Nesrin Hasirci

Incorporation of a sequential BMP-2/BMP-7 delivery system into chitosan-based scaffolds for bone tissue engineering

The aim of this study was to develop a 3-D construct carrying an inherent sequential growth factor delivery system. Poly(lactic acid-co-glycolic acid) (PLGA) nanocapsules loaded with bone morphogenetic protein BMP-2 and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nanocapsules loaded with BMP-7 made the early release of BMP-2 and longer term release of BMP-7 possible. 3-D fiber mesh scaffolds were prepared from chitosan and from chitosan-PEO by wet spinning. Chitosan of 4% concentration in 2% acetic acid (CHI4-HAc2) and chitosan (4%) and PEO (2%) in 5% acetic acid (CHI4-PEO2-HAc5) yielded scaffolds with smooth and rough fiber surfaces, respectively. These scaffolds were seeded with rat bone marrow mesenchymal stem cells (MSCs). When there were no nanoparticles the initial differentiation rate was higher on (CHI4-HAc2) scaffolds but by three weeks both the scaffolds had similar alkaline phosphatase (ALP) levels. The cell numbers were also comparable by the end of the third week. Incorporation of nanoparticles into the scaffolds was achieved by two different methods: incorporation within the scaffold fibers (NP-IN) and on the fibers (NP-ON). It was shown that incorporation on the CHI4-HAc2 fibers (NP-ON) prevented the burst release observed with the free nanoparticles, but this did not influence the total amount released in 25 days. However NP-IN for the same fibers revealed a much slower rate of release; ca. 70% released at the end of incubation period. The effect of single, simultaneous and sequential delivery of BMP-2 and BMP-7 from the CHI4-HAc2 scaffolds was studied in vitro using samples prepared with both incorporation methods. The effect of delivered agents was higher with the NP-ON samples. Delivery of BMP-2 alone suppressed cell proliferation while providing higher ALP activity compared to BMP-7. Simultaneous delivery was not particularly effective on cell numbers and ALP activity. The sequential delivery of BMP-2 and BMP-7, on the other hand, led to the highest ALP activity per cell (while suppressing proliferation) indicating the synergistic effect of using both growth factors holds promise for the production of tissue engineered bone.

EGF containing gelatin-based wound dressings.

In case of bulk loss of tissue or non-healing wounds such as burns, trauma, diabetic, decubitus and venous stasis ulcers, a proper wound dressing is needed to cover the wound area, protect the damaged tissue, and if possible to activate the cell proliferation and stimulate the healing process. In this study, synthesis of a novel polymeric bilayer wound dressing containing epidermal growth-factor (EGF) -loaded microspheres was aimed. For this purpose, a natural, nontoxic and biocompatible material, gelatin, was chosen as the underlying layer and various porous matrices in sponge form were prepared from gelatin by freeze-drying technique. As the external layer, elastomeric polyurethane membranes were used. Two different doses of EGF was added into the prepared gelatin sponges (1 and 15 microg/cm2) to activate cell proliferation. EGF addition was carried out either in free form or within microspheres to achieve prolonged release of EGF for higher efficiency. The prepared systems were tested in in vivo experiments on full-thickness skin defects created on rabbits. At certain intervals, wound areas were measured and tissues from wound areas were biopsied and processed for histological examinations. The wound areas decreased upon low-dose EGF application but the difference between the affects of free EGF and microsphere loaded EGF was not so distinct. Upon increasing the dose of EGF by a factor of 15, it was observed that controlled release of EGF from microspheres provided a higher degree of reduction in the wound areas. Histological investigations showed that the prepared dressings were biocompatible and did not cause any mononuclear cell infiltration or foreign body reaction. The structure of the newly formed dermis was almost the same as that of the normal skin.

Macroporous poly(3-hydroxybutyrate-co-3-hydroxyvalerate) matrices for bone tissue engineering

Macroporous poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) matrices were prepared after solvent evaporation and solute leaching. PHBV solutions with different concentrations were prepared in chloroform: dichloromethane (1:2, v/v). In order to create a matrix with high porosity and uniform pore sizes, sieved sucrose crystals (75-300 or 300-500 microm) were used. PHBV foams were treated with rf-oxygen plasma to modify their surface chemistry and hydrophilicity with the aim of increasing the reattachment of osteoblasts. Surface characteristics, pore sizes and their distribution on PHBV surface were studied by scanning electron microscopy (SEM) and Scion Image Analysis Program. Void volume, pore sizes and density of foams were found to be significantly affected by foam preparation conditions. Stability of PHBV foams in aqueous media was studied. Their weight and density were unchanged for a period of 120 days and then a significant decrease was observed for the rest of the study (60 days). Osteoblasts were seeded onto the foams and their proliferation inside the matrices was also determined by SEM. After 29 and 60 days of incubation, growth of osteoblasts on matrices was observed.

