In this study, we explored whether embryonic stem cell-derived mesenchymal stem cell (ES-MSC) cellular spheroids in combination with a closed chamber system could be used to create scaffold-free cartilage and endochondral graft tissues. ES-MSC cellular spheroids were cultured in chondrogenic medium for 3–4 days and seeded into a customizable Net Mold chamber system (NCS) and cultured in chondrogenic medium for an additional 18 days to fuse and form a single tissue construct. To assess potential for cartilage repair, cellular spheroids were matured in the NCS for only 7 days before implantation into ex vivo human cartilage defects. To engineer osteochondral tissues, cellular spheroids were initially cultured in chondrogenic medium for 14 days, seeded into one well of the NSC, and cultured together in osteogenic medium for 21 days. For the chondrogenic phase, cellular spheroids were initially cultured in chondrogenic medium for 14 days before seeding in an NCS chamber, adjacent to the osteogenic spheroids. The combined osteogenic and chondrogenic constructs were cultured in serum-free medium for an additional 3 weeks. Cellular spheroids cultured in the NCS developed into neocartilage tissues expressing cartilage-associated genes (COL2A1, ACAN, and COMP) and stained positive for cartilage matrix molecules (glycosaminoglycan and collagen type II). The cartilage-like constructs that were implanted into cartilage defects created in ex vivo osteoarthritic (OA) tissue resulted in repair tissue with an elastic modulus of 46 ± 6 kPa that was histologically integrated with the explant tissues. Spheroids cultured in osteogenic medium produced tissues that were positive for von Kossa stain and for osteopontin immunostaining. Pre-differentiation in chondrogenic and osteogenic medium before placing in the NCS resulted in fused cartilage and bone-like constructs with regional production of chondrogenic and mineralized matrix (Alizarin Red S, von Kossa, and osteopontin positive). Spheroids in stacked NCS chambers produced osteochondral neotissues up to 2 mm in thickness. Our results indicate the potential for cellular spheroids, from a clinically relevant ES-MSC source, to generate scaffold-free chondrogenic or osteochondrogenic graft tissues.
Impact Statement
This study demonstrates the possibility to use a clinically relevant embryonic stem cell-derived mesenchymal stem cell (ES-MSC) source to make cellular spheroids to fabricate scaffold-free cartilage and bone-like constructs, using a culture chamber system. Neocartilage tissues expressing cartilage-associated genes and matrix molecules, when implanted into ex vivo cartilage defects, integrated with existing tissue and showed promising repair potential. An osteochondral ossification differentiation approach generated scaffold-free osteochondral tissues with regional production of chondrogenic and mineralized matrix. Combining clinically relevant ES-MSC, cellular spheroids, specific culture environments within a chamber system offers a promising approach for cartilage and osteochondral repair.
LiangJ, LiuP, YangX, et al.Biomaterial-based scaffolds in promotion of cartilage regeneration: Recent advances and emerging applications. J Orthop Translat, 2023; 41:54–62; doi: 10.1016/j.jot.2023.08.006
2.
ChenR, PyeJS, LiJ, et al.Multiphasic scaffolds for the repair of osteochondral defects: Outcomes of preclinical studies. Bioact Mater, 2023; 27:505–545; doi: 10.1016/j.bioactmat.2023.04.016
3.
ChanBP, LeongKW. Scaffolding in tissue engineering: General approaches and tissue-specific considerations. Eur Spine J, 2008; 17(Suppl 4):467–479; doi: 10.1007/s00586-008-0745-3
4.
DuRaineGD, BrownWE, HuJC, et al.Emergence of scaffold-free approaches for tissue engineering musculoskeletal cartilages. Ann Biomed Eng, 2015; 43(3):543–554; doi: 10.1007/s10439-014-1161-y
5.
LeeJK, LinkJM, HuJCY, et al.The self-assembling process and applications in tissue engineering. Cold Spring Harb Perspect Med, 2017; 7(11):a025668; doi: 10.1101/cshperspect.a025668
6.
