Comparative evaluation of the antibacterial activity of bone allografts impregnated with various antibiotics

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INTRODUCTION

Chronic osteomyelitis represents one of the most complex and still unresolved problems in modern septic surgery and orthopedics [1, 2]. This disease, characterized by infectious inflammation of bone tissue with progressive destruction and formation of necrotic foci, involvement of the periosteum and surrounding soft tissues, is associated with prolonged treatment, high recurrence rate (20-35%), significant deterioration in patients’ quality of life, and substantial socio-economic burden [3, 4]. According to current concepts, chronic osteomyelitis develops as a result of ineffective treatment of acute (hematogenous) infection or as a complication of injuries, surgical interventions, and systemic diseases such as diabetes mellitus [2].

In recent years, research efforts have focused on a deeper understanding of the etiopathogenesis of chronic osteomyelitis, as well as on improving diagnostic and therapeutic approaches. A crucial prerequisite for successful treatment of patients with this pathology is radical surgical debridement aimed at excision of all nonviable tissues and sequestra and removal of infected implants. However, since even the most meticulous debridement cannot guarantee complete eradication of the pathogen, local antibacterial therapy becomes of key importance [5]. Achieving high local concentrations of antibacterial agents within the bone defect, far exceeding those attainable by systemic administration, makes it possible to overcome bacterial tolerance in residual microfoci of persistence, including bone marrow microabscesses, the lacunar-canalicular network of bone, and established biofilms. This ensures effective pathogen eradication and creates conditions for subsequent reparative regeneration [3, 6, 7, 8].

From a biological standpoint, bone grafts represent the most promising carriers for antibiotic delivery. Autologous bone, although considered the gold standard of osteoplasty, demonstrates unfavorable pharmacokinetics when simply mixed with antibiotic powder, with the release of the majority of the drug occurring within the first 1-3 days [9]. This creates the risk of both local cytotoxicity and rapid loss of therapeutic concentrations [10]. The use of allogeneic bone grafts modified with dedicated drug-delivery systems appears to be a more advanced approach. Studies have shown that the impregnation of allograft bone with biodegradable depot systems or the application of composite coatings enables controlled antibiotic release, approximating zero-order kinetics, over several weeks, thereby combining effective infection eradication with the stimulation of proper bone regeneration [11, 12]. In addition, there are various biomaterials with different properties, which can be adapted to achieve controlled antibiotic release and maintain concentrations above the minimum inhibitory level [13]. Local delivery systems based on polymers or calcium salts, despite a certain degree of effectiveness, have several significant limitations, including the lack of biodegradability and osteogenic potential, low mechanical strength, and unsatisfactory pharmacokinetic profiles. This underscores an urgent clinical need for the development of an osteoplastic biodegradable material with prolonged antimicrobial activity. Among the most promising options is a purified allogeneic bone-based material characterized by a standardized, preprogrammed duration of the elution of various antibacterial agents.

The aim of the study — to perform a com-parative evaluation of the duration of the antibacterial activity of allografts impregnated with different antibiotics against Gram-positive and Gram-negative bacteria.

METHODS

Bone tissue was manually cut under sterile conditions into cubes measuring 5×5×5 mm (Figure 1). Purified and delipidized bone allo-grafts were prepared using a patented method (RU 2722266 C1). Microphotographs of the tissue samples were obtained using a MINI SEM A5100 scanning electron microscope (PRC).

 

Figure 1. Osteoplastic material from purified allogeneic bone:

a — purified bone blocks;

b — micrograph of bone tissue after purification and lyophilization (scanning electron microscopy). Mag. ×400

 

The impregnation of the blocks was performed under standard conditions using a vacuum of 7-10 hPa for 60 mins. Four antimicrobial agents were used for comparative analysis: vancomycin (Kraspharma, Russia), aztreonam (ELFA, Russia), meropenem (ELFA, Russia), and fosfomycin (Kraspharma, Russia). Each group of samples (n = 4 per group) was treated in a solution with a concentration of 10%, i.e., 1 g of antibiotic was dissolved in 10 ml of distilled water.

The impregnated blocks were frozen at -80 °C for 24 hrs, which prevented the disruption of the carrier material structure and preserved the antibacterial properties of the samples. Lyophilization was performed under the following temperature conditions: from -40 °C (primary drying or sublimation) to +40 °C (secondary drying or desorption), with gradual temperature increases (+20 °C every 5-16 hrs), ensuring gentle moisture removal and preservation of structural integrity. The duration of lyophilization ranged from 24 to 48 hrs depending on sample volume and size, until a residual moisture content of less than 5% was achieved, as determined using a moisture analyzer.

