# Correlation Analysis between Chronic Osteomyelitis and Bacterial Biofilm

Authors: Kai Huang; Bingyuan Lin; Yiyang Liu; Haiyong Ren; Qiaofeng Guo
Journal: Stem Cells International
 (2022)

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## Abstract

Objective. To study the role of bacterial biofilm (BBF) in the formation of chronic osteomyelitis and its prevention and treatment. Methods. In this paper, a large amount of relevant literature was searched for analysis and summary, and the key words “chronic osteomyelitis,” “bacterial biofilm,” “infection,” and “debridement” were searched in databases, mainly CNKI, Wanfang, and Wipu. The search was conducted until December 2020. The role of bacterial biofilm formation in chronic osteomyelitis and its prevention were analyzed. Results. Chronic osteomyelitis is formed mainly due to poor blood supply and drug-resistant bacteria, of which cellular biofilm is the most important cause. BBF forms on the surface of necrotic soft tissue and bone tissue, which has a protective effect on bacteria and greatly enhances their resistance to antibiotics, leading to difficulties in complete bacterial clearance and recurrent infections in osteomyelitis. Conclusion. Through an in-depth study of the molecular biology and signal transduction of osteomyelitis biofilm, antibiotic biofilm treatment strategies and surgical debridement remain the focus of clinical translation of chronic osteomyelitis.


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## Body

## 1. Introduction

Most osteomyelitis is intraoperative vegetative osteomyelitis and open fractures with an incidence of 80%, of which about 30% develop chronic osteomyelitis [1]. In recent years, the incidence of chronic osteomyelitis is high and the number of patients is increasing [2]. According to incomplete statistics, about 25% of patients require more than two debridement procedures before bone transplantation and 6% of them cause infection which eventually leads to amputation and serious damage to the body [3]. Despite the continuous updating of antibiotics and the improvement of surgical techniques, the treatment of chronic osteomyelitis remains a major challenge [4].Osteonecrosis, sclerosis, and fistulas are common clinical signs of chronic osteomyelitis and patients are at risk of decay and recurrence of infection [5]. In addition to local scar proliferation, poor blood supply, and drug-resistant bacteria, the formation of BBF by bacterial adhesion on the surface of dead bone, internal fixation, and surrounding scar tissue is one of the main reasons for its refractory nature [6]. BBF is caused by a bacterial population that adheres to the surface of living or inanimate objects and secretes extracellular macromolecules to encase itself with a special internal ecology and different bacterial gene expression [7]. At present, the relationship between the work of biofilms, various pathways, and factors is unclear, but it is related to interleukins, toxin-antitoxin systems, and interleukins [8]. In this paper, a search was conducted with the keywords chronic osteomyelitis, bacterial biofilm, infection, and debridement, mainly HowNet, Wanfang, and VIP databases. The application is available until December 2020. The role of bacterial biofilms in the formation, prevention, and treatment of chronic osteomyelitis was analyzed.


