Single-cell RNA sequencing in orthopedic research

Abstract

Although previous RNA sequencing methods have been widely used in orthopedic research and have provided ideas for therapeutic strategies, the specific mechanisms of some orthopedic disorders, including osteoarthritis, lumbar disc herniation, rheumatoid arthritis, fractures, tendon injuries, spinal cord injury, heterotopic ossification, and osteosarcoma, require further elucidation. The emergence of the single-cell RNA sequencing (scRNA-seq) technique has introduced a new era of research on these topics, as this method provides information regarding cellular heterogeneity, new cell subtypes, functions of novel subclusters, potential molecular mechanisms, cell-fate transitions, and cell‒cell interactions that are involved in the development of orthopedic diseases. Here, we summarize the cell subpopulations, genes, and underlying mechanisms involved in the development of orthopedic diseases identified by scRNA-seq, improving our understanding of the pathology of these diseases and providing new insights into therapeutic approaches.

References

[1] Johnson VL, Hunter DJ. The epidemiology of osteoarthritis. Best. Pr. Res. Clin. Rheumatol., 2014, 28: 5-15

[2] Konstantinou K, Dunn KM. Sciatica: review of epidemiological studies and prevalence estimates. Spine, 2008, 33: 2464-2472

[3] Myasoedova E, Crowson CS, Kremers HM, Therneau TM, Gabriel SE. Is the incidence of rheumatoid arthritis rising?: results from Olmsted County, Minnesota, 1955-2007. Arthritis Rheum., 2010, 62: 1576-1582

[4] Chen W et al. National incidence of traumatic fractures in China: a retrospective survey of 512 187 individuals. Lancet Glob. Health, 2017, 5: e807-e817

[5] Bavin EP et al. Equine induced pluripotent stem cells have a reduced tendon differentiation capacity compared to embryonic stem cells. Front. Vet. Sci., 2015, 2: 55

[6] Cripps RA et al. A global map for traumatic spinal cord injury epidemiology: towards a living data repository for injury prevention. Spinal Cord., 2011, 49: 493-501

[7] Dey D et al. The traumatic bone: trauma-induced heterotopic ossification. Transl. Res., 2017, 186: 95-111

[8] Cho WH et al. Differential presentations, clinical courses, and survivals of osteosarcomas of the proximal humerus over other extremity locations. Ann. Surg. Oncol., 2010, 17: 702-708

[9] Hrdlickova, R., Toloue, M.&Tian, B. RNA-Seq methods for transcriptome analysis. Wiley Interdiscip. Rev. RNA. 8, https://doi.org/10.1002/wrna.1364 (2107).

[10] Wilhelm BT et al. Dynamic repertoire of a eukaryotic transcriptome surveyed at single-nucleotide resolution. Nature, 2008, 453: 1239-1243

[11] Olsen TK, Baryawno N. Introduction to Single-Cell RNA Sequencing. Curr. Protoc. Mol. Biol., 2018, 122: e57

[12] Hedlund E, Deng Q. Single-cell RNA sequencing: Technical advancements and biological applications. Mol. Asp. Med., 2018, 59: 36-46

[13] Shapiro E, Biezuner T, Linnarsson S. Single-cell sequencing-based technologies will revolutionize whole-organism science. Nat. Rev. Genet., 2013, 14: 618-630

[14] Saliba AE, Westermann AJ, Gorski SA, Vogel J. Single-cell RNA-seq: advances and future challenges. Nucleic Acids Res., 2014, 42: 8845-8860

[15] Suvà ML, Tirosh I. Single-Cell RNA Sequencing in Cancer: Lessons Learned and Emerging Challenges. Mol. Cell, 2019, 75: 7-12

[16] Rao DA, Arazi A, Wofsy D, Diamond B. Design and application of single-cell RNA sequencing to study kidney immune cells in lupus nephritis. Nat. Rev. Nephrol., 2020, 16: 238-250

[17] Yamada S, Nomura S. Review of single-cell rNA sequencing in the heart. Int. J. Mol. Sci., 2020, 21: 8345

[18] Cuevas-Diaz Duran R, Wei H, Wu JQ. Single-cell RNA-sequencing of the brain. Clin. Transl. Med., 2017, 6

[19] Edwards JJ et al. Quality indicators for the primary care of osteoarthritis: a systematic review. Ann. Rheum. Dis., 2015, 74: 490-498

[20] Jin X et al. Circulating C reactive protein in osteoarthritis: a systematic review and meta-analysis. Ann. Rheum. Dis., 2015, 74: 703-710

[21] Jiang Y, Tuan RS. Origin and function of cartilage stem/progenitor cells in osteoarthritis. Nat. Rev. Rheumatol., 2015, 11: 206-212

