Fabrication and Process Optimization of Poly(2-hydroxyethyl methacrylate) Nanofibers by Response Surface Methodology.

In this study, Response Surface Methodology (RSM) was used to model and optimize the electrospinning parameters to obtain poly(2-hydroxylethyl methacrylate) (pHEMA) nanofibers which is challenging in terms of evaluating the optimum conditions in nanofiber production. A second order (quadratic model) polynomial function was used for correlation between electrospinning parameters (flow rate, applied voltage, polymer/ethanol concentration) and average fiber diameter. An electro-spinning set-up was used to fabricate nanofibers and scanning electron microscopy (SEM) was used to determine the morphology and the size of the nanofibers with diameter ranging from 211 nm to 1661 nm. Results concluded that the concentration of polymer solution played an important role in distribution of fiber diameter. Based on RSM, the optimum pHEMA fibers with 245±35 nm diameter were collected at 13 µL/min flow rate, 12 kV applied voltage at an ethanol:pHEMA ratio of 1.76.

Combined delivery of PDGF-BB and BMP-6 for enhanced osteoblastic differentiation.

Natural microenvironment during bone tissue regeneration involves integration of multiple biological growth factors which regulate mitogenic activities and differentiation to induce bone repair. Among them platelet derived growth factor (PDGF-BB) and bone morphogenic protein-6 (BMP-6) are known to play a prominent role. The aim of this study was to investigate the benefits of combined delivery of PDGF-BB and BMP-6 on proliferation and osteoblastic differentiation of MC3T3-E1 preosteoblastic cells. PDGF-BB and BMP-6 were loaded in gelatin and poly (3-hydroxybutyric acid-co-3-hydroxyvaleric acid) particles, respectively. The carrier particles were then loaded into 3D chitosan matrix fabricated by freeze drying. The fast release of PDGF-BB during 7 days was accompanied by slower and prolonged release of BMP-6. The premising release of mitogenic factor PDGF-BB resulted in an increased MC3T3-E1 cell population seeded on chitosan scaffolds. Osteogenic markers of RunX2, Col 1, OPN were higher on chitosan scaffolds loaded with growth factors either individually or in combination. However, OCN expression and bone mineral formation were prominent on chitosan scaffolds incorporating PDGF-BB and BMP-6 as a combination.

Chitosan-based films grafted with citrus waste-derived antifungal agents: An innovative and sustainable approach to enhance post-harvest preservation of citrus fruit.

This paper reports the synthesis, characterization, and properties of chitosan films (CHI) grafted with a natural antifungal agent with the aim of developing active films of natural origin to prevent post-harvest losses of citrus fruit. The antifungal agent was prepared by fermentation using lemon peel (AntiFun-LM), a citrus waste, and grafted on chitosan using different coupling agents (CHI/AntiFun-LM). Bioactive films were prepared by solvent casting. FTIR-ATR and ToF-SIMS analyses provided compelling evidence of the successful grafting process. TGA-DSC demonstrated that the films are stable after grafting. SEM studies showed the continuous and compact surface of the films. WCA measurements proved that CHI/AntiFun-LM films are more hydrophilic than CHI films. Moreover, the CHI/AntiFun-LM films showed stronger UV shielding effect when compared to CHI. The biological evaluation demonstrated that CHI/AntiFun-LM films gained considerable antifungal properties against most fungi responsible for post-harvest decay. Cytotoxicity tests showed that CHI/AntiFun-LM films did not cause any toxic effect against L929 fibroblasts. This study highlights the great potential of chemical grafting of antifungal agents produced from citrus waste to chitosan and preparation of natural-based films to act as a powerful alternative in post-harvest protection of citrus fruit in a perspective of circular economy.

In vivo behavior of a collagen-coated nano-hydroxyapatite enriched polycaprolactone membrane in rat mandibular defects.

