Priming of adipose-derived stem cells with curcumin prior to cryopreservation preserves their functional potency: Towards an 'Off-the-shelf' therapy for burns.

Stem cells-based treatment for burn wounds require frozen cells as an off-the-shelf therapy; however, cryopreservation-induced oxidative stress resulted in post-thaw cell death or loss of cell functions, thus arrested their clinical practicality. Although antioxidant priming to stem cells increase their resistant to oxidative stress, but this strategy is still unexplored on cryopreserved cells. Herein, we investigated whether curcumin priming before cryopreservation could preserve the therapeutic potency of thawed stem cells. For this, unprimed and curcumin-primed adipose-derived stem cells (ASCs) were cryopreserved for one month. Post-thawing, cells were assessed for viability by trypan blue assay; metabolic activity by MTT assay; senescence by senescence-associated (SA)-ß-galactosidase activity staining assay; migration by scratch healing assay and; mRNA expression by real-time PCR. Subsequently, the healing potential was examined by injecting cells around the wound periphery of acidic burn in rats. Post-healing, skin architecture was histologically examined. Results demonstrated that, curcumin-primed frozen cells (Cryo/Cur-ASCs) showed better post-thaw viability, metabolic activity, migration ability and lower percent of senescence comparative to unprimed frozen cells (Cryo/ASCs). Curcumin priming alleviated the oxidative damage by activating the ROS-reducing cellular antioxidant system as shown by the evident increase in GSH levels and upregulated mRNA expression of glutathione peroxidase (GPx), superoxide dismutases (SOD1, SOD2), and catalase (CAT). Further, invivo findings revealed that Cryo/Cur-ASCs-treated wounds exhibited earlier wound closure with an improved architecture comparative to Cryo/ASCs and depicted healing capacity almost similar to Fresh/ASCs. Our findings suggested that curcumin priming could be effective to alleviate the cryo-induced oxidative stress in post-thawed cells.

Standardization of diethylnitrosamine-induced hepatocellular carcinoma rat model with time based molecular assessment.

This study was intended (1) to develop a robust animal model for hepatocellular carcinoma (HCC) research, in which HCC tumors develop in a background of fibrosis or cirrhosis; and (2) to explore time-dependent regulatory changes in key molecular markers during disease advancement and HCC development. With the aim of establishing such HCC model, male Sprague-Dawley rats were injected with diethylnitrosamine (DEN) at a dose of 30 mg/kg twice a week for 10 weeks then once a week from 12th to 16th weeks. The rats were kept under observation until 18th week. At defined time intervals (2nd, 4th, 12th, and 18th week), serum biomarkers and microscopic components of tissue samples were used to investigate the chronic progression of liver disease, while gene and protein analysis was used to monitor expression patterns during HCC development. DEN-intoxicated rats manifested inflammation at week 4, fibrosis at week 12 and cirrhosis with early HCC tumors at week 18. Molecular analysis revealed that key markers of inflammation (Il-1ß, Il-6, and Tnf-α), fibrosis (Tgf-ß1, Col1α1, Col3α1, and Timp-1), and angiogenesis (Hif1-α and Vegf) were promptly (P ≤ 0.001) up-regulated at week 4, week 12 and week 18, respectively. Oxidative stress (iNos, Cyp2e1, and Sod1) and pro-apoptotic (Bax) markers showed significant upregulation from week 4 to week 12. However, Sod1 and Bax expressions dropped after week 12 and reached a minimum at 18th week. Strikingly, expressions of anti-apoptotic (Bcl-2) and cell proliferation (Pcna, Hgf, and Afp) markers were abruptly increased at week 18. Collectively, we describe an 18-week HCC model in DEN-intoxicated rats that exhibit chronic inflammation, oxidative imbalance, advance fibrosis/cirrhosis, halted apoptosis, and angiogenic sprouting, progressively.

Adipose Tissue and Umbilical Cord Tissue: Potential Sources of Mesenchymal Stem Cells for Liver Fibrosis Treatment.

