Chondroitin Sulfate Enhances Proliferation and Migration via Inducing ß-Catenin and Intracellular ROS as Well as Suppressing Metalloproteinases through Akt/NF-κB Pathway Inhibition in Human Chondrocytes.

BACKGROUND: Chondroitin sulfate (CS) is found in humans' cartilage, bone, cornea, skin, and arterial wall. It consists of the foundation substance in the extracellular matrix (ECM) of connective tissue. The oral supplement form of CS is clinically used in treating osteoarthritis (OA). METHODS: Cell migration was observed by the transwell assay. The EMT, Akt/IKK/IκB pathways, TIMPs, collagen and MMPs in cell lysate were determined by Western blotting. The expression of MMP activity was determined by gelatin zymography. The production of reactive oxygen species (ROS) was determined by using a fluorescence spectrophotometer. RESULTS: In the current report, we demonstrated that CS can increase the cell proliferation and migration of chon-001 chondrocytes. Treatment with CS induced the epithelial-mesenchymal transition and increased the expression of type II collagen and TIMP-1/TIMP2 and inhibited the expressions and activities of metalloproteinase-9 (MMP-9) and metalloproteinase-2 (MMP-2). The phosphorylation of Akt, IκB kinase (IKK), IκB and p65 was decreased by CS. CS treatment resulted in ß-catenin production and XAV939, a ß-catenin inhibitor, and inhibited the cell proliferation by CS treatment. In addition, also significantly induced intracellular ROS generation. Treatment with antioxidant propyl gallate blocked cell migration induced by CS. CONCLUSION: We demonstrated that CS induced cell proliferation and migration of chondrocytes by inducing ß-catenin and enhancing ROS production. Moreover, our studies demonstrated that CS can increase the activity of chondrocytes and help patients with osteoarthritis to restore cartilage function.

Risk of osteoporosis and fracture in victims with burn injury.

Osteoporosis is a well-known bone disorder affecting people worldwide. Patients with osteoporosis have an increased risk of bone fracture. This study provides new information on the risk of developing osteoporosis post burn injury and the risk of fracture among those with osteoporosis developed. INTRODUCTION: The relationship between burn injury and hip fracture risk is unclear. Population-based evaluation on relationships between burn injury and osteoporosis development and subsequent fractures is limited. We conducted a retrospective cohort study as the investigation. METHODS: From the insurance data of Taiwan, we established a cohort of 43,532 patients with a burn injury in 2000-2012 and a comparison cohort of 174,124 individuals without such an injury, frequency matched by sex, age, and diagnosis date. Both cohorts were followed up to the end of 2013 to evaluate the occurrence of osteoporosis and hip fracture. RESULTS: The incidence of osteoporosis was greater in the burn cohort than in the comparison cohort (6.40 vs. 4.75 per 1,000 person-years) with an adjusted IRR of 1.35 (95% confidence interval = 1.32-1.39). The incidence rates in both cohorts were greater in women than in men, increased with age, income, and Charlson comorbidity index. Patients with burns involving 20%-49% of total body surface area and with burns confined to the lower/upper limbs had the greatest incidence rates, 8.32 and 8.58 per 1,000 person-years, respectively. Osteoporosis incidence increased further to 22.7 per 1,000 person-years for burn victims with comorbid diabetes. The risk of fracture was over five-fold greater for burn victims with osteoporosis developed than for comparisons without osteoporosis. CONCLUSION: Patients who have a burn injury deserve prevention intervention to reduce the risk of osteoporosis and fracture.

Hip fracture risk in patients with burn injury: a retrospective cohort study in Taiwan.