Sequential BMP-2/BMP-7 delivery from polyester nanocapsules

The aim of this study was to develop a nanosized, controlled growth factor release system to incorporate into tissue engineering scaffolds and thus activate the cells seeded in the scaffold. Nanocapsules of poly(lactic acid-co-glycolic acid) (PLGA) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) were loaded with the bone morphogenetic proteins BMP-2 and BMP-7, respectively, and with bovine serum albumin (BSA), the model protein. BSA-loading efficiency and release kinetics were used to determine the most appropriate nanocapsule pair to achieve the delivery of growth factors in a sequential manner, as occurs in natural processes. BSA-encapsulation efficiency was highest when the polymer concentration used in the preparation of PLGA and PHBV nanocapsules was 10% (w/v) (84.75% and 16.72%, respectively). Release of BSA was faster from PLGA than it was from PHBV. Based on the encapsulation efficiency and release data, 10% PLGA and 10% PHBV nanocapsules were chosen to provide the early BMP-2 and later BMP-7 release, respectively. Simultaneous, sequential delivery and individual release of the BMPs were studied for 7, 14, and 21 days, using rat bone marrow mesenchymal stem cells. Individual BMP-2 release suppressed cell proliferation while providing higher alkaline phosphatase activity with respect to BMP-7. The sequential delivery of BMP-2 and BMP-7 provided slightly lower proliferation than did simultaneous delivery, but the highest alkaline phosphatase activity of all indicated a synergistic effect on the osteogenic differentiation of mesenchymal stem cells caused by the use of the two growth factors in a sequential fashion.

Stability of α-amylase immobilized on poly(methyl methacrylate-acrylic acid) microspheres

Poly(methyl methacrylate-acrylic acid) microspheres were prepared and the acid groups were activated by using either carbodiimide (CDI) or thionyl chloride (SOCl2). alpha-Amylase was covalently bound on these activated microspheres. The properties of the immobilized enzyme were investigated and compared with those of the free enzyme. The relative activities were found to be 80.4 and 67.5% for carbodiimide and thionyl chloride bound enzymes, respectively. Maximum activities were obtained at lower pHs and higher temperatures upon immobilization compared to free enzyme. No change in Vmax and approximately 12-fold increase in K(m) were observed for immobilized enzymes. The enzyme activities, after storage for 1 month, were found to be 24.5 and 52.5% of the initial activity values for CDI and SOCl2 activated matrices, respectively. On the other hand the free enzyme lost its activity completely in 20 days. Immobilization, storage stability and repeated use capability experiments carried out in the presence of Ca2+ ions demonstrated higher stability, such as SOCl2 immobilized enzyme retained 83.7% and CDI immobilized enzyme retained 90.3% of the original activity of the enzyme. The immobilized enzymes that were used 20 times in 3 days in repeated batch experiments demonstrated that, in the absence of Ca2+ ions about 75% and in the presence of Ca2+ ions greater than 90% of the original enzyme activity was retained.

Cytotoxicity evaluation of gelatin sponges prepared with different cross-linking agents

Gelatin is a natural polymer used in pharmaceutical and medical applications, especially in the production of biocompatible and biodegradable wound dressings and drug delivery systems. Gelatin granules hydrate, swell and solubilize in water, and rapidly degrade in vivo. The durability of these materials could, however, be prolonged by cross-linking by aldehydes, carbodiimides, and aldose sugars, but the biocompatibility of collagenous biomaterials is profoundly influenced by the nature and extent of cross-linking. In this study, gelatin sponges were prepared by using various cross-linkers such as glutaraldehyde (GA), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDAC), and D-fructose. The effects of the type and the amount of cross-linker on thermal and mechanical properties, stability, and cytotoxicity were investigated. The mechanical analysis data showed that an increase in the amount of GA in the sponge structures caused a slight increase in the modulus of elasticity but had almost no effect on the tensile strength. Increase in the EDAC concentration produced a maximum in the modulus of elasticity and tensile strength values. The stability of the sponges and the time required for complete degradation in aqueous media increased in parallel with the cross-linker content. In vitro studies carried out with fibroblast cells demonstrated a higher cell viability for the samples cross-linked with low concentrations of GA than for those cross-linked with EDAC.

Controlled release of biologically active agents for purposes of agricultural crop management

Abstract Increasing attention is being directed to reducing the amount of pesticides, herbicides, and other biologically active agents used in modern agricultural crop management. One method for reducing the amount of such agents, while still maintaining effectiveness, is to encapsulate or otherwise incorporate the active agent into some from of plastic. Such ‘filled’ plastics, usually prepared by certain techniques for standard broadcast methods used in agriculture, may be sprayed, dusted, or spread as needed. By being incorporated into the plastic, the active agent diffuses slowly, but continuously, from the plastic matrix. It has been found in numerous instances that this use of controlled release delivery systems results in using less amount of the active agent. Further, with increasing attention being directed toward biologicals, rather than organic chemicals, for use in crop management, this incorporation of the biologicals into plastic serves the role of protection of the biological, as well as providing for slow release. One method of preparing an encapsulated polymeric controlled release system will be reviewed in depth, as well as field results.

