Development of nanocomposites based on chitosan/reduced graphene oxide for wound healing application.

Nanocomposites containing different effective materials have various effects, such as antioxidant, and anti-inflammatory activity, which are desirable for wound dressing. Herein, nanocomposites based on chitosan/reduced graphene oxide (CS/rGO) containing curcumin (CS/rGO/Cur), curcumin and papain (CS/rGO/Cur/Pa), curcumin, papain, and collagen peptide (CS/rGO/CP/Cur/Pa), prepared using ionic gelation method and characterized by Fourier Transform Infrared (FTIR), Differential Light Scattering (DLS), X-ray diffraction (XRD), and Scanning Electron Microscope (SEM). Subsequently, the nanocomposite's potential for wound healing was studied through parameters such as porosity, swelling, degradability, anti-inflammatory, antioxidant, antibacterial, cell viability, and in-vivo. The results of FTIR, XRD, SEM, and DLS showed that the nanocomposites synthesized properly with an almost spherical morphology, an average diameter of below 100 nm (mostly 40-85 nm), and a hydrodynamic diameter of 455-616 nm. The various tests demonstrated the nanocomposite's effectiveness in wound healing. The results showed that CS/rGO/CP/Cur/Pa increased the anti-inflammatory and cell viability up to 99.7 % and 395 %, respectively, which is higher than others. Animal tests on rats showed that CS/rGO/CP/Cur/Pa accelerated the wound healing rate up to 70 %. In conclusion, the results showed that the nanocomposites based on CS/rGO significantly improve wound healing, and the presence of collagen peptides boost their wound healing potency.

Hollow pollen grains as scaffolding building blocks in bone tissue engineering.

Introduction: The current study, for the first time, suggests nature-made pollen grains (PGs) of Pistacia vera L. as a potential candidate for using as scaffolding building blocks with encapsulation capability of bioactive compounds, such as bone morphogenetic protein 4 (BMP4). Methods: A modified method using KOH (5%, 25ºC) was developed to produce nonallergic hollow pollen grains (HPGs), confirmed by energy dispersive X-ray (EDX) analysis, field emission scanning electron microscopy (FESEM), and DNA and protein staining techniques. The in-vitro study was conducted on human adipose-derived mesenchymal stem cells (hAD-MSCs) to investigate the applicability of HPGs as bone scaffolding building blocks. Cytocompability was evaluated by FESEM, MTT assay, and gene expression analysis of apoptotic markers (BAX and BCL2). The osteoconductive potential of HPGs was assessed by alkaline phosphatase (ALP) activity measurement and gene expression analysis of osteogenic markers (RUNX2 and osteocalcin). Results: Findings demonstrated that HPGs can be considered as biocompatible compounds increasing the metabolic activities of the cells. Further, the bioactive nature of HPGs resulted in suitable cellular adhesion properties, required for a potent scaffold. The investigation of apoptotic gene expression indicated a reduced BAX/BCL2 ratio reflecting the protective effect of HPGs on hAD-MSCs. The increased ALP activity and expression of osteogenic genes displayed the osteoconductive property of HPGs. Moreover, the incorporation of BMP4 in HPGs initiated a synergistic effect on osteoblast maturation. Conclusion: Owing to the unique compositional and surface nanotopographical features of the Pistacia vera L. HPG, this microscale architecture provides a favorable microenvironment for the bottom-up remodeling of bone.

Fibronectin within Sodium Alginate Microcapsules Improved Osteogenic Differentiation of BMMSCs in Dose Dependent Manner by Targeting SP7, OCN, CDK1, ZBTB16, and Twist1 Expression.