Background/Aims: Mesenchymal stem cells (MSCs) are potential alternatives for liver fibrosis treatment; however, their optimal sources remain uncertain. This study compares the ex-vivo expansion characteristics of MSCs obtained from adipose tissue (AT) and umbilical cord (UC) and assesses their therapeutic potential for liver fibrosis treatment. Methods: Since MSCs from early to mid-passage numbers (P2-P6) are preferable for cellular therapy, we investigated the growth kinetics of AT-MSCs and UC-MSCs up to P6 and evaluated their therapeutic effects in a rat model of liver fibrosis induced by diethylnitrosamine. Results: Results from the expansion studies demonstrated that both cell types exhibited bona fide characteristics of MSCs, including surface antigens, pluripotent gene expression, and differentiation potential. However, AT-MSCs demonstrated a shorter doubling time (58.2 ± 7.3 vs. 82.3 ± 4.3 h; P < 0.01) and a higher population doubling level (10.1 ± 0.7 vs. 8.2 ± 0.3; P < 0.01) compared to UC-MSCs, resulting in more cellular yield (230 ± 9.0 vs. 175 ± 13.2 million) in less time. Animal studies demonstrated that both MSC types significantly reduced liver fibrosis (P < 0.05 vs. the control group) while also improving liver function and downregulating fibrosis-associated gene expression. Conclusion: AT-MSCs and UC-MSCs effectively reduce liver fibrosis. However, adipose cultures display an advantage by yielding a higher number of MSCs in a shorter duration, rendering them a viable choice for scenarios requiring immediate single-dose administration, often encountered in clinical settings.

Cassia Angustifolia Primed ASCs Accelerate Burn Wound Healing by Modulation of Inflammatory Response.

BACKGROUND: Thermal traumas impose a huge burden on healthcare systems. This merits the need for advanced but cost-effective remedies with clinical prospects. In this context, we prepared a regenerative 3D-construct comprising of Cassia angustifolia extract (SM) primed adipose-derived stem cells (ASCs) laden amniotic membrane for faster burn wound repair. METHODS: ASCs were preconditioned with SM (30 µg/ml for 24 h), and subsequently exposed to in-vitro thermal injury (51 °C,10 min). In-vivo thermal injury was induced by placing pre-heated copper-disc (2 cm diameter) on dorsum of the Wistar rats. ASCs (2.0 × 105) pre-treated with SM (SM-ASCs), cultured on stromal side of amniotic membrane (AM) were transplanted in rat heat-injury model. Non-transplanted heat-injured rats and non-heat-injured rats were kept as controls. RESULTS: The significantly upregulated expression of IGF1, SDF1A, TGFß1, VEGF, GSS, GSR, IL4, BCL2 genes and downregulation of BAX, IL6, TNFα, and NFkB1 in SM-ASCs in in-vitro and in-vivo settings confirmed its potential in promoting cell-proliferation, migration, angiogenesis, antioxidant, cell-survival, anti-inflammatory, and wound healing activity. Moreover, SM-ASCs induced early wound closure, better architecture, normal epidermal thickness, orderly-arranged collagen fibers, and well-developed skin appendages in healed rat-skin transplanted with AM+SM-ASCs, additionally confirmed by increased expression of structural genes (Krt1, Krt8, Krt19, Desmin, Vimentin, α-Sma) in comparison to untreated-ASCs laden-AM transplanted in heat injured rats. CONCLUSION: SM priming effectively enabled ASCs to counter thermal injury by significantly enhancing cell survival and reducing inflammation upon transplantation. This study provides bases for development of effective combinational therapies (natural scaffold, medicine, and stem cells) with clinical prospects for treating burn wounds.

Aloe vera and ofloxacin incorporated chitosan hydrogels show antibacterial activity, stimulate angiogenesis and accelerate wound healing in full thickness rat model.

