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Temperature-sensitive hydrogels are attractive alternatives to porous cell-seeded scaffolds and is minimally invasive through simple injection and in situ gelling. In this study, we compared the performance of two types of temperature-sensitive hydrogels on chondrocytes encapsulation for the use of tissue engineering of cartilage. The two hydrogels are composed of methoxy poly(ethylene glycol)- poly(lactic-co-valerolactone) (mPEG-PVLA), and methoxy poly(ethylene glycol)-poly(lactic- co-glycolide) (mPEG-PLGA). Osmolarity and pH were optimized through the manipulation of polymer concentration and dispersion medium. Chondrocytes proliferation in mPEG-PVLA hydrogels was observed as well as accumulation of GAGs and collagen. On the other hand, chondrocytes encapsulated in mPEG-PLGA hydrogels showed low viability and chondrogenesis. Also, mPEG-PVLA hydrogel, which is more hydrophobic, retained physical integrity after 14 days while mPEG-PLGA hydrogel underwent full degradation due to faster hydrolysis rate and more pronounced acidic self-catalyzed degradation. The mPEG-PVLA hydrogel can be furthered tuned by manipulation of molecular weights to obtain hydrogels with different swelling and degradation characteristics, which may be useful as producing a selection of hydrogels compatible with different cell types. Taken together, these results demonstrate that mPEG-PVLA hydrogels are promising to serve as three-dimensional cell carriers for chondrocytes and potentially applicable in cartilage tissue engineering. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 101A: 3311-3319, 2013.

Thermosensitive micelles composed of a copolymer of methoxy polyethylene glycol (mPEG), polylactic acid (PLA), and 1,6-bis (p-carboxyphenoxy) hexane (CPH), namely methoxy polyethylene glycol-co-polylactic acid-co-aromatic anhydride (mPEG-PLCPHA), were fabricated for application as a promising hydrophilic drug carrier. The copolymer can self-assemble into micelles in PBS by hydrophobic interaction. The diameters of these micelles increased as the environmental temperature increased. An increase in viscosity with sol-to-gel transition occurred as temperature increased from room temperature to body temperature. During the in vitro degradation process, hydrogels demonstrated a more stable degradation rate. Both in vitro and in vivo cytotoxicity results showed that the materials had excellent biocompatibility due to less acidic products formation. In vitro cefazolin release profiles showed a stable release for 30 days. The hydrogel encapsulated cefazolin exhibited a good antibacterial effect. Based on these results, mPEG-PLCPHA can serve as an injectable depot gel for drug delivery.In this study, thermosensitive hydrogel encapsulated cefazolin was found to exhibit good antibacterial effects with sustained levels for up to 30 days, enabling the development of an injectable depot gel for long-term drug delivery.

The clinical demand for cartilage tissue engineering is potentially large for reconstruction defects resulting from congenital deformities or degenerative disease due to limited donor sites for autologous tissue and donor site morbidities. Cartilage tissue engineering has been successfully applied to the medical field: a scaffold pre-cultured with chondrocytes was used prior to implantation in an animal model. We have developed a surgical approach in which tissues are engineered by implantation with a vascular pedicle as an in vivo bioreactor in bone and adipose tissue engineering. Collagen type II, chitosan, poly(lactic-co-glycolic acid) (PLGA) and polycaprolactone (PCL) were four commonly applied scaffolds in cartilage tissue engineering. To expand the application of the same animal model in cartilage tissue engineering, these four scaffolds were selected and compared for their ability to generate cartilage with chondrocytes in the same model with an in vivo bioreactor. Gene expression and immunohistochemistry staining methods were used to evaluate the chondrogenesis and osteogenesis of specimens. The result showed that the PLGA and PCL scaffolds exhibited better chondrogenesis than chitosan and type II collagen in the in vivo bioreactor. Among these four scaffolds, the PCL scaffold presented the most significant result of chondrogenesis embedded around the vascular pedicle in the long-term culture incubation phase.

The long term in vivo biocompatibility is an essential feature for the design and development of sustained drug release carriers. In the recent intraocular drug delivery studies, hydrogels were suggested as sustained release carriers. The biocompatibility test for these hydrogels, however, was commonly performed only through in vitro cell culture examination, which is insufficient before the clinical applications. We compared three thermosensitive hydrogels that have been suggested as the carriers for drugs by their gel-solution phase-change properties. A new block terpolymer (PEOz-PCL-PEOz, ECE) and two commercial products (Matrigel® and Pluronic F127) were studied. The results demonstrated that the ocular media remained translucent for ECE and Pluronic F127 in the first 2 weeks, but cataract formation for Matrigel occurred in 2 weeks and for Pluronic F127 in 1 month, while turbid media was observed for both Matrigel and Pluronic F127 in 2 months. The electrophysiology examinations showed significant neuroretinal toxicity of Matrigel and Pluronic F127 but good biocompatibility of ECE. The neuroretinal toxicity of Matrigel and Pluronic F127 and superior biocompatibility of ECE hydrogel suggests ECE as more appropriate biomaterial for use in research and potentially in intraocular application.

