UV-spectrometric study of antibiotic long-term release by different osteoplastic materials used in dentistry

Abstract

Nowadays, the development and improvement of osteoplastic materials containing antibiotics as long-term controlled-release systems represent a promising direction in bioactive dental practice. To evaluate the relative capacity of different osteoplastic materials used in oral surgery and clinical dental practice to accumulate and subsequently release antibiotics after exposure to antibiotic solutions, using spectrometric analysis. 3 osteoplastic materials "Biomin GT" ("Rapid", Ukraine), "easy-graft CLASSIC 150" ("Regenity Biosciences", USA), and "InterOss" (1–2 mm) ("SigmaGraft", USA) were immersed in 4 antibiotic aqueous solutions (5.0% ofloxacin, 5.0% metronidazole, 4.0% gentamicin sulfate, and 5.0% chloramphenicol) during 24 h and dried later. UV-spectrometric studies were performed to quantify the release of antibiotics into sterile saline on the 2nd, 5th, 10th, and 30th day. Evaluated osteoplastic materials released different volumes of the antibiotics. Samples of osteoplastic materials (after prior immersion in antibiotic solutions) can serve as systems for long-term release of antibacterial substances (up to 30 days). However, the levels of accumulation and release differ across materials and active substances. The highest levels of antibiotic release (and accumulation, respectively) were observed for "Easy GRAFT" – 6.07 ± 5.84 (M = 3.49) mg per 1 g of osteoplastic material. Among the antibiotics, the highest rate was for gentamicin sulfate – 10.67 ± 3.57 (M = 8.67) mg per 1 g, whereas chloramphenicol showed 3.11 ± 0.48 (M = 3.01) mg per 1 g of material. The interaction between osteoplastic material and various antibiotics requires further clarification and verification at both the experimental and clinical levels. Release curves for the studied antibiotics from various osteoplastic materials were obtained over a one-month period. The ability of dental osteoplastic materials to accumulate antibiotics within their mass has been demonstrated.

Introduction

Osteoplastic materials (bone substitutes) are widely used in oral and maxillofacial surgery for the reconstruction of bone defects and for supporting bone regeneration during implant therapy and reconstructive procedures. Dentistry ranks third among fields worldwide in overall consumption of these products. Typically, any surgical intervention involving the implantation of osteoplastic material into a bone defect is followed by a prophylactic course of antibacterial therapy, most often with broad-spectrum agents (Kwiecień et al. 2022; Posadowska et al. 2015; Tsuperyak and Mochalov 2022). Some routine procedures in oral surgery and dentistry (such as dental implantation, bone-grafting procedures, and sinus lifts) carry a high risk of bacterial contamination of the bone wound, which may lead to long-lasting inflammation and rejection of the implanted devices. That is why creating and improving bone osteoplastic materials with long-lasting antimicrobial qualities is the task for modern pharmaceutical chemistry and clinical practice. This concept is not entirely new; however, several aspects remain insufficiently understood and require further investigation before its widespread implementation in clinical practice. After impregnation or immersion of the material in antibiotics, the material must for a long time release the active substance in a sufficient volume, above/equal to the minimum inhibitory concentration, and even more, in the minimum concentration that suppresses the formation of bacterial biofilm. From the experimental studies we know that the introduction of osteoplastic material into a bone wound can dramatically change the metabolic conditions of the surrounding bone tissue, disrupt microcirculation and regeneration processes, create zones of potential colonization by various microorganisms, and as a result, prescribed antibiotics with a standard method of administration may not reach the bone wound. Importantly, pre-soaking/impregnation of graft granules in antibiotic solutions is used in some surgical settings as an off-label attempt to increase local antibiotic availability; however, there is no consensus regarding optimal drug concentration, loading time, or the most appropriate release medium for dental indications. Under current conditions, several osteoplastic materials incorporating the aforementioned modifications are already available on the medical market. However, there is limited research on the use of such medical products in dentistry. It has been established that osteoplastic materials of different origins and compositions exhibit significant differences in the properties of accumulation and long-term release of various antibacterial agents. The specified characteristics are significantly influenced by the composition of the material itself, its natural or artificial origin, the presence of organic/inorganic components, the chemical composition of the material itself, the operational properties of the product (hardening, plasticization, etc.), porosity and texture of the product, solubility in water and human body fluids, the ability to resorb over time, the presence of a protein component that is capable of binding to antibiotics of certain groups, etc. (Cyphert et al. 2021; Tuleubayev and Rudenko 2021).

Nowadays, some of wide-used antibiotics may present an interest for application for dental osteoplastic materials modifications. Gentamicin is a bactericidal aminoglycoside antibiotic with a broad spectrum of action. It is effective against a wide range of aerobic gram-negative bacteria and gram-positive species. Particular importance is its potential activity against pathogens of the conditional ESKAPE group. Due to its broad spectrum of action, gentamicin is used to treat more than 40 different clinical conditions, including bacterial sepsis, peritonitis, meningitis, urinary and respiratory tract infections, eyes, ears, bones, surgery and others, which confirms its importance. Results from studies on oral microbiota in infectious and inflammatory processes indicate its sensitivity to aminoglycosides, particularly gentamicin. Ofloxacin is a synthetic antibacterial agent belonging to the fluoroquinolone class. It is classified as a second-generation fluoroquinolone. It is commonly prescribed for the treatment of bacterial infections caused by both Gram-positive and Gram-negative bacteria, including, but not limited to, urinary tract infections, sexually transmitted diseases, tuberculosis, pneumonia, and skin and soft tissue infections. Ofloxacin exhibits both bactericidal and bacteriostatic activity against pathogenic and opportunistic microorganisms present in the oral cavity. Chloramphenicol is a broad-spectrum antibiotic that has bacteriostatic activity against many Gram-positive and Gram-negative cocci, as well as anaerobic bacteria. Today, chloramphenicol is used to treat typhoid fever, meningitis, yersiniosis, chlamydia, eye infections, purulent and burn wounds, and other diseases. It has been shown to effectively inhibit the growth of pathogenic and opportunistic microorganisms identified in the oral cavity. Metronidazole is a synthetic nitroimidazole, which is widely used in medicine and veterinary medicine due to its broad-spectrum antibacterial and antiprotozoal action. Metronidazole has been used to treat various infections for over 45 years and is still used to treat amebiasis, giardiasis, and trichomoniasis. It is especially effective against anaerobic bacterial infections caused by Fusobacteria, Bacteroides, and Clostridia. Metronidazole is included in treatment protocols for anaerobic bacterial infections and purulent oral lesions. Metronidazole is also included within the composition of certain collagen osteoplastic materials (for example, "Kolapan", Russian Federation).

