Hydroxyapatite containing superporous hydrogel composites: synthesis and in-vitro characterization

The synthesis of an acrylamide-based superporous hydrogel composite (SPHC) with hydroxyapatite (HA) was realized by solution polymerization technique. The characterization studies were performed by FTIR studies, determination of swelling kinetics, measurement of mechanical properties, SEM/EDAX studies and cytocompatibility tests. The FTIR and EDAX studies revealed the incorporation of HA in superporous hydrogel (SPH) structure. The results obtained from swelling experiments showed that, although the extent of swelling was decreased after incorporation of HA in SPH structure, the time to reach the equilibrium swelling was not affected for SPHC. This result indicated that, the presence of HA did not block the capillary channels and the interconnected pore structure was maintained which were consistent with the images obtained from SEM photographs. The results obtained from mechanical tests showed that, in the presence of HA, the compression strength of the hydrogel composite was improved significantly when compared to SPH structure. The compressive modulus for the SPHC increased to 6.59 ± 0.35 N/mm² whereas it was 0.63 ± 0.04 N/mm² for the SPH. The cytocompatibility test which was performed by using L929 fibroblasts showed that both the SPH and SPHC materials were cytocompatible towards fibroblasts. The synthesized superporous hydrogel composite possesses suitable properties especially for bone tissue engineering applications and shall be considered as a novel scaffold.     

PNIPAAm-grafted thermoresponsive microcarriers: Surface-initiated ATRP synthesis and characterization

In this study, we developed novel thermoresponsive microcarriers as a powerful tool for cell culture and tissue engineering applications. For this purpose, two types of commercially available spherical microparticles (approximately 100 μm in diameter), dextran-based Sephadex® and vinyl acetate-based VA-OH (Biosynth®), were used and themoresponsive poly(N-isopropylacrylamide) (PNIPAAm) was grafted to the beads' surfaces by surface-initiated atom transfer radical polymerization (SI-ATRP). Initially, hydroxyl groups of microbeads were reacted with 2-bromopropionyl bromide to form ATRP macroinitiator. Then, NIPAAm was successfully polymerized from the initiator attached microbeads by ATRP with CuBr/2,2′-dipyridyl, catalyst complex. Furthermore, grafted and ungrafted microbeads were characterized by attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, scanning electron microscope (SEM), atomic force microscopy (AFM) and electron spectroscopy for chemical analysis (ESCA). The results of characterization studies confirmed that PNIPAAm was successfully grafted onto both dextran and vinyl acetate-based beads by means of ATRP reaction and thus, grafted microbeads gained thermoresponsive characteristics which will be evaluated for cell harvesting in further studies.     

Evaluation of alginate-chitosan semi IPNs as cartilage scaffolds

In this study, alginate and alginate:chitosan semi interpenetrating polymer network (IPN) scaffolds were prepared by freeze-drying process. Alginate scaffolds were crosslinked with different concentrations of CaCl₂, i.e. 0.5, 1 or 3% (w/v), in 96% (v/v) ethanol solutions for two different periods, i.e. 4 and 24 h, after freeze-drying. Scanning electron microscope (SEM)/ Energy Dispersive Analysis by X-ray (EDAX) analysis and swelling studies indicated that crosslinking of scaffolds with 3% (w/v) CaCl₂ for 24 h was effectively created suitable alginate scaffolds in terms of optimum porosity and mechanical stability. This is why, alginate:chitosan semi IPN scaffolds were prepared at the crosslinking condition mentioned above in 70:30, 60:40 and 50:50% (v/v) alginate:chitosan ratios. Besides the attachment and proliferation abilities of ATDC5 murine chondrogenic cells on alginate, 70:30% (v/v) alginate:chitosan and 50:50% (v/v) alginate:chitosan scaffolds, their cellular responses were assessed for chondrogenic potential. These structural and cellular outcomes demonstrate potential utility of chitosan semi IPNs in alginate scaffolds. Comparative results found in relation to alginate scaffolds, support the necessity for alginate:chitosan scaffolds for improved cartilage tissue engineering.     

Magnetic poly(glycerol dimethacrylate) latex particles: synthesis, characterization and cellular interactions

Magnetic poly(glycerol dimethacrylate) (m-poly(GDMA)) latex particles were synthesized by precipitation polymerization in the presence of iron oxide nanoparticles obtained by co-precipitation of Fe²⁺ and Fe³⁺ salts. The mean size of iron oxide nanoparticles and m-poly(GDMA) latex particles was determined by dynamic light scattering as 57 and ~800?nm, respectively. Vibrating sample magnetometer results showed that iron oxide nanoparticles and m-poly(GDMA) latex particles are superparamagnetic, and their saturation magnetizations are 74.37 and 3.85 emu/g, respectively. MC3T3-E1 preosteoblasts were seeded in the presence of m-poly(GDMA) latex particles. Through the magnetic particles, magnetic field was applied onto the cells by an appropriate magnet (4500 G). Cell?Cparticle interactions were observed by optical microscope and scanning electron microscope. Mitochondrial activities of cells was measured by 3-[4,5-dimethylthiazol-2-y1]-2,5-diphenyl tetrazolium bromide test. The results show that the presence of m-poly(GDMA) particles in the culture medium did not affect the proliferation behaviour of MC3T3 cells and no toxicity against to L929 fibroblastic cell proliferation was observed when the particle concentration is 100?pg per cell.     

