Preparation and in vitro evaluation of cell adhesion and long-term proliferation of stem cells cultured on silibinin co-embedded PLGA/Collagen electrospun composite nanofibers.

The present research aims to evaluate the efficacy of Silibinin-loaded mesoporous silica nanoparticles (Sil@MSNs) immobilized into polylactic-co-glycolic acid/Collagen (PLGA/Col) nanofibers on the in vitro proliferation of adipose-derived stem cells (ASCs) and cellular senescence. Here, the fabricated electrospun PLGA/Col composite scaffolds were coated with Sil@MSNs and their physicochemical properties were examined by FTIR, FE-SEM, and TGA. The growth, viability and proliferation of ASCs were investigated using various biological assays including PicoGreen, MTT, and RT-PCR after 21 days. The proliferation and adhesion of ASCs were supported by the biological and mechanical characteristics of the Sil@MSNs PLGA/Col composite scaffolds, according to FE- SEM. PicoGreen and cytotoxicity analysis showed an increase in the rate of proliferation and metabolic activity of hADSCs after 14 and 21 days, confirming the initial and controlled release of Sil from nanofibers. Gene expression analysis further confirmed the increased expression of stemness markers as well as hTERT and telomerase in ASCs seeded on Sil@MSNs PLGA/Col nanofibers compared to the control group. Ultimately, the findings of the present study introduced Sil@MSNs PLGA/Col composite scaffolds as an efficient platform for long-term proliferation of ASCs in tissue engineering.

Cell sheet biofabrication by co-administration of mesenchymal stem cells secretome and vitamin C on thermoresponsive polymer.

Cell sheet technology aims at replacement of artificial extracellular matrix (ECM) or scaffolds, popular in tissue engineering, with natural cell derived ECM. Adipose tissue mesenchymal stem cells (ASCs) have the ability of ECM secretion and presented promising outcomes in clinical trials. As well, different studies found that secretome of ASCs could be suitable for triggering cell free regeneration induction. The aim of this study was to investigate the effect of using two bio-factors: secretome of ASCs (SE) and vitamin C (VC) for cell sheet engineering on a thermosensitive poly N-isopropyl acryl amide-Methacrylic acid (P(NIPAAm-MAA)) hydrogel. The results revealed that using thermosensitive P(NIPAAm-MAA) copolymer as matrix for cell sheet engineering lead to a rapid ON/OFF adhesion/deadhesion system by reducing temperature without enzymatic treatment (complete cell sheet release takes just 6 min). In addition, our study showed the potential of SE for inducing ASCs sheet formation. H&E staining exhibited the properties of a well-formed tissue layer with a dense ECM in sheets prepared by both SE and VC factors, as compared to those of VC or SE alone. Functional synergism of SE and VC exhibited statistically significant enhanced functionality regarding up-regulation of stemness genes expression, reduced ß-galactosidase associated senescence, and facilitated sheet release. Additionally, alkaline phosphatase activity (ALP), mineralized deposits and osteoblast matrix around cells confirmed a better performance of ostogenic differentiation of ASCs induced by VC and SE. It was concluded that SE of ASCs and VC could be outstanding biofactors applicable for cell sheet technology.

Development of Emu oil-loaded PCL/collagen bioactive nanofibers for proliferation and stemness preservation of human adipose-derived stem cells: possible application in regenerative medicine.

Adipose tissue-derived stem cells (ASCs) are promising candidate in stem cell therapies, and maintaining their stemness potential is vital to achieve effective treatment. Natural-based scaffolds have been recently attracted increasing attention in nanomedicine and drug delivery. In the present study, a polymeric nanofibrous scaffold was developed based on the polycaprolactone/Collagen (PCL/Coll) containing Emu oil as a bioactive material to induce the proliferation of ASCs, while simultaneously preserving the stemness property of those cells. Fabrication of the electrospun Emu oil-loaded PCL/Coll nanofibers was confirmed by using FE-SEM, FTIR, and tensile test. ASCs were seeded on two types of nanofibers (PCL/Coll and Emu oil-loaded PCL/Coll) and their proliferation, cell cycle progression, and stemness gene expressions were evaluated using MTT, propidium iodide staining, and qPCR during 14 days, respectively. The results indicated that ASCs displayed improved adhesion capacity with the higher rates of bioactivity and proliferation on the Emu oil-loaded nanofibers than the other groups. The proliferation capacity of ASCs on Emu oil-loaded PCL/Coll nanofibers was further confirmed by the cell cycle progression analysis. It was also found that Emu oil-loaded nanofibers significantly up-regulated the expression of stemness markers including sox-2, nanog, oct4, klf4, and c-Myc. The results demonstrated that the nanofibers containing Emu oil can reinforce the cell adhesion and enhance ASCs proliferation while preserving their stemness; therefore, using scaffolds containing natural products may have a great potential to enhance the in vitro expansion capacity of ASCs in the field of stem cell therapy and regenerative medicine.

