Monica Mattioli Belmonte

Effects of asbestiform antigorite on human alveolar epithelial A549 cells: A morphological and immunohistochemical study

The purpose of the study was to investigate the biological risk of asbestiform antigorite, which is a fibrous variety of antigorite, one of the natural mineral fibres of the serpentine group to which asbestos chrysotile belongs. Asbestiform antigorite is very abundant and commonly found associated with asbestos chrysotile in serpentinites, a kind of rock outcropping present in many geographical locations worldwide. In this study we evaluated the morphological, immunohistochemical and functional effects of antigorite fibres in alveolar epithelial cancer cells (A549), a standardized human cell line currently used as a model to study cytotoxicity induced by pharmacological agents. The antigorite fibres were identified and characterized morphologically and chemically by X-ray powder diffractometry, transmission and scanning electron microscopy, both with annexed energy dispersive spectrometry. The effects of 50 microg/ml of antigorite in A549 lung cells treated at 24 and 48 h resulted in increased synthesis of VEGF, Cdc42 and beta-catenin that represent potential risks for cancer development. Phalloidin labelling showed an irregular distribution of filamentous actin resulting from antigorite contact. Our studies indicate potential cellular toxicity of antigorite in vivo, providing the opportunity to elucidate the effect of asbestos on cancer induction and possible modes of therapy.

A biochemical-morphological study on microvillus plasma membrane development

The microvillus plasma membrane of the human placental syncytiotrophoblast at term has been extensively studied, while little is known about the characteristics of its development. The aim of the present work was to compare functional and structural properties of this membrane at early and term gestational age. Ten normal term placentas (40 weeks) and ten placentas at 10 weeks of gestational age were studied. The Na+/K+-ATPase activity is significantly decreased in the syncytiotrophoblast plasma membrane obtained from term placentas as compared to the early ones, with significant variation of maximum velocity (Vmax). The microviscosity, evaluated by the P parameter of DPH and Sn parameters of 5- and 16-NS, is increased in the term placentas compared to the early placentas. This alteration is accompanied by an increased cholesterol to phospholipids ratio in term placentas, while there is a decreased unsaturated to saturated fatty acid ratio. As follows from morphological studies, an increased mean diameter in the E face was observed in the term placenta with respect to the early placenta. The distribution factor DF, which indicates the particle aggregation state, decreased in the E face in the term placenta as compared to the early one. The present biochemical morphological study shows that a deep modification of the membrane is at the basis of the syncytiotrophoblast plasma membrane development.

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.

3D polysaccharide based hydrogel for bone tissue engineering

Hydrogels have attracted considerable attention in biomedical engineering applications due to their many favorable biomimetic properties. Hydrogels based on proteins or carbohydrates can also function as effective extracellular matrices to direct cellular behavior. Recently, polysaccharide based hydrogels have become particularly interesting 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 hydrogel with potential applications for bone tissue engineering. The aim of this study was to verify the biological potential of a new carboxymethyl cellulose—hydroxyapatite hybrid hydrogel in bone tissue regeneration. Mesenchymal stem cells were seeded on hydrogel scaffold, in presence and absence of hydroxyapatite, for 7, 14 and 21 days. Cell viability assay and morphological analysis were carried out to evaluate biocompatibility and cell adhesion of the materials. Real Time PCR for genes involved in tissue regeneration was carried out to assay the influence of the scaffold in cell differentiation. Results showed a high cell viability and biocompatibility of the tested material, confirmed by morphological analysis. The evaluation of osteoblast markers demonstrated the osteogenic induction of the 3D material enriched with hydroxyapatite in the production of mineralized extracellular matrix compared to the carboxymethyl cellulose based material. In conclusion, our data show that carboxymethyl cellulose—hydroxyapatite hybrid hydrogel may have great potential in bone tissue engineering applications.

Dental pulp stem cells senescence and regenerative potential relationship: IEZZI et al.

Uncomplicated treatments for pulpitis and periodontitis continues to be challenging and regenerative approaches could meet this contingency. Dental pulp stem cells (DPSCs) represent a good candidate for oral recovering therapies. Here, we investigated changes in morphology, proliferation, and in vitro differentiation toward mesenchymal and neuronal phenotypes of human DPSCs harvested from differently aged donors. Aging is a physiologic phenomenon occurring with time that hamper body’s capability to maintain homeostasis also affecting the functional reserve. Cytofluorimetric, immunohistochemical, quantitative reverse‐transcription polymerase chain reaction (qRT‐PCR), and western blot analyses were performed to gain insight for successful regenerative strategies in elderly. We observed a decline in DPSCs proliferation and differentiation potential with age. Interestingly, these cells behaved differently under osteogenic or odontogenic stimuli, showing different age‐related mineralization capabilities. Similarly, neurogenic differentiation decreased with age. In conclusion, our observations represent a valid tool for the development of tailored regenerative strategies in an aging 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.

