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.

A missense allele of PEX5 is responsible for the defective import of PTS2 cargo proteins into peroxisomes.

Peroxisomes, single-membrane intracellular organelles, play an important role in various metabolic pathways. The translocation of proteins from the cytosol to peroxisomes depends on peroxisome import receptor proteins and defects in peroxisome transport result in a wide spectrum of peroxisomal disorders. Here, we report a large consanguineous family with autosomal recessive congenital cataracts and developmental defects. Genome-wide linkage analysis localized the critical interval to chromosome 12p with a maximum two-point LOD score of 4.2 (θ = 0). Next-generation exome sequencing identified a novel homozygous missense variant (c.653 T > C; p.F218S) in peroxisomal biogenesis factor 5 (PEX5), a peroxisome import receptor protein. This missense mutation was confirmed by bidirectional Sanger sequencing. It segregated with the disease phenotype in the family and was absent in ethnically matched control chromosomes. The lens-specific knockout mice of Pex5 recapitulated the cataractous phenotype. In vitro import assays revealed a normal capacity of the mutant PEX5 to enter the peroxisomal Docking/Translocation Module (DTM) in the presence of peroxisome targeting signal 1 (PTS1) cargo protein, be monoubiquitinated and exported back into the cytosol. Importantly, the mutant PEX5 protein was unable to form a stable trimeric complex with peroxisomal biogenesis factor 7 (PEX7) and a peroxisome targeting signal 2 (PTS2) cargo protein and, therefore, failed to promote the import of PTS2 cargo proteins into peroxisomes. In conclusion, we report a novel missense mutation in PEX5 responsible for the defective import of PTS2 cargo proteins into peroxisomes resulting in congenital cataracts and developmental defects.

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.

Human neonatal stem cell-derived skin substitute improves healing of severe burn wounds in a rat model.

Conventional approaches can repair minor skin injuries; however, severe burn injuries require innovative approaches for efficient and better wound repair. Recent studies indicate that stem cell-based regenerative therapies can restore severe damaged skin both structurally and functionally. The current study aims to evaluate the wound healing potential of skin substitute derived from human neonatal stem cells (hNSCs) using a severe burn injury rat model. Amniotic epithelial cells (AECs) and mesenchymal stem cells (MSCs) were isolated from placenta (a source of neonatal stem cells) by explant culture method. After characterization, AECs and umbilical cord-MSCs were differentiated into keratinocyte and fibroblasts, respectively. Morphological changes, and expression of corresponding keratinocyte and fibroblast specific markers were used to verify differentiation into respective lineage. A skin substitute was developed by mixing hNSCs-derived skin cells (hNSCs-SCs) in plasma for transplantation in a rat model of severe burn injury. Results indicated that placenta-derived AECs and MSCs were efficiently differentiated into skin cells, that is, keratinocytes and fibroblasts, respectively, as indicated by morphological changes, immunostaining, and polymerase chain reaction analysis. Further, transplantation of hNSCs-SCs seeded in plasma significantly improved basic skin architecture, re-epithelization rate, and wound healing concurrent with reduced apoptosis. In conclusion, neonatal stem cell-derived skin substitute efficiently improved severe burn wounds in a rat model of burn injury. Unique properties of placenta-derived stem cells make them superlative candidates for the development of "off-the-shelf" artificial skin substitutes for future use.

Transplantation of stromal-derived factor 1α and basic fibroblast growth factor primed insulin-producing cells reverses hyperglycaemia in diabetic rats.

The defective insulin production is associated with severely reduced islet cell mass leading to diabetes. Growth factors preconditioned stem cells have arisen as an effective therapy to treat many diseases including diabetes. The current study was designed to assess the effect of pretreatment of ASCs derived IPCs with combination of stromal cell derived factor 1 alpha (SDF1α) and basic fibroblast growth factor (bFGF) in improving glucose tolerance in streptozotocin induced diabetic rats. The results showed maximally significant reduction in hyperglycaemia and fibrosis, while up-regulation of survival and pancreas-specific genes, insulin levels and homing of transplanted cells in SDF-1α + bFGF IPCs transplanted rats as compared with other groups. Moreover, increased expression of insulin, glucagon and Glut-2 in pancreas of the SDF-1α + bFGF IPCs transplanted group indicated more regeneration of pancreas. Hence, the use of IPCs preconditioned with SDF-1α + bFGF would be more effective for treating diabetes.

