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Abstract: meniscus injuries are widespread and the available treatments do not offer enough healing potential. Here, we provide critical support for using pigs as a biological model for meniscal degeneration and the development of cutting-edge therapies in orthopedics. We present a single-cell transcriptome atlas of the meniscus, consisting of cell clusters corresponding to four major cell types: chondrocytes, endothelial cells, smooth muscle cells, and immune cells. Five distinct chondrocyte subclusters (CH0‒CH4) were annotated, of which only one was widespread in both the red and white zones, indicating a major difference in the cellular makeup of the zones. Subclusters distinct to the white zone appear responsible for cartilage-specific matrix deposition and protection against adverse microenvironmental factors, while those in the red zone exhibit characteristics of mesenchymal stem cells and are more likely to proliferate and migrate. Additionally, they induce remodeling actions in other chondrocyte subclusters and promote the proliferation and maturation of endothelial cells, inducing healing and vascularization processes. Considering that they have substantial remodeling capabilities, these subclusters should be of great interest for tissue engineering studies. We also show that the cellular makeup of the pig meniscus is comparable to that of humans, which supports the use of pigs as a model in orthopedic therapy development.

猪半月板单细胞测序揭示了参与干性维持和血管化的高活性红区软骨细胞群

[1]AkiyamaH, ChaboissierMC, MartinJF, et al., 2002. The transcription factor Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is required for expression of Sox5 and Sox6. Genes Dev, 16(21):2813-2828.

[2]AlfaroMP, PagniM, VincentA, et al., 2008. The Wnt modulator sFRP2 enhances mesenchymal stem cell engraftment, granulation tissue formation and myocardial repair. Proc Natl Acad Sci USA, 105(47):18366-18371.

[3]AliY, Abd HamidS, 2016. Human topoisomerase II alpha as a prognostic biomarker in cancer chemotherapy. Tumor Biol, 37:47-55.

[4]Andersson-MolinaH, KarlssonH, RockbornP, 2002. Arthroscopic partial and total meniscectomy: a long-term follow-up study with matched controls. Arthroscopy, 18(2):183-189.

[5]BarretoG, SenturkB, ColomboL, et al., 2020. Lumican is upregulated in osteoarthritis and contributes to TLR4-induced pro-inflammatory activation of cartilage degradation and macrophage polarization. Osteoarthritis Cartilage, 28(1):92-101.

[6]BeaufilsP, BeckerR, KopfS, et al., 2017. The knee meniscus: management of traumatic tears and degenerative lesions. EFORT Open Rev, 2(5):195-203.

[7]BraceyDN, SeylerTM, JinnahAH, et al., 2019. A porcine xenograft‐derived bone scaffold is a biocompatible bone graft substitute: an assessment of cytocompatibility and the alpha‐Gal epitope. Xenotransplantation, 26(5):e12534.

[8]CajanderS, TinaE, BäckmanA, et al., 2016. Quantitative real-time polymerase chain reaction measurement of HLA-DRA gene expression in whole blood is highly reproducible and shows changes that reflect dynamic shifts in monocyte surface HLA-DR expression during the course of sepsis. PLoS ONE, 11(5):e0154690.

[9]ChakravartiS, 2002. Functions of lumican and fibromodulin: lessons from knockout mice. Glycoconj J, 19:287-293.

[10]ChistiakovDA, KillingsworthMC, MyasoedovaVA, et al., 2017. CD68/macrosialin: not just a histochemical marker. Lab Invest, 97(1):4-13.

[11]ChristmanKL, FangQZ, KimAJ, et al., 2005. Pleiotrophin induces formation of functional neovasculature in vivo. Biochem Biophys Res Commun, 332(4):1146-1152.

[12]de VegaS, IwamotoT, YamadaY, 2009. Fibulins: multiple roles in matrix structures and tissue functions. Cell Mol Life Sci, 66:1890-1902.

[13]DingY, PanYH, LiuS, et al., 2017. Elevation of MiR-9-3p suppresses the epithelial-mesenchymal transition of nasopharyngeal carcinoma cells via down-regulating FN1, ITGB1 and ITGAV. Cancer Biol Ther, 18(6):414-424.

[14]DoralMN, BilgeO, HuriG, et al., 2018. Modern treatment of meniscal tears. EFORT Open Rev, 3(5):260-268.

[15]DrecourtA, BabdorJ, DussiotM, et al., 2018. Impaired transferrin receptor palmitoylation and recycling in neurodegeneration with brain iron accumulation. Am J Hum Genet, 102(2):266-277.

