Are Chinese Patients with Type 2 Diabetes and a Body Mass Index of 27.5-32.5 kg/m2 Suitable for Metabolic Surgery? A One-Year Post-Surgery Study.

INTRODUCTION: The aim of this study was to clarify the efficacy and safety of metabolic surgery in Chinese patients with type 2 diabetes mellitus (T2DM) and a body mass index (BMI) of 27.5-32.5 kg/m2. METHODS: A total of 99 patients with T2DM were enrolled in this retrospective cohort study. Of these patients, 53 had a BMI of 27.5-32.5 kg/m2 and had undergone metabolic surgery (n = 21) or were on conventional antidiabetic therapy (n = 32)]; 46 had a BMI ≥ 32.5 kg/m2 and all had undergone metabolic surgery. Primary endpoints included the triple endpoint [hemoglobin A1c < 6.5%, low-density lipoprotein cholesterol (LDL-C) < 2.6 mmol/L, and systolic blood pressure (SBP) < 130 mmHg] and successful weight loss 1 year later. Remission of diabetes, glucose and lipid metabolism, medication usage, and adverse events were evaluated. RESULTS: Of patients with BMI 27.5-32.5 kg/m2 undergoing metabolic surgery, 33.33% achieved the composite endpoints, and 100% achieved successful weight loss. This result was similar to that in patients with BMI ≥ 32.5 and better than those with BMI 27.5-32.5 kg/m2 receiving conventional antidiabetic therapy. A significant and similar reduction in BMI, waist circumference, SBP, serum LDL-C, hemoglobin A1c, and uric acid, as well as similar frequency postoperative adverse events, were confirmed in both metabolic surgery groups. Patients with BMI 27.5-32.5 kg/m2 who had undergonemetabolic surgery showed more metabolic improvement than those only receiving medications but they experienced more adverse events. CONCLUSION: A BMI cutoff of 27.5 kg/m2 for metabolic surgery may be suitable for Chinese patients with T2DM.

Evaluation of a novel hybrid bioartificial liver based on a multi-layer flat-plate bioreactor.

AIM: To evaluate the efficacy and safety of a hybrid bioartificial liver (HBAL) system in the treatment of acute liver failure. METHODS: Canine models with acute liver failure were introduced with intravenous administration of D-galactosamine. The animals were divided into: the HBAL treatment group (n = 8), in which the canines received a 3-h treatment of HBAL; the bioartificial liver (BAL) treatment group (n = 8), in which the canines received a 3-h treatment of BAL; the non-bioartificial liver (NBAL) treatment group (n = 8), in which the canines received a 3-h treatment of NBAL; the control group (n = 8), in which the canines received no additional treatment. Biochemical parameters and survival time were determined. Levels of xenoantibodies, RNA of porcine endogenous retrovirus (PERV) and reverse transcriptase (RT) activity in the plasma were detected. RESULTS: Biochemical parameters were significantly decreased in all treatment groups. The TBIL level in the HBAL group was lower than that in other groups (2.19 ± 0.55 µmol/L vs 24.2 ± 6.45 µmol/L, 12.47 ± 3.62 µmol/L, 3.77 ± 1.83 µmol/L, P < 0.05). The prothrombin time (PT) in the BAL and HBAL groups was significantly shorter than the NBAL and control groups (18.47 ± 4.41 s, 15.5 ± 1.56 s vs 28.67 ± 5.71 s, 21.71 ± 3.4 s, P < 0.05), and the PT in the HBAL group was shortest of all the groups. The albumin in the BAL and HBAL groups significantly increased and a significantly higher level was observed in the HBAL group compared with the BAL group (27.7 ± 1.7 g/L vs 25.24 ± 1.93 g/L). In the HBAL group, the ammonia levels significantly decreased from 54.37 ± 6.86 to 37.75 ± 6.09 after treatment (P < 0.05); there were significant difference in ammonia levels between other the groups (P < 0.05). The levels of antibodies were similar before and after treatment. The PERV RNA and the RT activity in the canine plasma were all negative. CONCLUSION: The HBAL showed great efficiency and safety in the treatment of acute liver failure.

Microbiological safety of a novel bio-artificial liver support system based on porcine hepatocytes: a experimental study.

