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1.
Fibroblast chemotaxis is implicated in many physiological processes, including wound healing and morphogenesis. We present a novel assay for chemotaxis of fibroblasts (and other slow-moving tissue cells) in a direct-viewing chamber containing a physiologically relevant three-dimensional fibrin or collagen gel in which long-lasting, spatially continuous gradients have been sustained for at least 24 h, long enough for significant fibroblast migration. This combination of features is not available in any alternative assay of comparable setup simplicity. Using a putative fibroblast chemotactic factor, the fibronectin peptide GRGDSP, we measured human foreskin fibroblast alignment in the direction along the gradient, which followed a biphasic dependence on GRGDSP concentration with an optimal concentration of about 10 nM. Time-lapse video microscopy revealed that cell migration was up the soluble GRGDSP gradient, confirming positive chemotaxis to GRGDSP and rejecting the possibility of dominant haptotaxis down the soluble GRGDSP gradient, that is, up a putative gradient of integrin-mediated adhesion induced by the soluble GRGDSP gradient. 相似文献
2.
Masao Hara Masahiro Nakashima Takahiro Fujii Kazuki Uehara Chisako Yokono Ron Hashizume 《Bioscience, biotechnology, and biochemistry》2013,77(3):458-461
We designed a cyclic compression system using readily available six-well culture plates to investigate the influence of mechanical stress on skin-like structures. The effects of cyclic mechanical stress on protein expression by cells were easily examined, and hence, this system should be useful for further analysis of skin responses to mechanical stress. 相似文献
3.
Hajime Sugihara Shuji Toda Shinichi Miyabara Yasushi Kusaba Yohichi Minami 《In vitro cellular & developmental biology. Animal》1991,27(2):142-146
Summary The skin comprises three layers: epidermis, dermis, and hypodermis. We report here on a skin, reconstructed in vitro, that
is composed of all three layers. The topmost layer, epidermis, was exposed to air by a new method. The exposure induced an
extensive proliferation, and differentiation, i.e. keratinization was eventually observed in the cultured epidermal cells.
Skin thus cultured will be a useful graft of transplantation and provide an ideal model system in which to study diseases
of the skin. 相似文献
4.
Yuichi Adachi Tadashi Mio Keiichi Takigawa Ilja Striz Debra J. Romberger John R. Spurzem Stephen I. Rennard 《In vitro cellular & developmental biology. Animal》1998,34(3):203-210
Summary
In vivo, fibroblasts are distributed in a three-dimensional (3-D) connective tissue matrix. Fibronectin is a major product of fibroblasts
in routine cell culture and is thought to regulate many aspects of fibroblast biology. In this context, we sought to determine
if the interaction of fibroblasts with a 3-D matrix might affect fibronectin production. To examine this hypothesis, fibronectin
production by fibroblasts cultured in a 3-D collagen gel or on plastic dishes was measured by ELISA. Fibroblasts in 3-D gel
culture produced more fibronectin than those in monolayer culture. Fibroblasts in 3-D culture produced increasing amounts
of fibronectin when the collagen concentration of the gel was increased. The 3-D nature of the matrix appeared to be crucial
because plating the fibroblasts on the surface of a plastic dish underneath a collagen gel was not different from plating
them on a plastic dish in the absence of collagen. In addition to increased fibronectin production, the distribution of the
fibronectin produced in 3-D culture was different from that of monolayer culture. In monolayer culture, more than half of
the fibronectin was released into the culture medium. In 3-D culture, however, approximately two-thirds remained in the collagen
gel. In summary, the presence of a 3-D collagen matrix increases fibroblast fibronectin production and results in greater
retention of fibronectin in the vicinity of the producing cells. 相似文献
5.
