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Distraction-suppression effect on osteosarcoma
Medical Hypotheses 121 (2018) 4–5
Contents lists available at ScienceDirect
Medical Hypotheses journal homepage: www.elsevier.com/locate/mehy
Distraction-suppression effect on osteosarcoma b
a
a
a
Wei Liu , Hong-bo He , Can Zhang , Yu-peng Liu , Jun Wan a b
a,⁎
T
Department of Orthopaedics, Xiangya Hospital, Central South University, Changsha, Hunan Province, PR China Xiangya Nursing School, Central South University, Changsha, Hunan Province, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Epiphysis distraction Suppression Osteosarcoma
Osteosarcoma is the most common primary malignant bone tumor. It occurs mainly in children and adolescents. In patients with open growth plate, epiphyseal distraction is used to separate the uninvolved epiphysis from adjacent tumor. This helps preserve the growth potential and restore joint and limb function to a great extent. Interestingly, epiphyseal distraction also appears to inhibit the proliferation of osteosarcoma tumor cells and to increase sensitivity to chemotherapy. Tumor interstitial pressure (TIP) is often elevated in the microenvironment of most solid tumors, including osteosarcoma. Elevated TIP can promote the proliferation, invasion, and migration ability of osteosarcoma cells and also decrease the uptake and distribution of chemotherapeutic agents. Studies have confirmed that the sustained volumetric strain produced in distracted tissue decreases TIP; it stretches extracellular matrix, decreases interstitial density, and increases vessel diameter. We hypothesize that lowering of TIP during the period of epiphyseal distraction inhibits the proliferation and invasion of osteosarcoma cell and, at the same time, increases blood perfusion in the tumor and thus enhances uptake and distribution of chemotherapy agents. If the hypothesis is proved to be true, distraction of tumor segment could be a novel supplementary treatment for osteosarcoma by manipulation of TIP.
Introduction Osteosarcoma is the most common primary malignant bone tumor. It occurs mainly in the second to third decades of life and is an important cause of morbidity and mortality of teenagers the world over. It usually involves the metaphysis of the distal femur, the proximal tibia, or the proximal humerus. In patients with open growth plate not deconstructed by tumor invasion, epiphysis distraction is often applied to separate the uninvolved epiphysis from the adjacent tumor in the metaphysis so that the growth potential of the epiphysis can be preserved and joint and limb function restored as much as possible [1,2]. Interestingly, as Hu et al. [3] have demonstrated, sensitivity to chemotherapy increases during the period of distraction. Hu et al. proposed that some change in the biological environment occurs during distraction. In our department, we routinely use the Ilizarov technique to deal with orthopaedical problems such as bone nonunion, limb length discrepancy, large bone defect, and so on [4,5]. During epiphysis distraction, we have observed an obvious bone repair reaction in peripheral osteosarcoma tissue. According to Response Evaluation Criteria in Solid Tumors (RECIST), this indicates a suppressive effect on the tumor [6]. Thus the evidence suggests that epiphyseal distraction, to some extent, can inhibit the growth of osteosarcoma. ⁎
Recently, researchers have used three-dimensional finite element modeling analysis to explain the biological effect of distraction osteogenesis (DO) from the biomechanical point of view [7]. Morgan et al. [8] analyzed the different kinds of strains produced during the period of distraction and found that volumetric strain was the most sustained. It caused stretch-induced changes in cell morphology, promoted cell proliferation, and triggered osteogenic differentiation. This type of strain could also stretch extracellular matrix, dilate blood vessel, and increase blood perfusion, and result ultimately in tissue regeneration. If tumor and epiphysis are regarded as positing in a single anatomical space, this volumetric strain would also exist during the period of distraction. Hypotheses We hypothesized that volumetric strain during the period of epiphysis distraction alters the tumor microenvironment of osteosarcoma, influencing the biological behavior of osteosarcoma cells (seeds) and inducing changes in the extracellular matrix of osteosarcoma (soil), thus ultimately inhibiting osteosarcoma growth. We wished to clarify the mechanism by which this “distraction–suppression effect” acted to bring about the tumor microenvironment change.
