Furan-induced dose–response relationships for liver cytotoxicity,cell proliferation,and tumorigenicity (furan-induced liver tumorigenicity)

Rodent studies of furan are associated with liver cell necrosis, release of liver-associated enzymes, increased hepatocyte proliferation, and hepatocarcinogenesis. For carcinogens whose proposed mode of action is cytolethality, it is hypothesized that the dose–response curve for tumor development would parallel the dose–response curve for cell death with compensatory proliferation in the target organ. To prospectively test this hypothesis, female B6C3F₁ mice were exposed to furan at carcinogenic doses and lower for 3 weeks or 2 years. At 3 weeks and in the 2-year study, there were dose-dependent and significant increases in hepatic cytotoxicity at 1.0, 2.0, 4.0, and 8.0 mg furan/kg. For cell proliferation as measured by 5-bromo-2′-deoxyuridine (BrdU) labeling index (LI), there was a statistically significant trend with increasing dose levels of furan and increased LI at 8.0 mg/kg. There was an increased incidence of foci of altered hepatocytes, hepatocellular adenomas, and adenomas or carcinomas at 4.0 and 8.0 mg/kg and carcinomas at 8.0 mg/kg. The multiplicity of microscopic tumors was increased and latency was decreased in mice exposed to 8.0 mg/kg. Prevalence of hepatic nodules at necropsy was increased in mice exposed to 4.0 and 8.0 mg/kg. Data demonstrate an association among furan-induced hepatic cytotoxicity, compensatory cell replication, and liver tumor formation in mice; at high doses ?4.0 mg/kg, furan induced hepatotoxicity, compensatory cell replication and tumorigenesis in a dose-related manner, while furan did not produce tumors at cytotoxic doses of 1.0 and 2.0 mg/kg.     

Hepatocellular proliferation and hepatocarcinogen bioactivation in mice with diet-induced fatty liver and obesity

Human liver cancer is in part associated with obesity and related metabolic diseases. The present study was undertaken in a mouse model of diet-induced obesity (DIO) and hepatic steatosis, conditions which can be associated with hepatic neoplasia, to determine whether the rates of cell proliferation or hepatocarcinogen bioactivation were altered in ways which could facilitate hepatocarcinogenesis. DIO mice were generated by feeding C57BL/6 (B6) male mice a high-fat diet beginning at 4 weeks of age; age-matched conventional lean (LEAN) B6 mice fed a low fat diet (10% Kcal from fat) were used for comparison. Groups of 28 week old DIO and LEAN mice were dosed with the bioactivation-dependent DNA-reactive hepatocarcinogen 2-acetylaminofluorene (AAF), at 2.24 or 22.4 mg/kg, given by gavage 3 times per week for 31 days, or received no treatment (DIO and LEAN control groups). Compared with the LEAN control group, the DIO control group had a higher mean body weight (16.5 g), higher mean absolute (1.4 g) and mean relative (25.5%) liver weights, higher (394%) liver triglyceride concentrations, and an increased incidence and severity of hepatocellular steatosis at the end of the dosing phase. The DIO control group also had a higher mean hepatocellular replicating fraction (31% increase, determined by proliferating cell nuclear antigen immunohistochemistry). Hepatocarcinogen bioactivation, based on formation of AAF DNA adducts as measured by nucleotide ³²P-postlabeling, was similar in both DIO and LEAN AAF-dosed groups. Thus, hepatocellular proliferation, but not hepatocarcinogen bioactivation, was identified as an alteration in livers of DIO mice which could contribute to their susceptibility to hepatocarcinogenesis.     

