Cleft lip and palate surgery simulator: Open source simulation model.

Objective: Cleft lip and palate is the most common craniofacial birth anomaly and requires surgery in the first year of life. However, craniofacial surgery training opportunities are limited. The aim of this study was to present and evaluate an open-source cleft lip and palate hybrid (casting and three-dimensional (3D) printing) simulation model which can be replicated at low cost to facilitate the teaching and training of cleft surgery anatomy and techniques. Design: The soft tissue component of the cleft surgery training model was casted using a 3D printed 5-component mold and silicone. The bony structure was designed to simulate the facial anatomy and to hold the silicone soft tissue. Setting: Two groups, one group of trainees and one group of expert surgeons, at University Hospital Basel in Switzerland and Pontifical Catholic University of Chile in Santiago, Chile, tested the cleft lip and palate simulation model. Participants completed a Likert-based face and content validity questionnaire to assess the realism of the model and its usefulness in surgical training. Results: More than 70 % of the participants agreed that the model accurately simulated human tissues found in patients with unilateral cleft lip and palate. Over 60 % of the participants also agreed that the model realistically replicated surgical procedures. In addition, 80-90 % of the participants found the model to be a useful and appropriate tool for teaching the anatomy and surgical techniques involved in performing unilateral cleft lip and palate repair. Conclusion: This open-source protocol provides a cost-effective solution for surgeons to introduce the cleft morphology and surgical techniques to trainees on a regular basis. It addresses the current financial barrier that limits access to commercially available models during the early stages of surgeon training prior to specialization in the field.

Comparative study on cleft palate morphology after passive presurgical plate therapy in unilateral cleft lip and palate.

BACKGROUND: Presurgical plate therapy has been widely accepted as a treatment prior to palatal cleft closure. The effects of passive presurgical plate therapy on cleft morphology prior to single-stage unilateral cleft lip and palate (UCLP) repair were quantified. PATIENTS AND METHODS: We compared the dimensions of cleft width and cleft area (true cleft and palatal cleft) measured preoperatively at 2 European cleft centers. Center A performed single-stage UCLP repair in 8-month-old infants without any presurgical orthopedic treatment. Center B initiated passive presurgical plate therapy immediately after the birth of the neonates, followed by single-stage UCLP repair at 8 months of age. RESULTS: We included 28 patients with complete UCLP from Center A and 12 patients from Center B. The average anterior width of the true cleft before surgery was significantly smaller in infants at Center B than that in Center A (p = 0.001) with 95% confidence interval of (1.8, 5.7) mm, but the average posterior width was similar in the 2 groups. The mean presurgical true cleft area amounted to 106.8 mm2 (SD = 42.4 mm2) at Center A and 71.9 mm2 (SD = 32.2 mm2) at Center B, with a confidence interval for the difference being (9.8, 60.1) mm2. This corresponded to a 32.7% reduction of the true cleft area when passive presurgical plate therapy was used for the first 8 months of the infants' life. CONCLUSION: Passive presurgical plate therapy in UCLP significantly reduced the cleft area. Implications for the subsequent surgical outcome might depend on the surgical technique used.

Automated and data-driven plate computation for presurgical cleft lip and palate treatment.

PURPOSE: Presurgical orthopedic plates are widely used for the treatment of cleft lip and palate, which is the most common craniofacial birth defect. For the traditional plate fabrication, an impression is taken under airway-endangering conditions, which recent digital alternatives overcome via intraoral scanners. However, these alternatives demand proficiency in 3D modeling software in addition to the generally required clinical knowledge of plate design. METHODS: We address these limitations with a data-driven and fully automated digital pipeline, endowed with a graphical user interface. The pipeline adopts a deep learning model to landmark raw intraoral scans of arbitrary mesh topology and orientation, which guides the nonrigid surface registration subsequently employed to segment the scans. The plates that are individually fit to these segmented scans are 3D-printable and offer optional customization. RESULTS: With the distance to the alveolar ridges closely centered around the targeted 0.1 mm, our pipeline computes tightly fitting plates in less than 3 min. The plates were approved in 12 out of 12 cases by two cleft care professionals in a printed-model-based evaluation. Moreover, since the pipeline was implemented in clinical routine in two hospitals, 19 patients have been undergoing treatment utilizing our automated designs. CONCLUSION: The results demonstrate that our automated pipeline meets the high precision requirements of the medical setting employed in cleft lip and palate care while substantially reducing the design time and required clinical expertise, which could facilitate access to this presurgical treatment, especially in low-income countries.

