Callus organoids reveal distinct cartilage to bone transition mechanisms across donors and a role for biological sex

Abstract

Clinical translation of tissue-engineered advanced therapeutic medicinal products is hindered by a lack of patient-dependent and independent in-process biological quality controls that are reflective of in vivo outcomes. Recent insights into the mechanism of native bone repair highlight a robust path dependence. Organoid-based bottom-up developmental engineering mimics this path-dependence to design personalized living implants scaffold-free, with in-build outcome predictability. Yet, adequate (noninvasive) quality metrics of engineered tissues are lacking. Moreover, insufficient insight into the role of donor variability and biological sex as influencing factors for the mechanism toward bone repair hinders the implementation of such protocols for personalized bone implants. Here, male and female bone-forming organoids were compared to non-bone-forming organoids regarding their extracellular matrix composition, transcriptome, and secreted proteome signatures to directly link in vivo outcomes to quality metrics. As a result, donor variability in bone-forming callus organoids pointed towards two distinct pathways to bone, through either a hypertrophic cartilage or a fibrocartilaginous template. The followed pathway was determined early, as a biological sex-dependent activation of distinct progenitor populations. Independent of donor or biological sex, a cartilage-to-bone transition was driven by a common panel of secreted factors that played a role in extracellular matrix remodeling, mineralization, and attraction of vasculature. Hence, the secreted proteome is a source of noninvasive biomarkers that report on biological potency and could be the missing link toward data-driven decision-making in organoid-based bone tissue engineering.

Keywords

Engineering / Biomedical Engineering / Medical and Health Sciences / Clinical Sciences

References

[1] NegoroT, TakagakiY, OkuraH, MatsuyamaA. Trends in clinical trials for articular cartilage repair by cell therapy. NPJ Regen. Med., 2018, 3: 1-10.

[2] GerisL, PapantoniouI. The third era of tissue engineering: reversing the innovation drivers. Tissue Eng. Part A, 2019, 25: 821-826.

[3] HeathmanTR, et al. . The translation of cell-based therapies: clinical landscape and manufacturing challenges. Regen. Med., 2015, 10: 49-64.

[4] Pittenger, M. F. et al. Mesenchymal stem cell perspective: cell biology to clinical progress. NPJ Regen. Med.4, 22 (2019).

[5] Rousseau, C. F., Maciulaitis, R., Sladowski, D. & Narayanan, G. Cell and gene therapies: European view on challenges in translation and how to address them. Front. Med.5, 158 (2018).

[6] Jiang, Z. & Dalby, P. A. Challenges in scaling up AAV-based gene therapy manufacturing. Trends Biotechnol.0, 1268-1281 (2023).

[7] FukudaY, et al. . Layer-by-layer cell coating technique using extracellular matrix facilitates rapid fabrication and function of pancreatic β-cell spheroids. Biomaterials, 2018, 160: 82-91.

[8] LiuD, ChenS, NaingMW. A review of manufacturing capabilities of cell spheroid generation technologies and future development. Biotechnol. Bioeng., 2020, 118: 542-554.

[9] LenasP, LuytenFP. An emerging paradigm in tissue engineering: from chemical engineering to developmental engineering for bioartificial tissue formation through a series of unit operations that simulate the in vivo successive developmental stages. Ind. Eng. Chem. Res., 2011, 50: 482-522.

[10] LenasP, MoosMJ, 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, 2009, 15: 381-394.

[11] Clark, D. et al. Age-related decrease in periostin expression may be associated with attenuated fracture healing in old mice. J. Orthopaed. Res.41, 1–11 (2022).

[12] ClarkD, NakamuraM, MiclauT, MarcucioR. Effects of aging on fracture healing. Curr. Osteoporos. Rep., 2017, 15: 601.

[13] BahtGS, ViL, AlmanBA. The role of the immune cells in fracture healing. Curr. Osteoporos. Rep., 2018, 16: 138.

[14] KodamaJ, WilkinsonKJ, IwamotoM, OtsuruS, Enomoto-IwamotoM. The role of hypertrophic chondrocytes in regulation of the cartilage-to-bone transition in fracture healing. Bone Rep., 2022, 17: 101616.