D. A. Garzón-Alvarado J. C. Vanegas Acosta 《Computer methods in biomechanics and biomedical engineering》2013,16(6):571-583
The partial rupture of ligament fibres leads to an injury known as grade 2 sprain. Wound healing after injury consists of four general stages: swelling, release of platelet-derived growth factor (PDGF), fibroblast migration and proliferation and collagen production. The aim of this paper is to present a mathematical model based on reaction–diffusion equations for describing the repair of the medial collateral ligament when it has suffered a grade 2 sprain. We have used the finite element method to solve the equations of this. The results have simulated the tissue swelling at the time of injury, predicted PDGF influence, the concentration of fibroblasts migrating towards the place of injury and reproduced the random orientation of immature collagen fibres. These results agree with experimental data reported by other authors. The model describes wound healing during the 9 days following such injury. 相似文献
6.
探讨阿魏酸钠(SF)对增生性瘢痕成纤维细胞(HSFb)增殖及胶原合成的影响.体外培养HSFb,MTT法计算SF的LC50及最佳药物时间后,分为空白对照组、SF干预组(高、中、低浓度分别为0.3、0.03、0.003 mg/mL),培养72 h后,在倒置显微镜和透射电镜下观察HSFb微观形态学变化;MTT法、Wester... 相似文献
7.
The collagen gel contraction (CGC) assay is used frequently to study the cell-mediated reorganization of the extracellular natrix. In a typical CGC assay, cells embedded in a disk-shaped lattice (gel) of native type I collagen fibers compress the fibers and, consequently, reduce the diameter of the collagen disk within h or d. The degree to which the collagen is contracted is usually quantified by measurement of the diameter or the area of the disk. During CCC assays, friction or adhesion (or both) between gels and their culture containers can cause gels to be incompletely contracted or to acquire distorted shapes. Such occurrences degrade the reproducibility and reliability of measurements of gel dimensions. To address these problems, we developed an oil-supported collagen retraction (OSCR) assay that creates an environment of low friction and adhesion around the contracting collagen gel. The OSCR assay is accomplished with simple equipment and is easily performed, sensitive, and consistently yields fully contracted gels with minimal distortion. 相似文献
8.
Hisako Ohgawara Naomi Mochizuki Toshio Taira Sachiyo Nishijima Naoko Iwasaki Ryougo Yui Yukimasa Hirata Taro Hayakawa 《In vitro cellular & developmental biology. Plant》1990,26(4):348-352
Summary We describe a method for maintaining neonatal pig pancreatic isletlike cell clusters (as pseudo-islets) embedded in a collagen
gel matrix for long periods. The pseudo-islets were formed from single cells of pig pancreas maintained in a suspension culture
and then embedded in pepsin-solubilized type I collagen. When the pseudo-islets were cultured in the collagen matrix, the
amount of collagen in the culture decreased gradually during the culture period as soluble hydroxyproline-containing material
accumulated in the medium. A low concentration of collagen (0.16%) degraded the collagen gels more rapidly than did high concentrations
of collagen (0.64%). The degradation of collagen depended both on the number of pseudo-islets embedded in the gel matrix and
on the culture conditions used to maintain them. With added nicotinamide, the accumulation of hydroxyproline decreased in
the medium and the structure of the gel matrix was well maintained. Hydrocortisone or a specific inhibitor of collagenase
did not decrease the solubilization of embedded pseudo-islet cultures and did not help to maintain their structure. These
observation indicate the possible utility of long-term maintenance of pseudo-islets in collagen gel matrix in the presence
of nicotinamide.
This work was supported by a Grant-in-Aid for Scientific Research in Japan 相似文献
9.