Today, various methods are used to identify antibiotics in liquids, including liquid chromatography, mass spectrometry, UV–visible spectrometry, spectrophotometry, and fluorometry. Spectrometric and spectrophotometric detections of gentamicin and its compounds are carried out in the ultraviolet range from 200 to 260 nm, ofloxacin from 225 to 332 nm, chloramphenicol from 270 to 310 nm, and metronidazole from 230 to 319 nm.

The abovementioned modifications of osteoplastic materials can directly affect the physical and chemical properties of the products themselves, as well as the reactions of the patient’s body that occur during the resorption of the material and its replacement by its bone tissue (inflammatory response, migration of blood cells and inflammation, growth of blood vessels, proliferation and functioning of osteoblastic stem cells). Therefore, research aimed at the development and clinical implementation of osteoplastic materials with antibacterial properties is a relevant direction for modern medicine (Tsuperyak and Mochalov 2022; Wassif et al. 2021). However, there is still insufficient information regarding comparable modifications of osteoplastic materials specifically for dentistry, oral surgery, and maxillofacial surgery. Moreover, there are currently no data on the transformation of osteoplastic material, specialized for dentistry and oral surgery, into a controlled drug release system. This study was designed as an exploratory, comparative screening experiment in order of obtainin the new data about the relative capacity of different osteoplastic materials to accumulate antibiotics and subsequently release.

The study aims to evaluate the relative capacity of different osteoplastic materials used in oral surgery and clinical dental practice to accumulate and subsequently release antibiotics after exposure to antibiotic solutions, using spectrometric analysis.

Materials and Methods

The design of this experimental study represents a screening approach and it was dedicated to compare antibiotic loading and release behavior in contact with different osteoplastic matrices rather than to establish definitive pharmacokinetic models.

Preparation of the osteoplastic materials

To carry out the planned experiment, three osteoplastic materials used in dentistry were selected: synthetic biphasic material "Biomin GT" (LLC "Center for Scientific and Technical Services "Rapid", Ukraine), synthetic polylactide/polyglycolide-coated "easy-graft CLASSIC 150" ("Regenity Biosciences", USA), and natural bone material of bovine origin "InterOss" (1–2 mm) ("SigmaGraft", USA). "Biomin GT"—synthetic biphasic osteoplastic material, which contains calcium hydroxyapatite and beta-tricalcium phosphate in an equivalent ratio. The material was created for dental and oral surgical needs. It belongs to partially resorbable materials. It is extruded in granular form. Easy-graft CLASSIC 150—synthetic osteoplastic material, which contains 90% beta-tricalcium phosphate and 10% organic component (copolymer of polylactide and polyglycolide). The material is also available in granular form. Every granule is a porous, rounded structure covered with an organic layer, due to which the granules can stick together after using a special solvent and being introduced into biological fluids/water. The material is resorbable (up to a year). "InterOss"—dried natural deproteinized osteoplastic material of bovine origin in the form of crumbs of various sizes. The material exhibits high porosity. The material is resorbable (up to a year).

Selection of antibiotics

Four antibiotic aqueous solutions were selected for soaking of the materials: 5.0% solution of ofloxacin, 5.0% solution of metronidazole, 4.0% solution of gentamicin sulfate (LLC "VF "Bazalt", Ukraine), and 5.0% solution of chloramphenicol ("Levomican", "O.L.KAR.-AgroZooVet-Service", Ukraine). The selected list of antibacterial agents was justified by the fact that, for soaking osteoplastic materials, injectable forms were used, which do not degrade quickly at room or body temperature. Additionally, selected antibiotics exhibit both bactericidal and bacteriostatic effects against pathogenic microorganisms in the human oral cavity.

Weighed dose of each material (0.3 g) in plastic containers with a lid was immersed in 1.0 ml of antibiotic solution for 24 h at room temperature (20–24 °C), and control samples were filled with sterile saline (sterile isotonic sodium chloride solution in water for injection, Yuriya-Farm, Ltd, Ukraine) (Fig. 1). Each plastic container was tightly closed with a lid during the soaking time. The temperature regime in the laboratory was maintained by an automatic climate control system. After 24 h, the liquid contents of each container were aspirated using a micropipette, and the containers with moistened material were placed in a thermostat at 37.0 °C for 48 h to dry completely. Subsequently, 5.0 ml of sterile saline was added to each container with the material. The containers were stored in a dark environment at room temperature. On the 2nd, 5th, 10th, and 30th day of exposure, the liquid contents of the container were aspirated using a micropipette, and 5.0 ml of sterile saline was added again. The aspirated contents were sent for analysis of antibacterial residues. The collected liquid contents of the containers, after being appropriately labeled, were stored in a dark refrigerator at 4.0 °C to 6.0 °C. The total number of experimental systems was 60, and each system was prepared as a single experimental sample.

Fig. 1.

Scheme samples preparation during the experiment. Immersion of bone plastic material in different solutions: OFL – ofloxacin, MET – metronidazole, GEN – gentamicin, CHL – chloramphenicol, SAL – sterile saline

Experimental design and sample accounting

A total of 15 containers were prepared (3 materials × [4 antibiotic solutions + saline control]). Each container yielded eluates at four sampling points (days 2, 5, 10, and 30), resulting in 60 eluate samples in total. For each condition/time point, one eluate sample was available (n = 1 independent specimen per condition/time point); each eluate was measured in triplicate as technical (instrument) replicates.

Sink conditions

Elution was performed in 5.0 mL saline with complete medium replacement at each sampling point to maintain concentration gradients. The maximum measured eluate concentration across all groups was ≤ 0.454 mg/mL, which is far below the loading concentration of the antibiotic solutions (40–50 mg/mL for 4–5% solutions), supporting that sink conditions were reasonably approximated in this screening setup.

This stage of the study was performed at the Laboratory of the R&D Center of Jendental-Ukraine Ltd (Kyiv, Ukraine).