Serum from CCl4-induced acute rat injury model induces differentiation of ADSCs towards hepatic cells and reduces liver fibrosis.

Cellular therapies hold promise to alleviate liver diseases. This study explored the potential of allogenic serum isolated from rat with acute CCl4 injury to differentiate adipose derived stem cells (ADSCs) towards hepatic lineage. Acute liver injury was induced by CCl4 which caused significant increase in serum levels of VEGF, SDF1α and EGF. ADSCs were preconditioned with 3% serum isolated from normal and acute liver injury models. ADSCs showed enhanced expression of hepatic markers (AFP, albumin, CK8 and CK19). These differentiated ADSCs were transplanted intra-hepatically in CCl4-induced liver fibrosis model. After one month of transplantation, fibrosis and liver functions (alkaline phosphatase, ALAT and bilirubin) showed marked improvement in acute injury group. Elevated expression of hepatic (AFP, albumin, CK 18 and HNF4a) and pro survival markers (PCNA and VEGF) and improvement in liver architecture as deduced from results of alpha smooth muscle actin, Sirius red and Masson's trichome staining was observed.

Diazoxide preconditioning of endothelial progenitor cells from streptozotocin-induced type 1 diabetic rats improves their ability to repair diabetic cardiomyopathy.

Type 1 diabetes mellitus (DM) is a strong risk factor for the development of diabetic cardiomyopathy (DCM) which is the leading cause of morbidity and mortality in the type 1 diabetic patients. Stem cells may act as a therapeutic agent for the repair of DCM. However, deteriorated functional abilities and survival of stem cells derived from type 1 diabetic subjects need to be overcome for obtaining potential outcome of the stem cell therapy. Diazoxide (DZ) a highly selective mitochondrial ATP-sensitive K(+) channel opener has been previously shown to improve the ability of mesenchymal stem cells for the repair of heart failure. In the present study, we evaluated the effects of DZ preconditioning in improving the ability of streptozotocin-induced type 1 diabetes affected bone marrow-derived endothelial progenitor cells (DM-EPCs) for the repair of DCM in the type 1 diabetic rats. DM-EPCs were characterized by immunocytochemistry, flow cytometry, and reverse transcriptase PCR for endothelial cell-specific markers like vWF, VE cadherin, VEGFR2, PECAM, CD34, and eNOS. In vitro studies included preconditioning of DM-EPCs with 200 µM DZ for 30 min followed by exposure to either 200 µM H2O2 for 2 h (for oxidative stress induction) or 30 mM glucose media (for induction of hyperglycemic stress) for 48 h. Non-preconditioned EPCs with and without exposure to H2O2 and 30 mM high glucose served as controls. These cells were then evaluated for survival (by MTT and XTT cell viability assays), senescence, paracrine potential (by ELISA for VEGF), and alteration in gene expression [VEGF, stromal derived factor-1α (SDF-1α), HGF, bFGF, Bcl2, and Caspase-3]. DZ preconditioned DM-EPCs demonstrated significantly increased survival and VEGF release while reduced cell injury and senescence. Furthermore, DZ preconditioned DM-EPCs exhibited up-regulated expression of prosurvival genes (VEGF, SDF-1α, HGF, bFGF, and Bcl2) on exposure to H2O2, and VEGF and Bcl2 on exposure to hyperglycemia while down regulation of Caspase-3 gene. Eight weeks after type 1 diabetes induction, DZ preconditioned, and non-preconditioned DM-EPCs were transplanted into left ventricle of diabetic rats (at a dose of 2 × 10(6) DM-EPCs/70 µl serum free medium). After 4 weeks, DZ preconditioned DM-EPCs transplantation improved cardiac function as assessed by Millar's apparatus. There was decrease in collagen content estimated by Masson's trichrome and sirius red staining. Furthermore, reduced cell injury was observed as evidenced by decreased expression of Caspase-3 and increased expression of prosurvival genes Bcl2, VEGF, and bFGF by semi-quantitative real-time PCR. In conclusion, the present study demonstrated that DZ preconditioning enhanced EPCs survival under oxidative and hyperglycemic stress and their ability to treat DCM.

Diazoxide preconditioning of endothelial progenitor cells improves their ability to repair the infarcted myocardium.