[15] DudaGN, et al. . The decisive early phase of bone regeneration. Nat. Rev. Rheumatol., 2023, 19: 78-95.

[16] JefferyEC, MannTLA, PoolJA, ZhaoZ, MorrisonSJ. Bone marrow and periosteal skeletal stem/progenitor cells make distinct contributions to bone maintenance and repair. Cell Stem Cell, 2022, 29: 1547-1561.

[17] LongoniA, et al. . The impact of immune response on endochondral bone regeneration. NPJ Regen. Med., 2018, 3: 1-11.

[18] KönneckeI, et al. . T and B cells participate in bone repair by infiltrating the fracture callus in a two-wave fashion. Bone, 2014, 64: 155-165.

[19] OrtonaE, PaganoMT, CaposselaL, MalorniW. The role of sex differences in bone health and healing. Biology, 2023, 12: 993.

[20] MaesC. Signaling pathways effecting crosstalk between cartilage and adjacent tissues Seminars in cell and developmental biology: the biology and pathology of cartilage. Semin Cell Dev. Biol., 2017, 62: 16-33.

[21] KozhemyakinaE, LassarAB, ZelzerE. A pathway to bone: signaling molecules and transcription factors involved in chondrocyte development and maturation. Development, 2015, 142: 817-831.

[22] RochaB, et al. . Secretome analysis of human mesenchymal stem cells undergoing chondrogenic differentiation. J. Proteome Res., 2014, 13: 1045-1054.

[23] BolanderJ, et al. . Healing of a large long-bone defect through serum-free in vitro priming of human periosteum-derived cells. Stem Cell Rep., 2017, 8: 758-772.

[24] Nilsson HallG, et al. . Developmentally engineered callus organoid bioassemblies exhibit predictive in vivo long bone healing. Adv. Sci., 2019, 1902295: 1-16

[25] WangT, ZhangJ, LiaoJ, ZhangF, ZhouG. Donor genetic backgrounds contribute to the functional heterogeneity of stem cells and clinical outcomes. Stem Cells Transl. Med., 2020, 9: 1495-1499.

[26] KurapatySS, HsuWK. Sex-based difference in bone healing: a review of recent pre-clinical literature. Curr. Rev. Musculoskelet. Med., 2022, 15: 651-658.

[27] NandraR, GroverL, PorterK. Fracture non-union epidemiology and treatment. Trauma, 2016, 18: 3-11.

[28] Miguel-AliagaI, et al. . Voicing the need to consider sex-specific differences in research. Dev. Cell, 2022, 57: 2675-2678.

[29] StüdleC, et al. . Challenges toward the identification of predictive markers for human mesenchymal stromal cells chondrogenic potential. Stem Cells Transl. Med., 2019, 8: 194-204.

[30] CapelliC, et al. . Potency assays and biomarkers for cell-based advanced therapy medicinal products. Front. Immunol., 2023, 14. 1186224

[31] LipsitzYY, TimminsNE, ZandstraPW. Quality cell therapy manufacturing by design. Nat. Biotechnol., 2016, 34: 393-400.

[32] Committee for Advanced Therapies (CAT). Guideline on the Minimum Quality and Non-Clinical Data for Certification of Advanced Therapy Medicinal Products. EMA/CAT/852602/2018.

[33] European Medicines Agence (EMA). Reflection Paper on Clinical Aspects Related to Tissue Engineered Products EMA/CAT/573420/2009. 44 (2014).

[34] McCorryMC, et al. . Sensor technologies for quality control in engineered tissue manufacturing. Biofabrication, 2023, 15: 012001.

[35] MendesLF, et al. . Combinatorial analysis of growth factors reveals the contribution of bone morphogenetic proteins to chondrogenic differentiation of human periosteal cells. Tissue Eng. Part C. Methods, 2016, 22: 473-486.

[36] 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, (2022).

[37] DeckersT, HallGN, PapantoniouI, AertsJ-M, BloemenV. A platform for automated and label-free monitoring of morphological features and kinetics of spheroid fusion. Front. Bioeng. Biotechnol., 2022, 0: 1441

[38] AghajanianP, MohanS. The art of building bone: emerging role of chondrocyte-to-osteoblast transdifferentiation in endochondral ossification. Bone Res., 2018, 6: 1-9.