Determination of active substance residues using spectrometry

This stage of the research was carried out on the basis of the Laboratory of experimental optics at the Department of Optics of the Faculty of Physics of the State University "Uzhhorod National University" (responsible person – Senior Lecturer V. Yu. Biganych, Uzhhorod, Ukraine) using a compact two-channel scanning spectrometer SL40-2-1024USB (SOL Instruments, Germany), which operates in the light emission spectrum from 200 to 1070 nm. Specialized software, "SpectraSP: spectral instruments," was used to initially register the transparency spectra of the solutions; the spectra baseline was registered before every test.

The source of light was an ultraviolet lamp "DRT 240" (LLC "Lisma", RF) with an operating range of 200–400 nm, a radiation power of 240 W, and a light flux intensity of 24.6 lm. During the determination of the transparency of the tested solution, the latter was poured into a quartz (sapphire) cuvette with a diameter of 10.0 mm and a volume of 3.5 ml (JVLAB, China). After that, the cuvette was placed between the lamp and the spectrometer detector, and the solution’s transmission spectrum was scanned. Before each study, the cuvette was washed three times with bidistilled water, the transparent walls were wiped with a paper napkin moistened with 70.0% ethanol, and then dried. Each spectrometric measurement was performed three times, and the average values ​​were recorded for every liquid sample.

Obtaining the calibration curves of antibiotic solutions of different concentrations

For this purpose, a series of dilutions of the above-mentioned antibiotic solutions in bidistilled water was prepared using semi-automatic single-channel micropipette-dispensers "IKEME BIO" (China) with a variable volume, 20–200 μl and 100–1000 μl. The concentrations of the solutions were studied from 12.5 to 500 μg/ml. The study of the peaks of the light flux transmission by antibiotic solutions with different concentrations allowed us to determine the reference peaks of energy absorption and calculate calibration curves for further work.

Accordingly, for gentamicin sulfate solutions, transparency indicators in the range of 200 to 260 nm were used, with the decrease inversely dependent on the concentration of the active substance in the solution (Bidossi et al. 2020). Among the detected peaks, the value of the light wavelength of 250.54 nm was chosen as the reference, which allowed us to determine a linear dependence. Accordingly, a mathematical regression model was constructed (R² = 0,9587):

$$y=-\text{64,729}x+\text{7901,3}$$ 1

In chloramphenicol solutions, the localization of transparency peaks in the range from 270 to 315 nm was taken into account (Trivedi et al. 2015). In accordance with the transparency values ​​and data from previous studies, the value of the transparency of solutions for the wavelength of light 314.15 nm was chosen as a reference indicator. In this part of the spectrum, a linear dependence was observed. Accordingly, a mathematical linear model was constructed (R² = 0.9728), which was used to determine the concentration of the active substance:

$$y=-\text{205,19}x+29981$$ 2

In ofloxacin solutions, the difference in the light flux absorption peaks was investigated in the wavelength range of 285–300 nm (Olmo Alonso et al. 2024). To calculate the calibration graph, the solution transparency value at a wavelength of 286.40 nm was chosen, where a linear relationship was determined. According to the calculated transparency values, a mathematical model was calculated that reflected the detected dependence at (R² = 0,9813):

$$y=-\text{2,1106}x+\text{326,99}$$ 3

The optical properties of metronidazole solutions were investigated in the range from 240 to 280 nm (Czarnobaj and Sawicki 2012). To calculate the calibration curve, the value of the solution transparency at a light wavelength of 255.52 nm was chosen, where a linear dependence on concentration was determined (Fig. 2). According to the calculated transparency values, a mathematical model was calculated that reflected the detected dependence (R² = 0,9785):

Fig. 2.

Calibration curves of the studied antibiotic solutions

$$y=-\text{200,36}x+20483$$ 4

To assess background release and potential UV-interfering leachables, unloaded control samples of each osteoplastic material were prepared identically but immersed in saline instead of antibiotic solutions and then subjected to the same drying and elution protocol. Blank media (sterile saline) also analyzed as procedural controls. The methodology used is not standardized but is widely adopted in studies investigating the properties of medical devices that can release antimicrobial agents over a long period (Mabroum et al. 2025). UV-spectrometric quantification is practical for screening but is less analytically specific than chromatographic approaches (e.g., HPLC), particularly in complex matrices. To mitigate this limitation, analytical wavelengths were selected from calibration peaks. The matrix-blank testing/standard addition was used to evaluate non-specific interference.

Assessment of matrix interference and method sensitivity

Because UV measurements may be affected by non-specific absorbance/scattering, potential matrix effects were assessed using eluates from unloaded materials (matrix blanks) prepared under identical conditions. Matrix blank spectra were inspected at the analytical wavelengths used for quantification to detect possible non-specific contributions. Chromatographic confirmation (e.g., HPLC) was not performed in this screening study and is planned for follow-up work to strengthen analytical specificity.

Statistical analysis and mathematical calculations

Data processing and curve fitting were performed in Microsoft Excel 2019 and CurveExpert Basic; the online resource https://mycurvefit.com/, and Microsoft mathematical solver were also used. Statistical analysis was performed in Biostat LE using descriptive methods. Each eluate was scanned three times, and results are reported as mean ± SD of technical (instrument) replicates. Continuous variables are reported as mean ± standard deviation (SD) and, where appropriate, median (Me). Because the present study was designed as an exploratory in vitro screening experiment, each experimental system was prepared as a single sample. Because only one independently prepared specimen was available per material × antibiotic condition at each time point (n = 1), inferential statistics (t-tests/ANOVA) and multiplicity-controlled hypothesis testing were not applied; comparisons between materials and antibiotics are therefore descriptive and intended for screening-level interpretation. Descriptive statistics were primarily used to characterize trends in antibiotic release and to compare the relative elution behavior of osteoplastic materials. The study primarily provides a descriptive comparison of antibiotic release behavior.

