Stefano Focaroli

Stem cell origin differently affects bone tissue engineering strategies

Bone tissue engineering approaches are encouraging for the improvement of conventional bone grafting technique drawbacks. Thanks to their self-renewal and multi-lineage differentiation ability, stem cells are one of the major actors in tissue engineering approaches, and among these adult mesenchymal stem cells (MSCs) hold a great promise for regenerative medicine strategies. Bone marrow MSCs (BM-MSCs) are the first- identified and well-recognized stem cell population used in bone tissue engineering. Nevertheless, several factors hamper BM-MSC clinical application and subsequently, new stem cell sources have been investigated for these purposes. The fruitful selection and combination of tissue engineered scaffold, progenitor cells, and physiologic signaling molecules allowed the surgeon to reconstruct the missing natural tissue. On the basis of these considerations, we analyzed the capability of two different scaffolds, planned for osteochondral tissue regeneration, to modulate differentiation of adult stem cells of dissimilar local sources (i.e., periodontal ligament, maxillary periosteum) as well as adipose-derived stem cells (ASCs), in view of possible craniofacial tissue engineering strategies. We demonstrated that cells are differently committed toward the osteoblastic phenotype and therefore, taking into account their specific features, they could be intriguing cell sources in different stem cell-based bone/periodontal tissue regeneration approaches.

Calcium/Cobalt Alginate Beads as Functional Scaffolds for Cartilage Tissue Engineering

Articular cartilage is a highly organized tissue with complex biomechanical properties. However, injuries to the cartilage usually lead to numerous health concerns and often culminate in disabling symptoms, due to the poor intrinsic capacity of this tissue for self-healing. Although various approaches are proposed for the regeneration of cartilage, its repair still represents an enormous challenge for orthopedic surgeons. The field of tissue engineering currently offers some of the most promising strategies for cartilage restoration, in which assorted biomaterials and cell-based therapies are combined to develop new therapeutic regimens for tissue replacement. The current study describes the in vitro behavior of human adipose-derived mesenchymal stem cells (hADSCs) encapsulated within calcium/cobalt (Ca/Co) alginate beads. These novel chondrogenesis-promoting scaffolds take advantage of the synergy between the alginate matrix and Co+2 ions, without employing costly growth factors (e.g., transforming growth factor betas (TGF-βs) or bone morphogenetic proteins (BMPs)) to direct hADSC differentiation into cartilage-producing chondrocytes.

Thixotropic Peptide-Based Physical Hydrogels Applied to Three-Dimensional Cell Culture

Pseudopeptides containing the d-Oxd or the d-pGlu [Oxd = (4R,5S)-4-methyl-5-carboxyl-oxazolidin-2-one, pGlu = pyroglutamic acid] moiety and selected amino acids were used as low-molecular-weight gelators to prepare strong and thixotropic hydrogels at physiological pH. The addition of calcium chloride to the gelator solutions induces the formation of insoluble salts that get organized in fibers at a pH close to the physiological one. Physical characterization of hydrogels was carried out by morphologic evaluation and rheological measurements and demonstrated that the analyzed hydrogels are thixotropic, as they have the capability to recover their gel-like behavior. As these hydrogels are easily injectable and may be used for regenerative medicine, they were biologically assessed by cell seeding and viability tests. Human gingival fibroblasts were embedded in 2% hydrogels; all of the hydrogels allow the growth of encapsulated cells with a very good viability. The gelator toxicity may be correlated with their tendency to self-assemble and is totally absent when the hydrogel is formed.

Chondrogenic differentiation of human adipose mesenchimal stem cells: Influence of a biomimetic gelatin genipin crosslinked porous scaffold

Human adipose derived stem cells have shown chondrogenic differentiation potential in cartilage tissue engineering in combination with biomimetic materials. In this study, the chondrogenic potential of a porous gelatin based scaffold genipin (GNP) crosslinked was investigated in human mesenchymal stem cells obtained from adipose tissue. Cells were cultured up to 4 weeks on the scaffold and on monolayer, MTT assay was performed to evaluate cell viability, light, and transmission electron microscopy were carried out to demonstrate cell proliferation, scaffold adhesion, and cell colonization inside the porous architecture of the biomaterial. The expression of chondrogenic markers such as SOX9, collagen type II, aggregan, and versican was investigated by Real Time PCR. Results showed an high cell viability, adhesion, and colonization of the scaffold. Real Time PCR data demonstrated an upregulation of all the chondrogenic markers analyzed. In conclusion, 3D gelatin GNP crosslinked porous scaffold provides an improved environment for chondrogenic differentiation of stem cells compared with cell monolayer culture system. Microsc. Res. Tech. 77:928–934, 2014.

