Increase in gap-junctional intercellular communications (GJIC) of normal human dermal fibroblasts (NHDF) on surfaces coated with high-molecular-weight hyaluronic acid (HMW HA)

Normal human dermal fibroblast (NHDF) cells were used to detect differences in gap-junctional intercellular communication (GJIC) by hyaluronic acid (HA), a linear polymer built from repeating disaccharide units that consist of N-acetyl-D-glucosamine (GlcNa) and D-glucuronic acid (GlcA) linked by a beta 1-4 glycosidic bond. The NHDF cells were cultured with different molecular weights (MW) of HA for 4 days. The rates of cell attachment in dishes coated with high-molecular-weight (HMW; 310 kDa or 800 kDa) HA at 2 mg/dish were significantly reduced at an early time point compared with low-molecular-weight (LMW; 4.8 kDa or 48 kDa) HA with the same coating amounts. HA-coated surfaces were observed by atomic force microscopy (AFM) under air and showed that HA molecules ran parallel in the dish coated with LMW HA and had an aggregated island structure in the dish coated with HMW HA surfaces. The cell functions of GJIC were assayed by a scrape-loading dye transfer (SLDT) method using a dye solution of Lucifer yellow. Promotion of the dye transfer was clearly obtained in the cell monolayer grown on the surface coated with HMW HA. These results suggest that HMW HA promotes the function of GJIC in NHDF cells. In contrast, when HMW HA was added to the monolayer of NHDF cells, the functions of GJIC clearly were lowered in comparison with the cells grown in the control dish or with those grown on the surface of HMW HA. Therefore it is concluded that the MW size of HA and its application method are important factors for generating biocompatible tissue-engineered products because of the manner in which the GJIC participates in cell differentiation and cell growth rate.     

Mutagenicity and effect on gap-junctional intercellular communication of 4,4'-methylenebis(2-chloroaniline) and its oxidized metabolites

Oxidized metabolites of 4,4'-methylenebis(2-chloroaniline) (MBOCA) were tested for direct mutagenicity in a Salmonella typhimurium assay and for effects on gap-junctional communication of WB-F344 rat liver cells. The mutagenicities of the N-hydroxy, mononitroso and o-hydroxy (ring) metabolites of MBOCA were assayed without adding activating enzyme systems, using the frame-shift sensitive strain TA98 and the base pair substitution sensitive strain TA100. The mutagenicity of the hydroxylamine was demonstrated by a linear increase in the formation of mutant colonies in both strains, with a formation of two revertants/nmol by TA98 and 21 revertants/nmol by TA100. The mononitroso metabolite showed a slight positive effect on TA100, but effects were masked by its cytotoxicity towards this strain. This metabolite was neither mutagenic nor cytotoxic to TA98. The o-hydroxy and the dinitroso metabolites were negative for mutagenicity at concentrations up to 50 and 500 micrograms/plate, respectively. The effects of parent MBOCA and N-hydroxy, mononitroso and o-hydroxy metabolites on cell-cell communication were determined by a scrape loading/fluorescent dye transfer technique. Cytotoxicity was assessed by determination of colony-forming efficiency and lactate dehydrogenase release. MBOCA itself caused an inhibition of dye transfer at concentrations of 7.5, 11.3 and 15 nmol/ml, whereas measures of cytotoxicity were not seen until 15 and 30 nmol/ml for LDH release and plating efficiency, respectively. None of the oxidized metabolites were active in inhibiting dye transfer at non-cytotoxic concentrations.     

