Cytoprotective effect of hyaluronic acid and hydroxypropyl methylcellulose against DNA damage induced by thimerosal in Chang conjunctival cells.

BACKGROUND: To investigate genotoxicity of the preservative thimerosal (Thi), and the cytoprotective and antioxidant effects of hyaluronic Acid (HA) and hydroxypropyl methylcellulose (HPMC) on Chang conjunctival cells. METHOD: Cells were divided into three groups. One group was exposed to Thi at various concentrations (0.00001 %∼0.001 %) for 30 min; the other two groups were pretreated with 0.3 % HA or 0.3 % HPMC for 30 min before the Thi exposure. After cell viability was evaluated, alkaline comet assay and detection of the phosphorylated form of the histone variant H2AX (γH2AX) foci were used to determine DNA damage. Reactive oxygen species (ROS) production was assessed by the fluorescent probe, 2', 7'-dichlorodihydrofluorescein diacetate (DCFH-DA). RESULTS: A significant change of cell viability was observed after exposure to 0.001 % Thi for 30 min. DNA single- and double-strand breaks were significantly increased in a dose-dependent manner with Thi exposure. In addition, intracellular ROS induced by Thi was dose-dependent, except at 0.001 % less ROS was induced than at 0.0005 %. However, cells pretreated with 0.3 % HA or 0.3 % HPMC showed significantly increased cell survival, decreased DNA damage, and decreased ROS production compared to cells exposed to Thi alone. Pretreatment with 0.3 % HA was found to be even more protective than 0.3 % HPMC. CONCLUSION: Thi can induce DNA damage in human conjunctival epithelial cells, probably due to oxidative stress. HA and HPMC are protective agents that have antioxidant properties and can decrease DNA damage induced by Thi. Pretreatment of 0.3 % HA may be more protective of the ocular surface than 0.3 % HPMC.

Three-dimensional polycaprolactone scaffold-conjugated bone morphogenetic protein-2 promotes cartilage regeneration from primary chondrocytes in vitro and in vivo without accelerated endochondral ossification.

As articular cartilage is avascular, and mature chondrocytes do not proliferate, cartilage lesions have a limited capacity for regeneration after severe damage. The treatment of such damage has been challenging due to the limited availability of autologous healthy cartilage and lengthy and expensive cell isolation and expansion procedures. Hence, the use of bone morphogenetic protein-2 (BMP-2), a potent regulator of chondrogenic expression, has received considerable attention in cartilage and osteochondral tissue engineering. However, the exact role of BMP-2 in cartilage repair has been postulated to promote both cartilage formation and subsequent cartilage degradation through hypertrophy and endochondral ossification. Furthermore, it is likely that the manner in which BMP-2 is presented to chondrocytes will influence the physiologic pathway (repair vs. degeneration). This study investigates the relative influence of BMP-2 on cartilage matrix and potential subsequent bone matrix production using primary chondrocytes seeded on designed 3D polycaprolactone (PCL) scaffolds with chemically conjugated BMP-2. The results show that chemically conjugated BMP-2 PCL scaffolds can promote significantly greater cartilage regeneration from seeded chondrocytes both in vitro and in vivo compared with untreated scaffolds. Furthermore, our results demonstrate that the conjugated BMP-2 does not particularly accelerate endochondral ossification even in a readily permissible and highly vascular in vivo environment compared with untreated PCL scaffolds. This study not only reveals the potential use of the BMP-2 conjugation delivery method for enhanced cartilage tissue formation but also gives new insights for the effects of conjugated BMP-2 on cartilage regeneration and osteochondral ossification.

Direct electrochemistry of cytochrome c entrapped in agarose hydrogel in room temperature ionic liquids.

