Necrotizing soft-tissue infections (NSTIs) are aggressive and deadly. A major obstacle to prompt NSTI management is the lack of a definitive diagnostic test, causing treatment delays and increasing mortality. NSTI-affected tissues undergo prominent blood vessel thrombosis invisible to the naked eye that can be rapidly visualized using perfusion-based fluorescence imaging. To validate fluorescence imaging for rapid NSTI detection, we are developing a preclinical mouse model of NSTI using light-producing strains of causative bacteria that have biological equivalence to wild type. Co-registration of our wide-field fluorescence imagery with spectrally distinct, bacterial bioluminescence provides validation of infection presence and spatial extent.
Bacterial biofilms are a pervasive issue in orthopaedic surgery causing up to 80% of infections. Antimicrobial photodynamic therapy (aPDT) is a proposed technique for eradication of biofilms, clinical translation requires optimization of treatment parameters. This study assessed the effectiveness of three light spectra in activating photosensitizing porphyrins to kill a dual species biofilm of E. Coli and E. faecalis grown in a microfluidic device. Blue-red, amber, or blue-amber-red light sources were compared at either 30J/cm2 or 60J/cm2 doses given to activate endogenously produced porphyrins after one hour incubation with 10 or 20% 5-ALA in a saline solution. Changes in biomass 24 hours after treatment were measured using confocal microscopy and OCT to determine treatment effectiveness.
Well-organized ecosystems of bacteria colonize orthopaedic devices causing biofilm infections that are notoriously difficult to manage. Biofilms typically exhibit increased resistance to antibiotics leading to treatment failure, and tools for eradicating biofilms that do not increase antibiotic resistance are greatly needed. Antimicrobial photodynamic therapy (aPDT) is a promising form of treatment to combat clinically relevant biofilms. Exogenous provision of 5-aminolevulinic acid (5-ALA) to biofilm-forming clinical strains of E. coli, E. faecalis and S. aureus was recently shown by several research groups to result in the accumulation of sufficient quantities of endogenous photosensitizers porphyrins (protoporphyrin IX, coproporphyrin III and others), via the heme biosynthetic pathway, to produce a significant phototoxic effect when exposed to activating light. For clinical translation of this extremely promising approach, here we develop a portable light source for 5-ALA-based aPDT of orthopaedic implant biofilms, spectrally shaped for optimal porphyrin light absorption at wavelengths range approved by FDA for clinical use. After phantom calibration, we tested it on E.coli-E.faecalis biofilms grown in soft lithography-fabricated microfluidic chips and on methicillin-resistant S. aureus (MRSA) biofilms grown on titanium and stainless steel orthopaedic hardware in custom-designed macrofluidic devices. Successful in-vitro experiments allowed us to conduct a proof-of-concept validation study in a preclinical rat model of MRSA-contaminated open fracture. Following tibia fracture and two hours of wound infection development, a one hour incubation with 20% 5-ALA and treatment with either 90J/cm2 or three fractions of 30J/cm2 light doses demonstrated 94% and 99% overall reduction of MRSA, respectively, while the temperature of the tissue remained <39°C, below the threshold for thermal damage. The encouraging results suggest further preclinical testing of the developed light source for optimization of aPDT regimen and 5-ALA concentration to reduce the risk of long-term side effects in animal models of contaminated trauma surgery.
In orthopedic trauma surgery, traditional socket-based prostheses are associated with functionally limiting problems affecting 1.7 million amputees in the United States. To improve post-surgical performance and minimize socket-related complications, bone-anchored (osseointegrated) prostheses have been developed. Functionally superior, their widespread implementation has been limited due to infection. In an unacceptable number of patients well-organized biofilm ecosystems of bacteria colonize the osseointegrated implant (OI) and migrate into device-tissue interface, leading to superficial and deep infections, and implant failure. Since the OI implant protrudes through the skin, the site is easily contaminated by microbes. The problem is worsened by increased resistance to antibiotics contributing significantly to surgical outcome failure. Antimicrobial photodynamic therapy (aPDT)—which uses photosensitizers excited with visible light to disrupt biofilms and kill bacteria with produced reactive oxygen species—has been proposed to address this problem. To assess biofilm formation and aPDT effectiveness, we describe a rabbit OI model and steps to investigate the ability of aPDT using 5-Aminolevulinic acid (5-ALA)-based light therapy to control methicillin-resistant S. aureus (MRSA) bacterial infection. As part of an institutionally approved survival surgery, this model involves lower limb amputation at the tibia, OI installation and MRSA inoculation. Within a week of biofilm formation, the optimal aPDT regime of light and 5-ALA dose was applied to the implant-skin interface to eradicate migrating biofilms. We have built a circumferential light source spectrally shaped for optimal photoactivation and cooled without risk of bacteria dispersal. Optical coherence tomography (skin flap healing and side-effects), micro-computed tomography (OI-bone integrity) and bioluminescence (bacterial bioburden before and after aPDT) imaging were used to monitor outcome for up to three weeks post-treatment.
