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.

Osteoporosis stems from an unbalance between bone mineral resorption and deposition. Among the numerous cellular players responsible for this unbalance bone marrow (BM) monocytes/macrophages, mast cells, T and B lymphocytes, and dendritic cells play a key role in regulating osteoclasts, osteoblasts, and their progenitor cells through interactions occurring in the context of the different bone compartments (cancellous and cortical). Therefore, the microtopography of immune cells inside trabecular and compact bone is expected to play a relevant role in setting initial sites of osteoporotic lesion. Indeed, in physiological conditions, each immune cell type preferentially occupies either endosteal, subendosteal, central, and/or perisinusoidal regions of the BM. However, in the presence of an activation, immune cells recirculate throughout these different microanatomical areas giving rise to a specific distribution. As a result, the trabeculae of the cancellous bone and endosteal free edge of the diaphyseal case emerge as the primary anatomical targets of their osteoporotic action. Immune cells may also transit from the BM to the depth of the compact bone, thanks to the efferent venous capillaries coursing in the Haversian and Volkmann canals. Consistently, the innermost parts of the osteons and the periosteum are later involved by their immunomodulatory action, becoming another site of mineral reabsorption in the course of an osteoporotic insult. The novelty of our updating is to highlight the microtopography of bone immune cells in the cancellous and cortical compartments in relation to the most consistent data on their action in bone remodeling, to offer a mechanist perspective useful to dissect their role in the osteoporotic process, including bone damage derived from the immunomodulatory effects of endocrine disrupting chemicals.

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.

Brown adipose tissue (BAT) can provide a novel therapeutic to treat obesity. Using adult male rat, thyroid stem cells (TSC) recently isolated by our group we have studied their differentiation potential to BAT as a cell source to engineer metabolically-active biomaterial-based tissue implants. TSC were obtained as colony forming unit-like cultures [1] whereas adipose differentiation was reached based on a protocol with 4 cycles (6 d/cycle) of white adipogenic induction. Presence of lipid droplets was assessed by light microscopy (LM) using oil red O histochemistry, their ultrastructural morphology studied by TEM, and difference in the 3D cellular morphology screened by SEM. Finally, a morpho- metric analysis was conducted with LM to determine the contribution of different cell phenotypes at control and differentiated levels, and their relevant subcellular features. More than 90% of control, adult TSC displayed a multipolar morphology, and flattening at increasing times as opposed to less than 10% of cells that were fibroblastoid. Similar, adipoblast-like cells exhibited a multipolar and, less frequently fibroblastoid morphotypes characterized by the absence of intracellular triglycerides. In contrast, white preadipocytes were identified as multipolar and ovoid cells containing small lipid droplets fusing into bigger ones. Differently, brown preadipocytes displayed a multipolar filamen- tous-rich phenotype, with abundant lipid droplets around 50% less in size than those of the white counterpart. Finally, few mature brown adipocytes were observed, depicting a polygonal shape with a central nucleus surrounded by sizeable lipid droplets, and a nucleus/cytoplasm ratio lower than that of all preadipocytes. At 21 days of induction, 70% of adipoblast-like cells were replaced by white preadipocytes; however, brown preadipocytes increased in number throughout the differentiation time, reaching 14% of all cells at 28 days. Our results show that adult male rat TSC have a remark- able potential to differentiate in culture to the brown lineage even in the absence of specific browning stimuli, providing an innovative source to engineer metabolically-active bioimplants for the treatment of obesity.

Here we have developed and validated an original LC-MS/MS SRM procedure flexible enough to quantitatively screen collagen types I-V in copies of the same type of stromal matrix prepared with different protocols of cell removal to retain the native 3D architecture of the ECM. In a first step, identification of tryptic sequences exclusive to specific chains (either α1 or α2) of mammalian collagen standards types I-V was pursued using a combination of LC-LIT-Orbitrap XL and LC-MS/MS SRM analyses. In a second step, the adult male rat thyroid was decellularized using three different protocols specifically set for engineering of bioartificial 3D thyroid organoids. In a third step, DNA analysis of the decellularized 3D thyroid stroma was pursued to exclude contamination by cell nuclear debris. In a final step, collagen standards and 3D thyroid matrices were digested using the same mechanical / enzymatic protocol, and quantitative profiles of collagen types I-V ensued using comparisons of ionic intensities between tryptic peptides of collagen standards and matrices, as derived from targeted LC-MS/MS SRM analysis. Collectively, the procedure allowed for detection and quantitation of collagen types I-V at a femtomolar level in thyroid gland stromal matrices initially maintaining their original 3D architecture, tryptically digested through a method common to collagen standards and thyroid ECM, with satisfactory reproducibility of results, moderate procedural cost, and limited analytical time.

