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Application value of baicalein in the management of periprosthetic joint infection: a preliminary in vitro study

BMC Complementary Medicine and Therapies volume 25, Article number: 152 (2025) Cite this article

Abstract

Background

This study aims to evaluate the efficacy of baicalein, a flavonoid derived from Scutellaria baicalensis, against Staphylococcus aureus (S. aureus), focusing on its inhibitory and eradicative effects on biofilms, as well as its cellular cytotoxicity. The goal is to provide preliminary evidence for its potential application in the management of periprosthetic joint infection (PJI).

Methods

The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of baicalein against the standard strain of S. aureus ATCC 29213, a clinical strain of methicillin-sensitive S. aureus 115 (MSSA 115), and a clinical strain of methicillin-resistant S. aureus 49 (MRSA 49) were determined using broth microdilution and colony counting methods. Bactericidal kinetics over a 24-h period were evaluated using a time-kill assay. Biofilm inhibition and eradication were assessed on 96-well and titanium alloy plates, while the cellular cytotoxicity of baicalein was examined using the cell counting kit-8 (CCK-8) assay on human primary synovial fibroblasts and chondrocytes.

Results

The MIC of baicalein was 32 μg/mL for the ATCC 29213, and 64 μg/mL for both MSSA115 and MRSA49. Meanwhile, the MBC for all three strains was 128 μg/mL. Baicalein exhibited a time-dependent bactericidal activity, with maximum efficacy at 24 h. Biofilm inhibition was evident at concentrations equal to or exceeding the MIC, as confirmed by biofilm biomass and metabolic activity assays, along with scanning electron and confocal laser microscope. However, baicalein was unable to completely eradicate preformed biofilms. Baicalein demonstrated significant cytotoxic effects on both synovial fibroblasts and chondrocytes after exposure for 16 and 24 h.

Conclusions

Baicalein shows significant bactericidal effects and effectively inhibits S. aureus biofilm formation. These findings suggest its potential as a promising local antibacterial agent for postoperative continuous intra-articular lavage in the treatment of S. aureus-related early postoperative or acute hematogenous PJIs.

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Introduction

Periprosthetic joint infection (PJI) is a serious complication following joint arthroplasty, with an incidence of approximately 1–2% in primary procedures and 9.2–30% in revision surgeries [1,2,3]. The increasing number of primary arthroplasties will lead to a rise in PJI in the future. PJI not only exerts significant physiological and psychological burdens on patients but also presents a substantial economic challenge. Previous data indicates that within five years of total hip arthroplasty, the cost of revision surgeries due to PJI is more than five times higher than revisions performed for other reasons [4].

PJI is the result of a complex interplay of among factors, including bacterial load around the prosthesis, host immune response, surgical techniques, and perioperative management. The predominant causative microorganism is Gram-positive Staphylococcus, with Staphylococcus aureus (S. aureus) responsible for over 25% of PJI cases [5,6,7,8]. In PJI, bacteria typically adopt a non-planktonic form, adhering to the implant surface and becoming encased in an extracellular polymeric matrix, eventually forming a biofilm [9]. Biofilms benefit from implant surfaces that facilitate bacterial attachment and maturation, providing protection from host immune defenses [10, 11] and impeding the penetration of antibiotics [12,13,14]. This creates an ideal environment for bacterial survival and growth. Moreover, biofilms contain a population of metabolically dormant bacteria [15], further complicating treatment due to their inherent resistance to antibiotics [16,17,18]. Consequently, addressing PJI requires effective strategies for both eradicating biofilms and preventing their recurrence, underscoring the need for new antibacterial agents.

Recent research trends have highlighted natural plant-derived compounds as promising sources of antimicrobial agents, offering multiple active ingredients and diverse mechanisms of action—an advantage in combating rising antibiotic resistance [19]. Several studies have shown that plant-derived compounds inhibit bacterial biofilm formation by disrupting quorum sensing signals and effectively eradicate mature biofilms, especially those produced by drug-resistant bacteria. Among these, flavonoids and phenolic compounds have shown the most potent effects [20].

Scutellaria baicalensis, a medicinal plant featured in the Chinese Pharmacopoeia, has been used in China for over 2000 years due to its wide range of pharmacological effects, including antibacterial, anti-inflammatory, antiviral, anticancer, hepatoprotective, antioxidant, antihypertensive, anticonvulsant, and neuroprotective properties [21]. Baicalein, one of the most abundant flavonoids found in the root of Scutellaria baicalensis, has demonstrated strong antibacterial properties and the ability to inhibit biofilm formation in both in vitro and in vivo studies [22,23,24,25]. Given these findings, baicalein holds significant potential for therapeutic application in PJI and is worth further investigation.

In this study, we preliminarily investigated the in-vitro efficacy of baicalein against S. aureus and its biofilms. Additionally, we evaluated its cytotoxicity on intra-articular cells to determine the potential value of baicalein in PJI.