[39] DuX, CaiL, XieJ, ZhouX. The role of TGF-beta3 in cartilage development and osteoarthritis. Bone Res., 2023, 11: 1-16.

[40] AkashiS, et al. . Metabolic regulation of the CCN family genes by glycolysis in chondrocytes. J. Cell Commun. Signal, 2018, 12: 245-252.

[41] JulienA, et al. . FGFR3 in periosteal cells drives cartilage-to-bone transformation in bone repair. Stem Cell Rep., 2020, 15: 955-967.

[42] PrinsHJ, et al. . In vitro induction of alkaline phosphatase levels predicts in vivo bone forming capacity of human bone marrow stromal cells. Stem Cell Res., 2014, 12: 428-440.

[43] MazurCM, et al. . Osteocyte dysfunction promotes osteoarthritis through MMP13-dependent suppression of subchondral bone homeostasis. Bone Res., 2019, 7: 1-17.

[44] OmorphosNP, GaoC, TanSS, SanghaMS. Understanding angiogenesis and the role of angiogenic growth factors in the vascularisation of engineered tissues. Mol. Biol. Rep., 2021, 48: 941-950.

[45] Bian, Q. et al. A single cell transcriptional atlas of early synovial joint development. Development147, dev185777 (2020).

[46] OichiT, et al. . Nutrient-regulated dynamics of chondroprogenitors in the postnatal murine growth plate. Bone Res., 2023, 11: 1-20.

[47] DebnathS, et al. . Discovery of a periosteal stem cell mediating intramembranous bone formation. Nature, 2018, 562: 133-139.

[48] Zahn, G. et al. Targeting longevity gene SLC13A5: a novel approach to prevent age-related bone fragility and osteoporosis. Metabolites13, 1186 (2023).

[49] KuwaharaST, LiuS, ChareunsoukA, SerowokyM, MarianiFV. On the horizon: hedgehog signaling to heal broken bones. Bone Res., 2022, 10: 1-11.

[50] SpäterD, et al. . Wnt9a signaling is required for joint integrity and regulation of Ihh during chondrogenesis. Development, 2006, 133: 3039-3049.

[51] PesesseL, et al. . Bone sialoprotein as a potential key factor implicated in the pathophysiology of osteoarthritis. Osteoarthr. Cartil., 2014, 22: 547-556.

[52] IftikharM, et al. . Lysyl oxidase-like-2 (LOXL2) is a major isoform in chondrocytes and is critically required for differentiation. J. Biol. Chem., 2011, 286: 909-918.

[53] Long, J. T. et al. Hypertrophic chondrocytes serve as a reservoir for marrow-associated skeletal stem and progenitor cells, osteoblasts, and adipocytes during skeletal development. Elife11, e76932 (2022).

[54] ShenF, HuangX, HeG, ShiY. The emerging studies on mesenchymal progenitors in the long bone. Cell Biosci., 2023, 13: 1-22.

[55] DohertyL, et al. . A PDGFRβ-PI3K signaling axis mediates periosteal cell activation during fracture healing. PLoS One, 2019, 14: 0223846.

[56] KarsdalMA, et al. . The good and the bad collagens of fibrosis- their role in signaling and organ function. Adv. Drug Del. Rev., 2017, 121: 43-56.

[57] MurphyG, et al. . Matrix metalloproteinases in arthritic disease. Arthritis Res., 2002, 4: S39.

[58] StrackeJO, et al. . Matrix metalloproteinases 19 and 20 cleave aggrecan and cartilage oligomeric matrix protein (COMP). FEBS Lett., 2000, 478: 52-56.

[59] WangX, et al. . Comparison of the major cell populations among osteoarthritis, Kashin–Beck disease and healthy chondrocytes by single-cell RNA-seq analysis. Cell Death Dis., 2021, 12: 1-15

[60] KimJH, et al. . Lysosomal SLC46A3 modulates hepatic cytosolic copper homeostasis. Nat. Commun., 2021, 12: 1-12

[61] WamaithaSE, et al. . IGF1-mediated human embryonic stem cell self-renewal recapitulates the embryonic niche. Nat. Commun., 2020, 11: 1-16.