Direct electrochemistry of cytochrome c (cyt-c) entrapped in agarose hydrogel on gold electrode (Au), edge plane pyrolytic graphite electrode (EPPGE) and glassy carbon electrode (GC) in two room temperature ionic liquids was investigated. The effects of the addition of N,N-dimethylformamide (DMF) in the agarose-cyt-c film, water concentration in ionic liquids and exterior metal ions on the electrochemical behavior of cyt-c were monitored, and electrocatalytic properties of cyt-c were also done. Results showed that a good quasi-reversible redox behavior of cyt-c could be found after adding DMF in agarose-cyt-c film, and peak shape would not change after continuously scanning for 50 cycles. In addition, a certain amount of water in hydrophilic ionic liquids is necessary to maintain electrochemical activities of cyt-c, electrochemical performance of cyt-c is the best when the water content is 5.2% and 5.8% for 1-butyl-3-methylimidazolium bromide ([Bmim][Br]) and 1-butyl-3-methylimidazolium tetrafluoroborate([Bmim][BF(4)]) respectively. However, electrochemical activities of cyt-c are inhibited by exterior metal ions. Interestingly, cyt-c entrapped in agarose hydrogel on EPPGE and GC could catalyze the electroreduction of trichloroacetic acid (TCA) and tert-butyl hydroperoxide (t-BuOOH) in [Bmim][BF(4)], but could not in [Bmim][Br]. Reasons for above-mentioned differences of electrochemical properties of cyt-c in different ionic liquids were preliminarily discussed.

Three-dimensional poly(1,8-octanediol-co-citrate) scaffold pore shape and permeability effects on sub-cutaneous in vivo chondrogenesis using primary chondrocytes.

The objective of this study was to evaluate the coupled effects of three-dimensional poly(1,8-octanediol-co-citrate) (POC) scaffold pore shape and permeability on chondrogenesis using primary chondrocytes in vivo. Chondrogenesis was characterized as cartilage matrix formation by sulfated glycosaminoglycan (sGAG) quantification, relative mRNA expression of the cartilage-related proteins collagen types I, II and X, aggrecan and matrix metalloproteinases 13 and 3 and the compressive mechanical properties of the tissue/scaffold construct. A low permeability design with a spherical pore shape showed a significantly greater increase in cartilage matrix formation over 6 weeks in vivo than a high permeability design with a cubical pore shape. This increase in cartilage matrix synthesis corresponded with increases in mechanical compressive nonlinear elastic properties and histological data demonstrating darker red Safranin-O staining. There was higher mRNA expression for both cartilage-specific proteins and matrix degradation proteins in the high permeability design, resulting in overall less sGAG retained in the high permeability scaffold compared with the low permeability scaffold. Controlled POC scaffolds with a spherical pore shape and low permeability correlated with significantly increased cartilage matrix production using primary seeded chondrocytes. These results indicate that the low permeability design with a spherical pore shape provided a better microenvironment for chondrogenesis than the high permeability design with a cubical pore shape. Thus, scaffold architecture and material design may have a significant impact on the success of matrix-based clinical cartilage repair strategies.

Chemically-conjugated bone morphogenetic protein-2 on three-dimensional polycaprolactone scaffolds stimulates osteogenic activity in bone marrow stromal cells.

Poly(ε-caprolactone) (PCL) has received considerable attention in bone tissue engineering. However, the lack of osteoinductive ability of PCL limits its application. The aim of this study was to directly attach bone morphogenetic protein-2 (BMP-2) to PCL scaffolds by a crosslinking conjugation method and to investigate whether the bound BMP-2 maintained bioactivity in vitro. Immunofluorescent staining against BMP-2 and quantitative enzyme-linked immunosorbent assay measurements demonstrated that BMP-2 was successfully immobilized on the PCL three-dimensional scaffold by aminolysis and subsequent chemical conjugation. Conjugation produced much higher immobilization efficiency than the physical adsorption. Conjugated BMP-2 release from the PCL scaffolds was significantly slower than that from BMP-2-adsorbed PCL scaffolds over 15 days, which resulted in more BMP-2 locally retained on the conjugated scaffold. Further, the downstream Smads pathway was upregulated in bone marrow stromal cells cultured on the BMP-2-conjugated PCL scaffolds. Finally, gene expression of osteogenic markers (alkaline phosphatase, osteoclacin, and type I collagen) was upregulated in bone marrow stromal cells cultured on the PCL scaffolds with BMP-2 conjugation, but not on PCL scaffolds after BMP-2 adsorption. Therefore, our finding demonstrated that BMP-2 conjugation on polyester scaffolds is a feasible way to impart scaffolds with osteoinductive capability.

Comparison of bone marrow stromal cell behaviors on poly(caprolactone) with or without surface modification: studies on cell adhesion, survival and proliferation.

