mesЭкстремальная биомедицинаExtreme Medicine3033-89643033-8972Centre for Strategic Planning of the Federal Medical and Biological Agency10.47183/mes.2022.037mes-204Research ArticleОРИГИНАЛЬНОЕ ИССЛЕДОВАНИЕORIGINAL RESEARCHПолучение хрящеподобных структур из стволовых клеток с индуцированной плюрипотентностьюFabrication of cartilage tissue substitutes from cells with induced pluripotencyЕремеевА. В.EremeevA. V.

Артём Валерьевич Еремеев

ул. Малая Пироговская, д. 1а, 119435, г. Москва

Artem V. Eremeev

Malaya Pirogovskaya, 1a, 119435, Moscow

art-eremeev@yandex.ruПикинаА. С.PikinaA. S.

Москва

Moscow

РучкоЕ. С.RuchkoE. S.

Москва

Moscow

СидоровВ. С.SidorovV. S.

Москва

Moscow

РагозинА. О.RagozinA. O.

Москва

Moscow

Федеральный научно-клинический центр физико-химической медицины Федерального медико-биологического агентства; Институт биологии развития имени Н. К. КольцоваРоссияFederal State Budgetary Institution Federal Research and Clinical Center of Physical-Chemical Medicine of the Federal Medical Biological Agency; Koltzov Institute of Developmental BiologyRussian FederationФедеральный научно-клинический центр физико-химической медицины Федерального медико-биологического агентства; Московский государственный университет имени М. В. ЛомоносоваРоссияFederal State Budgetary Institution Federal Research and Clinical Center of Physical-Chemical Medicine of the Federal Medical Biological Agency; Lomonosov Moscow State UniversityRussian FederationФедеральный научно-клинический центр физико-химической медицины Федерального медико-биологического агентстваРоссияFederal State Budgetary Institution Federal Research and Clinical Center of Physical-Chemical Medicine of the Federal Medical Biological AgencyRussian Federation2022281020242443142Copyright © Еремеев А.В., Пикина А.С., Ручко Е.С., Сидоров В.С., Рагозин А.О., 20242024Еремеев А.В., Пикина А.С., Ручко Е.С., Сидоров В.С., Рагозин А.О.Eremeev A.V., Pikina A.S., Ruchko E.S., Sidorov V.S., Ragozin A.O.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.extrememedicine.ru/jour/article/view/204

Одним из подходов для решения проблемы восстановления хрящевой ткани является использование клеточных технологий с применением ИПСК, обладающих большим потенциалом к дифференцировке и являющихся неограниченным источником клеточного материала для тканевой инженерии. Однако стандартизированных протоколов хондрогенной дифференцировки ИПСК нет. Целью работы было получить хрящеподобные образцы ткани с помощью метода 3D-культивирования сфероидов с использованием четырех протоколов хондрогенной дифференцировки, сравнить характеристики хрящеподобных образцов, полученных с помощью разных протоколов, и определить наиболее эффективный способ дифференцировки. ИПСК дифференцировали по хондрогенному пути с помощью четырех протоколов («долгий», «короткий», «комбинированный», с кондиционированной средой от первичной культуры аутологичных хондроцитов) при различном сочетании факторов TGFβ1, BMP2, Chir 99021 и РК. Для получения сфероидов использовали планшеты с микролунками. Профили синтеза и экспрессии оценивали с помощью методов иммуноцитохимического окрашивания, ПЦР в реальном времени, а также гистологического окрашивания. Высокие показатели синтеза и экспрессии хондрогенных маркеров Sox9, аггрекана, коллагена II типа наблюдали в сфероидах «долгого», «комбинированного» протоколов и протокола с кондиционированной средой. Хондрогенез наиболее эффективно проходит при использовании «комбинированного» протокола дифференцировки. Высокую эффективность показало также использование кондиционированной среды для индукции и поддержания хондрогенной дифференцировки.

