Compact Linear Flow Phantom Model for Retinal Blood-Flow Evaluation.

Impaired retinal blood flow is associated with ocular diseases such as glaucoma, macular degeneration, and diabetic retinopathy. Among several ocular imaging techniques developed to measure retinal blood flow both invasively and non-invasively, adaptive optics (AO)-enabled scanning laser ophthalmoscopy (AO-SLO) resolves individual red blood cells and provides a high resolution with which to measure flow across retinal microvasculature. However, cross-validation of flow measures remains a challenge owing to instrument and patient-specific variability in each imaging technique. Hence, there is a critical need for a well-controlled clinical flow phantom for standardization and to establish blood-flow measures as clinical biomarkers for early diagnosis. Here, we present the design and validation of a simple, compact, portable, linear flow phantom based on a direct current motor and a conveyor-belt system that provides linear velocity tuning within the retinal microvasculature range (0.5-7 mm/s). The model was evaluated using a sensitive AO-SLO line-scan technique, which showed a <6% standard deviation from the true velocity. Further, a clinical SLO instrument showed a linear correlation with the phantom's true velocity (r2 > 0.997). This model has great potential to calibrate, evaluate, and improve the accuracy of existing clinical imaging systems for retinal blood flow and aid in the diagnosis of ocular diseases with abnormal blood flow.

Immediate inferior alveolar nerve reconstruction: Improving warfighter quality of life following mandibulectomy or traumatic avulsion of the mandible.

BACKGROUND: Mandibular reconstruction has historically been challenging due to the complex, highly functional, and esthetic nature of the anatomy. The most common etiologies of these defects requiring resection include trauma, benign tumors, and malignant pathology. Mandibular defects have been treated with little consideration for neural reconstruction, leaving patient's orally incompetent with associated social stigma. Although recent advances in reconstructive techniques improve oral rehabilitation, immediate inferior alveolar nerve (IAN) reconstruction has not been widely adapted. OBJECTIVE: Here-in we seek to discuss the innovations of neural reconstruction of large segment mandibular defects and associated IAN defects and present an example case performed at Naval Medical Center San Diego (NMCSD). METHODS: Pertinent literature discussing maxillofacial reconstruction and nerve repair using autogenous nerve harvest and allograft was queried from available online resources. RESULTS: Six patients have received immediate reconstruction of the IAN using processed nerve allograft over the past three years. All obtained sensation to S3 within six months of surgery. CONCLUSION: IAN repair using nerve allografts in conjunction with free flap reconstruction for large mandibular defects is a viable treatment and should be the new paradigm in maxillofacial reconstruction as it provides substantial quantifiable and qualitative improvements in social, functional, and esthetic outcomes of care.

Inhibition of Mac-1 allows human macrophages to migrate against the direction of shear flow on ICAM-1.

All immune cells must transit from the blood to distal sites such as the lymph nodes, bone marrow, or sites of infection. Blood borne monocytes traffic to the site of inflammation by adhering to the endothelial surface and migrating along endothelial intracellular adhesion molecule 1 (ICAM-1) by their ligand's macrophage 1 antigen (Mac-1) and lymphocyte functional antigen 1 (LFA-1) to transmigrate through the endothelium. Poor patient prognoses in chronic inflammation and tumors have been attributed to the hyper recruitment of certain types of macrophages. Therefore, targeting the binding of ICAM-1 to its respective ligands provides a novel approach to targeting the recruitment of macrophages. To that end, we determined whether the loss of Mac-1 expression could induce this upstream migration behavior by using blocking antibodies against Mac-1 to examine the effects of hydrodynamic flow on the migration of the human macrophage cell line U-937 on ICAM-1 surfaces. Blocking Mac-1 on U-937 cells led to upstream migration against the direction of shear flow on ICAM-1 surfaces. In sum, the ability of macrophages to migrate upstream when Mac-1 is blocked represents a new avenue to precisely control the differentiation, migration, and trafficking of macrophages.

Quantification of Human Photoreceptor-Retinal Pigment Epithelium Macular Topography with Adaptive Optics-Optical Coherence Tomography.