BACKGROUND: This research investigated the ability of fabricated collagen (COL) coated nano-hydroxyapatite (nHA) enriched polycaprolactone (PCL) membrane to facilitate new bone formation (NBF) and its biocompatibility. METHODS: Unilateral mandibular angulus defects of 28 female 12-week-old long Evans rats were created with a trephine bur with 5 mm in diameter and divided into two groups. While the test group was treated with the membrane (M-1, M-2), the control was left as self-healing (C-1, C-2) and sacrificed at 2nd (M-1, C-1) and 8th week (M-2, C-2) postoperatively. The mandibular bone of the rats was evaluated histopathologically. Density of the regenerated bone was evaluated with PET/CT. RESULTS: Histopathologically, NBF which started from the periphery of the defect had rich cellular character in M-1. Significantly higher NBF was found in M-2 when compared to M-1 (P=0.003). Furthermore, significantly lesser degree of inflammation was found in M-2 when compared to M-1 (P<0.05). CONCLUSION: This study suggests that the novel COL-coated nHA-enriched PCL membrane can serve a promising design for tissue engineering as guided bone regeneration in alveolar defects.

Optimizing Chitosan/Collagen Type I/Nanohydroxyapatite Cross-linked Porous Scaffolds for Bone Tissue Engineering.

Bio-composite scaffolds mimicking the natural microenvironment of bone tissue offer striking advantages in material-guided bone regeneration. The combination of biodegradable natural polymers and bioactive ceramics that leverage potent bio-mimicking cues has been an active strategy to achieve success in bone tissue engineering. Herein, a competitive approach was followed to point out an optimized bio-composite scaffold in terms of scaffold properties and stimulation of osteoblast differentiation. The scaffolds, composed of chitosan/collagen type I/nanohydroxyapatite (Chi/Coll/nHA) as the most attractive components in bone tissue engineering, were analyzed. The scaffolds were prepared by freeze-drying method and cross-linked using different types of cross-linkers. Based on the physicochemical and mechanical characterization, the scaffolds were eliminated comparatively. All types of scaffolds displayed highly porous structures. The cross-linker type and collagen content had prominent effects on mechanical strength. Glyoxal cross-linked structures displayed optimum mechanical and structural properties. The MC3T3-E1 proliferation, osteogenic-related gene expression, and matrix mineralization were better pronounced in collagen presence and triggered as collagen type I amount was increased. The results highlighted that glyoxal cross-linked scaffolds containing equal amounts of Chi and Coll by mass and 1% (w/v) nHA are the best candidates for osteoblast differentiation and matrix mineralization.

UiO-66 metal-organic framework as a double actor in chitosan scaffolds: Antibiotic carrier and osteogenesis promoter.

Metal-organic frameworks (MOFs) have recently emerged as a useful class of nanostructures with well-suited characteristics for drug delivery applications, due to the high surface area and pore size for efficient loading. Despite their use as a nano-carrier for controlled delivery of various types of drugs, the inherent osteo-conductive properties have stolen a great attention as a growing area of investigation. Here, we evaluated the double function of UiO-66 MOF structure as a carrier for fosfomycin antibiotic and also as an osteogenic differentiation promoter when introduced in 3D chitosan scaffolds, for the first time. Our results revealed that the wet-spun chitosan scaffolds containing fosfomycin loaded UiO-66 nanocrystals (CHI/UiO-66/FOS) possessed fiber mesh structure with integrated micro-scale fibers and increased mechanical strength. In vitro antibacterial studies indicated that CHI/UiO-66/FOS scaffolds showed bactericidal activity against Staphylococcus aureus. Moreover, the scaffolds were biocompatible to MC3T3-E1 pre-osteoblasts and significantly up-regulated the expression of osteogenesis-related genes and facilitated the extracellular matrix mineralization, in vitro. Taken together, our results demonstrate UiO-66 MOFs can present double functionality and CHI/UiO-66/FOS scaffolds hold a significant potential to be further explored as an alternative approach in treating infected bone defects like osteomyelitis.

Chitosan and silver nanoparticles are attractive auxin carriers: A comparative study on the adventitious rooting of microcuttings in apple rootstocks.

