Enhanced wound healing and hemostasis with exosome-loaded gelatin sponges from human umbilical cord mesenchymal stem cells

BACKGROUND Rapid wound healing remains a pressing clinical challenge,necessitating studies to hasten this process.A promising approach involves the utilization of human umbilical cord mesenchymal stem cells(hUC-MSCs)derived exosomes.The hypothesis of this study was that these exosomes,when loaded onto a gelatin sponge,a common hemostatic material,would enhance hemostasis and accelerate wound healing.AIM To investigate the hemostatic and wound healing efficacy of gelatin sponges loaded with hUC-MSCs-derived exosomes.METHODS Ultracentrifugation was used to extract exosomes from hUC-MSCs.Nanoparticle tracking analysis(NTA),transmission electron microscopy(TEM),and western blot techniques were used to validate the exosomes.In vitro experiments were performed using L929 cells to evaluate the cytotoxicity of the exosomes and their impact on cell growth and survival.New Zealand rabbits were used for skin irritation experiments to assess whether they caused adverse skin reactions.Hemolysis test was conducted using a 2%rabbit red blood cell suspension to detect whether they caused hemolysis.Moreover,in vivo experiments were carried out by implanting a gelatin sponge loaded with exosomes subcutaneously in Sprague-Dawley(SD)rats to perform biocompatibility tests.In addition,coagulation index test was conducted to evaluate their impact on blood coagulation.Meanwhile,SD rat liver defect hemostasis model and full-thickness skin defect model were used to study whether the gelatin sponge loaded with exosomes effectively stopped bleeding and promoted wound healing.RESULTS The NTA,TEM,and western blot experimental results confirmed that exosomes were successfully isolated from hUC-MSCs.The gelatin sponge loaded with exosomes did not exhibit significant cell toxicity,skin irritation,or hemolysis,and they demonstrated good compatibility in SD rats.Additionally,the effectiveness of the gelatin sponge loaded with exosomes in hemostasis and wound healing was validated.The results of the coagulation index experiment indicated that the gelatin sponge loaded with exosomes had significantly better coagulation effect compared to the regular gelatin sponge,and they showed excellent hemostatic performance in a liver defect hemostasis model.Finally,the full-thickness skin defect healing experiment results showed significant improvement in the healing process of wounds treated with the gelatin sponge loaded with exosomes compared to other groups.CONCLUSION Collectively,the gelatin sponge loaded with hUC-MSCs-derived exosomes is safe and efficacious for promoting hemostasis and accelerating wound healing,warranting further clinical application.

One-step cell biomanufacturing platform:porous gelatin microcarrier beads promote human embryonic stem cell-derived midbrain dopaminergic progenitor cell differentiation in vitro and survival after transplantation in vivo

Numerous studies have shown that cell replacement therapy can replenish lost cells and rebuild neural circuitry in animal models of Parkinson’s disease.Transplantation of midbrain dopaminergic progenitor cells is a promising treatment for Parkinson’s disease.However,transplanted cells can be injured by mechanical damage during handling and by changes in the transplantation niche.Here,we developed a one-step biomanufacturing platform that uses small-aperture gelatin microcarriers to produce beads carrying midbrain dopaminergic progenitor cells.These beads allow midbrain dopaminergic progenitor cell differentiation and cryopreservation without digestion,effectively maintaining axonal integrity in vitro.Importantly,midbrain dopaminergic progenitor cell bead grafts showed increased survival and only mild immunoreactivity in vivo compared with suspended midbrain dopaminergic progenitor cell grafts.Overall,our findings show that these midbrain dopaminergic progenitor cell beads enhance the effectiveness of neuronal cell transplantation.

Effect of porous titanium coated with IGF-1 and TGF-β_1 loaded gelatin microsphere on function of MG63 cells

Porous titanium with porosity of 60% was prepared by metal injection molding（MIM）,and coated with gelatin sustained-release microspheres which were made by improved emulsified cold condensation method.The effects of porous titanium coated with insulin-like growth factor-1（IGF-1） and transforming growth factor-β1（TGF-β1） gelatin microspheres on the function of MG63 cells were evaluated in vitro.The results show that porous titanium coated with gelatin sustained-release microspheres has no cytotoxicity.The IGF-1 and TGF-β1 loading concentrations are positively correlative with the proliferation and differentiation of MG63 after co-culturing with the concentrations of IGF-1 and TGF-β1 gelatin microspheres in the range of 0.1-10 ng/mg and 0.25-2.5 ng/mg,respectively.The MG63 cells exhibit the best proliferation and differentiation with the IGF-1 and TGF-β1 loading concentrations of 10 ng/mg and 2.5 ng/mg,respectively.The joint application of IGF-1 and TGF-β1 group,which promote adhesion,proliferation and differentiation of MG63 cells,is superior to a single application group.

