Hollow hydroxyapatite microspheres/chitosan composite as a sustained delivery vehicle for rhBMP-2 in the treatment of bone defects.

Composite scaffold comprised of hollow hydroxyapatite (HA) and chitosan (designated hHA/CS) was prepared as a delivery vehicle for recombinating human bone morphogenetic protein-2 (rhBMP-2). The in vitro and in vivo biological activities of rhBMP2 released from the composite scaffold were then investigated. The rhBMP-2 was firstly loaded into the hollow HA microspheres, and then the rhBMP2-loaded HA microspheres were further incorporated into the chitosan matrix. The chitosan not only served to bind the HA microspheres together and kept them at the implant site, but also effectively modified the release behavior of rhBMP-2. The in vitro release and bioactivity analysis confirmed that the rhBMP2 could be loaded and released from the composite scaffolds in bioactive form. In addition, the composite scaffolds significantly reduced the initial burst release of rhBMP2, and thus providing prolonged period of time (as long as 60 days) compared with CS scaffolds. In vivo bone regenerative potential of the rhBMP2-loaded composite scaffolds was evaluated in a rabbit radius defect model. The results revealed that the rate of new bone formation in the rhBMP2-loaded hHA/CS group was higher than that in both negative control and rhBMP2-loaded CS group. These observations suggest that the hHA/CS composite scaffold would be effective and feasible as a delivery vehicle for growth factors in bone regeneration and repair.

SOD mimetic improves the function, growth, and survival of small-size liver grafts after transplantation in rats.

BACKGROUND: Small-for-size syndrome (SFSS) may occur when graft volume is less than 45% of the standard liver volume, and it manifests as retarded growth and failure of the grafts and more mortality. However, its pathogenesis is poorly understood, and few effective interventions have been attempted. AIMS: The present study aimed to delineate the critical role of oxidant stress in SFSS and protective effects of a superoxide dismutase mimetic, Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), on graft function, growth, and survival in the recipient rats. METHODS: Small size graft liver transplantation (SSGLT) was performed to determine the survival, graft injury, and growth. MnTBAP was administered in SSGLT recipients (SSGLT+MnTBAP). RESULTS: Serum alanine aminotransferase levels were sustained higher in SSGLT recipients, which were correlated with an increased apoptotic cell count and hepatocellular necrosis in liver sections. Malondialdehyde content, gene expression of tumor necrosis factor α and interleukin 1ß, and DNA binding activity of nuclear factor-κB in the grafts were increased significantly in SSGLT recipients compared with sham-operated controls. Both phosphorylated p38 mitogen-activated protein kinase and nuclear c-Jun were increased in SSGLT. All these changes were strikingly reversed by the administration of MnTBAP, with an increase in serum superoxide dismutase activity. Moreover, in situ bromodeoxyuridine incorporation demonstrated that graft regeneration was much more profound in the SSGLT+MnTBAP group than in the SSGLT group. Finally, the survival of recipients with MnTBAP treatments was significantly improved. CONCLUSIONS: Enhanced oxidant stress with activation of the p38/c-Jun/nuclear factor-κB signaling pathway contributes to SFSS-associated graft failure, retarded graft growth, and poor survival. MnTBAP effectively reversed the pathologic changes in SFSS-associated graft failure.

Hepatic regenerative response in small-sized liver isografts in the rat.

BACKGROUND: To investigate hepatic regenerative response and associated mechanisms in different-size liver grafts in the rat. METHODS: Rat models of different-size-graft liver transplantation (whole, 50%-size, or 30%-size) were established, with a sham operation group serving as a control. Portal pressure, graft injury, interleukin 6 (IL-6), signal transducer and activator of transcription (Stat3), mitogen-activated protein kinase (MAPK), cyclin D1, and proliferating cell nuclear antigen (PCNA) were all assessed. RESULTS: The portal pressure was significantly higher and hepatic injury more severe in the smaller sized groups than in the whole graft group, especially in the 30%-size grafts. Hepatic IL-6 and tumor necrosis factor-alpha (TNF-alpha) levels in the two smaller sized groups were significantly higher than in the whole graft group, while IL-6 levels appeared to be negatively associated with graft sizes. Downstream markers of IL-6, Stat3 and MAPK phosphorylation, cyclin D1, and PCNA expression were also markedly increased in the small-sized grafts compared with the whole grafts, and appeared to positively correlate with early measurements of portal pressure and subsequent hepatic injury. CONCLUSION: Vigorous hepatic regeneration in small-for-size liver grafts may be associated with highly activated IL-6/Stat3 and MAPK signaling, which may in turn correlate with graft size, portal pressure, and hepatic injury.

[Surgical technique and concept in precise hepatectomy: experience of 338 cases of hepatectomy in single center].

