Comparison of four in vitro test methods to assess nucleus pulposus replacement device expulsion risk.

Background: Nucleus replacement devices (NRDs) are not routinely used in clinic, predominantly due to the risk of device expulsion. Rigorous in vitro testing may enable failure mechanisms to be identified prior to clinical trials; however, current testing standards do not specify a particular expulsion test. Multiple methods have therefore been developed, complicating comparisons between NRD designs. Thus, this study assessed the effectiveness of four previously reported expulsion testing protocols; hula-hoop (Protocol 1), adapted hula-hoop (Protocol 2), eccentric cycling (Protocol 3), and ramp to failure (Protocol 4), applied to two NRDs, one preformed and one in situ curing. Methods: Nucleus material was removed from 40 bovine tail intervertebral disks. A NRD was inserted posteriorly into each cavity and the disks were subjected to one of four expulsion protocols. Results: NRD response was dependent on both the NRD design and the loading protocol. Protocol 1 resulted in higher migration and earlier failure rates compared to Protocol 2 in both NRDs. The preformed NRD was more likely to migrate when protocols incorporated rotation. The NRDs had equal migration (60%) and expulsion (60%) rates when using unilateral bending and ramp testing. Combining the results of multiple tests revealed complimentary information regarding the NRD response. Conclusions: Adapted hula-hoop (Protocol 2) and ramp to failure (Protocol 4), combined with fluoroscopic analysis, revealed complimentary insights regarding migration and failure risk. Therefore, when adopting the surgical approach and animal model used in this study, it is recommended that NRD performance be assessed using both a cyclic and ramp loading protocol.

Can axial loading restore in vivo disc geometry, opening pressure, and T2 relaxation time?

Background: Cadaveric intervertebral discs are often studied for a variety of research questions, and outcomes are interpreted in the in vivo context. Unfortunately, the cadaveric disc does not inherently represent the LIVE condition, such that the disc structure (geometry), composition (T2 relaxation time), and mechanical function (opening pressure, OP) measured in the cadaver do not necessarily represent the in vivo disc. Methods: We conducted serial evaluations in the Yucatan minipig of disc geometry, T2 relaxation time, and OP to quantify the changes that occur with progressive dissection and used axial loading to restore the in vivo condition. Results: We found no difference in any parameter from LIVE to TORSO; thus, within 2 h of sacrifice, the TORSO disc can represent the LIVE condition. With serial dissection and sample preparation the disc height increased (SEGMENT height 18% higher than TORSO), OP decreased (POTTED was 67% lower than TORSO), and T2 time was unchanged. With axial loading, an imposed stress of 0.20-0.33 MPa returned the disc to in vivo, LIVE disc geometry and OP, although T2 time was decreased. There was a linear correlation between applied stress and OP, and this was conserved across multiple studies and species. Conclusion: To restore the LIVE disc state in human studies or other animal models, we recommend measuring the OP/stress relationship and using this relationship to select the applied stress necessary to recover the in vivo condition.

A missed opportunity: A scoping review of the effect of sex and age on osteoarthritis using large animal models.

OBJECTIVE: The objective was to critically analyze the published literature accounting for sex differences and skeletal age (open vs. closed physis) in preclinical animal models of OA, including the disaggregation of data by sex and skeletal maturity when data is generated from combined sex and/or multi-aged cohorts without proper confounding. METHOD: A scoping literature review of PubMed, Web of Science, EMBASE, and SCOPUS was performed for studies evaluating the effect of sex and age in experimental studies and clinical trials utilizing preclinical large animal models of OA. RESULTS: A total of 9727 papers were identified in large animal (dog, pig, sheep, goat, horse) models for preclinical OA research, of which 238 ex vivo and/or in vivo studies disclosed model type, animal species, sex, and skeletal age sufficient to analyze their effect on outcomes. Dogs, followed by pigs, sheep, and horses, were the most commonly used models. A paucity of preclinical studies evaluated the effect of sex and age in large animal models of naturally occurring or experimentally induced OA: 26 total studies reported some kind of analysis of the effects of sex or age, with 4 studies discussing the effects of sex only, 11 studies discussing the effects of age only, and 11 studies analyzing both the effects of age and sex. CONCLUSION: Fundamental to translational research, OARSI is uniquely positioned to develop recommendations for conducting preclinical studies using large animal models of OA that consider biological mechanisms linked to sex chromosomes, skeletal age, castration, and gonadal hormones affecting OA pathophysiology and treatment response.