The cartilage of the adult human nasal septum has recently been found as a source of neuroec- todermal-derived chondrocytes exhibiting capacity to regenerate the articular cartilage [1,2]. To shed light on the key cellular players of this differentiation, we studied the contribution of neural crest (NC)- and primary mesoderm (Mes)-derived stem cells to the development of the nasal septum in the human embryo. Ninety-nine sagittal and horizontal, paraffin-embedded and formaldehyde-fixed sec- tions of 8 human embryos (CRL 11, 19, 20, 24, 30) from the collection of the Museum and Historical Library of Biomedicine (BIOMED) of the University of Parma were used. After dewaxing and rehydra- tion, tissue epitope retrieval was achieved soaking sections in boiling citrate buffer pH 6.0 for 20 min, followed by immunocytochemical labelling with primary antibodies to Wnt1, Notch1, Msi1, Nestin, Sox10, and Chromogranin A (Chrom A) as NC markers, and Brachyury (T) as Mes marker. Immuno- reactive (IR) material was detected using either the peroxidase or alkaline phosphatase - ABC tech- niques, and DAB or Vector Red as chromogens, respectively and analyzed with light microscopy. A topographical arrangement was apparent for both Mes- and NC-derived stem cells: vertical stripes of Wnt1-IR cells segmented the lateral aspects of the septal primordium, moving from a posterior to an anterior direction. These cell fields alternated with either Notch1-IR, T-IR, or Nestin-IR cell columns / groups, the latter two diffusing also into the medial aspects of the septum. In contrast, Sox10-IR, T-IR, Chrom A-IR, and Msi1-IR cell groups contributed to the most anterior portion of the lateral aspects of the septal cartilage, giving rise to a caudally- to cranially- oriented pile of individual stem cell fields. These results raise the possibility that development of the human nasal septum is driven by a place- dependent, morphogenetic code provided by both NC- and Mes-derived stem cells. In addition, it sug- gests that different areas of the adult nasal septum may provide different types of stem cells, whose differentiation potential could be selectively exploited in bioengineering of cartilaginous grafts for the repair of a variety of mesodermal tissues including the articular and intervertebral disk cartilages.

We have recently characterized and differentiated towards endodermal and mesoder- mal lineages progenitor cells of the adult rat thyroid, expressing multipotency markers [1]. We have now assessed their clonogenicity, extent of side population, consistency of stem cell marker expression, and commitment to either follicular or hepatocyte-like lineages when in monolayer (2D), and suspension or Matrigel (3D). Colony forming unit (CFU)-like cultures were obtained by long-term subcultures of primary rat thyroid cells, under starvation conditions. CFU-like cultures seeded in Petri dishes by limiting dilution (1 cell / cm2) were observed to give rise to toluidine blue-positive, individual clones. In these cultures, quantitative densitometric analysis of immunoblotted Oct-3/4, Sca1, and GATA4 revealed an increase in stem cell markers ranging from 95% to 270% with respect to standard, primary thyroid cultures. In addition, using three different analytical techniques including DyeCycle Violet staining by flow cytometry, ABCG2 immunocytochemistry, and Hoechst 33342 histochemistry + the ABCG2 inhibitor, verapamil a side population involving 1-2% of CFU-like cultures was detected. Then, CFU-like cultures were differentiated using TSH, either in 2D or in 3D. Differentiated adherent cells resulted immunopositive for thyrocyte markers including thyroglobulin (TG), sodium-iodide symporter (NIS), and thyroperoxidase (TPO). Differentiation in suspension and in Matrigel gave rise to follicles with cells having ultrastructural features consistent with thyrocytes, and immunoreactivity (IR) for TG, NIS, and TPO. Finally, CFU-like cultures were differentiated in adherence to hepatocyte-like cells, resulting in pre-hepatocyte morphology, high periodic acid-Schiff reaction, and IR for α-fetoprotein and albumin. We conclude that our CFU-like thyroid cultures are enriched with a multipotent, stem cell population whose hepatic differentiation capacity has been revealed for the first time.