Microfiltration (MF) of a fermentation broth containing Escherichia coli is reported in this article. We used a ceramic membrane filter (zirconia on sintered carbon) having a nominal pore size of 0.2 μm. Our results indicate that the filtration resistance was mainly caused by the cake formed on the membrane surface. Both transmembrane pressure (TMP) and fluid sweeping velocity influenced this cake resistance. Resistances due to membrane itself and due to internal pore blockage by E. coli were less important and insensitive to both TMP and fluid sweeping velocity. Preliminary results also showed that the cell density could be significantly increased when we connected such a ceramic filter on-line with our fermentation system. In particular it was found that the gas bubbles entrained in the broth side of the filter could increase the filtration flux by as much as 80%.

Magnetic beads coated with monoclonal antibodies (MAb) were used to specifically remove cells carrying the common acute lymphoblastic leukemia antigen (cALLA). The target-cell elimination effectiveness (TEE) was found to be kinetically controled, and can be quantitatively described by a model where antigen-carrying cells and MAb-coated magnetic beads are described as colloidal particles. This model can correctly explain the temperature effect on the immunomagnetic cell adhesion where models based on antigen-antibody interaction failed.

In the recovery of cephalosporin C (CPC) from fermentation broth, the separation of desacetyl cephalosporin C (DAC) is a major concern. Multistage extraction in aqueous two-phase systems, mainly PEG/ammonium sulfate systems, proved to be promising. In preparative scale operation, high speed counter-current chromatography (HSCCC) with eccentric columns was used with aqueous two-phase systems to obtain baseline resolution of CPC and DAC. Solvents (e.g. 5% acetone) or neutral salts (e.g. 1.45% KSCN) added into aqueous two-phase systems enhanced the separation efficiency. Operation parameters of HSCCC such as rotational speed and mobile phase flow rate can affect the retention of the stationary phase and HETP.

Supplemental Digital Content is available in the text., Background: The application of bone tissue engineering for repairing bone defects has gradually shown some satisfactory progress. One of the concerns raising scientific attention is the poor supply of growth factors. A number of growth factor delivery approaches have been developed for promoting bone formation. However, there is no systematic comparison of those approaches on efficiency of neobone formation. In this study, the approaches using periosteum, direct supply of growth factors, or gene transfection of growth factors were evaluated to determine the osteogenic capacity on the repair of bone defect. Methods: In total, 42 male 21-week-old Sprague-Dawley rats weighing 250 to 400 g were used as the bone defect model to evaluate the bone repair efficiency. Various tissue engineered constructs of poly(ethylene glycol)-poly(l-lactic acid) (PEG-PLLA) copolymer hydrogel with periosteum, with external supply of bone morphogenetic protein-2 (BMP2), or with BMP2-transfected bone marrow–derived mesenchymal stem cells (BMMSCs) were filled in a 7-mm bone defect region. Animals were euthanized at 3 months, and the hydrogel constructs were harvested. The evaluation with histological staining and radiography analysis were performed for the volume of new bone formation. Results: The PEG-PLLA scaffold with BMMSCs promotes bone regeneration with the addition of periosteum. The group with BMP2-transfected BMMSCs demonstrated the largest volume of new bone among all the testing groups. Conclusions: Altogether, the results of this study provide the evidence that the combination of PEG-PLLA hydrogels with BMMSCs and sustained delivery of BMP2 resulted in the maximal bone regeneration.

Fractionation of various leukemic cells in aqueous two-phase systems, composed of poly(ethylene glycol) and dextran, was performed in a high speed counter-current chromatography (HSCCC) device with eccentric parallel coil orientation. Cells from different lineages or different culture conditions showed different elution patterns. Due to the instability of the stationary phase and the tendency of cells to concentrate at the phase interfaces, special modifications of HSCCC operation were developed to give satisfactory fractionation, these include optimal timing for sample injection, application of downward concentration gradient of mobile phase fluid and proper rotational speed.

Temperature-sensitive hydrogels are attractive alternatives to porous cell-seeded scaffolds and is minimally invasive through simple injection and in situ gelling. In this study, we compared the performance of two types of temperature-sensitive hydrogels on chondrocytes encapsulation for the use of tissue engineering of cartilage. The two hydrogels are composed of methoxy poly(ethylene glycol)- poly(lactic-co-valerolactone) (mPEG-PVLA), and methoxy poly(ethylene glycol)-poly(lactic- co-glycolide) (mPEG-PLGA). Osmolarity and pH were optimized through the manipulation of polymer concentration and dispersion medium. Chondrocytes proliferation in mPEG-PVLA hydrogels was observed as well as accumulation of GAGs and collagen. On the other hand, chondrocytes encapsulated in mPEG-PLGA hydrogels showed low viability and chondrogenesis. Also, mPEG-PVLA hydrogel, which is more hydrophobic, retained physical integrity after 14 days while mPEG-PLGA hydrogel underwent full degradation due to faster hydrolysis rate and more pronounced acidic self-catalyzed degradation. The mPEG-PVLA hydrogel can be furthered tuned by manipulation of molecular weights to obtain hydrogels with different swelling and degradation characteristics, which may be useful as producing a selection of hydrogels compatible with different cell types. Taken together, these results demonstrate that mPEG-PVLA hydrogels are promising to serve as three-dimensional cell carriers for chondrocytes and potentially applicable in cartilage tissue engineering.