Dogs can detect an odor profile associated with Staphylococcus aureus biofilms in cultures and biological samples.

Introduction: The study investigated the utilization of odor detection dogs to identify the odor profile of Staphylococcus aureus (S. aureus) biofilms in pure in vitro samples and in in vivo biosamples from animals and humans with S. aureus periprosthetic joint infection (PJI). Biofilms form when bacterial communities aggregate on orthopedic implants leading to recalcitrant infections that are difficult to treat. Identifying PJI biofilm infections is challenging, and traditional microbiological cultures may yield negative results even in the presence of clinical signs. Methods: Dogs were trained on pure in vitro S. aureus biofilms and tested on lacrimal fluid samples from an in vivo animal model (rabbits) and human patients with confirmed S. aureus PJI. Results: The results demonstrated that dogs achieved a high degree of sensitivity and specificity in detecting the odor profile associated with S. aureus biofilms in rabbit samples. Preliminary results suggest that dogs can recognize S. aureus volatile organic compounds (VOCs) in human lacrimal fluid samples. Discussion: Training odor detection dogs on in vitro S. aureus, may provide an alternative to obtaining clinical samples for training and mitigates biosecurity hazards. The findings hold promise for culture-independent diagnostics, enabling early disease detection, and improved antimicrobial stewardship. In conclusion, this research demonstrates that dogs trained on in vitro S. aureus samples can identify the consistent VOC profile of PJI S. aureus biofilm infections. The study opens avenues for further investigations into a retained VOC profile of S. aureus biofilm infection. These advancements could revolutionize infectious disease diagnosis and treatment, leading to better patient outcomes and addressing the global challenge of antimicrobial resistance.

Evaluating the Healing Potential of J-Plasma Scalpel-Created Surgical Incisions in Porcine and Rat Models.

Cold atmospheric plasma devices generate reactive oxygen and nitrogen species that can be anti-microbial but also promote cell migration, differentiation, and tissue wound healing. This report investigates the healing of surgical incisions created using cold plasma generated by the J-Plasma scalpel (Precise Open handpiece, Apyx Medical, Inc.) compared to a steel scalpel in in vivo porcine and rat models. The J-Plasma scalpel is currently FDA approved for the delivery of helium plasma to cut, coagulate, and ablate soft tissue during surgical procedures. To our knowledge, this device has not been studied in creating surgical incisions but only during deeper dissection and hemostasis. External macroscopic and histologic grading by blinded reviewers revealed no significant difference in wound healing appearance or physiology in incisions created using the plasma scalpel as compared with a steel blade scalpel. Incisions created with the plasma scalpel also had superior hemostasis and a reduction in tissue and blood carryover. Scanning electron microscopy (SEM) and histology showed collagen fibril fusion occurred as the plasma scalpel incised through the tissue, contributing to a sealing effect. In addition, when bacteria were injected into the dermis before incision, the plasma scalpel disrupted the bacterial membrane as visualized in SEM images. External macroscopic and histologic grading by blinded reviewers revealed no significant difference in wound healing appearance or physiology. Based on these results, we propose additional studies to clinically evaluate the use of cold plasma in applications requiring hemostasis or when an increased likelihood of subdermal pathogen leakage could cause surgical site infection (i.e., sites with increased hair follicles).

The 2023 Orthopaedic Research Society International Consensus Meeting on musculoskeletal infection.