Re-establishment of gap junctional intercellular communication (GJIC) between human endometrial carcinomas by prostaglandin E2

Reduced intercellular communication via gap junctions is correlated with carcinogenesis. Gap junctional intercellular communication (GJIC), between normal human endometrial epithelial cells is enhanced when endometrial stromal cells were present in culture. This enhancement of GJIC between normal epithelial cells also occurs when they are cultured in medium conditioned by stromal cells. This observation indicated that a soluble compound (or compounds) produced and secreted by stromal cells mediates GJIC in epithelial cells. Previous studies have shown that endometrial stromal cells release prostaglandin E₂ (PGE₂) and prostaglandin F_2α (PGF_2α) under physiological conditions. When we evaluated the response of normal endometrial epithelial cells to various concentrations of PGE_2, we found enhanced GJIC with 1 nM PGE₂. This is a smaller increase in GJIC than that induced by medium conditioned by stromal cells. When the extracellular concentration of PGE₂ was measured after incubation with stromal cells, it was found to be similar to the concentrations showing maximal GJIC between the normal epithelial cells. When indomethacin was used to inhibit prostaglandin synthesis by stromal cells, GJIC was reduced but not eliminated between normal endometrial epithelial cells. These observations suggest that although PGE₂ secreted by stromal cells is an important mediator of GJIC between the epithelial cells, it is not the sole mediator. Transformed endometrial epithelial cells did not demonstrate GJIC even in the presence of stromal cells. However, we were able to re-establish GJIC in transformed epithelial cells when we added PGE₂ to the cells. Our findings show that PGE₂ may serve as an intercellular mediator between stromal and epithelial cells that regulates GJIC in normal and malignant epithelial cells. This suggests that maintenance of GJIC by preserving or replacing PGE₂ secretion by endometrial stromal cells may have the potential to suppress carcinogenesis in endometrial epithelial cells.     

软性接触镜透氧系数测量方法研究

阐述利用极谱法(FATT)测量软性接触镜透氧系数(Dk)的原理,测试步骤和数据处理方法.并通过实验验证了该方法的可行性.     

Role of the water matric potential (psi(M)) and of equilibrium water content (EWC) on the water self-diffusion coefficient and on the oxygen permeability in hydrogel contact lenses

This paper illustrates a new thermodynamic and kinetic model that describes the relationship between the water self-diffusion coefficient, D*(w/g), in hydrogel contact lenses, in terms of water matric potential (psi(M)) and equilibrium water content (EWC). Experimental measurements on commercial contact lenses yield water thermodynamic activity values ranging between 0.996 and 0.999. The corresponding psi(M) matric potential is, respectively, between -8 and -2J/mol at temperature 35 degrees C. Comparison between water self-diffusion coefficients derived in this paper and those suggested by other authors shows that our values are greater (25%-50%) than the previous ones. The impact of this model on the nature of the oxygen permeability, pi, in the lenses has been evaluated and the changes of pi with psi(M) and EWC are predicted and compared with direct experimental measurements. For the contact lenses investigated, the oxygen permeability turns out to be only a quadratic function of equilibrium water content, despite the fact that the fraction of the "free" water molecules can be as high as 50%.     

A single-lens polarographic measurement of oxygen permeability (Dk) for hypertransmissible soft contact lenses

A novel polarographic apparatus is described that requires only a single soft contact lens (SCL) to ascertain oxygen permeabilities of hypertransmissible lenses. Unlike conventional methods where a range of lens thickness is needed for determining oxygen permeabilities of SCLs, the apparatus described here requires only a single-lens thickness. This is accomplished by minimizing (or completely eliminating) edge effects, boundary-layer resistances, and lens desiccation in the polarographic apparatus. By taking these effects into account, we measure reliable oxygen permeabilities of hypertransmissible SCLs (i.e., above 100 barrer). Results are reported for nine commercial SCLs ranging in permeability from 9 to 180 barrer. Measured single-lens oxygen permeabilities are in excellent agreement with those claimed by commercial manufacturers. Our new single-lens permeameter provides a reliable, efficient, and economical method for measuring oxygen permeabilities of commercial SCLs. The single-lens method offers a potential international standard for measuring oxygen permeabilities of SCLs up to 250 barrer.     

Effects of a corneal anesthetic on extinction of the classically conditioned nictitating membrane response in the rabbit (Oryctolagus cuniculus)

Rabbits (Oryctolagus cuniculus) were presented with 7 daily sessions of tone-alone training after conditioning. Before the beginning of each of the first 4 extinction sessions, an artificial tear solution or tetracaine hydrochloride was administered to the cornea of rabbits in the control group (n = 6) and experimental group (n = 7), respectively. There were no between-group differences in the percentage of conditioned responses between both groups. However, the amplitude of the conditioned response was notably reduced in the tetracaine group (M = 0.40, SEM +/- 0.216) relative to the control group (M = 1.32, SEM +/- 0.639) early in extinction. Results seem to suggest that although motor output has been found to play an important role in extinction, corneal sensory feedback is not necessary.     