[22] Decker RS, Koyama E, Pacifici M. Genesis and morphogenesis of limb synovial joints and articular cartilage. Matrix Biol., 2014, 39: 5-10

[23] Jeon OH et al. Local clearance of senescent cells attenuates the development of post-traumatic osteoarthritis and creates a pro-regenerative environment. Nat. Med., 2017, 23: 775-781

[24] Childs BG et al. Senescent cells: an emerging target for diseases of ageing. Nat. Rev. Drug Discov., 2017, 16: 718-735

[25] Ji Q et al. Single-cell RNA-seq analysis reveals the progression of human osteoarthritis. Ann. Rheum. Dis., 2019, 1: 100-110

[26] Chou CH et al. Synovial cell cross-talk with cartilage plays a major role in the pathogenesis of osteoarthritis. Sci. Rep., 2020, 10

[27] Sebastian A et al. Single-cell RNA-seq reveals transcriptomic heterogeneity and post-traumatic osteoarthritis-associated early molecular changes in mouse articular chondrocytes. Cells, 2021, 10: 1462

[28] Lv Z et al. Single cell RNA-seq analysis identifies ferroptotic chondrocyte cluster and reveals TRPV1 as an anti-ferroptotic target in osteoarthritis. EBioMedicine, 2022, 84: 104258

[29] Nanus DE et al. Synovial tissue from sites of joint pain in knee osteoarthritis patients exhibits a differential phenotype with distinct fibroblast subsets. EBioMedicine, 2021, 72: 103618

[30] Liu W et al. Single-Cell Profiles of Age-Related Osteoarthritis Uncover Underlying Heterogeneity Associated with Disease Progression. Front. Mol. Biosci., 2022, 8: 748360

[31] Sebastian A et al. Single-cell RNA-seq reveals changes in immune landscape in post-traumatic osteoarthritis. Front. Immunol., 2022, 13: 938075

[32] Huang ZY et al. Single cell transcriptomics in human osteoarthritis synovium and in silico deconvoluted bulk RNA sequencing. Osteoarthr. Cartil., 2022, 30: 475-480

[33] Bian Q et al. A single cell transcriptional atlas of early synovial joint development. Development, 2020, 147: dev185777

[34] Rickers KW, Pedersen PH, Tvedebrink T, Eiskjær SP. Comparison of interventions for lumbar disc herniation: a systematic review with network meta-analysis. Spine J., 2021, 21: 1750-1762

[35] Dowdell J et al. Intervertebral disk degeneration and repair. Neurosurgery, 2017, 80: s46-s54

[36] Feng Y, Egan B, Wang J. Genetic factors in intervertebral disc degeneration. Genes Dis., 2016, 3: 178-185

[37] Riester SM et al. RNA sequencing identifes gene regulatory networks controlling extracellular matrix synthesis in intervertebral disk tissues. J. Orthop. Res., 2018, 36: 1356-1369

[38] Hunter CJ, Matyas JR, Duncan NA. Cytomorphology of notochordal and chondrocytic cells from the nucleus pulposus: a species comparison. J. Anat., 2004, 205: 357-362

[39] Bagnall KM, Higgins SJ, Sanders EJ. The contribution made by cells from a single somite to tissues within a body segment and assessment of their integration with similar cells from adjacent segments. Development, 1989, 107: 931-943

[40] Cherif H et al. Single-Cell RNA-seq analysis of cells from degenerating and non-degenerating intervertebral discs from the same individual reveals new biomarkers for intervertebral disc degeneration. Int. J. Mol. Sci., 2022, 23: 3993

[41] Wang J et al. Novel biomarkers of intervertebral disc cells and evidence of stem cells in the intervertebral disc. Osteoarthr. Cartil., 2021, 29: 389-401

[42] Panebianco CJ, Dave A, Charytonowicz D, Sebra R, Iatridis JC. Single-cell RNA-sequencing atlas of bovine caudal intervertebral discs: Discovery of heterogeneous cell populations with distinct roles in homeostasis. FASEB J., 2021, 35: e21919

[43] Calió M, Gantenbein B, Egli M, Poveda L, Ille F. The cellular composition of bovine coccygeal intervertebral discs: a comprehensive single-cell RNAseq analysis. Int. J. Mol. Sci., 2021, 22: 4917

[44] Gao B et al. Discovery and application of postnatal nucleus pulposus progenitors essential for intervertebral disc homeostasis and degeneration. Adv. Sci., 2022, 9: e2104888

[45] Ling Z et al. Single-cell RNA-seq analysis reveals macrophage involved in the progression of human intervertebral disc degeneration. Front. Cell Dev. Biol., 2022, 9: 833420

[46] Tu J et al. Single-cell transcriptome profiling reveals multicellular ecosystem of nucleus pulposus during degeneration progression. Adv. Sci., 2022, 9: e2103631