Hepatocytic differentiation of mesenchymal stem cells in cocultures with fetal liver cells

AIM： To investigate the hepatocytic differentiation of mesenchymal stem cells （MSCs） in co-cultures with fetal liver cells （FLC） and the possibility to expand differentiated hepatocytic cells. METHODS： MSCs were marked with green fluorescent protein （GFP） by retroviral gene transduction. Clonal marked MSCs were either cultured under liver stimulating conditions using fibronectin-coated culture dishes and medium supplemented with stem cell factor （SCF）, hepatocyte growth factor （HGF）, epidermal growth factor （EGF）, and fibroblast growth factor 4 （FGF-4） alone, or in presence of freshly isolated FLC. Cells in co-cultures were harvested, and GFP＋ or GFP- cells were separated using fluorescence activated cell sorting. Reverse transcription-polymerase chain reaction （RT-PCR） for the liver specific markers cytokeratin-18 （CK-18）, albumin, and alpha-fetoprotein （AFP） was performed in different cell populations. RESULTS- Under the specified culture conditions, rat MSCs co-cultured with FLC expressed albumin, CK-18, and AFP-RNA over two weeks. At wk 3, MSCs lost hepatocytic gene expression, probably due to overgrowth of the cocultured FLC. FLC also showed a stable liver specific gene expression in the co-cultures and a very high growth potential. CONCLUSION： The rat MSCs from bone marrow can differentiate hepatocytic cells in the presence of FLC in vitro and the presence of MSCs in co-cultures also prorides a beneficial environment for expansion and differentiation of FLC.

Changes in the frequency of myeloid-derived suppressor cells after transarterial chemoembolization with gelatin sponge microparticles for hepatocellular carcinoma

Purpose: A series of clinical studies have established the safety and efficacy of transcatheter arterial chemoembolization(TACE) with gelatin sponge microparticles(GSMs) in treating hepatocellular carcinoma(HCC). HCC can lead to obvious necrosis inside tumors, especially larger ones, although it is unclear whether such necrotic tumor tissue can induce favorable immune reactions against the tumor. Myeloid-derived suppressor cells(MDSCs)have immunosuppressive functions and are currently considered a very important cell type affecting tumor immunity. This study observed changes in MDSC frequency in peripheral blood before and after GSM–TACE to evaluate the effect on the immune function of HCC patients.Methods: Eight patients diagnosed with HCC underwent GSM–TACE treatment in the Hepatobiliary Interventional Department of Beijing Tsinghua Chang Gung Hospital, Beijing, China;we followed up with the patients over a period of 30 days post-surgery. We used flow cytometry(FCM) to quantify the frequency of MDSCs in peripheral blood before TACE, 10 days after surgery and 30 days after surgery.Results: MDSC frequency after GSM–TACE had a significant downward trend. Pre-TACE, it was 30.73% ? 11.93%,decreasing to 18.60% ? 11.37% at 10 days after operation. This decrease was not statistically significant(P > 0.05). MDSC frequency was even lower 30 days after TACE(7.63% ? 7.32%) than at 10 days after TACE(P < 0.05), and there was a significant difference compared with pre-TACE(P < 0.001). We evaluated tumor response at 30 days after GSM–TACE according to the Modified Response Evaluation Criteria in Solid Tumors(mRECIST), and all eight patients showed partial response(PR).Conclusion: Our results confirmed that GSM–TACE was beneficial for improving anti-tumor immunity in the treatment of HCC.

