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Osteomyelitis is an inflammation of the bone and bone marrow that is most commonly caused by a Staphylococcus aureus infection. Much of our understanding of the underlying pathophysiology of osteomyelitis, from the perspective of both host and pathogen, has been revised in recent years, with notable discoveries including the role played by osteocytes in the recruitment of immune cells, the invasion and persistence of S. aureus in submicron channels of cortical bone, and the diagnostic role of polymorphonuclear cells in implant-associated osteomyelitis. Advanced in vitro cell culture models, such as ex vivo culture models or organoids, have also been developed over the past decade, and have become widespread in many fields, including infectious diseases. These models better mimic the in vivo environment, allow the use of human cells, and can reduce our reliance on animals in osteomyelitis research. In this review, we provide an overview of the main pathologic concepts in osteomyelitis, with a focus on the new discoveries in recent years. Furthermore, we outline the value of modern in vitro cell culture techniques, with a focus on their current application to infectious diseases and osteomyelitis in particular.
Osteomyelitis is an infectious disease affecting bone and bone marrow,
In addition, the following parameters are considered: i) signs of bone destruction and the presence of sequestra and purulent collection in soft tissue assessed with imaging, ii) occurrence of organisms in more than one deep specimen verified with microbiological cultures, and iii) leukocyte counts, erythrocyte sedimentation rate, and C-reactive protein levels.
Infection can be confirmed by histopathologic examination of deep tissue samples; the presence of microorganisms in deep tissue is examined using specific staining techniques for bacteria (eg, Gram stain or Ziehl-Neelsen stain for tuberculosis) or fungi (eg, Grocott methenamine silver stain).
For culture-negative patients, the diagnosis of osteomyelitis can be confirmed histologically if there are signs of active bone resorption and remodeling and the presence of acute and chronic inflammatory cells.
In contrast, for chronic and for implant-associated osteomyelitis, the success of antibiotic therapy alone is relatively low and requires debridement (ie, surgical removal of infected bone and implant components) to achieve satisfactory success rates.
These debridement or revision surgeries are often challenging, given that the extent of bone debridement can be difficult to judge, and the management of the resultant dead space can also require complex interventions and prolonged healing time.
Our understanding of the pathophysiology of osteomyelitis has evolved over recent years, and we now have a better insight into why chronicity and the presence of an implant require more vigorous treatment. For instance, we know that bacterial biofilm formation and bacterial invasion within the osteocyte lacuna-canalicular system are involved in chronic osteomyelitis.
Furthermore, the host response to the infection and subsequent changes in bone morphology (eg, sequester or involucrum formation) are also better understood at the present time. Much of this new understanding of the underlying pathophysiology of osteomyelitis (eg, bone turnover, osteolysis, and bacteriological changes over time) has been determined from histologic analyses of individual human specimens
In other fields, advanced in vitro cell culture models have been developed to reduce our overreliance on laboratory animals. In particular, three-dimensional (3D) cell culture has become a standard in the fields of tissue engineering and cancer biology, where the organotypic 3D constructs have been shown to i) have their own microenvironment, ii) resemble in vivo tissue organizations, and iii) have cellular behavior that more faithfully reflects the in vivo setting. When using human cells, these systems may be more reflective of the human situation than currently used preclinical in vitro two-dimensional culture systems.
In this review, we describe the pathophysiology of osteomyelitis and the currently used in vitro systems for osteomyelitis research, with the focus on the most recent discoveries and improved mechanistic understandings of the pathophysiology of osteomyelitis. Furthermore, we review published advanced multicellular in vitro models and their potential to further our understanding of human osteomyelitis, without requiring the use of experimental animals.
Pathophysiology of Osteomyelitis
Staphylococcus aureus arriving at the bone surface via iatrogenic or hematogenous routes can readily adhere to soft tissue, bone,
The bacterium may achieve this through binding to extracellular matrix (ECM) proteins via microbial surface components recognizing adhesive matrix molecules, such as collagen-binding protein and bone sialoprotein binding protein.
Specifically, S. aureus can form fibrin by promoting polymerization of fibrinogen and secreting enzymes, such as coagulase and von Willebrand factor–binding protein, which activate endogenous prothrombin and contribute to fibrin formation. This fibrin network surrounding the bacterial SACs protects the bacteria from invasion and clearance by immune cells, such as PMNs,
Furthermore, S. aureus resident in biofilm may secrete the so-called extracellular polymeric substance matrix consisting of self-produced polysaccharides and proteins and possibly extracellular DNA from dead bacterial cells, forming a matrix that functions as a physical barrier to immune cell infiltration.