Materials and methods

Bacterial strains, media, and reagents

The standard strain of S. aureus ATCC 29213 (American Type Culture Collection, USA) was kindly provided by Prof. Guobao Tian from the Department of Immunology and Microbiology, Zhongshan School of Medicine, Sun Yat-sen University (Guangzhou, China). The methicillin-sensitive S. aureus 115 (MSSA 115) and methicillin-resistant S. aureus 49 (MRSA 49) were isolated from patients with PJI at the First Affiliated Hospital of Sun Yat-sen University (Guangzhou, China). Strains were stored in 25% glycerol at -80 ℃ and used as needed.

Tryptic soy agar (TSA) and Tryptic soy broth (TSB) were purchased from Guangdong Huankai Biotechnology (Guangzhou, China). M-H agar (MHA) was sourced from Oxoid (Basingstoke, UK), and cation-adjusted M-H broth (CAMHB) from Qingdao Hope Biotechnology (Qingdao, China). Dimethyl sulfoxide (DMSO) was obtained from MP Biomedicals (USA) and crystal violet (CV) solution from Yuanye Bio-Technology (Shanghai, China). Glutaraldehyde (2.5%) was purchased from Servicebio Technology (Wuhan, China), and 2,3,5-triphenyl tetrazolium chloride (TTC) from Solarbio Science & Technology (Beijing, China). The LIVE/DEAD BacLight Bacterial Viability Kit, Minimum Essential Medium-Alpha (MEM-Alpha), Dulbecco’s Modified Eagle Medium/F-12 (DMEM/F-12), fetal bovine serum (FBS), and penicillin/streptomycin were sourced from Thermo Fisher Scientific (USA). The cell counting kit-8 (CCK-8) was obtained from Dojindo (Japan). The povidone-iodine was purchased from MeilunBio (Dalian, China).

Baicalein

Baicalein (purity ≥ 98%) was obtained from Chengdu Must Biotechnology (Chengdu, China). It has a molecular formula of C15H10O5 and a molecular weight of 270.24 g/mol, as shown in Fig. 1. Baicalein was dissolved in 100% DMSO, diluted to various concentrations with culture media as required, and stored at -80 ℃.

Fig. 1
figure 1

Chemical structure of baicalein

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Titanium alloy

Titanium alloy implants are commonly used in orthopedic applications. In this study, titanium alloy plates (Ti-6Al-4 V, 10 × 10 mm2 and 2 mm thickness) were obtained from Anhui Zhengying technology (China) to investigate the effect of baicalein on S. aureus biofilm. The plates were polished with 1200-grit metallographic abrasive sandpaper to achieve a uniform surface roughness. Prior to the experiments, the plates were autoclaved to ensure sterility.

Efficacy of baicalein against S. aureus

Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)

The MIC of baicalein was determined using the microbroth dilution method based on the Clinical and Laboratory Standards Institute (CLSI) guidelines [26]. S. aureus cultures were grown to the logarithmic phase and adjusted to 0.5 McFarland turbidity. This was followed by a 1:100 dilution in CAMHB. Bacterial suspensions (50μL) were mixed with varying concentrations of baicalein (50μL) in a 96-well plate, alongside growth controls, sterility controls, and DMSO controls. The final bacterial concentration was approximately 5 × 105 CFU/mL, with baicalein concentration ranging from 2 to 256 μg/mL. After incubation at 37℃ for 16–20 h, the MIC was defined as the lowest concentration without visible bacterial growth. Oxacillin was used as the quality-control antibiotic for validating the MIC determination of baicalein. This was to ensure the accuracy of the results and to determine the susceptibility and resistance of the three strains.

Following MIC determination, the MBC was assessed using the colony counting method. Cultures from wells with concentrations above the MIC were plated on MHA and incubated at 37 °C for 24–48 h. Colony-forming units (CFUs) were counted, and the MBC was defined as the lowest drug concentration that achieved a ≥ 3log10 (99.9%) reduction in CFU/mL compared to the initial bacterial load.

Time-kill assay

The time-kill assay was performed following National Committee for Clinical Laboratory Standards (NCCLS) guidelines [27]. Bacteria were grown to the logarithmic phase and adjusted to 0.5 McFarland turbidity. The bacterial suspension was used to inoculate glass tubes containing fresh CAMHB, with or without baicalein at concentrations ranging from 64 to 512 µg/mL (1/2 × MBC to 4 × MBC), achieving a final concentration of 1 × 106 CFU/mL in a 2 mL volume. The tubes were incubated at 37 °C. At 0, 4, 8, 16, and 24 h, 100 µL of the culture medium was removed from each tube and serially diluted in 900 µL of sterile saline. From each dilution, 100 µL was plated onto MHA, and colonies were counted after incubation at 37 °C for 24–48 h. A concentration was considered bactericidal if it achieved a ≥ 3log10 (99.9%) reduction in CFU/mL from the original inoculum.