Hepatitis C virus core proteins derived from different quasispecies of genotype 1b inhibit the growth of Chang liver cells

AIM: To investigate the influence of different quasispecies of hepatitis C virus (HCV) genotype 1b core protein on growth of Chang liver cells. METHODS: Three eukaryotic expression plasmids (pEGFP-N1/core) that contained different quasispecies truncated core proteins of HCV genotype 1b were constructed. These were derived from tumor (T) and non- tumor (NT) tissues of a patient infected with HCV and C191 (HCV-J6). The core protein expression plasmids were transiently transfected into Chang liver cells. At different times, the cell cycle and apoptosis was assayed by flow cytometry, and cell proliferation was assayed by methyl thiazolyl tetrazolium (MTT) assay. RESULTS: The proportion of S-phase Chang liver cells transfected with pEGFP-N1/core was significantly lower than that of cells transfected with blank plasmid at three different times after transfection (all P < 0.05). The proliferation ratio of cells transfected with pEGFP-N1/corewas significantly lower than that of cells transfected with blank plasmid. Among three different quasispecies, T, NT and C191 core expression cells, there was no significant difference in the proportion of S- and G0/G1-phase cells. The percentage of apoptotic cells was highest for T (T > NT > C191), and apoptosis was increased in cells transfected with pEGFP-N1/core as the transfection time increased (72 h > 48 h > 24 h). CONCLUSION: These results suggest that HCV genotype 1b core protein induces apoptosis, and inhibits cell- cycle progression and proliferation of Chang liver cells. Different quasispecies core proteins of HCV genotype 1b might have some differences in the pathogenesis of HCV persistent infection and hepatocellular carcinoma.

Metabolism of Mequindox in Isolated Rat Liver Cells

Mequindox （MEQ）, 3-methyl-2-quinoxalinacetyl-l,4-dioxide, is widely used in Chinese veterinary medicine as an antimicrobial agent and feed additive. Its toxicity has been reported to be closely related to its metabolism. To understand the pathways underlying MEQ＇s metabolism more clearly, we studied its metabolism in isolated rat liver cells by using liquid chromatography coupled with electrospray ionization hybrid linear trap quadrupole orbitrap （LC-LTQ-Orbitrap） mass spectrometry. The structures of MEQ metabolites and their product ions were readily and reliably characterized on the basis of accurate MS2 spectra and known structure of MEQ. Eleven metabolites were detected in isolated rat liver cells, two of which were detected for the first time in vitro. The major metabolic pathways reported previously for in vitro metabolism of MEQ in rat microsomes were confirmed in this study, including N O group reduction, carbonyl reduction, and methyl monohydroxylation. In addition, we fotmd that acetyl hydroxylation was an important pathway of MEQ metabolism. The results also demonstrate that cellular systems more closely simulate in vivo conditions than do other in vitro systems such as microsomes. Taken together, these data contribute to our understanding of the in vivo metabolism of MEQ.

Biocompatibility Studies on Bone Marrow Stromal Cells with Chitosan-gelatin Blends

To study the effect of chitosan-gelatin blends on the growth and proliferation of in vitro cultured bone marrow stromal cells(BMSCs) and explore a new carrier for the application of tissure engineering, cells from long bones of young rabbitsaged less than two weeks were expanded in vitro for one week and seeded onto the surface of pure chitosan and chitosan-gelatin blends. Cells attached to and proliferated on both pure chitosan and chitosan-gelatin blends were monitored with the aid of an inverted light microscope and a scanning electron microscope. The cell viability was monitored by MTT after 2, 4, 6, 8 days seeding. BMSCs could be attached to and proliferated on both pure chitosan and chitosan-gelatin blends and remain their morphologies seen in vivo. Chitosan-gelatin blends could promote BMSCs to proliferate(P<0.01). It is confirmed that chitosan-gelatin blends maintain the bioactivity feature of chitosan and even enhance the growth and proliferation of in vitro cultured BMSCs because of the adding of gelatin. It is a potential carrier for the delivery of cells tissue engineering.

Studies of the Kinetochore Proteins of the Regenerating Liver and the Liver Cells of Rats at Different Stages of Development

The kinetochore composition of rat liver cells was studied by indirect immunofluorescence andimmunoblotting using human anti-kinetochore/centromere autoantibodies(ACAs).Besides threemajor antigens(50kD,42 kD and 34 kD),ACAs used in this study could also identify those of 32-30 kD and 20 kD in newborn rat liver cells,90 kD in old rat liver cells,37 kD and 32-30 kD inregenerating liver cells.These results indicate that some kinetochore antigen(s)may be related to cellproliferation or specific for different stages of development.

