Gentamicin Sponge for Anti-infection, a Controversial Old Topic and Preliminary Exploration of the Solution, in Vitro and in Vivo Experiments

Purpose: Our study was to construct drug-loading and drug-release quantitative equation of gentamicin sponge, in addition, obtain the wound infection prevented and treated scheme of gentamicin sponge. Methods: Sterile sponge was cut into 1×1×0.5cm size and immersed into 40mg/ml, 16mg/ml, 8mg/ml, 4mg/ml, 1.6mg/ml, 0.8mg/ml or 0mg/ml gentamicin solution for 12h, 24h, 48h, 96h or 120h to evaluate gentamicin-loading of sponge. Sponge was immersed in gentamicin solution of different concentrations for 48h and then air-dried. Air-dried sponge was immersed into 10ml 0.9% physiological saline to evaluated gentamicin-release. Staphylococcus aureus (MSSA) and Pseudomonas aeruginosa (P. aeruginosa) were used to explore infection prevented scheme of gentamicin sponge. Besides, femur fractured with wound infection rat model was used to discuss the infection treated scheme. Results: The equation of gentamicin-loading of sponge was: z=(0.03718±0.01672)x+(-4.578e-4±0.06253)y+(-2.50935e-4±1.47521e-4)x2+ (0.00303±0.00149)y2+(0.00408±3.52827e-4)xy (R2 was 0.97) and drug-release equation was z=(4.37205±1.18048)x+(-7.05921±3.09628)y+ (-0.04596±0.01287)x2+(0.3309±0.07912)y2+(0.31559±0.02754)xy (R2 was 0.95). The antibacterial zone sizes of sponges immersed in 40mg/ml, 16mg/ml, 8mg/ml, 4mg/ml, 1.6mg/ml, 0.8mg/ml gentamicin solution were larger than 0mg/ml air-dried sponge, and the difference had statistically signicant (p<0.01). Rats of groups of 40mg/ml air-dried sponge, 16mg/ml air-dried sponge, 8mg/ml air-dried sponge had no wound suppuration in both MSSA and P. aeruginosa infection rat model. Conclusions: The quantied equation of gentamicin-loading and release of sponge was with high accuracy. 1.6 mg/ml air-dried sponge and 0.8mg/ml air-dried sponge were enough to prevent wound infection. Besides, if wound were conrmed to be sensitive bacteria infected, we recommended to use 40mg/ml air-dried sponge, 16mg/ml air-dried sponge or 8mg/ml air-dried sponge to treat. model. We give some ideas for clinical application of gentamycin sponge, especially in open fracture infection patients. contraposition as the previously study [10]. sterile bacterial muscle the skin using The mortality rate and wound tissue sections of rats were obtained following the establishment of the model to evaluate whether the model had succeeded and was stable. Finally, we chose 108 CFU/ml MSSA and 106 CFU/ml P. aeruginosa to establish the rat model. results are in the supplementary sponge group (5 rats, 1 rat with wound suppuration) had rats with wound suppuration. The differences between 1.6mg/ml air-dried sponge group and 40mg/ml air-dried sponge group were no statistically signicant (p>0.99). Wound suppuration rates of 0mg/ml air-dried sponge group rats) 100%, which was much higher than (all of two-groups were p>0.99).

