We correlate spatially resolved fluorescence (-lifetime) measurements with X-ray nanodiffraction to reveal surface defects in supercrystals of self-assembled cesium lead halide perovskite nanocrystals and study their effect on the fluorescence properties. Upon comparison with density functional modeling, we show that a loss in structural coherence, an increasing atomic misalignment between adjacent nanocrystals, and growing compressive strain near the surface of the supercrystal are responsible for the observed fluorescence blueshift and decreased fluorescence lifetimes. Such surface defect-related optical properties extend the frequently assumed analogy between atoms and nanocrystals as so-called quasi-atoms. Our results emphasize the importance of minimizing strain during the self-assembly of perovskite nanocrystals into supercrystals for lighting application such as superfluorescent emitters. 3  perovskite superlattices.By utilizing spatially resolved fluorescence (-lifetime) measurements and high precision X-ray nanodiffraction, the authors correlate the influence of structural misalignment and fluorescence (-lifetime) properties of all-inorganic CsPbX The individual NCs, which are the building blocks of a supercrystal, are often regarded as “artificial atoms”, and hence analogies between atomic crystals and such supercrystals have been made3. NC supercrystals are susceptible to doping4, and they can exhibit exceptional mechanical properties5, quasicrystal formation2, enhanced electronic coupling6, and engineered phonon modes7. In view of the recent progress in exploiting the massive structural coherence in NC supercrystals to generate collective optoelectronic properties10, a critical question remains whether this artificial atom analogy can be extended towards the optical properties of NC supercrystals. Due to surface dangling bonds and surface reconstruction, even the purest and most carefully prepared atomic crystals are not structurally perfect12. For atomic crystals, such surface defects strongly affect the fluorescence spectra, lifetime, and quantum yield17. For supercrystals, this is much less understood.Advances in the self-assembly of colloidal nanocrystals (NCs) from solution into three-dimensional arrays with long-range order have enabled the design of microscopic “supercrystals” that approach the structural precision of atomic single crystals19, CsPbBr2Cl and CsPbBr3 NC supercrystals exhibit structural distortions near their surfaces which significantly alter their fluorescence properties. This finding is of high relevance for the application of these materials as tunable, bright emitters with superfluorescent behavior10. Superfluorescence is a key property for the design of spectrally ultra-pure laser sources20 or highly efficient light-harvesting systems21. Recent quantum chemical simulations have suggested that structural disorder in CsPbBr3 supercrystals and its effect on the thermal decoherence plays a pivotal role in the efficiency of the superfluorescence22. Previous structural investigations of ensembles of CsPbBr3 supercrystals by grazing-incidence small angle X-ray scattering (SAXS) indicated a primitive unit cell with slight tetragonal distortion23, and wide-angle X-ray scattering (WAXS) showed a high degree of structural coherence24. Electron microscopy of individual supercrystals revealed a frequent occurrence of local defects in the supercrystals, such as isolated NC vacancies25. Confocal fluorescence microscopy of individual CsPbBr3 supercrystals displayed spatial variations in the fluorescence peak wavelength and intensity, indicating that local structural inhomogeneities may substantially affect the fluorescence properties of the entire supercrystal26. Our approach is based on simultaneous WAXS and SAXS measurements with a nano-focused beam to probe the structural defects and crystallographic orientation of the supercrystal and the constituting NCs on a local level with dimensions of ~3 µm and 7–9 nm, respectively29. By correlation with diffraction-limited confocal fluorescence microscopy and modeling with density functional theory (DFT) we present proof that compressive strain, a loss of structural coherence and an increasing atomic misalignment between adjacent nanocrystals at the edges of CsPbBr2Cl NC supercrystals are responsible for a blueshifted emission and decrease of the fluorescence lifetimes.In this work, we show that in close analogy to atomic crystals2Cl and CsPbBr3 NC supercrystals on glass substrates (see “Methods” for details on synthesis and self-assembly of NCs). Spatially resolved photoluminescence spectra of the NC supercrystals under 405 nm excitation in a confocal laser scanning microscope with a step size of 250 nm and 100 nm, respectively, are shown in Fig. 2Cl. We observe the same blueshifting behavior for supercrystals composed of CsPbBr3 NCs, although to a lesser extent (up to 12 meV).We study self-assembled CsPbBr2Cl and CsPbBr3 supercrystals measured on glass substrates with a lateral resolution of 200 nm under 405 nm excitation. For both supercrystal compositions, we obtain good fits of the experimental time-resolved fluorescence by pixel-by-pixel monoexponential reconvolution using an instrument response function acquired on a clean glass coverslip  around 2.1 ns in the center which decrease by approximately 20% when scanning from the center of a supercrystal towards its edges. Supercrystals composed of CsPbBr3 NCs exhibit typical lifetime values around 1.5 ns in the center, which shorten by approximately 30% when approaching the edges. We note that this holds true only for freshly prepared NC supercrystals. After several days of exposure to air, the trend in the spatially resolved τ-values is reversed in that such aged supercrystals exhibit longer lifetimes at the edges. However, the overall blueshift of the fluorescence peak wavelength towards the edges is preserved.In Fig. 2 X-ray beam, we perform a spatially resolved scan of a typical CsPbBr2Cl NC supercrystal on a Kapton substrate. While the results presented here are for one typical supercrystal, examples of more supercrystals are provided in the Supplementary information   and lattice distortion (g) using the Williamson–Hall method with d = 6.8 ± 0.1 nm and g = 2.3 ± 0.1% . All crystallographic axes of the NCs are aligned with the corresponding axes of the supercrystal (e.g. [100]AL||[100]SC and [010]AL||[010]SC), which is consistent with ref. 25.The SAXS pattern in Fig. a1 and a2, the angle γ between them, and the average azimuthal position φ, which are defined in Fig. a1 and a2 decrease at the edges , we observe that this lattice contraction is anisotropic. The ratio of the in-plane unit cell parameters a2/a1 differs from unity by ±20% in such a way that the NC spacing in the directions along the nearest supercrystal boundary is smaller than normal to it, as shown in Fig. a〉 = 9.4 ± 0.7 nm is slightly smaller than the unit cell parameters extracted from the average diffraction pattern. We attribute this to the low intensity of scattering from the supercrystal edges, which reduces their contribution to the average pattern. We do not observe a clear trend in the size of the SAXS Bragg peaks . The observed peak sizes are much larger and vary in the range from 0.05 nm−1 to 0.2 nm−1 and, as such, they depend mainly on the superlattice distortion. The characteristic length scale on which this distortion evolves is, most probably, smaller than the incident beam. Thus, the areas with different lattice parameters simultaneously illuminated by the incident beam lead to the peak broadening.Analyzing individual SAXS patterns from different locations on the supercrystal, we find substantial local deviations from the average structure , we find a difference in the radial width of the Bragg peaks at different locations. By the Williamson–Hall method, we extract the lattice distortion gq  at each spatial point (see “Methods” section for details). We find a clear trend of increasing atomic lattice distortion towards the edges of the supercrystal with a maximum of 2% at the edge, while it is about 1% at a distance 3 µm into the center, as shown in Fig. Despite the fact that the atomic lattice parameter δψ  is likely to have a similar effect on nearest-neighbor coupling as a reduced number of nearest neighbors. Second, we anticipate that the shorter interparticle spacing  of the NC. While axial stress applied to the CsCl-terminated surface of the CsPbBr2Cl particle results in a steady increase of the optical gap consistent with the experiment, similar stress on the CsBr-terminated surfaces of both particles are found to both increase or decrease Egap, depending on the magnitude of the applied stress.To rationalize the experimental trend of increased fluorescence energies at the edges of the supercrystal as compared to its center, we carry out density functional modeling of the system. We consider three individual contributions in this regard. First, we recognize that the number of nearest neighbors at the surface of the supercrystal is lower than that in the center, leading to stronger exciton confinement and hence increased fluorescence energies at the edges. Indeed, our DFT calculations confirm this trend in Fig. δψ Fig. . A largeing Fig.  should fOverall, our computational modeling suggests that the spectral blueshift of the fluorescence from the edges of the supercrystal can be caused mainly due to a reduced NC coordination number at the edges as well as the compressive atomic lattice strain in some cases, knowing that the third factor—the shorter interparticle distance—works in the opposite direction, facilitating electronic coupling between adjacent nanocrystals and decreasing the optical gap. However, since the experimentally measured spectral shift is seemingly a combination of all three effects discussed above, a fully quantitative prediction would require more detailed knowledge on their relative contributions as well as the relative orientation and positions of individual nanocrystals, which are currently not available.34. We hold such a strain responsible for the observed compression of the unit cell parameter by over 20% of the CsPbBr2Cl NC supercrystals in Fig. 12. The accompanying loss in the angular correlation of the constituting NCs with the superlattice fits a scenario where strain in the supercrystal is partially relieved by forming local structural defects. The comparison of the average 3 NC supercrystals with a similar fluorescence blueshift between the center and the edge26. As a main conclusion, gradual release of I2 gas under intense laser illumination led to the blueshift since lead bromide perovskites exhibit a larger bandgap than the corresponding lead iodide perovskites. The authors argued that the I2 loss commenced from the edges towards the center, which would explain the spatial fluorescence variations. In CsPbBr2Cl however, this mechanism is not easily applicable since the reduction potential of Br– is much lower than that of I–. In line with this, CsPbBr3 NC supercrystals without a halide mixture show a similar blueshift, indicating that a change in the mixed halide composition is not required to observe the effects reported here.We note a previous report on the spatially resolved fluorescence of CsPb in the