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Objective To explore the clinical application value of ultrasound-guided intrahepatic biliary puncture combined with internal double biliary stenting under DSA in elderly patients with malignant hilar biliary obstruction (MHBO).Methods Totally 108 elderly MHBO patients received interventional treatment were analyzed retrospectively.Half of patients were treated with ultrasound-guided intrahepatic biliary puncture (ultrasound group),and another 54 patients were treated with DSA intrahepatic biliary puncture (DSA group).After successful puncture,the patients received percutaneous transhepatic cholangial drainage (PTCD) with 4 methods under DSA guidance,namely internal double biliary stenting,contralateral external PTCD with single biliary stenting,complete external PTCD and external PTCD on the dominant side.The recent complications of intrahepatic biliary puncture at two groups and the curative effect with four methods were observed.Results The frequency of intrahepatic biliary puncture,the dosage of contrast agent,the incidence of pain at the puncture point and hemobilia in ultrasound group were all lower than those in DSA group (all P＜0.05),the successful rate of intrahepatic biliary puncture in first time was significantly higher compared with DSA group (P＜0.05).The liver function indicators at 14 days postoperation and total bilirubin at 21 days postoperation had statistical differences between any two biliary drainage methods (all P＜ 0.05).Conclusion Ultrasound-guide intrahepatic biliary puncture combined with internal double biliary stenting under DSA can significantly benefit elderly patients with MHBO.
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Objective To investigate the efficacy of absorbable screw rod system in the treatment of posterior hip dislocation complicated with femoral head fracture.Methods Between February 2009 and June 2014,20 patients were treated at our department for posterior hip dislocation complicated with femoral head fracture.They were 14 males and 6 females,with an average age of 38.2 years (range,from 27 to 60 years).Eight left hips and 12 right hips were affected.By the Pipkin classification,15 cases were type Ⅰ and 5 type Ⅱ.The time from injury to surgery ranged from 3 to 14 days (average,6 days).All of them were treated with absorbable screw rod system after Allis manual reduction.Results The operation time in this group ranged from 1 to 6 hours (average,1.8 hours).The intraoperative blood loss ranged from 70 to 400 mL (average,160 mL).They were followed up for 18 to 48 months (average,32 months).All the fractures united after an average time of 3.4 months (range,from 2.5 to 5.0 months).According to the Harris scores at 6 months postoperation,10 cases were rated as excellent,7 as good,2 as fair and one as poor,with a good to excellent rate of 85.0％.Total hip replacement was performed for 2 fair and one poor cases because their postoperative pain was not relieved and femoral avascular necrosis developed.Conclusion Absorbable screw rod system is an effective treatment of posterior dislocation with femoral head fracture,because it can simplify operative procedures,reduce trauma,fixate the fracture firmly,avoid secondary operation,and reduce postoperative complications.
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This study examined the effect of IKVAV peptide nanofiber on proliferation, adhesion and differentiation into neurocytes of bone marrow stromal cells (BMSCs). IKVAV Peptide-amphiphile was synthesized and purified. Then, hydrogen chloride was added to the diluted aqueous solutions of PA to induce spontaneous formation of nanofiber in vitro. The resultant samples was observed under transmission electron microscope. BMSCs were cultured with IKVAV peptide nanofiber. The effect of IKVAV nanofiber on the proliferation, adhesion and induction differentiation of BMSCs was observed by inverted microscopy, calcein-AM/PI staining, cell counting and immunofluorescence staining. The results demonstrated that IKVAV peptide-amphiphile could self-assemble to form nanofiber gel. BMSCs cultured in combination with IKVAV peptide nanofiber gel grew well and the percentage of live cells was over 90%. IKVAV peptide nanofiber gel exerted no influence on the proliferation of BMSCs and could promote the adhesion of BMSCs and raise the ratio of neurons when BMSCs were induced to differentiate into neurocytes. It is concluded that BMSCs could proliferate and adhere well and yield more neurons during when induced to differente into neurocytes on IKVAV peptide nanofiber gel.