This work aimed to evaluate the hip fracture risk for patients with burn injury. A total of 16,430 patients with burn injury had an adjusted hazard ratio of 1.54 to encounter a hip fracture, compared with controls without the injury. These results encourage future studies focusing on mechanisms leading to fracture associated with burn injury. INTRODUCTION: The relationship between burn injury and hip fracture risk is unclear. We conducted a retrospective cohort study to investigate this relationship. METHODS: From insurance data of Taiwan, we identified a cohort with 16,430 burn patients in 2000-2010 and a comparison cohort of 65,716 persons without the history of burn, frequency matched by sex, age, and diagnosis date. Both cohorts were followed up to the end of 2011 to evaluate the risk of hip fracture. RESULTS: Patients with burn injury were 1.62-fold more likely than comparisons to encounter a hip fracture (6.95 vs. 4.28 per 1000 person-years), with an adjusted hazard ratio (aHR) of 1.54 (95% confidence interval (CI) = 1.40-1.68). The fracture incidence increased with age and is slightly greater for women than for men in both cohorts. The fracture risk was greater for patients with burn in the eyes, face, and head with an incidence of 7.14 per 1000 person-years, or an aHR of 2.09 (95% CI = 1.53, 2.86). Diabetes and osteoporosis were also associated with an increased hip fracture risk. CONCLUSION: Burn injury is associated with an increased risk of hip fracture. Diabetes and osteoporosis are associated with an enhanced risk.

Nonconservative higher-order hydrodynamic modulation instability.

The modulation instability (MI) is a universal mechanism that is responsible for the disintegration of weakly nonlinear narrow-banded wave fields and the emergence of localized extreme events in dispersive media. The instability dynamics is naturally triggered, when unstable energy sidebands located around the main energy peak are excited and then follow an exponential growth law. As a consequence of four wave mixing effect, these primary sidebands generate an infinite number of additional sidebands, forming a triangular sideband cascade. After saturation, it is expected that the system experiences a return to initial conditions followed by a spectral recurrence dynamics. Much complex nonlinear wave field motion is expected, when the secondary or successive sideband pair that is created is also located in the finite instability gain range around the main carrier frequency peak. This latter process is referred to as higher-order MI. We report a numerical and experimental study that confirms observation of higher-order MI dynamics in water waves. Furthermore, we show that the presence of weak dissipation may counterintuitively enhance wave focusing in the second recurrent cycle of wave amplification. The interdisciplinary weakly nonlinear approach in addressing the evolution of unstable nonlinear waves dynamics may find significant resonance in other nonlinear dispersive media in physics, such as optics, solids, superfluids, and plasma.

Enhancing Survival of Human Hepatocytes by Neonatal Thymectomy and Partial Hepatectomy in Micro-miniature Pigs.

BACKGROUND: With the goal of in vivo cultivation of human hepatocytes that have not been sufficient in full differentiation in vitro, the advantage of neonatal thymectomy was verified on expansion of xenogeneic human hepatocyte in the micro-miniature pig (MMP). METHODS: The thymus was excised immediately after the birth of the MMPs via cesarean section. Newborns were fed by artificial feeding under specific pathogen-free conditions. The thymectomized and nonthymectomized littermates were transplanted with human hepatocytes via a portal vein with or without partial hepatectomy at the MMP adult stage. RESULTS: The growth of thymectomized MMPs and the sham operated littermates was not significantly different; the former weighed 1.98 ± 0.30 kg (average ± standard deviation, n = 4) and the latter weighed 2.28 ± 0.39 kg (n = 4) at 1 month of age, and 17.48 ± 1.92 kg and 16.75 ± 2.68 kg at 12 months of age. Blood thymosin α1 concentrations in the thymectomy group were significantly lower than in the control group (0.22 ± 0.05 ng/mL vs 0.46 ± 0.16 ng/mL; n = 4, 12 months old, P = .029). After human hepatocyte transplantation, human albumin levels were detectable on day 28 in the peripheral blood of the thymectomy plus hepatectomy group (14.3 ± 4.9 ng/mL [± range, n = 2]) but were not detectable even on day 21 in the control group. CONCLUSIONS: Neonatal thymectomy was successfully achieved in infantile MMPs born via cesarean section. These pigs were considered to be an ideal in vivo bioreactor for human hepatocytes.

Evaluation of nasal patency by visual analogue scale/nasal obstruction symptom evaluation questionnaires and anterior active rhinomanometry after septoplasty: a retrospective one-year follow-up cohort study.