The nding of lots of articles are inspiring. Han, J.-S. et al. analyzed the results of applying gentamicin-impregnated sponge during the spinal operation and they found that gentamicin-impregnated sponge can signi cantly decrease the SSI [4]. Chang, W. K. et al. analyzed whether gentamicin sponges decrease the incidence of SSI. They included 15 RCTs encompassing a total of 6979 patients and concluded that gentamicin sponge decreased the rate of SSI [5]. Schimmer, C. et al. rstly used a controlled, prospective, randomized, double-blind study to investigate the e cacy of gentamicin sponge in sternal wound complications after heart surgery. They enrolled 720 patients and found that gentamicin sponge can effectively reduce the infection complications [6].
However, some studies have demonstrated that gentamicin impregnated sponge cannot reduce the SSI and even some researches proposed that gentamicin sponge can increase the risk of infections. Wouthuyzen-Bakker, M. et al. discussed that the e cacy of applying local gentamicin-impregnated sponges during debridement in early acute periprosthetic joint infections. They found that local gentamicin impregnated sponges cannot reduce the incidence rate of infection, so they discouraged use gentamicin impregnated sponge [7]. Uçkay, I. et al. evaluated the bene t of the treatment of gentamicin sponge with mild diabetic foot ulcer infection. Regrettably, although gentamicin sponge had a very well tissue tolerated, it cannot improve the outcomes of infection [8]. Bennett-Guerrero, E. et al. randomly assigned 602 patients undergoing open or laparoscopically assisted colorectal surgery at 39 U.S. sites to undergo either gentamicin sponges or no intervention. They found that gentamicin sponges was not an effective method at preventing SSI, besides, it increased the rate of SSI [1]. Many other article have the similar results.
Over all, there are still debates about the effectiveness of gentamicin sponges for anti-infection, in additions, there are some leaks in present studies. First, the amount of gentamicin carried on sponges was inconsistent in each study and in each surgical implantation approved by different countries, thus may result in con icting conclusions. Second, the size of sponge is also different. We lack the uniform standards or guidelines. Third, whether there is local infection or not before using of gentamicin sponge is different. Gentamicin sponge in some studies are used in aseptic surgery to explore the preventive effect infection but in some studies are used in infected patients or animals to analyze the therapeutic effect of gentamicin sponge. Forth, the observation timepoint was not consistent.
Our study was to investigate some effects of gentamicin sponge in vitro and in vivo. First, we analyzed the gentamicin-loading of sponge to construct loading equation. Second, the release of gentamicin from air-dried gentamicin-saturated sponge was evaluated to get the release equation. Third, the antibacterial effect of gentamicin sponge was observed in vitro. Forth, the anti-infection effects and mortality-reduced effects of gentamicin sponge were explored in femur fractured with wound infection rat model. We give some ideas for clinical application of gentamycin sponge, especially in open fracture infection patients.

Reagents
Luria Broth (LB) powder (containing 10 g peptone, 5 g yeast powder and 10 g NaCl) was purchased from Beijing Solarbio Science & Technology Co., Ltd and phosphate buffer saline (PBS) was purchased from Merck KGaA. LB medium was made by LB powder dissolved in 1L sterile PBS and then ltered by 0.22 um, frozen at 4℃ to prepare to use. 0.9% sterile physiological saline was purchased from Baxter Medical Products Co. Ltd and sodium pentobarbital was provided by the Medical Research Center of Beijing Chaoyang Hospital a liated to Capital Medical University. Gentamicin ELISA kit was purchased from CUSABIO Bioengineering Co. Ltd (website: https://www.cusabio.com/food/Gentamicin-GEN-ELISA-kit-154817.html).