Purpose: Insoluble fibronectin as an extracellular matrix (ECM) protein has the potential to promote proliferation, differentiation, and migration of mesenchymal stem cells (MSCs). However, there is limited information about the effects of fibronectin various concentrations on bone marrow-derived MSCs (BMMSCs) function and differentiation. Methods: In this experimental study, using a gel injection device, BMMSCs were encapsulated in sodium alginate microcapsules containing 1.25% alginate, 1% gelatin, and fibronectin (0.01, 0.05, 0.1, and 0.2 µg/ml). MTT assay was used to examine the proliferation of BMMSCs. Also, BMMSCs apoptosis were analyzed using Annexin-V/PI staining and fluorescence activated cell sorting (FACS). Alkaline phosphatase (ALP) test was conducted to assess BMMSCs osteogenic differentiation potential. Finally, mRNA expression levels of the SP7, osteocalcin (OCN), Twist Family BHLH Transcription Factor 1 (Twist1), Peroxisome proliferator-activated receptor γ2 (PPARγ2), Cyclin-dependent kinase 1 (CDK1), and Zinc Finger and BTB Domain Containing 16 (ZBTB16), following exposure with fibronectin 0.1 µg/ml. Results: According to results, fibronectin had the potential to promote proliferation rates of the BMMSCs, in particular at 0.1 and 0.2 µg/ml concentrations. we showed that the fibronectin was not able to modify apoptosis rates of the BMMSCs. ALP test results approved the notable potential of the fibronectin, to trigger osteogenic differentiation of the BMMSCs. Also, RT-PCR results indicated that fibronectin 0.1 µg/ml could augment osteogenic differentiation of cultured BMMSCs through targeting of OCN, SP7, Twist1, CDK1, and ZBTB16, strongly or slightly. Conclusion: Results showed that fibronectin can improve proliferation and osteogenic differentiation of BMMSCs without any effect on these cells' survival.

Bioactive Chitosan-Based Organometallic Scaffolds for Tissue Engineering and Regeneration.

Captivating achievements in developing advanced hybrid biostructures through integrating natural biopolymers with inorganic materials (e.g., metals and metalloids) have paved the way towards the application of bioactive organometallic scaffolds (OMSs) in tissue engineering and regenerative medicine (TERM). Of various biopolymers, chitosan (CS) has been used widely for the development of bioactive OMSs, in large part due to its unique characteristics (e.g., biocompatibility, biodegradability, surface chemistry, and functionalization potential). In integration with inorganic elements, CS has been used to engineer advanced biomimetic matrices to accommodate both embedded cells and drug molecules and serve as scaffolds in TERM. The use of the CS-based OMSs is envisioned to provide a new pragmatic potential in TERM and even in precision medicine. In this review, we aim to elaborate on recent achievements in a variety of CS/metal, CS/metalloid hybrid scaffolds, and discuss their applications in TERM. We also provide comprehensive insights into the formulation, surface modification, characterization, biocompatibility, and cytotoxicity of different types of CS-based OMSs.

Synthesis and biological impacts of pollen shells/Fe3O4 nanoparticles composites on human MG-63 osteosarcoma cells.

INTRODUCTION: Cell-adhesive surfaces play a pivotal role in biomedical engineering, as most biological reactions take place on surfaces. Pollen shell (PSh) ofPistacia vera L., as a new medical device, has previously been reported to cause cytotoxicity and apoptosis in MG-63 bone cancer cells. METHODS: Iron oxide nanoparticles (Fe3O4NPs) were synthesized and their reaction to PShs was gauged at different concentrations, and then characterized using field emission scanning electron microscopy (FESEM), Fourier-transform infrared spectroscopy, energy dispersion X-ray spectrometer, X-ray diffraction spectra, dynamic light scattering, and vibrating sample magnetometer. Then, the biological impacts of PShs/Fe3O4NPs composites on MG-63 cells were investigated in-vitro using MTT assay, quantitative polymerase chain reaction (qPCR), Annexin V/propidium iodide, FESEM, and DAPI staining. RESULTS: Fe3O4NPs with a size range of 24-40 nm and a zeta potential value of -37.4 mV were successfully assembled on the PShs. The viability of MG-63 cells was significantly decreased when cultured on the magnetic PShs as compared to non-magnetic PShs, in Fe3O4 concentration and time-dependent manner. In contrast, magnetic PShs had a positive effect on the viability of normal human bone marrow-derived mesenchymal stem cells (hBM-MSCs). The analysis of apoptosis-related genes in cancer cells revealed that loading Fe3O4NPs on PShs increased expression of BAX/BCL2 and caspase-3 genes. The increased apoptotic activity of combined PShs/Fe3O4NPs was further confirmed by flow cytometric measurement, morphological analysis, and DAPI staining. CONCLUSION: The incorporation of Fe3O4NPs into PShs could effectively increase anticancer effects on MG-63 cells via the mitochondria-mediated apoptosis pathway, evident by upregulation of BAX/BCL2 ratio and caspase-3.