[16]DuprezJ, RomaLP, CloseAF, et al., 2012. Protective antioxidant and antiapoptotic effects of ZnCl₂ in rat pancreatic islets cultured in low and high glucose concentrations. PLoS ONE, 7(10):e46831.

[17]EyreDR, WeisMA, 2013. Bone collagen: new clues to its mineralization mechanism from recessive osteogenesis imperfecta. Calcif Tissue Int, 93(4):338-347.

[18]FangXY, ZhanYX, ZhouXM, et al., 2022. CXCL12/CXCR4 mediates orthodontic root resorption via regulating the M1/M2 ratio. J Dent Res, 101(5):569-579.

[19]FangYH, HuangH, ZhouG, et al., 2020. An animal model study on the gene expression profile of meniscal degeneration. Sci Rep, 10:21469.

[20]FisherMC, MeyerC, GarberG, et al., 2005. Role of IGFBP2, IGF-I and IGF-II in regulating long bone growth. Bone, 37(6):741-750.

[21]FuWL, ChenSJ, YangRZ, et al., 2022. Cellular features of localized microenvironments in human meniscal degeneration: a single-cell transcriptomic study. eLife, 11:e79585.

[22]FullerES, SmithMM, LittleCB, et al., 2012. Zonal differences in meniscus matrix turnover and cytokine response. Osteoarthritis Cartilage, 20(1):49-59.

[23]GautamR, ChintalaS, LiW, et al., 2004. The hermansky-pudlak syndrome 3 (Cocoa) protein is a component of the biogenesis of lysosome-related organelles complex-2 (BLOC-2). J Biol Chem, 279(13):12935-12942.

[24]GelseK, 2003. Collagens—structure, function, and biosynthesis. Adv Drug Deliv Rev, 55(12):1531-1546.

[25]GelseK, SöderS, EgerW, et al., 2003. Osteophyte development—molecular characterization of differentiation stages. Osteoarthritis Cartilage, 11(2):141-148.

[26]GibsonG, 2022. Perspectives on rigor and reproducibility in single cell genomics. PLoS Genet, 18(5):e1010210.

[27]GoetzJE, FredericksD, PetersenE, et al., 2015. A clinically realistic large animal model of intra-articular fracture that progresses to post-traumatic osteoarthritis. Osteoarthritis Cartilage, 23(10):1797-1805.

[28]GüllerM, Toualbi-AbedK, LegrandA, et al., 2008. c-Fos overexpression increases the proliferation of human hepatocytes by stabilizing nuclear Cyclin D1. World J Gastroenterol, 14(41):6339-6346.

[29]GuoL, ZhangHY, HouYL, et al., 2016. Plasmalemma vesicle-associated protein: a crucial component of vascular homeostasis. Exp Ther Med, 12(3):1639-1644.

[30]HalderA, KutznerI, GraichenF, et al., 2012. Influence of limb alignment on mediolateral loading in total knee replacement in vivo measurements in five patients. J Bone Joint Surg, 94(11):1023-1029.

[31]HaraT, TanegashimaK, 2012. Pleiotropic functions of the CXC-type chemokine CXCL14 in mammals. J Biochem, 151(5):469-476.

[32]HashimotoS, Creighton-AchermannL, TakahashiK, et al., 2002. Development and regulation of osteophyte formation during experimental osteoarthritis. Osteoarthritis Cartilage, 10(3):180-187.

[33]HeckmannTP, Barber-WestinSD, NoyesFR, 2006. Meniscal repair and transplantation: indications, techniques, rehabilitation, and clinical outcome. J Orthop Sports Phys Ther, 36(10):795-814.

[34]IzuharaK, ArimaK, OhtaS, et al., 2014. Periostin in allergic inflammation. Allergol Int, 63(2):143-151.

[35]JovicD, LiangX, ZengH, et al., 2022. Single‐cell RNA sequencing technologies and applications: a brief overview. Clin Transl Med, 12(3):e694.

[36]KaneshigeA, KajiT, ZhangLD, et al., 2022. Relayed signaling between mesenchymal progenitors and muscle stem cells ensures adaptive stem cell response to increased mechanical load. Cell Stem Cell, 29(2):265-280.e6.

[37]KimH, ChaJ, JangM, et al., 2019. Hyaluronic acid-based extracellular matrix triggers spontaneous M2-like polarity of monocyte/macrophage. Biomater Sci, 7(6):2264-2271.

[38]KoikeM, NojiriH, OzawaY, et al., 2015. Mechanical overloading causes mitochondrial superoxide and SOD2 imbalance in chondrocytes resulting in cartilage degeneration. Sci Rep, 5:11722.

