Osteogenic potential of adipose stem cells on hydroxyapatite-functionalized decellularized amniotic membrane.

Amniotic membrane (AM) is an attractive source for bone tissue engineering because of its low immunogenicity, contains biomolecules and proteins, and osteogenic differentiation properties. Hydroxyapatite is widely used as bone scaffolds due to its biocompatibility and bioactivity properties. The aim of this study is to design and fabricate scaffold based on hydroxyapatite-coated decellularized amniotic membrane (DAM-HA) for bone tissue engineering purpose. So human amniotic membranes were collected from healthy donors and decellularized (DAM). Then a hydroxyapatite-coating was created by immersion in 10X SBF, under variable parameters of pH and incubation time. Hydroxyapatite-coating was characterized and the optimal sample was selected. Human adipose-derived mesenchymal stem cell behaviors were assessed on control, amniotic membrane, and coated amniotic membrane. The results of the SEM, MTT assay, and Live-Dead staining showed that DAM and DAM-HA support cell adhesion, viability and proliferation. Osteogenic differentiation was evaluated by assessment of alkaline phosphatase activity and expression of osteogenic markers. Maximum gene expression values compared to control occurred in 14 days for alkalin phosphatase, while the highest values for osteocalcin and osteopontin in 21 days. These gene expression values in DAM and DAM-HA for alkalin phosphatase is 6.41 and 8.47, for osteocalcin is 3.95 and 5.94 and for osteopontin is 5.59 and 9.9 respectively. The results of this study indicated DAM supports the survival and growth of stem cells. Also, addition of hydroxyapatite component to DAM promotes osteogenic differentiation while maintaining viability. Therefore, hydroxyapatite-coated decellularized amniotic membrane can be a promising choice for bone tissue engineering applications.

Fabrication of a tailor-made conductive polyaniline/ascorbic acid-coated nanofibrous mat as a conductive and antioxidant cell-free cardiac patch.

Polyaniline (PANI) wasin-situpolymerized on nanofibrous polycaprolactone mats as cell-free antioxidant cardiac patches (CPs), providing electrical conductivity and antioxidant properties. The fabricated CPs took advantage of intrinsic and additive antioxidant properties in the presence of PANI backbone and ascorbic acid as a biocompatible dopant of PANI. The antioxidant nature of CPs may reduce the serious repercussions of oxidative stress, produced during the ischemia-reperfusion (I/R) process following myocardial infarction. The polymerization parameters were considered as aniline (60 mM, 90 mM, and 120 mM), ascorbic acid concentrations ([aniline]:[ascorbic acid] = 3:0, 3:0.5, 3:1, 3:3), and polymerization time (1 h and 3 h). Mainly, the more aniline concentrations and polymerization time, the less sheet resistance was obtained. 1,1 diphenyl-2-picrylhydrazyl (DPPH) assay confirmed the dual antioxidant properties of prepared samples. The advantage of the employedin-situpolymerization was confirmed by the de-doping/re-doping process. Non-desirable groups were excluded based on their electrical conductivity, antioxidant properties, and biocompatibility. The remained groups protected H9c2 cells against oxidative stress and hypoxia conditions. Selected CPs reduced the intracellular reactive oxygen species content and mRNA level of caspase-3 while the Bcl-2 mRNA level was improved. Also, the selected cardiac patch could attenuate the hypertrophic impact of hydrogen peroxide on H9c2 cells. Thein vivoresults of the skin flap model confirmed the CP potency to attenuate the harmful impact of I/R.

EMEMI: An interference-free mini-incubator with integrated electric and magnetic field exposure for real-time microscopic imaging of field effects.