[47] Sakai D, Andersson GB. Stem cell therapy for intervertebral disc regeneration: obstacles and solutions. Nat. Rev. Rheumatol., 2015, 11: 243-256

[48] Lyu FJ et al. IVD progenitor cells: a new horizon for understanding disc homeostasis and repair. Nat. Rev. Rheumatol., 2019, 15: 102-112

[49] Gan Y et al. Spatially defined single-cell transcriptional profiling characterizes diverse chondrocyte subtypes and nucleus pulposus progenitors in human intervertebral discs. Bone Res., 2021, 9: 37

[50] Sakai D et al. Exhaustion of nucleus pulposus progenitor cells with ageing and degeneration of the intervertebral disc. Nat. Commun., 2012, 3

[51] Mizrahi O et al. Nucleus pulposus degeneration alters properties of resident progenitor cells. Spine J., 2013, 13: 803-814

[52] Zhang Y et al. Single-cell RNA-seq analysis identifies unique chondrocyte subsets and reveals involvement of ferroptosis in human intervertebral disc degeneration. Osteoarthr. Cartil., 2021, 29: 1324-1334

[53] Han S et al. Single-cell RNA sequencing of the nucleus pulposus reveals chondrocyte differentiation and regulation in intervertebral disc degeneration. Front. Cell Dev. Biol., 2022, 10: 824771

[54] Fernandes LM et al. Single-cell RNA-seq identifies unique transcriptional landscapes of human nucleus pulposus and annulus fibrosus cells. Sci. Rep., 2020, 10

[55] Ellman MB et al. Fibroblast growth factor control of cartilage homeostasis. J. Cell. Biochem., 2013, 114: 735-742

[56] Brown EA et al. FGF4 retrogene on CFA12 is responsible for chondrodystrophy and intervertebral disc disease in dogs. Proc. Natl. Acad. Sci. USA, 2017, 114: 11476-11481

[57] Zhang J et al. TGF-β1 suppresses CCL3/4 expression through the ERK signaling pathway and inhibits intervertebral disc degeneration and inflammation-related pain in a rat model. Exp. Mol. Med., 2017, 49

[58] Chen S et al. TGF-β signaling in intervertebral disc health and disease. Osteoarthr. Cartil., 2019, 27: 1109-1117

[59] Hiyama A et al. The relationship between the Wnt/β-catenin and TGF-β/BMP signals in the intervertebral disc cell. J. Cell. Physiol., 2011, 226: 1139-1148

[60] Wang Z, Weitzmann MN, Sangadala S, Hutton WC, Yoon ST. Link protein N-terminal peptide binds to bone morphogenetic protein (BMP) type II receptor and drives matrix protein expression in rabbit intervertebral disc cells. J. Biol. Chem., 2013, 288: 28243-28253

[61] Paglia DN, Singh H, Karukonda T, Drissi H, Moss IL. PDGF-BB delays degeneration of the intervertebral discs in a rabbit preclinical model. Spine, 2016, 41: E449-E458

[62] McInnes IB, Schett G. The pathogenesis of rheumatoid arthritis. N. Engl. J. Med., 2011, 365: 2205-2219

[63] Rana AK, Li Y, Dang Q, Yang F. Monocytes in rheumatoid arthritis: Circulating precursors of macrophages and osteoclasts and, their heterogeneity and plasticity role in RA pathogenesis. Int. Immunopharmacol., 2018, 65: 348-359

[64] Guo Q et al. Rheumatoid arthritis: pathological mechanisms and modern pharmacologic therapies. Bone Res., 2018, 6: 15

[65] Aletaha D, Smolen JS. Diagnosis and management of rheumatoid arthritis: a review. JAMA, 2018, 320: 1360-1372

[66] Ten, Brinck RM et al. The risk of individual autoantibodies, autoantibody combinations and levels for arthritis development in clinically suspect arthralgia. Rheumatology, 2017, 56: 2145-2153

[67] Verheul MK et al. Mass-spectrometric identification of carbamylated proteins present in the joints of rheumatoid arthritis patients and controls. Clin. Exp. Rheumatol., 2021, 39: 570-577

[68] Sahlström P et al. Different hierarchies of anti-modified protein autoantibody reactivities in rheumatoid arthritis. Arthritis Rheumatol., 2020, 72: 1643-1657

[69] Bader L et al. Candidate markers for stratification and classification in rheumatoid arthritis. Front. Immunol., 2019, 10: 1488

[70] Saevarsdottir, S. et al. Multiomics analysis of rheumatoid arthritis yields sequence variants that have large effects on risk of the seropositive subset. Ann. Rheum. Dis. 81, 1085–1095 (2022).

