PURPOSE: The purpose of this study was to determine whether increasing the Ti6Al4V surface oxide negative charge through heat (600degrees C) or radiofrequency plasma glow discharge (RFGD) pretreatment, with or without a subsequent coating with fibronectin, stimulated osteoblast gene marker expression in the MC3T3 osteoprogenitor cell line. METHODS: Quantitative real-time polymerase chain reaction was used to measure changes over time in the mRNA levels for osteoblast gene markers, including alkaline phosphatase, bone sialoprotein, collagen type I (alpha1), osteocalcin, osteopontin and parathyroid hormone-related peptide (PTH-rP), and the osteoblast precursor genes Runx2 and osterix. RESULTS: Osteoprogenitors began to differentiate earlier on disks that were pretreated with heat or RFGD. The pretreatments increased gene marker expression in the absence of a fibronectin coating. However, pretreatments increased osteoblast gene expression for fibronectin-coated disks more than uncoated disks, suggesting a surface oxide-mediated specific enhancement of fibronectin's bioactivity. Heat pretreatment had greater effects on the mRNA expression of genes for PTH-rP, alkaline phosphatase and osteocalcin while RFGD pretreatment had greater effects on osteopontin and bone sialoprotein gene expression. CONCLUSIONS: The results suggest that heat and RFGD pretreatments of the Ti6Al4V surface oxide stimulated osteoblast differentiation through an enhancement of (a) coated fibronectin's bioactivity and (b) the bioactivities of other serum or matrix proteins. The quantitative differences in the effects of the two pretreatments on osteoblast gene marker expression may have arisen from the unique physico-chemical characteristics of each resultant oxide surface. Therefore, engineering the Ti6Al4V surface oxide to become more negatively charged can be used to accelerate osteoblast differentiation through fibronectin-dependent and independent mechanisms.
Alkaline Phosphatase ; Alloys ; Cell Differentiation ; Cell Line ; Collagen Type I ; Dental Implants ; Fees and Charges ; Fibronectins ; Gene Expression ; Hot Temperature ; Integrin alpha5beta1 ; Integrin-Binding Sialoprotein ; Osteoblasts ; Osteocalcin ; Osteopontin ; Parathyroid Hormone-Related Protein ; Plasma ; Proteins ; Real-Time Polymerase Chain Reaction ; RNA, Messenger ; Titanium

We made fusion protein of fastatin and FIII 9-10, termed tetra-cell adhesion molecule (T-CAM) that can interact simultaneously with alphavbeta3 and alpha5beta1 integrins, both playing important roles in tumor angiogenesis. T-CAM can serve as a cell adhesion substrate mediating adhesion and migration of endothelial cells in alphavbeta3 and alpha5beta1 integrin-dependent manner. T-CAM showed pronounced anti-angiogenic activities such as inhibition of endothelial cell tube formation, endothelial cell proliferation, and induction of endothelial cell apoptosis. T-CAM also inhibited angiogenesis and tumor growth in mouse xenograft model. The anti-angiogenic and anti-tumoral activity of molecule like fastatin could be improved by fusing it with integrin-recognizing cell adhesion domain from other distinct proteins. The strategy of combining two distinct anti-angiogenic molecules or cell adhesion domains could facilitate designing improved anticancer agent of therapeutic value.
Angiogenesis Inhibitors/chemistry/*pharmacology ; Animals ; Antineoplastic Agents/chemistry/*pharmacology ; Base Sequence ; Benzocaine/chemistry/*pharmacology ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Cells, Cultured ; Chloramphenicol/chemistry/*pharmacology ; DNA Primers ; Drug Combinations ; Factor VIII/chemistry/*pharmacology ; Humans ; Integrin alpha5beta1/*physiology ; Integrin alphaVbeta3/*physiology ; Male ; Mice ; Mice, Inbred BALB C ; Nitrofurazone/chemistry/*pharmacology ; Recombinant Fusion Proteins/chemistry/*pharmacology