Adsorption of bovine submaxillary mucin on silicone contact lenses grafted with poly(vinyl pyrrolidone)

Bovine submaxillary mucin is considered to be an analogue of the high molecular protein present in the conjunctival mucus. This mucin was isolated from fresh salivary glands and acetylated with [1-14C]acetic anhydride. In situ adsorption of the bovine submaxillary mucin on silicone contact lenses ungrafted and grafted with poly(vinyl pyrrolidone) was performed for the first time using an original radiotracer technique. The results show that the adsorbed amounts of mucin are higher on grafted samples and that thick layers are adsorbed when mucin concentration in the bulk solution is increased. Desorption experiments reveal that in addition to the tightly adsorbed protein layer, a loosely bound mucin layer of the same thickness exists on grafted and ungrafted silicones.     

机械牵张对兔角膜成纤维细胞细胞外基质基因表达的影响

目的 研究机械牵张与白介素-1β（IL-1β）联合作用对兔角膜成纤维细胞细胞外基质相关基因表达的影响。方法 对原代提取的兔角膜成纤维细胞进行牵张幅度15%、频率0.1 Hz的周期性牵张12、24、36 h,同时给予IL-1β处理,采用实时荧光定量PCR检测细胞基质金属蛋白酶（MMPs）、基质金属蛋白酶的组织抑制剂（TIMP-1）和I型胶原α1（Collagen Iα1）mRNA表达变化水平。结果 IL-1β单独作用可以诱导角膜成纤维细胞MMP-1、MMP-3和MMP-9 mRNA表达;MMP-1和MMP-3 mRNA表达随时间而降低,MMP-9、TIMP-1、Collagen Iα1 mRNA则随时间而增加;IL-1β与机械牵张联合作用使MMP-1、MMP-3、MMP-9 mRNA表达水平上调,TIMP-1和Collagen Iα1 mRNA表达下调,且具有时间依赖。单独机械牵张使Collagen Iα1 mRNA表达下降,IL-1β与机械牵张联合作用使其表达进一步下调,且具有时间依赖性。结论 机械牵张与炎性因子联合作用可加剧角膜组织破坏,促进角膜膨隆的发生发展。     

Effects of solvent dehydration on creep resistance of poly(vinyl alcohol) hydrogel

As a synthetic replacement material for osteochondral defect repair, poly(vinyl alcohol) (PVA) hydrogels offer a great potential due to their high water content and strong mechanical integrity. To survive the high stress environment in the joint space, high creep resistance becomes one of the key requirements for hydrogel implants. We hypothesized that reducing the equilibrium water content (EWC) of hydrogels would improve their creep resistance. We investigated the effect of dehydration of PVA theta-gels in various solvent/solution media followed by rehydration in saline solution. Decreasing EWC increased the creep resistance of PVA theta-gels. The most effective medium was isopropyl alcohol for reducing the EWC and increasing the creep resistance of PVA theta-gels.     

Combination of ascorbate and growth factor (TGF beta-3) in thermo-reversible hydrogel constructs embedded with rabbit chondrocytes for neocartilage formation

This study evaluated the potential of using poly(NiPAAm-co-AAc) as an injectable drug delivery carrier and a cell therapeutic agent in the form of a supporting matrix for the chondrogenic differentiation of rabbit chondrocytes. In particular, rabbit chondrocytes were embedded in hydrogels containing a combination of ascorbate and transforming growth factor beta-3 (TGF beta-3). Hydrogel constructs containing embedded cells either without ascorbate or a combination of ascorbate and TGF beta-3 were used as controls to determine the effects of ascorbate and TGF beta-3 on chondrogenic differentiation. The level of cartilage associated ECM proteins was examined using immunohistochemical staining for collagen type II as well as by Safranin-O and Alcian blue (GAG) staining. The results showed that ascorbate is an important factor for preparing cartilage constructs because of its action on chondrocyte phenotype modulation and proliferation.     