Effect of the protection layer formed by cross-linked gelatin on the stability and performance of glucose and oxygen fuel cells

A glucose oxidation catalyst comprising carbon nanotube,tetrathiafulvalene(TTF),gelatin,glutaraldehyde(GA)and glucose oxidase(GOx)(CNT/[TTF-GOx]/Gelatin+GA)is suggested to enhance the reactivity of glucose oxidation reaction(GOR),and the performance and stability of enzymatic biofuel cells(EBCs)using this catalyst.In this catalyst,TTF is used as mediator to transfer electron effectively,while GA is crosslinked to gelatin to form non-soluble network.The structure prevents the dissolution of gelatin from aqueous electrolyte and reduces the leaching-out of GOx and TTF molecules.To confirm the crosslinking effect of GA and gelatin,Fourier-transform infrared spectroscopy(FT-IR)and electrochemical evaluations are utilized.According to FT-IR analysis,it was observed that the amide I peak shifted after crosslinking.This is evidence showing the appropriate network formation and the reactivity of CNT/[TTFGOx]/Gelatin+GA is well preserved even after multiple potential cycling.In addition,its GOx activity is regularly monitored for one month and the measurements prove that the structure prevents the leaching out of GOx molecules.Based on that,EBC using the anodic catalyst shows excellent performances,such as open circuit voltage of 0.75 V and maximum power density of 184μW/cm^(2).

Malignant Transformation of Human Embryonic Liver Cells Induced by Hepatitis B Virus and Aflatoxin B_1

In order to investigate the effect of hepatitis B virus (HBV) and aflatoxin B 1 (AFB 1) on hepatocarcinogenesis, the human embryonic liver cells infected with HBV were transplanted to nude mice by subcutaneous route and the transplanted mice were divided into 4 groups for study, in which the group A of mice was injected with HBV-infected human embryonic liver cells and followed by injections of AFB 1 once a week (HBV+AFB 1); the group B was treated with HBV as group A, but no AFB 1 was given (HBV +); the group C was injected with normal human embryonic liver cells and AFB 1 was used as group (AFB 1 +) and the group D or control group was injected with normal embryonic liver cells without addition of AFB 1. The experimental results showed that the incidences of tumor formation in different groups were 27.3% (6/22) in group A; 0% (0/13) in group B; 13.3% (2/15) in group C and 0% (0/14) in group D respectively. All the tumors formed were proved to be human hepatocellular carcinoma (HCC) by pathological examinations and the tumor tissues were anthrogenetic as demonstrated by EMA monoclonal antibody. The HBV-X and HBV-S genes could be detected in the tumor tissues by means of slot hybridization and PCR amplification, indicating that the HBV-DNA genes had integrated into DNA of host cells. Thus, we have successfully induced the human HCC through HBV infection and introduction of AFB 1 with a synergistic effect between HBV and AFB 1 in hepatocarcinogenesis.

Comparative study on radiosensitivity of various tumor cells and human normal liver cells

AIM:To investigate the radiation response of various human tumor cells and normal liver cells. METHODS: Cell lines of human hepatoma cells (SMMC-7721), liver cells (L02), melanoma cells (A375) and cervical tumor (HeLa) were irradiated with 60Co γ-rays. Cell survive was documented by a colony assay. Chromatid breaks were measured by counting the number of chromatid breaks and isochromatid breaks immediately after prematurely chromosome condensed by Calyculin-A. RESULTS: Linear quadratic survival curve was observed in all of four cell lines, and dose-dependent increase in radiation-induced chromatid and isochromatid breaks were observed in GB2B phase. Among these four cell lines, A375 was most sensitive to radiation, while, L02 had the lowest radiosensitivity. For normal liver cells, chromatid breaks were easy to be repaired, isochromatid breaks were difficult to be repaired. CONCLUSION: The results suggest that the y-rays induced chromatid breaks can be possibly used as a good predictor of radiosensitivity, also, unrejoined isochromatid breaks probably tightly related with cell cancerization.