Pentraxin 3 as a potential biomarker of acetaminophen-induced liver injury

ObjectiveOverdose of acetaminophen (APAP) can lead to severe liver injury in humans and experimental animals. Pentraxin-3 (PTX-3) is produced and released by several cell types. In this study, we aimed to evaluate whether PTX-3 is a potential biomarker in the identification of APAP-induced liver injury.Materials and methodsThirty adult male Wistar rats were randomly divided into three groups: control, APAP-1 and APAP-2 groups. APAP-1 (1 g/kg) and APAP-2 (2 g/kg) group rats were given APAP by gastric tube. Liver tissues and blood samples were obtained for biochemical and histopathological analysis. Biochemical parameters, plasma and liver PTX-3 levels and degree of liver necrosis were measured in all groups.ResultsAPAP treatments caused necrosis in liver and accompanied by elevated liver PTX-3 levels after 48 h. In APAP-1 and APAP-2 groups when compared with control group (7.5 ± 3.3 ng/mg protein), mean liver PTX-3 concentrations were 14.1 ± 3.0 (p = 0.032) and 28.5 ± 8.2 (p < 0.001) ng/mg protein, respectively. All rats (100%) in the APAP-2 group had the degree 3 liver necrosis. However 10%, 40% and 50% of rats had the degree 1, the degree 2 and the degree 3 liver necrosis in the APAP-1 group, respectively. The degrees of liver necrosis of the APAP-1 and APAP-2 groups were higher than the group of control (p < 0.001 and p < 0.001, respectively).ConclusionsPTX-3 may have a role in the APAP-induced liver injury in the rats. The elevated liver PTX-3 in the APAP-induced hepatic necrosis might be a marker of acute histological liver damage. Further prospective studies are necessary to clarify the prognostic value of liver PTX-3 for prediction of histological hepatic necrosis in the APAP-induced liver injury.     

The age- and gender-dependent effects of desflurane and sevoflurane on rat liver

ObjectiveThis study aimed to investigate the age- and gender-dependent effects of desflurane and sevoflurane on the liver.Material and methodUpon the approval of ethics committee, 84 rats were divided into four groups as 21 young male, 21 young female, 21 old male, and 21 old female rats. Then, each group was further divided into three groups as desflurane, sevoflurane, and control groups. Maintaining the minimum alveolar concentration of 1, desflurane at 6 vol% and sevoflurane at 2 vol% in 6 L min^?1 100% O₂ were administered for 2 h in a transparent plastic container of 40 cm×40 cm×70 cm. Each liver preparation was evaluated for hydropic degeneration, nuclear polymorphism, portal neutrophile infiltration, portal lymphocyte infiltration, and focal necrosis, and each preparation was assigned injury points of 0–3; thus, the number of histopathologically injured cases, total injury scores of each preparation, and the mean injury scores of each group were determined.ResultsDesflurane and sevoflurane did not significantly increase hepatic injury in the young male rats, while both agents caused significantly more hepatic injury in the young female rats. In the old rats, both desflurane and sevoflurane inflicted more hepatic injury on both genders. In addition, desflurane caused more hepatic injury in the old female rats than in the young female or the old male rats.ConclusionHepatic injury associated with desflurane and sevoflurane was mild to moderate, suggesting that both agents can be safely used in routine anaesthesia procedures.     

Mechanical properties of bioactive glass (13-93) scaffolds fabricated by robotic deposition for structural bone repair

There is a need to develop synthetic scaffolds to repair large defects in load-bearing bones. Bioactive glasses have attractive properties as a scaffold material for bone repair, but data on their mechanical properties are limited. The objective of the present study was to comprehensively evaluate the mechanical properties of strong porous scaffolds of silicate 13-93 bioactive glass fabricated by robocasting. As-fabricated scaffolds with a grid-like microstructure (porosity 47%, filament diameter 330 μm, pore width 300 μm) were tested in compressive and flexural loading to determine their strength, elastic modulus, Weibull modulus, fatigue resistance, and fracture toughness. Scaffolds were also tested in compression after they were immersed in simulated body fluid (SBF) in vitro or implanted in a rat subcutaneous model in vivo. As fabricated, the scaffolds had a strength of 86 ± 9 MPa, elastic modulus of 13 ± 2 GPa, and a Weibull modulus of 12 when tested in compression. In flexural loading the strength, elastic modulus, and Weibull modulus were 11 ± 3 MPa, 13 ± 2 GPa, and 6, respectively. In compression, the as-fabricated scaffolds had a mean fatigue life of ～10⁶ cycles when tested in air at room temperature or in phosphate-buffered saline at 37 °C under cyclic stresses of 1–10 or 2–20 MPa. The compressive strength of the scaffolds decreased markedly during the first 2 weeks of immersion in SBF or implantation in vivo, but more slowly thereafter. The brittle mechanical response of the scaffolds in vitro changed to an elasto-plastic response after implantation for longer than 2–4 weeks in vivo. In addition to providing critically needed data for designing bioactive glass scaffolds, the results are promising for the application of these strong porous scaffolds in loaded bone repair.     