SchonBS, HooperGJ, WoodfieldTB. Modular tissue assembly strategies for biofabrication of engineered cartilage. Ann Biomed Eng, 2017; 45(1):100–114; doi: 10.1007/s10439-016-1609-3
7.
De PieriA, RochevY, ZeugolisDI. Scaffold-free cell-based tissue engineering therapies: Advances, shortfalls and forecast. NPJ Regen Med, 2021; 6(1):18; doi: 10.1038/s41536-021-00133-3
8.
GhoshS, LahaM, MondalS, et al.In vitro model of mesenchymal condensation during chondrogenic development. Biomaterials, 2009; 30(33):6530–6540; doi: 10.1016/j.biomaterials.2009.08.019
9.
LenasP, MoosM, LuytenFP. Developmental engineering: A new paradigm for the design and manufacturing of cell-based products. Part I: From three-dimensional cell growth to biomimetics of in vivo development. Tissue Eng Part B Rev, 2009; 15(4):381–394; doi: 10.1089/ten.TEB.2008.0575
10.
LuY, ZhangW, WangJ, et al.Recent advances in cell sheet technology for bone and cartilage regeneration: From preparation to application. Int J Oral Sci, 2019; 11(2):17; doi: 10.1038/s41368-019-0050-5
11.
ZhangW, YangG, WangX, et al.Magnetically controlled growth-factor-immobilized multilayer cell sheets for complex tissue regeneration. Adv Mater, 2017; 29(43); doi: 10.1002/adma.201703795
12.
ItoS, SatoM, YamatoM, et al.Repair of articular cartilage defect with layered chondrocyte sheets and cultured synovial cells. Biomaterials, 2012; 33(21):5278–5286; doi: 10.1016/j.biomaterials.2012.03.073
13.
MurdochAD, GradyLM, AblettMP, et al.Chondrogenic differentiation of human bone marrow stem cells in transwell cultures: Generation of scaffold-free cartilage. Stem Cells, 2007; 25(11):2786–2796; doi: 10.1634/stemcells.2007-0374
14.
MurdochAD, HardinghamTE, EyreDR, et al.The development of a mature collagen network in cartilage from human bone marrow stem cells in transwell culture. Matrix Biol, 2016; 50:16–26; doi: 10.1016/j.matbio.2015.10.003
15.
Mayer-WagnerS, SchiergensTS, SieversB, et al.Membrane-based cultures generate scaffold-free neocartilage in vitro: Influence of growth factors. Tissue Eng Part A, 2010; 16(2):513–521; doi: 10.1089/ten.TEA.2009.0326
16.
BianY, DuY, WangR, et al.A comparative study of HAMSCs/HBMSCs transwell and mixed coculture systems. IUBMB Life, 2019; 71(7):1048–1055; doi: 10.1002/iub.2074
17.
JoensuuK, UusitaloL, AlmJJ, et al.Enhanced osteoblastic differentiation and bone formation in co-culture of human bone marrow mesenchymal stromal cells and peripheral blood mononuclear cells with exogenous VEGF. Orthop Traumatol Surg Res, 2015; 101(3):381–386; doi: 10.1016/j.otsr.2015.01.014
18.
DikinaAD, StrobelHA, LaiBP, et al.Engineered cartilaginous tubes for tracheal tissue replacement via self-assembly and fusion of human mesenchymal stem cell constructs. Biomaterials, 2015; 52:452–462; doi: 10.1016/j.biomaterials.2015.01.073
19.
HuangGS, TsengCS, Linju YenB, et al.Solid freeform-fabricated scaffolds designed to carry multicellular mesenchymal stem cell spheroids for cartilage regeneration. Eur Cell Mater, 2013; 26:179–194; discussion 194; doi: 10.22203/ecm.v026a13
20.
BreathwaiteEK, WeaverJR, MurchisonAC, et al.Scaffold-free bioprinted osteogenic and chondrogenic systems to model osteochondral physiology. Biomed Mater, 2019; 14(6):065010; doi: 10.1088/1748-605X/ab4243
21.