Nanocarriers for encapsulation and sustained release of agrochemicals such as auxins have emerged as an attractive strategy to provide enhanced bioavailability and efficacy for improved crop yields and nutrition quality. Here, a comparative study was conducted on the effectiveness of chitosan-as a biopolymeric nanocarrier- and silver-as a metallic nanocarrier- on in vitro adventitious rooting potential of microcuttings in apple rootstocks, for the first time. Auxins indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) loaded silver (nAg) or chitosan nanoparticles (nChi) were synthesized. Scanning electron microscopy and transmission electron microscopy studies showed the spherical shape of the nanoparticles. The average particle size of IAA-nChi was 167.5 ± 0.1 nm while that of IBA-nChi was 123.2 ± 2.6 nm. The hydrodynamic diameter of the nAg-IAA and nAg-IBA particles were measured as 93.66 ± 5 nm and 71.41 ± 3 nm, respectively. Fourier transform infrared spectroscopy analyses confirmed the encapsulation of IAA or IBA in the chitosan nanoparticles. Meanwhile, the characteristic peaks of IAA or IBA were detected on silver nanoparticles. In-vitro adventitious rooting of microcuttings of Malling Merton 106 (MM 106) was significantly higher both in chitosan and silver nanoparticles loaded with IAA or IBA (91.7%-62.5%) compared to free IAA or IBA applications (50.0%-33.3%), except for 2.0 mg L-1 IBA (66.7%). However, the application of 2 mg L-1 IBA and IBA-nChi at all concentrations caused an undesirable large callus development.

BMP-6 carrying metal organic framework-embedded in bioresorbable electrospun fibers for enhanced bone regeneration.

Biomolecule carrier structures have attracted substantial interest owing to their potential utilizations in the field of bone tissue engineering. In this study, MOF-embedded electrospun fiber scaffold for the controlled release of BMP-6 was developed for the first time, to enrich bone regeneration efficacy. The scaffolds were achieved by first, one-pot rapid crystallization of BMP-6 encapsulated ZIF-8 nanocrystals-as a novel carrier for growth factor molecules- and then electrospinning of the blending solution composed of poly (ε-caprolactone) and BMP-6 encapsulated ZIF-8 nanocrystals. BMP-6 molecule encapsulation efficiency for ZIF-8 nanocrystals was calculated as 98%. The in-vitro studies showed that, the bioactivity of BMP-6 was preserved and the release lasted up to 30 days. The release kinetics fitted the Korsmeyer-Peppas model exhibiting a pseudo-Fickian behavior. The in-vitro osteogenesis studies revealed the superior effect of sustained release of BMP-6 towards osteogenic differentiation of MC3T3-E1 pre-osteoblasts. In-vivo studies also revealed that the sustained slow release of BMP-6 was responsible for the generation of well-mineralized, new bone formation in a rat cranial defect. Our results proved that; MOF-carriers embedded in electrospun scaffolds can be used as an effective platform for bone regeneration in bone tissue engineering applications. The proposed approach can easily be adapted for various growth factor molecules for different tissue engineering applications.

Regulation of chondrocyte hypertrophy in an osteochondral interface mimicking gel matrix.

Calcified cartilage extracellular matrix (ECM) is a critical interface at the osteochondral junction which plays an important role in maintaining the structural continuity between articular cartilage and subchondral bone. This mineralized network is primarily composed of glycosaminoglycans (GAGs) and collagen type II (col II) and hosts hypertrophic chondrocytes. This work aimed to investigate the effect of gel composition and collagen II content on the behavior and hypertrophic differentiation of ATDC5 cells for regeneration of calcified cartilage tissue. For this purpose, chitosan/collagen type II/nanohydroxyapatite (chi/col II/nHA) composite hydrogels were prepared to mimic the calcified cartilage ECM. ATDC5 cells were encapsulated within the composite gels and the viability, ECM production and hypertrophic gene expression were assessed during culture. All composites were favorable for ATDC5 viability and proliferation, whereas specific ECM production and hypertrophic differentiation were dependent on gel composition. Chitosan: collagen II ratio had an impact on ATDC5 cell fate. Hypertrophic differentiation was best pronounced in chi/col II/nHA 70:30 composition. The results obtained from this study offers a scaffold-based approach for calcified cartilage regeneration and provide an insight for biomimetic design and preparation of more complicated gradient osteochondral units.

Chitosan/collagen based biomimetic osteochondral tissue constructs: A growth factor-free approach.

