Initial therapeutic evidence of a borosilicate bioactive glass (BSG) and Fe3O4 magnetic nanoparticle scaffold on implant-associated Staphylococcal aureus bone infection

Abstract

Implant-associated Staphylococcus aureus (S. aureus) osteomyelitis is a severe challenge in orthopedics. While antibiotic-loaded bone cement is a standardized therapeutic approach for S. aureus osteomyelitis, it falls short in eradicating Staphylococcus abscess communities (SACs) and bacteria within osteocyte-lacuna canalicular network (OLCN) and repairing bone defects. To address limitations, we developed a borosilicate bioactive glass (BSG) combined with ferroferric oxide (Fe₃O₄) magnetic scaffold to enhance antibacterial efficacy and bone repair capabilities. We conducted comprehensive assessments of the osteoinductive, immunomodulatory, antibacterial properties, and thermal response of this scaffold, with or without an alternating magnetic field (AMF). Utilizing a well-established implant-related S. aureus tibial infection rabbit model, we evaluated its antibacterial performance in vivo. RNA transcriptome sequencing demonstrated that BSG + 5%Fe₃O₄ enhanced the immune response to bacteria and promoted osteogenic differentiation and mineralization of MSCs. Notably, BSG + 5%Fe₃O₄ upregulated gene expression of NOD-like receptor and TNF pathway in MSCs, alongside increased the expression of osteogenic factors (RUNX2, ALP and OCN) in vitro. Flow cytometry on macrophage exhibited a polarization effect towards M2, accompanied by upregulation of anti-inflammatory genes (TGF-β1 and IL-1Ra) and downregulation of pro-inflammatory genes (IL-6 and IL-1β) among macrophages. In vivo CT imaging revealed the absence of osteolysis and periosteal response in rabbits treated with BSG + 5%Fe₃O₄ + AMF at 42 days. Histological analysis indicated complete controls of SACs and bacteria within OLCN by day 42, along with new bone formation, signifying effective control of S. aureus osteomyelitis. Further investigations will focus on the in vivo biosafety and biological mechanism of this scaffold within infectious microenvironment.

Keywords: Biofilm; Borosilicate bioactive glass; Magnetic nanoparticles; Osteomyelitis; Staphylococcus aureus.

Graphical abstract

Fig. 1

A Schematic diagram of a model for the treatment of S. aureus related bone infection model with BSG + Fe₃O₄ magnetic scaffold. (1) To match the size of titanlnium alloy screws used for the establishment of S. aureus bone infection model, the size of BSG + Fe₃O₄ scaffold was prepared under the premise of full mixing. (2) BSG can promote the autoimmune regulation of M2 macrophage polarization and the bone-inducing properties of promoting osteogenesis. (3) Fe₃O₄ magnetic particles can eradicate S. aureus within OLCN when exposed to an AMF. (4) Establishment of an implant-related S. aureus bone infection model of rabbit and treatment process. In the untreated group, the infected screws were removed after challenged with S. aureus contaminated screw by day 7, only debridement was performed without scaffolds, as bacteria invasion and biofilm formation, the acute bone infections turned into chronic bone infections eventually. While, the treatment group suffered from debridement and implanted with BSG + Fe₃O₄ scaffolds at the original infection site, then the treatment last until 42 days under the AMF.

Fig. 2

Morphological characterization and biomechanical properties of BSG + Fe₃O₄magnetic scaffold. A) BSG + Fe₃O₄ scaffolds were compounded to match the size of the titanium screws use in the in vivo model. B) SEM images of the microarchitecture of BSG + Fe₃O₄ magnetic scaffolds, at 5000X and 20000X magnifications. C-D) FTIR spectra and XRD patterns of 3B3Sr, BSG, Fe₃O₄, and BSG with 4%, 5%, or 6%Fe₃O₄. E) Compressive strength (MPa) of each scaffold group (n = 3), presented as mean ± SD. F) Release of ions including BO₃³⁻, SiO₂²⁻, Ca²⁺, Sr²⁺ and Fe³⁺ from scaffolds incubated in PBS were quantified and plotted across multiple time points. Each data point represents the total released over two days. G) pH of PBS solution containing BSG + Fe₃O₄ scaffolds, measured to 28 days. H–I) FTIR spectra and XRD patterns of BSG + Fe₃O₄ scaffolds after immersion in PBS for 7, 14 and 28 days.

