Targeting angiogenesis for fracture nonunion treatment in inflammatory disease

Abstract

Atrophic fracture nonunion poses a significant clinical problem with limited therapeutic interventions. In this study, we developed a unique nonunion model with high clinical relevance using serum transfer-induced rheumatoid arthritis (RA). Arthritic mice displayed fracture nonunion with the absence of fracture callus, diminished angiogenesis and fibrotic scar tissue formation leading to the failure of biomechanical properties, representing the major manifestations of atrophic nonunion in the clinic. Mechanistically, we demonstrated that the angiogenesis defect observed in RA mice was due to the downregulation of SPP1 and CXCL12 in chondrocytes, as evidenced by the restoration of angiogenesis upon SPP1 and CXCL12 treatment in vitro. In this regard, we developed a biodegradable scaffold loaded with SPP1 and CXCL12, which displayed a beneficial effect on angiogenesis and fracture repair in mice despite the presence of inflammation. Hence, these findings strongly suggest that the sustained release of SPP1 and CXCL12 represents an effective therapeutic approach to treat impaired angiogenesis and fracture nonunion under inflammatory conditions.

References

[1] Hadjiargyrou M, O’Keefe RJ. The convergence of fracture repair and stem cells: interplay of genes, aging, environmental factors and disease. J. Bone Min. Res., 2014, 29: 2307-2322

[2] Einhorn TA. Enhancement of fracture-healing. J. Bone Jt. Surg. Am., 1995, 77: 940-956

[3] Megas P. Classification of non-union. Injury, 2005, 36: S30-S37

[4] Stewart SK. Fracture non-union: a review of clinical challenges and future research needs. Malays. Orthop. J., 2019, 13: 1-10

[5] Rupp M et al. Diaphyseal long bone nonunions—types, aetiology, economics, and treatment recommendations. Int. Orthop., 2018, 42: 247-258

[6] Claes L, Recknagel S, Ignatius A. Fracture healing under healthy and inflammatory conditions. Nat. Rev. Rheumatol., 2012, 8: 133-143

[7] Peel NF, Moore DJ, Barrington NA, Bax DE, Eastell R. Risk of vertebral fracture and relationship to bone mineral density in steroid treated rheumatoid arthritis. Ann. Rheum. Dis., 1995, 54: 801-806

[8] Zura R et al. Epidemiology of fracture nonunion in 18 human bones. JAMA Surg., 2016, 151: e162775

[9] Deodhar AA, Woolf AD. Bone mass measurement and bone metabolism in rheumatoid arthritis: a review. Br. J. Rheumatol., 1996, 35: 309-322

[10] Hoes JN, Bultink IE, Lems WF. Management of osteoporosis in rheumatoid arthritis patients. Expert Opin. Pharmacother., 2015, 16: 559-571

[11] Manara M, Sinigaglia L. Bone and TNF in rheumatoid arthritis: clinical implications. RMD Open, 2015, 1: e000065

[12] Xue AL et al. Bone fracture risk in patients with rheumatoid arthritis: a meta-analysis. Medicine, 2017, 96: e6983

[13] Inzana JA et al. Bone fragility beyond strength and mineral density: Raman spectroscopy predicts femoral fracture toughness in a murine model of rheumatoid arthritis. J. Biomech., 2013, 46: 723-730

[14] Okumura G et al. Experimental arthritis and Porphyromonas gingivalis administration synergistically decrease bone regeneration in femoral cortical defects. Sci. Rep., 2019, 9

[15] Wellen KE, Hotamisligil GS. Inflammation, stress, and diabetes. J. Clin. Investig., 2005, 115: 1111-1119

[16] Baker RG, Hayden MS, Ghosh S. NF-kappaB, inflammation, and metabolic disease. Cell Metab., 2011, 13: 11-22

[17] Ouchi N, Parker JL, Lugus JJ, Walsh K. Adipokines in inflammation and metabolic disease. Nat. Rev. Immunol., 2011, 11: 85-97

[18] Hollinger JO, Schmitt JM, Hwang K, Soleymani P, Buck D. Impact of nicotine on bone healing. J. Biomed. Mater. Res., 1999, 45: 294-301

[19] Brown ML et al. Delayed fracture healing and increased callus adiposity in a C57BL/6 J murine model of obesity-associated type 2 diabetes mellitus. PLoS ONE, 2014, 9: e99656