The Orthopaedic Research Society's Research Interest Group completed its international consensus meeting (ICM) on musculoskeletal infections (MSKI) following the 2023 Annual Meeting. The work products from this ICM include the 65 questions with recommendation and rationale, and the voting results from the 72 delegates. There are also five Consensus Articles in this issue of the Journal of Orthopaedic Research from the ICM Sections: Host Immunity, Established Infection-Treatment, Clinical Questions not addressed by the prior MSKI ICMs, In Vitro, and Animal Models. This Introduction summarizes the 3-year Delphi process used by the ICM with timelines and critical milestones. It also highlights several challenges that had to be addressed, and a large body of work that remains.

Targeting biofilm infections in humans using small scale robotics.

The eradication of drug-resistant microbial biofilms remains an unresolved global health challenge. Small-scale robotics are providing innovative therapeutic and diagnostic approaches with high precision and efficacy. These approaches are rapidly moving from proof-of-concept studies to translational biomedical applications using ex vivo, animal, and clinical models. Here, we discuss the fundamental and translational aspects of how microrobots target the infection sites to disrupt the structural and functional traits of biofilms and their antimicrobial resistance mechanisms. We emphasize current approaches of mechanochemical disruption and on-site drug delivery that are supported by in vivo models and preclinical testing, while also highlighting diagnostics potential. We also discuss clinical translation challenges and provide perspectives for development of microrobotics approaches to combat biofilm infections and biofouling in humans.

Fixation strength of swelling copolymeric anchors in artificial bone.

Fixation with suture anchors and metallic hardware for osteosynthesis is common in orthopedic surgeries. Most metallic commercial bone anchors achieve their fixation to bone through shear of the bone located between the threads. They have several deficiencies, including stress-shielding due to mechanical properties mismatch, generation of acidic by-products, poor osteointegration, low mechanical strength and catastrophic failure often associated with large bone defects that may be difficult to repair. To overcome these deficiencies, a swelling porous copolymeric material, to be used as bone anchors with osteointegration potential, was introduced. The purpose of this study was to investigate the fixation strength of these porous, swelling copolymeric bone anchors in artificial bone of various densities. The pull-out and subsidence studies indicate an effective fixation mechanism based on friction including re-fixation capabilities, and minimization of damage following complete failure. The study suggests that this swelling porous structure may provide an effective alternative to conventional bone anchors, particularly in low-density bone.

The 2023 Orthopedic Research Society's international consensus meeting on musculoskeletal infection: Summary from the in vitro section.

Antimicrobial strategies for musculoskeletal infections are typically first developed with in vitro models. The In Vitro Section of the 2023 Orthopedic Research Society Musculoskeletal Infection international consensus meeting (ICM) probed our state of knowledge of in vitro systems with respect to bacteria and biofilm phenotype, standards, in vitro activity, and the ability to predict in vivo efficacy. A subset of ICM delegates performed systematic reviews on 15 questions and made recommendations and assessment of the level of evidence that were then voted on by 72 ICM delegates. Here, we report recommendations and rationale from the reviews and the results of the internet vote. Only two questions received a ≥90% consensus vote, emphasizing the disparate approaches and lack of established consensus for in vitro modeling and interpretation of results. Comments on knowledge gaps and the need for further research on these critical MSKI questions are included.

Single cell RNA sequencing reveals emergent notochord-derived cell subpopulations in the postnatal nucleus pulposus.

Intervertebral disc degeneration is a leading cause of chronic low back pain. Cell-based strategies that seek to treat disc degeneration by regenerating the central nucleus pulposus (NP) hold significant promise, but key challenges remain. One of these is the inability of therapeutic cells to effectively mimic the performance of native NP cells, which are unique amongst skeletal cell types in that they arise from the embryonic notochord. In this study, we use single cell RNA sequencing to demonstrate emergent heterogeneity amongst notochord-derived NP cells in the postnatal mouse disc. Specifically, we established the existence of progenitor and mature NP cells, corresponding to notochordal and chondrocyte-like cells, respectively. Mature NP cells exhibited significantly higher expression levels of extracellular matrix (ECM) genes including aggrecan, and collagens II and VI, along with elevated transforming growth factor-beta and phosphoinositide 3 kinase-protein kinase B signaling. Additionally, we identified Cd9 as a novel surface marker of mature NP cells, and demonstrated that these cells were localized to the NP periphery, increased in numbers with increasing postnatal age, and co-localized with emerging glycosaminoglycan-rich matrix. Finally, we used a goat model to show that Cd9+ NP cell numbers decrease with moderate severity disc degeneration, suggesting that these cells are associated with maintenance of the healthy NP ECM. Improved understanding of the developmental mechanisms underlying regulation of ECM deposition in the postnatal NP may inform improved regenerative strategies for disc degeneration and associated low back pain.