[71] Zhang F et al. Defining inflammatory cell states in rheumatoid arthritis joint synovial tissues by integrating single-cell transcriptomics and mass cytometry. Nat. Immunol., 2019, 20: 928-942

[72] Liao L, Liang K, Lan L, Wang J, Guo J. Marker genes change of synovial fibroblasts in rheumatoid arthritis patients. Biomed. Res. Int., 2021, 2021: 5544264

[73] Culemann S et al. Locally renewing resident synovial macrophages provide a protective barrier for the joint. Nature, 2019, 572: 670-675

[74] Kuo D et al. HBEGF+ macrophages in rheumatoid arthritis induce fibroblast invasiveness. Sci. Transl. Med., 2019, 11: eaau8587

[75] Alivernini S et al. Distinct synovial tissue macrophage subsets regulate inflammation and remission in rheumatoid arthritis. Nat. Med., 2020, 26: 1295-1306

[76] Stephenson W et al. Single-cell RNA-seq of rheumatoid arthritis synovial tissue using low-cost microflfluidic instrumentation. Nat. Commun., 2018, 9

[77] Mizoguchi F et al. Functionally distinct disease-associated fibroblast subsets in rheumatoid arthritis. Nat. Commun., 2018, 9

[78] Croft AP et al. Distinct fibroblast subsets drive inflammation and damage in arthritis. Nature, 2019, 570: 246-251

[79] Wei K et al. Notch signaling drives synovial fibroblast identity and arthritis pathology. Nature, 2020, 582: 259-264

[80] Kelkka T et al. Adult-onset anti-citrullinated peptide antibody-negative destructive rheumatoid arthritis is characterized by a disease-specific CD8+ T lymphocyte signature. Front. Immunol., 2020, 11: 578848

[81] Rao DA et al. Pathologically expanded peripheral T helper cell subset drives B cells in rheumatoid arthritis. Nature, 2017, 542: 110-114

[82] Fonseka CY et al. Mixed-effects association of single cells identifies an expanded effector CD4+ T cell subset in rheumatoid arthritis. Sci. Transl. Med., 2018, 10: eaaq0305

[83] Han L et al. A comprehensive transcriptomic analysis of alternate interferon signaling pathways in peripheral blood mononuclear cells in rheumatoid arthritis. Aging, 2021, 13: 20511-20533

[84] Andreev D et al. Regulatory eosinophils induce the resolution of experimental arthritis and appear in remission state of human rheumatoid arthritis. Ann. Rheum. Dis., 2020, 2020: 218902

[85] Meednu N et al. Dynamic spectrum of ectopic lymphoid B cell activation and hypermutation in the RA synovium characterized by NR4A nuclear receptor expression. Cell Rep., 2022, 39: 110766

[86] Wu X et al. Single-cell sequencing of immune cells from anticitrullinated peptide antibody positive and negative rheumatoid arthritis. Nat. Commun., 2021, 12

[87] Cai S et al. Similar transition processes in synovial fibroblasts from rheumatoid arthritis and osteoarthritis: a single-cell study. J. Immunol. Res., 2019, 2019: 4080735

[88] Einhorn TA, Gerstenfeld LC. Fracture healing: mechanisms and interventions. Nat. Rev. Rheumatol., 2015, 11: 45-54

[89] Burge R et al. Incidence and economic burden of osteoporosis-related fractures in the United States, 2005–25. J. Bone Miner. Res., 2007, 22: 465-475

[90] Mills LA, Aitken SA, Simpson A. The risk of non-union per fracture: current myths and revised figures from a population of over 4 million adults. Acta Orthop., 2017, 88: 434-439

[91] Gruber R et al. Fracture healing in the elderly patient. Exp. Gerontol., 2006, 41: 1080-1093

[92] Xu R et al. Targeting skeletal endothelium to ameliorate bone loss. Nat. Med., 2018, 24: 823-833

[93] Wang R et al. miR-143 promotes angiogenesis and osteoblast differentiation by targeting HDAC7. Cell Death Dis., 2020, 11

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

[95] Hu DP 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: 221-234

[96] Baccin C et al. Combined single-cell and spatial transcriptomics reveal the molecular, cellular and spatial bone marrow niche organization. Nat. Cell Biol., 2020, 22: 38-48

[97] Baryawno N et al. A Cellular taxonomy of the bone marrow stroma in homeostasis and leukemia. Cell, 2019, 177: 1915-.e16

[98] Sivaraj KK et al. Regional specialization and fate specification of bone stromal cells in skeletal development. Cell Rep., 2021, 36: 109352

[99] Abou-Khalil R et al. Role of muscle stem cells during skeletal regeneration. Stem Cells, 2015, 33: 1501-1511

