INFLUENCE OF ANTITUMOR CHEMOTHERAPEUTICS ON THE STRUCTURE AND PHOSPHORUS-CALCIUM METABOLISM OF INJURED LONG TUBULAR SKELETAL BONES.

Objective. To study the structure and macronutrient composition of long tubular bones in rats under the influence of antitumor chemotherapeutics. Results. Antitumor chemotherapy slows down the formation of bone regenerate in the area of the defect and causes disorders of phosphorus-calcium metabolism in the injured bone. This is manifested by a decrease in the intensity of newly formed organic matrix mineralization in the area of the defect and a decrease in the level of calcium and phosphorus in the native bone and on its border with the regenerate. Doxorubicin and methotrexate provide the most negative impact on mineralization process among antitumor chemotherapeutic agents. Conclusions. The use of antitumor chemotherapeutic agents – The h igh frequency of fractures in cancer patients is due to a decrease in bone strength which is associated with bone metabolism disorders such as osteoporosis, metastatic bone disease, and pathological fractures. Anticancer chemotherapy is prescribed for long-term periods and affect s bone metabolism, in particular mineralization of bony tissue. doxorubicin, 5-fluorouracil and methotrexate – slows down the processes of reparative regeneration at all stages of recovery after injury and reduces the phosphorus-calcium metabolism of injured long tubular bones.