Regulation of collagen synthesis in fibroblasts within a three-dimensional collagen gel 总被引:7,自引:0,他引:7
Cornelia Mauch Atsushi Hatamochi Karin Scharffetter Thomas Krieg 《Experimental cell research》1988,178(2):493-503
Fibroblasts cultivated within a three-dimensional collagen gel display an elongated, spindle-like morphology, reduce their proliferation rate, contact the gel to a very dense tissue, and modify their metabolic activity as compared to monolayer cultures. Collagen synthesis measured as protein-bound hydroxyproline is reduced to 5% of the values found in monolayer culture. The reduction involving type I and type III collagen is due to decreased de novo synthesis and not to enhanced degradation. Dot blot hybridization, Northern blot analysis, and in situ hybridization using collagen I- and III-specific cDNA probes demonstrate that reduced biosynthesis rates are reflected by a marked reduction of pro alpha 1 (I), pro alpha 2 (I), and pro alpha 1 (III) collagen mRNA indicating pretranslational regulation. A similar reduction was observed for actin mRNA whereas levels of tubulin mRNA were similar for fibroblasts in monolayer culture or cultivated within the three-dimensional collagen gels. The data suggest a specific reprogramming of various cellular activities in response to contact with the reconstituted extracellular matrix. 相似文献
10.
Hodaka Ikeda 《In vitro cellular & developmental biology. Plant》1990,26(2):193-200
Summary Thecal cells isolated from bovine ovarian follicles were cultured with a serum-free basal medium or a serum-free complete
medium in the presence or absence of collagen gel matrix, and their cellular proliferation and steroidogenesis were compared
with those of cells cultured with a serum-containing medium. The cells cultured with the serum-free basal medium produced
larger amounts of progesterone, androstenedione, and estradiol than the cells cultured with the serum-containing medium, but
no appreciable cell proliferation was observed in the serum-free medium. Response of thecal cells to 8 bromo-cAMP, a steroidogenic
agent, varied according to the type of steroid production examined and the type of culture medium used. In a cultivation period
of 4 d, progesterone production was stimulated about five-fold by 8 bromo-cAMP in the serum-free complete medium on collagen
gel matrix and in the serum-free basal medium without collagen matrix, whereas androstenedione production was stimulated about
three- to fourfold in the serum-free complete medium on collagen gel matrix and in the serum-free basal medium with or without
collagen matrix. Estradiol production, however, was significantly suppressed by 8 bromo-cAMP in the serum-free complete medium
on collagen gel matrix and also in the serum-containing medium. Thus, among the conditions examined, the most suitable primary
culture media for steroidogenesis of thecal cells were the serum-free media, especially serum-free complete medium on collagen
gel matrix. 相似文献
11.
Morphological and functional differentiation of human thyroid cells in collagen gel culture 总被引:1,自引:0,他引:1
C Thomas-Morvan B Caillou M Schlumberger P Fragu 《Biology of the cell / under the auspices of the European Cell Biology Organization》1988,62(3):247-254
In order to study the expression of the morphological and functional characteristics of human thyroid cells, 3-dimensional cultures were carried out in collagen gel. This substrate allows the cells to retain their organization in follicles with a normal polarity. Cellular polarities appeared normal at the time of collagen embedding, but there was a delay of 4-5 days in culture before the maximal TSH stimulation of 125I- uptake and of cAMP accumulation occurred. In normal and adenoma-derived cells, 125I- uptake, which could be increased by TSH, was demonstrated. cAMP accumulated in the culture medium and thyroglobulin was secreted into the follicle lumen. Of the 4 differentiated carcinomas for which the 72-hr uptake of 125I- was measured, only 2 displayed slight 125I- uptake and response to TSH. Thus, human thyroid cells exhibit better morphological and functional differentiation in collagen gel culture than in monolayer culture. Furthermore, in a variety of pathological cases studied, the expression of specific characteristics in culture varied in a fashion similar to differences observed in vivo. 相似文献
12.
H Sugihara N Yonemitsu S Toda S Miyabara S Funatsumaru T Matsumoto 《Journal of lipid research》1988,29(5):691-697
Three-dimensional culture with collagen gel, developed recently for the in vitro study of some mammalian cells in a more physiological condition than a monolayer culture, was applied for a biological study of unilocular fat cells. Successfully embedded in the gel, the unilocular fat cells were shown to be able to keep their cellular function and actively proliferate. These findings confirm that unilocular fat cells do undergo proliferation under in vitro conditions as demonstrated in monolayer culture. 相似文献
13.