Animals
Female adult Sprague-Dawley (SD) rats (Charles River Laboratories, weighing 200±20 g) were kept in separate cages under a 12 h light/dark cycle at 23.6 ˚C and 35 % humidity. Animals were fed with sterilized chow diet and water. All procedures were complied with the ARRIVE guidelines and carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The present study was approved by the Capital Medical University Ethics Committee on the use of animals in research and education. At the end of the experiments, rats were euthanized by using an excessive dose of sodium pentobarbital (100 mg/kg, intraperitoneal injection).
femur fractured with wound infection rat model The speci c process was described in our other accepted article (The acceptance letter of the article is in the supplementary materials). Brie y, we built MSSA and P. aeruginosa wound infection rat model with femur fractured. LB medium was used to dissolved bacterial strain powders. Bacteria suspension was cultured at 37˚C, 200 rpm/min overnight. The precipitate was acquired at 10,000 rpm/min, 3 min, then resuspended in LB medium. After 3 times bacterial passage performed, bacterial suspension was diluted 10-fold by sequential transfer of 100 ml into 900 ml PBS. 100 μl of different diluted bacterial suspensions were inoculated in different LB agar media at 37˚C, overnight. The number of colonies yielded between 20 to 200 were counted and multiplied by the corresponding dilution times to obtain the bacterial quantity of the bacterial suspension. Finally, bacterial quantity of the bacterial suspension was adjusted to 10 6 , 10 7 , 10 8 and 10 9 CFU/ml by LB medium.
The rat femur fracture model was established on a small animal operating table in a super clean bench. We refer to Husodo, K. et al [9] for the rat femur fracture model. Brie y, the rats were fasted for 12 h and banned from drinking water for 3 h before anesthesia. Iso urane (concentration of 3%, oxygen ow 3L/min, small animal anesthesia machine) was used to induce anesthesia and sodium pentobarbital (0.4 ml/100 g, 40 mg/kg, intraperitoneal injection) was used to maintain anesthesia. The right hind limb of rats was shaved and disinfected by 75% alcohol 3 times. Right lateral position was adopted, the right hind limb was draped using a sterile surgical towel. The femoral lateral approach was used. The incision was 1-1.5 cm. Skin, subcutaneous tissue, super cial and deep fascia were opened, and middle part of the femur was revealed from the tensor fascia lata and femoral lateral muscle gap. A bone saw was used to make a femur shaft transverse fracture. Kirschner wire (1.0 mm) was used to x the fracture as an intramedullary nail. The movement of the hip and knee, the contraposition and alignment of the fracture, as well as the xation of the fracture, were examined. The method of causing bacterial infection was described as the previously study [10]. Brie y, the bacterial suspension was administered to the rats by gradual dripping onto the surface of the muscle and embrocation with a sterile bacterial inoculation needle. The volume of the bacterial suspension was 0.5 ml at a concentration of 10 6 , 10 7 , 10 8 and 10 9 CFU/ml. The muscle the skin incision was sutured using a 3-0 silk suture. Rats were kept in separate cage with sterilized chow diet and water.
The mortality rate and wound tissue sections of rats were obtained following the establishment of the model to evaluate whether the model had succeeded and was stable. Finally, we chose 108 CFU/ml MSSA and 106 CFU/ml P. aeruginosa to establish the rat model. (related results are in the supplementary materials: DOI:10.17632/vpj7vjkz86.1).

Evaluation of the gentamicin-loading of sponge
Gentamicin powder was dissolved into 0.9% physiological saline and we adjusted the concentration of gentamicin were 40mg/ml, 16mg/ml, 8mg/ml, 4mg/ml, 1.6mg/ml, 0.8mg/ml or 0mg/ml. Gelatin sponge was cut into 1×1×0.5cm size and sterilized with ethylene oxide (Fig.1). According to the concentration of gentamicin solution, gentamicin sponges were divided into 7 groups. The sponge was immersed in gentamicin solution of different concentrations for 12h, 24h, 48h, 96h or 120h. Then, sponge was air dried until the quality no longer changed (about 48h). The weight of sponge was measured by electronic balance to evaluate the gentamicin-loading of sponge.

Preparation of air-dried sponge
We used the similar methods to prepare the air-dried sponge. Brie y, gentamicin powder was dissolved into 0.9% physiological saline and the concentration of gentamicin were adjust to 40mg/ml, 16mg/ml, 8mg/ml, 4mg/ml, 1.6mg/ml, 0.8mg/ml or 0mg/ml. Gelatin sponge was cut into 1×1×0.5cm size and sterilized with ethylene oxide. According to the concentration of gentamicin solution, gentamicin sponges were divided into 7 groups. The sponge was immersed in gentamicin solution of different concentrations for 48h. Then, sponge was air dried until the quality no longer changed (about 48h). Finally, sponges were sterilized with ethylene oxide and kept in -20℃ until used.

