Overload of the Temporomandibular Joints Accumulates γδ T Cells in a Mouse Model of Rheumatoid Arthritis: A Morphological and Histological Evaluation

Recently, it has been reported that γδ T cells are associated with the pathology of rheumatoid arthritis (RA). However, there are many uncertainties about their relationship. In this study, we investigated the morphological and histological properties of peripheral as well as temporomandibular joints (TMJ) in a mouse model of rheumatoid arthritis with and without exposure to mechanical strain on the TMJ. Collagen antibody-induced arthritis (CAIA) was induced by administering collagen type II antibody and lipopolysaccharide to male DBA/1JNCrlj mice at 9−12 weeks of age, and mechanical stress (MS) was applied to the mandibular condyle. After 14 days, 3D morphological evaluation by micro-CT, histological staining (Hematoxylin Eosin, Safranin O, and Tartrate-Resistant Acid Phosphatase staining), and immunohistochemical staining (ADAMTS-5 antibody, CD3 antibody, CD45 antibody, RORγt antibody, γδ T cell receptor antibody) were performed. The lower jawbone was collected. The mandibular condyle showed a rough change in the surface of the mandibular condyle based on three-dimensional analysis by micro-CT imaging. Histological examination revealed bone and cartilage destruction, such as a decrease in chondrocyte layer width and an increase in the number of osteoclasts in the mandibular condyle. Then, immune-histological staining revealed accumulation of T and γδ T cells in the subchondral bone. The temporomandibular joint is less sensitive to the onset of RA, but it has been suggested that it is exacerbated by mechanical stimulation. Additionally, the involvement of γδ T cells was suggested as the etiology of rheumatoid arthritis.

Preparation and Properties of Decellularized Sheep Kidney Derived Matrix Scaffolds

Scaffolds from tissues or organs have nanoscale microstructures. Derived matrix scaffolds prepared by decellularized method can provide more cell attachment sites, which is conducive to cell adhesion, proliferation, differentiation and other physiological activities on scaffolds. In this study, the sheep kidney decellularized matrix scaffold was prepared by the method of decellularization. Due to the poor mechanical properties of the decellularized matrix, the cross linking method was adopted to enhance its mechanical properties. The decellularization efficiency of sheep renal matrix scaffolds was observed by scanning electron microscopy and histological staining, and the biocompatibility of the scaffolds was investigated by inoculating adipose derived stem cells. It was found that the scaffold had good decellularization effect and good pore structure.

A Framework for Falsifiable Explanations of Machine Learning Models with an Application in Computational Pathology

In recent years, deep learning has been the key driver of breakthrough developments in computational pathology and other image based approaches that support medical diagnosis and treatment. The underlying neural networks as inherent black boxes lack transparency, and are often accompanied by approaches to explain their output. However, formally defining explainability has been a notorious unsolved riddle. Here, we introduce a hypothesis-based framework for falsifiable explanations of machine learning models. A falsifiable explanation is a hypothesis that connects an intermediate space induced by the model with the sample from which the data originate. We instantiate this framework in a computational pathology setting using label-free infrared microscopy. The intermediate space is an activation map, which is trained with an inductive bias to localize tumor. An explanation is constituted by hypothesizing that activation corresponds to tumor and associated structures, which we validate by histological staining as an independent secondary experiment.

Iodine doping of a photocuring, one-component embedding medium for µCT and histology

Objective: Evaluation of μCT scans of bone implant complexes often shows a specific problem: if an implant material has a very similar radiopacity as the embedding medium (e.g. methacrylate resin), the implant is not visible in the μCT image. Segmentation is not possible, and especially osseointegration as one of the most important parameter for biocompatibility is not evaluable. Methods: To ensure μCT visualisation and contrast enhancement of the evaluated materials, the embedding medium Technovit® VLC7200 was doped with an iodine monomer for higher radiopacity in different concentrations and tested regarding to handling, polymerisation, and histological preparation, and visualisation in µCT. Six different µCT devices were used and compared with regard to scan conditions, contrast, artefacts, image noise, and spatial resolution for the evaluation of the bone-implant blocks. Results: Visualisation and evaluation of all target structures showed very good results in all μCT scans as well as in histology and histological staining, without negative effects caused by iodine doping. Subsequent evaluation of explants of in vivo experiments without losing important information was possible with iodine doped embedding medium. Conclusion: Visualisation of implants with a similar radiopacity as the embedding medium could be considerably improved. µCT scan settings should be selected with the highest possible resolution, and different implant materials should be scanned individually for optimal segmentation. µCT devices with higher resolutions should be preferred. Advances in knowledge: Iodine doped embedding medium is a useful option to increase radiopacity for better visualisation and evaluation of special target structures in µCT.