Development of nano-liposomal human growth hormone as a topical formulation for preventing uvb-induced skin damage.

Due to its involvement in skin maintenance and repair, topical administration of recombinant human growth hormone (rhGH) is an interesting strategy for therapeutic purposes. We have formulated and characterized a topical rhGH-loaded liposomal formulation (rhGH-Lip) and evaluated its safety, biological activity, and preventive role against UVB-induced skin damage. The rhGH-Lip had an average size and zeta potential of 63 nm and -33 mV, respectively, with 70 % encapsulation efficiency. The formulation was stable at 4 °C for at least one year. The SDS-PAGE and circular dichroism results showed no structural alterations in rhGH upon encapsulation. In vitro, studies in HaCaT, HFFF-2, and Ba/F3-rhGHR cell lines confirmed the safety and biological activity of rhGH-Lip. Franz diffusion cell study showed increased rhGH skin permeation compared to free rhGH. Animal studies in nude mice showed that liposomal rhGH prevented UVB-induced epidermal hyperplasia, angiogenesis, wrinkle formation, and collagen loss, as well as improving skin moisture. The results of this study show that rhGH-Lip is a stable, safe, and effective skin delivery system and has potential as an anti-wrinkle formulation for topical application. This study also provides a new method for the topical delivery of proteins and merits further investigation.

Hepatic cell-sheet fabrication of differentiated mesenchymal stem cells using decellularized extracellular matrix and thermoresponsive polymer.

PURPOSE: Liver tissue engineering via cell sheet technology would open new doors for treatment of patients with liver failure. Decellularized tissues could provide sufficient extracellular matrix (ECM) to support development of hepatocytes in in vivo niches. Besides, with the potential of temperature responsive polymer (pNIPAAm) as an intelligent surface for controlling the attachment/detachment of cell, we set out to generate three in vitro microenvironments models including I: pNIPAAm hydrogel (pN hydrogel), II: decellularized ECM incorporated into pNIPAAm hydrogel (dECM + pN hydrogel) and III: decellularized ECM scaffold (dECM scaffold) to investigate the structural and function cues of hepatocyte-like cells after differentiation of adipose tissue-derived mesenchymal stem cells (AT-MSCs) on the surface of these models. METHOD: dECM scaffold was obtained after decellularization of rat liver, and its efficiency was analyzed. pN hydrogel and dECM + pN hydrogel (1:3 and 2:3 ratios) of were fabricated, and scaffold architecture was characterized. Each well of culturing plates was coated separately with these three constructs and AT-MSCs were instructed to differentiate into hepatocyte-like cells (HLCs). After recellularization, patterns of differentiation, and expression of hepatogenic markers were investigated via biochemical assays and qRT-PCR at different time points. RESULTS: Multipotency of AT-MSCs, after their ability for osteogenesis and adipogenesis was documented. Production of dense and intact cell sheets was reported in dECM + pN hydrogel, as opposed to pN hydrogel and dECM scaffold. Also, statistically significant difference of HLCs functionality in dECM + pN hydrogel was confirmed after evaluation of the expression of hepatocyte markers including, alpha-fetoprotein, cytokeratin 18, cytochrome P450-2E1 and phosphoenolpyruvate carboxykinase. CONCLUSION: Our results proved dECM + pN hydrogel were able to preserve hepatocyte function in cell sheets owing to the high level of albumin, urea, hepatogenic markers, and glycogenesis potential of HLCs. Accordingly, dECM incorporated in pN hydrogel could remodel microenvironments to guide the AT-MSCs into conducive differentiation and proliferation to give rise to multilayer sheets of cells in their own ECM.