Photoreceptors (PRs) and retinal pigment epithelial (RPE) cells form a functional unit called the PR-RPE complex. The PR-RPE complex plays a critical role in maintaining retinal homeostasis and function, and the quantification of its structure and topographical arrangement across the macula are important for understanding the etiology, mechanisms, and progression of many retinal diseases. However, the three-dimensional cellular morphology of the PR-RPE complex in living human eyes has not been completely described due to limitations in imaging techniques. We used the cellular resolution and depth-sectioning capabilities of a custom, high-speed Fourier domain mode-locked laser-based adaptive optics-optical coherence tomography (FDML-AO-OCT) platform to characterize human PR-RPE complex topography across the temporal macula from eleven healthy volunteers. With the aid of a deep learning algorithm, key metrics were extracted from the PR-RPE complex of averaged AO-OCT volumes including PR and RPE cell density, PR outer segment length (OSL), and PR/RPE ratio. We found a tight grouping among our cohort for PR density, with a mean (±SD) value of 53,329 (±8106) cells/mm2 at 1° decreasing to 8669 (±737) cells/mm2 at 12°. We observed a power function relationship between eccentricity and both PR density and PR/RPE ratio. We found similar variability in our RPE density measures, with a mean value of 7335 (±681) cells/mm2 at 1° decreasing to 5547 (±356) cells/mm2 at 12°, exhibiting a linear relationship with a negative slope of -123 cells/mm2 per degree. OSL monotonically decreased from 33.3 (±2.4) µm at 1° to 18.0 (±1.8) µm at 12°, following a second-order polynomial relationship. PR/RPE ratio decreased from 7.3 (±0.9) µm at 1° to 1.5 (±0.1) µm at 12°. The normative data from this investigation will help lay a foundation for future studies of retinal pathology.

A microfluidic platform for the synthesis of polymer and polymer-protein-based protocells.

In this study, we demonstrate the fabrication of polymersomes, protein-blended polymersomes, and polymeric microcapsules using droplet microfluidics. Polymersomes with uniform, single bilayers and controlled diameters are assembled from water-in-oil-in-water double-emulsion droplets. This technique relies on adjusting the interfacial energies of the droplet to completely separate the polymer-stabilized inner core from the oil shell. Protein-blended polymersomes are prepared by dissolving protein in the inner and outer phases of polymer-stabilized droplets. Cell-sized polymeric microcapsules are assembled by size reduction in the inner core through osmosis followed by evaporation of the middle phase. All methods are developed and validated using the same glass-capillary microfluidic apparatus. This integrative approach not only demonstrates the versatility of our setup, but also holds significant promise for standardizing and customizing the production of polymer-based artificial cells.

Donor Limb Functional Restoration via a Novel Clinical Care Pathway following Fibula Free Flap Harvest for Head and Neck Reconstruction.

Patients undergoing head and neck skeletal reconstruction (HNR) often require free tissue transfer from the extremities to ensure proper restoration of form and function. This requires a team-based, highly reliable medical system centered around the patient needs. Surgical intervention across multiple sites and harvesting of donor tissue results in short- and long-term physical impairments. There is a paucity of research objectively measuring impairments resulting from the graft donor site. There is a lack of research that objectively measures impairments and protocols for the management of these patients postoperatively. Patients undergo little, if any, formal approach to dealing with the vast impairments, which are sequelae to this surgery. This leads to large discrepancies in proposed functional progressions, return to duty timelines, and utilization of rehabilitative resources. At a major military medical center, an innovative clinical care pathway for patients undergoing HNR using free tissue transfer was implemented using a multidisciplinary model that focuses on early engagement with rehabilitation. This model, paired with a single surgery, will attempt to return service members to duty months earlier than the traditional approach. This report describes the conceptual framework and implementation of a new criteria-based, multidisciplinary clinical care pathway for HNR patients. The collaboration amongst the multidisciplinary care team has optimized the holistic health of the patient and communication with their support network, yielding faster return to normalization of daily life activities. The long-term goal is to further develop and formalize this pathway to best serve this patient population.

Immediate dental rehabilitation in fibula free flaps for malignancy: Is it feasible?

BACKGROUND: Fibula free flap reconstruction has revolutionized maxillofacial reconstruction. While immediate dental rehabilitation with dental implants and teeth has shown benefits, it remains uncommon, especially for patients with malignancy. METHODS: A retrospective cohort study at a single institution explored immediate dental rehabilitation in fibula flaps for patients with malignant disease. Thirteen patients with malignancies that underwent immediate fibula free flap reconstruction with dental implants and dental prosthesis were included with a minimum of 3-month follow-up. RESULTS: Forty eight implants replaced 90 teeth in 13 patients. All implants were integrated at 3 months, with an overall success rate of 87.5%. Two patients experienced delayed (>3 months postoperatively) implant loss due to osteoradionecrosis and infection. Peri-mucositis occurred in three patients which resolved with treatment. Skin paddles were used in 11 patients and radiation therapy was not delayed for any patient. CONCLUSION: With proper patient selection, diagnosis of malignancy and the need for osteocutaneous flap reconstruction does not exclude the ability to place immediate implants and deliver an immediate dental prosthesis in head and neck reconstruction.