硝普钠促肺成纤维细胞增殖和凋亡的作用 总被引:4,自引:1,他引:4
用MTT测定、琼脂糖凝胶电泳和流式细胞术等方法,观察了一氧化氮供体硝普钠(sodium nitroprusside,SNP)对体外培养的肺成纤维细胞增殖和凋亡以及Bcl-2、Bax和p53蛋白含量的影响。结果发现:MTT吸光度、细胞数和增殖指数(proliferation index,PI)均较对照增加;凋亡细胞数也增加,但尚不足以出现明显的“梯形”凋亡电泳条带;同时,细胞内Bcl-2蛋白下调和Bax蛋白上调;而细胞内p53蛋白含量无明显变化。结果表明,外源性NO有增强肺成纤维细胞增殖和凋亡的双重作用,但以促细胞增殖为主;此作用的分子机制与Bcl-2蛋白下调和Bax蛋白上调有关。 相似文献
14.
A possible strategy for creating three-dimensional (3D) tissue-engineered organs in vitro with similar volumes to the primary organs is to develop a capillary network throughout the constructs to provide sufficient oxygenation and nutrition to the cells composing them. Here, we propose a novel approach for the creation of a capillary-like network in vitro, based on the spontaneous tube-forming activity of vascular endothelial cells (ECs) in collagen gel. We fabricated a linear tube of 500 microm in diameter, the inner surface of which was filled with bovine carotid artery vascular endothelial cells (BECs), in type I collagen gel as a starting point for the formation of a capillary-like network. The BECs exposed to a medium containing vascular endothelial growth factor (VEGF) migrated into the ambient gel around the tube. After 2 weeks of VEGF exposure, the distance of the migration into the ambient gel in the radial direction of the tube reached approximately 800 microm. Cross-sections of capillary-like structures composed of the migrating BECs, with a lumen-like interior space, were observed in slices of the gel around the tube stained with hematoxylin-eosin (H&E). These results demonstrate that this approach using a pre-established tube, which is composed of ECs, as a starting point for a self-developing capillary-like network is potentially useful for constructing 3D organs in vitro. 相似文献
15.
Contraction of fibroblast-containing collagen gels: Initial collagen concentration regulates the degree of contraction and cell survival 总被引:7,自引:0,他引:7
Zhu YK Umino T Liu XD Wang HJ Romberger DJ Spurzem JR Rennard SI 《In vitro cellular & developmental biology. Animal》2001,37(1):10-16
Remodeling of extracellular matrix involves a number of steps including the recruitment, accumulation, and eventual apoptosis of parenchymal cells as well as the production, organization, and rearrangement of extracellular matrix produced by these cells. The culture of fibroblasts in three-dimensional gels made of type I collagen has been used as a model of tissue contraction which characterizes both wound repair and fibrosis. The current study was designed to determine the effect of initial collagen concentration on the ability of fibroblasts to contract collagen gels and on cell survival. Native type I collagen was extracted from rat tail tendons and used to prepare collagen gels with varying collagen concentrations (0.75-2.0 mg/ml). Human lung fibroblasts (HFL-1) were cast into the gels and cultured in Dulbecco modified Eagle medium with 0.1% fetal calf serum for 2 wk. The gel size, collagen content, and deoxyribonucleic acid (DNA) content were determined. Gels prepared with an initial concentration of 0.75 mg/ml contracted more rapidly and to a smaller final size than gels prepared from 2 mg/ml initial collagen concentration (final size 7.1 versus 36.4% of initial size, P < 0.01). There was no significant degradation of the collagen in the gels under either condition. Hence, the dramatically increased contraction of the lower density gels resulted in a higher final density (P < 0.01). Cell density was estimated from DNA content. In low initial density gels, the final DNA content was significantly less than that in higher initial density gels (0.73 versus 1.88 microg/gel, P < 0.05). This was accompanied by an increased percentage of apoptotic cells at day 14 (43.3 versus 34.1%, P < 0.05). If the gels were maintained in the attached state which largely prevents contraction, apoptosis was significantly reduced, suggesting that contraction rather than matrix composition was a requirement for the increased apoptosis. In summary, these findings indicate that the initial matrix composition can lead to differing outcomes during fibroblast-mediated wound contraction. 相似文献
16.
