The aim of this study was to fabricate type II collagen fibrils with surface modified by long-chain hyaluronic acid. Monomeric type II collagen was isolated from bovine articular cartilage and reconstituted into collagen...The aim of this study was to fabricate type II collagen fibrils with surface modified by long-chain hyaluronic acid. Monomeric type II collagen was isolated from bovine articular cartilage and reconstituted into collagen fibrils followed by a reaction with EDC (1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide)-activated long-chain hyaluronic acid. The existence of the hyaluronan molecules on the fibrillar surface was confirmed by the specific bindings of gold nanoparticles labeled with wheat germ agglutinin. The topographic pattern of type II collagen fibrils revealed by AFM scanning changed significantly after the surface coupling of hyaluronic acid. Beneath the hyaluronan, the characteristic D-bandings of the reconstituted collagen fibrils remained intact as shown by the results of TEM observation. The collagen fibrils became more resistant to collagenase digestion after surface coupling of hyaluronic acid as compared with that without hyaluronic acid immobilization. In addition, human mesenchymal stem cells encapsulated and cultured within the matrix of HA-collagen fibrils have a higher proliferation rate than cells grown within the unmodified type II collagen fibrils. The newly synthesized material of HA-collagen II fibrils has a great potential for use in constructing scaffold for tissue repair.
There is a great demand for using phytases to reduce phytate content in animal feed stuffs and food for human consumption. Industrial application of phytase requires efficient methods to immobilize the enzyme, yielding a...There is a great demand for using phytases to reduce phytate content in animal feed stuffs and food for human consumption. Industrial application of phytase requires efficient methods to immobilize the enzyme, yielding a biocatalyst with high activity and stability compared to the free enzyme. In the present study, phytase was immobilized on Sepabead EC-EP and then used in the biodegradation of soymilk phytate. The immobilized enzyme exhibited an activity of 0.1 U per g of carrier and activity yield of 70.83%. Optimum temperature and pH for the immobilized enzyme were 55 degrees C and 5.5, respectively. Both the enzymes were stable between pH 3.0-8.0 and below 70 degrees C. Kinetic parameters(K(m) and V(max)) and the usability of the immobilized enzyme were determined. The immobilized enzyme hydrolyzed 65% of soymilk phytate in 8 h at 60 degrees C, as compared with 56% hydrolysis observed for the native enzyme over the same period of time.
To evaluate the changes of gel scaffold and spongy scaffold in fibroblast culture in vitro, two kinds of collagen dermis were constructed in this research. Type I collagenase and Dispase were used to isolate dermis fibro...To evaluate the changes of gel scaffold and spongy scaffold in fibroblast culture in vitro, two kinds of collagen dermis were constructed in this research. Type I collagenase and Dispase were used to isolate dermis fibroblast from neonate prepuce. The gel dermis was constructed by mixing the fibroblast and collagen swelling solution, and the collagen spongy dermis was also constructed. After histological and immunohistochemical staining, these two dermis' properties were compared. Compared with gel dermis, the spongy dermis possessed more superior tension strength, but it decreased significantly during the culture. The transition temperature in DSC reduced also. The rupture length of gel dermis increased, on the contrary. Gel dermis contained more moisture than spongy dermis. It had been observed that the collagen gel was more suitable for fibroblast in maintaining its morphology than spongy scaffold, though with less cell number. The reason might be due to the special structure of collagen gel. Water in gel is sub-bound water, which is different from free water, and it is suitable for fibroblasts to secrete extracellular matrix. It could be concluded that the collagen gel dermis is better than collagen spongy dermis in the biological property.
