Experimental study of impregnation conditions for sustained antimicrobial activity of the original osteoplastic material based on cancellous bone allograft

Introduction Local antibiotic therapy is used to prevent and treat periprosthetic joint infection, but the available antibiotic delivery systems have some limitations.
The objective was to determine optimal parameters of pressure, exposure time and type of solvent to ensure prolonged elution of vancomycin from the original osteosubstituting material based on cancellous allograft bone using an in vitro experiment.
Material and methods Seven impregnation techniques with different combinations of parameters were examined including pressure: from atmospheric to reduced (7–10 hPa), time: from 5 minutes to  24 hours, solvent (distilled water, 50 % ethanol solution, a combination of 50 % ethanol and 5 % polyvinylpyrrolidone (PVP)). The efficacy was assessed by changes in the diameter of the S. aureus ATCC 43300 inhibition zone using  the  bacteriological method and the dynamics of vancomycin concentration
in the eluate and high‑performance liquid chromatography (HPLC). Statistical analysis was performed using the ANOVA method, Tukey's post-hoc test, Spearman's rank correlation and calculation of the area under the pharmacokinetic curve.
Results The best efficiency was demonstrated by the method employing reduced pressure, 60-minute exposure and  an  alcohol solution with PVP, which provided prolonged release of  vancomycin for  14 days with the maximum area under the elution curve (301364.70) and a high correlation between the concentration of  the  antibiotic and the growth inhibition zone (r = 0.908, p < 0.001). The pressure was found to  be the  most  significant factor (F = 19.9916, p < 0.0001), followed by solvent type (F = 7.7485, p = 0.0006) and impregnation time (F = 6.8084, p = 0.0014).
Discussion The technique with use of reduced pressure and an alcohol solution with PVP provides prolonged release of vancomycin for 14 days as opposed to conventional local antibiotic therapy with limited effectiveness of  3 to  7  days. The advantage of the approach includes uniform elution kinetics compared to  polymethyl methacrylate and biodegradable carriers, which demonstrate a sharp initial release of the antibiotic. The complementary use of the microbiological method and HPLC indicated antimicrobial
activity of vancomycin maintained after impregnation being essential for the therapeutic effect.
Conclusion It has been experimentally established that reduced pressure (7–10 hPa), an exposure time of 60 min and the use of 50 % ethanol with 5 % PVP as a solvent appeared to be the optimal parameters for  ensuring prolonged elution of vancomycin from an osteosubstituting material based on cancellous allograft bone.

1. Masters EA, Ricciardi BF, Bentley KLM, et al. Skeletal infections: microbial pathogenesis, immunity and clinical management. Nat Rev Microbiol. 2022;20(7):385-400. doi: 10.1038/s41579-022-00686-0.

2. Kasimova AR, Tufanova OS, Gordina EM, et al. Twelve-Year Dynamics of Leading Pathogens Spectrum Causing Orthopedic Infection: A Retrospective Study. Traumatology and Orthopedics of Russia. 2024:30(1):66-75. doi: 10.17816/2311-2905‑16720.

3. Garcia-Moreno M, Jordan PM, Günther K, et al. Osteocytes Serve as a Reservoir for Intracellular Persisting Staphylococcus aureus Due to the Lack of Defense Mechanisms. Front Microbiol. 2022;13:937466. doi: 10.3389/fmicb.2022.937466.

4. Trouillet-Assant S, Lelievre L, Martins-Simoes P, et al. Adaptive processes of Staphylococcus aureus isolates during the progression from acute to chronic bone and joint infections in patients. Cell Microbiol. 2016;18(10):1405-1414. doi: 10.1111/cmi.12582.

5. Ricciardi BF, Muthukrishnan G, Masters E, et al. Staphylococcus aureus Evasion of Host Immunity in the Setting of Prosthetic Joint Infection: Biofilm and Beyond. Curr Rev Musculoskelet Med. 2018;11(3):389-400. doi: 10.1007/s12178-018-9501-4.

6. Paharik AE, Horswill AR. The Staphylococcal Biofilm: Adhesins, Regulation, and Host Response. Microbiol Spectr. 2016;4(2):10.1128/microbiolspec.VMBF-0022-2015. doi: 10.1128/microbiolspec.VMBF-0022-2015.

7. Coraca-Huber DC, Fille M, Hausdorfer J, et al. Staphylococcus aureus biofilm formation and antibiotic susceptibility tests on polystyrene and metal surfaces. J Appl Microbiol. 2012;112(6):1235-1243. doi: 10.1111/j.1365-2672.2012.05288.x.

8. Mankin HJ, Hornicek FJ, Raskin KA. Infection in massive bone allografts. Clin Orthop Relat Res. 2005;(432):210-216. doi: 10.1097/01.blo.0000150371.77314.52.