This study designed a series of polyanionic nanocarriers based on biodegradable and biocompatible poly (aspartic acid)s for oral administration. First, polysuccinimide (PSI) was synthesized from L-aspartic acid using acid-catalyzed bulk thermal polycondensation and acid-catalyzed thermal polycondensation in a mixture of mesitylene/sulfolane. PSI-C16 was then synthesized by aminolysis with nucleophile, hexadecylamine to react with PSI known as nucleophilic addition. Finally, a series of partially esterified poly (aspartic acid)s was produced by alkaline treatment to afford an amphiphilic polyanion, poly (sodium aspartate-g-hexadecyl aspartate) (Na-PASP-g-C16-PASP). 1HNMR, FTIR, DSC and GPC were utilized to demonstrate and characterize the polymers. The synthesized polyanion could be self-assembled into the nano-scaled micelles and be independent of pH in phosphoric buffer solutions. The hydrodynamic diameter and zeta potential were measured using the dynamic light scattering (DLS) method, and the critical micelle concentration (CMC) was determined using the fluorescence spectrophotometer. The micellar morphologies were examined using transmission electron microscopy (TEM), and atomic force microscopy (AFM) to present the nano-dimensional sphere. The stability of size transition at different pH levels, from strong acid to alkaline, proved that the micelles could stably transport from the stomach to intestinal lumen prior to arriving in the epithelium of the small intestine.

This study evaluates the crosslinkage effect of chondroitin sulphate C (CSC) and type II collagen (COL II) on chondrogenesis of mesenchymal stem cells (MSCs) in vitro and in vivo. In the in vitro studies, our results show that the weight ratio CSC:COL II that reaches 1.2:100 (CSC1.2/100 -COL II scaffold) can provide an optimal microenvironment for MSC chondrogenesis. When MSCs are cultured in this CSC1.2/100 -COL II scaffold, the chondrogenic gene expression of cultured cells is upregulated, while the osteogenic gene expression of these is downregulated. In addition, MSCs cultivated in the CSC1.2/100 -COL II scaffold are found to express the highest glycosaminoglycans:DNA ratio as compared to those in scaffolds of other CSC:COL II ratios. Histological and immunohistological evidence also supports the result. In the in vivo study, our results show that MSCs cultivated in the CSC1.2/100 -COL II scaffold demonstrate a better repair ability on cartilage lesions than does the COL II scaffold. After 1 month in vivo, the injected MSCs in the CSC1.2/100 -COL II scaffold show lacuna structures and stimulate the formation of type II collagen at the defective sites. Six months after transplantation, the generated cells in the CSC1.2/100 -COL II group show higher gene expressions of type II collagen and aggrecan but lower gene expression of type I collagen at the defective sites than those in the COL II group. The results strongly suggest that CSC1.2/100 -COL II scaffold can serve as a potential candidate for cartilage repair and improve the chondrogenesis of MSCs in general.

The feasibility of using genipin cross-linked type II collagen scaffold with rabbit bone marrow mesenchymal stem cells (RBMSCs) to repair cartilage defect was herein studied. Induction of RBMSCs into chondrocytic phenotype on type II collagen scaffold in vitro was conducted using TGF-β 3 containing medium. After 3-weeks of induction, chondrocytic behavior, including marker genes expression and specific extracellular matrix (ECM) secretion, was observed. In the in vivo evaluation experiment, the scaffolds containing RBMSCs without prior induction were autologous implanted into the articular cartilage defects made by subchondral drilling. The repairing ability was evaluated. After 2 months, chondrocyte-like cells with lacuna structure and corresponding ECM were found in the repaired sites without apparent inflammation. After 24 weeks, we could easily find cartilage structure the same with normal cartilage in the repair site. In conclusion, it was shown that the scaffolds in combination of in vivo conditions can induce RBMSCs into chondrocytes in repaired area and would be a possible method for articular cartilage repair in clinic and cartilage tissue engineering.

Chondroitin sulfate C (CSC) is a kind of glycosaminoglycans (GAGs) with molecular weights of 10,000 to 50,000 Da and a high charge density. GAGs are major components in extracellular matrix (ECM), which play important role in the regulation of cell proliferation, migration, and differentiation. In this study, we studied the effects of chondroitin sulfate C (CSC) on the differentiation of human mesenchymal stem cells (MSCs) toward the chondrocyte lineage. The MSCs were either cultured on type II collagen (COL II) scaffolds with high molecular weight CSC addition in the medium (free CSC) or with free oligosaccharide CSC. Special attention was given to the effects of MSCs cultured on CSC cross-linked type II scaffolds (cross-linked CSC). According to the analysis of histology stain, gene expression, and ECM secretion, our results showed that MSCs cultured with free CSC, free oligosaccharides CSC, and on the cross-linked CSC scaffolds all would be induced into chondrocytes. Moreover, free oligosaccharide CSC present in the microenvironment could significantly up-regulate MSC chondrogenesis gene expression and stimulate cartilage ECM accumulation more than free CSC with high molecular weight after 3-week induction. Importantly, cross-linked CSC had the most excellent effects on the MSC chondrogenesis. Thus, we believed that cross-linked CSC in the scaffold would play the similar roles with free oligosaccharide CSC in the medium. Cross-linked CSC would be a potential candidate for cartilage repair in the cell therapy and tissue engineering.