YKL40 expression in CD14^+ liver cells in acute and chronic injury

AIM:To demonstrate that CD14 + cells are an important source of the growth factor YKL40 in acute and chronic liver damage.METHODS:Rats were inoculated with one dose of CCl4 to induce acute damage.Liver biopsies were obtained at 0,6,12,24,48 and 72 h.For chronic damage,CCl4 was administered three days per week for 6 or 8 wk.Tissue samples were collected,and cellular populations were isolated by liver digestion and purified by cell sorting.YKL40 mRNA and protein expression were evaluated by realtime polymerase chain reaction and western blot.RESULTS:Acute liver damage induced a rapid increase of YKL40 mRNA beginning at 12 h.Expression peaked at 24 h,with a 26fold increase over basal levels.By 72 h however,YKL40 expression levels had nearly returned to control levels.On the other hand,chronic damage induced a sustained increase in YKL40 expression,with 7and 9fold higher levels at 6 and 8 wk,respectively.The pattern of YKL40 expression in different subpopulations showed that CD14+cells,which include Kupffer cells,are a source of YKL40 after acute damage at 72 h[0.09 relative expression units(REU)]as well as after chronic injury at 6 wk(0.11 REU).Hepatocytes,in turn,accounted for 0.06 and 0.01 REU after 72 h(acute)or 6 wk(chronic),respectively.The rest of the CD14cells(including T lymphocytes,B lymphocytes,natural killer and natural killer T cells) yielded 0.07 and 0.15 REU at 72 h and 6 wk,respectively.YKL40 protein expression in liver was detected at 72 h as well as 6 and 8 wk,with the highest expression relative to controls(11fold;P≤0.05)seen at 6 wk.Macrophages were stimulated by lipopolysaccharide.We demonstrate that under these conditions,these cells showed maximum expression of YKL40 at 12 h,with P<0.05 compared with controls.CONCLUSION:Hepatic CD14 + cells are an YKL40 mRNA and protein source in acute and chronic liver injury,with expression patterns similar to growth factors implicated in inflammationfibrogenesis.

A New Hematopoietic Stimulating Activity Produced by Fetal Liver Cells

Human fetal liver cells were cultured in vitro for 12h and the supernatant(Fetal liver cell conditioned medium,FLCM)was collected.The effects of FLCM ongranulopoiesis were studied.The results show that when combined with colonystimulating factor(CSF),FLCM could significantly stimulate the proliferation of normalmyctoid progenitor cells(CFU-e),and increase ~3H-TdR incorporation into bone mar-row cells.The data suggest that FLCM contains a CSF synergistic activity.

Endosulfan Causes Neoplastic Changes in the Liver Cells of Mice

The rapid growth in global population continues to challenge the world’s ability to provide enough food. As one of the most crucial issues for human development, food production must increase to offset hunger and poverty as well as social unrest. To augment the yield of crops a variety of pesticides like Endosulfan, Rogor, Aldrin, Chlorpyrifos, etc. are being used liberally by the farmers. In the present investigation, Endosulfan was administered orally (daily) by gavage method to female Swiss albino mice group for 4 weeks @ 3.0 mg/kg b.w. After that, they were left for 6 months and then sacrificed and liver tissues were fixed for light microscopy and Transmission Electron Microscopic study. The histopathological study of Endosulfan administered group liver showed hepatocytes with congestion in central vein with less dense cytoplasm, haemorrhaged bile duct, degenerated cytoplasm and central vein with vacuolations in sinusoidal spaces. Neoplastic changes in hepatocytes are the major finding of study. The ultrastuctural study revealed dilation in the nuclear pore complex and massive movement of cytoplasmic material from cytoplasm to the nucleus which is major finding which denotes neoplastic changes. Presence of abundant free lying polyribosomes in the cytoplasm, which denotes neoplastic changes in the cellis also one of theimportant finding observed. The present study thus deciphers that Endosulfan toxicity leads to onset of neoplasia thence carcinogenesis in liver cells in Swiss albino mice which is the novel finding in the field of toxicology.