Adsorbed fibrinogen leads to improved bone regeneration and correlates with differences in the systemic immune response

Designing new biomaterials that can modulate the inflammatory response instead of attempting just to reduce it constitutes a paradigm change in regenerative medicine. This work aimed to investigate the capacity of an immunomodulatory biomaterial to enhance bone regeneration. For that purpose we incorporated a molecule with well-established pro-inflammatory and pro-healing roles, fibrinogen, in chitosan scaffolds. Two different incorporation strategies were tested, leading to concentrations of 0.54 ± 0.10 mg fibrinogen g^?1 scaffold immediately upon adsorption (Fg-Sol), and 0.34 ± 0.04 mg fibrinogen g^?1 scaffold after washing (Fg-Ads). These materials were implanted in a critical size bone defect in rats. At two months post-implantation the extent of bone regeneration was examined by histology and the systemic immune response triggered was evaluated by determining the percentages of myeloid cells, T and B lymphocytes in the draining lymph nodes. The results obtained indicate that the fibrinogen incorporation strategy conditioned the osteogenic capacity of biomaterials. Fg-Ads scaffolds led to more bone formation, and the presence of Fg stimulated angiogenesis. Furthermore, animals implanted with Fg-Ads scaffolds showed significant increases in the percentages of B lymphocytes and myeloid cells in the draining lymph nodes, while levels of T lymphocytes were not significantly different. Finally, a significant increase in TGF-β1 was detected in the plasma of animals implanted with Fg-Ads. Taken together the results presented suggest a potential correlation between the elicited immune response and biomaterial osteogenic performance.     

Transient disruption of liver gap junctional intercellular communication and induction of apoptosis after administration of 1,4-bis[2-(3,5 dichloropyridyloxy)]benzene in mice

Gap junctional intercellular communication (GJIC) and connexin expression (Cx26 and Cx32) in mouse liver were studied after administration of 4-bis[2-(3,5 dichloropyridyloxy)]benzene (TCPOBOP), a phenobarbital-like enzyme inducer. Female C57Bl/6 mice were administered TCPOBOP (5.8 mg/kg BW) and euthanized 0, 24, 48 and 72 hours later. Liver samples were snap frozen, or fixed in formalin, or submitted to GJIC analysis. The proliferating cell nuclear antigen (PCNA) immunohistochemistry and the Western blotting for Cx26 and Cx32 were performed. After 48 and 72 h of drug administration the liver-to-body weight ratio was increased 70% and 117% (p<0.0001), respectively. There were temporal-dependent alterations in liver histopathology and a significant increase in cell proliferation was noted after 48 h and sustained after 72 h, though to a lesser extent (p<0.0001). In addition, TCPOBOP administration induced apoptosis, which appeared to be time-dependent showing statistical significance only after 72 h (p<0.0001). Interestingly, a transient disruption by nearly 50% of GJIC capacity was detected after 48 h of drug ingestion, which recovered after 72 h (p=0.003). These GJIC changes were due to altered levels of Cx26 and Cx32 in the livers of TCPOBOP-treated mice. We concluded that a single administration of TCPOBOP transiently disrupted the levels of GJIC due to decreased expression of connexins and increased apoptotic cell death in mouse liver.     