Tyrosinase is the first and rate limiting enzyme in the synthesis of melanin pigments for coloring hair, skin, and eyes. As reported in this study, a natural product, (-)-N-formylanonaine isolated from the leaves of Michelia alba D.C. (Magnolianceae), was found to inhibit mushroom tyrosinase with an IC50 of 74.3 microM and to have tyrosinase and melanin reducing activities in human epidermal melanocytes without apparent cytotoxicity to human cells, superior to the known tyrosinase inhibitors, such as kojic acid and 1-phenyl-2-thiourea (PTU). Based on homology modeling, the compound binds the active site by coordinating with two Cu2+ ions. In addition, the compound had antioxidation activities in tests for scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH), reducing power, and chelating metal ions. To our knowledge, this is the first study to reveal the bioactivities of (-)-N-formylanonaine from this plant species.

Osteomyelitis characterized by an inflammatory response often leads to bone loss and the spread of bacterial infection to surrounding soft tissues. To overcome the side effects induced by the systemic antibiotic treatment for osteomyelitis, recent investigations have explored the use of antibiotic-loaded undegradable or biodegradable delivery implants at the infected bone. Here, we show a novel biodegradable thermosensitive implant composed of poly(ethylene glycol) monomethyl ether (mPEG) and poly(lactic-co-glycolic acid) (PLGA) copolymer as a sol–gel drug delivery system for treating bone infection. The physical properties of a series of mPEG–PLGA nanocomposites, including the critical micelle concentration (CMC), particle size, polyindex (PI), sol–gel transition, viscosity and degradation rate, have been characterized in vitro. This sol-to-gel drug delivery system could provide several advantages in treating osteomyelitis, including easy preparation, 100% encapsulated rate, near-linear sustained release of drugs, injectable design and in situ gelling at the target tissue. Similar to the undegradable teicoplanin-impregnated polymethylmethacylate (PMMA) bone cements, we showed that implantation of the mPEG–PLGA hydrogel containing teicoplanin was effective for treating osteomyelitis in rabbits as detected by the histological staining and immunoblotting analyses. The use of the mPEG–PLGA-based biodegradable hydrogels may hold great promise as a therapeutic strategy for other infected diseases.

The mechanical stability of liquid-core alginate-poly-L-lysine (PLL) capsules used for encapsulation of hybridoma cells can be greatly enhanced by the inclusion of poly(ethylenimine) (PEI) in the hardening solution containing calcium chloride. The PEI can also reinforce the PLL-coated carboxymethyl-celluose liquid-core capsules. The cultivation of murein hybridoma CT04 in these two capsules was carried out. Cell concentrations higher than 10(8) cells/mL per capsule were obtained with ca. 80% of the specific antibody productivity as the freely suspended cells. These capsules could withstand severe agitation and aeration in an air-lift reactor over a period of 3 weeks with minimal damage.

The production of alkaline protease from Bacillus subtilis ATCC 14416 was studied. Inorganic nitrogen compounds in the medium were found to show inhibitory effects on the enzyme formation. The pH variations of the culture broth gave indications of the start and the end of the enzyme production period and thus provided a useful indicator for process control. The rapid deactivation of protease after the end of the production period was studied, and ways to stabilize the enzyme were discussed.

This study investigates a poly(epsilon-caprolactone)-graft-type II collagen-graft-chondroitin sulfate (PCL-g-COL-g-CS) biomaterial as a scaffold for cartilage tissue engineering. Biodegradable polyester, PCL, was utilized to fabricate three-dimensional (3D) porous scaffolds by particulate leaching. The PCL scaffold was then surface modified by chemical bonding of 1,6-hexanediamine and the grafting of a bioactive polymer layer of COL and CS with the help of 1-ethyl-3-(3-dimethyl- aminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) on the modified PCL surface to produce PCL-g-COL and PCL-g-COL-g-CS, respectively. The characteristics of these modified and grafted matrices were examined by ESCA, aminolysis, collagen and CS assay, porosity and water-binding capacity. Grafted COL and CS markedly increased water-binding capacity, and promoted the spreading and growth of chondrocytes. During a 4-week culture period, PCL-g-COL and PCL-g-COL-g-CS matrices both provided more cell proliferation, as determined by measuring the DNA assay. Additionally, a larger amount of secreted collagen and glycosaminoglycans (GAGs) appeared in the PCL-g-COL-g-CS matrices than in the control (PCL) as indicated by the histochemical sections via Hematoxylin and eosin (HE) stain, Masson trichrome stain and Safranin-O stain. The chondrocytes were induced to function normally; the cell phenotype was maintained, and the GAGs and collagen in the PCL-g-COL-g-CS scaffold were secreted in vitro. These results serve as a basis for future studies of the fabrication process and reveal the potential biocompatibility of the biomimetic matrix for regenerating articular cartilage or other organs.

Owing to of the limited repair capacity of articular cartilage, it is essential to develop tissue-engineered cartilage for patients suffering from joint disease. Chondroitin sulfate (CS) and hyaluronan (HA) are the components of the cartilage extracellular matrix (ECM) and are known to influence the proliferation and differentiation of chondrocytes. Scaffolds composed of type-II collagen, CS, and HA may create an environment that can preserve the normal phenotype of cells to promote regeneration of cartilage-like constructs. In this investigation, we prepared and characterized 3-dimensional type-II collagen scaffolds both with and without HA and CS. Porous composite scaffolds fabricated by freeze-drying showed interconnected pores with mean diameters of 140+/-30 microm and porosities of 92-95% after cross-linking with genipin. After a 14-day in vitro culture, morphologically round chondrocytes were found to be uniformly distributed throughout the sponges. Expression of genes of aggrecan, type-II collagen and cartilage oligomeric matrix protein (COMP) was statistically and significantly increased on scaffolds with CS and HA than those without CS and HA. Furthermore, there was a markedly greater accumulation of proteoglycans (PGs) on the scaffolds with CS and HA.