Reference strains of Gram-positive and Gram-negative bacteria used to assess the presence and duration of antibacterial activity of the obtained samples were selected according to the antimicrobial spectrum of the antibiotic used for impregnation: vancomycin — S. aureus ATCC 29213 (MSSA) and S. aureus ATCC 43300 (MRSA); aztreonam — K. pneumoniae ATCC 33495 and P. aeruginosa ATCC 27853; meropenem — S. aureus ATCC 29213 (MSSA), K. pneumoniae ATCC 33495, and P. aeruginosa ATCC 27853; fosfomycin — S. aureus ATCC 29213 (MSSA), S. aureus ATCC 43300 (MRSA), K. pneumoniae ATCC 33495, and P. aeruginosa ATCC 27853. For this purpose, antibiotic-loaded samples were placed in sterile tubes containing 3 ml of saline. Samples without antibiotics served as negative controls. Each sample was placed in a sepa-rate tube and incubated at 37 °C for 18-24 hrs. A suspension of reference bacterial strains adjusted to 0.5 McFarland was evenly spread over the surface of Mueller-Hinton agar using a cotton swab. For the assessment of antibacterial activity of fosfomycin-loaded samples, Mueller-Hinton agar supplemented with glucose-6-phosphate (25 mg/l) was used. Ten microliters of the 24-hour incubation supernatant from the tubes containing the samples were applied to the bacterial lawn in triplicate and incubated for 24 hrs at 37 °C. The formation of a zone of growth inhibition at the site of supernatant applica-tion was considered indicative of a sufficient antibiotic concentration to suppress bacterial growth. The supernatant was replaced daily with 3 ml of fresh sterile saline, and the procedure was repeated daily until antibacterial activity ceased and no growth inhibition zone was observed.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 9.0. Given the small sample size (n = 4 per group) and the associated limitations in the statistical power of normality tests, nonparametric methods were applied for intergroup comparisons. The Kruskal-Wallis test was used, followed by Dunn’s post-hoc test with Bonferroni correction for pairwise comparisons. Quantitative data are presented as mean ± standard deviation (M±SD). This format of descriptive statistics was chosen due to the low variability within the samples and to ensure the comparability of the results with data from other studies. A p value < 0.05 was considered statistically significant.

RESULTS

The analysis of changes in the mass of the tested specimens demonstrated that the applied standard vacuum impregnation protocol ensured reproducible saturation of bone tissue with antibiotics. The greatest increase in mass was observed after impregnation with aztreonam, whereas the smallest increase was noted after impregnation with fosfomycin (Table 1). The specimen groups differed significantly in terms of relative mass change (p = 0.003). Post-hoc analysis confirmed a statistically significant difference only between the aztreonam and fosfomycin groups (p = 0.00381). The difference between the aztreonam and vancomycin groups approached statistical significance (p = 0.069), while no statistically significant differences were observed in the remaining pairwise comparisons after correction.

 

Table 1

Changes in the mass of bone allografts impregnated with antimicrobial agents

Agent	n	Before loading, g	After loading, g	Δ mass (%)
Vancomycin	4	0.289±0.006	0.319±0.007	10.49±3.34
Aztreonam	4	0.205±0.004	0.303±0.007	47.65±4.31
Meropenem	4	0.245±0.006	0.309±0.006	26.05±3.89
Fosfomycin	4	0.185±0.004	0.195±0.003	5.23±3.22

Δ mass — relative mass change in percentage.

 

Impregnated samples of the bone substitute material exhibited antibacterial activity of varying duration against reference bacterial strains, in contrast to the control samples without additional antibiotic loading, which did not demon-strate activity against the tested pathogens.

During the first 24 hrs of incubation, a substantial amount of the antibacterial agent was released from the samples. Accordingly, the largest diameters of bacterial growth inhi-bition zones were observed after 24 hrs of incubation compared with subsequent incuba-tion periods (Figures 2, 3).

