Porcine sclera as a model of human sclera for in vitro transport experiments: histology, SEM, and comparative permeability

PURPOSE: To evaluate porcine sclera as a model of human sclera for in vitro studies of transscleral drug delivery of both low and high molecular weight compounds. METHODS: Human and porcine scleras were characterized for thickness and water content. The tissue surface was examined by scanning electron microscopy (SEM), and the histology was studied with hematoxylin-eosin staining. Comparative permeation experiments were performed using three model molecules, acetaminophen as the model compound for small molecules; a linear dextran with a molecular weight of 120 kDa as the model compound for high molecular weight drugs; and insulin, which was chosen as the model protein. Permeation parameters such as flux, lag time, and permeability coefficient were determined and compared. RESULTS: Human and porcine scleras have a similar histology and collagen bundle organization. The water content is approx 70% for both tissues while a statistically significant difference was found for the thickness, porcine sclera being approximately twofold thicker than human sclera. Differences in thickness produced differences in the permeability coefficient. In fact, human sclera was found to be two to threefold more permeable toward the three molecules studied than porcine sclera. CONCLUSIONS: The results obtained in the present paper prove that porcine sclera can be considered a good model for human sclera for in vitro permeation experiments of both low and high molecular weight compounds. In fact, if the different tissue thickness is taken into account, comparable permeability was demonstrated. This suggests a possible use of this model in the evaluation of the transscleral permeation of new biotech compounds, which currently represent the most innovative and efficient therapeutic options for the treatment of ocular diseases

Unusual clinical presentation of primary aortoduodenal fistula

Primary aorto-enteric fistula (PAEF) develops between the native aorta and the gastro-intestinal tract, in the presence of an abdominal aortic aneurysm. It is a rare, life-threatening condition and appears to be less frequent than secondary aorto- enteric fistula, which is associated with previous aortic prosthetic reconstruction. When untreated, the overall mortality rate is almost 100%. Diagnosis may be challenging until the occurrence of a massive haemorrhage. In the presence of gross contamination, patients tend to a worse prognosis. Extra-anatomical bypass and repair of the enteric tract is the treatment of choice in case of gross contamination. In situ reconstruction is often reported in cases of mild bacterial contamination. Endovascular treatment has recently become a valid option in haemodynamically unstable patients, but a staged approach, with delayed surgical treatment, seems advisable

Endocanalicular transendothelial crossing (ETC): A novel intravasation mode used by HEK-EBNA293-VEGF-D cells during the metastatic process in a xenograft model

In cancer metastasis, intravasation of the invasive tumor cell (TCi) represents one of the most relevant events. During the last years, models regarding cancer cell intravasation have been proposed, such as the "endocanalicular transendothelial crossing" (ETC) theory. This theory describes the interplay between two adjacent endothelial cells and the TCi or a leukocyte during intravasation. Two endothelial cells create a channel with their cell membranes, in which the cell fits in without involving endothelial cell intercellular junctions, reaching the lumen through a transendothelial passage. In the present study, ten SCID mice were subcutaneously xenotransplanted with the HEK-EBNA293-VEGF-D cell line and euthanized after 35 days. Post-mortem examinations were performed and proper specimens from tumors were collected. Routine histology and immunohistochemistry for Ki-67, pAKT, pERK, ZEB-1, TWIST-1, F-actin, E-cadherin and LYVE-1 were performed followed by ultrastructural serial sections analysis. A novel experimental approach involving Computed Tomography (CT) combined with 3D digital model reconstruction was employed. The analysis of activated transcription factors supports that tumor cells at the periphery potentially underwent an epithelial-to-mesenchymal transition (EMT)-like process. Topographical analysis of LYVE-1 immunolabeled lymphatics revealed a peritumoral localisation. TEM investigations of the lymphatic vessels combined with 3D digital modelling enhanced the understanding of the endotheliocytes behavior during TCi intravasation, clarifying the ETC theory. Serial ultrastructural analysis performed within tumor periphery revealed numerous cells during the ETC process. Furthermore, this study demonstrates that ETC is an intravasation mode more frequently used by the TCi than by leukocytes during intravasation in the HEK-EBNA293-VEGF-D xenograft model and lays down the potential basis for promising future studies regarding intravasation blocking therapy

The masks of Lorenzo Tenchini : their anatomy and surgical/bioengineering clues

An academic, anatomist, and Lombrosian psychiatrist active at the University of Parma in Italy at the end of the 19th century, Lorenzo Tenchini produced ceroplastic-like masks that are unique in the anatomical Western context. These were prepared from 1885 to 1893 with the aim of 'cataloguing' the behaviour of prison inmates and psychiatric patients based on their facial surface anatomy. Due to the lack of any reference to the procedure used to prepare the masks, studies were undertaken by our group using X-ray scans, infrared spectroscopy, bioptic sampling, and microscopy analysis of the mask constituents. Results showed that the masks were stratified structures including plaster, cotton gauze/human epidermis, and wax, leading to a fabrication procedure reminiscent of 'additive layer manufacturing'. Differences in the depths of these layers were observed in relation to the facial contours, suggesting an attempt to reproduce, at least partially, the three-dimensional features of the facial soft tissues. We conclude the Tenchini masks are the first historical antecedent of the experimental method for face reconstruction used in the early 2000s to test the feasibility of transferring a complete strip of face and scalp from a deceased donor to a living recipient, in preparation for a complete face transplant. In addition, the layering procedure adopted conceptually mimics that developed only in the late 20th century for computer-aided rapid prototyping, and recently applied to bioengineering with biomaterials for a number of human structures including parts of the skull and face. Finally, the masks are a relevant example of mixed ceroplastic-cutaneous preparations in the history of anatomical research for clinical purposes

