Safety Considerations for Lyophilized Human Amniotic Membrane Impregnated with Colistin and Silver Nanoparticles.

Lyophilized human amniotic membrane (HAM) and silver nanoparticles (AgNPs) have multispectral applications as a biological dressing. The present study focuses on the safety aspects of HAM coated with colistin and AgNPs (HACoN) dressing in relation to its structural and hematological changes. Four dressing groups were designed for the study, HAM, HAM coated with colistin (HACo), HAM coated with AgNPs (HAN), and HAM coated with colistin (HACo) and HACoN. Scanning electron microscopy (SEM) and Fourier-transform infrared spectroscopy (FTIR) were utilized for constitutional analysis. Biological safety was checked by applying HAM of all groups on open excisional burn wounds on Sprague-Dawley rats for 21 days. The skin, kidneys, liver, and spleen were removed, and histological analysis was performed for detailed structural analysis. Oxidative stress was assessed using homogenate from newly generated skin. No structural or biochemical change was observed in any of the study groups as observed by SEM and FTIR. After 21 days of grafting, wounds were healed properly with normal skin, and no anomaly was observed in related to kidneys, spleen, and liver. Some of antioxidant enzymes were increased, while malondialdehyde which is a reactive oxygen species was reduced in the skin tissue homogenate of HACoN group. Impregnation of colistin and AgNPs in combination on HAM has no effects on hematological and structural constitution of HAM. It leaves no obvious change in vital organs of rats and improves oxidative stress and inflammation. Hence, it can be claimed that HACoN is a biologically safe antibacterial dressing.

Synergistic efficacy of colistin and silver nanoparticles impregnated human amniotic membrane in a burn wound infected rat model.

Antimicrobials used to treat burn wound infections have become multidrug-resistant, thus delaying wound healing. When combined with silver nanoparticles, antibiotics create a multifaceted antibacterial mechanism of action to which bacteria are incapable of developing resistance. Similarly, the amniotic membrane has been found to lower the bacterial number. The purpose of the current study was to observe the antibacterial activity of combined topical colistin with silver nanoparticles and decellularized human amniotic membrane as a dressing in burn wounds infected with bacteria with the goal of promoting faster healing. Bacteria commonly isolated from burn wounds and the most sensitive topical antibiotic were identified. Colistin, silver nanoparticles and combined colistin with silver nanoparticles were impregnated into decellularized human amniotic membranes. These wound dressings were evaluated in third-degree multidrug-resistant bacterial infected thermal burns induced in rats. Out of a total of 708 pus samples from burn wounds, Pseudomonas aeruginosa was the most prevalent pathogen 308 (43.5%), followed by Klebsiella pneumoniae 300 (42.4%). Topical colistin was 100% sensitive for both bacteria. Overall, maximum wound contraction (p < 0.05), and increased collagen deposition (+++) with no isolation of bacteria from wound swabs were noted on day 21 for the combined colistin with silver nanoparticle-loaded human amniotic membrane dressing group. Our study concluded that the increased antimicrobial activity of the novel combination of colistin and silver nanoparticle-loaded decellularized human amniotic membrane manifested its potential as an effective burn wound dressing.

Oral Administration of Aloe vera Ameliorates Wound Healing through Improved Angiogenesis and chemotaxis in Sprague Dawley Rats.

BACKGROUND: Aloe vera has been reported as a topical antibiotic and healing agent for wounds, but advantages of oral administration and mechanisms of wound healing have not been reported. Present study focuses on the evaluation of effects of oral administration of Aloe vera for excisional cutaneous wounds in Sprague Dawley rats. METHODS: Sprague Dawley (SD) rats were inflicted with excisional wounds and were either treated with Aloe vera orally (Aloe vera) or kept untreated (wound). In contrast, healthy rats were kept as control group. Wound area was measured from day 7th to day 21st. Collagen content was estimated by hydroxyproline assay. Histology was analysed by hematoxylin and eosin staining. Angiogenesis was observed by indirect ELISA for Insulin like Growth Factor (IGF-1) and Vascular Endothelial Growth Factor (VEGF) protein from skin, serum and bone marrow. Chemotaxis was evaluated by RT-PCR analysis for Stromal cell-Derived Factor-1 (SDF-1) and C-X-C chemokine receptor type 4 (CXCR-4) from skin and bone marrow. RESULTS: Aloe vera healed wounds earlier than untreated rats with gradual improvement in wound areas and collagen content. Aloe vera also improved the expression of IGF-1 and VEGF in skin and bone marrow indicating an improvement in angiogenesis. RT- PCR analysis showed increased expression of genes for chemotaxis (SDF-1 and CXCR-4) in skin and bone marrow. CONCLUSION: Aloe vera improves healing by increasing collagen content, improving angiogenesis and chemotaxis.