We recently identified adult stem / progenitor cells in the male rat thyroid, based on expression of the multipotency maker, ATP-binding cassette subfamily G member 2 (ABCG2) (1). To characterize these cells, we have now determined their distribution as a side population of the adult thyroid gland, identified their epithelial vs mesenchymal commitment by the presence of cytoplasmic intermediate filaments, and enriched their number using long-term, in vitro expansion of adherent elements. Sprague-Dawley male rats (50-75 gr) were used as thyroid donors. Following penthobarbital anesthesia rats were thyroidectomised, and primary cells prepared using enzymatic breaking of the gland. After 72 hs in standard monolayer culture, adherent cells were trypsinized, and either incubated for 90 min with the vital dye, Hoechst 33342 (Hch) + the ABCG2 inhibitor, verapamil (VE, 150 mM) followed by cytospin (1300 RPM x 8 min) for single, double, and triple light microscopic immunocytochemistry (IC), or re-seeded (20 x103 / cm2) in monolayer and grown up to 4 months, using a starvation protocol based on a single weekly change of culture medium (low glucose DMEM / 15 % FBS-FHS serum). Co-localization of nuclear Hoch with immunoreactive (IR) ABCG2 (rabbit anti-human polyclonal antiserum, 1:300), IR-cytokeratin (CTK, mAb 1:200), and IR-vimentin (VIM, mAb 1:100) was assessed by the ABC and indirect fluorescence techniques, using DAB, FITC and TRITC as chromogens. Rat kidney, human keratinocyte cell line, NTCT 2544 (courtesy of C. Pellegrini), and primary mouse and human fibroblasts (courtesy of D. Mattioli) were used as positive controls for IC. A consistent increase in Hch-positive nuclei was observed in VE-treated cultures, as opposed to VE-untreaded monolayers. In addition, an inverse staining relationship occurred between nuclear Hch and IR-CTK, as opposed to a direct relationship between nuclear Hch and IR-VIM. Co-localization of IR-ABCG2 with IR-CTK was seen in some cells, whereas that of IR-ABCG2 with IR-VIM was only occasionally detected. Finally, longterm expansion of primary thyrocytes resulted in 30% increase in IR-ABCG2 cells, as opposed to less than 1% IR-ABCG2 elements in standard culture. We conclude that ABCG2-positive cells of the rat thyroid are a side population of stem / progenitor elements, they are primarily committed to the epithelial phenotype, and can be enriched in vitro as adherent cells, suggesting clonal expansion.

Few data are available on the effect of biomaterials on surface antigens of mammalian bone marrow-derived, adult mesenchymal stromal cells (MSCs). Since poly(L-lactic acid) or PLLA is largely used in tissue engineering of human bones, and we are developing a reverse engineering program to prototype with biomaterials the vascular architecture of bones for their bioartificial reconstruction, both in humans and animal models, we have studied the effect of porous, flat and smooth PLLA scaffolds on the immunophenotype of in vitro grown, rat MSCs in the absence of any coating, co-polymeric enrichment, and differentiation stimuli. Similar to controls on plastic, we show that our PLLA scaffold does not modify the distribution of some surface markers in rat MSCs. In particular, the maintained expression of CD73 and CD90 on two different subpopulations (small and large cells) is consistent with their adhesion to the PLLA scaffold through specialized appendages, and to their prominent content in actin. In addition, our PLLA scaffold favours retention of the intermediate filament desmin, believed a putative marker of undifferentiated state. Finally, it preserves all rat MSCs morphotypes, and allows for their survival, adhesion to the substrate, and replication. Remarkably, a subpopulation of rat MSCs grown on our PLLA scaffold exhibited formation of membrane protrusions of uncertain significance, although in a size range and morphology compatible with either motility blebs or shedding vesicles. In summary, our PLLA scaffold has no detrimental effect on a number of features of rat MSCs, primarily the expression of CD73 and CD90.

