Microglia Polarization and Antiglioma Effects Fostered by Dual Cell Membrane-Coated Doxorubicin-Loaded Hexagonal Boron Nitride Nanoflakes.

Microglial cells play a critical role in glioblastoma multiforme (GBM) progression, which is considered a highly malignant brain cancer. The activation of microglia can either promote or inhibit GBM growth depending on the stage of the tumor development and on the microenvironment conditions. The current treatments for GBM have limited efficacy; therefore, there is an urgent need to develop novel and efficient strategies for drug delivery and targeting: in this context, a promising strategy consists of using nanoplatforms. This study investigates the microglial response and the therapeutic efficacy of dual-cell membrane-coated and doxorubicin-loaded hexagonal boron nitride nanoflakes tested on human microglia and GBM cells. Obtained results show promising therapeutic effects on glioma cells and an M2 microglia polarization, which refers to a specific phenotype or activation state that is associated with anti-inflammatory and tissue repair functions, highlighted through proteomic analysis.

Effect of Combined Levothyroxine (L-T4) and 3-Iodothyronamine (T1AM) Supplementation on Memory and Adult Hippocampal Neurogenesis in a Mouse Model of Hypothyroidism.

Mood alterations, anxiety, and cognitive impairments associated with adult-onset hypothyroidism often persist despite replacement treatment. In rodent models of hypothyroidism, replacement does not bring 3-iodothyronamine (T1AM) brain levels back to normal. T1AM is a thyroid hormone derivative with cognitive effects. Using a pharmacological hypothyroid mouse model, we investigated whether augmenting levothyroxine (L-T4) with T1AM improves behavioural correlates of depression, anxiety, and memory and has an effect on hippocampal neurogenesis. Hypothyroid mice showed impaired performance in the novel object recognition test as compared to euthyroid mice (discrimination index (DI): 0.02 ± 0.09 vs. 0.29 ± 0.06; t = 2.515, p = 0.02). L-T4 and L-T4+T1AM rescued memory (DI: 0.27 ± 0.08 and 0.34 ± 0.08, respectively), while T1AM had no effect (DI: -0.01 ± 0.10). Hypothyroidism reduced the number of neuroprogenitors in hippocampal neurogenic niches by 20%. L-T4 rescued the number of neuroprogenitors (mean diff = 106.9 ± 21.40, t = 4.99, pcorr = 0.003), while L-T4+T1AM produced a 30.61% rebound relative to euthyroid state (mean diff = 141.6 ± 31.91, t = 4.44, pcorr = 0.004). We performed qPCR analysis of 88 genes involved in neurotrophic signalling pathways and found an effect of treatment on the expression of Ngf, Kdr, Kit, L1cam, Ntf3, Mapk3, and Neurog2. Our data confirm that L-T4 is necessary and sufficient for recovering memory and hippocampal neurogenesis deficits associated with hypothyroidism, while we found no evidence to support the role of non-canonical TH signalling.

A Novel Patient-Personalized Nanovector Based on Homotypic Recognition and Magnetic Hyperthermia for an Efficient Treatment of Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is the deadliest brain tumor, characterized by an extreme genotypic and phenotypic variability, besides a high infiltrative nature in healthy tissues. Apart from very invasive surgical procedures, to date, there are no effective treatments, and life expectancy is very limited. In this work, an innovative therapeutic approach based on lipid-based magnetic nanovectors is proposed, owning a dual therapeutic function: chemotherapy, thanks to an antineoplastic drug (regorafenib) loaded in the core, and localized magnetic hyperthermia, thanks to the presence of iron oxide nanoparticles, remotely activated by an alternating magnetic field. The drug is selected based on ad hoc patient-specific screenings; moreover, the nanovector is decorated with cell membranes derived from patients' cells, aiming at increasing homotypic and personalized targeting. It is demonstrated that this functionalization not only enhances the selectivity of the nanovectors toward patient-derived GBM cells, but also their blood-brain barrier in vitro crossing ability. The localized magnetic hyperthermia induces both thermal and oxidative intracellular stress that lead to lysosomal membrane permeabilization and to the release of proteolytic enzymes into the cytosol. Collected results show that hyperthermia and chemotherapy work in synergy to reduce GBM cell invasion properties, to induce intracellular damage and, eventually, to prompt cellular death.

