C Tonello

In vitro reconstruction of human dermal equivalent enriched with endothelial cells

Experiences coming from many cell-culture studies has brought about the concept that tissue and organ reconstruction should be performed in a three-dimensional environment as it normally occurs in vivo. As far as endothelial cell culture is concerned, it has been shown that angiogenesis can be successfully achieved only when cells are cultured in the presence of collagen-based matrices or basal membrane substrates. The aim of the present investigation is to demonstrate that human umbilical vein endothelial cells (HUVEC) can be grown and differentiated on an artificial dermis obtained by fibroblasts cultured on hyaluronic acid-based scaffolds. For this purpose, we have cultured HUVEC, retrieved by collagenase digestion of perfused human umbilical vein either alone and with fibroblast at 1/1 ratio into HYAFF-11 non-woven mesh. Cultures were maintained for up to 3 weeks. Samples were taken at different time points within this period for the MTT proliferation test and for immunohistochemical analysis. Our results demonstrate that hyaluronan-based biomaterials (HYAFF-11 NW mesh) represent a suitable substrate for HUVEC adhesion, proliferation and reorganization in microcapillary network.

In vitro reconstruction of an endothelialized skin substitute provided with a microcapillary network using biopolymer scaffolds

Successful in vitro reconstruction of skin requires the inclusion of several cell types that give rise in coculture to the specific elements present in native skin, and the appropriate scaffolding structure to house and support these cells. In addition to the two main structural components, epidermis and dermis, one critical apparatus of the skin is a capillary network that guarantees adequate perfusion of nutrients and oxygen. The aim of the present study was to develop an in vitro coculture system that assumed the human dermal‐epidermal architecture and included a microcapillary network in a three‐dimensional biomaterial that guaranteed ease of handling in a clinical setting. Endothelialized skin (ES) was prepared by coculturing three human cell types: keratinocytes, fibroblasts, and endothelial cells, obtained from human full‐thickness skin samples, in scaffolds produced from modified hyaluronic acid. Results were evaluated by histological and immunohistochemical analyses at different time points. In vitro, engineered skin obtained with this composite culture developed into a well‐differentiated upper layer of stratified keratinocytes lining a dermal‐like structure, in which fibroblasts, extracellular matrix and a microvascular network were present. Furthermore, the biodegradable fabric produced from hyaluronic acid and used as the scaffolding support for this in vitro constructed skin graft greatly facilitated handling in the perioperative period.

In vivo regeneration of small-diameter (2 mm) arteries using a polymer scaffold

The difficulty of obtaining significant long‐term patency and good wall mechanical strength in vivo has been a significant obstacle in achieving small‐diameter vascular prostheses. The aim of the present study was to develop a prosthetic graft that could perform as a small‐diameter vascular conduit. Tubular structures of hyaluronan (HYAFF‐11 tubules, 2 mm diameter, 1 cm length) were grafted in the abdominal aorta of 30 rats as temporary absorbable guides to promote regeneration of vascular structures. Performance was assessed by histology, immunohistochemistry, and ultra‐structural analysis. These experiments resulted in three novel findings: 1) complete endothelialization of the tubes luminal surface occurred; 2) sequential regeneration of vascular components led to complete vascular wall regeneration 15 days after surgery; and 3) the biomaterial used created the ideal environment for the delicate regeneration process during the critical initial phases, yet its biodegradability allowed for complete degradation of the construct four months after implantation, at which time, a new artery remained to connect the artery stumps. This study assesses the feasibility to create a completely biodegradable vascular regeneration guide in vivo, able to sequentially orchestrate vascular regeneration events needed for very small artery reconstruction.

Purification and characterization of a humoral opsonin, with specificity for D-galactose, in the colonial ascidian Botryllus schlosseri.

A humoral agglutinin from the hemolysate of the colonial ascidian Botryllus schlosseri was purified by affinity chromatography. This agglutinin does not require metal cations for its activity and is specific for derivatives of D-galactose. On SDS-PAGE analysis, it was resolved in two bands, of 17 and 19 kDa in reducing conditions and 15 and 16 kDa in non-reducing conditions. This behavior is due to the establishment of disulfide bridge between the thiols of cysteine, well represented in the molecule as revealed by amino acid analysis. The latter also indicated high percentages of hydrophilic residues, probably involved in sugar recognition. The lectin is an opsonin, as it increases both the phagocytic index and the number of phagocytized yeast cells. The hypothesis that this Botryllus agglutinin belongs to the galectin family of lectins is discussed.

Gland cell cultures into 3D hyaluronan-based scaffolds.

In this study we report a preliminary investigation of the feasibility of non-woven/sponge fabrics of a hyaluronan derived biomaterials (benzyl ester of HA (HYAFF-11™ FAB, Abano Terme, Italy) for the in vitro culture of rat hepatocytes and rat beta cells. Cell growth on hyaluronan derived biomaterials were tested in the presence of complete medium and in the presence of ECM (extracellular matrix) secreted by fibroblasts previously cultured into the scaffold. Hepatocytes and beta cells were extracted from rat liver/pancreas and seeded either on the HYAFF-11™ scaffold alone, or on HYAFF-11™ scaffold containing ECM. Direct assay of cell proliferation was performed with MTT test. For morphological observations samples were stained with hematoxylin and eosin. The results obtained by MTT test showed that hepatocytes cultivated in both the above described conditions were able to proliferate up to 14 days and Langerhans islet up to 21 days. After this time, cells started to undergo apoptosis. The morphological analyses showed cell aggregation in three-dimensional structures promoted by the fibers of the biomaterial. Our results confirmed that HYAFF-11™ meshes represent a suitable scaffold for hepatocyte adhesion/Langerhans islet organization and proliferation. In particular, the presence of a fibroblast secreted extracellular matrix improves the biological property of the scaffold.

Preliminary report of in vitro reconstruction of a vascularized tendonlike structure: a novel application for adipose-derived stem cells.

IntroductionA greater supply of tendinous tissue can be obtained through tissue engineering technology with increasing application of adult stem cells. It is well known that adipose-derived stem cells (ADSCs), found in abundance in adipose tissue, have the same differentiating capacity as mesenchymal stem cells yet have the advantage of being easily isolated. In the present study, we combined the great facility of ADSCs to differentiate with the application of an external mechanical stimulus to successfully create an in vitro reconstructed tendonlike structure with a microcapillary network. Materials and MethodsHyalonect meshes (Fidia Advanced Biopolymers, Abano Terme, Padova, Italy) were used as scaffold. Human ADSCs were seeded onto the biomaterials, and the cell/scaffold constructs were cultured under mechanical stress for up to 15 days. Human tenocytes were used in the same conditions as control. Performance was assessed by histology, immunochemistry, ultrastructure, and biomolecular analysis. ResultsAdipose-derived stem cells seeded onto Hyalonect adhered and differentiated along the entire surface of the biomaterial and began to infiltrate within its structure. Subsequently, endothelial cells migrated, forming a capillary in the new extracellular matrix. ConclusionsThis technique allowed for the creation of a vascularized tendon equivalent that could easily be detached from the bioreactor, thus facilitating its implant at the lesion site. These results highlight the biologic performance of biodegradable hyaluronic acid–based (HYAFF-11) scaffolds, which were shown to be suitable for deposition of the autologous extracellular matrix critical for ADSCs differentiation.