[100] Glass GE et al. TNF-α promotes fracture repair by augmenting the recruitment and differentiation of muscle-derived stromal cells. Proc. Natl. Acad. Sci. USA., 2011, 108: 1585-1590

[101] Harry LE et al. Comparison of the healing of open tibial fractures covered with either muscle or fasciocutaneous tissue in a murine model. J. Orthop. Res., 2008, 26: 1238-1244

[102] Liu R et al. Myogenic progenitors contribute to open but not closed fracture repair. BMC Musculoskelet. Disord., 2011, 12

[103] Byrd HS, Cierny G, Tebbetts JB. The management of open tibial fractures with associated soft–tissue loss: external pin fifixation with early flap coverage. Plast. Reconstr. Surg., 1981, 68: 73-79

[104] Richards RR, McKee MD, Paitich CB, Anderson GI, Bertoia JT. A comparison of the effects of skin coverage and muscle flap coverage on the early strength of union at the site of osteotomy after devascularization of a segment of canine tibia. J. Bone Jt. Surg. -Ser. A, 1991, 73: 1323-1330

[105] Willett K, Al-Khateeb H, Kotnis R, Bouamra O, Lecky F. Risk of mortality: the relationship with associated injuries and fracture treatment methods in patients with unilateral or bilateral femoral shaft fractures. J. Trauma - Inj. Infect. Crit. Care, 2010, 69: 405-410

[106] Chan JKK, Harry L, Williams G, Nanchahal J. Soft-tissue reconstruction of open fractures of the lower limb: muscle versus fasciocutaneous flaps. Plast. Reconstr. Surg., 2012, 130: 284e-295e

[107] Julien A et al. Direct contribution of skeletal muscle mesenchymal progenitors to bone repair. Nat. Commun., 2021, 12

[108] Al-Sebaei MO et al. Role of Fas and Treg cells in fracture healing as characterized in the fas-deficient (lpr) mouse model of lupus. J. Bone Min. Res., 2014, 29: 1478-1491

[109] Bragdon B et al. Earliest phases of chondrogenesis are dependent upon angiogenesis during ectopic bone formation in mice. Bone, 2017, 101: 49-61

[110] Zhang H et al. Single-cell RNA sequencing reveals B Cells are important regulators in fracture healing. Front. Endocrinol., 2021, 8: 666140

[111] Avin, K. G. et al. Single-cell RNAseq provides insight into altered immune cell populations in human fracture nonunions. J. Orthop. Res. (2022). https://doi.org/10.1002/jor.25452. Online ahead of print.

[112] Gumucio JP, Sugg KB, Mendias CL. TGF-β superfamily signaling in muscle and tendon adaptation to resistance exercise. Exerc. Sport Sci. Rev., 2015, 43: 93-99

[113] Kjaer M et al. Extracellular matrix adaptation of tendon and skeletal muscle to exercise. J. Anat., 2006, 208: 445-450

[114] Magnusson SP, Kjaer M. The impact of loading, unloading, ageing and injury on the human tendon. J. Physiol., 2019, 597: 1283-1298

[115] Mendias CL, Gumucio JP, Bakhurin KI, Lynch EB, Brooks SV. Physiological loading of tendons induces scleraxis expression in epitenon fibroblasts. J. Orthop. Res., 2012, 30: 606-612

[116] Paolillo C, Londin E, Fortina P. Single-cell genomics. Clin. Chem., 2019, 65: 972-985

[117] De Micheli AJ et al. Single-cell transcriptomic analysis identifies extensive heterogeneity in the cellular composition of mouse Achilles tendons. Am. J. Physiol. Cell Physiol., 2020, 319: C885-C894

[118] Still C et al. Single-cell transcriptomic profiling reveals distinct mechanical responses between normal and diseased tendon progenitor cells. Cell Rep. Med., 2021, 2: 100343

[119] Fan C et al. A Cd9+Cd271+ stem/progenitor population and the SHP2 pathway contribute to neonatal-to-adult switching that regulates tendon maturation. Cell Rep., 2022, 39: 110762

[120] Nakajima T et al. Grafting of iPS cell-derived tenocytes promotes motor function recovery after Achilles tendon rupture. Nat. Commun., 2021, 12

[121] Yoshimoto Y et al. Tenogenic induction from induced pluripotent stem cells unveils the trajectory towards tenocyte differentiation. Front. Cell Dev. Biol., 2022, 10: 780038

[122] Kaji DA, Montero AM, Patel R, Huang AH. Transcriptional profiling of mESC-derived tendon and fibrocartilage cell fate switch. Nat. Commun., 2021, 12

[123] Kult S et al. Bi-fated tendon-to-bone attachment cells are regulated by shared enhancers and KLF transcription factors. Elife, 2021, 10: e55361