OBJECTIVE: To assess the efficacy of septoplasty and the correlation between the subjective evaluations of a visual analogue scale (VAS) and the Nasal Obstruction Symptom Evaluation (NOSE) questionnaire and active anterior rhinomanometry of the nasal airway after septoplasty. DESIGN: A retrospective, individual cohort study. SETTING: Ear, Nose and Throat Department, Taipei City Hospital, Taipei, Taiwan. PARTICIPANTS: Fifty patients with chronic nasal obstruction were enrolled in the study. All 50 patients underwent septoplasty because of nasal septal deviation. Another 28 patients without nasal symptoms served as controls. MAIN OUTCOME MEASURES: VAS, NOSE and active anterior rhinomanometry were used to measure the sensation of nasal obstruction. All measurements were performed in both groups preoperatively and then repeated on three postoperative visits (3, 6 and 12 months). RESULTS: The mean VAS score, NOSE score and the nasal resistance in the narrow side of the nose in the study group showed reduced symptoms at 3, 6 and 12 months postoperatively compared with the respective preoperative measurements (P < 0.001, all). The VAS and NOSE scores did not significantly correlate with total nasal resistance preoperatively or postoperatively. The VAS and nasal resistance in the obstructed nasal cavity correlated significantly preoperatively (P < 0.05), but not postoperatively. CONCLUSIONS: The subjective and objective symptoms of nasal obstruction had improved 1 year after septoplasty. A significant correlation between VAS scores and nasal resistance in the narrow side of the nose was found before surgery. The subjective and objective measurements of nasal obstruction lacked significant correlation postoperatively.

Modulation Instability and Phase-Shifted Fermi-Pasta-Ulam Recurrence.

Instabilities are common phenomena frequently observed in nature, sometimes leading to unexpected catastrophes and disasters in seemingly normal conditions. One prominent form of instability in a distributed system is its response to a harmonic modulation. Such instability has special names in various branches of physics and is generally known as modulation instability (MI). The MI leads to a growth-decay cycle of unstable waves and is therefore related to Fermi-Pasta-Ulam (FPU) recurrence since breather solutions of the nonlinear Schrödinger equation (NLSE) are known to accurately describe growth and decay of modulationally unstable waves in conservative systems. Here, we report theoretical, numerical and experimental evidence of the effect of dissipation on FPU cycles in a super wave tank, namely their shift in a determined order. In showing that ideal NLSE breather solutions can describe such dissipative nonlinear dynamics, our results may impact the interpretation of a wide range of new physics scenarios.

Alveolar bone-loss area localization in periodontitis radiographs based on threshold segmentation with a hybrid feature fused of intensity and the H-value of fractional Brownian motion model.

BACKGROUND AND OBJECTIVE: Periodontitis involves progressive loss of alveolar bone around the teeth. Hence, automatic alveolar bone-loss (ABL) measurement in periapical radiographs can assist dentists in diagnosing such disease. In this paper, we propose an effective method for ABL area localization and denote it as ABLIfBm. METHOD: ABLIfBm is a threshold segmentation method that uses a hybrid feature fused of both intensity and texture measured by the H-value of fractional Brownian motion (fBm) model, where the H-value is the Hurst coefficient in the expectation function of a fBm curve (intensity change) and is directly related to the value of fractal dimension. Adopting leave-one-out cross validation training and testing mechanism, ABLIfBm trains weights for both features using Bayesian classifier and transforms the radiograph image into a feature image obtained from a weighted average of both features. Finally, by Otsu's thresholding, it segments the feature image into normal and bone-loss regions. RESULTS: Experimental results on 31 periodontitis radiograph images in terms of mean true positive fraction and false positive fraction are about 92.5% and 14.0%, respectively, where the ground truth is provided by a dentist. The results also demonstrate that ABLIfBm outperforms (a) the threshold segmentation method using either feature alone or a weighted average of the same two features but with weights trained differently; (b) a level set segmentation method presented earlier in literature; and (c) segmentation methods based on Bayesian, K-NN, or SVM classifier using the same two features. CONCLUSION: Our results suggest that the proposed method can effectively localize alveolar bone-loss areas in periodontitis radiograph images and hence would be useful for dentists in evaluating degree of bone-loss for periodontitis patients.