It is currently known that a number of human vascular systems have a fractal geometry. Since we have recently developed a technique to prototype single arterial branches of human soft tissue organs by additive layer manufacturing (AM), we have explored the possibility that auto-similarity in vessel branching represents a key variable for accurate computational modeling of the organ three-dimensional (3D) macro/microscopic anatomy, and its reproduction by inverse engineering. To this purpose, ramification features of the intralobar arteries of the human thyroidwere studied using injection-corrosion casts of the cadaveric gland. Vessel diameters, ramification angles, and branch lengths were measured by light microscopic, computer-aided optical metrology. Distribution of morphological variables was considered on a cumulative basis, and special focus was given to the branching laws. To reduce the bias of vascular distortion due to the pressure of intravascular resin injection, measures were made dimensionless through the use of a scaling parameter set on the vascular caliber of major afferent arteries. In addition, using high resolution microtomography (mCT Skyscan 1172, Bruker microCT) equipped with CTAn software and the Otsu algorithm for segmentation, spaces occupied by vascular branches (referred to as Volume of Interests, VOI) were selected, and their planar fractal dimension calculated. Finally, a computational simulation of the vascular tree was achieved using a mixed, stochastic/deterministic algorithm, based on diffusion limited aggregation (DLA), constrained by mean values of vascular variables. Ratios among decreasing cast calibers, ramification angles, and branch lengths, respectively, were found strictly interrelated, mCT-VOI depicted fractal dimensions, and DLA simulation led to a fractal-like organization consistent with real data morphometrics. In summary, thyroid arterial geometry reliably exhibited a degree of auto-similarity, suggesting that fractality is a key feature for computational modeling and eventual AM of 3D vascular networks of the human thyroid.

We recently identified adult progenitor cells expressing multipotency markers in the rat thyroid (1). We have now studied these markers in primary cultures, thyrospheres, and adherent cells exhibiting features of side population / multilineage differentiation. Primary rat thyroid monolayers were immunolabeled / immunoblotted for ABCG2, Oct-3/4, HNF4a and Sca1. Thyrospheres were cytospinned and immunolabeled for Oct-3/4. Long-term subcultures were obtained by re-seeding monolayers at very low density, and growing them up to 5 months, using a starvation protocol to obtain colony forming unit (CFU)-like cultures. The latter were incubated with Hoechst (Hch) 33342 + the ABCG2 inhibitor, verapamil (VE), to identify a side population, and immunostained for ABCG2, vimentin (VIM), and cytokeratin (CYT). Thyroid monolayers and CFU-like cultures were differentiated using TSH, adipogenic, and osteogenic media. Up to 1/4 cells from primary monolayers and thyrospheres resulted either ABCG2-, Oct-3/4-, HNF4a-, or Sca-1-positive. In contrast, in CFU-like cultures ABCG2 was detected in up to 1/3 cells, whereas VIM was ubiquitous, and CYT disappeared. Consistently, a side population was revealed by the Hch-VE staining. Finally, CFU-like cultures differentiated to cells containing either thyroglobulin, or red oil-, or alizarin red-positive deposits. We conclude that multilineage differentiation of our CFU-like thyroid cultures reveals enrichment of a thyroid stem cell population.

We are currently developing innovative methodologies for rapid prototyping of threedimensional (3D) replicas of the vascular matrix of soft and hard tissue organs, primarily the thyroid and bones, with the intent of obtaining organomorphic scaffolds for their ex situ reconstruction (1, 2). To identify biocompatible materials suitable for this application, we have studied the effect of poly-L-lactic acid (PLLA) and poly-ε-caprolactone (PCL) on survival, adhesion and hormonal secretory activity of primary rat thyroid cells. Sprague- Dawley male rats (225-250 g) 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 seeded (20 x103 / cm2) onto microporous scaffold sheets. Biomaterial scaffolds were prepared using either 3.5-5% PLLA (Lacea H100E, Mistui Chemicals) in anydrous dichloromethane / amylene (Sigma) or 3% PCL (Aldrich) in anydrous tetrahydrofurane / BHT (Aldrich). Solutions were dropped onto a wet glass support, and let polymerize as a thin layer at 25°C in the absence of forced air flow. Pattern of polymerization was assessed with thermogravimetry. Cells were let grow on biomaterials up to 8 days. Single biomaterial sheets without cells, and standard multiwell cultures were used as controls. Every 2 days, percentage of viable cells was assessed using a count chamber and Trypan blue exclusion, whereas morphology of growing cells was analyzed using a scanning electron microscopy (SEM) technique without critical point drying. Culture supernatants were collected every 48 hs, and free forms of thyroid hormone (FT3 and FT4) assessed with chemiluminescent immunoassays. PLLA and PCL scaffold sheets exhibited a quite homogeneous thickness (10-12 μm), a repetitive and regular pore geometry, and a crystalline pattern of polymerization. Both biomaterials promoted survival, adhesion and proliferation of primary thyroid elements. Secretion of FT3 and FT4 was variably maintained during the culture period, and resulted statistically higher with respect to monolayer cultures on standard multiwells. Our results indicate that PLLA and PCL are suitable for growth and differentiation of adult rat thyroid cells in culture, and suggests that they might be exploited as bioerodible materials for rapid prototyping of vascular-like, organomorphic scaffolds.