Invasion of S. aureus within the canalicular network could be a mechanism to promote persistence and chronic infection, with the potential to limit access to immune cells. This novel persistence mechanism was originally identified in a mouse model of implant-associated osteomyelitis, and was subsequently confirmed in a human S. aureus diabetic foot infection.
This discovery is particularly concerning in the context of S. aureus osteomyelitis, as these canalicular networks may be impenetrable by immune cells, and bacteria can possibly survive in this space for a long period of time, using bone matrix as a nutrient source.
This intracellular persistence provides the pathogen with crucial protection needed against the onslaught of the immune system and antibiotic treatments. Staphylococcus aureus–infected human osteoblasts may also mature into osteocytes and remain infected.
The numerous mechanisms by which S. aureus is able to survive intracellularly within the bone niche for long periods is a primary cause of chronic and recurrent osteomyelitis.
The presence of bacteria within the bone tissue triggers a host response, which encompasses an innate immune response primarily driven by PMNs, macrophages, and adaptive responses mediated by T cells, B cells, and pathogen-specific antibodies (Figure 1B).
First, on bacterial recognition, resident macrophages in bone,
PMNs can efficiently kill planktonic S. aureus via phagocytosis, oxidative bursts, and production of antimicrobial peptides, whereas secretion of proinflammatory cytokines and chemokines, such as tumor necrosis factor (TNF)-α, IL-1β, CXCL2, CXCL3, and others, activates and recruits PMNs, which ultimately leads to pathogen clearance.
As the infection persists and becomes chronic, bacteria tend to form a biofilm phenotype, and the influx of viable PMNs decreases drastically, as demonstrated in mice and observed in humans with chronic osteomyelitis.
The predominant M2 macrophages are inefficient in phagocytosing bacteria within the biofilm, and they tend to promote profibrotic environments and wound healing responses, generating abscesses during chronic osteomyelitis infections.
Adaptive immune responses against bone infections include both T- and B-cell responses. Unfortunately, pathogens, such as S. aureus, have evolved numerous evasion mechanisms toward these responses, resulting in chronic osteomyelitis. For instance, in a porcine osteomyelitis infection model, it was observed that the antibody responses against intracellular S. aureus in biofilms are skewed to a predominantly type 1 and type 17 helper T cell biased immune response, which cannot effectively clear intracellular pathogens.
Staphylococcus aureus can also efficiently manipulate B cells, affecting their survival and function via the secretion of staphylococcal protein A (SpA), which associates with the Fcγ and Fab domains of certain antibodies,
Although further studies are needed to fully understand this, it may be that due to SpA, interference antibodies secreted against S. aureus do not confer protection against reinfection or chronic musculoskeletal infections
Bone is a mineralized organic matrix containing osteocytes, bone-forming osteoblasts, and bone-resorbing osteoclasts. All three bone cells are impacted directly and indirectly by S. aureus (Figure 1C).
Directly, SpA binding to TNF receptor-1 on osteoblasts results in an increase in apoptosis and a decrease in differentiation and calcium deposition of the osteoblasts.
(Figure 2D). Indirectly, osteoclasts are activated and increase osteolysis activity by osteoblasts, osteocytes, and PMNs. These cell types secrete receptor activator of NF-κB ligand (RANK-L), which drives osteoclastogenesis and activates osteoclasts to resorb bone. Osteocytes do so in response to a neighboring osteocyte that underwent apoptosis
Another contributor to osteoclastogenesis and osteoclast activity is the persistent inflammatory environment itself. This occurs initially because of the secretion of the proresorptive cytokines IL-6, TNF-α, and IL-1β by immune cells and osteoblasts,
A crucial role of osteocytes is to mature and maintain the mineralized matrix, which is accomplished by their expression of enzymes capable of reversibly removing mineral and remodeling the organic phase of bone matrix, a process described as osteocytic osteolysis or perilacunar remodeling.
suggesting that osteocytic osteolysis is affected by S. aureus. Another interesting function of osteocytes is their potential role in the recruitment of immune cells. A recent study demonstrated that human osteocyte-like cultures exposed to S. aureus resulted in the differential expression of >1500 genes, including the robust induction of a large number of chemokines and cytokines.
Although classic PMN chemoattractants, such as CXCL1 and chemokine (C-C motif) ligand 5, were detected, CXC chemokine receptor 3 (CXCR3)-binding chemokines CXCL9, CXCL10, and CXCL11 were also expressed in abundance, suggesting the potential participation of osteocytes in the adaptive immune response to bacterial infection by recruiting cytotoxic and/or suppressive T-lymphocyte subsets to the infected sites.