[39]KorbeckiJ, GrochansS, GutowskaI, et al., 2020. CC chemokines in a tumor: a review of pro-cancer and anti-cancer properties of receptors CCR5, CCR6, CCR7, CCR8, CCR9, and CCR10 ligands. Int J Mol Sci, 21(20):7619.

[40]KozhemyakinaE, LassarAB, ZelzerE, 2015. A pathway to bone: signaling molecules and transcription factors involved in chondrocyte development and maturation. Development, 142(5):817-831.

[41]KrychAJ, BernardCD, KennedyNI, et al., 2020. Medial versus lateral meniscus root tears: is there a difference in injury presentation, treatment decisions, and surgical repair outcomes? Arthroscopy, 36(4):1135-1141.

[42]KwonH, BrownWE, LeeCA, et al., 2019. Surgical and tissue engineering strategies for articular cartilage and meniscus repair. Nat Rev Rheumatol, 15(9):550-570.

[43]LefebvreV, Dvir-GinzbergM, 2017. SOX9 and the many facets of its regulation in the chondrocyte lineage. Connect Tissue Res, 58(1):2-14.

[44]LiIMH, LiuK, NealA, et al., 2018. Differential tissue specific, temporal and spatial expression patterns of the Aggrecan gene is modulated by independent enhancer elements. Sci Rep, 8:950.

[45]LiZ, ChenZY, WangXT, et al., 2022. Integrated analysis of miRNAs and gene expression profiles reveals potential biomarkers for osteoarthritis. Front Genet, 13:814645.

[46]LiaoXC, WangW, YuBP, et al., 2022. Thrombospondin-2 acts as a bridge between tumor extracellular matrix and immune infiltration in pancreatic and stomach adenocarcinomas: an integrative pan-cancer analysis. Cancer Cell Int, 22:213.

[47]LinWZ, ZhuXH, GaoL, et al., 2021. Osteomodulin positively regulates osteogenesis through interaction with BMP2. Cell Death Dis, 12(2):147.

[48]LinYL, PersaudSD, NhieuJ, et al., 2017. Cellular retinoic acid-binding protein 1 modulates stem cell proliferation to affect learning and memory in male mice. Endocrinology, 158(9):3004-3014.

[49]LiuHR, LiDF, ZhangY, et al., 2018. Inflammation, mesenchymal stem cells and bone regeneration. Histochem Cell Biol, 149(4):393-404.

[50]LunneyJK, van GoorA, WalkerKE, et al., 2021. Importance of the pig as a human biomedical model. Sci Transl Med, 13(621):eabd5758.

[51]LynchJM, MailletM, VanhoutteD, et al., 2012. A thrombospondin-dependent pathway for a protective ER stress response. Cell, 149(6):1257-1268.

[52]MakrisEA, HadidiP, AthanasiouKA, 2011. The knee meniscus: structure-function, pathophysiology, current repair techniques, and prospects for regeneration. Biomaterials, 32(30):7411-7431.

[53]ManabeRI, TsutsuiK, YamadaT, et al., 2008. Transcriptome-based systematic identification of extracellular matrix proteins. Proc Natl Acad Sci USA, 105(35):12849-12854.

[54]MathieuM, IampietroM, ChuchanaP, et al., 2014. Involvement of angiopoietin-like 4 in matrix remodeling during chondrogenic differentiation of mesenchymal stem cells. J Biol Chem, 289(12):8402-8412.

[55]MitchellJ, GrahamW, BestTM, et al., 2016. Epidemiology of meniscal injuries in US high school athletes between 2007 and 2013. Knee Surg Sports Traumatol Arthrosc, 24(3):715-722.

[56]MousaviA, 2020. CXCL12/CXCR4 signal transduction in diseases and its molecular approaches in targeted-therapy. Immunol Lett, 217:91-115.

[57]NakamuraDS, HollanderJM, UchimuraT, et al., 2017. Pigment Epithelium-Derived Factor (PEDF) mediates cartilage matrix loss in an age-dependent manner under inflammatory conditions. BMC Musculoskelet Disord, 18:39.

[58]NaylorAJ, AzzamE, SmithS, et al., 2012. The mesenchymal stem cell marker CD248 (endosialin) is a negative regulator of bone formation in mice. Arthritis Rheum, 64(10):3334-3343.

[59]NeppleJJ, DunnWR, WrightRW, 2012. Meniscal repair outcomes at greater than five years. J Bone Joint Surg, 94(24):2222-2227.

[60]NishiyamaS, HiroseN, YanoshitaM, et al., 2021. ANGPTL2 induces synovial inflammation via LILRB2. Inflammation, 44(3):1108-1118.