Empagliflozin inhibits excessive autophagy through the AMPK/GSK3ß signalling pathway in diabetic cardiomyopathy.

AIMS: Sodium-glucose cotransporter 2 inhibitors have beneficial effects on heart failure and cardiovascular mortality in diabetic and non-diabetic patients, with unclear mechanisms. Autophagy is a cardioprotective mechanism under acute stress conditions, but excessive autophagy accelerates myocardial cell death leading to autosis. We evaluated the protective role of empagliflozin (EMPA) against cardiac injury in murine diabetic cardiomyopathy. METHODS AND RESULTS: Male mice, rendered diabetics by one single intraperitoneal injection of streptozotocin and treated with EMPA (30 mg/kg/day), had fewer apoptotic cells (4.9 ± 2.1 vs. 1 ± 0.5 TUNEL-positive cells %, P < 0.05), less senescence (10.1 ± 2 vs. 7.9 ± 1.2 ß-gal positivity/tissue area, P < 0.05), fibrosis (0.2 ± 0.05 vs. 0.15 ± 0.06, P < 0.05 fibrotic area/tissue area), autophagy (7.9 ± 0.05 vs. 2.3 ± 0.6 fluorescence intensity/total area, P < 0.01), and connexin (Cx)-43 lateralization compared with diabetic mice. Proteomic analysis showed a down-regulation of the 5' adenosine monophosphate-activated protein kinase (AMPK) pathway and upstream activation of sirtuins in the heart of diabetic mice treated with EMPA compared with diabetic mice. Because sirtuin activation leads to the modulation of cardiomyogenic transcription factors, we analysed the DNA binding activity to serum response elements (SRE) of serum response factor (SRF) by electromobility shift assay. Compared with diabetic mice [0.5 ± 0.01 densitometric units (DU)], non-diabetic mice treated with EMPA (2.2 ± 0.01 DU, P < 0.01) and diabetic mice treated with EMPA (2.0 ± 0.1 DU, P < 0.01) significantly increased SRF binding activity to SRE, paralleled by increased cardiac actin expression (4.1 ± 0.1 vs. 2.2 ± 0.01 target protein/ß-actin ratio, P < 0.01). EMPA significantly reversed cardiac dysfunction on echocardiography in diabetic mice and inhibited excessive autophagy in high-glucose-treated cardiomyocytes by inhibiting the autophagy inducer glycogen synthase kinase 3 beta (GSK3ß), leading to reactivation of cardiomyogenic transcription factors. CONCLUSION: Taken together, our results describe a novel paradigm in which EMPA inhibits hyperactivation of autophagy through the AMPK/GSK3ß signalling pathway in the context of diabetes.

Connexin Expression in Human Minor Salivary Glands: An Immunohistochemical Microscopy Study.

Connexins (Cxs) are transmembrane proteins involved in the formation of hemichannels and gap junctions (GJs). GJs are involved in various physiological functions, including secretion in glandular tissue. It has been demonstrated that Cx26, Cx32, and Cx43 are mainly expressed in glands, but no data are available in human salivary glands to date. The aim of our study was to investigate the presence and the localization of Cxs in human minor labial salivary glands. Immunofluorescence and immunoelectron microscopy were employed to evaluate the Cx26, Cx32, and Cx43 protein in human labial salivary gland biopsies (hLSGBs). RT-PCR was also used to detect their mRNA expression. Cx expression was found at both the mRNA and protein levels in all hLSGBs analysed. Cxs were observed at the level of the duct and acinar cells, as well as in myoepithelial cells. The localization of the three Cx types was very similar, suggesting colocalization of these Cxs in the same connexons. These results demonstrated the presence of Cxs in human salivary glands for the first time. Moreover, the few samples with primary Sjögren's Syndrome analysed only by immunofluorescence showed an alteration of the Cx expression, indicating that these proteins could be involved in salivary gland dysfunctions.