Evaluation of the release of air-dried gentamicin-saturated sponge
The air-dried sponge was immersed into 10ml 0.9% physiological saline 30min,2h,6h, 12h,24h,48h and 96h. Gentamicin ELISA kit was used to test the concentration of gentamicin in 10ml 0.9% physiological saline. The speci c operation steps are in accordance with the manual.
Evaluation of the antibacterial effect of gentamicin sponge in vitro After 3 times bacterial passage performed as described before, 100ul bacterial suspension was inoculated in LB agar media at 37˚C, overnight to make the surface of medium be full of colonies. The air-dried sponge was put on the center of the surface of the LB agar media at 37˚C, 48h. Then, the sponge was removed and the antibacterial area (area that bacteria do not grow) was measured to evaluate the antibacterial effect of gentamicin sponge. 70 rats were divided into 2 groups as MSSA infection (35 rats) and P. aeruginosa infection (35 rats) group. Then, each group was divided into 7 subgroups as: 40mg/ml air-dried sponge (5 rats), 16mg/ml air-dried sponge (5 rats), 8mg/ml air-dried sponge (5 rats), 4mg/ml air-dried sponge (5 rats), 1.6mg/ml air-dried sponge (5 rats), 0.8mg/ml air-dried sponge (5 rats) and 0mg/ml air-dried sponge (5 rats). As described before, we chose 10 7 CFU/ml MRSA and 10 6 CFU/ml P. aeruginosa to establish the femur fractured with wound infection rat model. After the bacterial suspension was administered to the rats, air-dried sponge was put in the bacteria inoculated muscle gap. Then, the muscle the skin incision was sutured using a 3-0 silk suture. Rats were kept in separate cage with sterilized chow diet and water. We recorded the mortality and wound suppuration rate of the rats at day 7.
Statistical analysis. SPSS 23.0 was used to analyze the data. Measurement data were reported as median (interquartile), Kruskal-Wallis H test was used to compare multiple groups and Mann-Whitney U test was used to compare two groups. Enumeration data were as a percentage, in addition, χ2 test and Fisher de nite probability methods was used to compare groups. P<0.05 was considered as statistically signi cant.
The equations of gentamicin-loading of sponge and release of air-dried gentamicin-saturated sponge was constructed by curve estimation of SPSS software.
R2 was used to evaluate the matching degree of the constructed equation. We used the median of each group at each timepoint to construct the surface estimation in Origin2019b software. The maximum number of iterations was set at 400, tolerance was 1e-9 and iterative algorithm was levenberg-Marquardt algorithm. Surface function was set to Poly2D and the coe cients of the surface equation are expressed by mean± standard deviation. R2 was also used to evaluate the matching degree of the constructed surface equation. Microsoft Excel 2016 was used to make the data visualization of wound suppuration rate of rats.

Evaluation of the gentamicin-loading of sponge
The gentamicin-loading of sponge was shown in table.1. The original weight of air-dried sponges was no statistical difference in multiple groups (p=0.92). However, when the time of sponge immersed in gentamicin solution was prolonged, the difference was statistically signi cant in multiple groups. Sponge immersed in 40mg/ml gentamicin solution had the largest weight. The difference of weight of 1.6mg/ml air-dried sponge and 0.8mg/ml air-dried sponge had no statistically signi cant in all observation time point (p>0.05). As the sponge was began to decompose when was immersed in the solution for 96 hours, the weight of 0mg/ml air-dried sponge at 96h, 120h was less than the original weight (p=0.01, p=0.01 respectively). z was gentamicin-loading of sponge, x was the immersed duration of sponge in gentamicin solution, y was the concentration of gentamicin solution that sponge was immersed in. R2 of the surface equation was 0.97 (Fig.2).
Evaluation of the release of air-dried gentamicin-saturated sponge The results of release of air-dried gentamicin-saturated sponge was shown in table.3. Since the sponge began to decompose after immersed in water for more than 96 hours, the longest observation time was set as 96 hours. The results showed that the amount of gentamicin released from sponge increased gradually with the extension of time, and the difference was statistically signi cant (p<0.01). Moreover, the amount of gentamicin released from sponges immersed in gentamicin solution of different concentrations was also different (p<0.01). The amount of gentamicin released from sponge immersed in 40 mg/ml gentamicin solution was the largest at each time point, and the difference was statistically signi cant (p<0.01).
The equation of gentamicin release of air-dried gentamicin-saturated sponge was shown in table.4. R2 of all constructed equations was above 0.9. The surface equation of gentamicin release of air-dried sponge was: Z=(4.37205±1.18048)x+(-7.05921±3.09628)y+(-0.04596±0.01287)x2+(0.3309±0.07912)y2+(0.31559±0.02754)xy z was gentamicin release of air-dried sponge, x was the immersed duration of sponge in gentamicin solution, y was the concentration of gentamicin solution that sponge was immersed in. R2 of the surface equation was 0.95 (Fig.3).