[20] Lu C et al. Effect of age on vascularization during fracture repair. J. Orthop. Res., 2008, 26: 1384-1389

[21] Naik AA et al. Reduced COX-2 expression in aged mice is associated with impaired fracture healing. J. Bone Min. Res., 2009, 24: 251-264

[22] Lienau J et al. Differential regulation of blood vessel formation between standard and delayed bone healing. J. Orthop. Res., 2009, 27: 1133-1140

[23] Scott DL, Kingsley GH. Tumor necrosis factor inhibitors for rheumatoid arthritis. N. Engl. J. Med., 2006, 355: 704-712

[24] Khoury M et al. Inflammation-inducible anti-TNF gene expression mediated by intra-articular injection of serotype 5 adeno-associated virus reduces arthritis. J. Gene Med., 2007, 9: 596-604

[25] Choy EH, Kavanaugh AF, Jones SA. The problem of choice: current biologic agents and future prospects in RA. Nat. Rev. Rheumatol., 2013, 9: 154-163

[26] Furman BD et al. Targeting pro-inflammatory cytokines following joint injury: acute intra-articular inhibition of interleukin-1 following knee injury prevents post-traumatic arthritis. Arthritis Res Ther., 2014, 16: R134

[27] Timmen M et al. Influence of antiTNF-alpha antibody treatment on fracture healing under chronic inflammation. BMC Musculoskelet. Disord., 2014, 15

[28] Beek KJ et al. Long-term treatment with TNF-alpha inhibitors improves bone mineral density but not vertebral fracture progression in ankylosing spondylitis. J. Bone Min. Res., 2019, 34: 1041-1048

[29] Deckers MM et al. Expression of vascular endothelial growth factors and their receptors during osteoblast differentiation. Endocrinology, 2000, 141: 1667-1674

[30] Cackowski FC et al. Osteoclasts are important for bone angiogenesis. Blood, 2010, 115: 140-149

[31] Huang B et al. Osteoblasts secrete Cxcl9 to regulate angiogenesis in bone. Nat. Commun., 2016, 7

[32] Chen JL et al. TGFbeta1 induces hypertrophic change and expression of angiogenic factors in human chondrocytes. Oncotarget, 2017, 8: 91316-91327

[33] Li Z et al. Angiogenic potential of human bone marrow-derived mesenchymal stem cells in chondrocyte brick-enriched constructs promoted stable regeneration of craniofacial cartilage. Stem Cells Transl. Med., 2017, 6: 601-612

[34] Yuasa M et al. Fibrinolysis is essential for fracture repair and prevention of heterotopic ossification. J. Clin. Investig., 2015, 125: 3723

[35] Hausman MR, Schaffler MB, Majeska RJ. Prevention of fracture healing in rats by an inhibitor of angiogenesis. Bone, 2001, 29: 560-564

[36] Glowacki, J. Angiogenesis in fracture repair. Clin Orthop Relat Res, S82–S89 (1998).

[37] Bell A, Templeman D, Weinlein JC. Nonunion of the Femur and Tibia: an update. Orthop. Clin. N. Am., 2016, 47: 365-375

[38] Murnaghan M, Li G, Marsh DR. Nonsteroidal anti-inflammatory drug-induced fracture nonunion: an inhibition of angiogenesis. J. Bone Jt. Surg. Am., 2006, 88: 140-147

[39] Fassbender M et al. Local inhibition of angiogenesis results in an atrophic non-union in a rat osteotomy model. Eur. Cell Mater., 2011, 22: 1-11

[40] Ding ZC, Lin YK, Gan YK, Tang TT. Molecular pathogenesis of fracture nonunion. J. Orthop. Transl., 2018, 14: 45-56

[41] Yamazaki M et al. Spatial and temporal distribution of CD44 and osteopontin in fracture callus. J. Bone Jt. Surg. Br., 1999, 81: 508-515

[42] Hirakawa K et al. Localization of the mRNA for bone matrix proteins during fracture healing as determined by in situ hybridization. J. Bone Min. Res., 1994, 9: 1551-1557

[43] Sodek J et al. Regulation of osteopontin expression in osteoblasts. Ann. N. Y. Acad. Sci., 1995, 760: 223-241

