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Early Osteogenic Differentiation of Mouse Preosteoblasts Induced by Collagen-Derived DGEA-Peptide

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rXXXX American Chemical SocietyA dx.doi.org/10.1021/bm1013475|Biomacromolecules XXXX, XXX, 000–000ARTICLEpubs.acs.org/BiomacEarly Osteogenic Differentiation of Mouse Preosteoblasts Inducedby Collagen-Derived DGEA-Peptide on Nanofibrous PhageTissue MatricesSo Young Yoo, Masae Kobayashi, Phin Peng Lee, and Seung-Wuk Lee*Bioengineering, University of California, Berkeley, Physical Biosciences Division, Lawrence Berkeley National Laboratory,Berkeley Nanoscience and Nanoengineering Institute, Berkeley, California 94720, United StatesbS Supporting InformationABSTRACT: Specific biochemical and physical cues in tissueextracellular matrices play a critical role in regulating cellulargrowth processes and their fate. We report initial responses ofbone stem cells induced by collagen-derived DGEA-peptideson nanofibrous M13 phage tissue matrices. We constructedgenetically engineered M13 phage with DGEA-peptide dis-played in high density on the major coat proteins and biomimeticnanofibrous tissue-like matrices in two and three dimensions.We investigated the effects of biochemical cues, specificallyDGEA-peptides on preosteoblast (MC3T3) morphologies. Thepreosteoblasts grown on the top of the DGEA-incorporatedphage matrices exhibited significant outgrown morphologywith early bone cell marker protein expression. Through solublepeptide competition assays and control experiments, we ver-ified that the observed cellular morphologies and osteogenicprotein m arker expression were specifically caused by theDGEA-peptides. We confirmed that the outgrown morphologies are linked with the early phase of osteogenic protein expressionthrough mRNA quantification and bone cell protein marker expression. Additionally, we demonstrated that the phage-based tissuematrix systems could work as a good cell culture platform to investigate the specificeffect of biochemical cues, which can be tunedprecisely at a single amino acid level with little change in other physical and chemical properties of the environment. Our studyadvances the understanding of osteogenic differentiation and our phage-based tissue matrices have the potential for future boneregeneration therapy and systemic investigation of specific cellular responses to biochemical ligand stimulation.1. INTRODUCTIONPrecisely designed active biomaterials with controlled struc-tural organization are of great interest in areas of regenerativemedicine, drug screening, and biosensors.1-7Many attemptshave been made to mimic natural tissue microenvironment invivo by using extracted extracellular matrix (ECM) or syntheticECM-mimetic macromolecules to manipulate the ECM’s biolog-ical, chemical, and mechanical properties.8-13The ECM ismainly composed of abundant nanomet er-scale fibrous proteinnetworks.14-17These nanofibrous structures provide cellularinstructive signals to regulate cell-matrix interactions by pre-senting biochemical ligands that interact specifically with cellularintegrins and other receptors to trigger cascading cellular signaltransduction processes18-20In addition, nanometer scale topo-graphy and textures presented by individual ECM molecules playan important role in supporting and controlling desired cellularfunctions and morphologies. For example, collagen 300 nm inlength and 1.5 nm in width21can form nanofibrils that extendfrom tens of nanometers to several micrometers in length anddiameter.22,23Together with these ECM nanofibrous structures,24,25neighboring proteins, various growth factors, and chemokinesprovide biochemical signaling for adhesion, migration, prolifera-tion, and differentiation.26-28Various ECM ligand peptidesequences have also been identified and demonstrated to reg-ulate the cellular functions. For example , RGD peptides fromfibr onectin induce focal adhesion through integrin binding;32DGEA from collagen type I induce bone cell differentiation;29,30IKVAV from laminin stimulates outgrowth of neurite;31,32and soon. Among these peptides, RGD has been the most well -character-ized to induce various cellular functions. However, collagen-derived DGEA-peptides have been relatively less investigated fortheir specific cellular re sponse compared to other ECM peptides,while collagen has bee n mostly applied for constructing tissueengineering scaffold.23,33-35In particular, collagen plays a criticalReceived: November 11, 2010Revised: January 22, 2011B dx.doi.org/10.1021/bm1013475 |Biomacromolecules XXXX, XXX, 000–000BiomacromoleculesARTICLErole in templating bone hard tissue formation in both cellular andcalcification processes.36If a biomaterial mimics the fibrousshape and chemical structure of collage n, it might be useful forengineering bone tissue. Direct incorporation of the various bio-chemical ligands into synthetic biomaterials or natural materialshas been used to design various tissue regenerating matrices.Although the resulting structures provide great promise to improvethe quality of materials to stimulate the cells, emulating complextissue microenvironments and precisely controlling biochemicaland physical cues using conventional approaches is still highlychallenging. Any modification of chemical structures requireslabor-intensive chemical synthesis and the resulting chemicalstructures affect other important physical and mechanical param-eters. A material that mimics the nanofibrous structure of theECM and whose biochemical structure can be convenientlymodified with little change in physical structure and assemblyprocesses will be very desirable for investigating various bio-chemical cues and their effects on cellular growth processes.Genetic engineering of phages provides novel opportunities tocreate functional nanomaterials for various applications in en-ergy, electronics, and biomedical engineering.37-42In particular,phage technology has recently been greatly expanded to biomed-ical applications including tissue eng ineering, drug delivery,43,44and bioimaging.45The M13 phage is a bacterial virus with atropism to infect only a specific bacterial strain; previous biomed-ical applications of phages showed that the phage is a safematerial that exhibits little inflammation at a targeted brain tissuesite, although its effect on other tissues have yet to be veri fied.46,47Endocytosed phages are also shown to degrade via the lysosomalpathway.48,49Recently, nanofibrous structu res of M13 phage have been usedafter chemical and genetic modification, with cell signaling pep-tides as novel


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