Fig. 3

Cytotoxicity, proliferation, viability, and cytoskeleton of MSCs when cultured with a 128-fold dilution of BSG + Fe₃O₄magnetic scaffold extracts. A-I) CCK8 results were obtained after culturing cells in extracts of different concentrations of BSG + Fe₃O₄ for 1 day. Cells exhibited lower survival ratio until the extract was diluted 128 times. J-M) MSCs proliferation was assessed using CCK8 after being culturing in 32-, 64-, 128-, and 256-fold dilutions of BSG + 5%Fe₃O₄ extract for 1 day. The 128-fold dilution of BSG + 5%Fe₃O₄ extract had no impact on cell proliferation. N) EdU cell proliferation staining, O) live/dead staining, and P) cytoskeleton staining of MSCs cultured with 128-fold dilutions of the scaffold extracts for 2 days. These results indicated that the extracts of each polymer had no significant effects on cell viability, skeleton and proliferation when diluted 128-fold. Results were shown as Mean ± SD; One-way ANOVA; n = 6; * indicates statistical difference (P < 0.05), ** means P < 0.01, *** means P < 0.001.

Fig. 4

Osteogenic effects of BSG extracts at 128-fold dilution on MSCs and macrophages in culture. A) ALP staining of MSCs (C3H10T1/2) cultured with scaffold extracts DMEM diluted 32-, 64-, 128-, and 256-fold in OIM for 7 days, and B) alizarin red S staining after 21 days of culture, C-D) with corresponding quantitative analysis (n = 3). Specifically, the 128-fold dilution of BSG + 5%Fe₃O₄ extract exerted the most substantial impact on ALP activity and late matrix mineralization. E-F) The expression of osteogenesis related mRNA (RUNX2, ALP, OCN) (n = 4) and G-H) proteins (RUNX2 and OCN) were assessed via RT-qPCR and Western blot, with quantification of the blot results displayed in I and J (n = 3). The results suggested that the 128-fold dilution of BSG + 5%Fe₃O₄ scaffold extracts exhibited the most significant upregulation of osteogenic genes. Results were shown as Mean ± SD; Two-way ANOVA; * indicates statistical difference (P < 0.05), ** means P < 0.01, *** means P < 0.001.

Fig. 5

BSG+5%Fe₃O₄enhances immune response to bacteria, osteogenic differentiation, and chemokine receptor gene expression in MSCs. A-B) MSCs (C3H10T1/2) were cultured in 128-fold dilution of BSG + 5%Fe₃O₄ extract for 7 days, and RNA transcriptome sequencing was performed on three separate samples, along with three unexposed MSCs as negative controls. Genes with significant changes in transcript numbers and high p-values were selected using a thresholding method. C-D) GO enrichment bubble map revealed enrichment of genes associated with the immune response of MSCs to viruses and bacteria (red boxes), osteoblast differentiation, extracellular matrix secretion, bone tissue mineralization (blue boxes), as well as chemokine receptor related pathways (such as CCR2) involved in mediating inflammation response (green boxes). E-F) The molecular pathways of differentially expressed genes were further investigated using KEGG enrichment, these findings revealed that BSG + 5%Fe₃O₄magnetic scaffold extract significantly upregulated the expression of genes associated with the NOD-like receptor (NLR) and TNF signaling pathways in MSCs (red boxes). This confirmation underscores the potential of the magnetic scaffold to modulate bone immunity and regulate bone homeostasis.

Fig. 6

BSG scaffolds regulate inflammation in macrophages in vitro. A-B) RAW 264.7 cells were cultured with 128-fold dilution of scaffold extract for 3 days, followed by flow cytometry for CD80 and CD206, the BSG+5%Fe₃O₄group showed the lowest expression (70.97 %) of CD80 and the highest expression (58.65 %) of CD206. C-D) The expression of anti-inflammatory (TGF-β1 and IL-1Ra) and pro-inflammatory (IL-6 and IL-1β) gene in macrophages were assessed by RT-qPCR. Compared to the control group, RAW 264.7 macrophages cultured with the128-fold dilution of BSG+5%Fe₃O₄had the significantly promotion effect on expression of TGF-β1and IL-1Ra, and the most obvious inhibition effect on IL-6 and IL-1β. Results were shown as Mean ± SD; Two-way ANOVA; * indicates statistical difference (P < 0.05), ** means P < 0.01, *** means P < 0.001.

Fig. 7

Magnetic-thermal-induced thermal efficiency and antibacterial properties of BSG + Fe₃O₄in vitro. A, D) Thermal images of each scaffold were captured over time-course using a far-infrared thermometer. B) When subjected to AMF for 180 s, the temperatures of BSG + 4%Fe₃O₄, BSG + 5%Fe₃O₄, and BSG + 6%Fe₃O₄ reached 46.1 ± 0.7 °C, 54.7 ± 1.1 °C, and 65.3 ± 1.2 °C, respectively. C) White light image of the tissue sample under AMF. E) Linear regression analysis of temperature change from the center of the interface of BSG + 5%Fe₃O₄ magnetic scaffold radially outward into the air when exposed to AMF for 180 s. F) In the fresh rabbit tibia, linear regression is drawn according to the attenuation of BSG + 5%Fe₃O₄ interface center temperature with distance. when exposed to AMF for 180 s. G) According to the linear regression of temperature attenuation from the center of BSG + 5%Fe₃O₄ interface, the effective sterilization temperature of 45 °C was achieved at an average radial distance of 0.1155 mm. H–I) Antibacterial properties of each scaffold in vitro and statistical analysis of diameter of bacteriostatic zone. inhibition zone of BSG + 5%Fe₃O₄ + AMF exhibited the largest zone of inhibition against S. aureus on TSA plate. Results are shown as Mean± SD; One-way ANOVA; *indicates statistical difference (P<0.05), *** means P<0.001.

