Rheological properties of binary and ternary protein-polysaccharide co-hydrogels and comparative release kinetics of salbutamol sulphate from their matrices

Rheological properties of binary (AgarGelA and AgarGelB) and ternary (AgarGelAB and GelABAgar) co-hydrogels of agar (polysaccharide) with gelatin A and gelatin B (proteins) were studied to investigate their differential viscoelastic behavior. Two sets of rheological experiments, isochronal temperature and isothermal frequency sweep, were performed and the storage modulii, G' was measured which could be correlated to the gel strengths. Two separate peaks at 70°C and 35°C, corresponding to melting temperatures of agar and gelatin gels respectively, were obtained when derivative of G' with respect to temperature, dG'/dT was plotted against temperature which clearly showed the presence of two separate networks of gelatin and agar interconnected to each other. The results revealed that AgarGelAB was the strongest and AgarGelA was the weakest gel among all the gels studied. In order to see the effect of gel microstructure on drug encapsulation and release behavior, a model drug salbutamol was encapsulated in various gel matrices and the release of the same was seen in phosphate buffer pH 7.4, in simulated gastric fluid pH 1.2 (SGF) and in simulated intestinal fluid pH 6.8 (SIF) media. The drug release behavior universally followed sigmoidal kinetics invariant of gel composition. It is concluded that the hydrogel microstructure influenced the release behavior and best release, in all the three media, could be found with binary gel, AgarGelB, and ternary gel, AgarGelAB. Finally, microstructure of these gels is proposed.     

The release kinetics of drug eluting stents containing sirolimus as coated drug: Role of release media

Prior understanding on the in vitro release profile of the drug from drug eluting devices such as stent (DES) is crucial in designing and optimizing the drug embedded coating or matrices. In fact, assessing in vitro release profile is a mandatory requirement prior to the clinical evaluation of DES. The in vitro release is also employed to estimate parameters such as T1/2. The release profile largely depends on the release medium selected for the studies. Normally PBS with a pH of 7.4 is used for assessing the release kinetics of the drug. Often drug undergoes irreversible changes such as hydrolysis in PBS leading to erroneous assessment of the release profile. This is particularly true in the case of sirolimus, one of the widely used drugs in various applications. We studied the influence of various media on the release profile of sirolimus from DES. The data generated suggested that a release medium consisting of 9:1 (v/v) of normal saline and isopropanol is a most suitable one for assessing in vitro the release kinetics of sirolimus from DES.     

Entrapment and in vitro release of delta-sleep inducing peptide from polymer hydrogels based on modified polyvinyl alcohol

The aim of this study was to entrap delta-sleep inducing peptide (DSIP) in cross-linked poly(vinyl alcohol)-based hydrogels of different structures and to determine kinetics of the peptide release from these hydrogels using an in vitro model. Isotropic and macroporous hydrogels based on poly(vinyl alcohol) acrylic derivative (Acr-PVA) and also macroporous epoxy groups containing hydrogels synthesized by copolymerization of this macromer and glycidyl methacrylate, have been used in this study. Isotropic hydrogels were prepared at positive temperatures while macroporous ones were obtained by formation in cryo-conditions. The peptide was entrapped into macroporous PVA hydrogels by adding the peptide solution onto preformed matrices, while peptide immobilization on PVA-GMA hydrogels, containing free epoxy groups, was carried out by sorption of peptide from its aqueous solution. In the case of DSIP entrapment into isotropic PVA gel the peptide solution was added into the polymer mixture at hydrogel formation. The kinetics of peptide release from hydrogels was studied by incubating matrices in PBS solution (pH 7.4), in physiological solution (0.9% NaCl) and in water. DSIP concentration in supernatants was determined by reverse-phase HPLC. Incubation of macroporous PVA gels in PBS, 0.9% NaCl, and water for 30 min caused release of 74, 70, and 64% DSIP, respectively, and this processes completed within 3 h. From hydrogel containing epoxy groups the release of neither peptide nor its degradation products was observed even after incubation for 48 h. For freshly prepared isotropic hydrogel the release kinetics was as follows: 27 and 78% DSIP were released within first 30 min and 33 h, relatively. For the lyophilized hydrogel samples the peptide release was 63% after incubation for 30 min, while drying of samples at room temperature for 3 days caused significant peptide loss because of its structure damage.     