GroganSP, DortheEW, GlembotskiNE, et al.Cartilage tissue engineering combining microspheroid building blocks and microneedle arrays. Connect Tissue Res, 2020; 61(2):229–243; doi: 10.1080/03008207.2019.1617280
22.
MurataD, TokunagaS, TamuraT, et al.A preliminary study of osteochondral regeneration using a scaffold-free three-dimensional construct of porcine adipose tissue-derived mesenchymal stem cells. J Orthop Surg Res, 2015; 10:35; doi: 10.1186/s13018-015-0173-0
23.
MurataD, AkiedaS, MisumiK, et al.Osteochondral regeneration with a scaffold-free three-dimensional construct of adipose tissue-derived mesenchymal stromal cells in pigs. Tissue Eng Regen Med, 2018; 15(1):101–113; doi: 10.1007/s13770-017-0091-9
24.
AnadaT, SatoT, KamoyaT, et al.Evaluation of bioactivity of octacalcium phosphate using osteoblastic cell aggregates on a spheroid culture device. Regen Ther, 2016; 3:58–62; doi: 10.1016/j.reth.2016.02.004
25.
ChatterjeaA, LaPointeVL, BarradasA, et al.Cell aggregation enhances bone formation by human mesenchymal stromal cells. Eur Cell Mater, 2017; 33:121–129; doi: 10.22203/eCM.v033a09
26.
KellerL, Idoux-GilletY, WagnerQ, et al.Nanoengineered implant as a new platform for regenerative nanomedicine using 3D well-organized human cell spheroids. Int J Nanomedicine, 2017; 12:447–457; doi: 10.2147/IJN.S116749
27.
BaptistaLS, KronembergerGS, SilvaKR, et al.Spheroids of stem cells as endochondral templates for improved bone engineering. Front Biosci (Landmark Ed), 2018; 23(10):1969–1986; doi: 10.2741/4683
28.
KronembergerGS, MatsuiRAM, MirandaG, et al.Cartilage and bone tissue engineering using adipose stromal/stem cells spheroids as building blocks. World J Stem Cells, 2020; 12(2):110–122; doi: 10.4252/wjsc.v12.i2.110
29.
ShenFH, WernerBC, LiangH, et al.Implications of adipose-derived stromal cells in a 3D culture system for osteogenic differentiation: An in vitro and in vivo investigation. Spine J, 2013; 13(1):32–43; doi: 10.1016/j.spinee.2013.01.002
30.
GurumurthyB, BierdemanPC, JanorkarAV. Spheroid model for functional osteogenic evaluation of human adipose derived stem cells. J Biomed Mater Res A, 2017; 105(4):1230–1236; doi: 10.1002/jbm.a.35974
31.
HildebrandtC, ButhH, ThieleckeH. A scaffold-free in vitro model for osteogenesis of human mesenchymal stem cells. Tissue Cell, 2011; 43(2):91–100; doi: 10.1016/j.tice.2010.12.004
32.
RuminskiS, KalaszczynskaI, DlugoszA, et al.Osteogenic differentiation of human adipose-derived stem cells in 3D conditions - comparison of spheroids and polystyrene scaffolds. Eur Cell Mater, 2019; 37:382–401; doi: 10.22203/eCM.v037a23
33.
WongSA, RiveraKO, MiclauT3rd, et al.Microenvironmental regulation of chondrocyte plasticity in endochondral repair-a new frontier for developmental engineering. Front Bioeng Biotechnol, 2018; 6:58; doi: 10.3389/fbioe.2018.00058
34.
ValentiMT, Dalle CarbonareL, MottesM. Osteogenic differentiation in healthy and pathological conditions. Int J Mol Sci, 2016; 18(1):41; doi: 10.3390/ijms18010041
35.
HuDP, FerroF, YangF, et al.Cartilage to bone transformation during fracture healing is coordinated by the invading vasculature and induction of the core pluripotency genes. Development, 2017; 144(2):221–234; doi: 10.1242/dev.130807
36.