Results

Gentamicin sulfate elution

At the first sampling point (day 2), the highest interval release was observed, followed by lower interval amounts at later time points (Table 1). Because n = 1 independent specimen was available per condition/time point, the results are presented descriptively without formal between-group hypothesis testing. The results of the spectrometry of the studied solutions showed that osteoplastic materials released different volumes of the active substance (antibiotic). Thus, the volumes of gentamicin sulfate release were the highest in numerical value, compared to other antibiotics. Among the osteoplastic materials tested, the artificial composite material "easyGRAFT" exhibited the highest numeric gentamicin sulfate release. The maximum volume was 2270.00 μg on the first day of observation, which gradually decreased to 75.35 μg by the 30th day of the experiment. "Biomin" released 1612.75 μg of gentamicin sulfate during the first day, and on the 30th day of the experiment, a volume of 741.05 μg was determined. The smallest amounts of the active substance were determined in the solution with the "InterOss" material – 1895.6 μg initially, and on the 30th day – 238.7 μg. The "InterOss" material ranked second in this indicator. Tabular and graphical analysis of the obtained data revealed a power-law dependence of antibiotic release levels throughout the observation period (Figs. 3 and 4). The kinetic modeling of cumulative antibiotic release from different osteoplastic materials is presented in Table 2. Model parameters are used for descriptive benchmarking only and should not be interpreted mechanistically.

Table 1.

Antibiotics release rates by different osteoplastic materials

Studied samples	Units of measure-ment	Gentamicin sulfate				Chloramphenicol				Ofloxacin				Metronidazole
	Day	2	5	10	30	2	5	10	30	2	5	10	30	2	5	10	30
Biomin	µg/ml	322.6	16.7	26.2	148.2	91.4	98.2	17.7	11.2	71.5	59.2	53.1	67.3	100.9	100.9	83.9	20.5
	µg	1612.8	83.3	131.1	741.1	456.8	491.0	88.5	55.8	357.7	296.0	265.3	336.4	504.6	504.5	419.5	102.3
	µg/day	1612.8	20.8	26.2	37.1	456.8	122.8	17.7	2.8	357.7	74.0	53.1	16.8	504.6	126.1	83.9	5.1
	µg/day per 1.00 g of material	5375.8	69.4	87.4	123.5	1522.5	409.2	59.0	9.3	1192.3	246.8	176.8	56.1	1681.8	420.4	279.6	17.0
EasyGRAFT	µg/ml	454.0	396.6	21.6	15.1	104.6	68.4	3.6	3.8	64.4	55.0	18.0	12.8	100.9	97.2	20.5	20.5
	µg	2270.0	1983.2	108.0	75.4	522.8	341.8	18.2	18.8	322.2	274.8	90.0	64.0	504.6	485.8	102.3	102.3
	µg/day	2270.0	495.8	21.6	3.8	522.8	85.4	3.6	0.9	322.2	68.70	18.0	3.2	504.6	121.5	20.5	5.1
	µg/day per 1.00 g of material	7566.7	1652.7	72.0	12.6	1742.5	284.8	12.1	3.1	1073.8	229.0	60.0	10.7	1681.8	404.8	68.2	17.0
InterOss	µg/ml	379.1	52.6	40.6	47.7	90.2	65.6	3.2	2.8	69.2	54.01	59.2	45.5	100.9	81.9	20.5	20.5
	µg	1895.6	262.8	203.1	238.7	450.8	327.9	16.2	14.0	345.9	270.1	296.1	227.4	504.7	409.5	102.3	102.3
	µg/day	1895.6	65.7	40.6	12.6	450.8	82.0	3.2	0.7	345.9	67.5	59.2	12.0	504.7	102.4	20.5	5.1
	µg/day per 1.00 g of material	6318.5	219.0	135.4	41.9	1502.5	273.2	10.8	2.3	1152.8	225.0	197.4	39.9	1514.1	307.1	61.4	17.0

Fig. 3.

Cumulative antibiotic release curves by different osteoplastic materials

Fig. 4.

Volumes of gentamicin sulfate elution per day during the study period

Table 2.

The empirical regression models of cumulative antibiotic release by different osteoplastic materials

Antibiotic	Osteoplastic material	Empirical regression model	R2
Gentamicin sulfate	Biomin	y* = 164,45x2—522,49x** + 1999	0,9724
	EasyGRAFT	y = −476,95x2 + 3045,5x—206,45	0,9480
	InterOss	y = 231,69x + 1674,7	0,9978
Chloramphenicol	Biomin	y = −108,8x2 + 743,44x—159,35	0,9730
	EasyGRAFT	y = −80,75x2 + 519,21x + 100,55	0,9464
	InterOss	y = −78,475x2 + 501,42x + 43,325	0,9461
Ofloxacin	Biomin	y = 295,84x + 56,85	0,9979
	EasyGRAFT	y = 310,67ln(x) + 342,47	0,9771
	InterOss	y = 267,69x + 84,15	0,9975
Metronidazole	Biomin	y = −100,55x2 + 852,59x—259,05	0,9959
	EasyGRAFT	y = −95,875x2 + 696,72x—77,075	0,9738
	InterOss	y = 440,45ln(x) + 538,61	0,9676

*y – cumulative antibiotic release rate (µg)

**x – day of observation

Chloramphenicol elution

At the first sampling point (day 2), the highest interval release was observed, followed by lower interval amounts at later time points (Table 1). Because n = 1 independent specimen was available per condition/time point, the results are presented descriptively without formal between-group hypothesis testing. Generally, chloramphenicol was released in smaller volumes. There was no such pronounced difference between the materials as was observed in the previous versions of the experiment. The greatest amount of chloramphenicol was determined in solutions containing the “easyGRAFT” on the second day of the experiment, 522.75 μg was released (which was even higher than in the previous version), and on the 30th, 18.75 μg was determined. Over a period of 30 days of observation, the “easyGRAFT” + chloramphenicol 5.00% system released 901.45 μg of the active substance into saline. In the solutions containing the “Biomin” on the second day of the experiment level of 456.75 μg was determined, and on the 30th day – 55.80 μg. Over a period of 30 days of observation, the “Biomin” material released 1092.05 μg of chloramphenicol. Less concentration of chloramphenicol was determined in solutions containing the “InterOss” bovine-derived xenograft. On the second day, 450.75 μg was determined in the system, and on the 30th, 14.00 μg. In just 30 days of the experiment, the “InterOss” + chloramphenicol 5.00% system released 808.75 μg of the active substance. A graphical analysis of the cumulative chloramphenicol release from all examined osteoplastic materials over a 30-day period revealed a nonlinear power-law relationship, as shown in Figs. 3 and 5. The kinetic modeling of cumulative antibiotic release from different osteoplastic materials is presented in Table 2. Model parameters are used for descriptive benchmarking only and should not be interpreted mechanistically.

Fig. 5.