Injectable Radiopaque Hyaluronic Acid Granular Hydrogels for Intervertebral Disc Repair.

Injectable hydrogels offer minimally-invasive treatment options for degenerative disc disease, a prevalent condition affecting millions annually. Many hydrogels explored for intervertebral disc (IVD) repair suffer from weak mechanical integrity, migration issues, and expulsion. To overcome these limitations, an injectable and radiopaque hyaluronic acid granular hydrogel is developed. The granular structure provides easy injectability and low extrusion forces, while the radiopacity enables direct visualization during injection into the disc and non-invasive monitoring after injection. The radiopaque granular hydrogel is injected into rabbit disc explants to investigate restoration of healthy disc mechanics following needle puncture injury ex vivo and then delivered in a minimally-invasive manner into the intradiscal space in a clinically-relevant in vivo large animal goat model of IVD degeneration initiated through degradation by chondroitinase. The radiopaque granular hydrogel successfully halted loss of disc height due to degeneration. Further, the hydrogel not only enhanced proteoglycan content and reduced collagen content in the nucleus pulposus (NP) region compared to degenerative discs, but also helped to maintain the structural integrity of the disc and promote healthy segregation of the NP and annulus fibrosus regions. Overall, this study demonstrates the great potential of an injectable radiopaque granular hydrogel for treatment of degenerative disc disease.

Tension-activated nanofiber patches delivering an anti-inflammatory drug improve repair in a goat intervertebral disc herniation model.

Conventional microdiscectomy treatment for intervertebral disc herniation alleviates pain but does not repair the annulus fibrosus, resulting in a high incidence of recurrent herniation and persistent dysfunction. The lack of repair and the acute inflammation that arise after injury can further compromise the disc and result in disc-wide degeneration in the long term. To address this clinical need, we developed tension-activated repair patches (TARPs) for annulus fibrosus repair and local delivery of the anti-inflammatory factor anakinra (a recombinant interleukin-1 receptor antagonist). TARPs transmit physiologic strain to mechanically activated microcapsules embedded within the patch, which release encapsulated bioactive molecules in direct response to spinal loading. Mechanically activated microcapsules carrying anakinra were loaded into TARPs, and the effects of TARP-mediated annular repair and anakinra delivery were evaluated in a goat model of annular injury in the cervical spine. TARPs integrated with native tissue and provided structural reinforcement at the injury site that prevented aberrant disc-wide remodeling resulting from detensioning of the annular fibrosus. The delivery of anakinra by TARP implantation increased matrix deposition and retention at the injury site and improved maintenance of disc extracellular matrix. Anakinra delivery additionally attenuated the inflammatory response associated with TARP implantation, decreasing osteolysis in adjacent vertebrae and preserving disc cellularity and matrix organization throughout the annulus fibrosus. These results demonstrate the therapeutic potential of TARPs for the treatment of intervertebral disc herniation.

Quantifying internal intervertebral disc strains to assess nucleus replacement device designs: a digital volume correlation and ultra-high-resolution MRI study.

