Germ Layers Research Articles

Inhibition of the serine protease HtrA1 by SerpinE2 suggests an extracellular proteolytic pathway in the control of neural crest migration.

We previously showed that SerpinE2 and the serine protease HtrA1 modulate fibroblast growth factor (FGF) signaling in germ layer specification and head-to-tail development of Xenopus embryos. Here we present an extracellular proteolytic mechanism involving this serpin-protease system in the developing neural crest (NC). Knockdown of SerpinE2 by injected antisense morpholino oligonucleotides did not affect the specification of NC progenitors but instead inhibited the migration of NC cells, causing defects in dorsal fin, melanocyte and craniofacial cartilage formation. Similarly, overexpression of the HtrA1 protease impaired NC cell migration and the formation of NC-derived structures. The phenotype of SerpinE2 knockdown was overcome by concomitant downregulation of HtrA1, indicating that SerpinE2 stimulates NC migration by inhibiting endogenous HtrA1 activity. SerpinE2 binds to HtrA1, and the HtrA1 protease triggers degradation of the cell surface proteoglycan Syndecan-4 (Sdc4). Microinjection of Sdc4 mRNA partially rescued NC migration defects induced both by HtrA1 upregulation and SerpinE2 downregulation. These epistatic experiments suggest a proteolytic pathway by a double inhibition mechanism: SerpinE2 ┤HtrA1 protease ┤Syndecan-4 → NC cell migration

Inhibition of the serine protease HtrA1 by SerpinE2 suggests an extracellular proteolytic pathway in the control of neural crest migration

We previously showed that SerpinE2 and the serine protease HtrA1 modulate fibroblast growth factor (FGF) signaling in germ layer specification and head-to-tail development of Xenopus embryos. Here, we present an extracellular proteolytic mechanism involving this serpin-protease system in the developing neural crest (NC). Knockdown of SerpinE2 by injected antisense morpholino oligonucleotides did not affect the specification of NC progenitors but instead inhibited the migration of NC cells, causing defects in dorsal fin, melanocyte, and craniofacial cartilage formation. Similarly, overexpression of the HtrA1 protease impaired NC cell migration and the formation of NC-derived structures. The phenotype of SerpinE2 knockdown was overcome by concomitant downregulation of HtrA1, indicating that SerpinE2 stimulates NC migration by inhibiting endogenous HtrA1 activity. SerpinE2 binds to HtrA1, and the HtrA1 protease triggers degradation of the cell surface proteoglycan Syndecan-4 (Sdc4). Microinjection of Sdc4 mRNA partially rescued NC migration defects induced by both HtrA1 upregulation and SerpinE2 downregulation. These epistatic experiments suggest a proteolytic pathway by a double inhibition mechanism:SerpinE2 ┤HtrA1 protease ┤Syndecan-4 → NC cell migration.

Assessing the role of surface layer and molecular probe size in diffusion within meniscus tissue.

Diffusion within extracellular matrix is essential to deliver nutrients and larger metabolites to the avascular region of the meniscus. It is well known that both structure and composition of the meniscus vary across its regions; therefore, it is crucial to fully understand how the heterogenous meniscal architecture affects its diffusive properties. The objective of this study was to investigate the effect of meniscal region (core tissue, femoral, and tibial surface layers) and molecular weight on the diffusivity of several molecules in porcine meniscus. Tissue samples were harvested from the central area of porcine lateral menisci. Diffusivity of fluorescein (MW 332 Da) and three fluorescence-labeled dextrans (MW 3k, 40k, and 150k Da) was measured via fluorescence recovery after photobleaching. Diffusivity was affected by molecular size, decreasing as the Stokes' radius of the solute increased. There was no significant effect of meniscal region on diffusivity for fluorescein, 3k and 40k dextrans (p>0.05). However, region did significantly affect the diffusivity of 150k Dextran, with that in the tibial surface layer being larger than in the core region (p = 0.001). Our findings contribute novel knowledge concerning the transport properties of the meniscus fibrocartilage. This data can be used to advance the understanding of tissue pathophysiology and explore effective approaches for tissue restoration.

Ventricular Epicardial Adipose Distribution on Human Hearts: 3-Dimensional Reconstructions and Quantitative Assessments.