Burns are potentially fatal and physically debilitating injuries, causing psychological and physical scars and result in chronic disabilities. A well vascularized wound bed is required to achieve complete and scar free wound closure. For many centuries, a variety of herbal plants have been used for wound healing, among these aloe vera (AV) has been found to be very effective in wound healing. Secondly, the main reason for delayed wound healing is bacterial infections. Ofloxacin (OX) has been reported as an active antibacterial drug for topical infections and it is effective against both positive and negative bacterial strains. In current research three different concentrations of OX (0.5, 2.5, and 5 mg) were loaded into chitosan (CS)/AV based hydrogels prepared by freeze gelation. The surface morphology of prepared CS/AV based OX loaded hydrogels were evaluated by scanning electron microscopy (SEM). In drug release analysis, 0.5 mg OX loaded hydrogel showed a sustained drug release behavior over 3 days period. An effective dose dependent antibacterial activity was exhibited by OX loaded hydrogels. Alamar Blue cells viability assay revealed that 0.5 mg OX hydrogel (CA 0.5 OX) showed comparatively better 3 T3 fibroblast cells proliferation as compared to CA 2.5 OX (2.5 mg OX) and CA 5 OX hydrogel (5 mg OX). Moreover, all OX loaded hydrogels showed good angiogenic activity in CAM bioassay while higher angiogenic potential was observed from CA 0.5 OX containing comparatively lower concentration of OX. These OX incorporated CS/AV based hydrogels are promising wound dressings for future clinical use.

Priming with caffeic acid enhances the potential and survival ability of human adipose-derived stem cells to counteract hypoxia.

The therapeutic effectiveness of stem cells after transplantation is hampered by the hypoxic milieu of chronic wounds. Prior research has established antioxidant priming as a thorough plan to improve stem cell performance. The purpose of this study was to ascertain how caffeic acid (CA) priming affected the ability of human adipose-derived stem cells (hASCs) to function under hypoxic stress. In order to study the cytoprotective properties of CA, hASCs were primed with CA in CoCl2 hypoxic conditions. Microscopy was used to assess cell morphology, while XTT, Trypan Blue, X-gal, LDH, Live Dead, scratch wound healing, and ROS assays were used to analyze viability, senescence, cell death, proliferation, and reactive oxygen species prevalence in the cells. According to our findings, CA priming enhances hASCs' ability to survive and regenerate in a hypoxic microenvironment more effectively than untreated hASCs. Our in-vitro research suggested that pre-treatment with CA of hASCs could be a unique way to enhance their therapeutic efficacy and ability to survive in hypoxic microenvironments.

Phase II trial of concurrent sunitinib, temozolomide, and radiotherapy with adjuvant temozolomide for newly diagnosed MGMT unmethylated glioblastoma.

Background: The overall prognosis of glioblastoma (GBM) remains dismal, particularly for patients with unmethylated O6-methylguanine-DNA-methyltransferase (MGMT) promoter. In this phase II trial, we tested the combination of the antiangiogenic agent sunitinib with radiotherapy and temozolomide (TMZ) for newly diagnosed unmethylated MGMT GBM patients. Methods: We enrolled 37 patients with unmethylated MGMT promoter GBM, age 18-70, and KPS ≥70. Patients received 12.5 mg of daily sunitinib for 7 days, followed by concurrent chemoradiation plus 12.5 mg sunitinib, then adjuvant TMZ. The primary endpoint was progression-free survival (PFS), and secondary endpoints were overall survival (OS), safety, and neutrophil-to-lymphocyte ratio (NLR) biomarker. Results: At a median follow-up time of 15.3 months (range: 3.1-71.3 months), the median PFS was 7.15 months (95% CI: 5.4-10.5) and the 6-month PFS was 54.0%. Median OS was 15.0 months (95% CI: 13.8-19.4) and 2-year OS rate was 17.1%. Patients receiving >3 cycles of adjuvant TMZ, undergoing surgery at progression, and presenting a post-concurrent NLR ≤6 experienced a significant improved OS with hazard ratios of 0.197 (P = .001), 0.46 (P = .049), and 0.38 (P = .021), respectively, on multivariable analysis. Age >65 years predicted for worse OS with hazard ratio of 3.92 (P = .037). Grade ≥3 thrombocytopenia occurred in 22.9%, grade ≥3 neutropenia in 20%, and grade ≥3 thromboembolic events in 14.3% of patients. There were no grade 5 events. Conclusion: Our findings suggest a potential benefit of combining sunitinib with chemoradiation in newly diagnosed GBM patients with unmethylated MGMT status and provide a strong rationale to test this combination in future studies.

Tumoricidal effects of unprimed and curcumin-primed adipose-derived stem cells on human hepatoma HepG2 cells under oxidative conditions.