OvsianikovA, KhademhosseiniA, MironovV. The synergy of scaffold-based and scaffold-free tissue engineering strategies. Trends Biotechnol, 2018; 36(4):348–357; doi: 10.1016/j.tibtech.2018.01.005
37.
LaschkeMW, MengerMD. Life is 3D: Boosting spheroid function for tissue engineering. Trends Biotechnol, 2017; 35(2):133–144; doi: 10.1016/j.tibtech.2016.08.004
YoussefJ, NurseAK, FreundLB, et al.Quantification of the forces driving self-assembly of three-dimensional microtissues. Proc Natl Acad Sci U S A, 2011; 108(17):6993–6998; doi: 10.1073/pnas.1102559108
40.
AndererU, LiberaJ. In vitro engineering of human autogenous cartilage. J Bone Miner Res, 2002; 17(8):1420–1429; doi: 10.1359/jbmr.2002.17.8.1420
41.
HochAI, MittalV, MitraD, et al.Cell-secreted matrices perpetuate the bone-forming phenotype of differentiated mesenchymal stem cells. Biomaterials, 2016; 74:178–187; doi: 10.1016/j.biomaterials.2015.10.003
HarvestineJN, VollmerNL, HoSS, et al.Extracellular matrix-coated composite scaffolds promote mesenchymal stem cell persistence and osteogenesis. Biomacromolecules, 2016; 17(11):3524–3531; doi: 10.1021/acs.biomac.6b01005
44.
HynesRO. The extracellular matrix: Not just pretty fibrils. Science, 2009; 326(5957):1216–1219; doi: 10.1126/science.1176009
45.
KelmJM, FusseneggerM. Scaffold-free cell delivery for use in regenerative medicine. Adv Drug Deliv Rev, 2010; 62(7–8):753–764; doi: 10.1016/j.addr.2010.02.003
46.
BartzC, MeixnerM, GiesemannP, et al.An ex vivo human cartilage repair model to evaluate the potency of a cartilage cell transplant. J Transl Med, 2016; 14(1):317; doi: 10.1186/s12967-016-1065-8
47.
SmithLJ, SilvermanL, SakaiD, et al.Advancing cell therapies for intervertebral disc regeneration from the lab to the clinic: Recommendations of the ORS spine section. JOR Spine, 2018; 1(4):e1036; doi: 10.1002/jsp2.1036
48.
EschenC, KapsC, WiduchowskiW, et al.Clinical outcome is significantly better with spheroid-based autologous chondrocyte implantation manufactured with more stringent cell culture criteria. Osteoarthr Cartil Open, 2020; 2(1):100033; doi: 10.1016/j.ocarto.2020.100033
49.
HoburgA, NiemeyerP, LauteV, et al.Matrix-associated autologous chondrocyte implantation with spheroid technology is superior to arthroscopic microfracture at 36 months regarding activities of daily living and sporting activities after treatment. Cartilage, 2021; 13(Suppl 1):437S–448S; doi: 10.1177/1947603519897290
50.
NiemeyerP, LauteV, ZinserW, et al.A prospective, randomized, open-label, multicenter, Phase III Noninferiority trial to compare the clinical efficacy of matrix-associated autologous chondrocyte implantation with spheroid technology versus arthroscopic microfracture for cartilage defects of the knee. Orthop J Sports Med, 2019; 7(7):2325967119854442; doi: 10.1177/2325967119854442
51.
GroganSP, GlembotskiNE, D’LimaDD. ALK-5 inhibitors for efficient derivation of mesenchymal stem cells from human embryonic stem cells. Tissue Eng Part A, 2023; 29(5–6):127–140; doi: 10.1089/ten.TEA.2022.0164
52.
TannenbaumSE, TuretskyTT, SingerO, et al.Derivation of xeno-free and GMP-grade human embryonic stem cells–platforms for future clinical applications. PLoS One, 2012; 7(6):e35325; doi: 10.1371/journal.pone.0035325
53.
KronenbergHM. Developmental regulation of the growth plate. Nature, 2003; 423(6937):332–336; doi: 10.1038/nature01657
54.