One of the approaches to cartilage tissue restoration problem relies on cellular technologies that use iPSCs, induced pluripotency stem cells that are an unlimited source of cellular material for tissue engineering with significant differentiation potential. However, there are no standardized protocols for chondrogenic differentiation of iPSCs. This study aimed to make cartilage tissue samples using 3D spheroid cultures and following four chondrogenic differentiation protocols, then compare characteristics of the cartilage samples made under different protocols and isolate the most effective way of differentiation. The iPSCs were differentiated chondrogenically, the four protocols were "long", "short", "combined" and with conditioned medium from a primary culture of autologous chondrocytes; the combinations of TGFβ1, BMP2, Chir 99021, and PK factors varied. Microwell plates were used to make spheroids. Immunocytochemical staining, real-time PCR and histological staining enabled assessment of the synthesis and expression profiles. High rates of synthesis and expression of chondrogenic markers Sox9, aggrecan, type II collagen were observed in spheroids experimented with under the "long", "combined" protocols and the conditioned medium protocol. The "combined" differentiation protocol made chondrogenesis most effective, and conditioned medium was highly efficient in inducing and supporting chondrogenic differentiation.

тканевая инженериясуставной хрящиндуцированные плюрипотентные стволовые клетки (ИПСК)сфероидыхондрогенезtissue engineeringarticular cartilageinduced pluripotency stem cells (iPSCs)spheroidschondrogenesisработа выполнена при поддержке РНФ #22-15-00250.the study was supported with an allocation #22-15-00250 by the Russian Science Foundation.ReferencesDiederichs S, Gabler J, Autenrieth J. Differential regulation of SOX9 protein during chondrogenesis of induced pluripotent stem cells versus mesenchymal stromal cells: a shortcoming for cartilage formation. Stem Cells and Development. 2016; (April): 1–34.Diederichs S, Gabler J, Autenrieth J. Differential regulation of SOX9 protein during chondrogenesis of induced pluripotent stem cells versus mesenchymal stromal cells: a shortcoming for cartilage formation. Stem Cells and Development. 2016; (April): 1–34.Tsumaki N, Okada M, Yamashita A. iPS cell technologies and cartilage regeneration. Bone. 2015; 70: 48–54. Available from: http://dx.doi.org/10.1016/j.bone.2014.07.011.Tsumaki N, Okada M, Yamashita A. iPS cell technologies and cartilage regeneration. Bone. 2015; 70: 48–54. Available from: http://dx.doi.org/10.1016/j.bone.2014.07.011.Yamashita A, Morioka M, Yahara Y, Okada M, Kobayashi T, Kuriyama S, et al. Generation of scaffoldless hyaline cartilaginous tissue from human iPSCs. Stem Cell Reports. 2015; 4 (3): 404–18.Yamashita A, Morioka M, Yahara Y, Okada M, Kobayashi T, Kuriyama S, et al. Generation of scaffoldless hyaline cartilaginous tissue from human iPSCs. Stem Cell Reports. 2015; 4 (3): 404–18.Kimura T, Yamashita A, Ozono K, Tsumaki N. Limited Immunogenicity of Human Induced Pluripotent Stem Cell-Derived Cartilages. Tissue Engineering — Part A. 2016; 22 (23–24): 1367–75.Kimura T, Yamashita A, Ozono K, Tsumaki N. Limited Immunogenicity of Human Induced Pluripotent Stem Cell-Derived Cartilages. Tissue Engineering — Part A. 2016; 22 (23–24): 1367–75.Lietman SA. Induced pluripotent stem cells in cartilage repair. World Journal of Orthopedics. 