supercrystals, we also consider a photon propagation effect, that is, multiple emission and (re-)absorption events, which become more likely with increasing thickness of the emitter material17. The structure-fluorescence correlations in supercrystals revealed here are thus another example for the analogy between atoms and NCs as so-called quasi-atoms.In conclusion, supercrystals of lead halide perovskite NCs self-assembled from solution exhibit a loss in structural coherence, an increasing atomic misalignment between adjacent NCs, and compressive strain near their surfaces. These structural distortions are strongly correlated with a blueshifted fluorescence and decreased radiative lifetimes. We note that structural distortion and surface defects have been shown to strongly affect the fluorescence properties in atomic crystals, such as transition metal dichalcogenides2CO3), 99.99% , Acros Organics; Lead(II)chloride (PbCl2), 99.999% , Sigma Aldrich; Lead(II)bromide (PbBr2), ≥98%, Sigma Aldrich; Toluene, 99.8%, extra dry, AcroSeal, Acros; Tetrachloroethylene (TCE), ≥99%, Acros Organics; Kapton® polyimide membranes (125 μm thickness) were purchased from DuPont; Si/SiOx wafers (200 nm SiOx thickness) were purchased from Siegert Wafer GmbH. All chemicals were used as purchased.1-Octadecene (ODE), technical grade, 90%, Sigma Aldrich; Oleic acid (OA), 97%, Acros Organics; Oleylamine (OAm), 80–90%, Acros Organics; Cesium carbonate (Cs2CO3 (0.625 mmol) was loaded into a 25 mL three-neck flask along with 10 mL 1-octadecene and 0.625 mL oleic acid, dried for 1 h at 120 °C and then heated to 150 °C under nitrogen atmosphere until all Cs2CO3 reacted with oleic acid. The mixture was kept in a glovebox and heated to 110 °C before injection.203.5 mg Cs3 NCs were made by a hot-Injection synthesis using a modified literature method46. To synthesize 9 nm CsPbBr3 or 7 nm CsPbBr2Cl NCs, 138 mg (0.38 mmol) PbBr2 or 92 mg (0.25 mmol) PbBr2 and 35 mg (0.125 mmol) PbCl2 were degassed in 10 mL ODE in a 25 mL three-neck flask under reduced pressure at 120 °C for 2 h. Then, 1 mL of dried oleylamine (OAm) and 0.5 mL of dried oleic acid (OA) were injected at 120 °C under nitrogen atmosphere with continuous stirring and the reaction mixture was heated to 160 °C. After the solubilization was completed, 0.8 mL of a previously prepared solution of Cs-oleate in ODE (0.125 M) was swiftly injected, and the reaction mixture was cooled to room temperature using an ice-bath.CsPbX3 NCs were collected by centrifuging the suspension , decanting the supernatant, and collecting the precipitate. The precipitate was centrifuged again without addition of a solvent , and the resulting supernatant was removed with a syringe, to separate the traces of residual supernatant. The precipitate was dissolved in 2 mL hexane and centrifuged again  to remove aggregates and larger particles. The resulting supernatant was filtered through a 0.2 µm PTFE syringe filter and stored as stock solution inside of a glovebox with a typically concentration of 16 mM following Maes et al.47.CsPbXFor the growth of supercrystals, different substrates  were used, depending on the desired experiment. The self-assembly experiment was set up in a glass Petri dish (with a 60 mm diameter), for this purpose three substrates each were placed in such a Petri dish together with a PTFE-lid filled with 1 mL tetrachloroethylene. To each of these substrates, 40 µL of a 1–3 mM solution of the perovskites in TCE was added. The lid of the Petri dish was closed, covered with aluminum foil, and allowed to stand for 24 h. After that, the lid was opened and left for another 5 h to dry completely. All self-assembly preparations were performed under inert atmosphere. The more monodisperse the size distribution of the perovskites, the better the resulting superlatticesAll spatially resolved optical measurements were performed using a home-built inverted confocal laser scanning microscope. The measurements were performed on glass substrates utilizing a high numerical aperture oil immersion objective (NA = 1.4) and a 405 nm pulsed diode laser (Picoquant LDH P-C-405) with variable repetition rates (Picoquant PDL 800-D laser driver) as the excitation source. Under these conditions the lateral resolution of the instrument is approximately 200 nm. A single photon avalanche diode (MPD PDM Series) was used in conjunction with the Picoquant HydraHarp 400 as a time-correlated single photon counting system to detect time-resolved fluorescence. Time-resolved data acquisition and analysis was performed using Picoquants SymPhoTime 64 software package. The spectral data was recorded using an Acton Spectra Pro 2300i spectrometer with a 300 grooves/mm grating. The detector temperature (Princeton PIXIS CCD) was kept steady at −45 °C.λ = 0.0898 nm (E = 13.8 keV) was focused down to a spot size of approximately 400 × 400 nm2 (FWHM) with a focal depth of about 0.5 mm at the GINIX nanodiffraction endstation48. The two-dimensional detector EIGER X 4 M (Dectris) with 2070 × 2167 pixels and a pixel size of 75 × 75 μm2 was positioned 412 mm downstream from the sample. The detector was aligned ~6 cm off-center in both directions normal to the incident beam to allow simultaneous detection of SAXS and WAXS. We performed a spatially resolved scan of the sample on a Kapton substrate by 25 × 25 spatial points with 500 nm step size and collected 625 diffraction patterns in transmission geometry. The exposure time was set to 0.5 s to prevent radiation damage of the sample. The background scattering pattern from a pure Kapton film was subtracted from every collected pattern.The nanodiffraction experiment was performed at the Coherence Applications beamline P10 of the PETRA III synchrotron source at DESY. An X-ray beam with the wavelength I0 is the integrated intensity, q0 and φ0 are the radial and azimuthal central positions, and σq and σφ are the corresponding root mean square (rms) values. The FWHMs of the Bragg peaks were evaluated according to relations: Each diffraction pattern was interpolated onto a polar coordinate grid with the origin at the direct beam position. The radial profiles were obtained by averaging along the azimuthal coordinate. To extract parameters of the WAXS and SAXS Bragg peaks separately, we fitted each of them by the 2D Gaussian functiona1 and a2, the angle γ between them and the average azimuthal position φ counted counterclockwise from an arbitrary horizontal axis .For the SAXS peaks, the parameters were pairwise averaged for the corresponding Friedel pairs of the Bragg peaks to improve statistics. The resulting momentum transfer values and angles were used to calculate the real-space parameters of the unit cell: the length of the basis vectors IAL and the azimuthal position ψ of the [010]AL axis. To obtain the average azimuthal position ψ, we averaged all four azimuthal positions for 010AL, 020AL, 110AL, and AL Bragg peaks, but corrected the last two values by +45° and −45°, respectively. We used the Williamson–Hall method49 to analyze the size of the WAXS Bragg peaks at each spatial point of the supercrystal. The FWHM of the Bragg peak is determined by the NC size and the lattice distortion as follows:wq,φ is the FWHM of the Bragg peak at q in radial or azimuthal direction, respectively, L is the NC size, gq,φ is the radial or angular lattice distortion of the atomic lattice, respectively, K is a dimensionless shape parameter that is about 0.86 for cubic NCs50. The radial lattice distortion gq calculated from the radial FWHM wq is equal to the ratio δaAL/aAL, where δaAL is the FWHM of the unit cell parameter distribution around the mean value aAL. The angular lattice distortion gφ calculated from the azimuthal FWHM wφ is equal to the FWHM δψ of the angular distribution of the NCs around their average azimuthal position ψ. For the spatially resolved analysis of the FWHMs, the NC size L was fixed at the value, obtained from the average radial profiles. For details of the analysis, see Supplementary materials.For the WAXS peaks, we calculated an average Bragg peak intensity x devices was performed with a HITACHI model SU8030 at 30 kV. To estimate the thickness of micro-crystals, samples were titled by 45° with respect to the incoming electron beam. AFM investigations were conducted with a Bruker MultiMode 8 HR in contact mode.SEM imaging of supercrystals on Si/SiO51. The PBE exchange correlation functional52, a dual basis of localized Gaussians and plane waves (GPW)53 with a 350 Ry plane-wave cutoff, double-ζ basis-set augmented with polarization functions (MOLOPT variant)54, and GTH pseudopotentials55 for core electrons are used for all calculations. The van der Waals (VDW) interaction was accounted for by employing Grimme’s DFT-D3 method56. SCF convergence criterion was set at 10−6 for all calculations.All computations are performed using the CP2K 5.1 program suite using the Quickstep module3  nanocrystals were obtained by cutting small cubes (~2.4 nm) from the bulk, exposing the CsX layer at the surface and maintaining overall charge neutrality of the particle57. All structures were then optimized in vacuum using the BFGS optimizer imposing non-periodic boundary conditions with a wavelet Poisson solver58, setting a maximum force of 5 meV·Å−1 (10–4 hartree/bohr) as convergence criteria. For the obtained cartesian coordinates, see Supplementary Data a × b × c, and stress is to be applied along the z-direction, “c” is fixed at some cʹ by constraining the z coordinates of both the top and bottom surface-atoms along the z-direction, with all other coordinates of all atoms relaxed. % Strain is reported as (cʹ − c)/c × 100%. For calculations involving dimers and tetramers, 2/4 monomers were explicitly considered, but periodic boundary condition was imposed with at least 10 Å vacuum above the surface of the nanocluster to avoid spurious interaction with its periodic image.Initial geometries of CsPbXSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1