Introduction/Вступ
Fractures are the most common lesions of the bone system. In connection with considerable growth of oncological diseases in present days, the percent of fractures among the patients of this group is also constantly increasing. This is due to the development of bone metabolism disorders such as osteoporosis, metastatic bone disease, and pathological fractures. Disorders of bone continuity are especially common in breast, prostate, thyroid, kidney, and lung cancers [1,2]. Fractures lead to long-term and significant disability of patients, and particularly in cancer patients, they cause cessation or postponing of the necessary anticancer chemotherapy.
An important factor in the occurrence of fractures is represented by a decrease in bone mineral density due to changes in its mineral composition [3]. The process of bone mineralization is important for bone hardness and strength. Bone metabolism and tissue properties depend on macronutrients, which can influence the regulation of mineral metabolism, as well as proliferation or activity of osteoblasts and osteoclasts.
According to the literature, extracellular calcium (Ca 2+ ) and inorganic phosphate (P) are the two important factors that determine bone mineralization [4]. Calcium is a macronutrient that is important for the development, growth, and maintenance of the bone tissue, as well as for cellular cytoskeleton stability. The total Ca content in the body of an adult is about 1200 g, which is about 2% of body weight. About 99% of Ca in the body is contained in the bones and teeth in the form of hydroxyapatite, which is responsible for tissue mineralization [5]. Insufficient Ca intake during the growth period can adversely affect bone maturity and contribute to the further increased risk of osteoporotic fractures.
Ca deficiency is a major factor in the development of osteoporosis [6]. The main factors that maintain blood Ca concentration at a constant level are 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ), parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF 23 ). Decreased serum Ca content leads to inactivation of the calcium-sensing receptor (CaSR) in the parathyroid glands, which thus stimulates the release of PTH. The latter binds to its receptor in the kidneys, which increases Ca reabsorption and production of 1,25(ОН) 2 D 3 . Circulating PTH and 1,25(ОН) 2 D 3 bind to respective receptors on osteoblasts, which enhances expression of the receptor activator of nuclear factor kappa-B ligand (RANKL). RANKL stimulates osteoclastic bone resorption and Ca and P release into the circulation. It restores Ca levels and triggers down-regulation mechanisms. They involve the release of calcitonin from the thyroid gland, which reduces Ca reabsorption in the kidneys and Ca absorption in the intestine, as well as inhibition of osteoclastic bone resorption, thus maintaining Ca level within optimal limits [7]. FGF23 (phosphatonin) is an endocrine hormone, which is produced by osteoblasts and osteocytes. FGF23 effect is due to the inhibition of renal phosphate reabsorption and vitamin D 3 (1,25(OH) 2 D 3 hormone synthesis, as well as to the maintenance of normal renal sensitivity to PTH and regulation of bone mineralization [8]. Phosphorus is the major component of bone tissue, being second only to Ca. It is present in the human body in the amount of 550-770 g, of which almost 85% is stored in bones and teeth in the form of phosphoproteins and hydroxyapatite crystals. Phosphorus homeostasis is regulated by three main hormones: PTH, 1,25(OH) 2 D 3 and FGF 23 . An appropriate level of inorganic P is crucial for osteoblasts and osteocytes activity in the process of matrix mineralization. Deficiency of P in most cases is due to impaired reabsorption of P in the kidneys rather than to its low content in food and leads to impaired mineral deposition and nonmineralized osteoid formation [9].
Bone tissue serves as a depot of calcium and phosphorus in the body and has an extremely high (up to 65%) content of calcium phosphate as compared to other body tissues. Calcium salts in the form of hydroxyapatite crystals are linked to collagen fibers (type I collagen). The effect of calcification (adhesion of calcium salts to the organic bone matrix) is ensured by specific proteins of bone tissue: osteonectin, osteocalcin, and bone sialoprotein. Osteoblasts, which play a major role in the formation of organic intercellular bone matrix, provide continuous growth of hydroxyapatite crystals and act as mediators in the binding of mineral crystals to the protein matrix [10].
It is known from the literature that decreased mineral content leads to a slowdown in the formation of mineralized bone matrix and significantly increases the risk of osteoporotic fractures [11].
The problem of reparative bone regeneration in cancer patients is underexplored today. Antitumor chemotherapy being one of the main methods of cancer treatment is prescribed for long-term courses and affects the mineral metabolism of the injured bone.
Objective: to study morphological features of reparative osteogenesis and macronutrient composition of long tubular bones in rats under the influence of antitumor chemotherapeutics.
Materials and methods. The study involved 96 white laboratory 7 month-old male rats weighing 230 ± 10 g. Throughout the experiment, the rats were kept in the vivarium of the Medical Institute of Sumy State University on a standard diet and water intake regime with free access to food and water. The rats were kept under standard light conditions ( Under ketamine anesthesia (50 mg/kg), all the rats were cut in a sterile operating room by a ballshaped dental burr to obtain a 2 mm diameter perforated defect to the medullary cavity in the middle third of the femoral shaft. The animals were divided into the control (n = 24) and three experimental groups (Group I, II, and III, n = 72). After being injured, the animals in experimental groups were given intraperitoneal antitumor chemotherapeutics, which are most often used in antitumor chemotherapy protocols; the therapy was repeated every 21 days of the study. Group I (n = 24) -doxorubicin (60 mg/m²), Group II (n = 24) -5fluorouracil (600 mg/m²), Group III (n = 24)methotrexate (40 mg/m²).
On the 15th, 30th, 45th, and 60th day after the injury, the animals were sacrificed by decapitation under ketamine anesthesia (100 mg/kg) with subsequent removal of the injured long tubular bones.
Bone samples were preliminarily fixed in 10% neutral buffered formalin. For further analysis of tissue-specific structures of the regenerate, a fracture was performed at the site of defect. The obtained samples were kept in PBS buffer (pH 7.4) for 2 hours to remove the formalin. Further, bone material was dehydrated through graded alcohols (50%, 70%, 90%, 96%, 100%; kept for 30 min in each). Then the test material was air-dried to constant weight and sprayed with silver in a standard vacuum device "ВУП-5М" (SELMI, Sumy, Ukraine). To study the morphology of regenerate surface, scanning electron microscopy (SEO-SEM Inspect S50-B Scanning Electron Microscope) was used [12]. Elemental analysis of the studied samples was performed by Xray energy dispersive spectroscopy (AZtecOne energy dispersive spectrometer with X-MaxN20 detector, Oxford Instruments PLC). Quantitative and qualitative distribution of chemical elements was determined in the following three areas: the bone regenerate, the border between the regenerate and native bone, and the native bone adjacent to the regenerate. Quantitative and qualitative assay of chemical elements was investigated by spot and linear analysis.
Statistical analysis of the obtained digital values was performed with the help of MX Excel XP 298 This work is licensed under Creative Commons Attribution 4.0 International License https://creativecommons.org/licenses/by/4.0/ statistical computer program using the Student's ttest. The difference was considered significant at p ˂ 0.05.