Calcification of poly(2-hydroxyethyl methacrylate) hydrogel sponges implanted in the rabbit cornea: A 3-month study

Poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogels have been used in the past as ocular implants. In a recent development, PHEMA sponges have shown suitable properties as materials for the peripheral component of an artificial cornea (keratoprosthesis). However, the propensity of PHEMA to calcify could threaten the long-term stability of the implanted devices. In an attempt to improve the understanding of the calcification mechanism, the dynamics, extent, and nature of calcified deposits within PHEMA sponges implanted in the cornea were investigated in this study, and the possible correlation between necrosis of cells and calcification was critically examined. Samples of a PHEMA sponge were implanted in rabbit corneas and explanted at predetermined time points (2, 4, and 12 weeks). The samples were examined by microscopy (light, transmission, scanning) and energy dispersive analysis of X-rays. Histological assessment and semiquantitative analysis of the amount of calcium deposited was performed using image analysis. An in vitro experiment was also performed by incubating sponge samples for 2 weeks in a solution of calcium and phosphate ions at a ratio similar to that in hydroxyapatite, in the absence of cells. Calcification was not seen in the 2- and 4-week explants, however, small deposits were detected in two of the 12-week explants, both within and on the sponge's constituent polymer particles. The deposit volumes represented 0.094% and 0.21%, respectively, of the total sponge volumes. Calcium deposits were present in large amounts both within the constituent polymer particles and on the surface of the sponges incubated in the abiotic calcifying solution. Cooperative mechanisms are suggested for the calcification of PHEMA sponges in vivo. The initial event may occur at a molecular level, when plasma proteins are adsorbed onto the polymer surface and bound through chelation to the calcium ions present in the medium. After their natural degradation, these structures may act as nucleation sites for calcium phosphate crystallization. Concurrently, the calcium ions can diffuse into the hydrogel particles and then the spontaneous precipitation of calcium phosphate may be caused by supersaturation due to the lower content of water in polymer, an effect which is likely predominant in vitro. The second event is the recruitment of phagocytic cells to clear calcium debris. Degeneration of these cells may then form nucleation sites for secondary calcification.     

Calcification of poly(2-hydroxyethyl methacrylate) hydrogel sponges implanted in the rabbit cornea: a 3-month study

Poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogels have been used in the past as ocular implants. In a recent development, PHEMA sponges have shown suitable properties as materials for the peripheral component of an artificial cornea (keratoprosthesis). However, the propensity of PHEMA to calcify could threaten the long-term stability of the implanted devices. In an attempt to improve the understanding of the calcification mechanism, the dynamics, extent, and nature of calcified deposits within PHEMA sponges implanted in the cornea were investigated in this study, and the possible correlation between necrosis of cells and calcification was critically examined. Samples of a PHEMA sponge were implanted in rabbit corneas and explanted at predetermined time points (2, 4, and 12 weeks). The samples were examined by microscopy (light, transmission, scanning) and energy dispersive analysis of X-rays. Histological assessment and semiquantitative analysis of the amount of calcium deposited was performed using image analysis. An in vitro experiment was also performed by incubating sponge samples for 2 weeks in a solution of calcium and phosphate ions at a ratio similar to that in hydroxyapatite, in the absence of cells. Calcification was not seen in the 2- and 4-week explants, however, small deposits were detected in two of the 12-week explants, both within and on the sponge's constituent polymer particles. The deposit volumes represented 0.094% and 0.21%, respectively, of the total sponge volumes. Calcium deposits were present in large amounts both within the constituent polymer particles and on the surface of the sponges incubated in the abiotic calcifying solution. Cooperative mechanisms are suggested for the calcification of PHEMA sponges in vivo. The initial event may occur at a molecular level, when plasma proteins are adsorbed onto the polymer surface and bound through chelation to the calcium ions present in the medium. After their natural degradation, these structures may act as nucleation sites for calcium phosphate crystallization. Concurrently, the calcium ions can diffuse into the hydrogel particles and then the spontaneous precipitation of calcium phosphate may be caused by supersaturation due to the lower content of water in polymer, an effect which is likely predominant in vitro. The second event is the recruitment of phagocytic cells to clear calcium debris. Degeneration of these cells may then form nucleation sites for secondary calcification.     