Biofilm inhibition effect of baicalein

Biofilm inhibition assay in 96-well plate

The inhibitory effect of baicalein on biofilm formation was evaluated in 96-well plates using CV staining method as previously described [28, 29]. Bacteria were grown to the logarithmic phase in TSB supplemented with 1% glucose, and the bacterial suspension was adjusted to 0.5 McFarland turbidity. The 1% glucose was added to promote biofilm formation on the bottom of the well plate. Bacterial suspensions (2 μL) were added to 96-well plates containing baicalein (198 μL) to achieve a final concentration of 1 × 106 CFU/ml, alongside growth and sterility controls. Following incubation at 37 °C for 24 h, culture medium was removed, and the wells were gently rinsed three times with sterile distilled water. The biofilms were fixed with methanol for 10 min, after which the methanol was removed, and 0.1% CV was added to stain the biofilm for 20 min. The plates were washed three times with distilled water and resuspended in 33% glacial acetic acid. The absorbance at 595 nm (A595) was measured using a microplate reader (Epoch 2, BioTek, USA). The percentage of biofilm biomass at different concentrations was determined by calculating the average A595 obtained from the growth and sterility controls.

Biofilm inhibition assay on titanium alloy

Scanning electron microscope (SEM)

Bacteria cultured in TSB supplemented with 1% glucose were diluted in different concentrations of baicalein to achieve a final concentration of 1 × 106 CFU/ml. A 1 mL aliquot of this mixture was added to a 24-well plate containing sterilized titanium alloy plates. The plates were incubated at 37 °C for 24 h, rinsed three times with sterile distilled water, and fixed overnight in 2.5% glutaraldehyde at 4 °C. The following day, the plates were washed with distilled water and dehydrated through a graded ethanol series (50%, 70%, 85%, 100%) for 10 min at each concentration. After critical point drying and gold sputtering, the morphology of the biofilms and baicalein's inhibitory effects were observed using a scanning electron microscope (Phenom Pharos, Phenom-World B.V., Netherlands).

Biofilm live/dead staining

Biofilm formation on titanium alloy plates followed the same procedure as above. The LIVE/DEAD BacLight Bacterial Viability Kit was used to stain the biofilm cells on the surface of the titanium alloy plates. The stain contains SYTO9 and propidium iodide (PI), which both bind to DNA. SYTO9 emits green fluorescence in live cells, while PI, which suppresses SYTO9 fluorescence, emits red fluorescence in dead cells. Each plate was incubated with 1 ml of diluted mixed dye for 15 min at room temperature in the dark. After staining, the plates were washed with sterile distilled water and visualized using a confocal laser scanning microscope (CLSM) (LSM 880, Carl Zeiss, Germany). Images were captured with 63x/1.4 oil-immersion objective and processed using ZEISS ZEN software, version 3.9 (Carl Zeiss).

Biofilm eradication effect of baicalein

Biofilm eradication assay in 96-well plate

The eradication effect of baicalein on preformed biofilms was evaluated using both CV staining and TTC staining methods [28, 30]. TTC, a tetrazolium salt, is reduced by metabolic activity in live cells to produce a red formazan product, enabling the quantification of biofilm metabolic activity and serving as an indicator of biofilm cell viability [31, 32].

Two 96-well plates were used for this experiment—one for CV staining to measure biofilm biomass and another for TTC staining to assess metabolic activity. In contrast to the inhibition assay, baicalein was applied to the biofilm after its formation. A uniform biofilm was generated by incubating a bacterial suspension in TSB supplemented with 1% glucose at 37 °C for 24 h. After incubation, the supernatants were carefully removed, and the wells were rinsed with sterile distilled water to remove non-adherent bacteria.

For the CV plates, 200 μL of different concentrations of baicalein were added to each well, along with growth and sterility controls. In the TTC plates, 195 µL of baicalein and 5 µL of a 2% TTC solution were added to each well, resulting in a final TTC concentration of 0.05%. Both plates were incubated for an additional 24 h. The following procedure for CV staining was carried out similarly to the biofilm inhibition assay, with CV used to stain the biofilm and absorbance measured at A595.

For the TTC plates, methanol was added to each well and incubated for 30 min. The metabolized TTC, which turned red, was resuspended in methanol, and absorbance at 500 nm (A500) was measured using a microplate reader. The percentage of biofilm biomass and biofilm metabolic activity at different concentrations was determined by calculating the average A595 and A500 obtained from the growth and sterility controls.

Biofilm eradication assay on titanium alloy

A bacterial suspension in TSB with 1% glucose was added to titanium alloy surfaces in 24-well culture plates and incubated at 37 °C for 24 h to allow biofilm formation. After incubation, the culture medium was aspirated, the plates were gently washed with sterile distilled water, and different concentrations of baicalein were added. The samples were incubated for an additional 24 h. Following incubation, SEM and biofilm live/dead staining were performed as outlined in Sect. 2.5.2.