Uninterrupted microscopic observation and real-time imaging of cell behavior during exposure to the stimulus, for example, electric and/or magnetic fields, especially for periods of several days, has been a challenge in experimental bioelectromagnetics due to a lack of proper gas/temperature conditions outside the incubator. Conventional mini-incubators might suffer from stray fields produced by heating elements. We report an in vitro electric and magnetic fields (EMF) exposure system embedded inside a novel under-the-microscope mini-CO2 -incubator with a unique design to avoid electromagnetic interference from the heating and circulation functions while ensuring the requisite temperature. A unique, reconfigurable array of electrodes and/or coils excited by calculated current distributions among array elements is designed to provide excellent field uniformity and controllable linear or circular polarization (even at very low frequencies) of the EMF within the cell culture. Using standard biochemical assays, long-term cell viability has been verified and compared with a conventional incubator. Cell orientation/migration in three-dimensional culture made of collagen-hydrogels has been successfully observed in vitro, in long-term, and in real-time under the influence of DC electric fields with the device.

High-efficient serum-free differentiation of trabecular meshwork mesenchymal stem cells into Schwann-like cells on polylactide electrospun nanofibrous scaffolds.

Cell-based therapies of the peripheral nerve injury (PNI) have provided satisfactory outcomes among which Schwann cells (SCs) are the most reliable candidate to improve repair of the damaged nerve, however, it is difficult to obtain sufficient amount of SCs for clinical applications. Trabecular meshwork-derived mesenchymal stem cells (TM-MSCs) are newly introduced neural crest originated MSCs, which may have a desirable potential for Schwann-like differentiation due to their common lineage. On the other hand, one of the challenges of cell-based therapies is usage of serum containing media which is inappropriate for clinical applications. In the present study, we investigated the differentiation potential of TM-MSCs into Schwann-like cells on polylactide (PLA) nanofibrous scaffolds in the presence or absence of serum. Our results revealed that PLA nanofibers had no negative effects on the cell growth and proliferation of TM-MSCs, and improved Schwann-like differentiation compared with tissue culture plates (TCPs). More importantly, when the cells cultured on the scaffold in the presence of serum-free media (SFM), expression mRNA levels of SC markers (S100B, GAP43, GFAP and SOX10) were significantly increased compared with those of serum-rich groups. Immunostaining of TM-MSCs cultured on serum-free PLA nanofibrous scaffolds also showed significant expression of GAP43, GFAP and SOX10 compared to those of control, indicating the efficient role of SFM in the differentiation of TM-MSCs into SCs lineage. Overall, the findings of this study revealed the differentiation potential of TM-MSCs to SC fate for the first time, and also showed the beneficial effects of SFM and PLA nanofibrous scaffolds as a promising approach for peripheral nerve regeneration.

Enhanced neural differentiation by applying electrical stimulation utilizing conductive and antioxidant alginate-polypyrrole/poly-l-lysine hydrogels.

The challenge of restoration from neurodegenerative disorder requires effective solutions. To enhance the healing efficiencies, scaffolds with antioxidant activities, electroconductivity, and versatile features to encourage neuronal differentiation are potentially useful. Herein, polypyrrole-alginate (Alg-PPy) copolymer was used to design antioxidant and electroconductive hydrogels through the chemical oxidation radical polymerization method. The hydrogels have antioxidant effects to combat oxidative stress in nerve damage thanks to the introduction of PPy. Additionally, poly-l-lysine (PLL) provided these hydrogels with a great differentiation ability of stem cells. The morphology, porosity, swelling ratio, antioxidant activity, rheological behavior, and conductive characteristics of these hydrogels were precisely adjusted by altering the amount of PPy. Characterization of hydrogels showed appropriate electrical conductivity and antioxidant activity for neural tissue applications. Cytocompatibility, live/dead assays, and Annexin V/PI staining by flow cytometry using P19 cells confirmed the excellent cytocompatibility and cell protective effect under ROS microenvironment of these hydrogels in both normal and oxidative conditions. The neural marker investigation in the induction of electrical impulses was assessed through RT-PCR and immunofluorescence assay, demonstrating the differentiation of P19 cells to neurons cultured in these scaffolds. In summary, the antioxidant and electroconductive Alg-PPy/PLL hydrogels demonstrated excellent potential as promising scaffolds for treating neurodegenerative disorders.

Size-adjustable self-assembled nanoparticles through microfluidic platform promotes neuronal differentiation of mouse embryonic stem cells.