Amino acids, including lysine, glutamic acid, and phenylalanine, in pure solution or in fermentation broth, were extracted with the aqueous two-phase system consisting of polyethylene glycol and salts, giving a very sharp separation. The partition is influenced by the type and the amount of salts used, pH and components of the broth.

A method of rapid determination of chloro- phyll a and lipid contents of microalgae based on colori- metric analysis of the digital images of the microalgae is proposed. The color variation of microalgae during cultiva- tion is evaluated by the brightness of the three primary colors (red, green, and blue). The brightness values of the three primary colors are modeled as two linear correlation functions (RGB model) for microalgal chlorophyll a and lipid contents, respectively. The chlorophyll a and lipid contents predicted by the proposed model are compared with that determined by the standard methods. The good agreement of the model predictions with experimental results is demonstrated with a squared correlation coeffi- cient (R 2 ) of 0.99 for chlorophyll a and lipid. The reliability of the RGB model was verified in real cultivations of the microalgae in a photobioreactor. Growth dynamics, con- tents of chlorophyll a and lipid corresponded very well with previously reported studies. Biotechnol. Bioeng. 2008;99: 1034-1039. 2007 Wiley Periodicals, Inc.

Tyrosinase is a key enzyme catalyzing the rate-limiting step for the biosynthesis pathway of melanin pigment, which is the most important determinant of the color of skin. Inhibiting tyrosinase and repressing melanocyte metabolism can reduce melanin production. Among the possible melanin reducing compounds, tyrosinase inhibitors are most promising for treating pigmentation and are used as skin-whitening agents in the cosmetic industry. In our investigation, some new tyrosinase inhibitors from plants have been identified to have high tyrosinase inhibitory activity. Specifically, p-hydroxybenzyl alcohol (4HBA) was found to inhibit the monophenolase activity of mushroom tyrosinase. When 4HBA binds with the enzyme, conformation of the enzyme is altered and its activity decreases. The inhibitory effect of 4HBA on melanogenesis has been studied using cultured mouse melanoma cells. Melanin synthesis in cell culture with 4HBA at 1.0 mM was decreased to 45% of control and below 1.0 mM there was no effect on cell growth. The inhibitory effects of 4HBA on melanogenesis are due to the direct inhibition of melanosomal tyrosinase activity, rather than to the suppression of tyrosinase gene. These results showed that 4HBA is a promising and safe agent for skin whitening.

Photocrosslinked nanogels with a hydrophobic core and hydrophilic shell are successfully fabricated with the goal of obtaining a biocompatible and biodegradable drug carrier for hydrophobic anticancer drugs. These nanogels are composed of amphiphilic triblock copolymers, poly(D,L-lactic acid)/poly(ethylene glycol)/poly(D,L-lactic acid) (PLA-PEG-PLA), with acrylated groups at the end of the PLA segments. The copolymers are synthesized by ring-opening polymerization and possess a low CMC (49.6 mg x L(-1)), which easily helps to form micelles by self-assembly. The acrylated end groups allow the micelles to be photocrosslinked by ultraviolet irradiation, which turn the micelles into nanogels. These nanogels exhibit excellent stability as a suspension in aqueous media at ambient temperature as compared to the micelles. Moreover, the size of the nanogels is easily manipulated in a range of 150 to 250 nm by changing the concentration of crosslinkers, e.g., ethylene glycol dimethacrylate, and ultraviolet light irradiation time. The nanogels achieve a high encapsulation efficiency and offer a steady and long-term release mechanism for the hydrophobic anticancer drug, CPT. It shows that these nanogels are useful for a hydrophobic anticancer drug-carrier system. [pictures: see text] Formation of the PLA-PEG-PLA nanogels.

The development of ex vivo expansion of hematopoietic stem cells (HSCs) is a promising approach to restore the required bone marrow function of patients with hematological disorders. Previously, we have reported the development of an optimized serum-free and cytokines-limited defined medium using statistic methodology for umbilical cord blood-derived HSC expansion. The aim of this study was to analyze further the characteristics and functions of cells in vitro and in vivo when cultured in this defined medium. After a 7-day batch culture, the average absolute fold expansions for CD133(+) cells, CD34(+)CD133(+) cells, CD34(+)CD38() cells, CD133(+)CD38(-) cells, CD34(+)CXCR4(+) cells, CD133(+)CXCR4(+) cells, and long-term culture-initiating cells were 21-, 20-, 723-, 618-, 160-, 384-, and 8-fold, respectively. The high enrichment of CD38(-) cells and CXCR4(+) cells of the CD34(+) subpopulation provided a very early uncommitted HSC proliferation and homing ability. Furthermore, the expanded cells showed a high level of telomerase activity to maintain their telomere length and repopulated the lethally irradiated NOD/SCID mice in vivo. These results indicated that the cytokines limited expanded cells from CD133(+) cells could substantially support simultaneous expansion of various stem/progenitor cells and engraft with the expanded cells from a low number of HSCs initially.

