Manuela Dicarlo

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.

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.

Gallium-modified chitosan/poly(acrylic acid) bilayer coatings for improved titanium implant performances

A gallium-modified chitosan/poly(acrylic acid) bilayer was obtained by electrochemical techniques on titanium to reduce orthopaedic and/or dental implants failure. The bilayer in vitro antibacterial properties and biocompatibility were evaluated against Escherichia coli and Pseudomonas aeruginosa and on MG63 osteoblast-like cells, respectively. Gallium loading into the bilayer was carefully tuned by the electrochemical deposition time to ensure the best balance between antibacterial activity and cytocompatibility. The 30min deposition time was able to reduce in vitro the viable cell counts of E. coli and P. aeruginosa of 2 and 3 log cfu/sheet, respectively. Our results evidenced that the developed antibacterial coating did not considerably alter the mechanical flexural properties of titanium substrates and, in addition, influenced positively MG63 adhesion and proliferation. Therefore, the gallium-modified chitosan/poly(acrylic acid) bilayer can be exploited as a promising titanium coating to limit bacterial adhesion and proliferation, while maintaining osseointegrative potential.

Liposome-modified titanium surface: A strategy to locally deliver bioactive molecules

Titanium and its alloys are widely employed materials for implants in orthopedic or dental surgery due to their mechanical properties, resistance to corrosion and osseointegration capability. However adverse reactions at the tissue/implant interface may occur, which limit the success of the osseointegration process. Therefore, different strategies have to be used to overcome these drawbacks. In this work, we developed two different liposome-based coatings on titanium surfaces as drug or bioactive molecule deposits for dental/orthopedic implant applications. The first one is a supported vesicular layer (SVL), obtained by liposome adhesion on passivated Ti surface, the second one is a covalently bonded vesicular layer (CBVL) grafted on properly functionalized Ti. Photoluminescence spectroscopy and atomic force microscopy investigations demonstrated the effective anchoring of intact liposomes in both systems. Cytotoxicity assays, performed after 48h, showed a MG63 cell viability higher than 75% and 70% on SVLs and CBVLs, respectively. Scanning electron microscopy investigation revealed numerous and spread MG63 cells after 48h on SVL modified Ti surface and a lower cell adhesion on samples coated with CBVL. The cellular uptake capability of liposome content was proved by fluorescence microscopy using carboxyfluorescein loaded SVLs and CBVLs. Finally, we demonstrated that these liposome-modified Ti surfaces were able to deliver a model bioactive molecule (phosphatidylserine) to adherent cells, confirming the potentiality of developed systems in bone related prosthetic applications.

Evidence Supporting a Paracrine Effect of IGF-1/VEGF on Human Mesenchymal Stromal Cell Commitment.

Healing of skeletal defects is strictly dependent on osteogenesis and efficient vascularization of engineered scaffolds. Insulin-like growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF) are both involved in these processes. The in vitro administration of IGF-1 in association with VEGF is able to modulate the osteoblastic or endothelial commitment of mesenchymal stromal cells (MSCs) of different origins (e.g. periosteum and skin). In the present study, in order to deepen a possible paracrine effect of IGF-1 and VEGF on periosteum-derived progenitor cells (PDPCs) and skin-derived MSCs (S-MSCs), a Transwell coculture approach was used. We explored the genes involved in endothelial and osteoblastic differentiation, those modulating mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3′-kinase (PI3K)-AKT signaling pathways as well as genes implicated in stemness (i.e. Sox2, Oct4, and Nanog). Periosteal cells, which are typically committed toward osteoblastogenesis, are driven in the direction of endothelial gene expression when influenced by S-MSCs. The latter, once influenced by PDPCs, lose their endothelial commitment and increase the expression of osteoblast-associated genes. PI3K/AKT and MAPK signaling pathways seem to be markedly involved in this behavior. Our results evidence that paracrine signals between MSCs may differently modulate their commitment in a bone microenvironment, opening stimulating viewpoints for skeletal tissue engineering strategies coupling angiogenesis and osteogenesis processes.

Exploiting a new glycerol-based copolymer as a route to wound healing: Synthesis, characterization and biocompatibility assessment.