Study results and discussion
On the 15th day of the experiment, raster electron microscopy in the control group showed irregular bone trabeculae with large-loop structure and areas of intertrabecular space at the site of the defect. This indicated the beginning of bone tissue formation in the regenerate. The surface of newly formed trabeculae presented with low calcium and phosphorus concentrations, which were 8.99 ± 0.15% and 4.35 ± 0.14%, respectively. The obtained values indicated the beginning of organic matrix calcification in the regenerate area. The surface of the native bone presented with Са concentration of 22.57 ± 0.16% and Р concentration of 13.05 ± 0.19%, which was lower than physiological parameters and was due to the bone reaction to mechanical trauma. At the border between the regenerate and native bone, the level of these macronutrients was also reduced due to the activation of remodeling processes in the newly formed bone regenerate and the use of endogenous Ca for ossification. Са and Р concentrations were 19.07 ± 0.22% and 9.3 ± 0.14%, respectively (Fig. 1).
On day 30 of the experiment, electron diffraction patterns showed that the main part of the defect was represented by reticulofibrous bone trabeculae and small areas of lamellar tissue located mainly on the periphery of the regenerate. There was some space between the regenerate and the native bone. A gradual increase in Ca and P concentrations in the newly formed regenerate was determined (9.87 ± 0.19% and 4.8 ± 0.18%, respectively). This indicated further active remodeling processes in the bone defect. In the native bone and on its border with the regenerate, the concentrations of Ca and P were almost unchanged vs. the preceding results.
On day 45 of the experiment, scintigram findings revealed that the vast majority of the bone defect was represented by lamellar and, to a lesser extent, by reticulofibrous bone tissue. There was no space between the regenerate and the native bone. Ca and P concentrations in the defect area were 11.2 ± 0.21% and 5.3 ± 0.18%, i.e. higher vs. the preceding results by 13.47% (p ˂ 0.005) and 10.42% (p ˂ 0.005), respectively. The increase in these values indicated active mineralization in the area of bone regeneration. Ca and P concentrations in the native bone (21.45 ± 0.19% and 12.55 ± 0.19%) and on its border with the regenerate (18.32 ± 0.17% and 9.04 ± 0.16%) remained almost unchanged. There was a decrease vs. the preceding results in Ca concentration in the native bone by 0.6% (p = 0.34) and on its border with the regenerate by 1.7% (p = 0.003); P concentration decreased by 2.33% (p = 0.019) and 1.09% (p = 0.33), respectively.
On day 60 of the experiment, electron diffraction patterns showed that the bone defect was represented by lamellar bone tissue tightly adhered to the native bone. Ca and P concentrations in the newly formed regenerate equaled 12.37 ± 0.21% and 6.15 ± 0.19%, i.e. higher by 10.45% (p ˂ 0.005) and 16  results. This indicated intensive mineralization in bone marrow and active processes of reparative regeneration. There was a further increase in Ca concentration in the native bone by 3.73% (p ˂ 0.005) and on the border with the regenerate by 2.89% (p ˂ 0.005); P concentration increased by 1.59% (p = 0.09) and 1.99% (p = 0.14), respectively. These changes indicated a pronounced remodeling activity in the native bone areas adjacent to the defect (Fig. 2).

Figure 2 -Changes in calcium content in bone defect area in mature rats from control and experimental groups at different points of reparative osteogenesis
Thus, starting from the 15th day and throughout the experiment, the animals of the control group presented with an active increase in calcium and phosphorus concentrations in the area of the newly formed bone defect and with a slight decrease in these trace elements at the border of regenerate and native bone, as well as in the native bone itself which was due to the compensatory reaction to injury. These changes indicated intensive mineralization processes in the newly formed bone regenerate and active remodeling processes in the native bone itself.
In the experimental groups, analysis of scintigrams on the 15th day of the experiment showed delayed formation of bone trabeculae in the defect area, reduced thickness of trabeculae, increased intertrabecular space, microcracks and empty osteocyte lacunae, lack of adhesion between the native bone and newly formed regenerate (Fig. 3). IIIby 36.04% (p ˂ 0.005) and 21.16% (p ˂ 0.005) (Fig. 4).
On the 15th day of the experiment, there was also a decrease in Ca and P concentrations on the native bone surface vs. the controls: Group Iby 14.79% (p ˂ 0.005) and 24.52% (p ˂ 0.005), respectively; Group IIby 18.52% (p ˂ 0.005) and 22.60% (p ˂ 0.005), Group IIIby 22.24% (p ˂ 0.005) and 13.64% (p ˂ 0.005). Thus, the obtained data of microanalysis in all experimental groups indicated a delay in the formation of bone regenerate in the area of the defect and reduced mineralization intensity, as well as slowdown of the remodeling activity of the native bone in response to injury (Fig. 5).