Fibroblasts alter their shape, direction of movement, cytoskeleton arrangement, and focal contact when placed upon square array pillars. We prepared pillars of 1 microm diameter, separated by 3 microm, and having 1, 5, and 10 microm heights using substrates displaying identical surface chemistry. When cells seeded initially onto the tops of the pillars, fibroblasts subsequently were immobilized in situ by several pillars that visibly protruded through, but did not pierce, the cell bodies. The cytoplasma then migrated outward with long straight lamella along the interval of the pillars and formed several discrete attachment zones at their side walls - the value of their form index (FI) was as high as 35 - which altered the cellular shape entirely. Most of the cells interacted with the pillar substrate by spreading preferentially in a particular direction, but some of them had the ability to undergo coincident two-direction (x and y) migration; right-angle turn orientations led to the growth of dramatic cellular morphologies. Interestingly, this fibroblast's behavior variation was gradually in proportion to the pillar height of substrate. Our results confirm that cellular migration and cellular shape are both strongly affected by the geometry of the growth microenvironment.

We used a hydrophobic micro-textured poly-dimethylsiloxane (PDMS) in the presence of serum protein at 37 degrees C to study the motility of mouse stromal fibroblast on variant (15-100microm) parallel ridge/groove with 30microm depth. In this paper, we observed the temporal changes in cell morphology and locomotion by using time-lapse phase-contrast microscopy. When fibroblasts seeded onto the micro-grooved substrate, almost all of cells concentrated at the bottom of the grooves. Sequentially, the fibroblasts attached and spread on the surface, migrated toward the walls of the grooves, climbed up and down the ridges frequently, apparently, the 30microm depth of groove did not hinder movement across the micro-grooves. Eventually, they stopped proliferating as a result of contact inhibition and formed a confluent monolayer on the ridges almost exclusively, with an orientation parallel to the direction of the ridge/groove. Cellular shape of fibroblast was enhanced with the micro-grooves, the form index of nucleus was 2.6-fold greater than that of cells on smooth surfaces. Further, we found that hydrophobic surfaces are more prone to direct cellular motility in comparison with hydrophilic surfaces.

We studied the influence of the substrate topography substrate on the cell respondence. Topographical cues, independent of biochemistry, generated by the polydimethylsiloxanes (PDMS) substrate may have significant effects on cellular behavior. Mouse bone marrow stromal fibroblasts were examined on grooved substrata of equal groove and ridge width (100 µm, 30 µm depth). The phase contrast light microscopy revealed the attachment and orientation of cells on the materials used and indicated that the fibroblasts prefer to grow to confluent on the top of the ridges. This showed that the topographical surface had a profound effect on cell alignment and migration.

Objective In this study, a serum-free, stroma-free, and chemically defined medium for hematopoietic stem cell (HSC) expansion was systematically developed and optimized using factorial design and the steepest ascent method. Materials and methods Mononuclear cells (MNCs) were isolated from umbilical cord blood (UCB). HSCs were stimulated to proliferate ex vivo in the MNC culture system with variable serum substitutes, cytokines, and basal media according to experimental design. The expanded cells were assessed for cellular characteristics by surface antigen analysis, colony-forming cell assay (CFC assay), and long-term culture-initiating cell assay (LTC-IC assay). Results The optimal compositions of serum substitutes and the cytokine cocktail for HSC expansion in the MNC culture system were BIT (4 g/L BSA, 0.71 μg/mL insulin, and 27.81 μg/mL transferrin), and CC-9 (5.53 ng/mL TPO, 2.03 ng/mL IL-3, 16 ng/mL SCF, 4.43 ng/mL FL, 2.36 ng/mL IL-6, 1.91 ng/mL G-CSF, 1.56 ng/mL GM-CSF, 2.64 ng/mL SCGF, and 0.69 ng/mL IL-11) in the Iscove's modified Dulbecco's medium. After 6-day culture, the absolute fold expansions for white blood cells, CD34 + cells, CD34 + CD38 − cells, CFC, and LTC-IC were 1.4-, 30.4-, 63.9-, 10.7-, 2.8-fold, respectively. Conclusion Using the statistic methodology to develop HSC medium, our formula had lower cytokine concentrations comparing to other literatures and commercial media, but had superior or comparable expansion ability on HSC growth.

In this research, poly(sebacic anhydride) was synthesized via melt-condensation. The polymer was then blended with poly(ethylene glycol) in various ratios by solvent casting, to obtain the desired polymer blends. A differential scanning calorimeter was employed to investigate the crystalline behavior of the blends. Blends with under 10% poly(ethylene glycol) were found to consist of two partially miscible polymers in the amorphous phase. This compatibility of the two polymers may induce the crystallization of the poly(sebacic anhydride) component from decreasing the glass transition temperature of the poly(sebacic anhydride) component in the blending system due to the presence of poly(ethylene glycol) chain segments. Furthermore, phase separation occurred and the crystallinity of poly(sebacic anhydride) diminished when at least 20% poly(ethylene glycol) was present in the blends. These results were verified by the variation in the absorptions of carbonyl groups in infrared spectra; these spectra exhibit the changes in crystallinity of poly(sebacic anhydride) in the blends.