Evaluation of the adhesion of human dental pulp stem cells to different endodontic biomaterials before and after setting.

Background. Stem cell-based treatment modalities have been potential strategies for tissue regeneration in many conditions. Several studies have evaluated the biologic properties of DPSCs and their efficacy in the treatment of a variety of diseases. The present study was undertaken to evaluate the adhesion behavior of DPSCs on different endodontic materials before and after setting. Methods. The crowns of the selected teeth were removed, and the root canals were prepared and obturated with gutta-percha and AH26 sealer. A retrograde cavity was prepared at root ends. Different materials were placed in the cavities. Then the samples were attached to the wells with the use of a chemical glue. Dental pulp stem cells were allowed to proliferate to reach a count of 2 million and transferred to -12well plates in association with a culture medium. Finally, the samples attached to the wells were exposed to the stem cells immersed in the culture medium before and after setting. Then adhesion of the stem cells was evaluated using SEM. Results. The SEM results showed cellular adhesion in the samples containing CEM cement both before and after setting. The samples containing MTA Angelus and ProRoot MTA exhibited cellular adhesion before setting, with no cellular adhesion after setting. The samples containing AH26 and MTA Fillapex sealers exhibited cellular adhesion after setting, with no adhesion before setting. The samples containing simvastatin exhibited no cellular adhesion before setting; this material had dissolved in the culture medium after setting evaluation. Conclusion. The results of the present study showed that of all the materials tested, CEM cement had the highest capacity for dental pulp stem cell adhesion.

Lysine-embedded cellulose-based nanosystem for efficient dual-delivery of chemotherapeutics in combination cancer therapy.

Combination therapy by two or multiple drugs with different mechanisms of action is a promising strategy in cancer treatment. In this regard, a wide range of chemotherapeutics has used simultaneously to achieve the synergistic effect and overcome the adverse side effects of single-drug therapy. Herein, we developed a biocompatible nanoparticle-based system composed of nanocrystalline cellulose (NCC) and amino acid l-lysine for efficient co-delivery of model chemotherapeutic methotrexate (MTX) and polyphenol compound curcumin (CUR) to the MCF-7 and MDA-MB-231 cells. The drugs could release in a sustained and acidic-facilitate manner. In vitro cytotoxicity results represented the superior anti-tumor efficacy of the dual-drug-loaded nanocarriers. Possible inhibition of cell growth and induction of apoptosis in the cells treated with different formulations of CUR and MTX were explored by cell cycle analysis and DAPI staining. Overall, the engineered nanosystem can be used as suitable candidates to achieve efficient multi-drug delivery for combination cancer therapy.

Macrophage repolarization using emu oil-based electrospun nanofibers: possible application in regenerative medicine.

In the regenerative medicine therapies, the availability of engineered scaffolds that modulate inflammatory states is highly required. The aim of this study was to evaluate the efficiency of electrospun nanofibrous scaffolds containing natural substances with anti-inflammatory properties such as Emu oil (EO) to control inflammation and re-polarization of macrophages toward M2 anti-inflammatory phonotype. For this purpose, bead free and smooth EO-blended PCL/PEG electrospun nanofibrous mats were successfully fabricated and characterized using FE-SEM, FTIR, and Universal Testing Machine. GC/MS findings of pure EO revealed the fatty acids composition. MTT results showed that macrophage viability on EO-PCL/PEG nanofibres was higher than on PCL/PEG nanofibres and control (p ≤ .05). Additionally, the presence of EO into nanofibres was found to influence on macrophage morphologies, using FE-SEM. qPCR results showed a reduction in iNOS-2 and an increase in Arg-1 levels of macrophages seeded on EO-PCL/PEG nanofibres, indicating the successfully polarization of the macrophages to M2 phenotype. The change in macrophage phenotype on EO-based nanofibres could suppress the inflammation in LPS/IFN-γ stimulated macrophages as evidenced by a major reduction in pro-inflammatory cytokine levels TNF-α, IL-1ß, and IL-6. Conclusively, the results demonstrated that EO-based nanofibres efficiently modulated RAW264.7 macrophage polarity toward an anti-inflammatory M2 phenotype.

Enhanced anticancer potency by thermo/pH-responsive PCL-based magnetic nanoparticles.