Tissue engineering approach offers alternative strategies to develop multi-layered/multi-component osteochondral mimetic constructs to meet the requirements of the heterogeneous and layered structure of native osteochondral tissue. Herein, an iterative overlaying process to fabricate a multi-layered scaffold with a gradient composition and layer specific structure have been developed by combining the natural extracellular matrix (ECM) components-chitosan, type I collagen, type II collagen, nanohydroxyapatite- of the osteochondral tissue in biomimetic compositions. Subchondral bone layer was prepared by using freeze-drying method to obtain 3D porous scaffolds. The calcified cartilage and cartilage layers were prepared by thermal gelation method in the hydrogel form. Osteochondral scaffolds fabricated by iterative overlaying of each distinct layer exhibited a porous, continuous gradient structure and supported cell proliferation in a co-culture of MC3T3-E1 preosteoblasts and ATDC5 chondrocytes. Histology and biochemical analysis showed enhanced extracellular matrix production and demonstrated collagen and glycosaminoglycan deposition. Expression of genes specific for bone, calcified cartilage and cartilage were improved in the osteochondral scaffold. Overall, these findings suggest that iterative overlaying of freeze-dried scaffolds and hydrogel matrices prepared by using ECM components in biomimetic ratios to fabricate gradient, multi-layered structures can be a promising strategy without the need for growth factors.

Synthesis of indole-3-acetic acid and indole-3-butyric acid loaded zinc oxide nanoparticles: Effects on rhizogenesis.

The aim of this work was to investigate the use of zinc oxide nanoparticles (nZnO) as nanocarriers for plant auxins indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) and determine the effects on rhizogenesis in micro cuttings of different Pyrus species. Auxin loaded nanoparticles (IAA-nZnO and IBA-nZnO) were characterized for particle size, morphology, thermal behavior and chemical structure. A high loading capacity was observed for both auxins (˜90%). Bioactivity assays were performed by using micro cuttings of Pyrus genotypes (Pyrus elaeagrifolia Pall and Pyrus communis L.) under aseptic conditions by dilute solution soaking method. In vitro rooting efficiency was increased at least two folds for the difficult-to-root wild pear (Pyrus elaeagrifolia Pallas) with IAA or IBA loaded ZnO nanoparticles. In this genotype, the highest rooting percentage was achieved for IBA-nZnO and IAA-nZnO at 400 mgL-1 concentration as 50.0% and 41.7%, respectively. Thus, auxin loaded ZnO nanoparticles could be used as efficient nanocarriers in agricultural applications.

Metal-organic frameworks for on-demand pH controlled delivery of vancomycin from chitosan scaffolds.

A potential bone substitute and drug carrier system is prepared to be used in treatment of serious bone infections like osteomyelitis. Vancomycin (VAN), as an antibiotic was loaded into ZIF8 nanocrystals for a pH responsive controlled release (ZIF8/VAN). Chitosan scaffolds loaded with ZIF8/VAN were prepared by wet-spinning to obtain 3D biocompatible scaffolds. Characterization of scaffolds were performed to determine the morphology, swelling behavior and pH controlled VAN release. Antibacterial activity studies were done to investigate the effectiveness of the carrier system against Staphylococcus aureus. VAN molecule encapsulation efficiency for nanosized ZIF8 crystals was calculated as 99.3%. The results showed that the VAN loaded to ZIF8 nanocrystals was released in a pH controlled manner from the chitosan scaffolds. About 70% of the VAN was released during 8 h at pH 5.4, while this value was 55% at pH 7.4. VAN release was increased with higher dissolution of ZIF8 in acidic conditions and reached a plateau value of ~77% at the end of 48 h at pH 5.4 conditions. ZIF8 and ZIF8/VAN chitosan scaffolds showed a strong effect in the reduction of S. aureus activity in comparison to chitosan scaffolds alone. This effect was best pronounced under pH 5.4 conditions which can mimic the environment of an inflamed tissue. MC3T3-E1 preosteoblasts showed high proliferation and osteogenic activities on ZIF8 loaded chitosan scaffolds.

Enhancement of fibroblastic proliferation on chitosan surfaces by immobilized epidermal growth factor.

