Alessandra Zamparelli

A combined additive layer manufacturing / indirect replication method to prototype 3D vascular-like structures of soft tissue and endocrine organs: A combined additive layer manufacturing (ALM)/ indirect replication method to prototype 3D vascular-like structures of soft tissue and endocrine organs is presented in this paper

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.

Growth on poly(l-lactic acid) porous scaffold preserves CD73 and CD90 immunophenotype markers of rat bone marrow mesenchymal stromal cells

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.

Thyrogenic, adipogenic, and osteogenic differentiation of adult rat, thyroid stem cells enriched by long-term adherent subculture

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.

Adult stem / progenitor cells of the rat thyroid: side population distribution, intermediate filament expression, and long-term in vitro expansion

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.

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].

Multipotent adult rat, thyroid stem cells can be differentiated to follicular thyrocyte, and hepatocyte- like cells in 2D and 3D culture systems

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.

Gli organi endocrini bioartificiali: prospettive della ricerca traslazionale applicata alla medicina rigenerativa in endocrinologia

RiassuntoRicostruire 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.

Poly-L-lactic acid and poly-ε-caprolactone as biomaterials for ex-situ bioengineering of the rat thyroid tissue

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-e-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.