2016; 7 (3): 149–55.Lietman SA. Induced pluripotent stem cells in cartilage repair. World Journal of Orthopedics. 2016; 7 (3): 149–55.Narsinh KH, Plews J, Wu JC. Comparison of human induced pluripotent and embryonic stem cells: Fraternal or identical twins? Molecular Therapy. 2011; 19 (4): 635–8. Available from: http://dx.doi.org/10.1038/mt.2011.41/nature06264.Narsinh KH, Plews J, Wu JC. Comparison of human induced pluripotent and embryonic stem cells: Fraternal or identical twins? Molecular Therapy. 2011; 19 (4): 635–8. Available from: http://dx.doi.org/10.1038/mt.2011.41/nature06264.Cheng B, Tu T, Shi X, Liu Y, Zhao Y, Zhao Y, et al. A novel construct with biomechanical flexibility for articular cartilage regeneration. Stem Cell Research and Therapy. 2019; 10 (1): 1–16.Cheng B, Tu T, Shi X, Liu Y, Zhao Y, Zhao Y, et al. A novel construct with biomechanical flexibility for articular cartilage regeneration. Stem Cell Research and Therapy. 2019; 10 (1): 1–16.Limraksasin P, Kosaka Y, Zhang M, Horie N, Kondo T, Okawa H, et al. Shaking culture enhances chondrogenic differentiation of mouse induced pluripotent stem cell constructs. Scientific Reports. 2020; 10 (1): 1–15. Available from: https://doi.org/10.1038/s41598-020-72038-y.Limraksasin P, Kosaka Y, Zhang M, Horie N, Kondo T, Okawa H, et al. Shaking culture enhances chondrogenic differentiation of mouse induced pluripotent stem cell constructs. Scientific Reports. 2020; 10 (1): 1–15. Available from: https://doi.org/10.1038/s41598-020-72038-y.Suchorska WM, Augustyniak E, Richter M, Trzeciak T. Comparison of Four Protocols to Generate Chondrocyte-Like Cells from Human Induced Pluripotent Stem Cells (hiPSCs). Stem Cell Reviews and Reports. 2017; 13 (2): 299–308.Suchorska WM, Augustyniak E, Richter M, Trzeciak T. Comparison of Four Protocols to Generate Chondrocyte-Like Cells from Human Induced Pluripotent Stem Cells (hiPSCs). Stem Cell Reviews and Reports. 2017; 13 (2): 299–308.Castro-Viñuelas R, Sanjurjo-Rodríguez C, Piñeiro-Ramil M, Hermida-Gómez T, Fuentes-Boquete IM, de Toro-Santos FJ, et al. Induced pluripotent stem cells for cartilage repair: Current status and future perspectives. European Cells and Materials. 2018; 36 (2006): 96–109.Castro-Viñuelas R, Sanjurjo-Rodríguez C, Piñeiro-Ramil M, Hermida-Gómez T, Fuentes-Boquete IM, de Toro-Santos FJ, et al. Induced pluripotent stem cells for cartilage repair: Current status and future perspectives. European Cells and Materials. 2018; 36 (2006): 96–109.Qu C, Puttonen KA, Lindeberg H, Ruponen M, Hovatta O, Koistinaho J, et al. Chondrogenic differentiation of human pluripotent stem cells in chondrocyte co-culture. International Journal of Biochemistry and Cell Biology. 2013; 45 (8): 1802–12.Qu C, Puttonen KA, Lindeberg H, Ruponen M, Hovatta O, Koistinaho J, et al. Chondrogenic differentiation of human pluripotent stem cells in chondrocyte co-culture. International Journal of Biochemistry and Cell Biology. 2013; 45 (8): 1802–12.Chijimatsu R, Ikeya M, Yasui Y, Ikeda Y, Ebina K, Moriguchi Y, et al. Characterization of Mesenchymal Stem Cell-Like Cells Derived from Human iPSCs via Neural Crest Development and Their Application for Osteochondral Repair. Stem Cells International. 