Volumes of chloramphenicol daily elution during the study period

Ofloxacin elution

At the first sampling point (day 2), the highest interval release was observed, followed by lower interval amounts at later time points (Table 1). Because n = 1 independent specimen was available per condition/time point, the results are presented descriptively without formal between-group hypothesis testing. Analysis of solutions showed that the levels of release from were different. However, the elution rates of the two materials ("Biomin" and "InterOss") differed slightly. During the first day of the experiment, 357.70 μg of the active substance was determined in a solution with "Biomin", and on the 30th day it was 336.35 μg. Over the entire observation period, the "Biomin" + ofloxacin 5.00%" system released 1255.45 μg of the active substance. Slightly lower levels of ofloxacin release were in the xenomaterial "InterOss"—on the first day of the experiment, 345.85 μg was determined, and on the 30th—227.40 μg. In total, during the month, the specified system released 1139.40 μg of active substance. The smallest amount of ofloxacin was determined in solutions containing the artificial osteoplastic material "easyGRAFT". During the first day of the experiment, such a system released a fairly rich amount of active substance—322.15 μg, and on the 30th day, 63.95 μg was determined in the studied solution. The subsequent graphical analysis of the daily release levels of ofloxacin by different osteoplastic materials also showed the presence of a nonlinear power dependence. However, in general, the elution levels of the active substance were higher than those of chloramphenicol, which resembled the “behavior” of gentamicin sulfate, but in the case of ofloxacin, the drop in antibiotic elution by osteoplastic materials was not so rapid (Figs. 3 and 6). The kinetic modeling of cumulative antibiotic release from different osteoplastic materials is presented in Table 2. Model parameters are used for descriptive benchmarking only and should not be interpreted mechanistically.

Fig. 6.

Volumes of ofloxacin elution per day during the study period

Metronidazole elution

At the first sampling point (day 2), the highest interval release was observed, followed by lower interval amounts at later time points (Table 1). Because n = 1 independent specimen was available per condition/time point, the results are presented descriptively without formal between-group hypothesis testing. The maximum amount of active substance was released during the first day of observation. In general, the release volumes of metronidazole from different materials were similar. In the system "Biomin" + metronidazole 5.00%" the maximum release of the active substance was noted during the first 5 days of the experiment—504.55 μg. On the 30th day, 102.25 μg was determined in the studied solution. In total, over the entire observation period, this system released 1530.70 μg of metronidazole. The material "easyGRAFT" released metronidazole in almost the same way. During the first day, 504.55 μg of the substance was also determined, on the 30th—also 102.25 μg. In general, the system "easyGRAFT" with metronidazole 5.00% released 1194.85 μg of the active substance during a 30-day observation period. Slightly lower volumes of metronidazole were released by the system "InterOss" with 5.00% metronidazole. In general, the elution rates matched those of the previous two systems on the first and last days of the experiment. So, they differed maximally on the fifth. The total amount of active substance released per month was slightly lower – 1118.70 μg, but in terms of the mass of the osteoplastic material used, the level of antibiotic release was higher – 5047.00 μg per 1.00 g. Subsequently, a graphical analysis of daily metronidazole elution from the three studied systems was performed, revealing a nonlinear power dependence (as shown in Figs. 3 and 7). The kinetic modeling of cumulative antibiotic release from different osteoplastic materials is presented in Table 2. Model parameters are used for descriptive benchmarking only and should not be interpreted mechanistically.

Fig. 7.

Volumes of metronidazole daily release during the study period

Evaluation of the hypothesis about the antibiotic deposition in osteoplastic materials

To evaluate the results and clarify the possible properties of the studied osteoplastic materials, a hypothetical concentration of the active substance (antibiotics) was calculated, assuming no accumulation. The basis for these calculations was the initial concentration and amount of the active substance in the initial solutions in which the osteoplastic materials were immersed, along with the total system volume. Accordingly, in the absence of accumulation of the active substance in the systems "Biomin + Chloramphenicol", "Biomin + Metronidazole", "Biomin + Ofloxacin", "easyGRAFT + Chloramphenicol", "easyGRAFT + Metronidazole" and "easyGRAFT + Ofloxacin" on the 2nd day of the experiment, the concentration of the active substance in the system should be 2177.07 μg/ml, on the 5th day—123.23 μg/ml, on the 10th day—6.98 μg/ml and on the 30th day—0.39 μg/ml (Table 3). However, the actual concentrations of the active substances within the experimental solutions on the second and fifth days of the experiment were lower – 64.40–104.55 μg/ml and 54.01–100.89 μg/ml, respectively. But on the 10th and 30th day of observation, the concentration of active substances was mostly higher than the calculated one, reaching levels of 3.23–83.89 μg/ml and 2.80–6.27 μg/ml, respectively.

Table 3.

Calculation of the hypothetical amount of active substance in the test solutions at the stages of the study

Day of observation	1	2	5	10	30
Antibiotic/material	Chloramphenicol, Metronidazole, Ofloxacin/Biomin, easyGRAFT
Amount of active substance, μg	50,000.00	11,538.46	653.12	36.97	2.09
System volume, ml	1.30	5.30	5.30	5.30	5.30
Concentration of active substance, μg/ml	38,461.54	2177.07	123.23	6.98	0.39
Antibiotic/material	Chloramphenicol, Metronidazole, Ofloxacin/InterOss
Amount of active substance, μg	50,000.00	1923.08	73.96	2.84	0.11
System volume, ml	1.20	5.20	5.20	5.20	5.20
Concentration of active substance, μg/ml	41,666.67	369.82	14.22	0.55	0.02
Antibiotic/material	Gentamicin sulfate/Biomin, easyGRAFT
Amount of active substance, μg	40,000.00	2264.15	128.16	7.25	0.41
System volume, ml	1.30	5.30	5.30	5.30	5.30
Concentration of active substance, μg/ml	38,461.54	427.20	24.18	1.37	0.08
Antibiotic/material	Gentamicin sulfate/InterOss
Amount of active substance, μg	40,000.00	1538.46	59.17	2.28	0.09
System volume, ml	1.30	5.30	5.30	5.30	5.30
Concentration of active substance, μg/ml	33,333.33	295.86	11.38	0.44	0.02

Calculation of hypothetical antibiotic concentrations in the systems "InterOss" + Chloramphenicol", "InterOss" + Metronidazole", and "InterOss" + Ofloxacin" showed that they should be 369.82 μg/ml on the second day of the experiment. 14.22 μg/ml—on the 5th day, 0.55 μg/ml—on the 10th day, and 0.02 μg/ml—on the 30th day. However, the actual concentrations of active substances on the second and fifth days were lower – 69.15–100.94 μg/ml and 54.01–81.90 μg/ml.