Evaluation of the antibacterial effect of gentamicin sponge in vitro
The antibacterial zone size of gentamicin sponge was shown in table.4. Air-dried sponge previously immersed in 40mg/ml gentamicin solution had the largest antibacterial zone size than others both in P. aeruginosa and MSSA (p<0.01). The antibacterial zone sizes of 1.6mg/ml air-dried sponge and 0.8mg/ml airdried sponge were no statistical differences both in P. aeruginosa and MSSA (both were p>0.99). Besides, antibacterial zone sizes of sponges previously immersed in 40mg/ml, 16mg/ml, 8mg/ml, 4mg/ml, 1.6mg/ml, 0.8mg/ml gentamicin solution were larger than 0mg/ml air-dried sponge, and the difference had statistically signi cant (p<0.01).
Evaluation of the effects of gentamicin sponge on femur fractured with wound infection rat model Rats of all groups were no died at day 7 after modeled in femur fractured with wound infection, so the mortality of each group was 0.

Discussion
Gentamicin-sponge has been studied for more than 30 years, but its e cacy is still controversial [1,[11][12][13][14][15]. Many researches have demonstrated that the application of gentamicin-sponge can signi cantly reduce the incidence of wound infections and kill pathogenic bacteria, whether in animal experiments or clinical studies [5,[16][17]. However, also many scholars believe that gentamicin-sponge has no effect on preventing or treating wound infection, and it maybe play an opposite role as increase the incidence of infection [1,[7][8][18][19]. Some randomized controlled studies with large sample size are used to con rm their opinion [1]. Therefore, it is unknown whether gentamicin-sponge is useful at present.
Gentamicin, as one of aminoglycoside antibiotics, has a clear spectrum of sensitive or resistant bacteria [20][21]. If wound is infected with bacteria, the bacterial resistance spectrum screening should be carried out rst. Gentamicin-sponge is considered to use only the bacteria are sensitive to gentamicin. In this study, we chose two kinds of bacteria which are sensitive bacteria to gentamicin. At present, the controversy about the e cacy of gentamicin sponge is related to the drug-loading and release of gentamicin-sponge. As there are few studies on the drug-loading and release of gentamicin-sponge, we rst quanti ed the drug-loading and release of gentamicin-sponge in vitro.
We cut the sponge into 1×1×0.5 cm to uniform the sponge size. Seven concentrations of gentamicin solution were used and we chose 5 timepoint to measure the air-dried sponge. We found that the amount of gentamicin absorbed by sponge was increased gradually within 96 hours. As sponge began to obviously decompose in water about 96 h, drug-loading of sponge was signi cantly decreased in 4 mg/ml air-dried sponge group, 1.6 mg/ml air-dried sponge group, 0.8 mg/ml air-dried sponge group after 96 h. For this reason, we advised that the immersed time of sponge in water should not be exceed 96 hours when studying the drug loading experiment of sponge. We constructed the sponge drug-loading equation of gentamicin through in vitro experiments, and tested the tting degree of the equation. The tting degree of the equation is more than 0.9, which means that the accuracy of the equation is very high. It can provide a reference for the future study of sponge drug loading.
In vitro release experiment of gentamicin-sponge, we mainly discussed whether the amount of gentamicin released from gentamicin-sponge can reach its therapeutic concentration. The effective treatment concentration of gentamicin is 4-10 µg/ml [20][21]. In group of 4 mg/ml air-dried sponge, 1.6 mg/ml airdried sponge, 0.8 mg/ml air-dried sponge, the amount of gentamicin released did not reach the effective therapeutic concentration of gentamicin at 2 hours, but at six hours. The results mean that the air-dried gentamicin-saturated sponge acquired from being immersed in 4 mg/ml, 1.6 mg/ml and 0.8 mg/ml gentamicin solution could not exert its bactericidal ability within 2 hours. Besides, the amount of gentamicin released from 40 mg/ml air-dried sponge and 16 mg/ml air-dried sponge were 16.3ug/ml and 4.7ug/ml so these two kinds sponges could exert the bactericidal ability at 30 min. We constructed the release equation of gentamicin-sponge in vitro to quantify the release of gentamicin-sponge. The tting degree of the equation was analyzed. R2 is more than 0.9, which means that the accuracy of the equation is high. We can use the equation to analyze the bactericidal ability of gentamicin sponge.
In drug release test of gentamicin sponges in vitro, the amount of gentamicin released by some sponges was lower than the therapeutic concentration within 2 hours, so we used bacteria and animal experiments to analyze whether gentamicin-sponges of different groups can play a similar bactericidal effect.
Although the bactericidal areas of 40 mg/ml air-dried sponge group and 16 mg/ml air-dried sponge group were signi cantly larger than other groups, 1.6 mg/ml air-dried sponge group and 0.8 mg/ml air-dried sponge group also had obvious bactericidal effects (compared with 0 mg / ml air-dried sponge group). Based on these, we think that 1.6 mg/ml air-dried sponge and 0.8 mg/ml air-dried sponge are enough to prevent wound infection.
In animal experiments, we rst established a rat model of femoral fracture combined with wound MSSA or P. aeruginosa infection. The stability of the model was analyzed by animal mortality and wound suppurative rate. Finally, we chose 10 8 CFU/ml MSSA and 10 6 CFU/ml P. aeruginosa to establish the rat model. By using different gentamicin-sponges, we found that 40 mg/ml air-dried sponge, 16 mg/ml air-dried sponge and 8 mg/ml air-dried sponge could reduce the wound infection rate to 0. Therefore, if the wound has been con rmed to be infected and the bacteria are identi ed as sensitive bacteria, it is recommended to use 40 mg/ml air-dried sponge, 16 mg/ml air-dried sponge and 8 mg/ml air-dried sponge to treat wound infection.
The innovation of this study is that drug-loading and drug-release of gentamicin-sponge were quanti ed for the rst time, and an equation with a tting degree of more than 0.9 was constructed. Secondly, we proposed which gentamicin-sponge should be used to prevent and treat wound infection. Thirdly, we provided the basis for the future study of wound infection model or gentamicin-sponge.
There are also many limitations in this study. Firstly, the sponges that we used were produced by the same factory, so the results may be different from sponges made by other manufacturers in drug-loading and drug-release experiments in vitro. Secondly, we chose MSSA and P. aeruginosa to do bacteriostatic test in vitro and infect rats. The results cannot represent the other bacteria, such as Escherichia coli, Staphylococcus epidermidis, and so on. Thirdly, the incidence of wound infection in rodents is signi cantly different from humans, so we need further study to analyze which gentamicin-sponge is enough for prevention or treatment of wound infection.

