A biomaterial formulation combining S53P4 bioactive glass with the synthetic antimicrobial peptide SAAP-148 successfully cleared bacterial biofilms in vitro and eradicated implant-adherent bacteria in mouse models, according to an experimental investigation published in Frontiers in Bioengineering and Biotechnology. Researchers at Amsterdam UMC and the University Hospital Regensburg evaluated whether embedding the peptide into bioactive glass putty and cream could overcome the clinical limitations of bioactive glass alone against high bacterial burdens. In a controlled in vivo mouse subcutaneous implant trial, bioactive glass putty carrying two percent SAAP-148 achieved complete bacterial eradication on all titanium implants after twenty-four hours, whereas unmodified bioactive glass putty failed to clear bacteria from any implant. The dual-action material also cleared viable bacteria from sixty-one percent of surrounding soft tissue sites and substantially lowered bacterial counts in the remaining tissue. These findings provide proof of concept that pairing synthetic peptides with bone graft substitutes offers immediate local bactericidal protection.

Orthopedic implant-associated infections represent a persistent and severe challenge in modern reconstructive surgery, frequently demanding prolonged systemic antibiotic therapy, surgical debridement, or complete device removal. The primary obstacle to successful treatment is the propensity of invading pathogens to form resilient biofilms upon abiotic implant surfaces and surrounding anatomical tissues. Within these multicellular communities, bacteria produce a protective extracellular matrix that impedes host immune defenses, fosters antibiotic tolerance, and harbors metabolically dormant persister cells capable of surviving standard antibiotic regimens. Among the microbial species implicated in orthopedic infections, Staphylococcus aureus and Acinetobacter baumannii pose exceptional clinical threats due to their extensive virulence repertoires and high rates of multidrug resistance. S. aureus accounts for the majority of orthopedic device infections, particularly through methicillin-resistant strains, while A. baumannii has increasingly emerged in traumatic wounds and open fractures. Both pathogens are recognized by the World Health Organization as high-priority targets requiring innovative therapeutic strategies.

Bioactive glass S53P4 is a resorbable bone graft substitute widely utilized in orthopedic procedures because of its demonstrated osteoconductive, osteostimulatory, and intrinsic antimicrobial characteristics. Its antibacterial performance stems from surface reactivity and the dissolution of glass constituents, which elevates local osmotic pressure and induces an alkaline microenvironment unfavorable for bacterial proliferation. Commercial formulations include granular glass particles and malleable putty, which blends granules measuring 500 to 800 micrometers with a polymeric binder consisting of polyethylene glycol and glycerol. In routine clinical use, these products are applied alongside surgical debridement and systemic antibiotics. However, experimental studies show that bioactive glass putty alone cannot reliably resolve established infections without concurrent systemic therapy. To enhance local antibacterial potency, researchers previously developed an experimental bioactive glass cream utilizing fine powder particles smaller than 45 micrometers, which increases surface area and ion exchange. Nevertheless, neither putty nor cream formulations consistently achieve total bacterial eradication under dense bacterial contamination.

To overcome these antimicrobial shortfalls, the research team incorporated SAAP-148, a synthetic antimicrobial and antibiofilm peptide that disrupts bacterial cell membranes independently of metabolic activity. This membrane-targeting mode of action enables the molecule to rapidly kill active bacteria alongside metabolically quiescent persister cells, presenting a low potential for resistance selection. For this study, two percent weight-per-volume SAAP-148 was integrated into both commercial putty and experimental cream matrices prepared by Bonalive Ltd. Preparing the peptide-loaded putty required warming the glass and binder mixture to 60 degrees Celsius under continuous mechanical agitation, followed by room-temperature cooling to ensure uniform distribution. Thermal stability testing confirmed that pre-incubating pure SAAP-148 at 60 degrees Celsius for thirty minutes caused no reduction in bactericidal strength. For the cream preparation, SAAP-148 was mixed directly into the polyethylene glycol and glycerol binder prior to introducing fine glass powder, avoiding viscosity-related mixing obstacles.

The scientists initially characterized the release and antibacterial effectiveness of the materials by collecting fluid eluates from formulations incubated in RPMI medium across intervals spanning five minutes to twenty-four hours. Bioactive glass dissolution rapidly shifted the eluates to alkaline conditions, reaching pH values between 10.5 and 10.9 within two hours, while incorporating SAAP-148 produced only a minimal, non-disruptive decrease in final pH. In standard killing assays using multidrug-resistant S. aureus and A. baumannii suspensions at one million colony-forming units per milliliter, undiluted cream eluates completely cleared both pathogens, whereas undiluted putty achieved an approximate three-log reduction. When diluted to twenty-five percent strength, however, eluates from unmodified glass products lost their bactericidal properties. In contrast, twenty-five percent eluates containing SAAP-148 completely eliminated both bacterial species, confirming that the peptide maintained robust microbicidal potency despite the alkaline environment created by the dissolving glass substrate.