OBJECTIVE: To evaluate the perioperative clinical outcome and predictive factors for perioperative complication morbidity and mortality. METHODS: From August 2003 to August 2008, the data of 338 cases of hepatectomy performed in the liver transplant center of the First Affiliated Hospital of Nanjing Medical University was collected in a prospective manner. The patients' perioperative clinical risk factors and results were analyzed. RESULTS: In the 338 hepatectomy cases, 255 patients (75.4%) underwent precise anatomical hepatectomy. The overall perioperative complication morbidity was 18.1%, while the perioperative mortality was 0.6%. In a total of 211 (62.4%) cases, the operation was carried out without blood transfusion. Univariate analysis revealed that cirrhotic liver, thrombocytopenia, blood loss in operation > 1000 ml, blood transfusion in operation and several other factors were closely related with the incidence rate of complication. Multivariate logistic regression analysis indicated that thrombocytopenia and perioperative blood transfusion were important independently predictive factors for the occurrence of perioperative complications in hepatectomy. CONCLUSIONS: Precise hepatectomy enables patients to obtain better clinical outcome with low complication morbidity and perioperative mortality. Reducing hemorrhage is an important factor that lead to good clinical results.

Adenoviral cardiotrophin-1 transfer improves survival and early graft function after ischemia and reperfusion in rat small-for-size liver transplantation model.

This study was to investigate the effect of donor liver adenoviral cardiotrophin-1 (CT-1) gene transfer on early graft survival and function in rat small-for-size liver transplantation. We constructed a recombinant murine CT-1 adenoviral vector. Donor rats were transduced in vivo with adenoviruses expressing CT-1 (AdCT-1) or control vector (AdEGFP). Livers were harvested 4 days later, reduced to 40% of weight, and transplanted. A syngeneic rat orthotopic liver transplantation model was performed using 40% small-for-size grafts. Graft survival, liver function, hepatic architecture change, the degree of necrosis and apoptosis, and cell survival signaling pathways were assessed. AdCT-1 pretreatment markedly improved liver function and the survival of small-for-size grafts. In the CT-1 treatment group, hepatic architecture was well protected, apoptotic and necrotic cells were reduced; anti-apoptotic protein bcl-2 was up-regulated and pro-apoptotic cleaved caspase-3 was down-regulated, cell survival signaling pathways were activated by phosphorylation of protein kinase B (Akt), extracellular-regulated kinase (ERK) and Signal transducer and activator of transcription-3 (Stat-3) after transplantation. In conclusion, donor liver adenoviral CT-1 transfer ameliorated ischemia/reperfusion injury by decreasing hepatic necrosis and apoptosis in small-for-size liver transplantation, mediated in part by activation of the Akt, ERK, and Stat-3 survival signaling pathways. These results may provide a potential clinical strategy to improve the outcome of small-for-size liver grafts.

Adoptive transfusion of ex vivo donor alloantigen-stimulated CD4(+)CD25(+) regulatory T cells ameliorates rejection of DA-to-Lewis rat liver transplantation.

BACKGROUND: Adoptive transfusion of splenocytes from long-term survivors of a tolerance model of rat orthotopic liver transplantation can induce acceptance of liver allografts in a rejection model preconditioned with donor gamma-irradiation before liver transplantation. Recent studies suggest that the regulatory T cells (Treg cells) in splenocytes from long-term survivors play an important role in the induction of liver graft tolerance, but this observation was made from a rejection model preconditioned with donor gamma-irradiation; little is known about the role of Treg cells in liver graft rejection using a naive rejection model. In this study, we examined the therapeutic potential of CD4(+)CD25(+) Treg cells in a naive rejection model of rat liver transplantation. METHODS: Freshly isolated or ex vivo alloantigen-stimulated CD4(+)CD25(+) Treg cells (1 x 10(6) cells) from naive Lewis RT(1) (LEW) rats were adoptively transferred into another LEW rat on days 1 and 7 after liver transplantation from a Dark Agouti RT1(a) (DA) rat. Recipients were treated with or without oral tacrolimus (FK506) (0.1 mg/kg/day) from days 1 to 7 after transplantation. For ex vivo alloantigen-stimulation, CD4(+)CD25(+) Treg cells from LEW rats were cocultured with mitomycin C-treated DA (donor alloantigen specific) or Brown Norway (BN)(RT1(n), third party) splenocytes for 72 hours. Ex vivo alloantigen-specific CD4(+)CD25(-) T-cell proliferation responses were assessed with fresh and stimulated CD4(+)CD25(+) Treg cells. RESULTS: Freshly isolated, donor alloantigen-stimulated and third-party alloantigen- stimulated CD4(+)CD25(+) Treg cells suppressed antigen-specific CD4(+)CD25(-) T-cell proliferation ex vivo, and adoptive transfusion of these 3 kinds of CD4(+)CD25(+) Treg cells prolonged survival of the liver allografts. The group transfused with the donor alloantigen-stimulated CD4(+)CD25(+) Treg cells had the greatest mean survival among the 3 groups (fresh Treg cells, 21 +/- 2 days, n = 6; third-party alloantigen-stimulated Treg cells, 20 +/- 2 days, n = 6; donor alloantigen-stimulated Treg cells, 30 +/- 2 days, n = 6). When combined with short-term tacrolimus administration, adoptive transfusion of donor antigen-stimulated Treg cells induced the greatest survival time in recipients (greater than 60 days; n = 6). CONCLUSION: Adoptive transfusion of ex vivo donor alloantigen-stimulated CD4(+)CD25(+) Treg cells combined with short-term tacrolimus treatment may represent a new strategy for preventing rejection after liver transplantation.