Digital impressions from newborns to preschoolers with cleft lip and palate: A two-centers experience.

BACKGROUND: Documenting cleft lip and palate morphology prior to surgery is standard care. Presurgical orthopedic treatment also requires a 3D cleft model. Endangering the airway, conventional impressions require additional safety measures and resources. We investigate the implementation and risks of digital impressions for the youngest patients with orofacial clefts. METHODS: We report a retrospective cohort study of patients with cleft lip and palate, aged up to 6 years, treated at two cleft centers in Europe (Basel (A), Warsaw (B)). We scanned with the Medit i500 (Medit Corp, Seoul, South Korea). Center A for presurgical orthopedics and prior surgery from June 2020 to March 2022. Center B prior surgery from December 2020 to May 2021. Scanning data were analyzed for adverse events and adverse device effects, scanning duration, and number of images according to cleft type and age. RESULTS: We analyzed 342 digital impressions in 190 patients (center A: 71, B: 119). The median age was 8.7 months with a range from the first day of birth (presurgical orthopedics) to six years of life (early alveolar bone grafting). No adverse events or adverse device effects were observed. The median scan duration was 85.5 s for cleft palate and 50 s for cleft lip and nose (IQR 56 s and 39 s, respectively). CONCLUSION: Digital impressions with intraoral scanners are safe in patients with cleft lip and palate from newborn to preschool age. Given the funding to purchase an intraoral scanner, interfaces to electronic patient records, and point-of-care 3D printing, cleft centers can successfully implement this technology.

Craniofacial Growth at Age 6-11 Years after One-Stage Cleft Lip and Palate Repair: A Retrospective Comparative Study with Historical Controls.

BACKGROUND: Primary alveolar bone grafting inhibits craniofacial growth. However, its effect on craniofacial growth in one-stage cleft lip and palate protocols is unknown. This study investigated whether primary alveolar bone grafting performed during one-stage unilateral cleft lip and palate repair negatively affects growth up to 6-11 years old. METHODS: The craniofacial growth, dental arch relationship and palatal morphology at 6-11 years old in children with unilateral cleft lip and palate were compared retrospectively. Two cohorts after a one-stage protocol without (Group A) and with (Group B) primary bone grafting at the same center were compared. Further, cephalometric measurements for growth were compared with an external cohort of a one-stage protocol and a heathy control. RESULTS: Group A comprised 16 patients assessed at 6.8 years (SD 0.83), and Group B comprised 15 patients assessed at 9 years (SD 2.0). Cephalometric measurements indicated similar sagittal maxillary growth deficits and a significant deviation in maxillary inclination in both groups compared to the healthy group. Moderate to severe changes in palatal morphology were observed in 70% of the members in both groups. CONCLUSION: Omitting primary alveolar bone grafting under the one-stage protocol with two-flap palatoplasty studied did not improve growth at 6-11 years. The results implicate two-flap palatoplasty with secondary healing as having greater adverse effects on growth than primary alveolar bone grafting. Dental and palatal morphology was considerably compromised regardless of primary alveolar bone grafting.

Confined Mobility of TonB and FepA in Escherichia coli Membranes.