INTRODUCTION: Adipose-derived stem cells (ASCs) have been proven to have tumoricidal effects against hepatic cancer cell lines. However, it appears that exposure to oxidative microenvironment compromises the potential outcome of ASCs in real hepatoma. Herein, we aimed to examine the tumoricidal effects of ASCs under oxidative conditions and to investigate the impact of curcumin priming on ASCs' therapeutic potential. METHODS: We used human hepatoma (HepG2) cells in a coculture system with unprimed or curcumin-primed ASCs (Cur-ASCs) under H2O2-induced oxidative conditions. To investigate HepG2 proliferation and death, MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) and annexin V staining assays were performed. To determine the HepG2 migration and invasion potential, the scratch healing and the transwell invasion assays were performed. To evaluate the expression of apoptosis-protein markers, Western blotting was performed. RESULTS: Cur-ASCs suppressed HepG2 proliferation, migration, and invasion as well as prompted apoptosis more significantly compared to unprimed ASCs under oxidative conditions. Expressional studies also revealed an obvious decline in the BCL-2/BAX ratio in HepG2 cocultured with Cur-ASCs. In addition, we noticed a marked elevation of apoptosis and senescence in unprimed ASCs compared to Cur-ASCs after coculture experiments, which demonstrated that curcumin priming preserved the survival and growth potential of ASCs; hence, Cur-ASCs performed better tumoricidal functions under oxidative conditions. CONCLUSION: Our findings suggest that ASCs have the intrinsic ability to induce cell death in HepG2 cells; however, their functions can be compromised under oxidative conditions. We believe that curcumin priming is an effective approach for improving the therapeutic effectiveness of ASCs in the cancerous microenvironment.

Vitamin E pretreated Wharton's jelly-derived mesenchymal stem cells attenuate CCl4-induced hepatocyte injury in vitro and liver fibrosis in vivo.

Oxidative microenvironment in fibrotic liver alleviates the efficacious outcome of mesenchymal stem cells (MSCs)-based cell therapy. Recent evidence suggests that pharmacological pretreatment is a rational approach to harness the MSCs with higher therapeutic potential. Here, we investigated whether Vitamin E pretreatment can boost the antifibrotic effects of Wharton's jelly-derived MSCs (WJMSCs). We used rat liver-derived hepatocytes injured by CCl4 treatment in co-culture system with Vitamin E pretreated-WJMSCs (Vit E-WJMSCs) to evaluate the hepatoprotective effect of Vit E-WJMSCs. After 24 h of co-culturing, we found that Vit E-WJMSCs rescued injured hepatocytes as hepatocyte injury-associated medium (AST, ALT, and ALP) and mRNA (Cyp2e1, Hif1-α, and Il-1ß) markers reduced to normal levels. Subsequently, CCl4-induced liver fibrosis rat models were employed to examine the antifibrotic potential of Vit E-WJMSCs. After 1 month of cell transplantation, it was revealed that Vit E-WJMSCs transplantation ceased fibrotic progression, as evident by improved hepatic architecture and functions, more significantly in comparison to naïve WJMSCs. In addition, Vit E-WJMSCs transplantation decreased the expressions of fibrosis-associated gene (Tgf-ß1, α-Sma, and Col1α1) markers in the liver parenchyma. Intriguingly, the results of tracing experiments discovered that more WJMSCs engrafted in the Vit E-WJMSCs treated rat livers compared to naïve WJMSCs treated livers. These findings implicate that pretreatment of WJMSCs with Vitamin E improves their tolerance to hostile niche of fibrotic liver; thereby further enhancing their efficacy for hepatic fibrosis.

Curcumin preconditioning enhances the efficacy of adipose-derived mesenchymal stem cells to accelerate healing of burn wounds.

