3D co-cultures of osteoblasts and endothelial cells in DegraPol foam: Histological and high field MRI analyses of pre-engineered capillary networks in bone grafts

Tissue engineering of bone grafts was addressed in a critical size model on the chick chorioallantoic membrane model (CAM assay), using DegraPol(R) (DP) foam as scaffold material. The scaffolds were seeded with cultures of human osteoblasts (OB) and human en notdo notthelial cells (EC), respectively, or with a co-culture of the two cell types (control: no cells). In vitro samples (7 days cultivation) and ex vivo CAM samples at incubation day 15 (ID 15) were analyzed by high field magnetic resonance imaging (MRI) and histology. The co-culture system performed best with respect to perfusion, as assessed by contrast-enhanced MRI using Gd-DTPA. The scaffold seeded by the co-culture supported an increased vascular ingrowth, which was confirmed by histological analysis. DP foam is a suitable scaffold for bone tissue engineering and the MRI technique allows for non-destructive and quantitative assessment of perfusion capability during early stages of bone forming constructs

Delineation of the healthy rabbit duodenum by immunohistochemistry - A short communication

The duodenum acts as a vital organ that performs fundamental physiological functions like digestion and nutrient absorption. Situated in the lower abdomen, the duodenum is located between the stomach and the jejunum. Usually, the duodenum is divided into four anatomical portions. We here compare paraffin embedded and cryosections of the healthy rabbit duodenum for research purposes. This analysis evaluates the differential outcomes resulting from the application of these fixation methodologies in conjunction with immunohistochemical assays targeting extracellular matrix markers collagen I, collagen III, fibronectin, α-smooth muscle actin (α-SMA), and proliferation marker ki67 as well as inflammatory marker PAR-2. Subsequent recommendations are provided based on our findings. Furthermore, the advantage of an antigen retrieval step in immunohistochemical labelling of paraffin sections was demonstrated and confirmed with an isotype negative control. Basic classical histological stainings as HE, GT and elastin were also performed. Comparison of different stainings and labellings was performed in serial sections, showing that adjacent to the circular muscle of the duodenum, the connective tissue was composed of collagen I and fibronectin, while the artery and vein walls were predominantly α-SMA positive. Moreover, PAR-2 immunohistochemical staining was performed, where particularly a type of gland adjacent to Brunner's glands showed prominent PAR-2 positive areas, while the Brunner's glands themselves were PAR-2 negative. Proliferating ki67 positive cells facing the lumen were highly abundant in all kinds of glands except for the Brunner's glands. This effort serves to furnish the research community with reference imagery pertinent to scientists opting for the rabbit duodenum model. The diversity of staining techniques employed herein establishes a foundational repository of images, primed for comparative analysis against pathological conditions. Furthermore, these images hold the potential to illustrate inter-species variations. For instance, they can be juxtaposed against murine or rat intestinal tracts, or even offer insights into the human context

Delineation of the healthy rabbit kidney by immunohistochemistry - A technical note.

Pre-clinical animal models are needed to investigate and study kidney injuries and diseases. The rabbit kidney model is frequently used because various important parameters can be assessed with it. For example, histology and immunohistochemistry are indispensable as tissue morphology and composition can be investigated qualitatively as well as quantitatively. Here, different histological and immunohistochemical stainings were performed in the rabbit healthy naïve kidney tissue. First, overnight formalin fixation followed by paraffin embedding and cryopreservation with a subsequent 10-minute formalin fixation prior to staining were compared. Cryosections showed a more pronounced staining pattern, with clear borders at low magnifications, but blurred borders at higher magnifications. Then, antigen retrieval (AR) for paraffin embedded sections resulted in more prominent corresponding signals compared to stainings without AR. Moreover, several advantages and disadvantages of chromogenic versus immunofluorescence stainings were considered. Chromogenic staining was advantageous compared to immunofluorescence for collagen I and III, and to a minor degree for fibronectin. Finally, distinct structures, such as the pelvis, the calices, the glomeruli and tubuli, were stained in serial sections with diverse immunohistochemical stainings in order to delineate their composition. The following stainings were performed: standard Haematoxylin&Eosin and Elastica van Gieson staining, collagen I, collagen III, fibronectin, α-SMA, ki-67 and protease-activated receptor-2 (PAR-2). While chromogenic stainings of collagen I and collagen III were particularly useful to depict kidney structures in paraffin sections compared with cryosections, cryosections immunofluorescently stained for α-SMA were superior to paraffin sections, particularly at higher magnifications. With regard to specific structures, we found renal vessel walls positive for fibronectin and α-SMA, while the Bowman's capsule was only positive for fibronectin and α-SMA showed only tiny spots. The mesangial cells of the glomeruli and the distal tubuli were PAR-2 positive, while the proximal tubuli were PAR-2 negative

