Development of an efficient vitrification method for chondrocyte sheets for clinical application.

INTRODUCTION: Regenerative therapy using chondrocyte sheets is effective for osteoarthritis. The clinical application of chondrocyte sheet therapy is expected to be further advanced by the use of a feasible cryopreservation technique. Previously, we developed a chondrocyte sheet vitrification method; however, it was too complex to be used for routine clinical application. Here, we aimed to develop a prototype method for vitrifying chondrocyte sheets for clinical practice. METHODS: We developed a "circulating vitrification bag" as a container to process cell sheets for vitrification in an efficient and sanitary fashion. Moreover, we invented the "vitrification storage box", which is useful for the vitrification of cell sheets, long-term preservation, and transportation. These devices were used to vitrify rabbit chondrocyte sheets, which were then assessed for their structural characteristics and the viability of the component cells after rewarming. RESULTS: In all cell sheet samples (n = 7) vitrified by the circulating vitrification bag method, the integrity of the sheet structure was maintained, and the cell survival rate was similar to that of non-vitrified samples (91.0 ± 2.9% vs. 90.0 ± 3.0%). Proteoglycan and type II collagen, which are major components of cartilage, were densely and evenly distributed throughout the chondrocyte sheet subjected to vitrification similarly to that observed in the non-vitrified sheet. After long-term storage using the vitrification storage box, the cell sheets maintained normal structure and cell viability (survival rate: 81.2 ± 1.0% vs. 84.3 ± 1.8%) compared to the non-vitrified sheet. CONCLUSION: Our results indicate that the circulating vitrification bag method is an effective approach for realizing the clinical application of vitrified chondrocyte sheets. The vitrification storage box is also useful for the long-term preservation of vitrified cell sheets, further enhancing the feasibility of the clinical application of cryopreserved chondrocyte sheets.

Distributions of endocrine cell clusters during porcine pancreatic development.

BACKGROUND: Pancreatic islet xenotransplantation is a potential treatment for diabetes mellitus, and porcine pancreas may provide a readily available source of islets. Islets in juvenile pigs are smaller than those in young adult pigs, but the insulin content is very similar. In addition, as juvenile pigs are more easily reared in uncontaminated conditions, many researchers have conducted studies using pancreatic islets from juvenile pigs. We aimed to analyze the distributions of endocrine cell clusters by comprehensively evaluating juvenile porcine pancreatic development and to propose an appropriate age at which islets could be isolated from the juvenile porcine pancreas. METHODS: Splenic (SL) and duodenal lobe (DL) samples were collected from the pancreases of pigs aged 0-180 days (n = 3/day after birth). The chronological changes in endocrine cell clustering were analyzed in relation to morphological changes, cell characterization, numbers, islet areas, and gene expression. RESULTS: In juvenile pigs aged 0-21 days, the pancreas contained numerous endocrine cells, and compact islets appeared from 21 days of age. Well-defined small islets were seen at 28 days of age, and the clusters were denser in the SL than in the DL. At 35 days of age, the islets were morphologically similar to those observed at 180 days of age, and the greater number of islets was similar to that seen at 90 days of age. The differences in the islets' cytoarchitecture between the lobes were negligible. The expression of ß-cell-related genes was higher in the juvenile pancreas than in the adult pancreas, and the expression of neurogenin-3 decreased dramatically over time. CONCLUSIONS: These findings may have implications for attempts to refine the most appropriate age for islet isolation from porcine donors. Focusing on porcine pancreatic islets isolated at around 35 days after birth may offer benefits regarding their xenotransplantation potential.

Effects of a bolus injection of 5-fluorouracil on dihydropyrimidine dehydrogenase activity in rats receiving continuous infusion of 5-fluorouracil.

PURPOSE: The options for improving the chemotherapeutic regimen consisting of bolus plus infusion of 5-fluorouracil (5-FU) include omitting the 5-FU bolus injection. We examined the effects of a 5-FU bolus injection on the activity of dihydropyrimidine dehydrogenase (DPD), which is the first and rate-limiting enzyme of 5-FU catabolism, in rats. METHODS: The rats were divided into three groups, and then continuous infusion (50 mg/m(2)/h) for 4 h was started with a bolus injection of saline, 20 mg/kg 5-FU, or 60 mg/kg 5-FU. Plasma 5-FU, uracil (Ura), dihydrouracil (UH2) levels, and hepatic DPD activity were determined after administration of 5-FU. RESULTS: The half-life after the end of the infusion (t 1/2, 4-8 h) of 5-FU in the rats given the bolus injection was significantly longer than in those that had been given saline, and it increased with increasing 5-FU bolus injection dosage (r = 0.801, p < 0.01). The plasma UH2/Ura ratio, an indirect biomarker of hepatic DPD activity, tended to be lower in the rats that had received a 5-FU bolus injection than in those that had not, and it remained low after infusion ended. The hepatic DPD activity in rats that had received a 5-FU bolus injection was significantly lower than in those that had not. Negative correlation was observed between DPD activity and bolus injection dosage (r = -0.691, p < 0.05). CONCLUSIONS: A bolus injection suppresses hepatic DPD activity and its effects are dependent on dosage, resulting in slower elimination of 5-FU from the blood and contributing to long-term systemic exposure to 5-FU.