Under normal conditions, the most significant expansion and differentiation of the adult mammary gland occurs in response to systemic reproductive hormones during pregnancy and lactation to enable milk synthesis and secretion to sustain the offspring. However, human mammary tissue remodelling that takes place during pregnancy and lactation remains poorly understood due to the challenge of acquiring samples. We report here single-cell transcriptomic analysis of 110,744 viable breast cells isolated from human milk or non-lactating breast tissue, isolated from nine and seven donors, respectively. We found that human milk largely contains epithelial cells belonging to the luminal lineage and a repertoire of immune cells. Further transcriptomic analysis of the milk cells identified two distinct secretory cell types that shared similarities with luminal progenitors, but no populations comparable to hormone-responsive cells. Taken together, our data offers a reference map and a window into the cellular dynamics that occur during human lactation and may provide further insights on the interplay between pregnancy, lactation and breast cancer. Human mammary tissue remodelling that takes place during pregnancy and lactation remains poorly understood. Here the authors characterize cells in human milk, identifying epithelial cells resembling luminal progenitors and immune cells, contributing insights into this process. Major changes in the architecture and cellular composition of the adult mammary gland are required for the synthesis and secretion of the complex bioactive fluid that is human milk2. Findings in murine models suggest that these changes have a lasting impact on the mammary epithelium at an epigenetic level3 and lead to the generation of parity-induced cell types5. These molecular and cellular changes occurring in the mammary gland may point toward a mechanism explaining the reduced long-term breast cancer risk associated with parity6 and extended periods of lactation7.The mammary gland undergoes cycles of tissue remodelling throughout a woman’s reproductive lifespan that are particularly pronounced during pregnancy, lactation and the return of the mammary gland to its resting state post involution. Determining the differentiation dynamics driving these developmental stages is not only essential to understanding normal mammary gland function but also the origins of breast cancer. The mammary gland consists of a bilayered ductal tree with an inner layer of luminal cells (LCs) that mature into secretory cells during lactation, and a basal network of contractile myoepithelial (MY) cells that support the transport of milk to the nipple during lactation. In the human mammary gland, this ductal tree is embedded in collagen-rich, specialized stroma containing different types of mesenchymal and immune (IM) cells9. Interestingly, findings from Bach et al. determined that the post-parous mammary gland contained primed parity-induced LP cells that upregulated lactation-associated genes5. In addition, recent findings have shown that ageing impacts the composition of the murine mammary gland with the rise of an age-dependent LC subpopulation co-expressing hormone-sensing and secretory-alveolar lineage markers10. These findings are of particular interest given that LP cells have been proposed as the cell of origin for different breast cancer subtypes11. Analogous to the murine mammary gland, an emerging number of studies have begun to characterize human mammary subpopulations using single-cell transcriptomics16. Normal mammary tissue is usually derived from aesthetic breast reductions from non-lactating women. Thus, compared to its resting state, tissue from lactating human mammary glands is difficult to obtain.Single-cell transcriptomic profiling of murine mammary epithelial cells has shed light on the differentiation dynamics of mammary epithelial cells. These studies described luminal progenitor (LP) cells in the virgin gland that gives rise to hormone-responsive (HR) mature LCs and, in the case of pregnancy, to secretory alveolar cellsWe report here a single-cell transcriptomic analysis of 110,744 viable breast cells isolated from human milk or non-lactating breast tissue, isolated from 9 and 7 donors, respectively. Our data set that comprised of 56,030 lactation-derived mammary cells (LMCs) or 54,714 non-lactation-derived mammary cells (NMCs) enable us to analyse and compare breast cells between these two states. We found that human milk largely contains epithelial cells belonging to the luminal lineage as well as a sophisticated repertoire of IM cells. Further transcriptomic analysis of the milk cells identified two distinct secretory cell types that share similarities with hormone receptor-negative LPs. Taken together, our data offer a reference map and a window into the cellular dynamics that occur during human lactation and may provide further insights into the interplay between pregnancy, lactation and breast cancer.17. While the profiles of NMCs fit with previously identified populations11, consisting of a CD45−/EpCAM−/CD49f+ basal MY, a CD45−/EpCAM+/CD49f+ LP and a CD45−/EpCAM+/CD49f− mature luminal subpopulation , keratin 17 (KRT17), metallopeptidase CD10 (MME), together with contractility genes encoding for alpha-smooth muscle actin (ACTA2), transgelin (TAGLN), myosin light chain kinase (MYLK) and tropomyosin (TPM2) demarked a single cluster as containing MY cells. The two remaining NMC clusters expressed key luminal markers encoding for keratin 18 (KRT18) and EPCAM (EPCAM). Upon closer examination, one cluster resembled the HR cluster previously described13 which expressed genes encoding for hormone receptors for oestrogen, progesterone and prolactin , in agreement with previous human mammary scRNA-seq studieslls Fig. . We founKRT18 and EPCAM), major human milk protein genes  and other secretory genes integral to milk fat secretion such as xanthine hydrogenase (XDH), CD36 (CD36) and mucin-1 (MUC1)19  from all (including nulliparous) donors Fig. . Due to −8) differentially expressed, where 1640 genes were found to be higher in LC1 and 1782 genes were found to be higher in LC2 , was significantly upregulated in LC2 cells X10 Fig.  enriched15, we identified GJPA4+  vascular accessory (VA) cells, PECAM1+ (encoding CD31) endothelial (EN) cells and DCN+/LUM+/COL1A1+ fibroblasts (FB) within all NMC samples  IM cluster. To better determine the different subtypes of IM cells isolated from LMC and NMC samples, we performed sub-clustering analysis on the cells and annotated them according to the expression of canonical IM subpopulation markers28. Thus, we identified 12 subclusters consisting of either myeloid or lymphocytic lineage hematopoietic cells from NMC or LMC samples  T cells or B cells (CD79A+/MS4A1+), with a small subset of JCHAIN+ plasma B cells consisting of both NMC and LMC samples  and osteopontin (SPP1) signalling pathways , LPs (n = 169 genes), MY (n = 128) or ST cells (n = 384) (Supplementary Dataset 9). However, delineating exact differentiation pathways would require sampling mammary cells as they were differentiating (i.e. taken during pregnancy).One major question arising from our data relates to the cell of origin of the milk-derived LCs. Which luminal population in the non-lactating, resting gland do they resemble most and likely arise from? To address this, we examined non-lactating cell signaturesLCs n = 18, LPs  using differential gene expression analysis. Thus, 1146 genes were found to be significantly upregulated in LC clusters and 922 genes were significantly upregulated in LP cells  that were stratified based on the PAM50 molecular subtype classification34. As has been observed previously, the LP score was found to be highest in the “basal”-like tumour samples , entering either the lumen of the alveoli or ducts and being transported through the nipple to the breastfeeding infant. Considering the cellular organization of the mammary gland2, it is therefore conceivable that cell subpopulations that reside distal to these sites (such as FBs or MY cells) do not enter the milk, compared to the more proximally localized LCs. Therefore, cells found in milk are unlikely to represent the full complement of populations in the lactating breast. Direct analysis of cells from human lactating breast tissue would be required to delineate further lactation-induced changes; however, obtaining such samples pose an ethical and logistical challenge.Curiously, we identified two secretory LC populations in our human samples (LC1 and LC2), unlike the single population that has been previously identified in mammary studies conducted in mice28. Even though our NMC samples were taken from donors with a range of parities and ages, our analysis did not reveal major shifts in mammary cell populations influenced by these factors. Current efforts of the human breast cell atlas on very large numbers of genetically diverse participants will have more power to address questions around how age and parity affect the mammary cell composition39. Nonetheless, comparing NMCs to LMCs provided an insight into the potential epithelial differentiation trajectories that occur over mammary maturation. Comparisons between the LC LMCs and LP NMCs identified DEGs that provide clues into the maturation pathways modified during lactation, as well as potential genes involved in milk component biosynthesis pathways. These pathways may be further explored in vitro using mammary organoids and may be used to determine therapeutic targets for women with breastfeeding difficulties.The cell clusters identified in our NMC samples were in concordance with those reported in the previous studiesBRCA1 mouse model40, suggesting a hijacking of normal mammary cell programming during cancer, which, if better understood, maybe leveraged as an early indicator of future breast cancer development. Therefore, cells in the milk provide an opportunity to explore this phenomenon further and could aid in the development of a pre-clinical tool for early detection of disease. Together, our study demonstrates the power of comparing mammary cells isolated from different stages of human mammary gland maturation and illustrates the luminal lineage remodelling that occurs during lactation.Finally, while mammary cells isolated from the milk do not directly contribute to breast cancer formation, they provide an opportunity to easily obtain and study human breast cells. These cells could also allow us to better understand changes in the functional mammary gland over the course of lactation or as a result of multiple parities, if sampled longitudinally or over consecutive lactations. Furthermore, we have recently identified aberrant differentiation of LPs towards the milk secretory lineage as one of the earliest steps of tumorigenesis in a Our research complies with all relevant ethical regulations as detailed in the below sections.41, however, in this case using a fast tissue digestion protocol . Briefly, the freshly collected mammary tissue was collected and minced using scalpels into <2–3 mm3 pieces. Twenty millilitres of minced tissue in digestion buffer (Dulbecco’s Modified Eagle Medium (DMEM)/F12 w/o phenol red, 2% w/v bovine serum albumin (BSA), 10 mM HEPES, 2 mM glutamine, 100 U/mL penicillin-streptomycin) supplemented with 1 µg/mL insulin  was added together with 800 U/mL collagenase  and 100 U/mL hyaluronidase  and made up to the 25 mL mark. Falcon tubes were then sealed with parafilm to ensure they were airtight and mixed at 100 r.p.m. at 37 °C for 3–4 h. Following digestion, the resulting dissociated organoid fragments were washed twice in washing buffer  and pelleted by 300 × g centrifugation for 5 min. Once the organoids were isolated, they were cryopreserved in 50% washing buffer, 40% foetal calf serum (FCS) and 10% dimethyl sulfoxide and stored in liquid nitrogen until required. When required, organoid fragments were gently defrosted in a 37 °C water bath for approximately 5 min before being treated with trypsin and dispase (Life Technology) to yield a highly viable single-cell suspension.Non-lactating human breast tissue was donated by participants who provided informed consent who were undergoing elective aesthetic reduction mammoplasty at the Nymphenburg Clinic for Plastic and Aesthetic Surgery in accordance with the regulations of the ethics committee of the Ludwig-Maximilian University, Munich, Germany . Limited demographic information on the participants was provided that included the age and parity of the participant . The pellet was washed by removing the supernatant and resuspending in 5–10 mL of cold