Figure 5 -Distribution of phosphorus and calcium content on the surface of the injured long bone (native bone, border between native bone and regenerate, regenerate) on the 15th day of the experiment after methotrexate use: Aphosphorus, Bcalcium
On the 30th day of the experiment, after two injections of appropriate antitumor chemotherapy with an interval of 21 days, most of the regenerate was represented by thinned irregular trabeculae of coarse-fibrous bone tissue with small areas of lamellar bone tissue (Fig. 6).
Microprobe analysis of the regenerate showed a gradual increase in the Ca and P concentrations, but they were still lower than in the control group. Thus, Ca and P values in Group I as compared to the controls were lower by 53.90% (p ˂ 0.005) and 37.91% (p ˂ 0.005), respectively; Group IIby Raster electron microscopy of rat bone samples on the 60th day of the experiment showed delayed formation of lamellar tissue in the defect area, the areas of thinned newly formed bone trabeculae, gaps between them, increased intertrabecular space and lack of close adhesion between the regenerate and native bone (Fig. 10). This indicates a delay in callus formation in the area of the defect under the action of chemotherapeutics.
The mineral content on the 60th day of the injury began to increase gradually, but was much lower than in the control group. Thus, there was a decrease in Ca and P concentrations in the area of the defect: Group Iby 45.68% (p ˂ 0.005) and 303 This work is licensed under Creative Commons Attribution 4.0 International License https://creativecommons.org/licenses/by/4.0/ 21.46% (p ˂ 0.005) vs control; Group IIby 26.03% (p ˂ 0.005) and 17.07% (p ˂ 0.005), Group IIIby 40.66% (p ˂ 0.005) and 25.20% (p ˂ 0.005). In the native bone and areas adjacent to the regenerate, in contrast to the preceding outcomes, there was a gradual increase in the level of these trace elements within 1-2%, which was still lower than in the control group. Thus, Ca and P levels in the native bone were lower than in the control group: Group Iby 15.60% (p ˂ 0.005) and 18.58% (p ˂ 0.005); Group IIby 20.45% (p ˂ 0.005) and 17.33% (p ˂ 0.005), Group IIIby 22.24% (p ˂ 0.005) and 14.59% (p ˂ 0.005) (Fig. 11). А В С who had found that chemotherapy with methotrexate inhibited bone formation by inhibiting the activation of Wnt/β-catenin signaling pathway. As a result, the differentiation of osteoblastic cells providing mineralization of the bone matrix [13,14,15] was inhibited. King (2012) also found that methotrexate treatment promoted the formation of osteoclasts in the long bones of rats by increasing the level of proinflammatory cytokines and enhancing the activation of NF-kβ transcription factor. This disrupted the processes of bone remodeling due to increased bone tissue loss, which reduced the activity of reparative regeneration [16].
The reduction in mineralization found in our experiment with the use of doxorubicin was consistent with the findings of Fan C., Georgiou K.R. at al. (2017) stating that treatment with doxorubicin induced osteoclast-mediated bone resorption and, by causing oxidative stress, inhibited osteoblast differentiation and survival. This, in turn, led to a decrease in the level of 25-hydroxyvitamin D 3 and alkaline phosphatase in the serum, which affected the process of calcium deposition in bone tissue. Bone specific alkaline phosphatase (BAP) is synthesized by osteoblasts, which provide bone mineralization, and reflects their activity. These changes in bone remodeling lead to a decrease in the volume of trabecular bone in metaphase and low bone mineralization, which with long-term use of doxorubicin chemotherapy increases the risk of fractures in such patients [17].
In our study, it was found that treatment with 5-fluorouracil adversely affects bone mineralization. This is confirmed by the work of Vyas D. at al. and Raghu Nadhanan et al. (2012) showing that 5-fluorouracil chemotherapy led to increased levels of proinflammatory cytokines such as NF-κB, TNF-α, IL-1β, and IL-6, as well as reduced osteoblast density and caused a significant increase in osteoclast activity. As a result, low bone mineral density developed and bone loss was enhanced [18,19]. However, it was found that after 5-fluorouracil cessation, there was a gradual improvement in bone metabolism, in contrast to the use of doxorubicin and methotrexate [20].
The use of chemotherapeutics in our experiment led to the development of hypophosphatemia. This is confirmed by the studies of Wigner N., where a decrease in phosphorus levels during chemotherapy caused the resistance to BMP-2 with subsequent inhibition of differentiation of the chondrogenic cells, namely chondrocytes and osteoblasts. This led to a decrease in the volume of mineralized tissue and the development of low bone mineral density, which increased the risk of fractures [21].
Although chemotherapeutic drugs are necessary for the treatment of cancer, they adversely affect mineral metabolism by causing hypocalcemia and hypophosphatemia, which disrupts mineralization processes and causes osteopenia and osteoporosis.

Conclusions/Висновки
Antitumor chemotherapy slows down the formation of callus in the area of the defect and causes disorders of phosphorus-calcium metabolism both in the native bone and regenerate. This is manifested by a decrease in the intensity of mineralization of the newly formed bone matrix and a slowdown in the remodeling activity of the native bone. Doxorubicin and methotrexate provide the most negative impact on mineralization process among antitumor chemotherapeutic agents.

Prospects for future research/Перспективи подальших досліджень
In the future, it is planned to study the dependence of changes in the strength and structural properties of bones on mineral metabolism disorders with the use of different groups of antitumor chemotherapeutics.