Evaluation of bone regeneration in implants composed of hollow HA microspheres loaded with transforming growth factor β1 in a rat calvarial defect model

Implants that serve simultaneously as an osteoconductive matrix and as a device for local growth factor delivery may be required for optimal bone regeneration in some applications. In the present study, hollow hydroxyapatite (HA) microspheres (106–150 μm) in the form of three-dimensional (3-D) scaffolds or individual (loose) microspheres were created using a glass conversion process. The capacity of the implants, with or without transforming growth factor β1 (TGF-β1), to regenerate bone in a rat calvarial defect model was compared. The 3-D scaffolds supported the proliferation and alkaline phosphatase activity of osteogenic MLO-A5 cells in vitro, showing their cytocompatibility. Release of TGF-β1 from the 3-D scaffolds into phosphate-buffered saline ceased after 2–3 days when ～30% of the growth factor was released. Bone regeneration in the 3-D scaffolds and the individual microspheres increased with time from 6 to 12 weeks, but it was significantly higher (23%) in the individual microspheres than in the 3-D scaffolds (15%) after 12 weeks. Loading with TGF-β1 (5 μg per defect) enhanced bone regeneration in the 3-D scaffolds and individual microspheres after 6 weeks, but had little effect after 12 weeks. 3-D scaffolds and individual microspheres with larger HA diameter (150–250 μm) showed better ability to regenerate bone. Based on these results, implants composed of hollow HA microspheres show promising potential as an osteoconductive matrix for local growth factor delivery in bone regeneration.     

Repairing a critical-sized bone defect with highly porous modified and unmodified baghdadite scaffolds

This is the first reported study to prepare highly porous baghdadite (Ca₃ZrSi₂O₉) scaffolds with and without surface modification and investigate their ability to repair critical-sized bone defects in a rabbit radius under normal load. The modification was carried out to improve the mechanical properties of the baghdadite scaffolds (particularly to address their brittleness) by coating their surfaces with a thin layer (～400 nm) of polycaprolactone (PCL)/bioactive glass nanoparticles (nBGs). The β-tricalcium phosphate/hydroxyapatite (TCP/HA) scaffolds with and without modification were used as the control groups. All of the tested scaffolds had an open and interconnected porous structure with a porosity of ～85% and average pore size of 500 μm. The scaffolds (six per scaffold type and size of 4 mm × 4 mm × 15 mm) were implanted (press-fit) into the rabbit radial segmental defects for 12 weeks. Micro-computed tomography and histological evaluations were used to determine bone ingrowth, bone quality, and implant integration after 12 weeks of healing. Extensive new bone formation with complete bridging of the radial defect was evident with the baghdadite scaffolds (modified/unmodified) at the periphery and in close proximity to the ceramics within the pores, in contrast to TCP/HA scaffolds (modified/unmodified), where bone tended to grow between the ulna adjacent to the implant edge. Although the modification of the baghdadite scaffolds significantly improved their mechanical properties, it did not show any significant effect on in vivo bone formation. Our findings suggest that baghdadite scaffolds with and without modification can serve as a potential material to repair critical sized bone defects.     

In vivo evaluation of microspheres containing the angiogenesis inhibitor,TNP-470, and the metastasis suppression with liver metastatic model implanted neuroblastoma

TNP-470 (AGM-1470, O-(chloroacetylcarbamoyl) fumagillol), which strongly inhibits the angiogenesis, is promising as a new drug for tumor dormancy therapy; however, TNP-470 is very unstable in vitro and in vivo. We previously prepared TNP-470 containing microspheres composed of poly (lactic acid) with medium-chain triglyceride, and demonstrated that the microspheres released TNP-470 over the long-term in vitro. The present study was undertaken to evaluate the release profile of TNP-470 in vivo and the inhibitory effect on hepatic metastasis of neuroblastoma. It was found that the microspheres could maintain high levels of TNP-470 in the blood plasma for over 4 weeks in vivo. In addition, hepatic metastasis of neuroblastoma was strongly inhibited at 2 weeks after intraperitoneal injection of the microspheres. Following 2 weeks of treatment, the liver weights of mice injected with TNP-DDS (TNP-DDS (H), and TNP-DDS (L) groups) and those injected with only physiological saline (C-1300 group) after implantation of neuroblastoma cells were 1.18 ± 0.13 g, 1.28 ± 0.10 g, and 2.54 ± 0.97 g, respectively (p < 0.05; C-1300 group compared with the TNP-DDS (H) and the TNP-DDS (L) groups, respectively). It was evident that microspheres containing TNP-470 have an excellent potential for clinical application in tumor dormancy therapy.     