A comparison of Thai silk fibroin-based and chitosan-based materials on in vitro biocompatibility for bone substitutes

The novel hybrid scaffolds fabricated from silk fibroin, gelatin, low deacetylation degree chitosan and hydroxyapatite were investigated for their in vitro biocompatibility and osteoconductivity to mouse pre-osteoblast cell line (MC3T3-E1) and rat bone marrow-derived stem cells (MSC). We found that gelatin-conjugated silk fibroin films and scaffolds dominantly promoted cell adhesion and proliferation. Film and scaffold prepared from gelatin-conjugated silk fibroin with hydroxyapatite grown crystals effectively enhanced osteogenic differentiation of both cell types, as evaluated by alkaline phosphatase activity and calcium content. However the blend of hydroxyapatite/low deacetylation degree chitosan hybrid materials did not support cell growth. Furthermore, the blended hydroxyapatite in the bulk scaffold was found to be less effective for osteogenic differentiation than the scaffold with hydroxyapatite grown crystals. The comparative study between MC3T3-E1 and MSC showed that both cell types had similar trend of proliferation and osteogenic differentiation on the same material. Also, higher proliferative rate of MC3T3-E1 than MSC was observed.     

EphA3基因稳定表达细胞模型的建立与分析

目的：建立稳定表达外源EphA3基因的小鼠成纤维细胞株模型,初步探讨EphA3基因表达对肿瘤发生、发展的影响。方法：通过脂质体介导的方法,将真核表达载体pcDNA3.1（-）/myc-his-EphA3转染NIH3T3细胞,用Western印迹确定外源EphA3基因表达;通过MTT实验、软琼脂集落形成实验,观察EphA3基因表达对NIH3T3细胞生物学特性的影响。结果：建立了稳定转染EphA3基因的NIH3T3细胞株;EphA3基因表达的小鼠成纤维NIH3T3细胞生长速度没有明显变化,但在软琼脂上锚着非依赖生长的能力加强。结论：建立了稳定表达外源EphA3基因的NIH3T3细胞株,EphA3基因稳定表达具有诱导正常NIH3T3细胞发生恶性转化的重要生物功能。     

Schistosoma mansoni: pH dependence of cercarial eicosanoid production, penetration, and transformation

The pH dependence of Schistosoma mansoni cercarial penetration and transformation was determined using a gelatin:agar matrix, containing 3mM linoleate, as a penetration substrate. Penetration was largely unaffected by pH, approaching 100% over the pH range of 5.4 to 8.2, while transformation had an optimum pH range between 6.2 and 7.4. Within this pH range, between 74 and 89% of cercariae lost their tails. Outside this range, transformation decreased to 0% above pH 7.8 and dropped to 57% at pH 5.4. Esculetin, a lipoxygenase inhibitor, also incorporated into the agar:gelatin plates at a concentration of 1 mM, had little effect on cercarial penetration, except between pH 6.5 and 6.65, where penetration rates fell to 50% at pH 6.63. Transformation, however, was inhibited by esculetin, except between pH 6.5 and 6.8, where transformation was statistically equivalent to controls (P = 0.064, two-tailed Student's t test). Cercarial eicosanoid production measured at pH 6.55 and 7.2 in the presence and absence of 1 mM esculetin has led to the tentative identification of a 5-lipoxygenase product associated with cercarial penetration: LTB4 or 6-trans LTB4, a breakdown product of LTB4. We discuss the importance of pH control in cercarial experiments as well as the possible modulatory role skin pH (surface, epidermal, and dermal) may have in regulating cercarial eicosanoid production.     

