Repair of extrahepatic bile duct defect using a collagen patch in a Swine model.

Extrahepatic bile duct (EBD) injury can happen during surgery. To repair a defect of the EBD and prevent postoperative biliary complications, a collagen membrane was designed. The collagen material was porous, biocompatible, and degradable and could maintain its shape in bile soaking for about 4 weeks. The goal was to induce rapid bile duct tissue regeneration. Twenty Chinese experimental hybrid pigs were used in this study and divided into a patch group and a control group. A spindle-shaped defect (20 mm × 6 mm) was made in the anterior wall of the lower EBD in the swine model, and then the defect was reconstructed using a collagen patch with a drainage tube and wrapped with greater omentum. Ultrasound was performed at 2, 4, 8, and 12 weeks postoperatively. Liver function tests and white blood cell count (WBC) were measured. Hematoxylin-eosin staining, cytokeratin 7 immunohistochemical staining, and Van Gieson's staining of EBD were used. The diameter and thickness of the EBD at the graft site were measured. There was no significant difference in liver function tests or WBC in the patch group compared with the control group. No evidence of leakage or stricture was observed, but some pigs developed biliary sludge or stone at 4 and 8 weeks. The drainage tube was lost within 12 weeks. The neo-EBD could withstand normal biliary pressure 2 weeks after surgery. Histological study showed the accessory glands and epithelial cells gradually regenerated at graft sites from 4 weeks, with increasing vessel infiltration and decreasing inflammation. The collagen fibers became regular with full coverage of epithelial cells. The statistical analysis of diameter and thickness showed no stricture formation at the graft site, but the EBD wall was slightly thicker than in the normal bile duct due to collagen fiber deposition. The structure of the neo-EBD was similar to that of the normal EBD. The collagen membrane patch associated with a drainage tube and wrapped with greater omentum effectively induced the regeneration of the EBD defect within 12 weeks.

Evaluation of two decellularization methods in the development of a whole-organ decellularized rat liver scaffold.

AIM: Hepatic tissue engineering is considered as a possible alternative to liver transplantation for end-stage liver disease. Several methods of decellularization of xenogeneic liver are available to produce three-dimensional organ scaffolds for engineering liver tissues. However, rare studies have examined and compared the effectiveness of different methods on the structure and composition of intact decellularized liver extracellular matrix. METHODS: Two decellularization methods were adopted herein. Their effects on collagen, elastin, glycosaminoglycans (GAGs), hepatocyte growth factor (HGF) content and influence to the function of hepatocytes cultured in scaffolds were examined and compared. RESULTS: The complete tissue decellularization was successfully achieved after treatment with sodium dodecyl sulphate (SDS) and Triton X-100. The total absence of nuclear structures and removal of viable cells were confirmed by haematoxylin-eosin staining and scanning electron microscopy. Collagen was preserved after both treatments. However, the elastin content decreased to about 20% and 60%, the GAGs content decreased to about 10% and 50% and the HGF content decreased to about 20% and 60% of the native liver level after SDS and Triton X-100 treatment respectively. The Triton X-100-treated scaffolds were much superior than SDS-treated scaffolds in supporting liver-specific function, including albumin secretion (P = 0.001), urea synthesis (P = 0.002), ammonia elimination (P = 0.007) and mRNA expression levels of drug metabolism enzymes. CONCLUSION: This study suggested that liver extracellular matrix scaffolds constructed using perfusion of Triton X-100 as described herein might provide a more effective and ideal material for the usage in tissue engineering and regenerative medicine approaches.

Superior antimetastatic effect of pemetrexed-loaded gelatinase-responsive nanoparticles in a mouse metastasis model.