The important process of nutrient uptake in Escherichia coli, in many cases, involves transit of the nutrient through a class of beta-barrel proteins in the outer membrane known as TonB-dependent transporters (TBDTs) and requires interaction with the inner membrane protein TonB. Here we have imaged the mobility of the ferric enterobactin transporter FepA and TonB by tracking them in the membranes of live E. coli with single-molecule resolution at time-scales ranging from milliseconds to seconds. We employed simple simulations to model/analyze the lateral diffusion in the membranes of E.coli, to take into account both the highly curved geometry of the cell and artifactual effects expected due to finite exposure time imaging. We find that both molecules perform confined lateral diffusion in their respective membranes in the absence of ligand with FepA confined to a region [Formula: see text] µm in radius in the outer membrane and TonB confined to a region [Formula: see text] µm in radius in the inner membrane. The diffusion coefficient of these molecules on millisecond time-scales was estimated to be [Formula: see text] µm2/s and [Formula: see text] µm2/s for FepA and TonB, respectively, implying that each molecule is free to diffuse within its domain. Disruption of the inner membrane potential, deletion of ExbB/D from the inner membrane, presence of ligand or antibody to FepA and disruption of the MreB cytoskeleton was all found to further restrict the mobility of both molecules. Results are analyzed in terms of changes in confinement size and interactions between the two proteins.

Energy-dependent motion of TonB in the Gram-negative bacterial inner membrane.

Gram-negative bacteria acquire iron with TonB-dependent uptake systems. The TonB-ExbBD inner membrane complex is hypothesized to transfer energy to outer membrane (OM) iron transporters. Fluorescence microscopic characterization of green fluorescent protein (GFP)-TonB hybrid proteins revealed an unexpected, restricted localization of TonB in the cell envelope. Fluorescence polarization measurements demonstrated motion of TonB in living cells, which likely was rotation. By determining the anisotropy of GFP-TonB in the absence and presence of inhibitors, we saw the dependence of its motion on electrochemical force and on the actions of ExbBD. We observed higher anisotropy for GFP-TonB in energy-depleted cells and lower values in bacteria lacking ExbBD. However, the metabolic inhibitors did not change the anisotropy of GFP-TonB in ΔexbBD cells. These findings demonstrate that TonB undergoes energized motion in the bacterial cell envelope and that ExbBD couples this activity to the electrochemical gradient. The results portray TonB as an energized entity in a regular array underlying the OM bilayer, which promotes metal uptake through OM transporters by a rotational mechanism.

Single-molecule imaging in live bacteria cells.

Bacteria, such as Escherichia coli and Caulobacter crescentus, are the most studied and perhaps best-understood organisms in biology. The advances in understanding of living systems gained from these organisms are immense. Application of single-molecule techniques in bacteria have presented unique difficulties owing to their small size and highly curved form. The aim of this review is to show advances made in single-molecule imaging in bacteria over the past 10 years, and to look to the future where the combination of implementing such high-precision techniques in well-characterized and controllable model systems such as E. coli could lead to a greater understanding of fundamental biological questions inaccessible through classic ensemble methods.

Single-molecule study of molecular mobility in the cytoplasm of Escherichia coli.

The cytoplasm of bacterial cells is filled with individual molecules and molecular complexes that rely on diffusion to bring them together for interaction. The mobility of molecules in the cytoplasm has been characterized by several techniques mainly using fluorescent probes and ensemble methods. In order to probe the microenvrionment inside the cytoplasm as viewed by an individual molecule, we have studied single green fluorescent proteins (GFPs) diffusing in the cytoplasm of Escherichia coli cells at observation at rates ranging from 60 to 1000 Hz. Over long times the diffusion shows confinement due to the geometry of the cells themselves. A simulation in model cells using the actual distribution of cell sizes found in the experiments describes accurately the experimental results as well as reveals a short time diffusion coefficient that agrees well with that determined by ensemble methods. Higher short time diffusion coefficients can be obtained by filling the simulated cell with small spheres modeling cytoplasmic molecules and, depending on the density of particles included in the modeled cytoplasm, can approach the diffusion coefficient of GFPs found in water. Thus, single-molecule tracking combined with analysis using simple simulation of Brownian motion is able to reveal the main contributors to the GFP mobility in the cytoplasm of E. coli.

Mobility of BtuB and OmpF in the Escherichia coli outer membrane: implications for dynamic formation of a translocon complex.