Conclusion
In this study, we constructed the drug-loading and drug-release equations of gentamicin sponge for the rst time, and the tting degree of the equations were more than 0.9. The equations can be used to analyze the gentamicin sponge for further. Meanwhile, we proposed that 1.6 mg/ml air-dried sponge and 0.8 mg/ml air-dried sponge are enough to prevent wound infection, in addition, 40 mg/ml air-dried sponge, 16 mg/ml air-dried sponge and 8 mg/ml air-dried sponge to treat wound infection.

Consent for publication
All authors agree for publication.

Availability of supporting data
The supplemental data used to support the ndings of this study have been deposited in the Mendeley Data repository (DOI:10.17632/vpj7vjkz86.1).

Competing interests
The authors have no con ict of interest or nancial disclosures.

Funding
This work is Supported by Beijing Natural Science Foundation (7202049) which was obtained by Junlin Zhou. The role of the funding was in experiments and collection.
Authors' contributions YDL and JLZ designed this study; YDL did the in vitro experiments and DW did the in vivo experiments; DW collected and analyzed the data; YDL wrote the article and JLZ revised the article. All the authors read and approved the nal manuscript.  * The data were expressed in the median (quartile). Each group was repeated 5 times at each time point. ug/ml was the concentration of gentamicin in 10 ml 0.9% physiological saline that air-dried gentamicin-saturated sponge immersed in.  Figure 1 Gelatin sponge was cut into 1×1×0.5cm size. Figure 4 wound suppuration rates of rats with wound P. aeruginosa infected. *: p<0.05, compared with 40mg/ml air-dried sponge group. Figure 5 wound suppuration rates of rats with wound MSSA infected. *: p<0.05, compared with 40mg/ml air-dried sponge group.