Conventional in Vitro Methods to Model Individual Aspects of Osteomyelitis
Although human biopsies and animal osteomyelitis models have contributed significantly to our understanding of osteomyelitis, conventional in vitro methods remain of value. Some of the mainstays of this approach include bacterial cultures and cocultures with host cells, which are described below.
Bacterial and Biofilm Cultures
Biofilm growth in vivo can be mimicked with conventional models, such as microtiter plate-based models or flow displacement biofilm models, as reviewed recently.
These models can be used for antimicrobial compound testing and measuring bacterial colonization/biofilm formation on various substrates. One of the most common methods currently used to assess anti-biofilm efficacy is the minimum biofilm eradication concentration assay, which is a 96-well biofilm system using polystyrene pegs. Bacterial biofilms grown on the pegs can be simultaneously challenged with multiple antibiotic combinations at different concentrations for assessing the bactericidal and/or bacteriostatic efficacies of these anitmicrobials.
Conventional bacterial models can also be used to examine bacterial colonization on materials such as polymethyl methacrylate and the efficacy of antibacterial coatings. Examples of orthopedic implant-related materials and coatings that have been tested for bacterial colonization have recently been reviewed.
Fibrin can form a layer on biomaterials and promote adherence of pathogens to the biomaterial. Fibrin is also a component of the S. aureus biofilm matrix that facilitates antibiotic resistance due to poor penetration of the antibiotic into the biofilm. It was shown that the tissue plasminogen activator-containing coating reduced bacterial adherence to the biomaterial.
In an effort to increase the complexity and relevance of in vitro studies, bacterial cocultures with host immune or bone cells identified as key players in osteomyelitis have also been performed. For this review, a coculture is defined as a culture that combines bacteria with at least one host cell type.
Multiple groups have examined the effects of bacteria, usually S. aureus, on osteoblasts using two-dimensional cell culture models.
To prevent bacterial overgrowth in static cultures, several techniques are routinely employed to remove extracellular bacteria. These include the use of antibiotics or the S. aureus–lysing enzyme lysostaphin, as well as rinsing to remove unbound bacteria. A variety of osteoblast coculture models have been developed using rodent and human cell lines, as well as human primary osteoblastic cells.
One study examined the host cell response to several S. aureus strains at a fixed multiplicity of infection and showed that human primary osteoblasts exposed acutely or for short periods of time did not undergo cell death.
Although relatively few intracellular bacteria were recovered, the primary cells secreted detectable levels of innate immune cell–relevant chemokines and cytokines, indicating the potential of osteoblasts to participate in innate immune responses and the utility of this model for studying this phenomenon.
A study using bone explant-derived cells from the femoral heads of patients undergoing hip replacement surgery found that infections for up to 48 hours generated only low-level chemokine and cytokine responses, which the authors interpreted as indicating that osteoblasts may serve to internalize bacteria but not contribute significantly to the innate immune response.
It is possible that matching the source of human primary cells and the pathology under investigation (osteomyelitis) may influence experimental outcomes. More specifically, periprosthetic joint infection most often occurs in patients treated for primary osteoarthritis, and osteoblastic cells derived from these donors display different phenotypic and behavioral qualities, such as aberrant in vitro mineralization, to those derived from nonosteoarthritis patients treated for fragility fractures of the hip.
Furthermore, in hip osteoarthritis patients, the femoral head is usually diseased and the cells derived from this site may, therefore, be aberrant in their responses ex vivo. Thus, a site more distal from the joint (eg, the intertrochanteric region of the proximal femur) may be a more suitable source of disease-naïve cells. In a study using human osteoarthritis proximal femur-derived osteoblasts differentiated to an osteocyte-like stage, no cell death effect was observed in response to S. aureus infection for up to 30 days.
Staphylococcus aureus formed small colony variant associated with the up-regulation of sigma B activity, consistent with establishment of a persistent intracellular infection. This correlated with observations of S. aureus bacteria inside viable osteocytes in clinical periprosthetic joint infection bone specimens.
This model allows the study of both the host response and adaptation of the bacteria to intracellular infection.
Other studies have incorporated foreign biomaterials into the infection model. A typical application is the coculture of S. aureus and osteoblasts on a biomaterial surface to model the so-called race for the surface.
Herein, the idea is that if host cells colonize the biomaterial first, bacterial adhesion is prevented. One way to study the race for the surface is by seeding a flow chamber with both staphylococci and osteoblasts.