2017: 1–18.Chijimatsu R, Ikeya M, Yasui Y, Ikeda Y, Ebina K, Moriguchi Y, et al. Characterization of Mesenchymal Stem Cell-Like Cells Derived from Human iPSCs via Neural Crest Development and Their Application for Osteochondral Repair. Stem Cells International. 2017: 1–18.Karen Niederreither JV, Vermot J, Schuhbaur B, Chambon P, Dollé P. Embryonic retinoic acid synthesis is required for forelimb growth andanteroposterior patterning in the mouse. Development. 2002: 129 (15): 3563–74.Karen Niederreither JV, Vermot J, Schuhbaur B, Chambon P, Dollé P. Embryonic retinoic acid synthesis is required for forelimb growth andanteroposterior patterning in the mouse. Development. 2002: 129 (15): 3563–74.Kawata M, Mori D, Kanke K, Hojo H, Ohba S, Chung U il, et al. Simple and Robust Differentiation of Human Pluripotent Stem Cells toward Chondrocytes by Two Small-Molecule Compounds. Stem Cell Reports. 2019; 13 (3): 530–44.Kawata M, Mori D, Kanke K, Hojo H, Ohba S, Chung U il, et al. Simple and Robust Differentiation of Human Pluripotent Stem Cells toward Chondrocytes by Two Small-Molecule Compounds. Stem Cell Reports. 2019; 13 (3): 530–44.Kreuser U, Buchert J, Haase A, Richter W, Diederichs S. Initial WNT/β-Catenin Activation Enhanced Mesoderm Commitment, Extracellular Matrix Expression, Cell Aggregation and Cartilage Tissue Yield From Induced Pluripotent Stem Cells. Frontiers in Cell and Developmental Biology. 2020; 8: 581331. DOI: 10.3389/fcell.2020.581331.Kreuser U, Buchert J, Haase A, Richter W, Diederichs S. Initial WNT/β-Catenin Activation Enhanced Mesoderm Commitment, Extracellular Matrix Expression, Cell Aggregation and Cartilage Tissue Yield From Induced Pluripotent Stem Cells. Frontiers in Cell and Developmental Biology. 2020; 8: 581331. DOI: 10.3389/fcell.2020.581331.Lee NH, Bayaraa O, Zechu Z, Kim HS. Biomaterials-assisted spheroid engineering for regenerative therapy. BMB Reports. 2021; 54 (7): 356–67.Lee NH, Bayaraa O, Zechu Z, Kim HS. Biomaterials-assisted spheroid engineering for regenerative therapy. BMB Reports. 2021; 54 (7): 356–67.Martin I, Suetterlin R, Baschong W, Heberer M, Vunjak-Novakovic G, Freed LE. Enhanced cartilage tissue engineering by sequential exposure of chondrocytes to FGF-2 during 2D expansion and BMP-2 during 3D cultivation. J Cell Biochem. 2001; 83 (1): 121–8. DOI: 10.1002/jcb.1203.Martin I, Suetterlin R, Baschong W, Heberer M, Vunjak-Novakovic G, Freed LE. Enhanced cartilage tissue engineering by sequential exposure of chondrocytes to FGF-2 during 2D expansion and BMP-2 during 3D cultivation. J Cell Biochem. 2001; 83 (1): 121–8. DOI: 10.1002/jcb.1203.Kim J, Tomida K, Matsumoto T, Adachi T. Spheroid culture for chondrocytes triggers the initial stage of endochondral ossification. Biotechnol Bioeng. 2022; 119 (11): 3311–8. DOI: 10.1002/bit.28203.Kim J, Tomida K, Matsumoto T, Adachi T. Spheroid culture for chondrocytes triggers the initial stage of endochondral ossification. Biotechnol Bioeng. 2022; 119 (11): 3311–8. DOI: 10.1002/bit.28203.Sulaiman S, Chowdhury SR, Fauzi MB, Rani RA, Mohamadyahaya NH, Tabata Y, et al. 3d culture of MSCS on a gelatin microsphere in a dynamic culture system enhances chondrogenesis. International Journal of Molecular Sciences. 