Covalent immobilization of α-amylase onto poly(2-hydroxyethyl methacrylate) and poly(styrene-2-hydroxyethyl methacrylate) microspheres and the effect of Ca2+ ions on the enzyme activity.

Abstract α-Amylase (1,4-α- d -glucan-glucanohydrolase; EC 3.2.1.1, Type VI-B from porcine pancreas, extra pure 29 units mg−1) was covalently immobilized on poly (2-hydroxyethyl methacrylate), p(HEMA), and poly (styrene- 2- hydroxyethyl methacrylate), p(St-HEMA) microspheres, which were activated by using epichlorohydrin (ECH). The properties of the immobilized enzyme were investigated and compared with those of the free enzyme. For the assays carried out at 25°C and pH 6.9, the relative activities were found to be 61.7 and 67.0% for ECH-activated P(HEMA) and P(St-HEMA) bound enzymes, respectively. The maximum activities were obtained at lower pH values and higher temperatures upon immobilization compared to free enzyme. Kinetic parameters were calculated as 2.51, 22.4 and 6.62 g dm−3 for Km and 1.67×10−3, 1.63×10−3 and 1.35×10−3 g dm−3 min−1 for Vmax in the case of free, P(HEMA) and P(St-HEMA) bound enzymes, respectively. Enzyme activity was found to be ca. 38.9% for ECH-activated P(HEMA) bound enzyme after storage for 1 month. On the other hand, free enzyme lost its activity completely in 20 days. Immobilization, storage stability and repeated use capability experiments that were carried out in the presence of Ca2+ ions demonstrated higher stability. The enzymes immobilized in the presence of Ca2+ ions retained 90.7 and 80.0% of their original activities even after 30 days for ECH-activated P(HEMA) and P(St-HEMA) systems, respectively. In repeated batch experiments, 20 uses in 3 days, in the absence of Ca2+ ions, retention of 79% of the original enzyme activities was observed for ECH-activated P(HEMA) immobilized enzymes. On addition of Ca2+ ions to the assay medium, 90.0 and 80.0% of retention was observed for ECH-activated P(HEMA) and P(St-HEMA) systems, respectively.

Synthesis and surface modification of polyurethanes with chitosan for antibacterial properties

Surface modification and providing antibacterial properties to the materials or devices are getting great attention especially in the last decades. In this study, polyurethane (PU) films were prepared by synthesizing them in medical purity from toluene diisocyanate and polypropylene ethylene glycol without using any other ingredients and then the film surfaces were modified by covalent immobilization of chitosan (CH) which has antibacterial activity. CH immobilized PU films (PU-CH) were found to be more hydrophilic than control PU films. Electron Spectroscopy for Chemical Analysis (ESCA) and Atomic Force Microscopy (AFM) analyses showed higher nitrogen contents and rougher surface topography for PU-CH compared to PU films. Modification with CH significantly increased antibacterial activity against Gram positive (Staphylococcus aureus) and Gram negative (Pseudomonas aeruginosa) bacteria. It was observed that the number of bacteria colonies were less about 10(2)-10(5) CFU/mL and number of attached viable bacteria decreased significantly after CH modification of PU films.

Development of porous chitosan-gelatin/hydroxyapatite composite scaffolds for hard tissue-engineering applications.

Composite scaffolds prepared from natural polymers and hydroxyapatite (HA) are expected to have enhanced osteoconductive properties and as a result gained much attention in recent years for use in bone tissue‐engineering applications. Although there are various natural polymers available for this purpose, chitosan (C) and gelatin (G) are commonly studied because of their inherent properties. The aim of this study was to prepare three‐dimensional (3D) scaffolds using these two natural polymers and to add either non‐sintered hydroxyapatite (nsHA) or sintered hydroxyapatite (sHA) to compare their influence on physical, chemical and mechanical properties of the scaffolds and on their affinities towards Saos‐2 cells. For this purpose, nsHA and sHA were synthesized and characterized by X‐ray diffraction (XRD), Fourier transform infrared (FTIR), scanning electron microscopy (SEM) and particle size analyses. Then nsHA and sHA particles, with average sizes of 16 µm and 6 µm, respectively, were added to the solutions of C and G during the preparation step and the resultant 3D scaffolds were characterized. Compression tests indicated that presence of nsHA or sHA increased the Youngs modulus and compressive strength of the scaffolds, and the values were very similar to those of human spongy bone. MTS assays, confocal microscopy and SEM analysis showed that cell attachment and proliferation were higher on CG/sHA composite scaffolds compared to the other scaffolds. It was shown that the scaffolds prepared from chitosan, gelatin and HA are appropriate cell carriers for bone tissue engineering, especially those with sHA incorporated. Copyright