Great efforts have been made to develop drug carriers with the aim of providing predictable therapeutic response. Moreover, combination therapies have become promising strategies for clinical cancer treatment with synergistic effects. The present study purposed to develop a new stimuli-responsive paramagnetic nanocarrier for the intracellular co-delivery of doxorubicin (DOX) and methotrexate (MTX) to the MCF7 cell line. A novel thermo/pH-sensitive amphiphilic paramagnetic nanocomposite comprised of hydrophobic and biodegradable PCL segments and a hydrophilic biocompatible P(NIPAAm-co-HEMA-co-MAA-co-TMSPMA) block was designed and synthesized by combining the ring opening and free radical polymerization methods. The structure and physic-chemical characterization of synthesized nanoparticles and intermediates were studied and revealed using FTIR, HNMR, CNMR, SEM, EDX, TGA, and VSM techniques. DOX and MTX on a nanocarrier achieved 95.04 and 97.29% encapsulation efficiency, respectively. The dual drug release profile revealed tumor niche-assisted release behavior (more drug release was observed at a temperature of 41 °C and pH ≤ 5.4). The antitumor ability of the DOX/MTX-loaded nanocomposite was significantly higher than that of free drugs, confirmed by MTT assay, DAPI staining, cell cycle, and real-time PCR analysis on MCF7 cell lines. Furthermore, the cytotoxicity assay of a nanocarrier to the MCF7 cell line revealed its suitability as an anticancer drug nanocarrier. The results indicated that this engineered dual anticancer drug delivery system ensures increased antitumor activity as well as decreased toxicity in comparison with the free drugs.

A Comparison of the Effects of Silica and Hydroxyapatite Nanoparticles on Poly(ε-caprolactone)-Poly(ethylene glycol)-Poly(ε-caprolactone)/Chitosan Nanofibrous Scaffolds for Bone Tissue Engineering.

BACKGROUND: The major challenge of tissue engineering is to develop constructions with suitable properties which would mimic the natural extracellular matrix to induce the proliferation and differentiation of cells. Poly(ɛ-caprolactone)-poly(ethylene glycol)-poly(ɛ-caprolactone) (PCL-PEG-PCL, PCEC), chitosan (CS), nano-silica (n-SiO2) and nano-hydroxyapatite (n-HA) are biomaterials successfully applied for the preparation of 3D structures appropriate for tissue engineering. METHODS: We evaluated the effect of n-HA and n-SiO2 incorporated PCEC-CS nanofibers on physical properties and osteogenic differentiation of human dental pulp stem cells (hDPSCs). Fourier transform infrared spectroscopy, field emission scanning electron microscope, transmission electron microscope, thermogravimetric analysis, contact angle and mechanical test were applied to evaluate the physicochemical properties of nanofibers. Cell adhesion and proliferation of hDPSCs and their osteoblastic differentiation on nanofibers were assessed using MTT assay, DAPI staining, alizarin red S staining, and QRT-PCR assay. RESULTS: All the samples demonstrated bead-less morphologies with an average diameter in the range of 190-260 nm. The mechanical test studies showed that scaffolds incorporated with n-HA had a higher tensile strength than ones incorporated with n-SiO2. While the hydrophilicity of n-SiO2 incorporated PCEC-CS nanofibers was higher than that of samples enriched with n-HA. Cell adhesion and proliferation studies showed that n-HA incorporated nanofibers were slightly superior to n-SiO2 incorporated ones. Alizarin red S staining and QRT-PCR analysis confirmed the osteogenic differentiation of hDPSCs on PCEC-CS nanofibers incorporated with n-HA and n-SiO2. CONCLUSION: Compared to other groups, PCEC-CS nanofibers incorporated with 15 wt% n-HA were able to support more cell adhesion and differentiation, thus are better candidates for bone tissue engineering applications.

Recent advances on biomedical applications of scaffolds in wound healing and dermal tissue engineering.