Increased risk of coronary heart disease in patients with hip fracture: a nationwide cohort study.

UNLABELLED: The study indicates that hip fracture is independently associated with increased risk of coronary heart disease. In addition, the highest risk of coronary heart disease following hip fracture appeared within the first year after hip fracture, indicating the need for multidisciplinary care for the patients. INTRODUCTION: Bone and vasculature are modulated through numerous common pathways. However, data on the risk of coronary heart disease (CHD) after hip fracture are scarce. Therefore, we investigated whether hip fracture increased the risk of CHD by conducting a large nationwide cohort study. METHODS: Using universal insurance claims data from 2000 to 2010, we identified a study cohort of 6013 participants newly diagnosed with hip fracture and a control cohort of 23,802 participants. Both cohorts were followed up to the end of 2011 to evaluate the risk of CHD. RESULTS: The overall incidence of CHD was 1.69-fold higher in the hip fracture cohort than it was in the control cohort (29.2 vs. 17.1 per 1000 person-years) with an adjusted hazard ratio of 1.51 (95 % confidence interval [CI] = 1.39-1.65). Sex-, age-, and comorbidity-specific analyses showed a higher relative risk of CHD for both women and men, all age groups, those with and without comorbidities, and patients with hip fracture compared with the control cohort. The highest risk of CHD was within the first year after hip fracture (adjusted HR = 1.72, 95 % CI = 1.45-2.04), and the risk remained high in the following years. CONCLUSION: Hip fracture was independently associated with a subsequent risk of CHD.

Social connections and happiness among the elder population of Taiwan.

OBJECTIVES: The purpose of this study was to examine the association between social connections and happiness among members of the elder population of Taiwan. METHODS: Longitudinal panel data collected in three waves from 1999 to 2007 that are selected from national samples of Taiwanese older people were used for the analysis (n = 4731 persons). Happiness was defined as a dichotomous variable. Social connection variables included living arrangements, contacts with children/grandchildren/parents/relatives/friends, telephone contacts, providing instrumental and informational support, receiving instrumental and emotional support, and social participation. We controlled for the variables demographics, physical and mental health, economic satisfaction, and lifestyle. A generalized linear model (GLM) was applied in the analysis. RESULTS: Happiness remained stable over time. Receiving more emotional support and participating in social events were related to happiness at the beginning, while the effect of social participation was offset over time. Living arrangements, telephone contacts, providing social support, and receiving instrumental support were not significant. CONCLUSION: The quality of social relationships experienced is possibly more important than the quantity of social interaction for older people, and having social relationships outside the informal social network may increase happiness.

Increased risk of stroke among hip fracture patients: a nationwide cohort study.

SUMMARY: Our study indicates that hip fracture is independently associated with increased risk of developing stroke. In addition, the risk of stroke following the incidence of hip fracture is more prominent in younger patients, men, those with cardiovascular comorbidities, and in patients using specific medication, such as diuretics and ABRs. INTRODUCTION: Hip fractures are associated with increased risk of major morbidity. However, few data are available on the risk of stroke after hip fracture. Therefore, we investigated whether hip fracture increases the risk of stroke in a large nationwide cohort study. METHODS: Using universal insurance claims data, we identified a study cohort comprising of 6013 newly diagnosed with hip fracture patients from 2000 to 2010 and a non-hip fracture cohort of 23,802 participants. Incidence and risk of stroke were estimated for both cohorts until the end of 2011. RESULTS: Stroke incidence was 1.69-fold (95% confidence interval [CI]=1.56-1.83) higher in the hip fracture cohort than in the comparison cohort with an adjusted hazard ratio (HR) of 1.54 (95% CI=1.42-1.67) for the hip fracture cohort. The hip fracture patients were at higher risk of developing ischemic stroke (HR=1.55, 95% CI=1.42-1.69) and hemorrhagic stroke (HR=1.55, 95% CI=1.16-1.89), respectively. At an incidence of 64.6 per 1000 person-years, the adjusted HR of stroke increases to 3.10 (95% CI=2.47-3.90) for patients with coexisting diabetes, hypertension, and heart failure compared with those without these three conditions. At an incidence of 60.4 per 1000 person-years, the adjusted HR of stroke increases to 2.92 (95% CI=2.43-3.51) for hip fracture patients prescribed with diuretics and angiotensin II receptor blockers (ARBs) compared with those without hip fracture or prescriptions for diuretics or ARBs. CONCLUSIONS: Hip fracture is independently associated with a subsequent risk of stroke.