PBS before transferring the sample to a new 15 mL tube  and centrifuging at 490 × g for 5 min at 4 °C. Following a second washing step, 100–550 µL of mammary epithelial cell growth medium (MECGM)  was added to the human milk cell aggregations according to the pellet size. Overall samples sequenced in this study ranged from 38 to 102.5 mL and yielded between 0.82 × 106 and 66.96 × 106 total membrane-enclosed structures (including cells) from milk . To examine the cells more closely, nuclear stain DRAQ5TM  and Nile red  were added to a final concentration of 0.4 µg/mL (1 mM) and 0.1 µg/mL, respectively, and the cells incubated for a further 5 min in the dark. Cells were then loaded onto a Neubauer Improved counting chamber and examined on an immunofluorescence microscope. Subsequently, single cells were either frozen or used immediately.Human milk donors were recruited from Pippagina English Prenatal and Postnatal classes or through the Helmholtz Zentrum München in accordance with regulations of the ethics committee of the Ludwig-Maximilian University, Munich, Germany . Participants provided written informed consent and filled out a detailed questionnaire to provide demographic information. Briefly, human milk was freshly collected using either a provided double electric breast pump  or participants personal pump, under aseptic conditions. Milk collections were obtained either within the lactation room at the Helmholtz Centre Munich, at the participants' homes or post-partum educational classes depending on the preferences of the participant. Fresh milk samples were immediately transported on ice to the laboratory and processed as soon as possible (<2 h after collection). Human milk cells were isolated by diluting milk samples in an equal volume of sterile phosphate-buffered saline (PBS)  and centrifuged at 870 × 44. Briefly, single cells were mixed with MECGM supplemented with 1% pen/strep (Invitrogen), 0.5% FCS (Pan Biotech), 3 μM Y-27632 (Biomol) and 10 μM forskolin (Biomol) and seeded onto polystyrene cell culture plates for 2D culture. After an establishment period of 5 days, the medium was changed to MECGM supplemented with 1% pen/strep and 10 μM forskolin. Where samples were cultured for immunofluorescence analysis, cells were seeded on 1% gelatine coated coverslips. Colonies of 2D-cultured cells were fixed using 4% paraformaldehyde and washed before staining the cells with a 1:100 primary CK8/18 antibody  in blocking buffer  with 10% normal donkey serum ) solution for 3 h at room temperature. Following washing, a 1:250 secondary antibody in  PBS 0.1% BSA solution was added and samples were incubated for a further 45 min at room temperature. After antibody incubation, 500 μL of a 0.4 μg/mL DAPI solution  were added and incubated for 2 min, followed by mounting of the slides. Images were acquired using a Zeiss “Axio Imager.M2” microscope in combination with the “Zeiss Zen 2.3 pro” software. For 3D culture, single cells were mixed with neutralizing solution and acidified rat tail collagen I (Corning) to generate collagen gels in siloxane coated 24-well plates. After allowing the gels to polymerize for an hour, supplemented media (as above) was added on top of the gels that were then gently encircled to generate floating collagen gels. Similar to 2D culture, after an establishment period of 5 days, the media on the floating collagen gels was changed to MECGM supplemented with pen/strep and forskolin only.Both NMC and LMCs were cultured in 2D and 3D using previously described methodsTM (as above) to a final concentration 1 µM. Flow cytometry was performed using a FACSAriaTM III cell sorter  with a 100 µm nozzle in combination with the FACSDivaTM 6.0 Software. Alternatively, flow cytometry was performed on LMCs to examine FR expression in live, nucleated epithelial cells. LMCs were prepared by staining the cells with CD45-PB  and anti-human FRs α and β (FR-αβ)-PE  for 45 min in the dark on ice. Subsequently, the samples were stained for DAPI  and DRAQ5 (same as above) for 10 min prior to analysis. In this case, flow cytometry was performed on a BD LSRFortessa machine  in combination with the FACSDivaTM 9.0 Software. In both cases, small volumes of cells from each sample were mixed prior to staining and used as comparisons and single stain controls. Both machines were used with a 100 µm nozzle. Laser settings were adjusted using unstained and single stain controls. Obtained data were analysed using the FlowJo_V10 Software .Flow cytometry was employed to determine the similarity of expression of mammary markers between LMC and NMCs. Cells were stained with CD45-PB , EpCAM-FITC  and CD49f-PE . After a 45-min incubation, stained cells were diluted in MECGM and filtered through 35 µm cell strainer caps of round-bottom tubes . Ten minutes prior to analysis, cells were further stained with DRAQ5n = 3) and parous (n = 4) females aged 19–65 years, as well as LMCs from uniparous or multiparous  females aged 27–44 years, with lactation stages ranging from 2 to 12 months . All lanes per sample were processed using the “cell ranger count” function. The output from different lanes was then aggregated using “cellranger aggr” with -normalise set to “none”. Batch 3 was prepared in a similar fashion, however, using version 2 chemistry, sequenced on an Illumina HiSeq 4000 and read processing was done using Cell Ranger Single-Cell Suite version 2.1.1.We examined cells that were prepared on three separate 10× genomics chips. Batch 1 contained four freshly dissociated NMCs (NMC1–4) and four samples of freshly collected and isolated LMCs (LMC1–4). Batch 2 contained three samples of NMCs (NMC5–7) and five viably frozen samples of LMCs (LMC5–8), including one sample that had been sequenced in the previous batch (LMC2B). Finally, batch 3 contained a new freshly collected and isolated LMC donor (LMC9) and a repeat sample of a freshly dissociated NMC donor (NMC1B), to examine batch effects  correction using the “fastMNN” function from the batchelor package. After performing quality control, combining, normalizing and performing MNN correction, we retained a total of high quality 54,714 NMCs and 56,030 LMCs  and Louvain clustering using scran47 was performed on the batch corrected data  or bottom 5/10% of the negative (downregulated) genes had gene set enrichment analysis performed on them using the “weight01” algorithm and “fisher” statistic using “runTest” in the “getSigGroups” function from topGO package. “GenTable” was used to generate a table with the top 50 biological process GO terms. Plots of selected GO terms were generated using ggplot2, plotting the resulting p value from the classic Fisher test and gene ratio, which is the number of significant genes for the term divided by the total number of significant genes used in the gene enrichment test.DEGs were identified between subsetted groups by first generating pseudobulk samples using “aggregateAcrossCells” function in the CellChat package version 1.1.0. First, LMC-derived cells were subsetted from the total cells and annotated according to their cell type: “LC1”, “LC2”, “Tcell”, “Mono”(cytes), “Macro”(phages), “Bcell_plasma” or “Myeloid” cells. A CellChat object was made using the “createCellChat” function. After annotating the object with relevant labels and identifying overexpressed genes, the communication probability was inferred using “computeCommunProb” function. Cell–cell communications for each cell signalling pathway were generated with “computeCommunProbPathway” function. Graphs were generated using the “netVisual_chord_gene” function.CellChat analysis was performed using the R 31 from previously published data11 from LP, mature luminal, MY and ST cells were investigated in our data using the “AddModuleScore” function from the Seurat package48 version 4.0.2. For each test, the overall expression of the genes/features from each signature was calculated after subtracting 20 randomly selected genes (from the same bin as the signature features) as a control feature per cell. The resulting signature score is unitless but is indicative of signature enrichment per cell, which was then compared between clusters. Few genes in the published score were47 not found in our data set and these have been reported in Supplementary Dataset Mammary signaturesscran package version 1.20.1. Genes that intersected between signature lists of multiple cell types were identified by the “intersect” function and were removed. Final gene lists for each cell type can be found in Supplementary Dataset TCGAbiolinks using the “GDCquery” function, where the parameters were set as follows project = “TCGA-BRCA”, platform = “Illumina HiSeq”, experimental.strategy = “RNA-Seq”, sample.type = “c. Only tumours that had been annotated for “paper_BRCA_Subtype_PAM50” were kept for analysis (n = 1083). Cell signature scores were then examined in the TCGA data using the “AddModuleScore” function as described above.Cell signatures were derived from each cell subtype by using the “findMarkers” from 21. First, the gene regulatory network was constructed using “grn” function in pyscenic (version 0.11.1) with standard settings. The regulons were then identified using the “ctx” (--mask_dropouts) function. Finally, the area under the curve was computed using the “aucell” function with standard settings. The regulon specificity score was computed per regulon and cell type to receive a cell type-specific ranking of regulons. For the visualization of regulons (FIGREF to the graphs of e.g. SOX10), the top 30 downstream targets (ranked by “importance”, see Supplementary Dataset For the sake of computational speed, a random sample of 20,000 cells was selected from all luminal epithelial cells. The analysis was performed as previously describedNo statistical methods were used to predetermine sample size. Unless stated otherwise, no data were excluded from the analyses. The experiments were not randomized and investigators were not blinded to allocation during experiments and outcome assessment.Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Supplementary DatasetsSupplementary Datasets 1-10Reporting Summary