Characterization of liver toxicity in F344/N rats and B6C3F1 mice after exposure to a flame retardant containing lower molecular weight polybrominated diphenyl ethers

Lower molecular weight polybrominated diphenyl ethers (PBDEs), components of flame retardants, are found in the environment and in human and animal tissues. Toxicity studies were conducted in F344/N rats and B6C3F1 mice by administering a flame retardant containing these lower molecular weight PBDEs (BDE-47, BDE-99, BDE-100, and BDE153) by oral gavage 5 days/week for 13 weeks at doses of 0.01, 5, 50, 100 or 500 mg/kg/day. Liver was the primary target organ in rats and mice. Treatment-related increases in liver weights, liver cytochrome P450 (1A1, 1A2, 2B) and UDPGT (rats only) levels, and liver lesions were seen in both rats and mice. Hepatocyte hypertrophy and vacuolization increased in incidence and severity with treatment, and occurred at levels of 50 mg/kg and above in rats, and at 100 mg/kg and above in mice. Liver Cyp 1A1, 1A2, and 2B levels were increased at exposure levels of 50 mg/kg and above in rats and mice. In addition, treatment-related thyroid lesions occurred particularly in rats. The most sensitive parameter for PBDE toxicity was the increase in liver weights which occurred at 5 mg/kg above in rats and 50 mg/kg and above in mice. These results suggest that liver may be a target organ for carcinogenesis processes after long-term administration of PBDEs. A chronic PBDE study is currently being conducted by the National Toxicology Program.     

Role of connexin 32 in acetaminophen toxicity in a knockout mice model

Gap junctional intercellular communication (GJIC), by which glutathione (GSH) and inorganic ions are transmitted to neighboring cells, is recognized as being largely involved in toxic processes of chemicals. We examined acetaminophen (APAP)-induced hepatotoxicity clinicopathologically using male wild-type mice and mice lacking the gene for connexin32, a major gap junction protein in the liver [knockout (Cx32KO) mice]. When APAP was intraperitoneally administered at doses of 100, 200, or 300 mg/kg, hepatic centrilobular necrosis with elevated plasma aminotransferase activities was observed in wild-type mice receiving 300 mg/kg, and in Cx32KO mice given 100 mg/kg or more. At 200 mg/kg or more, hepatic GSH and GSSG contents decreased significantly and the effect was more severe in wild-type mice than in Cx32KO mice. On the other hand, markedly decreased GSH staining was observed in the hepatic centrilobular zones of Cx32KO mice compared to that of wild-type mice. These results demonstrate that Cx32KO mice are more susceptible to APAP hepatotoxicity than wild-type mice, and indicate that the distribution of GSH of the centrilobular zones in the hepatic lobules, rather than GSH and GSSG contents in the liver, is important in APAP hepatotoxicity. In conclusion, Cx32 protects against APAP-induced hepatic centrilobular necrosis in mice, which may be through the GSH transmission to neighboring hepatocytes by GJIC.     

Preventive effect of a galactoglucomannan (GGM) from Dendrobium huoshanense on selenium-induced liver injury and fibrosis in rats

This study was carried out to investigate the preventive effects of galactoglucomannan (GGM), a homogeneous polysaccharide from Dendrobium huoshanense, on liver injury and fibrosis induced by sodium selenite. Sprague–Dawley rats injected subcutaneously with sodium selenite at the dosage of 3.28 mg kg^?1 b. wt. were set as the model groups. Rats treated with sodium selenite at the dosage of 3.28 mg kg^?1 b. wt. and GGM at 50–200 mg kg^?1 b. wt. were set as the prevention groups. Biochemical and histological analysis showed that GGM significantly ameliorated selenite-induced liver injury and fibrosis in rats. Oral administration of GGM effectively attenuated the toxicity of selenite to liver tissue, which was judged both by the decreased activities of serum hepatic enzymes, including alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH), and by liver histopathological examination. Meanwhile, GGM also reduced the levels of H₂O₂ and malondialdehyde (MDA), elevated the levels of GSH, restored the fluidity of hepatic plasma membrane, and retained the activities of endogenous enzymes including superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST). The prevention of selenite-induced liver injury and fibrosis by GGM was further supported by the reduced expression of transforming growth factor-β1 (TGF-β1) and type I collagen. These results suggested that GGM may be developed into a novel antifibrotic agent for the prevention of liver injury and fibrosis.     