Reduced survival and homing of the transplanted cells in the oxidative stressed ischemic environment limits the potential outcome of cell therapies for myocardial ischemia. Diazoxide (DZ), a highly selective mitochondrial ATP sensitive K(+) channel opener, is known to improve the survival and therapeutic ability of mesenchymal stem cells and skeletal myoblasts for the repair of heart failure. The current study explored the effect of DZ preconditioning in improving the ability of endothelial progenitor cells (EPCs) to counteract, in vitro oxidative stress, and to repair the infarcted myocardium. The EPCs were preconditioned by 30 min incubation with 200 µM DZ followed by exposure to 200 µM hydrogen peroxide (H2 O2 ) for 2 h. Non-preconditioned EPCs with and without exposure to H2 O2 were used as control. DZ preconditioning of EPCs resulted in significantly reduced cell injury as shown by reduced lactate dehydrogenase release and expression of annexin V-PE in comparison to untreated EPCs. Furthermore, DZ preconditioned EPCs exhibited upregulated expression of prosurvival genes (VEGF, SDF-1α, PCNA, and Bcl2 ), improved chemokines release (VEGF, IGF, and SDF-1α), viability, Akt phosphorylation and tube formation. In vivo experiments involved transplantation of DZ preconditioned and untreated EPCs in the left ventricle after permanent ligation of left anterior descending coronary artery in rats. The results demonstrated that DZ EPCs transplanted group showed significant reduction in infarct size along with robust cell proliferation, angiogenesis and improvement in cardiac function. The current study demonstrates that DZ preconditioning enhances EPCs survival under oxidative stress in vitro and their ability to treat myocardial infarction.

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.

Current progress of protein-based dressing for wound healing applications - A review.

Protein-based wound dressings have garnered increasing interest in recent years owing to their distinct physical, chemical, and biological characteristics. The intricate molecular composition of proteins gives rise to unique characteristics, such as exceptional biocompatibility, biodegradability, and responsiveness, which contribute to the promotion of wound healing. Wound healing is an intricate and ongoing process influenced by multiple causes, and it consists of four distinct phases. Various treatments have been developed to repair different types of skin wounds, thanks to advancements in medical technology and the recognition of the diverse nature of wounds. This review has literature reviewed within the last 3-5 years-the recent progress and development of protein in wound dressings and the fundamental properties of an ideal wound dressing. Herein, the recent strides in protein-based state-of-the-art wound dressing emphasize the significant challenges and summarize future perspectives for wound healing applications.

Chitosan-sodium percarbonate-based hydrogels with sustained oxygen release potential stimulated angiogenesis and accelerated wound healing.

The prolonged hypoxic conditions hinder chronic wounds from healing and lead to severe conditions such as delayed re-epithelialization and enhanced risk of infection. Multifunctional wound dressings are highly required to address the challenges of chronic wounds. Herein, we report polyurethane-coated sodium per carbonate-loaded chitosan hydrogel (CSPUO2 ) as a multifunctional dressing. The hydrogels (Control, CSPU, and CSPUO2 ) were prepared by freeze gelation method and the developed hydrogels showed high porosity, good absorption capacity, and adequate biodegradability. The release of oxygen from the CSPUO2 hydrogel was confirmed by the increase in pH and a sustained oxygen release was observed over the period of 21 days, due to polyurethane (CSPU) coating. The CSPUO2 hydrogel exhibited around 2-fold increased angiogenic potential in CAM assay when compared with Control and CSPU dressing. CSPUO2 also showed good level of antibacterial efficacy against E. coli and S. aureus. In a full-thickness rat wound model, CSPUO2 hydrogel considerably accelerated wound healing with exceptional re-epithelialization granulation tissue formation less inflammatory cells and improved skin architecture highlighting the tremendous therapeutic potential of this hydrogel when compared with control and CSPU to treat chronic diabetic and burn wounds.

Stromal cell derived factor-1alpha protects stem cell derived insulin-producing cells from glucotoxicity under high glucose conditions in-vitro and ameliorates drug induced diabetes in rats.

