Development of collagenous scaffolds for wound healing: characterization and in vivo analysis.

The development of wound dressings from biomaterials has been the subject of research due to their unique structural and functional characteristics. Proteins from animal origin, such as collagen and chitosan, act as promising materials for applications in injuries and chronic wounds, functioning as a repairing agent. This study aims to evaluate in vitro effects of scaffolds with different formulations containing bioactive compounds such as collagen, chitosan, N-acetylcysteine (NAC) and ε-poly-lysine (ε-PL). We manufactured a scaffold made of a collagen hydrogel bioconjugated with chitosan by crosslinking and addition of NAC and ε-PL. Cell viability was verified by resazurin and live/dead assays and the ultrastructure of biomaterials was evaluated by SEM. Antimicrobial sensitivity was assessed by antibiogram. The healing potential of the biomaterial was evaluated in vivo, in a model of healing of excisional wounds in mice. On the 7th day after the injury, the wounds and surrounding skin were processed for evaluation of biochemical and histological parameters associated with the inflammatory process. The results showed great cell viability and increase in porosity after crosslinking while antimicrobial action was observed in scaffolds containing NAC and ε-PL. Chitosan scaffolds bioconjugated with NAC/ε-PL showed improvement in tissue healing, with reduced lesion size and reduced inflammation. It is concluded that scaffolds crosslinked with chitosan-NAC-ε-PL have the desirable characteristics for tissue repair at low cost and could be considered promising biomaterials in the practice of regenerative medicine.

Jararhagin-C, a disintegrin-like protein, improves wound healing in mice through stimulation of M2-like macrophage, angiogenesis and collagen deposition.

Jararhagin-C (Jar-C) is a disintegrin-like protein, isolated from the venom of B. jararaca, with affinity for α2ß1 integrin and the ability to incite processes such as angiogenesis and collagen deposition in vivo. Thus, we raised the hypothesis that this protein could be used as a therapeutic strategy for stimulating the healing of excisional wounds in mice. Four wounds were made on the back of Swiss mice, treated with daily intradermal injections of PBS (control group) or Jar-C (200 ng). Ten animals from each experimental group were euthanized and the tissue from the wounds and skin around them were collected for further biochemical, histological and molecular analysis. Wounds treated with Jar-C showed a faster closure rate, accompanied by a reduction in neutrophil infiltrate (MPO), pro-inflammatory cytokine levels (TNF, CXCL1 and CCL2) and an accumulation of macrophages in the analyzed tissues. It was also observed a greater expression of genes associated with the phenotype of alternatively activated macrophages (M2). Concomitantly, the administration of Jar-C holds an angiogenic potential, increasing the density of blood vessels and the synthesis of pro-angiogenic cytokines (VEGF and FGF). We also observed an increase in collagen deposition, accompanied by higher levels of the pro-fibrogenic cytokine TGF-ß1. Our data suggests Jar-C stimulates wound healing through stimulation of M2-like macrophage, angiogenesis and collagen deposition. Jar-C may be explored as a therapeutic strategy for wound healing, including the treatment of chronic wounds, where processes such as inflammation, angiogenesis and the deposition / remodeling of the matrix constituents are unregulated.

Jararhagin-C, a disintegrin-like protein, improves wound healing in mice through stimulation of M2-like macrophage, angiogenesis and collagen deposition

Jararhagin-C (Jar-C) is a disintegrin-like protein, isolated from the venom of B. jararaca, with affinity for α2β1 integrin and the ability to incite processes such as angiogenesis and collagen deposition in vivo. Thus, we raised the hypothesis that this protein could be used as a therapeutic strategy for stimulating the healing of excisional wounds in mice. Four wounds were made on the back of Swiss mice, treated with daily intradermal injections of PBS (control group) or Jar-C (200 ng). Ten animals from each experimental group were euthanized and the tissue from the wounds and skin around them were collected for further biochemical, histological and molecular analysis. Wounds treated with Jar-C showed a faster closure rate, accompanied by a reduction in neutrophil infiltrate (MPO), pro-inflammatory cytokine levels (TNF, CXCL1 and CCL2) and an accumulation of macrophages in the analyzed tissues. It was also observed a greater expression of genes associated with the phenotype of alternatively activated macrophages (M2). Concomitantly, the administration of Jar-C holds an angiogenic potential, increasing the density of blood vessels and the synthesis of pro-angiogenic cytokines (VEGF and FGF). We also observed an increase in collagen deposition, accompanied by higher levels of the pro-fibrogenic cytokine TGF-β1. Our data suggests Jar-C stimulates wound healing through stimulation of M2-like macrophage, angiogenesis and collagen deposition. Jar-C may be explored as a therapeutic strategy for wound healing, including the treatment of chronic wounds, where processes such as inflammation, angiogenesis and the deposition / remodeling of the matrix constituents are unregulated.

The inhibition of adipogenesis via an in vitro assay can reduce animal use by more precisely estimating the starting dose for the acute toxic class method.