9. Michalak KA, Khoo PP, Yates PJ, et al. Iontophoresed segmental allografts in revision arthroplasty for infection. J Bone Joint Surg Br. 2006;88(11):1430-1437. doi: 10.1302/0301-620X.88B11.18335.

10. Elawady R, Aboulela AG, Gaballah A, et al. Antimicrobial Sub-MIC induces Staphylococcus aureus biofilm formation without affecting the bacterial count. BMC Infect Dis. 2024;24(1):1065. doi: 10.1186/s12879-024-09790-3.

11. Witsø E, Persen L, Løseth K, Bergh K. Adsorption and release of antibiotics from morselized cancellous bone. In vitro studies of 8 antibiotics. Acta Orthop Scand. 1999;70(3):298-304. doi: 10.3109/17453679908997812.

12. Mader JT, Calhoun J, Cobos J. In vitro evaluation of antibiotic diffusion from antibiotic-impregnated biodegradable beads and polymethylmethacrylate beads. Antimicrob Agents Chemother. 1997;41(2):415-418. doi: 10.1128/AAC.41.2.415.

13. Buttaro MA, Morandi A, Rivello HG, Piccaluga F. Histology of vancomycin-supplemented impacted bone allografts in revision total hip arthroplasty. J Bone Joint Surg Br. 2005;87(12):1684-1687. doi: 10.1302/0301-620X.87B12.16781.

14. Buttaro MA, Pusso R, Piccaluga F. Vancomycin-supplemented impacted bone allografts in infected hip arthroplasty. Two-stage revision results. J Bone Joint Surg Br. 2005;87(3):314-319. doi: 10.1302/0301-620x.87b3.14788.

15. Buttaro MA, Gimenez MI, Greco G, et al. High active local levels of vancomycin without nephrotoxicity released from impacted bone allografts in 20 revision hip arthroplasties. Acta Orthop. 2005;76(3):336-340.

16. Inzana JA, Schwarz EM, Kates SL, Awad HA. Biomaterials approaches to treating implant-associated osteomyelitis. Biomaterials. 2016;81:58-71. doi: 10.1016/j.biomaterials.2015.12.012.

17. Markov PА, Eremin PS, Berezkina ES, et al. Osteoplastic biomaterials from organic and mineral components of the bone matrix: a literature review. Bulletin of Rehabilitation Medicine. 2024;23(5):97-107. (In Russ.) doi: 10.38025/2078-1962-2024-23-5-97-107.

18. Mukhametov UF, Lyulin SV, Borzunov DY, et al. Alloplastic and implant materials for bone grafting: a literature review. Creative surgery and oncology. 2021;11(4):344. (In Russ.) doi: 10.24060/2076-3093-2021-11-4-343-353.

19. Bozhkova SA, Gordina EM, Markov MA, et al. The Effect of Vancomycin and Silver Combination on the Duration of Antibacterial Activity of Bone Cement and Methicillin-Resistant Staphylococcus aureusBiofilm Formation. Traumatology and Orthopedics of Russia. (In Russ.) 2021;27(2):54-64. doi: 10.21823/2311-2905-2021-27-2-54-64.

20. Melikova RE, Tsiskarashvili AV, Artyukhov AA, Sokorova NV. IIn vitro study of the dynamics in elution of antibacterial drugs impregnated into matrices based on polymer hydrogel. Genij Ortopedii. 2023;29(1):64-70. doi: 10.18019/1028-4427-2023-29-1-64-70.

21. Stogov MV, Shastov AL, Kireeva EA, Tushina NV. Release of antibiotics from the materials for postosteomyelitic bone defect filling. Genij Ortopedii. 2024;30(6):873-880. doi: 10.18019/1028-4427-2024-30-6-873-880.

22. McConoughey SJ, Howlin RP, Wiseman J, et al. Comparing PMMA and calcium sulfate as carriers for the local delivery of antibiotics to infected surgical sites. J Biomed Mater Res B Appl Biomater. 2015;103(4):870-877. doi: 10.1002/jbm.b.33247.

23. Janssen DMC, Willems P, Geurts J, Arts CJJ. Antibiotic release from PMMA spacers and PMMA beads measured with ELISA: Assessment of in vitro samples and drain fluid samples of patients. J Orthop Res. 2023;41(8):1831-1839. doi: 10.1002/jor.25510.

24. Bertazzoni Minelli E, Della Bora T, Benini A. Different microbial biofilm formation on polymethylmethacrylate (PMMA) bone cement loaded with gentamicin and vancomycin. Anaerobe. 2011;17(6):380-383. doi: 10.1016/j.anaerobe.2011.03.013.

25. Miclau T, Dahners LE, Lindsey RW. In vitro pharmacokinetics of antibiotic release from locally implantable materials. J Orthop Res. 1993;11(5):627-632. doi: 10.1002/jor.1100110503.

