In vitro and in vivo co-culture of chondrocytes and bone marrow stem cells in photocrosslinked PCL-PEG-PCL hydrogels enhances cartilage formation.

Chondrocytes (CH) and bone marrow stem cells (BMSCs) are sources that can be used in cartilage tissue engineering. Co-culture of CHs and BMSCs is a promising strategy for promoting chondrogenic differentiation. In this study, articular CHs and BMSCs were encapsulated in PCL-PEG-PCL photocrosslinked hydrogels for 4 weeks. Various ratios of CH:BMSC co-cultures were investigated to identify the optimal ratio for cartilage formation. The results thus obtained revealed that co-culturing CHs and BMSCs in hydrogels provides an appropriate in vitro microenvironment for chondrogenic differentiation and cartilage matrix production. Co-culture with a 1:4 CH:BMSC ratio significantly increased the synthesis of GAGs and collagen. In vivo cartilage regeneration was evaluated using a co-culture system in rabbit models. The co-culture system exhibited a hyaline chondrocyte phenotype with excellent regeneration, resembling the morphology of native cartilage. This finding suggests that the co-culture of these two cell types promotes cartilage regeneration and that the system, including the hydrogel scaffold, has potential in cartilage tissue engineering. Copyright © 2013 John Wiley & Sons, Ltd.

Glial cell line-derived neurotrophic factor gene delivery via a polyethylene imine grafted chitosan carrier.

Parkinson's disease is known to result from the loss of dopaminergic neurons. Direct intracerebral injections of high doses of recombinant glial cell line-derived neurotrophic factor (GDNF) have been shown to protect adult nigral dopaminergic neurons. Because GDNF does not cross the blood-brain barrier, intracerebral gene transfer is an ideal option. Chitosan (CHI) is a naturally derived material that has been used for gene transfer. However, the low water solubility often leads to decreased transfection efficiency. Grafting of highly water-soluble polyethylene imines (PEI) and polyethylene glycol onto polymers can increase their solubility. The purpose of this study was to design a non-viral gene carrier with improved water solubility as well as enhanced transfection efficiency for treating Parkinsonism. Two molecular weights (Mw =600 and 1,800 g/mol) of PEI were grafted onto CHI (PEI600-g-CHI and PEI1800-g-CHI, respectively) by opening the epoxide ring of ethylene glycol diglycidyl ether (EX-810). This modification resulted in a non-viral gene carrier with less cytotoxicity. The transfection efficiency of PEI600-g-CHI/deoxyribonucleic acid (DNA) polyplexes was significantly higher than either PEI1800-g-CHI/DNA or CHI/DNA polyplexes. The maximal GDNF expression of PEI600-g-CHI/DNA was at the polymer:DNA weight ratio of 10:1, which was 1.7-fold higher than the maximal GDNF expression of PEI1800-g-CHI/DNA. The low toxicity and high transfection efficiency of PEI600-g-CHI make it ideal for application to GDNF gene therapy, which has potential for the treatment of Parkinson's disease.

Oligoalanine-modified Pluronic-F127 nanocarriers for the delivery of curcumin with enhanced entrapment efficiency.

Curcumin is a naturally occurring compound that has been shown to have anti-oxidant, anti-inflammatory, and anti-carcinogenic activities. However, its pharmaceutical potential has been limited due to its low solubility in water. The use of amphiphilic nanocarriers is an attractive and simple method to solubilize curcumin. In this study, we modified Pluronic F-127 [poly(ethylene glycol)100-block-poly(propylene glycol)65-block-poly(ethylene glycol)100] (PF-127) with oligomers of alanine, an amino acid, to increase the drug entrapment efficiency of curcumin through core stabilization. Alanine-modified PF-127 exhibited lower critical micelle concentration and decreased molecular motion in both the hydrophilic and hydrophobic segments ((1)H NMR). Nanocarriers in the size range of 54.2-68.4 nm were observed. Entrapment efficiency of curcumin increased by at most 66% (from 25.3 to 91.3%) and the difference in solubility was clearly visualized by increased transparency of the nanocarrier solutions. Curcumin was released continuously up to 120 h from modified carriers, while drug release from unmodified carriers plateaued within 24 h. These modified nanocarriers exhibited no cytotoxicity and more efficiently delivered drugs to HeLa cells as confirmed by fluorescent microscopy. This study demonstrated that alanine modification of FDA-approved PF-127 affects copolymer nanoassembly and has a profound impact on curcumin loading and possibly on other hydrophobic drugs as well.

Evaluation of a mPEG-polyester-based hydrogel as cell carrier for chondrocytes.