Epicardial interventions have forged new frontiers in cardiac ablation and device therapies. Healthy human hearts typically present with significant adipose tissue layers superficial to the ventricular myocardium and may hinder success or increase the complexities of epicardial interventions. We quantitatively evaluated the distribution of epicardial adipose tissue on the surface of human hearts and provided high-fidelity 3-dimensional reconstructions of these epicardial adipose tissue layers. The regional thickness of adipose tissues was analyzed at 51 anatomical reference points surrounding both ventricles and compared to specific patient demographics. Adipose deposits on the human hearts displayed characteristic patterns, with the thickest accumulations along the interventricular septa (anterior, 9.01 ± 0.50mm; posterior, 6.78 ± 0.50mm) and the right ventricular margin (7.44 ± 0.57mm). We provide one of the most complete characterizations of human epicardial adipose location and relative layer thickness. These results are considered fundamental for an underlying anatomic understanding when performing procedures within the pericardial space.

A Novel Method for Fabricating the Undulating Structures at Dermal-Epidermal Junction by Composite Molding Process.

The dermal-epidermal junction (DEJ), located between the dermal-epidermal layers in human skin tissue, plays a significant role in its function. However, the limitations of biomaterial properties and microstructure fabrication methods mean that most current tissue engineered skin models do not consider the existence of DEJ. In this study, a nanofiber membrane that simulates the fluctuating structure of skin DEJ was prepared by the composite molding process. Electrospinning is a technique for the production of nanofibers, which can customize the physical and biological properties of biomaterials. At present, electrospinning technology is widely used in the simulation of customized natural skin DEJ. In this study, four different concentration ratios of poly (lactic-co-glycolic acid) (PLGA) and polycaprolactone (PCL) nanofiber membranes were prepared based on electrospinning technology. We selected a 15%PLGA + 5%PCL nanofiber membrane with mechanical properties, dimensional stability, hydrophilicity, and biocompatibility after physical properties and biological characterization. Then, the array-based microstructure model was prepared by three-dimensional (3D) printing. Subsequently, the microstructure was created on a 15%PLGA + 5%PCL membrane by the micro-imprinting process. Finally, the cell proliferation and live/dead tests of keratinocytes (HaCaTs) and fibroblasts (HSFs) were measured on the microstructural membrane and flat membrane. The results showed that 15%PLGA + 5%PCL microstructure membrane was more beneficial to promote the adhesion and proliferation of HaCaTs and HSFs than a flat membrane.

Analysis of muscle implanted antenna performance with the variation of implantation depth

Abstract Implantation depth of Implantable Medical Devices (IMDs) is an important factor of communication quality between implantable antennas in IMDs and external devices. Therefore, it is required to study the effect of implantation depth on implantable antenna performance. In this article, dependence of resonant frequency and scattering parameters of a two-antenna system where one miniaturized meander-line antenna is placed within human muscle tissue layer operating at 2.45 GHz ISM (industrial, scientific and medical) band and a rectangular patch antenna placed outside human body, are tested. Here 15 values of implantation depth ranging over 5–35 mm inside muscle layer are taken and for each case resonant frequency and scattering parameters (S 11 and S 21) of the system are recorded. Statistical analysis has been performed to observe how these performance parameters are dependent on depth of implantation which may be varied at the time of surgery.

Spatial and Depth Profiling of Agricultural Formulations in Leaf Tissue Using LAESI Mass Spectrometry.

Formulating agrochemical products involves combining several chemical components, including the active ingredient(s), to obtain a final product with desirable efficacy. A formulated product incorporates additional components to modulate properties that enhance the efficacy of the active(s) by modifying physical properties such as viscosity, hydrophobicity, miscibility, and others. In plants, understanding the formulation's ability to spread on tissues and penetrate through the outer layer is critical in evaluating the efficacy of the final product. We have previously demonstrated the use of mass spectrometry imaging to determine spreadability. In this study, we show that laser ablation electrospray mass spectrometry (LAESI-MS) can be a valuable tool to assess the penetrability of formulations into the leaf tissues by selectively sampling various layers of leaf tissue by manipulating the laser intensity and analyzing the ablated material using a mass spectrometer. Using this technique, we were able to identify a formulation composition that can improve the penetration and uptake of active ingredients.