Epithelial-Specific TLR4 Knockout Challenges Current Evidence of TLR4 Homeostatic Control of Gut Permeability

<b><i>Introduction:</i></b> Toll-like receptor 4 (TLR4) is a highly conserved immunosurveillance protein of innate immunity, displaying well-established roles in homeostasis and intestinal inflammation. Current evidence shows complex relationships between TLR4 activation, maintenance of health, and disease progression; however, it commonly overlooks the importance of site-specific TLR4 expression. This omission has the potential to influence translation of results as previous evidence shows the differing and distinct roles that TLR4 exhibits are dependent on its spatiotemporal expression. <b><i>Methods:</i></b> An intestinal epithelial TLR4 conditional knockout (KO) mouse line (<i>Tlr4</i>^<i>ΔIEC</i>, <i>n</i> = 6–8) was utilized to dissect the contribution of epithelial TLR4 expression to intestinal homeostasis with comparisons to wild-type (WT) (<i>n</i> = 5–7) counterparts. Functions of the intestinal barrier in the ileum and colon were assessed with tissue resistance in Ussing chambers. Molecular and structural comparisons in the ileum and colon were assessed via histological staining, expression of tight junction proteins (occludin and zonular occludin 1 [ZO-1]), and presence of CD11b-positive immune cells. <b><i>Results:</i></b> There was no impact of the intestinal epithelial TLR4 KO, with no differences in (1) tissue resistance–ileum (mean ± standard error of mean [SEM]): WT 22 ± 7.2 versus <i>Tlr4</i>^<i>ΔIEC</i> 20 ± 5.6 (Ω × cm²) <i>p</i> = 0.831, colon WT 30.8 ± 3.6 versus <i>Tlr4</i>^<i>ΔIEC</i> 45.1 ± 9.5 <i>p</i> = 0.191; (2) histological staining (overall tissue structure); and (3) tight junction protein expression (% area stain, mean ± SEM)–ZO-1: ileum–WT 1.49 ± 0.155 versus <i>Tlr4</i>^<i>ΔIEC</i> 1.17 ± 0.07, <i>p</i> = 0.09; colon–WT 1.36 ± 0.26 versus <i>Tlr4</i>^<i>ΔIEC</i> 1.12 ± 0.18 <i>p</i> = 0.47; occludin: ileum–WT 1.07 ± 0.12 versus <i>Tlr4</i>^<i>ΔIEC</i> 0.95 ± 0.13, <i>p</i> = 0.53; colon–WT 1.26 ± 0.26 versus <i>Tlr4</i>^<i>ΔIEC</i> 1.02 ± 0.16 <i>p</i> = 0.45. CD11b-positive immune cells (% area stain, mean ± SEM) in the ileum were mildly decreased in WT mice: WT 0.14 ± 0.02 versus <i>Tlr4</i>^<i>ΔIEC</i> 0.09 ± 0.01 <i>p</i> = 0.04. However, in the colon, there was no difference in CD11b-positive immune cells between strains: WT 0.53 ± 0.08 versus <i>Tlr4</i>^<i>ΔIEC</i> 0.49 ± 0.08 <i>p</i> = 0.73. <b><i>Conclusions:</i></b> These data have 2 important implications. First, these data refute the assumption that epithelial TLR4 exerts physiological control of intestinal physiology and immunity in health. Second, and most importantly, these data support the use of the <i>Tlr4</i>^<i>ΔIEC</i> line in future models interrogating health and disease, confirming no confounding effects of genetic manipulation.