In the systems "Biomin + Gentamicin sulfate" and "easyGRAFT + Gentamicin sulfate", the hypothetical concentration of the active substance on the 2nd day of the experiment should have been 427.20 μg/ml, on the 5th day—24.18 μg/ml, on the 10th – 1.37 μg/ml and on the 30th day – 0.08 μg/ml. However, the determined real concentrations of gentamicin sulfate in the experimental solutions differed. On the second and fifth days of the experiment, they were 322.55–445.00 μg/ml and 16.65–396.64 μg/ml, respectively. But on the 10th and 30th days, the real concentrations exceeded the calculated ones—21.60–26.21 μg/ml and 15.07–148.21 μg/ml, respectively. In the “InterOss” + Gentamicin sulfate” system, the hypothetical concentrations of the active substance on the 2nd day of the experiment should have been 295.86 μg/ml, on the 5th day – 11.38 μg/ml, on the 10th day – 0.44 μg/ml, and on the 30th day – 0.02 μg/ml. The actual concentrations of the active substance on the second and fifth days were lower – 126.37 μg/ml and 17.52 μg/ml, respectively. But on the 10th and 30th days of the experiment, the concentrations were 13.54 μg/ml and 15.91 μg/ml, respectively.

These findings suggest that the applied osteoplastic materials, which are used in varying volumes for the restoration of bone tissue defects and for preparing the oral cavity for dental implantation and prosthetic rehabilitation, are also able to act as systems of long-term release of antibacterial substances (up to 30 days) with prior immersion in appropriate solutions. However, the level of accumulation and release differs in different materials and antibiotics. Thus, spectrometric studies showed that in general, the highest levels of antibiotic release (and accumulation, respectively) were observed in "InterOss" – 9.83 ± 1.70 (M = 9.93) mg of antibiotic per 1 g of osteoplastic material. The most contrasting indicators were in the synthetic material "Easy GRAFT" – 6.07 ± 5.85 (M = 3.49) mg of antibiotic per 1 g of osteoplastic material. Among the antibiotics used, the highest accumulation was in gentamicin sulfate – 10.67 ± 3.57 (M = 8.67) mg of antibiotic per 1 g of material, the lowest – in chloramphenicol – 4.91 ± 2.77 (M = 3.64) mg of antibiotic per 1 g of material (Table 4). Overall, the mean cumulative antibiotic release normalized to material mass (mg/g) was 5.37 ± 2.21 for Biomin, 6.07 ± 5.84 for easyGRAFT, and 4.72 ± 2.68 for InterOss (median values: 4.64, 3.49, and 3.76 mg/g, respectively; Table 4). Across materials, gentamicin showed the highest cumulative release (10.67 ± 3.57 mg/g), whereas chloramphenicol showed the lowest (3.11 ± 0.48 mg/g). Given the screening design with n = 1 independent specimen per condition/time point, these differences should be interpreted as descriptive trends rather than confirmed statistically significant effects.

Table 4.

Volumes of active substances released by the studied osteoplastic materials over 30 days

Material	Gentamicin sulfate		Chloramphenicol		Metronidazole		Ofloxacin		M ± m; median. mg/g
	Total, mg	mg/g	Total, mg	mg/g	Total, mg	mg/g	Total, mg	mg/g
Biomin	2.57	8.56	1.09	3.64	1.53	5.10	1.26	4.19	5.37 ± 2.21 (Median = 4.64)
Easy GRAFT	4.44	14.79	0.90	3.01	1.20	3.98	0.75	2.50	6.07 ± 5.84 (Median = 3.49)
InterOss	2.60	8.67	0.81	2.70	1.12	3.73	1.14	3.80	4.72 ± 2.68 (Median = 3.76)
Across materials (mg/g), M ± m; median	-	10.67 ±  3.57 (M = 8.67)	-	3.11 ± 0.48 (M = 3.01)	-	4.27 ± 0.73 (M = 3.98)	-	3.50 ± 0.88 (M = 3.80)

Discussion

Therefore, the results should be interpreted primarily as comparative in vitro screening data rather than as definitive mechanistic characterization of drug release kinetics. The incorporation of an antibiotic into a ceramic biomaterial appeared sufficient to make it effective in both treating osteomyelitis and preventing the progression of a contaminated bone cavity into osteomyelitis, while promoting bone formation and osteointegration across all animal models. Future studies should evaluate the in vivo release profile of antibiotics under conditions of infection. Although the release profiles of most tested biomaterials have been studied in vitro or in animal models, there is limited understanding of the drug release profiles under conditions closer to reality. This is important data to ensure that low antibiotic concentrations do not persist for extended periods in the infected area, which would facilitate the development of bacterial resistance. In conclusion, the addition of antibiotics to bioactive osteoplastic materials appears to be a promising solution for treating infected bone defects while facilitating bone regeneration; however, it is not yet possible to determine which biomaterial is most effective for this dual purpose. The relatively high antibiotic concentrations were intentionally selected to ensure measurable loading of the osteoplastic matrices and to simulate a near-saturation loading condition in this screening experiment.

From a perspective of development and impovement of biomaterials for the dental usage, the observed antibiotic elution profiles in our experiment likely reflect the coaction between the physicochemical properties of the osteoplastic matrices (bone grafts) and the adsorption–desorption processes which of drug molecules within their porous structures. High porosity and large surface area of the osteoplastic materials may provide more adsorption sites for antibiotic molecules. It enables greater initial loading but also promotes an early burst release associated with surface-bound fractions. Subsequently, the sustained phase of release is expected to be primarily governed by diffusion from internal pores and by partial desorption from the mineral or organic components of the matrix. The differences found between synthetic ceramics and the natural animal-derived material in the present study may therefore be explained by microstructural variations, mineral composition, and due to the possible presence of residual organic components capable to interact with antibiotic molecules (electrostatic or hydrogen-bond interactions). These mechanisms are consistent with general principles described for antibiotic-loaded bone graft substitutes and porous bioceramics used as local drug delivery systems in orthopedic and maxillofacial applications.

