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.     

Enhanced Expression of Sodium Hydrogen Exchanger (NHE)-1, 2 and 4 in the Cervix of Ovariectomised Rats by Phytoestrogen Genistein

Restoring the pH of cervicovaginal fluid is important for the cervicovaginal health after menopause. Genistein, which is a widely consumed dietary health supplement to overcome the post-menopausal complications could help to restore the cervicovaginal fluid pH. We hypothesized that genistien effect involves changes in expression of NHE-1, 2 and 4 proteins and mRNAs in the cervix. This study investigated effect of genistein on NHE-1, 2 and 4 protein and mRNA expression in the cervix in order to elucidate the mechanisms underlying possible effect of this compound on cervicovaginal fluid pH after menopause. Methods: Ovariectomised adult female rats received 25, 50 and 100 mg/kg/day genistein for seven consecutive days. At the end of the treatment, animals were sacrificed and cervix was harvested. Expression of Nhe-1, 2 and 4 mRNA were analyzed by Real-time PCR while distribution of NHE-1, 2 and 4 protein were observed by immunohistochemistry. Results: Treatment with 50 and 100 mg/kg/day genistein caused marked increase in the levels of expression and distribution of NHE-1, 2 and 4 proteins in the endocervical epithelia. Levels of Nhe-1, 2 and 4 mRNA in the cervix were also increased. Coadministration of ICI 182 780 and genistein reduced the expression levels of NHE-1, 2 and 4 proteins and mRNAs in the cervix. Conclusions: Enhanced expression of NHE-1, 2 and 4 proteins and mRNAs expression in cervix under genistein influence could help to restore the cervicovaginal fluid pH that might help to prevent cervicovaginal complications related to menopause.     

Nitroaromatic compounds: Environmental toxicity,carcinogenicity, mutagenicity,therapy and mechanism

Vehicle pollution is an increasing problem in the industrial world. Aromatic nitro compounds comprise a significant portion of the threat. In this review, the class includes nitro derivatives of benzene, biphenyls, naphthalenes, benzanthrone and polycyclic aromatic hydrocarbons, plus nitroheteroaromatic compounds. The numerous toxic manifestations are discussed. An appreciable number of drugs incorporate the nitroaromatic structure. The mechanistic aspects of both toxicity and therapy are addressed in the context of a unifying mechanism involving electron transfer, reactive oxygen species, oxidative stress and antioxidants. Copyright © 2014 John Wiley & Sons, Ltd.     

Toxicity of imine–iminium dyes and pigments: electron transfer,radicals, oxidative stress and other physiological effects

Although conjugation is well known as an important contributor to color, there is scant recognition concerning involvement of imine and iminium functions in the physiological effects of this class of dyes and pigments. The group includes the dyes methylene blue, rhodamine, malachite green, fuchsin, crystal violet, auramine and cyanins, in addition to the pigments consisting of pyocyanine, phthalocyanine and pheophytin. The physiological effects consist of both toxicity and beneficial aspects. The unifying theme of electron transfer–reactive oxygen species–oxidative stress is used as the rationale in both cases. Toxicity is frequently prevented or alleviated by antioxidants. The apparent dichotomy of methylene blue action as both oxidant and antioxidant is rationalized based on similar previous cases. This mechanistic approach may have practical benefit. This review is important in conveying, for the first time, a unifying mechanism for toxicity based on electron transfer–reactive oxygen species–oxidative stress arising from imine–iminium. Copyright © 2014 John Wiley & Sons, Ltd.     

Quercetin induces morphological and proliferative changes of rat’s uteri under estrogen and progesterone influences

This study investigated the effect of 10 or 100 mg/kg/day quercetin on the uterus of ovariectomized adult female rats receiving sex-steroid replacement regime mimicking changes in hormonal profiles during the reproductive cycle. Following seven days of treatment with estrogen and progesterone with or without quercetin, uteri were harvested for histological and proliferative cell nuclear antigen (PCNA) protein and mRNA expression and PCNA protein distribution analyses. Our findings indicated that co-administration of 10 mg/kg/day quercetin with estrogen and progesterone caused a significant decrease in the size of uterine lumen and epithelial heights with lower PCNA protein and mRNA expression as compared to estrogen plus progesterone-only treatment (P < 0.05). Concomitant treatment with estrogen and progesterone with 100 mg/kg/day quercetin resulted in a marked increase in the number of glands with increased PCNA protein and mRNA expression. Significantly higher PCNA distribution was observed in the stroma and glands as compared to estrogen plus progesterone-only treatment (P < 0.05). In conclusion, at 10 mg/kg/day, quercetin affects uterine morphology but not proliferation, however at 100 mg/kg/day, quercetin induced significant stromal and glandular proliferation which could predispose the uterus towards neoplastic development.     

Thrombophilic mutations in pre-eclampsia and pregnancy-induced hypertension

AIM: The aim of the present study was to determine the existence or prevalence of thrombophilic markers such as Factor V Leiden, prothrombin G20210A, protein S, protein C, activated protein C and anti-thrombin in pre-eclampsia and pregnancy-induced hypertensive patients. METHODS: Blood samples were collected from a total number of 124 women at the maternity unit, University of Malaya Medical Center. These included 49 patients with pre-eclampsia, 63 patients with pregnancy-induced hypertension and 12 normal pregnant women. DNA was extracted from the blood samples. Factor V Leiden (Taq I) and prothrombin G20210A (Hind III) genotyping was done on polymerase chain reaction-restriction fragment length polymorphism. Anti-thrombin activity and the concentrations of protein C, protein S and activated protein C were measured using the IL Coagulation System (Hemosil). RESULTS: Of the 124 subjects, one pre-eclampsia patient was homozygous for Factor V Leiden mutation but prothrombin G20210A mutation was not present in any of the subjects. The subject with Factor V Leiden mutation also had a low activated protein C resistance and a low protein S concentration. CONCLUSIONS: Factor V Leiden mutation is present in the Asian population and may very well serve as one of the genetic factors responsible for pre-eclampsia and other adverse pregnancy outcomes.     

A re-evaluation of plasma sialic acid determination using the periodate resorcinol method versus the enzymatic method

The concentration of plasma sialic acid was estimated using the modified chemical method and the more sensitive enzymatic method in 20 subjects with impaired glucose tolerance and 20 control subjects. The mean sialic acid concentration values of the control subjects and subjects with impaired glucose tolerance using the enzymatic method were 1.747 +/- 0.047 and 2.583 +/- 0.070 mmole/l and 1.753 +/- 0.067 and 2.591 +/- 1.02 mmole/l for the chemical method. The intra-assay coefficient of variation for the control subjects and for the subjects with impaired glucose tolerance were 1.963% and 1.583%, respectively, for the enzymatic assay and 2.728% and 2.431%, respectively, for the chemical assay. The inter-assay coefficient of variation for the control subjects and for the subjects with impaired glucose tolerance were 2.686% and 2.723% for the enzymatic assay, and 3.819% and 3.95% for the chemical assay. Since the values do not differ significantly, the chemical assay is a cost effective method that can be used in large epidemiological studies.     

Histoplasmosis in pregnancy: case series and report of transplacental transmission

Although a healthy pregnancy seems to confer an increased susceptibility to systemic fungal infections, to our knowledge, no known association exists in the case of Histoplasma capsulatum. In addition, to our knowledge, transplacental transmission of H capsulatum has not been previously described. We describe a series of patients treated at our institutions between 1997 and 2000 with evidence of transplacental transmission of H capsulatum. Mechanisms of increased risk of histoplasmosis during pregnancy are proposed, although this risk has yet to be proved.