Engineering bone/cartilage organoids: strategy, progress, and application

Abstract

The concept and development of bone/cartilage organoids are rapidly gaining momentum, providing opportunities for both fundamental and translational research in bone biology. Bone/cartilage organoids, essentially miniature bone/cartilage tissues grown in vitro, enable the study of complex cellular interactions, biological processes, and disease pathology in a representative and controlled environment. This review provides a comprehensive and up-to-date overview of the field, focusing on the strategies for bone/cartilage organoid construction strategies, progresses in the research, and potential applications. We delve into the significance of selecting appropriate cells, matrix gels, cytokines/inducers, and construction techniques. Moreover, we explore the role of bone/cartilage organoids in advancing our understanding of bone/cartilage reconstruction, disease modeling, drug screening, disease prevention, and treatment strategies. While acknowledging the potential of these organoids, we discuss the inherent challenges and limitations in the field and propose potential solutions, including the use of bioprinting for organoid induction, AI for improved screening processes, and the exploration of assembloids for more complex, multicellular bone/cartilage organoids models. We believe that with continuous refinement and standardization, bone/cartilage organoids can profoundly impact patient-specific therapeutic interventions and lead the way in regenerative medicine.

References

[1] Tagliaferri C, Wittrant Y, Davicco M-J, Walrand S, Coxam V. Muscle and bone, two interconnected tissues. Ageing Res. Rev., 2015, 21: 55-70

[2] Fang EF, et al. A research agenda for ageing in China in the 21st century: focusing on basic and translational research, long-term care, policy and social networks. Ageing Res. Rev., 2020, 64: 101174

[3] Sharma S, Basu BJB. Biomaterials assisted reconstructive urology: the pursuit of an implantable bioengineered neo-urinary bladder. Biomaterials, 2022, 281

[4] Alshangiti DM, El-Damhougy TK, Zaher A, Madani M. Revolutionizing biomedicine: advancements, applications, and prospects of nanocomposite macromolecular carbohydrate-based hydrogel biomaterials: a review. RSC Adv., 2023, 13: 35251-35291

[5] Chen KG, Park K, Spence JR. Studying SARS-CoV-2 infectivity and therapeutic responses with complex organoids. Nat. Cell Biol., 2021, 23: 822-833

[6] Kim W, Gwon Y, Park S, Kim H, Kim J. Therapeutic strategies of three-dimensional stem cell spheroids and organoids for tissue repair and regeneration. Bioact. Mater., 2023, 19: 50-74

[7] Schutgens F, Clevers H. Human organoids: tools for understanding biology and treating diseases. Annu. Rev. Pathol. Pathol. Mech. Dis., 2020, 15: 211-234

[8] Dhawan A, Kennedy PM, Rizk EB, Ozbolat IT. Three-dimensional bioprinting for bone and cartilage restoration in orthopaedic surgery. J. Am. Acad. Orthop. Surg., 2019, 27: e215-e226

[9] Hu Y, et al. Bone/cartilage organoid on-chip: construction strategy and application. Bioact. Mater., 2023, 25: 29-41

[10] Cox B, et al. Application of high-throughput screening techniques to drug discovery. Prog. Med. Chem., 2000, 37: 83-134

[11] Huey DJ, Hu JC, Athanasiou KA. Unlike bone, cartilage regeneration remains elusive. Science, 2012, 338: 917-921

[12] Veninga V, Voest EE. Tumor organoids: opportunities and challenges to guide precision medicine. Cancer Cell, 2021, 39: 1190-1201

[13] Sakalem, M. E., De Sibio, M. T., da Costa, F. A. D. & de Oliveira, M. Historical evolution of spheroids and organoids, and possibilities of use in life sciences and medicine. Biotechnol. J. 16, e2000463 (2021).

[14] Kaushik G, Ponnusamy MP, Batra SK. Concise review: current status of three-dimensional organoids as preclinical models. Stem Cells, 2018, 36: 1329-1340

[15] Shankaran, A., Prasad, K., Chaudhari, S., Brand, A. & Satyamoorthy, K. Advances in development and application of human organoids. 3 Biotech 11, 257 (2021).

