EXPERIMENTAL MODELS OF OSTEONECROSIS IN PRECLINICAL STUDIES: LITERATURE REVIEW

ABSTRACT

Nowadays, in some segments of the population, there is an increased risk of developing osteonecrosis due to a lack of a healthy lifestyle, insufficient intake of  comorbidities. At the same time, statistical data indicate a significant increase in the risk of developing osteonecrosis in people experiencing post-covid syndrome after the COVID-19 pandemic [1]. The cause of this pathological process is explained, on the one hand, by the direct destruction of bone and cartilage tissue by the SARS-CoV-2 virus, and on the other hand, it is also considered as a consequence of long-term pharmacotherapy with steroids. These and other examples indicate the current relevance of the studied pathology, the need for an etiopathogenetic complex approach to diagnosis, and the need for personalized pharmacotherapeutic treatment. In finding a solution to this, specialists conducting medical and biological research face the urgent task of not only experimentally correctly modeling osteonecrosis, but also identifying the pathogenetic factor in its development, improving diagnostic criteria and pathogenetic treatment methods. The article provides a scientific overview of methods for modeling osteonecrosis in laboratory animals, markers of osteonecrosis, and an analysis of the obtained results.

АННОТАЦИЯ

В настоящее время у некоторых групп населения наблюдается повышенный риск развития остеонекроза, обусловленный отсутствием здорового образа жизни, недостаточным потреблением сопутствующих заболеваний. В то же время статистические данные свидетельствуют о значительном увеличении риска развития остеонекроза у лиц, перенесших постковидный синдром после пандемии COVID-19 [1]. Причину этого патологического процесса объясняют, с одной стороны, прямым разрушением костной и хрящевой ткани вирусом SARS-CoV-2, а с другой – также рассматривают как следствие длительной фармакотерапии стероидами. Эти и другие примеры свидетельствуют об актуальности изучаемой патологии, необходимости комплексного этиопатогенетического подхода к диагностике и необходимости персонализированного фармакотерапевтического лечения. В поисках решения этой проблемы перед специалистами, проводящими медико-биологические исследования, стоит актуальная задача не только экспериментально корректного моделирования остеонекроза, но и выявления патогенетического фактора его развития, совершенствования диагностических критериев и патогенетических методов лечения. В статье представлен научный обзор методов моделирования остеонекроза у лабораторных животных, маркеров остеонекроза, а также анализ полученных результатов.

Keywords. Osteonecrosis, corticosteroids, lopopolysaccharide, methylprednisolone, non-traumatic osteonecrosis.

Ключевые слова: остеонекроз, кортикостероиды, лопополисахарид, метилпреднизолон, нетравматический остеонекроз.

Introduction. Osteonecrosis, also known as avascular necrosis, represents a degenerative disorder of bone tissue characterized by the death of bone cells. Recent investigations indicate that the prevalence of osteonecrosis has increased noticeably following the COVID-19 pandemic, significantly diminishing patients’ quality of life. The condition most frequently affects the femoral head and is often bilateral or multifocal in nature. Major risk factors include trauma, chronic corticosteroid therapy, excessive alcohol consumption, and various systemic disorders [1].

Epidemiological analyses focused on the occurrence of osteonecrosis across different populations demonstrate a consistent year-by-year rise in its incidence. For instance, a Swedish cohort study reported a 10-year risk of 0.4% for osteonecrosis, with a morbidity rate of 0.17%. The disparity between these two indicators highlights the dynamic nature of the disease and suggests that the overall burden is likely to increase over the next decade. In the United Kingdom, from 1989 to 2003, the annual incidence ranged between 1.4 and 3.0 cases per 100,000 people, while in the Netherlands, it remained below 1.0 case. In the United States, an estimated 10,000 to 30,000 new cases of osteonecrosis are diagnosed each year, with the average patient age ranging between 33 and 38 years. In Japan, the 2004 incidence of femoral head avascular necrosis was 89 cases per 100,000 population, corresponding to approximately 11,400 diagnosed individuals. Similarly, studies from China estimate between 75,000 and 150,000 new femoral head avascular necrosis cases annually. In South Korea, the average annual incidence was 14,103 cases, or roughly 20 per 100,000 individuals [2,3].

Materials and Methods. The primary objective of animal modeling in osteonecrosis research is to reproduce the full spectrum of bone degeneration—from vascular disruption and necrosis to subsequent reparative processes. When selecting animal species, researchers consider factors such as anatomical similarity to humans, susceptibility to osteonecrosis, and the reproducibility of pathological features. Models used for osteonecrosis induction include traumatic, corticosteroid-induced, alcohol-induced, and other variants.

Traumatic Models. Traumatic models of femoral head osteonecrosis are achieved by acutely interrupting the blood supply to the femoral head. Common procedures include experimental hip dislocation, surgical transection of the round ligament, or placement of ligatures around the femoral neck. For instance, in porcine studies, osteonecrosis was induced by cutting the round ligament and applying dual ligatures on the femoral neck. Microangiographic and tomographic imaging revealed markedly reduced or absent perfusion in the epiphysis, indicating the onset of avascular necrosis. In rabbits, decompression drilling of the distal medial femoral condyle has been utilized, primarily to evaluate osteochondral regeneration implants. Although such traumatic models are valuable, they fail to fully replicate the complex metabolic, cellular, and microcirculatory mechanisms underlying non-traumatic human osteonecrosis [4].

