Composition and toxicity of damaging fragments in gunshot and mine-blast spine injuries

Vladimir P. Orlov; Орлов Владимир Петрович; Vladimir P. Orlov; Yuliya A. Nashchekina; Нащекина Юлия Александровна; Yuliya A. Nashchekina; Alexey V. Nashchekin; Нащекин Алексей Викторович; Alexey V. Nashchekin; Saidmirze D. Mirzametov; Мирзаметов Саидмирзе Джамирзоевич; Saidmirze D. Mirzametov; Sergey M. Idrichan; Идричан Сергей Михайлович; Sergey M. Idrichan; Maxim N. Kravtsov; Кравцов Максим Николаевич; Maxim N. Kravtsov; Dmitry V. Svistov; Свистов Дмитрий Владимирович; Dmitry V. Svistov

doi:10.17816/brmma634519

Composition and toxicity of damaging fragments in gunshot and mine-blast spine injuries

Authors: Orlov V.P.¹, Nashchekina Y.A.², Nashchekin A.V.³, Mirzametov S.D.¹, Idrichan S.M.¹, Kravtsov M.N.¹, Svistov D.V.¹
Affiliations:
1. Kirov Military Medical Academy
2. Institute of Cytology of the Russian Academy of Sciences
3. Ioffe Physical-Technical Institute
Issue: Vol 26, No 4 (2024)
Pages: 559-568
Section: Original Study Article
Submitted: 22.07.2024
Accepted: 01.10.2024
Published: 16.12.2024
URL: https://journals.eco-vector.com/1682-7392/article/view/634519
DOI: https://doi.org/10.17816/brmma634519
ID: 634519

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Abstract

The feasibility of removing damaging fragments from the spine in gunshot and mine-blast injuries is assessed based on the data of their composition and cytotoxicity. Four damaging fragments removed from the spine and paravertebral tissues were analyzed. Elemental analysis was performed using a scanning electron microscope. The composition of the damaging fragments was studied using spectral analysis. The cytotoxicity of the medium with damaging fragments was evaluated using the methyl tetrazolium test, comparing to the control medium. Morphological changes in cells were assessed using optical light microscopy, comparing to the control. Elemental analysis showed that all studied fragments consisted of alloys of various metals and other chemical elements. During the first few weeks of incubation in a complete nutrient medium, metals underwent fairly active oxidation, producing an orange precipitate. During further incubation, the oxidation of metals continued quite intensively, leading to a change in the nutrient medium and reducing cell proliferation. Moreover, morphological examination showed that cells exposed to metal oxides were rounded, while control sample cells were elongated and spindle-shaped. The methyl tetrazolium test revealed high cytotoxicity of all the fragments studied. All fragments were found to release toxic metal oxides into the nutrient medium, significantly reducing cell viability, regardless of their elemental composition. To prevent complications associated with possible local and/or systemic toxicity of metal fragments, as well as early and late infections, it is recommended to remove projectiles to the maximum extent feasible.

Keywords

mine-blast injuries, gunshot spine injuries, wounding projectile, fragment toxicity, methyl tetrazolium test, elemental analysis of wounding projectiles, scanning electron microscopy, spectral analysis

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Introduction

Gunshot wounds to the spine and spinal cord are a devastating combat injury, characterized by high mortality rates at all stages of spinal cord injury and significant long-term disability in most affected individuals [1]. However, gunshot wounds to the spine and spinal cord are relatively rare in combat settings. During the Great Patriotic War, their incidence depended on the type of military operations and ranged from 0.5% to 3% [2]. Data from localized conflicts, such as military operations in Afghanistan and the Chechen Republic, show an incidence of 4.7%–5.1% for gunshot wounds to the spine and spinal cord [3]. Recent high-tech localized armed conflicts demonstrated that most injuries are caused by mine explosions and shrapnel, often presenting as combined and multiple traumas, which exacerbates the severity of the wounded. in recent years, surgeons have adopted a highly effective active treatment approach. Additionally, treatment should be comprehensive, particularly for combined injuries [4, 5].

Modern gunshot wounds differ from those in past wars owing to their increased variety and the extensive tissue damage that extends beyond the wound canal [6–8]. in contemporary localized conflicts, combat operations frequently involve advanced weaponry, with each region exhibiting its own combat characteristics. Shrapnel are metallic fragments from aerial bombs, artillery shells, rockets, grenades, or landmines.