 

Figure 2. Antibacterial effect of allografts with vancomycin, Day 1. Zones of growth inhibition of staphylococcal reference strains: S. aureus ATCC 29213 (MSSA) and S. aureus ATCC 43300 (MRSA)

 

Figure 3. Antibacterial effect of allografts with aztreonam, Day 1. Zones of growth inhibition of reference Gram-negative strains: P. aeruginosa ATCC 27853 and K. pneumoniae ATCC 33495

 

With each subsequent replacement of the saline solution, a progressive daily reduction in the diameter of the inhibition zones was recorded until their complete disappearance. Differences in the duration of antibacterial activity of bone allografts were observed depending on the antibiotic used for impregnation (Table 2).

 

Table 2

Duration of antibacterial activity of impregnated bone allografts

Agent	Strain	Duration, d
Vancomycin	S. aureus ATCC 29213 (MSSA)	8
	S. aureus ATCC 43300 (MRSA)	8
Aztreonam	K. pneumoniae ATCC 33495	6
	P. aeruginosa ATCC 27853	2
Meropenem	S. aureus ATCC 29213 (MSSA)	3
	K. pneumoniae ATCC 33495	4
	P. aeruginosa ATCC 27853	2
Fosfomycin	S. aureus ATCC 29213 (MSSA)	8
	S. aureus ATCC 43300 (MRSA)	7
	K. pneumoniae ATCC 33495	6
	P. aeruginosa ATCC 27853	6

 

Samples impregnated with vancomycin demonstrated the longest antibacterial activity against S. aureus, showing comparable efficacy against both methicillin-susceptible and methi-cillin-resistant strains. Allografts loaded with meropenem exhibited the longest antibacterial effect against K. pneumoniae ATCC 33495, lasting up to 4 days. Impregnation with aztreonam pro-vided antibacterial activity against K. pneumoniae ATCC 33495 for up to 6 days. Samples impregnated with fosfomycin were characterized by a broad spectrum of antibacterial activity, with the maximum duration observed against S. aureus ATCC 29213; activity against Gram-negative pathogens persisted for up to 6 days. Notably, samples impregnated with aztreonam and meropenem showed shorter durations of activity against P. aeruginosa compared with fosfomycin.

DISCUSSION

The fundamental mechanisms underlying the pathogenesis of chronic osteomyelitis, inclu-ding the formation of bacterial biofilms, intracellular persistence of pathogens, and impairment of local blood supply with the development of avascular sequestra, create almost insurmountable barriers to systemic antibacterial therapy [6, 14, 15]. This indicates that, in addition to radical surgical debridement, a key prerequisite for the successful treatment of chronic osteomyelitis is the achievement of high local concentrations of antimicrobial agents directly at the site of infection.

At present, both experimental studies and clinical practice focus on the impregnation of bone substitute materials with well-proven antibiotics, such as glycopeptides, cephalospo-rins, and others [16, 17, 18]. The majority of published studies have been conducted using vancomycin. However, its rapid elution results in limited retention within bone tissue [19, 20]. Similarly, teicoplanin has been evaluated in bone grafts, but the amount released was insufficient, with drug concentrations falling below 10 µg/ml by day 5. In addition, cancellous bone allografts have been shown to provide adequate elution of fusidic acid, but not of teicoplanin or dalbavancin [21, 22].

Additional processing of the material, for example, polymer surface coating or the use of co-adjuvants mixed with the allograft, may affect antibiotic release [23, 24]. The author-developed impregnation technique is based on a deliberately “minimalist” approach: standard vacuum loading in an aqueous medium (7-10 hPa for 60 mins) without polymer coatings or co-adjuvants. This protocol ensures:

• uniform penetration of the solution into the intertrabecular network through air displacement and capillary filling;
• preservation of open porosity and the native three-dimensional architecture, which are critical for osteoconduction;
• absence of extraneous matrix components that could modify the local biological tissue response or unpredictably alter elution kinetics.

As a result, even basic vacuum impregnation produces a high initial drug release (as evidenced by maximal inhibition zones on day 1) and an antibiotic-specific duration of antibacterial activity: up to 8 days for vancomycin against MSSA/MRSA and up to 6 days against several Gram-negative pathogens when fosfomycin or aztreonam is used. Notably, the greatest mass loading of the bone substitute material (aztreonam) did not always result in the longest duration of antibacterial activity, highlighting that not only the amount of the loaded drug but also pathogen-specific susceptibility plays a crucial role. In addition, the molecular weight of the antibacterial agent should be taken into account, as well as potential differences in drug-bone matrix binding, which may further influence the final degree of bone material saturation.

