| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 5, 2003 as doi:10.1096/fj.02-0564fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
,2
Division of Bioscience, Graduate School of Environmental Earth Science, Hokkaido University, Sapporo 060-0810, Japan; and
Department of Anatomy, Kitasato University School of Medicine, Sagamihara 228-8555, Japan
2Correspondence: Laboratory of Bio-Material Chemistry, Division of Bioscience, Graduate School of Environmental Earth Science Hokkaido University, Kita 10 Nishi 5, Kita-ku, Sapporo 060-0810, Japan. E-mail: nomizu{at}ees.hokudai.ac.jp
SPECIFIC AIM
In this study, we designed laminin-1 derived peptide-conjugated chitosan membranes as a basement membrane mimetic and investigated their biological activities to show a potential use of the membrane as a bioadhesive/scaffold for tissue regeneration and cell engineering.
PRINCIPAL FINDINGS
1. Preparation of the peptide-conjugated chitosan membrane and their cell attachment activities with cell type specificity
Four biologically active peptides (A13: RQVFQVAYIIIKA, A99: AGTFALRGDNPQG, AG73: RKRLQVQLSIRT, and C16: KAFDITYVRLKF), previously identified from the adhesive protein laminin-1, were synthesized with a cysteine residue at the amino terminus. Chitosan, a polysaccharide, was mixed with N-(maleimidobenzoyloxy)-succinimide (MBS) and the obtained MB-chitosan was coated on tissue culture plates. The peptides were then added into the wells and covalently coupled to the MB-chitosan. The peptide-conjugated chitosan membranes showed cell attachment activity in a dose-dependent manner, whereas unmodified chitosan membranes were inactive. When > 1.0 nmol/well of peptides were reacted with the MB-chitosan membrane (10 mg/well), the number of attached cells did not increase. These results indicate that the peptides quantitatively react with MB groups on the chitosan membrane and are active for cell adhesion. The peptide-chitosan membranes promoted cell attachment and cell spreading of HT-1080 human fibrosarcoma cells and human foreskin fibroblasts in a cell-type specific manner. Fibroblasts strongly attached and spread on the A99-chtosan membrane whereas HT-1080 cells showed weak cell attachment. The AG73-, A13-, and C16-chitosan membranes were active with both cells.
2. Peptide-conjugated chitosan membrane enhances biological activities
Cell attachment activity of fibroblasts to the peptide-chitosan membranes was quantified. The peptide-chitosan membranes showed higher cell attachment activity at low doses compared with attachment to the peptide alone. The biological activities of laminin-1 peptides, not only their cell attachment activity but cell spreading and differentiation, were clearly enhanced after conjugating to chitosan membranes. Morphological differences of fibroblasts were observed with these peptide-chitosan membranes (Fig. 1
). These differences were not observed on peptide alone. The most dramatic differences have been observed between cell morphology on the A99- and the AG73-chitosan membranes (Fig. 1)
. Cell morphology on the A13- and C16-chitosan membranes was between that of A99 and AG73. We also found that the A99-chitosan membrane interacted with an integrin and the AG73-chitosan membrane promoted syndecan-mediated cell attachment. These data demonstrate that physiological interactions with the appropriate cellular receptors take place when the peptides are coupled to chitosan.
|
We tested neurite outgrowth activity using PC12 rat pheochromocytoma cells. We previously showed that the AG73 peptide promoted neurite outgrowth with PC12 cells. When the peptides (A13, A99, AG73, or C16) were conjugated on chitosan membranes, AG73- and A99-chitosan membranes strongly promoted neurite outgrowth with PC12 cells and the others showed weak activity (Fig. 2
). These data confirm that the chitosan membrane enhances the biological activity of the peptides.
|
CONCLUSIONS AND SIGNIFICANCE
Although the laminin-1-derived peptides have diverse biological activities, their application in the biomedical field has been limited due to their short half-life in the body. It would be useful to have stable peptides of long-lasting activity that directly affect cells at sites of injury. Chiotsan alone has been used as a dressing for wounds. Although chitosan was shown to adhere strongly to tissues in vivo, we found that chitosan membrane has no effect on cells in vitro. By conjugating laminin-1-derived synthetic peptides, we designed the peptide-chitosan membrane as a basement membrane mimetic for application to tissue engineering and have generated a chitosan-based material that has cell adhesion activity. Peptide-chitosan membranes promoted strong cell attachment with cell-type specificity, which activities are consistent with the known activities of the peptide. Moreover, biological activities were clearly enhanced after conjugating to the chitosan membrane. The enhancement of these biological activities is likely because the peptides bound to chitosan membrane are allowed to change their conformation more easily than those bound to plastic surface.
Two major possible uses are described in Fig. 3
. One is for a better understanding of the molecular interaction between ECM proteins and cellular receptors. Peptides coupled to chitosan are more active than peptide alone. A99-chitosan membrane promotes integrin-mediated cell adhesion with well-organized actin stress fiber formation, and AG73-chitosan membrane induced well-developed membrane ruffles via cell surface syndecans. We found that fibroblast cell morphology can be controlled by the amount of the peptides. Moreover, we found that a combination of peptides that recognize different receptors can regulate biological functions including cell attachment and morphology. The multifunctional chitosan membrane may serve as a basement membrane mimetic. This strategy will help to further our understanding of the molecular mechanism of cell-surface receptors, signaling, and morphologenesis.
|
Another possible application is as a bioadhesive for tissue engineering. The peptide-chitosan membranes have a number of advantages for biomedical uses. In wounds, cell migration and initial adhesion are crucial for survival and tissue organization. The presence of a scaffold might help cells to function during wound repair. The peptide-chitosan membranes can be prepares as a scaffold for cells in wounds. We found that these matrices can regulate and promote peptide activity depending on the cell types and cellular receptors. The peptide-chitosan membranes could also be prepared as a membrane sheet (M. Mochizuki and M. Nomizu, unpublished results). Using membranes with keratinocytes already attached may promote wound healing. We conclude that these peptide-conjugated chitosan membranes have a potential use for cell and tissue engineering and for therapeutic applications.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0564fje; to cite this article, use FASEB J. (March 5, 2003) 10.1096/fj.02-0564fje 
This article has been cited by other articles:
|
|
 |
|
  N. Suzuki, H. Nakatsuka, M. Mochizuki, N. Nishi, Y. Kadoya, A. Utani, S. Oishi, N. Fujii, H. K. Kleinman, and M. Nomizu Biological Activities of Homologous Loop Regions in the Laminin {alpha} Chain G Domains J. Biol. Chem., November 14, 2003; 278(46): 45697 - 45705. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