[44] Morinobu M et al. Osteopontin expression in osteoblasts and osteocytes during bone formation under mechanical stress in the calvarial suture in vivo. J. Bone Min. Res., 2003, 18: 1706-1715

[45] Toupadakis CA et al. Long-term administration of AMD3100, an antagonist of SDF-1/CXCR4 signaling, alters fracture repair. J. Orthop. Res., 2012, 30: 1853-1859

[46] Murata K et al. Stromal cell-derived factor 1 regulates the actin organization of chondrocytes and chondrocyte hypertrophy. PLoS ONE, 2012, 7: e37163

[47] Jung Y et al. Regulation of SDF-1 (CXCL12) production by osteoblasts; a possible mechanism for stem cell homing. Bone, 2006, 38: 497-508

[48] Christopher MJ, Liu F, Hilton MJ, Long F, Link DC. Suppression of CXCL12 production by bone marrow osteoblasts is a common and critical pathway for cytokine-induced mobilization. Blood, 2009, 114: 1331-1339

[49] Fujio M et al. Stromal cell-derived factor-1 enhances distraction osteogenesis-mediated skeletal tissue regeneration through the recruitment of endothelial precursors. Bone, 2011, 49: 693-700

[50] Duvall CL, Taylor WR, Weiss D, Wojtowicz AM, Guldberg RE. Impaired angiogenesis, early callus formation, and late stage remodeling in fracture healing of osteopontin-deficient mice. J. Bone Min. Res., 2007, 22: 286-297

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

[52] Angelotti F et al. One year in review 2017: pathogenesis of rheumatoid arthritis. Clin. Exp. Rheumatol., 2017, 35: 368-378

[53] Choy E. Understanding the dynamics: pathways involved in the pathogenesis of rheumatoid arthritis. Rheumatology, 2012, 51: v3-v11

[54] Yamamoto Y, Turkiewicz A, Wingstrand H, Englund M. Fragility fractures in patients with rheumatoid arthritis and osteoarthritis compared with the general population. J. Rheumatol., 2015, 42: 2055-2058

[55] Bastian O et al. Systemic inflammation and fracture healing. J. Leukoc. Biol., 2011, 89: 669-673

[56] Wang C, Abu-Amer Y, O’Keefe RJ, Shen J. Loss of Dnmt3b in chondrocytes leads to delayed endochondral ossification and fracture repair. J. Bone Min. Res., 2018, 33: 283-297

[57] Buettmann EG et al. VEGFA from early osteoblast lineage cells (Osterix+) is required in mice for fracture healing. J. Bone Min. Res., 2019, 34: 1690-1706

[58] Hu K, Olsen BR. Osteoblast-derived VEGF regulates osteoblast differentiation and bone formation during bone repair. J. Clin. Investig., 2016, 126: 509-526

[59] Pesesse L et al. Consequences of chondrocyte hypertrophy on osteoarthritic cartilage: potential effect on angiogenesis. Osteoarthr. Cartil., 2013, 21: 1913-1923

[60] Geris L, Gerisch A, Sloten JV, Weiner R, Oosterwyck HV. Angiogenesis in bone fracture healing: a bioregulatory model. J. Theor. Biol., 2008, 251: 137-158

[61] Street J et al. Vascular endothelial growth factor stimulates bone repair by promoting angiogenesis and bone turnover. Proc. Natl Acad. Sci. USA, 2002, 99: 9656-9661

[62] Obayemi JD et al. Degradable porous drug-loaded polymer scaffolds for localized cancer drug delivery and breast cell/tissue growth. Mater. Sci. Eng. C. Mater. Biol. Appl., 2020, 112: 110794

[63] Guo X et al. Creating 3D angiogenic growth factor gradients in fibrous constructs to guide fast angiogenesis. Biomacromolecules, 2012, 13: 3262-3271

[64] Mills LA, Simpson AH. In vivo models of bone repair. J. Bone Jt. Surg. Br., 2012, 94: 865-874

[65] Garcia P et al. Development of a reliable non-union model in mice. J. Surg. Res., 2008, 147: 84-91

[66] Dickson KF, Katzman S, Paiement G. The importance of the blood supply in the healing of tibial fractures. Contemp. Orthop., 1995, 30: 489-493