SignificanceIndocyanine green-based dynamic contrast-enhanced fluorescence imaging (DCE-FI) can objectively assess bone perfusion intraoperatively. However, it is susceptible to motion artifact due to patients’ involuntary respiration and mechanical disturbance. Reducing motion artifacts would significantly improve DCE-FI for orthopedic surgical guidance.AimOur primary objective is to develop an automated correction method to reduce motion artifacts in DCE-FI and improve the accuracy of bone perfusion assessment.ApproachWe developed an automated motion correction approach based on frame-by-frame mutual information (MI) and validated the effectiveness of this approach in various phantom studies and patient images from 45 imaging sessions of fifteen amputees.ResultsThe MI-based correction reduced motion artifacts by 93% for mechanical disturbances and 76% for simulated respiration in phantom studies. Patient images show improved alignment, improved kinetic curves, and restored bone perfusion-related parameters with an average correction of 4.3 and 9.6 mm in x- and y-axes per session.ConclusionsThe automated MI-based motion correction was able to eliminate motion artifacts effectively and significantly improved the quantitative assessment of bone perfusion by DCE-FI.
Following orthopaedic trauma, bone devitalization is a critical determinant of complications such as infection or nonunion. Intraoperative assessment of bone perfusion has thus far been limited. Furthermore, treatment failure for infected fractures is unreasonably high, owing to the propensity of biofilm to form and become entrenched in poorly vascularized bone. Fluorescence-guided surgery and molecularly-guided surgery could be used to evaluate the viability of bone and soft tissue and detect the presence of planktonic and biofilm-forming bacteria. This proceedings paper discusses the motivation behind developing this technology and our most recent preclinical and clinical results.
We have co-developed a first-in-kind model of fluorophore testing in freshly amputated human limbs. Ex vivo human tissue provides a unique opportunity for the testing of pre-clinical fluorescent agents, collection of imaging data, and histopathologic examination in human tissue prior to performing in vivo experiments. Existing pre-clinical fluorescent agent studies rely primarily on animal models, which do not directly predict fluorophore performance in humans and can result in wasted resources and time if an agent proves ineffective in early human trials. Because fluorophores have no desired therapeutic effect, their clinical utility is based solely on their safety and ability to highlight tissues of interest. Advancing to human trials even via the FDA’s phase 0/microdose pathway still requires substantial resources, single-species pharmacokinetic testing, and toxicity testing. In a recently concluded study using amputated human lower limbs, we were able to test successfully a nerve-specific fluorophore in pre-clinical development. This study used systemic administration via vascular cannulization and a cardiac perfusion pump. We envision that this model may assist with early lead agent testing selection for fluorophores with various targets and mechanisms.
Indocyanine green (ICG)-based dynamic contrast-enhanced fluorescence imaging (DCE-FI) can objectively assess bone perfusion intraoperatively. However, it is susceptible to motion artifacts due to patient’s involuntary respiration during the 4.5-minute DCE-FI data acquisition. An automated motion correction approach based on mutual information (MI) frame-by-frame was developed to overcome this problem. In this approach, MIs were calculated between the reference and the adjacent frame translated and the maximal MI corresponded to the optimal translation. The images obtained from eighteen amputation cases were utilized to validate the approach and the results show that this correction can significantly reduce the motion artifacts and can improve the accuracy of bone perfusion assessment.