Novel PLA microspheres with hydrophilic and bioadhesive surfaces for the controlled delivery of fenretinide

Abstract Novel polylactide (PLA) microspheres endowed with hydrophilic and bioadhesive surfaces as injectable formulations for the controlled release of fenretinide were prepared, using a novel technique based on the co-precipitation of PLA with gelatin, at the interface of a liquid dispersion formed by the addition of N-methylpyrrolidone containing PLA and dextrin (DX), towards an aqueous solution of gelatin (G). The resulting PLA–G–DX microspheres were compared with others prepared by the same technique using polylactide-co-glycolide (PLGA), with or without DX, and with or without phosphatidylcholine. Of the different systems, the PLA–G–DX microspheres had the best morphological, dimensional and functional characteristics. They had the highest drug loading, and their drug release was the most efficient over time without any burst effect. Their in vitro anti-tumoural activity was strongly enhanced with respect to the pure fenretinide. This paralleled the increased drug concentration inside the cells due to their marked bioadhesion to the tumour cell membranes as indicated by scanning electron microscope images.

In vitro osteogenic and odontogenic differentiation of human dental pulp stem cells seeded on carboxymethyl cellulose-hydroxyapatite hybrid hydrogel

Stem cells from human dental pulp have been considered as an alternative source of adult stem cells in tissue engineering because of their potential to differentiate into multiple cell lineages. Recently, polysaccharide based hydrogels have become especially attractive as matrices for the repair and regeneration of a wide variety of tissues and organs. The incorporation of inorganic minerals as hydroxyapatite nanoparticles can modulate the performance of the scaffolds with potential applications in tissue engineering. The aim of this study was to verify the osteogenic and odontogenic differentiation of dental pulp stem cells (DPSCs) cultured on a carboxymethyl cellulose—hydroxyapatite hybrid hydrogel. Human DPSCs were seeded on carboxymethyl cellulose—hydroxyapatite hybrid hydrogel and on carboxymethyl cellulose hydrogel for 1, 3, 5, 7, 14, and 21 days. Cell viability assay and ultramorphological analysis were carried out to evaluate biocompatibility and cell adhesion. Real Time PCR was carried out to demonstrate the expression of osteogenic and odontogenic markers. Results showed a good adhesion and viability in cells cultured on carboxymethyl cellulose—hydroxyapatite hybrid hydrogel, while a low adhesion and viability was observed in cells cultured on carboxymethyl cellulose hydrogel. Real Time PCR data demonstrated a temporal up-regulation of osteogenic and odontogenic markers in dental pulp stem cells cultured on carboxymethyl cellulose—hydroxyapatite hybrid hydrogel. In conclusion, our in vitro data confirms the ability of DPSCs to differentiate toward osteogenic and odontogenic lineages in presence of a carboxymethyl cellulose—hydroxyapatite hybrid hydrogel. Taken together, our results provide evidence that DPSCs and carboxymethyl cellulose—hydroxyapatite hybrid hydrogel could be considered promising candidates for dental pulp complex and periodontal tissue engineering.

HEMA but not TEGDMA induces autophagy in human gingival fibroblasts

Polymerized resin-based materials are successfully used in restorative dentistry. Despite their growing popularity, one drawback is the release of monomers from the polymerized matrix due to an incomplete polymerization or degradation processes. Released monomers are responsible for several adverse effects in the surrounding biological tissues, inducing high levels of oxidative stress. Reactive oxygen species are important signaling molecules that regulate many signal-trasduction pathways and play critical roles in cell survival, death, and immune defenses. Reactive oxygen species were recently shown to activate autophagy as a mechanism of cell survival and cell death. Although the toxicity induced by dental resin monomers is widely studied, the cellular mechanisms underlying these phenomena are still unknown. The aim of the study was to investigate the behavior of human gingival cells exposed to 2-hydroxy-ethyl methacrylate (HEMA) and triethylene glycol dimethacrylate (TEGDMA) to better elucidate the mechanisms of cell survival and cell death induced by resin monomers. Primary culture of human gingival cells were exposed to 3 mmol/L of HEMA or 3 mmol/L of TEGDMA for 24, 48, and 72 h. Morphological investigations were performed by transmission electron microscopy to analyze the ultrastructure of cells exposed to the monomers. The expression of protein markers for apoptosis (caspase – 3 and PARP) and autophagy (beclin – 1 and LC3B I/II) were analyzed by western blot to investigate the influence of dental resin monomers on mechanisms underlying cell death. Results showed that HEMA treatment clearly induced autophagy followed by apoptosis while the lack of any sign of autophagy activation is observed in HGFs exposed to TEGDMA. These data indicate that cells respond to monomer-induced stress by the differential induction of adaptive mechanisms to maintain cellular homeostasis.