Novel pemetrexed-loaded gelatinase-responsive nanoparticles were prepared as a targeted delivery system to determine its potential for clinical therapy of malignant melanoma. The pemetrexed-loaded poly(ethylene glycol)(PEG)-peptide-poly(ε-caprolactone) (PCL) nanoparticles included a gelatinase-cleavage peptide and a PEG-PCL-based structure. The pemetrexed-loaded PEG-peptide-PCL nanoparticles have shown the best antimetastatic effect in experimental lung metastasis models. The expressions of CD133 and thymidylate synthetase of metastatic tumors were also evaluated in our studies. Our results showed that pemetrexed-loaded gelatinase-responsive nanoparticles may represent a potent drug delivery system for inhibiting pulmonary metastasis and our preclinical results can provide new avenues for clinical therapy of malignant melanoma.

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.

Iron-based nanoscale coordination polymers synergistically induce immunogenic ferroptosis by blocking dihydrofolate reductase for cancer immunotherapy.

Iron is indispensable for cancer cell survival and cancer cells are more vulnerable to ferroptosis than normal cells. Ferroptosis holds promise for overcoming chemoresistance and inducing tumor immunogenic cell death, which offers new possibilities for cancer immunotherapy. However, the prevalence of immunogenic ferroptosis in cancer cells is diminished because of the high levels of reducing substances within tumor microenvironments. Ferroptosis-needed iron is overdose for livings, which is also an obstacle for effective immune responses. In this study, we construct self-assembled carrier-free nanoscale coordination polymers based on iron and methotrexate (MFe-NCPs). The low-dose-iron-induced immunogenic ferroptosis is obviously enhanced by methotrexate via inhibiting dihydrofolate reductase and abating substance reduction, respectively. Of note, MFe-NCPs sequentially promoted antigen presentation, immune activation, T cell infiltration and boosted the therapeutic effect of immune checkpoint blockade therapy.

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.

Superior antitumor efficiency of cisplatin-loaded nanoparticles by intratumoral delivery with decreased tumor metabolism rate.

cis-Dichlorodiamminoplatinum (II) (cisplatin) has demonstrated extraordinary activities against a variety of solid tumors. However, the clinical efficacy is contrasted by its toxicity profile. To reduce the toxicity and enhance the circulation time of cisplatin, core-shell structure nanoparticles were prepared from block copolymer of methoxy poly(ethylene glycol)-polycaprolactone (mPEG-PCL). Cisplatin was incorporated into the nanoparticles with high encapsulation efficiency more than 75%. Controlled release of cisplatin was observed in a sustained manner. In vitro cytotoxicity studies proved the efficacy of cisplatin-loaded nanoparticles against BGC823 and H(22) cells in a dose and time-dependent manner. Furthermore, intratumoral administration was applied to improve the tumor-targeted delivery in the in vivo evaluation. Compared with free cisplatin, cisplatin-loaded nanoparticles exhibited superior antitumor effect by delaying tumor growth when delivered intratumorally, while no significant improvement was observed when they were administrated intraperitoneally. Positron emission tomography/computed tomography (PET/CT) imaging was utilized for the first time to detect the declined (18)F-labeled 2-fluoro-2-deoxy-d-glucose ((18)F-FDG) uptake of the tumor in mice receiving cisplatin-loaded nanoparticles intratumorally. These results suggest that polymeric nanoparticles with core-shell structures are promising for further studies as drug delivery carriers, and intratumoral delivery of drug-loaded nanoparticles could be a probable clinically useful therapeutic regimen.

Novel thermosensitive polymeric micelles for docetaxel delivery.

Targeted delivery of antitumor drugs triggered by hyperthermia has significant advantages in clinical applications, since it is easy to implement and side effects are reduced. To release drugs site-specifically upon local heating often requires the drugs to be loaded into a thermosensitive polymer matrix with a low critical solution temperature (LCST) between 37 and 42 degrees C. However, the LCSTs of most thermosensitive materials were below 37 degrees C, which limits their application in clinic because they would precipitate once injected into human body and lost thermal targeting function. Herein, we prepared a novel thermosensitive copolymer (poly(N-isopropylacrylamide-co-acrylamide)-b-poly (DL-lactide)) that exhibits no obvious physical change up to 41 degrees C when heated. Docetaxel loaded micelles made of such thermosensitive polymer were prepared by dialysis method and the maximum loading content was found to be up to 27%. The physical properties, such as structure, morphology, and size distribution of the micelles with and without docetaxel were investigated by NMR, X-ray diffraction, dynamic light scattering, atomic force microscopy, etc. The efficacy of this drug delivery system was also evaluated by examining the proliferation inhibiting activity against different cell lines in vitro. After hyperthermia, the cytotoxicity of docetaxel-loaded micelles increased prominently. Our results demonstrated that this copolymer could be an ideal candidate for thermal targeted antitumor drug delivery.