[16] Wilson HV. A new method by which sponges may be artificially reared. Science, 1907, 25: 912-915

[17] Sato T, et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature, 2009, 459: 262-265

[18] Spence JR, et al. Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature, 2011, 470: 105-109

[19] Eiraku M, et al. Self-organizing optic-cup morphogenesis in three-dimensional culture. Nature, 2011, 472: 51-56

[20] Nakano T, et al. Self-formation of optic cups and storable stratified neural retina from human ESCs. Cell Stem Cell, 2012, 10: 771-785

[21] Lancaster MA, et al. Cerebral organoids model human brain development and microcephaly. Nature, 2013, 501: 373-379

[22] Jamieson PR, et al. Derivation of a robust mouse mammary organoid system for studying tissue dynamics. Development, 2017, 144: 1065-1071

[23] Kessler M, et al. The Notch and Wnt pathways regulate stemness and differentiation in human fallopian tube organoids. Nat. Commun., 2015, 6

[24] Sakaguchi H, et al. Generation of functional hippocampal neurons from self-organizing human embryonic stem cell-derived dorsomedial telencephalic tissue. Nat. Commun., 2015, 6

[25] Lee J, et al. Hair-bearing human skin generated entirely from pluripotent stem cells. Nature, 2020, 582: 399-404

[26] Iordachescu A, et al. An in vitro model for the development of mature bone containing an osteocyte network. Adv. Biosyst., 2017, 2

[27] Akiva A, et al. An organoid for woven bone. Adv. Funct. Mater., 2021, 31

[28] Park, Y. et al. Trabecular bone organoid model for studying the regulation of localized bone remodeling. Sci. Adv. 7, eabd6495 (2021).

[29] O’Connor SK, Katz DB, Oswald SJ, Groneck L, Guilak F. Formation of osteochondral organoids from murine induced pluripotent stem cells. Tissue Eng. Part A, 2021, 27: 1099-1109

[30] Nilsson Hall G, et al. Developmentally engineered callus organoid bioassemblies exhibit predictive in vivo long bone healing. Adv. Sci., 2019, 7

[31] Wen, Y. et al. Hyperplastic human macromass cartilage for joint regeneration. Adv. Sci. 10, e2301833 (2023).

[32] Yang Z, et al. In situ self-assembled organoid for osteochondral tissue regeneration with dual functional units. Bioact. Mater., 2023, 27: 200-215

[33] Kanton, S. & Paşca, S. P. Human assembloids. Development 149, dev201120 (2022).

[34] Kadoshima T, et al. Self-organization of axial polarity, inside-out layer pattern, and species-specific progenitor dynamics in human ES cell-derived neocortex. Proc. Natl. Acad. Sci. USA, 2013, 110: 20284-20289

[35] Kim J, Koo B-K, Knoblich JA. Human organoids: model systems for human biology and medicine. Nat. Rev. Mol. Cell Biol., 2020, 21: 571-584

[36] Panoutsopoulos AA. Organoids, assembloids, and novel biotechnology: steps forward in developmental and disease-related neuroscience. Neuroscientist, 2021, 27: 463-472

[37] Birey F, et al. Dissecting the molecular basis of human interneuron migration in forebrain assembloids from Timothy syndrome. Cell Stem Cell, 2022, 29: 248-264 e247

[38] Andersen J, et al. Generation of functional human 3d cortico-motor assembloids. Cell, 2020, 183: 1913-1929

[39] Miura Y, et al. Generation of human striatal organoids and cortico-striatal assembloids from human pluripotent stem cells. Nat. Biotechnol., 2020, 38: 1421-1430

[40] Zhang Z, Mu Y, Zhou H, Yao H, Wang DA. Cartilage tissue engineering in practice: preclinical trials, clinical applications, and prospects. Tissue Eng. Part B Rev., 2023, 29: 473-490

[41] Wang XH, Liu N, Zhang H, Yin ZS, Zha ZG. From cells to organs: progress and potential in cartilaginous organoids research. J. Transl. Med., 2023, 21

[42] Vinatier C, et al. Cartilage tissue engineering: towards a biomaterial-assisted mesenchymal stem cell therapy. Curr. Stem Cell Res. Ther., 2009, 4: 318-329

[43] Doss, M. X. & Sachinidis, A. Current challenges of iPSC-based disease modeling and therapeutic implications. Cells 8, 403 (2019).

[44] Yen BL, et al. Three-dimensional spheroid culture of human mesenchymal stem cells: offering therapeutic advantages and in vitro glimpses of the in vivo state. Stem Cells Transl. Med., 2023, 12: 235-244

[45] Zhao, D., Saiding, Q., Li, Y., Tang, Y. & Cui, W. Bone organoids: recent advances and future challenges. Adv. Healthc. Mater. 13, e2302088 (2023).