A 3D bioprinted hydrogel gut-on-chip with integrated electrodes for transepithelial electrical resistance (TEER) measurements

Conventional gut-on-chip (GOC) models typically represent the epithelial layer of the gut tissue, neglecting other important components such as the stromal compartment and the extracellular matrix (ECM) that play crucial roles in maintaining intestinal barrier integrity and function. These models often employ hard, flat porous membranes for cell culture, thus failing to recapitulate the soft environment and complex 3D architecture of the intestinal mucosa. Alternatively, hydrogels have been recently introduced in GOCs as ECM analogs to support the co-culture of intestinal cells in in vivo-like configurations, and thus opening new opportunities in the organ-on-chip field. In this work, we present an innovative GOC device that includes a 3D bioprinted hydrogel channel replicating the intestinal villi architecture containing both the epithelial and stromal compartments of the gut mucosa. The bioprinted hydrogels successfully support both the encapsulation of fibroblasts and their co-culture with intestinal epithelial cells under physiological flow conditions. Moreover, we successfully integrated electrodes into the microfluidic system to monitor the barrier formation in real time via transepithelial electrical resistance measurements.

Evaluation of the impact of iPSC differentiation protocols on transcriptomic signatures

Human induced pluripotent stem cells (iPSC) have the potential to produce desired target cell types in vitro and allow for the high-throughput screening of drugs/chemicals at population level thereby minimising the cost of drug discovery and drug withdrawals after clinical trials. There is a substantial need for the characterisation of the iPSC derived models to better understand and utilise them for toxicological relevant applications. In our study, iPSC (SBAD2 or SBAD3 lines obtained from StemBANCC project) were differentiated towards toxicologically relevant cell types: alveolar macrophages, brain capillary endothelial cells, brain cells, endothelial cells, hepatocytes, lung airway epithelium, monocytes, podocytes and renal proximal tubular cells. A targeted transcriptomic approach was employed to understand the effects of differentiation protocols on these cell types. Pearson correlation and principal component analysis (PCA) separated most of the intended target cell types and undifferentiated iPSC models as distinct groups with a high correlation among replicates from the same model. Based on PCA, the intended target cell types could also be separated into the three germ layer groups (ectoderm, endoderm and mesoderm). Differential expression analysis (DESeq2) presented the upregulated genes in each intended target cell types that allowed the evaluation of the differentiation to certain degree and the selection of key differentiation markers. In conclusion, these data confirm the versatile use of iPSC differentiated cell types as standardizable and relevant model systems for in vitro toxicology.

Generation of induced pluripotent stem cells (CSSi017-A)(12862) from an ALS patient carrying a repeat expansion in the C9orf72 gene

Genetic expansions of the hexanucleotide repeats (GGGGCC) in the C9orf72 gene appear in approximately 40% of patients with familial ALS and 7% of patients with sporadic ALS in the European population, making this mutation one of the most prevalent genetic mutations in ALS. Here, we generated a human induced pluripotent stem cell (hiPSC) line from the dermal fibroblasts of a patient carrying a 56-repeat expansion in an ALS disease-causing allele of C9orf72. These iPSCs showed stable amplification in vitro with normal karyotype and high expression of pluripotent markers and differentiated spontaneously in vivo into three germ layers.

Generation of two induced pluripotent stem cell lines (LVPEIi007-B, LVPEIi008-B) from patients harboring homozygous mutation in ABCA4 (c.6088C>T) using non-integrative Sendai virus-based approach

Mutations in ABCA4 gene leads to the most common form of an inherited retinal disease namely, the Stargardt disease, type 1. Here, we report the generation of two different patient-specific induced pluripotent stem cell lines (LVPEIi007-B and LVPEIi008-B), carrying an identical homozygous mutation, (c.6088C>T) within the exon 44 of ABCA4 gene. These lines were generated by the reprogramming of patient-specific dermal fibroblasts, using the integration-free, Sendai viral vectors. Both lines were stably expanded and expressed the stemness and pluripotency markers, differentiated into cell types of all three germ layers, and maintained a normal karyotype.

Comparison of three methods for collecting interstitial fluid from subcutaneous tissue in mini pigs

Interstitial fluid, owing to its similarity to blood components and higher sensitivity and specificity, finds widespread application in disease diagnosis and tumor marker detection. However, collecting interstitial fluid, particularly from the deep subcutaneous connective tissue, remains challenging.•This study aimed to compare three different collection methods - push-pull perfusion, multi-filament nylon thread implantation, and tissue centrifugation - for collecting interstitial fluid from the subcutaneous connective tissue layer of mini-pigs. High-performance ion chromatography was employed to analyze the conventional cation components in the samples and compare ion composition analysis between the different methods.•Results indicated that while the distribution of conventional cations in the interstitial fluid collected by the three methods was generally consistent, there were slight variations in the detection rates and concentrations of different ions. Hence, suitable collection methods should be selected based on the ions or collection sites of interest.