Diffusion of two Escherichia coli outer membrane proteins-the cobalamin (vitamin B12) receptor (BtuB) and the OmpF porin, which are implicated in the cellular import pathways of colicins and phages-was measured in vivo. The lateral mobility of these proteins is relevant to the mechanism of formation of the translocon for cellular import of colicins such as the rRNase colicin E3. The diffusion coefficient (D) of BtuB, the primary colicin receptor, complexed to fluorescent antibody or colicin, is 0.05±0.01 µm2/s and 0.10±0.02 µm2/s, respectively, over a timescale of 25-150 ms. Mutagenesis of the BtuB TonB box, which eliminates or significantly weakens the interaction between BtuB and the TonB energy-transducing protein that is anchored in the cytoplasmic membrane, resulted in a fivefold larger value of D, 0.27±0.06 µm2/s for antibody-labeled BtuB, indicating a cytoskeletal-like interaction of TonB with BtuB. OmpF has a diffusion coefficient of 0.006±0.002 µm2/s, ∼10-fold smaller than that of BtuB, and is restricted within a domain of diameter 100 nm, showing it to be relatively immobile compared to BtuB. Thus, formation of the outer membrane translocon for cellular import of the nuclease colicins is a demonstrably dynamic process, because it depends on lateral diffusion of BtuB and collisional interaction with relatively immobile OmpF.

MacMARCKS interacts with the metabotropic glutamate receptor type 7 and modulates G protein-mediated constitutive inhibition of calcium channels.

We have previously shown that the interaction of Ca2+/calmodulin with the metabotropic glutamate receptor type 7 (mGluR7) promotes the G-protein-mediated inhibition of voltage-sensitive Ca2+ channels (VSCCs) seen upon agonist activation. Here, we performed a yeast two-hybrid screen of a new-born rat brain cDNA library using the cytoplasmic C-terminal tail of mGluR7 as bait and identified macrophage myristoylated alanine-rich c-kinase substrate (MacMARCKS) as a binding protein. The interaction was confirmed in vitro and in vivo by pull-down assays, immunoprecipitation, and colocalization of mGluR7 and MacMARCKS in transfected HEK293 cells and cultured cerebellar granule cells. Binding of MacMARCKS to mGluR7 was antagonized by Ca2+/calmodulin. In neurons, cotransfection of MacMARCKS with mGluR7, but not mGluR7 mutants unable to bind MacMARCKS, reduced the G-protein-mediated tonic inhibition of VSCCs in the absence of mGluR7 agonist. These results suggest that competitive interactions of Ca2+/calmodulin and MacMARCKS with mGluR7 control the tonic inhibition of VSCCs by G-proteins.

Single hepatitis-B virus core capsid binding to individual nuclear pore complexes in Hela cells.

We investigate the interaction of hepatitis B virus capsids lacking a nuclear localization signal with nuclear pore complexes (NPCs) in permeabilized HeLa cells. Confocal and wide-field optical images of the nuclear envelope show well-spaced individual NPCs. Specific interactions of capsids with single NPCs are characterized by extended residence times of capsids in the focal volume which are characterized by fluorescence correlation spectroscopy. In addition, single-capsid-tracking experiments using fast wide-field fluorescence microscopy at 50 frames/s allow us to directly observe specific binding via a dual-color colocalization of capsids and NPCs. We find that binding occurs with high probability on the nuclear-pore ring moiety, at 44 +/- 9 nm radial distance from the central axis.

Kinetics of the initial steps of G protein-coupled receptor-mediated cellular signaling revealed by single-molecule imaging.

We report on an in vivo single-molecule study of the signaling kinetics of G protein-coupled receptors (GPCR) performed using the neurokinin 1 receptor (NK1R) as a representative member. The NK1R signaling cascade is triggered by the specific binding of a fluorescently labeled agonist, substance P (SP). The diffusion of single receptor-ligand complexes in plasma membrane of living HEK 293 cells is imaged using fast single-molecule wide-field fluorescence microscopy at 100 ms time resolution. Diffusion trajectories are obtained which show intra- and intertrace heterogeneity in the diffusion mode. To investigate universal patterns in the diffusion trajectories we take the ligand-binding event as the common starting point. This synchronization allows us to observe changes in the character of the ligand-receptor-complex diffusion. Specifically, we find that the diffusion of ligand-receptor complexes is slowed down significantly and becomes more constrained as a function of time during the first 1000 ms. The decelerated and more constrained diffusion is attributed to an increasing interaction of the GPCR with cellular structures after the ligand-receptor complex is formed.