Dysregulated insulin secretion is associated with pancreatic ß-cell hyperplasia and direct acinar-ß-cell trans-differentiation in partially eNOS-deficient mice.

eNOS-deficient mice were previously shown to develop hypertension and metabolic alterations associated with insulin resistance either in standard dietary conditions (eNOS-/- homozygotes) or upon high-fat diet (HFD) (eNOS+/- heterozygotes). In the latter heterozygote model, the present study investigated the pancreatic morphological changes underlying the abnormal glycometabolic phenotype. C57BL6 wild type (WT) and eNOS+/- mice were fed with either chow or HFD for 16 weeks. After being longitudinally monitored for their metabolic state after 8 and 16 weeks of diet, mice were euthanized and fragments of pancreas were processed for histological, immuno-histochemical and ultrastructural analyses. HFD-fed WT and eNOS+/- mice developed progressive glucose intolerance and insulin resistance. Differently from WT animals, eNOS+/- mice showed a blunted insulin response to a glucose load, regardless of the diet regimen. Such dysregulation of insulin secretion was associated with pancreatic ß-cell hyperplasia, as shown by larger islet fractional area and ß-cell mass, and higher number of extra-islet ß-cell clusters than in chow-fed WT animals. In addition, only in the pancreas of HFD-fed eNOS+/- mice, there was ultrastructural evidence of a number of hybrid acinar-ß-cells, simultaneously containing zymogen and insulin granules, suggesting the occurrence of a direct exocrine-endocrine transdifferentiation process, plausibly triggered by metabolic stress associated to deficient endothelial NO production. As suggested by confocal immunofluorescence analysis of pancreatic histological sections, inhibition of Notch-1 signaling, likely due to a reduced NO availability, is proposed as a novel mechanism that could favor both ß-cell hyperplasia and acinar-ß-cell transdifferentiation in eNOS-deficient mice with impaired insulin response to a glucose load.

Sodium-glucose cotransporter type 2 inhibitors prevent ponatinib-induced endothelial senescence and disfunction: A potential rescue strategy.

BACKGROUND: Ponatinib (PON), a third-generation tyrosine kinase inhibitor (TKI), has proven cardiovascular toxicity, with no known preventing agents usable to limit such side effect. Sodium-glucose cotransporter type 2 (SGLT2) inhibitors are a new class of glucose-lowering agents, featuring favorable cardiac and vascular effects. AIMS: We assessed the effects of the SGLT2 inhibitors empagliflozin (EMPA) and dapagliflozin (DAPA) on human aortic endothelial cells (HAECs) and underlying vasculo-protective mechanisms in an in vitro model of PON-induced endothelial toxicity. METHODS AND RESULTS: We exposed HAECs to PON or vehicle (DMSO) in the presence or absence of EMPA (100 and 500 nmol/L) or dapagliflozin (DAPA) for 0-48 h exposure times. Compared with vehicle, incubations of HAECs with PON significantly reduced cell viability (0.56 ± 0.11 vs 0.23 ± 0.05 absorbance units, p < 0.01), increased the number of senescent cells at ß-gal-assay (PON 9 ± 4 vs basal DMSO 3 ± 1 ß-Gal+ cells/field, p < 0.01), decreased tubulization in Matrigel (PON PON: 6 ± 1 vs basal DMSO 12 ± 1 tubuli number/field, p < 0.05) with a non-statistically significant trend of PON to decrease the number of autophagic cells at immunofluorescence assay and flow cytometry. EMPA reverted the effects of PON on cell viability (E 500 + PON 0.24 ± 0.05 vs PON 0.56 ± 0.11 absorbance units, p < 0.01) and induced autophagy (E 500 7 ± 4.3 vs basal DMSO 2.6 ± 2.3 mean fluorescence vs PON 2.6 ± 2.4 mean fluorescence, p < 0.05). EMPA and DAPA also reversed the effects of PON on cell senescence (E 500 + PON 4 ± 1 and DAPA 100 4 ± 2 vs PON 9 ± 4 ß-Gal+ cells/field, p < 0.01) and improved cell tubulization (E 500 + PON 21 ± 3 vs PON 6 ± 1 tubuli number/field, p < 0.05; DAPA 100 + PON 16 ± 2 vs PON 6 ± 1 tubuli number/field, p < 0.05). CONCLUSION: EMPA and DAPA attenuate the vasculo-toxic effect exerted by PON by reverting endothelial cell senescence and dysfunction. These findings support the design of clinical studies exploring the vasculo-protective effects of EMPA or DAPA on PON-induced vascular toxicity.

Subcellular Localization of Connexin 26 in Cardiomyocytes and in Cardiomyocyte-Derived Extracellular Vesicles.