The use of biocompatible materials based on naturally derived monomers plays a key role in pharmaceutical and cosmetic industries. In this paper we describe the synthesis of a new low molecular weight copolymer, based on glycerol and l-tartaric acid, useful to develop biocompatible dermal patches with drug delivery properties. The copolymers chemical composition was assessed by FT-IR (Fourier transform infrared spectroscopy), (1)H NMR ((1)H Nuclear Magnetic Resonance) and XPS (X-ray photoelectron spectroscopy), while its molecular weight distribution was estimated by SEC (size exclusion chromatography). Copolymer thermal properties were studied by TGA (thermogravimetric analysis). Biological evaluations by MTT assay and SEM (scanning electron microscopy) observations performed with murine fibroblasts and human keratinocytes (HaCaT) revealed a good compatibility of the proposed copolymer. Ciprofloxacin was selected as model drug and its release was evaluated by HPLC (high performance liquid chromatography), showing that the new copolymer supplied promising results as drug delivery system for wound healing applications. Furthermore, investigations on Skin-Mesenchymal stem cells (S-MSCs) behaviour and gene expression showed that the copolymer and its combination with ciprofloxacin did not affect their stemness. In this regard, the fabrication of dermal patches with new, low cost materials for local treatment of skin infections represents an attractive strategy in order to bypass the worrying side effects of systemic antibiotic therapy. Overall, the performed physico-chemical characterization, drug release test and biological evaluations showed that this new copolymer could be a promising tool for the in situ delivery of bioactive molecules during skin lesions treatment.

Detecting senescent fate in mesenchymal stem cells: a combined cytofluorimetric and ultrastructural approach

Abstract Senescence can impair the therapeutic potential of stem cells. In this study, senescence-associated morphofunctional changes in periosteum-derived progenitor cells (PDPCs) from old and young individuals were investigated by combining cytofluorimetry, immunohistochemistry, and transmission electron microscopy. Cell cycle analysis demonstrated a large number of G0/G1 phase cells in PDPCs from old subjects and a progressive accumulation of G0/G1 cells during passaging in cultures from young subjects. Cytofluorimetry documented significant changes in light scattering parameters and closely correlated with the ultrastructural features, especially changes in mitochondrial shape and autophagy, which are consistent with the mitochondrial-lysosomal axis theory of ageing. The combined morphological, biofunctional, and ultrastructural approach enhanced the flow cytometric study of PDPC ageing. We speculate that impaired autophagy, documented in replicative senescent and old PDPCs, reflect a switch from quiescence to senescence. Its demonstration in a tissue with limited turnover—like the cambium layer of the periosteum, where reversible quiescence is the normal stem cell state throughout life—adds a new piece to the regenerative medicine jigsaw in an ageing society.

Aging of periosteal-derived stem cells during expansion: an alternative tool for a customized bone regenerative strategy

Increased in life expectancy points out the necessity for tailored strategies to restore bone loss due to trauma and/or disease in elderly. Moreover, there is a compelling need for improved cell systems to test scaffolds interfacing with an “aging” tissue. For skeletal tissue regeneration, periosteal-derived stem cells (PDPCs) could represent an easily recruited source of Mesenchymal stromal cells (MSCs) [1,2]. This study investigated the effects of long-term in vitro expansion on the stability and function of PDPCs, since extensive culture expansion is usually performed to obtain clinically relevant cell numbers, but its impact on cell behaviour is still unclear. An integrated approach based on flow cytometry, ultrastructural and quantitative Real time PCR (qRT-PCR) analyses was adopted. Senescent cell data were compared with those of cells isolated from differently aged subjects. Both replicative-senescent PDPCs and cells isolated from old donors were permanently blocked in G1 phase of cell cycle, through a pathway that seemed to involve nitric oxide (NO) production and the expression of tumour suppressor proteins p16 or p53, respectively. Changes in the expression of MSC surface markers were detected in PDPCs during subculturing, whilst it was superimposable in young and aged PDPCs. Cytofluorimetric analysis of the physical parameters (i.e. FSC and SSC) showed a trend toward an increase in cell dimension and internal complexity in both populations analysed. This data was consistent with morphological observation that also evidenced similar alterations in mitochondrial shape. In addition, an intense autophagic activity in early passage PDPCs was observed, whilst in the late passages cells had a robust protein synthesis activity that could be related with “senescence-associated secretory phenotype” (SASP). In conclusion, the morphofunctional similarities detected in replicative-senescent and aged PDPCs suggest that their long-term expansion could be a reproducible and useful tool to mimic in vivo ageing.