Thickened submucosal adipose tissue layer during endoscopic submucosal dissection of inflammatory bowel disease dysplasia

Thickened submucosal adipose tissue layer during endoscopic submucosal dissection of inflammatory bowel disease dysplasia

In vivo validation of highly customized cranial Ti-6AL-4V ELI prostheses fabricated through incremental forming and superplastic forming: an ovine model study

Cranial reconstructions are essential for restoring both function and aesthetics in patients with craniofacial deformities or traumatic injuries. Titanium prostheses have gained popularity due to their biocompatibility, strength, and corrosion resistance. The use of Superplastic Forming (SPF) and Single Point Incremental Forming (SPIF) techniques to create titanium prostheses, specifically designed for cranial reconstructions was investigated in an ovine model through microtomographic and histomorphometric analyses. The results obtained from the explanted specimens revealed significant variations in bone volume, trabecular thickness, spacing, and number across different regions of interest (VOIs or ROIs). Those regions next to the center of the cranial defect exhibited the most immature bone, characterized by higher porosity, decreased trabecular thickness, and wider trabecular spacing. Dynamic histomorphometry demonstrated differences in the mineralizing surface to bone surface ratio (MS/BS) and mineral apposition rate (MAR) depending on the timing of fluorochrome administration. A layer of connective tissue separated the prosthesis and the bone tissue. Overall, the study provided validation for the use of cranial prostheses made using SPF and SPIF techniques, offering insights into the processes of bone formation and remodeling in the implanted ovine model.

Optical design of a tunable microneedle array for photodynamic therapy of metastatic melanomas

The need for photodynamic therapy is increasing with the rise of skin cancer melanoma detection. However, low penetration depth of light in the UV range and higher scattering of skin tissues cause harsh clinical practices and reduced irradiance for the activation of chemicals such as photosensitizers to facilitate cell necrosis of malignant tumors. In this paper, a tunable microlens integrated on a microneedle array is designed for variable focusing to reduce melanomas at multiple sites with an improved intensity for reactive oxygen species production. A light distribution of 36% percent of the input intensity is achieved at the targeted distance of 4.35 mm for blue light. Red light attained a light distribution of 13% of the input intensity at a targeted distance of 6.5 mm. Designing such a light, delivery-based in-vivo biomedical device is of extreme importance for photodynamic therapy of skin cancer on and beyond the epidermal tissue layer.

Reducing fistula rate in hypospadias repair: A comparative study between standard tubularized incised plate repair and incorporation of a layer of spongiosum tissue

The most prevalent complication in hypospadias repair is fistula formation. Adhering to the principle of providing urethroplasty coverage to mitigate fistula occurrence, we implemented a modification by integrating a spongiosum layer between the urethroplasty and the dartos flap. Our hypothesis posited that this approach would result in a reduced fistula formation rate. This study is a comparative analysis involving patients under 18 years of age who underwent primary hypospadias repair. The cases were allocated into two groups. In the study group, in addition to performing tubularized incised plate (TIP) repair, a spongiosum layer harvested from both sides of the urethra was used to cover the urethroplasty (modification of the Y-to-I urethroplasty). The control group underwent a standard TIP repair. The primary outcome was to assess urethrocutaneous fistula formation. Secondary outcomes included evaluating the development of meatal stenosis and glans dehiscence, along with assessing cosmetic results by the Hypospadias Objective Penile Evaluation (HOPE) score. Overall, 154 hypospadias cases were included in the study. Eighty-seven patients (56%) were allocated to the study group, and 67 (44%) were allocated to the control group. Urethrocutaneous fistula developed in 3 (3.4%) and 11 (16.4%) patients in the study and control group, respectively (p=0.006). Glans dehiscence occurred in 2 (2.3%) patients in the study group and 4 (6%) patients in the control group (p=0.198). Meatal stenosis was observed in 4 (4.6%) patients in the study group and 5 (7.5%) patients in the control (p=0.452). The mean HOPE score was 59 in both groups (p=0.36). This study details our encounters with the novel modification and compares the results with standard TIP. Our data suggest that adding a layer of spongiosum tissue over the urethroplasty reduces the complication of urethrocutaneous fistula without increasing the occurrence of dehiscence, significant meatal stenosis, or compromising cosmetic outcomes. We advocate for implementing the described surgical modification as a viable option for hypospadias repair.