Effects of tacrolimus (FK506) on the development of osteonecrosis in a rabbit model

The present study examined the effects of tacrolimus (FK506) on the development of osteonecrosis in rabbits. In Experiment A, rabbits were given FK506, and also given a single dose of steroid. Control rabbits were given the same dose of steroid only. In Experiment B, rabbits were given FK506 and a reduced dose of steroid. The results showed that addition of FK506 did not change the number of rabbits with osteonecrosis when an identical steroid dose was given. When the steroid dose was reduced, the osteonecrosis incidence significantly decreased (p < 0.01). These results suggest that the clinically reported decrease in the osteonecrosis incidence following the introduction of FK506 is most likely attributable to the lower doses of steroids.     

Effects of ATP and its analogues on [Ca2+]i dynamics in the rabbit corneal epithelium.

Adenosine-5'-triphosphate (ATP) plays a pivotal role in various tissues as an extracellular transmitter. ATP released from nerve endings and/or damaged cells may elicit reactions in adjacent cells. To identify such reactions, we investigated the dynamics of the intracellular calcium ion concentrations ([Ca2+]i) in the rabbit corneal epithelium during ATP-stimulation. Intact epithelial sheets isolated from corneal tissue were loaded with Fura-2, and [Ca2+]i dynamics in each cell layer were analyzed using a digital imaging system (Argus 50/CA). Normal architecture was preserved, suggesting that functional integrity remained intact. Perfusion with HEPES-buffered Ringer's solution containing ATP (10 microM) and uridine-5'-triphosphate (UTP; 10 microM) caused a biphasic [Ca2+]i increase in the superficial layer that manifested itself as a rapid initial spike followed by a long-lasting plateau phase. Adenosine-5'-diphate (10 microM) elevated the [Ca2+]i level, but induced only the initial spike, which was smaller than those induced by ATP and UTP. Adenosine (10 microM) did not elicit any [Ca2+]i changes in the epithelial cells. Suramin (10 microM; a P2 receptor antagonist) blocked the ATP-induced [Ca2+]i increase, whereas the P2X receptor agonists, alpha, beta-methylene ATP (10 microM), 2-methyl-thio ATP (10 microM) and Benzoylbenzoyl ATP (10 microM), did not elicit any increases in [Ca2+]i. In the basal cell layer, ATP-induced [Ca2+]i dynamics were biphasic, while oscillatory fluctuations of [Ca2+]i were induced in the wing cells of the mid layer of the corneal epithelium by ATP stimulation. Ca2+ oscillations were sometimes synchronized among adjacent wing cells, but these waves did not propagate to other cell layers. These results suggest that extracellular ATP elicits a [Ca2+]i increase mainly via P2Y receptors. In addition, synchronized Ca2+ oscillation in the wing cell layer indicates that intracellular events may spread to neighboring cells within the layer.     

Effects of age and communication level on eye contact in fragile X males and non-fragile X autistic males

Dyadic social gaze and eye contact were examined in fragile X [fra(X)] males and in non-fra(X) autistic males as a function of age and level of communicative ability. Lag sequential analysis showed that responsive eye contact was highly correlated with age and communicative ability in non-fra(X) autistic males but not in fra(X) males. Older, more communicative non-fra(X) autistic males exhibited more responsive eye contact than their fra(X) cohorts. Implications of these observations for theory and intervention are discussed.     

