Decellularization and genipin crosslinking of amniotic membrane suitable for tissue engineering applications

Abstract

Amniotic membrane has the potential to be used as scaffold in various tissue engineering applications. However, increasing its biostability at the same time maintaining its biocompatibility is important to enhance its usage as a scaffold. This studied characteristics genipin-crosslinked amniotic membrane as a bioscaffold. Redundant human amniotic membranes (HAM) divided into native (nAM), decellularized (dAM) and genipin-crosslinked (clAM) groups. The dAM and clAM group were decellularized using thermolysin (TL) and sodium hydroxide (NaOH) solution. Next, clAM group was crosslinked with 0.5% and 1.0% (w/v) genipin. The HAM was then studied for in vitro degradation, percentage of swelling, optical clarity, ultrastructure and mechanical strength. Meanwhile, fibroblasts isolated from nasal turbinates were then seeded onto nAM, dAM and clAM for biocompatibility studies. clAM had the slowest degradation rate and were still morphologically intact after 30 days of incubation in 0.01% collagenase type 1 solution. The dAM had a significantly highest percentage of swelling than other groups (p?<?0.05). Besides, the dAM retained the collagen content at similar level of nAM. Although the dAM had highest mechanical strength compared to the rest of the groups, the differences were statistically insignificant. Cell attachment on dAM and 0.5% clAM was higher compared to that on nAM and 1.0% clAM. In conclusion, clAM have better biostability and biocompatibility compared to the nAM and dAM. Together with other suitable characteristics of the clAM such as percentage of swelling, structural integrity and ECM content, clAM is suitable as scaffold for various tissue engineering applications.     

Oral silibinin inhibits in vivo human bladder tumor xenograft growth involving down-regulation of survivin.

PURPOSE: Chemoprevention is an upcoming approach to control bladder cancer, which is one of the commonly diagnosed malignancies showing recurrence rate of 70% or even higher. Recently, we observed the in vitro efficacy of silibinin, a flavanolignan, in human bladder transitional cell papilloma RT4 cells. Here, we investigated the antitumor efficacy and associated mechanisms of silibinin in RT4 tumor xenograft. EXPERIMENTAL DESIGN: RT4 tumor xenograft was implanted s.c. in athymic nude mice, and then animals were oral gavaged with silibinin at 100 and 200 mg/kg doses, 5 days/week for 12 weeks. Tumor growth, body weight, and diet consumption were recorded, and tumors were analyzed for proliferation, apoptosis, and angiogenesis biomarkers and molecular alterations by immunohistochemistry, immunoblot analysis, and ELISA. p53 small interfering RNA was used in cell culture to examine the role of p53 in survivin expression. RESULTS: Silibinin feeding inhibited tumor xenograft growth without any gross signs of toxicity. Silibinin decreased tumor volume by 51% to 58% (P 相似文献   

Mechanical Properties of Aramid/Carbon Hybrid Fiber-Reinforced Concrete

In this study, aramid fiber (Kevlar^® 29 fiber) and carbon fiber were added into concrete in a hybrid manner to enhance the static and impact mechanical properties. The coupling agent presence on the surface of carbon fibers was spotted in Scanning Electron Microscope (SEM) and energy-dispersive X-ray spectroscopy (EDS) graphs. The carbon fiber with a coupling agent affected the mechanical strength of the reinforced concrete. At 1% fiber/cement weight percentage, the hybrid fiber-reinforced concrete (HFRC) prepared using Kevlar fiber and carbon fiber of 12 and 24 mm in length under different mix proportions was investigated to determine the maximum mechanical strengths. From the test results, the mechanical strength of the HFRC attained better performance than that of the concrete with only Kevlar or carbon fibers. Foremost, the mix proportion of Kevlar/carbon fiber (50–50%) significantly improved the compressive, flexural, and splitting tensile strengths. Under different impact energies, the impact resistance of the HFRC specimen was much higher than that of the benchmark specimen, and the damage of the HFRC specimens was examined with an optical microscope to identify slippage or rupture failure of the fiber in concrete.     

Studies on bioprospecting potential of a gastropod mollusc Cantharus tranquebaricus (Gmelin, 1791)

Objective

To study the biological activities of the tissue extract of Cantharus tranquebaricus (C. tranquebaricus).

Methods

Crude extract of gastropod was tested for inhibition of bacterial growth. Antibacterial assay was carried out by disc diffusion method and the activity was measured accordingly based on the inhibition zone around the disc impregnated with gastropod extract. Molecular weight of the extract was determined by using SDS-PAGE. Plasma coagulation, Fibrin plate assay and substrate SDS-PAGE were used to determine the effect of sample on plasma coagulation, fibrin (ogen) olytic and proteolytic activity.