Cellular neoplastic transformation induced by 916 MHz microwave radiation

There has been growing concern about the possibility of adverse health effects resulting from exposure to microwave radiations, such as those emitted by mobile phones. The purpose of this study was to investigate the cellular neoplastic transformation effects of electromagnetic fields. 916 MHz continuous microwave was employed in our study to simulate the electromagnetic radiation of mobile phone. NIH/3T3 cells were adopted in our experiment due to their sensitivity to carcinogen or cancer promoter in environment. They were divided randomly into one control group and three microwave groups. The three microwave groups were exposed to 916 MHz EMF for 2 h per day with power density of 10, 50, and 90 w/m(2), respectively, in which 10 w/m(2) was close to intensity near the antenna of mobile phone. The morphology and proliferation of NIH/3T3 cells were examined and furthermore soft agar culture and animal carcinogenesis assay were carried out to determine the neoplastic promotion. Our experiments showed NIH/3T3 cells changed in morphology and proliferation after 5-8 weeks exposure and formed clone in soft agar culture after another 3-4 weeks depending on the exposure intensity. In the animal carcinogenesis study, lumps developed on the back of SCID mice after being inoculated into exposed NIH/3T3 cells for more than 4 weeks. The results indicate that microwave radiation can promote neoplastic transformation of NIH/3T3cells.     

Polyomavirus transforms rat F111 and mouse NIH 3T3 cells by different mechanisms.

Polyomavirus middle tumor antigen (mT) was expressed in a line of mouse NIH 3T3 cells under control of the dexamethasone-regulatable mouse mammary tumor virus promotor. Contrary to rat F111 cells which were rendered anchorage independent by mT expression alone (L. Raptis, H. Lamfrom, and T.L. Benjamin, Mol. Cell. Biol. 5:2476-2487, 1985), mT-producing NIH 3T3 cells were unable to grow in agar even after full mT induction. The mT:pp60c-src-associated phosphatidylinositol kinase was activated in these cells to a degree similar to that in fully transformed cells expressing the small and large T antigens, in addition to mT. We therefore propose that the stimulation of this phosphatidylinositol kinase, although apparently necessary, is not sufficient for transformation of NIH 3T3 cells by polyomavirus.     

Resistance to oncogenic transformation in revertant R1 of human ras-transformed NIH 3T3 cells.

A flat revertant, R1, was isolated from human activated c-Ha-ras-1 (hu-ac-Ha-ras) gene-transformed NIH 3T3 cells (EJ-NIH 3T3) treated with mutagens. R1 contained unchanged transfected hu-ac-Ha-ras DNA and expressed high levels of hu-ac-Ha-ras-specific mRNA and p21 protein. Transfection experiments revealed that NIH 3T3 cells could be transformed by DNA from R1 cells but R1 cells could not be retransformed by Kirsten sarcoma virus, DNA from EJ-NIH 3T3 cells, hu-ac-Ha-ras, v-src, v-mos, simian virus 40 large T antigen, or polyomavirus middle T antigen. Somatic cell hybridization studies showed that R1 was not retransformed by fusion with NIH 3T3 cells and suppressed anchorage independence of EJ-NIH 3T3 and hu-ac-Ha-ras gene-transformed rat W31 cells in soft agar. These results suggest that the reversion and resistance to several oncogenes in R1 is due not to cellular defects in the production of the transformed phenotype but rather to enhancement of cellular mechanisms that suppress oncogenic transformation.     

