Injectable bone-graft substitute for in vivo tissue regeneration

We have demonstrated that using growth factors we can induce the proliferation of human primary osteoblasts but it isn’t enough to form new bone that need also the differentiation of the proliferated osteoblasts [1]. Being these two steps regulated by different pathways and different stimuli, in the same work we have found the combination of a proliferating growth factor (FGF2) with a differentiating stimulus (1,25Vit D3) as an optimal solution. With the aim to develop an injectable medicated scaffold which speeds bone formation in sinus lift augmentation, in bony void and in fracture repair, we have tested in vitro osteoblasts’ behavior in a 3D jelly collagen model (1mg/ml) using soluble native collagen prepared from rat tail tendons [2]. We have seen an osteoblasts’ Rho-kinase mediated contraction of the collagen that causes an approach of bone fragments within a week of culture with the formation of a fibrous bone tissue within 3 weeks of culture. FGF2 addition to the collagen fastened this result by increasing cell proliferation rate while the addition of 1,25Vit D3 to collagen at a concentration of 0,1 mg/mL that shows at HPLC analysis a release of 0,26 mcg/ ml/day during the incubation time studied, favors the mineralization of the new formed tissue that shows also increased tensile strenght. We think that this combination of factors could be used in vivo to accelerate bone growth and fracture healing

Enhanced Gene Silencing through Human Serum Albumin-Mediated Delivery of Polyethylenimine-siRNA Polyplexes

<div><p>Small interfering RNA (siRNA) targeted therapeutics (STT) offers a compelling alternative to tradition medications for treatment of genetic diseases by providing a means to silence the expression of specific aberrant proteins, through interference at the expression level. The perceived advantage of siRNA therapy is its ability to target, through synthetic antisense oligonucleotides, any part of the genome. Although STT provides a high level of specificity, it is also hindered by poor intracellular uptake, limited blood stability, high degradability and non-specific immune stimulation. Since serum proteins has been considered as useful vehicles for targeting tumors, in this study we investigated the effect of incorporation of human serum albumin (HSA) in branched polyethylenimine (bPEI)-siRNA polyplexes in their internalization in epithelial and endothelial cells. We observed that introduction of HSA preserves the capacity of bPEI to complex with siRNA and protect it against extracellular endonucleases, while affording significantly improved internalization and silencing efficiency, compared to bPEI-siRNA polyplexes in endothelial and metastatic breast cancer epithelial cells. Furthermore, the uptake of the HSA-bPEI-siRNA ternary polyplexes occurred primarily through a caveolae-mediated endocytosis, thus providing evidence for a clear role for HSA in polyplex internalization. These results provide further impetus to explore the role of serum proteins in delivery of siRNA.</p></div

Characterization of HSA-bPEI-siRNA complexes.

<p>A) Schematic representation of the two steps formulation protocol, where bPEI-siRNA native interaction was preserved and HSA was used for interacting with the positive charged amino-groups of bPEI. B) Transmission electron microscopy (TEM) images showing the increase in size upon addition of HSA. C) Gel retardation assay, demonstrating siRNA complexation on the top of the gel. From left, 10 base pair ladder (L10); free siRNA (-); PEI-siRNA (PEI); HSA-PEI-siRNA (+HSA); 100 base pair ladder (L100). D) RNAse protection assay, indicating efficent siRNA protection from RNAse degradation. Complexes are unaltered on the top of the gel, while free RNA is completely degraded. From left, 10 base pair ladder (L10); free siRNA (-); samples incubated with RNAse: free siRNA (+), PEI-siRNA (PEI) and HSA-PEI-siRNA (+HSA); 100 base pair ladder (L100).</p

Protein knockdown efficiency in cells expressing TurboGFP.

<p>Cell fluorescence reduction was estimated to evaluate the percentage of TurboGFP silencing. Both cell lines showed improved siRNA efficiency compared to bPEI-siRNA control. A) MDA-MB-231 ***p<0.0005. B) HPMEC ***p<0.0005. The silencing efficiency resulted comparable with Lipofectamine2000-siRNA lipoplexes (LFA-siRNA). Results are shown as mean ± SD (n = 6).</p

Size—zeta potential characterization and polydispersity index value.