Erkki Tarpila Reza M. Ghassemifar Lennart E. Franzén 《In vitro cellular & developmental biology. Animal》1998,34(8):640-645
Summary In this study we assessed the behavior of fibroblasts during contraction of collagen lattices. We applied a new technique
for three-dimensional time-lapse studies of movements of living cells using phase-contrast laser scanning microscopy. Five
anchored and five floating collagen lattices were studied regarding the activity of cells during a 7-h period of active contraction.
Three-dimensional reconstructions of the fibroblasts and their extensions were made from datasets of 16–26 “optical sections”
5 μm apart recorded hourly during the period of measurements. The distance between fibroblast nuclei in the floating lattices
decreased by a mean of 6.8 μm, but remained constant in the anchored group. Only minor variations were found in the angle
between a line connecting any two nuclei and the tangent of the lattice margin. The lengths of the cellular extensions continuously
changed by shortening and extending, and an increasing number of intercellular contacts were established with time. The angle
between the extensions and the periphery of the lattice varied continually, and no distinct pattern of arrangement of the
extensions was seen. In conclusion, we have shown in living cells in vitro that fibroblasts do not appear to move around within lattices during contraction but rather send out and withdraw cellular
extensions continuously. This speaks against cellular locomotion or movement as a main feature of contraction. Time-lapse
scanning laser microscopy has also been shown to be a suitable method to study cellular behavior quantitatively in three dimensions
during lattice contraction. 相似文献
17.
A balance is maintained between matrix synthesis and degradation, and a prolonged increase in matrix metalloproteinases (MMPs) affects healing. Photobiomodulation (PBM) speeds up healing and alters wound environment. The study aimed to determine changes in protein and gene expression of collagen type 1 (Col‐I), MMP‐3 and ‐9 and TIMP‐1 in fibroblasts irradiated at 660 or 830 nm. Commercially purchased human skin fibroblast cells were modeled into five groups namely, normal, normal wounded, diabetic wounded, hypoxic wounded and diabetic hypoxic wounded. Control cells were sham irradiated. Laser irradiation was conducted at 660 or 830 nm (108/or 94 mW, 9.1 cm2, 420/or 483 s) with 5 J/cm2. Forty‐eight hours post‐irradiation, protein expression of TIMP‐1, MMP‐3, ?9 and Col‐I was determined by flow cytometry and immunofluorescence, and gene expression by real‐time RT‐PCR. There was an increase in TIMP‐1 and Col‐I, and a decrease in MMP‐3 and ‐9, as well as an alteration in mRNA expression of MMP3, MMP9, TIMP1 and COL1A1 in irradiated cells. Due to the responsiveness of the diabetic hypoxic wounded model, the findings propose this model as appropriate for wound healing studies and suggest that PBM promotes the remodeling phase of wound healing by decreasing matrix degradation and upregulating synthesis. 相似文献
18.
Takata M Maniwa Y Doi T Tanaka Y Okada K Nishio W Ohbayashi C Yoshimura M Hayashi Y Okita Y 《Cell communication & adhesion》2007,14(4):157-167
Although various methods for collagen gel-based cell invasion assays have been described, there continues to be a need for a simpler and more objective assay. Here, we describe an easy-to-prepare double-layered collagen gel hemisphere (DL-CGH) system that satisfies these requirements, and we demonstrate the advantages of this new system for visualizing cell movements during invasion. DL-CGH consists of a central core collagen layer surrounded by an outer cover collagen layer. A droplet of collagen I solution (containing cells to be examined) naturally forms a small hemisphere on the bottom of the culture dish. After this central core layer gels, a second droplet is placed atop the first gel, encapsulating it completely. The hemisphere is submerged in the medium and cultured. The invasive activity of cells that infiltrate from the inner to the outer layer can be evaluated optically. Using this in vitro system, we measured the inhibitory effect of E-cadherin expression on cancer cell invasion. DL-CGH also allowed visualization of interactions between invading cancer cells and the stroma. Cancer cells, which lack the proteases required for direct entrance into the three-dimensional collagen matrix, were seen to slip like amoebas through matrix gaps generated by the pericellular proteolytic activity of fibroblasts. [Supplementary materials are available for this article. Go to the publisher's online edition of Cell Communication and Adhesion for the following free supplemental resources: Movies 1-3; 4a and b]. 相似文献
19.