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KLD-12 peptide with a sequence of AcN-KLDLKLDLKLDL-CNH(2) was synthesized and its biocompatibility was assessed in animals. Rabbit MSCs were cultured in the hydrogel for 2 weeks. Live cells were counted by using Calcein-AM/PI fluorescence staining. MTT was employed to assess the viability of MSCs cultured in KLD-12 peptide solution of 0.01%, 0.03%, and 0.05%. Hemolysis test, skin irritation test and implantation test were conducted to evaluate its biocompatibility with host tissues. Our results demonstrated that the MSCs in hydrogel grew well and maintained round shape. Cell survival rate was 92.15% (mean: 92.15%+/-1.17%) at the 7th day and there was no difference in survival rate between day 7 and day 14. Cell proliferation test showed that the A value of the KLD-12 solutions was not significantly different from that of control groups (complete culture media) (P>0.05) at the 24th and 48th h. The hemolysis rate of KLD-12 solution was 0.112%. Skin irritation test showed that the skin injected with KLD-12 solution remained normal and the score of skin irritation was 0. The histological examination with HE staining exhibited that the skin layers were clear and there was no infiltration with neutrophilic granulocytes and lymphocytes. It is concluded that KLD-12 peptide hydrogel had a good biocompatibility with host rabbit and MSCs, and KLD-12 peptide hydrogel can provide an appropriate microenvironment for MSCs.
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The neotype of amphiphilic oligopeptide (C16 H31 O-AAAGGGGDDIKVAV) was synthesized. The framework of three-dimensional and porous hydrogel self-assembly from the amphiphilic oligopeptide on different conditions was explored. The peptide, whose molecular weight (MW) and purity were detected by Mass Spectrometer (MS) and High Performance Liquid Chromatograph (HPLC) respectively, was synthesized in solid phase methods. Peptide was dissolved in 0.1 mol/L Sodium Hydroxide (NaOH) solution. 200 microl of 10, 2, 1, 0.5 wt% peptide solutions, which were prepared respectively, were added into the same volume of DMEM/F12, or placed into the vapor of 10 mol/L Hydrochloric acid (HCl), or were used to coat in the surface of coverslip and set into the baking oven at 37 degrees C. The self-assembly hydrogel was examined with transmission electron microscope (TEM) and scanning electron microscope (SEM). MS showed that peptide MW was 1438.31. HPLC testified that the peptide purity was 96%. The peptide solution was self-supported into hydrogel triggered with DMEM/F12 in few seconds, or the thin hydrogel after two hours in the vapor of 10 mol/L HCl, or not hydrogel in the baking oven at 37 degrees C. SEM showed that the hydrogel self-assembly from 10 wt% peptide solution was composed of nanofibers that ranked in layers where there were thick voids. TEM showed that the hydrogel self-assembly from 2, 1, 0.5 wt% peptide solution comprised woven network nanofibers, that the nanofibers of hydrogel self-supported from 1 wt% peptide solution varied from 3 to 6 nm in diameter and 100 nm to 1.5 um in length, that the nanofibers of hydrogel self-supported from 2 wt% peptide solution ranked closely, and there were big voids within the thin nanofibers of hydrogel self-supported from 0.5 wt% peptide solution. The amphiphilic oligopeptide was synthesized and self-organized successfully into porous hydrogel characterized as "intelligent" tissue engineering scaffolds containing the bioactive ligand, which was triggered by DMEM/F12.