At the same time, the short duration of activity against P. aeruginosa (2 days for meropenem and aztreonam) indicates the need for further optimization of the impregnation process within the framework of the basic approach (through variations in exposure time and pressure, solution concentration, and selection of molecular combinations) as well as the importance of rational antibiotic selection for empirical therapy. In an in vitro study, H. Winkler et al. demonstrated that vancomycin elutes from cancellous bone significantly more effectively than tobramycin [25]. E. Witsø et al. investigated the release kinetics of antibiotics from cancellous bone and found that rifampicin was released from allografts for up to 21 days, whereas benzylpenicillin, cephalothin, and dicloxacillin were released for up to 7 days [26].

It is important to note that rational selection of antibacterial therapy is possible only after the identification of the causative pathogen and assessment of its antibiotic susceptibility. Therefore, in the absence of microbiological data, only empirical antibacterial therapy can be prescribed, targeting a broad spectrum of the most common orthopedic infection pathogens [27]. Furthermore, treatment is complicated by the increasing prevalence of strains resistant to multiple classes of antibacterial agents, making the search for new approaches to antibiotic application increasingly relevant [28]. Although achieving high local antibiotic concentrations is one of the key factors for successful eradication of infection, it should be kept in mind that high drug doses may exert cytotoxic effects [29, 30].

Published experimental and clinical data reported by international researchers demonstrate high efficacy of such materials in the treatment of orthopedic infections. Previously, in our clinical study, we identified significant differences in vancomycin concentrations in drainage fluid between patient groups treated with different bone substitute materials (group 1: β-tricalcium phosphate and hydroxyapatite; group 2: the original osteoplastic material developed by the authors). The study showed that the use of the original osteoplastic material impregnated with vancomycin resulted in higher local antibiotic concentrations [31].

Study perspectives

The development of new bone substitute materials based on antibiotic-impregnated bone allografts appears highly promising. Such materials should be biodegradable and biocompatible, capable of stimulating new bone regeneration, providing mechanical support to the affected long bone during the healing period, and ensuring prolonged drug release. The results of the present study indicate the need to develop methods for prolonging the release of antibacterial agents from bone substitute materials, as well as to test other potentially effective antimicrobial agents in combination with antibiotics in order to overcome possible pathogen resistance.

Study limitations

Antibacterial activity of the obtained material samples was tested only against reference strains; the in vitro model does not fully reproduce in vivo conditions; and the kinetics of changes in antibacterial drug concentrations in the incubation solutions were not assessed.

CONCLUSION

The developed technique was shown to ensure the antibiotic loading of the bone substitute material and drug release over several days, with the most prolonged effect observed following impregnation with vancomycin and fosfomycin.

DISCLAIMERS

Author contribution

Gordina E.M. — conducting research, statistical data processing, drafting and editing the manuscript.

Bozhkova S.A. — study concept and design, draftingand editing the manuscript.

Antipov A.P. — data acquisition, analysis and interpretation, conducting research.

All authors have read and approved the final version of the manuscript of the article. All authors agree to bear responsibility for all aspects of the study to ensure proper consideration and resolution of all possible issues related to the correctness and reliability of any part of the work.

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

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

Ethics approval. Not applicable.

Consent for publication. Not required.

Use of artificial intelligence. No generative artificial intelligence technologies were used in the preparation of this manuscript.

About the authors

Ekaterina M. Gordina

Vreden National Medical Research Center of Traumatology and Orthopedics

Author for correspondence.
Email: emgordina@win.rniito.ru
ORCID iD: 0000-0003-2326-7413
SPIN-code: 9647-8565

Cand. Sci. (Med.)

Russian Federation, St. Petersburg

Svetlana A. Bozhkova

Vreden National Medical Research Center of Traumatology and Orthopedics

Email: clinpharm-rniito@yandex.ru
ORCID iD: 0000-0002-2083-2424
SPIN-code: 3086-3694

Dr. Sci. (Med.), Professor

Russian Federation, St. Petersburg

Alexander P. Antipov

Vreden National Medical Research Center of Traumatology and Orthopedics

Email: a-p-antipov@ya.ru
ORCID iD: 0000-0002-9004-5952
SPIN-code: 8997-8235
Russian Federation, St. Petersburg

References

Supplementary files