[61]OntsukaK, KotobukiY, ShiraishiH, et al., 2012. Periostin, a matricellular protein, accelerates cutaneous wound repair by activating dermal fibroblasts. Exp Dermatol, 21(5):331-336.

[62]PrivratskyJR, NewmanPJ, 2014. PECAM-1: regulator of endothelial junctional integrity. Cell Tissue Res, 355(3):607-619.

[63]QuYK, WangYG, WangSX, et al., 2023. A comprehensive analysis of single-cell RNA transcriptome reveals unique SPP1+ chondrocytes in human osteoarthritis. Comput Biol Med, 160:106926.

[64]RahmaniM, WongBW, AngL, et al., 2006. Versican: signaling to transcriptional control pathways. Can J Physiol Pharmacol, 84:77-92.

[65]RaiMF, McNultyAL, 2017. Meniscus beyond mechanics: using biology to advance our understanding of meniscus injury and treatment. Connect Tissue Res, 58(3-4):221-224.

[66]RajagopalA, HomanEP, JoengK, et al., 2016. Restoration of the serum level of SERPINF1 does not correct the bone phenotype in Serpinf1 null mice. Mol Genet Metab, 117(3):378-382.

[67]RapleyJ, NicolàsM, GroenA, et al., 2008. The NIMA-family kinase Nek6 phosphorylates the kinesin Eg5 at a novel site necessary for mitotic spindle formation. J Cell Sci, 121(23):3912-3921.

[68]RodriguesM, YatesCC, NuschkeA, et al., 2013. The matrikine tenascin-C protects multipotential stromal cells/mesenchymal stem cells from death cytokines such as FasL. Tissue Eng Part A, 19(17-18):1972-1983.

[69]Salazar-NorattoGE, de NijsN, StevensHY, et al., 2019. Regional gene expression analysis of multiple tissues in an experimental animal model of post-traumatic osteoarthritis. Osteoarthritis Cartilage, 27(2):294-303.

[70]SalvadorJM, Brown-ClayJD, FornaceAJJr, 2013. Gadd45 in stress signaling, cell cycle control, and apoptosis. In: Liebermann DA, Hoffman B (Eds.), Gadd45 Stress Sensor Genes. Springer, New York, p.1-19.

[71]SantoroA, CondeJ, ScoteceM, et al., 2015. SERPINE2 inhibits IL-1α-induced MMP-13 expression in human chondrocytes: involvement of ERK/NF-κB/AP-1 pathways. PLoS ONE, 10(8):e0135979.

[72]SchmidtMB, ChenEH, LynchSE, 2006. A review of the effects of insulin-like growth factor and platelet derived growth factor on in vivo cartilage healing and repair. Osteoarthritis Cartilage, 14(5):403-412.

[73]SembaJA, MielochAA, RybkaJD, 2020. Introduction to the state-of-the-art 3D bioprinting methods, design, and applications in orthopedics. Bioprinting, 18:e00070.

[74]SidneyLE, BranchMJ, DunphySE, et al., 2014. Concise review: evidence for CD34 as a common marker for diverse progenitors. Stem Cells, 32(6):1380-1389.

[75]SmitsP, LiP, MandelJ, et al., 2001. The transcription factors L-Sox5 and Sox6 are essential for cartilage formation. Dev Cell, 1(2):277-290.

[76]SommaggioR, Uribe-HerranzM, MarquinaM, et al., 2016. Xenotransplantation of pig chondrocytes: therapeutic potential and barriers for cartilage repair. Eur Cells Mater, 32:24-39.

[77]SunH, WenXZ, LiHY, et al., 2020. Single-cell RNA-seq analysis identifies meniscus progenitors and reveals the progression of meniscus degeneration. Ann Rheum Dis, 79(3):408-417.

[78]SunJ, QiaoYN, TaoT, et al., 2020. Distinct roles of smooth muscle and non-muscle myosin light chain-mediated smooth muscle contraction. Front Physiol, 11:593966.

[79]SunY, WangTL, TohWS, et al., 2017. The role of laminins in cartilaginous tissues: from development to regeneration. Eur Cells Mater, 34:40-54.

[80]TagliaferriC, WittrantY, DaviccoMJ, et al., 2015. Muscle and bone, two interconnected tissues. Ageing Res Rev, 21:55-70.

[81]TakanoM, HiroseN, SumiC, et al., 2021. ANGPTL2 promotes inflammation via integrin α5β1 in chondrocytes. Cartilage, 13(S2):885S-897S.