2020; 21 (8): 1–17.Sulaiman S, Chowdhury SR, Fauzi MB, Rani RA, Mohamadyahaya NH, Tabata Y, et al. 3d culture of MSCS on a gelatin microsphere in a dynamic culture system enhances chondrogenesis. International Journal of Molecular Sciences. 2020; 21 (8): 1–17.Gionet-Gonzales MA, Leach JK. Engineering principles for guiding spheroid function in the regeneration of bone, cartilage, and skin. Biomedical Materials (Bristol). 2018; 13 (3): 034109. DOI: 10.1088/1748-605X/aab0b3.Gionet-Gonzales MA, Leach JK. Engineering principles for guiding spheroid function in the regeneration of bone, cartilage, and skin. Biomedical Materials (Bristol). 2018; 13 (3): 034109. DOI: 10.1088/1748-605X/aab0b3.Sridharan BP, Laflin AD, Detamore MS. Generating Chondromimetic Mesenchymal Stem Cell Spheroids by Regulating Media Composition and Surface Coating. Cellular and Molecular Bioengineering. 2018; 11 (2): 99–115.Sridharan BP, Laflin AD, Detamore MS. Generating Chondromimetic Mesenchymal Stem Cell Spheroids by Regulating Media Composition and Surface Coating. Cellular and Molecular Bioengineering. 2018; 11 (2): 99–115.Wang G, An Y, Zhang X, Ding P, Bi H, Zhao Z. Chondrocyte Spheroids Laden in GelMA/HAMA Hybrid Hydrogel for TissueEngineered Cartilage with Enhanced Proliferation, Better Phenotype Maintenance, and Natural Morphological Structure. Gels. 2021; 7 (4): 247. Available from: https://www.mdpi.com/2310-2861/7/4/247.Wang G, An Y, Zhang X, Ding P, Bi H, Zhao Z. Chondrocyte Spheroids Laden in GelMA/HAMA Hybrid Hydrogel for TissueEngineered Cartilage with Enhanced Proliferation, Better Phenotype Maintenance, and Natural Morphological Structure. Gels. 2021; 7 (4): 247. Available from: https://www.mdpi.com/2310-2861/7/4/247.Jang Y, Jung H, Ju JH. Chondrogenic differentiation induction of adipose-derived stem cells by centrifugal gravity. Journal of Visualized Experiments. 2017; 2017 (120). DOI: 10.3791/54934.Jang Y, Jung H, Ju JH. Chondrogenic differentiation induction of adipose-derived stem cells by centrifugal gravity. Journal of Visualized Experiments. 2017; 2017 (120). DOI: 10.3791/54934.Jang J, Lee J, Lee E, Lee EA, Son Y. Disc-type hyaline cartilage reconstruction using 3D-cell sheet culture of human bone marrow stromal cells and human costal chondrocytes and maintenance of its shape and phenotype after transplantation. Tissue Engineering and Regenerative Medicine. 2016; 13 (4): 352–63.Jang J, Lee J, Lee E, Lee EA, Son Y. Disc-type hyaline cartilage reconstruction using 3D-cell sheet culture of human bone marrow stromal cells and human costal chondrocytes and maintenance of its shape and phenotype after transplantation. Tissue Engineering and Regenerative Medicine. 2016; 13 (4): 352–63.Salonius E, Kontturi L, Laitinen A, Haaparanta AM, Korhonen M, Nystedt J, et al. Chondrogenic differentiation of human bone marrow-derived mesenchymal stromal cells in a three-dimensional environment. Journal of Cellular Physiology. 2020; 235 (4): 3497–507.Salonius E, Kontturi L, Laitinen A, Haaparanta AM, Korhonen M, Nystedt J, et al. Chondrogenic differentiation of human bone marrow-derived mesenchymal stromal cells in a three-dimensional environment. Journal of Cellular Physiology. 2020; 235 (4): 3497–507.Markway BD, Tan GK, Brooke G, Hudson JE, Cooper-White JJ, Doran MR. Enhanced chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells in low oxygen environment micropellet cultures. Cell Transplantation. 