Results

The maximum inhibition zone (10 mm) was observed against Vibrio cholera (V. cholera) and minimum inhibition zone (2 mm) was noticed against Proteus mirablis (P. mirablis). The molecular weight was determined as 47-106 kDa. The tissue extract shows proteolytic activity above 48 kDa. SDS-PAGE analysis of fibrinogen after incubation with the tissue extract showed fibrinogenolytic activity. In plasma coagulation assay C. tranquebaricus tissue extract showed procoagulant property and it coagulated chicken plasma within 150 s, while control took 5 min to clot. The 9 HU hemolytic units were found against chicken blood and also exhibit high level of brine shrimp lethality.

Conclusions

This study suggests that C. tranquebaricus could be used as potential source for isolating bioactive compounds, since it is explored first time and found with promising results.     

Differential effect of silibinin on E2F transcription factors and associated biological events in chronically UVB-exposed skin versus tumors in SKH-1 hairless mice

UVB radiation-induced DNA damage in skin activates cellular pathways involved in DNA repair, cell cycle regulation, and apoptosis, important events that prevent conversion of damaged skin cells into cancer. We reported recently the efficacy of silibinin against photocarcinogenesis along with altered molecular events in tumors (Cancer Research, 64:6349-56, 2004). The molecular and biological events modulated by silibinin in chronically UVB-irradiated skin leading to cancer prevention, however, are not known. Herein, we describe effect of silibinin on skin 15 and 25 weeks after UVB exposure and compared them with molecular alterations in skin tumors. UVB decreased E2F1 but increased E2F2 and E2F3 protein levels in skin, and these were reversed by silibinin treatment. Silibinin-induced E2F1 was accompanied by an inhibition of apoptosis and decreases in p53 and cyclin-dependent kinase inhibitors. Silibinin-caused decrease in E2F2 and E2F3 was accompanied by reduced levels of cyclin-dependent kinases, cyclins, CDC25C, and mitogen-activated protein kinases and Akt signaling and inhibition of cell proliferation. In tumorigenesis protocols, topical or dietary silibinin significantly inhibited tumor appearance and growth. As opposed to UVB-exposed skin, UVB-induced tumors showed elevated levels of E2F1, but these were reduced in silibinin-treated tumors without any effect on E2F2 and E2F3. Contrary to the inhibition of apoptosis and p53 expression in UVB-exposed skin cells, silibinin increased these variables in tumors. These differential effects of silibinin on E2F1 versus E2F2 and E2F3 and their associated molecular alterations and biological effects in chronic UVB-exposed skin suggest their role in silibinin interference with photocarcinogenesis.     

Silibinin inhibits ultraviolet B radiation-induced mitogenic and survival signaling, and associated biological responses in SKH-1 mouse skin

Ultraviolet B (UVB) radiation is a complete skin carcinogen causing DNA damage as a tumor-initiating event and activating signaling cascades that play a critical role in its tumor-promoting potential. Recently we reported that a naturally occurring flavonoid, silibinin, protects UVB-induced skin damages and prevents photocarcinogenesis. Here we examined silibinin efficacy on acute and chronic UVB-caused mitogen-activated protein kinases (MAPKs) and AKT activation and associated biological responses in SKH-1 hairless mouse skin. A single UVB exposure at 180 mJ/cm2 dose resulted in varying degrees of ERK1/2, JNK1/2, MAPK/p38 and AKT phosphorylation at various time-points in mouse skin; however, topical application of silibinin prior to or immediately after UVB exposure, or its dietary feeding strongly inhibited the activation of these molecules at all the time-points examined. Stronger effects of silibinin towards inhibition of UVB-caused phosphorylation of MAPKs and AKT were also observed in a chronic UVB (180 mJ/cm2/day for 5 days) exposure protocol. Immunohistochemical analysis of chronically exposed skin sections showed that silibinin treatment in all three protocols increases UVB-induced p53-positive cells and decreases UVB-caused cell proliferation, apoptotic and sunburn cells. These findings suggest that silibinin inhibits UVB-induced MAPK and AKT signaling and increases p53 in mouse skin, and that these effects of silibinin possibly lead to a decrease in UVB-caused proliferation and apoptosis, which might, in part, be responsible for its overall efficacy against photocarcinogenesis.     