A Comparative Study on the Effect of Exosomes Secreted by Mesenchymal Stem Cells Derived from Adipose and Bone Marrow Tissues in the Treatment of Osteoarthritis-Induced Mouse Model

Background. Exosomes as extracellular vesicles (EVs) are nanoscale intercellular messengers secreted from cells to deliver biological signals. Today, exosomes have become a new field of research in regenerative medicine and are considered as potential therapies to control inflammation and wound healing and enhance and improve healing in many diseases. Given the global burden of osteoarthritis (OA) as the fastest-growing health condition and one of the major causes of physical disability in the aging population, research to establish EVs as therapeutic products can meet the basic clinical needs in the management of osteoarthritis and provide a therapeutic solution. Objectives. The present study is aimed at evaluating the regenerative potentials of the exosomes secreted from adipose and bone marrow tissue-derived mesenchymal stem cells (AD- and BM-MSCs) in ameliorating the symptoms of OA. Method. In this experimental study, AD- and BM-MSCs were isolated and cultured in the laboratory until passage 3. Finally, these cells’ secreted exosomes were isolated from their conditioned medium. Ciprofloxacin-induced OA mouse models underwent intra-articular injection of exosomes from AD-MSCs and BM-MSCs. Finally, the expression levels of collagen I and II, sox9, and aggrecan genes using real-time PCR, histological analysis, and immunohistochemical (IHC) studies were performed. Results. Real-time PCR data showed that although the expression level of collagen type II was lower in both exosome-treated groups than the normal, but it was significantly increased in comparison with the sham and OA, with higher expression in BM-Exo rather than AD-Exo group. Similarly, the histological staining and IHC results have provided almost identical data, emphasizing on better therapeutic effect of BM-MSCs-exosome than AD-MSCs-exosome. Conclusion. BM-MSCs secreted exosomes in comparison with AD-MSCs could be considered as a better therapeutic option to improve osteoarthritis and exhibit potential as a disease-modifying osteoarthritis cell-free product.

Exosomes from Adipose-Derived Stem Cells Regulate M1/M2 Macrophage Phenotypic Polarization to Promote Bone Healing via miR-451a/MIF

Objectives: Bone defects caused by diseases and trauma are usually accompanied by inflammation, and the implantation of biomaterials as a common repair method has also been found to cause inflammatory reactions, which affect bone metabolism and new bone formation. This study investigated whether exosomes from adipose-derived stem cells (ADSC-Exos) plays an immunomodulatory role in traumatic bone defects and elucidated the underlying mechanisms.Methods:ADSC-Exos were loaded by a biomaterial named gelatine nanoparticles (GNPs), physical and chemical properties were analyzed by zeta potential, surface topography and rheology. A rat model of skull defect was used for our in vivo studies, micro-CT and histological staining were used to analyse histological changes in the bone defect area. RT-qPCR and western blotting were performed to verify that ADSC-Exos could regulate M1/M2 macrophage polarization. MicroRNA (miRNA) array analysis was conducted to determine the miRNA expression profiles of ADSC-Exos. After macrophages were treated with a miR-451a mimic, miR-451a inhibitor and ISO-1, the relative expression of genes and proteins was measured by RT-qPCR and western blotting.Results: In vivo, micro-CT and histological staining showed that exosome-loaded GNPs(GNP-Exos) hydrogel, with good biocompatibility and strong mechanical adaptability,exhibited immunomodulatory effect mainly by regulating macrophage immunity and promoting bone tissue healing. Immunofluorescence further indicated that ADSC-Exos reduced M1 marker (iNOS) expression and increased M2 marker (CD206) expression. Moreover, in vitro studies, western blotting and RT-qPCR showed that ADSC-Exos inhibited M1 macrophage marker expression and upregulated M2 macrophage marker expression. MiR-451a was enriched in ADSC-Exos and targeted macrophage migration inhibitory factor (MIF). Macrophages treated with the miR-451a mimic showed lower expression of M1 markers. In contrast, miR-451a inhibitor treatment upregulated the expression of M1 markers and downregulated the expression of M2 markers, while ISO-1 (a MIF inhibitor) treatment upregulated miR-451a expression and downregulated M1 macrophage marker expression.Conclusion: GNP-Exos can effectively regulate bone immune metabolism and futher promote bone healing partly through immune regulation of miR-451a,which may provide a therapeutic direction for bone repair.

