An experimental study on the effects of splenectomy on bone fracture healing

Bone fracture healing is a complex process involving multiple mechanisms where immune system has vital role. The spleen is a part of this process and its effect on bone metabolism is not completely understood. Herein, the effects of simultaneous bone fractures and splenectomy were evaluated regarding early and late periods of fracture healing in rats. The closed fracture model was applied in sixty-four healthy male Wistar-albino rats. Rats were randomly selected and divided into four groups. The splenectomy was carried out in two groups. The early period was declared as three weeks and late period as five weeks for evaluating the fracture healing in rats,(Groups A: splenectomized 3rd week, Groups B: non-splenectomized 3rd week, Groups C: splenectomized 5th week, and Groups D: non-splenectomized 5th week). The groups were compared regarding radiological, biomechanical, and histopathological aspects at 3rd and 5th weeks of fracture healing. The callus/bone volume ratios of Groups C and D were lower than those of Groups A and B (A–C, p ≤ 0.001; B–D, p ≤ 0.001). Radiographic Union Score for Tibial Fracture scores of Groups C and D were higher than those for Groups A and B (A–C, p ≤ 0.001; B–D, p ≤ 0.001). Bone mineral density measurements of Groups C and D were higher than those for Groups A and B (A–C, p ≤ 0.001; A–D, p ≤ 0.001; B–C, p ≤ 0.001 and B–D, p ≤ 0.001). The histological scoring of Groups A was lower than Groups C and D (A–C, p = 0.004; A–D, p = 0.001). Splenectomy may thus adversely affect the early phase fracture healing.

Splenectomy; Fracture healing; Experimental rat study

[1] Fransvea P, Costa G, Massa G, Frezza B, Mercantini P, BaIducci G. Non-operative management of blunt splenic injury: is it really so extensively feasible? A critical appraisal of a single-center experience. The Pan African Medical Journal. 2019; 32: 52.

[2] Bagaria D, Kumar A, Ratan A, Gupta A, Kumar A, Kumar S, et al. Changing aspects in the management of splenic injury patients: experience of 129 isolated splenic injury patients at level 1 trauma center from India. Journal of Emergencies, Trauma, and Shock. 2019; 12: 35–39.

[3] Xiao W, Hu Z, Li T, Li J. Bone fracture healing is delayed in splenectomic rats. Life Sciences. 2017; 173: 55–61.

[4] Cirocchi R, Boselli C, Corsi A, Farinella E, Listorti C, Trastulli S, et al. Is non-operative management safe and effective for all splenic blunt trauma?A systematic review. Critical Care. 2013; 17: R185.

[5] Coccolini F, Montori G, Catena F, Kluger Y, Biffl W, Moore EE, et al. Splenic trauma: WSES classification and guidelines for adult and pediatric patients. World Journal of Emergency Surgery. 2017; 12: 40.

[6] Podda M, De Simone B, Ceresoli M, Virdis F, Favi F, Wiik Larsen J, et al. Follow-up strategies for patients with splenic trauma managed non-operatively: the 2022 world society of emergency surgery consensus document. World Journal of Emergency Surgery. 2022; 17: 52.

[7] Takayanagi H. Osteoimmunology: shared mechanisms and crosstalk between the immune and bone systems. Nature Reviews Immunology. 2007; 7: 292–304.

[8] Rapp AE, Bindl R, Recknagel S, Erbacher A, Müller I, Schrezen-meier H, et al. Fracture healing is delayed in immunodeficient NOD/scid‑IL2Rγcnull mice. PLOS ONE. 2016; 11: e0147465.

[9] Baht GS, Vi L, Alman BA. The role of the immune cells in fracture healing. Current Osteoporosis Reports. 2018; 16: 138–145.

[10] Clement ND, Beauchamp NJF, Duckworth AD, McQueen MM, Court-Brown CM. The outcome of tibial diaphyseal fractures in the elderly. The Bone & Joint Journal. 2013; 95-B: 1255–1262.

[11] Claes L, Recknagel S, Ignatius A. Fracture healing under healthy and inflammatory conditions. Nature Reviews Rheumatology. 2012; 8: 133–143.

[12] Theill LE, Boyle WJ, Penninger JM. RANK-L and RANK: T cells, bone loss, and mammalian evolution. Annual Review of Immunology. 2002; 20: 795–823.

[13] Wang X, Zhang Y, Ji W, Ao J. Categorising bone defect hematomas—enhance early bone healing. Medical Hypotheses. 2018; 113: 77–80.

[14] Bahney CS, Zondervan RL, Allison P, Theologis A, Ashley JW, Ahn J, et al. Cellular biology of fracture healing. Journal of Orthopaedic Research. 2019; 37: 35–50.

[15] Reinke S, Geissler S, Taylor WR, Schmidt-Bleek K, Juelke K, Schwachmeyer V, et al. Terminally differentiated CD8⁺ T cells negatively affect bone regeneration in humans. Science translational medicine. 2013; 5: 177ra36.

[16] Könnecke I, Serra A, El Khassawna T, Schlundt C, Schell H, Hauser A, 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.

[17] Toben D, Schroeder I, El Khassawna TN, Mehta M, Hoffmann JE, Frisch JT, et al. Fracture healing is accelerated in the absence of the adaptive immune system. Journal of bone and mineral research. 2011; 26: 11324.