The tissue engineering field has developed in response to the shortcomings related to the replacement of the tissues lost to disease or trauma: donor tissue rejection, chronic inflammation and donor tissue shortages. The driving force behind the tissue engineering is to avoid the mentioned issues by creating the biological substitutes capable of replacing the damaged tissue. This is done by combining the scaffolds, cells and signals in order to create the living, physiological, three-dimensional tissues. A wide variety of skin substitutes are used in the treatment of full-thickness injuries. Substitutes made from skin can harbour the latent viruses, and artificial skin grafts can heal with the extensive scarring, failing to regenerate structures such as glands, nerves and hair follicles. New and practical skin scaffold materials remain to be developed. The current article describes the important information about wound healing scaffolds. The scaffold types which were used in these fields were classified according to the accepted guideline of the biological medicine. Moreover, the present article gave the brief overview on the fundamentals of the tissue engineering, biodegradable polymer properties and their application in skin wound healing. Also, the present review discusses the type of the tissue engineered skin substitutes and modern wound dressings which promote the wound healing.

Towards optimization of odonto/osteogenic bioengineering: in vitro comparison of simvastatin, sodium fluoride, melanocyte-stimulating hormone.

Tissue engineering has emerged as a potential therapeutic option for dental problems in recent years. One of the policies in tissue engineering is to use both scaffolds and additive factors for enhancing cell responses. This study aims to evaluate and compare the effect of three types of biofactors on poly-caprolactone-poly-ethylene glycol-poly caprolactone (PCL-PEG-PCL) nanofibrous scaffold on human dental pulp stem cell (hDPSCs) engineering. The PCL-PEG-PCL copolymer was synthesized with ring opening polymerization method, and its nanofiber scaffold was prepared by electrospinning method. Nanofibrous scaffold-seeded hDPSCs were treated with sodium fluoride (NaF), melanocyte-stimulating hormone (MSH), or simvastatin (SIM). Non-treated nanofiber seeded cells were utilized as control. The viability, biocompatibility, adhesion, proliferation rate, morphology, osteo/odontogenic potential, and the expression of tissue-specific genes were studied. The results showed that significant higher results demonstrated significant higher adhesive behavior, viability, alizarin red activity, and dentin specific gene expression in MSH- and SIM-treated cells (p < 0.05). This study is unique; in that, it compares the effects of different treatments for optimization of dental tissue engineering.

An overview on different strategies for the stemness maintenance of MSCs.

Recent evidence suggests that mesenchymal stem cells (MSCs) have promising therapeutic potential for a broad range of diseases. Because the percentage of MSCs obtained from tissues is very low for cell therapy applications, ex vivo expansion of MSCs is necessary, but aging, loss of stemness and undesired differentiation of them during in vitro cultivation reduces their effectiveness. For achieving ideal therapeutic potential of MSCs in tissue regenerative purposes, it is necessary to retain their stemness properties in vitro. This review emphasis on the last updates in preserving the self-renewal capability of stem cells through in vitro expansion with different parameters.

Novel dual stimuli-responsive ABC triblock copolymer: RAFT synthesis, "schizophrenic" micellization, and its performance as an anticancer drug delivery nanosystem.

A novel pH- and thermo-responsive ABC triblock copolymer {poly[(2-succinyloxyethyl methacrylate)-b-(N-isopropylacrylamide)-b-[(N-4-vinylbenzyl),N,N-diethylamine]]} [P(SEMA-b-NIPAAm-b-VEA)] was successfully synthesized via reversible addition of fragmentation chain transfer (RAFT) polymerization technique. The molecular weights of PHEMA, PNIPAAm, and PVEA segments in the synthesized triblock copolymer were calculated to be 10,670, 6140, and 9060gmol-1, respectively, from proton nuclear magnetic resonance (1H NMR) spectroscopy. The "schizophrenic" self-assembly behavior of the synthesized P(SEMA-b-NIPAAm-b-VEA) triblock copolymer under pH and thermal stimulus were investigated by means of 1H NMR and ultraviolet-visible (UV-vis) spectroscopies as well as dynamic light scattering (DLS) and zeta potential (ξ) measurements. The doxorubicin hydrochloride (DOX)-loading capacity, and stimuli-responsive drug release ability of the synthesized triblock copolymer were also investigated. The biocompatibility of the synthesized triblock copolymer was confirmed through the assessing survival rate of breast cancer cell line (MCF7) using MTT assay. In contrast, DOX-loaded triblock copolymer exhibited an efficient anticancer performance in comparison with free DOX verified by MTT and DAPI staining assays. As the results, we envision that the synthesized P(SEMA-b-NIPAAm-b-VEA) triblock copolymer can be applied as an enhanced anticancer drug delivery nanosystem, mainly due to its smart physicochemical and biocompatibility properties.