Neuronal differentiation from stem cells is one of the most potent therapeutic approaches for recovering neurological function in individuals with neurodegenerative disorders. Herein, an on-demand intracellular retinoic acid released nanoparticles with tunable size and accurately controlled physico-biological properties have been prepared for achieving efficient neuronal differentiation. The amphiphilic chitosan oligosaccharide-cholesterol copolymers were synthesized by varying cholesterol content and self-assembled into spherical micelle in a microfluidic chip with different flow rates. Notably, the results indicated that by increasing the lipophilicity of the chitosan chain as well as mixing rate, the size of micelles was decreased. Retinoic acid (RA) was efficiently encapsulated in the core of micelles. The retinoic acid-containing nanoparticles could escape lysosome, accumulate in the cytoplasm, and release payload with a sustained pattern. The cytotoxicity assay of free retinoic acid and retinoic acid-loaded formulations against P19 embryonic stem cells confirmed the desirable safety of micelles. The result obtained from the uptake study showed that internalization of micelles occurs predominantly via lipid-raft endocytosis in the presence of higher cholesterol content. Moreover, the intracellular RA release upregulated the expression levels of neuronal factors. The micelles described here offer a promising nanomedicine strategy for neuronal differentiation of stem cells.

A hybrid oxygen-generating wound dressing based on chitosan thermosensitive hydrogel and decellularized amniotic membrane.

Amniotic membrane (AM) has been utilized as a wound dressing extensively. Given the importance of oxygen in wound healing, here we have reported the fabrication and characterization of an oxygen-generating wound dressing based on AM. This construct was composed of H2O2-loaded polylactic acid (PLA) microparticles embedded within a chitosan/ß-glycerophosphate (ß-GP) thermosensitive hydrogel covered with a layer of decellularized human-AM. The microparticles had a diameter of 4.48 ± 1.8 µm, an encapsulation efficiency of 44.172 ± 4.49%, and generated oxygen for at least 7 days. The hybrid construct was formed at 32.4 ± 2 °C, had a porous structure (84.69 ± 8.34%) with a pore size of 46.72 ± 26.21 µm. The hydrogel/dAM extract was non-toxic after 7 days based on our MTT results, and the final composite supported cell growth and adhesion. This sample had the most negligible blood cell adhesion with less than 5% hemolysis. Our results indicate the proposed structure's desirable biological, chemical, and physical properties as an active wound dressing.

Enhanced mechanical properties and electrical conductivity of Chitosan/Polyvinyl Alcohol electrospun nanofibers by incorporation of graphene nanoplatelets.

The subject of this paper is to develop a highly conductive Graphene nanoplatelets (GNPs)-Chitosan (CS)/Polyvinyl Alcohol (PVA) (GNPs-CP) nanofibers with excellent mechanical properties. An experimental study was designed to produce nanofibers based on CP nanofibers as matrix and GNPs as reinforcement materials. The microstructure and the surface morphology of the electrospun nanofibers along with their electrical and mechanical properties were examined to study the effect of GNPs content. The SEM results showed that the gradual increase in GNPs content led to a porous web like morphology with no bead. There is a decrease in the diameter of nanofibers by increasing the concentration of GNPs to 1 wt% GNPs from 370 ± 40 nm for CP blend to 144 ± 18 nm for 1 wt% GNPs. Transmission electron microscopy results depicted that GNPs were dispersed uniformly confirmed by the absence of characteristic peak of graphite at 2θ = 26.5°. Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy results indicate the occurrence of a few interactions between GNPs and CP matrix. Nitrogen adsorption/desorption measurement demonstrated that increasing GNPs content increased the specific surface area of nanofibers from 238.377 to 386.708 m2/g for 0 and 1 wt% GNPs content. The test results also show that the presence of GNPs considerably enhances tensile strength, elastic modulus and electrical conductivity. Furthermore, the toughness of GNPs-CP nanofibers including 1 wt% GNPs significantly improved (12-fold) compared to the one for CP nanofibers. So, the proposed composite provides a decent functionality for nanofibers as scaffolds in tissue engineering applications.

Enhancing Cellular Infiltration on Fluffy Polyaniline-Based Electrospun Nanofibers.