Currently, localized military conflicts heavily rely on artillery and strike drones, which function as explosive devices. The use of depleted uranium munitions has been reported. Concerns about the health and environmental effects of depleted uranium have prompted several nations to seek alternative materials for armor-piercing munitions, leading to the development of tungsten-based substitutes. However, experimental studies on laboratory rodents have demonstrated that highly aggressive malignant rhabdomyosarcomas developed after implantation of military-grade composite granules (e.g., tungsten, nickel, and cobalt) into limb muscles [9]. Furthermore, during the Gulf War, inhalation of desert dust particles led to outbreaks of respiratory diseases of unknown etiology, referred to as “severe acute pneumonitis.” Detailed analysis of Iraqi desert dust revealed that these particles contained a clay or quartz core surrounded by an inorganic calcium carbonate layer, incorporating various metals such as (in descending order of concentration) aluminum, iron, uranium, nickel, cobalt, copper, lead, and chromium [10].

Lead, which is commonly present in bullets, is a heavy metal classified as a chemical element that causes damage through ionic mimicry, intracellular calcium homeostasis disruption, nitric oxide synthesis inhibition, oxidative stress production, and gene transcription alterations, including the formation of subacute and delayed abscesses [11].

This STUDY AIMED to evaluate the feasibility of extracting metallic fragments resulting from gunshot and mine-blast spinal injuries by analyzing their composition and cytotoxicity in relation to mesenchymal stromal cells (MSCs).

Materials and methods

Damaging fragments removed from the spine and paravertebral tissues were studied. Indications for the removal of metallic fragments in spinal and spinal cord injuries included blind penetrating wounds of the spine and spinal cord, cauda equina root injuries, and blind nonpenetrating and paravertebral spinal wounds. Accessible metallic fragments were extracted using minimally invasive techniques, such as tubular retractors and endoscopic procedures (Fig. 1).

Four samples were selected for composition and cytotoxicity analysis. The first stage of the cytological studies involved preparing the fragments. The metallic fragments extracted from the spinal cord and vertebrae were washed in running water and mechanically cleaned of organic material adhering to their surfaces. Subsequently, oxides were removed from the fragment surfaces using a metal brush and were washed in running water, dried, and prepared for analysis. Elemental analysis was performed using a JSM-7001F scanning electron microscope (Jeol, Japan). The composition of the fragments was determined through spectral analysis at the A.F. Ioffe Physical-Technical Institute of the Russian Academy of Sciences (St. Petersburg, Russia).

For cytotoxicity testing, the fragment samples were sterilized in 70% ethanol and incubated in a complete nutrient medium (Dulbecco’s Modified Eagle Medium [DMEM/F12], Gibco, USA), supplemented with 1% essential amino acids, 10% heat-inactivated fetal bovine serum (HyClone, USA), 1% L-glutamine, 50 IU/ml penicillin, and 50 µg/ml streptomycin.

For cytotoxicity assessment, the FetMSCs human mesenchymal stromal cell line (Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia) was used. The cells were cultured in a CO₂ incubator at 37 °C in a humidified atmosphere containing 5% CO₂ in DMEM/F12 medium. The methyl tetrazolium (MTT) assay was conducted using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (0.1 mg/ml) to quantitatively assess the cytotoxicity of metal oxides.

For the experiment, 5×10³ cells per 100 µl per well were seeded in 96-well plates and cultured for 24 hours to allow cell attachment. After 24 hours, the medium was removed, and the wells were replenished with complete nutrient medium containing the metal fragments, which were incubated for 3 weeks. After 3 days, the medium was removed, and 50 µl of DMEM/F12 containing MTT was added to each well [12]. The cells were incubated in a CO₂ incubator for 2 hours at 37 °C. After removing the supernatant, formazan crystals, which were produced by metabolically active cells, were dissolved in 50 µl of dimethyl sulfoxide per well and transferred to clean wells. Then, MSC viability was assessed by measuring the optical density at 570 nm using a plate spectrophotometer. Similar experiments were conducted with the medium after an incubation period of 3 months. Polynomial regression analysis was performed using Microsoft Excel (Microsoft Corporation, USA) to calculate the MSC viability.