Fabrication of gelatin-hyaluronic acid hybrid scaffolds with tunable porous structures for soft tissue engineering

The development of three-dimensional (3-D) scaffolds with highly open porous structure is one of the most important issues in tissue engineering. In this study, 3-D macroporous gelatin/hyaluronic acid (GE/HA) hybrid scaffolds with varying porous morphology were prepared by freeze-drying their blending solutions and subsequent chemical crosslinking by using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). The resulting scaffolds were characterized by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). Their swelling, in vitro degradation properties and compressive strength were also investigated. To evaluate in vitro cytocompatibility of scaffolds, mouse L929 fibroblasts were seeded onto the scaffolds for cell morphology and cell viability studies. It was found that the porous structure of scaffolds can be tailored by varying the ratios of gelatin to HA, both the swelling ratios and degradation rate increased with the increase of HA content in hybrid scaffolds, and crosslinking the scaffolds with EDC improved the degradation resistance of the scaffold in culture media and increased the mechanical strength of scaffolds. The in vitro results revealed that the prepared scaffolds do not induce cytotoxic effects and suitable for cell growth, especially in the case of scaffolds with higher gelatin content. The combined results of the physicochemical and biological studies suggested that the developed GE/HA hybrid scaffolds exhibit good potential and biocompatibility for soft tissue engineering applications.     

Down-modulation of an oncogene protein product and reversion of the transformed phenotype by monoclonal antibodies

Exposure of neu-oncogene-transformed NIH 3T3 cells to monoclonal antibodies reactive with the neu gene product, p185, results in the rapid and reversible loss of both cell-surface and total cellular p185. Although not directly cytotoxic, monoclonal anti-p185 antibody treatment causes neu-transformed NIH 3T3 cells to revert to a nontransformed phenotype, as determined by anchorage-independent growth. Isotype matched control antibodies of an unrelated specificity do not affect p185 levels or colony formation in soft agar by neu-transformed NIH 3T3 cells. Soft agar colony formation by NIH 3T3 cells transformed by ras oncogenes is not affected by anti-p185 antibody treatment. Anchorage-independent growth of cells from the ethylnitrosourea-induced rat neuroblastoma line in which neu was originally detected by DNA transfection is also inhibited in the presence of anti-p185 monoclonal antibodies. Collectively, these results suggest that p185 is required to maintain transformation induced by the neu oncogene.     

V-fos基因对NIH3T3细胞转化的研究

本文报道了用含v-fos基因的pFBJ-2质粒转染NIH 3 T 3细胞,获得了转化细胞株。对细胞株的研究结果表明:(1)Southern杂交检测到细胞基因组中有v-fos基因的整合;(2)点杂交测得有v-fos mRNA的表达;(3)出现一系列转化表型,包括细胞形态的改变,异常的增长速率,在软琼脂上的贴壁不依赖性生长,对低血清浓度培养液的适应性以及细胞膜表而超微结构的变化等,提示v-fos基因能使NIH 3 T 3细胞发生转化,并在体外转化过程中起决定性作用。     

Normal mouse serum contains peptides which induce fibroblasts to grow in soft agar

The untransformed mouse fibroblast cells NIH/3T3, C3H/10T1/2, and rat NRK cells do not grow in soft agar in medium supplemented with 10% fetal calf serum. When fetal calf serum in the growth medium was supplemented with less than 1% of sera from mice or other vertebrates, however, these cells responded, forming large colonies. The morphology of soft agar colonies was a function of the treated cell type. In the presence of 10% serum from C57BL/6 mice, NRK cells grew to smooth-surfaced spherical colonies, while NIH/3T3 colonies showed individual round cells on their surface and C3H/10T1/2 cells grew as extended cells forming columns of end to end connected fibroblasts. Mus Musculus Castaneus-Epithelial (MMC- E) cells were not stimulated to grow in soft agar under these conditions. The major fibroblast colony-inducing factor (F-CIF) was partially purified from mouse serum by acid/ethanol-extraction, gel permeation chromatography, and reverse-phase high-pressure liquid chromatography. F-CIF is a polypeptide, which does not compete for binding to epidermal growth factor (EGF) receptors, but stimulates normal fibroblasts to form small colonies in semisolid medium and very large colonies in the presence of added EGF (2 ng/ml). In contrast to unfractionated mouse serum, purified F-CIF did not induce C3H/10T1/2 cells to grow in soft agar, suggesting that serum contains additional cell type-specific agar growth-stimulating activities.     