Despite the unique properties of polyaniline (PANI), the processability of this smart polymer is associated with challenges. Particularly, it is very difficult to prepare PANI nanofibers due to poor solubility, high charge density, and rigid backbone. The most common approach for solving this problem is blending PANI with a carrier polymer. Furthermore, the major limitations of nanofibers for tissue engineering applications are their low porosity and two-dimensional (2D) structure. In this study, conductive nanofibers were fabricated through electrospinning of PANI/poly(ether sulfone) (PES) with different solvents including dimethyl sulfoxide (DMSO), N-methyl-2-pyrrolidone (NMP), and hexafluoroisopropanol (HFIP). The effect of solvent, carrier polymer (PES), and PANI content on formation of 3D conductive nanofibers with appropriate porosity were investigated. It was shown that a solvent with suitable properties should be selected in such a way that the composite nanofibers can be electrospun at the lowest concentration of PES. In this way, the ratio of PANI increased in the scaffold, the electrical conductivity of nanofibers enhanced, and the flat 2D structure of scaffold changed to a fluffy 3D structure. Among the three studied solvents, HFIP with the lowest boiling point and the lowest surface tension was the best solvent for the fabrication of PANI/PES nanofibers. PES could be electrospun at a concentration of 9% w/w in HFIP, while the optimum percentage of PES in DMSO and NMP was above 23% w/w to produce uniform nanofibers. 3D nanofibrous scaffold obtained from 0.5% PANI/9% PES/HFIP solution with electrical conductivity of 3.7 × 10-5 S/Cm and porosity of 92.81 ± 1.23%. Cell infiltration into the 3D nanofibers with low packing density improved compared to densely packed 2D nanofibers.

Microfluidic fabrication of alendronate-loaded chitosan nanoparticles for enhanced osteogenic differentiation of stem cells.

AIMS: In this study, we used a cross-junction microfluidic device for preparation of alendronate-loaded chitosan nanoparticles with desired characteristics to introduce a suitable element for bone tissue engineering scaffolds. MAIN METHODS: By controlling the reaction condition in microfluidic device, six types of alendronate-loaded chitosan nanoparticles were fabricated which had different physical properties. Hydrodynamic diameter of synthetized particles was evaluated by dynamic light scattering (102 to 215 nm). Nanoparticle morphology was determined by SEM and AFM images. The osteogenic effects of prepared selected nanoparticles on human adipose stem cells (hA-MSCs) were evaluated by assessment of alkaline phosphatase (ALP) activity, calcium deposition, ALP and osteopontin gene expression. KEY FINDINGS: The highest loading efficiency percentage (%LE) was %32.42 ± 2.02. Based on MTT assessment, two samples which had no significant cytotoxicity were chosen for further studies (particle sizes and %LE were 142 ± 6.1 nm, 198 ± 16.56 nm, %16.76 ± 3.91 and %32.42 ± 2.02, respectively). In vitro release behavior of nanoparticles displayed pH responsive characteristics. Significant faster release was seen in acidic pH = 5.8 than neutral pH = 7.4. The selected nanoparticles demonstrated higher ALP activity at 14 days in comparison to selected blank sample and osteogenic differentiation media (ODM) and a downregulation at 21 days in comparison to 14 days. Calcium content assay at 21 days displayed significant differences between alendronate-loaded nanoparticles and ODM. ALP and osteopontin mRNA expression was significantly higher than the cells cultured in ODM at 14 and 21 days. SIGNIFICANCE: We concluded that our prepared nanoparticles significantly enhanced osteogenic differentiation of hA-MSCs and can be a suitable compartment of bone tissue engineering scaffolds.

Nanofibrous Scaffolds Containing Hydroxyapatite and Microfluidic-Prepared Polyamidoamin/BMP-2 Plasmid Dendriplexes for Bone Tissue Engineering Applications.