The aberrant vascular architecture in solid tumors is the key limiting factor known to ameliorate hypoxia and increase circulating anticancer drugs, thus resulting in resistance to radiotherapy and chemotherapy in tumor...The aberrant vascular architecture in solid tumors is the key limiting factor known to ameliorate hypoxia and increase circulating anticancer drugs, thus resulting in resistance to radiotherapy and chemotherapy in tumor treatment. Previous experiments have reported hemoglobin-based oxygen carriers (HBOCs) that are effective to improve tumor oxygenation, thereby serving as potential agents target-oriented to the hypoxic tumor. Herein, we draw the hypothesis that HBOCs combined with an anticancer drug may increase oxygen bioavailability and anticancer drug retention in solid tumors and in turn contribute to enhanced sensitivity of radiotherapy and chemotherapy. This novel drug will bring a new breakthrough in the field of the development of anticancer drugs and reveal the alternative clinical use of HBOCs in tumor treatment.
The Tie-2 receptor has been shown to play a role in angiogenesis in atherosclerosis. The conventional method assaying the level of soluble Tie-2 (sTie-2) was ELISA. However, this method has some disadvantages. The aims o...The Tie-2 receptor has been shown to play a role in angiogenesis in atherosclerosis. The conventional method assaying the level of soluble Tie-2 (sTie-2) was ELISA. However, this method has some disadvantages. The aims of this research are to establish a more simple detection method, the optical protein-chip based on imaging ellipsomtry (OPC-IE) applying to Tie-2 assay. The sTie-2 biosensor surface on silicon wafer was prepared first, and then serum levels of sTie-2 in 38 patients with AMI were measured on admission (day 1), day 2, day 3 and day 7 after onset of chest pain and 41 healthy controls by ELISA and OPC-IE in parallel. Median level of sTie-2 increased significantly in the AMI patients when compared with the controls. Statistics showed there was a significant correlation in sTie-2 results between the two methods (r=0.923, P<0.01). The result of this study showed that the level of sTie-2 increased in AMI, and OPC-IE assay was a fast, reliable, and convenient technique to measure sTie-2 in serum.
The purpose is to prepare 2% w/v emulsions of dodecafluoropentane, perfluorodecalin, and perfluoroctylbromide and compare them for their ability to absorb oxygen. The oxygen uptake capability and volume expansion of each...The purpose is to prepare 2% w/v emulsions of dodecafluoropentane, perfluorodecalin, and perfluoroctylbromide and compare them for their ability to absorb oxygen. The oxygen uptake capability and volume expansion of each emulsion and the blank vehicle were evaluated in water at 21 degrees C and 37 degrees C. The average particle size of the dodecafluoropentane emulsion is < 400 nm stored at room temperature for 6 months. In comparison to water treated with either the blank vehicle, the perfluorodecalin emulsion, or the perfluoroctylbromide emulsion, the dodecafluoropentane emulsion absorbs 3 times more oxygen at 21 degrees C and 7 times more oxygen at 37 degrees C. Furthermore, a significantly higher in vitro expansion (5 times) is observed with the dodecafluoropentane emulsion at 37 degrees C. As such, DDFP has been hypothesized to be a better oxygen carrier and delivery agent in vivo. This may be applicable to a variety of hypoxic medical conditions where oxygen delivery might be therapeutically beneficial.
Alginate has a unique property of gel formation by chelating with divalent cations such as Ca(+2) in aqueous solution. The sol-gel characteristic of alginate has been utilized to fabricate both microsphere and microcapsu...Alginate has a unique property of gel formation by chelating with divalent cations such as Ca(+2) in aqueous solution. The sol-gel characteristic of alginate has been utilized to fabricate both microsphere and microcapsule for cell immobilization. In this study, a wet spinning process was employed to prepare fibers comprised of gelatin and sodium alginate. Gelatin fibers containing interconnected porous structure were fabricated by the extraction of alginate from these gelatin/alginate composite fibers with phosphate buffer. The application of these porous gelatin fibers for enzyme immobilization was demonstrated by its usage as the base material of glucose biosensor.