[62] HollanderJM, et al. . A critical bioenergetic switch is regulated by IGF2 during murine cartilage development. Commun. Biol., 2022, 5: 1-13.

[63] WuM, WuS, ChenW, LiYP. The roles and regulatory mechanisms of TGF-β and BMP signaling in bone and cartilage development, homeostasis and disease. Cell Res., 2024, 34: 101-123.

[64] TranDQ, et al. . GARP (LRRC32) is essential for the surface expression of latent TGF-β on platelets and activated FOXP3⁺ regulatory T cells. Proc. Natl Acad. Sci. USA, 2009, 106: 13445-13450.

[65] ChanCKF, et al. . Identification of the human skeletal stem cell. Cell, 2018, 175: 43-56.

[66] CapulliM, et al. . The C-terminal domain of chondroadherin: a new regulator of osteoclast motility counteracting bone loss. J. Bone Miner. Res., 2014, 29: 1833-1846.

[67] HatfieldJT, AndersonPJ, PowellBC. Retinol-binding protein 4 is expressed in chondrocytes of developing mouse long bones: implications for a local role in formation of the secondary ossification center. Histochem. Cell Biol., 2013, 139: 727-734.

[68] Akhter, S., Rahman, M., Lee, H. S., Kim, H. & Hong, S. Dynamic roles of angiopoietin-like proteins 1, 2, 3, 4, 6 and 7 in the survival and enhancement of ex vivo expansion of bone-marrow hematopoietic stem cells. Protein Cell. 4, 220–230 (2013).

[69] WilsonR, et al. . Changes in the chondrocyte and extracellular matrix proteome during post-natal mouse cartilage development. Mol. Cell Proteom., 2012, 11: 1-18.

[70] LinX, ChangW, WangY, TianM, YuZ. SRPX2, an independent prognostic marker, promotes cell migration and invasion in hepatocellular carcinoma. Biomed. Pharmacother., 2017, 93: 398-405.

[71] Duchamp De LagenesteO, et al. . Periosteum contains skeletal stem cells with high bone regenerative potential controlled by Periostin. Nat. Commun., 2018, 9: 1-15.

[72] HodaxJK, et al. . Aggrecan is required for chondrocyte differentiation in ATDC5 chondroprogenitor cells. PLoS One, 2019, 14: 1-14.

[73] BessueilleL, et al. . Tissue-nonspecific alkaline phosphatase is an anti-inflammatory nucleotidase. Bone, 2020, 133. 115262

[74] GenetosDC, WongA, WeberTJ, KarinNJ, YellowleyCE. Impaired osteoblast differentiation in annexin A2- and -A5-deficient cells. PLoS One, 2014, 9: 107482.

[75] ZaykovV, ChaqourB. The CCN2/CTGF interactome: an approach to understanding the versatility of CCN2/CTGF molecular activities. J. Cell Commun. Signal., 2021, 15: 567-580.

[76] HashiguchiS, TanakaT, ManoR, KondoS, KodamaS. CCN2-induced lymphangiogenesis is mediated by the integrin αvβ5–ERK pathway and regulated by DUSP6. Sci. Rep., 2022, 12: 1-14.

[77] SiJ, et al. . Osteopontin in bone metabolism and bone diseases. Med. Sci. Mon., 2020, 26919159

[78] KobayashiT, et al. . Impaired fracture healing in macrophage migration inhibitory factor-deficient mice. Osteoporos. Int., 2011, 22: 1955-1965.

[79] You, Y., Yuan, H., Min, H., Li, C. & Chen, J. Fibroblast-derived CXCL14 aggravates crystalline silica-induced pulmonary fibrosis by mediating polarization and recruitment of interstitial macrophages. J. Hazard Mater.460, 132489 (2023).

[80] Eldridge, S. E. et al. Agrin induces long-term osteochondral regeneration by supporting repair morphogenesis. Sci. Transl. Med.12, eaax9086 (2020).

[81] DetelaG, LodgeA. EU Regulatory Pathways for ATMPs: standard, accelerated and adaptive pathways to marketing authorisation. Mol. Ther. Methods Clin. Dev., 2019, 13: 205-232.