Anodic spark deposition method(ASD) surface treated titanium implant possesses a considerable osteoconductive potential that promoting a high level of implant osseointegration in normal bone. The purpose of this study was to observe the ASD implant's osseointegration in the osteoporosis-induced animal model. Twenty four rats, 10 weeks of age, were ovarectomized and 5 weeks later divided into two groups : ASD implant group and control implant group. Titanium screw implants (diameter; 2.0 mm, length, 3.5 mm; pitch-height, 0.4 mm) were designed for this study. Experimental implants were ASD treated and no treatment on control implants. ASD implants and control implants were placed in to left tibiae of rats. The rats were sacrificed at different time interval(1, 2, 4 and 8 weeks after implantation) for histopathologic observation and immunohisto -chemistrical observation, with collagen type I, fibronectin, integrin alpha2beta1 and integrin alpha5beta1 antibodies. The results obtained from this study were as follow: 1. Histopathologic findings, overall tissue response and the pattern of bone formation in both groups were similar. In ASD group, more newly formed bone was seen at 1 week and 2weeks than control group. 2. The levels of type I collagen and fibronectin expression were the most abundant at 2weeks and decreased gradually in both groups. Fibronectin and type I collagen expression in ASD group were stronger than control group but no significance. 3. The levels of integrin alpha2beta1 and Integrin alpha5beta1 expression were most abundant at 2 weeks and decreased gradually in both groups. No significant difference was observed in both groups. From this results, anodic oxidized titanium implants were more advantages in early stage of bone formation than control group, but have no significance in tissue responses and late bone formations. It could be stated that although anodic oxidized titanium implant possesses considerable osteoconductive potential but in osteoporotic bone condition dental implant procedure should performed after improving or treating the osteoporotic bone condition.
Animals ; Antibodies ; Collagen Type I ; Dental Implants ; Fibronectins ; Implants, Experimental ; Integrin alpha2beta1 ; Integrin alpha5beta1 ; Models, Animal ; Osseointegration ; Osteogenesis ; Osteoporosis ; Rats ; Tibia ; Titanium

OBJECTIVETo investigate the molecular mechanism of epidermal growth factor (EGF) signal pathway on the expression of integrin alpha5 beta1 in prostate cancer cell line DU145.

METHODSUsing flow-cytometry, the effects of EGF and the mitogen-activated protein kinase (MAPK) signal pathway inhibitor PD98059 on the expression of integrin alpha5 and beta1 subunits on DU145 cell surface were analyzed. RT-PCR and Western blot methods were used to examined the expression of mRNA and cell total protein of integrin alpha5 and beta1 subunits. And the metastatic phenotypes in DU145 cell were investigated.

RESULTSThe expression levels of integrin alpha5 beta1, which was the receptor for fibronectin, were changed. EGF up-regulated the protein and mRNA expression of beta1 subunit on DU145 cell surface, 231% and 248% (P < 0.01) compared to the control respectively, and it could significantly promote the ability of DU145 cell adhesion to fibronectin and migration. However PD98058, which was the inhibitor of MAPK signal pathway, down-regulated the protein and mRNA expression of beta1 subunit, 60% and 63% (P < 0.01) compared to the control respectively, and it had the contrary function on the adhesion and migration ability of DU145 cell. But both had no effect the expression of alpha5 subunit.

CONCLUSIONSEGF might promote the metastatic ability mainly by up-regulating the expression of beta1 subunit by activating MAPK signal pathway in DU145 cells. Their regulation effects are on the mRNA transcriptional level.

Cell Adhesion ; drug effects ; Cell Line, Tumor ; Epidermal Growth Factor ; pharmacology ; Flavonoids ; pharmacology ; Humans ; Integrin alpha5beta1 ; biosynthesis ; genetics ; Male ; Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; Prostatic Neoplasms ; metabolism ; pathology ; RNA, Messenger ; genetics ; Receptors, Fibronectin ; biosynthesis ; genetics ; Signal Transduction ; drug effects ; Up-Regulation ; drug effects

OBJECTIVETo investigate the relationship between the expressions of integrin alpha5 beta1 and E-CD, and clinicopathological characteristics and prognosis of patients with non-small cell lung carcinoma (NSCLC).

METHODSThe expression of integrin alpha5 beta1 and E-CD were analyzed in 53 NSCLC and 12 control specimens by immunohistochemical assay.

RESULTSThe expression of integrin alpha5 beta1 was significantly higher in NSCLC (58.5%) than that in normal lung tissue (16.7%), and also positively related with pathological characteristics (P = 0.021), lymph node metastasis (P = 0.006), and clinical stage (P = 0.002). The 3-year survival rate in NSCLC group was significantly lower than that in control group (22.3% vs 40.6% , P = 0.041). The positive expression of E-CD in NSCLC and control group was 32.1% and 91.7%, respectively, and negatively correlated with pathological characteristics (P = 0.010) and lymph node metastasis (P = 0.002). The 3-year survival rate in control group was 19.9%, lower than that in NSCLC group (41.2%, P > 0.05), but the difference is not significant.