OBJECTIVE: The aim of this study is to fabricate functional scaffolds to gene delivery bone morphogenetic protein-2 (BMP-2) plasmid for bone formation in bone tissue engineering. METHODS: Dendriplexes (DPs) of generation 4 polyamidoamin (G4-PAMAM)/BMP-2 plasmid were prepared through microfluidic (MF) platform. The physiochemical properties and toxicity of DPs were evaluated by DLS, AFM, FESEM and MTT assay. In order to create a suitable environment for stem cell growth and differentiation, poly-l-lactic acid (PLLA) and poly-l-lactic acid/poly (ethylene oxide) (PLLA/PEO) scaffolds containing hydroxyapatite nanoparticles (HA) and DPs were fabricated by the electrospinning method. The osteogenic potency of the scaffolds on human adipose tissue-derived mesenchymal stem cells (hASCs) was investigated. RESULTS: The results revealed that tuning the physical properties of DPs by adjusting flow parameters in microfluidic platform can easily improve the cell viability compared to conventional bulk mixing method. Also, the result showed that the presence of HA and DPs in PLLA/PEO scaffold enhanced alkaline phosphatase (ALP) activity and increased the amount of deposited Ca, as well as, related to osteogenesis gen markers. CONCLUSION: This study indicated that on using the MF platform in preparation of DPs and loading them along with HA in PLLA/PEO scaffold, the osteogenic differentiation of hASCs could be tuned.

A Review on Modifications of Amniotic Membrane for Biomedical Applications.

The amniotic membrane (AM) is the innermost layer of the fetal placenta, which surrounds and protects the fetus. Its unique structure, in addition to its physical and biological properties, makes it a useful substance in many applications related to regenerative medicine. The use of this fantastic substance with a century-old history has produced remarkable results in vivo, in vitro, and even in clinical studies. While the intact or preserved AM is widely used for these purposes, the addition of further modifications to AM can be considered as a relatively new subject in its applications. These modifications are applied to improve AM properties, ease of handling, and durability. Here, we will discuss the cases in which AM has undergone additional modifications besides the required processes for sterilization and preservation. In this article, we have categorized these modifications and discussed their applications and results.

Surface mineralized hybrid nanofibrous scaffolds based on poly(l-lactide) and alginate enhances osteogenic differentiation of stem cells.

Surface mineralized nanofibrous scaffolds hold great potential for bone tissue engineering applications. In this study, a new hybrid nanofibrous scaffold composed of alginate/poly(l-lactide) nanofibers was fabricated using electrospinning method and then crosslinking process was employed. Hydroxyapatite crystal formation took place using in situ precipitation by immersion of the scaffolds in simulated body fluid solution for 10 days at 37°C. The morphologies of the scaffolds were observed using scanning electron microscope. Hydroxyapatite crystal formation on the surface of nanofibers was confirmed using scanning electron microscopy, energy-dispersive X-ray spectroscopy, Fourier transform infrared spectroscopy and X-ray diffraction analysis. The biocompatibility of the fabricated scaffolds was evaluated using mesenchymal stem cell culture and MTT assay. According to alkaline phosphatase activity and calcium content assays, it was concluded that the osteogenic differentiation of stem cells was considerable on scaffolds containing hydroxyapatite crystals in comparison with other specimens. The results showed biocompatibility of the scaffolds and their support from stem cell adhesion, growth and osteogenic differentiation, so the hydroxyapatite-coated poly(l-lactide)/alginate hybrid nanofibrous scaffolds hold suitable characteristics for bone tissue engineering applications. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 586-596, 2019.

L. inermis-loaded nanofibrous scaffolds for wound dressing applications.

Since ancient times, some herbal medicines have been extensively used for burn and wound treatments, showing preference to the common synthetic medications by virtue of having less side effects and faster healing rate. In this study, hybrid nanofibrous scaffolds of poly-l-lactic-acid (PLLA) and gelatin incorporated L. inermis were fabricated via electrospinning technique. Morphology and characteristics of the scaffolds were studied by scanning electron microscopy (SEM) and Fourier transform infrared (FTIR), respectively. The release profile of the L. inermis from the nanofibers was also assessed in vitro. Moreover, the structural stability of the released L. inermis from the nanofibers was evaluated using high-performance liquid chromatography (HPLC). The nanofibers showed a gradual release of L. inermis up to two days while the intact structure was preserved. Furthermore, antibacterial assay demonstrated that L. inermis-loaded nanofibrous scaffolds could effectively kill E. coli and S. aureus within 2 h. Finally, biocompatibility of the nanofibers was proven on 3T3 fibroblasts. Therefore, the L. inermis loaded PLLA-Gelatin nanofibers showed a potential application as a wound dressing in order to control wound infections.