Subject(s)
Biocompatible Materials , Hydrogel, Polyethylene Glycol Dimethacrylate , Chemistry , Peptides , Chemistry , Porosity , Tissue Engineering , Methods , Tissue Scaffolds , ChemistryABSTRACT
We observed the effects of IKVAV self-assembling peptide nanofiber scaffold (SAPNS) on olfactory ensheathing cells (OECs). The IKVAV molecules were triggered to self-assemble to interconnected nanofibers hydrogel by adjusting pH of solution and adding of DMEM/F12 culture medium. Atomic Force Microscopy (AFM) showed that self-assembly hydrogel was consisted of the interconnected nanofibers, which varied from three nm to five nm in diameter and hundreds nanometer in length. The primary OECs were isolated from rat olfactory bulb and purified by differential adhesion twice. At days 12, the purity of OECs was 85% according to immunostaining of P75 NGFR antibody. OECs were cultured with IKVAV peptide. The adhesion, viability and proliferation of OECs were observed with inverted microscope, Calcein-AM/PI staining and Cell Counting Kit-8. OECs cultured on IKVAV SAPNS grew well and the viable cell count was 95%. IKVAV SAPNS can promote the adhesion of OECs and did no hinder the proliferation of OECs. IKVAV SAPNS nanofiber gel has good biocompatibility and bioactivity for OECs. It can serve as a good nerve tissue engineering scaffold.
Subject(s)
Animals , Humans , Rats , Biocompatible Materials , Chemistry , Hydrogels , Chemistry , Laminin , Chemistry , Materials Testing , Nanofibers , Chemistry , Olfactory Bulb , Cell Biology , Olfactory Nerve , Cell Biology , Peptide Fragments , Chemistry , Rats, Sprague-Dawley , Tissue Engineering , Methods , Tissue Scaffolds , ChemistryABSTRACT
A systematic method of isolating and culturing human bone mesenchymal stem cells (hMSCs), and inducing them to differentiate into neuron-like cells in vitro was established. The hMSCs were isolated from bone marrow with the lymphocyte-separating medium, cultured and expanded in vitro, and induced after addition of compound neuro-revulsants. The morphological changes of hMSCs were observed, and the expression of surface markers in induced hMSCs was immunocytochemically identified during induction period. The hMSCs could be separated, cultured and expanded in vitro. After induction by compound neuro-revulsants for 48 h, the changes of neuron-like cells, such as cellular shrinkage and neurite growth, were observed in some cells. The immunochemical staining revealed nestin (+) or NF (+), and GFAP (-). It was concluded that hMSCs were successfully cultured and induced to differentiate into neuron-like cells.
Subject(s)
Bone Marrow Cells/cytology , Cell Culture Techniques , Cell Differentiation/physiology , Cells, Cultured , Mesenchymal Stem Cells/cytology , Neurons/cytologyABSTRACT
BACKGROUND:IKVAV-containing peptide sequence is an active region that promotes the adhesion, growth, and differentiation of neural cells in the laminin. It can be fabricated into a novel tissue-engineered scaffold material by self-assembly into hydrogel.OBJECTIVE: This study was designed to synthesize IKVAV-containing (C16H31O-AAAA GGGEIKVAV) peptide. The peptide was observed self-assembly into three-dimensional and porous hydrogel after triggered with phosphate buffered saline (PBS).DESIGN, TIME AND SETTING: Single-sample experiment, performed in the Laboratory of Department of Orthopedics,Union Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology between September 2004 and January 2005.MATERIALS: IKVAV-containing peptide was synthesized by solid phase method.METHODS: Peptide purity and relative molecular mass were detected by high performance liquid chromatogram and mass-spectrometry, respectively. 1% peptide, whose pH value was equal to 9.5, was self-assembled into hydrogel with the addition of PBS. The ultramicrostructure of the hydrogei was observed through the use of transmission electron microscope (TEM).MAIN OUTCOME MEASURES: Peptide purity and relative molecular mass were detected. Moreover, gross observation of the peptide after self-assembly and TEM observation of peptide ultramicrostructure were performed.RESULTS: Peptide relative molecular mass was 1351.6, which was in accordance with its theoretical value. Peptide purity was 95%. After triggered with PBS, 1% peptide was self-supported into hydrogel in a few seconds. TEM results showed that self-assembled hydrogel consisted of the interconnected nanofibers which varied from 3 to 6 nm in diameter and 100 to 1 500 nm m in length.CONCLUSION: The IKVAV-containing peptide was synthesized and self-organized successfully into porous hydrogel,which was triggered with PBS solution.