Connexins (Cxs) are a family of membrane-spanning proteins, expressed in vertebrates and named according to their molecular weight. They are involved in tissue homeostasis, and they function by acting at several communication levels. Cardiac Cxs are responsible for regular heart function and, among them, Cx26 and Cx43 are widely expressed throughout the heart. Cx26 is present in vessels, as well as in cardiomyocytes, and its localization is scattered all over the cell aside from at the intercalated discs as is the case for the other cardiac Cxs. However, having been found in cardiomyocytes only recently, both its subcellular localization and its functional characterization in cardiomyocytes remain poorly understood. Therefore, in this study we aimed to obtain further data on the localization of Cx26 at the subcellular level. Our TEM immunogold analyses were performed on rat heart ventricles and differentiated H9c2 cardiac cell sections as well as on differentiated H9c2 derived extracellular vesicles. The results confirmed the absence of Cx26 at intercalated discs and showed the presence of Cx26 at the level of different subcellular compartments. The peculiar localization at the level of extracellular vesicles suggested a specific role for cardiac Cx26 in inter-cellular communication in an independent gap junction manner.

Ezrin Is a Novel Protein Partner of Aquaporin-5 in Human Salivary Glands and Shows Altered Expression and Cellular Localization in Sjögren's Syndrome.

Sjögren's syndrome (SS) is an exocrinopathy characterized by the hypofunction of salivary glands (SGs). Aquaporin-5 (AQP5); a water channel involved in saliva formation; is aberrantly distributed in SS SG acini and contributes to glandular dysfunction. We aimed to investigate the role of ezrin in AQP5 mislocalization in SS SGs. The AQP5-ezrin interaction was assessed by immunoprecipitation and proteome analysis and by proximity ligation assay in immortalized human SG cells. We demonstrated, for the first time, an interaction between ezrin and AQP5. A model of the complex was derived by computer modeling and in silico docking; suggesting that AQP5 interacts with the ezrin FERM-domain via its C-terminus. The interaction was also investigated in human minor salivary gland (hMSG) acini from SS patients (SICCA-SS); showing that AQP5-ezrin complexes were absent or mislocalized to the basolateral side of SG acini rather than the apical region compared to controls (SICCA-NS). Furthermore, in SICCA-SS hMSG acinar cells, ezrin immunoreactivity was decreased at the acinar apical region and higher at basal or lateral regions, accounting for altered AQP5-ezrin co-localization. Our data reveal that AQP5-ezrin interactions in human SGs could be involved in the regulation of AQP5 trafficking and may contribute to AQP5-altered localization in SS patients.

Unraveling Human AQP5-PIP Molecular Interaction and Effect on AQP5 Salivary Glands Localization in SS Patients.

Saliva secretion requires effective translocation of aquaporin 5 (AQP5) water channel to the salivary glands (SGs) acinar apical membrane. Patients with Sjögren's syndrome (SS) display abnormal AQP5 localization within acinar cells from SGs that correlate with sicca manifestation and glands hypofunction. Several proteins such as Prolactin-inducible protein (PIP) may regulate AQP5 trafficking as observed in lacrimal glands from mice. However, the role of the AQP5-PIP complex remains poorly understood. In the present study, we show that PIP interacts with AQP5 in vitro and in mice as well as in human SGs and that PIP misexpression correlates with an altered AQP5 distribution at the acinar apical membrane in PIP knockout mice and SS hMSG. Furthermore, our data show that the protein-protein interaction involves the AQP5 C-terminus and the N-terminal of PIP (one molecule of PIP per AQP5 tetramer). In conclusion, our findings highlight for the first time the role of PIP as a protein controlling AQP5 localization in human salivary glands but extend beyond due to the PIP-AQP5 interaction described in lung and breast cancers.

Connexin 43 and Connexin 26 Involvement in the Ponatinib-Induced Cardiomyopathy: Sex-Related Differences in a Murine Model.