Corresponding author at: Department of Orthopedics, Xiangya Hospital, Central South University, Xiangya Road 87#, Changsha 410008, Hunan, PR China. E-mail address: [email protected] (J. Wan).
https://doi.org/10.1016/j.mehy.2018.09.004 Received 17 August 2017; Accepted 5 September 2018 0306-9877/ © 2018 Elsevier Ltd. All rights reserved.
Medical Hypotheses 121 (2018) 4–5
W. Liu et al.
Evaluation of the hypotheses
continued to generate sustainable volume in both epiphyseal segment and adjacent osteosarcoma segment during the period of distraction.
Tumor interstitial pressure (TIP) is often raised in solid tumors, including osteosarcoma [9]. Studies have shown that TIP has two components: solid phase pressure or solid stress (SS) and interstitial hydraulic pressure or interstitial fluid pressure (IFP). SS in the tumor microenvironment usually shows a sustained increase due to the rapid proliferation of tumor cells, the thickening of extracellular matrix, and the decrease in tissue compliance. Meanwhile, vascular hyperplasia in the basement membrane leads to high vascular permeability and transfer of serum from intratumoral blood vessels into the interstitial space, resulting in increased IFP. Moreover, SS increases the compressive force on veins and lymph vessels and hinders drainage of the accumulated interstitial fluid, which contributes to increase in IFP as well. Thus, SS and IFP promote each other, raising the TIP in the tumor. This elevation of TIP has been postulated to be a self-protection mechanism evolved by the body [10–12]. Tensile stress produced by epiphyseal distraction may produce sustained volumetric strain in osteosarcoma tissue and lower the TIP. Epiphysis distraction increases the volume of the anatomical compartment containing the osteosarcoma and the epiphysis. The volumetric strain reduces the compressive effect of peritumoral tissue on the osteosarcoma, stretches tumor stroma, decreases interstitial density, and breaks down the boundary of tumor interstitial, thus reducing SS from both outside and inside the tumor. Many studies have provided evidence supporting this theory. Hofmann et al. [13] showed that lowering of TIP could reduce the proliferation, invasion, and migration ability of solid epithelial tumor cells (A431 and A549). Tse et al. [14] found that compressive stress could promote malignant phenotype expression in “leading cells.” Jain et al. [15] proved that application of mechanical forces could lead to tumor development and metastasis. Yang et al. [16] demonstrated obvious inhibition of proliferation and migration of pancreatic cancer cell line SW1990 when cultured under negative pressure. We believe that reduction of TIP by distraction could be a feasible way to inhibit proliferation and migration of osteosarcoma cells (seeds). High TIP may also alter the sensitivity of osteosarcoma cells to adjuvant treatment. Nathan et al. [17] reported that osteosarcoma cells cultured in a pressurized environment have higher proliferation activity than those cultured in a conventional system; moreover, cells cultured in the high-pressure environment showed lower chemosensitivity. The authors concluded that changes in TIP had the potential to alter the efficacy of chemotherapy. In another study, Nathan et al. [18] showed that osteosarcoma angiogenesis could be regulated by changing the TIP in the tumor microenvironment. They concluded that this could be a new way to normalize blood perfusion in the tumor and improve sensitivity to adjuvant treatment. Eikenes et al. [19] used hyaluronidase to degrade extracellular matrix and reported improvement in the distribution and uptake of liposomal doxorubicin in human osteosarcoma xenografts. They stated that rebuilding of the transcapillary pressure gradient was responsible for this effect. Gade et al. [20] observed that delivery of low-molecular-weight chemotherapeutic agents was enhanced after TIP was lowered by collagenase injection. They suggested that lowering of TIP resulted in an increase in tumor microcirculation and consequent improved uptake and distribution of chemotherapeutic agents. To summarize, increased volume strain during the period of epiphyseal distraction reduces the compressive force on peritumoral and intratumoral microvasculature, leading to improved blood perfusion. The end result is a lower TIP and better uptake and distribution of chemotherapeutic agents.