Cytotoxicity assay

Synovial and cartilage tissues were obtained from osteoarthritis patients undergoing total knee arthroplasty at the First Affiliated Hospital of Sun Yat-sen University. Written informed consent was obtained from all patients prior to tissue collection. The study was approved by the institutional review board and ethics committee of the hospital. Primary synovial fibroblasts and chondrocytes were aseptically isolated from the synovial and cartilage tissues, respectively. Synovial fibroblasts were cultured in MEM-Alpha supplemented with 10% FBS and 1% penicillin/streptomycin. Chondrocytes were cultured in DMEM/F-12 with the same supplements. Cells were cultured in standard 25 or 75 cm2 tissue culture flasks at 37 °C with 5% CO2 and passaged 2–4 times before being used for the CCK-8 assay to assess the effect of baicalein on cell viability. Furthermore, in order to assess the biocompatibility and clinical translational potential of baicalein, a 0.1% povidone-iodine solution, which is a commonly used concentration in surgery, was included in the cytotoxicity assay.

For the assay, 100 μL of cells were seeded in 96-well plates at a density of 10,000 cells/well and cultured to 80% confluence at 37 °C with 5% CO2. The culture medium was then aspirated, and the cells were exposed to 100 μL of different concentrations of baicalein as well as corresponding DMSO controls for 16 or 24 h. In the povidone-iodine group, cells were treated for 1 min. The control groups also included a growth control and a blank control. After incubation, the cells were washed twice with PBS, and 100μL of medium containing 10% CCK-8 reagent was added, followed by an incubation period of 2–4 h. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated according to the manufacturer's protocol.

Statistical analysis

Statistical analyses were conducted using GraphPad Prism (Version 9.5.0, GraphPad Software, San Diego, USA). The normality of data distributions was assessed using the Shapiro–Wilk and Kolmogorov–Smirnov tests. For normally distributed data, one-way analysis of variance was used for comparisons between groups, followed by Bonferroni correction for multiple comparisons. Cytotoxicity of povidone-iodine was assessed using an independent-samples t-test. For non-normally distributed data, the Kruskal–Wallis test followed by Dunn's test for multiple comparisons was applied. A p-value of less than 0.05 was considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). All experiments were repeated at least three times.

Results

Efficacy of baicalein against S. aureus

MIC and MBC determination

The antimicrobial susceptibility results of S. aureus strains are summarized in Table 1. Baicalein demonstrated both inhibitory and bactericidal activity against planktonic forms of S. aureus. The MIC of the quality control antibiotic oxacillin for ATCC 29213 and MSSA 115 was 0.25 μg/mL, whereas the MIC for MRSA 49 was 16 μg/mL. Both values within the range recommended by the CLSI, confirming that strain MRSA 49 is methicillin-resistant Staphylococcus aureus.

Table 1 Antimicrobial susceptibility of S. aureus strains
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Time-kill assay

The time-kill curve indicated a time-dependent bactericidal effect of baicalein (Fig. 2). In the growth control group, the number of colonies peaked between 8–24 h. Baicalein exhibited a delayed bactericidal action, with no significant reduction in bacterial counts observed within the first 8 h. At 16 h, concentrations of 512 µg/mL (4 × MBC) and 256 µg/mL (2 × MBC) demonstrated bactericidal effects against ATCC 29213 and MSSA 115, leading to a ≥ 3log10 reduction in CFU/mL compared to the initial bacterial count (Fig. 2A,B). At 128 µg/mL, bactericidal effect was observed after 24 h. The maximum bactericidal effect was observed at 24 h for these three concentrations. For MRSA 49, bactericidal effect was observed only at a concentration of 512 µg/mL after 16 h of exposure, while at 128 and 256 µg/mL, bactericidal efficacy was achieved after 24 h (Fig. 2C). The maximum bactericidal effect was observed at concentrations of 128–512 µg/mL after 24 h. At 64 µg/mL (1/2 × MBC), baicalein exhibited inhibition but no bactericidal effect against the three strains, with CFU/mL reductions of 2.3 log10, 2.4 log10, and 2.1 log10 for ATCC 29213, MSSA115, and MRSA 49, respectively.

Fig. 2
figure 2

Time-kill curves of Baicalein against ATCC 29213 (A), MSSA 115 (B), and MRSA 49 (C)

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Biofilm inhibition effect of baicalein

Biofilm inhibition assay in 96-well plate

The biofilm inhibition effect of baicalein against S. aureus strains in 96-well plates is shown in Fig. 3. Baicalein at concentrations equal to or above the MIC significantly reduced biofilm formation, indicating effective prevention of planktonic bacteria from proliferating and adhering, thus hindering biofilm maturation (Fig. 3A-C). At 32 µg/mL, biofilm biomass was reduced to 14% for ATCC 29213 (p < 0.0001) (Fig. 3A). At 64 µg/mL, the biomass of MSSA 115 and MRSA 49 biofilms was reduced to 1% and 33%, respectively (p < 0.001) (Fig. 3B, C).

