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Prostate-specific antigen (PSA) test is a commonly used clinical examination to evaluate the risk of prostate cancer, with the antibodies used normally as the recognition molecules for measuring PSA levels in serum. Alternatively, aptamers that are able to bind target molecules with high affinity and specificity similar to antibodies could be generated much easier and cheaper than the production of antibodies. In this study, we used computaional and experimental approaches to select truncated PSA-binding aptamers generated from the sequence information of PSA-binding aptamers previously reported in a literature. Genetic algorithm, the analysis of secondary structure, and molecular simulation were utilized in the in silico analysis. The top 4 ranked sequecnes in silico analysis were evaluated through their PSA-binding ability on the quartz crystal microbalance (QCM) biosensor. Finally, We identified a truncated aptamer obtained from the selection showing a nearly 3.5-fold higher measured signal than the response produced by the best known DNA sequence in the QCM measurement.

Encapsulation of nucleus pulposus (NP) cells within in situ forming hydrogels is a novel biological treatment for early stage intervertebral disc degeneration. The procedure aims to prolong the life of the degenerating discs and to regenerate damaged tissue. In this study we developed an injectable oxidized hyaluronic acid–gelatin–adipic acid dihydrazide (oxi-HAG–ADH) hydrogel. High molecular weight (1900 kDa) hyaluronic acid was crosslinked with various concentrations of gelatin to synthesize the hydrogels and their viscoelastic properties were analyzed. Interactions between the hydrogels, NP cells, and the extracellular matrix (ECM) were also evaluated, as were the effects of the hydrogels on NP cell gene expression. The hydrogels possess several clinical advantages, including sterilizability, low viscosity for injection, and ease of use. The viscoelastic properties of the hydrogels were similar to native tissue, as reflected in the complex shear modulus (∼11–14 kPa for hydrogels, 11.3 kPa for native NP). Cultured NP cells not only attached to the hydrogels but also survived, proliferated, and maintained their round morphology. Importantly, we found that hydrogels increased NP cell expression of several crucial ECM-related genes, such as COL2A1, AGN, SOX-9, and HIF-1A.

To develop an effective ocular drug delivery carrier, we prepared two different charged gelatin nanoparticles (GPs) and evaluated particle size, surface charge, and morphology. Thein vitrobiocompatibility of GPs was assessed using human corneal epithelium (HCE) cells andin vivosafety by administering them as eye drops to New Zealand rabbits. The GPs prepared using type A gelatin were positively charged (GP(+), +33 mV; size,180.6±45.7 nm). Water-soluble tetrazolium salt (WST)-1 assay showed that both GPs were nontoxic to HCE cells. The fluorescence intensity of HCE cells cultured with cationic GPs conjugated with a fluorescent dye was higher than that of the anionic GP-treated HCE cells.In vivoexamination showed no serious irritation to the rabbit eyes. Furthermore, corneal thickness and ocular pressure in the eyes of the treated rabbits were similar to those in the eyes of normal rabbits. Microscopic examination of corneal cryosections showed widely distributed fluorescent nanocarriers, from the anterior to the posterior part of the cornea of the GP(+) group, and higher fluorescence intensity in the GP(+) group was also observed. In conclusion, GPs as cationic colloidal carriers were efficiently adsorbed on the negatively charged cornea without irritating the eyes of the rabbits and can be retained in the cornea for a longer time. Thus, GPs(+) have a great potential as vehicles for ocular drug delivery.

Osteomyelitis therapy is a long-term and inconvenient procedure for a patient. Antibiotic-loaded bone cements are both a complementary and alternative treatment option to intravenous antibiotic therapy for the treatment of osteomyelitis. In the current study, the biphasic calcium phosphate cement (CPC), calledα-TCP/HAP (α-tricalcium phosphate/hydroxyapatite) biphasic cement, was prepared as an antibiotics carrier for osteomyelitis. The developed biphasic cement with a microstructure ofα-TCP surrounding the HAP has a fast setting time which will fulfill the clinical demand. The X-ray diffraction and Fourier transform infrared spectrometry analyses showed the final phase to be HAP, the basic bone mineral, after setting for a period of time. Scanning electron microscopy revealed a porous structure with particle sizes of a few micrometers. The addition of gentamicin inα-TCP/HAP would delay the transition ofα-TCP but would not change the final-phase HAP. The gentamicin-loadedα-TCP/HAP supplies high doses of the antibiotic during the initial 24 hours when they are soaked in phosphate buffer solution (PBS). Thereafter, a slower drug release is produced, supplying minimum inhibitory concentration until the end of the experiment (30 days). Studies of growth inhibition ofStaphylococcus aureusandPseudomonas aeruginosain culture indicated that gentamicin released after 30 days fromα-TCP/HAP biphasic cement retained antibacterial activity.