BACKGROUND: Following recent findings from our group that curcumin preconditioning augments the therapeutic efficacy of adipose-derived stem cells in the healing of diabetic wounds in rats, we aimed to investigate the regenerative effects of curcumin preconditioned adipose-derived mesenchymal stem cells (ASCs) for better recovery of acid inflicted burns in this study. METHODS: ASCs were preconditioned with 5 µM curcumin for 24 hours and assessed for proliferation, migration, paracrine release potential and gene expression comparative to naïve ASCs. Subsequently, the healing capacity of curcumin preconditioned ASCs (Cur-ASCs) versus naïve ASCs was examined using acidic wounds in rats. For this, acid inflicted burns of 20 mm in diameter were made on the back of male Wistar rats. Then, 2 × 106 cells of Cur-ASCs and naïve ASCs were intradermally injected in the wound periphery (n = 6) for comparison with an untreated saline control. Post-transplantation, wounds were macroscopically analysed and photographed to evaluate the percentage of wound closure and period of re-epithelization. Healed wound biopsies were excised and used for histological evaluation and expression analysis of wound healing markers at molecular level by quantitative PCR and western blotting. RESULTS: We found that Cur-ASCs exhibited greater proliferation, migration and paracrine potential in vitro. Further, Cur-ASCs showed more effective recovery than naïve ASCs as exhibited by gross morphology, faster wound closure and earlier re-epithelialization. Masson's trichrome and hematoxylin and eosin staining demonstrated the improved architecture of the healing burns, as evidenced by reduced infiltration of inflammatory cells, compact collagen and marked granulation in Cur-ASC treated rats. Corroborating these findings, molecular assessment showed significantly reduced expressions of pro-inflammatory factors (interleukin-1 beta, interleukin-6, tumor necrosis factor alpha) a with striking upsurge of an oxidative marker (superoxide dismutase 1), pro-angiogenic factors (vascular endothelial growth factor, hepatocyte growth factor, hypoxia-inducible factor-1 alpha) and collagen markers (transforming growth factor beta 1, fibroblast growth factor-2, collagen type 1 alpha 1), verifying that Cur-ASCs modulate the regulation of pro-inflammatory and healing markers at burn sites. CONCLUSIONS: Treatment with Cur-ASCs resulted in faster re-epithelization of acid inflicted burns compared to the treatment with naïve ASCs. Based on observed findings, we suggest the transplantation of Cur-ASCs is a valuable therapy for the potent clinical management of acidic burns.

Curcumin preconditioned human adipose derived stem cells co-transplanted with platelet rich plasma improve wound healing in diabetic rats.

AIM: Inflammatory and oxidative microenvironment at diabetic' wound site hinder the therapeutic efficacy of cell-based therapies in diabetic patients. The purpose of this study is to explore the competence of curcumin preconditioned human adipose derived cells (hASCs) in combination with platelet rich plasma (PRP) for the repair of wounds in diabetic rats. MAIN METHODS: The cytoprotective effect of curcumin preconditioning for hASCs against hyperglycemic stress was evaluated through analysis of cell morphology, viability, cytotoxicity, senescence, and scratch wound healing assays. Subsequently, the healing capacity of curcumin preconditioned hASCs (Cur-hASCs) added to PRP was examined in excisional wounded diabetic rat model. Healed skin biopsies were excised to analyze gene and protein expression of wound healing markers by qPCR and western blotting. Histopathological changes were observed through hematoxylin and eosin staining. KEY FINDINGS: We found that Cur-hASCs counteract the glucose stress much better than non-preconditioned hASCs by maintaining their cellular morphology and viability as well as metabolic potential. Further in vivo results revealed that, Cur-hASCs co-injected with PRP resulted in faster wound closure, improved fibroblast proliferation, increased neovascularization, marked reduction in inflammatory cells, and compact extracellular matrix with completely covered thick epithelium. Moreover, Cur-hASCs + PRP treatment significantly improved the expression of key healing markers such as pro-angiogenic (Vegf), dermal matrix deposition (Col1α1), cell migration (bFgf) and cell proliferation (Pcna) at wound site. SIGNIFICANCE: Our findings propose a combinatorial therapy (Cur-hASCs + PRP) as a novel modality to improve the efficacy of hASCs-based therapy for diabetic wounds.

Oxygen Generating Polymeric Nano Fibers That Stimulate Angiogenesis and Show Efficient Wound Healing in a Diabetic Wound Model.