Cobalt chloride supplementation differently affects human mesenchymal stem cells isolated from dental pulp, umbilical cord and adipose tissues in their chondrogenic potential

Articular cartilage is an avascular tissue without innervations, characterized by low cell density and abundant extracellular matrix (ECM). These characteristics leave articular cartilage with very limited capacity of repair and regeneration. Multipotent stem/stromal cells (MSC) are considered promising for cartilage tissue engineering. Stem cells are resided in a special microenvironment known as the stem-cell niche, characterized by the presence of low oxygen concentration. Previous studies have reported that hypoxic conditions could enhance the chondrogenic differentiation of mesenchymal stem cells in the presence of an inductive medium. Cobalt chloride (CoCl2) imitates hypoxia in vitro by preventing hypoxia-inducible factor-alpha (HIF-a) from being destroyed by oxygen. However, the long-term hypoxic culture of stem cells is difficult and requires special attention to avoid cell death due to cobalt treatment. In this study we investigated if CoCl2 affected MSCs isolated from dental pulp, umbilical cord and adipose tissue in their potential to differentiate toward the chondrogenic phenotype. Cells were treated with concentrations of CoCl2 ranging from 50 to 400 uM. Cell proliferation, mRNA expression of stem-cell marker and chondrogenic associated genes were analyzed by RT-PCR and Real-time PCR. The results showed that the CoCl2 supplementation had no effect on the proliferation of all the three type of cells analyzed, while the up-regulation of chondrogenic markers such as aggrecan, sox9, and type II collagen, was dependent on the cellular source. This study shows that hypoxia induced by CoCl2 treatment can differently influence the behavior of MSCs of different sources in their chondrogenic potential. These findings should be taken into consideration in the treatment of cartilage repair and regeneration based on stem cell therapies.

Bone regeneration strategies in the elderly: the role of ageing and replicative senescence in periosteal-derived stem cells

Periosteum contains resident progenitor cells (PDPCs) representing an attractive alternative source of mesenchymal stem cells (MSCs) for skeletal tissue engineering approaches based on cell recruitment (1). Increased in life expectancy point out the necessity for customized strategies to restore bone loss due to trauma and/or disease in elderly. Aim of the present research was the evaluation of the ageing impact on PDPCs isolated from differently aged subjects. Moreover, since long-term culture could lead MSCs to senescence, the effects of culture expansion method on young PDPC through sequential serial passages were examined. Age-related increase of p53 expression and impairment in proliferating capacity were observed; those findings were strictly related to nitric oxide (NO) release. Moreover, qRT-PCR analysis showed a greater expression of genes involved in bone remodelling in elderly donors. As far as replicative in vitro expansion was concerned, we observed that later PDPC passages exhibited the typical “replicative senescence” features (i.e. flattened and enlarged morphology, prolonged population doubling time and increased SA-βgal activity). In these cells, p16 rather than p53 seemed to be involved in senescence processes. Similarly to the elderly, the decrease in proliferating ability of in vitro senescent PDPCs was concomitant with a higher NO production, and the changes in the expression of genes involved in bone resorption and RANKL/OPG ratio were superimposable. Interestingly, the relationship between NO release and ageing could represent a cutting edge “replicative senescence index” as emerged by our System Biology approach. In conclusion, our findings suggest that in vivo cell ageing and in vitro subculturing must be taken into account when testing regenerative tissue strategies that use progenitor cells. Indeed, cells (e.g. MSCs and PDPCs) from the earliest subculture passages could be useful to validate any bone tissue engineering strategies, whilst the later ones could be used to test in vitro scaffolds for regenerative medicine approaches in elderly.This work was supported by grants from MIUR (Project PRIN 2010, MIND-2010J8RYS7).