Improvement in Therapeutic Ability of Wharton's Jelly Derived Mesenchymal Stem Cells with Vitamin E in Breast Cancer.

OBJECTIVE: To assess the role of Vitamin E to improve the survival of Wharton's jelly derived mesenchymal stem cells (WJMSCs) in breast cancer conditions. STUDY DESIGN: An experimental study. PLACE AND DURATION OF STUDY: Centre for Research in Molecular Medicine, University of Lahore, from November 2016 to March 2017. METHODOLOGY: WJMSCs were obtained from umbilical cord tissue with enzyme digestion method. Isolated cells were characterized for CD90 and CD45 by immunocytochemistry. Pretreatment and conjugation therapies of vitamin E in 50mM and 100mM concentration were used on WJMSCs and breast cancer plasma was provided to mimic the cancer conditions, while WJMSCs provided with normal plasma were considered control. Cells' viability, proliferation and death were evaluated by crystal violet staining, MTT assay and LDH assay, respectively. Oxidative stress was observed by activity of anti-oxidant enzymes (GSH, catalase, SOD) and reactive oxygen species (MDA). RESULTS: The isolated cells expressed mesenchymal stem cells marker CD90 and lacked hematopoietic marker CD45. Vitamin E improved the viability and proliferation of WJMSCs in normal plasma, in conjugation with breast cancer plasma and in pretreatment groups but conjugation group showed even better results with concentration of 100mM as compared to the pretreatment group and opposite was observed for LDH assay for cells death analysis. Vitamin E also reduced the oxidative stress in 100mM more pronounced in conjugation group as compared to pretreatment group while left no harmful effects on WJMSCs in normal plasma. CONCLUSION: Vitamin E conjugation with breast cancer conditions significantly improved growth of WJMSCs. Thus vitamin E treated WJMSCs are better therapeutic options for breast cancer.

Effect of type 2 diabetic serum on the behavior of Wharton's jelly-derived mesenchymal stem cells in vitro.

OBJECTIVE: Wharton jelly-derived mesenchymal stem cells (WJMSCs) exhibit multilineage differentiation potential and can be used to treat multiple organs. However, diabetes affects the repair capability of MSCs. The aim of this study was to evaluate the effect of diabetic patient-derived serum on WJMSC behavior. METHODS: WJMSCs at passage 3 were treated with serum derived from type 2 diabetic patients. WJMSCs were characterized for surface markers expression by using immunocytochemistry technique. The effects on cell viability, proliferation, cell death rate, and vascular endothelial growth factor level were assessed by crystal violet staining, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, lactate dehydrogenase assay, and enzyme-linked immuno-sorbent assay, respectively. Oxidative stress was assessed by the estimation of free radical species malondialdehyde (MDA) and enzymes glutathione (GSH), catalase, and superoxide dismutase (SOD). RESULTS: WJMSCs isolated in this study were positive for CD29, CD49, CD73, CD90, CD105, and SSEA4 and negative for CD45 and CD34. Under diabetic stress conditions, WJMSCs showed low viability and high lactate dehydrogenase release. A low level of vascular endothelial growth factor was also observed after diabetic serum treatment. Antioxidant level was also lower in diabetic serum-treated WJMSCs compared to in normal serum-treated WJMSCs. CONCLUSION: The results of the present study suggest that pre-treatment of WJMSCs with type 2 diabetic serum decreases the survival of WJMSCs. The findings of this study provide insight into diabetes-induced harmful effects on WJMSCs.

Comparative analysis of biochemical parameters in diabetic and non-diabetic acute myocardial infarction patients.