Introduction: Nucleus replacement has been proposed as a treatment to restore biomechanics and relieve pain in degenerate intervertebral discs (IVDs). Multiple nucleus replacement devices (NRDs) have been developed, however, none are currently used routinely in clinic. A better understanding of the interactions between NRDs and surrounding tissues may provide insight into the causes of implant failure and provide target properties for future NRD designs. The aim of this study was to non-invasively quantify 3D strains within the IVD through three stages of nucleus replacement surgery: intact, post-nuclectomy, and post-treatment. Methods: Digital volume correlation (DVC) combined with 9.4T MRI was used to measure strains in seven human cadaveric specimens (42 ± 18 years) when axially compressed to 1 kN. Nucleus material was removed from each specimen creating a cavity that was filled with a hydrogel-based NRD. Results: Nucleus removal led to loss of disc height (12.6 ± 4.4%, p = 0.004) which was restored post-treatment (within 5.3 ± 3.1% of the intact state, p > 0.05). Nuclectomy led to increased circumferential strains in the lateral annulus region compared to the intact state (-4.0 ± 3.4% vs. 1.7 ± 6.0%, p = 0.013), and increased maximum shear strains in the posterior annulus region (14.6 ± 1.7% vs. 19.4 ± 2.6%, p = 0.021). In both cases, the NRD was able to restore these strain values to their intact levels (p ≥ 0.192). Discussion: The ability of the NRD to restore IVD biomechanics and some strain types to intact state levels supports nucleus replacement surgery as a viable treatment option. The DVC-MRI method used in the present study could serve as a useful tool to assess future NRD designs to help improve performance in future clinical trials.

Single Cell RNA Sequencing Reveals Emergent Notochord-Derived Cell Subpopulations in the Postnatal Nucleus Pulposus.

Intervertebral disc degeneration is a leading cause of chronic low back pain. Cell-based strategies that seek to treat disc degeneration by regenerating the central nucleus pulposus hold significant promise, but key challenges remain. One of these is the inability of therapeutic cells to effectively mimic the performance of native nucleus pulposus cells, which are unique amongst skeletal cell types in that they arise from the embryonic notochord. In this study we use single cell RNA sequencing to demonstrate emergent heterogeneity amongst notochord-derived nucleus pulposus cells in the postnatal mouse disc. Specifically, we established the existence of early and late stage nucleus pulposus cells, corresponding to notochordal progenitor and mature cells, respectively. Late stage cells exhibited significantly higher expression levels of extracellular matrix genes including aggrecan, and collagens II and VI, along with elevated TGF-ß and PI3K-Akt signaling. Additionally, we identified Cd9 as a novel surface marker of late stage nucleus pulposus cells, and demonstrated that these cells were localized to the nucleus pulposus periphery, increased in numbers with increasing postnatal age, and co-localized with emerging glycosaminoglycan-rich matrix. Finally, we used a goat model to show the Cd9+ nucleus pulposus cell numbers decrease with moderate severity disc degeneration, suggesting that these cells are associated with maintenance of the healthy nucleus pulposus extracellular matrix. Improved understanding of the developmental mechanisms underlying regulation of ECM deposition in the postnatal NP may inform improved regenerative strategies for disc degeneration and associated low back pain.

An in vitro comparison of three nucleus pulposus removal techniques for partial intervertebral disc replacement: An ultra-high resolution MRI study.

Background: Nuclectomy, also known as nucleotomy, is a percutaneous surgical procedure performed to remove nucleus material from the center of the disc. Multiple techniques have been considered to perform a nuclectomy, however, the advantages and disadvantages of each are not well understood. Aims: This in vitro biomechanical investigation on human cadaveric specimens aimed to quantitatively compare three nuclectomy techniques performed using an automated shaver, rongeurs, and laser. Material & Methods: Comparisons were made in terms of mass, volume and location of material removal, changes in disc height, and stiffness. Fifteen vertebra-disc-vertebra lumbar specimens were acquired from six donors (40 ± 13 years) and split into three groups. Before and after nucleotomy axial mechanical tests were performed and T2-weighted 9.4T MRIs were acquired for each specimen. Results: When using the automated shaver and rongeurs similar volumes of disc material were removed (2.51 ± 1.10% and 2.76 ± 1.39% of the total disc volume, respectively), while considerably less material was removed using the laser (0.12 ± 0.07%). Nuclectomy using the automated shaver and rongeurs significantly reduced the toe-region stiffness (p = 0.036), while the reduction in the linear region stiffness was significant only for the rongeurs group (p = 0.011). Post-nuclectomy, 60% of the rongeurs group specimens showed changes in the endplate profile while 40% from the laser group showed subchondral marrow changes. Discussion: From the MRIs, homogeneous cavities were seen in the center of the disc when using the automated shaver. When using rongeurs, material was removed non-homogeneously both from the nucleus and annulus regions. Laser ablation formed small and localized cavities suggesting that the technique is not suitable to remove large volumes of material unless it is developed and optimized for this application. Conclusion: The results demonstrate that both rongeurs and automated shavers can be used to remove large volumes of NP material but the reduced risk of collateral damage to surrounding tissues suggests that the automated shaver may be more suitable.