Consequences of the hypothesis Volumetric strain produced in osteosarcoma by tensile stress during the period of epiphyseal distraction lowers the elevated TIP in the tumor microenvironment. This decrease in TIP inhibits the proliferation and invasion ability of osteosarcoma cell (seeds) and, at the same time, increases blood perfusion in the tumor, leading to enhanced uptake and distribution of chemotherapy agents (soil). If the hypothesis is proved to be true, distraction could be a novel supplementary treatment for osteosarcoma by manipulation of TIP. Conflict of interest statement None. Funding This study is supported by National Natural Science Foundation of China (No. 81301671). Acknowledgements We’d like to thank Mrs. Jing Wan (the wife of Dr. Jun Wan) for her hard work in language editing and selfless supporting during the period of this study. References [1] Betz M, Dumont CE, Fuchs B, et al. Physeal distraction for joint preservation in malignant metaphyseal bone tumors in children. Clin Orthop Relat Res 2012;470(6):1749–54. [2] Gao S, Zheng Y, Cai Q, et al. Preliminary clinical research on epiphyseal distraction in osteosarcoma in children. World J Surg Oncol. 2014;12:251. [3] He X, Hu YC, Huang HC, et al. Ilizarov technique in bone tumor of extremities. J Orthop China 2014(03):236–40. [4] Wan J, Zhang XS, Ling L, et al. Femoral bone transport by a monolateral external fixator with or without the use of intramedullary nail: a single-department retrospective study. Eur J Orthop Surg Traumatol 2013;23(4):457–64. [5] Wan J, Zhang XS, Ling L, et al. Tibial lengthening for unilateral Crowe type-IV developmental dysplasia of the hip. Indian J Orthop 2014;48(4):415–20. [6] Eisenhauer EA, Therasse P, Bogaerts J, et al. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer 2009;45(2):228–47. [7] Isaksson H. Recent advances in mechanobiological modeling of bone regeneration. Mech Res Commun 2012;42:22–31. [8] Morgan Elise F, Longaker Michael T, Carter Dennis R. Relationships between tissue dilatation and differentiation in distraction osteogenesis. Matrix Biol 2006;25(2):94–103. [9] Ariffin AB, Forde PF, Jahangeer S, et al. Releasing pressure in tumors: what do we know so far and where do we go from here? A Rev Cancer Res 2014;74(10):2655–62. [10] Nagelkerke A, Bussink J, Rowan AE, et al. The mechanical microenvironment in cancer: how physics affects tumors. Semin Cancer Biol 2015;35:62–70. [11] Stylianopoulos T. The solid mechanics of cancer and strategies for improved therapy. J Biomech Eng 2017;139(2). [12] Heldin CH, Rubin K, Pietras K, et al. High interstitial fluid pressure – an obstacle in cancer therapy. Nature 2004;4:806–13. [13] Hofmann M, Guschel M, Bernd A, et al. Lowering of tumor interstitial fluid pressure reduces tumor cell proliferation in a xenograft tumor model. Neoplasia 2006;8(2):89–95. [14] Tse JM, Cheng G, Tyrrell JA, et al. Mechanical compression drives cancer cells toward invasive phenotype. Proc Natl Acad Sci USA 2012;109(3):911–6. [15] Jain RK, Martin JD, Stylianopoulos T. The role of mechanical forces in tumor growth and therapy. Annu Rev Biomed Eng 2014;16:321–46. [16] Yang X, Sun B, Zhu H, et al. Suppression effects of negative pressure on the proliferation and metastasis in human pancreatic cancer cells. J Cancer Res Ther 2015;11(1):195–8. [17] Nathan SS, DiResta GR, Casas-Ganem JE, et al. Elevated physiologic tumor pressure promotes proliferation and chemosensitivity in human osteosarcoma. Clin Cancer Res 2005;11(6):2389–97. [18] Nathan SS, Huvos AG, Casas-Ganem JE, et al. Tumor interstitial fluid pressure may regulate angiogenic factors in osteosarcoma. J Orthop Res 2008;26(11):1520–5. [19] Eikenes L, Tari M, Tuffo I, et al. Hyaluronidase induces a transcapillary pressure gradient and improves the distribution and uptake of liposomal doxorubicin (Caelyx) in human osteosarcoma xenografts. Br J Cancer 2005;93(1):81–8. [20] Gade TP, Buchanan IM, Motley MW. Imaging intratumoral convection: pressure-dependent enhancement in chemotherapeutic delivery to solid tumors. Clin Cancer Res 2009;15(1):247–55.