Four kinds of chitosan membranes, ( Ch, C-G, C-N, and C-GN ), were prepared in this study. Ch system of chitosan membranes were prepared by solution casting method without any treatment. C-G system of chitosan membranes were prepared by solution casting method with adding glutaraldehyde as crosslinking agent. C-N system of chitosan membranes were prepared by solution casting method with the treatment of NaOH solution. C-GN system of chitosan membranes were crosslinked with glutaraldehyde and treated with 1 M NaOH solution. Infrared spectroscopy and differential scanning calorimetry (DSC) were conducted to characterize the structure and thermal properties of chitosan membrane. To evaluate the usage of chitosan for the carrier of drug, water content and degradation of the chitosan membrane were determined. It was found that the values of equilibrated water content in chitosan membranes increased with the chitosan content in the stock chitosan solution. The assessment of 5-FU permeability through the chitosan membranes was determined. The linear relationship between the permeability of 5-FU through the membrane and the wt.% of chitosan content in stock chitosan solution was found. Both values of intercept and slope were determined. The value of intercept means that the permeability of 5-FU through the chitosan membrane, which is assumed to be prepared from stock chitosan solution containing infinite dilute chitosan content. The value of slope indicates that the effect of the treatment method on the permeability of 5-FU through the chitosan membrane. From the evaluation of normalized flux, diffusivity and saturation concentration of 5-FU in various chitosan membrane, it was found that more number of 5-FU molecules were trapped in the chitosan membrane of C-GN system than that in C-N system and resulted in the decrease of diffusivity of 5-FU molecules through the chitosan membrane.

Vitrectomy is a common procedure for treating ocular-related diseases. The surgery involves removing the vitreous humor from the center of the eye, and vitreous substitutes are needed to replace the vitreous humor after vitrectomy. In the present study, we developed a colorless, transparent and injectable hydrogel with appropriate refractive index as a vitreous substitute. The hydrogel is formed by oxidated hyaluronic acid (oxi-HA) cross-linked with adipic acid dihydrazide (ADH). Hyaluronic acid (HA) was oxidized by sodium periodate to create aldehyde functional groups, which could be cross-linked by ADH. The refractive index of this hydrogel ranged between 1.3420 and 1.3442, which is quite similar to human vitreous humor (1.3345). The degradation tests demonstrated that the hydrogel could maintain the gel matrix over 35 days, depending on the ADH concentration. In addition, the cytotoxicity was evaluated on retina pigmented epithelium (RPE) cells cultivated following the ISO standard (tests for in vitro cytotoxicity), and the hydrogel was found to be non-toxic. In a preliminary animal study, the oxi-HA/ADH hydrogel was injected into the vitreous cavity of rabbit eyes. The evaluations of slit-lamp observation, intraocular pressure, cornea thickness and histological examination showed no significant abnormal biological reactions for 3 weeks. This study suggests that the injectable oxi-HA/ADH hydrogel should be a potential vitreous substitute.

Injectable hydrogel allows irregular surgical defects to be completely filled, lessens the risk of implant migration, and minimizes surgical defects due to the solution-gel state transformation. Here, we first propose a method for preparing oxidized hyaluronic acid/adipic acid dihydrazide (oxi-HA/ADH) injectable hydrogel by chemical cross-linking under physiological conditions. Fourier transform infrared spectrometry and trinitrobenzene sulfonate assay were used to confirm the oxidation of hyaluronic acid. Rheological properties were measured to evaluate the working ability of the hydrogel for further clinical application. The oxi-HA/ADH in situ forming hydrogel can transform from liquid form into a gel-like matrix within 3-8 min, depending on the operational temperature. Furthermore, hydrogel degradation and cell assessment is also a concern for clinical application. Injectable oxi-HA/ADH8 hydrogel can maintain its gel-like state for at least 5 weeks with a degradation percentage of 40%. Importantly, oxi-HA/ADH8 hydrogel can assist in nucleus pulposus cell synthesis of type II collagen and aggrecan mRNA gene expression according to the results of real-time PCR analysis, and shows good biocompatibility based on cell viability and cytotoxicity assays. Based on the results of the current study, oxi-HA/ADH hydrogel may possess several advantages for future application in nucleus pulposus regeneration.