Gelatin crosslinked with dehydroascorbic acid as a novel scaffold for tissue regeneration with simultaneous antitumor activity

A porous scaffold was developed to support normal tissue regeneration in the presence of residual tumor disease. It was prepared by gelatin crosslinked with dehydroascorbic acid (DHA). A physicochemical characterization of the scaffold was carried out. SEM and mercury porosimetry revealed a high porosity and interconnection of pores in the scaffold. Enzymatic degradation provided 56% weight loss in ten days. The scaffold was also evaluated in vitro for its ability to support the growth of normal cells while hindering tumor cell development. For this purpose, primary human fibroblasts and osteosarcoma tumor cells (MG-63) were seeded on the scaffold. Fibroblasts attached the scaffold and proliferated, while the tumor cells, after an initial attachment and growth, failed to proliferate and progressively underwent cell death. This was attributed to the progressive release of DHA during the scaffold degradation and its cytotoxic activity towards tumor cells.

The tumor microenvironment promotes cancer progression and cell migration

The tumor microenvironment contributes to cancer progression, in part through interactions between tumor and normal stromal cells. This study analyzed morphological and molecular changes induced in co-cultured human fibroblasts (HFs) and the MG-63 osteosarcoma cell line. Co-cultured cell monolayers were morphologically analyzed using high resolution scanning electron microscopy (HR-SEM), and trans-well assays were performed to assess cell migration and invasion. Proteins involved in inflammatory responses, cancer cell invasion, and angiogenesis were assessed using western blotting. HR-SEM showed progressive spatial orientation loss by fibroblasts in contact with MG-63s, while MG-63s proliferated rapidly and invaded HF space. Trans-well assays showed enhanced MG-63 migration in the presence of HFs. IL-6 expression was increased in co-cultured HFs, possibly stimulated by TNF-α. HFs do not normally express YKL-40 but did so in co-culture. Band densitometric analyses showed that increasing YKL-40 expression was followed by VEGF overexpression, especially in MG-63s. Finally, our results confirmed fibroblasts as the main matrix metalloproteinase source in this tumor microenvironment. Our study sheds new light on how tumor-stroma interactions promote tumor development and progression, and may support identification of novel anti-cancer therapeutics.

Matricellular Protein Expression and Cell Ultrastructure as Parameters to Test In Vitro Cytotoxicity of a Biomimetic Scaffold

Following scaffold implantation, cell sufferance, in-vivo encapsulation, foreign body reaction and inflammatory response has been reported and the up- regulation of matricellular proteins is often connected with this condition. Cytotoxicity of biomaterials is generally tested according to ISO standard 10993-5 based mainly on viability tests. Additional assays, based on improved cytotoxicity knowledge, are suggested in order to better analyze the biocompatibility of implant materials. The purpose of the study was to evaluate the matricellular protein expression as biomarker for in vitro-testing the biocompatibility of implant materials. Tenascin-C, osteocalcin and osteopontin belong to the matricellular protein family and were chosen as cytotoxicity markers. Mesenchymal stem cells were seeded on collagen/hydroxyapatite scaffold and on carboxymethyl cellulose based hydrogel in order to evaluate gene/protein expression by cell viability test, Real Time PCR and western blot. Electron microscopy was carried out to evaluate the morphological changes induced by cell/scaffold interactions. A low expression of tenascin-c, osteonectin and osteopontin was demonstrated in collagen/hydroxyapatite scaffold compared to the cells cultured on tissue flasks and on hydrogel scaffold. Based on our results, we propose matricellular protein expression as parameter for testing in vitro biocompatibility of implant materials.