CONCLUSIONThe overexpression of integrin alpha5 beta1 may contribute to lymph node metastasis and play an inverse role, while E-CD may be a beneficial prognostic factor in patients with NSCLC.

Adult ; Aged ; Cadherins ; metabolism ; Carcinoma, Non-Small-Cell Lung ; metabolism ; pathology ; Female ; Humans ; Immunohistochemistry ; Integrin alpha5beta1 ; metabolism ; Lung Neoplasms ; metabolism ; pathology ; Lymphatic Metastasis ; Male ; Middle Aged ; Neoplasm Staging ; Smoking ; Survival Analysis

OBJECTIVETo explore the correlation between the expression of integrin subunits alpha5 and beta1 in prostate cancer (PCa) and its clinicopathological data including tumor grade and clinical stage.

METHODSExpressions of integrin subunits alpha5 and beta1 were examined in 30 cases of PCa and 30 cases of normal prostatic tissues by immunohistochemical assay.

RESULTSExpressions of integrin subunits alpha5 and beta1 in PCa were lower than those in normal prostatic tissues (P <0.05) respectively.

CONCLUSIONCompared with normal prostatic tissues, expressions of integrin subunits alpha5 and beta1 in PCa were rather weaker or even faded. Expressions of integrin subunits alpha5 and beta1 revealed an positive correlation with tumor's Gleason grade and negative with clinical TNM stage. The results indicate that integrin subunits alpha5 and beta1 have potential values in the diagnosis and are predictable indices in the proliferation of PCa.

Aged ; Aged, 80 and over ; Animals ; Humans ; Integrin alpha5 ; biosynthesis ; Integrin alpha5beta1 ; biosynthesis ; Male ; Mice ; Middle Aged ; Neoplasm Staging ; Prostatic Hyperplasia ; metabolism ; Prostatic Neoplasms ; metabolism ; pathology

PURPOSE: We investigated the effects of recombinant 9-10th type III repeat of fibronectin (rhFNIII9-10) on the adhesion, proliferation, and the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells(hMSCs). MATERIALS AND METHODS: Adhesion and blocking assay for hMSCs were performed on the plates which had been coated with 100 microgram/ml rhFNIII9-10 or fibronectin. hMSCs seeded on the precoated plates were cultured in the osteogenic media for 3 weeks. MTS(Dimethylthiazole carboxymethoxyphenyl sulfophenyl tetrazolium compound) assay for the cell number, [Methyl-3H] thymidine incorporation study, alkaline phosphatase activity assay, calcium content assay and RT-PCR for alkaline phosphatase, osteopontin, cbfa-1, and type I collagen were performed during the osteogenic differentiation. RESULTS: hMSCs showed significantly increased adhesion to rhFNIII9-10-coated plates and fibronectin-coated plates. A monoclonal antibody to the integrin alpha 5 beta 1 inhibited adhesion to rhFNIII9-10-coated plates and fibronectin-coated plates in dose-dependent manner. hMSCs seeded on the rhFNIII9-10-coated plates showed increased proliferation during the osteogenic differentiation. However, there was no significant difference in the alkaline phosphatase activity, calcium content and expression levels of mRNAs for alkaline phosphatase, osteopontin, cbfa-1, and type I collagen of hMSCs seeded on the rhFNIII9-10-coated plates. CONCLUSION: rhFNIII9-10 stimulates hMSCs adhesion and increases hMSCs proliferation during the osteogenic differentiation. Although osteogenic differentiation is not promoted, adsorption of rhFNIII9-10 onto appropriate biomaterials can enhance integrin-mediated hMSCs adhesion and proliferation. This biomolecular engineering strategy represents a robust approach to increase biofunctional activity and integrin specificity of hMSCs.
Adsorption ; Alkaline Phosphatase ; Biocompatible Materials ; Bone Marrow ; Calcium ; Cell Count ; Collagen Type I ; Fibronectins* ; Humans* ; Integrin alpha5beta1 ; Mesenchymal Stromal Cells* ; Osteopontin ; RNA, Messenger ; Sensitivity and Specificity ; Thymidine

OBJECTIVETo study the expression of alpha5beta1 integrin in the abnormal scars and its role and significance in the formation and development of abnormal scars.