Bioactivity and Antibacterial Behaviors of Nanostructured Lithium-Doped Hydroxyapatite for Bone Scaffold Application.

The material for bone scaffold replacement should be biocompatible and antibacterial to prevent scaffold-associated infection. We biofunctionalized the hydroxyapatite (HA) properties by doping it with lithium (Li). The HA and 4 Li-doped HA (0.5, 1.0, 2.0, 4.0 wt.%) samples were investigated to find the most suitable Li content for both aspects. The synthesized nanoparticles, by the mechanical alloying method, were cold-pressed uniaxially and then sintered for 2 h at 1250 °C. Characterization using field-emission scanning electron microscopy (FE-SEM) revealed particle sizes in the range of 60 to 120 nm. The XRD analysis proved the formation of HA and Li-doped HA nanoparticles with crystal sizes ranging from 59 to 89 nm. The bioactivity of samples was investigated in simulated body fluid (SBF), and the growth of apatite formed on surfaces was evaluated using SEM and EDS. Cellular behavior was estimated by MG63 osteoblast-like cells. The results of apatite growth and cell analysis showed that 1.0 wt.% Li doping was optimal to maximize the bioactivity of HA. Antibacterial characteristics against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were performed by colony-forming unit (CFU) tests. The results showed that Li in the structure of HA increases its antibacterial properties. HA biofunctionalized by Li doping can be considered a suitable option for the fabrication of bone scaffolds due to its antibacterial and unique bioactivity properties.

Osteogenesis Promotion of Selenium-Doped Hydroxyapatite for Application as Bone Scaffold.

The combined bioceramic of selenium (Se) and hydroxyapatite (HA) has been considered as a moderate bone scaffold biomaterial. In the present work, Se was doped into the HA structure using the mechano-chemical alloying (MCA) method for the improvement of osteogenic properties of HA. HA extracted from fish bone and Se-doped hydroxyapatite (Se-HA) were analyzed using X-ray diffraction spectra (XRD), scanning electron microscope (SEM), energy dispersion X-ray spectrometer (EDX), and Fourier transform infrared spectroscopy (FT-IR). In-vitro cell responses on the Se-HA bioceramic scaffold were investigated using human adipose-derived mesenchymal stem cells (hAD-MSCs). The effect of Se on cell proliferation was studied by MTT assay, and cell adhesion responses were analyzed by optical microscopy and SEM. Furthermore, the effect of Se on osteogenic properties of HA was studied by alkaline phosphatase (ALP) activity, alizarin red S (ARS) staining, and Western blot tests. The MTT results showed that the Se dopant synergistically increases the proliferation of hAD-MSCs. Moreover, good cell-adhesive and osteoblast-shaped behaviors were observed on the Se-HA scaffold. The results of osteogenic differentiation demonstrated synergistically enhanced ALP activity and calcification on the Se dopant compared to HA. Also, the results of Western blot test presented that the differentiation of hAD-MSCs toward being a bone tissue was increased by up to 50% while selenium doping. Additional MTT analysis using Human Bone Osteosarcoma cell line (KHOS-240S) revealed the antiproliferative activity of the Se-HA scaffold against bone cancerous cells. Therefore, it has been concluded that Se-HA bioceramic can be employed as a scaffold with simultaneous anticancer and bone regenerative properties.

Barium-cross-linked alginate-gelatine microcapsule as a potential platform for stem cell production and modular tissue formation.