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The characteristics for the ex vivo expansion of the endothelial progenitor cells (EPCs)were explored. CD34+ cells were selected from umbilical cord blood mononuclear cells (MNC) by MiniMACS system, expanded under the same conditions as those for total MNC, coincubation of CD34+ and CD34- from the same donor for EPCs. In addition, the effects of vessel endothelial growth factor (VEGF) and passage on cell differentiation, expansion kinetics and apoptosis were examined.EPCs were determined and quantified by immunocytochemistry and flow cytometry. The results showed that both coculture of CD34+ and CD34 and total MNC led to a significant increase in the expansion of CD34+ cells as compared with CD34 enrichment (P＜0.05). There was a tendency toward decreased apoptosis in cultures when early passage was performed immediately after cord like structures appeared. VEGF had no significant effect on apoptosis (P＞0.05). These differentiated EPCs were positive for CD34+, von Willebrand factor (vWF), KDR, CD31 staining and phagocytized acetylated low-density lipoprotein (LDL). CD34+ cells accounted for (68.2±6.3) % of attaching (AT)cells at day 7 of culture. It was suggested the most efficient method to ex vivo expansion of EPCs was coculture of CD34+ and CD34- or total MNC. Early passage makes cell apoptosis rate decrease.VEGF had no significant effect on ex vivo expansion of EPCs.
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Lack of biocompatibility and bioactivity is a big problem for the synthetic materials that have been generated for neural tissue engineering. To get around the problem and generate better scaffold for neural tissue repair, we intended to generate nano-fibers by self-assembly of polypeptide IKVAV. Bioactive IKVAV Peptide-Amphiphile (IKVAV-PA) was first synthesized and purified, the property of which was analyzed and determined by high-performance liquid chromatography (HPLC)and mass spectrometry (MS). Then, by addition of hydrogen chloride (HCl), self-assembly of IKVAV-PA was induced in vitro and nano-fibers formed as shown by transmission electron microscopy (TEM). The effect of IKVAV nanofibers on adherence of PC12 cells was assayed in cell culture and the results showed that the rates of adherence of PC12 increased significantly when the density of IKVAV was within a certain range (0.58 μg/cm2 to 15.6 μg/cm2). However, its effect on the rates of adherence did not significantly alter with time, whether after 1 hour or 3 hours of culture. In general,we showed that IKVAV-PA can successfully self-assemble to form nanofiber, and promote rapid and stable adherence of PC12 cells, and the effect of the self-assembled IKVAV to promote PC12 cells adherence is dosage-dependent within a certain range of densities.
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Lack of biocompatibility and bioactivity is a big problem for the synthetic materials that have been generated for neural tissue engineering. To get around the problem and generate better scaffold for neural tissue repair, we intended to generate nano-fibers by self-assembly of polypeptide IKVAV. Bioactive IKVAV Peptide-Amphiphile (IKVAV-PA) was first synthesized and purified, the property of which was analyzed and determined by high-performance liquid chromatography (HPLC) and mass spectrometry (MS). Then, by addition of hydrogen chloride (HCl), self-assembly of IK-VAV-PA was induced in vitro and nano-fibers formed as shown by transmission electron microscopy (TEM). The effect of IKVAV nanofibers on adherence of PC12 cells was assayed in cell culture and the results showed that the rates of adherence of PC12 increased significantly when the density of IKVAV was within a certain range (0.58 microg/cm2 to 15.6 microg/cm2). However, its effect on the rates of adherence did not significantly alter with time, whether after 1 hour or 3 hours of culture. In general, we showed that IKVAV-PA can successfully self-assemble to form nanofiber, and promote rapid and stable adherence of PC12 cells, and the effect of the self-assembled IKVAV to promote PC12 cells adherence is dosage-dependent within a certain range of densities.