Enhanced Cardiac Differentiation of Human Cardiovascular Disease Patient-Specific Induced Pluripotent Stem Cells by Applying Unidirectional Electrical Pulses Using Aligned Electroactive Nanofibrous Scaffolds.

In the embryonic heart, electrical impulses propagate in a unidirectional manner from the sinus venosus and appear to be involved in cardiogenesis. In this work, aligned and random polyaniline/polyetersulfone (PANI/PES) nanofibrous scaffolds doped by Camphor-10-sulfonic acid (ß) (CPSA) were fabricated via electrospinning and used to conduct electrical impulses in a unidirectional and multidirectional fashion, respectively. A bioreactor was subsequently engineered to apply electrical impulses to cells cultured on PANI/PES scaffolds. We established cardiovascular disease-specific induced pluripotent stem cells (CVD-iPSCs) from the fibroblasts of patients undergoing cardiothoracic surgeries. The CVD-iPSCs were seeded onto the scaffolds, cultured in cardiomyocyte-inducing factors, and exposed to electrical impulses for 1 h/day, over a 15-day time period in the bioreactor. The application of the unidirectional electrical stimulation to the cells significantly increased the number of cardiac Troponin T (cTnT+) cells in comparison to multidirectional electrical stimulation using random fibrous scaffolds. This was confirmed by real-time polymerase chain reaction for cardiac-related transcription factors (NKX2.5, GATA4, and NPPA) and a cardiac-specific structural gene (TNNT2). Here we report for the first time that applying electrical pulses in a unidirectional manner mimicking the unidirectional wave of electrical stimulation in the heart, could increase the derivation of cardiomyocytes from CVD-iPSCs.

The synergistic effect of nano-hydroxyapatite and dexamethasone in the fibrous delivery system of gelatin and poly(l-lactide) on the osteogenesis of mesenchymal stem cells.

Recently, electrospun nanofibrous scaffolds are vastly taken into consideration in the bone tissue engineering due to mimicking the natural structure of native tissue. In our study, surface features of nanofibers were modified through simultaneous electrospining of the synthetic and natural polymers using poly l-lactide (PLLA) and gelatin to fabricate the hybrid scaffold (PLLA/gelatin). Then, hydroxyapatite nanoparticles (nHA) were loaded in electrospun PLLA nanofibers (PLLA,nHA/gelatin) and also dexamethasone (DEX) was incorporated in these fibers (PLLA,nHA,DEX/gelatin) in the second experiment. Fabricated nanofibrous composite scaffolds were characterized via SEM, FTIR spectroscopy, contact angle, tensile strength measurements, DEX release profile and MTT assay. After seeding adipose derived mesenchymal stem cells, osteoinductivity and osteoconductivity of fabricated scaffolds were analyzed using common osteogenic markers such as alkaline phosphatase activity, calcium depositions and gene expression. These results confirmed that all properties of nanofibers were improved by modifications. Moreover, osteogenic differentiation of stem cells increased in PLLA,nHA/gelatin group in comparison with PLLA/gelatin. The sustained release of DEX was obtained from PLLA,nHA,DEX/gelatin which subsequently led to more osteogenic differentiation. Taken together, PLLA,nHA,DEX/gelatin showed significant potential to support the stem cell proliferation and ostogenic differentiation, and can be a good candidates for tissue engineering and regenerative medicine applications.

Polymer/metal nanocomposites for biomedical applications.

Polymer/metal nanocomposites consisting of polymer as matrix and metal nanoparticles as nanofiller commonly show several attractive advantages such as electrical, mechanical and optical characteristics. Accordingly, many scientific and industrial communities have focused on polymer/metal nanocomposites in order to develop some new products or substitute the available materials. In the current paper, characteristics and applications of polymer/metal nanocomposites for biomedical applications are extensively explained in several categories including strong and stable materials, conductive devices, sensors and biomedical products. Moreover, some perspective utilizations are suggested for future studies.