2010; 19 (1): 29–42.Markway BD, Tan GK, Brooke G, Hudson JE, Cooper-White JJ, Doran MR. Enhanced chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells in low oxygen environment micropellet cultures. Cell Transplantation. 2010; 19 (1): 29–42.Rogan H, Ilagan F, Yang F. Comparing Single Cell Versus Pellet Encapsulation of Mesenchymal Stem Cells in Three-Dimensional Hydrogels for Cartilage Regeneration. Tissue Engineering — Part A. 2019; 25 (19–20): 1404–12.Rogan H, Ilagan F, Yang F. Comparing Single Cell Versus Pellet Encapsulation of Mesenchymal Stem Cells in Three-Dimensional Hydrogels for Cartilage Regeneration. Tissue Engineering — Part A. 2019; 25 (19–20): 1404–12.Zhang M, Shi J, Xie M, Wen J, Niibe K, Zhang X, et al. Recapitulation of cartilage/bone formation using iPSCs via biomimetic 3D rotary culture approach for developmental engineering. Biomaterials. 2020; 260. DOI: 10.1016/j.biomaterials.Zhang M, Shi J, Xie M, Wen J, Niibe K, Zhang X, et al. Recapitulation of cartilage/bone formation using iPSCs via biomimetic 3D rotary culture approach for developmental engineering. Biomaterials. 2020; 260. DOI: 10.1016/j.biomaterials.Zhang K, Yan S, Li G, Cui L, Yin J. In-situ birth of MSCs multicellular spheroids in poly(L-glutamic acid)/chitosan scaffold for hyaline-like cartilage regeneration. Biomaterials. 2015; 71: 24–34.Zhang K, Yan S, Li G, Cui L, Yin J. In-situ birth of MSCs multicellular spheroids in poly(L-glutamic acid)/chitosan scaffold for hyaline-like cartilage regeneration. Biomaterials. 2015; 71: 24–34.Stuart MP, Matsui RAM, Santos MFS, Côrtes I, Azevedo MS, Silva KR, et al. Successful low-cost scaffold-free cartilage tissue engineering using human cartilage progenitor cell spheroids formed by micromolded nonadhesive hydrogel. Stem Cells International. 2017. 2017; DOI: 10.1155/2017/7053465. Epub 2017 Dec 20.Stuart MP, Matsui RAM, Santos MFS, Côrtes I, Azevedo MS, Silva KR, et al. Successful low-cost scaffold-free cartilage tissue engineering using human cartilage progenitor cell spheroids formed by micromolded nonadhesive hydrogel. Stem Cells International. 2017. 2017; DOI: 10.1155/2017/7053465. Epub 2017 Dec 20.STEMCELL Technologies. Reproducible and Uniform Embryoid Bodies Using AggreWellTM Plates. 2011. Available from: https://www.stemcell.com/media/files/brochure/BR29150-AggreWell_Reproducible_Uniform_Embryoid_Bodies.pdf.STEMCELL Technologies. Reproducible and Uniform Embryoid Bodies Using AggreWellTM Plates. 2011. Available from: https://www.stemcell.com/media/files/brochure/BR29150-AggreWell_Reproducible_Uniform_Embryoid_Bodies.pdf.Eremeev AV, Volovikov EA, Shuvalova LD, Davidenko AV, Khomyakova EA, Bogomiakova ME, Lebedeva OS, Zubkova OA, Lagarkova MA. “Necessity is the Mother of Invention” or Inexpensive, Reliable, and Reproducible Protocol for Generating Organoids. Biochemistry (Moscow). 2019; 84 ( 3): 321–28. DOI: 10.1134/S0006297919030143.Eremeev AV, Volovikov EA, Shuvalova LD, Davi denko AV, Khomyakova EA, Bogomiakova ME, Lebedeva OS, Zubkova OA, Lagarkova MA. “Necessity is the Mother of Invention” or Inexpensive, Reliable, and Reproducible Protocol for Generating Organoids. Biochemistry (Moscow). 2019; 84 ( 3): 321–28. DOI: 10.1134/S0006297919030143.QIAGEN. RNeasy ® Plus Mini Handbook. 