Temperature-sensitive hydrogels are attractive alternatives to porous cell-seeded scaffolds and is minimally invasive through simple injection and in situ gelling. In this study, we compared the performance of two types of temperature-sensitive hydrogels on chondrocytes encapsulation for the use of tissue engineering of cartilage. The two hydrogels are composed of methoxy poly(ethylene glycol)- poly(lactic-co-valerolactone) (mPEG-PVLA), and methoxy poly(ethylene glycol)-poly(lactic- co-glycolide) (mPEG-PLGA). Osmolarity and pH were optimized through the manipulation of polymer concentration and dispersion medium. Chondrocytes proliferation in mPEG-PVLA hydrogels was observed as well as accumulation of GAGs and collagen. On the other hand, chondrocytes encapsulated in mPEG-PLGA hydrogels showed low viability and chondrogenesis. Also, mPEG-PVLA hydrogel, which is more hydrophobic, retained physical integrity after 14 days while mPEG-PLGA hydrogel underwent full degradation due to faster hydrolysis rate and more pronounced acidic self-catalyzed degradation. The mPEG-PVLA hydrogel can be furthered tuned by manipulation of molecular weights to obtain hydrogels with different swelling and degradation characteristics, which may be useful as producing a selection of hydrogels compatible with different cell types. Taken together, these results demonstrate that mPEG-PVLA hydrogels are promising to serve as three-dimensional cell carriers for chondrocytes and potentially applicable in cartilage tissue engineering.

Poly(ethylene imine)-g-chitosan using EX-810 as a spacer for nonviral gene delivery vectors.

Polyelectrolyte complexes have been widely studied as gene carriers in recent years. In this study, poly (ethylene imine) was grafted onto chitosan (PEI-g-CHI) as a nonviral gene carrier in order to improve the water solubility as well as the inherent transfection efficiency of chitosan. We present a novel method to conjugate the amine or hydroxyl groups of chitosan (CHI) and the amine groups of PEI through opening the epoxide rings of ethylene glycol diglycidyl ether (EX-810), which also brings the merits as mentioned in PEGylation chemistry. The degree of substitution of PEI onto CHI was characterized by NMR. The preliminarily cellular mechanisms, from the cellular entry to the endosomal release, were investigated by the correlations among the physicochemical properties of the DNA-polymer complexes, the buffering capacity of the modified polymer, the cytotoxicity, and the efficiency of the transgene expression. The cytotoxicity assayed by MTT shows that cell viability of PEI-g-CHI is higher than CHI especially noticeable at high concentrations using human kidney 293T cells. The efficiency of transgene expression and the amount of intracellular plasmid were monitored using green fluorescent protein (GFP) and visualized by fluorescence microscopy. The transfection efficiency of PEI-g-CHI/DNA polyplex is significantly better than CHI/DNA polyplex when using the weight ratios higher than 2.5.

Characterization of hydrogels prepared from copolymerization of the different degrees of methacrylate-grafted chondroitin sulfate macromers and acrylic acid.

Different degrees (between 20 and 75%) of methacrylate-grafted chondroitin sulfate (CS-MA) were synthesized. These CS-MA macromers were further copolymerized with acrylic acid (AA) at the molar ratio of 1 to 5 to form hydrogels. The sol percents of these CS-MA-AA hydrogels decreased and the cross-linking densities were studied with respect to the degrees of MA substitution onto CS-MA. The cytotoxicity with the increase in degree of MA substitution (DS) onto CS-MA as well as their hydrogels prepared from the corresponding macromers was tested using 293T cells. The cell viability of human dermal fibroblast and mescenchymal stem cells was further tested upon exposure to 75% CS-MA for 1-, 3-, and 7-day incubation period. The hydrogels maintained degradability for long periods of time as evidenced by SEM. A model protein, BSA, demonstrated the prolong-release behaviors of these hydrogels in simulated gastric fluids and pH 7.4 phosphate buffer solutions and a faster release rate in the presence of chondroitinase and esterase at pH 7.4.

Chondroitin sulfate-based anti-inflammatory macromolecular prodrugs.

Macromolecular prodrugs of three non-steroidal anti-inflammatory drugs (NSAIDs), ibuprofen, ketoprofen, and naproxen, were prepared by the covalent attachment of the drugs onto chondroitin sulfate (ChS) using PEG 1000 as a spacer. Drug-PEG adducts were synthesized using 1,1'-carbonyl diimidazole as a coupling agent in dimethyl sulfoxide, followed by the reaction with ChS in highly dilute aqueous solution at pH 6.8 via N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDAC) as a conjugation agent. The drug-ChS conjugates were confirmed by FTIR, 1H NMR and 13C NMR and the molar percent of drug substitution onto ChS was characterized by 1H NMR using the peak areas of the three protons of -PhiCHCH3 on the drugs to those of -NHCOCH3 on ChS. All drug-ChS conjugates are water-soluble. The release amounts of the free drugs from their corresponding drug-ChS conjugates were evaluated in the presence or absence of either esterase or chondroitinase, and the both enzymes in pH 7.4 Tris-buffer solutions at 37 degrees C by high performance liquid chromatography (HPLC). Keto-ChS conjugates released approximately 100% ketoprofen within 12h in the presence of esterase, but the combination with chondroitinase did not accelerate the release rate. The degradation of Keto-ChS conjugates by chondroitinase was confirmed by gel permeation chromatography (GPC). The Keto-ChS conjugates still retained the enzymatic recognition even at the substitution of ketoprofen as high as 56 mol%. The inhibition percent of carrageenan-induced edema of Keto-ChS-56 was comparable to that of a simple blend of ChS and ketoprofen, suggesting that biologically active ChS and ketoprofen could be liberated from the conjugate.