In the present work, we established an adipogenesis inhibition assay as an adequate and sensitive in vitro model for reducing animal use by estimating the starting dose for the acute toxic class (ATC) method. First, human adipose-derived stem cells (ADSCs) underwent adipogenic differentiation induction for 14 days. Then, by high-content imaging analysis, we determined the percentage and area of cell differentiation that we considered suitable for negative and positive internal control according to the quality control criteria strictly standardized mean difference (SSMD) and robust SSMD. Moreover, we established sodium dodecyl sulfate (SDS) as an external positive control in this assay. To measure reduction in animal use to estimate the starting dose for the ATC method, we evaluated 10 chemicals representing Globally Harmonized System of Classification and Labeling of Chemicals (GHS) toxicity categories 1-5 and unclassified toxicity and determined the dose-response curves for percentage and area of cell differentiation by using the Hill function with an R2 ≥ 0.85. The resulting IC50 values were used for LD50 prediction and for estimating the starting dose for the ATC method. Our results indicated that use of the inhibition of adipogenesis assay to estimate the starting dose for the ATC method would decrease animal use for 7 out of 10 tested substances, possibly all substances if we consider the more toxic test substances in GHS categories 1, 2, and 3. We can conclude that the present assay is a suitable alternative to reduce animal testing in the first steps of predicting highly toxic substances. Moreover, this method also presents internal and external controls as differentials, which guarantee the quality of the assay as well as the results. These features are important for suggesting a methodology for regulatory purposes.

Human adipose-derived stem cells (ADSC) and human periodontal ligament stem cells (PDLSC) as cellular substrates of a toxicity prediction assay.

With the increasing need to develop in vitro assays to replace animal use, human stem cell-derived methods are emerging and showing outstanding contributions to the toxicological screening of substances. Adult human stem cells such as adipose-derived stem cells (ADSC) and periodontal ligament stem cells (PDLSC) were used as cell substrates for a cytotoxicity assay and toxicity prediction using the neutral red uptake (NRU) assay. First, primary cell cultures from three independent donors, from each tissue source, were characterized as mesenchymal stem cells (MSC) by plastic adherence and appropriate immunophenotype for MSC markers (positive for CD90, CD73, and CD105 and negative for CD11b, CD34, CD45, HLADR, and CD19). Furthermore, ADSC and PDLSC were able to differentiate into adipocytes and osteoblasts when maintained under the same culture conditions previously established for the NRU assay. NRU assays for three reference test substances were performed. R2 was higher than 0.85 for all conditions, showing the feasibility to calculate IC50 values. The IC50 values were then used to predict the LD50 of the test substances, which were comparable to previous results and the ICCVAM standard test report. Primary ADSC and PDLSC showed the potential to be considered as additional models for use in cytotoxicity assays.

Bombesin Encapsulated in Long-Circulating pH-Sensitive Liposomes as a Radiotracer for Breast Tumor Identification.

Bombesin (BBN) is a tetradecapeptide that binds specifically to gastrin-releasing peptide receptors in humans. These receptors are over-expressed in several forms of cancer; radiolabeled BBN could therefore be used to detect such cancers. However, the degradation of peptides is a critical issue in the development of tumor tracers. Liposomes can be used to overcome this problem and improve the uptake of tracers by tumors. Therefore, the purpose of this study was to prepare and characterize long-circulating and pH-sensitive liposomes (SpHL) containing 99mTc-HYNIC-ßAla-Bombesin(7-14) (99mTc-BBN(7-14). In addition, the ability of this system to identify human breast cancer tissue was evaluated using biodistribution studies and scintigraphic images. Long-circulating and pH-sensitive liposomes (SpHL) were prepared and freeze-dried in the presence of cryoprotectants (glucose, mannitol, and trehalose). They were subsequently reconstituted with a solution of 99mTc-HYNIC-ßAla-Bombesin(7-14) (99mTc-BBN(7-14)). The liposomes were evaluated for size, encapsulation percentage, radiotracer leakage, and storage stability. In addition, in vivo studies were performed in breast tumor-bearing nude mice. Liposomes in the presence of glucose (SpHLG), exhibited a mean diameter of 164.5 ± 6.5 nm and exhibited a 99mTc-BBN(7-14) encapsulation percentage of 30%. In addition, they remained highly stable for up to 120 days of storage. SpHLG- 99mTc-BBN(7-14) showed longer blood circulation than free 99mTc-BBN(7-14), did. The tumor-to-muscle and tumor-to-blood ratios for SpHLG-99mTc-BBN(7-14 were high at 4 h post-injection (9.31%ID/g and 7.93%ID/g, respectively). Furthermore, scintigraphic images revealed a strong signal in the tumor area, indicating tumor specificity of SpHLG-99mTc-BBN(7-14). In summary, SpHLG-99mTc-BBN(7-14) presented characteristics suitable for a diagnostic agent, and is a potential tool for tumor identification.

Cytoplasmic-targeted parvalbumin blocks the proliferation of multipotent mesenchymal stromal cells in prophase.