Yu-Shiang Peng,1,* Po-Liang Lai,2,* Sydney Peng,1 His-Chin Wu,3 Siang Yu,1 Tsan-Yun Tseng,4 Li-Fang Wang,5 I-Ming Chu1 1Department of Chemical Engineering, National Tsing Hua University, Hsinchu, 2Department of Orthopedic Surgery, Chang Gung Memorial Hospital at Linkou, Chang Gung University College of Medicine, Taoyuan, 3Department of Materials Engineering, Tatung University, Taipei, 4Graduate School of Biotechnology and Bioengineering, College of Engineering, Yuan Ze University, Chung-Li, 5Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung, Taiwan *Yu-Shiang Peng and Po-Liang Lai contributed equally to this work Abstract: Parkinson’s disease is known to result from the loss of dopaminergic neurons. Direct intracerebral injections of high doses of recombinant glial cell line-derived neurotrophic factor (GDNF) have been shown to protect adult nigral dopaminergic neurons. Because GDNF does not cross the blood–brain barrier, intracerebral gene transfer is an ideal option. Chitosan (CHI) is a naturally derived material that has been used for gene transfer. However, the low water solubility often leads to decreased transfection efficiency. Grafting of highly water-soluble polyethylene imines (PEI) and polyethylene glycol onto polymers can increase their solubility. The purpose of this study was to design a non-viral gene carrier with improved water solubility as well as enhanced transfection efficiency for treating Parkinsonism. Two molecular weights (Mw =600 and 1,800 g/mol) of PEI were grafted onto CHI (PEI600-g-CHI and PEI1800-g-CHI, respectively) by opening the epoxide ring of ethylene glycol diglycidyl ether (EX-810). This modification resulted in a non-viral gene carrier with less cytotoxicity. The transfection efficiency of PEI600-g-CHI/deoxyribonucleic acid (DNA) polyplexes was significantly higher than either PEI1800-g-CHI/DNA or CHI/DNA polyplexes. The maximal GDNF expression of PEI600-g-CHI/DNA was at the polymer:DNA weight ratio of 10:1, which was 1.7-fold higher than the maximal GDNF expression of PEI1800-g-CHI/DNA. The low toxicity and high transfection efficiency of PEI600-g-CHI make it ideal for application to GDNF gene therapy, which has potential for the treatment of Parkinson’s disease. Keywords: chitosan, gene transfection, glial cell, Parkinson’s disease, polyethylene imine

Poly (ethylene glycol) (PEG) was copolymerized with E-caprolactone and then acrylated to produce photo-crosslinked cell encapsulation hydrogels. The osteogenesis of encapsulated umbilical mesenchymal stem cells (UMSCs) was induced in these hydrogels and PEG hydrogels for comparison. The physical properties of the hydrogel, including the swelling ratio and compressive modulus, were measured. Gel degradation in phosphate buffered saline was also tested. Changes in cell morphology in the hydrogel were examined. Collagen type I gene expression, alkaline phosphatase activity, and calcium deposition were used to evaluate the degree of osteogenesis in the hydrogels. It was found that hydrogels composed of PEG copolymerized with e-caprolactone are suitable for inducing osteogenesis of UMSCs and thus helpful for bone regeneration.

The accumulation of beta-2-microglobulin (beta2M) in collagen-rich tissues has been proven to be the main cause of dialysis related amyloidosis. However, it remains uncertain which technique for the removal of beta2M can be used without compromising the advantages of other dialysis strategies. A new concept, an immunoadsorption wall, which combines the principles of immunoisolation and immunoadsorption is proposed to remove beta2M. The present investigations suggested that the application of the concept to clinical use is feasible and worthwhile. The concept, if validated, will help shape a novel multitask type of artificial kidney based on the combination of different separation technologies.

Hydrogels are of interest as scaffolds for cartilaginous tissue engineering due to their biocompatibility, swelling ratio, mechanical strength, and degradative behavior. This study was conducted to establish the most suitable poly(e-caprolactone)–poly(ethylene glycol)–poly(e-caprolactone) (PCL–PEG–PCL) triblock copolymer hydrogel for the optimal proliferation and differentiation of encapsulated chondrocytes for cartilage regeneration. PEG was copolymerized with PCL and then acrylated to confer photocrosslinking capacity. Chondrocytes were encapsulated within photocrosslinked triblock PEG-co-PCL hydrogels prepared with varying compositions. The effects of composition on the hydrogel properties and behavior of embedded cells were studied by varying the molecular weights, and thus the segment length, of the PEG and PCL blocks. Hydrogels with high-molecular-weight (10 000 Da) PEG were associated with higher swelling ratios (9.6 ± 0.2) and a reduced elastic modulus (0.223 ± 0.007 N mm−2). Biochemical analysis indicated a positive correlation between the swelling ratio and expression levels of glycosaminoglycans and total collagen. There was a 1.8-fold increase in glycosaminoglycan and 2.4-fold increase in total collagen content in hydrogels with the highest-molecular-weight (10 000 Da) PEG when compared to hydrogels with the low-molecular-weight (2000 Da) PEG after four weeks of culture. Histological examinations revealed more extensive collagen type II secretion and accumulation around chondrocytes when the molecular weight of the hydrophobic PCL segment increased. The lengths of the hydrophobic (PCL) and hydrophilic (PEG) segments resulted in changes in the properties of hydrogels that improved cellular proliferation and the production and distribution of extracellular matrix for cartilage regeneration.