Delineation of the healthy rabbit tonsil by immunohistochemistry - A short communication

Situated in the oral cavity, the rabbit palatine tonsils are part of the mucosal immune system and help to defend the body against foreign pathogens. Expressed as two oval protrusions in the wall of the oropharynx, the rabbit palatine tonsils are characterized by excretory ducts and trabeculae. We here compare paraffin embedded and cryosections of the healthy rabbit tonsils. This analysis centers on evaluating the differential outcomes resulting from the application of these fixation methodologies in conjunction with immunohistochemical assays targeting collagen I, collagen III, fibronectin, α-smooth muscle actin (α-SMA), and ki67. Subsequent recommendations are provided based on our findings. Furthermore, we demonstrate the advantage of an antigen retrieval step in immunohistochemical labeling of paraffin sections. Basic classical histological stainings as HE, GT and elastin were also performed. Comparison of different stainings and labelings was furthermore performed in serial sections, showing that adjacent to the excretory ducts, the tonsillar tissue was particularly composed of collagen I and fibronectin, while the vessel walls were predominantly α-SMA positive. Moreover, PAR-2 immunohistochemical staining was performed, where a small fraction of the cells found in the tonsillar connective tissue were PAR-2 positive (probably a subpopulation of mast cells), as well as the lumen of some excretory ducts and trabeculae. Collagen III on the other hand was only weakly expressed in the tonsils. Proliferating ki67 positive cells were rare. This endeavor serves to furnish the scientific community with reference imagery pertinent to researchers opting for the rabbit palatine tonsil model. The diversity of staining techniques employed herein establishes a foundational repository of images, primed for comparative analysis against pathological conditions. Furthermore, these images hold the potential to illustrate inter-species variations. For instance, they can be juxtaposed against murine or rodent tonsils, or even offer insights into the human context

Delineation of the healthy rabbit heart by immunohistochemistry - A technical note

Heart failure poses a big health problem and may result from obesity, smoking, alcohol and/or growing age. Studying pathological heart tissue demands accurate histological and immunohistochemical stainings in animal models, including chromogenic and fluorescent approaches. Moreover, a reliable set of healthy heart stainings and labeling are required, in order to provide a reference for the pathological situation. Heart and brain tissue of a healthy rabbit were collected, and different histological key steps were compared, such as paraffin embedding after formalin fixation versus cryopreservation; an antigen retrieval (AR) step in processing paraffin sections versus the same procedure without AR; or a chromogenic with a fluorescent detection system, respectively. Using serial sections, we stained the same morphological structure with classic approaches (HE, Masson Goldner Trichrome (GT) and Elastica van Gieson (EL)) and with different markers, including collagen I, collagen III, fibronectin, α-SMA, protease-activated receptor-2 (PAR-2) which is an inflammation-related marker, and ki67 for proliferating cells. Differences between conditions were quantitatively assessed by measuring the color intensity. Generally, cryosections exhibited a more prominent signal intensity in immunohistochemically labeled sections than in paraffin sections, but the strong staining was slurry, which sometimes impeded proper identification of morphological structures, particularly at higher magnifications. In addition, the advantage of an AR step was observed when compared to the condition without AR, where signal intensities were significantly lower. Different stainings of the heart arteries and the myocardium revealed a clear distribution of extracellular matrix components, with prominent collagen III in the artery wall, but an absence of collagen III in the myocardium. Moreover, paraffin-embedded sections provided more distinct structures compared to cryosections after collagen III, ki67, fibronectin, and α-SMA labeling. As for the Purkinje cells that were depicted in the heart and the cerebellum (Purkinje neurons), we found GT staining most suitable to depict them in the heart, while HE as well as EL staining was ideal to depict Purkinje neurons in the cerebellum. In sum, we provide useful reference images with different stainings for researchers using the rabbit heart or brain model. Such images can help to decide which of the immunohistochemical protocols are valuable to reach a specific aim. Recommendations are given for the best visualization of the target structures and specific (immunohistochemical) staining