INTRODUCTION: Multipotent mesenchymal stromal cells (MSCs) have gained considerable interest because of their potential use in the treatment of a variety of diseases and injuries. Although remarkable advancements have been made in clinical studies, substantial concerns still regard the safety of MSCs. Some evidence suggests that MSCs can spontaneously generate a population of cells with tumorigenic potential. Thus, studying the molecular mechanisms that control the proliferation of MSCs may be a necessary step toward the development of strategies for safe clinical practice. Ca(2+) is a second messenger that mediates a wide range of cellular responses, including the regulation of cell proliferation, but little is known about its function in MSCs. The aim of this study was to investigate the effects of targeted Ca(2+) buffering on MSCs proliferation in vitro. METHODS: Here, we used an adenoviral (Ad) vector encoding the Ca(2+) chelator protein parvalbumin (PV) fused to a nuclear exclusion signal (NES) and the Discosoma red fluorescent protein (DsRed) to investigate the function of cytoplasmic Ca(2+) signals on MSC proliferation. Confocal microscopy was used to demonstrate that PV-NES-DsRed was expressed in the cytoplasm. Ca(2+) signaling was monitored by using Fluo-4-AM. Fluorescence-activated cell sorting (FACS) analysis of cells that were stained with propidium iodide was used as a quantitative measure of cell death. The mitotic index was assessed by immunofluorescence, and the expression of cyclins was examined with Western blot. RESULTS: Our results show that the Ad-PV-NES-DsRed fusion protein decreased serum-induced Ca(2+) signaling and blocked the proliferation of rat adipose-derived MSCs (AT-MSCs) in prophase. FACS analysis revealed that Ad-PV-NES-DsRed did not induce cell death in AT-MSCs. Furthermore, Western blot analysis demonstrated that Ad-PV-NES-DsRed reduced extracellular signal-regulated kinase (Erk1/2) phosphorylation and cyclin B1 expression. Buffering cytosolic Ca(2+) did not alter the expression of cyclins A/D1/D2/D3/E and E2. CONCLUSIONS: Our results show that cytoplasmic Ca(2+) signals are important for AT-MSCs progression beyond prophase because of their effects on Erk phosphorylation and cyclin B1 expression.

SET domain-containing Protein 4 (SETD4) is a Newly Identified Cytosolic and Nuclear Lysine Methyltransferase involved in Breast Cancer Cell Proliferation.

Cancer is comprised of a multitude of epigenetic abnormalities, including the global loss and regional gain of DNA methylation as well as alterations in histone methylation. Here, we characterize a new methyltransferase, SET domain-containing protein 4 (SETD4), which is involved in breast carcinogenesis. Quantitative real-time PCR (qPCR) showed elevated expression levels of SETD4 in several breast cancer cell lines. SETD4 overexpression was confirmed by western blot analysis suggesting a correlation between high expression of SETD4 and a lack of the estrogen receptor (ER) in breast cancer. In addition, cell fractionation studies and confocal immunofluorescence revealed the nuclear and non-nuclear localization of this new protein. SETD4 knockdown in breast cancer cell lines significantly suppressed their proliferation and delayed the G1/S cell cycle transition without affecting apoptosis. Furthermore, western blot analysis showed that knockdown of SETD4 decreased cyclin D1 expression, revealing the involvement of SETD4 in cell cycle regulation. These data imply that SETD4 plays a crucial role in breast carcinogenesis and could be a novel molecular target for the development of new strategies for the diagnosis and treatment of breast cancer.

Formation of ion pairing as an alternative to improve encapsulation and anticancer activity of all-trans retinoic acid loaded in solid lipid nanoparticles.

This work aims to develop solid lipid nanoparticles (SLNs) loaded with retinoic acid (RA) to evaluate the influence of two lipophilic amines, stearylamine (SA) and benethamine (BA), and one hydrophilic, triethylamine (TA), on drug-encapsulation efficiency (EE) and cytotoxicity in cancer cell lines. The SLNs were characterized for EE, size, and zeta potential. The mean particle size decreased from 155 ± 1 nm (SLNs without amine) to 104 ± 4, 95 ± 1, and 96 ± 1 nm for SLNs prepared with SA, BA, and TA, respectively. SA-RA-loaded SLNs resulted in positively charged particles, whereas those with TA and BA were negatively charged. The EEs were significantly improved with the addition of the amines, and they increased from 36% ± 6% (without amine) to 97% ± 2%, 90% ± 2%, and 100% ± 1% for SA, TA, and BA, respectively. However, stability studies showed higher EE for BA-RA-loaded SLNs than TA-RA-loaded SLNs after 30 days. The formulations containing SA loaded or unloaded (blank SLNs) with RA were cytotoxic in normal and cancer cell lines. In contrast, the blank SLNs containing TA or BA did not show cytotoxicity in human breast adenocarcinoma cells (MCF-7), while RA-loaded SLNs with the respective amines were significantly more cytotoxic than free RA. Furthermore, the cytotoxicity of BA-RA-loaded SLNs was significantly higher than TA-RA-loaded SLNs. These findings are in agreement with the data obtained in the evaluation of subdiploid DNA content and cell-cycle analysis, which showed better anticancer activity for BA-RA-loaded SLNs than TA-RA-loaded SLNs and free RA. Taken together, these findings suggest that the BA-RA-loaded SLN formulation is a promising alternative for the intravenous administration of RA in the treatment of cancer.