<p>Reported values are an average (n = 12)</p><p>Size—zeta potential characterization and polydispersity index value.</p

Uptake mechanism of HSA-bPEI-siRNA complexes in MDA-MB-231 and HPMEC.

<p>The uptake inhibition was quantified in presence of Nystatin (Nyst), Filipin III (Fil III) and Chlorpromazine (CPZ). Fluorescence reduction was evaluated in FACS. A) MDA-MB-231; B) HPMEC. Results are shown as mean ± SD (n = 3). From the study, partial involvement of clathrin-mediated pathway was observed, with around 30% inhibition by CPZ, and low colocalization with Tf. Predominant activation of caveolae-mediated endocytosis resulted in the internalization of HSA-bPEI-siRNA complexes, in both cell lines. Colocalization study in fluorescence microscopy with uptake markers cholera toxin b (CTB), marker of caveolae-mediated pathway, and transferrin (Tf), marker of clathrin-mediated pathway, is shown in C) MDA-MB-231 cells; D) Colocalization results in HPMEC. White arrows indicate points of colocalization. Scale bar: 20μm.</p

Uptake of HSA-bPEI-siRNA and PEI-siRNA complexes in MDA-MB-231 and HPMEC.

<p>A) Representative fluorescence micrographs showing siRNA labeled complexes in cells (DAPI nuclear stain, blue; Alexa-fluor 555 siRNA labeled complexes, red) Scale bar: 20μm. B) FACS quantitative measurements, expressed as mean fluorescence value for cell,reveald increased uptake by HSA-bPEI-siRNA complexes, compared to bPEI-siRNA control, in both cell lines. Results are shown as mean ± SD (n = 3). MDA-MB-231 p<0.001, HPMEC p<0.005.</p

Uptake efficiency comparing complexes formed with BSA and HSA.

<p>Uptake efficiency was evaluated by FACS. (A) Study in MDA-MB-231, p<0.005; B) Study in HPMEC, p<0.01. Results are shown as mean ± SD (n = 3). Complexes formed with BSA showed reduced internalization compared with HSA, confirming the role of HSA in improving siRNA delivery in both cell lines.</p

Colocalization of HSA and bPEI-siRNA polyplexes in MDA-MB-231.

<p>Fluorescence microscopy revealed that Alexa-fluor 488 labeled HSA and Alexa-fluor 555 siRNA access in the same intracellular trafficking. (DAPI nuclear stain, blue; Alexa-fluor 555 siRNA labeled complexes, red; Alexa-fluor 488 labeled HSA, green). White arrows indicate points of colocalization. Scale bar: 20μm.</p

Delta Radiomic Analysis of Mesorectum to Predict Treatment Response and Prognosis in Locally Advanced Rectal Cancer

Background: The aim of this study is to evaluate the delta radiomics approach based on mesorectal radiomic features to develop a model for predicting pathological complete response (pCR) and 2-year disease-free survival (2yDFS) in locally advanced rectal cancer (LARC) patients undergoing neoadjuvant chemoradiotherapy (nCRT). Methods: Pre- and post-nCRT MRIs of LARC patients treated at a single institution from May 2008 to November 2016 were retrospectively collected. Radiomic features were extracted from the GTV and mesorectum. The Wilcoxon–Mann–Whitney test and area under the receiver operating characteristic curve (AUC) were used to evaluate the performance of the features in predicting pCR and 2yDFS. Results: Out of 203 LARC patients, a total of 565 variables were evaluated. The best performing pCR prediction model was based on two GTV features with an AUC of 0.80 in the training set and 0.69 in the validation set. The best performing 2yDFS prediction model was based on one GTV and two mesorectal features with an AUC of 0.79 in the training set and 0.70 in the validation set. Conclusions: The results of this study suggest a possible role for delta radiomics based on mesorectal features in the prediction of 2yDFS in patients with LARC