在NIH3T3细胞中高表达EDAG—1基因导致细胞恶性转化

探讨一种新近克隆的特异表达在造血系统组织和细胞中的胚胎发育相关新基因 (EDAG 1)的生物学功能。构建EDAG 1真核表达载体pcDNA3.1( ) EDAG 1,以脂质体法将其稳定转染入NIH3T3细胞中 ,结果发现高表达EDAG 1的NIH3T3细胞失去了正常的平行走向及接触抑制现象 ,侵袭力较正常增强约 10倍并获得锚定不依赖生长能力。将该细胞株皮下接种裸鼠 ,受鼠在 4周左右 10 0 % (6 / 6 )成瘤。这些结果表明EDAG 1是一种具有转化活性的新基因 ,可能与肿瘤发生、发展密切相关     

Isolation and characterization of NIH 3T3 cells expressing polyomavirus small T antigen.

The polyomavirus small T-antigen gene, together with the polyomavirus promoter, was inserted into a retrovirus vector pGV16 which contains the Moloney sarcoma virus long terminal repeat and neomycin resistance gene driven by the simian virus 40 promoter. This expression vector, pGVST, was packaged into retrovirus particles by transfection of psi 2 cells which harbor packaging-defective murine retrovirus genome. NIH 3T3 cells were infected by this replication-defective retrovirus containing pGVST. Of the 15 G418-resistant cell clones, 8 express small T antigen at various levels as revealed by immunoprecipitation. A cellular protein with an apparent molecular weight of about 32,000 coprecipitates with small T antigen. Immunofluorescent staining shows that small T antigen is mainly present in the nuclei. Morphologically, cells expressing small T antigen are indistinguishable from parental NIH 3T3 cells and have a microfilament pattern similar to that in parental NIH 3T3 cells. Cells expressing small T antigen form a flat monolayer but continue to grow beyond the saturation density observed for parental NIH 3T3 cells and eventually come off the culture plate as a result of overconfluency. There is some correlation between the level of expression of small T antigen and the growth rate of the cells. Small T-antigen-expressing cells form small colonies in soft agar. However, the proportion of cells which form these small colonies is rather small. A clone of these cells tested did not form tumors in nude mice within 3 months after inoculation of 10(6) cells per animal. Thus, present studies establish that the small T antigen of polyomavirus is a second nucleus-localized transforming gene product of the virus (the first one being large T antigen) and by itself has a function which is to stimulate the growth of NIH 3T3 cells beyond their saturation density in monolayer culture.     

过表达Gankyrin基因细胞模型的建立及其成瘤性分析

目的：Gankyrin作为一个新的癌基因,在细胞周期调控和肿瘤发生过程中有重要功能。建立Gankyrin过表达的细胞和动物模型,以便进一步研究其在肿瘤形成过程中的作用机制。方法：采用逆转录病毒感染细胞的方法构建Gankyrin稳定表达的细胞株,采用Western-blot检测Gankyrin的表达,采用软琼脂和裸鼠成瘤实验验证该细胞株的恶性转化效应。结果：构建了Gankyrin稳定过表达的NIH3T3细胞株,且该细胞株具有成瘤性。结论：采用逆转录病毒感染细胞的方法可以有效建立Gankyrin转化细胞株,为进一步研究Gankyrin的作用机制及其在肿瘤形成中的功能提供了基础。     

Expression of phosphophoryn is sufficient for the induction of matrix mineralization by mammalian cells