METHODSThe expression of alpha5beta1 integrin was observed in hypertrophic scar (15 samples), keloid (15 samples) and normal skin (10 samples) with SP immunohistochemical method and colloidal gold immuno-electron microscopic technique. The data were semi-quantitatively analyzed.

RESULTSThe expression levels of alpha5beta1 integrin in the fibroblasts of keloids and hypertrophic scars were higher than normal skin; the expression of alpha5beta1 integrin in the fibroblasts of keloids was higher than hypertrophic scars (P < 0.01).

CONCLUSIONThe alpha5beta1 integrin appears to have close relation to the formation and development of abnormal scars. To find a way to decrease the expression level of alpha5beta1 integrin in fibroblasts may be a new approach to inhibit scar hypertrophy.

Cicatrix ; metabolism ; pathology ; Humans ; Immunohistochemistry ; Integrin alpha5beta1 ; analysis ; metabolism ; Keloid ; metabolism ; pathology ; Microscopy, Immunoelectron ; Skin ; chemistry ; pathology ; ultrastructure

We assessed the cytokine combinations that are best for ex vivo expansion of cord blood (CB) and the increment for cell numbers of nucleated cells, as well as stem cells expressing homing receptors, by an ex vivo expansion of cryopreserved and unselected CB. Frozen leukocyte concentrates (LC) from CB were thawed and cultured at a concentration of 1x10(5)/mL in media supplemented with a combination of SCF (20 ng/mL)+TPO (50 ng/mL)+FL (50 ng/mL)+/-IL-6 (20 ng/mL)+/-G-CSF (20 ng/mL). After culturing for 14 days, the expansion folds of cell numbers were as follows: TNC 22.3+/-7.8~26.3+/-4.9, CFU-GM 4.7+/-5.1~11.7+/-2.6, CD34+CD38- cell 214.0+/-251.9~464.1+/-566.1, CD34+CXCR4+ cell 4384.5+/-1664.7~7087.2+/-4669.3, CD34+VLA4+ cell 1444.3+/-1264.0~2074.9+/-1537.0, CD34+VLA5+ cell 86.2+/-50.9~ 113.2+/-57.1. These results revealed that the number of stem cells expressing homing receptors could be increased by an ex vivo expansion of cryopreserved and unselected CB using 3 cytokines (SCF, TPO, FL) only. Further in vivo studies regarding the engraftment after expansion of the nucleated cells, as well as the stem cells expressing homing receptors will be required.
ADP-ribosyl Cyclase/metabolism ; Antigens, CD/metabolism ; Antigens, CD34/metabolism ; Cryopreservation ; Fetal Blood/*cytology ; Humans ; Integrin alpha4beta1/metabolism ; Integrin alpha5beta1/metabolism ; Membrane Proteins ; Receptors, CXCR4/metabolism ; Receptors, Lymphocyte Homing/*metabolism ; Research Support, Non-U.S. Gov't ; Stem Cell Factor ; Stem Cells/*cytology/*metabolism ; Thrombopoietin

Ullrich's disease is a congenital muscular dystrophy clinically characterized by generalized muscle weakness, multiple contractures of the proximal joints, and hyperextensibility of the distal joints. All the patients develop rigidity of spine, often assoicated with scoliosis, failure to thrive, and early and severe respiratory involvement, irrespective of their levels of motor function. Intellectual development is normal. The biopsied muscles show dystrophies including remarkable variation in the fiber size, notably proliferated endomysial connective tissues, and a lot of degenerated and regenerated fibers. The expression of merosin and dytrophin is normal. Recent studies have demonstrated that collagen VI is deficient in the muscles of the patients with Ullrich's disease, and some result from recessive mutations of the collagen VIalpha 2 gene(COL6A2). And a marked reduction of fibronectin receptors in the extracellular matrix of skin and cultured fibroblasts of these patients is also reported. These results suggest that collagen VI deficiency may lead to the reduction of fibronectin receptors and that any abnormalities of cell adhesion may be involved in the pathogenesis of the disease. A case of Ullrich's disease has not been reported yet in Korea. So, we describe a male patient with Ullrich's disease with a brief review of the literature.
Cell Adhesion ; Collagen ; Connective Tissue ; Contracture ; Extracellular Matrix ; Failure to Thrive ; Fibroblasts ; Humans ; Integrin alpha5beta1 ; Joints ; Korea ; Laminin ; Male ; Muscle Weakness ; Muscles ; Muscular Dystrophies ; Receptors, Fibronectin ; Scoliosis ; Skin ; Spine