BACKGROUND: Our institute has developed a novel bio-artificial liver (BAL) support system, based on a multi-layer radial-flow bioreactor carrying porcine hepatocytes and mesenchymal stem cells. It has been shown that porcine hepatocytes are capable of carrying infectious porcine endogenous retroviruses (PERVs) into human cells, thus the microbiological safety of any such system must be confirmed before clinical trials can be performed. In this study, we focused on assessing the status of PERV infection in beagles treated with the novel BAL. METHODS: Five normal beagles were treated with the novel BAL for 6 hours. The study was conducted for 6 months, during which plasma was collected from the BAL and whole blood from the beagles at regular intervals. DNA and RNA in both the collected peripheral blood mononuclear cells (PBMCs) and plasma samples were extracted for conventional PCR and reverse transcriptase (RT)-PCR with PERV-specific primers and the porcine-specific primer Sus scrofa cytochrome B. Meanwhile, the RT activity and the in vitro infectivity of the plasma were measured. RESULTS: Positive PERV RNA and RT activity were detected only in the plasma samples taken from the third circuit of the BAL system. All other samples including PBMCs and other plasma samples were negative for PERV RNA, PERV DNA, and RT activity. In the in vitro infection experiment, no infection was found in HEK293 cells treated with plasma. CONCLUSIONS: No infective PERV was detected in the experimental animals, thus the novel BAL had a reliable microbiological safety profile.

[New evidence of porcine endogenous retrovirus transmission with new bio-artificial liver system: a experimental study].

OBJECTIVE: To investigate the potential transmissibility of porcine endogenous retrovirus (PERV) from a newly-developed porcine hepatocyte bioartificial liver (BAL) system prior to human clinical trial by using a live canine model. METHODS: Five normal beagles were treated with the new BAL support system for six hours. Samples of plasma from the BAL system and whole blood from the beagles were collected at regular intervals over the six month study period. DNA and RNA were isolated from both the peripheral blood mononuclear cells (PBMCs) and plasma for evaluation by polymerase chain reaction (PCR) and reverse transcription (RT)-PCR, respectively, to detect PERV and the Sus scrofa cytochrome B normalization standard. In addition, RT activity and the in vitro infectivity of the plasma were detected in HEK293 cells. RESULTS: All five beagles remained in stable physical health throughout the treatment and survived until the end of the study. PERV RNA-positivity and RT activity were only detected in the plasma samples from the 3rd BAL treatment cycle. All other samples, including PBMCs and plasma, were negative for PERV RNA, PERV DNA, and RT activity. In addition, none of the sera samples showed in vitro infectivity. CONCLUSION: Application of our BAL system does not lead to PERV transmission.

Immunosafety evaluation of a multilayer flat-plate bioartificial liver.

INTRODUCTION: To study and evaluate the immunosafety of our newly developed multilayer flat-plate bioartificial liver (BAL) in treatment of canines with acute liver failure. METHODS: Fresh porcine hepatocytes and bone marrow mesenchymal stem cells were cocultured in new BAL. Ten canine models with acute liver failure were set up through D-galactosamine administration; 24 hours after administration, the beagles were randomly allocated to a 6-hour treatment with the BAL. The beagles were divided into 2 groups by treatment times. Group 1 beagles (n = 5) received a single BAL treatment. Group 2 beagles (n = 5) received 3 BAL treatments. The hemodynamic, hematologic response and humoral immune responses to BAL therapy were studied before and after treatments. RESULTS: All beagles remained hemodynamically and hematologically stable during BAL treatments. The levels of IgG and IgM were similar before and after treatment after a single treatment. In addition, the level of CH50 in group 1 slightly decreased after the initiation of BAL treatment, and then the level recovered to baseline quickly after treatments. Time-course changes of the levels of antibodies and CH50 after 3 treatments in group 2 were similar to group 1. Only trace levels of IgG were detected in BAL medium after treatments. CONCLUSION: The multilayer flat-plate BAL showed a great immunosafety in the treatment of canines with acute liver failure and exhibited a good prospect of its use in clinic.

The influence of membrane molecular weight cutoff on a novel bioartificial liver.

Given the xenogeneic immune reaction relevant to the molecular weight cutoff of the membrane of a bioartificial liver (BAL) system, we investigated the influence of membrane molecular weight cutoff in our BAL system in this study. Acute liver failure in beagles was induced by d-galactosamine administration. Eight beagles were divided into two groups by the membrane molecular weight cutoff of the plasma component separator. Group 1 beagles were treated with BAL containing 200 kDa retention rating membrane. Group 2 beagles were treated with BAL containing 1200 kDa retention rating membrane. Each group underwent two 6-h BAL treatments that were performed on day 1 and day 21. The hemodynamic and hematologic response, humoral immune responses, and cytotoxic immune response to BAL therapy were studied before and after treatments. All beagles remained hemodynamically and hematologically stable during BAL treatments. BAL treatment was associated with a significant decline in levels of complement; however, a longer time of level maintenance was observed in Group 2. Group 2 beagles experienced a significant increase in levels of IgG and IgM after two BAL treatments. Significant levels of canine proteins were detected in BAL medium from Group 2; only trace levels of canine proteins were detected in BAL medium from Group 1. The posttreatment viability of co-culture cells in Group 2 was lower compared with Group 1, and the viability of co-culture cells after treatments was associated with deposition of canine proteins on the cells. Xenogeneic immune response was influenced by membrane molecular weight cutoff in the BAL.