[46] Schaffler MB, Kennedy OD. Osteocyte signaling in bone. Curr. Osteoporos. Rep., 2012, 10: 118-125

[47] Freyria A-M, Mallein-Gerin F. Chondrocytes or adult stem cells for cartilage repair: the indisputable role of growth factors. Injury, 2012, 43: 259-265

[48] Bahney, C. et al. Transdifferentiation of hypertrophic chondrocytes during endochondral bone repair by activation of pluripotent stem cell programs. FASEB J. 28, 216.211 (2014).

[49] Leibbrandt A, Penninger JM. RANK/RANKL: regulators of immune responses and bone physiology. Ann. N. Y. Acad. Sci., 2008, 1143: 123-150

[50] Hong J, Zheng W, Wang X, Hao Y, Cheng G. Biomedical polymer scaffolds mimicking bone marrow niches to advance in vitro expansion of hematopoietic stem cells. J. Mater. Chem. B, 2022, 10: 9755-9769

[51] Ye S, et al. A chemically defined hydrogel for human liver organoid culture. Adv. Funct. Mater., 2020, 30

[52] Tam WL, et al. Human pluripotent stem cell-derived cartilaginous organoids promote scaffold-free healing of critical size long bone defects. Stem Cell Res. Ther., 2021, 12: 513

[53] Zheng M, et al. A review of recent progress on collagen-based biomaterials. Adv. Healthc. Mater., 2023, 12

[54] Kleuskens MWA, et al. Neo-cartilage formation using human nondegenerate versus osteoarthritic chondrocyte-derived cartilage organoids in a viscoelastic hydrogel. J. Orthop. Res., 2023, 41: 1902-1915

[55] Wilson RL, et al. Protein-functionalized poly(ethylene glycol) hydrogels as scaffolds for monolayer organoid culture. Tissue Eng. Part C Methods, 2020, 27: 12-23

[56] Zustiak SP, Leach JB. Hydrolytically degradable poly(ethylene glycol) hydrogel scaffolds with tunable degradation and mechanical properties. Biomacromolecules, 2010, 11: 1348-1357

[57] Vallmajo-Martin Q, Broguiere N, Millan C, Zenobi-Wong M, Ehrbar M. PEG/HA hybrid hydrogels for biologically and mechanically tailorable bone marrow organoids. Adv. Funct. Mater., 2020, 30: 1910282

[58] Jansen LE, et al. A poly(ethylene glycol) three-dimensional bone marrow hydrogel. Biomaterials, 2022, 280

[59] Han Y, et al. Heterogeneous DNA hydrogel loaded with Apt02 modified tetrahedral framework nucleic acid accelerated critical-size bone defect repair. Bioact. Mater., 2024, 35: 1-16

[60] Shen C, et al. Boosting cartilage repair with silk fibroin-DNA hydrogel-based cartilage organoid precursor. Bioact. Mater., 2024, 35: 429-444

[61] Galibert L, Tometsko ME, Anderson DM, Cosman D, Dougall WC. The involvement of multiple tumor necrosis factor receptor (TNFR)-associated factors in the signaling mechanisms of receptor activator of NF-kappaB, a member of the TNFR superfamily. J. Biol. Chem., 1998, 273: 34120-34127

[62] Valenta T, et al. Wnt ligands secreted by subepithelial mesenchymal cells are essential for the survival of intestinal stem cells and gut homeostasis. Cell Rep., 2016, 15: 911-918

[63] Yuasa M, et al. Dexamethasone enhances osteogenic differentiation of bone marrow- and muscle-derived stromal cells and augments ectopic bone formation induced by bone morphogenetic protein-2. PLoS ONE, 2015, 10: e0116462

[64] Chen J, et al. Topical combined application of dexamethasone, vitamin C, and β-sodium glycerophosphate for healing the extraction socket in rabbits. Int. J. Oral. Maxillofac. Surg., 2015, 44: 1317-1323

[65] Khan AO, et al. Human bone marrow organoids for disease modeling, discovery, and validation of therapeutic targets in hematologic malignancies. Cancer Discov., 2023, 13: 364-385

[66] Stoltz JF, et al. Stem cells and vascular regenerative medicine: a mini review. Clin. Hemorheol. Microcirc., 2016, 64: 613-633

[67] Huang, J., Zhang, L., Lu, A. & Liang, C. Organoids as innovative models for bone and joint diseases. Cells 12, 1590 (2023).