Debridement of the surgical site during open fracture reduction and internal fixation is important for preventing surgical site infection; the risk of subsequent fracture-associated infection for a particular area of tissue is assessed by the surgeon based on multi-level variables, including demographics and laboratory results. Intraoperative fluorescence imaging can contribute additional information at a more localized level. Here we present a fluorescence-based predictive model using features from dynamic contrast enhanced-fluorescence imaging (DCE-FI), as well as patient-level variables associated with infection risk. Regions-of-interest were selected from thirty-eight enrolled open fracture patients. Spatial and kinetic features were extracted from DCE-FI, and combined with patient infection risk factor describing the possibility of getting surgical-site-infection. The model was evaluated for ability to predict composite outcome scores—intra-operative surgeon assessment coupled with post-operative confirmed infection outcome. This proposed model demonstrates high predictive performance with an accuracy of 0.86, evaluated with a cross-validation approach, and is a promising approach for early and quick identification of tissue prone to infection.
Iatrogenic nerve injury is a common complication across all surgical specialties. Better nerve visualization and identification during surgery will improve outcomes and reduce nerve injuries. The Gibbs Laboratory at Oregon Health and Science University has developed a library of near-infrared, nerve-specific fluorophores to highlight nerves intraoperatively and aid surgeons in nerve identification and visualization; the current lead agent is LGW16-03. Prior to this study, testing of LGW16-03 was restricted to animal models; therefore, it was unknown how LGW16-03 performs in human tissue. To advance LGW16-03 to clinic, we sought to test this current lead agent in ex vivo human tissues from a cohort of patients and determine if the route of administration affects LGW16-03 fluorescence contrast between nerves and adjacent background tissues (muscle and adipose). LGW16-03 was applied to ex vivo human tissue from lower limb amputations via two strategies: (1) systemic administration of the fluorophore using our first-in-kind model for fluorophore testing, and (2) topical application of the fluorophore. Results showed no statistical difference between topical and systemic administration. However, in vivo human validation of these findings is required.
Necrotizing soft-tissue infections (NSTIs) are aggressive and deadly. Immediate surgical debridement is standard-ofcare, but patients often present with non-specific symptoms, thereby delaying treatment. Because NSTIs cause microvascular thrombosis, we hypothesized that perfusion imaging using indocyanine green (ICG) would show diminished fluorescence signal in NSTI-affected tissues, particularly compared to non-necrotizing, superficial infections. Through a first-in-kind clinical study, we performed first-pass ICG fluorescence perfusion imaging of patients with suspected NSTIs. Early results support our hypothesis that ICG signal voids occur in NSTI-affected tissues and that dynamic contrast-enhanced fluorescence parameters reveal tissue kinetics that may be related to disease progression and extent.
SignificanceThis first-in-kind, perfused, and amputated human limb model allows for the collection of human data in preclinical selection of lead fluorescent agents. The model facilitates more accurate selection and testing of fluorophores with human-specific physiology, such as differential uptake and signal in fat between animal and human models with zero risk to human patients. Preclinical testing using this approach may also allow for the determination of tissue toxicity, clearance time of fluorophores, and the production of harmful metabolites.AimThis study was conducted to determine the fluorescence intensity values and tissue specificity of a preclinical, nerve tissue targeted fluorophore, as well as the capacity of this first-in-kind model to be used for lead fluorescent agent selection in the future.ApproachFreshly amputated human limbs were perfused for 30 min prior to in situ and ex vivo imaging of nerves with both open-field and closed-field commercial fluorescence imaging systems.ResultsIn situ, open-field imaging demonstrated a signal-to-background ratio (SBR) of 4.7 when comparing the nerve with adjacent muscle tissue. Closed-field imaging demonstrated an SBR of 3.8 when the nerve was compared with adipose tissue and 4.8 when the nerve was compared with muscle.ConclusionsThis model demonstrates an opportunity for preclinical testing, evaluation, and selection of fluorophores for use in clinical trials as well as an opportunity to study peripheral pathologies in a controlled environment.
ICG-based dynamic contrast-enhanced fluorescence imaging (DCE-FI) and intraoperative DCE- magnetic resonance imaging (MRI) have been carried out nearly simultaneously in three lower extremity bone infection cases to investigate the relationship between these two imaging modalities for assessing bone blood perfusion during open orthopedic surgeries. Time-intensity curves in the corresponding regions of interest of two modalities were derived for comparison. The results demonstrated that ICG-based DCE-FI has higher sensitivity to perfusion changes while DCE-MRI provides superior and supplemental depth-related perfusion information. Research applying the depth-related perfusion information derived from MRI to improve the overall analytic modeling of intraoperative DCE-FI is ongoing.
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