BACKGROUND: Diabetes is a metabolic disorder characterized by enhanced production of free radicals hence oxidative stress. The aim of this study was to evaluate the activity of cardiac and antioxidant enzymes in diabetic and non-diabetic acute myocardial infarction (AMI) patients. METHODS: This case-control study was conducted on 450 subjects (70-85 years). Subjects were divided into three groups (Normal, N; Non-diabetic AMI, N-AMI; and Diabetic AMI, D-AMI). Each individual was subjected to a detailed history, clinical examination, and cardiovascular parameters analysis (fasting blood sugar, HbA1c, systolic and diasystolic blood pressure, total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL), high-density lipoprotein (HDL), TC/HDL and LDL/HDL ratios). Cardiac markers (Troponin-I, creatine phosphokinase (CPK), creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH), C-reactive protein (CRP) and aspartate aminotransferase (AST)) and oxidative stress markers (superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), catalase (CAT)) were also assessed. All these parameters were compared between diabetic and non-diabetic AMI patients. RESULTS: D-AMI individuals had high level of TC, TG, LDL, and low level of HDL in comparison to N-AMI individuals. Study suggests that cardiac markers such as Troponin I, CPK, CK-MB, AST, LDH, and CRP levels were significantly increased in patients suffering from myocardial infarction with diabetes mellitus (DM) compared to patients of myocardial infarction without DM. The activity levels of antioxidant SOD and GSH were lower in D-AMI patients than in N-AMI. However, levels of MDA and CAT were higher in D-AMI than in N-AMI controls. CONCLUSION: Study suggests elevated cardiac markers and reduced antioxidants in D-AMI patients compared to N-AMI patients.

Chronic Myeloid Leukemia Blood Inflicted Injury in Cord Derived Wharton's Jelly Mesenchymal Stem Cells.

OBJECTIVE: To determine the effects of blood from CML patients on human umbilical cord derived Wharton's jelly mesenchymal stem cells (WJMSCs) for evaluation of their therapeutic potential. STUDY DESIGN: An experimental study. PLACE AND DURATION OF STUDY: Centre for Research in Molecular Medicine, University of Lahore, from September 2013 to December 2014. METHODOLOGY: Possible behavior of WJMSCs in CMLpatients was assessed by culturing these cells in their plasma. WJMSCs at passage 3 were cultured in plasma isolated from 9 CMLpatients as well as 9 normal subjects. Effects on cell viability, proliferation, LDH release, paracrine factors (p38 and p53) and oxidative stress were evaluated. RESULTS: WJMSCs cultured in plasma of CML patients showed decreased viability, slow proliferation, high LDH release, high expression of p38 and p53 and a high oxidative stress compared to normal subjects. CONCLUSION: Stressed environment of CML patients' blood/plasma induced injury to WJMSCs as well as reduced their viability. Effectiveness of these cells for therapeutics of CML is, therefore, likely to be reduced.

Characterization of lipid parameters in diabetic and non-diabetic atherosclerotic patients.

BACKGROUND & OBJECTIVE: The relationship between lipid profile perturbation and diabetes associated complications has long been an area of interest. Dyslipidemia is a potent predictor of cardiovascular morbidity and mortality in diabetic patients. The aim of present study was to investigate relationship between aging and lipid profiles in diabetic and non-diabetic atherosclerotic patients. METHODS: Five hundred and seventy six individuals (45-75 year age) participated in this study. Among these, 192 were having history of diabetes mellitus and atherosclerosis. Individuals are categorized on the base of health (normal, non-diabetic atherosclerosis, diabetic atherosclerosis) and age (45-55 years, 56-65 years, and 66-75 years). All the participants were subjected to the procedures like a detailed history, biochemical analysis for fasting blood sugar, hemoglobin A1c, total cholesterol (TC), triglycerides (TG), low-density lipoprotein-(LDL), very low-density lipoprotein (VLDL) and high-density lipoprotein (HDL). All these parameters were compared between diabetic and non-diabetic atherosclerotic patients of all three age groups. TC/HDL and LDL/HDL were also calculated. RESULTS: Diabetic atherosclerotic individuals (both males and females) had high level of TC, TG, LDL, VLDL and low level of HDL in comparison to non-diabetic atherosclerotic and normal control individuals. Among all three age groups, lipoprotein abnormality was observed to be more frequent in females than males. There was a significant increase in TC/HDL and LDL/HDL ratio in diabetic atherosclerotic subjects compared to age and sex matched non-diabetic atherosclerotic and normal control. CONCLUSIONS: Degree of dyslipidemia increases with increase in age in both genders. Female are more prone to diabetic dyslipidemia and hence have more risk of developing atherosclerosis with increasing age.

The effect of gestational diabetes on proliferation capacity and viability of human umbilical cord-derived stromal cells.