[68] Shen S, et al. Three dimensional printing-based strategies for functional cartilage regeneration. Tissue Eng. Part B Rev., 2019, 25: 187-201

[69] Pievani A, et al. Human umbilical cord blood-borne fibroblasts contain marrow niche precursors that form a bone/marrow organoid in vivo. Development, 2017, 144: 1035-1044

[70] Sacchetti B, et al. No identical “mesenchymal stem cells” at different times and sites: human committed progenitors of distinct origin and differentiation potential are incorporated as adventitial cells in microvessels. Stem Cell Rep., 2016, 6: 897-913

[71] Abraham DM, et al. Self‐assembling human skeletal organoids for disease modeling and drug testing. J. Biomed. Mater. Res. Part B Appl. Biomater., 2021, 110: 871-884

[72] Iordachescu A, et al. Trabecular bone organoids: a micron-scale ‘humanised’ prototype designed to study the effects of microgravity and degeneration. npj Microgravity, 2021, 7

[73] Wang, J. et al. Engineering large-scale self-mineralizing bone organoids with bone matrix-inspired hydroxyapatite hybrid bioinks. Adv. Mater. 36, e2309875 (2024).

[74] Zhang, J. et al. Long-term mechanical loading is required for the formation of 3D bioprinted functional osteocyte bone organoids. Biofabrication 14, 035018 (2022).

[75] Giger S, et al. Microarrayed human bone marrow organoids for modeling blood stem cell dynamics. APL Bioeng., 2022, 6: 036101

[76] Li A, et al. Vascularization of a bone organoid using dental pulp stem cells. Stem Cells Int., 2023, 2023: 1-9

[77] Hall, G. N. et al. Patterned, organoid-based cartilaginous implants exhibit zone specific functionality forming osteochondral-like tissues in vivo. Biomaterials 273, 120820 (2021).

[78] Crispim JF, Ito K. De novo neo-hyaline-cartilage from bovine organoids in viscoelastic hydrogels. Acta Biomater., 2021, 128: 236-249

[79] Abe, K. et al. Engraftment of allogeneic iPS cell-derived cartilage organoid in a primate model of articular cartilage defect. Nat. Commun. 14, 804 (2023).

[80] Sun, Y., Wu, Q., Dai, K., You, Y. & Jiang, W. Generating 3D-cultured organoids for pre-clinical modeling and treatment of degenerative joint disease. Signal Transduct. Tar. 6, 380 (2021).

[81] Burdis, R. et al. Spatial patterning of phenotypically distinct microtissues to engineer osteochondral grafts for biological joint resurfacing. Biomaterials 289, 121750 (2022).

[82] Mehboob A, Chang S-H. Effect of composite bone plates on callus generation and healing of fractured tibia with different screw configurations. Compos. Sci. Technol., 2018, 167: 96-105

[83] Xie C, et al. High-efficient engineering of osteo-callus organoids for rapid bone regeneration within one month. Biomaterials, 2022, 288

[84] Decoene, I., Herpelinck, T., Geris, L. & Luyten, F. P., Papantoniou, I. Engineering bone-forming callus organoid implants in a xenogeneic-free differentiation medium. Front. Chem. Eng. 4, 892190 (2022).

[85] Abraham DM, et al. Self-assembling human skeletal organoids for disease modeling and drug testing. J. Biomed. Mater. Res. Part B Appl. Biomater., 2022, 110: 871-884

[86] Caire R, et al. YAP/TAZ: key players for rheumatoid arthritis severity by driving fibroblast like synoviocytes phenotype and fibro-inflammatory response. Front. Immunol., 2021, 12

[87] Chen S, Chen X, Geng Z, Su J. The horizon of bone organoid: a perspective on construction and application. Bioact. Mater., 2022, 18: 15-25

[88] Yu Y, Wang J, Li Y, Chen Y, Cui W. Cartilaginous organoids: advances, applications, and perspectives. Adv. NanoBiomed. Res., 2023, 3: 2200114

[89] Lin W, Wang M, Xu L, Tortorella M, Li G. Cartilage organoids for cartilage development and cartilage-associated disease modeling. Front. Cell Dev. Biol., 2023, 11

[90] Xu H, et al. Organoid technology in disease modelling, drug development, personalized treatment and regeneration medicine. Exp. Hematol. Oncol., 2018, 7: 30

[91] Mikael PE, Golebiowska AA, Kumbar SG, Nukavarapu SP. Evaluation of autologously derived biomaterials and stem cells for bone tissue engineering. Tissue Eng. Part A, 2020, 26: 1052-1063

[92] Vainieri ML, Wahl D, Alini M, van Osch GJVM, Grad S. Mechanically stimulated osteochondral organ culture for evaluation of biomaterials in cartilage repair studies. Acta Biomater., 2018, 81: 256-266