Excitons and biexcitons in InGaN quantum dot like localization centers.

Indium segregation in a narrow InGaN single quantum well creates quantum dot (QD) like exciton localization centers. Cross-section transmission electron microscopy reveals varying shapes and lateral sizes in the range ∼1-5 nm of the QD-like features, while scanning near field optical microscopy demonstrates a highly inhomogeneous spatial distribution of optically active individual localization centers. Microphotoluminescence spectroscopy confirms the spectrally inhomogeneous distribution of localization centers, in which the exciton and the biexciton related emissions from single centers of varying geometry could be identified by means of excitation power dependencies. Interestingly, the biexciton binding energy (E(b)xx) was found to vary from center to center, between 3 to -22 meV, in correlation with the exciton emission energy. Negative binding energies are only justified by a three-dimensional quantum confinement, which confirms QD-like properties of the localization centers. The observed energy correlation is proposed to be understood as variations of the lateral extension of the confinement potential, which would yield smaller values of E(b)xx for reduced lateral extension and higher exciton emission energy. The proposed relation between lateral extension and E(b)xx is further supported by the exciton and the biexciton recombination lifetimes of a single QD, which suggest a lateral extension of merely ∼3 nm for a QD with strongly negative E(b)xx = -15.5 meV.

Increased cancer risk in patients with haemophilia A: a nationwide population-based 14-year study in Taiwan.

Haemostasis is associated with the development and dissemination of cancer. Whether cancer incidence is increased in haemophiliacs remains uncertain; thus, we aimed to further examine this issue. By using data from the National Health Insurance Research Database in Taiwan, we obtained a cohort of 683 patients with haemophilia A, and compared the incidence rate ratio (IRR) of cancer in this cohort with an age- and sex-matched control of 6830 patients. The log-rank test was used to compare Kaplan-Meier curve of the cumulative cancer incidence between two cohorts. Cox regressions were used to identify independent risk factors of cancer in the study patients. The cancer incidence of patients with haemophilia A was significantly higher compared to the control group (IRR 1.95, 95% CI 1.18-3.09, P = 0.008) during the 14-year follow-up period. The non-lymphoma and non-liver cancer incidence in the haemophilia A cohort remained higher than that of the matched control (P = 0.050 by the log-rank test). The multivariate Cox proportional hazards analysis indicated that age (per year, HR 1.09, 95% CI 1.06-1.12, P < 0.001) was the only significant risk factor for cancer development in haemophilia patients. Patients with haemophilia A had higher cancer incidence than the age- and sex-matched patients, especially for the elderly. With increasing life expectancy for haemophiliacs, physicians should be aware of their cancer development.

Greater risk of hip fracture in hemodialysis than in peritoneal dialysis.

UNLABELLED: Several differences may have existed between patients treated with peritoneal dialysis and hemodialysis because of the difference in dialysis modality. This nationwide population-based cohort study demonstrated that patients on hemodialysis had an increased risk of hip fracture compared to patients on peritoneal dialysis; the hazard ratio was 1.52. INTRODUCTION: Numerous debates on which dialysis modality is "superior" have taken place in recent decades. However, no large-scale study has ever mentioned about the relationship between dialysis modality and risk of hip fracture. METHODS: We identified 64,124 incident end-stage renal disease patients from the National Health Insurance Research Database in Taiwan between 1998 and 2008, including 59,457 (92.72%) hemodialysis (HD) and 4,667 (7.28%) peritoneal dialysis (PD) patients. After 8:1 propensity score matching, 31,554 patients, of whom 28,048 were HD and 3,506 were PD patients, were included in the study. We conducted the Cox proportional hazards model to examine the effects of dialysis modality and other variables on hip fracture risk. RESULTS: A total of 2,587 hip fractures were identified in 64,124 dialysis patients. The incidence rate of hip fracture was 13.60 per 1000 patient-years in the HD group and 6.25 in the PD group. Dialysis modality, sex, age, presence of cardiovascular disease, diabetes, medication with antiepileptic drugs, diuretics, steroids, and vitamin D had statistically significant associations with hip fracture. Patients on HD had an increased risk of hip fracture compared to patients on PD; the hazard ratio (HR) was 1.52 (95% CI: 1.09-2.12, P = 0.02). CONCLUSIONS: In this population-based cohort study, HD had a greater hip fracture risk compared to PD; the HR was 1.52. We should focus more on reducing the risk of hip fractures in hemodialysis patients.

