Macrophage cell morphology-imprinted substrates can modulate mesenchymal stem cell behaviors and macrophage M1/M2 polarization for wound healing applications.

Mesenchymal stem cells and macrophages (MQ) are two very important cells involved in the normal wound healing process. It is well understood that topological cues and mechanical factors can lead to different responses in stem cells and MQ by influencing their shape, cytoskeleton proliferation, migration, and differentiation, which play an essential role in the success or failure of biomaterial implantation and more importantly wound healing. On the other hand, the polarization of MQ from proinflammatory (M1) to prohealing (M2) phenotypes has a critical role in the acceleration of wound healing. In this study, the morphology of different MQ subtypes (M0, M1, and M2) was imprinted on a silicon surface (polydimethylsiloxane [PDMS]) to prepare a nano-topography cell-imprinted substrate with the ability to induce anti-inflammatory effects on the mouse adipose-derived stem cells (ADSCs) and RAW264.7 monocyte cell line (MO). The gene expression profiles and flow cytometry of MQ revealed that the cell shape microstructure promoted the MQ phenotypes according to the specific shape of each pattern. The ELISA results were in agreement with the gene expression profiles. The ADSCs on the patterned PDMS exhibited remarkably different shapes from no-patterned PDMS. The MOs grown on M2 morphological patterns showed a significant increase in expression and section of anti-inflammatory cytokine compared with M0 and M1 patterns. The ADSCs homing in niches heavily deformed the cytoskeletal, which is probably why the gene expression and phenotype unexpectedly changed. In conclusion, wound dressings with M2 cell morphology-induced surfaces are suggested as excellent anti-inflammatory and antiscarring dressings.

Chitosan-Placental ECM Composite Thermos-Responsive Hydrogel as a Biomimetic Wound Dressing with Angiogenic Property.

Attempts are being made to develop an ideal wound dressing with excellent biomechanical and biological properties. Here, a thermos-responsive hydrogel is fabricated using chitosan (CTS) with various concentrations (1%, 2.5%, and 5% w/v) of solubilized placental extracellular matrix (ECM) and 20% ß-glycerophosphate to optimize a smart wound dressing hydrogel with improved biological behavior. The thermo-responsive CTS (TCTS) alone or loaded with ECMs (ECM-TCTS) demonstrate uniform morphology using SEM. TCTS and ECM1%-TCTS and ECM2.5%-TCTS show a gelation time of 5 min at 37 °C, while no gel formation is observed at 4 and 25 °C. ECM5%-TCTS forms gel at both 25 and 37 °C. The degradation and swelling ratios increase as the ECM content of the hydrogel increase. All the constructs show excellent biocompatibility in vitro and in vivo, however, the hydrogels with a higher concentration of ECM demonstrate better cell adhesion for fibroblast cells and induce expression of angiogenic factors (VEGF and VEGFR) from HUVEC. Only the ECM5%-TCTS has antibacterial activity against Acinetobacter baumannii ATCC 19606. The data obtained from the current study suggest the ECM2.5%-TCTS as an optimized smart biomimetic wound dressing with improved angiogenic properties now promises to proceed with pre-clinical and clinical investigations.

Anti-inflammatory effect of mesenchymal stem cells on hepatocellular carcinoma in the xenograft mice model.

BACKGROUND: Hepatocellular carcinoma (HCC) is the fifth most diagnosed cancer and the second leading cause of cancer-related deaths worldwide. Sorafenib is the standard treatment used in the advanced stages of HCC. Cell therapy with mesenchymal stem cells (MSCs)-based cell therapy has proven effective in immune regulation and tumour growth inhibition. OBJECTIVES: In this study, we investigated the anti-inflammatory effect of MSCs on HCC xenografts. METHODS: Human HepG2 cell lines were subcutaneously implanted into the flank of 12 nude mice, divided into three groups: the control group, the IV group (intravenous MSCs injection) and the local group (local MSCs injection). Mice were sacrificed 6 weeks after tumour implantation, and tumours were resected entirety. Quantitative real-time polymerase chain reaction (qRT-PCR) measured the gene expression of inflammatory markers, including tumour necrosis factor-α (TNF-α), interleukin (IL)-1α and IL-10. Aspartate transaminase (AST), alanine transaminase (ALT) and urea levels were measured using spectrophotometry to ensure the safety of MSC therapy. RESULTS: Gene expressions for all three inflammatory markers were reduced in both MSCs groups compared to the control group. AST, ALT and urea levels remained in normal ranges. CONCLUSIONS: MSC therapy can reduce inflammation in HCC xenograft mouse models.

Sorafenib and Mesenchymal Stem Cell Therapy: A Promising Approach for Treatment of HCC.