Poly(caprolactone) (PCL) is a promising biodegradable polymer for tissue engineering. However, intrinsically poor cell-adhesive properties of PCL may limit its application. In this study, the PCL film surface was modified with RGDC peptide by a chemical immobilization procedure. Furthermore, bone marrow stromal cell (BMSC) behaviors including attachment, spreading, focal adhesion formation, focal adhesion kinase (FAK) activation, apoptosis and proliferation when cultured on the modified PCL films were investigated. Our results demonstrated that PCL with RGD modification promoted initial BMSC attachment, spreading and focal adhesion formation. At a later time point (12 h), BMSC attachment on both RGD peptide-modified PCL and PCL-NH(2) films significantly increased compared to untreated PCL films. Importantly, FAK phosphorylation was significantly increased only on the films with RGD-modified films, not on the PCL-NH(2) films, demonstrating that PCL with RGD modification had an advantage in initiating the specific integrin-mediated signal transduction and might play an important role in the subsequent retardation in cell death and enhancement in cell proliferation. The present results provide more evidence that functionalizing PCL with RGD peptides may be a feasible way to improve the interaction between BMSC and PCL substrate, which is important in tissue engineering.

The interaction between bone marrow stromal cells and RGD-modified three-dimensional porous polycaprolactone scaffolds.

We previously established a simple method to immobilize the Arg-Gly-Asp (RGD) peptide on polycaprolactone (PCL) two-dimensional film surfaces that significantly improved bone marrow stromal cell (BMSC) adhesion to these films. The current work extends this modification strategy to three-dimensional (3D) PCL scaffolds to investigate BMSC attachment, cellular distribution and cellularity, signal transduction and survival on the modified PCL scaffold compared to those on the untreated ones. The results demonstrated that treatment of 3D PCL scaffold surfaces with 1,6-hexanediamine introduced the amino functional groups onto the porous PCL scaffold homogenously as detected by a ninhydrin staining method. Followed by the cross-linking reaction, RGDC peptide was successfully immobilized on the surface of PCL scaffold. Although the static seeding method used in this study caused heterogeneous cell distribution, the RGD-modified PCL scaffold still demonstrated the improved BMSC attachment and cellular distribution in the scaffold. More importantly, the integrin-mediated signal transduction FAK-PI3K-Akt pathway was significantly up-regulated by RGD modification and a subsequent increase in cell survival and growth was found in the modified scaffold. The present study introduces an easy method to immobilize RGD peptide on the 3D porous PCL scaffold and provides further evidence that modification of 3D PCL scaffolds with RGD peptides elicits specific cellular responses and improves the final cell-biomaterial interaction.

Intradiscal injection of simvastatin retards progression of intervertebral disc degeneration induced by stab injury.

INTRODUCTION: Earlier work indicates that the cholesterol-lowering drug, simvastatin, is anabolic to chondrogenic expression of rat intervertebral disc (IVD) cells, which suggests a potential role for simvastatin in IVD regeneration. In this study, we expand on our earlier work to test the effectiveness of simvastatin on disc degeneration utilizing a rat tail disc degeneration model. METHODS: 30 rats that underwent 21 G needle-puncture at rat tail discs were injected with simvastatin-loaded poly(ethylene glycol)-poly(lactic acid-co-glycolic acid)-poly(ethylene glycol) (PEG-PLGA-PEG) gel (5 mg/ml) or vehicle control at 4 weeks after needle injury. All animals were sacrificed 2 weeks after simvastatin injection. Bone morphogenetic protein-2 (BMP-2), aggrecan, collagen type II, and collagen type I messenger ribonucleic acid (mRNA) expression in the rat nucleus pulposus (NP) were measured by real-time polymerase chain reaction (PCR). In vivo magnetic resonance imaging (MRI) was performed to monitor changes in disc degeneration. Rat discs were also assessed by histology using hematoxylin and eosin (H&E) and safranin O staining. In addition, the NP weight, glycosaminoglycan (sGAG) and DNA content were also measured. RESULTS: A single dose of simvastatin loaded in thermo-sensitive PEG-PLGA-PEG gel injected into the NP had the trend to increase aggrecan expression and sGAG content, and significantly increased mRNA levels of BMP-2, collagen type II, and the differentiation index (the ratio of collagen type II to collagen type I). The decreased NP weight, T2 intensity, as well as MRI index in the rat tail discs induced by needle puncture were significantly reversed after 2 weeks of simvastatin treatment. In addition, simvastatin treatment also improved histological changes induced by needle puncture. CONCLUSIONS: A single injection of simvastatin loaded in PEG-PLGA-PEG gel into rat tail discs had the potential to retard or regenerate the degenerative disc.