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The characteristics for the ex vivo expansion of the endothelial progenitor cells (EPCs) were explored. CD34+ cells were selected from umbilical cord blood mononuclear cells (MNC) by MiniMACS system, expanded under the same conditions as those for total MNC, coincubation of CD34+ and CD34- from the same donor for EPCs. In addition, the effects of vessel endothelial growth factor (VEGF) and passage on cell differentiation, expansion kinetics and apoptosis were examined. EPCs were determined and quantified by immunocytochemistry and flow cytometry. The results showed that both coculture of CD34+ and CD34- and total MNC led to a significant increase in the expansion of CD34+ cells as compared with CD34 enrichment (P 0.05). These differentiated EPCs were positive for CD34+, von Willebrand factor (vWF), KDR, CD31 staining and phagocytized acetylated low-density lipoprotein (LDL). CD34+ cells accounted for (68.2 +/- 6.3)% of attaching (AT) cells at day 7 of culture. It was suggested the most efficient method to ex vivo expansion of EPCs was coculture of CD34+ and CD34- or total MNC. Early passage makes cell apoptosis rate decrease. VEGF had no significant effect on ex vivo expansion of EPCs.
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The expression of Smad2 and Smad3 and the influence of exogenous transforming growth factorβ1 (TGFβ1) on them in rat bone marrow-derived mesenchymal stem cells (MSCs) cultured in vitro were investigated. The effects of different concentrations of TGFβ1 on cell proliferation and ALP activity were detected by MTT and PNPP in MSCs respectively. The expression of Smad2 and Smad3 and the influence of exogenous TGFβ1 on them were also examined by immunocytochemistry and Western blot assays. The exogenous TGFβ1 induced a dose-dependent decrease in cell proliferation and a dose-dependent increase in ALP activity, which plateaued at 5 ng/ml. Smad2 and Smad3 proteins were detected only in the cytoplasm in the absence of TGFβ1 and TGFβ1 could stimulate the translocation of them from the cytoplasm to the nucleus. The total amount of Smad2 protein remained unchanged before and after TGFβ1 treatment (P＞0.05). The expression levels of Smad3 remained unchanged after 3 h and 6 h treatment (P＞0. 05), but decreased markedly after 24 h treatment (P＜0.05). It was concluded that TGFβ1 is a latent osteoinductive factor involved in osteoblastic differentiation. Both Samd2 and Smad3 mediate TGFβ1 signaling as downstream mediators in MSCs. The biological output of TGFβ1 triggering the osteoblastic differentiation could be entirely determined by Smad3 in MSCs.
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To explore the possibility and condition of differentiation of bone marrow mesenchymal cells (BMSCs) to neural cells in vitro, BMSCs from whole bone marrow of rats were cultured. The BMSCs of passage 3 were identified with immunocytochemical staining of CD44 ( + ), CD71 ( + )and CD45(-). There were type Ⅰ and type Ⅱ cells in BMSCs. Type Ⅰ BMSCs were spindleshaped and strong positive in immunocytochemical staining of CD44 and CD71, whereas flat and big type Ⅱ BMSCs were lightly stained. The BMSCs of same passage were induced to differentiate into neural cells by β-mercaptoethanol (BME). After induction by BME, the type Ⅰ BMSCs withdrew to form neuron-like round soma and axon-like and dendrite-like processes, and were stained positively for neurofilament (NF). The type Ⅱ BMSCs did not change in the BME medium and were negatively or slightly stained of NF.
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The cortexes were obtained from new-born rats and dissociated to single cells by triturating. The cells were cultured in neural stem cell (NSC) culture medium (DMEM supplemented with bFGF, EGF and B27) and formed primary neurospheres after 7 days. Single cells dissociated from neurosphere were cultured in 96-well plates and formed single-cell cloning neurosphere 7 days later. The primary and single-cell cloning neurospheres were both positive for the immunofluorescent staining of nestin and were identified as NSC. It was proved that NSC can be expanded in vitro and provide seed cells for neural tissue engineering.