## 2. Structure and Formation Mechanism of Biofilm

The concept of biofilm was first introduced in 1978 [9]. It is a colonized colony that is colonized on the mucosal surface or endophyte in vivo [10]. The composition of colonies is mainly bacteria attached to the mucosal or endophytic surface and their autocrine polymeric matrix, as opposed to planktonic bacteria in body fluids, with a special internal ecology and phenotypic variation of bacterial genes [11]. The composition of biofilms mainly contains 10%-25% of bacteria wrapped in 75%-90% of self-dividing bacteria [12]. In staphylococcal biofilms, the polysaccharide component is an intracellular lipopolysaccharide antigen (PIA) [13]. Bacterial infections are usually divided into two stages: docking and locking. The first requires reversal because the bacteria accidentally come into contact with glue or are under the influence of chemicals. When the distance between them is less than 1 nm, bacteria spread to the carrier through various forces such as hydrophobic effect, electrostatic effect, van der Waals force, and sterility resistance. When bacteria stick to the surface of the carrier, BBF begins to develop until it matures. First, the expression of bacterial genes changes during the same period of growth and reproduction: it secretes a large number of extracellular polysaccharides, which can combine with homologous bacteria or various bacteria to form a flora, and a large number of microaggregates thicken BBF. The nutrient-producing environment affects the development of BBF, and the smooth transport of metabolites directly affects the maturation of BBF. In addition, the pH, osmotic pressure, and oxygen content of bacteria are very important, and the maturation of BBF also plays an important role. At this stage, changes in bacterial drug resistance, UV resistance, and gene exchange efficiency are often observed. Biofilm formation is a dynamic process, including cell adhesion formation of microbial communities and extracellular encapsulation of polymer matrix. Before adhesion, there is a conditional membrane formation process when aseptic drug after implantation with implant devices (mainly biomaterial polymers). Its surface immediately surrounds saliva, blood, urine, and other liquids. Glycoproteins, mucopolysaccharides, metal ions, and other components are after a few minutes. It penetrates and adsorbs to its surface, forming a conditional film network. It also covers the surface of the substrate so bacteria can identify membrane components and enter one step adsorption into it. Cationic metal ions such as sodium and magnesium are often used as negative metal ions, and bacterial surface-specific adhesion factors recognize host surface receptors for adhesion, a process that is specific and selective. The transcription of some specific genes is active during the adhesion phase, such as the enhanced transcription of Pseudomonas aeruginosa algc and algu genes, which promotes the subsequent production of extracellular polysaccharides [14]. Once the bacteria adhere to the surface, gene expression is immediately regulated. During growth and reproduction, it secretes large amounts of extracellular polysaccharides and adheres to monocyte cells to form microbial colonies [15]. Encapsulated by an extracellular polymer matrix, this is the mature stage of the biofilm [16]. The polymer matrix secreted by the body of the bacteria floating on the surface forms a well-organized structure. Laser copolymer microscopy observations showed that biofilms are not formed by microfilms of the same generation. Biological colonies form a monolayer of cellular structures but depending on time and space colonies alternate between previous generations. What factors influence the formation of bacterial biofilm in a specific environment during biofilm formation, quorum detection system, and regulation of specific gene expression plays an important role. It varies according to the type of bacteria, solid object, material, place, and difficulty of nutrition. The literature [17] found that the mature biofilm model contains the bulk of the biofilm, 1 ink membrane, processing membrane, and matrix from the outside to the inside. It was found that the living biofilm contained approximately 15% hydrate and 85% matrix. Bacteria colonize the matrix in the form of fungi. There are scattered water channels or intermittent springs between adherent cells containing microclones. E fills the environmental test between bacterial communities, a channel for obtaining bacterial nutrients, and excreting metabolic wastes. From these bacteria, extracellular polymers are produced including extracellular polysaccharides (EPS) and glycoproteins which are wrapped outside the bacterial community and affect the survival of bacteria in the biofilm. Life is important. The structure of biofilms is widely heterogeneous with marked differences in bacterial volume and metabolism between the deeper and lower layers. Some researchers believe that bacteria infect and grow on the surface of objects. In order to be considered a true biofilm, it must reach a certain number and be resistant to the action of antibiotics or biocides [18]. Studies have shown that bacteria in biofilms differ significantly from free or floating bacteria in morphological and physiological properties [19]. There are also significant differences between surface bacteria and bacteria within biofilms. Surface bacteria are similar to planktonic bacteria in that they have easy access to oxygen and nutrients, and metabolites are easily excreted [20]. Therefore, they have an active metabolism, rapid distribution, large cell volume, and sensitivity to antibiotics. Internal bacteria are different, they are not easy to obtain nutrients, and the excretion of metabolites occurs only through the surrounding interstitial waterways. Metabolism is low. Most internal bacteria are dormant, usually not distributed often, and the cell volume is small. Mah believes that due to the different metabolic forms of surface and internal bacteria, they also react differently to the environment.Osteomyelitis due to biofilm infection is mostly subacute or chronic and usually occurs 3-10 weeks after trauma or surgery; bacteria in biofilm of osteomyelitis originate from planktonic or endophytic bacteria on the surface of open wound contaminants [21]. As a result of trauma, surgery, and implantation of endophytic bacteria at the fracture site, the periosteum and soft tissue are disrupted leading to poor local circulation and reduced soft tissue value; as a foreign body, endophytic bacteria activate the immune response leading to neutropenia and the formation of a local immunocompromised fibrous inflammatory zone. In the initial phase, planktonic bacteria colonize and form microcolonies on the inner surface of bones or plants in the area by reversible passive adhesion (hydrostatic/hydrophobic forces). As bacterial density increases, bacterial quota detection systems regulate bacterial production of large amounts of extracellular polysaccharides and adhesion factors (collagen adhesion/fibronectin adhesion) to form irreversible actively adherent colonies, i.e., biofilms, a BBF may consist of one or more sites, and in a multifaceted BBF, different sites develop at different times and spaces, and these sites develop alternately [22].