INTRODUCTION: Diabetic wounds are challenging to treat due to a wide range of pathophysiological changes. Hypoxia is one of the predominant contributing factors of poor vascularization and chronicity in diabetic wounds. This study was designed to develop polycaprolactone (PCL)-based oxygen-releasing electrospun wound dressings and evaluate their efficacy for improved full thickness wound healing in diabetic rats. METHODS: PCL-based oxygen releasing wound dressings were made using electrospinning technology. The developed dressings were characterized in terms of physical as well as biological properties both in vitro and in vivo. E-spun nanofibrous dressings were physically characterized with scanning electron microscopy, Fourier-transform infrared spectroscopy, and Energy-dispersive X-ray spectroscopy. To study the likely impact of the fabricated wound dressings in hypoxic conditions, HIF-1α expression analysis was carried out both at gene and protein levels. Wound dressings were further evaluated for their healing potential for extensive wounds in diabetic rat models. RESULTS: The experimental results showed that the developed dressings were capable of continuously generating oxygen for up to 10 days. Cell studies further confirmed pronounced expression of HIF-1α at gene and protein levels in cells seeded on PCL-sodium percarbonate (SPC) and PCL scaffolds compared with the cells cultured on a tissue culture plate. Chorioallantoic membrane assay revealed the supportive role of oxygen releasing dressings on angiogenesis compared to the control group. Histological assessment of the regenerated skin tissues proved that full thickness wounds covered with SPC loaded PCL dressings had a comparatively better vascularized and compact extracellular matrix with completely covered thick epithelium. DISCUSSION: The developed oxygen generating polymeric nanofibrous wound dressings could potentially be used as an envisioned approach for the efficient recovery of chronic diabetic wounds.

Sodium benzoate and potassium sorbate preservatives in food stuffs in Iran.

A high-performance liquid chromatography method was applied for the determination of the levels of benzoate and sorbate in 400 food samples, including pickled cucumbers, canned tomato pastes, sour cherry jams, soft drinks, fruit juices and dairy products (UF-Feta cheeses, Lighvan cheeses, lactic cheeses, yogurts and doogh). The results showed that 270 (67.5%) of all samples contained benzoate ranging from 11.9 to 288.5 mg kg(-1) in lactic cheese and fruit juice, respectively. The levels of sorbate in 98 (24.5%) of the samples were 20.1 to 284.3 mg kg(-1) in doogh and fruit juice, respectively. Moreover, benzoate was detected in all dairy products ranging from 11.9 mg kg(-1) in lactic cheese to 91.2 mg kg(-1) in UF-Feta cheese. A low concentration of benzoate could originate naturally, due to specific biochemical mechanisms during cheese, yogurt and doogh maturation. In conclusion, a minimum level for benzoate in dairy products should be defined in the legislation.

Dietary iron status has little effect on expression of ceruloplasmin but alters that of ferritin in rats.

Evidence supports a role for ceruloplasmin (ferroxidase I) in the release of iron to the blood from mammalian cells. However, recent studies with cultured cells have suggested that it has the opposite effect, and that iron deficiency enhances expression of ceruloplasmin. We therefore examined in rats how nutritional iron status would affect expression of ceruloplasmin. Groups of male Sprague-Dawley rats were reared on a low iron, starch-based diet for 6-8 wk; half were supplemented by injection of iron dextran. At killing, hematocrits of deficient rats were half normal. Supplemented rats had normal liver concentrations of ferritin and ferritin iron. No ferritin was detected in the livers of the deficient rats. Northern analysis showed that ferritin L and H mRNAs were present in the deficient livers, but expression was half that of the normal rats. There was also twice as much copper. Levels of circulating ceruloplasmin (measured by rocket immunoelectrophoresis) were not altered by iron deficiency, although p-phenylenediamine oxidase activity and plasma copper were reduced approximately 30%. In repeated studies, no differences in the expression of hepatic ceruloplasmin mRNA were detected. Treatment of rats of both sexes with additional iron (25 mg as iron dextran) 5-14 d before killing increased liver ferritin but did not alter liver ceruloplasmin mRNA expression or levels of circulating ceruloplasmin. We conclude that iron status is not an important factor in the expression of plasma ceruloplasmin made by the liver. However, it does have modest effects on steady-state levels of liver ferritin mRNA.