Fig. 8

Representative gross images and CT images of infected tibiae with untreated group, BSG+5%Fe₃O₄or BSG+5%Fe₃O₄+AMF as far as 42 days. A) The design of 1-stage revision bone infection model. B) Gross images of the superior tibial metaphysis on days 7. All specimens exhibited well-maintained scaffold positioning without displacement or prolapse, while soft tissue swelling and pus moss formation were observed in all groups. C) Gross images of the superior tibial metaphysis on days 42. By day 42, pus (yellow arrow) and soft tissue infection (orange arrow) were present at the screw tract in the untreated and BSG + 5%Fe₃O₄ groups, and with a noticeable reduction in the diameter of the screw tract compared to 7 days. In contrast, pus was absent in the BSG + 5%Fe₃O₄ + AMF group, and the screw tract had nearly disappeared (red arrow) by day 42. D) CT images of rabbit tibiae on days 7, 14, 28, and 42 post revision. On days 28 and 42, the untreated group displayed evident periosteal response (green arrow) and osteolytic destruction or formation of dead bone (orange arrow) in the medullary cavity. Periosteal response was also observed in cortical bone on days 7, 14 and 28 in the BSG + 5%Fe₃O₄ group (green arrows), while a noticeable periosteal reaction was absent in the BSG + 5%Fe₃O₄ + AMF group. On day 42, the BSG + 5%Fe₃O₄ and BSG + 5%Fe₃O₄ + AMF groups exhibited cortical bone healing (red arrows) at the implant site.

Fig. 9

Histological evidence of the antibacterial effects of BSG+5%Fe₃O₄in vivo. A) H&E and Gram staining after infection surgery for 7 days, histological evidence of bone infection could be observed in both the untreated and the BSG + 5%Fe₃O₄ groups (red arrows). SACs were observed around the scaffolds of BSG + 5%Fe₃O₄ group, and undegraded iron was also seen in BSG + 5%Fe₃O₄ group (black arrows). C-E) H&E and Gram staining at 28 days after infection. On day 28, the infection sites had progressed to diffuse inflammatory lesions in both the untreated and BSG + 5%Fe₃O₄ groups (green arrow), and osteolysis occurred in the two groups (blue arrow). Substantial SACs formation was observed in the pulp cavity of the untreated group (red arrows). In the BSG + 5%Fe₃O₄ group, S. aureus was detected around and within the scaffold (red arrow). In contrast, the scaffold and pulp cavity of the BSG + 5%Fe₃O₄ + AMF group exhibited significantly reduced signs of bone infection and early bone formation. F-G) At 42 days post-treatment, H&E and Gram staining revealed partial degradation of the BSG + 5%Fe₃O₄ scaffold and a reduction in the diffuse medullary lesions observed on day 28 (green arrows). However, some bacteria persisted within the scaffolds (red arrows). Remarkably, the BSG + 5%Fe₃O₄ + AMF group exhibited significant new bone deposition (yellow arrows) and pronounced scaffold degradation.

Fig. 9

Histological evidence of the antibacterial effects of BSG+5%Fe₃O₄in vivo. A) H&E and Gram staining after infection surgery for 7 days, histological evidence of bone infection could be observed in both the untreated and the BSG + 5%Fe₃O₄ groups (red arrows). SACs were observed around the scaffolds of BSG + 5%Fe₃O₄ group, and undegraded iron was also seen in BSG + 5%Fe₃O₄ group (black arrows). C-E) H&E and Gram staining at 28 days after infection. On day 28, the infection sites had progressed to diffuse inflammatory lesions in both the untreated and BSG + 5%Fe₃O₄ groups (green arrow), and osteolysis occurred in the two groups (blue arrow). Substantial SACs formation was observed in the pulp cavity of the untreated group (red arrows). In the BSG + 5%Fe₃O₄ group, S. aureus was detected around and within the scaffold (red arrow). In contrast, the scaffold and pulp cavity of the BSG + 5%Fe₃O₄ + AMF group exhibited significantly reduced signs of bone infection and early bone formation. F-G) At 42 days post-treatment, H&E and Gram staining revealed partial degradation of the BSG + 5%Fe₃O₄ scaffold and a reduction in the diffuse medullary lesions observed on day 28 (green arrows). However, some bacteria persisted within the scaffolds (red arrows). Remarkably, the BSG + 5%Fe₃O₄ + AMF group exhibited significant new bone deposition (yellow arrows) and pronounced scaffold degradation.