## 3. Pathogenesis, Diagnosis, and Treatment of Chronic Osteomyelitis

Bacterial infection can destroy bone and cause chronic inflammation, the clinical symptoms of which are ulcers and rot in the limbs, swelling, and pigmentation in the skin tissue. Chronic osteomyelitis occurs mostly in the tibia and femur of young men. The most common cause is fracture and multiple surgeries. It often occurs repeatedly and is difficult to cure. The most common pathogenic bacteria are Gram-positive bacteria, Gram-negative bacteria, andStaphylococcus. There is a certain correlation between infection and soft tissue damage. Bacteria are often not enough to cause osteomyelitis. Therefore, the treatment of chronic osteomyelitis should focus on soft tissues. Acute infection is difficult to diagnose at an early stage, but early and correct antibiotic therapy can eliminate acute bone marrow infection in about 4 weeks, and the likelihood of acute infection developing into chronic osteomyelitis is high.Early diagnosis and treatment of chronic osteomyelitis is the primary key which can usually be detected by appropriate imaging or laboratory measurements. Chronic osteomyelitis can be diagnosed if the number of white blood cells increases in routine blood tests. Imaging can be done by X-ray, CT, MRI, and radionuclide examination. Radionuclide examination combined with CT has a high diagnostic value in the diagnosis of early osteomyelitis. X-ray can detect a periosteal reaction, osteosclerosis, and sinus canal, but it can be found only 2 weeks after bone destruction. It has low sensitivity in the early stage of chronic osteomyelitis and cannot distinguish fracture from chronic osteomyelitis. Ultrasound can detect signs of osteomyelitis at an early stage such as soft tissue swelling, periosteal thickening, and effusion. It also has certain advantages of directing needle subperiosteal injection, low cost, low radiation, and easy to accept patients. Compared to X-ray imaging, CT can show periosteal reaction, bone destruction, and necrosis around the focus in more detail. MRI is better than X-ray and CT diagnosis of chronic osteomyelitis. It can be detected at 3 to 5 days of osteomyelitis and has a sensitivity and specificity of more than 90% [23, 24].It is increasingly used in clinical practice because it can better differentiate between the bone and surrounding soft tissue and muscle infections, soft tissue, fascia and conjunctiva providing more information to inform the development of surgical plans. However, this technique has some drawbacks: image quality is easily affected by internal implants produced by the object; incorrect diagnosis is due to its oversensitivity to tissue swelling and scar tissue; PET-CT is indicated for osteomyelitis associated with internal fixation [25–27]. It can provide the user with a precise focus point prior to surgery and is not affected by the internal implant. It can distinguish between a bone healing reaction and a bone infection that cannot be distinguished by MRI. However, the method has a high cost, and therefore, its usability is limited; Technetium 99m-dimethyl bisphosphonate (99 mTc) bone scanning and leukocyte radionuclide scanning are commonly used radionuclide scanning methods; the first of which is highly sensitive to the acceleration of bone metabolism, while the latter uses leukocyte aggregation properties at the infected site. Sex is very specific for identifying an infection. The combination of these two methods can improve the diagnostic sensitivity of chronic osteomyelitis. Currently, these two methods are not routinely used in clinical practice and are usually used only when patients have contraindications to MRI. Studies have shown that an IL-13 α MRI imaging system with 2 receptor-labeled probes can distinguish between aseptic and infectious inflammation, but this technique has not been applied clinically since S. aureus is the most important pathogen of chronic osteomyelitis in clinical practice. Raman spectroscopy has been reported for the diagnosis of chronic osteomyelitis caused by S. aureus, which provides an opportunity to develop an early and rapid diagnosis of this disease; this diagnosis provides an opportunity to develop a diagnostic method for early and rapid diagnosis of this disease [28, 29]. The clinic usually uses surgical treatment in combination with systemic or local antibiotic therapy, and drug therapy alone cannot completely improve it. In chronic osteomyelitis, due to a large amount of necrotic cortical bone and insignificant bone, antibiotics cannot fully reach and cannot remove the base of bacteria biofilm, and the use of antibiotic therapy can only temporarily eliminate the symptoms. Therefore, we must choose surgical treatment for thorough debridement to remove the carrier bacteria biofilm. Surgical debridement leads to soft tissue and bone tissue damage which can be repaired by skin transplantation. Most researchers believe that the risk of primary bone graft infection is high, but the use of antibiotics in primary transplantation can also contribute to heal the fracture in curing the infection. The use of antibiotics improves the clinical treatment of chronic osteomyelitis, but it cannot be completely cured. Pregnancy is often treated with a fixed stent, but it is easy to cause postoperative infection. A serious infection leads to large bone damage which must be corrected by osteotomy.


## 4. Characteristics of Biofilm

Bacterial biofilm contains bacteria, a large amount of water, macromolecular polymers, metabolites, and bacterial lysates. The formation of a multicellular structure is a dynamic process involving the adhesion, development, and maturation of bacteria. Bacterial biofilm formation is associated with many factors such as bacterial species, surrounding environment, surface components of adhesion vectors, and expression of essential genes. Bacterial biofilm has different physiological and biochemical properties at each stage.