[124] Garcia-Melchor E et al. Novel self-amplificatory loop between T cells and tenocytes as a driver of chronicity in tendon disease. Ann. Rheum. Dis., 2021, 80: 1075-1085

[125] Jiang Y et al. Enhanced tenogenic differentiation and tendon-like tissue formation by tenomodulin overexpression in murine mesenchymal stem cells. J. Tissue Eng. Regen. Med., 2017, 11: 2525-2536

[126] Jo CH, Lim H-J, Yoon KS. Characterization of tendon-specific markers in various human tissues, tenocytes and mesenchymal stem cells. Tissue Eng. Regen. Med., 2019, 16: 151-159

[127] Tran AP, Warren PM, Silver J. The biology of regeneration failure and success after spinal cord injury. Physiol. Rev., 2018, 98: 881-917

[128] Guimarães-Camboa N et al. Pericytes of multiple organs do not behave as mesenchymal stem cells in vivo. Cell Stem Cell, 2017, 20: 345-359

[129] Riew TR, Jin X, Kim S, Kim HL, Lee MY. Temporal dynamics of cells expressing NG2 and platelet-derived growth factor receptor-β in the fibrotic scar formation after 3-nitropropionic acid-induced acute brain injury. Cell Tissue Res., 2021, 385: 539-555

[130] Li Y et al. Microglia organized scar-free spinal cord repair in neonatal mice. Nature, 2020, 587: 613-618

[131] Ramotowski C, Qu X, Villa-Diaz LG. Progress in the use of induced pluripotent stem cell-derived neural cells for traumatic spinal cord injuries in animal populations: meta-analysis and review. Stem Cells Transl. Med., 2019, 8: 681-693

[132] Nori S et al. Grafted human-induced pluripotent stem-cell-derived neurospheres promote motor functional recovery after spinal cord injury in mice. Proc. Natl. Acad. Sci. USA, 2011, 108: 16825-16830

[133] Zengeler KE, Lukens JR. Innate immunity at the crossroads of healthy brain maturation and neurodevelopmental disorders. Nat. Rev. Immunol., 2021, 21: 454-468

[134] Wahane S et al. Diversified transcriptional responses of myeloid and glial cells in spinal cord injury shaped by HDAC3 activity. Sci. Adv., 2021, 7: eabd8811

[135] Wang J et al. Single-cell transcriptome analysis reveals the immune heterogeneity and the repopulation of microglia by Hif1α in mice after spinal cord injury. Cell Death Dis., 2022, 13

[136] Hakim R et al. Spinal cord injury induces permanent reprogramming of microglia into a disease-associated state which contributes to functional recovery. J. Neurosci., 2021, 41: 8441-8459

[137] Milich LM et al. Single-cell analysis of the cellular heterogeneity and interactions in the injured mouse spinal cord. J. Exp. Med., 2021, 218: e20210040

[138] Hugnot JP, Franzen R. The spinal cord ependymal region: a stem cell niche in the caudal central nervous system. Front. Biosci., 2011, 16: 1044-1059

[139] Shu M et al. Single-cell RNA sequencing reveals Nestin+ active neural stem cells outside the central canal after spinal cord injury. Sci. China Life Sci., 2022, 65: 295-308

[140] Stenudd M et al. Identification of a discrete subpopulation of spinal cord ependymal cells with neural stem cell properties. Cell Rep., 2022, 38: 110440

[141] Shimono K et al. Potent inhibition of heterotopic ossification by nuclear retinoic acid receptor-γ agonists. Nat. Med., 2011, 17: 454-460

[142] Regard JB et al. Activation of hedgehog signaling by loss of GNAS causes heterotopic ossification. Nat. Med., 2013, 19: 1505-1512

[143] Ranganathan K et al. Heterotopic ossification: basic-science principles and clinical correlates. J. Bone Jt. Surg., 2015, 97: 1101-1111

[144] Wang G, Kang Y, Chen F, Wang B. Cervical intervertebral disc calcification combined with ossification of posterior longitudinal ligament in an-11-year old girl: case report and review of literature. Childs Nerv. Syst., 2016, 32: 381-386

[145] Xu Y et al. Heterotopic ossification: clinical features, basic researches, and mechanical stimulations. Front. Cell Dev. Biol., 2022, 10: 770931

[146] C. Meyers J et al. Heterotopic ossification: A comprehensive review. JBMR ., 2019, 3: e10172

[147] Agarwal S et al. Scleraxis-lineage cells contribute to ectopic bone formation in muscle and tendon. Stem Cells, 2017, 35: 705-710

[148] Agarwal S et al. Inhibition of Hif1 prevents both trauma-induced and genetic heterotopic ossification. Proc. Natl. Acad. Sci. USA, 2015, 113: E338-E347