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).

Loss of nuclear BAP1 protein expression is a marker of poor prognosis in patients with clear cell renal cell carcinoma

BAP1 is a gene situated on chromosome 3p in a region that is deleted in over 90% of Renal Cell Carcinomas (RCCs) (1,2). In the present study we studied BAP1 immunohistochemical expression in a large series of conventional clear cell RCCs (ccRCCs) treated with radical nephrectomy and we assessed the prognostic value of their expression in terms of patients survival at long-term follow-up. 154 consecutive patients with ccRCC were selected from a prospective database and considered for the study purpose; all patients were treated with radical nephrectomy and lymphadenectomy at our Institute of Urology between 1983 and 1985. The features considered in this study were tumor size, grade and stage, vascular and capsular invasion, incidence of metastasis and patient specific survival; all these parameters were correlated with immunohistochemical cytoplasmic and nuclear expression of BPA-1 in tumoral tissue. Median follow-up was 196.18 months (range 5 to 274); median survival was 125.34 months (range 5 to 274 months). We found that nuclear BAP1 expression showed a high frequency of loss in tumoral cells; nuclear BAP1 negative tumors had higher tumor size, higher Fuhrman grade, and higher stage, a greater amount of vascular and capsular invasion and a higher incidence of metastases. We have demonstrated that nuclear BAP1 expression is a marker of prognosis in ccRCC, having an impact on cancer-specific survival. The clinical importance for BAP1 will be realized with the identification and application of targeted therapies and with individualized approaches in the adjuvant and/or metastatic setting.

Age-related changes in human periosteal derived stem cells: a matter for effective bone regeneration strategies

Possible age-related changes in Mesenchymal Stem Cells (MSCs) are of great inter- est in view of their use for regenerative medicine approaches also in the elderly. Con- sidering the primary role of periosteum in bone biology and to acquire data for a cell- based therapy stimulating graft osseointegration, we tried to identify specific aging markers or pattern of expression in human periosteal precursor cells (PDPCs). To this aim periosteal tissue was obtained from differently aged healthy subjects, gender matched with a mean age of 16, 28, 63 and 92 years. Immunohistochemical detection of Ki67 and p53, Nitric Oxide (NO) production and qRT- PCR of a selected gene pan- el for early osteoblastic differentiation (bmp2 and runx2) and bone remodelling (IL-6, RANKL and OPG) were evaluated. Our data evidenced that both Ki67 and p53 rep- resent striking markers of cell-cycle arrest in these cells and that their expression cor- relates with NO production. In addition, age affects genes involved in bone remodel- ling, with a significant increase in IL-6 mRNA expression as well as in RANKL/OPG ratio. As far as NO release is concerned, our data showed higher levels in PDPCs iso- lated from the elderly and a good correlation with the immunohistochemical analysis Moreover, mathematical modelling, in silico simulations and biochemical experiments were combined to investigate about possible underlying quantitative correlations. A clear one-term exponential relationship emerged from a comparison of involved marker trends against age of donors concerning measured NO concentration / Ki67 ratio. This analytical approach confirmed Ki67 as a senescence marker to be focused on. We believe that this study, taking into account age-related changes in human PDPCs, opens up new regenerative medicine strategies for aged bone and/or bone metabolic diseases.

Influence of a biomimetic gelatin porous scaffold in chondrogenic and osteogenic differentiation of mesenchymal stem cells

Recently, tissue engineering has merged with stem cell technology to develop new sources of transplantable material for injury or disease treatment. Eminently interesting, are bone and joint injuries/disorders because of the low self-regenerating capacity of the matrix secreting cells, particularly chondrocytes (1). Gelatin based scaffolds are considered to be a highly suitable 3D material for tissue regeneration, due to high biocompatibility and adaptation to native tissues. In the present study, the chondrogenic and osteogenic potential of a porous gelatin based scaffold (2), alone or in combination with hydroxyapatite crystals, was investigated in human mesenchymal stem cells. Cells were culture 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 colonization inside the porous architecture of the biomaterial and scaffold adhesion. The expression of chondrogenic markers such as SOX9, collagen type II, aggregan and versican and osteogenic markers such as Collagen type I, Runx -2, osteopontin and bone matrix protein, were investigated by Real Time PCR. Results showed an high cell viability, adhesion and colonization of the scaffold. Real Time PCR data demonstrated an up-regulation of all the chondrogenic and osteogenic markers analyzed. In conclusion, gelatin porous scaffold provides an improved environment for chondrogenic and osteogenic differentiation of stem cells compared to cell monolayer culture system.