The effects of royal jelly on liver damage induced by paracetamol in mice

The present study was undertaken to investigate the protective effect of royal jelly against paracetamol-induced liver damage. The study was conducted in 90 female Swiss Albino mice, and six groups were established. While the first group was maintained as control, Groups 2–6 were administered 200 mg/kg RJ for 1 day, 200 mg/kg RJ for 7 days, 400 mg/kg PAR for 1 day, 200 mg/kg RJ plus 400 mg/kg PAR for 1 day and 200 mg/kg RJ for 7 days and then second 400 mg/kg PAR on the 7th day, orally, respectively. It was shown that PAR significantly increased serum ALT, AST, ALP, liver MDA levels and significantly decreased liver GSH-Px activity, when compared to the control group (Group 1). On the other hand, meaningful changes were observed in the biochemical parameters of the group which was administered long-term RJ (Group 6). The aforementioned parameters which were statistically significant were determined to have drawn closer to values of the control group, and among these, the existing statistical differences for MDA level and GSH-Px activity between the trial group (Group 6) and the control group disappeared (Group 1). Compared to the pathological changes observed in the liver parenchyma, remark cords, sinusoids and hepatocytes in the group which was administered paracetamol alone (Group 4), lesions were determined to be less severe particularly in the group (Group 6) which received royal jelly for 7 days prior to paracetamol. In conclusion, the administration of royal jelly as a hepatoprotective agent for 7 days against paracetamol-induced liver damage was determined to exhibit marked protective effect on liver tissue.     

A biaxial rotating bioreactor for the culture of fetal mesenchymal stem cells for bone tissue engineering

The generation of effective tissue engineered bone grafts requires efficient exchange of nutrients and mechanical stimulus. Bioreactors provide a manner in which this can be achieved. We have recently developed a biaxial rotating bioreactor with efficient fluidics through in-silico modeling. Here we investigated its performance for generation of highly osteogenic bone graft using polycaprolactone–tricalcium phosphate (PCL–TCP) scaffolds seeded with human fetal mesenchymal stem cell (hfMSC). hfMSC scaffolds were cultured in either bioreactor or static cultures, with assessment of cellular viability, proliferation and osteogenic differentiation in vitro and also after transplantation into immunodeficient mice. Compared to static culture, bioreactor-cultured hfMSC scaffolds reached cellular confluence earlier (day 7 vs. day 28), with greater cellularity (2×, p < 0.01), and maintained high cellular viability in the core, which was 2000 μm from the surface. In addition, bioreactor culture was associated with greater osteogenic induction, ALP expression (1.5× p < 0.01), calcium deposition (5.5×, p < 0.001) and bony nodule formation on SEM, and in-vivo ectopic bone formation in immunodeficient mice (3.2×, p < 0.001) compared with static-cultured scaffolds. The use of biaxial bioreactor here allowed the maintenance of cellular viability beyond the limits of conventional diffusion, with increased proliferation and osteogenic differentiation both in vitro and in vivo, suggesting its utility for bone tissue engineering applications.     

Expression patterns of heat shock protein 25 in carbon tetrachloride-induced rat liver injury