[82] Salazar-Fontana, L. I. A regulatory risk-based approach to ATMP/CGT development: integrating scientific challenges with current regulatory expectations. Front. Med.9, 855100 (2022).

[83] Silva, D. N. et al. ATMP development and pre-GMP environment in academia: a safety net for early cell and gene therapy development and manufacturing. Immuno-Oncol. Technol.16, 100099 (2022).

[84] HeathmanTRJ, et al. . Characterization of human mesenchymal stem cells from multiple donors and the implications for large scale bioprocess development. Biochem. Eng. J., 2016, 108: 14-23.

[85] KriegerJ, NießingB, KönigN, SchmittRH. High-speed-microscopy for scalable quality control in automated production of stem cell spheroids for tissue engineering. Procedia CIRP, 2023, 120: 303-307.

[86] Zhu, J., Xiong, J. & Ji, W. A systematic review of bone marrow stromal cells and periosteum-derived cells for bone regeneration. Tissue Eng. Part B0, 103-122 (2022).

[87] PitaccoP, SadowskaJM, O’BrienFJ, KellyDJ. 3D bioprinting of cartilaginous templates for large bone defect healing. Acta Biomater., 2023, 156: 61-74.

[88] OsingaR, et al. . Generation of a bone organ by human adipose-derived stromal cells through endochondral ossification. Stem Cells Transl. Med., 2016, 5: 1090-1097.

[89] StortiG, et al. . Adipose-derived stem cells in bone tissue engineering: useful tools with new applications. Stem Cells Int., 2019, 2019. 3673857

[90] KasamkattilJ, et al. . Human 3D nucleus pulposus microtissue model to evaluate the potential of pre-conditioned nasal chondrocytes for the repair of degenerated intervertebral disc. Front. Bioeng. Biotechnol., 2023, 11. 1119009

[91] Kim, S. H. L. et al. Ectopic transient overexpression of OCT-4 facilitates BMP4-induced osteogenic transdifferentiation of human umbilical vein endothelial cells. J. Tissue Eng.11, 2041731420909208 (2020).

[92] ArakuraM, et al. . Endochondral bone tissue engineering using human induced pluripotent stem cells. Tissue Eng. Part A, 2022, 28: 184-195.

[93] Kim, J.-K. et al. Enhancement of cartilage regeneration of synovial stem cells/hydrogel by using transglutaminase-4. Tissue Eng. Part A.27, 1–26 (2020).

[94] ChungJE, et al. . Cultured human periosteum-derived cells can differentiate into osteoblasts in a perioxisome proliferator-activated receptor gamma-mediated fashion via bone morphogenetic protein signaling. Int. J. Med Sci., 2016, 13: 806-818.

[95] Pranskunas, M. et al. Assessment of the bone healing process mediated by periosteum-derived mesenchymal stem cells’ secretome and a xenogenic bioceramic-an in vivo study in the rabbit critical size calvarial defect model. Materials14, 3512 (2021).

[96] PerrinS, ColnotC. Periosteal skeletal stem and progenitor cells in bone regeneration. Curr. Osteoporos. Rep., 2022, 20: 334-343.

[97] TenreiroMF, et al. . Advancing organoid design through co-emergence, assembly, and bioengineering. Trends Biotechnol., 2023, 41: 923-938.

[98] MarcucioRS, MiclauT, BahneyCS. A shifting paradigm: transformation of cartilage to bone during bone repair. J. Dent. Res., 2023, 102: 13.

[99] LiuYL, TangXT, ShuHS, ZouW, ZhouBO. Fibrous periosteum repairs bone fracture and maintains the healed bone throughout mouse adulthood. Dev. Cell, 2024, 59: 1-18.

[100] PerrinS, et al. . Single nuclei transcriptomics reveal the differentiation trajectories of periosteal skeletal/stem progenitor cells in bone regeneration. Elife, 2024, 13. RP92519

[101] El KhassawnaT, et al. . T lymphocytes influence the mineralization process of bone. Front. Immunol., 2017, 8: 256924.

[102] EmmanuelleNE, et al. . Critical role of estrogens on bone homeostasis in both male and female: from physiology to medical implications. Int. J. Mol. Sci., 2021, 22: 1-18

[103] LiC, ZhengZ. Males and females have distinct molecular events in the articular cartilage during knee osteoarthritis. Int. J. Mol. Sci., 2021, 22: 7876.