Biofunctionalization of magnetic poly(glycidyl methacrylate) microspheres with protein A: Characterization and cellular interactions

Monodisperse poly(glycidyl methacrylate) (m-PGMA) microspheres which show superparamagnetic behaviour were synthesized by dispersion polymerization. Bioligand protein A was covalently immobilized onto glutaraldehyde activated microspheres (3.12 mg protein A per gram of microspheres). Cell culture studies denoted that 61% of total L929 mouse fibroblasts were bound to the m-PGMA microspheres while 84% of total cells were bounding to the protein A immobilized (m-PGMA-PrA) microspheres. Interactions between m-PGMA-PrA microspheres and L929 cells were stronger than that of m-PGMA microspheres due to the non-specific interactions between protein A and cell surface. The cells interacted with m-PGMA-PrA microspheres keep their round form rather than attaching to the tissue culture polystyrene (TCPS) surface. In conclusion, this study consists a basis for the fractionation of blood lymphocytes bearing IgG antibodies on their surfaces by using protein A immobilized m-PGMA microspheres.     

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.     

Surface characteristics of ionically crosslinked chitosan membranes

In this study, homogenous dense chitosan membranes were prepared by solution‐casting procedure. Then the membranes were ionically crosslinked by sulfuric acid. The surfaces of chitosan membranes before and after crosslinking were characterized by using FTIR‐ATR, X‐ray photoelectron spectroscopy (XPS), and atomic‐force microscopy (AFM) techniques. The XPS data suggest that the surface composition of crosslinked membrane does not change significantly with respect to uncrosslinked membrane and the most important evidence is a certain amount of sulfur, coming from the crosslinker. The result from FTIR‐ATR data shows the effectiveness of the crosslinking procedure by the shift in amide I and amide II bands. The investigation of membrane surfaces by AFM indicates that the crosslinking procedure modifies the surface morphology of chitosan. After crosslinking, the surface topography becomes more homogenous and relatively flat. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 2007     

Osteogenic activities of polymeric soybean oil-g-polystyrene membranes

A novel biocompatible copolymer membrane was synthesized and characterized for use in guided bone regeneration using polymeric soybean oil-g-polystyrene (PSO-g-PS) graft copolymer which was successfully obtained by free radical polymerization of styrene initiated by PSO peroxide as a macroinitiator at 80 °C. Osteoblastic cellular activities of MC3T3-E1 cells on PSO-g-PS membranes with different soybean oil composition (PSO-g-PS1, PSO-g-PS2, and PSO-g-PS3) were evaluated. Nuclear magnetic resonance (¹H NMR) spectra showed that PSO inclusion (mol%) was found to be 27, 69, and 51 % for PSO-g-PS1, PSO-g-PS2, and PSO-g-PS3 membranes, respectively. Superior biocompatibility of the PSO-g-PS membranes was determined compared to polystyrene tissue culture plates (TCPS) as positive control. Cell proliferation was enhanced on PSO-g-PS2 and PSO-g-PS3 membranes compared to PSO-g-PS1 membranes (p < 0.001), and a statistically significant higher ALP value of MC3T3-E1 cells on PSO-g-PS2 membranes (p < 0.05) suggested that proliferation and differentiation of preosteoblastic on PSO-g-PS membranes were enhanced with regard to soybean oil content within the membranes. Thus, the present study suggests that PSO-g-PS2 membranes, which showed a favorable biological environment for the preosteoblastic cells, can be well suited for bone tissue engineering applications.     

Superporous polyacrylate/chitosan IPN hydrogels for protein delivery

In this study, poly(acrylamide), poly(AAm), and poly(acrylamide-co-acrylic acid), poly(AAm-co-AA) superporous hydrogels (SPHs) were synthesized by radical polymerization in the presence of gas blowing agent, sodium bicarbonate. In addition, ionically crosslinked chitosan (CH) superporous hydrogels were synthesized to form interpenetrating superporous hydrogels, i.e. poly(AAm)-CH and poly(AAm-co-AA)-CH SPH-IPNs. The hydrogels have a structure of interconnected pores with pore sizes of approximately 100-150 μm. Although the extent of swelling increased when AA were incorporated to the poly(AAm) structure, the time to reach the equilibrium swelling (~30 s) was not affected so much. In the presence of chitosan network mechanical properties significantly improved when compared with SPHs, however, equilibrium swelling time (~30 min) was prolonged significantly as due to the lower porosities and pore sizes of SPH-IPNs than that of SPHs. Model protein bovine serum albumin (BSA) was loaded into SPHs and SPH-IPNs by solvent sorption in very short time (<1 h) and very high capacities (~30-300 mg BSA/g dry gel) when compared to conventional hydrogels. BSA release profiles from SPHs and SPH-IPNs were characterized by an initial burst of protein during the first 20 min followed by a completed release within 1 h. However, total releasable amount of BSA from SPH-IPNs was lower than that of SPHs as due to the electrostatic interactions between chitosan and BSA.