The impact of Hypoglycemic Ziyabiti Tablets (HZT) on diabetes mellitus rats and its activity in cultured rat liver cells

Objective Hypoglycemic Ziyabiti Tablets(HZT)is a traditional multicomponent treatment for diabetes in Xinjiang Uyghur Traditional Medicine.The present study aimed to investigate the effect of HZT in diabetic rats and its activity in cultured liver cells to investigate the relative mechanisms.Methods 10 days high-fat diet fed rats were intraperitoneally injected with alloxan(ALX)at next two subsequent days to induce diabetes mellitus(DM).Then were divided into 5 groups:saline,positive DM control and DM groups treated with different doses of HZT.Fasting blood glucose(FBG),total cholesterol(TC),total triglycerides(TG),high-density lipoprotein(HDL-C),fasting insulin(FI),insulin secretion(IS)and insulin sensitivity index(ISI)were measured.The IC_(50) of HZT in L-02 cells was determined by MTT assay,in intact and in paracetamol-induced liver injury(Par),on lactate dehydrogenase(LDH)activity and on glucose consumption.Results HZT decreased FBG and TC(P <0.05),increased IS(P <0.05)and at 440 mg·kg^(-1)·d^(-1) increased FI(P < 0.01).In vitro,HZT at 0.1,0.2,and 0.4 mg/mL decreased LDH activity and promoted glucose consumption.Conclusion The hypoglycemic mechanism of HZT is possibly related to increased insulin secretion from the pancreas and increased utilization of glucose by the liver.

Current perspectives on mesenchymal stem cells as a potential therapeutic strategy for non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease(NAFLD)has emerged as a significant health challenge,characterized by its widespread prevalence,intricate natural progression and multifaceted pathogenesis.Although NAFLD initially presents as benign fat accumulation,it may progress to steatosis,non-alcoholic steatohepatitis,cirrhosis,and hepatocellular carcinoma.Mesenchymal stem cells(MSCs)are recognized for their intrinsic self-renewal,superior biocompatibility,and minimal immunogenicity,positioning them as a therapeutic innovation for liver diseases.Therefore,this review aims to elucidate the potential roles of MSCs in alleviating the progression of NAFLD by alteration of underlying molecular pathways,including glycolipid metabolism,inflammation,oxidative stress,endoplasmic reticulum stress,and fibrosis.The insights are expected to provide further understanding of the potential of MSCs in NAFLD therapeutics,and support the development of MSC-based therapy in the treatment of NAFLD.

Synthesis and Bio-activities of Poly(3,4-ethylenedioxythiophene)(PEDOT)/Poly(styrene sulfonate)(PSS)/Gelatin Layer on Indium Tin Oxide(ITO)-coated Glass

Poly （3, 4-ethylenedioxythiophene） （PEDOT）, together with its dopes, such as poly （styrene sulfonate） （PSS）, has been acknowledged to have a wide range of biomedical applications as an important conducting polymer. In this study, gelatin can be polymerized into PEDOT/PSS polymers on indium tin oxide （ITO）-coated glass. PEDOT/PSS/gelatin layer on ITO-coated glass significantly decreases electrochemical impedance spectroscopy （EIS） and increases charge delivery capacity relative to the gelatin layer and bare ITO- coated glass, comparable to the PEDOT/PSS layer on ITO-coated glass. PEDOT/PSS/gelatin layer on ITO- coated glass enhances pheochromocytoma （PC 12） cell affinity, possesses a high biocompatibility and promotes PC 12 cell growth by delivery of electrical stimulation. These results suggest that gelatin can be incorporated into the PEDOT/PSS polymers through electrochemical polymerization and the PEDOT/PSS/gelatin layer on ITO-coated glass possesses high electrochemical and biological activities.

Comprehensive Understanding of Immune Cells in The Pathogenesis of Non-alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease(NAFLD)is the most common chronic liver disease,defined by several phases,ranging from benign fat accumulation to non-alcoholic steatohepatitis(NASH),which can lead to liver cancer and cirrhosis.Although NAFLD is a disease of disordered metabolism,it also involves several immune cell-mediated inflammatory processes,either promoting and/or suppressing hepatocyte inflammation through the secretion of pro-inflammatory and/or anti-inflammatory factors to influence the NAFLD process.However,the underlying disease mechanism and the role of immune cells in NAFLD are still under investigation,leaving many open-ended questions.In this review,we presented the recent concepts about the interplay of immune cells in the onset and pathogenesis of NAFLD.We also highlighted the specific non-immune cells exhibiting immunological properties of therapeutic significance in NAFLD.We hope that this review will help guide the development of future NAFLD therapeutics.