Osteogenic differentiation of rabbit mesenchymal stem cells in thermo-reversible hydrogel constructs containing hydroxyapatite and bone morphogenic protein-2 (BMP-2)

The aim of this study was to assess the efficacy of ectopic bone formation in a three-dimensional hybrid scaffold in combination with hydroxyapatite (HA) and poly(NiPAAm-co-AAc) as an injectable vehicle in the form of a supporting matrix for the osteogenic differentiation of rabbit mesenchymal stem cells (MSCs). Osteogenic differentiation of MSCs in the hybrid scaffold was greatly influenced by the addition of growth factors. When the osteoinduction activity of hybrid scaffold was studied following implantation into the back subcutis of nude mouse in terms of histological and biochemical examinations, significantly homogeneous bone formation was histologically observed throughout the hybrid scaffolds containing growth factor (BMP-2: bone morphogenic protein-2). The level of alkaline phosphatase activity and osteocalcin content at the implanted sites of hybrid scaffolds were significantly high for the perfusion group compared with those in static culture group. We conclude that combination of MSC-seeded hybrid scaffold containing BMP-2 was a promising method by which to enhance in vitro osteogenic differentiation of MSC and in vivo ectopic bone formation.     

Delivery of dexamethasone, ascorbate, and growth factor (TGF beta-3) in thermo-reversible hydrogel constructs embedded with rabbit chondrocytes

The aim of this study was to assess the efficacy of poly(N-isopropylacrylamide-co-acrylic acid) (p(NiPAAm-co-AAc)) as an injectable drug delivery vehicle and a cell therapeutic agent in the form of a supporting matrix for the chondrogenic differentiation of rabbit chondrocytes. The p(NiPAAm-co-AAc) hydrogel itself without specific differentiation-inducing drugs was used as a control in order to determine the effects of these materials on chondrogenic differentiation. The level of cartilage associated extracellular matrix (ECM) proteins was examined by immunohistochemical staining for collagen type II as well as Safranin-O and Alcian blue (GAG) staining. These results highlight the potential of a thermo-reversible hydrogel mixed with chondrocytes and differentiation materials as an injectable delivery vehicle for use in neocartilage formation.     

Lesions of the dorsal spinal cord decrease the duration of contact defensive immobility (animal hypnosis) in the rabbit

Rabbits received either bilateral dorsal or unilateral dorsolateral spinal cord lesions. The duration and incidence of contact defensive immobility (CDI; animal hypnosis) were tested in these rabbits and in intact controls. Neither of the spinal cord lesions affected the number of CDI inductions, but rabbits with lesions of the dorsal spinal cord exhibited significantly shorter durations of CDI than either of the other groups which did not differ from each other. These results are interpreted to indicate that the somesthetic systems that ascend in the dorsal spinal cord are important for the maintenance, but not the initiation, of CDI.     

The use of green fluorescence gene (GFP)-modified rabbit mesenchymal stem cells (rMSCs) co-cultured with chondrocytes in hydrogel constructs to reveal the chondrogenesis of MSCs

This study was conducted to reveal the chondrogenesis of mesenchymal stem cells that had been genetically modified with the green fluorescence protein (GFP) gene and then co-cultured with chondrocytes in vitro and in vivo. Subsequent mixing of chondrocytes in the hydrogel constructs induced increased chondrogenic differentiation of the transfected hMSCs. The proliferation and differentiation of MSCs that were transfected with the GFP gene and co-cultured with chondrocytes (1:1 and 1:3) or chondrocytes alone were evaluated by a live/dead assay, MTT assay, GAG & DNA assay, RT-PCR, real time-PCR, and histological and immunochemical analysis in vitro and in vivo. Real-time PCR revealed that the expression of aggrecan and COMP by genetically modified hMSCs co-cultured with chondrocytes was 2 or 3 times greater than that of genetically modified MSCs alone. Moreover, the expression of collagen type II was more than 3.5 times greater than that of genetically modified MSCs alone. 3-D hydrogel constructs co-cultured with chondrocytes and genetically modified MSCs showed a significantly higher number of specific lacunae phenotypes at the end of the 4 week study, regardless of whether they were co-cultured in the presence of chondrocytes. These findings indicate that co-culture with chondrocytes and genetically modified MSCs can be used to engineer well designed implants for the formation of neocartilage by transplanted genetically modified MSCs.