Poly (PVA) in multifilament and braided yarns (BY) forms presents great potential for the design of numerous applications. However, such solutions fail to accomplish their requirements if the chemical and thermomechanical behaviour is not sufficiently known. Hence, a comprehensive characterisation of PVA multifilament and three BY architectures  was performed involving the application of several techniques to evaluate the morphological, chemical, thermal, and mechanical features of those structures. Scanning electron microscopy (SEM) was used to reveal structural and morphological information. Differential thermal analysis (DTA) pointed out the glass transition temperature of PVA at 76 °C and the corresponding crystalline melting point at 210 °C. PVA BY exhibited higher tensile strength under monotonic quasi-static loading in comparison to their multifilament forms. Creep tests demonstrated that 6BY structures present the most deformable behaviour, while 8BY structures are the least deformable. Relaxation tests showed that 8BY architecture presents a more expressive variation of tensile stress, while 10BY offered the least. Dynamic mechanical analysis (DMA) revealed storage and loss moduli curves with similar transition peaks for the tested structures, except for the 10BY. Storage modulus is always four to six times higher than the loss modulus. The most commonly used techniques to repair bone injuries involve the use of mechanical fixation systems that employ wires, screws, grids, or metal plates  . OsteosyPVA is a water-soluble and biocompatible synthetic homopolymer ,9,10,11,PVA can be used in different ways, the most common being in the form of membrane ,17 and hBraided textiles have achieved fast-growing importance in the reinforcement of composite materials and biomedical applications nowadays ,19. BraiPVA scaffolds have been studied by Teixeira et al. , offerinuσ, and ultimate strain uε. Viscoelastic characterisation was also executed through creep and relaxation tests and dynamic mechanical analysis (DMA).The former works do not cover the mechanical behaviour of PVA-braided structures neither for the quasi-static nor the viscoelastic response. Taking into account this gap in the literature, monotonic quasi-static tensile tests were performed in different PVA structures to evaluate the material strength, the Young modulus, ultimate stress ®, Kuraray, Tokio, Japan) was obtained by the melt spinning process of Exceval® polymer from Kuraray . All the other reagents used for characterisation were analytical grade purchased from Sigma–Aldrich®, St. Louis, MO, USA and used without further purification.PVA six-filament yarn  winding machine, model PR/810, serial number 00/2311  is the building block material of this work. PVA multifilament yarns were transformed into PVA multifilament yarn bobbins. Six of them were joined to form a six-yarn bobbin . Both stages were executed in a TRENZ-EXPORT 00/2311 .®  vertical braiding machine, model 16/100, serial number 00/2295. Three types of braided yarns  were formed from 6 PVA yarn . This prThe morphology of each PVA architecture was analysed by scanning electron microscopy (SEM), an ultra-high resolution field emission gun scanning electron microscopy (FEG-SEM), NOVA 200 Nano SEM, FEI Company . Secondary electron images were performed at an acceleration voltage of 10 kV. Samples were covered with a thin film (25 nm) of Au-Pd (80–20 weight %), using a high-resolution sputter coater, 208HR Cressington Company , coupled to an MTM-20 Cressington High-Resolution Thickness Controller. The braiding angle of each fibrous architecture, a fundamental physical property, was accurately measured from images obtained by SEM, using the ImageJ software.®  in which TGA and DTA are shown simultaneously. PVA yarn (1Y) was submitted to a single heating step within the range of 25–500 °C under nitrogen atmosphere (200 mL min−1) at 3 °C∙min−1 using an aluminium pan. The initial mass was measured prior to testing. TGA data were plotted as weight loss (WL) (in percentage) versus temperature. The derivative thermogravimetric (DTG) analysis was performed to identify the thermal transformation events (namely the maximum peaks). DTA data were plotted with ∆T versus temperature.Thermal analyses were carried out in an STA 7200 Hitachi® spectrometer, model FTIR-8400S, IRAffinity-1 , coupled with an attenuated total reflectance (ATR) accessory, the PIKE MIRacleTM single reflection with a diamond/ZnSe crystal . Spectra were obtained in the range of 4000–700 cm−1 from 30 scans at a resolution of 4 cm−1. All measurements were performed in triplicate. FTIR spectra of the PVA yarn (1Y) was collected using a ShimadzuSix PVA yarn architectures were subjected to uniaxial tensile testing to rupture: one yarn (1Y), six parallel yarns (6PY), six PVA yarn (6Y), six, eight, and ten braided yarns  to evaluate quasi-static properties. A total of 28 specimens were tested per PVA fibrous architecture.K0), ultimate load (Pu), and displacement at rupture (δu). Before testing, a low tare load (0.1 N) was applied to establish a consistent zero position. Then, the adjusted distance between grips was used as the initial length. The gauge length was set to 100 mm, except for the 6PY, with 170 mm  with a 250 N load cell at a 100 mm/min crosshead velocity. Load–displacement curves were obtained to evaluate the initial stiffness . The stress magnitude was set equal to 16% of the tensile strength σu  previously evaluated in uniaxial tensile tests for each braid geometry. A total of 10 specimens were tested per braided architecture .Three PVA braided yarn architectures—6BY, 8BY, and 10BY—were subjected to creep tensile tests to evaluate their viscoelastic properties. A step function with magnitude ε0 was fixed for a relaxation time of 2700 s and an acquisition frequency of 5 Hz. The strain magnitude was set equal to the strain obtained when stress attained 16% of the tensile strength σu . A total of 10 specimens were also tested per braided architecture .Tensile relaxation tests were performed in the braided configurations  to evaluate viscoelastic properties. A step function with magnitude ®  in the programmed tension (tensile) method. These analyses were carried out in an atmosphere of nitrogen (200 mL min−1) to ensure an inert environment. The presented values for tension moduli were recorded over a range of frequencies from 0.1 to 2 Hz in synthetic oscillation. Temperature dependence of storage and loss moduli , and corresponding loss tangent, tanδ, was measured in the range of 25 to 160 °C at 3 °C min−1 .Dynamic mechanical analyses were performed in a 7100 DMA from HitachiThe braiding angles of the three braided architectures studied in this work were measured from the SEM images and using the ImageJ software, as shown in TGA results for PVA 1Y configuration revealed a first small weight loss (WL) up to 100 °C , which iTg) of PVA in accordance with the DMA data [Tg temperature. Only DMA analysis is sufficiently sensitive to depict beta transitions (see Tm) of PVA was observed at 210 °C. The degree of crystallinity of PVA 1Y was estimated to be 30% and was determined as the ratio between the calculated heat of fusion  and the thermodynamic enthalpy of melting of a 100% crystalline PVA  [Four main endothermic peaks were observed in the DTA of PVA fibre . The DTADMA data . No otheions see . A secon0 J g−1) . The thi0 J g−1) . However0 J g−1) ,34.−1 attributed to the stretching ν(O–H), 2910 and 2880 cm−1 are related to the stretching of ν(C–H2) and ν(C–H), respectively, 1736 cm−1 is attributed to the stretching of ν(C=O), 1420 cm−1 is due to the bending of δ(CH–O–H), 1320 cm−1 is attributed to the wagging π(C–H), 1080 cm−1 is related to the stretching of ν(C–O), and 830 cm−1 is due to the stretching of ν(C–C) [−1 could be attributed to water adsorbed in the PVA fibre. Nonetheless, it can also be related to a partial interaction of the OH groups of PVA with a crosslinker [−1 attributed to the stretching of ν(C=O) and ν(C–O–C), respectively, could be attributed to a low degree of hydrolysis or to a possible crosslinking of the PVA [Tβ) peak observed in the DMA analysis  ,28. The sslinker . Moreove the PVA ,36. The ysis see  due to tK0), ultimate load (Pu), and displacement at rupture (δu) of the studied PVA architectures , the tensile strength (σu), and the ultimate strain (εu), which are both true values. Average true stress–strain curves were also obtained  from 28 σ0) over a period . To carry out those tests, input σ0 stress values of 63.76 MPa, 67.12 MPa, and 56.45 MPa  were imposed to each braided architecture 6BY, 8BY, and 10BY, respectively. The analysis of the attained mean curves for true tensile strain vs. time in εu (ε) obtained in the creep test (time of creep), it can be concluded that this difference is smaller in the 6BY configuration (13.7%) and larger in 8BY (29.4%). This means that the braided architecture that allows a smaller margin for further deformation until final rupture is 6BY, while the highest margin is attained for the 8BY configuration. Taking this argument into account, it turns out that the behaviour of 10BY, although identical to the 8BY architecture in the tensile creep test  over a period of time. The applied strain has been set to 5%, 8.5%, and 8%, respectively, for 6BY, 8BY, and 10BY architectures (σu) for each braided structure. Relaxation can be defined as the decrease in the stress under constant applied strain (tectures . These vδ), storage, and loss moduli  were measured in the temperature range of 30–160 °C at a heating rate of 3 °C min−1. Both E′ and E″ curves present similar transition peaks for all the tested braided yarns , DMA tests were performed for the analysed PVA braided yarns. The presented values for tensile modulus were collected over a range of frequencies from 0.1 to 2 Hz in synthetic oscillation mode for 6BY , 8BY Fi, and 10Bed yarns , except E′ between 30 and 37 °C can be attributed to a cold crystallisation effect of the PVA Mintval® fibres obtained by the melt spinning process of Exceval® polymer from Kuraray. A cold crystallisation peak is not a surprise, since spun fibres have a lower degree of crystallisation than drawn or staples fibres. Consequently, the modulus of a semicrystalline polymer will result in a higher value than the analogous amorphous polymer [E′ rapidly decreases, due to melting of crystal form, after reaching a peak that is concomitant with a negative peak in tanδ. This indicates a clear elastic and solid-like behaviour [δ at these physiological temperatures varies from 0.19 to 0.25, which confirms that the system is basically elastic under tensile load, presenting a small, though not negligible, viscosity. After this peak, a dramatic decrease E′ and E″ is observed, corresponding to peaks at 41 °C and 47 °C in tanδ of the 6BY and 8BY, respectively. These peaks could be associated with the beta relaxation Tβ, which is related to the motion of backbone segments of PVA side chains where acetate groups or crosslinking species are present, or to the glass transition temperature Tg of the non-hydrolysed poly(vinyl acetate). The unusual growth in the  polymer . A cold ehaviour . The valδ peak shift to higher temperature only at the highest frequency of 2 Hz, suggesting that the peak can be associated with the Tg of poly(vinyl acetate). However, that selected frequency is too low to show an explicit Arrhenius behaviour as a function of oscillation frequency, not allowing the determination of the activation energy [Tg of PVA, which is an alpha transition (Tα) that represents the primary motion of the PVA main chain in the amorphous areas in the polymer matrix [Tg is barely visible, and the Tβ appears at a lower temperature in both moduli. The fibre structural effect can explain this behaviour. The reduction of peak amplitude and the increase in peak within tanδ could be associated with a decrease in polymer chain mobility by restricting both side-chains rotations and main backbone movements, which could also be attributed to the more rigid 10BY architecture. Since PVA is usually obtained by the hydrolysis of poly(vinyl acetate) ,40, thesn energy . The secr matrix . In the δ peak amplitude. The higher rigidity of the system is also proved by the fact that no shift in temperature peak in tanδ at the highest frequency of 2 Hz is detected. DMA, in this case, is not only able to provide information about the behaviour of the amorphous and crystalline regions