Mineralized tissues such as dentin and bone assemble extracellular matrices uniquely rich in a variety of acidic phosphoproteins. Although these proteins are presumed to play a role in the process of biomineralization, key questions regarding the nature of their contributions remain unanswered. First, it is not known whether highly phosphorylated proteins alone can induce matrix mineralization, or whether this activity requires the involvement of other bone/dentin non-collagenous proteins. Second, it remains to be established whether the protein kinases that phosphorylate these acidic proteins are unique to cells responsible for producing mineralized tissues. To begin to address these questions, we consider the case of phosphophoryn (PP), due to its high content of phosphate, high affinity for Ca(2+), and its potential role in hydroxyapatite nucleation. We have created a model system of biomineralization in a cellular environment by expressing PP in NIH3T3 fibroblasts (which do not produce a mineralized matrix); as a positive control, PP was expressed in MC3T3-E1 osteoblastic cells, which normally mineralize their matrices. We show that expression of PP in NIH3T3 cells is sufficient for the induction of matrix mineralization. In addition, assessment of the phosphorylation status of PP in these cells reveals that the transfected NIH3T3 cells are able to phosphorylate PP. We suggest that the phosphorylation of PP is essential for mineral formation. The principle goal of this study is to enrich the current knowledge of mineralized tissue phosphorylation events by analyzing them in the context of a complete cellular environment.     

3D-printed PLA/Gel hybrid in liver tissue engineering: Effects of architecture on biological functions

The liver is one of the vital organs in the body, and the gold standard of treatment for liver function impairment is liver transplantation, which poses many challenges. The specific three-dimensional (3D) structure of liver, which significantly impacts the growth and function of its cells, has made biofabrication with the 3D printing of scaffolds suitable for this approach. In this study, to investigate the effect of scaffold geometry on the performance of HepG2 cells, poly-lactic acid (PLA) polymer was used as the input of the fused deposition modeling (FDM) 3D-printing machine. Samples with simple square and bioinspired hexagonal cross-sectional designs were printed. One percent and 2% of gelatin coating were applied to the 3D printed PLA to improve the wettability and surface properties of the scaffold. Scanning electron microscopy pictures were used to analyze the structural properties of PLA–Gel hybrid scaffolds, energy dispersive spectroscopy to investigate the presence of gelatin, water contact angle measurement for wettability, and weight loss for degradation. In vitro tests were performed by culturing HepG2 cells on the scaffold to evaluate the cell adhesion, viability, cytotoxicity, and specific liver functions. Then, high-precision scaffolds were printed and the presence of gelatin was detected. Also, the effect of geometry on cell function was confirmed in viability, adhesion, and functional tests. The albumin and urea production of the Hexagonal PLA scaffold was about 1.22 ± 0.02-fold higher than the square design in 3 days. This study will hopefully advance our understanding of liver tissue engineering toward a promising perspective for liver regeneration.     

Comparison of chondrogensis in static and perfused bioreactor culture

As a result of the low yield of cartilage from primary patient harvests and a high demand for autologous cartilage for reconstructive surgery and structural repair, primary explant cartilage must be augmented by tissue engineering techniques. In this study, chondrocytes seeded on PLLA/PGA scaffolds in static culture and a direct perfusion bioreactor were biochemically and histologically analyzed to determine the effects of fluid flow and media pH on matrix assembly. A gradual media pH change was maintained in the bioreactor within 7.4-6.96 over 2 weeks compared to a more rapid decrease from 7.4 to 6.58 in static culture over 3 days. Seeded scaffolds subjected to 1 microm/s flow demonstrated a 118% increase (p < 0.05) in DNA content, a 184% increase (p < 0.05) in GAG content, and a 155% (p < 0.05) increase in hydroxyproline content compared to static culture. Distinct differences were noted in tissue morphology, including more intense staining for proteoglycans by safranin-O and alignment of cells in the direction of media flow. Culture of chondrocyte seeded matrices thus offers the possibility of rapid in vitro expansion of donor cartilage for the repair of structural defects, tracheal injury, and vascularized tissue damage.