### 4.1. Bacterial Transmission

Bacterial biofilm formation first requires bacteria to stick to the surface of living organisms or plants. The adhesin between bacteria and plants is not specific, whereas the adhesin protein of living organisms requires specific adhesin proteins on the surface of bacteria to identify host surface receptors that are selective and specific. Infection of bacteria is usually divided into two stages: docking and locking. The first case must be the opposite, because bacteria can accidentally come in contact with the adhesive or be driven by the chemical agent. When the distance between the two is less than 1 nm, bacteria are transmitted to the carrier by various forces such as hydrophobic effect, electrostatic effect, van der Waals force, and sterile resistance [30]. Bacteria and tissues in the body are usually negatively charged so electrostatic effects are an objectionable force while hydrophobic effects.It is the main force of bacterial transmission. It should be noted that the hydrophobic effect of the carrier may be influenced by other factors such as the adhesion ofStaphylococcus epidermidis to polyethylene is significantly increased in the presence of surfactant platelets, while adhesion is weakened in the presence of plasma proteins. The bacterial adhesion lock is irreversible from time to time, and when the bacteria dock on the carrier, they secrete polysaccharides which transmit a close combination of the bacteria and the carrier. Different bacteria can secrete different polysaccharides, and one bacterium can secrete different polysaccharides; for example, Staphylococcus epidermidis can secrete polysaccharide adhesin between cells. Through this adhesion, the same or different types of bacteria can also be combined, and the adhesion of one bacterium can improve the adhesion of another bacterium.


### 4.2. Development

When the bacteria stick to the surface of the carrier, the bacterial biofilm begins to develop until it matures. First, the expression of bacterial genes changes, and a large number of extracellular polysaccharides are excreted during growth and reproduction. External polysaccharides can bind one or several bacteria to form bacterial clusters, and a large number of microcolonies thicken the biofilm of the bacteria. The amount of nutrients produced by the environment and the smooth transport of metabolic products directly affect the maturity of the bacterial biofilm. In addition, the pH value, osmotic pressure, and oxygen content of bacteria play an important role in the maturation of bacterial biofilm. At this stage, changes in antibiotic resistance, ultraviolet radiation resistance, and the effectiveness of gene exchange are often observed.


### 4.3. Maturity

The structure of mature bacterial biofilm is heterogeneous. Bacteria near the surface of the carrier grow slowly due to lack of nutrients, oxygen, and accumulation of metabolites. For example, the oxygen content of the outer layer of the bacterial biofilm can be 30 times higher than the middle; bacteria on the surface of the grain mill grow actively and metabolize extensively, and when the number of bacteria reaches a certain level, they are released from the bacterial biofilm and form planktonic bacteria [31]. Bacterial biofilms have many water channels to transport nutrients, metabolites, etc. Recent studies have shown that the maturation of bacterial biofilms is associated with a density detection system [32]. This system regulates gene expression by monitoring bacterial density to ensure nutrient transport and waste discharge. For example, most Gram-negative bacteria use N-acylhomoserine lactone (AHL) as a signaling molecule that binds to transcriptional activators to induce expression of target genes when N-acylhomoserine lactone reaches a threshold. Bacterial biofilms may consist of one or more strains, and in a pluralistic bacterial biofilm, different strains develop in different times and spaces, and strains develop differently.


## 4.1. Bacterial Transmission

Bacterial biofilm formation first requires bacteria to stick to the surface of living organisms or plants. The adhesin between bacteria and plants is not specific, whereas the adhesin protein of living organisms requires specific adhesin proteins on the surface of bacteria to identify host surface receptors that are selective and specific. Infection of bacteria is usually divided into two stages: docking and locking. The first case must be the opposite, because bacteria can accidentally come in contact with the adhesive or be driven by the chemical agent. When the distance between the two is less than 1 nm, bacteria are transmitted to the carrier by various forces such as hydrophobic effect, electrostatic effect, van der Waals force, and sterile resistance [30]. Bacteria and tissues in the body are usually negatively charged so electrostatic effects are an objectionable force while hydrophobic effects.It is the main force of bacterial transmission. It should be noted that the hydrophobic effect of the carrier may be influenced by other factors such as the adhesion ofStaphylococcus epidermidis to polyethylene is significantly increased in the presence of surfactant platelets, while adhesion is weakened in the presence of plasma proteins. The bacterial adhesion lock is irreversible from time to time, and when the bacteria dock on the carrier, they secrete polysaccharides which transmit a close combination of the bacteria and the carrier. Different bacteria can secrete different polysaccharides, and one bacterium can secrete different polysaccharides; for example, Staphylococcus epidermidis can secrete polysaccharide adhesin between cells. Through this adhesion, the same or different types of bacteria can also be combined, and the adhesion of one bacterium can improve the adhesion of another bacterium.