The immobilization of hepatocytes on 24 nm-sized gold colloid for enhanced hepatocytes proliferation.

Bioartificial liver and hepatocyte transplantation is anticipated to supply a temporary metabolic support for candidates of liver transplantation or for patients with fulminant liver failure. An essential restriction of this form is the inability to acquire an enough amount of hepatocytes. Enhancement of the proliferation and differentiated function of hepatocytes is becoming a pursued target. Here, porcine hepatocytes were successfully immobilized on nano-sized gold colloid particles to construct a "hepatocyte/gold colloid" interface at which hepatocytes can be quickly proliferated. The properties of this resulting interface were characterized and confirmed by scanning electron microscopy and atomic force microscopy. The proliferative mechanism of hepatocytes was also discussed. The proliferated hepatocytes could be applied to the clinic based on their excellent functions for the synthesis of protein, glucose and urea as well as lower lactate dehydrogenase release.

[Effect of sustained-release basic fibroblast growth factor on healing of bile duct defect in pigs].

OBJECTIVE: To investigate the effects of sustained-release basic fibroblast growth factor (bFGF) on healing of bile duct defect. METHODS: A model of bile duct wall defect (2 cm in length and 1/3-2/3 of the bile duct circumference in width) was made in 24 pigs (male or female, weighing 15-30 kg), and then defect was repaired with sustained-release bFGF collagen membrane (2.0 cm x 1.0 cm x 0.5 cm in size) in the experimental group (n=12) or with collagen membrane (2.0 cm x 1.0 cm x 0.5 cm in size) alone in the blank control group (n=12). Another 4 healthy pigs were used to obtain normal bile duct as normal control group. The survival condition of pigs was observed after operation; at 1, 2, and 3 months after operation, the blood sampling was collected to test the changes of liver function, and the bile duct specimens were harvested to count the microvessel density (MVD) and submucosal gland by HE staining and immunohistochemistry staining; and at 3 months after operation, cholangiography examination was done. RESULTS: All the animals survived to completion of the experiment. Intra-abdominal adhesion was serious in the experimental and blank control groups at 1 week after operation, but the adhesion was markedly improved in the experimental group when compared with the blank control group with time passing. The liver function test showed that alkaline phosphatase in the experimental group was significantly lower than that in the blank control group at 2 and 3 months (P < 0.05), but no significant difference in aspartate aminortransferase, total bilirubin, and albumin was found among 3 groups (P > 0.05). The histology and immunohistochemistry staining observations showed that the regeneration rates of submucosal glands and epithelium in the experimental group were faster than those in the blank control group; defect was covered with the epithelium at 2 months, and the structure was similar to that of normal control group at 3 months; and the edema and inflammation infiltration were reduced when compared with the blank control group. The counts of MVD and submucosal gland were significantly higher than those in blank control group and normal control group at 1 month after operation (P < 0.05), and then decreased and remained at normal levels at 2 months after operation. There was a positive correlation between submucosal gland counting and MVD counting in 3 groups after operation (P < 0.01). The cholangiography examination showed no biliary dilatation or cholelithiasis after 3 months in experimental group and blank control group. CONCLUSION: Sustained-release bFGF can promote healing of bile duct defect by accelerating the vascularization, gland regeneration, and epithelialization.

Factors influencing the transfer of porcine endogenous retroviruses across the membrane in bioartificial livers.