Stomal cells derived from Wharton's jelly of human umbilical cord (WJMSCs) are considered as the potential therapeutic agents for regeneration and are getting famous for stem cell banking. Our study aims to evaluate the effects of gestational diabetes on proliferation capacity and viability of WJMSCs. Mesenchymal stromal cells were isolated from Wharton's jelly of human umbilical cords from normal and gestational diabetic (DWJMSCs) mothers. Growth patterns of both types of cells were analyzed through MTT assay and population doubling time. Cell survival, cell death and glucose utilization were estimated through trypan blue exclusion assay, LDH assay and glucose detection assay respectively. Angiogenic ability was evaluated by immunocytochemistry and ELISA for VEGF A. Anti-cancerous potential was analyzed on HeLa cells. DWJMSCs exhibited low proliferative rate, increased population doubling time, reduced cell viability and increased cell death. Interestingly, DWJMSCs were found to have a reduced glucose utilization and anti-cancerous ability while enhanced angiogenic ability. Gestational diabetes induces adverse effects on growth, angiogenic and anti-cancerous potential of WJMSCs.

Vitamin E protects chondrocytes against hydrogen peroxide-induced oxidative stress in vitro.

OBJECTIVE AND DESIGN: This study evaluated the effect of an antioxidant, Vitamin E, on cultured chondrocytes against H2O2-induced damage in vitro. MATERIAL: Rat chondrocytes isolated from articular cartilage. TREATMENT: Chondrocytes were pretreated with either 50 or 100 µM Vitamin E or serum-free medium for 24 h followed by their exposure to 200 µM H2O2 for 3 h. Chondrocytes without exposure to H2O2 served as control group. METHODS: The effect of Vitamin E pretreatment was evaluated by examining proteoglycan contents, nitrite levels, viability, apoptosis, and senescence of cultured chondrocytes. RESULTS: Proteoglycan contents increased in groups treated with Vitamin E. Semi-quantitative real-time PCR data also correlated with these results and demonstrated that Vitamin E up-regulated expression of Agc1, Col2a1, and PCNA genes along with down-regulation in the expression of Col1a1 and Casp3 genes. The differentiation index improved after Vitamin E pretreatment. Nitrite levels were reduced with a corresponding increase in cell viability. Reduction in apoptosis and senescence was also observed after Vitamin E pretreatment. Moreover, a dose-dependent effect of Vitamin E was seen. In contrast to 50 µM Vitamin E, 100 µM was more potent in inducing protection of chondrocytes from H2O2-induced oxidative damage. CONCLUSION: Vitamin E reversed the oxidant-induced alterations in chondrocytes and may be a good option to pretreat chondrocytes before transplantation.

Lovastatin protects chondrocytes derived from Wharton's jelly of human cord against hydrogen-peroxide-induced in vitro injury.

Our aim was to improve the survival and reduce the apoptosis of chondrocytes derived from mesenchymal stem cells from Wharton's jelly of human umbilical cord (WJMSCs) by Lovastatin supplementation under hydrogen-peroxide-induced injury conditions to simulate the osteoarthritic micro-environment. Chondrocytes were differentiated in vitro from WJMSCs. The cultured WJMSCs expressed CD90 (84.07%), CD105 (80.84%), OCT4 (26.90%), CD45 (0.42%) and CD34 (0.48%) as determined by flow cytometry. Increased aggregation of proteoglycans observed by Safranin-O staining accompanied by increased expression of COL2A1, ACAN, SOX9 and BGN shown by immunocytochemistry and reverse transcription with the polymerase chain reaction (PCR) confirmed the chondrogenic differentiation of the WJMSCs. The in vitro differentiated chondrocytes were subjected to oxidative stress by exposure to 200 µM hydrogen peroxide, either in the presence or absence of Lovastatin (2 µM) for 5 h. Lovastatin treatment resulted in decreased apoptosis, senescence and LDH release and in increased viability and proliferation of WJMSC-derived chondrocytes. Real time PCR analysis showed markedly up-regulated expression of prosurvival, proliferation and chondrogenic genes (BCL2L1, BCL2, AKT, PCNA, COL2A1, ACAN, SOX9 and BGN) and significantly down-regulated expression of pro-apoptotic genes (BAX, FADD) in the Lovastatin-treated group in comparison with injured cells. The reduced expression of VEGF and p53 as determined by enzyme-linked immunosorbent assay and PCR suggests the suitability of the use of Lovastatin in adjunct to WJMSC-derived chondrocytes for the treatment of osteoarthritis. We conclude that Lovastatin protects WJMSC-derived chondrocytes from hydrogen-peroxide-induced in vitro injury.