Fig. 3
figure 3

Inhibitory effect of baicalein on biofilm formation of Staphylococcus aureus ATCC 29213 (A), MSSA 115 (B), and MRSA 49 (C) detected by the crystal violet staining method. The upper are the quantitative results of the absorbance of biofilm biomass at 595 nm, and the below are representative images of crystal violet staining in 96-well plates

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Baicalein inhibits biofilm formation on titanium alloy surface

The biofilm inhibition on titanium alloy surfaces was further assessed using SEM (Fig. 4). In the control group, a compact biofilm structure formed on the surface of the titanium alloy. Baicalein exhibited a concentration-dependent inhibition of biofilm formation in both strains. For ATCC 29213, at 4 µg/mL, the biofilm structure resembled that of the control group, but at 8–16 µg/mL, a reduction in biofilm density and relaxation of structure was observed. At concentrations of 32 µg/mL and above, the biofilm was largely eradicated, leaving only sparse bacterial colonies and greatly reduced biofilm density. For MSSA 115, no significant inhibition was observed at 4–8 µg/mL. However, at 16–32 µg/mL, a slight reduction in biofilm density and structural relaxation occurred. At higher concentrations (64–128 µg/mL), significant biofilm reduction and complete inhibition were achieved. These findings aligned with the results from the CV staining in the 96-well plates, indicating a consistent biofilm inhibition pattern for both strains.

Fig. 4
figure 4

SEM images of the inhibitory effects of baicalein at different concentrations on biofilm formation of ATCC 29213 and MSSA115 on titanium alloy surfaces. Original magnification × 1500, Scale bar = 50 μm

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To further assess the inhibitory effect of baicalein on biofilm viability on titanium alloy surfaces, a LIVE/DEAD viability kit was employed for staining, with results observed using CLSM. (Fig. 5). The staining results indicated a consistent trend for both strains. The control group, which was not treated with baicalein, displayed green fluorescence, signifying a high bacterial density and intact biofilm structure. At concentrations ranging from 4 to 32 µg/mL, the majority of cells exhibited green fluorescence, suggesting a predominance of viable bacteria within the biofilm. However, at a concentration of 32 µg/mL, a noticeable decline in bacterial density was observed, leading to structural relaxation of the biofilm. Notably, at concentrations of 64 to 128 µg/mL, particularly at 128 µg/mL, a significant number of cells showed red fluorescence, indicating a substantial increase in bacterial death.

Fig. 5
figure 5

CLSM images of the inhibitory effects of baicalein at different concentrations on biofilm formation of ATCC 29213 and MSSA115 on titanium alloy surfaces. The live and dead bacteria showed green and red fluorescence, respectively. Scale bar = 10 μm

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Biofilm eradication effect of baicalein

Biofilm eradication assay in 96-well plate

The eradication effect of baicalein on the biofilm growth of S. aureus strains in 96-well plates is illustrated in Fig. 6. For ATCC 29213, baicalein reduced CV staining starting at a concentration of 64 µg/mL, with 62% of biofilm biomass detected (p < 0.05). The most pronounced effects were observed at concentrations of 128, 256, and 512 µg/mL, resulting in corresponding biofilm biomasses of 41%, 33%, and 30%, respectively (p < 0.0001) (Fig. 6A). For MSSA 115, biofilm biomass was measured with 75% at 64 µg/mL of baicalein (p < 0.05), further decreasing to 55%, 44%, and 45% at 128, 256, and 512 µg/mL respectively (p < 0.0001) (Fig. 6B). For MRSA 49, baicalein significantly reduced CV staining at 32 µg/mL, reducing the biofilm biomass to 69%. As the concentrations increased further, from 64 to 512 µg/mL, the measured biofilm biomass values were 73%, 70%, 63%, and 71%, respectively (Fig. 6C). However, baicalein had minimal impact on the metabolic activity of the biofilm bacteria. The metabolic activity of the biofilm of ATCC 29213 was significantly lower than that of the control group only at concentrations of 256 and 512 µg/mL, recorded at 66% (p < 0.001) and 54% (p < 0.0001), respectively (Fig. 6D). The metabolic activity of the clinical strains MSSA 115 showed no significant reduction compared to the control group (Fig. 6E). However, at a concentration of 512 μg/mL, baicalein significantly reduced the metabolic activity of MRSA 49 biofilms to 58% (Fig. 6F).