The empirical power-law decrease observed in daily release is consistent with an initial surface desorption (‘burst’) followed by diffusion-controlled depletion from porous matrices. Classical mechanistic models (e.g., Higuchi or Korsmeyer–Peppas) are typically fitted to cumulative fractional release data under defined sink conditions; in the present design, discrete medium replacement and limited sampling points motivated the use of an empirical power-law description. Future work with denser sampling and cumulative-release fitting is warranted to better infer mechanisms (Medarametla et al. 2024).

Benchmarking against commercially available antibiotic-loaded bone substitutes and coated allografts is essential for interpretation. Published data show that industrially prepared antibiotic-loaded grafts and ceramics can maintain high local antibiotic levels for weeks with a pronounced initial burst followed by sustained release (e.g., gentamicin- or vancomycin/tobramycin-loaded graft systems). In the present study we did not include a commercial antibiotic-loaded graft as a positive control, which limits direct performance ranking; however, the cumulative amounts and month-long detectability observed here support the concept that dental graft matrices can function as reservoirs after simple ex vivo loading.

The best way to promote a controlled-release formulation capable of maintaining concentrations above the minimum inhibitory concentration for a sufficiently prolonged period to eradicate bacteria locally has not yet been defined. The preferred first-line antibiotic and the ideal ceramic delivery system (pure or in association with other substances) are still under investigation. It is essential to follow consistent guidelines and develop suitable models to reduce the time lag between animal investigation and human application (Alegrete et al. 2023; Bormann et al. 2023).

Antibiotic release from osteoplastic materials (also known as antibiotic carriers or local antibiotic delivery systems) is an important approach for the treatment and prevention of various bone infections. Nowadays, the traditional systemic administration of antibiotics often fails to provide sufficient concentrations at the site of infection due to the poor vascularization of bone tissue, while causing systemic side effects. Local delivery of antibiotics from osteoplastic materials allows: to achieve high concentrations of antibiotic directly at the infection site, significantly higher than those that may be achieved systemically (with minimal systemic toxicity); to provide sustained release of antibiotic, maintaining therapeutic concentrations over a long period of time, which is critical for complete suppression of infection; to reduce an antibiotic resistance, as high local concentrations promote more efficient bacterial elimination; to promote the bone regeneration due to osteoinductive or osteoconductive properties of antibiotic carriers (Freischmidt et al. 2022; Hong et al. 2024; Sebastian et al. 2022).

A major limitation of the present screening design is that elution was primarily quantified in saline under static conditions, which does not reproduce the ionic strength, protein binding, enzymatic activity, and dynamic fluid exchange present in vivo (saliva/blood/tissue fluids). These factors may alter both the apparent release kinetics and the effective free antibiotic concentration. Future work should therefore replicate the release experiments in PBS and standardized artificial saliva under dynamic flow to improve clinical translatability.

Antibiotics may be released from osteoplastic materials by several mechanisms: diffusion (the antibiotic dissolved in the material gradually diffuses into the surrounding tissue); erosion/degradation of the material (biodegradable materials may break down, leading to the release of the entrapped antibiotic); surface desorption (rapid release of the antibiotic adsorbed on the surface of the material, often observed in the initial period). Key aspects and challenges in the development of bone-grafts with antibacterial properties are well presented and may be limited in the next: controlled release – it is important to ensure a therapeutic concentration of the antibiotic over the required time, avoiding both too rapid a "burst" release (which can lead to toxicity and rapid depletion of the antibiotic) and too slow (ineffective); preservation of antibiotic activity – mixing and sterilization processes should not deactivate the antibiotic; biocompatibility and osseointegration – the material should not cause undesirable reactions in the body and should promote bone healing; mechanical properties—for some applications (e.g. fixation of prostheses) the material must have sufficient mechanical strength; antibiotic spectrum – the choice of antibiotic depends on the suspected pathogen (vancomycin, gentamicin, tobramycin, clindamycin are often used) (Wassif et al. 2021; Winkler and Haiden 2016; Guardia et al. 2021).

Clinical interpretation requires relating the measured eluate concentrations to minimum inhibitory concentration (MIC) values of relevant oral pathogens. In our study, eluate concentrations at the first sampling point ranged from 126 to 454 µg/mL for gentamicin (Table 1), whereas reported gentamicin MICs for Aggregatibacter actinomycetemcomitans range from 0.25 to 4 mg/L (µg/mL). For Fusobacterium nucleatum, metronidazole MIC₉₀ values around 0.5 µg/mL and chloramphenicol MIC₉₀ values around 1 µg/mL have been reported, which are markedly below the concentrations still observed at day 30 in our eluates (metronidazole ≈20 µg/mL; chloramphenicol ≈2.8–11.2 µg/mL). For Prevotella intermedia/Prevotella nigrescens, metronidazole has been reported as highly active (e.g., MIC₉₀ ≈0.125 µg/mL, with resistant isolates reported at much higher values). For Porphyromonas gingivalis, published MIC₉₀ ranges for metronidazole (< 0.016–0.75 mg/L) and fluoroquinolones (0.032–2.00 mg/L) suggest that the concentrations measured in our eluates are within or above inhibitory ranges for many strains. Nevertheless, in vivo conditions (fluid turnover, protein binding, pH shifts) can reduce the free drug concentration and may create sub-MIC exposure windows. This fact is relevant for resistance risk and should be addressed in future studies of dynamic-release (CLSI, 2025). The calculated antibiotic concentrations in the eluates exceeded reported MICs for several oral pathogens obtained in vitro. However, the present experiment was performed under simplified laboratory conditions and does not reproduce physiological factors such as protein binding, fluid turnover, or tissue diffusion. Therefore, these values should not be interpreted as direct predictors of clinical antimicrobial efficacy. It should be noted that the antibiotic concentrations measured in this study were obtained under laboratory conditions and were not evaluated in biological models; therefore, it cannot be concluded that such concentrations would necessarily be achieved in clinical conditions.

Similar studies were conducted by Rihawy et al. (2019), who investigated the long-term release of chloramphenicol from a carboxymethylcellulose/polyvinyl alcohol/2-hydroxyethyl acrylate hydrogel system. Results showed a significant release of chloramphenicol over 20 h after loading and possible control of this process using FTIR spectroscopy.