but also quantify the degree of the PVA chain confinement due to the fibre architecture and filament numbers observing the reduction of the tanδ is characterised by a frequency-dependent with a more elastic response (lower tanδ) at high frequencies. Since the rate of deformation and the sample response is strongly related to the relaxation phenomenon, this indicates the clear viscoelastic nature of PVA fibre. At higher temperatures, up to 160 °C, tanδ continues to decrease. However, the response tends to converge to a single frequencies-independent value, which indicates that the polymer is becoming more molecular-oriented even if its elasticity continues to increase.The region between 90 and 130 °C in tanThe performed extensive analysis of polyvinyl alcohol (PVA) is a fundamental task to understand the potentialities of this material in applications requiring the employment of PVA yarns (Y) and braided yarns (BY). PVA architectures were highlighted by scanning electron microscopy (SEM), rendering it possible to show the morphology and surface texture of those structures. This technique allowed verification that the transversal section for one PVA yarn (1Y) is constituted by six filaments. A thermogravimetric analysis (TGA) was applied to 1Y PVA with a range of temperatures from 25 to 500 °C to investigate the corresponding mass decomposition. The results showed that the degradation of this polymer suffers from 80% of mass loss at 385 °C peak corresponding to the primary chain dehydration reaction, after which tiny residues of 5% remain at the end of the analysis (500 °C). Tg) of PVA was reached at 76 °C. The corresponding crystalline melting temperature (Tm) was observed at 210 °C. The crosslinking degradation was observed at two expressive peaks at 330 and 370 °C. Differential thermal analysis (DTA) was executed to measure the amount of heat required to increase the temperature of the 1Y PVA sample. The glass transition temperature (Fourier-transform infrared spectroscopy (FTIR) analysis was performed, confirming the identity of the chemical compounds present in the PVA 1Y structure. σu) for the PVA braided yarns structures. Monotonic tests proved that PVA braided yarns are more viable than non-braided, since an enhancement of their mechanical properties has been attained. Monotonic tensile tests were executed to evaluate quasi-static properties of the PVA structures with a different number of fibres (6 to 360), showing higher tensile strength . According to creep tests, the 6BY PVA structure presents the most deformable behaviour, while the 8BY PVA structure is the least deformable. On the order hand, the results from relaxation tests showed that the 8BY PVA structure presents the most strength loss and the 10BY PVA architecture verified the minimum loss strength. E′ and E″, respectively) revealed similar transition peaks for all braided yarns, except for the 10BY system. In addition, the E′ value is always four to six times higher than E″ in the same order of magnitude.Dynamic mechanical analysis (DMA) characterisation was executed to determine the viscoelastic properties of the PVA braided yarns. The graphic representing the evolution of the storage and loss moduli (Among the possible applications of such fibrous systems are bone fracture immobilisation devices, which could be customised with a proper fibrous architecture of PVA according to bone fracture configuration, fragment size, and location. Those solutions would benefit from intrinsic PVA features such as biocompatibility, biodegradability, and odourlessness, while preserving the required mechanical performance of the fastening system to allow the regeneration of bone tissue throughout the clinical recovery period. This would give the opportunity for the development of less-intrusive bone fixation systems to offer an adequate response to temporary injuries, dispensing further revision processes.


Oscillometric, non-invasive blood pressure measurement (NIBP) is the first choice of blood pressure monitoring in the majority of low and moderate risk surgeries. In patients with morbid obesity, however, it is subject to several limitations. The aim was to compare arterial pressure monitoring by NIBP and a non-invasive finger-cuff technology (Nexfin®) with the gold-standard invasive arterial pressure (IAP).In this secondary analysis of a prospective observational, single centre cohort study, systolic (SAP), diastolic (DAP) and mean arterial pressure (MAP) were measured at 16 defined perioperative time points including posture changes, fluid bolus administration and pneumoperitoneum (PP) in patients undergoing laparoscopic bariatric surgery. Absolute arterial pressures by NIBP, Nexfin® and IAP were compared using correlation and Bland Altman analyses. Interchangeability was defined by a mean difference ≤ 5 mmHg (SD ≤8 mmHg). Percentage error (PE) was calculated as an additional statistical estimate. For hemodynamic trending, concordance rates were analysed according to the Critchley criterion.2) were enrolled and data from 56 finally analysed. Pooled blood pressure values of all time points showed a significant positive correlation for both NIPB and Nexfin® versus IAP. Pooled PE for NIBP versus IAP was 37% (SAP), 35% (DAP) and 30% (MAP), for Nexfin versus IAP 23% (SAP), 26% (DAP) and 22% (MAP). Correlation of MAP was best and PE lowest before induction of anesthesia for NIBP versus IAP  and after intraoperative fluid bolus administration for Nexfin® versus IAP . Concordance of MAP trending was 90%  for NIBP and 91%  for Nexfin®. MAP trending was best during intraoperative ATP positioning for NIBP (97%) and at induction of anesthesia for Nexfin® (97%).Sixty patients  on June 12, 2017.The non-interventional, observational study was registered retrospectively at ( The standard of care in most low to medium risk surgeries is the oscillometric, non-invasive, intermittent blood pressure measurement (NIBP), which is associated with a lower rate of perioperative complications compared with invasive arterial pressure (IAP) measurements , 2. Howe2) patients are more likely to have a history of type 2 diabetes, arterial hypertension or obstructive sleep apnea syndrome [Obese and morbidly obese  class ≥ II and written informed consent for study participation. Since not only morbidly obese patients defined as a BMI ≥ 40.0 kg/m2 or 35.0 and 39.9 kg/m2 with comorbidities, but also obese patients with BMI > 30 and < 35 kg/m2 and type 2 diabetes may have been scheduled for bariatric surgery [2 criterion was selected. Pre-existing cardiac arrhythmias, peripheral arterial vascular disease Fontaine stadium > 2, pre-existing aortic aneurysm > 4,5 cm and cognitive or linguistic barriers were defined as exclusion criteria.This is a planned secondary analysis of a single-center prospective observational cohort study where the comparison of continuous cardiac index measurements by Nexfin® and a semi-invasive reference method was previously published . The sin surgery , the BMIA description of the study participants’ instrumentation, hemodynamic monitoring and anesthesia was already described previously .NIBP measurement using a forearm cuff on both arms was used for all study participants in order to detect physiological as well as pathological blood pressure differences. Non-invasive blood pressure monitoring  was conducted on the right arm along with relaxometry and pulse oxymetrically measured oxygen saturation. The appropriately sized finger-cuff of the Nexfin® system   was placIn the pre-, intra- and postoperative phase, the arterial pressure was measured at 16 predefined measurement time points at which hemodynamic changes were likely expected.The first measurement was performed in the awake, spontaneously breathing patient in neutral position (baseline measurement time point 1). Directly before the induction of general anesthesia, the patients were placed in 30° anti-Trendelenburg positioning (ATP) as per clinical standard for oxygenation improvement and aspiration prophylaxis (measurement time point 2). The next measurements were taken under general anesthesia, but still in ATP (measurement time point 3), and re-positioned to baseline (measurement time point 4). The following three measurement pairs  were taken during 30° passive leg raising (PLR), again in neutral position and after administration of a 500 ml balanced crystalloid solution bolus infusion . After this preoperative period the patients were transferred to the operating room, where the measurement devices were reconnected and a new zero balance was performed . Further measurements were taken intraoperatively after the pneumoperitoneum (PP) had been applied to 15 mbar , during intraoperative ATP positioning (measurement time point 10) and in that position after another fluid bolus of 500 ml crystalloid solution (measurement time point 11). The last three intraoperative measurements (measurement time points 12–14) were carried out after termination of PP in neutral position, and again in ATP by the end of general anesthesia, followed by a last measurement in neutral position. Measurement time points 15 and 16 were carried out in the post-anesthesia care unit (PACU) upon arrival and after 2 h in ATP posture, before the patient was discharged from PACU. Demographic data including gender, age, height and weight, ASA classification, comorbidities, and type of surgery and anesthesia were collected from all study participants.N = 60 study participants, followed by an intermediate evaluation. For a Bland-Altman analysis, the width w of the confidence interval for the limits of agreement (LOA) was calculated as w = 6.79•σ•1/√n, where n is number of cases and σ is the standard deviation. For a case count of N = 60, the result is w = 0.88 • σ and thus considered a sufficiently large number. Normal distribution of the outcomes was checked and verified by visual inspection of the histogram analysis. First, a Spearman correlation analysis of measurement pairs for SAP, DAP and MAP between the monitoring devices was performed. Furthermore, Bland-Altman analysis was used for the comparison of the pressure variables with calculation of the mean difference (bias) and limits of agreement (LOA) defined as the standard deviation (SD) of the bias ±1.96 accounting for repeated measurements [The primary objective of the study was to test interchangeability of the test (NIPB or Nexfin®) against the invasive reference method (IAP). As there is no global definition of interchangeability, we chose two commonly accepted criteria to allow approximation: 1) the recommended Association for the Advancement of Medical Instrumentation (AAMI) criterion of a mean difference ≤ 5 mmHg and associated SD of ≤8 mmHg , 2) the urements , 33. Theurements . We furtP value of < 0.05 was considered as statistically significant. Statistical analysis was performed using GraphPad Prism 5, GraphPad Software Inc., San Diego, CA, USA.A N = 832) of all planned measurements could be realized. Reasons for lack of measurement data pairs (N = 128) were mostly attributed to intraoperative events preventing measurements (N = 90) and challenges in function and handling of the measurement device, of which the NIBP device failed significantly more frequently (N = 31) than the IAP (N = 2) or Nexfin® monitoring (N = 5). All measurements were pooled and included in the statistical analysis.Sixty patients were enrolled in the study with four patients being excluded from the statistical analysis due to insufficient data acquisition Fig. . Table 1Table The pooled correlation coefficient between NIBP and IAP for MAP was 0.77 . Bland-Altman plots corroborated a correlation with a PE of 30% and a bias of − 3.94 mmHg for MAP . Pooled correlation between Nexfin® and IAP over all measurement time points was 0.88 for MAP . Bland Altman analysis showed a PE of 22% and a bias of 1.43 mmHg for MAP .With respect to hypotension, PE was comparable between NIBP 30%) and Nexfin® (29%) vs. IAP whereas during hypertension, PE was lower for NIBP (28%) and Nexfin® (21%) vs. IAP  and DAP , but only moderate for SAP , missing the predefined AAMI criterion of interchangeability. Trending capability was good with concordance rates of 88–93%. In contrast to our study, measurements were recorded continuously using an interface cable and recording software for 45 min independently of intraoperative events and cleared of obvious artifacts subsequently by visual inspection [2 was unable to show interchangeability of intraoperatively measured absolute arterial pressure values with an observed bias (SD) of 14.3 mmHg (14.1) for SAP, 2.6 mmHg (10.8) for DAP and 5.2 mmHg (10.9) for MAP as compared to IAP [2) with bariatric surgery. In their study, measurements were obtained within 15 min before start of PP in the horizontal position, 3, 15, 30, and 45 min after the beginning of PP in the 30° reverse Trendelenburg position and 3 min after end of PP in horizontal posture. Furthermore, NIBP and Nexfin® were recorded at the ipsilateral arm as opposed to the contralateral measurement in our study. DAP showed best precision (mean difference 0 ± 11 mmHg), followed by MAP (mean difference − 1 ± 11 mmHg) and SAP (mean difference − 7 ± 14 mmHg) between finger-cuff and IAP measurements while NIBP measurement was significantly less precise [2) and young adults during robotic, laparoscopic-assisted bariatric surgery and were able to only show a clinically useful trend ability of the arterial pressure values for CNAP while accuracy for absolute values failed the AAMI criterion [2) undergoing laparoscopic bariatric surgery correspondingly found good trending ability for the CNAP® system compared to continuous IAP measurements obtained with a radial arterial catheter, but again no interchangeability for absolute values [With regard to morbidly obese patients undergoing bariatric surgery, four observational studies have been previously published focussing on the performance of the Nexfin® (Clearsight®) finger-cuff device for arterial pressure monitoring , 26, 27.surement . A furthspection . The pro precise , 25 in pe values .In essence, while results from our and the 5 published studies on interchangeability for absolute values remain inconclusive with a notion that Nexfin is slightly more precise, overall trend behavior of the finger-cuff technology was consistently shown to be clinically useful. The latter was also shown for various subsequent measurement time points in our study where hemodynamic changes become evident in the perioperative phases of laparoscopic bariatric surgery, providing useful clinical information on the presence of arterial pressure variation and its management. Finally, we further analyzed precision in the range of hypo- and hypertension as this is clinically even more relevant than information on “global performance”. With focus on hypotension, Nexfin® and NIBP tended to overestimate arterial pressure, possibly leading to false security in clinical practice while during hypertensive episodes no difference was observed. However, data pairs for hypotension in our study were too limited to be conclusive, most likely because a direct therapeutic intervention was performed for patient safety. In a previous study in 30 awake carotid endarterectomy patients, Noto and coworkers showed that precision of Clearsight® measurements were not interchangeable below an average MAP cut-off value of 69 mmHg .A limitation of our study is that the sample size of 60 patients deviates from the recently updated standard for the validation of blood pressure measuring devices by the AAMI, the European Society of Hypertension (ESH) and the International Organization for Standardization (ISO) collaboration where where a minimum sample size of 85 participants is recommended for studies validating non-invasive sphygmomanometers . HoweverStrengths of our study include the examination of different hemodynamic stresses typically occurring throughout the perioperative course of laparoscopic surgery, particularly due to different postures and on/off-set of PP. Mimicking those physiological stresses using a structured protocol in the pre-, intra- and postoperative phase enhances the “real-life” comparison of the device performance.In the perioperative management of patients undergoing laparoscopic bariatric surgery, our study indicates that NIBP and Nexfin® derived absolute arterial pressure recordings were not interchangeable with IAP, but Nexfin® was more precise than NIBP. However, a good trending ability even under different hemodynamic stresses was found. Thus, Nexfin® may serve clinically useful to detect arterial pressure changes and render perioperative hemodynamic treatment, particularly in those individuals where NIBP cannot be reliably established.


To assess the prognostic value of the ChOLE classification in predicting the severity of acquired cholesteatoma.A, FU > 52 weeks after last-look surgery or MRI-DWI; recurrent cholesteatoma in group B, FU > 52 weeks after last outpatient visit; and adverse events (AE) in group C, FU > 12 weeks after surgery. Cholesteatomata were staged according to the ChOLE classification. Kaplan–Meier curves were used to determine the prognostic value of the classification in predicting cholesteatoma severity, while correcting for FU.A retrospective chart review of patients undergoing primary cholesteatoma surgery in our tertiary referral center. The primary outcome measures were analyzed in three groups of follow up (FU): residual cholesteatoma in group No significant differences were observed between the various stages of the ChOLE classification and residual or recurrent cholesteatoma rate, nor the occurrence of AE. Cholesteatoma extension to the sinus tympani or widespread in the mastoid, as well as absence of the stapes superstructure were predictive of residual disease. Sclerotic mastoids had a lower risk of residual disease than mastoids with good or poor pneumatization and ventilation. Poorly ventilated and poorly pneumatized mastoids were associated with increased risk of recurrence. Widespread cholesteatoma in the mastoid as well as presence of preoperative extracranial complications were correlated with an increased risk of AE.The ChOLE classification does not predict residual nor recurrent disease, nor the occurrence of AE, in our study population. Risk factors for severe cholesteatoma were identified, potentially useful for the development of future classifications. Chronic otitis media with cholesteatoma formation is a common otologic disease, requiring surgery in majority of cases and still causing severe intracranial complications if not treated effectively . The priIn 2018, the ChOLE classification was introduced by Linder et al. . This syClassification of cholesteatoma can be of great clinical value if it aids an otologic surgeon in determining treatment strategy. A classification should therefore be predictive of cholesteatoma severity, further defined as the occurrence of residual and recurrent disease and the occurrence of adverse events (AE). In this study, we evaluate the prognostic value of the ChOLE classification on these primary outcome measures.A retrospective chart analysis was done of all patients undergoing primary cholesteatoma surgery between January 1, 2004 and October 1, 2019 in a tertiary otologic referral center. The following data were collected: age, unilateral or bilateral disease, type of surgery, follow up (FU), occurrence of residual and recurrent disease, and occurrence of AE. Extension of cholesteatoma (“Ch”), ossicular chain status (“O”), presence of life-threatening complications (“L”), and mastoid pneumatization and ventilation (“E”) were defined retrospectively according to the ChOLE classification . All choA, studying residual cholesteatoma, FU > 52 weeks since last-look surgery or MRI-DWI upon primary surgery; group B, studying recurrent disease, FU > 52 weeks since last outpatient visit upon primary surgery; and group C, studying AE, FU > 12 weeks since last outpatient visit upon primary surgery. Based on this categorization, patients could be placed in more than one group.Participants were categorized in three groups based on FU: group Cholesteatoma surgery was classified into six types of surgery Table . The corFollowing Clavien et al , AE werep < 0.05 was used.Statistical analyses were performed using SPSS 27.0 . Data are expressed as number (%) and median [IQR]. Normal distribution of data was tested with a Shapiro–Wilk test. To correct for FU, Kaplan–Meier survival curves and corresponding log-rank analyses were performed to evaluate the prognostic value of the ChOLE classification on residual and recurrent disease. Only the first event of recurrent or residual disease was included. Cox regression was applied for continuous variables. Only when overall analyses of categories were significant, sub analyses were performed in which both general categories and sub categories were tested . Chi-square and Fisher's exact tests were used to determine correlations between the ChOLE classification and AE. Sub analyses were performed to identify possible factors independently influencing outcome parameters, for example surgery type. Analyses were repeated while excluding paired ears, to rule out possible confounders . A significance level of A total of 404 patients were eligible to enroll in this study based on the inclusion criteria. This corresponded to 440 ears, as 36 patients underwent bilateral primary surgery in our center. The clinicopathologic characteristics are presented in Table A included 317 ears, in which 88 residual cholesteatomata were found (27.8%). Group B consisted of 368 ears, of which 112 ears (30.4%) had recurrent cholesteatoma. Group C included 428 ears, as 12 ears were lost to otoscopic FU. In this group, in 147 ears one or more AE was observed (34.3%), mainly presenting with one pre- or postoperative AE (79.6%). The most common AE was preoperative iatrogenic defect of mastoid borders, not needing any intervention , followed by postoperative wound infection  and prolonged healing of the wound longer than 12 weeks  . This effect was also observed when excluding all TCA surgeries (p < 0.01). When further subdividing extension of disease, localization of cholesteatoma in the sinus tympani led to additional risk of residual disease as “Ch1b” had a higher risk than “Ch1a,” “Ch2a” and “Ch2b” . Extensive spread of cholesteatoma in the mastoid cavity beyond the lateral canal and/or extensive canal wall destruction was also a risk factor, as “Ch3” had a higher risk of residual disease than “Ch1a,” “Ch2a,” and “Ch2b” . Supra- or