[149] Chen Y et al. Single-cell integration analysis of heterotopic ossification and fibrocartilage developmental lineage: endoplasmic reticulum stress effector Xbp1 transcriptionally regulates the notch signaling pathway to mediate fibrocartilage differentiation. Oxid. Med. Cell Longev., 2021, 2021: 7663366

[150] J.Sahlman R et al. Premature vertebral endplate ossification and mild disc degeneration in mice after inactivation of one allele belonging to the Col2a1 gene for type II collagen. Spine, 2001, 26: 2558-2565

[151] Mutsuzaki H, Nakajima H. Development of fibrocartilage layers in Achilles tendon enthesis in rabbits. J. Rural Med., 2021, 16: 160-164

[152] De Kretser DM, O’Hehir RE, Hardy CL, Hedger MP. The roles of activin A and its binding protein, follistatin, in inflammation and tissue repair. Mol. Cell. Endocrinol., 2012, 359: 101-106

[153] Xia Y, Schneyer AL. The biology of activin: Recent advances in structure, regulation and function. J. Endocrinol., 2009, 202: 1-12

[154] Hwang C et al. Activin A does not drive post-traumatic heterotopic ossification. Bone, 2020, 138: 115473

[155] Mundy, C. et al. Activin A promotes the development of acquired heterotopic ossification and is an effective target for disease attenuation in mice. Sci. Signal. 14 , eabd0536 (2021).

[156] Barruet E et al. Modeling the ACVR1R206H mutation in human skeletal muscle stem cells. Elife, 2021, 10: e66107

[157] Hsu GC et al. Endogenous CCN family member WISP1 inhibits trauma-induced heterotopic ossification. JCI Insight, 2020, 5: e135432

[158] Tan L et al. Discovery of type II inhibitors of TGFβ-activated kinase 1 (TAK1) and mitogen-activated protein kinase kinase kinase kinase 2 (MAP4K2). J. Med. Chem., 2015, 58: 183-196

[159] Kim SI et al. TGF-beta-activated kinase 1 and TAK1-binding protein 1 cooperate to mediate TGF-beta1-induced MKK3-p38 MAPK activation and stimulation of type I collagen. Am. J. Physiol. Ren. Physiol., 2007, 292: F1471-F1478

[160] Lee KS, Hong SH, Bae SC. Both the Smad and p38 MAPK pathways play a crucial role in Runx2 expression following induction by transforming growth factor-beta and bone morphogenetic protein. Oncogene, 2002, 21: 7156-7163

[161] Strong AL et al. Small molecule inhibition of non-canonical (TAK1-mediated) BMP signaling results in reduced chondrogenic ossification and heterotopic ossification in a rat model of blast-associated combat-related lower limb trauma. Bone, 2020, 139: 115517

[162] Lin J et al. Single cell analysis reveals inhibition of angiogenesis attenuates the progression of heterotopic ossification in Mkx-/- mice. Bone Res., 2022, 10: 4

[163] Qin Q et al. Neuron-to-vessel signaling is a required feature of aberrant stem cell commitment after soft tissue trauma. Bone Res., 2022, 10: 43

[164] Sorkin M et al. Regulation of heterotopic ossification by monocytes in a mouse model of aberrant wound healing. Nat. Commun., 2020, 11

[165] Mujtaba B et al. Heterotopic ossifification: radiological and pathological review. Radio. Oncol., 2019, 53: 275-284

[166] Shehab D, Elgazzar AH, Collier D. Heterotopic ossification. J. Nucl. Med., 2002, 43: 346-353

[167] Zagarella A, Impellizzeri E, Maiolino R, Attolini R, Castoldi MC. Pelvic heterotopic ossifification: when CT comes to the aid of MR imaging. Insights Imaging, 2013, 4: 595-603

[168] Edwards NJ et al. High frequency spectral ultrasound imaging detects early heterotopic ossification in rodents. Stem Cells Dev., 2021, 30: 473-484

[169] Pingping B et al. Incidence and mortality of sarcomas in Shanghai, China, during 2002–14. Front. Oncol., 2019, 9: 662

[170] Kyle RA et al. Monoclonal gammopathy of undetermined signifcance (MGUS) and smoldering (asymptomatic) multiple myeloma: IMWG consensus perspectives risk factors for progression and guidelines for monitoring and management. Leukemia, 2010, 24: 1121-1127

[171] Kansara M, Teng MW, Smyth MJ, Thomas DM. Translational biology of osteosarcoma. Nat. Rev. Cancer, 2014, 14: 722-735

[172] Zhou Y et al. Single-cell RNA landscape of intratumoral heterogeneity and immunosuppressive microenvironment in advanced osteosarcoma. Nat. Commun., 2020, 11