Suprasellar Immature Teratoma in A 20-year-old Male: A Case Report

Background: Immature teratomas are very rare in adults, especially in the suprasellar area, but they should be considered in the differential diagnosis, particularly when dealing with tumors located in the midline structure. Case presentation: In this study, we present a rare case of an immature teratoma in the suprasellar region of a 20-year-old male. A contrast-enhanced brain MRI revealed a multilocular cystic mass in the suprasellar area, accompanied by non-communicating hydrocephalus. To alleviate intracranial pressure, a shunting procedure was performed to redirect the cerebrospinal fluid. Moreover, tumor resection was carried out using an interhemispheric transcallosal approach. Conclusion: Histopathological examinations unveiled the presence of all three germ layers, followed by immature tissue, consistent with the features of an immature teratoma.

Dental pulp stones and their correlation with metabolic diseases.

Dental pulp calcifications or pulp stones are calcified structures found in dental pulp, mostly around or enclosing a blood vessel. The formation of these calcifications begins with concentric layers of calcified tissue within which remnants of necrotic and calcified cells may be present. The calcifications of thrombi in blood vessels, called phleboliths, may also serve as nidi for denticles. In metabolic diseases such as diabetes, hypertension or poor periodontal health, there are obvious changes in blood vessels and vascularization. In our study, we observed histopathological sections of dental pulp and correlated systemic diseases such as diabetes and hypertension with poor periodontal health and dental pulp stones. The aim of our study was to evaluate the histopathology of dental pulp stones, their distribution among various age groups and sexes and to identify any correlations between pulp stone formation and systemic diseases such as type II diabetes and hypertension. Samples from 100 patients with metabolic diseases such as type II diabetes and hypertension were collected. The pulp was extirpated from the teeth that were undergoing root canal treatment, and the teeth were extracted. The collected pulp sample was fixed in 10% formaline neutral buffer, subjected to routine histopathological procedures and stained with haematoxylin and eosin. The pulp of teeth extracted for orthodontic treatment was considered a control for patients with no metabolic disease. There was a definite relationship between increased pulp stones and metabolic diseases such as type II diabetes and hypertension; likewise, poor periodontal health was significantly related to pulp stones.

Optimization of energy parameters for laser-induced photodynamic therapy of cervical tissues using numerical simulation and fluorescent navigation

SignificanceThe main problem in the photodynamic therapy of tumors is insufficient light exposure to tissue depth or the appearance of undesirable surface effects. It is required to investigate the influence of energy density and radiation spot diameter on the photosensitizer photobleaching efficiency by depth. ApproachOptimization of energy parameters for laser-induced photodynamic therapy of cervical tissues was conducted using numerical simulation and fluorescent navigation. ResultsThe laser radiation fluence rate in the near-surface layer of cervical tissue increases relative to the incident radiation fluence rate with increasing spot diameter in the range from 0.2 to 15 mm. The photosensitizer photobleaching increases with an increase in the spot diameter from 5 to 15 mm. ConclusionsThe developed method will improve the efficiency of photodynamic therapy by providing a therapeutic effect throughout the entire depth of tumor invasion without superficial thermal damage.

Validating human induced pluripotent stem cell-specific quality control tests for the release of an intermediate drug product in a Good Manufacturing Practice quality system

One of the challenges in Good Manufacturing Practice (GMP)-compliant human induced pluripotent stem cell (hiPSC) production is the validation of quality control (QC) tests specific for hiPSCs, which are required for GMP batch release. This study presents a comprehensive description of the validation process for hiPSC-specific GMP-compliant QC assays; more specifically, the validation of assays to assess the potential presence of residual episomal vectors (REVs), the expression of markers of the undifferentiated state and the directed differentiation potential of hiPSCs. Critical aspects and specific acceptance criteria were formulated in a validation plan prior to assay validation. Assay specificity, sensitivity and reproducibility were tested, and the equipment used for each assay was subjected to performance qualification. A minimum input of 20 000 cells (120 ng of genomic DNA) was defined for accurate determination of the presence of REVs. Furthermore, since vector loss in hiPSC lines is a passage-dependent process, we advocate screening for REVs between passages eight and 10, as testing at earlier passages might lead to unnecessary rejection of hiPSC lines. The cutoff value for assessment of markers of the undifferentiated state was set to the expression of at least three individual markers on at least 75% of the cells. When multi-color flow cytometry panels are used, a fluorescence minus one control is advised to ensure the control for fluorescent spread. For the assay to assess the directed differentiation potential, the detection limit was set to two of three positive lineage-specific markers for each of the three individual germ layers. All of our assays proved to be reproducible and specific. Our data demonstrate that our implemented analytical procedures are suitable as QC assays for the batch release of GMP-compliant hiPSCs.