Denis O. Pakhmurin

Antibiotic therapy is losing effectiveness against resistant pathogens like Staphylococcus aureus (SA) strains with reduced susceptibility to vancomycin (VMN), particularly in bone infections where drug delivery is difficult. Hyperthermia has emerged as a potential alternative, yet its specific impact on infected bone remains poorly understood. This study investigated the systemic and local effects of combining VMN with local thermal exposure on inflammation and regeneration in rat femurs locally infected with SA strain 209 P insensitive to VMN at an in vitro dose of 10 µg/mL. In vitro, SA growth was assessed after heating at 45 °C and 60 °C for up to 60 min. In vivo experiments involved six groups of rats that received local hyperthermia (45 °C) or thermal ablation (60 °C) for 20 min, with or without systemic VMN administration. At 45 °C, a bacteriostatic effect was achieved after 45–60 min, whereas 60 °C inhibited growth completely within 15 min. In post-hyperthermia at 45 °C, bone tissue showed a regenerative effect comparable to VMN alone and significantly reduced local inflammation. Unexpectedly, combined treatment partially weakened this regenerative benefit. Thermal ablation at 60 °C, while inducing bone regeneration, also elevated markers of systemic inflammation like blood leukocytosis and weight loss, which were partially mitigated by VMN. However, antibiotic showed a negligible effect on local inflammation and reparative processes at this higher temperature. The findings indicate that a single hyperthermia session has more biological promise than thermal ablation as a targeted approach to limit local and systemic inflammation in SA-complicated trauma. Nevertheless, the complex interactions between high temperature and antibiotics warrant further investigation to determine optimal combined therapeutic regimens for osteomyelitis.

Aim. To evaluate the response of mesenchymal stromal/stem cells (MSCs) in vitro and the status of bone cells (osteoblasts, osteocytes) during regeneration of the femoral bone after local thermoablation at 55–60 °C. Materials and methods. Morphology and viability (MTT assay) of human adipose tissue-derived MSCs were analyzed after incubation at 37 °C or 56 °C for 0–60 minutes. In vivo , a heating cuff was applied to the femur of anesthetized rabbits, and intraoperative thermoablation was performed for 30 minutes (bone marrow canal temperature: 55–60 °C). Bone tissue was histologically examined (hematoxylin – eosin and Einarson staining) immediately and 14 days post-treatment. Quantitative morphometry was implemented in ImageJ, with subsequent statistical analysis performed in R. Results . In vitro , signs of massive MSC death were observed after 15 minutes of heating; at 30 minutes, viable fibroblast-like cells were nearly absent. In vivo , local thermoablation caused direct death of osteoblasts and osteocytes, evidenced by morphological signs of apoptosis and necrosis, as well as impaired DNA/RNA synthesis. Morphological and molecular markers of cellular damage significantly increased by day 14 post-treatment. Conclusion. Moderate thermoablation (55–60 °C) exerts significant direct and delayed damaging effects on osteogenic cells, from stem to mature forms. These findings are relevant for combined treatment of tumor and metastatic bone lesions.

Introduction: Microbial biofilm formation on surgical implants significantly ex-acerbates implant-associated infections (IAIs) and often leads to implant failure. This study aimed to investigate the ex vivo effects of elevated temperature and antibiotics, or zinc in-corporation, on methicillin-resistant Staphylococcus aureus (MRSA) biofilm growth on ti-tanium (Ti)-based 3D plates fabricated by selective laser melting (SLM) and coated with a rough calcium phosphate (CaP) layer using the micro-arc oxidation technique. Materials and Methods: MRSA strain 43300 broth cultures were exposed to heating at 45°C for 60 min or 56°C for 30 min, with or without vancomycin (VMN). The control culture (250 microbial cells per 1 ml of isotonic sodium chloride) was maintained at 37°C. Ti-based discs (10 mm in diameter and 1 mm in thickness) with structured surfaces were coated with a CaP layer via micro-arc oxidation. The Ti-based samples were added to MRSA broth cul-tures for 2 hours, then transferred to fresh serum-free medium and incubated for 48 hours at 37 °C. Bacterial biofilm optical density on the CaP coating was quantified by computer mor-phometry after fixation and Gram staining. Results: A single ex vivo administration of VMN at its minimum inhibitory concentration (MIC, 2 μg/ml) was ineffective against MRSA biofilm growth. The therapeutic VMN con-centration (20 μg/ml) reduced the biofilm optical density by 17%. Heating of MRSA broth cultures showed no significant bacteriostatic effect, and combining hyperthermia with VMN did not enhance antimicrobial efficacy. In contrast, Zn-doped (~0.31 at.%) CaP coatings inhibited MRSA biofilm development at 37°C. The bacteriostatic effect (5%) exceeded that of VMN at MIC (0%) but was lower than that observed for the therapeutic VMN dose (17%). Discussion: The negatively charged CaP coating on titanium implants may promote MRSA biofilm formation through electrostatic interactions with bacterial cell surfaces. Hyperther-mia at 45°C or 56°C, either alone or in combination with vancomycin, showed limited effi-cacy, possibly due to bacterial thermotolerance and antibiotic resistance. Incorporation of zinc into CaP coatings demonstrated a modest bacteriostatic effect, suggesting potential as an adjunct strategy alongside conventional antibiotics such as vancomycin. Conclusion: Zn-doped rough CaP coatings on Ti-based implants show promise for use in bone tissue engineering, where there is a risk of implant-associated infection, owing to the bacteriostatic activity of zinc at low concentrations.