The effect of S. aureus on osteoblast-induced osteoclastogenesis has also been studied in cocultures. These studies revealed that bacterial surface proteins could drive osteoclast formation because formaldehyde-fixed S. aureus induced RANK-L expression
to assess phagocytosis of biofilm-resident bacteria and the migration of PMNs to the biofilm. PMNs migrated toward the biofilm and engaged in phagocytosis of the biofilm, especially when the biofilm was in an immature state (<6 days old). Mature biofilm was less sensitive to PMN attack than immature biofilm because 15-day–old biofilm was subjected to significantly less phagocytosis by PMNs than 2- and 6-day–old biofilms.
To our knowledge, only one multicellular model involving bacteria cocultured with both bone and host immune cells has been reported. To investigate competition for the surface of a polymethyl methacrylate plate, bacteria (S. aureus, Staphylococcus epidermidis, or Pseudomonas aeruginosa) were cultured in a flow chamber with an osteoblast cell line in the presence or absence of macrophages. It was shown that colonization of the polymethyl methacrylate plate by osteoblasts did not increase in the presence of macrophages, and it was primarily colonized by bacteria.
This is in line with clinical observations where, despite host cell presence, bacteria win the race for the surface. The presence of macrophages did prolong the survival of osteoblasts in the multicellular cultures with either S. aureus or P. aeruginosa, and osteoblasts were able to grow and spread in the presence of low-virulence S. epidermidis.
Future studies using such a multicellular model and testing different biomaterials would be of interest.
Although conventional models are of great use, they can only model the following aspects of osteomyelitis: biofilm formation and interactions of bacteria with one or multiple host cell types (Table 1). To resemble bone tissue, fibrous encapsulation, complex interactions between bacteria and multiple host cells, and osteomyelitis-induced bone abscesses with a necrotic core, more sophisticated systems, such as 3D in vitro systems, will be of immense value.
Table 1Aspects of Osteomyelitis That Are Achievable in Conventional or Theoretically Achievable in 3D Models
Aspects of osteomyelitis
Coculturing bacteria with one other cell type
Coculturing bacteria with multiple cell types
Modeling in vivo bone tissue
Generation of a fibrous encapsulation around a 3D structure
Complex interactions between bacteria and multiple cell types
Because the cells grow in a 3D environment composed of an ECM, cells in 3D in vitro models can have complex interactions not only with each other but also with the ECM. Therefore, cells in 3D culture do not lose their cell polarity,
Furthermore, an advantage of 3D cell culture models over animal models, including humanized mice, is that human cells and fluids can be used. This is specifically of interest because S. aureus has some human-specific functions (eg, it was shown that staphylokinase has little activity toward murine plasminogen compared with the activity toward human plasminogen).
3D models developed for other infections may contain relevant information for the development of an in vitro osteomyelitis model. Below, recent examples of 3D in vitro infection models based on organoids, rotating wall vessel (RWV) bioreactors, microcolonies in collagen gels, bacteria-containing printable inks, human skin equivalents, ex vivo models, microfluidic 3D models, and a 3D osteomyelitis model are discussed. Figure 3 illustrates these 3D in vitro infection models.
Infected organoid cultures have been used to study host-microbe interactions for multiple pathogens. Organoids are simplified versions of organs, which in a matrix with appropriate environmental cues grow from single stem cells given their self-organizing capacity. Figure 3A illustrates a gastric organoid culture with Helicobacter pylori. The gastric organoids are grown from gastric stem cells, and this model has been used to study infection-induced changes in gastric epithelial cells.
It has been demonstrated that H. pylori infection causes up-regulation of the NF-κB pathway in infected gastric organoids and, subsequently, an increase in IL-8, a neutrophil chemoattractant that promotes inflammation.
In a more complex model, intestinal organoids from a human embryonic stem cell line were used to simulate Escherichia coli intestinal infection. The E. coli infected organoid was subsequently challenged with PMNs to thoroughly examine innate immune responses, such as reactive oxygen species production.
Cells are first grown in a monolayer and left to either aggregate onto a scaffold, such as ECM-coated microcarrier beads, then transferred into the RWV bioreactor, or self-aggregate by directly transferring the cells into the RWV bioreactor.
(Figure 3B). In this study, either RWV bioreactor-generated 3D intestinal aggregates or a monolayer culture of small intestinal epithelial cells (standardly used) was infected with S. enterica serovar typhimurium. Salmonella was less able to adhere to and invade the intestinal 3D aggregates compared with the monolayer of cells.
Similar results were observed for RWV bioreactor-formed lung aggregates infected with P. aeruginosa. In sharp contrast, monolayer cells were easily penetrable by P. aeruginosa, demonstrating that the RWV bioreactor-formed aggregates allowed for more in vivo–like infection by the bacterium given that the aggregates had more in vivo–like tight-junction complexes.