Identification of putative adult stem cells in the rat thyroid and their use in ex situ bioengineering

Adult stem cells have been recently isolated from the human and mouse thyroid. Identification has been possible by their capacity to form floating cell spheroids or thyrospheres when primary cells are cultured in the absence of serum but presence of epidermal growth factor and basic fibroblast growth factor, as well as by the presence of stem cell markers like the breast cancer-resistant protein 1 (Bcrp1)/ATP-binding cassette subfamily G member 2 (ABCG2). Using this strategy, and an innovative in vitro growing system, we have attempted identification of stem / progenitor elements from the adult rat thyroid. Sprague-Dawley male rats (50-75 gr) were used as thyroid donors. After penthobarbital anesthesia rats were thyroidectomised, thyroids surgically excised, and primary cells prepared using enzymatic breaking. After 72 hs in standard monolayer culture, cells were trypsinized and either seeded (20 x103/cm2) and grown for 8 days in a 3D Matrigel (12.5-50%) system using low-glucose DMEM and serum, or immediately cytospinned (1200 RPM x 5 min) for immunocytochemistry, or harvested and frozen with lysis buffer for Western blotting (WB). Bcrp1/ ABCG2-immunoreactivity (IR) was detected using a rabbit anti-human, polyclonal antibody (1:500, Cell Signalling), and visualized either with the ABC technique and DAB as a chromogen, or with a chemiluminescence-based staining. The human plasmocytoma cell line, RPMI 8226 (B lymphocytes) and the acute lymphoblastic leukemia cell line CCRF-CEM (T lymphocytes) were used as positive and negative controls, respectively. Thyrosphere-like aggregates were transiently observed after initial monolayer expansion and, more consistently, at day 3 in 50% Matrigel culture, followed by rapid cell differentiation (days 4-8), including epithelial-mesenchymal transitions, formation of follicles and pavment layering. Similar differentiation changes were also detected after seeding of primary thyroid cells onto decellularized rat thyroid matrixes, as previously reported [1]. Less than 0.4% of cytospinned thyroid cells exhibited cytoplasmic Bcrp1/ABCG2-IR, and a band of around 72kD was detected by WB in cell lysates. We conclude that the thyroid of the adult rat contains a small population of stem / progenitor-like elements, likely contributing to the regenerative processes that occur during ex situ recellularization of acellular thyroid matrixes [1, 2]

Ex situ bioengineering of the rat thyroid using as a scaffold the three-dimensional (3D) decellularized matrix of the glandular lobe: clues to the organomorphic principle

Recently, we designed a bioreactor system for bioengineering ex situ (i.e. on the laboratory bench) a bioartificial thyroid gland suitable for transplantation. It is based on the organomorphic principle, i.e. the bioreactor mimics the macro-microscopic architecture of the thyroid stromal-vascular scaffold (SVS). To prove the reliability of this approach, we have initiated a pilot study using as a model the rat thyroid, and its natural decellularized 3D matrix to be eventually recellularized up to formation of a viable 3D thyroid lobe ex situ. Sprague-Dawley male rats (220-240 g) were used as thyroid donors. After penthobarbital anesthesia, rats were thyroidectomised and thyroid matrixes obtained by decellularization of the native SVS. Initially, we applied a sequence of liquid N2 freezing at - 80°C / thawing at 4°C for a total of 72 h, various washings with 0.02% trypsin / 0.05% EDTA for 1 h at 37°C, 3% Triton X-100 for 72 h at 4°C, and 4% deoxycholic acid for 24 h at 4°C, followed by sterilization with 0.1% peracetic acid, and 1% penicillin / streptomycin / fungizone for 24 h. Test matrixes were made electrondense with uranium / bismuth / lead counterstaining, and analyzed by microtomography (microTC). Primary thyroid cultures were prepared using enzymatic breaking of the native thyroid tissue. Cells were seeded at 19.000 / cm 2, and grown 72 h in low-glucose DMEM supplemented with 10% FBS / 5% FCS. Following trypsinization, 450.000 cells were harvested to coat the inner surface of the matrix. After 7 and 14 days, colonized matrixes were fixed in aldheydes and processed for light (LM), transmission (TEM) and scanning electron (SEM) microscopy. Culture supernatants were collected every 48 h, and thyroid hormones assessed with chemiluminescent immunoassays. Complete decellularization and maintenance of the 3D architecture of the thyroid SVS were achieved. Thyroid-derived cells were found to aggregate, link and give rise to intracytoplasmic cavities up to follicular coating, whereas secretory de-differentiation occurred. These results show that the 3D matrix of the rat thyroid can be used as a natural scaffold to recellularize the thyroid lobe with progenitor-like elements, supporting the validity of the organomorphic principle for ex situ bioengineering of a bioartificial thyroid gland. Grants FIL09, PRIN082008ZCCJX4, FIRB2010RBAP10MLK