rev Rheumatol, 2015; 11(4):206–212; doi: 10.1038/NRRHEUM.2014.200
8.
BekkersJEJ, CreemersLB, DhertWJA, et al.Diagnostic modalities for diseased articular cartilage—from defect to degeneration: A review. Cartilage, 2010; 1(3):157–164; doi: 10.1177/1947603510364539
9.
ThomasAC, Hubbard-TurnerT, WikstromEA, et al.Epidemiology of posttraumatic osteoarthritis. J Athl Train, 2017; 52(6):491–496; doi: 10.4085/1062-6050-51.5.08
10.
AndersonDD, ChubinskayaS, GuilakF, et al.Post-traumatic osteoarthritis: Improved understanding and opportunities for early intervention. J Orthop Res, 2011; 29(6):802–809; doi: 10.1002/JOR.21359
11.
BankNC, SanghviP, HechtCJ, et al.The epidemiology of posttraumatic osteoarthritis of the knee in the United States: An analysis of 948,853 patients from 2000 to 2022. J Am Acad Orthop Surg, 2024; 32(7):E313–E320; doi: 10.5435/JAAOS-D-23-00662
12.
ŻylińskaB, SilmanowiczP, Sobczyńska-RakA, et al.Treatment of articular cartilage defects: Focus on tissue engineering. In Vivo, 2018; 32(6):1289–1300; doi: 10.21873/INVIVO.11379
13.
VisweswaranM, PohlS, ArfusoF, et al.Multi-lineage differentiation of mesenchymal stem cells—To Wnt, or not Wnt. Int J Biochem Cell Biol, 2015; 68:139–147; doi: 10.1016/J.BIOCEL.2015.09.008
14.
MackayAM, BeckSC, MurphyJM, et al.Chondrogenic differentiation of cultured human mesenchymal stem cells from marrow. Tissue Eng, 1998; 4(4):415–428; doi: 10.1089/TEN.1998.4.415
15.
JohnstoneB, HeringTM, CaplanAI, et al.In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res, 1998; 238(1):265–272; doi: 10.1006/excr.1997.3858
16.
WakitaniS, ImotoK, YamamotoT, et al.Human autologous culture expanded bone marrow mesenchymal cell transplantation for repair of cartilage defects in osteoarthritic knees. Osteoarthritis Cartilage, 2002; 10(3):199–206; doi: 10.1053/JOCA.2001.0504
17.
ReissisD, TangQO, CooperNC, et al.Current clinical evidence for the use of mesenchymal stem cells in articular cartilage repair. Expert Opin Biol Ther, 2016; 16(4):535–557; doi: 10.1517/14712598.2016.1145651
18.
LinYM, LamATL, LeeJ, et al.Human early mesenchymal stromal cells delivered on porous lightweight biodegradable polycaprolactone-based microcarriers result in improved cartilage formation. Materialia (Oxf), 2020; 13:100851; doi: 10.1016/J.MTLA.2020.100851
19.
PelttariK, WinterA, SteckE, et al.Premature induction of hypertrophy during in vitro chondrogenesis of human mesenchymal stem cells correlates with calcification and vascular invasion after ectopic transplantation in SCID mice. Arthritis Rheum, 2006; 54(10):3254–3266; doi: 10.1002/ART.22136
20.
FarrellMJ, FisherMB, HuangAH, et al.Functional properties of MSC-based engineered cartilage are unstable with very long term in vitro culture. J Biomech, 2014; 47(9):2173–2182; doi: 10.1016/J.JBIOMECH.2013.10.030
21.
SorrellJM, SomozaRA, CaplanAI. Human mesenchymal stem cells induced to differentiate as chondrocytes follow a biphasic pattern of extracellular matrix production. J Orthop Res, 2018; 36(6):1757–1766; doi: 10.1002/JOR.23820
22.
MakrisEA, GomollAH, MalizosKN, et al.Repair and tissue engineering techniques for articular cartilage. Nat Rev Rheumatol, 2015; 11(1):21–34; doi: 10.1038/NRRHEUM.2014.157
23.
ArmientoAR, AliniM, StoddartMJ. Articular fibrocartilage—Why does hyaline cartilage fail to repair? Adv Drug Deliv Rev, 2019; 146:289–305; doi: 10.1016/J.ADDR.2018.12.015
24.