Preparation and characterization of PLGA-ß-CD polymeric nanoparticles containing methotrexate and evaluation of their effects on T47D cell line.

Among all cancers that affect women, breast cancer has most mortality rate. It is essential to attain more safe and efficient anticancer drugs. Recent advances in medical nanotechnology and biotechnology have caused in novel improvements in breast and other cancer drug delivery. Methotrexate is an anticancer drug that prevents the dihydrofolate reductase enzyme, which inhibits in the formation of DNA, RNA and proteins which have poor water-solubility. For enhancing the solubility and stability of drugs in delivery systems, we used methotrexate-loaded PLGA- beta-cyclodextrin nanoparticles. The PLGA- beta-cyclodextrin nanoparticles were synthesized by a double emulsion method and characterized with FT-IR and SEM. T47D breast cancer cell lines were treated with equal concentrations of methotrexate-loaded PLGA- beta-cyclodextrin nanoparticles and free methotrexate. MTT assay confirmed that methotrexate-loaded PLGA- beta-cyclodextrin nanoparticles enhanced cytotoxicity and drug delivery in T47D breast cancer cells. These results indicate that encapsulated drugs could be effective in controlled drug release for a sustained period would serve the purpose for long-term treatment of many diseases such as breast cancer.

Novel antibacterial polymeric nanocomposite for smart co-delivery of anticancer drugs.

Here a novel antibacterial nanocomposite was developed for combination cancer therapy. The synthesized nanocarrier was characterized by FTIR, 1H NMR, thermogravimetric analysis (TGA), and FESEM-EDX. Its antibacterial activity was assessed by determining minimum inhibitory concentration (MIC) values. Doxorubicin (DOX) and methotrexate (MTX) conjugation with nanocarrier sustained the release of both drugs with apparent pH-triggered manner. Co-administration of DOX with MTX leads to an efficient anticancer performance to MCF7 cell lines verified by qRT-PCR and MTT assay tests. It was concluded that this novel drug delivery vehicle makes antibacterial and anticancer therapeutic processes proceed spontaneously, representing more efficient drug delivery system in nanomedicine. [Formula: see text].

Osteogenic/Odontogenic Bioengineering with co-Administration of Simvastatin and Hydroxyapatite on Poly Caprolactone Based Nanofibrous Scaffold.

Purpose: Statin is an effective factor for promoting osteogenesis. The aim of the present study was to evaluate the effect of simvastatin (SIM) and/or HA addition on changes in osteogenesis levels by human DPSCs transferred onto three-dimensional (3D) nanofibrous Poly (ε-caprolactone) (PCL)/Poly lactic acide (PLLA) polymeric scaffolds. Methods: For this purpose, a 3D nanofibrous composite scaffold of PCL/PLLA/HA was prepared by electrospinning method. SIM was added to scaffolds during DPSCs culturing step. Cell proliferation and osteogenic activity levels were assessed by using MTT assay and Alizarin Red assay methods. In addition, the expression of genes responsible for osteogenesis, including BMP2, Osteocalcin, DSPP and RUNX2, were determined before and 2 weeks after incorporation of SIM. Results: The MTT assay showed that PCL/PLLA/HA scaffolds seeded with DPSCs has significant (p<0.05) more proliferative effect than PCL/PLLA or DMEM cultured cells, additionally SIM administration improved this result over the PCL/PLLA/HA scaffolds without SIM treatment. SEM imaging revealed improved adhesion and probably osteogenic differentiation of DPSCs on PCL/PLLA/HA nanofibers treated with SIM, moreover the alizarin red assay ensured significant (p<0.05) higher mineralization of this group. Finally, real time PCR confirmed the positive regulation (P<0.05) of the expression of osteo/odontogenesis markers BMP2, Osteocalcin, DSPP and RUNX2 genes in PLLA-PCL-HA (0.1)-SIM group. Conclusion: As a result, addition of simvastatin with incorporation of hydroxyapatite in PCL-PLLA scaffolds might increase the expression of osteogenesis markers in the DPSCs, with a possible increase in cell differentiation and bone formation.