Mesenchymal stem cells over-expressing hepatocyte growth factor improve small-for-size liver grafts regeneration.

Ischemia-reperfusion (I/R) associated with small-for-size liver transplantation (SFSLT) impairs liver graft regeneration. Mesenchymal stem cells (MSCs) have the capability, under specific conditions, of differentiating into hepatocytes. Hepatocyte growth factor (HGF) has potent anti-apoptotic and mitogenic effects on hepatocytes during liver injury, and has been utilized in many experimental and clinical applications. In this study, we implanted HGF-expressing MSCs into liver grafts via the portal vein, using a 30% small-for-size rat liver transplantation model. HGF, c-met expression, hepatic injury and liver regeneration were assessed after liver transplantation. Our study demonstrated that MSCs over-expressing HGF prevented liver failure and reduced mortality in rats after SFSLT. These animals also exhibited improved liver function and liver weight recovery during the early post-transplantation period. Using green fluorescent protein (GFP) gene as a marker, we demonstrated that the engrafted cells and their progeny incorporated into remnant livers and produced albumin. These findings suggest that MSCs genetically modified to over-express HGF and implanted in the liver graft, may offer a novel approach to promoting liver regeneration after small-for-size transplantations.

[Isolation and function analysis of rat CD4+ CD25+ regulatory T cells].

AIM: To investigate the isolation method and to analyze the function of rat CD4(+) CD25(+) regulatory T cells. METHODS: Lymphocytes were isolated from the rat spleens and then CD4(+) CD25(+) T cells were sorted by magnetic bead cell sorting (MACS) system. The purity and Foxp3 expression of CD4(+) CD25(+) T cells were analyzed by flow cytometry(FCM) and RT-PCR, respectively. The suppressive effect of CD4(+) CD25(+) T cells on the proliferation of CD4(+) CD25(-) T cells was analyzed by mixed lymphocyte reaction. IL-2, IFN-gamma and IL-10 levels in culture supernatant were detected by ELISA. RESULTS: The purity of CD4(+) CD25(+) T cells sorted by MACS was 86%-93%. The CD4(+) CD25(+) T cells could specifically express the Foxp3 gene as compared with CD4(+) CD25(-) T cells. In vitro CD4(+) CD25(+) T cells could suppress the proliferation of CD4(+) CD25(-) T cells and IFN-gamma, IL-2 production, but they themselves could secrete IL-10. CONCLUSION: We established an effective procedure for enrichment of CD4(+) CD25(+) regulatory T cells by MACS with satisfactory cell purity, viability and function. CD4(+) CD25(+) T cells can suppress CD4(+) CD25(-) T cells and specifically express Foxp3 gene.

[CD4(+) CD25(+) Tr cells and transcription factor Foxp3 in the naturally tolerance of rat liver transplantation].

OBJECTIVE: To investigate the role of intrahepatic CD4(+)CD25(+) T regulatory cells and Foxp3 gene in the natural tolerance in rat liver transplantation. METHODS: The orthotopic liver transplantation models of inbred rats (LEW and DA rats) were established with double-sleeve technique and the models were divided into two groups: tolerance group (TOL group, LEW-to-DA) and rejection group (REJ group, DA-to-LEW). The intrahepatic lymphocytes from each group were isolated by using density gradient centrifugation. CD4(+)CD25(+) T cells were isolated by magic cell sorting system (MACS) and identified by flow cytometry (FCM). CD4(+)CD25(+) Tr cells suppression on the proliferation of CD4(+)CD25(-) T effector cells were analyzed by cell proliferation assay in vitro. Western blot was used to detect Scurfin protein expression of CD4(+)CD25(+) Tr cells. RESULTS: CD4(+)CD25(+) Tr cells developed significantly greater in the TOL group than in the REJ group. In vitro, the spleen cells from LEW rats can irritate the proliferation of CD4(+)CD25(+) T cells more obviously than the syngeneic spleen cells. CD4(+)CD25(+) T cells could suppress the proliferation of CD4(+)CD25(-) T cells, but the inhibition was reversed by exogenous IL-2 (200 U/ml). CONCLUSIONS: The immune suppression function of CD4(+)CD25(+) Tr cell, mediated by Foxp3 gene, is one of the mechanisms in liver transplantation tolerance.