PaulJ, SagstetterA, KrinerM, et al.Donor-site morbidity after osteochondral autologous transplantation for lesions of the talus. J Bone Joint Surg Am, 2009; 91(7):1683–1688; doi: 10.2106/JBJS.H.00429
25.
WangW, RigueurD, LyonsKM. TGFβ signaling in cartilage development and maintenance. Birth Defects Res C Embryo Today, 2014; 102(1):37–51; doi: 10.1002/BDRC.21058
26.
CoricorG, SerraR. TGF-β regulates phosphorylation and stabilization of Sox9 protein in chondrocytes through p38 and Smad dependent mechanisms. Sci Rep, 2016; 6(1):38616–38611; doi: 10.1038/srep38616
27.
LiJ, ZhaoZ, LiuJ, et al.MEK/ERK and p38 MAPK regulate chondrogenesis of rat bone marrow mesenchymal stem cells through delicate interaction with TGF-β1/SMADs pathway. Cell Prolif, 2010; 43(4):333–343; doi: 10.1111/J.1365-2184.2010.00682.X
28.
KhanWS, AdesidaAB, HardinghamTE. Hypoxic conditions increase hypoxia-inducible transcription factor 2alpha and enhance chondrogenesis in stem cells from the infrapatellar fat pad of osteoarthritis patients. Arthritis Res Ther, 2007; 9(3):R55; doi: 10.1186/AR2211
29.
KanichaiM, FergusonD, PrendergastPJ, et al.Hypoxia promotes chondrogenesis in rat mesenchymal stem cells: A role for AKT and hypoxia-inducible factor (HIF)-1alpha. J Cell Physiol, 2008; 216(3):708–715; doi: 10.1002/JCP.21446
30.
MurphyCL, PolakJM. Control of human articular chondrocyte differentiation by reduced oxygen tension. J Cell Physiol, 2004; 199(3):451–459; doi: 10.1002/JCP.10481
31.
BroweDC, ColemanCM, BarryFP, et al.Hypoxia Activates the PTHrP–MEF2C pathway to attenuate hypertrophy in mesenchymal stem cell derived cartilage. Sci Rep, 2019; 9(1):13274–13212; doi: 10.1038/S41598-019-49499-X
32.
HuangYC, ZhuHM, CaiJQ, et al.Hypoxia inhibits the spontaneous calcification of bone marrow-derived mesenchymal stem cells. J Cell Biochem, 2012; 113(4):1407–1415; doi: 10.1002/JCB.24014
33.
MonfortJ, Garcia-GiraltN, López-ArmadaMJ, et al.Decreased metalloproteinase production as a response to mechanical pressure in human cartilage: A mechanism for homeostatic regulation. Arthritis Res Ther, 2006; 8(5):R149; doi: 10.1186/AR2042
34.
ShiQ, QianZ, LiuD, et al.Maintaining the phenotype stability of chondrocytes derived from MSCs by C-type natriuretic peptide. Front Physiol, 2017; 8(MAR):143; doi: 10.3389/fphys.2017.00143
35.
GuilakF, PferdehirtL, RossAK, et al.Designer stem cells: Genome engineering and the next generation of cell-based therapies. J Orthop Res, 2019; 37(6):1287–1293; doi: 10.1002/JOR.24304
36.
KaurG, WuB, MuraliS, et al.A synthetic, closed-looped gene circuit for the autonomous regulation of RUNX2 activity during chondrogenesis. Faseb J, 2024; 38(4):e23484; doi: 10.1096/FJ.202300348RR
37.
El-SappahAH, YanK, HuangQ, et al.Comprehensive mechanism of gene silencing and its role in plant growth and development. Front Plant Sci, 2021; 12:705249; doi: 10.3389/FPLS.2021.705249
38.
LiaoL, ZhangS, GuJ, et al.Deletion of Runx2 in articular chondrocytes decelerates the progression of DMM-Induced osteoarthritis in adult mice. Sci Rep, 2017; 7(1):2371–2312; doi: 10.1038/s41598-017-02490-w
39.
LittleCJ, BawolinNK, ChenX. Mechanical properties of natural cartilage and tissue-engineered constructs. Tissue Eng Part B Rev, 2011; 17(4):213–227; doi: 10.1089/TEN.TEB.2010.0572
40.