Revealing speckle obscured living human retinal cells with artificial intelligence assisted adaptive optics optical coherence tomography.

BACKGROUND: In vivo imaging of the human retina using adaptive optics optical coherence tomography (AO-OCT) has transformed medical imaging by enabling visualization of 3D retinal structures at cellular-scale resolution, including the retinal pigment epithelial (RPE) cells, which are essential for maintaining visual function. However, because noise inherent to the imaging process (e.g., speckle) makes it difficult to visualize RPE cells from a single volume acquisition, a large number of 3D volumes are typically averaged to improve contrast, substantially increasing the acquisition duration and reducing the overall imaging throughput. METHODS: Here, we introduce parallel discriminator generative adversarial network (P-GAN), an artificial intelligence (AI) method designed to recover speckle-obscured cellular features from a single AO-OCT volume, circumventing the need for acquiring a large number of volumes for averaging. The combination of two parallel discriminators in P-GAN provides additional feedback to the generator to more faithfully recover both local and global cellular structures. Imaging data from 8 eyes of 7 participants were used in this study. RESULTS: We show that P-GAN not only improves RPE cell contrast by 3.5-fold, but also improves the end-to-end time required to visualize RPE cells by 99-fold, thereby enabling large-scale imaging of cells in the living human eye. RPE cell spacing measured across a large set of AI recovered images from 3 participants were in agreement with expected normative ranges. CONCLUSIONS: The results demonstrate the potential of AI assisted imaging in overcoming a key limitation of RPE imaging and making it more accessible in a routine clinical setting.

The retinal pigment epithelium (RPE) is a single layer of cells within the eye that is crucial for vision. These cells are unhealthy in many eye diseases, and this can result in vision problems, including blindness. Imaging RPE cells in living human eyes is time consuming and difficult with the current technology. Our method substantially speeds up the process of RPE imaging by incorporating artificial intelligence. This enables larger areas of the eye to be imaged more efficiently. Our method could potentially be used in the future during routine eye tests. This could lead to earlier detection and treatment of eye diseases, and the prevention of some causes of blindness.

Determinants that enable disordered protein assembly into discrete condensed phases.

Cells harbour numerous mesoscale membraneless compartments that house specific biochemical processes and perform distinct cellular functions. These protein- and RNA-rich bodies are thought to form through multivalent interactions among proteins and nucleic acids, resulting in demixing via liquid-liquid phase separation. Proteins harbouring intrinsically disordered regions (IDRs) predominate in membraneless organelles. However, it is not known whether IDR sequence alone can dictate the formation of distinct condensed phases. We identified a pair of IDRs capable of forming spatially distinct condensates when expressed in cells. When reconstituted in vitro, these model proteins do not co-partition, suggesting condensation specificity is encoded directly in the polypeptide sequences. Through computational modelling and mutagenesis, we identified the amino acids and chain properties governing homotypic and heterotypic interactions that direct selective condensation. These results form the basis of physicochemical principles that may direct subcellular organization of IDRs into specific condensates and reveal an IDR code that can guide construction of orthogonal membraneless compartments.

Free flap jaw reconstruction with dental implantation: A single-institution experience.

BACKGROUND: We sought to review our institution's experience with dental implant placement in free flap jaw reconstruction to determine factors impacting restoration of dental occlusion. METHODS: Exactly 48 patients underwent free flap jaw reconstruction with or without dental restoration from 2017 to 2022. Primary outcome was achievement of restored dental occlusion after jaw free flap reconstruction. RESULTS: A total of 48 patients with a mean age of 59.8 ± 16.4 years underwent jaw reconstruction from 2017 to 2022. Ten patients (20.8%) received osteointegrated dental implants. Two patients received a temporary dental prosthesis, 12 ± 4 months after initial reconstruction. Three patients received a final prosthesis, with a mean time to final prosthesis of 17.7 ± 12.4 months. Five patients did not receive any prosthesis despite placement of implants. CONCLUSION: A minority of patients received dental implant placement with free flap jaw reconstruction and only a small subset of these received a definitive dental prosthesis.

Dental prosthesis extraoral indexing and pick-up technique for mandibular full arch immediate rehabilitation in fibula free flap reconstruction.