Subject(s)
Animals , Rats , Animals, Newborn , Cell Separation , Cells, Cultured , Cerebral Cortex , Cell Biology , Culture Media , Neurons , Cell Biology , Rats, Sprague-Dawley , Stem Cells , Cell Biology , Tissue EngineeringABSTRACT
The cortexes were obtained from new-born rats and dissociated to single cells by triturating. The cells were cultured in neural stem cell (NSC) culture medium (DMEM supplemented with bFGF, EGF and B27) and formed primary neurospheres after 7 days. Single cells dissociated from neurosphere were cultured in 96-well plates and formed single-cell cloning neurosphere 7 days later. The primary and single-cell cloning neurospheres were both positive for the immunofluorescent staining of nestin and were identified as NSC. It was proved that NSC can be expanded in vitro and provide seed cells for neural tissue engineering.
Subject(s)
Animals, Newborn , Cell Separation , Cells, Cultured , Cerebral Cortex/cytology , Culture Media , Neurons/cytology , Rats, Sprague-Dawley , Stem Cells/cytology , Tissue EngineeringABSTRACT
To investigate the effect of basic fibroblast growth factor(bFGF) gene transfection on the proliferation and differentiation of mesenchymal stem cells (MSCs) and to provide basis for accelerating bone defect repairing using gene-enhanced tissue engineering technology, Rabbit periosteum-derived MSCs were transfected with the full-length rat bFGF cDNA in vitro. The transient and stable gene expression of bFGF were determined by immunohistochemistry. The proliferation and the synthesis alkaline phosphatase (ALP) and osteocalcin(OC) of the transfected MSCs were also examined. The results showed that bFGF cDNA could be transferred into osteoblasts and expressed stably at least 4 weeks. The proliferation and OC content of genetically modified MSCs were increased significantly, whereas the ALP activity remained no change. In conclusion, transfer of gene encoding bFGF to MSCs increases its proliferation and osteogenesis property. Based on the successful conjunction of the existing techniques of tissue engineering with the novel possibilities offered by modern gene transfer technology, an innovative concept, molecular tissue engineering, was put forward for the first time. As a new branch of tissue engineering, it represents both a new area and an important trend in tissue engineering research.
Subject(s)
Animals , Rabbits , Alkaline Phosphatase , Cell Differentiation , Physiology , Cell Division , Physiology , Cells, Cultured , Fibroblast Growth Factor 2 , Genetics , Physiology , Osteocalcin , Stem Cells , Cell Biology , Physiology , Stromal Cells , Cell Biology , Physiology , Tissue Engineering , Methods , TransfectionABSTRACT
AIM: To explore the ex vivo expansion characteristics of the endothelial progenitor cells (EPCs). METHODS: CD34+ cells were selected from umbilical cord blood mononuclear cells (MNC) by MiniMACS system, expanded at the same conditions as that for total MNC, coincubation of CD34+ and CD34- from the same donation for EPCs. In addition, we tested the effect of vessel endothelial growth factor (VEGF) and passage on cell differentiation, expansion kinetics and apoptosis. EPCs were determined and quantified by immunocytochemistry and flow cytometry. RESULTS: Coculture of CD34+ and CD34-,total MNC led to a significant increase in the expansion of CD34+ cells compared with CD34 enrichment (P0.05). These differentiated EPCs were stained positive for CD34+, von Willebrand factor (vWF), KDR, CD31 and incorporate acetylated low-density lipoprotein (LDL). CD34+ and AC133+cells accounted for 68.2%?6.3% (n=6) and 57.2%?9.8% (n=6) of attaching (AT) cells at day 7 of culture, respectively. CONCLUSIONS: Coculture of CD34+ and CD34- or culture of MNC enhances ex vivo expansion of EPCs. Early passage decreases apoptosis rate, VEGF has no significant effect on ex vivo expansion of EPCs.