[93] Roberto de Barros N, et al. Engineered organoids for biomedical applications. Adv. drug Deliv. Rev., 2023, 203

[94] Araújo R, et al. NMR metabolomics to study the metabolic response of human osteoblasts to non-poled and poled poly (L-lactic) acid. Magn. Reson Chem., 2019, 57: 919-933

[95] Teriyapirom I, Batista-Rocha AS, Koo BK. Genetic engineering in organoids. J. Mol. Med., 2021, 99: 555-568

[96] Li H, Liu H, Chen K. Living biobank-based cancer organoids: prospects and challenges in cancer research. Cancer Biol. Med., 2022, 19: 965-982

[97] Jacob F, et al. A patient-derived glioblastoma organoid model and biobank recapitulates inter- and intra-tumoral heterogeneity. Cell, 2020, 180: 188-204.e122

[98] Yang S, et al. Organoids: the current status and biomedical applications. MedComm, 2023, 4

[99] Matsiko A, Levingstone TJ, O’Brien FJ. Advanced strategies for articular cartilage defect repair. Materials, 2013, 6: 637-668

[100] Huang C, et al. The application of organs-on-a-chip in dental, oral, and craniofacial research. J. Dent. Res., 2023, 102: 364-375

[101] Salewskij K, Penninger JM. Blood vessel organoids for development and disease. Circ. Res., 2023, 132: 498-510

[102] Murphy SV, Atala A. 3D bioprinting of tissues and organs. Nat. Biotechnol., 2014, 32: 773-785

[103] Renner H, Schöler HR, Bruder JM. Combining automated organoid workflows with artificial intelligence-based analyses: opportunities to build a new generation of interdisciplinary high-throughput screens for Parkinson’s disease and beyond. Mov. Disord., 2021, 36: 2745-2762

[104] Takebe T, et al. Vascularized and complex organ buds from diverse tissues via mesenchymal cell-driven condensation. Cell Stem Cell, 2015, 16: 556-565

[105] Long B, Xiangfei L, Jiacan S. ChatGPT: the cognitive effects on learning and memory. Brain-X, 2023, 1

[106] Long B, Jiacan S. Brain-inspired intelligence-driven scientific research. Brain-X, 2024, 2

[107] Bai L, et al. AI-enabled organoids: construction, analysis, and application. Bioact. Mater., 2024, 31: 525-548

[108] Renner, H. et al. A fully automated high-throughput workflow for 3D-based chemical screening in human midbrain organoids. eLife 9, e52904 (2020).

[109] Ma C, et al. A novel surgical planning system using an AI model to optimize planning of pedicle screw trajectories with highest bone mineral density and strongest pull-out force. Neurosurg. Focus, 2022, 52: E10

[110] Yang C, Yang L, Gao G-D, Zong H-Q, Gao D. Assessment of artificial intelligence-aided reading in the detection of nasal bone fractures. Technol. Health Care, 2023, 31: 1017-1025

[111] Zhu J, Lun W, Feng Q, Cao X, Li Q. Mesenchymal stromal cells modulate YAP by verteporfin to mimic cartilage development and construct cartilage organoids based on decellularized matrix scaffolds. J. Mater. Chem. B, 2023, 11: 7442-7453

[112] Kale S, et al. Three-dimensional cellular development is essential for ex vivo formation of human bone. Nat. Biotechnol., 2000, 18: 954-958

[113] Serafini M, et al. Establishment of bone marrow and hematopoietic niches in vivo by reversion of chondrocyte differentiation of human bone marrow stromal cells. Stem Cell Res., 2014, 12: 659-672

[114] Torisawa YS, et al. Bone marrow-on-a-chip replicates hematopoietic niche physiology in vitro. Nat. Methods, 2014, 11: 663-669

[115] Papadimitropoulos A, et al. A 3D in vitro bone organ model using human progenitor cells. Eur. Cells Mater., 2011, 21: 445-458

[116] Bloks, N. G. et al. A high-impact COL6A3 mutation alters the response of chondrocytes in neo-cartilage organoids to hyper-physiologic mechanical loading. BioRxiv, 520461 (2022).

Funding

Integrated Project of Major Research Plan of National Natural Science Foundation of China (92249303), Shanghai Committee of Science and Technology (23141900600, Laboratory Animal Research Project), Shanghai Clinical Research Plan of SHDC2023CRT01

National Natural Science Foundation of China (National Science Foundation of China)(82172098)

Young Elite Scientist Sponsorship Program by China Association for Science and Technology (YESS20230049)