Kinetic evaluations of the fluid eluates demonstrated exceptionally rapid release of bactericidal activity from both peptide-loaded preparations within minutes of liquid contact. Lethal concentration assays determining the minimum dose required for 99.9 percent bacterial reduction indicated that pure SAAP-148 killed both pathogens at 0.94 micromolar. Eluates taken after just five minutes of elution from peptide-loaded putty and cream achieved 99.9 percent killing at 64-fold and 128-fold dilutions, respectively, expanding to 1024-fold dilutions by thirty minutes. This corresponded to calculated SAAP-148-equivalent bactericidal concentrations rising from 60.16 micromolar at five minutes to 962.56 micromolar from thirty minutes through twenty-four hours. Formal checkerboard testing confirmed that combining SAAP-148 with bioactive glass produced neither synergy nor antagonism, demonstrating that the observed high potency represents an additive dual-action mechanism uniting immediate peptide bactericidal activity with delayed alkaline glass effects.

The antibiofilm capabilities of two-hour eluates were subsequently examined against young twenty-four-hour and mature seventy-two-hour biofilms grown in ninety-six-well plates. Following treatment periods of two, eight, or twenty-four hours, biofilms were rinsed, resuspended with sodium polyanethol sulphonate to neutralize residual peptide, and mechanically dispersed by ultrasonic sonication to quantify surviving colony-forming units. Unloaded putty eluates produced no significant reduction in viable biofilm bacteria at any incubation interval, whereas unmodified cream eluates yielded significant but incomplete reductions of one to two logs. In striking contrast, eluates containing SAAP-148 eradicated all viable cells in twenty-four-hour biofilms across all treatment durations. When applied to mature seventy-two-hour biofilms, peptide-loaded eluates achieved an approximate six-log reduction within two hours and produced complete bacterial eradication after eight and twenty-four hours of exposure across both bacterial species.

To evaluate performance in complex biological matrices, the researchers established an intermediate cadaver mouse subcutaneous implant infection model using titanium plates inserted into dorsal tissue pockets. Implants were inoculated with 800,000 colony-forming units of S. aureus before receiving fifty milligrams of test formulation and undergoing overnight room-temperature incubation. Both formulations remained localized around the titanium devices. Standard quantitative cultures showed that unloaded putty and cream decreased implant-associated bacteria by approximately two and three logs, respectively, but failed to sterilize surfaces. In tissue, unloaded putty yielded no significant bacterial reduction. Conversely, both SAAP-148-loaded putty and cream achieved complete bacterial clearance across both implant surfaces and adjacent soft tissues. This intermediate validation confirmed that the antimicrobial peptide effectively diffused through local tissue spaces and retained bactericidal capacity amidst host tissue components.

Building upon the cadaver results, the team conducted an in vivo study utilizing immunocompetent female C57BL/6J mice carrying bilateral subcutaneous titanium implants pre-inoculated with S. aureus. Bioactive glass putty was selected for this live evaluation because the formulation already possesses regulatory approval for human clinical use. Animals received fifty milligrams of either unmodified putty or peptide-loaded putty per pocket, with eighteen implants evaluated across nine mice in each experimental cohort. After twenty-four hours, all eighteen implants and tissue sites treated with unmodified putty remained culture-positive, matching initial inoculation densities. By comparison, peptide-loaded putty achieved complete bacterial eradication on all eighteen titanium implants. Within surrounding tissue, eleven of eighteen samples were completely culture-negative, while the remaining seven showed an average two-log reduction in viable bacteria. The animals displayed no macroscopic signs of abnormal tissue irritation or inflammation.

The study highlights how formulation engineering and peptide delivery can address current gaps in local biomaterial infection control. In existing clinical literature, bioactive glass S53P4 achieves osteomyelitis eradication rates of roughly 85 to 88 percent, but relies on concurrent systemic antibiotic therapy and debridement. The researchers emphasized that incorporating SAAP-148 provides rapid bactericidal action during the vulnerable early postoperative period, complementing the delayed, long-term activity of bioactive glass. Several limitations were documented, including the use of polystyrene rather than metal plates for in vitro biofilm assays, the short one-day in vivo follow-up, and the reliance on a subcutaneous rather than an orthotopic bone model. The authors also noted that unquantified local peptide availability might explain incomplete bacterial clearance in peri-implant tissue, warranting longer-term studies to assess histocompatibility, pharmacokinetic release, and efficacy against Acinetobacter baumannii in vivo.

Subject of Research: Antibacterial efficacy of SAAP-148 peptide loaded bioactive glass against implant infections

Article Title: Prevention of murine implant-associated infection by SAAP-148-loaded bioactive glass S53P4 formulation

Article References: Rajkumar, D., Balraadjsing, P. P. S., de Boer, L., Riool, M., & Zaat, S. A. J. (2026). Prevention of murine implant-associated infection by SAAP-148-loaded bioactive glass S53P4 formulation. Frontiers in Bioengineering and Biotechnology, 14, Article 1959395. https://doi.org/10.3389/fbioe.2026.1959395

Image Credits: AI Generated

DOI: 10.3389/fbioe.2026.1959395

Keywords: bioactive glass S53P4, SAAP-148, antimicrobial peptides, orthopedic implant infections, bacterial biofilms, Staphylococcus aureus, Acinetobacter baumannii, biomaterials, Prevention, murine, implant-associated, infection