The gelatin-tricalcium phosphate membranes were cross-linking with low concentration glutaraldehyde solution (GTG). This material has good mechanical property, biocompatibility, and is feasible for surgical manipulation. For axonal regeneration, nerve growth factors (NGF) were immobilized onto the composite (GTG) with carbodiimide. The purpose of this study was to evaluate the release characteristics and bioactivity of NGF after covalent immobilization onto the GTG membranes (GEN). NGF immobilized onto and released from the composite was quantified using ELISA method. PC 12 cells were cultured on the GTG and GEN composites. Cell survival, cytotoxicity, and cellular activity were evaluated by total protein content, LDH activity, and MTT assay respectively. Neurite outgrowth assay was used to evaluate the biological activity of NGF released from GEN composite. From ELISA measurement, the releasing curve for NGF showing two distinctive parts with different slopes indicated that NGF were released from the composite in diffusion-controlled mechanism and degradation-controlled mechanism respectively. While culturing with PC 12 cells, LDH leakage results implied that whether GTG composite cross-linked with NGF or not showed little cytotoxicity. The total protein content and cellular activity of PC 12 cells were lower on GTG and GEN membranes than control group. However, 56%±3.98 of PC 12 cells showed significant neurite outgrowth on GEN membranes which was statistically higher than GTG without NGF immobilization. In addition, sustained release of bioactive NGF for two months had been demonstrated by neurite outgrowth assay. From these experiments, it can be concluded that the technique used in the present study is capable of immobilizing NGF onto GTG membranes covalently and remaining the bioactivity of NGF. Therefore, GEN composite can be materials for sustained release of bioactive NGF and a candidate for future therapeutic application in nerve repair.

The thermal and degradable properties of carbodiimide (EDC) or glutaraldehyde (GTA) cross-linked gelatin membranes have been investigated in order to evaluate the effects of different concentrations of two kinds of cross-linking reagent on the stability of membranes. In the thermogram recorded from a gelatin membrane cross-linked with EDC solution, the endothermic peak of 0.8% EDC cross-linking gelatin was centered at about 61°C that was higher than other samples treated with EDC solutions. Denaturation temperature (Td) of gelatin samples increased on increasing EDC concentration (0.2% to 0.8%), in agreement with the simultaneous increased of the extent of cross-linking. But increasing GTA concentration from 0.05% to 0.6%, the Td values of gelatin samples were decreased from 66.2°C to 56.3°C . In addition, two endothermic peaks were observed in 0.4% and 0.6% GTA cross-linking groups because of the GTA concentration was too high to complete cross-linking reaction. Therefore, partial of gelatin membrane was cross-linked completely but others were not. In the thermogravimetric analysis, the proportion of cracking endothermic peak of 0.6% GTA cross-linking gelatin (g15G0.6) was higher than the peak of 0.6% EDC cross-linking gelatin (g15C0.6). Therefore, g15G0.6 cracked to smaller molecules has to absorb more calorific capacity than g15C0.6. The increase in the strength of covalent binding on increasing the proportion of endothermic peak was evident. The results of degradable rate were in agreement with the lower concentration of cross-linked reagent the faster degraded rate of gelatin membrane. The MTT assay showed that 15% gelatin cross-linked by 0.8% EDC has the least cytotoxicity, and cell activity of this group was similar to control group (blank dish). As the concentration of GTA in gelatin membranes was down to 0.05% or 0.1% the cell viability was returned to approach the value of control group.

Blended membranes of chitosan and poly(vinyl alcohol) (PVA) in various ratios were prepared and treated with formaldehyde. Electron spectroscopy for chemical analysis of the membrane showed that the NH 2 group in chitosan changed into NC group after membrane treatment with formaldehyde. From the spectral change of FTIR, the hydroxyl groups disappeared and an acetal ring and ether linkage were formed for the reaction between the hydroxyl groups of PVA and formaldehyde. The results of differential scanning calorimetry (DSC), dynamic mechanical analysis (DMA) and thermogravimetric analysis (TGA) showed that (1) the crystalline area in PVA decreased after treatment with formaldehyde, (2) chitosan and PVA are not very compatible in the chitosan/PVA blended hydrogel membrane and (3) the thermostability of the membranes is enhanced by formaldehyde as crosslink agent. The effect of chitosan content on the water content and water vapor transmission rates on the blended hydrogel membrane were determined. It was found that the water content and water vapor transmission rates on the blended hydrogel membrane increased with increasing chitosan content. In antibacterial assessment, it appears that the antibacterial activity of all chitosan/PVA blended hydrogel membranes is similar. The viable cell number of aurococcus on the various chitosan/PVA blended hydrogel membranes was about (2.5±0.5)×10 7 cells/ml. The permeation of creatinine, 5-FU, uric acid and vitamin B 12 through the chitosan/PVA blended hydrogel membranes were conducted. The linear relationship between permeability of creatinine, 5-FU and vitamin B 12 molecules and chitosan content in the chitosan/PVA blended hydrogel membranes were found. As the electrostatic attraction between the uric acid and chitosan in the membrane, the permeation of uric acid through the chitosan/PVA blended hydrogel membranes is higher for the membranes with chitosan content higher than 80% in the blended hydrogel membranes.