Hepatocellular carcinoma (HCC) is the fifth most commonly diagnosed cancer and the second most common cause of cancer-related death worldwide. Sorafenib (Sora) is used as a targeted therapy for HCC treatment. Mesenchymal stem cells (MSCs) are applied as a new approach to fight malignancies. Drug resistance and side effects are the major concerns with Sora administration. The effect of using the combination of sorafenib and MSCs on tumor regression in xenograft HCC models was evaluated in this study. Methods and Materials. Human hepatocellular carcinoma cell lines (HepG2) were subcutaneously implanted into the flank of 18 nude mice. The animals were randomly divided into six groups (n = 3); each received Sora (oral), MSCs (IV injection), MSCs (local injection), Sora + MSCs (IV injection), Sora + MSCs (local injection), or no treatment (the control group). Six weeks after tumor implantation, the mice were scarified and tumoral tissues were resected in their entirety. Histopathological and immunohistochemical evaluations were used to measure tumor proliferation and angiogenesis. Apoptotic cells were quantified using the TUNEL assay. Results. No significant difference was found in the tumor grade among the treatment groups. Differentiation features of the tumoral cells were histopathologically insignificant in all the groups. Tumor necrosis was highest in the hpMSC (local) + Sora group. Tumor cell proliferation was reduced in hpMSC (local) + Sora-treated and hpMSC (IV) + Sora-treated mice compared with the other groups. Apoptotic-positive cells occupied a greater proportion in the Sora, hpMSC (IV) + Sora, and hpMSC (local) + Sora groups. Conclusion. A combination of chemotherapy and MSC can yield to more favorable results in the treatment of HCC.

Fabrication of chitosan-polyethylene glycol nanocomposite films containing ZIF-8 nanoparticles for application as wound dressing materials.

Biocompatible nanocomposite films based on chitosan (CS) and polyethylene glycol (PEG) polymers containing cephalexin (CFX) antibiotic drug and zeolitic imidazolate framework-8 (ZIF-8) nanoparticles (NPs) were designed and fabricated to develop wound dressing materials capable of controlled drug release. Swelling experiment was performed in three acidic, neutral, and alkaline solutions. The tensile strength test reflected that upon increasing the NPs loading within the films, the tensile strength was enhanced but the elongation at break was diminished. The release of the CFX was intensively increased within approximately 3, 8, and 10 h (burst release) in acidic, neutral, and alkaline media, respectively while after that the CFX was smoothly released over time (sustained release). The antibacterial activities of all films were examined against Gram-positive (S. aureus, B. cereus) and Gram-negative (E. coli, P. aeruginosa, and Acinetobacter) bacteria frequently found in the infected wounds. Moreover, the MTT assay revealed that all films had high cell viabilities towards the L929 fibroblast cells confirming these nanocomposites could be used as favorable wound dressing materials. Finally, the film containing 4% ZIF-8 NPs (film 5) was chosen as the best sample due to it revealed appropriate mechanical properties, swelling, drug release and cell viability among all samples examined.

Characterization and Validation of Hepatocellular Carcinoma (HCC) Xenograft tumor as a Suitable Liver Cancer Model for Preclinical Mesenchymal Stem Cell Studies

Background: Hepatocellular carcinoma (HCC) is the fifth most diagnosed cancer and the third leading cause of cancer-related death. sorafenib is used as a standard therapy to treat HCC. mesenchymal stromal cells (MSCs) have also been used to suppress HCC. Here we investigate the development of a xenograft model of liver cancer to study the homing of hpMSC-GFP cells, tumor kinetics and molecular characterizations of HCC. Methods: To create xenograft models of HCC, HepG2 cell lines were inoculated into the flanks of 9 nude mice bilaterally. Animals were then divided into three groups: the first group received hpMSC-GFP systemically, the second received intra-tumoral hpMSC-GFP and the third received PBS. The first two groups were sacrificed after 72 hours of MSCs injection but the third group was followed up for forty days. One tumor from each animal was then transferred to formalin buffer for H&E staining and immunohistochemistry analysis (KI67 and CD34), and the other tumor was used for ex-vivo imaging. Blood samples were taken from all subjects before sacrificing them. Results: Histopathological fidelity of heterotopic HePG2 xenograft models to human HCC tumors was demonstrated. Biochemical evaluation suggested the health of the animal's liver and kidneys. Ex-vivo imaging illustrated homing of more hpMSC-GFP cells in tumor tissues derived from the group receiving intra-tumoral hpMSC-GFP. Conclusion: A standard method was used to inoculate tumor cells and the intervention was shown to be safe to liver and kidneys. Local injection of MSCs can be used as cell therapy to fight neoplasms.

IgG1 and IgG2a profile of serum antibodies to Leishmania major amastigote in BALB/c and C57BL/6Mice.