Cardiac connexins (Cxs) are proteins responsible for proper heart function. They form gap junctions that mediate electrical and chemical signalling throughout the cardiac system, and thus enable a synchronized contraction. Connexins can also individually participate in many signal transduction pathways, interacting with intracellular proteins at various cellular compartments. Altered connexin expression and localization have been described in diseased myocardium and the aim of this study is to assess the involvement of Cx43, Cx26, and some related molecules in ponatinib-induced cardiac toxicity. Ponatinib is a new multi-tyrosine kinase inhibitor that has been successfully used against human malignancies, but its cardiotoxicity remains worrisome. Therefore, understanding its signaling mechanism is important to adopt potential anti cardiac damage strategies. Our experiments were performed on hearts from male and female mice treated with ponatinib and with ponatinib plus siRNA-Notch1 by using immunofluorescence, Western blotting, and proteomic analyses. The altered cardiac function and the change in Cxs expression observed in mice after ponatinib treatment, were results dependent on the Notch1 pathway and sex. Females showed a lower susceptibility to ponatinib than males. The downmodulation of cardiac Cx43, Cx26 and miR-122, high pS368-Cx43 phosphorylation, cell viability and survival activation could represent some of the female adaptative/compensatory reactions to ponatinib cardiotoxicity.

Delivery of Thyronamines (TAMs) to the Brain: A Preliminary Study.

Recent reports highlighted the significant neuroprotective effects of thyronamines (TAMs), a class of endogenous thyroid hormone derivatives. In particular, 3-iodothyronamine (T1AM) has been shown to play a pleiotropic role in neurodegeneration by modulating energy metabolism and neurological functions in mice. However, the pharmacological response to T1AM might be influenced by tissue metabolism, which is known to convert T1AM into its catabolite 3-iodothyroacetic acid (TA1). Currently, several research groups are investigating the pharmacological effects of T1AM systemic administration in the search of novel therapeutic approaches for the treatment of interlinked pathologies, such as metabolic and neurodegenerative diseases (NDDs). A critical aspect in the development of new drugs for NDDs is to know their distribution in the brain, which is fundamentally related to their ability to cross the blood-brain barrier (BBB). To this end, in the present study we used the immortalized mouse brain endothelial cell line bEnd.3 to develop an in vitro model of BBB and evaluate T1AM and TA1 permeability. Both drugs, administered at 1 µM dose, were assayed by high-performance liquid chromatography coupled to mass spectrometry. Our results indicate that T1AM is able to efficiently cross the BBB, whereas TA1 is almost completely devoid of this property.

Nanoparticles Based on Quaternary Ammonium Chitosan-methyl-ß-cyclodextrin Conjugate for the Neuropeptide Dalargin Delivery to the Central Nervous System: An In Vitro Study.

Peptide oral administration is a hard goal to reach, especially if the brain is the target site. The purpose of the present study was to set up a vehicle apt to promote oral absorption of the neuropeptide dalargin (DAL), allowing it to cross the intestinal mucosal barrier, resist enzymatic degradation, and transport drugs to the brain after crossing the blood-brain barrier. Therefore, a chitosan quaternary ammonium derivative was synthesized and conjugated with methyl-ß-cyclodextrin to prepare DAL-medicated nanoparticles (DAL-NP). DAL-NP particle size was 227.7 nm, zeta potential +8.60 mV, encapsulation efficiency 89%. DAL-NP protected DAL from degradation by chymotrypsin or pancreatin and tripled DAL degradation time compared to non-encapsulated DAL. Use of DAL-NP was safe for either Caco-2 or bEnd.3 cells, with the latter selected as a blood-brain barrier model. DAL-NP could also cross either the Caco-2 or bEnd.3 monolayer by the transepithelial route. The results suggest a potential DAL-NP ability to transport to the brain a DAL dose fraction administered orally, although in vivo experiments will be needed to confirm the present data obtained in vitro.

Heart and liver connexin expression related to the first stage of aging: A study on naturally aged animals.

Connexins are membrane-spanning proteins that form membrane channels and hemichannels. They are involved in the cellular communication and in the maintenance of tissue homeostasis. Recent studies in humans and animals have demonstrated that the expression and distribution of Cx43, the most studied connexin, can change during aging. However, the research on the involvement of the other connexins in cardiac and hepatic aging is, at present, still very poor. Hence, the aim of this study is to evaluate the expression of Cx43 and Cx26 in the heart as well as Cx26 and Cx32 in the liver of a rat model that aged naturally, rather than prematurely because of genetic mutations or age-related diseases. The results obtained in the present study have demonstrated that these connexins decrease in rat cardiomyocytes and in rat hepatocytes as they age. This change was revealed only at protein level, as connexin-mRNAs remained unchanged during aging. Moreover, the aged rats showed an increase in body fat, whose subcutaneous layer tended to be higher. Finally, how these changes could represent signs of physiological adaptation in successful aging was discussed.