Fig. 6
figure 6

Eradication effect of baicalein on preformed biofilm of ATCC 29213, MSSA 115, and MRSA 49 detected by the crystal violet staining method (A-C) and TTC staining method (D-F). The upper of A-C are the quantitative results of the absorbance of biofilm biomass at 595 nm, and the below are representative images of crystal violet staining in 96-well plates. The upper of D-F are the quantitative results of the absorbance of biofilm metabolic activity at 500 nm, and the below are representative images of TTC staining in 96-well plates

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Limited efficacy of baicalein on titanium alloy surface biofilm

Results from SEM and CLSM revealed that baicalein had limited efficacy in eradicating preformed biofilm on titanium alloy surfaces. The SEM results (Fig. 7A) indicated that the control group displayed a compact biofilm structure on the titanium alloy surface. Treatments at concentrations of 128 µg/mL and 256 µg/mL did not show significant differences compared to the control group, with the biofilm structure remaining intact. However, at a concentration of 512 µg/mL, a noticeable decrease in biofilm density and structural relaxation were observed. The CLSM results (Fig. 7B) showed that all treatment concentrations exhibited similar green fluorescence to the control group, with only a few instances of red fluorescence detected in the MSSA 115 biofilm, indicating a limited lethal effect of baicalein on bacteria within the biofilm.

Fig. 7
figure 7

Baicalein had little effect on the eradication of biofilm on the titanium alloy surface. (A) SEM images of preformed ATCC 29213 and MSSA115 biofilms treated with different concentrations of baicalein. Original magnification × 1500, Scale bar = 50 μm. (B) CLSM images of preformed ATCC 29213 and MSSA115 biofilms on titanium alloy surfaces treated with different concentrations of baicalein. The live and dead bacteria showed green and red fluorescence, respectively. Scale bar = 10 μm

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Cytotoxicity assay

Synovial fibroblasts and chondrocytes were treated with baicalein at concentrations ranging from 16 to 256 µg/mL for 16 or 24 h. Additionally, the cells were treated with 0.1% povidone-iodine for 1 min. The findings indicated that baicalein exhibited significant cytotoxic effects. Following a 24-h treatment, cell viability decreased below 70% for all the concentrations tested, showing a dose- and time-dependent reduction (Fig. 8A-D). For synovial fibroblasts (Fig. 8A-B), there was a significant decrease in cell viability at both time points. Specifically, at bactericidal concentrations of 128 µg/mL and 256 µg/mL, cell viability dropped to 9.5% and 8.6% after 16 h, respectively (p < 0.0001), further decreasing to as low as 6% and 5% after 24 h (p < 0.0001). Chondrocytes also exhibited reduced viability. Specifically, at bactericidal concentrations of 128 µg/mL and 256 µg/mL, the cell viabilities were 39% and 32% at 16 h, respectively (p < 0.0001) (Fig. 8C). These rates further declined to 21% and 17% at 24 h (p < 0.0001) (Fig. 8D). Notably, chondrocytes displayed a higher tolerance to the cytotoxicity effects of baicalein compared to synovial fibroblasts. However, when compared to baicalein, the DMSO control group showed significantly lower cytotoxicity. In both synovial fibroblasts and chondrocytes exposed to DMSO, cell viability was sustained above 92%, except at the 1.7% concentration (Fig. 8E-H). At a 1.7% DMSO concentration, the viability of synovial fibroblasts decreased from 88.7% at 16 h to 80.8% at 24 h (Fig. 8E-F). Likewise, the viability of chondrocytes decreased from 82.9% at 16 h to 77.9% at 24 h under the same conditions (Fig. 8G-H). Similar to baicalein, 0.1% povidone-iodine exhibited substantial cytotoxic effects on both cell types, reducing cell viability to less than 2% (Fig. 8I, J).

Fig. 8
figure 8

Cytotoxicity of baicalein and 0.1% povidone-iodine on synovial fibroblasts and chondrocytes. The viability of synovial fibroblasts and chondrocytes was evaluated using the CCK-8 assay after 16- and 24-h treatments with different concentrations of baicalein (A-D), DMSO controls (E–H), and 0.1% povidone-iodine (I, J)

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Discussion

The rising severity of antibiotic resistance in PJI underscores the need for effective antibacterial agents derived from natural sources. This study preliminarily investigates the potential application of baicalein in managing S. aureus-related PJI. The results indicate that baicalein exhibits a strong antibacterial effect against S. aureus, with MIC values significantly lower than those reported in previous studies [22]. Bactericidal activity was observed at a concentration of 128 µg/mL, which is 2–4 times higher than its MIC. According to established classifications, antimicrobial agents are considered bactericidal when the MBC to MIC ratio is ≤ 4; conversely, they are termed bacteriostatic when this ratio exceeds 4 [27, 33]. Thus, baicalein qualifies as a bactericide. The time-kill curves demonstrated a moderate bactericidal rate, with complete sterilization requiring 16 to 24 h. Therefore, baicalein is a promising antibacterial agent for PJI management.