Structural stability and sustained release of protein from a multilayer nanofiber/nanoparticle composite.

The cellular microenvironment can be engineered through the utilization of various nano-patterns and matrix-loaded bioactive molecules. In this study, a multilayer system of electrospun scaffold containing chitosan nanoparticles was introduced to overcome the common problems of instability and burst release of proteins from nanofibrous scaffolds. Bovine serum albumin (BSA)-loaded chitosan nanoparticles was fabricated based on ionic gelation interaction between chitosan and sodium tripolyphosphate. Suspension electrospinning was employed to fabricate poly-ɛ-caprolacton (PCL) containing protein-loaded chitosan nanoparticles with a core-shell structure. To obtain the desired scaffold mechanical properties with enough elasticity for expansion and contraction, a hybrid mono and multilayer electrospun scaffold was fabricated using PCL containing protein-loaded chitosan nanoparticles and poly-L-lactic acid (PLLA). According to the BSA release profile, the multi-layered structure of nanofibers with two barrier layers provided a programmable release pattern of the loaded protein. Moreover, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and circular dichroism spectra results showed that the electrospinning process had no significant effect on the primary and secondary structure of the protein. The results indicated a desirable biocompatibility and mechanical cues of the multilayer nanofibrous scaffolds supporting structural stability and controlled release of the protein, which can offer diverse applications in hollow organ tissue engineering.

A home-brew real-time PCR assay for reliable detection and quantification of mature miR-122.

miR-122 is a liver-specific miRNA that has significant gene expression alterations in response to specific pathophysiological circumstances of liver such as drug-induced liver injury, hepatocellular carcinoma, and hepatitis B and C virus infections. Therefore, accurate and precise quantification of miR-122 is very important for clinical diagnostics. However, because of the lack of in vitro diagnostics assays for miR-122 detection and quantification of the existence of an open-source assay could inevitably provide external evaluation by other researchers and the chance of promoting the assay when required. The aim of this study was to develop a Taqman real-time polymerase chain reaction assay, which is capable of robust and reliable quantification of miR-122 in different sample types. We used stem loop methodology to design a specific Taqman real-time polymerase chain reaction assay for miR-122. This technique enabled us to reliably and reproducibly quantify short-length oligonucleotides such as miR-122. The specificity, sensitivity, interassay and intra-assay, and the dynamic range of the assay were experimentally determined by their respective methodology. The assay had a linear dynamic range of 3E to 4.8E miR-122 copies/reaction and the limit of detection was determined to be between 960 and 192 copies/reaction with 95% confidence interval. The assay gave a coefficient of variation for the Ct values of <1.4% and 0.78% for intra-assay and interassay, respectively. Taking into account that miR-122 is expressed in >50,000 copies per hepatocyte, this assay is able to suffice the need for reliable detection and quantification of this miRNA. Therefore, this study can be considered as a start point for standardizing miR-122 quantification.

PLGA/gelatin hybrid nanofibrous scaffolds encapsulating EGF for skin regeneration.

The novel strategies of skin regenerative treatment are aimed at the development of biologically responsive scaffolds capable of delivering multiple bioactive agents and cells to the target tissues. In this study, nanofibers of poly(lactic-co-glycolic acid) (PLGA) and gelatin were electrospun and the effect of parameters viz polymer concentration, acid concentration, flow rate and voltage on the morphology of the fibers were investigated. PLGA nanofibers encapsulating epidermal growth factor were also prepared through emulsion electrospinning. The core-sheath structure of the nanofibers was verified by transmission electron microscopy. The hemostatic attributes and the biocompatibility of the scaffolds for human fibroblast cell were scrutinized. Furthermore, gene expression of collagen type I and type III by the cells on the scaffolds was quantified using real-time reverse transcriptase polymerase chain reaction. The results indicated desirable bioactivity and hemostasis of the scaffolds with the capability of encapsulation and controlled release of the protein which can be served as skin tissue engineering scaffolds and wound dressings.