Aim. To study the ex vivo effect of high temperature exposure (55–56 °C) combined with vancomycin on culture behavior of pathogenic Staphylococcus aureus (S. aureus).Materials and methods. Liquid cultures of methicillin-resistant S. aureus (MRSA) strain 43300 were heated ex vivo at 55–56 °C for 0–60 min, either with or without vancomycin (20 μg/ml), followed by incubation at 37 °C up to 120 min. A control suspension (100 or 250 microbial cells per 1 ml of isotonic saline) was maintained at 37 °C. Then, cultures were seeded on solid agar medium, and colony-forming units (CFU) were calculated using computer morphometry after 48h growth. Each experimental subgroup (growth control, thermal ablation, antibiotic, and thermal ablation + antibiotic) included at least three replicates.Results. A semi-lethal heat exposure time (LD50) of 12.25 min was determined for a liquid microbial culture at 100 cells/ml. When the density was increased to 250 cells/ml, 30 min thermal ablation (55–56 °C) was insufficient for MRSA growth suppression. Vancomycin (20 μg/ml) alone did not affect CFU output. However, combined heat and antibiotic treatment resulted in 28% bacteriostatic effect (p < 0.001) on agar medium.Conclusion. The study revealed a bacteriostatic effect of combined use of high-temperature exposure with vancomycin, which were ineffective when used separately. The obtained results have practical significance for reconstructive surgery of bone tissue, but require additional studies to clarify the mechanisms of the discovered phenomenon.

Introduction. Bone tumors are heterogeneous group of skeletal neoplasms characterized by frequent recurrences, aggressive clinical course and low survival rates. The development of new treatment methods continues to pose pressing challenges. Radical intraoperative thermal ablation (RIT) using high-temperature exposure is emerging as a new and promising strategy for organ-preserving treatment. This study focuses on the effect of thermal ablation (TA) on tumors.Objective of the Study to assess the impact of TA (using the RIT method) on the viability of tumor cells.Material and Methods. The study included 8 patients with bone tumors. Tumors underwent TA at a temperature of 60 °C for 30 minutes ex vivo. Apoptosis was studied in tumor tissue samples before and after TA. Apoptosis was assessed using two methods: flow cytometry and the TUNEL assay.Results. The TA procedure developed by our scientific group represents a promising organ-preserving approach for treating malignant bone tumors. A temperature regime of 60 °C for 30 minutes was effective in initiating tumor cells death. This was confirmed by two independent methods – flow cytometry and TUNEL assay – which demonstrated a significant increase in the number of apoptotic cells immediately following the procedure and a notable rise in the number of cells exhibiting signs of late apoptosis one hour post thermal ablation. Therefore, the collected data confirm a pronounced antitumor effect immediately after implementing RIT.Conclusion. The findings confirm that RIT is a viable organ-preserving method for treating bone tumors, meriting further investigation to expand its application in clinical practice.

BACKGROUND: Thermal ablation is a promising method for the treatment of bone tumors. However, to maximize the potential of this method, it is important to select the optimal dose/time ratio of high temperature exposure. AIM: The aim of the study was to evaluate in vivo the response of rabbit bone tissue during early recovery (days 3 and 7) after local intraoperative hyperthermic ablation at 55–60°C in the medullary canal. MATERIALS AND METHODS: The study used 6 outbred rabbits aged 15 weeks and weighing 3–4 kg. Animals were removed from the experiment on days 3 and 7 after local thermal ablation of the femoral diaphysis. Microscopic visual examination of bone tissue sections was performed after standard hematoxylin and eosin staining. In addition, the morphometric measurements were performed to determine area of non-mineralized bone matrix (Mallory staining); absorbance and area of osteoblast cytoplasm; absorbance and area of osteocyte nuclei (Einarson staining for nucleic acid detection). The results obtained were compared with those of the femur of the contralateral limb, which was not subjected to direct hyperthermia and served as a control. R-Studio, a free development environment for R programming language, was used to statistically process the data. RESULTS: After high temperature exposure, histological sections of bone tissue stained with hematoxylin and eosin showed evidence of cellular abnormalities such as denucleated osteocytes and empty bone lacunae. Mallory staining showed no evidence of a negative effect of local thermal ablation on the intercellular bone matrix. Morphometry of Einarson stained sections showed an increase in osteoblast area on day 7 after exposure, with a decrease in their synthetic activity, signs of which were observed as early as day 3 of the experiment. There was also a decrease in the area and absorbance of osteocyte nuclei in the diaphysis of thermally ablated bones by day 3 after exposure. However, on day 7 after exposure, the area of nuclei in mature bone cells did not differ from the corresponding value in the contralateral limb. Considering the decrease in nuclei absorbance, the described changes may indicate colliquative necrosis of osteocytes. CONCLUSIONS: Local intraoperative thermal ablation of the femoral diaphysis in rabbits at a marrow canal temperature of 55–60℃ reduces the absorbance of osteoblast cytoplasm and osteocyte nuclei in the early recovery period, reflecting damage to the organelles of bone cells and disruption of metabolic processes in these cells. However, there was evidence of remodeling of the damaged area, presumably caused by migration of endosteal and periosteal cells from the metaphysis, which had not been exposed to direct hyperthermia. The results obtained may be useful for choosing the optimal regime (dose and duration) of high temperature exposure to bone tissue tumors, given the higher sensitivity of malignant cells to heating.