Delineation of the healthy rabbit tongue by immunohistochemistry - A technical note

In the oral cavity the tongue is an important muscular organ that supports the swallowing of food and liquids. It is responsible for the sense of taste, based on the many different taste buds it contains. Research in the field of tongue diseases demands for suitable preclinical models. The healthy rabbit tongue may therefore serve as baseline and reference for the pathological situation. With this consideration, we covered the fixation and histological stainings as well as the immunohistochemical labelling of the healthy rabbit tongue. In this technical note, initial choice of the fixative is discussed, with a comparison of formalin fixation and subsequent paraffin embedding versus cryopreservation. Moreover, we delineate the effect of an antigen retrieval step for formalin fixation by several examples. Finally, we provide ECM markers collagen I, collagen III, fibronectin, α-SMA and elastin staining as well as ki67 for proliferative status and PAR-2 protein expression as a marker for inflammatory status and nociception in tongue sections, mainly from the tongue body. Technically, we found superiority of paraffin sections for collagen I, collagen III, fibronectin, ki67 and α-SMA labelling, for selected detections systems. As for ECM components, the lamina propria was very rich in collagen and fibronectin, while the muscular body of the tongue showed only collagen and fibronectin positive areas between the muscle fibers. Moreover, α-SMA was clearly expressed in the walls of arteries and veins. The inflammatory marker PAR-2 on the other hand was prominently expressed in the salivary glands and to some extent in the walls of the vessels. Particular PAR-2 expression was found in the excretory ducts of the tongue. This technical note has the aim to provide baseline images that can be used to compare the pathological state of the diseased rabbit tongue as well as for inter-species comparison, such as mouse or rat tongue. Finally, it can be used for the comparison with the human situation

Sensitization and desensitization of burn patients as potential candidates for vascularized composite allotransplantation

Sensitization describes the acquired ability of the immune system to react to foreign human leukocyte antigens (HLA) by producing antibodies and developing memory cells. In the field of transplantation, recipient preformed HLA antibodies due to previous sensitization have been identified - beneath ABO incompatibility - as a major factor for acute graft rejection. Several reasons for sensitization have largely been studied, such as previous blood transfusions, pregnancies or former transplants. Recent studies indicate that the use of assist devices (e.g. ECMO) or cadaveric skin allotransplantation providing temporary coverage in burn patients may lead to additional sensitization. As vascularized composite allotransplantation (VCA) has become a rapidly advancing therapeutic option for reconstruction of complex tissue defects in burns, it seems even more important to become familiar with immunological principles and to be cautiously aware of both sources of sensitization and therapeutic concepts in burns avoiding sensitization. This may also include emergency VCAs in burn patients as potential strategy for early definitive reconstruction avoiding procedures triggering HLA antibody formation. We hereby provide an overview on current evidence in the field of pre- and peritrans-plant sensitization, followed by posttransplant strategies of desensitization and their potential impact on future treatments of burn patients. (C) 2015 Elsevier Ltd and ISBI. All rights reserved.Peer reviewe