Fig. 1. Minimally invasive removal of a fragment from paravertebral soft tissues; а, b, c, d — stages of preparing access using tubular retractors and installation of a working sheath (21 mm in diameter); e — X-ray guided adjustment of the sheath position; f — removed wounding projectile

Рис. 1. Минимально инвазивное удаление осколка из паравертебральных мягких тканей: а, b, c, d — этапы выполнения доступа с применением тубулярных ретракторов и установки рабочего тубуса (диаметр 21 мм); e — коррекция положения тубуса под контролем рентгенографии; f — внешний вид удаленного ранящего снаряда

Statistical processing was conducted using recommended methods for medical, pharmaceutical, and biomedical research, and Microsoft Excel 2010 (Microsoft Corporation, USA) was utilized. The sample size was not precalculated.

Results and discussion

The examined fragments were alloys composed of various metals and other chemical elements (Fig. 2, Table 1).

Each fragment contained oxygen (O₂) in the form of oxides, along with iron (Fe) (samples 1–4) or copper (Cu) (samples 1, 3, and 4). Moreover, a significant amount of carbon (C) was detected in all the samples. The magnetic properties of the fragments varied depending on the metal composition. Fragments with a higher iron content demonstrated stronger magnetic properties.

During the incubation of the fragments in the complete nutrient medium, active oxidation of the metals occurred within the first few weeks, as evidenced by a change in medium color and formation of an orange precipitate (Fig. 3a). Metal oxidation remained intense as the incubation period continued, leading to a more pronounced color change and accumulation of a dense orange precipitate (Fig. 3b).

Fig. 2. Scanning electron microscopy of the surface of four fragment samples; а — Sample 1; b — Sample 2; c — Sample 3; d — Sample 4

Рис. 2. Сканирующая электронная микроскопия поверхности четырех образцов осколков: а — образец 1; b — образец 2; c — образец 3; d — образец 4

Table 1. Elemental composition of four fragment samples

Таблица 1. Элементный состав четырех образцов осколков

Sample 1		Sample 2		Sample 3		Sample 4
Element	Mass %	Element	Mass %	Element	Mass %	Element	Mass %
C	35.2	C	43.69	C	47.77	C	38.49
O	22.25	O	26	O	17.79	O	14.87
F	4.53	Na	2.41	F	1.75	F	4.43
Mg	0.19	Al	0.18	P	0.74	Na	2.01
Al	0.29	Si	0.59	Ca	1.1	P	0.27
Si	1.88	P	0.65	Mn	0.9	Cl	0.21
P	0.19	K	0.3	Fe	23.82	Ca	0.27
S	0.16	Ca	0.76	Cu	3.64	Cr	0.43
K	0.15	Cr	0.36	Zn	2.15	Fe	36.79
Ca	0.69	Fe	24.3	Mo	0.34	Co	0.08
Mn	0.45	Cu	0.37	–	–	Tb	2.15
Fe	27.26	Mo	0.38	–	–	–	–
Cu	4.16	–	–	–	–	–	–
Zn	2.6	–	–	–	–	–	–

To assess the potential cytotoxicity of the fragments and metal oxides formed during incubation, the conditioned nutrient medium was added to the MSCs and cultured for 3 days. Then, cell morphology was evaluated through light microscopy (Fig. 4).

Figure 4 shows that all four samples contained metal oxide precipitates in the form of orange deposits. This precipitate reduced cell proliferation, as indicated by the lower cell density in the experimental samples compared with the control, where a monolayer of cells had formed by day 3. Additionally, cell morphology differed between the experimental and control samples. in contrast to the control sample, wherein cells exhibited their characteristic elongated, spindle-like shape, the experimental samples showed more rounded cells. Sample 1 exhibited a higher number of adherent cells compared with samples 2–4.