Aging and biomarkers: Transcriptional levels evaluation of Osteopontin/miRNA-181a axis in hepatic tissue of rats in different age ranges.

Osteopontin (OPN), a novel hepatic damage marker, as well as several non-coding RNA seem to be associated with liver aging, a little studied and not yet defined process. The aim of the study was to evaluate the expression variations of OPN and miRNA-181a, the transcriptional profiling of long non coding (lnc) RNA GAS-5/miRNA-222 axis and lncRNA NEAT-1 in liver tissue of rats. In addition, to monitor the senescence process, the telomere shortening and TERT/TERC gene expression were also measured. Three groups of male Wistar rats were studied: A (n = 6, young); B (n = 13, adult); C (n = 10, old). Total RNA, including miRNAs, was extracted from liver and analysed by Real-Time PCR. Ultrasound and biochemical evaluation were performed in all rats as well as the histological analysis. OPN mRNA resulted lower in C with respect to A and B while miRNA-181a expression was significantly increased as a function of age. An increasing of both NEAT-1 and miRNA-222 expression as a function of age in parallel with a decreasing of GAS-5 expression in young and old rats, but not in the adults, was observed. A positive correlation was detected between miRNA-181a and miRNA-222. The hepatic ultrasound analysis revealed areas of hyperechogenicity distributed as a function of age. A significant telomere shortening was measured as a function of age while the two subunits TERT and TERC expressions showed an opposite trend. This work could provide a valid starting point to better understand the physiopathological changes during aging, pinpointing in the OPN/miRNA-181a axis significant predictors of successful aging.

Nutlin-loaded magnetic solid lipid nanoparticles for targeted glioblastoma treatment.

AIM: Glioblastoma multiforme is one of the deadliest forms of cancer, and current treatments are limited to palliative cares. The present study proposes a nanotechnology-based solution able to improve both drug efficacy and its delivery efficiency. MATERIALS & METHODS: Nutlin-3a and superparamagnetic nanoparticles were encapsulated in solid lipid nanoparticles, and the obtained nanovectors (nutlin-loaded magnetic solid lipid nanoparticle [Nut-Mag-SLNs]) were characterized by analyzing both their physicochemical properties and their effects on U-87 MG glioblastoma cells. RESULTS: Nut-Mag-SLNs showed good colloidal stability, the ability to cross an in vitro blood-brain barrier model, and a superior pro-apoptotic activity toward glioblastoma cells with respect to the free drug. CONCLUSION: Nut-Mag-SLNs represent a promising multifunctional nanoplatform for the treatment of glioblastoma multiforme.

Liver Cancer: Current and Future Trends Using Biomaterials.

Hepatocellular carcinoma (HCC) is the fifth most common type of cancer diagnosed and the second leading cause of death worldwide. Despite advancement in current treatments for HCC, the prognosis for this cancer is still unfavorable. This comprehensive review article focuses on all the current technology that applies biomaterials to treat and study liver cancer, thus showing the versatility of biomaterials to be used as smart tools in this complex pathologic scenario. Specifically, after introducing the liver anatomy and pathology by focusing on the available treatments for HCC, this review summarizes the current biomaterial-based approaches for systemic delivery and implantable tools for locally administrating bioactive factors and provides a comprehensive discussion of the specific therapies and targeting agents to efficiently deliver those factors. This review also highlights the novel application of biomaterials to study HCC, which includes hydrogels and scaffolds to tissue engineer 3D in vitro models representative of the tumor environment. Such models will serve to better understand the tumor biology and investigate new therapies for HCC. Special focus is given to innovative approaches, e.g., combined delivery therapies, and to alternative approaches-e.g., cell capture-as promising future trends in the application of biomaterials to treat HCC.

Ex Vivo and in Vivo Study of Sucrosomial® Iron Intestinal Absorption and Bioavailability.