Empirical data In a previous experiment using three-dimensional finite element modeling of the osteosarcoma segment and epiphysis segment we simulated epiphyseal distraction and showed that the tensile stress–strain 5
Contents lists available at ScienceDirect
Medical Hypotheses journal homepage: www.elsevier.com/locate/mehy
Distraction-suppression effect on osteosarcoma b
a
a
a
Wei Liu , Hong-bo He , Can Zhang , Yu-peng Liu , Jun Wan a b
a,⁎
T
Department of Orthopaedics, Xiangya Hospital, Central South University, Changsha, Hunan Province, PR China Xiangya Nursing School, Central South University, Changsha, Hunan Province, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Epiphysis distraction Suppression Osteosarcoma
Osteosarcoma is the most common primary malignant bone tumor. It occurs mainly in children and adolescents. In patients with open growth plate, epiphyseal distraction is used to separate the uninvolved epiphysis from adjacent tumor. This helps preserve the growth potential and restore joint and limb function to a great extent. Interestingly, epiphyseal distraction also appears to inhibit the proliferation of osteosarcoma tumor cells and to increase sensitivity to chemotherapy. Tumor interstitial pressure (TIP) is often elevated in the microenvironment of most solid tumors, including osteosarcoma. Elevated TIP can promote the proliferation, invasion, and migration ability of osteosarcoma cells and also decrease the uptake and distribution of chemotherapeutic agents. Studies have confirmed that the sustained volumetric strain produced in distracted tissue decreases TIP; it stretches extracellular matrix, decreases interstitial density, and increases vessel diameter. We hypothesize that lowering of TIP during the period of epiphyseal distraction inhibits the proliferation and invasion of osteosarcoma cell and, at the same time, increases blood perfusion in the tumor and thus enhances uptake and distribution of chemotherapy agents. If the hypothesis is proved to be true, distraction of tumor segment could be a novel supplementary treatment for osteosarcoma by manipulation of TIP.
Introduction Osteosarcoma is the most common primary malignant bone tumor. It occurs mainly in the second to third decades of life and is an important cause of morbidity and mortality of teenagers the world over. It usually involves the metaphysis of the distal femur, the proximal tibia, or the proximal humerus. In patients with open growth plate not deconstructed by tumor invasion, epiphysis distraction is often applied to separate the uninvolved epiphysis from the adjacent tumor in the metaphysis so that the growth potential of the epiphysis can be preserved and joint and limb function restored as much as possible [1,2]. Interestingly, as Hu et al. [3] have demonstrated, sensitivity to chemotherapy increases during the period of distraction. Hu et al. proposed that some change in the biological environment occurs during distraction. In our department, we routinely use the Ilizarov technique to deal with orthopaedical problems such as bone nonunion, limb length discrepancy, large bone defect, and so on [4,5]. During epiphysis distraction, we have observed an obvious bone repair reaction in peripheral osteosarcoma tissue. According to Response Evaluation Criteria in Solid Tumors (RECIST), this indicates a suppressive effect on the tumor [6]. Thus the evidence suggests that epiphyseal distraction, to some extent, can inhibit the growth of osteosarcoma. ⁎
Recently, researchers have used three-dimensional finite element modeling analysis to explain the biological effect of distraction osteogenesis (DO) from the biomechanical point of view [7]. Morgan et al. [8] analyzed the different kinds of strains produced during the period of distraction and found that volumetric strain was the most sustained. It caused stretch-induced changes in cell morphology, promoted cell proliferation, and triggered osteogenic differentiation. This type of strain could also stretch extracellular matrix, dilate blood vessel, and increase blood perfusion, and result ultimately in tissue regeneration. If tumor and epiphysis are regarded as positing in a single anatomical space, this volumetric strain would also exist during the period of distraction. Hypotheses We hypothesized that volumetric strain during the period of epiphysis distraction alters the tumor microenvironment of osteosarcoma, influencing the biological behavior of osteosarcoma cells (seeds) and inducing changes in the extracellular matrix of osteosarcoma (soil), thus ultimately inhibiting osteosarcoma growth. We wished to clarify the mechanism by which this “distraction–suppression effect” acted to bring about the tumor microenvironment change.
Corresponding author at: Department of Orthopedics, Xiangya Hospital, Central South University, Xiangya Road 87#, Changsha 410008, Hunan, PR China. E-mail address: [email protected] (J. Wan).
https://doi.org/10.1016/j.mehy.2018.09.004 Received 17 August 2017; Accepted 5 September 2018 0306-9877/ © 2018 Elsevier Ltd. All rights reserved.