Application of dissolving microneedles to glucose monitoring through dermal interstitial fluid.

Dissolving microneedles (DMs) were applied to glucose monitoring in the dermal interstitial fluid (ISF) of rats and their potential as an alternative blood glucose monitoring device was evaluated. Sodium chondroitin sulfate was used to prepare DM array chips, which consisted of 300 DMs/cm(2). The mean length of the DMs was 475±18 µm and the mean diameter of the basement was 278±8 µm. After DMs were inserted into the skin of the hair-removed rat abdomen, a wet unwoven cloth containing 10-30 µL of water was placed on the skin and ISF was extracted. By increasing the absorbed amount of water on the unwoven cloth from 10 to 30 µL, the extracted amount of glucose increased from 1.66±0.35 µg to 2.75±0.61 µg. Increasing the adhesion time of the wet unwoven cloth to the skin from 0.1 to 5.0 min, increased the amount of ISF glucose from 1.99±0.13 µg to 5.04±0.38 µg. The relation between the amount of glucose in ISF and blood glucose concentrations was examined. With increase in the adhesion time, the coefficient of determination, r(2), increased from 0.501 to 0.750. The number of DMs also affected the relationship and values of the coefficient of determinations, r(2) were: 0.340 (25 DMs), 0.758 (50 DMs), 0.763 (100 DMs), 0.774 (200 DMs), and 0.762 (300 DMs). These results indicate the usefulness of DMs as an alternative blood glucose monitoring device.

Is estrogen effective for full-thickness cutaneous wound healing in young male mice?

The aim of this study is to show the effects of estrogen upon its topical application on the wound healing process in young male mice. Fifty-six male mice aged 7 weeks old were divided into 4 groups: sham operation, castration, estrogen treatment after sham operation, and estrogen treatment after castration. Wound healing was observed daily until day 14 after wounding. Specimens were harvested on days 3, 7, 10, and 14, and stained to evaluate reepithelialization, inflammation, contraction, and collagenaccumulation. Wound healing periods of all groups were almost the same, although the concentration of serum estrogen in the estrogen-applied mice was very high, and that in the nonapplied groups was low. The numbers of macrophages in the castrated, estrogen-treated after sham operation, and estrogen-treated after castration groups were significantly decreased compared with that in the sham group in the inflammatory phase; however, the ratio of wound area in these groups did not decrease, and other histological data did not reveal any effects of estrogen. These results indicate that estrogen may show limited effectiveness for full-thickness cutaneous wound healing in young male mice, and decreased inflammation may not always be associated with decreased wound area. .

Molecular and catalytic properties of monoacetylphloroglucinol acetyltransferase from Pseudomonas sp. YGJ3.

Monoacetylphloroglucinol (MAPG) acetyltransferase, catalyzing the conversion of MAPG to 2,4-diacetylphloroglucinol (DAPG), was purified from Pseudomonas sp. YGJ3 grown without Cl(-). Cl(-) and pyoluteorin repressed expression of the enzyme. SDS-polyacrylamide gel electrophoresis showed that the purified enzyme (M(r)=330 kDa) was composed of three subunits of 17, 38, and 43 kDa, and protein sequencing identified these as PhlB, PhlA, and PhlC respectively. The enzyme catalyzed the reversible disproportionation of 2 moles of MAPG to phloroglucinol (PG) and DAPG. The equilibrium constant K (=[DAPG][PG]/[MAPG](2)) was estimated to be about 1.0 at 25 °C. A KpnI 20-kb DNA fragment was cloned from the genomic DNA of strain YGJ3, and a 12,598-bp long DNA region containing the phl gene cluster phlACBDEFGHI was sequenced. PCR cloning and expression of the phl genes in Escherichia coli confirmed that expression of phlACB genes produced MAPG ATase.

Pharmacological profile of celecoxib, a specific cyclooxygenase-2 inhibitor.

The pharmacological profile of celecoxib (CAS 169590-42-5, SC-58635), a specific cyclooxygenase-2 (COX-2) inhibitor, was investigated. Celecoxib inhibited COX-2-mediated prostaglandin E2 (PGE2) production in human dermal fibroblasts (IC50 = 91 nmol/l), whereas it was a weak inhibitor of COX-1-mediated PGE2 production in human lymphoma cells (IC50 = 2800 nmol/l). In in vivo studies, the effects of celecoxib were compared with those of nonsteroidal anti-inflammatory drugs (NSAIDs) in acute rat models of hyperalgesia and pyrexia. Celecoxib abrogated carrageenan-induced hyperalgesia in the hind paw accompanied by a decrease in PGE2 content in paw exudates and cerebrospinal fluid in a dose-related manner, with an ED30 = 0.81 mg/kg. Its analgesic potency was comparable to those of NSAIDs. In lipopolysaccharide-induced pyrexia, the anti-pyretic potency of celecoxib was equal to that of NSAIDs. On the other hand, in a gastric toxicity study in rats, single oral administration of celecoxib had no effect on gastric mucosa or mucosal PGE2 content at doses up to 200 mg/kg. Additionally, celecoxib did not inhibit thromboxane B2 production of calcium ionophore-stimulated peripheral blood of rats or arachidonic acid-induced aggregation of human platelets. These findings suggest that celecoxib might be a safe and effective alternative to NSAIDs for clinical use.