Chitosan membranes were modified with mouse epidermal growth factor (EGF) by a photochemical technique. Photochemical immobilization was performed via a two-step process, in which EGF was first reacted with a heterobifunctional cross-linker sulfo-SANPAH (sulfosuccinimidyl 6(4'-azido-2'-nitrophenyl-amino)hexanoate) and then immobilized on the chitosan membrane by UV irradiation. The success of immobilization process was checked by Fourier transform infrared attenuated total reflection spectroscopy and X-ray photoelectron spectroscopy. Atomic force microscopy was used to evaluate the surface topography. The mitogenic effect of the EGF-modified chitosan membrane was investigated using mouse fibroblasts (L929 cell line), and cell proliferation was investigated by MTT and crystal violet assays. The results obtained from cell culture experiments showed that immobilized EGF stimulated fibroblast growth on chitosan membranes, and a considerable difference in cell proliferation was detected on EGF-modified chitosan membranes.

Physico-chemical and thermodynamic aspects of fibroblastic attachment on RGDS-modified chitosan membranes.

In the present study, the cell attachment/spreading behaviour of L929 mouse fibroblasts on chitosan membranes was evaluated by using physico-chemical properties. For this purpose chitosan membranes were prepared and then photochemically modified with the cell adhesive peptide RGDS (Arg-Gly-Asp-Ser). The physico-chemical properties of unmodified (CHI) and RGDS-modified chitosan (CHI-RGDS) membranes were evaluated by calculating surface free energy (gamma(sv)) and interfacial free energy (gamma(sw)) values using captive bubble contact angle measurements and harmonic mean equation. The cell attachment experiments were performed both in 10% FBS containing and serum-free media with CHI and CHI-RGDS membranes. Eventually, it was not possible to predict a direct relationship between the change in physico-chemical properties and L929 cell attachment behaviour. The experimental results obtained from cell attachment agree with the theoretical prediction for the free energy of adhesion except for the cell attachment on CHI membrane in serum-free medium. Although a negative interfacial free energy of adhesion was calculated for CHI membrane in serum-free medium (DeltaF(adh)=-2.19 ergs/cm(2)), the cell attachment was poor (approximately 70%) compared to CHI-RGDS (approximately 90%) and none of the cells were spread on CHI surface to gain a fibroblastic morphology. Negative energy of adhesion was calculated for CHI and CHI-RGDS in 10% FBS medium, in which approximately 100% of cells were attached on the membranes correlating with the thermodynamic approach. It can be suggested that, adsorption of serum proteins strongly affected the cell attachment meanwhile the presence of biosignal RGDS molecules triggered the cell spreading in serum medium.

A direct tissue-grafting approach to increasing endogenous brown fat.

There is widespread evidence that increasing functional mass of brown adipose tissue (BAT) via browning of white adipose tissue (WAT) could potentially counter obesity and diabetes. However, most current approaches focus on administration of pharmacological compounds which expose patients to highly undesirable side effects. Here, we describe a simple and direct tissue-grafting approach to increase BAT mass through ex vivo browning of subcutaneous WAT, followed by re-implantation into the host; this cell-therapy approach could potentially act synergistically with existing pharmacological approaches. With this process, entitled "exBAT", we identified conditions, in both mouse and human tissue, that convert whole fragments of WAT to BAT via a single step and without unwanted off-target pharmacological effects. We show that ex vivo, exBAT exhibited UCP1 immunostaining, lipid droplet formation, and mitochondrial metabolic activity consistent with native BAT. In mice, exBAT exhibited a highly durable phenotype for at least 8 weeks. Overall, these results enable a simple and scalable tissue-grafting strategy, rather than pharmacological approaches, for increasing endogenous BAT and studying its effect on host weight and metabolism.

Evaluation of L929 fibroblast attachment and proliferation on Arg-Gly-Asp-Ser (RGDS)-immobilized chitosan in serum-containing/serum-free cultures.