OBJECT: To evaluate the correlation between prothrombin gene G20210A mutation and deep vein thrombosis after total joint replacement through detecting distribution frequency of single nucleotide polymorphism of the gene...OBJECT: To evaluate the correlation between prothrombin gene G20210A mutation and deep vein thrombosis after total joint replacement through detecting distribution frequency of single nucleotide polymorphism of the gene in patients undergoing total joint replacement. METHOD: PCR and direct sequencing of DNA are used to analyze the frequency of prothrombin gene G20210A mutation in 55 patients undergoing total joint replacement, and the relationship between the mutation and deep vein thrombosis after total joint replacement is evaluated. RESULT: Morbidity of prothrombin gene G20210A mutation in 55 patients is 0, both in the DVT group and non-DVT group. There is no significant difference between the two groups (p > 0.05). CONCLUSION: Mutation of prothrombin gene G20210A in Chinese patients is rare, and there is no correlation between the genetic mutation and deep vein thrombosis after total joint replacement.
A solid phase conjugation process was developed for attachment of polyethylene glycol to hemoglobin molecule. Bovine hemoglobin was loaded onto an ion exchange chromatography column and adsorbed by the solid medium. Succ...A solid phase conjugation process was developed for attachment of polyethylene glycol to hemoglobin molecule. Bovine hemoglobin was loaded onto an ion exchange chromatography column and adsorbed by the solid medium. Succinimidyl carbonate mPEG was introduced in the mobile phase after the adsorption. Pegylation took place between the hemoglobin on the solid phase, and the pegylation reagent in the liquid phase. A further elution was carried out to separate the pegylated and the unpegylated protein. Analysis by HPSEC, SDS-PAGE, and MALLS demonstrated that the fractions eluted from the solid phase contained well-defined components. Pegylated hemoglobin with one PEG chain was obtained with the yield of 75%, in comparison to the yield of 30% in the liquid phase pegylation. The P(50) values of the mono-pegylated hemoglobin, prepared with SC-mPEG 5 kDa, 10 kDa and 20 kDa, were 19.97, 20.23 and 20.54 mmHg, which were much closer to the value of red blood cells than that of pegylated hemoglobin prepared with the conventional method.
This work reports for the first time the expression of a soluble B2 globin chain that is part of the extracellular hexagonal-bilayer haemoglobin from Arenicola marina. Two recombinant B2 globins were produced, one fused...This work reports for the first time the expression of a soluble B2 globin chain that is part of the extracellular hexagonal-bilayer haemoglobin from Arenicola marina. Two recombinant B2 globins were produced, one fused with gluthatione S-tranferase (B2-GST) and the other without a fusion tag (RecB2) and requiring a different purification procedure. We also describe a new method for the expression of globin that uses Studier's auto-induction medium together with the heme precursor delta-aminolevulinic acid. Media supplementation with the heme precursor delta-aminolevulinic acid in the culture increased heme synthesis by E. coli leading to the expression of the recombinant B2 globins in their active form. RecB2 and B2-GST were expressed with a yield of up to 105 mg/l of E. coli culture. Our approach is rapid and requires only one chromatographic purification step for B2-GST and three purification steps for RecB2. The overall results on RecB2 and B2-GST show that the recombinant globins exhibit similar properties to those of Arenicola marina native HBL-Hb with a great stability and a strong oxygen binding. The results and methodologies described in this paper are the beginning of a work aiming at reconstituting a recombinant HBL-Hb by genetic engineering in order to produce an innovative oxygen carrier for therapeutic applications.
Of all the food additives for which the FDA has received adverse reaction reports, the ones that most closely resemble true allergens are sulfur-based preservatives. Sulfites are used primarily as antioxidants to prevent...Of all the food additives for which the FDA has received adverse reaction reports, the ones that most closely resemble true allergens are sulfur-based preservatives. Sulfites are used primarily as antioxidants to prevent or reduce discoloration of light-colored fruits and vegetables, such as dried apples and potatoes, and to inhibit the growth of microorganisms in fermented foods such as wine. This work aims to prepare an electrochemical biosensor based on bay leaf tissue homogenate that contains polyphenol oxidase enzyme abundantly for sulfite detection in foods. The principle of the biosensor is based on the inhibition effect of sulfites on polyphenol oxidase in the bioactive layer. Optimum conditions for the biosensor, such as temperature and pH, were investigated. Some stability parameters of the biosensor were also identified. The biosensor showed a linear calibration graph in the range of 25-100 microM sulfite. The biosensor presents a very simple, economical, reliable, and feasible method for sulfite detection in foods.