Background Polymethylmethacrylate (PMMA) is commonly used for clinical applications. However, the short handling time increases the probability of a surgeon missing the crucial period in which the cement maintains its ideal viscosity for a successful injection. The aim of this article was to illustrate the effects a reduction in temperature would have on the cement handling time during percutaneous vertebroplasty. Methods The injectability of bone cement was assessed using a cement compressor. By twisting the compressor, the piston transmits its axial load to the plunger, which then pumps the bone cement out. The experiments were categorized based on the different types of hypothermic manipulation that were used. In group I (room temperature, sham group), the syringes were kept at 22°C after mixing the bone cement. In group 2 (precooling the bone cement and the container), the PMMA powder and liquid, as well as the beaker, spatula, and syringe, were stored in the refrigerator (4°C) overnight before mixing. In group 3 (ice bath cooling), the syringes were immediately submerged in ice water after mixing the bone cement at room temperature. Results The average liquid time, paste time, and handling time were 5.1 ± 0.7, 3.4 ± 0.3, and 8.5 ± 0.8 min, respectively, for group 1; 9.4 ± 1.1, 5.8 ± 0.5, and 15.2 ± 1.2 min, respectively, for group 2; and 83.8 ± 5.2, 28.8 ± 6.9, and 112.5 ± 11.3 min, respectively, for group 3. The liquid and paste times could be increased through different cooling methods. In addition, the liquid time (i.e. waiting time) for ice bath cooling was longer than for that of the precooling method (p < 0.05). Conclusions Both precooling (i.e. lowering the initial temperature) and ice bath cooling (i.e. lowering the surrounding temperature) can effectively slow polymerization. Precooling is easy for clinical applications, while ice bath cooling might be more suitable for multiple-level vertebroplasty. Clinicians can take advantage of the improved injectability without any increased cost.

Cephalosporin C was extracted from diluted or whole broth by PEG/salt aqueous two-phase systems. Parameters such as PEG molecular weight, salt type, pH, and salt concentration were investigated for finding a suitable extraction system. In PEG 600/ammonium sulfate or phosphate systems, K(c) (partition coefficienct of cephalosporin C) was observed to be larger than 1, with K(d) (partition coefficient of desacetyl cephalosporin C) being smaller than 1. The particular values of these coefficients would imply that the difficult separation of cephalosporin C and desacetyl cephalosporin C could possibly be achieved via the aqueous two-phase extraction. The addition of surfactants, water-miscible solvents, and neutral salts for enhancement of the separation efficiency was also investigated. The addition of surfactants to the system did not affect the separation efficiency substantially. K(c) would increase whereas K(d) decreased as a result of the addition of acetone, MeOH, EtOH, IPA, and n-BuOH. Meanwhile both K(c) and K(d) would decrease whenever neutral salts, NaCl, KCl, Kl, or KSCN, were added. The partitioning behavior of cephalosporin C and desacetyl cephalosporin C in filtered, whole, and different batches of broth was notably quite similar to that of diluted broth. The recovery yield of cephalosporin C in whole broth extraction was observed to be a function of centrifugal force used in phase separation. (c) 1994 John Wiley & Sons, Inc.

Objective A complete process for mass generation of megakaryocytes from hematopoietic stem cells under serum-free conditions has great clinical potential for rapid platelet reconstruction in thrombocytopenia patients. We have previously reported on the generation of an optimized serum-free medium (serum-free hematopoietic stem cell medium) for ex vivo expansion of CD34 + cells. Here, we further generated large amounts of functional megakaryocytes from serum-free expanded CD34 + cells under a complete and optimal serum-free condition for complying with clinical regulations. Materials and Methods Serum substitutes and cytokines were screened and optimized for their concentration for megakaryocyte generation by systemically methods. Serum-free induced megakaryocytes were characterized by surface antigens, gene expression, ex vivo megakaryocyte activation ability, and ability of megakaryocyte and platelet recovery in nonobese diabetic/severe combined immunodeficient mice. Results The optimal serum-free megakaryocyte induction medium was Iscove's modified Dulbecco's medium containing serum substitutes (i.e., human serum albumin, human insulin, and human transferrin) and a cytokine cocktail (i.e., thrombopoietin, stem cell factor, Fms-like tyrosine kinase 3 ligand, interleukin-3, interleukin-6, interleukin-9, and granulocyte-macrophage colony-stimulating factor). After induction, induced megakaryocytes expressed CD41a and CD61 surface antigens, nuclear factor erythroid-derived 2 and GATA-1 transcription factors and megakaryocyte activation ability. Importantly, transplantation of induced megakaryocytes could accelerate megakaryocyte and platelet recovery in irradiated nonobese diabetic/severe combined immunodeficient mice. Conclusion In conclusion, we have developed a serum-free megakaryocyte induction medium, and the combination of serum-free megakaryocyte and serum-free hematopoietic stem cell media can generate a large amount of functional megakaryocytes efficiently. Our method represents a promising source of megakaryocytes and platelets for future cell therapy.

Pure potassium penicillin-G solution and penicillin-G fermentation broth were extracted using the aqueous two-phase systems of poly-ethylene glycol and salts. The partition coefficients of penicillin-G were above 10 in both pure solution and broth systems. The partition coefficients of phenyl acetic acid were about 0.25. Cell mass and solid residue in the broth system were partly concentrated around the interfacial region and partly settled to the bottom when under 1000 × g centrifugal force. Accordingly, this technique is a promising alternative for the recovery of penicillins.