Teeth segmentation of dental periapical radiographs based on local singularity analysis.

Teeth segmentation for periapical raidographs is one of the most critical tasks for effective periapical lesion or periodontitis detection, as both types of anomalies usually occur around tooth boundaries and dental radiographs are often subject to noise, low contrast, and uneven illumination. In this paper, we propose an effective scheme to segment each tooth in periapical radiographs. The method consists of four stages: image enhancement using adaptive power law transformation, local singularity analysis using Hölder exponent, tooth recognition using Otsu's thresholding and connected component analysis, and tooth delineation using snake boundary tracking and morphological operations. Experimental results of 28 periapical radiographs containing 106 teeth in total and 75 useful for dental examination demonstrate that 105 teeth are successfully isolated and segmented, and the overall mean segmentation accuracy of all 75 useful teeth in terms of (TP, FP) is (0.8959, 0.0093) with standard deviation (0.0737, 0.0096), respectively.

Relationship between Frailty and Cognitive Function among Older Adults in Taiwan.

BACKGROUND: The causal relationship between physical frailty and cognitive function is not yet completely confirmed. OBJECTIVES: This study aimed at exploring the reciprocal relationship of frailty and cognitive function among Taiwanese older adults. PARTICIPANTS AND SETTING: The study evaluated data from a five-wave survey conducted in Taiwanese older persons (n=3,155 respondents, 10,631 observations). MEASUREMENTS: Frailty was defined as low physical activity, exhaustion, poor appetite, poor grip strength, and difficulty in carrying 10-pound objects; individuals were defined as frail if they met three or more of the above criteria. Cognitive function was assessed using five items from the Short Portable Mental Status Questionnaire (SPMSQ) scale. Time-lagged and time-concurrent models were performed to examine the reciprocal relationship between cognitive function and frailty. Growth-curve hierarchical linear modeling was applied. RESULTS: Concurrent frailty was related to low cognitive function at the intercept and time slope and vice versa. Previous frailty also predicted later decline of cognitive function. Nevertheless, previous cognitive function was not found to have a significant relationship with subsequent frailty. Education, physical function and social support represented shared factors to both frailty and cognitive function. CONCLUSIONS: Frailty and cognitive function are correlated, and frailty may occur prior to cognitive impairment.

Bovine lactoferricin B induces apoptosis of human gastric cancer cell line AGS by inhibition of autophagy at a late stage.

Gastric cancer is one of the most common malignant cancers, with poor prognosis and high mortality rates worldwide. Therefore, development of an effective therapeutic method without side effects is an urgent need. It has been reported that cationic antimicrobial peptides can selectively bind to negatively charged prokaryotic and cancer cell membranes and exert cytotoxicity without causing severe drug resistance. In the current study, we prepared a series of peptide fragments derived from bovine lactoferrin and evaluated their anticancer potency toward the gastric cancer cell line AGS. Cell viability assay revealed that a 25-AA peptide fragment, lactoferricin B25 (LFcinB25), exhibited the most potent anticancer capability against AGS cells. Lactoferricin B25 selectively inhibited AGS cell growth in a dose-dependent manner, exhibiting a half-maximal inhibitory concentration (IC50) value of 64 µM. Flow cytometry showed a notable increment of the sub-G1 populations of the cell cycle, indicating the induction of apoptosis by LFcinB25. Western blot analysis further revealed that upon LFcinB25 treatment for 2 to 6h, apoptosis-related caspases-3, 7, 8, 9, and poly(ADP-ribose) polymerase (PARP) were cleaved and activated, whereas autophagy-related LC3-II and beclin-1 were concomitantly increased. Thus, both apoptosis and autophagy are involved in the early stage of LFcinB25-induced cell death of AGS cells. However, upon treatment with LFcinB25 for 12 to 24h, LC3-II began to decrease, whereas cleaved beclin-1 increased in a time-dependent manner, suggesting that consecutive activation of caspases cleaved beclin-1 to inhibit autophagy, thus enhancing apoptosis at the final stage. These findings provide support for future application of LFcinB25 as a potential therapeutic agent for gastric cancer.