## 4.2. Development

When the bacteria stick to the surface of the carrier, the bacterial biofilm begins to develop until it matures. First, the expression of bacterial genes changes, and a large number of extracellular polysaccharides are excreted during growth and reproduction. External polysaccharides can bind one or several bacteria to form bacterial clusters, and a large number of microcolonies thicken the biofilm of the bacteria. The amount of nutrients produced by the environment and the smooth transport of metabolic products directly affect the maturity of the bacterial biofilm. In addition, the pH value, osmotic pressure, and oxygen content of bacteria play an important role in the maturation of bacterial biofilm. At this stage, changes in antibiotic resistance, ultraviolet radiation resistance, and the effectiveness of gene exchange are often observed.


## 4.3. Maturity

The structure of mature bacterial biofilm is heterogeneous. Bacteria near the surface of the carrier grow slowly due to lack of nutrients, oxygen, and accumulation of metabolites. For example, the oxygen content of the outer layer of the bacterial biofilm can be 30 times higher than the middle; bacteria on the surface of the grain mill grow actively and metabolize extensively, and when the number of bacteria reaches a certain level, they are released from the bacterial biofilm and form planktonic bacteria [31]. Bacterial biofilms have many water channels to transport nutrients, metabolites, etc. Recent studies have shown that the maturation of bacterial biofilms is associated with a density detection system [32]. This system regulates gene expression by monitoring bacterial density to ensure nutrient transport and waste discharge. For example, most Gram-negative bacteria use N-acylhomoserine lactone (AHL) as a signaling molecule that binds to transcriptional activators to induce expression of target genes when N-acylhomoserine lactone reaches a threshold. Bacterial biofilms may consist of one or more strains, and in a pluralistic bacterial biofilm, different strains develop in different times and spaces, and strains develop differently.


## 5. Biofilm Barrier Effect in Chronic Osteomyelitis

### 5.1. Antibiotics

The EPS secreted by bacteria can realize the biomembrane barrier effect on antibiotics and improve antibiotic drug resistance through dormant bacteria. The number required for bacterial infection can be reduced through the biofilm barrier, the entry of antibiotics, and the role of sleeping bacteria. Some researchers found that the minimum inhibitory biofilm of the same type of bacteria was 1000 times the minimum inhibitory concentration. However, in vitro experiments have formed a similar infection state; when there is no biofilm on the inner surface of the plant, 104 times more bacteria are required than biofilm, see Figure1.Figure 1
Barrier antibiotics.The presence of biofilm can significantly reduce the penetration of antibiotics such as vancomycin and teicoplanin. The principle of special barrier can be divided into the following categories: (1) hydrophobic structure of the biofilm: the main component of the extracellular polymer of the biofilm is water, but the extracellular polymer is not hydrophilic. An extracellular polymer that converts free water and gathers together through polysaccharides, proteins, and other components. The molecular weight of the water mass increases with the maturity of the biofilm. The properties of the water mass are also changing. The water mass with a high molecular weight is usually hydrophobic, and the hydrophobic macromolecular mass is usually formed a hydrophobic film on the surface of the biofilm. It is stable in body fluids. Adding heavy metal ions destroys macromolecular water mass and eliminates hydrophobicity, and the biofilm further decomposes. Low-molecular-weight water is usually hydrophilic in biofilms. This internal hydrophilic and external hydrophobic amphoteric structure complicates the penetration of most hydrophilic antibiotics released from the kidneys and is unable to achieve an effective concentration in the biofilm. (2) Negative charge and acidic environment of the biofilm: because the extracellular polymer of the biofilm and the cell wall of bacteria contain a large amount of polysaccharide and wall phosphate, the biofilm is negatively charged, resulting in positively charged molecules such as aminoglycoside antibiotics and combine with EPS by electrostatic action to prevent further penetration of antibiotics. Experimental results indicate that the difference in pH between the surface and inside of the biofilm may be greater than 1 which may be due to accumulation of H absorbed by the negative charge or accumulation of acid metabolites of bacteria. A significant pH gradient inside and outside the biofilm directly affects antibiotic activity. (3) Biofilm directly inactivates antibiotics: in addition to structural proteins, biofilms also contain functional proteins such as protein secreted by bacteria. In theβ-EPS-protected biofilm, bacterial efflux enzymes can maintain concentration and activity [33]. In vitro antibiotic degradation and inactivation, however, the inhibitory effect of biofilm is not effective for all antibiotics and is not evident for rifampicin, clindamycin, and macrolide antibiotics. (4) Neutrophil extracellular traps (NETs) thicken the biofilm barrier: NETs are a retinal fibrous structure released by extracellular neutrophils—their basic structure is DNA and a large number of protein structures attached to the gaps [34]. As a cellular immune defense mechanism in the body, mosquito nets can effectively control the spread of microbial infections and prevent the spread of bacteria to the brain, testes, and other vital organs. Studies have shown that γ-hemolysin AB bacterial biofilm leads to the release of a network of neutrophils, but the network accelerates and thickens biofilm formation [35]. This positive feedback culminates in the formation of a thick and compact biofilm. This barrier prevents the spread of bacteria but also makes the bacterial biofilm resistant to antibiotics and leukocytes, i.e., the bacterial biofilm EPS in vivo is not secreted by the bacteria themselves but is formed together with the host’s internal environment.