Medical Hypotheses 121 (2018) 4–5
W. Liu et al.
Evaluation of the hypotheses
continued to generate sustainable volume in both epiphyseal segment and adjacent osteosarcoma segment during the period of distraction.
Tumor interstitial pressure (TIP) is often raised in solid tumors, including osteosarcoma [9]. Studies have shown that TIP has two components: solid phase pressure or solid stress (SS) and interstitial hydraulic pressure or interstitial fluid pressure (IFP). SS in the tumor microenvironment usually shows a sustained increase due to the rapid proliferation of tumor cells, the thickening of extracellular matrix, and the decrease in tissue compliance. Meanwhile, vascular hyperplasia in the basement membrane leads to high vascular permeability and transfer of serum from intratumoral blood vessels into the interstitial space, resulting in increased IFP. Moreover, SS increases the compressive force on veins and lymph vessels and hinders drainage of the accumulated interstitial fluid, which contributes to increase in IFP as well. Thus, SS and IFP promote each other, raising the TIP in the tumor. This elevation of TIP has been postulated to be a self-protection mechanism evolved by the body [10–12]. Tensile stress produced by epiphyseal distraction may produce sustained volumetric strain in osteosarcoma tissue and lower the TIP. Epiphysis distraction increases the volume of the anatomical compartment containing the osteosarcoma and the epiphysis. The volumetric strain reduces the compressive effect of peritumoral tissue on the osteosarcoma, stretches tumor stroma, decreases interstitial density, and breaks down the boundary of tumor interstitial, thus reducing SS from both outside and inside the tumor. Many studies have provided evidence supporting this theory. Hofmann et al. [13] showed that lowering of TIP could reduce the proliferation, invasion, and migration ability of solid epithelial tumor cells (A431 and A549). Tse et al. [14] found that compressive stress could promote malignant phenotype expression in “leading cells.” Jain et al. [15] proved that application of mechanical forces could lead to tumor development and metastasis. Yang et al. [16] demonstrated obvious inhibition of proliferation and migration of pancreatic cancer cell line SW1990 when cultured under negative pressure. We believe that reduction of TIP by distraction could be a feasible way to inhibit proliferation and migration of osteosarcoma cells (seeds). High TIP may also alter the sensitivity of osteosarcoma cells to adjuvant treatment. Nathan et al. [17] reported that osteosarcoma cells cultured in a pressurized environment have higher proliferation activity than those cultured in a conventional system; moreover, cells cultured in the high-pressure environment showed lower chemosensitivity. The authors concluded that changes in TIP had the potential to alter the efficacy of chemotherapy. In another study, Nathan et al. [18] showed that osteosarcoma angiogenesis could be regulated by changing the TIP in the tumor microenvironment. They concluded that this could be a new way to normalize blood perfusion in the tumor and improve sensitivity to adjuvant treatment. Eikenes et al. [19] used hyaluronidase to degrade extracellular matrix and reported improvement in the distribution and uptake of liposomal doxorubicin in human osteosarcoma xenografts. They stated that rebuilding of the transcapillary pressure gradient was responsible for this effect. Gade et al. [20] observed that delivery of low-molecular-weight chemotherapeutic agents was enhanced after TIP was lowered by collagenase injection. They suggested that lowering of TIP resulted in an increase in tumor microcirculation and consequent improved uptake and distribution of chemotherapeutic agents. To summarize, increased volume strain during the period of epiphyseal distraction reduces the compressive force on peritumoral and intratumoral microvasculature, leading to improved blood perfusion. The end result is a lower TIP and better uptake and distribution of chemotherapeutic agents.