Introduction. Difficult diagnosis, aggressive progression, and absence of an increase in treatment effectiveness (especially in patients with osteosarcoma and Ewing sarcoma) make malignant tumors of the bones a significant medical problem. Use of traditional therapy methods is often associated with development of drug resistance and frequent recurrences. Therefore, there is a need for new treatment strategies and overcoming of the limitations of traditional methods through their improvement, combination or substitution to achieve higher survival, lower recurrence rate, and wider availability for patients with bone tumors.Aim. To present a clinical observation of the use of a new method of radical intraoperative thermal ablation for bone tumors, assess exposure tolerance, long-term oncological and functional results.Clinical observation. This article presents a clinical case of the use of a new radical treatment method for a patient with tibial osteosarcoma who underwent intraoperative thermal ablation using a new domestic complex of local hyperthermia “Phoenix-2”.Results. A session of intraoperative thermal ablation was performed in the operating room under anesthesia directly in the wound for 48 minutes. Access to the tumor-affected bone was performed with the standard oncological approach (en-block), the bone was not transected or removed. A constant heating mode of 90 °С was used, assuming a gradual increase in temperature. Thus, 60 °С reached 20 minutes, and the temperature at the end of high-temperature exposure was 78 °С (inside the medullary canal). The operation time was 2 hours 30 minutes. The postoperative period proceeded without complications. After 38 months of follow-up, examinations did not reveal any recurrence or progression in the patient.Conclusion. The technique of radical intraoperative thermal ablation using a local hyperthermia complex Phoenix-2 is effective and allows to widen the toolkit of treatment methods for patients with bone tumors. It can serve as an alternative or addition to organ-preserving surgical treatment without costly reconstruction stages.

Purpose: to study the changes in the temperature outside and inside the long tubular bones of animals under the influence of different temperature regimes for a given time.Material and Methods. The experiments were conducted using fresh frozen pig long bones. The heating was carried out using surface heaters, the temperature of which was determined by the selected mode for 1 hour; fixation of temperature values was carried out every minute. Four heating modes were used: 3 modes of constant heating (60, 70, 80 °C) and the maximum heating mode, in which no special temperature limit was set, so heating was carried out up to 120 °C.Results. During the first 10 min of heating, a rapid rise in temperature occurred. The temperature increase rate on the outer surface outstripped the temperature increase rate on the inner surface of the bone, thus leading to a significant temperature difference. Further, there was a slowdown in the rate of temperature increase, which led to a gradual convergence of the temperature values inside and outside the bone, followed by temperature stabilization at a stable level (plateau), which was different for the studied areas. During this period, the temperature difference was 3.5–6 °C and it remained at this level until the end of the study. At a constant heating mode (60/70/80 °C), temperature stabilization occurred at the level of 55/61/70 °C in the center of the medullary canal and at the level of 58/67/75 °C under the heater, respectively. The period before reaching the stabilization temperature was 30–40 min. The stable temperature levels both inside and outside the bone were below the temperature stabilization level of the heater. Therefore, to achieve the planned temperature in the center of the bone to its outer surface, it is necessary to apply a high temperature, i.e., a downward temperature gradient is formed: the heater stabilization temperature – the temperature on the outer surface – the temperature inside the medullary canal. Increasing the exposure temperature can shorten the heating period, but increase the temperature difference during the heating period (up to 25 °C in the fifth minute of heating when using the maximum heating mode).Conclusion. To ensure reaching the required temperature (60°C) within a short time (15–20 min) while maintaining optimal temperature parameters, it was proposed to develop variable temperature modes that would combine the initial use of the maximum heating mode until reaching the desired temperature in medullary cavity, followed by switching to a constant temperature mode, which allowed maintaining the achieved temperature level during therapeutic exposure.