The early diagnosis of oral squamous cell carcinoma (OSCC) is crucial to prevent deformity and malfunction post-surgery, as well as to allow clinicians to make a rapid decision about treatment. The aim of this study was...The early diagnosis of oral squamous cell carcinoma (OSCC) is crucial to prevent deformity and malfunction post-surgery, as well as to allow clinicians to make a rapid decision about treatment. The aim of this study was to search a serum diagnostic model at a molecular level for OSCC. After collection and processing of serum from 28 OSCC patients and 32 healthy volunteers in the Department of Stomatology at the university hospital, samples were detected using Surface Enhanced Laser Desorption Ionization Time of Flight Mass Spectrometry (SELDI-TOF-MS) technology with CM10 protein chip and bioinformatics. Seven protein mass peaks were screened out to build a serum diagnosis model with a significant P value, respectively, and the sensitivity, specificity, and total accuracy were 93.75%, 92.86%, and 93.33%. The use of serum protein fingerprint provides a promising approach for early diagnostics, which could benefit determining preventative and therapeutic stages of patients with OSCC.
We established a HPLC method for content determination of aniracetam in aniracetam inclusion complex. The chromato column was Agilent ODS (4.6mm x 150mm, 5 microm), the mobile phase was methanol-0.01 mol/L Potassium dihy...We established a HPLC method for content determination of aniracetam in aniracetam inclusion complex. The chromato column was Agilent ODS (4.6mm x 150mm, 5 microm), the mobile phase was methanol-0.01 mol/L Potassium dihydrogen phosphate buffer solution (25:75, pH 3.0), with the flow rate of 1.0 ml/min, column temperature of 30 degrees and the detection wave at 280 nm, the sample size was 20microL. A good linear relationship was obtained between the peak areas and the concentrations of aniracetam in the range from 5- 80microg/ml (r=0.9998), the mean recovery was 100.1% (n=15), RSD=0.19%. This method is convenient, rapid, accurate, and brings about good recovery; it can be used for content determination of aniracetam in aniracetam inclusion complex.
Lethal dose experiments in animals have demonstrated that second-generation perfluorocarbon oxygen carriers are remarkably non-toxic. However, this non-toxicity has not previously been demonstrated in a liver failure sce...Lethal dose experiments in animals have demonstrated that second-generation perfluorocarbon oxygen carriers are remarkably non-toxic. However, this non-toxicity has not previously been demonstrated in a liver failure scenario. A surgical liver damage and regeneration model in rats was selected using a well-controlled cross tabulated study design. A large number of physiological, biochemical, and hematological parameters were measured. No indications were found that intravenously injected perfluorooctyl bromide emulsion was toxic at the concentrations employed, in either healthy or severe liver injury scenarios. Neither was there any significant impact on the rate of liver regeneration following the injuries. Bearing in mind prior human clinical studies, it is therefore safe to assume that perfluorocarbon emulsions are also non-toxic in bioartificial liver treatments.
Batch cultivation of Candida rugosa for the production of lipase and esterase enzymes was carried out by employing two different inoculation strategies. The effects of triolein (2, 3 and 5 g/l) and oleic acid (3 g/l) as...Batch cultivation of Candida rugosa for the production of lipase and esterase enzymes was carried out by employing two different inoculation strategies. The effects of triolein (2, 3 and 5 g/l) and oleic acid (3 g/l) as carbon sources of the enzyme production medium on the activity, productivity, and yield of enzymes were also compared for both strategies. Inoculation of the cells into the enzyme production medium either directly or after cultivation in a pre-culture medium rich in glucose affected the activity and yield of esterase more than those of lipase for both carbon sources. In both strategies, triolein and oleic acid yielded the same lipase activity (16.67 U/ml) whereas triolein provided higher esterase activity (0.0035 U/ml). Time courses of the extracellular and intracellular lipase and esterase enzymes indicated that lipase activity was growth-associated and the cells secreted esterase into the medium after a considerable level of extracellular lipase activity was reached. The role of protease in the enzyme activities was also discussed.