### 5.2. Resting Bacteria Improve Drug Resistance

Biofilm can resist host-specific and nonspecific immunity and plays an important role in bacterial immunity. Studies have shown thatStaphylococcus aureus is a restricted immune system based mainly on biofilm. Experiments show that there is an asymmetric relationship between biofilm and host immunity, that is, biofilm can trigger a corresponding immune response, but the immune response cannot effectively remove biofilm. From the point of view of biofilm structure, EPS biofilm is closely bound to bacteria, and bacteria secrete macrophage-permeable biofilm α-toxin and Leukocidin AB, and disappeared from immune monocytosis. Pseudomonas aeruginosa PSL polysaccharide can also protect bacteria from conditional effects. Proteins in biofilms such as serine protease and cysteine protease can break down immunoglobulins and antimicrobial peptides that are secreted by the immune system. From a molecular biology point of view, biofilms pass through interleukin-12 and interleukin-1 β, CCL5, and other cytokines and chemokines attract bone marrow-derived suppressor cells affect macrophage polarization, transfer congenital immunity, promote inflammation, sterilization (type M1), inflammation, and fibrosis immune response (type M2) leading to local immunosuppression, excessive fibrosis, and scarring.


## 5.1. Antibiotics

The EPS secreted by bacteria can realize the biomembrane barrier effect on antibiotics and improve antibiotic drug resistance through dormant bacteria. The number required for bacterial infection can be reduced through the biofilm barrier, the entry of antibiotics, and the role of sleeping bacteria. Some researchers found that the minimum inhibitory biofilm of the same type of bacteria was 1000 times the minimum inhibitory concentration. However, in vitro experiments have formed a similar infection state; when there is no biofilm on the inner surface of the plant, 104 times more bacteria are required than biofilm, see Figure1.Figure 1
Barrier antibiotics.The presence of biofilm can significantly reduce the penetration of antibiotics such as vancomycin and teicoplanin. The principle of special barrier can be divided into the following categories: (1) hydrophobic structure of the biofilm: the main component of the extracellular polymer of the biofilm is water, but the extracellular polymer is not hydrophilic. An extracellular polymer that converts free water and gathers together through polysaccharides, proteins, and other components. The molecular weight of the water mass increases with the maturity of the biofilm. The properties of the water mass are also changing. The water mass with a high molecular weight is usually hydrophobic, and the hydrophobic macromolecular mass is usually formed a hydrophobic film on the surface of the biofilm. It is stable in body fluids. Adding heavy metal ions destroys macromolecular water mass and eliminates hydrophobicity, and the biofilm further decomposes. Low-molecular-weight water is usually hydrophilic in biofilms. This internal hydrophilic and external hydrophobic amphoteric structure complicates the penetration of most hydrophilic antibiotics released from the kidneys and is unable to achieve an effective concentration in the biofilm. (2) Negative charge and acidic environment of the biofilm: because the extracellular polymer of the biofilm and the cell wall of bacteria contain a large amount of polysaccharide and wall phosphate, the biofilm is negatively charged, resulting in positively charged molecules such as aminoglycoside antibiotics and combine with EPS by electrostatic action to prevent further penetration of antibiotics. Experimental results indicate that the difference in pH between the surface and inside of the biofilm may be greater than 1 which may be due to accumulation of H absorbed by the negative charge or accumulation of acid metabolites of bacteria. A significant pH gradient inside and outside the biofilm directly affects antibiotic activity. (3) Biofilm directly inactivates antibiotics: in addition to structural proteins, biofilms also contain functional proteins such as protein secreted by bacteria. In theβ-EPS-protected biofilm, bacterial efflux enzymes can maintain concentration and activity [33]. In vitro antibiotic degradation and inactivation, however, the inhibitory effect of biofilm is not effective for all antibiotics and is not evident for rifampicin, clindamycin, and macrolide antibiotics. (4) Neutrophil extracellular traps (NETs) thicken the biofilm barrier: NETs are a retinal fibrous structure released by extracellular neutrophils—their basic structure is DNA and a large number of protein structures attached to the gaps [34]. As a cellular immune defense mechanism in the body, mosquito nets can effectively control the spread of microbial infections and prevent the spread of bacteria to the brain, testes, and other vital organs. Studies have shown that γ-hemolysin AB bacterial biofilm leads to the release of a network of neutrophils, but the network accelerates and thickens biofilm formation [35]. This positive feedback culminates in the formation of a thick and compact biofilm. This barrier prevents the spread of bacteria but also makes the bacterial biofilm resistant to antibiotics and leukocytes, i.e., the bacterial biofilm EPS in vivo is not secreted by the bacteria themselves but is formed together with the host’s internal environment.