The present study aimed to demonstrate that Sideral® RM (SRM, Sucrosomial® Raw Material Iron) is transported across the excised intestine via a biological mechanism, and to investigate the effect that this transport route may produce on oral iron absorption, which is expected to reduce the gastrointestinal (GI) side effects caused by the bioavailability of non-absorbed iron. Excised rat intestine was exposed to fluorescein isothiocyanate (FITC)-labeled SRM in Ussing chambers followed by confocal laser scanning microscopy to look for the presence of fluorescein-tagged vesicles of the FITC-labeled SRM. To identify FITC-labeled SRM internalizing cells, an immunofluorescence analysis for macrophages and M cells was performed using specific antibodies. Microscopy analysis revealed the presence of fluorescein positive particulate structures in tissues treated with FITC-labeled SRM. These structures do not disintegrate during transit, and concentrate in macrophage cells. Iron bioavailability was assessed by determining the time-course of Fe3+ plasma levels. As references, iron contents in liver, spleen, and bone marrow were determined in healthy rats treated by gavage with SRM or ferric pyrophosphate salt (FP). SRM significantly increased both area under the curve (AUC) and clearance maxima (Cmax) compared to FP, thus increasing iron bioavailability (AUCrel = 1.8). This led to increased iron availability in the bone marrow at 5 h after single dose gavage.

Pooled human serum: A new culture supplement for bioreactor-based cell therapies. Preliminary results.

BACKGROUND: Bone Marrow MSCs are an appealing source for several cell-based therapies. Many bioreactors, as the Quantum Cell Expansion System, have been developed to generate a large number of MSCs under Good Manufacturing Practice conditions by using Human Platelet Lysate (HPL). Previously we isolated in the human bone marrow a novel cell population, named Mesodermal Progenitor Cells (MPCs), which we identified as precursors of MSCs. MPCs could represent an important cell source for regenerative medicine applications. As HPL gives rise to a homogeneus MSC population, limiting the harvesting of other cell types, in this study we investigated the efficacy of pooled human AB serum (ABS) to provide clinically relevant numbers of both MSCs and MPCs for regenerative medicine applications by using the Quantum System. METHODS: Bone marrow aspirates were obtained from healthy adult individuals undergoing routine total hip replacement surgery and used to generate primary cultures in the bioreactor. HPL and ABS were tested as supplements to culture medium. Morphological observations, cytofluorimetric analysis, lactate and glucose level assessment were performed. RESULTS: ABS gave rise to both heterogeneous MSC and MPC population. About 95% of cells cultured in HPL showed a fibroblast-like morphology and typical mesenchymal surface markers, but MPCs were scarcely represented. DISCUSSION: The use of ABS appeared to sustain a large scale MSC production, as well as the recovery of a subset of MPCs, and resulted a suitable alternative to HPL in the cell generation based on the Quantum System.

Bovine bone matrix/poly(l-lactic-co-ε-caprolactone)/gelatin hybrid scaffold (SmartBone®) for maxillary sinus augmentation: A histologic study on bone regeneration.

The ideal scaffold for bone regeneration is required to be highly porous, non-immunogenic, biostable until the new tissue formation, bioresorbable and osteoconductive. This study aimed at investigating the process of new bone formation in patients treated with granular SmartBone® for sinus augmentation, providing an extensive histologic analysis. Five biopsies were collected at 4-9 months post SmartBone® implantation and processed for histochemistry and immunohistochemistry. Histomorphometric analysis was performed. Bone-particle conductivity index (BPCi) was used to assess SmartBone® osteoconductivity. At 4 months, SmartBone® (12%) and new bone (43.9%) were both present and surrounded by vascularized connective tissue (37.2%). New bone was grown on SmartBone® (BPCi=0.22). At 6 months, SmartBone® was almost completely resorbed (0.5%) and new bone was massively present (80.8%). At 7 and 9 months, new bone accounted for a large volume fraction (79.3% and 67.4%, respectively) and SmartBone® was resorbed (0.5% and 0%, respectively). Well-oriented lamellae and bone scars, typical of mature bone, were observed. In all the biopsies, bone matrix biomolecules and active osteoblasts were visible. The absence of inflammatory cells confirmed SmartBone® biocompatibility and non-immunogenicity. These data indicate that SmartBone® is osteoconductive, promotes fast bone regeneration, leading to mature bone formation in about 7 months.