MackieEJ, AhmedYA, TatarczuchL, et al.Endochondral ossification: How cartilage is converted into bone in the developing skeleton. Int J Biochem Cell Biol, 2008; 40(1):46–62; doi: 10.1016/j.biocel.2007.06.009
55.
MartinI, JakobM, SchaferD, et al.Quantitative analysis of gene expression in human articular cartilage from normal and osteoarthritic joints. Osteoarthritis Cartilage, 2001; 9(2):112–118; doi: 10.1053/joca.2000.0366
56.
HayesWC, KeerLM, HerrmannG, et al.A mathematical analysis for indentation tests of articular cartilage. J Biomech, 1972; 5(5):541–551; doi: 10.1016/0021-9290(72)90010-3
57.
PatilS, SteklovN, SongL, et al.Comparative biomechanical analysis of human and caprine knee articular cartilage. Knee, 2014; 21(1):119–125; doi: 10.1016/j.knee.2013.03.009
58.
CleriesR, GalvezJ, EspinoM, et al.BootstRatio: A web-based statistical analysis of fold-change in qPCR and RT-qPCR data using resampling methods. Comput Biol Med, 2012; 42(4):438–445; doi: 10.1016/j.compbiomed.2011.12.012
59.
FreemanFE, HaughMG, McNamaraLM. Investigation of the optimal timing for chondrogenic priming of MSCs to enhance osteogenic differentiation in vitro as a bone tissue engineering strategy. J Tissue Eng Regen Med, 2016; 10(4):E250–E262; doi: 10.1002/term.1793
60.
FreemanFE, McNamaraLM. Endochondral priming: A developmental engineering strategy for bone tissue regeneration. Tissue Eng Part B Rev, 2017; 23(2):128–141; doi: 10.1089/ten.TEB.2016.0197
61.
LianJB, McKeeMD, ToddAM, et al.Induction of bone-related proteins, osteocalcin and osteopontin, and their matrix ultrastructural localization with development of chondrocyte hypertrophy in vitro. J Cell Biochem, 1993; 52(2):206–219; doi: 10.1002/jcb.240520212
62.
GerstenfeldLC, ShapiroFD. Expression of bone-specific genes by hypertrophic chondrocytes: Implication of the complex functions of the hypertrophic chondrocyte during endochondral bone development. J Cell Biochem, 1996; 62(1):1–9; doi: 10.1002/(SICI)1097-4644(199607)62:1%3C1::AID-JCB1%3E3.0.CO;2-X
63.
RyuNE, LeeSH, ParkH. Spheroid culture system methods and applications for mesenchymal stem cells. Cells, 2019; 8(12):1620; doi: 10.3390/cells8121620
64.
ZhangL, SuP, XuC, et al.Chondrogenic differentiation of human mesenchymal stem cells: A comparison between micromass and pellet culture systems. Biotechnol Lett, 2010; 32(9):1339–1346; doi: 10.1007/s10529-010-0293-x
65.
TevlekA, KeciliS, OzcelikOS, et al.Spheroid engineering in microfluidic devices. ACS Omega, 2023; 8(4):3630–3649; doi: 10.1021/acsomega.2c06052
66.
MartinezI, ElvenesJ, OlsenR, et al.Redifferentiation of in vitro expanded adult articular chondrocytes by combining the hanging-drop cultivation method with hypoxic environment. Cell Transplant, 2008; 17(8):987–996; doi: 10.3727/096368908786576499
67.
KondoS, NakagawaY, MizunoM, et al.Transplantation of aggregates of autologous synovial mesenchymal stem cells for treatment of cartilage defects in the femoral condyle and the femoral groove in microminipigs. Am J Sports Med, 2019; 47(10):2338–2347; doi: 10.1177/0363546519859855
68.
WehlandM, SteinwerthP, AleshchevaG, et al.Tissue engineering of cartilage using a random positioning machine. Int J Mol Sci, 2020; 21(24):9596; doi: 10.3390/ijms21249596
69.