Biofilm formation on prosthetic surfaces involves several sequential steps: initial reversible adhesion, irreversible adhesion and aggregation, biofilm maturation, and diffusion to initiate further infections [34]. We propose that biofilm management should be divided into inhibiting formation during the early stages and eradicating mature biofilms. Our findings from 96-well plate assays demonstrated that baicalein completely inhibited biofilm formation at MIC and above; even at a sub-inhibitory concentration of 1/2 × MIC, baicalein significantly reduced biofilm growth by at least 30%. These results align with those of Chen et al., who reported concentration-dependent inhibition of bacterial adhesion and reduction in biofilm biomass with baicalein when cultured with S. aureus for 3 and 7 days at concentrations of 16, 32, and 64 µg/mL [22]. Our SEM and CLSM analyses further confirmed that baicalein effectively inhibits S. aureus adhesion and subsequent biofilm formation on titanium alloy surfaces in a concentration-dependent manner. At MIC and above, only a few colonies remained on the titanium alloy, with minimal aggregation into biofilms; the number of dead bacteria began to increase, and most bacteria were dead at MBC (128 µg/mL). These findings indicate that baicalein can prevent initial bacterial adhesion and intervene in early-stage biofilm formation in PJI.

Effective treatment of PJI requires the eradication of mature biofilms. However, bacteria protected by biofilms present a greater challenge compared to planktonic bacteria [35]. The results from 96-well plates indicated that baicalein significantly reduced biofilm biomass of three strains at MBC and above. However, the reduction in TTC staining, which reflects the metabolic activity of bacteria within the biofilm, was less pronounced than that of CV staining. This suggests that baicalein has a limited effect on bacterial metabolism within the biofilm. Furthermore, no significant differences were observed between the TTC staining results for MSSA 115 and those of the control group, indicating potentially higher bacterial activity in clinical strain biofilms. Integrating CLSM and SEM results revealed that only at a high concentration of 512 µg/mL did baicalein lead to a decrease in biofilm density and some relaxation of its structure, but it did not exhibit bactericidal effects on the bacteria within the biofilms. Conversely, Luo et al. demonstrated that baicalein effectively dispersed both early (1-day) and mature (5-day) Pseudomonas aeruginosa biofilms in a concentration-dependent manner [23].

The concentration of antibiotics required to effectively eradicate bacteria in biofilms is 100–1000 times higher than the MIC [36]. However, systemic intravenous administration of antibiotics can achieve only two to three times higher concentrations in infected joint tissues than the MIC [37]. Consequently, local antibacterial agents are crucial in this context. While our data suggest that baicalein is ineffective in eradicating mature biofilms, it shows promise as a local antibacterial agent in managing early postoperative or acute hematogenous PJI, where mature biofilms have not yet formed. Combined with the results of the time-kill assay, we believe that baicalein is not suitable for short-term intraoperative irrigation; however, it may be more appropriate for postoperative continuous intra-articular lavage. This approach has the potential to effectively control infections, enhance treatment success rates, reduce antibiotic-related adverse effects, and offer additional therapeutic options for PJI.

An ideal antibacterial solution should demonstrate potent bactericidal activity while minimizing toxicity to host tissues, thereby requiring a balance between efficacy and safety. In assessing the cytotoxicity of baicalein, we evaluated the cell viability of primary synovial fibroblasts and chondrocytes to simulate in vivo conditions more closely. At 16 and 24 h, baicalein exhibited greater toxicity to both cell types at effective bactericidal concentrations of 1 × MBC (128 µg/mL) and 2 × MBC (256 µg/mL). The cytotoxicity assay results in the DMSO control group showed that although DMSO had some cytotoxicity, the cytotoxic effects of baicalein were mainly due to its own properties, with little contribution from DMSO. Since povidone-iodine is the main chemical debridement agent in PJI surgery and surgical sites are usually irrigated with it for at least one minute in clinical procedures, we chose one minute as the exposure time of povidone-iodine to the cells. Our findings are in line with existing literature on the in-vitro cytotoxicity of commercial povidone-iodine, which has been shown to significantly impact the viability and functionality of various cell types in vitro, such as fibroblasts, chondrocytes, and osteoblasts [38,39,40]. However, this has not hindered its extensive clinical use. Based on clinical experience and available in vitro results, we propose that tissue cells in vivo exhibit superior tolerance to various antibacterial agents compared to in vitro cultured cells, which lack tissue perfusion and are more fragile. Therefore, future in vivo experiments are essential to verify whether the relative antiseptic benefits of baicalein outweigh any cytotoxicity it may produce.