(Figure 3C). Supplementation of the collagen gel with fibrinogen was performed to facilitate fibrin-dependent formation of an inner pseudocapsule around the staphylococcal microcolony and an outer dense microcolony-associated mesh surrounding the pseudocapsule.
Acetobacter xylinum, which produce bacterial cellulose, has been incorporated into hydrogel ink with hyaluronic acid, k-carrageenan, and fumed silica (Figure 3D). This bacteria-laden hydrogel ink was not toxic for the bacteria and has successfully been 3D printed into various shapes. In addition, because the bacteria present within the hydrogel retained their metabolic capacity, this technology resulted in functional materials that may be used for biomedical applications.
Infection models examining the interactions between skin commensals, such as staphylococci and the epidermis, have been performed with human skin equivalents (HSEs). HSE cultures are developed by layering fibroblasts and keratinocytes, and then promoting their differentiation via air exposure.
(Figure 3E). A similar approach is used to study airway infection; a bronchial epithelial model was used to clarify changes occurring to the bronchial epithelium in response to nontypeable Hemophilus influenzae infection, which was applied apically.
Ex vivo models have been established to investigate inflammatory bone destruction. For this model, 1-mm–thick murine mandibular slices were cultured in an air-liquid interface. Cells continued to proliferate, and protein synthesis was unaltered. Tissue was not infected with bacteria, but inflammation was achieved by supplementing media with lipopolysaccharide from Porphyromonas gingivalis, resulting in an increased number of osteoclasts in the ex vivo culture.
(Figure 3F). Fresh bone fragments without bone marrow, obtained from patients with femoral fracture (1 mm3 in size) were cultured with S. aureus for 12 hours to achieve infection. Interestingly, S. aureus invaded osteocytes and lacunae of the ex vivo bone fragment, and the host cells responded in a manner similar to that of an in vitro differentiated two-dimensional culture of human primary osteocyte-like cells exposed to S. aureus.
A microfluidic 3D model has also been generated that promotes cells to form a 3D structure given the confined space and the microcirculation of nutrients and waste products in this system. For its development, a layer of human fibronectin, S. epidermidis, and osteoblasts were applied into the microfluidic device.
(Figure 3G). This model allows the testing of treatments, such as antibiotics or wound-healing accelerators, by placing the microfluidic 3D model on inkjet-printed micropatterns containing the treatment.
This model was used to test rifampicin-eluting biphasic calcium phosphate–containing beads, and it was demonstrated that these beads promoted osteoblast proliferation and ECM production, while simultaneously preventing biofilm formation.
(Figure 3H). More specifically, this model is a bone marrow analog that consists of a cationized bovine serum albumin scaffold resembling trabecular bone seeded with hematopoietic stem cells and mesenchymal stromal cells to mimic bone marrow. To infect this bone marrow analog, it was cocultured with a biofilm of methicillin-resistant S. aureus or P. aeruginosa grown on a titanium plate as a clinically relevant implant material. Pseudomonas aeruginosa caused cell death of both hematopoietic stem cells and mesenchymal stromal cells, whereas methicillin-resistant S. aureus stimulated IL-6 secretion by mesenchymal stromal cells and impaired differentiation of hematopoietic stem cells.
To the best of our knowledge, this is the only reported 3D in vitro model realistically mimicking osteomyelitis pathophysiology. This model serves as an excellent starting point for further 3D osteomyelitis in vitro model development.
Outlook for in Vitro 3D Osteomyelitis Model Development
The previously described 3D models in other areas of infectious diseases offer great opportunities to translate the technological possibilities of 3D models to more faithfully model osteomyelitis.
in which osteoblasts could form bone, and osteoclasts could resorb bone. Furthermore, it would be interesting to adapt a long-term ex vivo mechanically loading culture, such as the Zetos system, for the study of osteomyelitis.
Using the Zetos system in combination with micro–computed tomography (or equivalent imaging technique), bone remodeling in response to infection could be monitored that would enable longitudinal observations of bone changes over time and response to therapy. Another interesting option would be to use the RWV bioreactor to culture sequestra from bone or bone mimics, and coculture with host cells to 3D model osteomyelitis. Once a source of the infection is present, the model may be exposed to different immune cells at multiple time points. Poor diffusion of nutrients, waste, and oxygen are traditionally considered complications for 3D models,
Multicellular, 3D in vitro models of osteomyelitis have now also emerged as an exciting option to study the pathology of osteomyelitis using human cells, which offers promise in the advancement of our understanding of this disease, while also reducing animal use.