Related studies were performed by Posadowska et al. (2016), who studied vancomycin release from loaded nanoparticles and from a gellan gum matrix. Results showed ensured local antibiotic delivery with an advantageously high burst phase, a long-sustained phase, and a positive effect on the proliferation of osteoblast-like cells.

In the results presented by Alegrete et al. (2023), the studies of antibiotic release from the impregnated osteoconductive materials (calcium sulfate, hydroxyapatite, and tricalcium phosphate) were conducted. Authors showed that bioceramics potentially can release antibiotics for a period long enough to treat the infection, but we also have a need to stop the drug delivery at a certain time point to avoid a low antibiotic concentration and the emergence of bacterial resistance.

In the review prepared by Barzdina et al. (2024), actual literature data on the prolonged release of gentamicin from different types of gentamicin-loaded polylactide-polyglycolide nanoparticles were presented. The duration of gentamicin release from such a kind of delivery system. Data included the following types of duration: 10 h, 120 h, 216 h, over 16 days, 28 days, and 35 days. Also, the authors inform that the possible encapsulation of gentamicin–polyphenol combinations in drug delivery systems should be further investigated.

Czarnobaj and Sawicki (2012) in their studies of metronidazole release from SiO₂-CaO-P₂O₅ composite materials showed that maximal antibiotic release was detected during the first 15–30 min and gradually decreased during the 5 h of the experiment. The process also depends on the quality of the tested matrix.

Studies of Tuleubayev and Rudenko (2021) showed the antibiotic release from bone allograft among animals. Authors used the “PerOssal” medium and whole bone allograft was soaked in antibiotics – gentamicin with different mechanical preparations of bone allograft. The results showed a significant release of gentamicin over the 7 days of the experiment; the antibiotic concentrations were sufficient for effective inhibition of pathogenic microorganisms.

The studies of Berglund et al. (2024), dedicated to bone allograft impregnated with tobramycin and vancomycin delivery of antibiotic (bone chips) showed that allografts release the active substances in high concentrations (1,200 mg/L post-application) which is more than needed for eradication of testing strains of pathogenic bacteria.

Winkler and Haiden (2016) showed that modified allografts (OSTEOmycin™V – with vancomycin and Osteomycin™ T – with tobramycin) work as effective local delivery systems during 4 weeks. Authors detected extremely high local concentrations with a logarithmic decrease over the following weeks.

Witsø (2007) presented the results of clinical study based on cancellous bone impregnation with tobramycin and vancomycin. Author confirmed the subtoxic serum levels of antibiotics (6 and 9 µg/mL, respectively) at 12 h after the implantation. And the local concentrations were extremely high 185–1690 µg/mL of tobramycin and 230–2345 µg/mL of vancomycin. 26 patients treated with cancellous bone impregnated with tobramycin and vancomycin, subtoxic serum levels (6 and 9 µg/mL, respectively) were registered at 12 h postoperatively. Antibiotic concentration in the drain fluid was very high: 185–1690 µg/mL of tobramycin and 230–2345 µg/mL of vancomycin.

Stravinskas et al. (2019), studied the new biphasic ceramic osteoplastic material consisting of hydroxyapatite and calcium sulphate for local antibiotic delivery in combination with bone regeneration (gentamicin). Results showed that 95% of the antibiotic was released from osteoplastic material over 14 days and 100% at 28 days.

Hornyák et al. (2014), studied the release time of antibiotics from bone allografts through a novel biodegradable coating and showed that lyophilization with or without chitosan usually caused the short-time release (up to 48 h). Alginate coatings provided long-lasting release: 8 days for amoxicillin, 28 days with ciprofloxacin coating, and 50 days with vancomycin.

In the present study, we calculated the daily release rates of various antibiotics from different osteoplastic materials. Moreover, our results support the hypothesis that osteoplastic materials intended for dental applications are capable of antibiotic deposition and can be transformed, through simple modification, into medical devices providing prolonged elution of antimicrobial agents. The levels of deposition and elution of antibiotics differ significantly between materials of different origins (synthetic and natural), which may be explained by differences in structure and the presence of organic tissue residues. Therefore, in the present work, the immersion procedure was intentionally used as a standardized screening model and maximum-loading approach to compare the deposition and long-term release (elution) capacities of different dental osteoplastic materials, rather than as a proposed clinical protocol. High enough loading concentrations (4–5%) and immersion for 24 h were chosen to ensure measurable deposition and to approximate a near-saturation loading condition for inter-material comparison in an in vitro screening setting. We highlight that selecting the concentration and time for clinical use (including lower concentrations and shorter loading times feasible intraoperatively) requires separate additional studies. Additionally, the static batch-release design with complete medium replacement at discrete time points does not capture dynamic in vivo transport. Therefore, the presented curves should be interpreted as comparative in vitro elution profiles rather than direct predictors of clinical tissue concentrations.

An additional limitation of this study may be the absence of chromatographic confirmation (e.g., HPLC) of antibiotic concentrations, which could further improve analytical specificity and exclude potential matrix interference. Because the release medium was replaced at each sampling point, the system approximated sink conditions during the experiment. However, verifying sink conditions precisely requires further studies.

Conclusions

The interaction of osteoplastic materials with antibiotics from various groups is a relatively understudied issue in medicinal and pharmaceutical chemistry. Given modifications to modern materials and the inclusion of antimicrobial substances in their composition, existing recommendations and expert opinions on the necessity of immersing osteoplastic materials in antibiotic solutions before application require further clarification and verification at the experimental and clinical levels. Spectrometric measurements demonstrated detectable antibiotic release from both natural and synthetic osteoplastic materials over a 30-day observation period under in vitro conditions. The presence of antibiotic accumulation in the osteoplastic mass has been established. Release curves of the studied antibiotics were obtained from different osteoplastic materials over a one-month period.

Author contributions

Mochalov I. - Conceptualization, Project administration, Supervision, Writing - review and editing. Myhajlichenko B. - Data collection, Writing - original draft. Biganych V. - Data collection, Data analysis. Karpchuk L. - Conceptualization, Writing - review and editing. Kizim A. - Literature review, Writing - original draft (methodology and discussion).

Funding

This research received no external funding.

Data availability

Data are available upon request.

Declarations

Informed consent statement

Not applicable.

Institutional review board statement

Not applicable.

Conflicts of interest

The authors declare no conflict of interest.