The glutathione-s-transferases are a family of multifunctional enzymes involved in the detoxification of electrophilic xenobiotics primarily through conjugation to reduce glutathione. A form of the enzyme, designated GSH...The glutathione-s-transferases are a family of multifunctional enzymes involved in the detoxification of electrophilic xenobiotics primarily through conjugation to reduce glutathione. A form of the enzyme, designated GSH-S transferase rho, was purified chicken erythrocyte by acetone precipitation, ethanol-chloroform treatment, DEAE-Cellulose, Q-Sepharose, Sephadex G-100 chromatography. The molecular weight of GST purified from chicken erythrocyte was estimated as 47,500 Da by gel filtration. The subunit molecular weight of chicken erythrocyte GST as determined by electrophoresis in the presence of sodium dodecyl sulfate was predicted as 24,000 Da. The specific activity was found to be 20.39 U/mg. The km for CDNB calculated from Lineweaver-Burk plot was 0.71 mM. Optimum temperature of maximum GST activity was 28 degrees C for CDNB. The maximal activity of the enzyme was observed at pH 7.5. The activity of purified GST is inhibited by DDT, urea, CDNB, Triton X-100, DTNB.
To investigate the changes of calcitonin gene-related peptide (CGRP) in rat's blood plasma, spinal anterior motorneuron, and dorsal root ganglion (DRG) after fractures combined with central or peripheral nerve injuries a...To investigate the changes of calcitonin gene-related peptide (CGRP) in rat's blood plasma, spinal anterior motorneuron, and dorsal root ganglion (DRG) after fractures combined with central or peripheral nerve injuries and its influence on fracture healing, 72 healthy adult SD rats (male or female) were divided into 4 groups (18 rats in each group): group A, simple(left) tibial fracture; group B, left tibial fracture combined with left sciatic nerve injury; group C, left tibial fracture combined with T9-11 spinal cord transection injury; group D, left tibial fracture combined with right cerebral cortex injury. Group A was the control group. The concentration of serum CGRP was measured immediately, 1w, 2w, and 4w after injury using radio immunoassay. X-ray photograph was taken at 1w, 2w, and 4w after injury to assess fracture healing. The concentration of serum CGRP in spinal anterior motorneuron and dorsal root ganglion was measured 1w, 2w, and 4w after injury. Bony callus at 2w after injury using H.E.staining was observed. 1w and 2w after injury, the fracture line was still clear on the X-ray of all groups, but 4w after injury the fracture line disappeared with complete healing except the peripheral nerve injury group. By H.E. staining, we found lesser bony callus contents in the peripheral nerve injury group than the simple fracture group at 2w after injury; irregular bone trabecula and healing defect were found in the former group. While the spinal injury group and cerebral cortex injury group represented more bony callus than the simple fracture group, increased bone trabecula and regularity, medullary cavity occluded and finally solid bony connections were found. CGRP concentration in blood plasma and spinal anterior motorneuron represented no apparent differences among all groups during each observation period. For the dorsal root ganglion group, 1w after fracture, there was no apparent difference of CGRP concentration in the peripheral nerve injury group and cerebral cortex group compared with the control group (P > 0.05), but the spinal injury group showed more CGRP than the control group (P < 0.01). 2w after injury, the peripheral nerve injury group and cerebral cortex group also showed no difference compared with the control group, but the cerebral cortex group had more CGRP contents than the peripheral nerve injury group (P < 0.05), and the spinal injury group showed more CGRP than the control group (P < 0.01). 4w after injury, the peripheral nerve group, spinal injury group, and cerebral cortex injury group all showed higher concentration of CGRP than the control group. Among the 3 groups, the spinal injury group is the highest (P < 0.01). When fracture combined with peripheral nerve injury, the healing process can be slowed down. In contrast, fracture combined with spinal injury and cerebral cortex injury will accelerate the healing process. The CGRP in dorsal root ganglion in spinal injury group and cerebral cortex injury group increased, which may have positive effects on fracture healing.