Influence of chitosan nanofiber scaffold on porcine endogenous retroviral expression and infectivity in pig hepatocytes.

AIM: To investigate the influence of chitosan nanofiber scaffold on the production and infectivity of porcine endogenous retrovirus (PERV) expressed by porcine hepatocytes. METHODS: Freshly isolated porcine hepatocytes were cultured with or without chitosan nanofiber scaffold (defined as Nano group and Hep group) for 7 d. The daily collection of culture medium was used to detect reverse transcriptase (RT) activity with RT activity assay kits and PERV RNA by reverse transcription-polymerase chain reaction (PCR) and real time PCR with the PERV specific primers. And Western blotting was performed with the lysates of daily retrieved cells to determine the PERV protein gag p30. Besides, the in-vitro infectivity of the supernatant was tested by incubating the human embryo kidney 293 (HEK293) cells. RESULTS: The similar changing trends between two groups were observed in real time PCR, RT activity assay and Western blotting. Two peaks of PERV expression at 10H and Day 2 were found and followed by a regular decline. No significant difference was found between two groups except the significantly high level of PERV RNA at Day 6 and PERV protein at Day 5 in Nano group than that in Hep group. And in the in-vitro infection experiment, no HEK293 cell was infected by the supernatant. CONCLUSION: Chitosan nanofiber scaffold might prolong the PERV secreting time in pig hepatocytes but would not obviously influence its productive amount and infectivity, so it could be applied in the bioartificial liver without the increased risk of the virus transmission.

Effects of membrane molecular weight cutoff on performance of a novel bioartificial liver.

Immunoisolation using semipermeable membranes has been incorporated into bioartificial liver (BAL) devices to separate cellular components of the recipient's immune system from the cells within the BAL device. This study was designed to explore the influence of membrane molecular weight cutoff on performance of the multilayer radial-flow BAL using porcine hepatocytes cocultured with mesenchymal stem cells. In this study, healthy beagles underwent 6-h treatment with a BAL containing membrane with 200 kDa retention rating or 1200 kDa retention rating. Functional markers of BAL performance were monitored before and after treatment, as well as cytotoxic immune response to BAL therapy. The results showed that hepatocyte performance levels such as albumin secretion, urea synthesis, and viability were all significantly higher in 200 kDa retention rating group compared with the 1200 kDa retention rating group after treatment (P < 0.05). Significant levels of canine proteins were detected in BAL medium from the 1200 kDa retention rating group. Fluorescence microscopy further verified that heavy deposition of canine IgG, IgM, and complement (C3) on coculture cells was obtained after BAL treatment in the 1200 kDa retention rating group. However, only trace deposits of canine immunoproteins were observed on coculture cells obtained from BAL in the 200 kDa retention rating group. Small membrane molecular weight cutoff of the BAL could reduce the transfer of xenoreactive antibodies into the BAL medium and improve the performance of the BAL.

[The efficacy research of multi-layer flat-plate bioartificial liver on acute liver failure].

OBJECTIVE: To evaluate the efficacy of newly developed multi-layer flat-plate bioartificial liver in treatment of canines with acute liver failure. METHODS: Porcine hepatocytes and bone marrow mesenchymal stem cells were cocultured in newly developed multi-layer flat-plate bioreactor. Acute liver failure in canine models was induced by D-galactosamine administration.Sixteen canine models were divided into two groups: treatment group (n = 8) and control group (n = 8). Biochemical parameters were determined for 7 days after treatment and liver specimens were collected for histological analysis. RESULTS: Hepatic encephalopathy and general conditions were significantly improved in the treatment group, but no changes in the control group. Alanine aminotransferase was significantly decreased from (1512 ± 183) U/L to (86 ± 25) U/L in the treatment group, aspartate aminotransferase was significantly decreased from (1472 ± 365) U/L to (46 ± 11) U/L, lactate dehydrogenase was significantly decreased from (463 ± 76) U/L to (312 ± 84) U/L, total bilirubin was significantly decreased from (28.8 ± 6.2) µmol/L to (12.5 ± 3.6) µmol/L, ammonia was significantly decreased from (56 ± 15) µmol/L to (34 ± 10) µmol/L, and prothrombin time were significantly decreased in the treatment group but increased in the control group, albumin was improved in the treatment group but decreased in the control group. There were 5 canines survived in the treatment group but only 3 in the control group. But there was no difference on survival rates between the two group (P = 0.294). CONCLUSION: The application of newly developed multi-layer flat-plate bioartificial liver system was effective in the treatment of canines with acute liver failure.