Numerical analysis of the mechanical response of novel swelling bone implants in polyurethane foams.

In this study, a numerical framework was developed in order to analyze the swelling properties, mechanical response and fixation strength of swelling bone anchors. Using this framework, fully porous and solid implants, along with a novel hybrid design (consisting of a solid core and a porous sleeve), were modeled and studied. Free swelling experiments were conducted to investigate their swelling characteristics. The finite element model of swelling was validated using the conducted free swelling. Compared with the experimental data, results obtained from the finite element analysis proved the reliability of this frame-work. Afterwards, the swelling bone anchors were studied embedded in artificial bones with different densities with two different interface properties: considering frictional interface between the bone anchors and artificial bones (simulating the stages prior to osteointegration, when the bone and implant are not fully bonded and the surface of the implant can slide along the interface), and perfectly bonded (simulating the stages subsequent to osteointegration, when the bone and implant are fully bonded). It was observed that the swelling considerably decreases while the average radial stress on the lateral surface of the swelling bone anchor surges in the denser artificial bones. Ultimately, the pull-out experiments and simulations of the swelling bone anchors from the artificial bones were conducted to look into the fixation strength of the swelling bone anchors. It was found that the hybrid swelling bone anchor exhibits mechanical and swelling properties close to those of solid bone anchors, while also bone in-growth is expected to happen, which is an integral factor to these bone anchors.

Acute Repair of Meniscus Root Tear Partially Restores Joint Displacements as Measured With Magnetic Resonance Images and Loading in a Cadaveric Porcine Knee.

The meniscus serves important load-bearing functions and protects the underlying articular cartilage. Unfortunately, meniscus tears are common and impair the ability of the meniscus to distribute loads, increasing the risk of developing osteoarthritis. Therefore, surgical repair of the meniscus is a frequently performed procedure; however, repair does not always prevent osteoarthritis. This is hypothesized to be due to altered joint loading post-injury and repair, where the functional deficit of the meniscus prevents it from performing its role of distributing forces. The objective of this study was to quantify joint kinematics in an intact joint, after a meniscus root tear, and after suture repair in cadaveric porcine knees, a frequently used in vivo model. We utilized an magnetic resonance images-compatible loading device and novel use of a T1 vibe sequence to measure meniscus and femur displacements under physiological axial loads. We found that anterior root tear led to large meniscus displacements under physiological axial loading and that suture anchor repair reduced these displacements but did not fully restore intact joint kinematics. After tear and repair, the anterior region of the meniscus moved posteriorly and medially as it was forced out of the joint space under loading, while the posterior region had small displacements as the posterior attachment acted as a hinge about which the meniscus pivoted in the axial plane. Methods from this study can be applied to assess altered joint kinematics following human knee injuries and evaluate repair strategies aimed to restore joint kinematics.

In Vitro and In Vivo Evaluation of Ultrasound-Triggered Release From Novel Spinal Device.