## 5.2. Resting Bacteria Improve Drug Resistance

Biofilm can resist host-specific and nonspecific immunity and plays an important role in bacterial immunity. Studies have shown thatStaphylococcus aureus is a restricted immune system based mainly on biofilm. Experiments show that there is an asymmetric relationship between biofilm and host immunity, that is, biofilm can trigger a corresponding immune response, but the immune response cannot effectively remove biofilm. From the point of view of biofilm structure, EPS biofilm is closely bound to bacteria, and bacteria secrete macrophage-permeable biofilm α-toxin and Leukocidin AB, and disappeared from immune monocytosis. Pseudomonas aeruginosa PSL polysaccharide can also protect bacteria from conditional effects. Proteins in biofilms such as serine protease and cysteine protease can break down immunoglobulins and antimicrobial peptides that are secreted by the immune system. From a molecular biology point of view, biofilms pass through interleukin-12 and interleukin-1 β, CCL5, and other cytokines and chemokines attract bone marrow-derived suppressor cells affect macrophage polarization, transfer congenital immunity, promote inflammation, sterilization (type M1), inflammation, and fibrosis immune response (type M2) leading to local immunosuppression, excessive fibrosis, and scarring.


## 6. Role of Biofilm in Chronic Osteomyelitis

The clinical symptoms of chronic osteomyelitis are mainly due to the release of planktonic bacteria after biofilm maturation [36]. The separation and diffusion of biofilms are an active process regulated by the detection of queering between bacteria [37]. In the early stages of biofilm formation, the number of bacteria in the membrane is very low. Bacteria mainly produce surface adhesion proteins and polysaccharides that help bacteria to adhere to bones or plant surfaces. When the bacterial density reaches a certain level, bacteria mainly secrete proteases and esterases to split and break down the biofilm, and the released S. aureus can penetrate the vascular endothelium. Therefore, osteoblasts can lead to apoptosis. Biofilms also have the ability to absorb directly into the bone, which can destroy the bone without immunity and osteoclasts in vitro. Gram-negative bacteria can also produce endotoxin and trigger an immune response. Bacteria can change the composition of EPS polysaccharide in the biofilm to control the formation and diffusion of the biofilm to meet the needs of the biofilm at different times.In the biofilm bacterial proliferation process, secondary metabolites are formed into signal molecules. When signal molecules reach the threshold level, they can bind to bacterial receptors and regulate the transcription of target genes so that bacteria can play an important physiological role as a whole at the multicellular level. This regulatory framework is called quorum recognition (QS). In Gram-negative bacteria, the signal molecule is acyl hypericin lactone (AHL). In Gram-positive bacteria, the signal molecule is a self-inducing peptide, and different bacteria can also exchange the self-inducing factor-2 (AI-2), which is a byproduct of bacteria’s metabolic methyl cycle. Most bacteria contain two or more groups of quarium detection systems. Changes in the expression profile of bacterial genes in biofilm are regulated by a regulatory network consisting of several quarium detection systems. For example, the LuxS/AI-2 population detection system regulates bacterial polysaccharide expression via the kdpde binary signaling system, while the age population detection system regulates virulence factors inS. aureus, sufficiently inducing phenotypic changes in bacterial biofilms. Biofilm EPS can also bind to plasmids to promote horizontal transfer of resistance genetic information among bacteria and accelerate the transition from drug-resistant to drug-resistant bacteria. Bacteria are better able to adapt to changes in the external environment than individual planktonic bacteria by establishing biofilms to communicate, coordinate, and cooperate among bacteria, see Figure 2.Figure 2
Toxin and genetic information transmission of biofilm in osteomyelitis.