Rat hepatocyte aggregate formation on discrete aligned nanofibers of type-I collagen-coated poly(L-lactic acid).

Primary hepatocytes cultured in three dimensional tissue constructs composed of multicellular aggregates maintain normal differentiated cellular function in vitro while cultured monolayers do not. Here, we report a technique to induce hepatocyte aggregate formation using type-I collagen-coated poly(L-lactic acid) (PLLA) discrete aligned nanofibers (disAFs) by providing limited cell-substrate adhesion strength and restricting cell migration to uniaxial movement. Kinetics of aggregate formation, morphology and biochemical activities of rat hepatocyte aggregates were tested over a 15 day culture period. Evidence was provided that physical cues from disAFs quickly induced the formation of aggregates. After 3 days in culture, 88.3% of free hepatocytes on disAFs were incorporated into aggregates with an average diameter of 61 +/- 18 microm. Hepatocyte aggregates formed on disAFs displayed excellent cell retention, cell activity and stable functional expression in terms of albumin secretion, urea synthesis and phase I and II (CYP1A and UGT) metabolic enzyme activity compared to monolayer culture of hepatocytes on tissue culture plastic (TCP) with type-I collagen as well as on meshes of type-I collagen-coated PLLA random nanofibers (meshRFs). These results suggest that disAFs may be a suitable method to maintain large-scale hepatic cultures with high activity for tissue engineering research and potential therapeutic applications, such as bioartificial liver devices.

Electrospun chitosan nanofibers for hepatocyte culture.

In this study, we developed a method to obtain high surface area nanofiber meshes composed of chitosan of a number of molecular weights. This method required decreasing the viscosity and surface tension of the chitosan solution as well as optimization of the electrospinning parameters such as applied voltage and environmental humidity. These chitosan nanofiber meshes were developed as a culture substrate for hepatocytes. The fibers exhibited a uniform diameter distribution (average diameter: 112 nm) and FTIR results indicate that the chemical structure of chitosan is stable during the electrospinning process. The attachment, morphology and activity of hepatocytes cultured on the chitosan nanofiber meshes were tested. The results showed that the chitosan nanofibers are biocompatible with hepatocytes and that these chitosan nanofiber meshes could be useful tissue culture substrates for various applications, including bioartificial liver-assist devices and tissue engineering for liver regeneration.

In vitro evaluation of a multi-layer radial-flow bioreactor based on galactosylated chitosan nanofiber scaffolds.

Clinical use of bioartificial livers (BAL) strongly relies on the development of bioreactors. In this study, we developed a multi-layer radial-flow bioreactor based on galactosylated chitosan nanofiber scaffolds and evaluated its efficacy in vitro. The bioreactor contains 65 layers of stacked flat plates, on which the nanofiber scaffolds were electrospinned for hepatocyte immobilization and aggregation. Culture medium containing pig red blood cells (RBCs) was perfused from the center to periphery, so that exchange materials are sufficient to afford enough oxygen. We determined the parameters for hepatocyte-specific function and general metabolism and also measured the oxygen consumption rate (OCR). Microscope and scanned electron microscopy observation showed a tight adhesion between cells and scaffolds. Compared with the control (bioreactors without nanofiber scaffolds), the number of adhered cells in our bioreactor was 1.59-fold; the protein-synthesis capacity of hepatocytes was 1.73-fold and urea was 2.86-fold. Moreover, the OCR of bioreactors with RBCs was about 1.91-fold that of bioreactors without RBCs. The galactosylated chitosan nanofiber scaffolds introduced into our new bioreactor greatly enhanced cell adhesion and function, and the RBCs added into the culture medium were able to afford enough oxygen for hepatocytes. Importantly, our new bioreactor showed an exciting efficiency, and it may afford the short-term support of patients with hepatic failure.

Chitosan nanofiber scaffold enhances hepatocyte adhesion and function.

To enhance cell attachment and promote liver functions of hepatocytes cultured in bioreactors, a chitosan nanofiber scaffold was designed and prepared via electrospinning. Effects of the scaffold on hepatocyte adhesion, viability and function were then investigated. Data showed that hepatocytes on chitosan nanofiber scaffold exhibited better viability and tighter cell-substrate contact than cells on regular chitosan film. In addition, urea synthesis, albumin secretion and cytochrome P450 activity of hepatocytes on chitosan nanofiber scaffold were all 1.5 to 2 folds higher than the controls. Glycogen synthesis was also increased as compared with the controls. These results suggested the potential application of this chitosan nanofiber scaffold as a suitable substratum for hepatocyte culturing in bioreactors.