LeeJI, SatoM, KimHW, et al.Transplantatation of scaffold-free spheroids composed of synovium-derived cells and chondrocytes for the treatment of cartilage defects of the knee. Eur Cell Mater, 2011; 22:275–290; discussion 290; doi: 10.22203/ecm.v022a21
70.
YoonHH, BhangSH, ShinJY, et al.Enhanced cartilage formation via three-dimensional cell engineering of human adipose-derived stem cells. Tissue Eng Part A, 2012; 18(19–20):1949–1956; doi: 10.1089/ten.TEA.2011.0647
71.
MoldovanNI, HibinoN, NakayamaK. Principles of the Kenzan Method for robotic cell spheroid-based three-dimensional bioprinting. Tissue Eng Part B Rev, 2017; 23(3):237–244; doi: 10.1089/ten.TEB.2016.0322
72.
MurataD, FujimotoR, NakayamaK. Osteochondral regeneration using adipose tissue-derived mesenchymal stem cells. Int J Mol Sci, 2020; 21(10):3589; doi: 10.3390/ijms21103589
73.
IshiharaK, NakayamaK, AkiedaS, et al.Simultaneous regeneration of full-thickness cartilage and subchondral bone defects in vivo using a three-dimensional scaffold-free autologous construct derived from high-density bone marrow-derived mesenchymal stem cells. J Orthop Surg Res, 2014; 9:98; doi: 10.1186/s13018-014-0098-z
74.
GriffithLA, ArnoldKM, SengersBG, et al.A scaffold-free approach to cartilage tissue generation using human embryonic stem cells. Sci Rep, 2021; 11(1):18921; doi: 10.1038/s41598-021-97934-9
75.
YamasakiA, KunitomiY, MurataD, et al.Osteochondral regeneration using constructs of mesenchymal stem cells made by bio three-dimensional printing in mini-pigs. J Orthop Res, 2019; 37(6):1398–1408; doi: 10.1002/jor.24206
76.
NakamuraA, MurataD, FujimotoR, et al.Bio-3D printing iPSC-derived human chondrocytes for articular cartilage regeneration. Biofabrication, 2021; 13(4); doi: 10.1088/1758-5090/ac1c99
77.
FrisbieDD, CrossMW, McIlwraithCW. A comparative study of articular cartilage thickness in the stifle of animal species used in human pre-clinical studies compared to articular cartilage thickness in the human knee. Vet Comp Orthop Traumatol, 2006; 19(03):142–146.
78.
MengX, ZiadlouR, GradS, et al.Animal models of osteochondral defect for testing biomaterials. Biochem Res Int, 2020; 2020:9659412; doi: 10.1155/2020/9659412
WangX, GroganSP, RieserF, et al.Tissue engineering of biphasic cartilage constructs using various biodegradable scaffolds: An in vitro study. Biomaterials, 2004; 25(17):3681–3688; doi: 10.1016/j.biomaterials.2003.10.102
81.
DaH, JiaSJ, MengGL, et al.The impact of compact layer in biphasic scaffold on osteochondral tissue engineering. PLoS One, 2013; 8(1):e54838; doi: 10.1371/journal.pone.0054838
82.
LiJJ, KimK, Roohani-EsfahaniSI, et al.A biphasic scaffold based on silk and bioactive ceramic with stratified properties for osteochondral tissue regeneration. J Mater Chem B, 2015; 3(26):5361–5376; doi: 10.1039/C5TB00353A
83.
BhumiratanaS, EtonRE, OungoulianSR, et al.Large, stratified, and mechanically functional human cartilage grown in vitro by mesenchymal condensation. Proc Natl Acad Sci U S A, 2014; 111(19):6940–6945; doi: 10.1073/pnas.1324050111
84.
TrompetD, MelisS, ChaginAS, et al.Skeletal stem and progenitor cells in bone development and repair. J Bone Miner Res, 2024; 39(6):633–654; doi: 10.1093/jbmr/zjae069
85.
ChanWCW, TanZ, ToMKT, et al.Regulation and Role of Transcription Factors in Osteogenesis. Int J Mol Sci, 2021; 22(11):5445; doi: 10.3390/ijms22115445
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