Heat shock protein 25 (Hsp25) is a molecular chaperone playing roles in cytoprotection. We investigated the distribution and localization of Hsp25 expression in CCl₄-induced rat hepatic lesions; liver samples were obtained from 3 h to 10 days after a single oral administration of CCl₄. Immunohistochemically, Hsp25-positive hepatocytes started to appear in the perivenular area at 6 h after CCl₄ administration. Their number and strength increased till day 1. Expression of Hsp25 mRNA significantly increased after 3 h and proceeded to increase with time till day 1. Apoptotic hepatocytes were detected around the perivenular area after 6 h. The area where Hsp25-positive hepatocytes were observed till day 1 corresponded to the area where apoptotic hepatocytes were seen. On days 2 and 3, degenerative and/or necrotic hepatocytes in the perivenular area were replaced by macrophages reacting to ED1 (for CD68) and ED2 (for CD163); Hsp25 expression was seen in hepatocytes around the perivenular area and there was a close relationship of reactive macrophages with Hsp25-positive hepatocytes, suggesting a potential role for Hsp25 in suppressing injury by inflammation. The mRNA expression of tumor necrosis factor-α, monocyte chemoattractant protein-1 and osteopontin, which can be produced by infiltrating macrophages, corresponded to that of Hsp25 from day 1 to day 3; these factors might be related to the induction of Hsp25 expression. The shift of the Hsp25 expression pattern in the liver lesion might have depended on microenvironmental conditions evoked by interactions between necrobiotic hepatocytes and infiltrating macrophages. Thus, Hsp25 expression analyses should be beneficial for evaluations of hepatotoxicants.     

Digital micromirror device projection printing system for meniscus tissue engineering

Meniscus degeneration due to age or injury can lead to osteoarthritis. Although promising, current cell-based approaches show limited success. Here we present three-dimensional methacrylated gelatin (GelMA) scaffolds patterned via projection stereolithography to emulate the circumferential alignment of cells in native meniscus tissue. Cultured human avascular zone meniscus cells from normal meniscus were seeded on the scaffolds. Cell viability was monitored, and new tissue formation was assessed by gene expression analysis and histology after 2 weeks in serum-free culture with transforming growth factor β1 (10 ng ml^?1). Light, confocal and scanning electron microscopy were used to observe cell–GelMA interactions. Tensile mechanical testing was performed on unseeded, fresh scaffolds and 2-week-old cell-seeded and unseeded scaffolds. 2-week-old cell–GelMA constructs were implanted into surgically created meniscus defects in an explant organ culture model. No cytotoxic effects were observed 3 weeks after implantation, and cells grew and aligned to the patterned GelMA strands. Gene expression profiles and histology indicated promotion of a fibrocartilage-like meniscus phenotype, and scaffold integration with repair tissue was observed in the explant model. We show that micropatterned GelMA scaffolds are non-toxic, produce organized cellular alignment, and promote meniscus-like tissue formation. Prefabrication of GelMA scaffolds with architectures mimicking the meniscus collagen bundle organization shows promise for meniscal repair. Furthermore, the technique presented may be scaled up to repair larger defects.     

Response of liver to lipopolysaccharide treatment in male and female rats

Gender is considered to be an important factor in endotoxin-induced tissue damage. Our aim was to examine the role of sex on the prooxidant–antioxidant status, necrotic and apoptotic events in the liver of lipopolysaccharide (LPS)-treated rats. We determined levels of lipid peroxides, non-enzymatic and enzymatic antioxidants, and expressions of apoptosis-related proteins, antiapoptotic B cell lymphoma-2 (Bcl-2) and proapoptotic Bax, caspase-3 activity and apoptotic cell numbers in the liver. Hepatic histopathology and serum alanine transaminase (ALT) and aspartate transaminase (AST) activities were also investigated. Male and female Wistar rats (180–200 g) were injected with LPS (10 mg/kg, i.p.) and examinations were performed 6 h after the injection. Significant increases in hepatic thiobarbituric acid reactive substances and diene conjugate levels were observed in male and female rats following LPS treatment. However, there were no changes in hepatic glutathione, vitamin E and vitamin C levels together with superoxide dismutase, glutathione peroxidase and glutathione transferase activities. LPS treatment caused significant increases in serum ALT and AST activities and lymphocyte infiltration and necrotic changes in the livers. Bcl-2 and Bax expressions, caspase-3 activity and apoptotic cell numbers were also found to be increased in both groups. In conclusion, no sex-dependent difference was observed in the changed hepatic prooxidant–antioxidant status of rats following LPS treatment. Besides, the process leading to apoptosis and necrosis in the liver showed a similar pattern in both gender of rats.     