[173] Liu Y et al. Single-cell transcriptomics reveals the complexity of the tumor microenvironment of treatment-naive osteosarcoma. Front. Oncol., 2021, 11: 709210

[174] Akiyama T et al. Novel therapeutic strategy for osteosarcoma targeting osteoclast differentiation, bone-resorbing activity, and apoptosis pathway. Mol. Cancer Ther., 2008, 7: 3461-3469

[175] Pelon F et al. Cancer-associated fifibroblast heterogeneity in axillary lymphnodes drives metastases in breast cancer through complementary mechanisms. Nat. Commun., 2020, 11

[176] Smeland S et al. Survival and prognosis with osteosarcoma: outcomes in more than 2000 patients in the EURAMOS-1 (European and American Osteosarcoma Study) cohort. Eur. J. Cancer, 2019, 109: 36-50

[177] Chiu DK et al. Hepatocellular carcinoma cells up-regulate PVRL1, stabilizing PVR and inhibiting the cytotoxic T-cell response via TIGIT to mediate tumor resistance to PD1 inhibitors in mice. Gastroenterology, 2020, 159: 609-623

[178] Hoogi S et al. A TIGIT-based chimeric co-stimulatory switch receptor improves T-cell anti-tumor function. J. Immunother. Cancer, 2019, 7: 243

[179] Zhang Q et al. Blockade of the checkpoint receptor TIGIT prevents NK cell exhaustion and elicits potent anti-tumor immunity. Nat. Immunol., 2018, 19: 723-732

[180] Stamm H et al. Targeting the TIGIT-PVR immune checkpoint axis as novel therapeutic option in breast cancer. Oncoimmunology, 2019, 8: e1674605

[181] Qin Z et al. ATG16L1 is a potential prognostic biomarker and immune signature for osteosarcoma: a study based on bulk rna and single-cell RNA-sequencing. Int. J. Gen. Med., 2022, 15: 1033-1045

[182] Manoharan M et al. A computational approach identifies immunogenic features of prognosis in human cancers. Front. Immunol., 2018, 9: 3017

[183] Gharibi A et al. ITGA1 is a pre-malignant biomarker that promotes therapy resistance and metastatic potential in pancreatic cancer. Sci. Rep., 2017, 7

[184] Yim DH et al. ITGA1 polymorphisms and haplotypes are associated with gastric cancer risk in a Korean population. World J. Gastroenterol., 2013, 19: 5870-5876

[185] Li H et al. Integrin alpha1 promotes tumorigenicity and progressive capacity of colorectal cancer. Int. J. Biol. Sci., 2020, 16: 815-826

[186] Liu L et al. Novel genetic variants of SYK and ITGA1 related lymphangiogenesis signaling pathway predict non-small cell lung cancer survival. Am. J. Cancer Res., 2020, 10: 2603-2616

[187] Rao A, Barkley D, França GS, Yanai I. Exploring tissue architecture using spatial transcriptomics. Nature, 2021, 596: 211-220

[188] Rai MF et al. Single cell omics for musculoskeletal research. Curr. Osteoporos. Rep., 2021, 19: 131-140

[189] Sarmiento P, Little D. Tendon and multiomics: advantages, advances, and opportunities. NPJ Regen. Med., 2021, 6: 61

[190] Buenrostro JD, Wu B, Chang HY, Greenleaf WJ. ATAC-seq: a method for assaying chromatin accessibility genome-wide. Curr. Protoc. Mol. Biol., 2015, 109: 2191-2199

[191] Buenrostro JD, Giresi PG, Zaba LC, Chang HY, Greenleaf WJ. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleo some position. Nat. Methods, 2013, 10: 1213-1218

[192] Llorens-Bobadilla E et al. A latent lineage potential in resident neural stem cells enables spinal cord repair. Science, 2020, 370: 6512

[193] Ståhl P et al. Visualization and analysis of gene expression in tissue sections by spatial transcriptomics. Science, 2016, 353: 78-82

[194] Stephenson E, Webb S, Haniffa M. Multiomics uncovers developing immunological lineages in human. Eur. J. Immunol., 2021, 51: 764-772

[195] Ballantyne KN et al. Decreasing amplification bias associated with multiple displacement amplification and short tandem repeat genotyping. Anal. Biochem., 2007, 368: 222-229

[196] Buttner M, Miao Z, Wolf FA, Teichmann SA, Theis FJ. A test metric for assessing single-cell RNA-seq batch correction. Nat. Methods, 2019, 16: 43-49

[197] Hicks SC, Townes FW, Teng M, Irizarry RA. Missing data and technical variability in single-cell RNA-sequencing experiments. Biostatistics, 2018, 19: 562-578

[198] Shaham U et al. Removal of batch effects using distribution-matching residual networks. Bioinformatics, 2017, 33: 2539-2546