Delayed enzymatic debridement in severe burns: Proof of concept

Introduction Enzymatic debridement (ED) is a novel powerful therapy for debridement of severe burns. Standard ED is usually performed within 72 h after injury following a presoaking phase. Little evidence exists on the effectiveness of ED later than 72 h after trauma. In this retrospective study, we compared outcomes of burn patients treated within versus later than 72 h after injury. Patients and Methods 110 patients with severe burns treated with ED between 2016 and 2020 were evaluated. Patients treated later than 72 h after trauma were identified and matched to a control group treated within 72 h. Matching criteria included age, area treated with ED, and localization of ED. Exclusion criteria were abbreviated burn severity index (ABSI) greater than 12 and death within the first 10 days after burn injury. Primary outcomes were time to full epithelialization and number of secondary surgical interventions. Results 16 patients (11 female, 5 male) matched the inclusion criteria and were assigned to the late treatment group. Mean age was 54.0 ± 19.0 years, the = and mean ABSI score 6.3 ± 3.2. 16 matched patients were assigned to the early ED group. Secondary surgical procedures were performed in 62.5% of cases in both groups with a mean of 1.7 (late treatment) vs. 2.2 (control; p = 0.29) secondary procedures in each group, respectively. No significant difference between groups regarding time to complete epithelialization (28.2 days vs. 27.3 days, p = 0.45) was observed. Infection rate was higher (18.8% vs. 6.3%, p = 0.28) in the delayed group. Conclusion Delayed ED is a feasible procedure as part of personalized care in burn surgery. In our retrospective study, we could not identify r safety issues except a slightly higher infection rate. This may however be attributed to delayed initiation of burn treatment itself

Probing Vasoreactivity and Hypoxic Phenotype in Different Tumor Grafts Grown on the Chorioallantoic Membrane of the Chicken Embryo In Ovo Using MRI

Tumor grafts grown on the chorioallantoic membrane (CAM) of chicken embryos represent a transition between cell culture and mammalian in vivo models. Magnetic resonance imaging (MRI) started to harness this potential. Functional gas challenge is feasible on the CAM. Using quantitative T1 and T2* mapping, we characterized the response of MC-38 colon, A549, and H460 adeno-carcinoma cell grafts to hypercapnic (HC) and hypercapnic-hyperoxic (HCHO) gas challenges, pertaining to the grafts' vascular and oxygenation phenotypes. MR imaging revealed that larger T1 and T2* were located in the center of H460 and MC-38 tumors. Quantitative analysis showed a significant reduction in T1 and a significant increase in T2* in response to HCHO for A549 grafts, while H460 and MC-38 tumors did not respond to either gas challenge. Different tumor grafts respond differentially to HC and HCHO conditions. A549 tumor grafts, with higher vessel density and smaller tumor diameter compared with H460 and MC-38 grafts, had a significant response in T1 for HCHO and T2* increased slightly during HC and significantly under HCHO, consistent with a normoxic phenotype and functional vasoreactivity. Therefore, gas challenges enable differential characterization of tumor grafts with respect to their vascular and oxygenation status

Tumor grafts grown on the chicken chorioallantoic membrane are distinctively characterized by MRI under functional gas challenge

Recently, a tumor model based on the chorioallantoic membrane (CAM) was characterized structurally with Magnetic Resonance Imaging (MRI). Yet, capability of MRI to assess vascular functional reserve and potential of oxygenation-sensitive MRI remain largely unexplored in this model. For this purpose, we compared MC-38 colon and A549 lung adenocarcinoma cell grafts grown on the CAM, using quantitative T1 and T2* MRI readouts as imaging markers. These are associated with vascular functionality and oxygenation status when compared between periods of air and carbogen exposure. Our data show that in A549 lung adenocarcinoma cell grafts T2* values increased significantly upon carbogen exposure (p < 0.004, Wilcoxon test; no change in T1), while MC-38 grafts displayed no changes in T1 and T2*), indicating that the grafts differ in their vascular response. Heterogeneity with regard to T1 and T2* distribution within the grafts was noted. MC-38 grafts displayed larger T1 and T2* in the graft centre, while in A549 they were distributed more towards the graft surface. Finally, qualitative assessment of gadolinium-enhancement suggests that A549 grafts display more prominent enhancement compared to MC-38 grafts. Furthermore, MC-38 grafts had 65% larger volumes than A549 grafts. Histology revealed distinct underlying phenotypes of the two tumor grafts, pertaining to the proliferative status (Ki-67) and cellularity (H&E). In sum, a functional gas challenge with carbogen is feasible through gas exchange on the CAM, and it affects MRI signals associated with vascular reactivity and oxygenation status of the tumor graft planted on the CAM. Different grafts based on A549 lung adenocarcinoma and MC-38 colon carcinoma cell lines, respectively, display distinct phenotypes that can be distinguished and characterized non-invasively in ovo using MRI in the living chicken embryo