Consequences of the hypothesis Volumetric strain produced in osteosarcoma by tensile stress during the period of epiphyseal distraction lowers the elevated TIP in the tumor microenvironment. This decrease in TIP inhibits the proliferation and invasion ability of osteosarcoma cell (seeds) and, at the same time, increases blood perfusion in the tumor, leading to enhanced uptake and distribution of chemotherapy agents (soil). If the hypothesis is proved to be true, distraction could be a novel supplementary treatment for osteosarcoma by manipulation of TIP. Conflict of interest statement None. Funding This study is supported by National Natural Science Foundation of China (No. 81301671). Acknowledgements We’d like to thank Mrs. Jing Wan (the wife of Dr. Jun Wan) for her hard work in language editing and selfless supporting during the period of this study. References [1] Betz M, Dumont CE, Fuchs B, et al. Physeal distraction for joint preservation in malignant metaphyseal bone tumors in children. Clin Orthop Relat Res 2012;470(6):1749–54. [2] Gao S, Zheng Y, Cai Q, et al. Preliminary clinical research on epiphyseal distraction in osteosarcoma in children. World J Surg Oncol. 2014;12:251. [3] He X, Hu YC, Huang HC, et al. Ilizarov technique in bone tumor of extremities. J Orthop China 2014(03):236–40. [4] Wan J, Zhang XS, Ling L, et al. Femoral bone transport by a monolateral external fixator with or without the use of intramedullary nail: a single-department retrospective study. Eur J Orthop Surg Traumatol 2013;23(4):457–64. [5] Wan J, Zhang XS, Ling L, et al. Tibial lengthening for unilateral Crowe type-IV developmental dysplasia of the hip. Indian J Orthop 2014;48(4):415–20. [6] Eisenhauer EA, Therasse P, Bogaerts J, et al. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer 2009;45(2):228–47. [7] Isaksson H. Recent advances in mechanobiological modeling of bone regeneration. Mech Res Commun 2012;42:22–31. [8] Morgan Elise F, Longaker Michael T, Carter Dennis R. Relationships between tissue dilatation and differentiation in distraction osteogenesis. Matrix Biol 2006;25(2):94–103. [9] Ariffin AB, Forde PF, Jahangeer S, et al. Releasing pressure in tumors: what do we know so far and where do we go from here? A Rev Cancer Res 2014;74(10):2655–62. [10] Nagelkerke A, Bussink J, Rowan AE, et al. The mechanical microenvironment in cancer: how physics affects tumors. Semin Cancer Biol 2015;35:62–70. [11] Stylianopoulos T. The solid mechanics of cancer and strategies for improved therapy. J Biomech Eng 2017;139(2). [12] Heldin CH, Rubin K, Pietras K, et al. High interstitial fluid pressure – an obstacle in cancer therapy. Nature 2004;4:806–13. [13] Hofmann M, Guschel M, Bernd A, et al. Lowering of tumor interstitial fluid pressure reduces tumor cell proliferation in a xenograft tumor model. Neoplasia 2006;8(2):89–95. [14] Tse JM, Cheng G, Tyrrell JA, et al. Mechanical compression drives cancer cells toward invasive phenotype. Proc Natl Acad Sci USA 2012;109(3):911–6. [15] Jain RK, Martin JD, Stylianopoulos T. The role of mechanical forces in tumor growth and therapy. Annu Rev Biomed Eng 2014;16:321–46. [16] Yang X, Sun B, Zhu H, et al. Suppression effects of negative pressure on the proliferation and metastasis in human pancreatic cancer cells. J Cancer Res Ther 2015;11(1):195–8. [17] Nathan SS, DiResta GR, Casas-Ganem JE, et al. Elevated physiologic tumor pressure promotes proliferation and chemosensitivity in human osteosarcoma. Clin Cancer Res 2005;11(6):2389–97. [18] Nathan SS, Huvos AG, Casas-Ganem JE, et al. Tumor interstitial fluid pressure may regulate angiogenic factors in osteosarcoma. J Orthop Res 2008;26(11):1520–5. [19] Eikenes L, Tari M, Tuffo I, et al. Hyaluronidase induces a transcapillary pressure gradient and improves the distribution and uptake of liposomal doxorubicin (Caelyx) in human osteosarcoma xenografts. Br J Cancer 2005;93(1):81–8. [20] Gade TP, Buchanan IM, Motley MW. Imaging intratumoral convection: pressure-dependent enhancement in chemotherapeutic delivery to solid tumors. Clin Cancer Res 2009;15(1):247–55.
Empirical data In a previous experiment using three-dimensional finite element modeling of the osteosarcoma segment and epiphysis segment we simulated epiphyseal distraction and showed that the tensile stress–strain 5