Cultivation of auricular chondrocytes in poly(ethylene glycol)/poly(ε-caprolactone) hydrogel for tracheal cartilage tissue engineering in a rabbit model.

Tissue engineering has the potential to overcome the limitations of tracheal reconstruction. To tissue-engineer a tracheal cartilage, auricular chondrocytes were encapsulated in a photocurable poly(ethylene glycol)/poly(ε-caprolactone) (PEG/PCL) hydrogel. Chondrogenic genes, including Sox9, Acan and Col2a1, were up-regulated in auricular chondrocytes after 2 weeks of in vitro cultivation in the PEG/PCL hydrogel. Co-cultivation of 70 % auricular chondrocytes and 30 % bone marrow mesenchymal stem cells (BMSCs) accelerated the chondrogenic genes' expression in the PEG/PCL hydrogel. Cartilaginous matrix markers, including proteoglycans and collagen type II, were detected in the chondrocytes-encapsulated PEG/PCL hydrogel after 4 weeks of in vitro cultivation. The higher expression level of cartilaginous matrix markers was observed in the PEG/PCL hydrogel with co-cultivation of 70 % chondrocytes and 30 % BMSCs. After 4 weeks of ectopic cultivation in rabbits, the cylindrical PEG/PCL structure was sustained with the use of a luminal silicon stent. However, without the stent, the construct collapsed under a compression force. No fibrosis or vessel ingrowth were found in the PEG/PCL hydrogel after 4 weeks of ectopic cultivation, whereas the auricular chondrocytes showed proteoglycans' accumulation and collagen type II production. Rabbit auricular chondrocytes could survive and retain chondrogenic ability in the PEG/PCL hydrogel under both in vitro and in vivo conditions. While the PEG/PCL hydrogel did not show sufficient mechanical properties for supporting the cylindrical shape of the construct, the high chondrogenesis level of chondrocytes in the PEG/PCL hydrogel displayed the potential of this material for tracheal tissue engineering.

Evaluation of saliva and plasma cytokine biomarkers in patients with oral squamous cell carcinoma.

The aim of this study was to investigate potential biomarkers in human saliva and plasma to aid in the early diagnosis of oral squamous cell carcinoma (OSCC). Saliva and plasma samples obtained from OSCC patients (n=41) and non-oral cancer patients (n=24) were analyzed by Luminex Bead-based Multiplex Assay. Data were analyzed using the non-parametric Mann-Whitney U-test, Kruskal-Wallis test, and receiver operating characteristics curve (ROC) to evaluate the predictive power of 14 biomarkers individually for OSCC diagnosis. The plasma level of IP-10 in early OSCC differed significantly from that in controls. Among the salivary biomarkers, IL-1ß, IL-6, IL-8, MIP-1ß, eotaxin and IFN-γ and TNF-α showed significant differences between OSCC patients and controls. With respect to carcinogenesis, significant differences in plasma levels of eotaxin, G-CSF, and IL-6 were found between OSCC stages III/IV and OSCC stages I/II. The area under the curve (AUC) for OSCC vs. control was greater than 0.7 for plasma IP-10 and saliva IL-1ß, IL-6, IL-8, and TNF-α. The study findings indicate that salivary biomarkers may serve a useful role as a complementary adjunct for the early detection of oral OSCC. With regard to the evaluation of tumour progression, plasma eotaxin, G-CSF, and IL-6 may help in the detection of advanced OSCC. However, the correlation between saliva and plasma biomarkers in OSCC was weak.

Enzymatic synthesis of L-cysteine.

O-acetylserine sulfhydrase in the form of a crude extract from Salmonella typhimurium LT2 was used for the production of L-cysteine from L-O-acetylserine and sodium hydrosulfide at pH 7.0 and 25 degrees C. The two substrates have quite different pH stability relationships. O-Acetylserine readily rearranges to N-acetylserine and the rate of this O --> N acyl transfer reaction increases at higher pH, temperature, and concentration of O-acetylserine. On the other hand, sodium hydrosulfide is more soluble at a higher pH. A stirred-tank bioreactor with a continuous substrate feed was employed to overcome this problem. The O-acetylserine feed was stored at its saturation level (2.05M) at pH 5.0, and the sodium hydrosulfide feed was dissolved at 2.05-2.3M without pH adjustment (pH >or= 11.5). Both substrates were simultaneously introduced into the bioreactor. The performance of the bioreactor was optimized by employing an automatic feedback control system to regulate the concentration of O-acetylserine in the bioreactor. This feedback control system was based on the fact that as the bioconversion proceeds, protons are produced along with cysteine. A pH controller thus detected the decrease in pH and activated the substrate pumps. After mixing in the bioreactor, these two substrate solutions behaved as a base due to the high alkalinity of sodium hydrosulfide. Thus, substrate infusion started when the pH was lower than the set point, i.e., the reaction pH, and stopped when the pH was raised higher than the set point. The amount of substrate introduced was determined by the alkalinity of the mixture of the two substrates, which in turn was controlled by the concentration of sodium hydrosulfide. After optimizing the sodium hydrosulfide concentration and the substrate feed rate, the bioconversion gave a productivity of 3.6 g L-cysteine/h/g dry cell weight S. typhimurium, an L-cysteine titer of 83 g/L and a molar yield based on O-acetylserine of 94%.

Enzymatic production of L-serine with a feedback control system for formaldehyde addition.

Serine hydroxymethyltransferase (SHMT) in the form of crude extract from a recombinant strain of Klebsiella aerogenes was used for the production of L-serine from glycine and formaldehyde (HCHO). A stirred tank bio-reactor with a continuous feed of HCHO (37%) was employed. Since the performance of the serine bioreactor was heavily dependent on how HCHO was fed, an automatic feedback control system was developed for HCHO delivery utilizing the phenomenon of formol titration. This control procedure was based on the following circumstance: as a bioconversion proceeded, if the rate of HCHO feed was balanced by the rate of serine synthesis so that HCHO concentration was maintained near zero, then there was no pH change in the bioreactor. Once the rate of HCHO addition exceeded that of serine synthesis, the HCHO concentration built up and the excess HCHO reacted with the amino group of an amino acid (e.g. glycine or serine) to produce a Schiff base and a proton which lowered the pH. A pH controller detected and relayed this pH change to the on-off switch of the HCHO feed pump. Thus, HCHO infusion stopped when the pH was lower than the set point, which was the initial pH of the reaction. With this control system, the maximum concentration of HCHO that was reached in the bioreactor was only 1mM-3.3mM depending on the pH and amino acid composition in the bioreactor. Moreover, a decrease in pH also signaled the use of a slower feed rate at which HCHO was to be, delivered once the pH resumed its initial value after excess HCHO was consumed by the reaction. Employing this control system, we have optimized the performance of the serine bioreactor to give a serine titer of 450 g/L with an 88% molar conversion of glycine at a volumetric serine productivity of 8.9 g/L/h.

Use of immobilized carboxypeptidase Y (I-CPY) as a catalyst for deblocking in peptide synthesis.

CPY is a metal-free carboxypeptidase from yeast with broad specificity [1]. In addition to exopeptidase activity at acid pH, the enzyme is an effective esterase at alkaline pH. N-alpha-acetyl-L-tyrosine ethyl ester is hydrolyzed faster by CPY than by chymotrypsin. These observations suggested that the immobilized form of the enzyme would be of value in removing ester groups from the C-terminal ends of peptides. In this report we describe sequential synthesis using I-CPY and alpha-COOH deblocking of peptides made by conventional methods.