BACKGROUND: Diabetes mellitus is affecting more than 300 million people worldwide. Current treatment strategies cannot prevent secondary complications. Stem cells due to their regenerative power have long been the attractive target for the cell-based therapies. Mesenchymal stem cells (MSCs) possess the ability to differentiate into several cell types and to escape immune recognition in vitro. MSCs can be differentiated into insulin-producing cells (IPCs) and could be an exciting therapy for diabetes but problems like poor engraftment and survivability need to be confronted. It was hypothesized that stromal cell derived factor- 1alpha (SDF-1alpha) will enhance therapeutic potential of stem cell derived IPCs by increasing their survival and proliferation rate. METHODS: Novel culture conditions were developed to differentiate bone marrow derived mesenchymal stem cells (BMSCs) into IPCs by using endocrine differentiation inducers and growth factors via a three stage protocol. In order to enhance their therapeutic potential, we preconditioned IPCs with SDF-1alpha. RESULTS: Our results showed that SDF-1alpha increases survival and proliferation of IPCs and protects them from glucotoxicity under high glucose conditions in vitro. SDF-1alpha also enhances the glucose responsive insulin secretion in IPCs in vitro. SDF-1alpha preconditioning reverses hyperglycemia and increase serum insulin in drug induced diabetic rats. CONCLUSIONS: The differentiation of BMSCs into IPCs and enhancement of their therapeutic potential by SDF-1alpha preconditioning may contribute to cell based therapies for diabetes.

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.

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.

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.

Zinc oxide loaded chitosan-elastin-sodium alginate nanocomposite gel using freeze gelation for enhanced adipose stem cell proliferation and antibacterial properties.

Hydrogels have been the material of choice for regenerative medicine applications due to their biocompatibility that can facilitate cellular attachment and proliferation. The present study aimed at constructing a porous hydrogel composite scaffold (chitosan, sodium alginate and elastin) for the repair of chronic skin wounds. Chitosan-based hydrogel incorporating varying concentrations of zinc oxide nanoparticles i.e. ZnO-NPs (0, 0.001, 0.01, 0.1 and 1 % w/w) as the antimicrobial agent tested against Escherichia coli (E.coli) and Staphylococcus aureus (S. aureus) exhibited good antibacterial activities. ZnO-NPs were characterized by UV visible spectroscopy, Scanning electron microscopy (SEM) analysis, Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) analysis. Fabricated gels were characterized by SEM analysis, FTIR, XRD, swelling ratio, degradation behavior and controlled release kinetics of ZnO-NPs. In vitro cytocompatibility of the composite was investigated using human adipose stem cells (ADSCs) by MTT and lactate dehydrogenase (LDH) assay, further assessed by SEM analysis and PKH26 staining. The SEM and XRD analysis confirmed the successful loading of ZnO-NPs into these scaffolds. Fluorescence PKH26 stained images and SEM analysis of ADSCs seeded scaffolds revealed biocompatible nature. The findings suggested that the developed composite gels have potential clinically for tissue engineering and chronic wound treatment.

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.

Pre-conditioned mesenchymal stem cells ameliorate renal ischemic injury in rats by augmented survival and engraftment.

BACKGROUND: Ischemia is the major cause of acute kidney injury (AKI), associated with high mortality and morbidity. Mesenchymal stem cells (MSCs) have multilineage differentiation potential and can be a potent therapeutic option for the cure of AKI. METHODS: MSCs were cultured in four groups SNAP (S-nitroso N-acetyl penicillamine), SNAP + Methylene Blue (MB), MB and a control for in vitro analysis. Cultured MSCs were pre-conditioned with either SNAP (100 µM) or MB (1 µM) or both for 6 hours. Renal ischemia was induced in four groups (as in in vitro study) of rats by clamping the left renal padicle for 45 minutes and then different pre-conditioned stem cells were transplanted. RESULTS: We report that pre-conditioning of MSCs with SNAP enhances their proliferation, survival and engraftment in ischemic kidney. Rat MSCs pre-conditioned with SNAP decreased cell apoptosis and increased proliferation and cytoprotective genes' expression in vitro. Our in vivo data showed enhanced survival and engraftment, proliferation, reduction in fibrosis, significant improvement in renal function and higher expression of pro-survival and pro-angiogenic factors in ischemic renal tissue in SNAP pre-conditioned group of animals. Cytoprotective effects of SNAP pre-conditioning were abrogated by MB, an inhibitor of nitric oxide synthase (NOS) and guanylate cyclase. CONCLUSION: The results of these studies demonstrate that SNAP pre-conditioning might be useful to enhance therapeutic potential of MSCs in attenuating renal ischemia reperfusion injury.