[18] Lewis SM, Williams A, Eisenbarth SC. Structure and function of the immune system in the spleen. Science Immunology. 2019; 4: eaau6085.

[19] Mick P, Fischer C. Delayed fracture healing. Seminars in Musculoskeletal Radiology. 2022; 26: 329–337.

[20] Tyagi AM, Srivastava K, Sharan K, Yadav D, Maurya R, Singh D. Daidzein prevents the increase in CD4+CD28null T cells and B lymphopoesis in ovariectomized mice: a key mechanism for anti-osteoclastogenic effect. PLOS ONE. 2011; 6: e21216.

[21] Klavert J, van der Eerden BCJ. Fibronectin in fracture healing: biological mechanisms and regenerative avenues. Frontiers in Bioengineering and Biotechnology. 2021; 9: 663357.

[22] Bonnarens F, Einhorn TA. Production of a standard closed fracture in laboratory animal bone. Journal of Orthopaedic Research. 1984; 2: 97–101.

[23] Histing T, Garcia P, Holstein JH, Klein M, Matthys R, Nuetzi R, et al. Small animal bone healing models: standards, tips, and pitfalls results of a consensus meeting. Bone. 2011; 49: 591–599.

[24] Whelan DB, Bhandari M, Stephen D, Kreder H, McKee MD, Zdero R, et al. Development of the radiographic union score for tibial fractures for the assessment of tibial fracture healing after intramedullary fixation. The Journal of trauma. 2010; 68: 629–632.

[25] Morgan EF, Mason ZD, Chien KB, Pfeiffer AJ, Barnes GL, Einhorn TA, et al. Micro-computed tomography assessment of fracture healing: Relationships among callus structure, composition, and mechanical function. Bone. 2009; 44: 335–344.

[26] Huo MH, Troiano NW, Pelker RR, Gundberg CM, Friedlaender GE. The influence of ibuprofen on fracture repair: biomechanical, biochemical, histologic, and histomorphometric parameters in rats. Journal of Orthopaedic Research. 1991; 9: 383–390.

[27] Turner CH, Burr DB. Basic biomechanical measurements of bone: a tutorial. Bone. 1993; 14: 595–608.

[28] Park S, Silva M, Bahk W, McKellop H, Lieberman JR. Effect of repeated irrigation and debridement on fracture healing in an animal model. Journal of Orthopaedic Research. 2002; 20: 1197–1204.

[29] Lin H, Cottrell J, O’Connor JP. Variation in lipid mediator and cytokine levels during mouse femur fracture healing. Journal of Orthopaedic Research. 2016; 34: 1883–1893.

[30] Menger MM, Manuschewski R, Ehnert S, Rollmann MF, Maisenbacher TC, Tobias AL, et al. Radiographic, biomechanical and histological characterization of femoral fracture healing in aged CD-1 mice. Bioengineering. 2023; 10: 275.

[31] Dong Y, Zhou H, Alhaskawi A, Wang Z, Lai J, Abdullah Ezzi SH, et al. Alterations in bone fracture healing associated with TNFRSF signaling pathways. Frontiers in Pharmacology. 2022; 13: 905535.

[32] Boyce BF, Xing L. Biology of RANK, RANKL, and osteoprotegerin. Arthritis Research & Therapy. 2007; 9: S1.

[33] Ardeniz FÖ, Yargucu ZF, Erdoğan AP, Yıldırım ŞI. The roles of the RANKL/RANK/OPG in the immune system. Turkiye Klinikleri J Immun Allergy-Special Topics. 2016; 9: 1–9.

[34] Wright HL, McCarthy HS, Middleton J, Marshall MJ. RANK, RANKL and osteoprotegerin in bone biology and disease. Current Reviews in Musculoskeletal Medicine. 2009; 2: 56–64.

[35] Holstein JH, Klein M, Garcia P, Histing T, Culemann U, Pizanis A, et al. Rapamycin affects early fracture healing in mice. British Journal of Pharmacology. 2008; 154: 1055–1062.

[36] Xiao WA, Wang Y, Li JJ, Yang XX, Li ZW, Zheng W. Effect of immune function changes after splenectomy on fracture healing in patients with compound injury. International Journal of Clinical and Experimental Medicine. 2016; 9: 1716–1723.

[37] Schlundt C, El Khassawna T, Serra A, Dienelt A, Wendler S, Schell H, et al. Macrophages in bone fracture healing: their essential role in endochondral ossification. Bone. 2018; 106: 78–89.

[38] Sun D, Zheng X, Chen Y, Jia C, Xu S, Lin C, et al. Enhancement of osteogenesis post-splenectomy does not attenuate bone loss in ovariectomized rats. Journal of Orthopaedic Research. 2015; 33: 1356–1363.

[39] Bucay N, Sarosi I, Dunstan CR, Morony S, Tarpley J, Capparelli C, et al. Osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification. Genes & Development. 1998; 12: 1260–1268.

[40] Gültekin MZ, Yüksel S, Alagöz E, Özsoy Ş, Avcı A, Güleç MA, et al. Investigation of the Effects of Splenectomy on Bone Healing (Experimental Study). Istanbul Medical Journal. 2021; 22: 45–49.

[41] Hao Y, Ma Y, Wang X, Jin F, Ge S. Short-term muscle atrophy caused by botulinum toxin—a local injection impairs fracture healing in the rat femur. Journal of Orthopaedic Research. 2012; 30: 574–580.