Regulation of cell invasion and morphogenesis in a three-dimensional type I collagen matrix by membrane-type matrix metalloproteinases 1, 2, and 3 总被引:20,自引:0,他引:20
During tissue-invasive events, migrating cells penetrate type I collagen-rich interstitial tissues by mobilizing undefined proteolytic enzymes. To screen for members of the matrix metalloproteinase (MMP) family that mediate collagen-invasive activity, an in vitro model system was developed wherein MDCK cells were stably transfected to overexpress each of ten different MMPs that have been linked to matrix remodeling states. MDCK cells were then stimulated with scatter factor/hepatocyte growth factor (SF/HGF) to initiate invasion and tubulogenesis atop either type I collagen or interstitial stroma to determine the ability of MMPs to accelerate, modify, or disrupt morphogenic responses. Neither secreted collagenases (MMP-1 and MMP-13), gelatinases (gelatinase A or B), stromelysins (MMP-3 and MMP-11), or matrilysin (MMP-7) affected SF/HGF-induced responses. By contrast, the membrane-anchored metalloproteinases, membrane-type 1 MMP, membrane-type 2 MMP, and membrane-type 3 MMP (MT1-, MT2-, and MT3-MMP) each modified the morphogenic program. Of the three MT-MMPs tested, only MT1-MMP and MT2-MMP were able to directly confer invasion-incompetent cells with the ability to penetrate type I collagen matrices. MT-MMP-dependent invasion proceeded independently of proMMP-2 activation, but required the enzymes to be membrane-anchored to the cell surface. These findings demonstrate that MT-MMP-expressing cells can penetrate and remodel type I collagen-rich tissues by using membrane-anchored metalloproteinases as pericellular collagenases. 相似文献
20.
Influence of serum on adult and fetal dermal fibroblast migration adhesion, and collagen expression 总被引:1,自引:0,他引:1
Brink HE Stalling SS Nicoll SB 《In vitro cellular & developmental biology. Animal》2005,41(8-9):252-257
Summary The wound healing response to injury can be affected by many factors such as cell migration and extracellular matrix elaboration.
The objective of this study was to examine the serum- and age-dependent effects on cell migration, adhesion, and collagen
expression by skin fibroblasts. Dermal fibroblasts were isolated and plated with and without serum for up to 7 d. Cell migration
was determined by quantitative image analysis, adhesion was quantified using a centrifugation assay, and collagen expression
was assessed by PCR and immunohistochemical staining. Both adult and fetal fibroblasts migrated significantly faster in serum-containing
medium compared to serum-free medium. There was no significant difference in migration between the two cell types in either
serum-containing or serum-free medium. There was no significant difference in adhesion in the presence of serum, although
there was a greater faction of adherent fetal skin fibroblasts than adult fibroblasts in serum-free medium. Moreover, the
adherent fraction of fetal fibroblasts in serum-free medium was not significantly different from that in serum-containing
medium, suggesting that fetal skin fibroblasts possess serum-independent adhesion properties. Collagen mRNA expression was
significantly up-regulated in serum-free compared to serum-containing medium for both cell types. With respect to collagen
immunohistochemistry, both dermal fibroblast populations exhibited greater type I collagen compared to type III collagen staining.
Quantitative assessment of collagen staining indicated significantly enhanced type I collagen secretion in the presence of
serum by fetal skin fibroblasts. These findings suggest that intrinsic cellular characteristics may govern the observed differences
in adult and fetal wound healing. 相似文献