Improvement of hepatocyte recovery from rat liver subjected to 1-hour warm ischemic injury by using citrate phosphate dextrose added to euro-collins perfusion solution.

Warm ischemia (WI)-related injury interferes with recovery of primary hepatocyte after collagenase digestion of surgically resected or non-heart-beating donor livers as human cell sources. We speculated that digestion is impaired due to reduced microcirculation, caused by microembolism after WI. We sought to improve hepatocyte recovery after WI using a rat model. Anesthetized 9-week-old male Sprague-Dawley rats underwent a midline abdominal incision to insert a 22-gauge cannula into the portal vein. WI was initiated by ligating both the cannula and the hepatic artery. We compared Euro-Collins (EC) perfusion solution with 2 anticoagulants-heparin or citrate phosphate dextrose (CPD)-versus ethylene glycol tetraacetic acid (EGTA) combined with Ca(2+)- and Mg(2+)-free Hank's solution (CM-free Hank's solution). Use of CM-free Hank's solution yielded only 0.75 ± 0.15 × 10(8) and 0.82 ± 0.20 × 10(8) cells at 30 and 60 minutes WI respectively. However, CPD, but not heparin, added to the EC solution produced the best cell recovery (CPD: 2.15 ± 0.38 × 10(8); heparin: 1.63 ± 0.31 × 10(8)). During macroscopic observation, CPD added to EC solution also demonstrated best blood flushing. CPD added to EC solution achieved greater hepatocyte recovery than CM-free Hank's solution by restoring microcirculation during flushing of blood from liver tissue subjected to WI.

Transgenic pig expressing the red fluorescent protein kusabira-orange as a novel tool for preclinical studies on hepatocyte transplantation.

INTRODUCTION: Research on hepatocyte transplantation as an alternative or supplementary treatment for liver transplantation is progressing. However, to advance to clinical trials, confidence in the technique must be established and its safety must be validated by conducting experiments using animals of comparable sizes to humans, such as pigs. We used transgenic pigs expressing red fluorescence protein for investigating the distribution and survival of transplanted cells. MATERIALS AND METHODS: Donor hepatocytes were isolated from transgenic Kusabira-Orange (KO)-expressing pigs (age, 41 days; weight, 10 kg) created by in vitro fertilization using sperm from a transgenic-cloned KO pig by Matsunari et al. and ova from a domestic pig. The hepatocyte transplant recipients were the nontransgenic, KO-negative littermates. In these recipient pigs, double lumen cannulae were inserted into the supramesenteric veins to access the hepatic portal region. KO-positive donor hepatocytes from the transgenic male pig were isolated using collagenase perfusion. Hepatocytes (1 × 10(9) cells) were transplanted through the cannula. For estimating allogeneic immunogenicity, full-thickness skin (3 × 3 cm) from the same donor was grafted orthotopically on the neck region of the recipients. Immunosuppressive treatment was not implemented. The recipient pigs were humanely killed at 7 and 39 days after transplantation, and the organs were harvested, including the lungs, heart, liver, pancreas, and kidneys. RESULTS: Strong red fluorescence was detected in both the parenchymal and nonparenchymal hepatocytes of the transgenic male donor pig by fluorescent microscopy. Transplanted cells were detected in the liver and lung of the recipient pigs at 7 days after perfusion. Hepatocytes remained in the liver and lung of recipients on day 39, with lower numbers than that on day 7. CONCLUSION: Transgenic pigs expressing the fluorescent protein KO serve as a useful model of cell transplantation in preclinical studies.