Several limitations exist in our study. Firstly, due to its inherent poor water solubility, baicalein can only be dissolved in the organic solvent DMSO. Since DMSO itself has antibacterial activity and cytotoxic properties, it is crucial to control the solvent concentration strictly to avoid any potential effects on experimental outcomes. Moreover, exceeding a certain concentration threshold causes baicalein to precipitate from the medium, which affects the actual drug concentration. Consequently, the highest tested concentration was limited to 512 µg/mL, further restricting its applicability in biofilm eradication experiments. Secondly, the present study only focused on S. aureus. In future research, it would be necessary to include more strains with different types and resistance patterns to comprehensively evaluate the antibacterial activity of baicalein. Thirdly, further in vivo experiments were not conducted to evaluate the efficacy and cytotoxicity of baicalein. Future studies will systematically assess the effects and potential toxicity of baicalin on joint tissues and cells through in vivo experiments, providing detailed data to support its clinical translation. Lastly, this study did not investigate the specific mechanisms responsible for the bactericidal and biofilm inhibition effects of baicalein.

Conclusion

Baicalein exhibits significant inhibitory and bactericidal effects against S. aureus, effectively preventing biofilm formation. These findings suggest its potential as a promising local antibacterial agent for postoperative continuous intra-articular lavage in the treatment of S. aureus-related early postoperative or acute hematogenous PJIs. Additionally, we are exploring the application of nanotechnology to enhance the water solubility of baicalein. This approach aims to enable the eradication of mature biofilms, reduce cytotoxicity, and achieve optimal efficacy and safety in vivo, thereby establishing reliable experimental evidence for future clinical translation.

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Abbreviations

CAMHB:

Cation-adjusted M-H broth

CCK-8:

Cell counting kit-8

CLSI:

Clinical and Laboratory Standards Institute

CLSM:

Confocal laser scanning microscope

CV:

Crystal violet

DMEM/F12:

Dulbecco’s Modified Eagle Medium/F-12

DMSO:

Dimethyl sulfoxide

FBS:

Fetal bovine serum

MBC:

Minimum bactericidal concentration

MEM-Alpha:

Minimum Essential Medium-Alpha

MHA:

M-H agar

MIC:

Minimum inhibitory concentration

MSSA:

Methicillin-sensitive Staphylococcus aureus

PJI:

Periprosthetic joint infection

PI:

Propidium iodide

S. aureus :

Staphylococcus aureus

SEM:

Scanning electron microscope

TSA:

Tryptic soy agar

TSB:

Tryptic soy broth

TTC:

Triphenyl tetrazolium chloride

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Acknowledgements

The authors would like to thank Prof. Guobao Tian from the Department of Immunology and Microbiology, Zhongshan School of Medicine, Sun Yat-sen University (Guangzhou, China), for kindly providing the standard strain of Staphylococcus aureus ATCC 29213.

Funding

This work was supported by the National Natural Science Foundation of China (Grant number: 82372424, to P. Sheng; Grant number: 82203677, to L. Long), the Postdoctoral Fellowship Program of China Postdoctoral Science Foundation (Grant number: GZC20233275, to X. W). The study sponsors were not involved in the study design, in the collection, analysis and interpretation of data, in writing of the article, and in the decision to submit the article for publication.

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Author notes

  1. Aerman Alimu, Xiaoyu Wu, and Dongwei Huang contributed equally to this work.

Authors and Affiliations

  1. Department of Joint Surgery, The First Affiliated Hospital of Sun Yat-Sen University, NO.58, Zhongshan 2nd Road, Guangzhou, 510080, China

    Aerman Alimu, Xiaoyu Wu, Dongwei Huang, Chenghan Chu, Baiqi Pan, Yang Xing, Weishen Chen & Puyi Sheng

  2. Guangdong Provincial Key Laboratory of Orthopaedics and Traumatology, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, 510080, China

    Aerman Alimu, Xiaoyu Wu, Dongwei Huang, Chenghan Chu, Baiqi Pan, Yang Xing, Weishen Chen & Puyi Sheng

  3. Research Center of Translational Medicine, The First Affiliated Hospital of Sun Yat-Sen University, NO.58, Zhongshan 2nd Road, Guangzhou, 510080, China

    Lingli Long

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Contributions

AA, XW, WC, LL and PS contributed to the conception and design of the study. AA carried out the experiments. AA, XW, CC, BP and YX collected the data, while AA and DH performed the statistical analysis. AA and DH wrote the manuscript. XW, WC, LL and PS critically revised the manuscript. All authors reviewed and approved the final manuscript.

Corresponding authors

Correspondence to Weishen Chen, Lingli Long or Puyi Sheng.

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The isolation of primary cells from human samples was performed in accordance with the ethical standards of the Declaration of Helsinki and approved by the institutional review board and ethics committee of the First Affiliated Hospital of Sun Yat-sen University (Approval Number: [2023]740). Informed consent was obtained from the patients prior to the collection of tissue samples.

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Alimu, A., Wu, X., Huang, D. et al. Application value of baicalein in the management of periprosthetic joint infection: a preliminary in vitro study. BMC Complement Med Ther 25, 152 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12906-025-04888-w

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BMC Complementary Medicine and Therapies

ISSN: 2662-7671

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