2020, 47 p. https://www.qiagen.com/us/resources/resourcedetail?id=16b8f578-d192-4613-ae32-8e02e0b0fa77&lang=enQIAGEN. RNeasy ® Plus Mini Handbook. 2020, 47 p. https://www.qiagen.com/us/resources/resourcedetail?id=16b8f578-d192-4613-ae32-8e02e0b0fa77&lang=enЕвроген. MMLV RT kit. 2021. Available from: www.evrogen.ru.Евроген. MMLV RT kit. 2021. Available from: www.evrogen.ru.Molecular Cellular and Developmental Biology. Cryostat Procedure. 2012; 5 p. Available from: https://labs.mcdb.ucsb.edu/fisher/steven/Website/protocols/Cryosectioning.pdf.Molecular Cellular and Developmental Biology. Cryostat Procedure. 2012; 5 p. Available from: https://labs.mcdb.ucsb.edu/fisher/steven/Website/protocols/Cryosectioning.pdf.Shuvalova LD, Eremeev AV, Bogomazova AN, Novosadova EV, Zerkalenkova EA, Olshanskaya YV, et al. Generation of induced pluripotent stem cell line RCPCMi004-A derived from patient with Parkinson's disease with deletion of the exon 2 in PARK2 gene. Stem Cell Res. 2020; 44: 101733. DOI: 10.1016/j.scr.2020.101733.Shuvalova LD, Eremeev AV, Bogomazova AN, Novosadova EV, Zerkalenkova EA, Olshanskaya YV, et al. Generation of induced pluripotent stem cell line RCPCMi004-A derived from patient with Parkinson's disease with deletion of the exon 2 in PARK2 gene. Stem Cell Res. 2020; 44: 101733. DOI: 10.1016/j.scr.2020.101733.Goldring MB, Tsuchimochi K, Ijiri K. The control of chondrogenesis. Journal of Cellular Biochemistry. 2006; 97: 33–44.Goldring MB, Tsuchimochi K, Ijiri K. The control of chondrogenesis. Journal of Cellular Biochemistry. 2006; 97: 33–44.Rutgers M, Saris DB, Vonk LA, van Rijen MH, Akrum V, Langeveld D, et al. Effect of collagen type I or type II on chondrogenesis by cultured human articular chondrocytes. Tissue Engineering — Part A. 2013; 19 (1–2): 59–65.Rutgers M, Saris DB, Vonk LA, van Rijen MH, Akrum V, Langeveld D, et al. Effect of collagen type I or type II on chondrogenesis by cultured human articular chondrocytes. Tissue Engineering — Part A. 2013; 19 (1–2): 59–65.Theodoropoulos JS, DeCroos AJN, Petrera M, Park S, Kandel RA. Mechanical stimulation enhances integration in an in vitro model of cartilage repair. Knee Surgery, Sports Traumatology, Arthroscopy. 2016; 24 (6): 2055–64.Theodoropoulos JS, DeCroos AJN, Petrera M, Park S, Kandel RA. Mechanical stimulation enhances integration in an in vitro model of cartilage repair. Knee Surgery, Sports Traumatology, Arthroscopy. 2016; 24 (6): 2055–64.Endo K, Fujita N, Nakagawa T, Nishimura R. Effect of Fibroblast Growth Factor-2 and Serum on Canine Mesenchymal Stem Cell Chondrogenesis. Tissue Engineering — Part A. 2019; 25 (11–12): 901–10.Endo K, Fujita N, Nakagawa T, Nishimura R. Effect of Fibroblast Growth Factor-2 and Serum on Canine Mesenchymal Stem Cell Chondrogenesis. Tissue Engineering — Part A. 2019; 25 (11–12): 901–10.Wu CL, Dicks A, Steward N, Tang R, Katz DB, Choi YR, et al. Single cell transcriptomic analysis of human pluripotent stem cell chondrogenesis. Nature Communications. 2021; 12 (1): 1–18.Wu CL, Dicks A, Steward N, Tang R, Katz DB, Choi YR, et al. Single cell transcriptomic analysis of human pluripotent stem cell chondrogenesis. Nature Communications. 2021; 12 (1): 1–18.

The authors declare that there are no conflicts of interest present.