Same-day ablative and reconstructive surgeries for the treatment of head and neck pathologies are gaining in popularity with the recognition that single-day surgeries reduce morbidity and increase quality of life. Implant-borne prosthetics on the donor graft provide immediate dental reconstruction. This report describes a novel technique for extraoral pickup of a full arch immediate prosthesis from the donor site free flap. This technique minimizes intraoperative occlusal adjustments, saves intraoperative time, prevents undesirable "rolling" of a fibula segment, and immediately rehabilitates patients with dental prosthetics.

Cellular-Level Visualization of Retinal Pathology in Multiple Sclerosis With Adaptive Optics.

Purpose: To apply adaptive optics-optical coherence tomography (AO-OCT) to quantify multiple sclerosis (MS)-induced changes in axonal bundles in the macular nerve fiber layer, ganglion cell somas, and macrophage-like cells at the vitreomacular interface. Methods: We used AO-OCT imaging in a pilot study of MS participants (n = 10), including those without and with a history of optic neuritis (ON, n = 4), and healthy volunteers (HV, n = 9) to reveal pathologic changes to inner retinal cells and structures affected by MS. Results: We found that nerve fiber layer axonal bundles had 38% lower volume in MS participants (1.5 × 10-3 mm3) compared to HVs (2.4 × 10-3 mm3; P < 0.001). Retinal ganglion cell (RGC) density was 51% lower in MS participants (12.3 cells/mm2 × 1000) compared to HVs (25.0 cells/mm2 × 1000; P < 0.001). Spatial differences across the macula were observed in RGC density. RGC diameter was 15% higher in MS participants (11.7 µm) compared to HVs (10.1 µm; P < 0.001). A nonsignificant trend of higher density of macrophage-like cells in MS eyes was also observed. For all AO-OCT measures, outcomes were worse for MS participants with a history of ON compared to MS participants without a history of ON. AO-OCT measures were associated with key visual and physical disabilities in the MS cohort. Conclusions: Our findings demonstrate the utility of AO-OCT for highly sensitive and specific detection of neurodegenerative changes in MS. Moreover, the results shed light on the mechanisms that underpin specific neuronal pathology that occurs when MS attacks the retina. The new findings support the further development of AO-based biomarkers for MS.

Not all (cells) who wander are lost: Upstream migration as a pervasive mode of amoeboid cell motility.

Leukocytes possess the ability to migrate upstream-against the direction of flow-on surfaces of specific chemistry. Upstream migration was first characterized in vitro for T-cells on surfaces comprised of intracellular adhesion molecule-1 (ICAM-1). Upstream migration occurs when the integrin receptor αLß2 (also known as lymphocyte function-associated antigen-1, or LFA-1) binds to ICAM-1. LFA-1/ICAM-1 interactions are ubiquitous and are widely found in leukocyte trafficking. Upstream migration would be employed after cells come to arrest on the apical surface of the endothelium and might confer an advantage for both trans-endothelial migration and tissue surveillance. It has now been shown that several other motile amoeboid cells which have the responsibility of trafficking from blood vessels into tissues, such as Marginal zone B cells, hematopoietic stem cells, and neutrophils (when macrophage-1 antigen, Mac-1, is blocked), can also migrate upstream on ICAM-1 surfaces. This review will summarize what is known about the basic mechanisms of upstream migration, which cells have displayed this phenomenon, and the possible role of upstream migration in physiology and tissue homeostasis.

Asymmetry-Enhanced Motion of Urease-Powered Micromotors from Double Emulsion-Templated Microcapsules.

Autonomous motion of enzyme-powered motors has important implications for drug delivery, cell-cell communication, and protocell engineering. Although many of these systems are inspired by the motion of biological cells, most of them lack key structural features, like micrometer-sized boundaries and aqueous compartments, and rely on bubble propulsion to generation motion. In this study, we use droplet microfluidics to generate large populations of cell-sized microcapsules with poly(lactic-co-glycolic acid) shells and functionalize their surfaces with the enzyme urease to drive their motion. We adjust the number of surface functional groups for urease conjugation by preparing microcapsules with two different surfactants, poly(vinyl alcohol) (PVA) and poly(ethylene-alt-maleic anhydride) (PEMA). We also tune the surface roughness of the microcapsules by varying the concentration of silica nanoparticles in the droplet middle phase. We find that PEMA plays a crucial role in increasing the grafting density of urease on the surface of smooth microcapsules, leading to active motion in the presence of urea. In addition, rough microcapsules prepared with PEMA and loaded with comparable amounts of urease move up to three times faster than their smooth counterparts, which we believe is due to an asymmetric distribution of urease on the surface, giving rise to a preferred direction of motion. Taken together, these results provide new insights into the role that various stabilizing agents play in the induction of motion by enzymatic motors prepared from microfluidics, which is a potentially powerful tool for future preparation of motile protocells in biomedicine.