We describe an innovative methodology combining Additive Layer Manufacturing (ALM) and indirect replication to reconstruct reticular-like, three-dimensional (3D) structures mimicking the vascular network of soft tissue and endocrine organs. Using a fractal-like algorithm capable of modelling the intraparenchymal vascular distribution of these viscera, single intraglandular branches of the human thyroid arteries were prototyped with synthetic resin, based on the algorithmic standard to layer (STL) output and ALM techniques. Satisfactory dimensional accuracy was obtained for these models, which were used as masters to evaluate protocols for their indirect replication, through both single and double procedures. Additional studies were conducted using casts of the human kidney arteries, obtained by injection/corrosion of the isolated organ. Satisfactory 3D reproduction of the external morphology of the kidney vessels was achieved. We conclude that our approach has the potential to develop up to the reconstruction with biomaterials of an entire, intraparenchymal vascular tree of soft tissue and endocrine organs.

Ricostruire in laboratorio, ossia ex situ, ghiandole endocrine bioartificiali è una prospettiva innovativa della ricerca traslazionale applicata alla medicina rigenerativa dei disturbi endocrino-metabolici. Utilizzando cellule staminali o primarie è possibile ingegnerizzare organoidi funzionali mediante ricellularizzazione sia di matrici tridimensionali (3D) acellulari allogeniche o xenogeniche, derivate da una ghiandola endocrina come quella che si vuole riprodurre, sia di supporti reticolari 3D biocompatibili amorfi o che mimano la morfologia originaria delle ghiandola, cioè organomorfi. Con questo metodo sono stati ricostruiti in modelli animali insule pancreatiche, follicoli ovarici e tiroidei, tubuli seminiferi del testicolo e parte della corteccia surrenale. Utilizzando cellule umane è stato riprodotto un abbozzo di ovaio e microaggregati di paratiroide. Le ghiandole endocrine bioartificiali promettono di fornire un’alternativa alla terapia sostitutiva con ormoni di sintesi, che richiede compliance da parte del paziente, integrandosi nei circuiti naturali di feed-back. Inoltre, quando generate con cellule autologhe e supporti organomorfi individualizzati, è plausibile riproducano una condizione secretiva simile a quella originale del soggetto donatore (terapia personalizzata). Infine, il loro uso potrebbe ridurre i costi sostenuti dal Servizio Sanitario Nazionale per le terapie croniche, superando i limiti etici e farmacologici connessi al trapianto cellulare/tissutale da cadavere e donatore vivente allogenico o xenogenico e promuovendo il mercato della salute attraverso lo sviluppo delle biotecnologie mediche, analogamente a quanto accadde, oltre un trentennio addietro, per quello delle tecnologie informatiche, con l’avvento dei semiconduttori e dei calcolatori elettronici.

We describe an innovative methodology combining Additive Layer Manufacturing (ALM) and indirect replication to reconstruct reticular-like, three-dimensional (3D) structures mimicking the vascular network of soft tissue and endocrine organs. Using a fractal-like algorithm capable of modelling the intraparenchymal vascular distribution of these viscera, single intraglandular branches of the human thyroid arteries were prototyped with synthetic resin, based on the algorithmic standard to layer (STL) output and ALM techniques. Satisfactory dimensional accuracy was obtained for these models, which were used as masters to evaluate protocols for their indirect replication, through both single and double procedures. Additional studies were conducted using casts of the human kidney arteries, obtained by injection / corrosion of the isolated organ. Satisfactory 3D reproduction of the external morphology of the kidney vessels was achieved. We conclude that our approach has the potential to develop up to the reconstruction with biomaterials of an entire, intraparenchymal vascular tree of soft tissue and endocrine organs.