[82]TakigawaM, 2013. CCN2: a master regulator of the genesis of bone and cartilage. J Cell Commun Signal, 7(3):191-201.

[83]TaoTQ, LiY, GuiC, et al., 2018. Fibronectin enhances cartilage repair by activating progenitor cells through Integrin α5β1 receptor. Tissue Eng Part A, 24(13-14):1112-1124.

[84]TarquiniC, PucciS, ScioliMG, et al., 2020. Clusterin exerts a cytoprotective and antioxidant effect in human osteoarthritic cartilage. Aging, 12(11):10129-10146.

[85]ThadaniR, KamenzJ, HeegerS, et al., 2018. Cell-cycle regulation of dynamic chromosome association of the condensin complex. Cell Rep, 23(8):2308-2317.

[86]TheinR, HershkovichO, GordonB, et al., 2017. The prevalence of cruciate ligament and meniscus knee injury in young adults and associations with gender, body mass index, and height a large cross-sectional study. J Knee Surg, 30(6):565-570.

[87]TheocharidisG, ConnellyJT, 2019. Minor collagens of the skin with not so minor functions. J Anat, 235(2):418-429.

[88]TóthF, JohnsonCP, MillsB, et al., 2019. Evaluation of the suitability of miniature pigs as an animal model of juvenile osteochondritis dissecans. J Orthop Res, 37(10):2130-2137.

[89]UngsudechachaiT, HonsawekS, JittikoonJ, et al., 2021. Clusterin is associated with systemic and synovial inflammation in knee osteoarthritis. Cartilage, 13(S1):1557S-1565S.

[90]WangF, DingPW, LiangX, et al., 2022. Endothelial cell heterogeneity and microglia regulons revealed by a pig cell landscape at single-cell level. Nat Commun, 13:3620.

[91]WangS, XieLJ, XieZX, et al., 2021. Dynamic changes in the expression of interferon-stimulated genes in joints of SPF chickens infected with avian reovirus. Front Vet Sci 8:618124.

[92]WenBQ, WangGL, LiEH, et al., 2022. In vivo generation of bone marrow from embryonic stem cells in interspecies chimeras. eLife, 11:e74018.

[93]WilsonR, NorrisEL, BrachvogelB, et al., 2012. Changes in the chondrocyte and extracellular matrix proteome during post-natal mouse cartilage development. Mol Cell Proteomics, 11(1):M111.014159.

[94]WonY, YangJI, ParkS, et al., 2021. Lipopolysaccharide binding protein and CD14, cofactors of toll‐like receptors, are essential for low‐grade inflammation‒induced exacerbation of cartilage damage in mouse models of posttraumatic osteoarthritis. Arthritis Rheumatol, 73(8):1451-1460.

[95]WuPT, SuWR, LiCL, et al., 2019. Inhibition of CD44 induces apoptosis, inflammation, and matrix metalloproteinase expression in tendinopathy. J Biol Chem, 294(52):20177-20184.

[96]XuC, LiuXW, FangXX, et al., 2022. Single-cell RNA sequencing reveals smooth muscle cells heterogeneity in experimental aortic dissection. Front Genet, 13:836593.

[97]XuL, LiZ, LiuSY, et al., 2015. Asporin and osteoarthritis. Osteoarthritis Cartilage, 23(6):933-939.

[98]YagamiK, SuhJY, Enomoto-IwamotoM, et al., 1999. Matrix Gla protein is a developmental regulator of chondrocyte mineralization and, when constitutively expressed, blocks endochondral and intramembranous ossification in the limb. J Cell Biol, 147(5):1097-1108.

[99]YanD, ZhouGD, ZhouX, et al., 2009. The impact of low levels of collagen IX and pyridinoline on the mechanical properties of in vitro engineered cartilage. Biomaterials, 30(5):814-821.

[100]YapL, TayHG, NguyenMTX, et al., 2019. Laminins in cellular differentiation. Trends Cell Biol, 29(12):987-1000.

[101]ZhangT, MaC, ZhangZQ, et al., 2021. NF‐κB signaling in inflammation and cancer. MedComm, 2(4):618-653.

[102]ZhangYW, MarmorsteinLY, 2010. Focus on molecules: fibulin-3 (EFEMP1). Exp Eye Res, 90(3):374-375.

[103]ZhaoT, SuZP, LiYC, et al., 2020. Chitinase-3 like-protein-1 function and its role in diseases. Sig Transduct Target Ther, 5:201.

[104]ZhouYC, ChenXQ, TianQL, et al., 2022. Deletion of ApoE leads to intervertebral disc degeneration via aberrant activation of adipokines. Spine, 47(12):899-907.