[104] Haffner-LuntzerM, FischerV, IgnatiusA. Differences in fracture healing between female and male C57BL/6J mice. Front. Physiol., 2021, 0: 1227

[105] NiemannT, et al. . Analyzing sex-specific dimorphism in human skeletal stem cells. Cells, 2023, 12: 2683.

[106] AndrewTW, et al. . Sexually dimorphic estrogen sensing in skeletal stem cells controls skeletal regeneration. Nat. Commun., 2022, 13: 1-13.

[107] XuJ, et al. . PDGFRα reporter activity identifies periosteal progenitor cells critical for bone formation and fracture repair. Bone Res., 2022, 10: 1-15.

[108] TrompetD, MelisS, ChaginAS, MaesC. Skeletal stem and progenitor cells in bone development and repair. J. Bone Miner. Res., 2024, 39: 633-654.

[109] LanKC, et al. . Targeted activation of androgen receptor signaling in the periosteum improves bone fracture repair. Cell Death Dis., 2022, 13: 1-13.

[110] KimHK, et al. . A subset of paracrine factors as efficient biomarkers for predicting vascular regenerative efficacy of mesenchymal stromal/stem cells. Stem Cells, 2019, 37: 77-88.

[111] Zhou, K. et al. A “best-in-class” systemic biomarker predictor of clinically relevant knee osteoarthritis structural and pain progression. Sci. Adv.9, eabq5095 (2023).

[112] LiangW, et al. . An integrated multi-omics analysis reveals osteokines involved in global regulation. Cell Metab., 2024, 36: 1-20.

[113] KhangM, et al. . Manufacturing innovation to drive down cell therapy costs. Trends Biotechnol., 2023, 41: 1216-1219.

[114] SharmaA, et al. . Sexing bones: improving transparency of sex reporting to address bias within preclinical studies. J. Bone Miner. Res., 2023, 38: 5-13.

[115] MielkeMM, MillerVM. Improving clinical outcomes through attention to sex and hormones in research. Nat. Rev. Endocrinol., 2021, 17: 625-635.

[116] IwobiN, SparksNR. Endocrine disruptor-induced bone damage due to hormone dysregulation: a review. Int. J. Mol. Sci., 2023, 24: 8263.

[117] Cases-PereraO, et al. . Development of secretome-based strategies to improve cell culture protocols in tissue engineering. Sci. Rep., 2022, 12: 1-14.

[118] EyckmansJ, RobertsSJ, SchrootenJ, LuytenFP. A clinically relevant model of osteoinduction: a process requiring calcium phosphate and BMP/Wnt signalling. J. Cell Mol. Med, 2010, 14: 1845-1856.

[119] Babraham Bioinformatics - FastQC A Quality Control tool for High Throughput Sequence Data. https://www.bioinformatics.babraham.ac.uk/projects/fastqc/.

[120] BolgerAM, LohseM, UsadelB. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics, 2014, 30: 2114-2120.

[121] DobinA, et al. . STAR: ultrafast universal RNA-seq aligner. Bioinformatics, 2013, 29: 15-21.

[122] LiaoY, SmythGK, ShiW. The Subread aligner: fast, accurate and scalable read mapping by seed-and-vote. Nucleic Acids Res, 2013, 41: e108-e108.

[123] Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol.15, 550 (2014).

[124] LandiniG, MartinelliG, PiccininiF. Colour deconvolution: stain unmixing in histological imaging. Bioinformatics, 2021, 37: 1485-1487.

[125] BergS, et al. . Ilastik: interactive machine learning for (bio)image analysis. Nat. Methods, 2019, 16: 1226-1232.

[126] ShevchenkoA, WilmM, VormO, MannM. Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal. Chem., 1996, 68: 850-858.

Funding

Hercules Foundation(AKUL/13/47)

EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)(874837)

Fonds Wetenschappelijk Onderzoek (Research Foundation Flanders)(G.0929.15)

Regenerative Medicine Crossing Borders (REGMED XB)

RIGHTS & PERMISSIONS

The Author(s)