Influence of gelatine concentration and cross-linker ions of Ca2+ and Ba2+ was evaluated on characteristics of alginate hydrogels and proliferation behaviours of model adherent and suspendable stem cells of fibroblast and U937 embedded in alginate microcapsules. Increasing gelatine concentration to 2.5% increased extent of swelling to 15% and 25% for barium- and calcium-cross-linked hydrogels, respectively. Mechanical properties also decreased with increasing swelling of hydrogels. Both by increasing gelatine concentration and using barium ions increased considerably the proliferation of encapsulated model stem cells. Barium-cross-linked alginate-gelatine microcapsule tested for bone building block showed a 13.5 ± 1.5-fold expansion for osteoblast cells after 21 days with deposition of bone matrix. The haematopoietic stem cells cultured in the microcapsule after 7 days also showed up to 2-fold increase without adding any growth factor. The study demonstrates that barium-cross-linked alginate-gelatine microcapsule has potential for use as a simple and efficient 3D platform for stem cell production and modular tissue formation.

Composite electrospun nanofibers of reduced graphene oxide grafted with poly(3-dodecylthiophene) and poly(3-thiophene ethanol) and blended with polycaprolactone.

In this paper, an effective method was employed for preparation of nanofibers using conducting polymer-functionalized reduced graphene oxide (rGO). First, graphene oxide (GO) was obtained from graphite by Hommer method. GO was reduced to rGO by NaBH4 and covalently functionalized with a 3-thiophene acetic acid (TAA) by an esterification reaction to reach 3-thiophene acetic acid-functionalized reduced graphene oxide macromonomer (rGO-f-TAAM). Afterward, rGO-f-TAAM was copolymerized with 3-dodecylthiophene (3DDT) and 3-thiophene ethanol (3TEt) to yield rGO-f-TAA-co-PDDT (rGO-g-PDDT) and rGO-f-TAA-co-P3TEt (rGO-g-PTEt), which were confirmed by Fourier transform infrared spectra. The grafted materials depicted better electrochemical properties and superior solubilities in organic solvents compared to GO and rGO. The soluble rGO-g-PDDT and rGO-g-PTEt composites blended with polycaprolactone were fabricated by electrospinning, and then cytotoxicity, hydrophilicity, biodegradability and mechanical properties were investigated. The grafted rGO composites exhibited a good electroactivity behavior, mainly because of the enhanced electrochemical performance. The electrospun nanofibers underwent degradation about 7 wt% after 40 days, and the fabricated scaffolds were not able to induce cytotoxicity in mouse osteoblast MC3T3-E1 cells. The soluble conducting composites developed in this study are utilizable in the fabrication of nanofibers with tissue engineering application.

Development of novel electrically conductive scaffold based on hyperbranched polyester and polythiophene for tissue engineering applications.

A novel electrically conductive scaffold containing hyperbranched aliphatic polyester (HAP), polythiophene (PTh), and poly(ε-caprolactone) (PCL) for regenerative medicine application was succesfully fabricated via electrospinning technique. For this purpose, the HAP (G4; fourth generation) was synthesized via melt polycondensation reaction from tris(methylol)propane and 2,2-bis(methylol)propionic acid (bis-MPA). Afterward, the synthesized HAP was functionalized with 2-thiopheneacetic acid in the presence of N,N-dicyclohexyl carbodiimide, and N-hydroxysuccinimide as coupling agent and catalyst, respectively, to afford a thiophene-functionalized G4 macromonomer. This macromonomer was subsequently used in chemical oxidation copolymerization with thiophene monomer to produce a star-shaped PTh with G4 core (G4-PTh). The solution of the G4-PTh, and PCL was electrospun to produce uniform, conductive, and biocompatible nanofibers. The conductivity, hydrophilicity, and mechanical properties of these nanofibers were investigated. The biocompatibility of the electrospun nanofibers were evaluated by assessing the adhesion and proliferation of mouse osteoblast MC3T3-E1 cell line and in vitro degradability to demonstrate their potential uses as a tissue engineering scaffold. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2673-2684, 2016.