26. Kwong JW, Abramowicz M, Kühn KD, et al. High and Low Dosage of Vancomycin in Polymethylmethacrylate Cements: Efficacy and Mechanical Properties. Antibiotics (Basel). 2024;13(9):818. doi: 10.3390/antibiotics13090818.

27. Tsiskarashvili AV, Melikova RE, Volkov AV, et al. In vivo effectiveness of polymer hydrogels impregnated with an antibacterial drug in chronic osteomyelitis. Genij Ortopedii. 2023;29(5):535-545. doi: 10.18019/1028-4427-2023-29-5-535-545.

28. Wahl P, Guidi M, Benninger E, et al. The levels of vancomycin in the blood and the wound after the local treatment of bone and soft-tissue infection with antibiotic-loaded calcium sulphate as carrier material. Bone Joint J. 2017;99-B(11):1537-1544. doi: 10.1302/0301-620X.99B11.BJJ-2016-0298.R3.

29. Luo S, Jiang T, Yang Y, et al. Combination therapy with vancomycin-loaded calcium sulfate and vancomycin-loaded PMMA in the treatment of chronic osteomyelitis. BMC Musculoskelet Disord. 2016;17(1):502. doi: 10.1186/s12891-016-1352-9.

30. Giavaresi G, Bertazzoni Minelli E, Sartori M, et al. New PMMA-based composites for preparing spacer devices in prosthetic infections. J Mater Sci Mater Med. 2012;23(5):1247-1257. doi: 10.1007/s10856-012-4585-7.

31. Kang DG, Holekamp TF, Wagner SC, Lehman RA Jr. Intrasite vancomycin powder for the prevention of surgical site infection in spine surgery: a systematic literature review. Spine J. 2015;15(4):762-770. doi: 10.1016/j.spinee.2015.01.030.

32. Lawrie CM, Kazarian GS, Barrack T, et al. Intra-articular administration of vancomycin and tobramycin during primary cementless total knee arthroplasty : determination of intra-articular and serum elution profiles. Bone Joint J. 2021;103-B(11):1702-1708. doi: 10.1302/0301-620X.103B11.BJJ-2020-2453.R1.

33. Kamra P, Lamba AK, Faraz F, Tandon S. Effect of antibiotic impregnation time on the release of gentamicin from cryopreserved allograft bone chips: an in vitro study. Cell Tissue Bank. 2019;20(2):267-273. doi: 10.1007/s10561-019-09765-8.

34. Berglund B, Wezenberg D, Nilsson M, et al. Bone allograft impregnated with tobramycin and vancomycin delivers antibiotics in high concentrations for prophylaxis against bacteria commonly associated with prosthetic joint infections. Microbiol Spectr. 2024;12(12):e0041424. doi: 10.1128/spectrum.00414-24.

35. Coraça-Huber DC, Ammann CG, Nogler M, et al. Lyophilized allogeneic bone tissue as an antibiotic carrier. Cell Tissue Bank. 2016;17(4):629-642. doi: 10.1007/s10561-016-9582-5.

36. Coraça-Huber DC, Steixner SJM, Najman S, et al. L Lyophilized Human Bone Allograft as an Antibiotic Carrier: An In Vitro and In Vivo Study. Antibiotics (Basel). 2022;11(7):969. doi: 10.3390/antibiotics11070969.

37. Edmondson MC, Day R, Wood D. Vancomycin iontophoresis of allograft bone. Bone Joint Res. 2014;3(4):101-7. doi: 10.1302/2046-3758.34.2000223.

38. Karageorgiou V, Kaplan D. Porosity of 3D biomaterial scaffolds and osteogenesis. Biomaterials. 2005;26(27):5474-91. doi: 10.1016/j.biomaterials.2005.02.002.

39. Ketonis C, Barr S, Adams CS, et al. Bacterial colonization of bone allografts: establishment and effects of antibiotics. Clin Orthop Relat Res. 2010;468(8):2113-2121. doi: 10.1007/s11999-010-1322-8.

40. Dolete G, Purcăreanu B, Mihaiescu DE, et al. Comparative Loading and Release Study of Vancomycin from a Green Mesoporous Silica. Molecules. 2022;27(17):5589. doi: 10.3390/molecules27175589.

41. Kato A. Atmospheric impregnation behavior of calcium phosphate materials for antibiotic therapy in neurotrauma surgery. PLoS One. 2020;15(3):e0230533. doi: 10.1371/journal.pone.0230533.

42. Kato A. Antibiotic Impregnation, Release, Activity, and Interaction With Porous Hydroxyapatite for Infectious Control in Neurotrauma Surgery. J Pharm Sci. 2022;111(8):2389-2396. doi: 10.1016/j.xphs.2022.04.017.

43. Edin ML, Miclau T, Lester GE, et al. Effect of cefazolin and vancomycin on osteoblasts in vitro. Clin Orthop Relat Res. 1996;(333):245-251.