Fig. 3. External appearance of the nutrient medium; а — after 3-week incubation with fragments; b — after 3-month incubation with fragments

Рис. 3. Внешний вид питательной среды: а — после 3 нед. инкубирования осколков; b — после 3 мес. инкубирования осколков

Fig. 4. Light microscopy of mesenchymal stromal cells cultured in the medium after incubation with fragments for 3 days; а — Sample 1; b — Sample 2; c — Sample 3; d — Sample 4: e — control medium

Рис. 4. Световая микроскопия МСК при культивировании в среде после инкубирования с осколками в течение 3 сут: а — образец 1; b — образец 2; c — образец 3; d — образец 4: e — контрольная среда

Figure 5 presents a viability assessment diagram for MSCs cultured in the conditioned medium after 3 weeks of incubation with metal fragments. The MTT assay results was consistent with the light microscopy findings. The number of viable cells in samples 2–4 was significantly lower than that in the control sample. Although the number of viable cells was higher in sample 1 than in samples 2–4, it was still lower than that in the control group.

With longer incubation (3 months), metal oxide formation remained active. Metal oxides were observed in the culture medium after 3 days of cell culturing (Fig. 6).

In all four samples, no complete monolayer formation was observed. Moreover, the morphology of the cells in samples 1–3 differed significantly from that of the control group. The number of viable cells in these media was <50% compared with the control, as confirmed by the MTT assay (Fig. 7).

Fig. 5. Methyl tetrazolium test of mesenchymal stromal cells cultured for 3 days in the medium after incubation with fragments for 3 weeks

Рис. 5. МТТ МСК после 3 сут культивирования в присутствии питательной среды после инкубирования с осколками в течение 3 нед.

Fig. 6. Light microscopy of mesenchymal stromal cells cultured in the medium after incubation with fragments for 3 days; а — Sample 1; b — Sample 2; c — Sample 3; d — Sample 4; e — control medium

Рис. 6. Световая микроскопия МСК при культивировании МСК в среде после инкубирования с осколками в течение 3 сут: а — образец 1; b — образец 2; c — образец 3; d — образец 4; e — контрольная среда

Fig. 7. Methyl tetrazolium test after 3-day cultivation in the medium after incubation with fragments for 3 months

Рис. 7. МТТ после 3 сут культивирования в присутствии питательной среды после инкубирования с осколками в течение 3 мес

All examined fragments consisted of multiple chemical elements and various metal alloys. A comparison of the elemental analysis and MTT assay results demonstrated that all fragments released toxic metal oxides into the nutrient medium, significantly reducing the viability of surrounding tissues, regardless of their elemental composition.

No adverse events were observed during the study. However, a limitation of the present study was the lack of a comparative cytotoxicity analysis between wounding projectiles and bioinert materials (e.g., implants).

Conclusion

The study results reveal that oxides of various metal alloys present in damaging fragments removed from the spine and paravertebral tissues exhibit toxicity to the human body, regardless of their elemental composition. It is crucial to maximize the removal of wounding projectiles whenever feasible to prevent complications associated with the potential local and/or systemic toxicity of metallic fragments and early and late infectious complications.

Additional information

Authors’ contribution. Thereby, all authors made a substantial contribution to the conception of the study, acquisition, analysis, interpretation of data for the work, drafting and revising the article, final approval of the version to be published and agree to be accountable for all aspects of the study.

The contribution of each author. V.P. Orlov — development of a general concept; writing an article; Yu.A. Nashchekina — determination of the toxicity of fragments, data analysis; A.V. Nashchekin — assessment of the composition of wounding shells, data analysis; S.D. Mirzametov — removal of fragments, data analysis; S.M. Idrichan — removal of fragments; M.N. Kravtsov — removal of fragments; D.V. Svistov — study design, data analysis.

Competing interests. The authors declare that they have no competing interests.

Funding source. This study was not supported by any external sources of funding.

Дополнительная информация

Вклад авторов. Все авторы внесли существенный вклад в разработку концепции, проведение исследования и подготовку статьи, прочли и одобрили финальную версию перед публикацией.

Вклад каждого автора. В.П. Орлов — разработка общей концепции; написание статьи; Ю.А. Нащекина — определение токсичности осколков, анализ данных; А.В. Нащекин — оценка состава ранящих снарядов, анализ данных; С.Д. Мирзаметов — удаление осколков, анализ данных; С.М. Идричан — удаление осколков; М.Н. Кравцов — удаление осколков; Д.В. Свистов — дизайн исследования, анализ данных.

Конфликт интересов. Авторы декларируют отсутствие явных и потенциальных конфликтов интересов, связанных с публикацией настоящей статьи.

Источник финансирования. Авторы заявляют об отсутствии внешнего финансирования при проведении исследования.

About the authors