It is well accepted that in experimental model of Leishmania major infection, BALB/c mice mount a Th2 response and produce IgG1 predominantly whereas C57BL/6mice enhance Th1 response with the biased production of IgG2a antibodies. Therefore, screening for parasite antigens on the basis of reactivity with sera from infected susceptible or resistant mice might be used for the identification of Th1- or Th2-inducing antigens.In this study, the antigenic profile of Leishmania major amastigote that induce IgG1 or IgG2a isotypes in infected BALB/c or C57BL/6 mice were compared.Western blot analyses revealed that 27, 40, 43, 45 and 114 kDa proteins elicited IgG1 and not IgG2a in both BALB/c and C57BL/6 mice and 55 kDa protein was recognized exclusively by IgG1 of BALB/c mice sera. On the contrary, the bands corresponding to proteins with molecular weights (MW) of 30, 35, 52, 58 and 66 were intensively immunostained with IgG2a from C57BL/6 mice which made them potential candidates for eliciting Th1 response.In conclusion, the results showed that the generation of the immune responses depended on the mouse strain and some leishmanial antigens had an intrinsic potency to elicit Th2 responses.

Toll-like receptor 4 polymorphisms predispose to cutaneous leishmaniasis.

The clinical spectrum of cutaneous leishmaniasis (CL) is extremely variable. Studies in experimental leishmaniasis have revealed a role for TLR4 in control of infection. In the present study the associations between TLR4 mutations (Asp299Gly and Thr399Ile) with outcome of CL have been investigated. Genotyping for Asp299Gly and Thr399Ile was performed in patients with chronic (N = 22) and acute (N = 61) CL, asymptomatic (N = 45) and healthy leishmanin skin test negative individuals (N = 75). The results showed the frequency of the Asp299Gly genotype was increased in patients with chronic disease (OR 25.3, 95% CI 5.2-115.6, P < 0.001) and patients with acute disease (OR 8.03, 95% CI 1.7-37.7, P = 0.006) compared to LST negative subjects. Thr399Ileu genotype was significantly over represented among patients with chronic disease (27.3%, P < 0.001), patients with acute disease (13.1%, P = 0.016), and asymptomatic donors (15.6%, P = 0.008) in comparison with LST negative normal group (1.3%). Both variants were found together more frequently in patients with chronic disease compared to the patients with acute disease (P = 0.045), and asymptomatic donors (P = 0.045). The results provide evidence that polymorphisms of TLR4 gene may lead to the increased susceptibility to and severity of infection by Leishmania major. The concomitant carriage of both mutations increases the susceptibility of individuals to CL.

Lack of association of Toll-Like Receptor 2 Arg753Gln with cutaneous leishmaniasis.

The aim of the present study was to investigate the frequency of Arg753Gln and Arg677Trp polymorphisms of TLR2 in patients with cutaneous leishmaniasis (CL) compared to healthy controls. The polymorphisms were genotyped by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and amplification refractory mutation system assay (ARMS-PCR). The results showed that the frequency of Arg753Gln genotype was 14.3% and 10.1% in CL patients and normal controls, respectively. No one in either group was homozygous for the mutation. There was no significant difference in the genotype frequency. In contrast to the results for Arg753Gln polymorphism, we did not detect any case with Arg677Trp polymorphism in either control or patient group. In conclusion the TLR2 mutations are found equally in CL patients and healthy subjects.

Immune response to Leishmania antigen in anthroponotic cutaneous leishmaniasis.

BACKGROUND: Leishmania (L.) tropica is the causative agent of anthroponotic cutaneous leishmaniasis (ACL) in Iran. The disease often heals within a year; however, the non-healing forms of disease are also known. The immunologic responses to L. major infection have been studied in depth, however little is known about the immune status of L. tropica-infected patients. MATERIALS AND METHODS: This study was conducted to evaluate T-cell responses to Leishmania antigen in non-healing patients, patients with acute lesion, and healthy donors. Peripheral blood mononuclear cells (PBMC) were cultured with antigen and lymphoproliferative responses were determined. Cytokine profile including gamma interferon (IFN-gamma), interleukin (IL)-5, and IL-13 in supernatants of stimulated cells was also determined. RESULTS: The results showed PBMC from both groups of patients proliferated vigorously in response to Leishmania antigens. The levels of IFN-gamma and IL-13 were comparable between patients with acute lesions and non-healing patients. Non-healing patients had significantly higher median levels of IL-5 than patients with acute lesions. The cells from healthy individuals did not respond to Leishmania antigens. CONCLUSIONS: High levels of IFN-gamma, IL-5, and IL-13 in non-healing patients suggest a mixed Th1/Th2 response, whereas patients with acute lesion respond to infection by Th1-type response.