[67] Pretre R, Peter RE, Kursteiner K. Limb revascularization to stimulate bone fracture healing. Am. Surg., 1997, 63: 836-838

[68] Hu K, Olsen BR. The roles of vascular endothelial growth factor in bone repair and regeneration. Bone, 2016, 91: 30-38

[69] Wein M et al. Differential osteopontin expression in human osteoblasts derived from iliac crest and alveolar bone and its role in early stages of angiogenesis. J. Bone Min. Metab., 2019, 37: 105-117

[70] Rowe GC et al. PGC-1alpha induces SPP1 to activate macrophages and orchestrate functional angiogenesis in skeletal muscle. Circ. Res., 2014, 115: 504-517

[71] Liekens S, Schols D, Hatse S. CXCL12-CXCR4 axis in angiogenesis, metastasis and stem cell mobilization. Curr. Pharm. Des., 2010, 16: 3903-3920

[72] Petrow PK et al. Expression of osteopontin messenger RNA and protein in rheumatoid arthritis: effects of osteopontin on the release of collagenase 1 from articular chondrocytes and synovial fibroblasts. Arthritis Rheum., 2000, 43: 1597-1605

[73] Kim KW et al. Up-regulation of stromal cell-derived factor 1 (CXCL12) production in rheumatoid synovial fibroblasts through interactions with T lymphocytes: role of interleukin-17 and CD40L-CD40 interaction. Arthritis Rheum., 2007, 56: 1076-1086

[74] Anborgh PH, Mutrie JC, Tuck AB, Chambers AF. Role of the metastasis-promoting protein osteopontin in the tumour microenvironment. J. Cell Mol. Med., 2010, 14: 2037-2044

[75] Sun X et al. CXCL12/CXCR4/CXCR7 chemokine axis and cancer progression. Cancer Metastasis Rev., 2010, 29: 709-722

[76] Critchley S et al. 3D printing of fibre-reinforced cartilaginous templates for the regeneration of osteochondral defects. Acta Biomater., 2020, 113: 130-143

[77] Zhang W et al. 3D printed composite scaffolds with dual small molecule delivery for mandibular bone regeneration. Biofabrication, 2020, 12: 035020

[78] Uchiyama E, Yamakoshi K, Sasaki T. Measurement of mechanical characteristics of tibial periosteum and evaluation of local differences. J. Biomech. Eng., 1998, 120: 85-91

[79] Kusumbe AP, Ramasamy SK, Adams RH. Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone. Nature, 2014, 507: 323-328

[80] Shevde LA, Samant RS. Role of osteopontin in the pathophysiology of cancer. Matrix Biol., 2014, 37: 131-141

[81] Guyon A. CXCL12 chemokine and its receptors as major players in the interactions between immune and nervous systems. Front .Cell. Neurosci., 2014, 8: 65

[82] Tzeng YS et al. Imbalanced osteogenesis and adipogenesis in mice deficient in the chemokine Cxcl12/Sdf1 in the bone mesenchymal stem/progenitor cells. J. Bone Min. Res., 2018, 33: 679-690

[83] Zhu W, Liang G, Huang Z, Doty SB, Boskey AL. Conditional inactivation of the CXCR4 receptor in osteoprecursors reduces postnatal bone formation due to impaired osteoblast development. J. Biol. Chem., 2011, 286: 26794-26805

[84] Everhart MB et al. Duration and intensity of NF-kappaB activity determine the severity of endotoxin-induced acute lung injury. J. Immunol., 2006, 176: 4995-5005

[85] Swarnkar G, Zhang K, Mbalaviele G, Long F, Abu-Amer Y. Constitutive activation of IKK2/NF-kappaB impairs osteogenesis and skeletal development. PLoS ONE, 2014, 9: e91421

Funding

U.S. Department of Health & Human Services | NIH | National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)(AR075860)

U.S. Department of Health & Human Services | NIH | National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)

U.S. Department of Health & Human Services | NIH | National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)

U.S. Department of Health & Human Services | NIH | National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)

U.S. Department of Health & Human Services | NIH | National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)

Shriners Hospitals for Children(85160)

U.S. Department of Health & Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (NIBIB)(EB022018)

U.S. Department of Health & Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (NIBIB)

U.S. Department of Health & Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (NIBIB)

U.S. Department of Health & Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (NIBIB)