OBJECTIVES: to investigate the factors influencing the transfer of porcine endogenous retroviruses (PERVs) across the membrane in a new bioartificial liver (BAL). METHODS: A new BAL containing 2 circuits was constructed using plasma component separators with membrane pore sizes of 10 nm, 20 nm, 30 nm, and 35 nm, or a plasma filter with a membrane pore size of 500 nm. Cocultured cells of porcine hepatocytes and mesenchymal stem cells or single porcine hepatocytes were incubated in the bioreactors, and the BAL worked for 72 hours, with supernatant samples in internal and external circuits collected every 12 hours. PERV RNA, reverse transcriptase (RT) activity, and in vitro infectivity of the supernatant were detected. RESULTS: With the plasma filters, the results of PERV detection were the same in both circuits. With plasma component separators, PERV RNA was found in the external circuits, but no positive RT activity or HEK293 cell infection was found. The time at which the PERV RNA was first detected varied with the pore size of membrane; the larger the membrane pore size was, the earlier the RNA was detected. The PERV RNA level in the external circuits was reduced significantly compared with that in the internal circuits at any detecting time. CONCLUSIONS: The plasma component separators with membrane pore size =35 nm could significantly reduce the passage of infectious PERVs. And the membrane pore size, the treatment duration, and the viral level in the internal circuit were potential factors influencing the transfer of PERVs across the membrane in a BAL. In addition, a low risk of PERV transmission from porcine hepatocytes to human cells was found.

Reversion of pH-induced physiological drug resistance: a novel function of copolymeric nanoparticles.

AIMS: The extracellular pH of cancer cells is lower than the intracellular pH. Weakly basic anticancer drugs will be protonated extracellularly and display a decreased intracellular concentration. In this study, we show that copolymeric nanoparticles (NPs) are able to overcome this "pH-induced physiological drug resistance" (PIPDR) by delivering drugs to the cancer cells via endocytosis rather than passive diffussion. MATERIALS AND METHODS: As a model nanoparticle, Tetradrine (Tet, Pka 7.80) was incorporated into mPEG-PCL. The effectiveness of free Tet and Tet-NPs were compared at different extracellular pHs (pH values 6.8 and 7.4, respectively) by MTT assay, morphological observation and apoptotic analysis in vitro and on a murine model by tumor volume measurement, PET-CT scanning and side effect evaluation in vivo. RESULTS: The cytotoxicity of free Tet decreased prominently (P<0.05) when the extracellular pH decreased from 7.4 to 6.8. Meanwhile, the cytotoxicity of Tet-NPs was not significantly influenced by reduced pH. In vivo experiment also revealed that Tet-NPs reversed PIPDR more effectively than other existing methods and with much less side effects. CONCLUSION: The reversion of PIPDR is a new discovered mechanism of copolymeric NPs. This study emphasized the importance of cancer microenvironmental factors in anticancer drug resistance and revealed the superiority of nanoscale drug carrier from a different aspect.

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.

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.

Preparation and evaluation of PEG-PCL nanoparticles for local tetradrine delivery.

To establish a satisfactory delivery system for local delivery of Tetradrine (Tet), four kinds of core-shell nanoparticles were prepared from di-block copolymer of methoxy poly(ethylene glycol)-polycaprolactone (MePEG-PCL) and tri-block copolymer of polycaprolactone-poly(ethylene glycol)-polycaprolactone (PCL-PEG-PCL). The physiochemical traits of the four kinds of nanoparticles including morphology, particle size, zeta-potential, drug-loading content, stability, and in vitro release profile were studied. We also evaluated the four kinds of nanoparticles by in vitro cellular uptake experiment, cytotoxicity assay against LoVo cells, and biocmpatibility study. Histoculture Drug Response Assay (HDRA), a more predictive method usually used to evaluate chemosensitivity was firstly applied in our study to evaluate the antitumor potency of polymeric nanoparticles. The current study showed that all the four kinds of copolymers exhibited remarkable in vitro antitumor effects, especially in HDRA assay. The configuration and composition of the copolymers were important for the properties and functions of the nanoparticles. Nanoparticles prepared from the di-block copolymer with a particle size around 300nm and the hydrophobic composition about 80% was determined as the most effective drug carrier for further studies.