In this study, chitosan membranes prepared by the solvent casting method were modified with the Arg-Gly-Asp-Ser (RGDS) sequence of fibronectin using the photochemical immobilization technique. The results obtained from attenuated total reflection-Fourier transform infrared spectra and X-ray photoelectron spectroscopy studies confirmed the successful immobilization of RGDS on chitosan membranes. The immobilized peptide concentration was determined by ninhydrin analysis on the order of 10(-7) mol/cm(2). In vitro cell culture studies were performed with L929 mouse fibroblasts to investigate the effect of biomodification on fibroblast cell behaviour in serum-free and 10% serum-containing media. The results obtained from cell culture studies pointed out the specific interactions between biosignal RGDS molecules and fibroblast cells. A triggered cell attachment and proliferation were observed on RGDS-modified chitosan membranes that were more distinguishable in serum-free medium. In addition, the photochemical immobilization technique was realized in the presence of a photomask that was used to immobilize the RGDS molecules in a defined micropattern. L929 mouse fibroblasts attached on the RGDS-micropatterned areas indicating integrin-mediated interactions.

Synthesis and characterization of Fe3O4-MPTMS-PLGA nanocomposites for anticancer drug loading and release studies.

Magnetic nanocomposites (Fe3O4-MPTMS-PLGA) were synthesized by single oil emulsion method and characterized by transmission electron microscopy (TEM), X-Ray diffraction (XRD), and vibrating sample magnetometer (VSM). Particle size of nanocomposites was between 117 nm and 246 nm. High performance liquid chromatography (HPLC) was used to investigate drug loading (paclitaxel, PTX) and release from Fe3O4-MPTMS-PLGA-PTX nanocomposites. The percentages of drug loading and encapsulation efficiency onto nanocomposites were found as 7.35 and 68.58, respectively. Cytotoxities of free anticancer drug and anticancer drug-loaded nanocomposites were determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. In vitro cell culture studies indicated that Fe3O4-MPTMS-PLGA-PTX had significant toxicity on MG-63 cancer cells.

Effect of emulsification-diffusion parameters on the formation of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) particles.

The aim of this work was to evaluate the effect of various production parameters on the formation and particle size of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) PHBV particles prepared by the emulsification-diffusion technique. The increase in homogenization time and speed caused a decrease in particle size. No particle formation was observed below 2% (w/v) PHBV in the organic phase. Smaller particle size and narrower size distribution were observed when polyvinyl alcohol (PVA) was used as a stabilizer, when compared to didodecyldimethylammonium bromide. Submicron particles of 531 ± 150 nm size were obtained with 2% (w/v) PVA at 17 500 rpm and 15 min homogenization conditions with dichloromethane as the organic solvent.

Preparation of bioactive and antimicrobial PLGA membranes by magainin II/EGF functionalization.

Development of dual functional materials that are capable of both reducing bacterial interaction and encouraging host tissue integration has gained importance in design of biomaterials. In this study, we prepared a bilayer poly (lactide co-glycolide) fibrous membrane with antibacterial and bioactive properties by electrospinning. The antibacterial layer was produced by covalent immobilization of antimicrobial peptide, Magainin II. The bioactive layer incorporating epidermal growth factor (EGF) molecules was subsequently electrospun on the antibacterial layer. The membranes were characterized by X-ray photoelectron spectroscopy, scanning electron microscopy and fluorescence microscopy. EGF release was detected by enzyme-linked immunosorbent assay. The antibacterial activity was tested against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The ability to support tissue cell integration was detected by using L-929 mouse fibroblasts. The dual functional membranes established enhanced antibacterial properties and increased tissue cell compatibility. This combined approach suggests a promising strategy for wound dressings, vascular grafts and dental membranes as well as catheters and fixation devices.

Antibacterial activity on electrospun poly(lactide-co-glycolide) based membranes via Magainin II grafting.

An antimicrobial peptide (AMP), Magainin II (Mag II) was covalently immobilized on poly(lactide-co-glycolide) (PLGA) and PLGA/gelatin electrospun fibrous membranes. The surface immobilization was characterized by X-ray Photoelectron Spectroscopy (XPS). Scanning Electron Microscopy (SEM) and Atomic Force Microscopy studies showed that the surface morphology of the fibers at micron scale was not affected by the immobilization process. The antibacterial activity of the bound Mag II was tested against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. Bacterial adhesion tests, SEM and confocal analyses revealed that the attachment and survival of bacteria were inhibited on Mag II functionalized membranes. AMP immobilization strategy was introduced as a new perspective for the modulation of antibacterial properties on PLGA based materials prepared by electrospinning.