Steroid-Induced Models. In corticosteroid-based models, intramuscular injections of methylprednisolone at repeated high doses (40 mg/kg) have induced osteonecrosis in approximately 83% of rabbits, with an 86% survival rate. Similar models in rats demonstrated that administration of dexamethasone at a concentration of 1 μmol/L for 24 hours elevated reactive oxygen species levels, increased osteoclast activity, and led to femoral head necrosis.

A practical and cost-effective experimental method for inducing steroid-associated osteonecrosis involves the combined use of lipopolysaccharide (LPS) and methylprednisolone. In this protocol, 24-month-old rats receive intravenous LPS (0.2 mg/kg) followed by intraperitoneal methylprednisolone (100 mg/kg) every 24 hours, two to three times per week for 2–6 weeks at 40 mg/kg. Mortality in these models can reach up to 15%, yet osteonecrosis occurs in 95–100% of surviving animals [6,7,8,9].

Recent findings indicate that Toll-like receptor 4 (TLR4) signaling contributes to oxidative stress, which may influence osteonecrosis development. In one study, Wistar rats were divided into four groups: (1) saline control, (2) saline + methylprednisolone, (3) LPS + saline, and (4) LPS + methylprednisolone. After 14 days, osteonecrosis was detected in rats receiving LPS and methylprednisolone but not in control groups. Glutathione peroxidase activity in the liver increased one day after LPS administration but was suppressed by methylprednisolone. No significant changes were observed in femoral glutathione activity. The results demonstrated that TLR4 activation enhances corticosteroid-induced osteonecrosis but oxidative stress alone via TLR4 is insufficient to induce osteonecrosis.

Alcohol-Induced Models. In alcohol-based models, Wistar rats are typically fed a 5% ethanol-containing Lieber-DeCarli liquid diet for 1–24 weeks. In another variant, 10 ml of pure ethanol (>99.7%) was directly injected into both femoral heads. Researchers such as Abeles (1979), Mont (2006), and Hirota (1993) established that prolonged alcohol consumption increases the risk of non-traumatic osteonecrosis, often paralleling corticosteroid-related pathology. Lieber and colleagues (1989) explored alcohol-induced hepatic lipolysis and osteonecrosis using rodent models maintained on ethanol diets [12].

Comparative analyses revealed that both alcoholic and corticosteroid-induced osteonecrosis share common pathogenetic mechanisms mediated by TLR4 signaling. In one experiment, male Wistar rats were given a Lieber-DeCarli diet containing 5% ethanol (experimental group) or dextran (control group) for up to 24 weeks. Ethanol-fed rats developed hepatic steatosis, hyperlipidemia, and non-traumatic osteonecrosis within seven days, confirmed via histopathology and biochemical assays. In contrast, control animals exhibited no signs of necrosis [16-20].

Bisphosphonate-Induced Models. Another widely adopted method involves oral administration of bisphosphonates following ovariectomy in rats. After eight weeks post-surgery, rats receive oral alendronate (1.0 mg/kg) or saline once weekly for four weeks. Bone turnover is assessed via serum collagen type I C-telopeptide measurements. Lipopolysaccharide or saline is then injected into the bone marrow of the mandible and femur, and necrotic regions are quantified histomorphometrically.

Serum-Induced Models. In a different protocol, osteonecrosis is induced by two intraperitoneal injections of sterile human serum (10 ml/kg) spaced two weeks apart, followed by three consecutive daily injections of methylprednisolone (40 mg/kg/day) two weeks after the final serum dose.

Diagnostic Confirmation Methods. Several diagnostic techniques are applied to confirm osteonecrosis in experimental models.

Radiological Approaches. Radiographic imaging, computed tomography, and bone scintigraphy are commonly used to visualize disease progression. Magnetic resonance imaging (MRI) remains the most sensitive method for detecting early osteonecrosis, particularly through identification of the characteristic “double-line sign.”

Histopathological Evaluation. Researchers such as Yamamoto (1997), Okazaki (2009), and Tateda (2012) utilized histopathological analysis of decalcified femoral bone tissue processed with Kalki-toxTM and neutralized with sodium sulfate buffer. Samples fixed in 10% formalin-phosphate buffer (pH 7.4) were stained with hematoxylin and eosin. The presence of necrosis was determined by identifying empty lacunae or pyknotic osteocytes in trabeculae adjacent to marrow necrosis.

Biochemical assays measuring serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglycerides (TG), total cholesterol (TC), and high-density lipoproteins (HDL) are also conducted. Cytokine quantification (IL-1α, IL-1β, IL-17, IFNγ, adiponectin) is performed using rat-specific ELISA kits to assess TLR4-related inflammatory signaling.

Microvascular Perfusion Assessment. Laser Doppler flowmetry is employed to evaluate microvascular blood flow and intraosseous pressure in femoral heads affected by osteonecrosis. In experimental groups, perfusion levels measured 165 mV in intertrochanteric regions, 430 mV near wound margins, and only 35 mV in necrotic zones. Corresponding intraosseous pressures were 38, 61, and 55 mmHg, respectively. These findings confirm that blood perfusion in osteonecrotic femoral heads is heterogeneous and markedly reduced.

Conclusion. Analysis of the studied literature showed that the incidence of osteonecrosis is increasing year by year, and treatment with corticosteroids and alcoholism are among the leading factors in the pathogenesis of osteonecrosis. Reliable models for modeling osteonecrosis in animals allow comparing the effectiveness of chemical, biological, and physical methods of treatment and prevention of osteonecrosis. It was established that all the several experimental models of osteonecrosis development in laboratory animals presented by the researchers are caused by various factors that have their own significance. However, in terms of convenience and effectiveness, experimental methods induced by corticosteroids and ethanol are of particular importance.

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