The link between the sphingolipid rheostat and obstructive sleep apnea.

Chronic inflammation induced by hypoxia during sleep is an important mechanism of microvascular damage in OSA patients. In this study, we investigated the role of the sphingosine rheostat, which has diverse inflammatory effects. Thirty-seven healthy subjects and 31 patients with OSA were recruited. We collected data on demographics and comorbidities. Plasma sphingosine-1-phosphate and ceramide antibody concentrations were measured by ELISA. The results were compared between the OSA and control groups, and the correlations between these measurements and markers of disease severity and comorbidities were explored. Ceramide antibody levels were significantly elevated in OSA patients (892.17 ng/ml) vs. controls (209.55 ng/ml). S1P levels were also significantly higher in patients with OSA (1760.0 pg/ml) than in controls (290.35 pg/ml, p < 0.001). The ceramide antibody concentration showed correlations with BMI (ρ = 0.25, p = 0.04), CRP (ρ = 0.36, p = 0.005), AHI (ρ = 0.43, p < 0.001), ODI (ρ = 0.43, p < 0.001), TST90% (ρ = 0.35, p = 0.004) and the lowest oxygen saturation (ρ =  0.37, p = 0.001) in the whole study population but not when patients with OSA were analyzed separately. The elevated ceramide antibody and sphingosine-1-phosphate concentrations in patients suffering from OSA suggests their involvement in the pathomechanism of OSA and its comorbidities.

Tenants of Mandibular Reconstruction in Segmental Defects.

The premises of mandibular reconstruction are the restoration of occlusion and mandibular contour for the purpose of preserving the facial identity, oral airway, and effective speech and mastication. Establishing functional occlusion is the primary tenant in all mandibular reconstruction. In cases of segmental defects, particularly in dentate regions of the mandible, there has been a paradigm shift over the past two decades in how surgeons are approaching the restoration of load-bearing mandibular continuity with capacity for dental implantation. Here we discuss considerations for deciding the most effective method of reconstruction in segmental defects.

Development of polarization-sensitive optical coherence tomography imaging platform and metrics to quantify electrostimulation-induced peripheral nerve injury in vivo in a small animal model.

Significance: Neuromodulation devices are rapidly evolving for the treatment of neurological diseases and conditions. Injury from implantation or long-term use without obvious functional losses is often only detectable through terminal histology. New technologies are needed that assess the peripheral nervous system (PNS) under normal and diseased or injured conditions. Aim: We aim to demonstrate an imaging and stimulation platform that can elucidate the biological mechanisms and impacts of neurostimulation in the PNS and apply it to the sciatic nerve to extract imaging metrics indicating electrical overstimulation. Approach: A sciatic nerve injury model in a 15-rat cohort was observed using a newly developed imaging and stimulation platform that can detect electrical overstimulation effects with polarization-sensitive optical coherence tomography. The sciatic nerve was electrically stimulated using a custom-developed nerve holder with embedded electrodes for 1 h, followed by a 1-h recovery period, delivered at above-threshold Shannon model k -values in experimental groups: sham control (SC, n = 5 , 0.0 mA / 0 Hz ), stimulation level 1 (SL1, n = 5 , 3.4 mA / 50 Hz , and k = 2.57 ), and stimulation level 2 (SL2, n = 5 , 6.8 mA / 100 Hz , and k = 3.17 ). Results: The stimulation and imaging system successfully captured study data across the cohort. When compared to a SC after a 1-week recovery, the fascicle closest to the stimulation lead showed an average change of + 4 % / - 309 % (SL1/SL2) in phase retardation and - 79 % / - 148 % in optical attenuation relative to SC. Analysis of immunohistochemistry (IHC) shows a + 1 % / - 36 % difference in myelin pixel counts and - 13 % / + 29 % difference in axon pixel counts, and an overall increase in cell nuclei pixel count of + 20 % / + 35 % . These metrics were consistent with IHC and hematoxylin/eosin tissue section analysis. Conclusions: The poststimulation changes observed in our study are manifestations of nerve injury and repair, specifically degeneration and angiogenesis. Optical imaging metrics quantify these processes and may help evaluate the safety and efficacy of neuromodulation devices.

Introduction to the Feature Issue on Adaptive Optics for Biomedical Applications.

The guest editors introduce a feature issue commemorating the 25th anniversary of adaptive optics in biomedical research.