OBJECTIVES: Bacterial infection following spinal fusion is a major clinical concern with up to 20% incidence. An ultrasound-triggered bulk-release system to combat postsurgical bacterial survival was designed and evaluated. METHODS: Polylactic acid (PLA) clips were loaded with vancomycin (VAN) and microbubbles (Sonazoid, GE HealthCare) in vitro. Stability was determined over 14 days. VAN-loaded clips were submerged in water and insonated using a Logiq E10 scanner (GE HealthCare) with a curvilinear C6 probe. Doppler-induced VAN release was quantified using spectrophotometry. For in vivo testing, clips were loaded with methylene blue (MeB) solution and Sonazoid. These clips were implanted into a rabbit along the spine at L2 and L5, as well as a pig at L1 and L3, then insonated in Doppler mode using the C6 probe. RESULTS: Sonazoid microbubbles were better preserved when incubated in VAN compared with distilled water at 4°C, 25°C, and 37°C incubation temperatures (P = .0131). Contrast enhancement was observed from both solutions when incubated at 4°C storage conditions. Insonated clips achieved average cumulative VAN release of 101.8 ± 2.8% (81.4 ± 2.8 mg) after 72 hours. Uninsonated clips had only 0.3 ± 0.1% (0.3 ± 0.1 mg) average cumulative VAN release (P < .0001). Clips retrieved from the rabbit did not rupture with insonation nor produce MeB staining of surrounding tissues. In the pig, the PLA film was visibly ruptured and MeB tissue was observed following insonation, whereas the uninsonated clip was intact. CONCLUSION: These results demonstrate ultrasound-triggered release of an encapsulated prophylactic solution and provide an important proof-of-concept for continuing large animal evaluations for translational merit.

Berberine disrupts staphylococcal proton motive force to cause potent anti-staphylococcal effects in vitro.

The presence of antibiotic resistance has increased the urgency for more effective treatments of bacterial infections. Biofilm formation has complicated this issue as biofilm bacteria become tolerant to antibiotics due to environmental factors such as nutrient deprivation and adhesion. In septic arthritis, a disease with an 11% mortality rate, bacteria in synovial fluid organize into floating, protein-rich, bacterial aggregates (mm-cm) that display depressed metabolism and antibiotic tolerance. In this study, Staphylococcus aureus (S. aureus), which is the most common pathogen in septic arthritis, was tested against different inhibitors that modulate bacterial surface protein availability and that should decrease bacterial aggregation. One of these, berberine, a quaternary ammonium compound, was found to reduce bacterial counts by 3-7 logs in human synovial fluid (aggregating medium) with no effect in tryptic soy broth (TSB, non-aggregating). Unlike traditional antibiotics, the bactericidal activity of berberine appeared to be independent of bacterial metabolism. To elucidate the mechanism, we used synovial fluid fractionation, targeted MRSA transposon insertion mutants, dyes to assess changes in membrane potential (DiSC3(5)) and membrane permeability (propidium iodide (PI)), colony counting, and fluorescence spectroscopy. We showed that berberine's activity was dependent on an alkaline pH and berberine killed both methicillin-sensitive S. aureus and MRSA in alkaline media (pH 8.5-9.0; p < 0.0001 vs. same pH controls). Under these alkaline conditions, berberine localized to S. aureus where berberine was isolated in cytoplasmic (∼95%) and DNA (∼5%) fractions. Importantly, berberine increased bacterial cell membrane permeability, and disrupted the proton motive force, suggesting a mechanism whereby it may be able to synergize with other antibacterial compounds under less harsh conditions. We suggest that berberine, which is cheap and readily available, can be made into an effective treatment.

Microbubble cavitation restores Staphylococcus aureus antibiotic susceptibility in vitro and in a septic arthritis model.

Treatment failure in joint infections is associated with fibrinous, antibiotic-resistant, floating and tissue-associated Staphylococcus aureus aggregates formed in synovial fluid (SynF). We explore whether antibiotic activity could be increased against Staphylococcus aureus aggregates using ultrasound-triggered microbubble destruction (UTMD), in vitro and in a porcine model of septic arthritis. In vitro, when bacterially laden SynF is diluted, akin to the dilution achieved clinically with lavage and local injection of antibiotics, amikacin and ultrasound application result in increased bacterial metabolism, aggregate permeabilization, and a 4-5 log decrease in colony forming units, independent of microbubble destruction. Without SynF dilution, amikacin + UTMD does not increase antibiotic activity. Importantly, in the porcine model of septic arthritis, no bacteria are recovered from the SynF after treatment with amikacin and UTMD-ultrasound without UTMD is insufficient. Our data suggest that UTMD + antibiotics may serve as an important adjunct for the treatment of septic arthritis.