## 7. Characteristics ofStaphylococcus Biofilm

The main pathogenic bacteria of chronic osteomyelitis areStaphylococcus, including Staphylococcus aureus and Staphylococcus epidermidis. When Staphylococcus biofilm grows in the dead bone, scar, and other tissues, it leads to repeated attacks of infection. Staphylococcus can cause serious infections when the epidermis and mucous membrane are damaged. It can attach to the surface of inactive tissues and medicinal plants, secrete polysaccharides, form a biofilm, and cause infection. Recent studies have confirmed that this polysaccharide component is an intracellular lipopolysaccharide binder. The synthesis of intracellular lipopolysaccharide binder (PIA) depends on the activity of the icaabcd gene, which encodes an intracellular lipopolysaccharide binder synthetase, and its activity is regulated by genes such as ICAR, sigB, and RsbU, while the external environment such as glucose, ethanol, osmolality, temperature, and antibiotics can affect the above regulatory genes. For example, glucose can induce the expression of icaabcd, which is partially dependent on sigB and RsbU genes. RsbU genes are activators of sigB genes, and these two genes can induce biofilm formation. icar genes encode inhibitors of icaabcd genes and ethanol and NaCl can inhibit the expression of icar genes and activate sigB and RsbU genes and induce biofilm formation. However, studies have shown that 30% Staphylococcus epidermidis is intracellular lipopolysaccharide adhesin negative. Another study confirmed that only 1 in 3 Staphylococcus epidermidis isolated from the surface of infected hip prostheses was ICA gene positive. The literature [38] found that knockdown of the ICA gene of Staphylococcus isolated from infected pseudopodia did not reduce biofilm formation. The literature [39] also found that intracellular lipopolysaccharide of methicillin-resistant Staphylococcus aureus was not associated with biofilm formation, but intracellular lipopolysaccharide of methicillin-resistant Staphylococcus aureus was associated with biofilm formation. Therefore, it can be concluded that the formation of staphylococcal biofilms caused by intracellular lipopolysaccharide is related to the strain and the surrounding environment. Without the role of intracellular lipopolysaccharide, protein adhesins play an important role in the formation of biofilms. The literature [40] identified a large number of aggregation-associated proteins in the biofilm of Staphylococcus epidermidis. However, the mechanism of staphylococcal biofilm formation in the absence of intracellular lipopolysaccharide duplexes needs further investigation.The literature [41] confirms that extracellular DNA plays an important role in the dissemination and biofilm formation of S. aureus. atlE mutation results in reduced release of bacterial DNA and reduced biofilm formation.Many factors including regulatory proteins are involved in the formation of staphylococcal biofilm, and its mechanism needs to be further investigated.


## 8. Bacterial Biofilm Treatment

The inability of antibiotics to completely remove bacterial biofilms is mainly due to the fact that bacterial biofilms target mainly planktonic bacteria. In recent years, most researchers have studied the relationship between staphylococci and biofilms, mainly focusing on (1) reducing the synthesis of intracellular lipopolysaccharide adhesins and affecting their adhesion and (2) some researchers found that dspb can effectively remove intracellular lipopolysaccharide adhesins and prevent bacterial transmission. However, not all bacteria are completely positive. Some researchers have suggested that bacterial biofilms can inhibit growth through RNA inhibitory peptides and confirmed that the consistency of density detection systems can facilitate the purification of bacterial biofilms from antibiotics [42]. However, some scientists have expressed the opposite opinion, so we need to look into it.In conclusion, in recent years, the treatment of osteomyelitis has changed from previous debridement to debridement, masquelet bone reconstruction or osteotomy, ilizarov bone handling, or negative pressure coagulation. The therapeutic effect has been significantly improved, but the number of surgeries, the length of the treatment process, the secondary trauma, the poor adaptability of patients, and the presence of biofilm make osteomyelitis infection very persistent. It is difficult to resolve with surgical techniques and antibiotics alone. Studying the role of biofilms in osteomyelitis and determining the mechanisms and pathways of biofilm action in osteomyelitis is a trend for future research. Antibiotic biofilm therapy and surgical debridement are hot spots for clinical changes in chronic osteomyelitis. For example, antibiotic transport materials are used to achieve effective concentrations in biofilms or to form a coating on the inner surface of the plant to prevent biofilm adhesion. Interfering with the detection of bacterial populations leading to self-degradation of biofilms; adding antibiotic adjuvants to dormant bacteria to remove bacterial residues along with conventional antibiotics; modulating immune activity to prevent immune polarization; adding Edna or polysaccharidases or phagocytic enzymes to break down EPS components and networks in biofilms to break down and release them. The research and treatment of chronic osteomyelitis has been enhanced by in-depth molecular biology studies and osteomyelitis signaling.



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