TreppoS, KoeppH, QuanEC, et al.Comparison of biomechanical and biochemical properties of cartilage from human knee and ankle pairs. J Orthop Res, 2000; 18(5):739–748; doi: 10.1002/JOR.1100180510
41.
KorhonenRK, LaasanenMS, TöyräsJ, et al.Comparison of the equilibrium response of articular cartilage in unconfined compression, confined compression and indentation. J Biomech, 2002; 35(7):903–909; doi: 10.1016/S0021-9290(02)00052-0
42.
ParkH, TemenoffJS, TabataY, et al.Injectable biodegradable hydrogel composites for rabbit marrow mesenchymal stem cell and growth factor delivery for cartilage tissue engineering. Biomaterials, 2007; 28(21):3217–3227; doi: 10.1016/j.biomaterials.2007.03.030
43.
XuJ, LiuJ, GanY, et al.High-Dose TGF-β1 Impairs Mesenchymal Stem Cell–Mediated Bone Regeneration via Bmp2 Inhibition. J Bone Miner Res, 2020; 35(1):167–180; doi: 10.1002/jbmr.3871
44.
CalsFLJ, HellingmanCA, KoevoetW, et al.Effects of transforming growth factor-β subtypes on in vitro cartilage production and mineralization of human bone marrow stromal-derived mesenchymal stem cells. J Tissue Eng Regen Med, 2012; 6(1):68–76; doi: 10.1002/TERM.399
45.
FutregaK, RobeyPG, KleinTJ, et al.A single day of TGF-β1 exposure activates chondrogenic and hypertrophic differentiation pathways in bone marrow-derived stromal cells. Commun Biol, 2021; 4(1):29–12; doi: 10.1038/s42003-020-01520-0
46.
MuellerMB, FischerM, ZellnerJ, et al.Hypertrophy in mesenchymal stem cell chondrogenesis: Effect of TGF-β isoforms and chondrogenic conditioning. Cells Tissues Organs, 2010; 192(3):158–166; doi: 10.1159/000313399
47.
FurumatsuT, TsudaM, TaniguchiN, et al.Smad3 induces chondrogenesis through the activation of SOX9 via CREB-binding protein/p300 recruitment. J Biol Chem, 2005; 280(9):8343–8350; doi: 10.1074/JBC.M413913200
48.
van der KraanPM, Blaney DavidsonEN, BlomA, et al.TGF-beta signaling in chondrocyte terminal differentiation and osteoarthritis. Modulation and integration of signaling pathways through receptor-Smads. Osteoarthritis Cartilage, 2009; 17(12):1539–1545; doi: 10.1016/J.JOCA.2009.06.008
49.
BallockRT, HeydemannA, WakefieldLM, et al.TGF-β1 prevents hypertrophy of epiphyseal chondrocytes: Regulation of gene expression for cartilage matrix proteins and metalloproteases. Dev Biol, 1993; 158(2):414–429; doi: 10.1006/DBIO.1993.1200
50.
FergusonCM, SchwarzEM, ReynoldsPR, et al.Smad2 and 3 mediate transforming growth Factor-β1-Induced inhibition of chondrocyte maturation*The work was supported by National Health Services Grant AR-38945 (to R.J.O.) and an orthopaedic research education foundation award (to C.M.F.). Endocrinology, 2000; 141(12):4728–4735; doi: 10.1210/ENDO.141.12.7848
51.
SomozaRA, CorreaD, LabatI, et al.Transcriptome-Wide analyses of human neonatal articular cartilage and human mesenchymal stem cell-derived cartilage provide a new molecular target for evaluating engineered cartilage. Tissue Eng Part A, 2018; 24(3–4):335–350; doi: 10.1089/TEN.TEA.2016.0559
52.
CarrionB, SouzanchiMF, WangVT, et al.The synergistic effects of matrix stiffness and composition on the response of chondroprogenitor cells in a 3D precondensation microenvironment. Adv Healthc Mater, 2016; 5(10):1192–1202; doi: 10.1002/ADHM.201501017
53.
RauhJ, JacobiA, StiehlerM. Identification of stable reference genes for gene expression analysis of three-dimensional cultivated human bone marrow-derived mesenchymal stromal cells for bone tissue engineering. Tissue Eng Part C Methods, 2015; 21(2):192–206; doi: 10.1089/ten.tec.2014.0230