The effect of BMP-2 on micro- and macroscale osteointegration of biphasic calcium phosphate scaffolds with multiscale porosity

It is well established that scaffolds for applications in bone tissue engineering require interconnected pores on the order of 100 μm for bone in growth and nutrient and waste transport. As a result, most studies have focused on scaffold macroporosity (>100 μm). More recently researchers have investigated the role of microporosity in calcium phosphate -based scaffolds. Osteointegration into macropores improves when scaffold rods or struts contain micropores, typically defined as pores less than ～50 μm. We recently demonstrated multiscale osteointegration, or growth into both macropores and intra-red micropores (<10 μm), of biphasic calcium phosphate (BCP) scaffolds. The combined effect of BMP-2, a potent osteoinductive growth factor, and multiscale porosity has yet to be investigated. In this study we implanted BCP scaffolds into porcine mandibular defects for 3, 6, 12 and 24 weeks and evaluated the effect of BMP-2 on multiscale osteointegration. The results showed that given this in vivo model BMP-2 influences osteointegration at the microscale, but not at the macroscale, but not at the macroscale. Cell density was higher in the rod micropores for scaffolds containing BMP-2 compared with controls at all time points, but BMP-2 was not required for bone formation in micropores. In contrast, there was essentially no difference in the fraction of bone in macropores for scaffolds with BMP-2 compared with controls. Additionally, bone in macropores seemed to have reached steady-state by 3 weeks. Multiscale osteointegration results in bone-scaffold composites that are fully osteointegrated, with no ‘dead space’. These composites are likely to contain a continuous cell network as well as the potential for enhanced load transfer and improved mechanical properties.     

Preparation and characterization of collagen-based ADSC-carrier sheets for cardiovascular application

The use of scaffolds composed of natural biodegradable matrices represents an attractive strategy to circumvent the lack of cell engraftment, a major limitation of stem cell therapy in cardiovascular diseases. Bovine-derived non-porous collagen scaffolds with different degrees of cross-linking (C0, C2, C5 and C10) were produced and tested for their mechanical behavior, in vitro biocompatibility with adipose-derived stem cells (ADSCs) and tissue adhesion and inflammatory reaction. Uniaxial tensile tests revealed an anisotropic behavior of collagen scaffolds (2 × 0.5 cm) and statistically significant differences in the mechanical behavior between cross-linked and non-cross-linked scaffolds (n = 5). In vitro, ADSCs adhered homogenously and showed a similar degree of proliferation on all four types of scaffolds (cells × 10³ cm^?2 at day 7: C0: 94.7 ± 37.1; C2: 91.7 ± 25.6; C5: 88.2 ± 6.8; C10: 72.8 ± 10.7; P = n.s.; n = 3). In order to test the in vivo biocompatibility, a chronic myocardial infarction model was performed in rats and 1.2 × 1.2 cm size collagen scaffolds implanted onto the heart 1 month post-infarction. Six animals per group were killed 2, 7 and 30 days after transplant. Complete and long-lasting adhesion to the heart was only observed with the non-cross-linked scaffolds with almost total degradation 1 month post-transplantation. After 7 and 30 days post-implantation, the degree of inflammation was significantly lower in the hearts treated with non-cross-linked scaffolds (day 7: C0: 10.2 ± 2.1%; C2: 16.3 ± 2.9%; C5: 15.9 ± 4.8%; C10: 17.4 ± 4.1%; P < 0.05 vs. C0; day 30: C0: 1.3 ± 1.3%; C2: 9.4 ± 3.0%; C5: 7.0 ± 2.1%; C10: 9.8 ± 2.5%; P < 0.01 vs. C0). In view of the results, the non-cross-linked scaffold (C0) was chosen as an ADSC-carrier sheet and tested in vivo. One week post-implantation, 25.3 ± 7.0% of the cells transplanted were detected in those animals receiving the cell-carrier sheet whereas no cells were found in animals receiving cells alone (n = 3 animals/group).We conclude that the biocompatibility and mechanical properties of the non-cross-linked collagen scaffolds make them a useful cell carrier that greatly favors tissue cell engraftment and may be exploited for cell transplantation in models of cardiac disease.