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The crucial role of collagen-binding integrins in maintaining the mechanical properties of human scleral fibroblasts-seeded collagen matrix.

ABSTRACT: PURPOSE: The aim of this study was to identify the presence of collagen-binding integrin subunits in human scleral fibroblasts (HSFs) and investigate their actual functions in maintaining the mechanical creep properties of the HSFs-seeded collagen matrix. METHODS: Primary HSFs were cultured in vitro. Reverse- transcription PCR was used to detect mRNA expression of integrin ?1, ?2, and ?1 subunits in HSFs. In addition, western blot analysis and immunofluorescence were used to detect their protein in HSFs. Monoclonal antibodies were applied directly against the extracellular domains of integrin subunits in HSFs cultured in the three-dimensional collagen gels to block the interaction between HSFs and the extracellular collagen matrix. The effects of anti-integrin antibodies on HSFs morphology in collagen gel were observed. The effects of the added antibodies on fibroblast-mediated collagen gels' contraction were evaluated. Furthermore, the changes in mechanical creep properties of collagen gel were measured by a biomechanics test instrument. RESULTS: The mRNA and protein expressions of collagen-binding integrin ?1, ?2, and ?1 subunits were present in HSFs. The elongated bipolar cells converted to spherical shapes after 6 h after the addition of integrin ?1?1 and ?2?1 antibody. The blocking of integrin ?1?1 and ?2?1 subunits noticeably decreased the contraction in the collagen gels. In addition, all samples were subjected to a constantly applied load of 0.03 N for 600 s. The blocking of integrin ?1?1 and ?2?1 subunits also induced increases in the values of final extension, creep extension, and creep rate, compared to those of the controls (p<0.01). Furthermore, the creep elements were significantly increased with the augmentation of the integrin antibody dose (p<0.01). The final extension of the integrin ?2?1 antibody (1 ?g/ml or 4 ?g/ml) group was significantly higher compared to that of the integrin ?1?1 antibody (1 ?g/ml or 4 ?g/ml) group (p<0.01). However, the creep extension and creep rate of the integrin ?2?1 antibody (1 ?g/ml or 4 ?g/ml) group were not significantly different from those in the integrin ?1?1 antibody (1 ?g/ml or 4 ?g/ml) group (p>0.05). CONCLUSIONS: Our findings suggested that HSF integrin ?1?1 and ?2?1 participated in maintaining the mechanical creep properties of the HSFs-seeded collagen matrix. Furthermore, integrin ?2?1 might play a more crucial role in maintaining the mechanical creep properties of the collagen matrix than does integrin ?1?1.

SUBMITTER: Hu S

PROVIDER: S-EPMC3107998 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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The crucial role of collagen-binding integrins in maintaining the mechanical properties of human scleral fibroblasts-seeded collagen matrix.

Hu Shoulong S Cui Dongmei D Yang Xiao X Hu Jianmin J Wan Wenjuan W Zeng Junwen J

Molecular vision 20110520

<h4>Purpose</h4>The aim of this study was to identify the presence of collagen-binding integrin subunits in human scleral fibroblasts (HSFs) and investigate their actual functions in maintaining the mechanical creep properties of the HSFs-seeded collagen matrix.<h4>Methods</h4>Primary HSFs were cultured in vitro. Reverse- transcription PCR was used to detect mRNA expression of integrin α1, α2, and β1 subunits in HSFs. In addition, western blot analysis and immunofluorescence were used to detect ...[more]

PMID: 21647271

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Project description:The development of living heart valves that grow with the patient is a promising strategy for heart valve replacements in pediatric patients. Despite active research in the field of tissue engineered heart valves there have been limited efforts to optimize the balance between biodegradation of the scaffolds and de novo extracellular matrix (ECM) synthesis by cells and study their consequences on the mechanical properties of the cell-seeded construct. This study investigates the effect of in vitro degradation and ECM secretion on the mechanical properties of hybrid polyester scaffolds. The scaffolds were synthesized from blends of fast degrading polyglycerol sebacate (PGS) and slowly degrading polycaprolactone (PCL). PGS-PCL scaffolds were electrospun using a 2:1 ratio of PGS to PCL. Accelerated hydrolytic degradation in 0.1 mM sodium hydroxide revealed 2-fold faster degradation of PGS-PCL scaffolds compared with PCL scaffolds. Thermal analysis and scanning electron microscopy demonstrated marginal change in PCL scaffold properties, while PGS-PCL scaffolds showed preferential mass loss of PGS and thinning of the individual fibers during degradation. Consequently, the mechanical properties of PGS-PCL scaffolds decreased gradually with no significant change for PCL scaffolds during accelerated degradation. Valvular interstitial cells (VICs) seeded on PGS-PCL scaffolds showed higher ECM protein secretion compared with PCL. Thus the mechanical properties of the cell-seeded PCL scaffolds did not change significantly compared with acellular scaffolds, probably due to slower degradation and ECM deposition by VICs. In contrast, the PGS-PCL scaffolds exhibited a gradual decrease in the mechanical properties of the acellular scaffolds due to degradation, which was compensated for by new matrix secreted by VICs seeded on the scaffolds. Our study demonstrated that the faster degrading PGS component of PGS-PCL accelerated the degradation rate of the scaffolds. VICs, on the other hand, were able to remodel the synthetic scaffold, depositing new matrix proteins and maintaining the mechanical properties of the scaffolds.

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The crucial role of collagen-binding integrins in maintaining the mechanical properties of human scleral fibroblasts-seeded collagen matrix.

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The crucial role of collagen-binding integrins in maintaining the mechanical properties of human scleral fibroblasts-seeded collagen matrix.

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