AMP-Activated Protein Kinase Signaling Protects Oligodendrocytes that Restore Central Nervous System Functions in an Experimental Autoimmune Encephalomyelitis Model

AMP-activated protein kinase (AMPK) signaling is reported to protect neurons under pathologic conditions; however, its effect on oligodendrocytes (OLs) remains to be elucidated. We investigated whether AMPK signaling protects OLs to restore central nervous system (CNS) functions in an experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis. Increased inflammation and demyelination in the CNS and peripheral immune responses were consistent with the observed clinical impairments in EAE animals, which were attenuated by treatment with metformin compared with vehicle. In addition, expressions of neurotrophic factors and of signatory genes of OL lineages were increased in the CNS of metformin-treated EAE animals. Likewise, metformin attenuated inflammatory response and enhanced expressions of neurotrophic factors, thereby protecting OLs via AMPK activation in mixed glial cultures stimulated with lipopolysaccharide/interferon γ in vitro, as evidenced by analysis of the expression of signatory genes of O1⁺/MBP⁺ OLs and their cellular populations. Metformin also attenuated oxidative stress and malondialdehyde-containing protein levels, with corresponding induction of antioxidative defenses in OLs exposed to cytokines via AMPK activation. These effects of metformin were evident in the CNS of EAE animals. These data provide evidence that AMPK signaling is crucial to protect OLs and, thus, CNS functions in EAE animals. We conclude that AMPK activators, including metformin, have the potential to limit neurologic deficits in multiple sclerosis and related neurodegenerative disorders.The critical role of oligodendrocytes (OLs) in producing and maintaining the myelin sheath around axons that supports rapid salutatory conduction in central nervous system (CNS) neurons is well established.¹ In addition, OLs are reported to provide trophic factors and metabolic support for neurons.^2–4 The CNS has the inherent capacity to generate myelin-forming OLs after episodes of demyelination under various pathologic conditions.⁵ Persistent demyelination is one of the pathologic hallmarks of multiple sclerosis (MS) and spinal cord (SC) trauma, which is responsible for neurologic deficits in affected individuals.^6,7 Therefore, a major question in neurobiology today is how to protect OLs in CNS demyelinating diseases, including MS, to limit neurologic deficits in affected individuals.Pro- and anti-inflammatory cytokines are known to play critical roles in the survival and generation of myelin-forming OLs that are lost after a demyelination attack.^8,9 OLs are required for the induction of remyelination to restore the structural integrity of the axon-myelin unit and axonal conduction properties in the MS brain.⁹ Proinflammatory cytokine signaling is reported to induce OL death and is antagonized by anti-inflammatory cytokine signaling mechanisms.^10–13 In MS brain lesions, spontaneous remyelination follows the demyelination attack in acute lesions.⁷ In contrast, remyelination fails in chronic lesions, irrespective of the presence of OL progenitor cells (OPCs).¹⁴ OPCs transplanted into demyelinated SC lesions, however, are reported to enhance remyelination in adult rats.¹ Consistently, we earlier documented that lovastatin enhances the differentiation of OPCs into myelin-forming OLs that eventually enhance remyelination in experimental autoimmune encephalomyelitis (EAE), a murine model of MS.^15,16 In light of this information, we assessed whether a pharmacologic agent that can influence OL survival holds promise to restore CNS integrity and functions in MS.Accumulating evidence suggests that AMP-activated protein kinase (AMPK)–mediated restoration of the CNS energy balance is critical to protect the brain under pathologic conditions.^17,18 We earlier documented that AMPK activators, ie, 5-amino-4-imidazole carboxamide riboside (AICAR) and metformin, attenuate clinical impairments in EAE animals.^19,20 The protective effects of these AMPK activators were ascribed to their immunomodulatory activities and the restoration of blood-brain barrier integrity in EAE animals.^19–21 AMPK activity was reduced in the brain at the onset and peak of EAE disease.²² Consistently, we earlier documented that the genetic ablation of AMPK causes severe clinical impairments in EAE animals.²³ In addition, we documented that AICAR inhibits the expressions of inflammatory mediators in astrocytes, microglia, and peritoneal macrophages that are known to play critical roles in EAE/MS pathogenesis.²⁴ Recently, AMPK signaling was reported to interfere with interferon γ (IFN-γ)–mediated activation of astrocytes and microglia²² as well as induction of the inflammatory response in liver cells.^25,26 These studies provide evidence that CNS bioenergetics and inflammation are linked, thus understanding of their regulatory mechanisms has the scope to identify new therapeutic interventions for MS.Recently, AMPK signaling was reported to protect neurons under pathologic conditions.^27,28 In addition, we earlier documented that AMPK activity was reduced in OLs exposed to psychosine, a galactosylsphingosine lipid that accumulates in the Krabbe brain.²⁹ Based on this knowledge, we investigated whether AMPK signaling can protect OLs in the CNS of EAE animals. By using in vitro and in vivo approaches, we demonstrated that AMPK signaling influences OL survival and, thereby, restores CNS integrity and functions in EAE animals treated with metformin (first-line drug of choice for type 2 diabetes mellitus).