The Cortical Spectrum: a robust structural continuum in primate cerebral cortex revealed by histological staining and magnetic resonance imaging

High-level characterizations of the primate cerebral cortex sit between two extremes: on one end the cortical mantle is seen as a mosaic of structurally and functionally unique areas, and on the other it is seen as a uniform six-layered structure in which functional differences are defined solely by extrinsic connections. Neither of these extremes captures the crucial neuroanatomical finding: that the cortex exhibits systematic gradations in architectonic structure. These gradations have been shown to predict cortico-cortical connectivity, which in turn suggests powerful ways to ground connectomics in anatomical structure, and by extension cortical function. A challenge to more widespread use of this concept is the labor-intensive and invasive nature of histological staining, which is the primary means of recognizing anatomical gradations. Here we show that a novel computational analysis technique can be used to derive a coarse-grained picture of cortical variation. For each of 78 cortical areas spanning the entire cortical mantle of the rhesus macaque, we created a high dimensional set of anatomical features derived from captured images of cortical tissue stained for myelin and SMI-32. The method involved semi-automated de-noising of images, and enabled comparison of brain areas without hand-labeling of features such as layer boundaries. We applied nonmetric multidimensional scaling (NMDS) to the dataset to visualize similarity among cortical areas. This analysis shows a systematic variation between weakly laminated (limbic) cortices and sharply laminated (eulaminate) cortices. We call this smooth continuum the ‘cortical spectrum’. We also show that this spectrum is visible within subsystems of the cortex: the occipital, parietal, temporal, motor, prefrontal, and insular cortices. We compared the NMDS-derived spectrum with a spectrum produced using T1- and T2-weighted magnetic resonance imaging (MRI) data derived from macaque, and found close agreement of the two coarse-graining methods. This evidence suggests that T1/T2 data, routinely obtained in human MRI studies, can be used as an effective proxy for data derived from high-resolution histological methods. More generally, this approach shows that the cortical spectrum is robust to the specific method used to compare cortical areas, and is therefore a powerful tool to understand the principles of organization of the primate cortex.

Mesenchymal Stromal Cells Enhance Vascularization and Epithelialization within 7 Days after Gingival Augmentation with Collagen Matrices in Rabbits

Soft gingival tissue deficiency remains a severe problem leading to postoperative recession, peri-implantitis, and bone resorption. The use of collagen matrices does not always lead to complete rebuilding of the gingiva volume. The application of mesenchymal stromal cells (MSCs) simultaneously with collagen materials represents a promising approach for the restoration of soft gingival tissues. However, short-term effects of MSCs-enriched collagen grafts after gingival augmentation have not yet been studied properly. Mucograft and Mucoderm matrices were implanted in rabbits (n = 12) simultaneously with the intraoperative injection of rabbit bone marrow-derived mesenchymal stromal cells (BM-MSCs) or without cells. Collagen matrices were implanted under the flap or by the surface technique without intentional primary closure. The samples were harvested seven days after implantation, histological staining with hematoxylin and eosin, and immunohistochemical staining for VEGF, IGF1, and TGF were performed. The use of Mucoderm led to better augmentation outcomes on day 7 compared with Mucograft (p < 0.0001). Gingival augmentation in combination with the local administration of BM-MSCs led to better regeneration of the soft gingival tissues independently of the type of implanted collagen matrices (p < 0.0001). Furthermore, injection of BM-MSCs significantly enhanced gingival vascularization and epithelization with a clear positive correlation between vascular growth and epithelial response. Administration of BM-MSCs in combination with various collagen materials may potentially improve gingiva regeneration.