infralabyrinthine extension (“Ch4a”) did not lead to a higher risk of residual disease compared to almost all other categories of extension (“Ch1b” to “Ch3”). Also, petrous bone cholesteatoma (“Ch4b”) did not have a higher risk of residual disease than any of the other categories. Choice of surgical approach was not significantly different for supra- or infralabyrinthine cholesteatoma (“Ch4a”), as well as apical cholesteatoma (“Ch4b”), compared to other cholesteatoma (supplementary Table 1). Pediatric patients had a higher residual rate of cholesteatoma than adult patients (p < 0.01). This could not be evidently correlated to a greater extension of pediatric cholesteatoma, as both “Ch1” and “Ch4” cholesteatoma were significantly more common in the pediatric group .Cholesteatoma extension significantly influenced residual rate Table . Sub anap < 0.01). Absence of the stapes superstructure  was correlated with cholesteatoma localization in the sinus tympani  (p < 0.01). Life-threatening complications did not have any effect on residual disease.Ossicular chain status was correlated with residual rate: sub analyses showed “O2” had a significantly higher risk of residual disease than “O1” . This effect could not be explained by choice of surgical approach as sub analyses showed no relevant differences in the distribution of this effect across the various surgical techniques (supplementary Table 1).Mastoid pneumatization did influence residual disease as a sclerotic mastoid (“E2”) had a significantly lower risk of residual disease compared to both poorly (“E1”) and well-pneumatized and well-ventilated mastoids (“E0”) . As there were no relevant differences between choice of surgical approach and mastoid pneumatization, surgical approach was not a confounder in this analysis (supplementary Table 1).Cholesteatoma extension, ossicular chain status and life-threatening complications did not influence recurrent disease Table . Mastoidp < 0.01). Ossicular chain status was not correlated with occurrence of AE. Life-threatening complications were correlated with AE, as ears with extracranial complications had a significantly higher chance of AE than ears without extracranial complications (p < 0.05). More specifically, there was increased risk of sensorineural hearing loss (p < 0.05), persistent drainage (p < 0.01), postoperative vertigo (p < 0.05) and postoperative wound infection (p < 0.05). It is possible choice of surgical approach plays a role as CWD and CWD-CWR were preferred when extracranial complications were present  and CWD was associated with a higher rate of AE (p < 0.05), while STP was preferred in patients with intracranial complications (p < 0.05).Cholesteatoma extension did influence the chance of occurrence of AE Table , where “p < 0.01 and p < 0.05, respectively). Staging did not lead to significant differences in recurrence after TCA, CWD, CWD-CWR, and STP surgeries. For residual cholesteatoma and AE, ChOLE stage did not differ significantly between any of the surgical approaches.The ChOLE stage was partially predictive of recurrent disease in selectively CWU patients, and in CWU and CWUO patients combined Fig. C, as botp < 0.001) and “Ch3” cholesteatoma were preferably operated with CWUO (p < 0.05) (supplementary Table 1).Choice of CWU, CWD, CWD-CWR, and STP in our population was not influenced by cholesteatoma extension. Only “Ch1” cholesteatoma were preferably operated with TCA (p < 0.01) and significantly more well-pneumatized and well-ventilated mastoids were found in the TCA group (p < 0.001). As the residual and recurrence rates of cholesteatoma in these groups do not significantly differ from the other surgical approaches, this is not expected to be a confounder in analyses.There was no significant correlation between poorly pneumatized or sclerotic mastoids and choice of CWD, CWU, CWUO, or CWD-CWR (supplementary Table 1). Significantly more sclerotic mastoids were found in the STP group  mastoids, introducing the possibility of a tipping point in pathology. The authors therefore hypothesize that if the mastoid is completely dense, no sick mucosa is present to maintain inflammation and/or a negative middle-ear pressure, resulting in lower recurrence rates. This could warrant a more aggressive surgical approach including obliteration of the epitympanum and mastoid bowl, aiming to eradicate sick mucosa and drill out remaining poorly ventilated mastoid cell tracts.Sclerotic mastoids were associated with a lower risk of residual disease than mastoids with good and poor pneumatization and ventilation, also regardless of surgical approach. Surgically removing disease from widely pneumatized mastoid cells is more challenging, especially in noncyst like cholesteatomata that easily infiltrate remaining air cell tracts, than removing it from dense bone. The combination of a poorly pneumatized mastoid and widespread cholesteatoma into the “high risk area” mastoid could demand an even more meticulous approach to eradicating disease during surgery.Apart from preventing residual and recurrent disease, cholesteatoma surgery aims to create a hygienic and waterproof ear, while maintaining or improving hearing. In our study, we did not look into the value of the ChOLE classification in predicting these secondary outcome measures. The presence of preoperative extracranial complications was associated with more postoperative AE in the short-term. The classification could be used to counsel patients on a potentially more challenging recovery after surgery, especially after CWD. More intensive outpatient clinic appointments could be justified to follow-up on wound healing, postoperative vertigo or sensorineural hearing loss and prophylactic antibiotics could be prescribed to prevent postoperative wound infections. In a recent study, the health-related quality of life as measured by the Zurich Chronic Middle Ear inventory, was not associated with ChOLE staging in the long-term . On the A limitation of this study could be the fact that ChOLE stage II cholesteatoma were overrepresented in our population, diminishing the discriminative ability of the classification. A previous study confirmed preponderance of ChOLE stage II cholesteatoma in a population with comparable state of health care . FurtherA strength of the ChOLE classification is the use of a scoring system, summing up to a certain cholesteatoma stage. The classification assigns points to, for instance, extra- and intracranial complications, rather than automatically staging the cholesteatoma in the severest category when intracranial complications are present, as in the EAONO-JOS classification . This alIn our opinion, the ChOLE classification in its current form is a registration system, providing a valid basis for standard reporting of cholesteatoma. However, there is a fundamental difference between a registration system and a classification. The ChOLE staging does not have the power to differentiate between less aggressive and more severe cholesteatoma. Therefore, it has no consequences for choice of appropriate treatment strategy and FU scheme and therefore does not measure up to our previously described definition of a classification with clinical value. The current classification could be tested in subgroups stratified by SAMEO-ATO classification of tympanomastoid surgery, however the clinical value of such a classification is also limited in our opinion. In the future, a classification could be considered including only previously identified risk factors for residual and recurrent cholesteatoma as well as AE. We challenge the need to categorize all aspects of cholesteatoma, if these aspects have limited influence on the primary outcome measures. Such a classification could include the risk factors identified in this study: localization of cholesteatoma in the “high risk area” sinus tympani or widespread into the mastoid cavity, and poorly pneumatized and ventilated mastoid cells. Additional risk factors such as age, previously reported in other studies and also confirmed in our data, could also be included ,26. OnceThe ChOLE classification does not predict residual and recurrent cholesteatoma, nor the occurrence of AE in our population. Risk factors for severe cholesteatoma were identified: localization of cholesteatoma in the sinus tympani and widespread cholesteatoma into the mastoid cavity, as well as poorly pneumatized and ventilated mastoid cells. In the future, a classification could be developed including these specific risk factors, potentially defining stages of cholesteatoma with different prognoses, benefitting from different treatment strategies.


Extrapolating data from H1N1 influenza pandemic, ELSO guidelines for VV-ECMO [The previously published cohorts of CADRS treated with VV-ECMO report a 65% survival rate with a strict patients’ selection . We repoData are presented as median value (interquartile range) or number (percentage). Comparisons were made using Wilcoxon–Mann–Whitney or Fisher’s exact tests as appropriate. Logistic models were used to evaluate associations after adjustment on covariates. According to French regulation, the study was approved by the Angers University Hospital ethics committee and information letters were sent to the patients.2 < 80 mmHg for 6 h or < 60 mmHg for 3 h or pH < 7.25 for 3 h, despite appropriate ventilatory settings and prone positioning) [Fifty-six patients  aged 24 to 71 years were treated with ECMO from March 2020 to June 2021 (Table p = 0.014). There was no significant association between the duration of mechanical ventilation before VV-ECMO and the mortality , even after adjustment on age and SOFA score at cannulation .The patients received VV-ECMO support for 17.5 (10–31.2) days and 27/56 (48%) discharged hospital. Patients’ survival according to age, SOFA score at cannulation and duration of mechanical ventilation before ECMO are reported in Fig. The mortality observed in our cohort was higher than previously reported in patients treated with VV-ECMO during the first COVID-19 pandemic wave , includiThe relative contraindication for VV-ECMO in patients mechanically ventilated for more than 7 days in ELSO guidelines remains questionable. It is mainly based on ELSO registry data referring to patients cannulated before 2012, particularly during the H1N1 influenza pandemic, reporting a strong correlation between mortality and duration of mechanical ventilation before cannulation , but hasIn the COVID-19 context with a very high pressure on available resources, most of the ECMO centers seem to have strongly limited their indications of late cannulation. To the best of our knowledge, our cohort is the first specific report of late cannulation experience in this indication and our results suggest that it is feasible and may have benefit some patients .Our study has several limitations. It is a retrospective cohort with a limited number of patients, and some subjective criteria may have been considered in decisions of VV-ECMO initiation even if a predefined common protocol was used.In conclusion, a late initiation of VV-ECMO for CARDS was not associated with an increased mortality in our cohort. The criterion of duration of mechanical ventilation for the decision of VV-ECMO initiation in this indication should be carefully considered.