Injectable hydrogel is one of the great interests for tissue engineering and cell encapsulation. In the study, the gelatin molecules were added to the thermosensitive chitosan/beta-glycerol phosphate (C/GP) disodium salt hydrogels to form chitosan/gelatin/beta-glycerol phosphate (C/G/GP) disodium salt hydrogels which were applied as a cell carrier for nucleus pulposus (NP) regeneration. The gelation temperature, gelation time, and gel strength of the C/G/GP hydrogels were analyzed by the rheometer. NP cells were then harvested from the intervertebral discs of the adult New Zealand white rabbits and cultured in monolayer or in C/G/GP hydrogel, respectively. The cell viability, material-mediated cytotoxicity, cell proliferation, production of sulfated glycosaminoglycans, anabolic/catabolic gene expressions, and extracellular matrix-related gene expressions of the NP cells were demonstrated. The results show that the sol/gel transition temperature of the C/G/GP hydrogel was in the range of 31.1-33.8 degrees C at neutral pH value, the gelation time was shortened, and the gel strength also improved at body temperature when compared with the C/GP hydrogel. Among those, C/GP with 1% gelatin addition showed the most promising gelation time and gel strength and were utilized in the later experiments. From the results of cell activity, cytotoxicity, and cell proliferation assays, NP cells cultured in C/G/GP hydrogel had normal cell viability and cell proliferation that indicated the hydrogel was noncytotoxicity. The amounts of sulfated glycosaminoglycans of NP cells cultured in C/G/GP hydrogels were significantly higher than monolayer cultured. Considering the extracellular matrix-related gene expression, type II collagen and aggrecan of NP cells cultured in the hydrogels greatly increased than those in monolayer culture. On the contrary, the unfavorable gene expression, such as that of type I collagen, was decreased significantly. The results reveal that gelatin added into C/GP hydrogel significantly shortened the gelation time and improved the gel strength without influencing the biocompatibility. NP cells cultured in the C/G/GP hydrogel also displayed better gene expressions when compared with the monolayer culture. This study indicates that using chitosan/gelatin hydrogel for NP cell culture is feasible and may apply in minimal invasive intervertebral disc surgery in the future.

An organic–inorganic hybrid material, epoxy–SiO2, was prepared by incorporating epoxy structure units covalently into a SiO2 glass network via the sol–gel approach. The precursor was obtained by the reaction of diglycidyl ether of bisphenol A (DGEBA) with 3-aminopropyl trimethoxysilane (APTS). The precursor was then hydrolyzed and co-condensated with tetraethyl orthosilicate (TEOS) in tetrahydrofuran (THF) at room temperature to yield epoxy–SiO2 hybrid sol–gel material having a 50 wt % SiO2 content. Thermal properties of the hybrid material were characterized by differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). The hybrid sol–gel material epoxy–SiO2 was the solid, powder component of bone cement. The liquid component contains bis-phenol-A glycidyl methacrylate (Bis-GMA), triethyleneglycol dimethacrylate (TEGDMA), and methyl methacrylate (MMA) with 25, 55, and 20 vol %, respectively. We discuss the comparison between the new epoxy–SiO2 bone cement and the commercial Simplex® P bone cement. Mechanical properties such as Young's modulus, compressive strength, hardness, and impact strength of the new epoxy–SiO2 bone cement exceeded those of SimplexR P bone cement. The tensile and bending strengths of the new epoxy–SiO2 bone cement were approximately the same as those of Simplex® P bone cement. In order to evaluate the biocompatibility of the new bone cement, an MTT test and optical microscopy were conducted in cell culture. Results indicated that the new epoxy–SiO2 bone cement exhibits very low cytotoxicity compared with Simplex® P bone cement. © 2002 Wiley Periodicals, Inc. J Biomed Mater Res 64A: 138–146, 2003