Platelet gels (PG), activated by bovine thrombin (BT), have increasingly been used in orthopedic surgery. However, BT may induce immunological reactions and carry potential viral and prion risks. To avoid these side effe...Platelet gels (PG), activated by bovine thrombin (BT), have increasingly been used in orthopedic surgery. However, BT may induce immunological reactions and carry potential viral and prion risks. To avoid these side effects, thrombin derived from human plasma (human thrombin, HT) is becoming the preferred platelet activator to prepare PG. However, limited experience and data on the clinical benefits of HT-generated PG (HTPG) in orthopedic surgery is reported. Consequently, we designed and performed a series of studies in dogs to compare the impacts of promotion of bone growth by an artificial bone substitute (Osteoset) in combination with HTPG or without it in the spinal repair experiments. X-ray observations and histological studies were performed at predetermined periods post-operation. The preliminary results revealed the preparation of HTPG was easy and required less than 30 minutes. HTPG was capable of embedding the artificial bone substitute Osteoset to prepare a sticky and easily manipulated composite for the application into spinal defect. We found HTPG exhibited enhancement of grafting capacity in consolidation of bone mass. After 12 weeks, tissue reconstruction reached approximately 80% of the injury defects when treated by HTPG/Osteoset combination, but only 30 approximately 40% in the absence of HTPG. The physiological activity of artificial bone substitute combined with PG activated by HT may therefore open beneficial prospects for more successful and safer bone formation in spine procedures in the near future.
During extracorporeal procedures like hemodialysis, heparin is administered to patients to prevent clotting. Unfractionated heparin has side effects such as excessive bleeding. It would be advantageous if the blood could...During extracorporeal procedures like hemodialysis, heparin is administered to patients to prevent clotting. Unfractionated heparin has side effects such as excessive bleeding. It would be advantageous if the blood could be deheparinased before it returns to the patient. Previous work has indicated that poly-L-lysine/alginate beads can efficiently remove heparin from saline solutions 1. Heparin is irreversibly absorbed onto the beads. This article explores ways of optimizing the absorption process by performing in vitro rate experiments with varying physical parameters of the beads. Fetal calf serum and blood are also used in experiments to investigate the possibility of designing a safe and efficient reactor to absorb heparin. All the experiments were performed to obtain the required parameters for optimal reactor design. The results indicate that the absorption could be optimized by controlling the membrane thickness of the beads. The beads also showed efficient removal of heparin from whole blood.
Based on the research on small gap bridging peripheral nerve injury in SD rats, we decided to investigate the histological analysis possibility of bridging peripheral nerve injury with small gap using a de-acetyl chitin...Based on the research on small gap bridging peripheral nerve injury in SD rats, we decided to investigate the histological analysis possibility of bridging peripheral nerve injury with small gap using a de-acetyl chitin conduit in primate. Median nerves of 8 rhesus monkeys were cut at 2 cm above the elbow, and the right sides were subjected to small gap (2 mm) bridging to repair the nerve with chitin conduit (conduit inner diameter 4 mm; length 10 mm); the left sides were subjected to traditionary epineurium suture. Histology detections were conducted after 6th month. The conduit was almost absorbed and the conduit cast contour disappeared after 6 months. The histological analysis displayed that the regenerated nerve fibers in conduit grew forward in fasciculation. The re-myelinated nerve axons number per unit area in the conduit group distal segment was higher than that of traditionary epineurium suture group. The biocompatibility of biological chitin conduit in primate rhesus monkeys was quite good. The regenerated nerve fibers in conduit grew forward in fasciculation. The histological analysis results of biological conduit in primate rhesus monkeys were better than the traditionary epineurium suture. The biological conduit can be used in primate rhesus monkeys to substitute the traditionary epineurium suture methods.